Yu Guo PDF

©2012 by Guo Yu. All rights reserved.
HYDROTHERMAL LIQUEFACTION OF LOW-LIPID MICROALGAE TO

PRODUCE BIO-CRUDE OIL
BY
GUO YU
DISSERTATION
Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy in Agricultural and Biological Engineering
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2012
Urbana, Illinois
Doctoral Committee:
Professor Yuanhui Zhang, Chair

Assistant Professor Ted L. Funk
Assistant Professor Lance C. Schideman
Professor Hans P. Blaschek
ABSTRACT
Microalgae are considered as suitable feedstocks for next-generation biofuel production
because of their fast growth rates and prolific yields. Moreover, growing algae has less impact on
land-use for food production compared with grain and other lignocellulosic biomass. However,
current lipid-to-biodiesel technology primarily focuses on utilizing high-lipid algae, which
usually has lower biomass productivities than low-lipid algal strains. In addition, biodiesel
production from algae often requires drying of the algal biomass followed by solvent and/or
mechanical extraction, which are both energy intensive processes.
Hydrothermal liquefaction (HTL) has been demonstrated to be an effective technology to
produce bio-crude oil from different biowastes with high moisture content. Therefore, it is
suitable to convert algae into bio-crude oil. In this study, two fast-growing, low-lipid, high-
protein microalgal species, Chlorella pyrenoidosa and Spirulina platensis, were converted into
four products via HTL: bio-crude oil, aqueous product, gaseous product, and solid residue.
Effects of the operating parameters, including reaction temperature, retention time and
initial pressure on HTL product yields and bio-crude oil quality were investigated. Bio-crude oil
yield increased with temperature: it increased from 0.4% to 39.8% as temperature increased from
100°C to 320°C for Chlorella, and increased from 9.7% to 37.3% as temperature increased from
200°C to 300°C for Spirulina. Gaseous product yield, mainly consisting of CO2, also increased
with temperature, but the rate of increase was low at temperatures from 240°C to 300°C.
Aqueous product yield, which represents the water-soluble fraction of HTL products, first
increased and then slightly decreased as temperature increased. Higher reaction temperature
resulted in substantially lower yield of solid residue. Solid residue yields were both lower than 5%
for Chlorella and Spirulina at 300°C. At five temperature levels (200°C, 220°C, 240°C, 260°C,
and 280°C), bio-crude oil yield increased by prolonging retention time. The highest bio-crude oil
yield was obtained at 280°C with 120 minutes retention time. Initial pressure provided by
nitrogen had a negligible effect on HTL product yields. At 240°C and 280°C with 30 minutes
retention time, the effects of five metal catalysts and two alkaline catalysts on HTL of
microalgae were investigated. Effects of catalysts at 280°C were more prominent than at 240°C.
The highest increase of bio-crude oil yield by adding catalysts compared with uncatalyzed tests
was 10% at 280°C. Carbon deposition and mineral mixing was found on the surface of metal
catalysts after hydrothermal treatment.
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Carbon recovery (CR), nitrogen recovery (NR) and energy recovery (ER) in the bio-crude
oil fraction increased with reaction temperature and retention time. For instance, from 100°C to
300°C with 30 minutes retention time, CR of bio-crude oil produced from Chlorella increased
from 0.1% to 55.3%, NR increased from 0.4% to 23.4%, and ER increased from 0% to 64.1%.
Both carbon and nitrogen tended to preferentially accumulate in the bio-crude oil as temperature
and retention time increased, but the opposite was true for the solid residual product. The NR
value of the aqueous product also increased with reaction temperature and retention time. 65-70%
of nitrogen and 35-40% of carbon in the original material were converted into water-soluble
fraction when reaction temperature was higher than 220°C and retention time was longer than 10
minutes. At 240°C, with the presence of alkaline catalysts, the CR of bio-crude oil and aqueous
product both increased, but the increase of CR of bio-crude oil was mainly due to the increase of
its yield. At the same time, NR of bio-crude oil also increased with additions of alkaline catalysts.
At 280°C, CR of bio-crude oil increased with the addition of different catalysts. The addition of
metal catalysts could decrease the NR of the aqueous product. The energy consumption ratio
(ECR), decreased from 0.42 to 0.30 for Chlorella, and from 20.20 to 0.29 for Spirulina, as the
temperature increased from 200°C to 300°C, which implies that HTL becomes more
energetically favorable at higher temperatures. The fossil energy balance (FEB) value of
producing algae bio-crude oil via HTL is higher than that of corn ethanol, algae-biodiesel and
petroleum gasoline. The energy balance of algae bio-crude oil is comparable with lignocellulosic
ethanol in terms of similar FEB values.
The fraction of hydrocarbons, cyclic oxygenates and heterocyclic compounds in bio-crude
oil increased with reaction temperature. Some intermediates produced from protein hydrolysis,
which can be dissolved in water, degraded at higher temperatures due to their instabilities under
hydrothermal conditions. Fraction of amides and N&O heterocyclic compounds in bio-crude oil
increased as retention time was prolonged. At the same time, the fraction of organic acids
decreased. Additions of heterogeneous catalysts increased the fraction of hydrocarbons as well as
decreased the fraction of organic acids in bio-crude oil. The fraction of N&O heterocyclic
compounds in bio-crude oil was largely increased with additions of alkaline catalysts.
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A first-order kinetic model was established for microalgae decomposition under
hydrothermal conditions. The activation energy for Chlorella and Spirulina decomposition was
29.8 kJ∙mol-1 and 24.0 kJ∙mol-1, respectively. A simplified reaction pathway including
interactions among intermediates produced from protein, lipid and carbohydrates was proposed.
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To my mother, WEN-JU JI and my father, ZHEN-BANG YU
For their love, support and understanding
and
To my wife, YAN-JU CHEN
Who loves me, and whom I dearly love.
v
ACKNOWLEDGEMENTS
This dissertation would not have been possible without the help and support from many
people. I dedicate this page to the people who provided help and advice to my research and
helped me overcome all of the difficulties.
First, I would like to express my sincere appreciation to my advisor, Dr. Yuanhui Zhang for
his trust, encouragement, support and patience, without which the completion of this dissertation
would not have been possible. His brilliant ideas and optimistic attitude always encouraged and
helped me overcome difficulties in my research. I would like to thank Dr. Lance Schideman, Dr.
Ted Funk and Dr. Hans Blaschek for their valuable advice and for serving on my Doctoral
Committee. Appreciation is also extended to Dr. John Regalbuto for serving on my prelim exam
committee.
I would like to thank my fellow researchers in the Bioenvironmental Engineering group,
including Dr. Brian He, Dr. Rong Dong, Dr. Zhichao Wang, Dr. Yigang Sun, Bin Guo, Mitchell
J. Minarick, Steve Ford, Yan Zhou, Dr. Xufei Yang, Dr. Wei Yan, Dr. Hai Wu, Chi-Ting Kuo,
Peng Zhang, Wan-Ting Chen, Jixiang Zhang, Zhongzhong Zhang, Douglas Barker, Dr. Les
Christianson, and anyone else who has worked with me and provided valuable support.
I have enjoyed being a member of the Department of Agricultural and Biological
Engineering. The great atmosphere here benefits my research and makes my life joyful. I
enjoyed the discussions we had in the lunch room every day, with topics covering almost every
area in our life. This is a great department!
I deeply appreciate my parents, who support and understand me all the time. Their love
always encourages me and helps me to face and eventually to overcome the difficulties in my
life. Although I do not say I love you often in accordance with our Chinese culture and tradition,
I love and miss you every day. Finally, I would like to thank my wife, Yanju Chen, who
accompanies and listens to me all the time. She is a great soul mate and listener and makes my
life meaningful and colorful. I truly appreciate your endless love, support and encouragement
every day in my life.
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TABLE OF CONTENTS
LIST OF FIGURES ..................................................................................................................... xi
LIST OF TABLES ..................................................................................................................... xvi
LIST OF ABBREVIATIONS AND ACRONYMS ............................................................... xviii
CHAPTER 1. INTRODUCTION............................................................................................. 1
1.1 BACKGROUND ............................................................................................................................. 1
1.1.1 Feedstocks for biofuel production ........................................................................................... 3
1.1.2 Bio-crude oil produced from thermochemical conversion ...................................................... 5
1.2 ALGAE AS FEEDSTOCK FOR BIO-CRUDE OIL PRODUCTION ............................................. 7
1.2.1 Classification of algae ............................................................................................................. 8
1.2.2 Bottlenecks for producing biodiesel from microalgae ............................................................ 9
CHAPTER 2. LITERATURE REVIEW .............................................................................. 12
2.1 WATER PROPERTIES AT HIGH TEMPERATURE .................................................................. 12
2.2 DEVELOPMENT OF HTL ........................................................................................................... 14
2.3 HTL OF DIFFERENT BIOMASS INTO BIO-CRUDE OIL ......................................................... 16
2.3.1 Lignocellulosic biomass and biowaste .................................................................................. 16
2.3.2 Algae as feedstock for bio-crude oil production ................................................................... 20
2.4 REACTION MECHANISMS AND KINETICS OF HTL ............................................................. 24
2.4.1 Lipids/Fats ............................................................................................................................. 24
2.4.2 Proteins/Amino acids ............................................................................................................ 25
2.4.3 Carbohydrates and lignocellulose ......................................................................................... 27
2.5 MIMICKING MOTHER NATURE’S OIL FORMATION VIA HTL .......................................... 32
2.5.1 Formation of conventional petroleum ................................................................................... 32
2.5.2 Similarities between HTL and natural geochemical processes ............................................. 35
CHAPTER 3. OBJECTIVES ................................................................................................. 37
CHAPTER 4. EXPERIMENTAL DESIGNS AND PROCEDURES ................................. 39
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4.1 ALGAL FEEDSTOCKS ................................................................................................................ 39
4.2 HYDROTHERMAL LIQUEFACTION PROCEDURE ............................................................... 40
4.2.1 Reactor types and experiment procedure .............................................................................. 40
4.2.2 Recovery procedures of HTL products ................................................................................. 42
4.3 EXPERIMENTAL DESIGN ......................................................................................................... 44
4.3.1 Effect of temperature............................................................................................................. 44
4.3.2 Effect of retention time ......................................................................................................... 47
4.3.3 Effect of initial pressure ........................................................................................................ 48
4.3.4 Effects of catalysts/additives on HTL process ...................................................................... 48
4.3.5 Elemental balance of HTL process ....................................................................................... 52
4.3.6 Characterization of HTL products......................................................................................... 54
4.3.7 Study reaction kinetics and mechanism of HTL process ...................................................... 57
CHAPTER 5. SCREENING ALGAL SPECIES FOR HTL EXPERIMENTS................. 59
5.1 CHARACTERISTICS OF DIFFERENT ALGAL SPECIES ........................................................ 59
5.2 HTL RESULTS OF DIFFERENT ALGAE SPECIES ................................................................... 61
5.2.1 Bio-crude oil yield of different algae species ........................................................................ 61
5.2.2 Relationship between oil quality and feedstock characteristics ............................................ 62
CHAPTER 6. EFFECTS OF OPERATING CONDITIONS ON PRODUCT YIELDS

FROM HTL OF MICROALGAE ............................................................................................. 64
6.1 HTL OF CHLORELLA PYRENOIDOSA .................................................................................... 64
6.1.1 Replicates of experiments ..................................................................................................... 64
6.1.2 Effect of reaction temperature ............................................................................................... 65
6.1.4 Effect of initial pressure ........................................................................................................ 71
6.1.5 Effect of heating period ......................................................................................................... 74
6.2 HTL OF SPIRULINA PLATENSIS .............................................................................................. 76
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6.2.1 Effect of reaction temperature ............................................................................................... 76
CHAPTER 7. EFFECTS OF CATALYSTS ON HTL PRODUCT YIELDS .................... 84
7.1 CATALYST EFFECTS ON BIO-CRUDE OIL YIELD ................................................................ 85
7.1.1 Effects of catalysts on bio-crude oil yield of Chlorella ......................................................... 86
7.1.2 Effects of catalysts on bio-crude oil yield of Spirulina ......................................................... 88
7.2 CATALYST EFFECTS ON BOILING POINT DISTRIBUTION OF BIO-CRUDE OIL ............ 89
7.3 MORPHOLOGY CHANGE OF CATALYST DURING HTL PROCESS ................................... 93
CHAPTER 8. ELEMENTAL AND ENERGY BALANCE OF HTL OF LOW-LIPID

MICROALGAE ........................................................................................................................ 100
8.1 ELEMENTAL DISTRIBUTION OF HTL OF CHLORELLA .................................................... 102
8.1.1 Effects of reaction temperature on C and N recovery of HTL products from Chlorella ..... 102
8.1.2 Effects of retention time on C and N recovery of HTL products from Chlorella ............... 112
8.1.3 Effects of catalysts on C and N recovery of HTL products from Chlorella ........................ 115
8.2 ELEMENTAL DISTRIBUTION OF HTL OF SPIRULINA ....................................................... 119
8.2.1 Effects of reaction temperature on C and N recovery of HTL products from Spirulina ..... 119
8.2.2 Effects of retention time on C and N recovery of HTL products from Spirulina................ 123
8.2.3 Effects of catalysts on C and N recovery of HTL products from Spirulina ........................ 125
8.3 ENERGY BALANCE OF HTL OF MICROALGAE .................................................................. 127
8.3.1 Energy consumption ratio of HTL of microalgae ............................................................... 127
8.3.2 Comparison of energy balances of different biofuel productions ....................................... 133
CHAPTER 9. CHARACTERIZATION OF HTL PRODUCTS AND EXPLORATION

OF REACTION KINETICS AND MECHANISMS ............................................................. 136
9.1 EFFECT OF REACTION TEMPERATURE .............................................................................. 136
9.1.1 Bio-crude oil produced at different temperatures................................................................ 136
9.1.2 Aqueous product produced at different temperatures ......................................................... 142
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9.2 EFFECT OF RETENTION TIME ............................................................................................... 148
9.3 EFFECT OF CATALYSTS ......................................................................................................... 154
9.4 EXPLORATION OF HTL KINETICS AND MECHANISMS ................................................... 161
9.4.1 Kinetic model for HTL of microalgae................................................................................. 161
9.4.2 Reaction mechanism for HTL of microalgae ...................................................................... 165
CHAPTER 10. SUMMARY AND RECOMMENDATIONS .......................................... 168
10.1 SUMMARY AND FINDINGS .................................................................................................... 168
10.2 RECOMMENDATIONS AND FUTURE WORK ...................................................................... 172
10.2.1 Upgrading and refinery of bio-crude oil ............................................................................. 172
10.2.2 Characterization of HTL products....................................................................................... 173
10.2.3 Challenges for series HTL experimental design ................................................................. 173
10.2.4 Kinetic study of HTL .......................................................................................................... 174
10.2.5 Life cycle assessment (LCA) .............................................................................................. 174
REFERENCES .......................................................................................................................... 176
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LIST OF FIGURES
Figure 1-1 Share of total energy consumption and crude oil price in U.S. (EIA, 2010b) ........... 2
Figure 1-2 Biofuel shares of renewable energy consumption and total energy consumption in
U.S. (EIA, 2010b) ...................................................................................................... 3
Figure 1-3 Operating conditions (temperature and pressure) of thermochemical conversion

processes .................................................................................................................... 6
Figure 1-4 Schematic view of the tree of life based on rDNA sequence comparisons [adapted
from (Sogin et al., 1996)]........................................................................................... 8
Figure 1-5 Relationship of biomass productivities and lipid content for 30 microalgae strains.
................................................................................................................................. 10
Figure 1-6 General procedure of producing biodiesel from algae [adapted and modified from
(Demirbaş, 2008)] .................................................................................................... 11
Figure 2-1 Biomass conversion technologies and products ...................................................... 12
Figure 2-2 Pressure-temperature phase diagram of water [adapted from (Peterson et al., 2008)]
................................................................................................................................. 13
Figure 2-3 Hydrolysis of peptide bond [adapted from (Tissot & Welte, 1984)] ....................... 26
Figure 2-4 Origin and maturation of petroleum [adapted from (Hunt, 1996)] .......................... 32
Figure 4-1 Picture of the 4534 floor stand reactor..................................................................... 40
Figure 4-2 Picture of the 4593 floor stand reactor..................................................................... 42
Figure 4-3 Recovery procedures for products from a hydrothermal liquefaction ..................... 43
Figure 4-4 Profile of temperature and pressure during an HTL experiment ............................. 45
Figure 4-5 General structure of amino acids ............................................................................. 50
Figure 4-6 Environment-Enhancing-Energy (E2-Energy) technology roadmap ...................... 53
Figure 5-1 Bio-crude oil yield and lipid content of different algae and biowastes ................... 61
Figure 5-2 Solubility of raw oil in toluene: (a) vs. ash content; and (b) vs. crude protein content
in algae ..................................................................................................................... 62
Figure 6-1 Replicates of HTL of Chlorella obtained at 280°C, 30 min reaction time with 0.69
MPa initial pressure of nitrogen............................................................................... 65
xi
Figure 6-2 Effects of the reaction temperature on product yields with 30 min reaction time
(Chlorella) ................................................................................................................ 66
Figure 6-3 Effects of retention time on bio-crude oil yield (Chlorella) .................................... 68
Figure 6-4 Effects of retention time on solid residue yield (Chlorella)..................................... 69
Figure 6-5 Solubility of raw oil in toluene with different retention time and temperature
(Chlorella) ................................................................................................................ 70
Figure 6-6 Effects of the initial N2 pressure on the system maximum pressure and partial
pressure of gaseous product (Chlorella) .................................................................. 72
Figure 6-7 Effects of the initial N2 pressure on the HTL product yields at 280°C with 30 min
reaction time ............................................................................................................ 73
Figure 6-8 Effect of heating period on bio-crude oil yield from Chlorella ............................... 74
Figure 6-9 Effects of the reaction temperature on product yields with 30 min reaction time
(Spirulina) ................................................................................................................ 77
Figure 6-10 Comparison of HTL of Spirulina results using different reactors ........................... 80
Figure 6-11 Effects of retention time on bio-crude oil yield (Spirulina)..................................... 81
Figure 6-12 Effects of retention time on solid residue yield (Spirulina) ..................................... 82
Figure 6-13 Solubility of raw oil in toluene with different reaction time and temperature
(Spirulina) ................................................................................................................ 83
Figure 7-1 Catalyst effects on bio-crude oil yield from Chlorella at 240°C with 30 min
retention time ........................................................................................................... 86
Figure 7-2 Catalyst effects on bio-crude oil yield from Chlorella at 280°C with 30 min
retention time ........................................................................................................... 87
Figure 7-3 Catalyst effects on bio-crude oil yield from Spirulina at 240°C with 30min retention
time .......................................................................................................................... 88
Figure 7-4 Catalyst effects on bio-crude oil yield from Spirulina at 280°C with 30 min
retention time ........................................................................................................... 89
Figure 7-5 Comparison of BP distribution of bio-crude oil and shale oil ................................. 90
Figure 7-6 Catalyst effects on BP distribution of bio-crude oil produced at 240°C ................. 91
Figure 7-7 Catalyst effects on BP distribution of bio-crude oil produced at 280°C ................. 92
Figure 7-8 ESEM photograph of Chlorella ............................................................................... 93
xii
Figure 7-9 ESEM photograph of Spirulina................................................................................ 94
Figure 7-10 ESEM photograph of catalyst sample with EDAX spectra: (a) Pt/C without HTL
process; (b) Chlorella with Pt/C at 240°C with 30 min; (c) Chlorella with Pt/C at
280°C with 30 min; (d) EDAX spectra of representative spot on (b); (e) EDAX
spectra of representative spot on (c) ........................................................................ 95
Figure 7-11 ESEM photograph of catalyst sample with EDAX spectra: (a) Spirulina with
Pd/Al2O3 at 240°C with 30 min; (b) Spirulina with Pd/Al2O3 at 280°C with 30 min;
(c) EDAX spectra of representative spot on (a); (d) EDAX spectra of representative
spot on (b). ............................................................................................................... 96
Figure 7-12 ESEM photograph of catalyst sample with EDAX spectra: (a) Raney Ni without
HTL process; (b) Spirulina with Raney Ni at 280°C with 30 min; (c) EDAX spectra
of (a); (d) EDAX spectra of (b). .............................................................................. 98
Figure 8-1 CR in different phases vs. reaction temperatures with 30 min retention time
(Chlorella) .............................................................................................................. 103
Figure 8-2 CR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention
time (Chlorella) ...................................................................................................... 105
Figure 8-3 Van Krevelen diagram of raw oil and bio-crude oil (Chlorella)............................ 106
Figure 8-4 CR of CO and CH4 from Chlorella ........................................................................ 108
Figure 8-5 pH of post-HTL water (Chlorella) ......................................................................... 109
Figure 8-6 NR of raw oil and aqueous product vs. reaction temperature with 30 min retention
time (Chlorella) ...................................................................................................... 111
Figure 8-7 NR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention
time (Chlorella) ...................................................................................................... 112
Figure 8-8 CR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Chlorella) 113
Figure 8-9 NR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Chlorella) 114
Figure 8-10 CR of HTL product at 240°C with different catalysts (Chlorella) ........................ 116
Figure 8-11 CR of HTL product at 280°C with different catalysts (Chlorella) ........................ 117
Figure 8-12 NR of HTL product at 240°C with different catalysts (Chlorella) ........................ 118
Figure 8-13 NR of HTL product at 280°C with different catalysts (Chlorella) ........................ 119
Figure 8-14 CR in different phases vs. reaction temperatures with 30 min retention time
(Spirulina) .............................................................................................................. 120
xiii
Figure 8-15 CR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention
time (Spirulina) ...................................................................................................... 121
Figure 8-16 NR of raw oil and aqueous product vs. reaction temperature with 30 min retention
time (Spirulina) ...................................................................................................... 122
Figure 8-17 NR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention
time (Spirulina) ...................................................................................................... 123
Figure 8-18 CR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Spirulina). 124
Figure 8-19 NR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Spirulina) 125
Figure 8-20 CR of HTL product with different catalysts (Spirulina): (a) 240°C; and (b) 280°C
............................................................................................................................... 125
Figure 8-21 NR of HTL product with different catalysts (Spirulina): (a) 240°C; and (b) 280°C
............................................................................................................................... 126
Figure 8-22 Energy recovery (%) of bio-crude oil produced from Chlorella ........................... 130
Figure 8-23 ECR of HTL at different temperature: (a) Chlorella; (b) Spirulina ....................... 130
Figure 8-24 ECR of HTL vs. initial moisture content of feedstock (Chlorella) ........................ 132
Figure 8-25 Fossil energy balance of different biofuels ............................................................ 134
Figure 9-1 Total ion chromatogram of bio-crude oil (Chlorella, 200°C, 30 min)................... 137
Figure 9-2 Total ion chromatogram of bio-crude oil (Chlorella, 300°C, 30 min)................... 138
Figure 9-3 Temperature effect on relative percentages of major compound classes in bio-crude
oil ........................................................................................................................... 141
Figure 9-4 Total ion chromatogram of aqueous product at different temperature (Chlorella) 143
Figure 9-5 Effect of retention time on bio-crude oil characteristics (Chlorella, 240°C) ......... 148
Figure 9-6 Effect of retention time on bio-crude oil characteristics (Chlorella, 280°C) ......... 149
Figure 9-7 Effect of retention time on TIC of aqueous product at 240°C (Chlorella) ............ 150
Figure 9-8 Effect of retention time on relative concentrations of 5 compounds in aqueous

product (Chlorella, 240°C) .................................................................................... 151
Figure 9-9 Effect of retention time on TIC of aqueous product at 280°C (Chlorella) ............ 153
Figure 9-10 Effect of retention time on relative concentrations of 5 compounds in aqueous

product (Chlorella, 280°C) .................................................................................... 154
xiv
Figure 9-11 Effects of catalysts on relative concentration of major compound classes in bio-
crude oil (Chlorella, 240°C) .................................................................................. 155
Figure 9-12 Effects of catalysts on relative concentration of major compound classes in bio-
crude oil (Chlorella, 280°C) .................................................................................. 156
Figure 9-13 Effect of catalysts on TIC of aqueous product at 240°C (Chlorella) ..................... 159
Figure 9-14 Effect of catalysts on TIC of aqueous product at 280°C (Chlorella) ..................... 160
Figure 9-15 Determinations of Arrhenius kinetic parameters ................................................... 163
Figure 9-16 Simplified reaction pathway scheme for HTL of microalgae................................ 167
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LIST OF TABLES
Table 1-1 Comparison of first, second and third generations of biofuel.....................................4
Table 1-2 Typical properties of bio-crude oil produced from liquefaction, fast pyrolysis, and
heavy fuel oil ..............................................................................................................7
Table 2-1 Properties of water at different conditions ................................................................14
Table 2-2 Summary of HTL studies using different feedstock excluding algae .......................17
Table 2-3 Summary of HTL studies using algae as the feedstock ............................................22
Table 2-4 Elemental composition of fossil fuels, kerogen, natural substances and algae ........33
Table 2-5 Comparison of HTL and natural process of oil formation........................................35
Table 4-1 Characteristics of algal powder (% dry weight except as noted) ..............................39
Table 4-2 Measurements and equations for calculating product yields ....................................44
Table 4-3 Experimental design for effects of reaction temperature ..........................................46
Table 4-4 Experimental design for effects of heating period ....................................................46
Table 4-5 Experimental design for effects of reaction time ......................................................47
Table 4-6 Experimental design for effect of initial pressure of N2 ...........................................48
Table 4-7 Experimental design for effect of catalysts/additives ...............................................52
Table 5-1 Characteristics of different algae species, swine manure and sludge used in this
study ..........................................................................................................................60
Table 7-1 Compositions of catalysts used in this study ............................................................85
Table 7-2 Elemental composition on the surface of Pd/Al2O3 after HTL of Spirulina .............97
Table 8-1 Appearance of raw oil products from different temperatures (Chlorella) ..............103
Table 8-2 Element analysis and HHV of Chlorella and bio-crude oil ....................................128
Table 9-1 Comparisons of different methods to analyze lipid content in algae ......................138
xvi
Table 9-2 Major compounds in bio-crude oil produced from Chlorella (300°C, 30 min) ......139
Table 9-3 Major compounds in aqueous product produced from Chlorella ...........................146
Table 9-4 Reaction rate constants at different temperatures (Chlorella) ................................162
Table 9-5 Apparent kinetic parameters for HTL of microalgae..............................................163
xvii
LIST OF ABBREVIATIONS AND ACRONYMS
A pre-exponential factor in Arrhenius law

BP boiling point
CHN the content of carbon, hydrogen and nitrogen
CR carbon recovery
C. pyrenoidosa Chlorella pyrenoidosa (microalga)
BSE back-scattered electron
EC energy recovery of HTL product
ECR energy consumption ratio
EDS energy-dispersive spectroscopy
E2-Energy Environment-Enhancing Energy
ESEM environmental scanning electron microscope
FEB fossil energy balance
GC gas chromatography
GC-MS gas chromatography-mass spectrometry
GHGs greenhouse gases
HHV higher heating value
HTL hydrothermal liquefaction
NFC non-fibrous carbohydrate
NR nitrogen recovery
R ideal gas constant, 8.314 Jmol-1K-1
S. platensis Spirulina platensis (microalga)
TCC thermochemical conversion
TIC total ion current chromatogram
xviii
CHAPTER 1. INTRODUCTION
1.1 BACKGROUND
Two of the most important issues closely related to sustained human society development
in 21st century are energy sustainability and environmental protection. Dependence on fossil
fuels has not only threatened national energy security but has also led to severe environmental
problems, including global climate change. Therefore, developing alternative energy to diverse
the current energy portfolio and eventually replace fossil fuel usage is both important and
necessary. Clean, renewable energy is the first choice as the alternative to replace traditional
fossil fuel and could be more widely acceptable compared with nuclear power especially for the
nations without uranium supply (Sørensen, 1975). Biomass materials are treated as renewable
sources as they can be regenerated through photosynthesis. Biofuel refers to the fuel derived
from biomass, which usually includes solid, liquid and gas fuels. Liquid fuel derived from
biomass is the major substitute for transportation fuel. This is one of the reasons why both
research and industrial applications for producing biofuel from different kinds of biomass
materials have been conducted extensively. Carbon dioxide, one of the major greenhouse gases
(GHGs) can be captured by plant, algae and microorganisms through photosynthesis so that
biofuel produced from biomass can be treated as carbon neutral.
As a petroleum substitute, the interests in biofuel research, industrial application and usage
were affected largely by the petroleum prices in the past several decades (Gowen, 1989). Many
efforts for developing biofuels were conducted during the energy crisis during the 1970s and
1980s and then went into decline when petroleum prices slumped. Today, more nations realize
the importance of alternative energy to national energy security and environmental protection,
and their requests for biofuels were motivated after the petroleum prices reached a record high in
2008. Figure 1-1 shows the trends of different energy shares of total energy consumption in the
United States (U.S.) from 1949 to 2008. Note that the real crude oil price is not in nominal U.S.
dollars but adjusted by inflation in chained (2000) dollars.
It is observed from Figure 1-1 that the share of total energy consumption by renewable
energy was less than 8%, which indicated fossil fuels are still the dominant energy source in the
U.S. The biofuel share of total energy consumption and total renewable energy consumption are
1
summarized in Figure 1-2. The biofuel in Figure 1-2 primarily consisted of bioethanol and
biodiesel, plus losses and co-products from the production of these two kinds of biofuel. All of
the data in Figure 1-1 and Figure 1-2 were adapted from the U.S. Energy Information
Administration (EIA, 2010b). From Figure 1-2 it was shown that biofuel contributed less than
1.5% of the total energy consumption in the U.S. in 2008, although biofuel consumption already
contributed 19.4% of the total renewable energy consumption.
Figure 1-1 Share of total energy consumption and crude oil price in U.S. (EIA, 2010b)
Therefore, one of the key problems in biofuel development is how the biofuel production
can be economically scaled up in the future. And at the same time, the successful biofuel
production should not be achieved by sacrificing our ecological system and environment. From
this point of view, selecting and producing sustainable biomass materials that can supply enough
feedstocks for biofuel production in the future would be a critical criterion.
2
Figure 1-2 Biofuel shares of renewable energy consumption and total energy consumption in U.S.
(EIA, 2010b)
1.1.1 Feedstocks for biofuel production
Biomass was used as the main energy source for thousands of years in human history.
Since fossil fuels have much higher energy densities than raw biomass materials, they started to
serve as the main energy sources since the industrial revolution. Although there are existing
debates, many people believe that fossil fuel production will peak in the near future and we are
‘lucky’ to be living in the oil age (Greene et al., 2006; Hirsch et al., 2006; Hubbert, 1956; Kerr,
2011). Since the shortage of fossil fuel is expected to occur in the near future, interest is again
focused on biomass and bioenergy. From this aspect, the concept of producing alternative fuel
from biomass materials is not new but renewed.
There are several major classes of biomass resources for biofuel production, which include
traditional food crops (such as corn, soybeans, and sugarcane), agricultural or industrial waste
(such as corn stover, straw, municipal waste, animal wastes, and food processing residuals), and
energy crops (such as perennial grasses, fast growing trees and microalgae) (Alfonso et al., 2009;
Miao et al., 2004). Total world liquid fuels consumption in 2008 was 31×109 barrels per year and
3
was predicted to be 41×109 barrels per year in 2035 (EIA, 2010a). The worldwide raw biomass
energy potential has been estimated to be 150-450 EJ year-1 or 25×109 to 76×109 barrels of oil
energy equivalent (IEA, 2004). Therefore energy from biomass shows great potential to meet a
large portion of the energy consumption. The evolution of liquid biofuel is summarized in Table
1-1. From the comparison, the main difference between different generations of biofuel is the
feedstock. The choice of feedstock to produce biofuel has gradually been focused on those that
have more environmental benefits and less impact on ecological system.
Table 1-1 Comparison of first, second and third generations of biofuel
1st generation 2nd generation 3rd generation
Energy crops such as

Feedstocks Sugar, starch, vegetable Agricultural and forest
perennial grasses, fast
oil, animal fat from food residue
growing trees and algae
Hydrolysis Fermentation
Conversion Process Fermentation Fermentation Transesterification
Transesterification Thermochemical Thermochemical
conversion conversion
Bio-alcohols
Products Bio-alcohols Bio-alcohols
Biodiesel
Biodiesel Biodiesel
Bio-crude oil
Benefits Commercialized Not compete with food; Not compete with food;
Utilize biowastes Abundant feedstock
Advanced technology still
Limited feedstock (Food Limited feedstock; Low
Limitations under development to
vs. Fuel); Blended partly conversion efficiency
reduce the cost
with conventional fuel
The productions of many biomass materials have to rely on fertile land especially for the
first and second generation of biofuels. For example, 4-13% of the agricultural land in the
European Union (EU) was used for biofuel production if 5.75% of the transportation fuels in the
EU was provided by biofuels (Bendz, 2005). In fact, biofuel production from food crops and
lignocellulosic biomass could lead to land clearing and have a great impact on current land-use
for food production systems if the existing agricultural land is diverted to biofuel production,
unless they are grown on abandoned agricultural lands (Fargione et al., 2008). Furthermore,
land-use change from croplands for biofuels will not only trigger high crop prices but also
increase the GHGs emission because the carbon storage and sequestration by the cropland could
be sacrificed by diverting land from its existing uses (Searchinger et al., 2008). Therefore, there
4
are concerns current land-based biomass cultivation for biofuel production may compete for
arable land with food production, increase environmental pollution from fertilizers and pesticides,
and may potentially threaten biodiversity through land-use changes, pollution, soil degradation,
and climate impacts from cultivation, transportation, refining, and burning (Tilman et al., 2006).
Expansion of agricultural lands for biofuels may also reduce availability of habitats suitable for
many species and reduce the ecosystem services offered by more complex ecological systems
(Groom et al., 2008).
1.1.2 Bio-crude oil produced from thermochemical conversion

Among different forms of biofuels, liquid fuel attracts more attention because it is easily
stored, transported and has the potential to replace the petroleum-based transportation fuel. Bio-
crude oil or bio-oil was produced through thermochemical conversion (TCC) of biomass. Not
like bioethanol from sugar- or starch-based processes or biodiesel from oil crops and animal fats,
feedstocks for bio-crude oil production are usually from agricultural or industrial waste and
energy crops. Thus, producing bio-crude oil from these feedstocks usually has less impact on
current land-use systems and does not threaten biodiversity. Desirable biofuels substituting for
fossil fuels should be produced from sustainable feedstocks that neither compete with food crops
nor cause land-clearing and that can offer advantages in reducing GHGs emissions (Tilman et al.,
2009). From this point of view, bio-crude oil produced from sustainable biomass feedstocks
exists in the “best biofuels” category and could be the potential substitute for transportation fuel.
Generally, the biomass conversion process can be divided into two broad pathways:
thermochemical decomposition and biological digestion. Thermochemical conversion processes
can be divided into three categories: gasification, pyrolysis and direct liquefaction (Demirbaş,
2001). Gasification has also been named as indirect liquefaction since its product “syngas” could
be further converted into liquid fuel through the “Fischer-Tropsch-type” (FT) reaction. The terms
of pyrolysis and direction liquefaction are sometimes confused with each other because both
their final products are bio-crude oil and the process conditions are similar to each other.
However, pyrolysis requires higher reaction temperature compared to direct liquefaction and its
feedstock must contain much less water, which indicates a drying process is usually necessary
for the pyrolysis process. On the other hand, the operation pressures of pyrolysis are closed to
atmospheric pressure and the operation pressures of direct liquefaction can be up to 200 times of
atmospheric pressure. The capital cost of the direct liquefaction could be higher than a pyrolysis
5
process due to its high pressure requirement (Elliott et al., 1991). Figure 1-3 shows the
temperature and pressure operating conditions for the three thermochemical conversion
processes.
Figure 1-3 Operating conditions (temperature and pressure) of thermochemical conversion processes
Since biomass feedstocks usually contain 40-60 wt% oxygen content, one overall objective
to producing fuels from biomass is to remove oxygen (Peterson et al., 2008). Bio-crude oil is
generally produced from liquefaction and fast pyrolysis process. The name “bio-crude” has two
conventional meanings and implications: it means the product is derived from biomass materials
and it implies the oil quality is not comparable with conventional petroleum. Table 1-2 compares
typical properties of bio-crude oil produced from the TCC processes and heavy fuel oil. It shows
the oxygen content in bio-crude oil is much higher than that in heavy fuel oil. The oils produced
from pyrolysis usually have higher moisture and oxygen content compared to oils from
liquefaction. In that regard, liquefaction oils typically have more desirable quantities than
pyrolysis oils and can be produced with higher energetic efficiency.
Although there are many discussions on which thermochemical process is the “best choice”
for producing biofuel (Demirbaş, 2000b; Elliott et al., 1991; Huber et al., 2006; Peterson et al.,
6
2008), it is believed that feedstock properties will be a decisive factor in the selection of
conversion methods. Since bio-crude oils are usually produced from crop residue, animal waste,
municipal waste, food processing waste, and algae, and most of these feedstocks have significant
amounts of water content, a process without drying feedstock will have a huge advantage
because the drying process consumes a large amount of energy for the water vaporization. For
example, in the traditional microalgae-to-biodiesel technology, more than 75% of the total
consumed energy were used for the drying process (Lardon et al., 2009). Among the three
thermochemical conversion processes, direct liquefaction does not require a drying process for
the feedstock. Hydrothermal liquefaction (HTL) belongs to the classification of direct
liquefaction since the liquefaction occurs in a water medium. It could be more suitable for bio-
crude oil production from wet biomass materials, such as microalgae.
Table 1-2 Typical properties of bio-crude oil produced from liquefaction, fast pyrolysis, and heavy fuel oil
Property Liquefaction oil Pyrolysis oil a,b Heavy fuel oil a
Moisture (wt%) 5-10 15-30 0.1

pH n.a. 2.5 n.a.
Specific gravity 1.1 1.2 0.94
Elemental composition (wt%)
C 70-77 54-58 85
H 8-10 5-7 11
O 10-16 35-40 1.0
N 5-7 0-0.2 0.3
Ash 5 0-0.2 0.1
Higher heating value (MJ/kg) 34-38 16-23 40-49
Viscosity (cps) a 15000 @ 61°C 40-100@ 50°C 180
Distillation residue (wt%) a n.a. Up to 50 1
n.a.: not available; a (Huber et al., 2006); b (Peterson et al., 2008)
1.2 ALGAE AS FEEDSTOCK FOR BIO-CRUDE OIL PRODUCTION
Among the potential sustainable biomass, algae production does not require soil and can
occur on marginal lands and in different bodies of water; therefore, it has less impact on current
land-use for food production systems. Algae have faster growth rates, shorter growing cycles and
higher photosynthetic efficiencies when compared with other terrestrial lignocellulosic plants
(Pirt, 1986). They are viewed as one of the most suitable feedstocks to produce the next
generation biofuel (Chisti, 2007; Chisti, 2008; Luque et al., 2008; Luque et al., 2010).
7
1.2.1 Classification of algae
Algae is the plural form of the word alga; they are a diverse group of organisms that are
classified in different kingdoms by different people at different times. Traditionally, most of
algae are considered as plant subkingdom with the 5-kingdom classification (Monera, Protista,
Plants, Animals, and Fungi). However, this classification excludes the cyanobacteria, which have
been well known for a long time as blue-green algae. Although in the new definition of algae,
they are loosely defined as photosynthetic eukaryotes excluding the land plants, some algal
species are closely related to cyanobacteria in common ancestors of the chlorophytes,
rhodophytes, and glaucocystophytes (Bhattacharya, 1998).
Figure 1-4 Schematic view of the tree of life based on rDNA sequence comparisons
[adapted from (Sogin et al., 1996)]
Figure 1-4 shows the three domains of life with emphasis on the phylogeny of the
eukaryotes. It indicates that although cyanobacteria are in different domains of life compared to
most of the other algae, there is a relationship between the cyanobacteria and some algae species.
This is the reason that many studies treated cyanobacteria as one of microalgae to study the
feasibility of biofuel production from microalgae (Biller and Ross, 2011; Jena et al., 2011; Ross
et al., 2010). In this study, we expanded our definition of algae to cyanobacteria, such as
8
Spirulina. From this point of view, the large number of algal species can be subdivided into 10
taxonomic groups: Chlorephyceae (green algae), Bacillariophyceae (diatoms), Xanthophyceae
(yellow-green), Chrysophyceae (golden algae), Rhodophyceae (red algae), Phaeophyceae (brown
algae), Dinophyceae (dinoflagellates), Prasinophyceae, Eustingmatophyceae, and Cynophyceae
(cyanobacteria) (Williams and Laurens, 2010).
The size of algae differs from each other, some of them may be up to 30 m in length (kelps,
which are in the Heterokonta), and some of them may be bacteria-sized (1-5 µm). From their size,
algae can be distinguished as microalgae and macroalgae. Generally, microalgae refer to the
algae that can only be seen with the aid of a microscope, while macroalgae refer to large aquatic
photosynthetic plants that can be seen by naked human eyes. Macroalgae usually have higher
cellulose and ash content but lower lipid and protein content than microalgae. Although
macroalgae were converted via HTL in this study, the primary goal is focused on microalgae.
1.2.2 Bottlenecks for producing biodiesel from microalgae

Microalgae are single cell organisms, which can be found in colonies or individual cells.
Microalgae are usually comprised of lipids, proteins, nucleic acids, and carbohydrates. For a
traditional algae-to-biodiesel technology, lipid content was the only criterion for screening
suitable algal species. However, the lipids storage, which are predominantly triglycerides in the
microalgae cells, may gain a greater portion of the overall lipid fraction as the metabolic rate
slows down (Williams and Laurens, 2010). Figure 1-5 shows the relationship of lipid contents of
microalgae and their biomass productivities generated from literature data (Rodolfi et al., 2009).
It indicates that high lipid content of microalgae is usually associated with low productivities.
Other researchers also observed the similar relationship (Mata et al., 2010).
To improve the economic feasibility of biodiesel production from algae, two research
strategies are employed: first, screen and isolate suitable algae for a high lipid producer; and
second, identify the rate limiting enzyme and modify the cell’s genetics to increase its expression
and so increase the yield (Williams and Laurens, 2010). However, because the lipid content is
negatively proportional to biomass productivity, neither of these two tasks is easily accomplished.
Especially, a well-maintained production system is usually required for growing pure algal
species with high lipid content, which could tremendously increase the production cost.
From 1978 to 1996, the Office of Fuels Development in the U.S. Department of Energy
funded an Aquatic Species Program to develop biodiesel and other renewable transportation
9
fuels from algae. The program was discontinued due to the low crude oil price in the late 1990’s
but it successfully developed two algae growing facilities: a raceway pond and a photobioreactor.
Meanwhile, many microalgae samples were collected, screened and characterized in order to find
the fast-growing, high-lipid content microalgal species. However, in the close-out report, the
researcher admitted that focusing on lipid accumulation in microalgae under a stressed condition
such as nitrogen depletion, sacrifices biomass productivity, might reduce the net energy yield,
and make the process very sensitive to contamination, and therefore, could cause a net reduction
in lipid productivity. Or in other words, high productivity and high lipid content could be
mutually exclusive (Sheehan et al., 1998).
Figure 1-5 Relationship of biomass productivities and lipid content for 30 microalgae strains
A typical procedure of producing biodiesel from algae is illustrated in Figure 1-6. This
procedure usually involves several energy intensive processes, such as drying, grinding and oil
extraction. Drying could consume 75% of the total energy input for the whole procedure (Lardon
et al., 2009) and make the entire process energetically unfavorable.
To summarize, three major bottlenecks for current algae-to-biodiesel technology include:
10
1. Screen and genetically modify algal genes in order to obtain fast-growing and high-
lipid species, which are negatively proportional to each other.
2. In order to maintain an environment for growing the pure algal stains, operating cost
will be increased to prevent contamination.
3. An energy intensive drying process is often required to pretreat the algal biomass
before oil extraction.
High-lipid algae
Harvest & Drying
Grinding
Oil extraction
Transesterification
Separation of biodiesel and glycerine
Water washing soap and glycerine
Biodiesel product
Figure 1-6 General procedure of producing biodiesel from algae

[adapted and modified from (Demirbaş, 2008)]
Using HTL to convert low-lipid, fast-growing, and wet algal biomass into biofuel could be
another feasible way to produce enough alternative transportation fuel for the future. Although
many studies have been conducted for HTL of lignocellulosic or biowastes feedstock in literature,
research on HTL of algae, especially for low-lipid algae, is sparse. More research efforts are
needed to explore how to convert low-lipid algae into bio-crude oil, especially for those strains
that can be grown prolifically in wastewater.
11
CHAPTER 2. LITERATURE REVIEW
Energy contained in biomass can be utilized via biological digestion, biochemical process
and thermochemical conversion. Figure 2-1 summarizes current technologies for utilizing and
converting biomass materials into different types of renewable energies. The different operating
conditions of the three thermochemical conversion (TCC) processes have been discussed in
CHAPTER 1 (Figure 1-3). This study is focused on hydrothermal liquefaction (HTL), which
belongs to the direct liquefaction process. HTL uses water as the reaction medium while other
direct liquefaction processes use solvents or other reaction mediums.
Figure 2-1 Biomass conversion technologies and products
2.1 WATER PROPERTIES AT HIGH TEMPERATURE
High-temperature water (HTW) usually refers to liquid water at temperature above 200°C
(Akiya and Savage, 2002). In the natural process of conventional petroleum formation, HTW is
the reaction medium, in which kerogen can be converted into petroleum with the presence of
clay minerals (Marshak, 2008; Simoneit, 1993; Siskin and Katritzky, 1991). Additionally, when
12
water is near its critical point (374°C, 218 atm), its properties such as dielectric constant and
density are much different with those of ambient liquid water. It behaves like organic solvents so
that organic compounds can be dissolved or completely miscible in the water phase (Savage,
1999). In that regard, HTL can be treated as a green process to mimic nature’s millions-of-years
process of crude oil formation, while the HTL conversion can be completed in 10-120 minutes.
Figure 2-2 indicates HTL usually occurs between 200°C to 374°C, with pressure between 4
to 20 MPa. Unlike gasification and pyrolysis, HTL requires much lower temperatures and the
water is still in a liquid state under these conditions. Additionally, gases can be miscible in the
supercritical water (SCW). HTL provides an environment in which the chemical reactions can
occur in a single fluid phase. Under this single phase reaction environment, higher
concentrations of reactants can be obtained than in a multiphase system therefore the mass
transfer efficiency could be enhanced (Savage, 1999).
Pressure (MPa)
Critical point
Temperature ( C)
Figure 2-2 Pressure-temperature phase diagram of water [adapted from (Peterson et al., 2008)]
Properties of water under a normal condition (25°C, 1 atm), subcritical condition and
supercritical condition are compared in Table 2-1. As the temperature increases, water has a
13
lower dynamic viscosity, dielectric constant but higher heat capacity. Meanwhile, the properties
of water are significantly changed when the temperature is further increased under the
supercritical condition. For example, at 400°C and 0.5 gcm-3 (ρ, density), water retains 30-45%
of hydrogen bonds that exist at ambient conditions, whereas water at 500°C and 0.1 gcm-3
retains only 10-14% of hydrogen bonds (Hoffmann and Conradi, 1997). With increased
temperatures, the changes of water characteristics benefit the small organic compounds produced
from HTL and become increasing soluble and eventually miscible in SCW.
Table 2-1 Properties of water at different conditions
Properties Normal Water Subcritical Water a Supercritical Water b
Temperature (°C) 25 250 400

Pressure (MPa) 0.1 5 25
Density, ρ (g∙cm-3) 0.997 0.80 0.17
Dynamic viscosity, η (mPa s) 0.89 0.11 0.03
Heat capacity, Cp (kJ∙kg-1∙K-1) 4.22 4.86 13.0
Dielectric constant, ε 78.5 27.1 5.9
Ionic product, pKw 14.0 11.2 19.4
Heat conductivity, λ (m∙W∙m-1∙K-1) 608 620 160
a
(Bröll et al., 1999); b (Krammer and Vogel, 2000).
Since HTW have these advantages, it was treated as an attractive reaction medium and
there has been much previous research in this area with applications in biomass conversion,
biofuels production, and waste treatment (Peterson et al., 2008; Savage, 1999).
2.2 DEVELOPMENT OF HTL
The original idea about producing “renewable petroleum” from biomass was probably
motivated by the arguments about the biological origins of conventional petroleum in the early
20th century (Peterson et al., 2008). Direct liquefaction processes started from efforts to try to
liquefy coal to produce liquid fuel in the 1920s (Moffatt and Overend, 1985). The first HTL
study was reported by E. Berl in 1934 based on the knowledge of the author. A “proto product”
containing aliphatic, naphthenic and aromatic substances was produced from cellulose and
carbohydrates together with alkaline materials in the presence of water at temperatures higher
than 230°C. These results was used to explain the formation of natural gas, asphalts, oils and
bituminous coals and confirmed oils could be produced from biomass (Berl, 1934, 1935). About
ten years later, Berl further pointed out that many plants, such as cornstalks, corncobs, sugarcane,
and other biomass could be converted into “proto product” materials. The product contained 60%
14
of carbon in the feedstock and it was semi-liquid at room temperature and liquid at higher
temperatures. Berl also calculated that about 7.37×106 acres land area with a climate similar to
that of Hawaii was needed to grow these “carbohydrate containing material” (mainly referred to
as sugar cane) if all of the gasoline consumed by cars at that time were replaced by the liquid
fuel produced from HTL (Berl, 1944).
Since the crude oil price was very low and relatively stable from the 1940s to the early
1970s (Figure 1-1), the ambitions of using HTL to produce renewable energy slumped until the
crude oil price started to soar in the middle of the 1970s.
The U.S. Bureau of Mines started to pursue the possibility of converting wood into bio-
crude oil with carbon monoxide as the process gas, and many efforts were conducted in 1969
(Appell et al., 1969a; Appell et al., 1969b, 1969c) and early 1970s (Appell et al., 1971; Appell et
al., 1975) at the Pittsburgh Energy Research Center (PERC). Although most of the previous
HTL studies were laboratory scale batch tests, the Lawrence Berkeley Laboratory (LBL)
developed a continuous HTL unit to convert wood chips into bio-crude oil with carbon monoxide,
hydrogen or mixed gas as the process gas. The bio-crude oil product was described as a bitumen-
like black liquid material with high viscosity (Davis et al., 1982). Compared to the PERC process
with an oil recycle set-up, the LBL process with a single pass water slurry process, avoided the
high oil and process water recycles, which consumed a lot of carbon monoxide. However, the oil
yield from the LBL process was lower than that from the PERC process.
The efforts from both PERC and LBL led to the construction of a continuous pilot plant at
Albany, Oregon and this pilot plant, which mainly used the LBL process, operated continuously
for over 500 hours at about 20% of its nominal design capacity and finally produced 30 barrels
of bio-crude oil during 1980~1981 (Thigpen and Berry, 1982). The purpose of their work was to
demonstrate the feasibility of direct, continuous, thermochemical liquefaction of biomass (Klass,
1998). However, plugging occurred due to high wood chip content in the feedstock slurry in both
the lab scale experiment and the pilot plant, and finally interrupted the process. To support the
Albany project, more laboratory work was conducted at the U.S. DOE Pacific Northwest
National Laboratories (PNNL, Richland, WA) (Elliott and Walkup, 1977). Since then, the PNNL
conducted many studies related to HTL and Elliott et al. authored comprehensive reviews on
different kinds of thermochemical conversion processes (Beckman and Elliott, 1985; Elliott et al.,
1991; Elliott et al., 1988). In the early 1980s, the research of PERC was shifted to the University
15
of Arizona (Tucson, AZ) and focused on improving the solids content of the biomass slurry fed
into the high-pressure reactor (Elliott et al., 1991).
The difficulties in the feeding system for liquefaction of wood, such as plugging, seemed to
be overcome by an approach called HTU, or hydrothermal upgrading process (Rezzoug and
Capart, 1996) conducted at Shell Corp. Although Shell, which originally developed the HTU,
abandoned the process in 1989, carried on to some research study the economic feasibility of
HTU and the conversion efficiency of the process. Goudriaan and Peferoen (1990) also
concluded that the HTU process was more attractive than pyrolysis or gasification because HTU
avoided the drying process and the reaction medium, water could have the unique
thermophysical and chemical properties under supercritical or near critical conditions (Goudriaan
and Peferoen, 1990; Goudriaan et al., 2001).
2.3 HTL OF DIFFERENT BIOMASS INTO BIO-CRUDE OIL
2.3.1 Lignocellulosic biomass and biowaste

The HTL process was used to convert many kinds of biomass feedstocks into bio-crude oil.
Bio-crude oil from HTL is usually defined as the solvent soluble fraction of the oil/solid product
after conversion. Although different studies used different solvents to extract the bio-crude oil
fraction and this may introduce uncertainties for comparing results of bio-crude oil yields, using
solvents to extract the solvent soluble fraction of the oil/solid product is still the primary method
to define the bio-crude oil in HTL studies. One study suggests using nonpolar solvents may
achieve higher gravimetric yields of bio-crude oil, but also lower carbon content, and thus, lower
energy density (Valdez et al., 2011). However, the method has not been standardized yet.
There are many studies about producing bio-crude oil from different kinds of biomass
materials via HTL. Table 2-2 summarizes the operating conditions, reactor type and bio-crude oil
yield of some remarkable studies in the literature about converting lignocellulosic biomass and
biowaste feedstock into bio-crude oil via HTL. Note that studies about converting algae into bio-
crude oil are excluded in Table 2-2 because they are specifically discussed in Section 2.3.2.
16
Table 2-2 Summary of HTL studies using different feedstock excluding algae
Temperature Retention Catalyst/ Bio-crude oil

Feedstock Process Gas Reactor Reference
(°C) Time (min) Additives yield (% d.w.)
Batch, (Panayotova-
Peat 275~350 120 CO K2CO3 25~60
Autoclave Björnbom et al., 1979)
Batch,
Poplar 340 60~120 H2 Raney nickel 33.8 (Boocock et al., 1980)
Autoclave
Continuous,
Wood chips 300~370 20~90 CO Na2CO3 40 (Elliott, 1980)
Autoclave
Batch,
Cellulose 271~407 20~100 CO Na2CO3 8~37 (Donovan et al., 1981)
Autoclave
Continuous,
Wood chips 340~360 >10~30 H2/CO n.a. 27~33 (Davis et al., 1982)
Tubular
Cellulose/ 279~407 20~100 4~37
Batch,
Sewage sludge 250~320 20~60 N2 Na2CO3 3~37 (Nelson et al., 1984)
Autoclave
/hop residue 250~300 20 35~41
Batch,
Sewage sludge 250~340 0 N2 Na2CO3 26~50 (Suzuki et al., 1986)
Autoclave
CO, N2 Batch,
Pine wood 300~350 45 NaOH 35.2~56.4 (Alen et al., 1989)
H2 Autoclave
Wood pellet/ 42.5 Continuous, (Goudriaan and

260~400 >5 n.a n.a.
Eucalyptus chips 48.6 Tubular Peferoen, 1990)
Batch,
Barley stillage 250~300 0~120 N2 Na2CO3 16~38 (Dote et al., 1991)
Autoclave
Hydrolytic Continuous, (Schuchardt et al.,

200~300 27.4 Argon HCOONa 18.3~44.8
eucalyptus lignin Tubular 1993)
17
Table 2-2(cont.)

Municipal cellulosic (Gharieb et al.,

260~340 0 H2 Boric acid 26~50 Batch, Autoclave
wastes 1993)
(Minowa et al.,
Artificial garbage 250~350 10~120 N2 Na2CO3 5~27.6 Batch, Autoclave
1995b)
Egg albumin 150~340 30~120 N2 Na2CO3 <10 Batch, Autoclave (Dote et al., 1996)
Batch, (Holliday et al.,

Vegetable oils 260~280 15~69 n.a. n.a. >97*
Reaction vessel 1997)
Na2CO3 Batch,
Turkish biomass 380 30 n.a. 43.2~59.3 (Demirbaş, 1998)
K2CO3 Autoclave
Amino acids 300 60 N2 n.a. 0.2~66.4 Batch, Autoclave (Dote et al., 1998b)
(Minowa et al.,
Biomass residues 300 30 N2 Na2CO3 21~36 Batch, Autoclave
1998)
Cellulose and
300 60 N2 n.a. 10.5~23.7 Batch, Autoclave (Inoue et al., 1999)
ammonia
Continuous,
Vegetable oils 270~340 7~15 n.a. n.a. 22~100* (King et al., 1999)
Autoclave
Sawdust 302 30 N2 KOH 38.6~92.5 Batch, Autoclave (Demirbaş, 2000a)
Swine manure 275~350 5~180 CO n.a. 51.7~66.5 Batch, Autoclave (He et al., 2000a)
18
Table 2-2(cont.)

CO, H2
Swine manure 275~315 120 n.a. 49.8~67.8 Batch, Autoclave (He et al., 2001b)
CO2, N2
Cunninghamia
280~360 10~30 n.a. n.a. 12.6~23.8 Batch, Autoclave (Qu et al., 2003)
lanceolata
Rb2CO3 (Karagöz et al.,

Pine sawdust 280 15 N2 8.6~25.2 Batch, Autoclave
Cs2CO3 2004)
Plant biomass 277~377 25 n.a. n.a. 12.2~28.4 Batch, Autoclave (Demirbaş, 2005)
Continuous, (Ocfemia et al.,

Swine manure 285~325 40~80 N2 n.a. 31.2~61.1
Autoclave 2006b)
Silver birch 280~420 n.a. H2 Na2CO3 <53.3 Batch, Autoclave (Qian et al., 2007)
Eucalyptus + (Sugano et al.,

150~350 0 N2 NaOH <38 Batch, Autoclave
wastewater 2008)
* Yields of free fatty acids; n.a. Not available
19
From the summary of Table 2-2, the HTL process is a low temperature but high pressure
thermochemical conversion process compared with pyrolysis. The operating temperature of HTL
was usually in the range of 200-400C and the reaction time (retention time or hold time) was
usually less than 2 hours. Although the operating pressures in many studies of the HTL process
ranged from 7 to 30 MPa (Peterson et al., 2008), few of the studies really controlled the pressure
during the process. In other words, most of the time the high operating pressures were self-built-
up and primarily attributed to water vapor, process gas and the gaseous product produced via
HTL (Yu et al., 2011a). Additionally, most of the previous laboratory studies were still
conducted in the batch reactors instead of continuous ones.
2.3.2 Algae as feedstock for bio-crude oil production

Compared with the long research history of HTL, using algae as energy sources is relatively
new. In 1955, a concept of producing methane from algae was proposed (Meier, 1955). This
concept only received attention due to the energy crisis in the 1970s. From 1978 to 1996, the U.S.
Department of Energy (USDOE) founded a program named “Aquatic species program: biodiesel
from algae” to investigate the possibility of massively producing algae as energy sources for
biodiesel production (Sheehan et al., 1998). However, this program was shut down due to low
crude oil prices in the late of 1990s. Since the crude oil price started to increase at the beginning
of the 21st century, interest in using algae as an energy source were raised again.
The first study for using HTL to convert microalgae into bio-crude oil was published in
1994 based on the knowledge of the author. Dote et al. (1994) found the yield of bio-crude oil
produced from Botryococcus branunii via HTL was higher than the original oil content in the
algal feedstock. This important result suggested that producing bio-crude oil via HTL was not
only extracting the naturally occurring oils in the algae, but most importantly it can produce oils
from the non-lipid components of the algae (Dote et al., 1994; Peterson et al., 2008). Therefore,
unlike producing biodiesel from algae, which relies on the lipid content of the algae, producing
bio-crude oil via HTL will not just focus on the high-lipid algae. Other algal species with high
biomass productivities could also be suitable feedstocks for the HTL process.
Remarkable studies on producing bio-crude oil via HTL are summarized in Table 2-3. Most
of the HTL studies of converting algae into bio-crude oil focused on microalgae instead of
macroalgae. In particular, bio-crude oil yields from microalgae were higher than that from
macroalgae. On the other hand, ash contents of macroalgae are usually high, and lipid, protein
20
contents are usually low. Renaud and Luong-Van (2006) studied the seasonal variation in the
chemical composition of tropical marine macroalgae in Australia and found the mean value of
ash content could be as high as 38.8% of the dry weight. On the other hand, neither lipid nor
protein content exceeded 8% (Renaud and Luong-Van, 2006). Since the ash content can
significantly influence the bio-crude oil yield and quality, although macroalgae could still be the
candidates for feedstock for producing bio-crude oil via HTL, the attention of this study is
primarily focused on microalgae.
From Table 2-3, a majority of previous studies about converting algae into bio-crude oil via
HTL were conducted under subcritical conditions. This is due to the high moisture content of
algae and much less lignocellulosic content in microalgal cells than in terrestrial plants.
Therefore a much lower temperature is needed to break down the lipid, protein and hydrocarbon
in the microalgal feedstock. It should be noted that the algal species used in previous studies
were mainly high-lipid content algae. This is probably due to the availability of these algal
feedstocks from algae-biodiesel industry and that previous researchers wanted to compare their
results with algae-to-biodiesel application. For that reason, low-lipid algal species were
overlooked in the literature.
In the period of 1997 to 2009, there were only a few studies in literature about using algae
as feedstock to produce bio-crude oil via HTL. Many more studies in this area were published
after 2010, probably due to the high price of petroleum. The research interest in producing
biofuel from algae is apparently highly related to conventional fuel price. As in the past (even
USDOE shut down the Aquatic Species Program due to the low petroleum price), we have to
find a way to avoid the same up-and-down situation happening in the future if the price of oil
slumps again—which is not completely impossible. A different justification is needed to support
such research efforts regardless of oil prices.
21
Table 2-3 Summary of HTL studies using algae as the feedstock
Lipid content Temperature Retention Catalyst/ Bio-crude oil

Algae species Process Gas Reference
(% d.w.) (°C) Time (min) Additives yield (% d.w.)
Botryococcus
50 200~340 60 N2 Na2CO3 57~64 (Dote et al., 1994)
branunii
Botryococcus
36 200~340 n.a. n.a. Na2CO3 n.a. (Inoue et al., 1994)
branunii
Dunaliella (Minowa et al.,

20.5 250~340 5~60 N2 Na2CO3 30.9~42.6
tertiolecta 1995a)
Spirulina 12.0 300~450 60 N2, H2, CO Fe(CO)5-S 61 (Matsui et al., 1997)
Microcystis viridis n.a. 300~340 30~60 N2 Na2CO3 24~33 (Yang et al., 2004)
Chaetomorpha
linum a n.a. 250~395 60 N2 n.a. 2~8 (Aresta et al., 2005)
Dunaliella
2.87 120~200 4.77~60 n.a. H2SO4 n.a. (Zou et al., 2009)
tertiolecta b
Enteromorpha
n.a. 220~320 5~60 N2 Na2CO3 9.6~23.0 (Zhou et al., 2010b)
prolifera a
Na2CO3, KOH,
Chlorella vulgaris/
n.a. 300/350 60 n.a. CH3COOH, 11.6~27.3 (Ross et al., 2010)
Spirulina
HCOOH
Nannochloropsis 28 200~500 60 Helium n.a. 16~43 (Brown et al., 2010)
Co/Mo/Al2O3,
Chlorella vulgaris/ 25 (Biller and Ross,
350 120 n.a. Ni/Al2O3, 18.1~38.9
Nannochloropsis 32 2011)
Pt/Al2O3
Laminaria (Anastasakis and
n.a. 250~370 15~120 n.a. KOH 3.8~19.3
Saccharina a Ross, 2011)
22
Table 2-3(cont.)
Lipid content Temperature Retention Catalyst/ Bio-crude oil

Algae species Process Gas Reference
(% d.w.) (°C) Time (min) Additives yield (% d.w.)
Pd/C, Pt/C,
Ru/C, Ni/SiO2-
Helium (Duan and Savage,
Nannochloropsis 28 350 60 Al2O3, 35~57
H2 2011b)
CoMo/γ-Al2O3
Zeolite
Chlorella
0.1 200~300 0~120 N2 n.a. 24.0~39.4 (Yu et al., 2011a)
pyrenoidosa
Spirulina platensis 13.3 200~380 0~120 N2 n.a. 17~39.9 (Jena et al., 2011)
(Garcia Alba et al.,

Desmodesmus sp. 10~14 175~450 5~60 Helium n.a. 8.6~49.4
2011)
a
macroalgae; b hydrothermal liquefaction in ethylene glycol medium.
23
2.4 REACTION MECHANISMS AND KINETICS OF HTL
Although different biomass feedstocks have been converted via HTL, studies on the HTL
reaction mechanism were primarily conducted using model compounds instead of the biomass
feedstocks. Reports on possible reaction pathways of those model compounds such as glucose,
xylose, protein, and triacylglycerides (TAGs) were copious in literature. Most of conclusions on
HTL reaction mechanisms and kinetics are derived for feedstocks which have relatively simpler
structures than real biomass materials. In other words, the chemical pathways of, kinetics of, and
interactions among different biomass components under HTL conditions are still largely
uncharacterized (Peterson et al., 2008). In this section, current knowledge of HTL reaction
mechanisms and kinetics are summarized.
2.4.1 Lipids/Fats
Lipids and fats are non-polar compounds and can be applied to all organism-produced
substances that are practically insoluble in water, but are extractable by one or another of the so-
called fat solvents (Tissot and Welte, 1984). The term of lipids encompass fat substances, which
include animal fat, vegetable oils, and wax.
Generally, the increasing temperatures causes fat to become increasingly soluble in water,
ultimately becoming completely miscible when the temperature of water reaches supercritical
state. This is because the dielectric constant of water will be significantly lowered with the
increasing temperature (Table 2-1). There is a study observing that the temperature for the
completely miscible of fatty acids derived from coconut oil and beef tallow with water was
293°C and 321°C, respectively (Mills and McClain, 1949).
Triacylglycerides (TAGs) are the most common form of lipids in biological systems and the
mixture of various TAGs make up naturally occurring fats and are usually classified chemically
as esters. TAGs are readily hydrolyzed in hot compressed water and catalysts are normally not
required (Toor et al., 2011). The products from hydrolysis of fat, fatty acids are relatively stable
in the subcritical water. Holliday et al. (1997) and King et al. (1999) achieved rapid hydrolysis
of vegetable oil under a hydrothermal environment and the highest fatty acids yields in these two
studies were both close to 100% (Holliday et al., 1997; King et al., 1999).
24
Fatty acids can be further degraded to produce long-carbon-chain hydrocarbons, which
have excellent fuel properties with the presence of catalysts. Watanabe et al. (2006) reported the
decarboxylation of stearic acid could be accelerated with the addition of KOH and the
conversion of the stearic acid can be increased from 2% to 32%. When metal oxide catalysts
were applied, the conversion of stearic acid could be higher than 60% (Watanabe et al., 2006).
As the backbone of TAGs, glycerol is one of the important products from TAGs hydrolysis.
Since glycerol is also the byproduct for biodiesel production, the crude glycerol from biodiesel
production usually contains fatty acids, glycerol, methanol, and salts. Therefore, it could be co-
liquefied via HTL to produce bio-crude oil (Xiu et al., 2010). However, pure glycerol is not a
suitable feedstock for HTL to produce bio-crude oil because it is converted into a water-soluble
product rather than an oily product under the hydrothermal condition (Toor et al., 2011).
A previous study demonstrates that the kinetics of fatty acid esters hydrolysis under
subcritical water conditions (210°C-270°C) obey the first-order reaction rate (Khuwijitjaru et al.,
2004). Fu et al. (2010) also reported that the kinetics of palmitic acid deoxygenation with the
presence of a Pt/C catalyst under hydrothermal conditions (with 290°C-380°C as reaction
temperature) showed a first-order reaction rate following the Arrhenius equation. The pre-
exponential factor (A) was determined as 103.20±0.41 s-1 and the activation energy Ea was 79±5 kJ
mol-1 (Fu et al., 2010).
2.4.2 Proteins/Amino acids

Proteins are polymers made from amino acids and they account for most of the nitrogen
compounds in organisms. Generally, amino acids can be produced via conventional acid
hydrolysis of proteins. However, yields of amino acids are much lower and generally below 10
wt% by HTL due to the subsequently degraded amino acids produced in the hydrothermal
environment (Peterson et al., 2008).
Figure 2-3 shows the general pathway of peptide bond hydrolysis. Peptide bond is a C-N
bond between the carboxyl and amine groups present in all amino acids. After the amino acids
are produced, decarboxylation and deamination are the two main mechanisms occurring in the
hydrothermal environment. Sato et al. (2004) reported the reaction kinetics of amino acid
decomposition under hydrothermal conditions. Five selected amino acids: alanine, leucine,
phenylalanine, serine and aspartic acid were hydrolyzed in a continuous-flow tubular reaction
system as temperatures ranged from 200-340°C with a constant pressure of 20 MPa. The two
25
main reaction pathways were observed: deamination to produce ammonia and organic acids, and
decarboxylation to produce carbonic acid and amines. The Arrhenius equation was used to study
the kinetics of alanine decomposition. It was found the overall decomposition rate of amino acids
can be described to be a first-order reaction and the activation energy for alanine decomposition
is 154 kJ mol-1 (Sato et al., 2004).
Figure 2-3 Hydrolysis of peptide bond [adapted from (Tissot and Welte, 1984)]
Decarboxylation and deamination are beneficial reactions in HTL since they can remove
oxygen and nitrogen content from biomass materials (Toor et al., 2011). However, deamination
could also lead to the loss of hydrogen associated with the nitrogen atom. Therefore, the
comprehensive effects of deamination on the HTL product quality need to be studied. Amino
acids are degraded quickly in hydrothermal conditions. For example, at 350°C more than 70% of
alanine and glycine are degraded less than 30 s and 20 s, respectively (Klingler et al., 2007).
Although ammonia is often the product from deamination, considerable fraction of nitrogen in
the aqueous phase was organic nitrogen compounds when amino acids were converted under
hydrothermal conditions (Dote et al., 1998b; Yu et al., 2011b).
Other than studying the HTL reaction pathways and kinetics by using pure amino acids as
the feedstock, model compounds representing proteins were also used to study the behavior of
proteins in the hydrothermal environment. Rogalinski et al. (2005) used bovine serum albumin
26
(BSA) as the model compound to study the production of amino acids in a continuous sub-
critical water hydrolysis reactor. It was found the highest amino acid yield was obtained at 290°C
with retention time of 65 s. Increasing retention time could lower the optimal temperature but
there was no significant influence of operating pressure. Adding CO2 resulted in higher amino
acid yields due to acid hydrolysis of peptide bonds (Rogalinski et al., 2005). Dote et al. (1996)
used egg albumin as a model protein to study the nitrogen distribution in HTL products. The
bio-crude oil yield from egg albumin (10%) was much lower than that obtained from actual
feedstocks. The nitrogen recovery in the bio-crude oil was also lower (only about 5% of the
nitrogen in the original feedstock is recovered in bio-crude oil) compared with the real biomass
feedstock and majority of nitrogen (80%) was remained in the aqueous phase after HTL (Dote et
al., 1996).
2.4.3 Carbohydrates and lignocellulose

Carbohydrate is a collective name for individual sugars and their polymers, which generally
includes the monosaccharides, oligosaccharides and polysaccharides. The name carbohydrate is
derived from the empirical formula of hydrated carbon, Cn(H2O)n (Tissot and Welte, 1984). All
carbohydrates, including sugars, starches, cellulose, hemicellulose and chitin are fundamentally
polymers of monosaccharides.
Cellulose/Glucose
Cellulose has a structure consisting basically of glucose, linked by β-(1→4)-glycosidic

bonds. Higher plants synthesize the largest amount of cellulose since it is the source of energy
and forms the supporting tissues of plants. Compared to starch, which is a polysaccharide
consisting of glucose monomers bound with α-(1→4) and α-(1→6) bonds, cellulose has a higher
degree of crystallinity because the straight chains in cellulose enable formation of strong intra-
and inter-molecule hydrogen bonds (Delmer and Amor, 1995; Peterson et al., 2008). Therefore,
cellulose is insoluble in water and resistant to attack by enzymes.
In contrast, many algae, such as some seaweeds appear to be devoid of cellulose (Percival,
1966). But some algae, especially brown algae (Phaeophyceae), can contain as much as 40%
(dry weight basis) of alginic acid, which is a carbohydrate derivative. And some bacteria and
higher plants contain pectin, a similar carbohydrate like alginic acid (Tissot and Welte, 1984).
27
Cellulose from different biological sources usually has different properties. From this
aspect, although cellulose can be rapidly solubilized and hydrolyzed under sub- or super-critical
hydrothermal conditions, the constituents of cellulose hydrolysis may vary. As the basic structure
unit of cellulose, glucose is the main product from cellulose hydrolysis. Under hydrothermal
conditions, both water at elevated temperatures and pressures can hydrolyze the β-(1→4)-
glycosidic bond and break up the hydrogen-bound crystalline structure, resulting in the
production of glucose monomers. However, glucose itself is hydrolyzed or degraded under
hydrothermal conditions to produce fragmentation products, such as glycolaldehyde,
pyruvaldehyde and glyceraldehyde, etc. On the other hand, fructose, which is a glucose isomer,
will react to a higher amount of the dehydration product 5-hydrozymethylfurfural (5-HMF)
(Bonn and Bobleter, 1983; Srokol et al., 2004). Therefore, these competing reactions can hinder
high glucose yields from cellulose hydrothermal degradation. Although glucose yield may be
increased by conducting high-temperature, short-time reforming of cellulose, since cellulose
destruction starts to proceed faster than glucose degradation at temperatures roughly at and
above the critical point of water (Mok et al., 1992), its yield could still be limited by the
oligomers produced from some cellulose hydrolyzed because these oligomers cannot be further
hydrolyzed into glucose (Peterson et al., 2008).
The reaction kinetics of cellulose hydrothermal degradation was widely studied in literature
(Adschiri et al., 1993; Mochidzuki et al., 2000; Sasaki et al., 2004; Sasaki et al., 2000; Schwald
and Bobleter, 1989). Most of these studies proposed the first-order Arrhenius kinetics for the
cellulose degradation. However, the activation energies from different studies were considerably
different. For example, in the temperature range of 215°C to 274°C, the activation energy for
cotton cellulose degradation was 129.1 kJ mol-1 (Schwald and Bobleter, 1989). However, the
activation energy for de-ashed microcrystalline cellulose degradation could be as high as
547.9±27.8 kJ mol-1 when the temperature is above 370°C (Sasaki et al., 2004). This significant
difference of activation energy of cellulose degradation probably is due to the different structures
of cellulose, types of reactor, and temperature range used in previous studies. Peterson et al.
(2008) summarizes the results from the previous studies and obtained the activation energy,
which is derived from the best-fit line, to be 215 kJ mol-1 (Peterson et al., 2008).
28
Starch
As mentioned before, starch is a polysaccharide consisting of glucose monomers bound

with α-(1→4) and α-(1→6) bonds. The degradation pathway for starch is similar with that of
cellulose. Nagamori and Funazukuri (2004) conducted hydrolysis of sweet potato starch under
hydrothermal conditions in the temperature range of 180°C to 240°C using a small bomb-type
reactor. The major product was found to be glucose, oligomers having various degrees of
polymerization, maltose, fructose, 5-HMF and furfural. The maximum glucose yield (63% on
carbon basis) was obtained at 200°C with 30 minutes retention time. When the temperature
increased or retention time prolonged, the glucose was hydrothermal degraded to 5-HMF and
other byproducts, and therefore, its yield was decreased (Nagamori and Funazukuri, 2004).
The glucose yield from starch could be significantly increased with the addition of CO2. At
a temperature of 200°C with 15 minutes as the retention time, there was a linear relationship
between the glucose yield from starch hydrolysis and the loaded CO2. The highest glucose yield
(53% on carbon basis) was achieved with 0.32 g CO2 (or 0.106 g CO2 per g H2O) loaded
(Miyazawa and Funazukuri, 2005). The reason that addition of CO2 can increase glucose yield
was because it lowered the pH value of the water, therefore, the carbonic acid enhances the
hydrolysis of starch.
Hemicellulose/Xylose
Hemicellulose is a heteropolymer composed of various monosaccharides, such as xylose,

mannose, glucose, galactose, and others, which also have side chains. Hemicelluloses can make
up 20%-40% of the potential raw plant materials (Maki-Arvela et al., 2011; Toor et al., 2011).
The compositions of hemicellulose in different plant types are usually different. For example, the
most important hemicelluloses in softwoods are galactoglucomannans (20%) and
arabinoglucurunoxylans (10%), while the most important hardwood hemicellulose is xylan,
especially O-acetyl-4-O-methylglucuronoxylan, making up to 80-90% of the hardwood
hemicelluloses. Xylan is also the main hemicellulose in agricultural waste (Garrote et al., 1999).
Gum arabic, consisting of a highly branches polysaccharide β-(1,3)-linked galactose backbone
with branches of arabinose and rhamnose, is the main hemicellulose in plant gums (Maki-Arvela
et al., 2011).
29
Hemicellulose has a much lower degree of crystallinity than cellulose due to the abundance
of side-groups and less uniform structure and it is easily solubilized and hydrolyzed in water
when the temperature of the water is above 180°C with acidic and basic catalysts presences
(Bobleter, 1994). Hemicellulose can be easily extracted as monomeric under hydrothermal
conditions with a temperature range of 200-230°C and a retention time range of several minutes
(Mok and Antal Jr, 1992). Previous studies have reviewed the reaction kinetics for hemicellulose
degradation into sugars and subsequent degradation of the sugars into furfurals and other
degradation products (Garrote et al., 1999; Maki-Arvela et al., 2011).
Xylose is a five-carbon sugar and is one of the most common monosaccharide residues
contained in hemicellulose. It is a very important feedstock for producing furfural. Industrially,
most of the furfural (2-furaldehyde) is produced from hemicellulose-derived xylose (Peterson et
al., 2008). Xylose can exist in water as a pyranose ring, a furanose ring or as an open-chain
structure. Under hydrothermal conditions, the furanose ring was relatively stable, whereas
furfural can be produced from the pyranose ring form of xylose; at the same time the open-chain
form of xylose can be degraded into fragmentation byproducts in furfural production, such as
glyceraldehyde, pyruvaldehyde, lactic acid, glycolaldehyde, formic acid, and acetol (Antal et al.,
1991). However, in acidic solutions, furfural can be produced from both the open-chain and the
pyranose ring forms of xylose (Nimlos et al., 2006). Although furfural can also degrade under
hydrothermal conditions, it was found the rate for furfural degradation is much lower than that of
furfural production from xylose (Jing and LÜ, 2007). Therefore, furfural can be accumulated
during the hydrothermal pretreatment of lignocellulose and becomes the inhibitor for the bio-
ethanol fermentation process. Other than producing furfural, xylose can also be degraded into
aromatic compounds in the acidic solution under hydrothermal conditions (Nelson et al., 1988).
Lignin
Together with cellulose and hemicellulose, lignin constitutes one of the three major
components of lignocellulosic biomass and it is a complex high molecular-weight compound.
Lignin is a complicated polymer with a three-dimensional amorphous structure and the structure
varies with different sources. But the three most prevalent monomers in lignin include p-
coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Peterson et al., 2008). The carbon and
oxygen content in lignin are typically more than 60% and 30%, respectively. Although in the
30
plant materials the amount of lignin is smaller than cellulose, it can represent about 50% of the
available combustible energy in naturally occurring sources (Glasser, 1985).
Due to the complexity of lignin structure, it is relatively resistant to chemical or enzymatic
degradation (Bobleter, 1994). However, lignin can be degraded under hydrothermal conditions to
produce phenols and methoxy phenols by hydrolysis of ether-bonds in lignin. At the same time,
the benzene rings in lignin seem to be stable under hydrothermal conditions (Toor et al., 2011).
Lignin with other mixtures or the biomass feedstock containing lignin were widely used to
produced valuable chemicals (Quitain et al., 2003; Wu et al., 1994; Xiang and Lee, 2001), bio-
crude oil (Funazukuri et al., 1990; Karagöz et al., 2006; Karagoz et al., 2004; Saisu et al., 2003)
and gas product (mainly methane and hydrogen) (Furusawa et al., 2007; Osada et al., 2007; Sato
et al., 2006; Yoshida and Matsumura, 2001) via hydrothermal process. However, studies on
hydrothermal degradation of pure lignin are not as common as those of cellulose (Toor et al.,
2011). In a batch reactor, a pure model compound for lignin, guaiacol, was treated in
supercritical water with the temperature ranging from 380-400°C and 25-40 MPa pressures under
an argon atmosphere. It was found the main degradation products in the aqueous phase included
40.73 wt% catechol, 14.18 wt% phenol and 4.45 wt% o-cresol. First-order Arrhenius kinetics
was derived from the results and the activation energy for guaiacol decomposition was estimated
to be 39.35 kJ mol-1. However, the dehydration reactions of some low molecular weight
compounds resulted in the char formation (Kanetake et al., 2007). It should be noted that there
was no oil-phase products produced during this HTL study, and the results were consistent with
another study, which found the aqueous product yield was between 58%-79%, whereas the solid
residue yield was between 12%-37% when Kraft pine lignin was used as the feedstock for
hydrothermal treatment (Zhang et al., 2008).
Since the char formation is one of the important pathways in lignin degradation under
hydrothermal treatment, some studies found there were strong correlation between the solid
residue yield and the lignin content in the biomass feedstock (Demirbaş, 2000a; Minowa et al.,
1998). Especially, with the slow heating and cooling the solid residue yield could be increased
(Karagoz et al., 2005).
31
2.5 MIMICKING MOTHER NATURE’S OIL FORMATION VIA HTL
2.5.1 Formation of conventional petroleum

Two kinds of theories were used to describe the formation of the fossil fuels: the biogenic
theory and the abiogenic theory. The abiogenic theory claims that oil forms from the reduction of
primordial carbon or its oxidized form at elevated temperatures deep in the earth (Porfir'ev,
1974). However, the overwhelming geochemical and geological evidences from both sediment
and petroleum studies clearly indicates most of the petroleum originated from organic matter
buried with the sediments in sedimentary basins (Hunt, 1996).
Figure 2-4 shows the origin and mature of petroleum, which is adapted and modified
according to previous studies (Hunt, 1996; Marshak, 2008). It should be pointed out that only
about 10%-20% of the petroleum is directly formed from the hydrocarbons in organisms, while
the rest was formed from kerogen, which was produced from lipids, proteins and carbohydrates.
PLANKTON & ALGAE
Sink and accumulate in O2-poor water
Hydrocarbons & Lipids, proteins,

proto-hydrocarbons carbohydrates
DIAGENESIS
Biological degradation and low-temperature chemical reactions (15 C~50 C)
Increasing 50 C~80 C KEROGEN

Temperature &
Pressure
BITUMEN
High-temperature
Chemical Reactions
(80 C~120 C) CATAGENESIS
PETROLEUM
(Maturation)
Light oil & gas Heavy oil
Gas ~200 C Pyrobitumens
METAGENESIS
Methane ~250 C Graphite
Figure 2-4 Origin and maturation of petroleum [adapted from (Hunt, 1996)]
32
From the formation pathway of petroleum, it is assumed that the organic compounds in
organisms (mainly algae and other plankton) are degraded by bacteria first and then those
degraded materials are converted into kerogen via chemical reaction at low temperature. When
kerogen is formed, it is further converted into petroleum at a relatively higher temperature. It
should be noted that when the temperature is about 200°C, petroleum might be further converted
into gas and graphite.
Table 2-4 compares the elemental compositions of fossil fuels, kerogen, natural substances,
two algal species used in this study and one representative of high-lipid microalga (Botryococcus
braunii). Kerogen is one of the most important origins of petroleum. The hydrogen content of
kerogen can determine the petroleum quality (Hunt, 1996). However, although the hydrogen
content in petroleum is higher than that in kerogen, the difference of carbon content between
kerogen and petroleum is very small. On the other hand, nitrogen, sulfur and oxygen contents in
petroleum are much lower than those in kerogen. This indicates that increase of hydrogen
content in petroleum is related to substituting nitrogen, sulfur and oxygen by hydrogen.
Table 2-4 Elemental composition of fossil fuels, kerogen, natural substances and algae
Elemental composition (% of dry weight)
C H N S Oa
Petroleum 85 13 0.5 1 0.5
Natural Gas 76 24 n.a. n.a. n.a.
Coal 83 5 1 1 10
Asphalt 84 10 1 3 2
Kerogen 79 6 2 5 8
Carbohydrates 44 6 n.a n.a. 50
Proteins 53 7 17 1 22
Lipids 76 12 n.a. n.a. 12
Lignin 63 5 0.3 0.1 31.6
Chlorella pyrenoidosa 51.4 6.6 11.1 0.7 30.2
Spirulina platensis 49.3 6.4 11.0 0.8 32.5
Botryococcus braunii b 63.1 11.7 2.8 n.a. 22.4
a b
Calculated by difference; (Dote et al., 1994); n.a. Not available
33
The elemental composition of the two algal species used in this study indicates that a
substantial amount of oxygen and nitrogen should be removed if algae are the main origins to
form fossil fuel. The high-lipid microalga Botryococcus braunii has higher carbon and hydrogen
content, but lower nitrogen content than the two low-lipid microalgal strains used in this study.
Both the oxygen and nitrogen contents in kerogen are much lower than those in natural
substances and algae. Since kerogen is generated at a low temperature via both biological and
chemical reaction according to Figure 2-4, it could be speculated that either the lipids, which has
almost no nitrogen and relatively low oxygen is released under this temperature and then further
converted into kerogen, or a considerable fraction of nitrogen and oxygen in other components in
algae are removed under this process.
From the comparison of elemental composition, lipids can be converted to petroleum by
losing a small amount of oxygen, whereas a large amount of oxygen needs to be removed from
carbohydrate, lignin and even protein to form petroleum. Although all of the natural substances
listed in Table 2-4 may be the origins of petroleum, less than 1% of the organic matter deposited
in sediments over geologic time is required to form all the known petroleum deposits, or in other
words, the lipid content of all forms of living matter is more than enough to account for the
origin of oil (Hunt, 1996). It is found that the nitrogen content in most of the natural substances,
such as carbohydrates, lipids and lignins were extremely low. Therefore, the nitrogen in kerogen
is mainly originated from proteins, because the building blocks of protein are amino acids and
they are joined together by the peptide bonds. From this aspect, in the natural process of
petroleum formation, the deoxygenation and denitrogen process should both be involved, and
perhaps is the major pathway of fossil fuel formation.
Nitrogen is ubiquitous in petroleum but only as small amount. About 90% of crude oils
contain less than 0.2% of nitrogen and the average nitrogen fraction in crude oils is 0.094% by
weight. The nitrogen compounds is usually found in the high molecular weight and high boiling
point fractions in the crude oil (Tissot and Welte, 1984). Typically, the nitrogen compounds can
be classified into neutral pyrrolic types and basic pyridinic type. Most of the nitrogen in
petroleum is present as heterocyclic aromatic structures (Baxby et al., 1994; Richter et al., 1952;
Snyder and Buell, 1965). Since the composition of plankton and algae is predominantly protein
(>50%), amino acids constitute the principal nitrogen-bearing structures in living organisms.
Although Snyder proposed that many of the alkylcarbazoles and alkylbenzocarbazole in
34
petroleum may be derived from plant alkaloids (Snyder, 1965), a later study (Li et al., 1992)
showed that plant alkaloids could not be the major source of pyrrolic nitrogen compounds and
proteins or pigments may also be the origin of the nitrogen compounds in petroleum. There is no
agreement about how the nitrogen becomes incorporated into the macromolecular organic matter
in sediments during early diagenesis (Bennett et al., 2004).
2.5.2 Similarities between HTL and natural geochemical processes

As discussed in the last section, there are several prerequisites for nature’s process of
petroleum formation: abundant of organisms (primarily from plankton and algae) as the
feedstock, oxygen-poor environment and increasing temperature and pressure to provide the
reaction environment for the biological and chemical reactions.
Table 2-5 compares the parameters of HTL and the natural process of petroleum formation.
In the natural process, the formation of petroleum can be divided into three stages: Diagenesis,
Catagenesis and Metagenesis. The product from a previous stage is the feedstock of the next one.
Obviously, the temperature of HTL is more intensive than the natural process; therefore, the
retention time for producing bio-crude oil could be shortened from the geological time scale to
less than several minutes to hours. If algae are chosen as the feedstock, HTL could mimic
nature’s millions-of-years process of turning deceased living matters buried beneath the ground
into petroleum, and it only takes less than 2 hours.
Table 2-5 Comparison of HTL and natural process of oil formation
HTL Natural process

Diagenesis Catagenesis Metagenesis
 Algae
 Plankton
Feedstock  Biowaste Kerogen Petroleum
 Algae
 Lignocellulose
 Kerogen  Methane
Product Bio-crude oil Petroleum
 Methane  Graphitic carbon
Temperature 200~350°C 15~50°C 50~200°C >200°C
Pressure 20~30MPa <30MPa 30~150 MPa >150MPa
 Microbial activity
Thermochemical
Reaction type  Chemical Chemical conversion Thermal cracking
Conversion
rearrangement
Retention time 10~120 minutes Geological time for petroleum formation ranges from 5~100 million years
35
Claiming to use HTL to mimic oil formation in nature may sound too idealistic and
ambitious, given that the three stages in the natural process of oil formation happen in tandem
and the conditions for each stage are different. Nevertheless, HTL does provide such an
opportunity for researchers to investigate the oil formation pathways by mimicking nature’s
petroleum formation process. We hope this is enough of a justified reason to keep studying HTL,
especially using abundant biomass such as algae and biowaste as feedstocks.
36
CHAPTER 3. OBJECTIVES
The ultimate goal of this study is to provide scientific justifications to develop a viable
technology to convert fast-growing, low-lipid content microalgae into bio-crude oil via a
hydrothermal liquefaction (HTL) process in the future. Two algal species (Chlorella pyrenoidosa
and Spirulina platensis) were chosen as representative low-lipid content algal feedstocks in this
study based on the results from the property screening and the availability.
Although the effects of operating conditions on converting some microalgal species into
bio-crude oil were extensively studied, research reported in the literature has been primarily
focused on high-lipid content algal species. The reaction mechanisms of HTL remain unclear for
the low-lipid content algae. Reaction pathways and kinetics of algae conversion under
hydrothermal conditions are still largely uncharacterized, even though reaction mechanisms of
model compounds under hydrothermal conditions were copiously documented in the literature.
The lack of study is primarily due to extremely complicated interactions among the reaction
intermediates, which are produced from different components in the real biomass. Available
mechanisms and kinetics are probably only suitable for pure model compounds with relatively
simpler molecular structures. It is necessary to investigate the reaction pathways and kinetics of
typical biomass materials, which are low-lipid content microalgae in this study.
To achieve the research goal, the following objectives were established for this study:
1. Determine representative low-lipid content algal species for this HTL study by screening
available algal species based on the bio-crude oil quality and conversion efficiency in the
preliminary HTL research.
2. Investigate the effects of operating conditions, including reaction temperature, retention
time and initial pressure on HTL product yields and bio-crude oil quality.
3. Examine the effects of catalysts and conduct preliminary experimental evaluation of
selected catalysts on the bio-crude oil conversion efficiency and bio-crude oil quality.
4. Study the elemental distribution and energy balance in the HTL process. The distribution of
essential elements, such as carbon and nitrogen, will not only provide support to improve
oil conversion efficiency, product upgrade or refinery technologies and developing
associated wastewater treatment, but also offer essential information for the life-cycle
assessment of the entire HTL process.
37
5. Characterize HTL products to better understand the reaction mechanisms so as to predict
the reaction pathways in the HTL process under different operating conditions.
Characterization of bio-crude oil includes boiling points distribution, higher heating values
and molecular identification. Analyzing the organic compounds in aqueous product is also
necessary to explore the reaction mechanisms.
6. Explore the reaction kinetics and mechanisms of HTL of microalgae. This objective will be
realized based on the information obtained from Objectives 1-5. The outputs include the
kinetic model and pathways of essential components, such as protein, lipid and
carbohydrates in algae, under hydrothermal conditions.
38
CHAPTER 4. EXPERIMENTAL DESIGNS AND PROCEDURES
4.1 ALGAL FEEDSTOCKS
Both commercially produced and naturally grown algal species (collected from open ponds,
recreational areas and wastewater treatment plants) were investigated in this study. Two kinds of
commercialized produced microalgae were finally chosen as the feedstock. Chlorella
pyrenoidosa (green microalgae) was purchased from a health food store and Spirulina platensis
(cyanobacteria) was provided by algae farm companies collaborating with our research group.
The results of the screen tests are reported in CHAPTER 5.
Before conducting the HTL experiment, the characteristics of the feedstock were analyzed.
The dry solid content and ash content of algae were analyzed as the dry residue at 105ºC and the
combustion residue at 600ºC. The crude protein was measured using the Kjeldahl method and the
crude fat was measured using Soxhlet extraction method. The other chemical compositions of
alga, such as acid detergent fiber, neutral detergent fiber and lignin were analyzed using the
methods of the Association of Official Analytical Chemists (AOAC). The elemental composition
of the algae was measured using a CHN analyzer (CE-440, Exeter Analytical Inc., North
Chelmsford, MA). Table 4-1 summarizes the analytical data. The crude fat in the table represents
the lipid content of microalgae.
Table 4-1 Characteristics of algal powder (% dry weight except as noted)
Properties C. pyrenoidosa S. platensis

a
Moisture content (wt %) 6.3 6.5
Dry solid content (wt %) a 93.7 93.5
Volatile solid content 94.4 90.5
Ash content 5.6 9.5
Chemical composition
Crude protein 71.3 64.4
Crude fat 0.1 5.1
Acid detergent fiber 0.5 0.7
Neutral detergent fiber 1.0 2.1
Lignin 0.2 0.2
Non-fibrous carbohydrate b 22.0 18.9
Elemental composition
C 51.4 49.3
H 6.6 6.4
N 11.1 11.0
Ob 30.9 33.3
a b
On the total weight basis; Calculated by difference
39
4.2 HYDROTHERMAL LIQUEFACTION PROCEDURE
4.2.1 Reactor types and experiment procedure

The experiment of HTL was performed according to previously reported methods (Dong,
2008). Two types of reactors were used in this study:
4534 2 L batch reactor
The 4534 Reactor is a stainless steel cylinder-shaped reactor with a magnetic drive stirrer
(Model 4534, Parr Instrument Co., Moline, IL) in a batch mode. Figure 4-1 shows a picture of
the 4534 floor stand reactor. The volume of the reactor vessel is 2000 mL and the maximum
pressure is 13.1MPa (1900 psi) at 350°C. A 1500W heater associated with the controller was
used to heat the reactor during the experiment. An internal cooling coil was equipped to cool the
reactor by running water through it. The operating pressure in this study was monitored and
indirectly controlled by controlling the temperature. A digital tachometer is used to monitor the
agitation speed of the impeller, which is controlled through the controller panel.
Figure 4-1 Picture of the 4534 floor stand reactor
In a typical test, tap water was mixed with algae powder to make a slurry feedstock
containing 20% dry matter and 80% water by weight, and 800 g of the slurry feedstock was
loaded into the reactor. After the reactor was sealed, nitrogen was added into the reactor to purge
40
residual air out through the gas outlet. This procedure was repeated three times before nitrogen
was built to a predetermined initial pressure inside the reactor. The reactor was then heated up to
the designated experimental temperature. The heating period usually takes about 20-50 minutes.
Then the temperature was kept constant for the designated retention time. At the end of the
reaction, the reactor was rapidly cooled by running water through the cooling coil inside the
reactor. When the temperature in the reactor returned to room temperature, the gas phase in the
reactor was carefully released through a control valve and collected into the Tedlar® gas
sampling bags for further analysis.
4593 100 mL batch reactor
For studying the effects of the catalysts on HTL of microalgae, a smaller batch reactor was
used. The 4593 reactor is a stainless steel cylinder-shaped reactor of 100 mL capacity with a
magnetic drive stirrer and moveable vessel (Model 4593, Parr Instrument Co., Moline, IL). An
aluminum block heater with cooling channel was used to heat and cool the reactor during the
experiment. The maximum pressure for this reactor is 34.5 MPa (5000 psi) at 350°C. Figure 4-2
shows a picture of the 4593 reactor. During the cooling period, tap water is run through the water
cooling channel associated with the aluminum block heater. It was found that the ways to cool
the two types of reactors were different from each other. For the 2 L reactor, the cooling water
coil is directly contacting the reacting materials inside the reactor, whereas for the 100 mL
reactor, the reactor is cooled down using the cooling water channel contacting the outside surface
of the reactor.
In a typical test, Nanopure® water was mixed with algae powder to make a slurry feedstock
containing 30% dry matter and 70% water by weight, and 70 g of the slurry feedstock was
loaded into the reactor. Nanopure® water is used to eliminate any effects caused by minerals in
the tap water. After the reactor was sealed, nitrogen was added into the reactor to purge residual
air out through the gas outlet. This procedure was repeated three times before nitrogen was built
to a predetermined initial pressure inside the reactor. The reactor was then heated up to the
designated experimental temperature, which took about 20-50 minutes. Then the temperature
was kept constant for the designated retention time. At the end of the reaction, the reactor was
rapidly cooled by running water through the cooling channel associated with the aluminum block
heater outside the reactor. When the temperature in the reactor returned to room temperature, the
41
gas phase in the reactor was carefully released through a control valve and collected into the
Tedlar® gas sampling bags for further analysis.
Figure 4-2 Picture of the 4593 floor stand reactor
4.2.2 Recovery procedures of HTL products

After the gas was removed from the reactor, the reactor was opened and the remaining
mixture was separated and analyzed in the lab. Most of the raw oil can be self-separated from the
reaction mixture. Some raw oil droplets mixed with water can be separated from the aqueous
phase by filtration. The recovery procedure is shown in Figure 4-3. The moisture content in the
raw oil phase was determined using a distillation apparatus based on ASTM Standard D95-99
(ASTM, 2004a). Solid residue content of the raw oil product was measured using Soxhlet
extraction, according to ASTM Standards D473-02 (ASTM, 2004b) and D4072-98 (ASTM,
2004c).
Based on the product recovery procedure outlined above, the feedstock included both water
and dry matter of algae. Excluding the moisture content, the water insoluble product was defined
as raw oil. The toluene soluble fraction of raw oil is defined as bio-crude oil in this study.
42
Feedstock
Hydrothermal Liquefaction
Reaction Mixture
Filtration
Water Insoluble
Moisture
Measurement
Aqueous Aqueous
Raw Oil Product 2 Product 1
Toluene
Extraction
Gas Bio-crude Solid Aqueous

Product Oil Residue Product
Figure 4-3 Recovery procedures for products from a hydrothermal liquefaction
Table 4-2 lists the measurements and symbols used in the product recovery process and
mass distribution analysis. After the gas product is collected, it is analyzed using Varian CP-
3800 Gas Chromatography equipped with an Alltech Hayesep D 100/120 column and a thermal
conductivity detector (TCD). The carrier gas was helium with a flow rate at 30 ml min-1. The
temperature of both the injector and detector were 120°C.
43
Table 4-2 Measurements and equations for calculating product yields
Measurement (g) Symbol

Mass of Feedstock F
Mass of dry matter of microalgae C
Mass of reaction mixtures RM
Mass of water insoluble product WI
Mass of moisture in the water insoluble product MWI
Mass of toluene insoluble fraction in raw oil SR
Product Equation
F  RM
Gas yield (%)   100
C
WI  MWI
Raw oil yield (%)   100
C
SR
Solid residue yield (%)   100
C
WI  MWI  SR
Bio-crude oil yield (%)   100
C
RM  WI  MWI  (F  C)
Aqueous product yield (%)   100
C
SR
Toluene solubility (%)  (1  )  100
WI  MWI
4.3 EXPERIMENTAL DESIGN
The key operating parameters in the HTL process include reaction temperature, reaction
time (retention time), catalyst/additives dosage, and initial pressure of process gas. The effects of
these operating parameters on HTL product yields and characteristics are essential for
understanding the reaction mechanisms of HTL. In this study, the effects of operating conditions
and catalysts are both investigated.
4.3.1 Effect of temperature

Reaction temperature is the designated experimental temperature. Theoretically, the heating
and cooling process of the reactor should be as short as possible to eliminate the byproducts such
as the char production. However, due to the capacity of the heater for the batch reactor and the
heat transfer limitation, the long heating time problem occurs in all of the HTL experiments.
Heating periods range from 20-50 minutes during which the temperature was increased from
room temperature to the designated reaction temperature. One of the alternative ways to shorten
the heating time is to use a small reactor and a sand/salt bath instead of a traditional furnace
heater (Brown et al., 2010). However, measuring and monitoring the temperature in those small
reactors are very difficult; therefore, most of the previous studies assumed the temperature inside
44
the reactor would reach the equilibrium with the sand/salt bath in a short time, which may
introduce some uncertainties.
Figure 4-4 shows the profile of the temperature and pressure during a typical HTL
experiment. It can be found that the entire experimental process can be divided into three periods:
heating period, reacting period and cooling period. For the heating period, it usually takes 20 to
50 minutes to increase the temperature to the designated temperature. This limitation is due to
the heating capacity of the heater and cannot be completely avoided due to the current reactor
design.
Reaction Temperature
Heating Reaction Time Cooling
Figure 4-4 Profile of temperature and pressure during an HTL experiment
Therefore, for investigating the effects of temperature, two series of experiments were
performed.
(1) Effects of reaction temperature with the same retention time
For investigating the effects of reaction temperature, HTL tests were conducted with the
same retention time (30 minutes) but with a different reaction temperature for both C.
45
pyrenoidosa and S. platensis. Table 4-3 shows the details of the experimental design. Note that in
each test, pure nitrogen gas was used to purge the residue air inside the reactor and then build the
initial pressure of the reactor to 0.69 MPa (gauge pressure) before starting to heat the reactor.
Table 4-3 Experimental design for effects of reaction temperature
Reaction temperature (°C) Reaction time (minutes)

100 30
120 30
140 30
160 30
180 30
200 30
220 30
240 30
260 30
280 30
300 30
(2) Effects of the heating period

Since the reaction of bio-crude oil formation may occur during the heating period of the
HTL experiments, it is necessary to investigate how the heating period affects the products yield.
One approach to investigate the effect of the heating period is to study how different reaction
temperatures with zero retention time can affect the HTL process. Zero minutes of retention time
means that as soon as the feedstock reached the designated reaction temperature, the batch
reactor was immediately cooled down, with no additional retention time. Studying the HTL
product yields with zero minute retention at different temperatures is helpful to estimate the
accumulated effects of the entire heating period. Table 4-4 shows the experimental design.
Table 4-4 Experimental design for effects of heating period
Reaction temperature (°C) Retention time (minutes)

180 0
200 0
220 0
240 0
260 0
280 0
300 0
46
4.3.2 Effect of retention time
The retention time used in this study refers to the elapsed time starting from the time when
the reaction temperature is first achieved to the time the reactor starts to be cooled down. Effects
of retention time are investigated at different reaction temperature levels. Table 4-5 summarizes
the experimental design. It should be noted that this experimental design has covered the
experimental design for studying the effects of the heating period.
Table 4-5 Experimental design for effects of reaction time
Reaction temperature (°C) Reaction time (minutes)

200 0
200 10
200 30
200 60
200 120
220 0
220 10
220 30
220 60
220 120
240 0
240 10
240 30
240 60
240 120
260 0
260 10
260 30
260 60
260 120
280 0
280 10
280 30
280 60
280 120
The chosen reaction temperature ranges from 200ºC to 280ºC. The reason to choose this
range is to primarily focus on temperatures at which bio-crude oil could be produced. Based on
our preliminary results, bio-crude oil formation of C. pyrenoidosa could occur at a reaction
temperature of 200ºC. Therefore, we chose the temperature range from 200ºC to 280ºC to study
the impacts of reaction time since the reaction has already started at these temperatures, but
proceeds a little slower making it easier to observe the influence of retention time. At higher
temperatures, the reaction could proceed too fast to isolate differences due to retention time.
47
4.3.3 Effect of initial pressure
The purpose of using nitrogen as process gas includes: (1) purge the residual air out from
the reactor; and (2) provide initial pressure to the HTL to prevent the intensive water boiling
process during the experiment, which usually is an energy intensive process. In this study, the
effects of initial pressure provided by nitrogen were investigated at an operating condition that
the formation of bio-crude oil already occurs (280°C, 30 minutes). Table 4-6 summarizes the
experimental design.
Table 4-6 Experimental design for effect of initial pressure of N2
Reaction temperature (°C) Retention time (min) Initial pressure of N2 (MPa)

280 30 0.000
280 30 0.138
280 30 0.276
280 30 0.414
280 30 0.552
280 30 0.689
4.3.4 Effects of catalysts/additives on HTL process

Effects of homogeneous catalysts and heterogeneous catalysts on bio-crude oil conversion
efficiency and oil quality are both investigated in this study. Sodium carbonate and sodium
hydroxide are chosen as the representatives for the homogeneous catalyst. Metal catalysts such
as Raney nickel, Pd, Pt are chosen as the representatives of the heterogeneous catalyst.
Effects of homogeneous catalysts
(1) Sodium carbonate (Na2CO3)

Alkaline metal carbonates, such as Na2CO3 were widely used in HTL of different biomass
feedstocks to produce bio-crude oil (Table 2-2 & Table 2-3). Appell (1977) proposed the
following mechanism for sodium carbonate-catalyzed conversion of carbohydrate to bio-crude
oil (Appell, 1977):
i. Reaction of sodium carbonate and water with carbon monoxide to yield sodium formate:
ii. Dehydration of vicinal hydroxyl groups in a carbohydrate to an enol, followed by

isomerization to ketone:
48
iii. Reduction of newly formed carbonyl group to the corresponding alcohol with formate ion
and water:
iv. Then finally the hydroxyl ion reacts with additional carbon monoxide to regenerate the
formate ion:
In this reaction mechanism, sodium carbonate or other carbonates can act as catalysts for
hydrolysis of macromolecules into smaller fragments. Sodium carbonate reacts with carbon
monoxide, which could be produced during the HTL process. Another study found reaction (i)
could proceed without an addition of carbon monoxide (Ogi et al., 1985). Since organic
compounds with vicinal hydroxyl groups could be the key reactants in this mechanism, many
studies used sodium carbonate as a catalyst for HTL of lignocellulosic biomass (Demirbaş, 1998;
Elliott, 1980; Minowa et al., 1995b; Nelson et al., 1984; Qian et al., 2007).
From Table 2-3, it was found sodium carbonate was also widely used in hydrothermal
liquefaction of microalgae to produce bio-crude oil. Since many algal species contain
carbohydrates, sodium carbonate could also be helpful in converting carbohydrates in algal
feedstock into bio-crude oil. From the characteristics of the algal powder used in this study, the
carbohydrate contents of C. pyrenoidosa and S. platensis are 22.0% and 18.9%, respectively
(Table 4-1). Therefore, it is necessary to investigate whether sodium carbonate can improve the
bio-crude oil yield in the HTL process in this study.
49
(2) Sodium hydroxide (NaOH)
The two algal species chosen for this study both have very high protein contents (Table 4-1).
It is reasonable to expect the primary source material for the production of bio-crude oil to be
protein and it is necessary to investigate the pathway of protein in the HTL process. The building
blocks of protein are amino acids, which are joined together by the peptide bonds. The general
structure of an amino acid is shown in Figure 4-5.
There are two functional groups in an amino acid molecule: amine group and carboxyl
group. Amino acids and other short chain organic acids can be produced through protein
hydrolysis during the HTL process. Then two processes, deamination and decarboxylation could
occur and remove the nitrogen and oxygen content from the original organic compounds.
Figure 4-5 General structure of amino acids
Deamination is a process to remove nitrogens atom from organic compounds with amine
groups. The end-product of the nitrogen is expected to be ammonia. The process of deamination
is illustrated below:
Decarboxylation is a process to remove carbon atoms from organic compounds with

carboxylic groups. In this process, oxygen atoms are also removed at the same time and form
carbon dioxide with the removed carbon atoms. The decarboxylation process is illustrated below:
50
From the two reaction pathways of amino acids under HTL conditions, some of the reaction
intermediates from deamination and decarboxylation could react to each other to form
complicated organic compounds. On the other hand, some products could further decompose into
small molecules. Those molecules from recombination or decomposition could form the bio-
crude oil in the HTL process.
Decarboxylation could be enhanced in the presence of alkali hydroxide catalysts, such as
NaOH and KOH (Watanabe et al., 2006). It is possible that decarboxylation will be favorable in
alkaline conditions so that it is worth studying the effects of strong alkali on HTL of C.
pyrenoidosa and S. platensis, which are both high-protein content microalgae. Based on this
consideration, NaOH is chosen to represent the strong alkali and its effect on the HTL process is
investigated.
Effects of heterogeneous catalysts
Metal catalysts are used for upgrading or refining the bio-crude oil from the HTL (Elliott,
2007; Grange and Vanhaeren, 1997) with the presence of hydrogen. However, decarboxylation
could also be enhanced by metal oxides (Watanabe et al., 2006) and metal with catalyst supports
such as Al2O3, silica-alumina and activated carbon (Mäki-Arvela et al., 2008; Ping et al., 2010;
Simakova et al., 2010). Decarboxylation and deoxygenation of fatty acids and amino acids in a
hydrothermal media were widely studied (Fu et al., 2010; Fu et al., 2009; Li and Brill, 2003a; Li
and Brill, 2003b). Study of investigating the effects of metal catalysts on HTL of high-protein
microalgae is sparse in literature. Therefore, it is necessary to explore their effects on producing
bio-crude oil from the high-protein algal species under the hydrothermal conditions.
In this study, the effects of different commercialized metal catalysts, such as Raney nickel,
Pd and Pt, on the HTL process were investigated. HTL tests with different catalysts at different
reaction temperatures were performed. Since catalysts do not change the reaction equilibrium but
lower the activation energy of favorable reactions, the catalysts effects at low and high
temperatures, at which the bio-crude oil formation already occurs, are both investigated. Based
on our preliminary results (CHAPTER 5), bio-crude oil formation of both C. pyrenoidosa and S.
platensis could occur under the reaction temperature of 240C. Therefore, 240C and 280C
were chosen as the two temperature levels to study the effects of the catalysts.
51
Table 4-7 summarizes the experimental design to investigate the effects of homogeneous
and heterogeneous catalysts/additives. The dosage of catalysts is 5% based on the total dry mass
of feedstock.
Table 4-7 Experimental design for effect of catalysts/additives
Reaction time Catalyst

Reaction temperature (°C)
(min) (5% of dry weight of algae)
30 Pd/Al2O3
30 Pd/C
30 Pt/Al2O3
240 30 Pt/C
30 Raney Ni
30 NaOH
30 Na2CO3
30 Pd/Al2O3
30 Pd/C
30 Pt/Al2O3
280 30 Pt/C
30 Raney Ni
30 NaOH
30 Na2CO3
4.3.5 Elemental balance of HTL process

Bio-crude oil from HTL usually needs to refining or upgrading to be converted into
transportation fuel as the oxygen and nitrogen contents of the product are higher than the
conventional petroleum (He et al., 2000a; Minowa et al., 1995a; Peterson et al., 2008). Another
study reported during the HTL process, that only around 40% of carbon and 35% of hydrogen in
algal feedstock were converted into oil, which means most of the organic compounds in the
feedstock were converted into water soluble and gas products (Yang et al., 2004). Although most
of the nitrogen in the feedstock was converted into the water soluble products via HTL (Dote et
al., 1996), the nitrogen content in bio-crude oil derived from protein can still influence the
quality of the oil, thus the concerns of NOx emission to the atmosphere were raised. The carbon
and nitrogen remaining in the aqueous product after the HTL cannot be discharged into the
environment without further treatment. On the other hand, the nutrients in post-HTL water can
be used to grow algae. So study of the elemental balance of HTL process is very useful for
tracing the footprints of different elements and providing information for the life-cycle
assessment (LCA) of HTL.
52
From the product recovery procedure of HTL (Figure 4-3), the feedstock is converted into
four kinds of products via HTL: bio-crude oil, solid residue, aqueous product, and gaseous
product. Carbon dioxide contributes more than 98% of the total mass for the gas product. On the
other hand, most of the nitrogen and phosphorous in the algal feedstock remain in the aqueous
phase. Both the carbon in the gas and the nutrients in the aqueous phase can be utilized to grow
algae if they are recycled after the HTL process. Through this recycle process, environmental
quality is enhanced because carbon dioxide and nutrients in the HTL gaseous and aqueous
products are not emitted into the environment but utilized for algal biomass production, which
can provide additional feedstock to produce bio-crude oil.
This idea of energy production is named as Environment-Enhancing-Energy (E2-Energy)
and the roadmap of E2-Energy is illustrated as Figure 4-6.
Figure 4-6 Environment-Enhancing-Energy (E2-Energy) technology roadmap
For investigating the carbon and nutrient recycles, carbon, hydrogen and nitrogen content of
the feedstock and HTL products were analyzed using a CHN analyzer (CE-440, Exeter
Analytical Inc., North Chelmsford, MA), and the oxygen content were calculated by difference.
Two parameters, carbon recovery (CR) and nitrogen recovery (NR) are used to reflect the
distributions of the two essential elements in HTL products. The equations for calculating carbon
recovery and nitrogen recovery are listed below:
mass of product  C% of the product
Carbon Recoveries of Products (%)   100
mass of carbon in dry matter of feedstock
53
mass of product  N% of the product
Nitrogen Recoveries of Products (%)   100
mass of nitrogen in dry matter of feedstock
4.3.6 Characterization of HTL products
Simulated boiling point distribution of bio-crude oil
The toluene soluble fraction of the raw oil product is defined as bio-crude oil. The toluene
dissolved bio-crude oil sample was filtered using Whatman® 0.45 µm filter to remove any
suspended particulates. The boiling points distribution of the bio-crude oil sample was simulated
using ASTM D7169-05 method (ASTM, 2005) and performed using a HP 5890 Series II FID gas
chromatograph and a Durabond DB-HT-SimDis GC column by Agilent-J&W Scientific (5 m ×
0.53 mm i.d., 0.15 µm film). The carrier gas was helium with a flow rate of 18 mlmin-1. The
oven temperature was initially set to 0°C, using CO2 cryogenic cooling to improve solvent
separation from low-boiling peaks. Then the temperature program is followed by an oven
temperature ramp of 10°Cmin-1 to 420°C for the final 10 minutes. The detector temperature is
set to 450°C, H2 gas is set to 32 mlmin-1, with the airflow of 400 mlmin-1 and nitrogen makeup
of 24 mlmin-1.
The boiling points were determined by comparison against a series of reference standards
purchased from Accustandard, including a DRH-2887 calibration mix, DRH-008S calibration
mix, decane, hexadecane, PolyWax 850, and D-65352 Reference Gas Oil. Data were collected at
a sampling rate of 1 Hz using Varian-STAR software and signal integration and distribution
analysis was performed using a MATLAB program. For this boiling point simulation analysis,
FID detector intensities were assumed to be directly proportional to the weight percent of
samples eluted (Vardon et al., 2011).
Higher heating value (HHV) of bio-crude oil
There are two methods to obtain the HHVs of bio-crude oil: (1) calculated using empirical
equations, such as Dulong formula and (2) the HHVs of raw oil and solid residue were first
analyzed using a bomb calorimeter (Model 1281, Parr Instrument Co., Moline, IL), then the
HHV of the bio-crude oil was calculated using the percentage of solid residue in the raw oil and
their HHVs.
54
First, HHVs of selected oil samples will be measured using method (2). Then the
comparison and evaluation of HHVs achieved from both method (1) and method (2) will be
performed. If empirical equations can give reasonable HHVs with small errors, it will be used to
calculate HHVs for all samples.
Bio-crude oil molecules identification using GC-MS analysis
Molecular information of selected bio-crude oil samples are tested using GC-MS. After the
toluene dissolved bio-crude oil sample was filtered using Whatman® 0.45 µm filter, 1 µl filtered
bio-crude oil sample dissolved in toluene is injected in a split mode (5:1) into the GC-MS system
consisting of an Agilent 6890 (Agilent Inc, Palo Alto, CA) gas chromatograph, an Agilent 5973
mass selective detector and Agilent 7683B autosampler.
Gas chromatography was performed on a non-polar 30 m DB5 column with 0.32 mm inner
diameter (I.D.) and 0.25 m film thickness (Agilent Inc, Palo Alto, CA, USA) with an injection
temperature of 250°C, MSD transfer line of 250°C, and the ion source adjusted to 230°C. The
helium carrier gas was set at a constant flow rate of 4 mlmin-1. The temperature program was 5
minutes at 50°C, followed by an oven temperature ramp of 5°Cmin-1 to 315°C for the final 5
minutes. The mass spectrometer was operated in positive electron impact mode (EI) at 69.9 eV
ionization energy in m/z 30-800 scan range.
The spectra of all chromatogram peaks were evaluated using the HP Chemstation (Agilent,
Palo Alto, CA, USA) and AMDIS (NIST, Gaithersburg, MD, USA) programs. The spectra of all
chromatogram peaks were compared with the electron impact mass spectra from NIST Mass
Spectral Database (NIST08) and W8N08 library (John Wiley & Sons, Inc., USA). To allow
comparison between samples all data were normalized to the internal standard (methyl-heptanoic
400 M acid) in each chromatogram.
Aqueous product molecules identification using GC-MS analysis
The two algal species investigated in this study are both low-lipid but high-protein content
species. It is already known that protein is hydrolyzed at low temperature to produce amino acids
and some organic acids via the HTL process. The sensitivity of amino acids and organic acids
without derivatization are usually low. Thus, derivatization of the aqueous product was first
performed, and then the molecules in the aqueous product was analyzed using GC-MS
55
depending on its high sensitivity for separating molecular peaks and the molecules were
identified in the mass spectral database.
Dried aqueous samples (100 μl) were derivatized by the following procedure: 60 minutes at
50°C with 80 μl of methoxyamine hydrochloride in pyridine (20 mgml-1) followed by a 120
minutes treatment at 70°C with 80 μl N-methy-N-(trimethylsilyl) trifluoroacetamide (MSTFA).
A 10 μl of an internal standard (hentriacontanoic acid, 10 mgml-1) was added prior to
trimethylsilylation.
A sample volume of 1 μl was injected with a split ratio of 5:1. The GC-MS system consisted
of an Agilent 7890A (Agilent Inc, Palo Alto, CA, USA) gas chromatograph, an Agilent 5975C
mass selective detector and Agilent 7683B autosampler. Gas chromatography was performed on
a 60 m HP-5MS column with 0.25 mm inner diameter and 0.25 μm film thickness (Agilent Inc,
Palo Alto, CA, USA) with an injection temperature of 250°C, the interface set to 250°C, and the
ion source adjusted to 230°C. The helium carrier gas was set at a constant flow rate of 1.5
mlmin-1. The temperature program was 5 minutes isothermal heating at 70°C, followed by an
oven temperature increase of 5°Cmin-1 to 310°C and a final 20 minutes at 310°C. Mass spectra
were recorded in the m/z 50-800 scanning range.
The spectra of all chromatogram peaks were compared with electron impact mass spectrum
libraries NIST08 (NIST, MD, USA), WILEY08 (Palisade Corporation, NY, USA), and a custom
library. To allow comparison between samples all data were normalized to the internal standard
in each chromatogram. The chromatograms and mass spectra were evaluated using the MSD
ChemStation (Agilent, Palo Alto, CA, USA) and AMDIS (NIST, Gaithersburg, MD, USA)
programs.
Gas product molecules identification using GC analysis
After the HTL test, the gas phase in the reactor was carefully released through a control
valve and collected into the Tedlar® gas sampling bag and then analyzed using Varian CP-3800
Gas Chromatography equipped with an Alltech Hayesep D 100/120 column and a thermal
conductivity detector (TCD). The carrier gas was helium with a flow rate at 30 mlmin-1. The
temperature of inject and detect were both 120°C. A standard gas with composition of hydrogen,
nitrogen, oxygen, carbon monoxide, methane, and carbon dioxide are used to quantify the
gaseous product.
56
4.3.7 Study reaction kinetics and mechanism of HTL process
Reaction kinetics of HTL could be investigated with the assumption of reaction order and
Arrhenius equation. Since HTL tests are performed at different reaction temperatures with
different retention times, the data is sufficient to calculate the reaction constant k with the
Arrhenius parameters: activation energy Ea and pre-exponential factor A. The general procedure
of this calculation follows the steps below:
i. Assumption of reaction order. For example if we assume the HTL process is a first-order
reaction, there is a relationship between the concentration of an objective material A and
reaction time t:
d [ A]
  k[ A]
dt
Integrated this equation, we have a relationship of:

ln[ A]  kt  C
where C is a constant and represent the initial value of ln[A]. Since the values of [A] is known at
different reaction temperatures with different reaction times, reaction rate constant k values at
different temperatures can be derived by linear regression of ln[A] against t.
ii. The expression of Arrhenius equation is:
⁄
where Ea is activation energy and A is pre-exponential factor. Since reaction rate constants at
different reaction temperatures are already known, both Ea and A values can be determined by
linear regression of ln k vs. 1/T.
For the reaction mechanism of the HTL process, the reaction pathway of high protein
biomass under hydrothermal conditions often involves the steps listed below:
(1) Protein hydrolysis into amino acids.
(2) Carboxyl group removal from amino acids via decarboxylation.
(3) Amine group removal from amino acids via deamination.
(4) Recombination/repolymerization of reaction intermediates from steps (2) and (3).
(5) Further degradation of intermediates from steps (2) and (3).
57
The number of side reactions among reaction intermediates from procedures (2) and (3)
could be humongous. On the other hand, other compositions such as carbohydrates and lipids in
microalgae can also be converted into bio-crude oil. Therefore, the comprehensive reaction
pathway of HTL of microalgae is expected to be very complicated. However, based on the
molecular level information achieved from bio-crude oil, aqueous product and reaction kinetics
data, it is possible to elucidate a main pathway of bio-crude oil formation from HTL of high-
protein microalgae.
58
CHAPTER 5. SCREENING ALGAL SPECIES FOR HTL EXPERIMENTS
There are thousands of algal species living on earth but not all of them are suitable
feedstocks for biofuel production. Other than conversion efficiency, availability of feedstock
should also be considered for algal species selection.
5.1 CHARACTERISTICS OF DIFFERENT ALGAL SPECIES
Before C. pyrenoidosa and S. platensis were finally chosen as the two low-lipid microalgae
to study the feasibility of bio-crude oil production via HTL, more than 10 different algal species
were converted via HTL. The characteristics of all of the algal feedstocks were analyzed and
summarized in Table 5-1. For comparison, the characteristics of swine manure and sewage
sludge are also listed in the table.
From the comparison, it was found the lipid content of algae, which is represented by crude
fat in Table 5-1, is generally lower than that of swine manure. It should be noted that the crude
fat of the algal species were analyzed using Soxhlet extraction with petroleum ether as the
solvent. This is an approximation of the lipid content in the algal cell because some of the lipids
cannot be extracted if the algal cells are not broken thoroughly. Folch extraction was one of the
classic methods to determine the total lipid content in organism tissue (Folch et al., 1957) and
widely used in algae biodiesel research and industrial applications. However, the Folch reagent
is considered to be toxic and mutagenetic due to its component of CHCl3 (Lin et al., 2004) and
the extraction efficiency for some fatty acids in specific organism tissue is low and requires
pretreatment of the samples, such as freeze-drying (Sattler et al., 1991). Additionally, other
components in algae, such as pigment, could also be co-extracted using the Folch method so that
led to the overestimation of lipid content. To sum up, the methodology for algal lipid analysis
has not been standardized; therefore, the lipid values in literature should be approached with a
healthy level of skepticism (Pienkos and Darzins, 2009).
The ash contents in macroalgae, such as seaweed, Red Alga, Alga collected from retention
ponds and KELP are much higher than those in microalgae. And the lignocellulose content
(cellulose, hemicellulose and lignin) in macroalgae is also higher compared with microalgae.
The reaction pathways for macroalgae and microalgae under hydrothermal conditions are
expected to be different.
59
Table 5-1 Characteristics of different algae species, swine manure and sludge used in this study
Chemical Composition Elemental Composition
Species VS a Ash Crude Crude Cellulose Hemi- Lignin Non-fibrous C H N S P Ob

Fat Protein cellulose carbohydrate b
Seaweed 73.5 26.5 5.6 12.9 15.8 21.1 4.4 13.7 n.a. n.a. n.a. 0.51 0.29 n.a.
Red Alga 58.4 41.6 0.36 21.5 5.6 14.0 2.1 14.9 42.22 5.99 3.86 2.79 0.24 44.9
c
RT Alga 50.4 49.6 5.18 5.6 16.0 20.4 0.2 2.2 30.09 5.17 0.00 0.52 0.06 64.16
d
Algae UCSD 71.5 28.6 4.15 33.6 23.0 0.5 7.3 3.0 n.a. n.a. n.a 0.55 0.70 n.a.
KELP 78.6 21.4 4.33 5.46 11.8 1.7 27.9 27.4 n.a. n.a. n.a. 2.52 0.19 n.a.
Algae_API_1 e 78.4 21.6 1.13 54.6 2.5 13.5 0.2 6.6 39.59 5.65 7.20 0.63 1.87 45.06
e
Algae_API_2 95.2 4.8 1.58 19.5 13.4 27 7.0 26.8 45.13 9.22 1.12 0.27 0.64 43.62
f
Algae SWP 84.1 15.9 0.1 36.3 1.2 23.3 2.7 20.5 56.17 6.87 7.69 0.44 0.59 28.24
g
Algae HTLW 99.9 0.1 15.4 66.1 0.4 0.3 0.1 17.6 51.09 7.19 10.12 0.69 1.12 29.79
h
Algae GOM 77.1 22.9 n.a. n.a. n.a. n.a. n.a. n.a. 38.24 5.35 7.48 n.a. n.a. 48.94
Diatom 67.4 32.6 5.57 40.0 6.4 0.5 3.1 11.9 33.65 4.90 5.51 0.89 1.10 53.95
Chlamydomonas 94.4 5.6 3.77 58.0 0.3 0.5 0.2 31.7 46.10 6.70 8.44 0.6 1.54 36.62
Spirulina 90.5 9.5 5.1 64.4 0.5 1.4 0.2 18.9 49.3 6.4 11.0 0.76 1.13 31.41
Chlorella 94.4 5.6 0.1 71.3 0.3 0.5 0.2 22.0 51.4 6.6 11.1 0.73 1.68 28.49
Swine Manure 88.6 11.4 18.8 26.9 1.8 27.9 5.3 7.9 44.22 6.27 3.55 0.32 1.87 43.77
i
Sewage Sludge 73.7 26.3 0.1 41.6 26.0 15.1 9.9 n.a. 37.97 7.79 7.29 1.23 2.44 43.28
n.a. Not available; a Volatile solid; b calculated by difference; c Algae collected from a retention pond in Champaign, IL; d Algal mixture collected from Urbana
Champaign Sanitary District (Urbana, IL); e Algae provided by API company; f Algae grown in an open swimming pool in the Department of Agricultural and
Biological Engineering (ABE), University of Illinois at Urbana-Champaign, Urbana, IL; g Algae grown in continuous test bioreactor feeding 1% HTL wastewater;
h
Algae collected from the Gulf of Mexico and grown in the ABE lab; i Sewage sludge sample collected from the Urbana Champaign Sanitary District (Urbana,
IL).
60
5.2 HTL RESULTS OF DIFFERENT ALGAE SPECIES
5.2.1 Bio-crude oil yield of different algae species

Different algae and biowastes were converted into bio-crude oil via HTL at a reaction
temperature of 280°C with 30 minutes retention time and 0.69 MPa of initial pressure provided
by N2. Figure 5-1 shows the results of bio-crude oil yield and crude fat, which represents lipid
content of the feedstocks.
Figure 5-1 Bio-crude oil yield and lipid content of different algae and biowastes
It was found the average bio-crude oil yield from microalgae (32.33%) is much higher than
that from macroalgae (18.44%). Therefore microalgae are more suitable feedstocks for bio-crude
oil production via HTL. Additionally, the results indicate that high lipid content algae are not the
exclusive choices for algae-to-biofuel applications if HTL is chosen as the conversion
technology. Since bio-crude oil yields from different feedstocks were all higher than their lipid
contents in Figure 5-1, there must be some other components in the feedstock contributing to the
bio-crude oil formation.
61
5.2.2 Relationship between oil quality and feedstock characteristics
Toluene solubility is the percentage of raw oil that can be dissolved in toluene. It can
provide an indicator of the raw oil quality related to the upgradability and refinability of the bio-
crude oil product.
100% 100%
(a) (b)
Solubility in Toluene (% of Raw oil)
Solubility in Toluene (% of Raw oil)

80% 80% R²= 0.87
60% 60%
40% 40%
20% R²= 0.78 20%
0% 0%
0% 10% 20% 30% 40% 50% 60% 0% 10% 20% 30% 40% 50% 60% 70% 80%
Ash content of algae (wt%) Crude protein of algae (wt%)
Figure 5-2 Solubility of raw oil in toluene: (a) vs. ash content; and (b) vs. crude protein content in algae
Figure 5-2 shows the relationship of solubility of raw oil in toluene with the ash and crude
protein contents in algae. The relationships between the oil quality with the ash and protein
content are both linear. The quality of bio-crude oil generally decreases with the increase of ash
content in algae. From Table 5-1 it was found the average ash content of macroalgae is higher
than that of microalgae. This finding is consistent with literature which demonstrates macroalgae
have lower lipid and protein but higher ash content than microalgae (Renaud and Luong-Van,
2006). The qualities of the bio-crude oil produced from macroalgae are lower than the qualities
of bio-crude oils produced from microalgae.
On the other hand, crude protein content of algae can benefit the quality of bio-crude oil
due to its organic derivatives under hydrothermal conditions being dissolved in solvent.
Although some microalgal species used in our study, such as algae grown in HTL wastewater,
seem to be suitable HTL feedstocks, the availability of algae must also be considered. For
instance, in each HTL test, about 160 g or 21 g of dry alga is needed if the 2 L or 100 mL reactor
is used. Since the two microalgae C. pyrenoidosa and S. platensis are already commercially
grown in large scale, which demonstrates the feasibility of large amounts of feedstock production
in the future; these two low lipid content microalgae were finally chosen as the feedstocks to
62
study the feasibility of converting low-lipid microalgae into bio-crude oil via HTL. It should be
noted that the selection of the two algal species in this study has not excluded the possibilities of
other algal strains being used as feedstock for biofuel production. The purpose of selecting C.
pyrenoidosa and S. platensis as feedstock is to represent the low-lipid content algal strains,
which can be grown in wastewater in the future.
63
CHAPTER 6. EFFECTS OF OPERATING CONDITIONS ON PRODUCT
YIELDS FROM HTL OF MICROALGAE
6.1 HTL OF CHLORELLA PYRENOIDOSA
Chlorella pyrenoidosa is a green unicellular microalga with about 2% lipid content based
on the dry matter basis (Becker, 1994) that is found in both fresh and marine waters. The
physiological and biochemical properties as well as photosynthetic apparatus of C. pyrenoidosa
are similar to higher terrestrial plants, e.g., containing chloroplasts and having the ability to
absorb carbon dioxide to generate oxygen via photosynthesis (Hörcsik et al., 2007), but its
biomass productivity is much higher than that of many microalgal species with high lipid content
(Mata et al., 2010). Most importantly, since C. pyrenoidosa is not highly sensitive to water
quality (Lin et al., 2007), it can be cultured in a variety of wastewaters (Shen et al., 2008; Yang
et al., 2008), making it a promising choice for both biofuel production and wastewater treatment
in the future.
6.1.1 Replicates of experiments

As mentioned before, 160g of dry algal feedstock is required in a typical HTL experiment
conducted in the 2 L reactor. In the lab, this scale of consumption of feedstock is considered to
be large so that a reliable experimental procedure and product recovery are both important and
necessary. For studying the repeatability of hydrothermal liquefaction experiments, five
replications were conducted at 280°C with 0.69 MPa initial pressure of nitrogen for 30 minutes
retention time. The whiskers in the box-whisker plot (Figure 6-1) represent the maximum and
minimum values. As shown in Figure 6-1, the average yields and associated standard deviations
were 34.4%1.24% for bio-crude oil, 4.1%1.00% for solid residue, 45.7%1.5% for aqueous
product, and 15.3%0.8% for gas product. The small deviations of the results among the five test
replicas demonstrate that the experimental results from HTL were reliable, and the repeatability
of the HTL experiments was acceptable.
64
Figure 6-1 Replicates of HTL of Chlorella obtained at 280°C, 30 min reaction time with 0.69 MPa initial
pressure of nitrogen
6.1.2 Effect of reaction temperature

To investigate the effects of reaction temperature on product yields, HTL tests were
conducted at different reaction temperatures with the same retention time of 30 minutes and 0.69
MPa initial pressure of nitrogen. The results are summarized in Figure 6-2. The bio-crude oil
yield increased from 0.4% to 39.8% and the solid residue yield decreased dramatically from 98.5%
to 1.7% when the reaction temperature increased from 100°C to 320°C. When the temperature
increased further from 320°C to 340°C, the bio-crude oil yield slightly decreased to 35.0%. The
gas yields were negligible when reaction temperature was lower than 160°C. When the
temperature was above 160°C, the gas yields were least affected by the reaction temperature,
with all gas yields falling in the range of 11.0% to 17.3%. This indicates that, at high reaction
temperature, the algal feedstock was preferentially converted into oil and aqueous products,
rather than to the gaseous product, although the gaseous product yield slightly increased with the
reaction temperature. However, with the temperature further increasing to supercritical
conditions, the gaseous product yield might be significantly increased (Brown et al., 2010).
65
100%
90% Bio-crude oil Aqueous product

Gas product Solid residue
80%
70%
Product Yields (%)
60%
50%
40%
30%
20%
10%
0%
100 120 140 160 180 200 220 240 260 280 300 320 340
Reaction Temperature ( C)
Figure 6-2 Effects of the reaction temperature on product yields with 30 min reaction time (Chlorella)
It was found the bio-crude oil formation is substantial when the reaction temperature is
above 180°C and then increasing temperature can benefit the bio-crude oil yield. On the other
hand, the aqueous product yield increased with the reaction temperature when the temperature
increased from 100°C to 240°C. After the reaction temperature was increased to higher than
240°C, the yield of aqueous product started to decrease. On the other hand, the yield of solid
residue substantially decreased with the reaction temperature. This phenomenon indicated when
the temperature was above 240°C but below 320°C, some of the water soluble product could be
converted into bio-crude oil.
The raw oil product is the combination of bio-crude oil and solid residue based on our
definition. When the temperature was lower than 240°C, the raw oil product was more like
asphalt or char instead of flowable oil. It was observed that the viscosity of the raw oil decreased
as reaction temperature increased so that the fluidity of the produced raw oil at room temperature
increased with the reaction temperature. Therefore, increasing temperature will make the oil
conversion process faster and complete and can significantly improve the fluidity of the product,
which is important for the handling and transport of the bio-crude oil product.
66
Reaction temperature also showed strong effects on the bio-crude oil yield in other
hydrothermal conversion research (He et al., 2000b; Minowa et al., 1994, 1995b; Minowa et al.,
1995a). For example, Minowa et al. (1995a) used Dunaliella tertiolecta as the feedstock to
produce oil through HTL without the presence of a catalyst and showed that the oil yield
increased from 30.9% to 43.8% when the reaction temperature increased from 250°C to 300°C.
The oil yield of Chaetomorpha linum Kützing was also found to increase with increasing
reaction temperature and reached a plateau at 350°C to 395°C (Aresta et al., 2005). Results of the
present study agree with data in the literature, indicating that more organic matter in the
feedstock will be broken down and formed into oil compounds with the increase of temperature
in the hydrothermal liquefaction of C. pyrenoidosa. Since C. pyrenoidosa has an initial lipid
content of 0.1% and a protein content of 71%, the primary source material for the production of
bio-crude oil is expected to be protein. The work of Dote et al. (1998b) supports this proposed
pathway as they used an HTL process to convert tryptophan (one of the common amino acids
building block of protein) and achieved an oil yield as high as 66% (Dote et al., 1998b). Larger
protein molecules could be hydrolyzed to amino acids at a relatively low temperature first, and
then the amino acids could be converted into bio-crude oil at high temperatures. This potential
reaction mechanism is discussed in detail in CHAPTER 9. The important implication of these
findings is that high-lipid content algal strains will not necessarily be the only choices for biofuel
production via HTL.

Reaction time is a kinetic parameter affecting the organic matter conversion and product
distribution. Insufficient reaction time may lead to an incomplete conversion process, while
excessive reaction time can result in decomposition of desirable products. To study the effect of
reaction time on product yield, HTL experiments with different retention times (hold time) and
different reaction temperatures were conducted, and the experimental results are summarized in
Figure 6-3 and Figure 6-4. The retention time defined in this study refers to the elapsed time
starting from the time when the reaction temperature is first achieved to the time when the
reactor starts to cool down. Zero minutes of retention time means that as soon as the feedstock
reached the designated reaction temperature, the batch reactor was cooled down immediately,
with no additional hold time.
67
Figure 6-3 Effects of retention time on bio-crude oil yield (Chlorella)
Bio-crude oil yields kept increasing with retention time at the five reaction temperatures
(200°C, 220°C, 240°C, 260°C, 280°C) (Figure 6-3), and the highest bio-crude oil yield (39.4%)
was achieved at a temperature of 280°C with a retention time of 120 minutes. These results
indicate that the oil formation must start to occur at a reaction temperature of 200°C; otherwise,
the oil yield would not have increased with the retention time. It is also found the effect of
retention time at lower temperature was more pronounced than its effect at higher temperatures.
For instance, at 200°C, when the retention time increased from 0 to 120 minutes, bio-crude oil
yield increased from 3.2% to 22.6%, while the yield of bio-crude oil only increased from 29.6%
to 39.4% at a temperature of 280°C. He et al. (2000b) observed a strong relationship between
retention time and oil product yield from the conversion of swine manure at 285°C through HTL.
The oil yield increased from 12% to 57% when the retention time increased from 30 to 120
minutes (He et al., 2000b). Dote et al. (1996) found that oil yield increased with retention time
when they employed HTL to convert protein into biofuel (Dote et al., 1996). Minowa et al.
(1995a) observed that when the retention time increased from 5 to 60 minutes at 250°C, the oil
yield from HTL increased from 30.9% to 43.3% based on the organic basis of the D. tertiolecta
(Minowa et al., 1995a). These earlier studies provide evidence that we can produce the same
amount of oil from C. pyrenoidosa at a high temperature with a short reaction time or at a low
temperature with a long reaction time.
68
Figure 6-4 summarizes the effect of retention time on solid residue yield. The decrease of
the solid residue with retention time implied that the solid matter of C. pyrenoidosa was active
and subjected to decomposition in HTL, and further decomposition could be achieved by
increasing the retention time at the same reaction temperature. At 220°C, 240°C, 260°C and
280°C, the solid residue yields substantially decreased as the retention time increased from 0 to
30 minutes and then decreased more slowly from 30 to 120 minutes. Additionally, a high
reaction temperature with the same retention time also resulted in a lower yield of solid residue
than at a low reaction temperature. When the retention time was 0 minutes, the solid residue
yields were 52.2% (200°C), 31.7% (220°C), 27.8% (240°C), 17.3% (260°C), and 10.1% (280°C).
When the retention time was 120 minutes, the solid residue yields at 240°C, 260°C and 280°C
were all lower than 5%. It can be concluded from Figure 6-4 that the retention time had a more
pronounced effect on the solid residue yield at low temperature than at high temperature.
Extending retention time can increase the organic conversion and thus the oil yield.
Figure 6-4 Effects of retention time on solid residue yield (Chlorella)
69
Toluene solubility is the percentage of the raw oil that can be dissolved in toluene. It
provides an indicator of the quality of raw oil related to the upgradability and refinability of the
bio-crude oil product. Figure 6-5 shows the relationship between the toluene solubility of the raw
oil products and the retention time at different temperatures. The toluene solubility value of the
dry C. pyrenoidosa powder was 0.15% before HTL, which means that the feedstock was not
toluene soluble. Therefore, the toluene-soluble fraction in the raw oil product, which is defined
as bio-crude oil in this study, was confirmed to be produced from the HTL process.
Figure 6-5 Solubility of raw oil in toluene with different retention time and temperature (Chlorella)
At first, the toluene solubility increased sharply with increasing retention time. The rate of
increase slowed and started to plateau as the retention time further increased for reaction
temperatures higher than 260°C. With the same retention time, higher solubility was achieved at
a higher reaction temperature. In a previous study with swine manure as the feedstock, it was
also found that increasing the operating temperature could help to increase the oil's solubility in
solvent (He et al., 2000b). This means that even though the same bio-crude oil yield can be
achieved at low temperature with a long reaction time, the high reaction temperature may still be
preferable due to the better quality of the oil product, which makes it more suitable for transport
and upgrading.
70
6.1.4 Effect of initial pressure
One purpose of elevating the initial pressure in the HTL reactor is to prevent the intensive
water boiling process. Different initial pressures of nitrogen in the headspace of the batch reactor
can lead to different system pressures during the process. The total operating pressure of the
HTL system consisted of several partial pressures: (1) the initial pressure contributed by the
nitrogen, (2) the water vapor pressure generated during the heating process, and (3) the pressure
contributed by the gas produced from the biomass during HTL. All of the experiments in Figure
6-6 were conducted under the conditions of 280°C reaction temperature and 30 minutes of
retention time. The water vapor pressure and partial pressure of nitrogen were estimated by the
Antoine equation and ideal gas law. The gas product partial pressure was then calculated by the
difference between the total pressure and the sum of the water vapor and initial gas pressure at
the reaction conditions.
As shown in Figure 6-6, the maximum pressure of the system during the reaction exhibited
an approximately linear relationship with the initial headspace gas pressure. The partial pressure
of the gas product did not change as the initial pressure increased, and had an average value of
2.430.07 MPa, which implied that the gas product yields at different initial pressure conditions
should remain the same. All of the pressures in Figure 6-6 are gauge pressures. For the 0.0 MPa
nitrogen initial pressures, nitrogen was also introduced into the reactor to purge the residual air
and then released from the reactor before starting the test. The system maximum pressures at
different initial nitrogen pressures ranged from 8.9 to 10.3 MPa, which were higher than the
pressures achieved under the same reaction conditions using swine manure as feedstock (Dong,
2008). The symbols Ps and Pn in Figure 6-6 represent the increase of system maximum
pressure and partial pressure of nitrogen, respectively, when the initial pressure increased from 0
to 0.69 MPa. The value of Pn was about 93.4% of Ps, which indicates that the increase of total
system pressure was mainly contributed to by the increase of partial pressure of nitrogen.
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Figure 6-6 Effects of the initial N2 pressure on the system maximum pressure and partial pressure of
gaseous product (Chlorella)
Figure 6-7 shows the product yields at different initial pressures under the conditions of
280°C reaction temperature and 30 minutes of retention time and indicates that the initial
pressure of the nitrogen had no significant effect on the hydrothermal conversion of C.
pyrenoidosa. Under the above experimental conditions, the aqueous product yield, gas yield, bio-
crude oil yield, and solid residue yield were about 45%, 15%, 35%, and 5%, respectively, based
on the dry mass of the feedstock. The standard deviations of the product yields at different initial
pressure were all below 2%. Of the products, the aqueous product had the highest yield, which
means that about 45% of the feedstock was converted into water-soluble substances. On the
other hand, the product that could not dissolve in either water or toluene, which was defined as
the solid residue, had the lowest yield of about 5%. Gas product yields at different initial
pressure conditions were all around 15%, which was consistent with the conclusion that the
partial pressure of the gas product did not change with the increase in initial pressure.
72
60%
Aqueous Gas Bio-crude Oil Solid Residue

50%
40%
Product Yields (%)
30%
20%
10%
0%
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Initial pressure (MPa)
Figure 6-7 Effects of the initial N2 pressure on the HTL product yields at 280°C with 30 min reaction time
These results agree with a previous study (Goudriaan and Peferoen, 1990), in which
additional initial pressure above the saturation pressure of water had no effect on the
hydrothermal liquefaction for wood pellets conversion. However, other studies showed that a
process gas, either reducing or inert, should be added to achieve an oil product when swine
manure was used as feedstock (He et al., 2001b). There is another report (Yang et al., 2009)
about bio-crude yields from forestry waste conversion through HTL increased sharply when the
initial pressure of hydrogen increased from 0.0 to 2.0 MPa. These different observations indicate
that the type or composition of the feedstock and the process gas may have pronounced impacts
on how operating parameters affect the HTL process. Therefore, different feedstocks should be
expected to have different optimal conversion conditions, including initial process gases. In this
study, the nitrogen probably was not a reactant in the HTL process but only provided pressure to
prevent the water from boiling. Because the amount of nitrogen introduced into the reactor vessel
did not affect the product yields, it was unlikely that the oil formation from C. pyrenoidosa
utilized a pathway for nitrogen gas formation or consumption.
73
6.1.5 Effect of heating period
For all of the HTL experiments conducted in a batch reactor, the effect of the heating period
is very difficult to be completely avoided. As shown in Figure 4-4, it usually takes 20-50 minutes
to heat the reactor from room temperature to the designated temperature. Obviously, the time for
heating the reactor to the designated temperature could be even longer than the retention time.
Most importantly, the effect of the heating period can have significant influence on the
study of HTL reaction kinetics. For example, if we want to explore how much bio-crude oil is
produced at 280°C with 30 minutes reaction time, ideally the temperature should be increased
from room temperature to 280°C as fast as possible to guarantee the reaction time is strictly 30
minutes. However, in a batch reactor using an electronic furnace as a heating apparatus, it is very
difficult to achieve this goal. For example, if the bio-crude oil from C. pyrenoidosa is 33.2%
(Figure 6-2) at 280°C with 30 minutes retention time, this yield is not the “real” bio-crude oil
yield under this operating condition, because as soon as the temperature reached 280°C, 29.6%
of the bio-crude oil yield (bio-crude oil yield obtained at 280°C with 0 minutes retention time)
has already been obtained. So the “real” bio-crude oil yield obtained in the subsequent 30
minutes (retention time) was only 3.6%.
Figure 6-8 Effect of heating period on bio-crude oil yield from Chlorella
74
From this analysis, it was found that the difference between the retention time and the
reaction time (which is often needed for kinetics study) is the obstacle for exploring the HTL
reaction kinetics. It must be known how much HTL products have been produced as soon as the
reactor reaches the reaction temperature, and then the “real” HTL product yield with certain
retention time can be derived. This approach provides a compromise way to study the reaction
kinetics of experiments conducted in the batch reactor when long heating period cannot be
avoided.
Figure 6-8 shows the bio-crude oil yield obtained during the heating period of the
experiments and their contributions to the bio-crude oil yield obtained at the same temperature
with 30 minutes retention time. To investigate the bio-crude oil yield achieved during the heating
period, the reaction is quenched as soon as the temperature reached the designated reaction
temperature. Therefore, the bio-crude oil yield achieved during the heating period equals the bio-
crude oil yield at a different temperature with zero minute retention time. It is found that the
effects of the heating period at a higher temperature are distinctly more substantial. For example,
at 200°C with 30 minutes retention time the bio-crude oil yield was 22.4%, and during the
heating period (from room temperature to 200°C) the bio-crude oil yield was only 3.2%, which
means 14.3% of the bio-crude oil was produced in the heating period. However, about 89.3% of
bio-crude oil obtained at 280°C with 30 minutes retention time, was actually produced during the
period of heating the reactor from room temperature to 280°C.
In other words, the value of retention time is much closer to real reaction time at lower
temperature. When reaction temperature is higher than 200°C, more than 67% of the bio-crude
oil produced with 30 minutes retention time was actually produced before the temperature
reached the designated temperature. This finding explains one of the drawbacks of the
experiment set-up which uses an electronic furnace as the heating apparatus of the batch reactor.
The low heat transfer efficiency influences the accuracy to study the reaction kinetics. From this
aspect, it is very challenging to explore the intrinsic kinetics of HTL under this scenario;
therefore, an apparent kinetics study is discussed in CHAPTER 9.
In order to avoid the effect of the long heating period and study the intrinsic kinetics, self-
design mini batch reactors with a very small volume (1.67 mL) associated with a sand bath as
heating apparatus (Fu et al., 2010) or an assumed ideal plug flow tubular reactor (Rogalinski et
al., 2008) were used in the literature. Although these designs could decrease the effects of the
75
heating period by improving the heat transfer efficiency, some new problems could also be
introduced. For example, for the mini-batch reactor, system error could be enlarged due to the
small volume of feedstock and the temperature inside the reactor is usually unable to be
monitored during the experiment. For the tubular reactor, the plugging could interrupt the
experiment due to the high viscosity of the feedstock and the fouling of the process equipment is
difficult to clean after the experiment.
6.2 HTL OF SPIRULINA PLATENSIS
Spirulina (Arthrospira) platensis is a filamentous cyanobacterium which has been produced

largely in China, India, and the United States (Ferreira et al., 2011). However, other than to
provide feedstock for biofuel production, the original reason to largely produce S. platensis is to
utilize its intracellular compounds, such as protein, fatty acids and pigments, which have great
commercial interests as feed and food additives in agriculture, the food industry, pharmaceutical
feedstock, and medicine (Mosulishvili et al., 2002).
Since S. platensis is non-toxic and has a fast growth rate and high organic matter content, it
shows potential to be the feedstock for biofuel production. Some previous studies have
investigated the possibility of utilizing HTL to convert S. platensis into bio-crude oil (Biller and
Ross, 2011; Jena et al., 2011). However, most of the knowledge on the effects of HTL operating
conditions and the addition of catalysts, mass, element and energy balance of the HTL process,
are still uncharacterized. Consequently, the reaction mechanism and kinetics has not been
explored in literature based on the knowledge of the author.
6.2.1 Effect of reaction temperature

From the results of HTL of C. pyrenoidosa, it was found reaction temperatures have
significant effect on HTL product yields. Therefore, similar tests were conducted to investigate
the effects of reaction temperature on product yields from HTL of S. platensis. All the tests at
different reaction temperature levels were conducted with the same retention time of 30 minutes
and 0.69 MPa initial pressure of nitrogen. It was found that as temperature increased from 200°C
to 300°C, the bio-crude oil yield increased from 9.7% to 37.3%. When temperature was lower
than 200°C, the yield of bio-crude oil was negligible. As shown in Figure 6-9, when the reaction
temperature was lower than 160°C, the hydrothermal liquefaction process had not occurred
76
because no aqueous product and bio-crude oil were produced at this temperature level. When the
temperature increased from 160°C to 200°C, only a small amount of gas was produced. Most of
the biomass materials remained as solid residue after the experiment. However, when
temperature increased from 200°C to 220°C, a large portion (65.9%) of the S. platensis was
converted into water soluble product. At the same time, bio-crude oil started to form with a yield
of 9.7%.
With further increase of the temperature, aqueous product yield decreased and the bio-crude
oil yield increased, which is consistent with the findings in the literature (Jena et al., 2011). On
the other hand, the gas yield in this study was found to be around 10% and did not change much
when temperature increased from 220°C to 300°C. This gas product yield is lower than the
literature data (around 20%). Since the gas yields in literature for studying the temperature
effects on HTL products from S. platensis was calculated based on the weight difference of the
reactor and contents in it before and after the experiment, it is possible that some system errors
could be introduced due to low accuracies for products collected and recovered after the HTL
experiment. For example, not completely recovering the products attached to the mixer blades
after the experiment could lead to a higher gaseous product yield but a lower bio-crude oil yield.
100%
90% Bio-crude oil Aqueous product

Gas product Solid residue
80%
70%
Product Yields (%)
60%
50%
40%
30%
20%
10%
0%
160 180 200 220 240 260 280 300 320
Reaction Temperature ( C)
Figure 6-9 Effects of the reaction temperature on product yields with 30 min reaction time (Spirulina)
77
Compared with the results in Section 6.1.2, the effects of reaction temperature on HTL of C.
pyrenoidosa and S. platensis are similar to each other. But the lowest temperature range for bio-
crude oil formation is 180-200°C for C. pyrenoidosa and 200-220°C for S. platensis. This
implies that C. pyrenoidosa is easier to be hydrothermal liquefied compared with S. platensis.
When the reaction temperature reaches 300°C, the solid residue for S. platensis is 3.2%, which is
also closed to the solid residue yield of C. pyrenoidosa at this temperature (4.3%). Meanwhile,
the high aqueous product yield for both microalgae implies that most of the algal biomass is
converted into a water soluble product rather than water insoluble bio-crude oil product.
From the summary in Table 5-1, it was found more than 60% of the total components in C.
pyrenoidosa and S. platensis is protein. The second largest fraction in these two algal species is a
non-fibrous carbohydrate (NFC). NFC often includes starches, sugars, pectins, and β-glucans
(Van Soest et al., 1991). Lignocellulose contributes less than 2.5% of the total weight of
microalgae. Under hydrothermal conditions, the possible pathway for protein hydrolysis involves
amino acids production first and then the amino acids would be degraded into organic acids or
amines via deamination and decarboxylation. On the other hand, the NFC can be degraded into
aldehyde compounds. Most of the products from protein and NFC hydrolysis are water soluble.
Therefore the aqueous product from HTL of C. pyrenoidosa and S. platensis primarily consists
of the derivatives from hydrolysis of protein and NFC. It should be noted that the interactions
between protein and NFC hydrolysis products, such as the Maillard reaction, may occur under
the hydrothermal condition, so that some desirable reaction pathways may be quenched
(Peterson et al., 2010). Some of the water soluble products may be converted into bio-crude oil
or gas product with further increase of reaction temperature. This could explain why the aqueous
product yields from C. pyrenoidosa and S. platensis both decreased with further increase of
reaction temperature. More details about this reaction mechanism hypothesis are discussed in
CHAPTER 9.

After the 100 mL reactor became available to our research group in early 2011, the HTL
experiments were conducted in the 100 mL reactors to study the effect of retention time on HTL
of S. Platensis and the effects of catalysts on HTL of different microalgal species. Compared
with the 2 L reactor, less amounts of feedstock is needed for each HTL test if the experiment is
conducted in the 100 mL reactor. For example, 160 g of dry algal biomass is needed for each test
78
if the 2 L reactor is used; however, only 21 g of dry algal biomass is required if the experiment is
conducted in the 100 mL reactor. On the other hand, the smaller reactor is much easier to handle
and clean after the experiments.
The product recovery procedures for the 100 mL and 2 L reactors are the same except for
the gas yield calculation. As discussed previously, the gas yield for the experiments conducted in
the 2 L reactor is calculated based on the weight difference of the reactor and contents in it
before and after the experiment. However, a large system error will be introduced if the same
method is applied for the experiments conducted in the 100 mL reactor. For instance, if the mass
of the un-recovered product after the HTL experiment is 1 g, it only introduces 0.625% (1g/160g)
error into the result if the experiment is conducted in the 2 L reactor. However, it will introduce
4.8% (1g/21g) error into the result if the experiment is conducted in the 100 mL reactor. This
indicates the system errors may be amplified up to several times if the same method is used to
calculate the gas yield. It is clear that the previous method for calculating the gas yield is not
suitable if the experiments are conducted in the 100 mL reactor. This is the reason that some of
the previous studies, which used small reactors (75 mL) to perform the HTL experiments,
calculated the gas yield from the ideal gas law by using the residual pressure and the estimated
average molecular weight of the gases (Anastasakis and Ross, 2011; Biller and Ross, 2011).
In this study, the compositions of the gaseous product are analyzed using gas
chromatography and CO2 is found to contribute more than 98% total mass of the gaseous product.
Therefore, the gas yield is calculated by following the procedures below:
(1) The partial pressure of nitrogen is calculated based on an ideal gas law using the
residual pressure (system pressure after the reactor is cooled down to room
temperature).
(2) Considering the effect of water vapor generated during the experiment, the Antoine
equation is used to estimate the water vapor partial pressure.
(3) The partial pressure of the gaseous product generated by HTL equals to the total system
pressure minus the sum of partial pressure of nitrogen and water vapor.
(4) Assume the average molecular weight of the gas equals to the molecular weight of CO2
so that the mass of the gaseous product can be calculated based on the ideal gas law.
79
To compare the difference of gas yield obtained from two types of reactors, S. platensis was
converted in the two types of reactors under the same experimental conditions (reaction
temperature: 300°C; retention time: 30 minutes; initial N2 pressure: 0.69 MPa). The results are
shown in Figure 6-10. It was found the differences of the yield of bio-crude oil, solid residue and
aqueous products obtained from different reactors were less than 5% based on the dry matter of
feedstock. At the same time, the gas yield obtained from the 100 mL reactor was about 5% (dry
matter of feedstock) lower than that obtained from the 2 L reactor. It is confirmed that the results
generated from two types of reactors are comparable to each other.
60%
2L reactor
50%
100mL reactor
Product Yields ( wt %)
40%
30%
20%
10%
0%
Bio-crude oil Aqueous Gas product Solid residue
Product
Figure 6-10 Comparison of HTL of Spirulina results using different reactors
Figure 6-11 shows the effects of retention time on bio-crude oil yield from S. platensis.
With the increase of retention time, the bio-crude oil yield increased. At 260°C and 280°C,
substantially increasing the bio-crude oil was achieved in 10 minutes retention time. On the other
hand, the increase of bio-crude oil yield at lower temperature was achieved with a longer
retention time. However, considering the entire retention time range (0-120 minutes) the effect of
retention time on bio-crude oil yield was more pronounced at a lower temperature than at a
higher temperature. This finding indicates, if the reaction temperature is further increased, the
effects of retention time on bio-crude oil yield could be less notable because the bio-crude oil
formation was completed in a very short retention time period or even in the heating period when
80
the reaction temperature is high. This conclusion is consistent with a previous study, in which the
retention time showed little effect on bio-crude oil yield at a higher reaction temperature of
350°C (Jena et al., 2011).
Figure 6-11 Effects of retention time on bio-crude oil yield (Spirulina)
Figure 6-12 shows the effect of retention time on solid residue yield from HTL of S.
platensis. It was found that increasing the retention time could decrease the solid residue yield.
At 200°C solid residue did not decrease substantially when the retention time increased from 0 to
30 minutes. However, with further increase of retention time, the solid residue yield substantially
decreased. This phenomenon implies that the degradation of solid matter of S. platensis should
already occur at 200°C with 0 minutes retention time, but the change in solid residue yield was
too small to be observed. Otherwise, the solid residue yield could not decrease with additional
increase in retention time.
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Similar to its effect on the bio-crude oil yield, the effects of retention time on solid residue
yield at higher temperature was less substantial than at a lower temperature. For instance, the
solid residue yield decreased from 14.4% to 6.0% and from 95.8% to 33.9% with the retention
time increasing from 0 to 120 minutes at 280°C and 200°C, respectively. Compared with the
results shown in Figure 6-4, the lowest temperature required for liquefying S. platensis into bio-
crude oil (200°C) was higher than that of C. pyrenoidosa (180°C).
Figure 6-12 Effects of retention time on solid residue yield (Spirulina)
Figure 6-13 shows the toluene solubility of the raw oil product from HTL of S. platensis.
The toluene solubility of S. platensis feedstock is about 5%, therefore, the bio-crude oil fraction,
which is toluene soluble, was mainly produced from the HTL process.
At 200°C, 220°C and 240°C, the solubility of raw oil increased with retention time.
However, at 260°C and 280°C, the solubility of raw oil first increased with retention time, and
then reached a plateau when the retention time was longer than 30 minutes. This indicates the
effects of retention time on toluene solubility of raw oil was more pronounced at lower
temperature that at a higher temperature.
82
Figure 6-13 Solubility of raw oil in toluene with different reaction time and temperature (Spirulina)
The highest toluene solubility of raw oil produced from S. platensis was 93.0%, which was
less than the highest toluene solubility of raw oil produced from C. pyrenoidosa (97.0%). From
the analysis of the compositions of the two algal species (Table 4-1), the ash content of S.
platensis is higher than that of C. pyrenoidosa. Therefore, the previous conclusion that the ash
content of algal feedstock can influence the HTL raw oil quality is confirmed again.
83
CHAPTER 7. EFFECTS OF CATALYSTS ON HTL PRODUCT YIELDS
For a long time, the studies using microalgae as feedstock to produce bio-crude oil via HTL
were conducted without the presence of a heterogeneous catalyst. On the other hand, the effects
of some water soluble catalysts, such as Na2CO3 (Dote et al., 1994; Minowa et al., 1995a; Yang
et al., 2004), organic acids and KOH (Ross et al., 2010) were investigated in literature.
Heterogeneous catalysts were first applied in the gasification of microalgae to produce methane
rich gas (Chakinala et al., 2009; Minowa and Sawayama, 1999; Stucki et al., 2009).
In recent years, a few studies applied heterogeneous catalysts in the HTL process to convert
microalgae into bio-crude oil (Biller et al., 2011; Duan and Savage, 2011b). Although these
studies investigated the potential effects of some commonly used heterogeneous catalysts on
HTL products properties such as elemental composition of bio-crude oil, some important issues,
such as the comparisons of heterogeneous and homogeneous catalysts, effects of heterogeneous
catalysts on element and energy recoveries, and how the HTL product qualities could be changed
with the addition of catalysts, have not been discussed in the literature. In CHAPTER 7, the
effects of catalysts on HTL product yields are discussed. Effects of the catalysts on element
balance, energy balance and HTL product compositions are discussed in CHAPTER 8 and
CHAPTER 9, respectively.
In order to eliminate any effects caused by the minerals in the tap water, Nanopure® water
was used to mix with the algal powder and make a slurry feedstock containing 30% dry matter
and 70% water by weight. In a typical test, 70 g of the slurry feedstock was loaded into the
reactor. Then 5% of different catalysts were loaded into the reactor and completely mixed with
the wet feedstock.
Two noble catalysts with supporting materials of activated carbon and alumina (Pd/C,
Pd/Al2O3, Pt/C, and Pt/Al2O3), one nickel-aluminum alloy (Raney nickel), and two homogeneous
catalysts – sodium hydroxide and sodium carbonate – were selected to study their effects of HTL
of microalgae. The noble-metal catalysts (Pd and Pt) are selected based on their reduction
activity which may be helpful for the deoxygenation and hydrogenation of the bio-crude oil.
Raney nickel was selected based on its wide application in hydrogenation process. The two
homogeneous catalysts were selected because they can promote the disassociation of amino acids
and organic acids and were demonstrated to benefit the HTL process in literature.
84
All metal catalysts were obtained from Acros Organics® and the two homogenous catalysts
were purchased from Fisher Scientific® in high purity and used as received. It should be pointed
out, in this study the scope of studying catalysts effects on the HTL process is to investigate
whether these commercially available catalysts could benefit the production of bio-crude oil and
improve its quality in a short term, therefore, they are all used as received and we did not pretreat
the catalysts or disqualify them because of some of the catalysts lacking long-term stability under
hydrothermal conditions. Table 7-1 summarizes the compositions of different catalysts used in
this study.
Table 7-1 Compositions of catalysts used in this study
Catalyst Composition (from supplier)

Pd/C Pd (5 wt %)
Pd/Al2O3 Pd (5 wt %)
Pt/C Pt (5 wt %)
Pt/Al2O3 Pt (5 wt %)
Raney-type Alloy Al-Ni (50 wt%-50 wt%)
NaOH >99.5 wt%
Na2CO3 >99.5 wt%
7.1 CATALYST EFFECTS ON BIO-CRUDE OIL YIELD
To investigate the catalyst effects on bio-crude oil yields, 5 wt% of different catalysts were
loaded into the reactor and mixed with algal slurry before the reaction. In this study, all of the
catalysts were used as received, which indicates there is no pre-reduction for the metal catalysts.
A previous study implied that the pre-reduction of metal catalysts before HTL was not necessary
because the catalysts would be exposed to the oxidizing hydrothermal environment when in use,
and the pre-reduction has no significant effects on the deoxygenation of fatty acids (Fu et al.,
2010).
Hydrogenation is widely used in the conventional petroleum upgrading process to remove
oxygen content from the fuel. In the HTL process the oxygen atom is removed as CO2 rather
than H2O via decarboxylation; therefore, no H2 was added in our experiments. In fact, H2
addition was not required for promoting decarboxylation under hydrothermal conditions (Fu et
al., 2010; Fu et al., 2011a). On the other hand, although the addition of H2 could improve the
H/C ratio of bio-crude oil produced from algae, the bio-crude oil yield with the presence of metal
catalysts and the addition of H2 could be lower than the yield achieved with added helium. At the
same time, under an uncatalyzed HTL condition, the addition of H2 could promote the bio-crude
85
oil yield (Duan and Savage, 2011b). Since large variables of the addition of H2 exist in literature,
no H2 addition was applied in this study because the objective of this chapter is to conduct
preliminary evaluation of different catalysts on the HTL of microalgae.
7.1.1 Effects of catalysts on bio-crude oil yield of Chlorella

Figure 7-1 shows the bio-crude oil yield from the HTL of C. pyrenoidosa at 240°C with 30
minutes retention time. Through the student’s t-test it was found that compared with bio-crude
oil yield from an uncatalyzed test, the bio-crude oil yield was significantly increased from 31.1%
to 41.0% when sodium hydroxide was applied. However, all of the other catalysts did not show
significant effects on the bio-crude oil yield under this temperature level.
Figure 7-1 Catalyst effects on bio-crude oil yield from Chlorella at 240°C with 30 min retention time
Note that the effects of the catalysts on HTL were usually investigated at a high
temperature (350°C) in literature (Biller et al., 2011; Duan and Savage, 2011b), the reason we
conducted the HTL experiment at the mild temperature level is to study whether the effects of
the catalysts can be observed at low temperatures, under which the formation of bio-crude oil
already starts to occur. From discussion in CHAPTER 6, it was found the lowest temperature for
the bio-crude oil formation should be in the range of 200-240°C, therefore, we conducted a series
86
of tests at 240°C to investigate the effects of different catalysts. However, it was found the
effects of catalysts addition at this mild temperature were not significant. In fact, at 240°C the
formation of bio-crude oil could not be completed in 120 minutes (Figure 6-3), and it is expected
that some unreacted algal biomass could remain after 30 minutes of the HTL process. The
unreacted algal biomass could mix with the catalysts and even deposit on the surface of a catalyst
so that the surface area of the catalysts decreases, and clogs the porous structure so that the
catalysts are deactivated.
When similar HTL experiments were conducted at a higher reaction temperature level
(280°C) with the same retention time, all of the catalysts increased the bio-crude oil yield. Figure
7-2 shows the bio-crude oil yield from C. pyrenoidosa with different catalysts at 280°C. This
finding is consistent with the literature which shows under an inert condition, different metal
catalysts could promote the formation of bio-crude oil (Duan and Savage, 2011b). It should be
pointed out that although significant differences of bio-crude oil yield were achieved at 280°C,
the difference between the highest yield (50.0% with 5% Raney nickel) and the lowest yield
(39.0%, uncatalyzed) was only about 10% based on the dry matter of the feedstock. The bio-
crude oil yields obtained under this condition are comparable with previous studies.
Figure 7-2 Catalyst effects on bio-crude oil yield from Chlorella at 280°C with 30 min retention time
87
The bio-crude oil yields with the presence of Pd/Al2O3 and Pd/C were 46.6±1.6% and
42.6±0.7%, respectively. Although the difference between these two values was not pronounced,
through statistical analysis it was found there was significant difference between these two series
of experimental results. On the other hand, the bio-crude oil yield was 45.8±2.7% and 45.2±2.4%
with additions of Pt/Al2O3 and Pt/C, respectively. But no significant difference was found
between the two series of data. To investigate the effects of the catalyst dosage on bio-crude oil
yield, 30% of Pt/Al2O3 based on the total dry mass of feedstock was applied into the test of HTL
of Chlorella at 280°C with 30 minutes retention time. The bio-crude oil yield was 41.8%, which
was close to the bio-crude oil yield obtained with the addition of 5% catalyst. Therefore, it can
be concluded at this temperature level, the effect of catalyst dosage was not substantial.
7.1.2 Effects of catalysts on bio-crude oil yield of Spirulina

The results of the effects of the catalysts on bio-crude oil yield produced from HTL of S.
platensis are shown in Figure 7-3 and Figure 7-4. Similar to the results of C. pyrenoidosa, at
240°C although the bio-crude oil yield obtained with the presence of several catalysts showed
significant difference compared with the uncatalyzed test results, the difference between the
highest oil yield (32.7±1.86% with 5% Na2CO3) and lowest oil yield (24.3±1.2% with 5% NaOH)
were less than 10%.
Figure 7-3 Catalyst effects on bio-crude oil yield from Spirulina at 240°C with 30min retention time
88
At 280°C, except for Pt/C, addition of all the other catalysts increased the bio-crude oil
yield. But again the difference between the highest oil yield (40.4%±1.9% with 5% Na2CO3) and
the lowest oil yield (33.9±2.4%, uncatalyzed) was less than 10%.
Figure 7-4 Catalyst effects on bio-crude oil yield from Spirulina at 280°C with 30 min retention time
7.2 CATALYST EFFECTS ON BOILING POINT DISTRIBUTION OF BIO-CRUDE

OIL
Boiling point distribution of bio-crude oil reflects the potential how bio-crude oil can be
cracked or refined into transportation fuel in the future. In this study the boiling point distribution
of bio-crude oil was generated using the GC simulation method (ASTM, 2005). The boiling
point distribution of bio-crude oil was compared with Illinois shale oil, which is a low-grade
petroleum crude.
Figure 7-5 compares the simulated boiling point (BP) distribution of bio-crude oil produced
from C. pyrenoidosa and the Illinois shale oil. It was found with the increase of reaction
temperature, the BP distribution of bio-crude oil shifted to the low temperature range, which
indicates the quality of bio-crude oil improved. Generally, organic compounds with complicated
89
structures, such as the polycyclic structures and longer carbon chain are more difficult to be
eluted from the GC column. Therefore Figure 7-5 implies that the large organic molecules in the
bio-crude oil could degrade or be cracked into small molecules when temperature increased from
240°C to 280°C.
Meanwhile, it was found the quality of bio-crude oil was still much lower than the low
grade petroleum—shale oil. For example, the fraction of shale oil with BP lower than 200°C is
about 19.5%. However, the fractions with BP lower than 200°C are 1.5% and 2.7% for the bio-
crude oil produced at 240°C and 280°C, respectively.
100
90
80
70
Distillate Fraction (%)
60
50
40 Shale Oil
30 240°C, 30min
280°C, 30min
20
10
0
0 100 200 300 400 500 600 700
Boiling Temperature (°C)
Figure 7-5 Comparison of BP distribution of bio-crude oil and shale oil
Figure 7-6 compares the BP distribution of bio-crude oil produced at 240°C with the
presence of different catalysts. It shows that with the addition of catalysts, the BP distribution
curve of bio-crude oil was shifted to the low temperature range. For instance, without the
addition of a catalyst, the fraction of bio-crude oil with BP higher than 500°C was about 25.5%.
When sodium carbonate was applied to the HTL process, this fraction was lowered to 11.6%.
Compared with a heterogeneous metal catalyst, a homogeneous catalyst has more substantial
effects on the bio-crude oil BP distribution at this temperature.
90
On the other hand, different supporting materials seem to have little effect on the BP
distribution of bio-crude oil. For instance, the fraction of bio-crude oil with BP higher than
500°C were 22.9% and 20.6%, when 5% of Pd/Al2O3 and Pd/C were applied during the HTL
process, respectively. The percentages of this fraction with the heterogeneous catalysts were also
close to the value of 25.5%, which was obtained from an uncatalyzed HTL process. From this
aspect, metal catalysts had less substantial effects on the BP distribution of bio-crude oil than the
alkaline catalysts.
100
Shale Oil
No Catalyst
90
Pd/Al2O3
Pd/C
80
Pt/Al2O3
Pt/C
70
Raney Ni
NaOH
60 Na2CO3
50
40
30
20
10
0
50 150 250 350 450 550 650
Figure 7-6 Catalyst effects on BP distribution of bio-crude oil produced at 240°C
Figure 7-7 shows the effects of different catalysts on the BP distribution of bio-crude oil
produced at 280°C from C. pyrenoidosa. It was found that the addition of heterogeneous
catalysts had little effect on bio-crude oil BP distribution. At the same time, the addition of
alkaline catalysts made the bio-crude oil BP distribution shift to the high temperature range,
which indicates more complicated molecules were produced, probably through the
recombination of small molecules.
When no catalyst was applied to the HTL process, the fraction of bio-crude oil with BP
ranging from 300°C to 400°C was 51.4%. It was found the fraction with BP in this range was
significantly lowered to 34.2% and 34.6%, when NaOH and Na2CO3 were added into the HTL
91
process, respectively. At the same time, the fraction of bio-crude oil with BP higher than 600°C
increased from less than 1% to about 10% when the alkaline catalysts were applied. Additionally,
the fraction of bio-crude oil with BP ranging from 400°C to 500°C was not changed much.
Therefore it could imply that some of the small molecules, which BP are between 300°C to
400°C, were recombined together to produce the large molecules with BP higher than 600°C
when alkaline catalysts were applied at 280°C.
100
Shale Oil
No Catalyst
90
Pd/Al2O3
Pd/C
80 Pt/Al2O3
Pt/C
70 Raney Ni
NaOH
60 Na2CO3
50
40
30
20
10
0
0 100 200 300 400 500 600 700
Figure 7-7 Catalyst effects on BP distribution of bio-crude oil produced at 280°C
The Illinois shale oil has a higher percentage of H content and much lower percentage of N
and O contents compared with bio-crude oil produced form algae (Vardon et al., 2012). In
summary, although adding of catalysts could change the BP distribution of bio-crude oil, the
quality of bio-crude oil was still much lower than the conventional low grade petroleum.
Therefore, additional refining or upgrading of bio-crude oil from HTL of algae is necessary to
produce substitutes for the transportation fuel in the future.
Previous results show that direct addition of catalysts in the HTL process did not
significantly improve the BP distribution of bio-crude oil. Since the percentage of C, H, N and O
were not significantly changed with the addition of different catalysts under hydrothermal
conditions (Duan and Savage, 2011b; Ross et al., 2010), the change of BP distribution implies
92
that the degradation and recombination of compounds in bio-crude oil would occur if catalysts
were applied during the HTL process. This process could be treated as a re-distribution process
of C and H atoms among different compounds in the bio-crude oil phase and aqueous phase.
7.3 MORPHOLOGY CHANGE OF CATALYST DURING HTL PROCESS
Microalgae are biological feedstocks, therefore, their compositions are very complicated,
which indicates other than organic compounds, such as protein, lipid and carbohydrates, they
contain a lot of inorganic elements such as calcium, magnesium, phosphorus, sulfur, and so on.
Some of these inorganic elements could poison catalysts and cause rapid catalyst deactivation
under hydrothermal conditions. On the other hand, carbon deposition is another issue which can
decrease the porous structure and the pore size of catalysts. Therefore it is necessary to
investigate the morphology change of the metal catalysts during the HTL process.
The morphology of catalyst before and after HTL process were obtained using a high-
resolution environmental scanning electron microscope (ESEM) equipped with a back-scattered
electron (BSE) detector (Philips XL30 ESEM-FEG). Since the intensity of the BSE signal is
related to the atomic number of the specimen, BSE images can provide the element information
in the samples. An EDAX light-element energy-dispersive spectroscopy (EDS) was also used to
obtain the semi-quantitative elemental compositions of the specimen. An electrically conductive
coating process was applied to increase the conductivity of specimen using a turbo-pumped
sputter coater, with rotating/tilting specimen holder and film thickness monitor (Denton Desk II
TSC). The coating material was gold/palladium target and the thickness of coating layer was
around 7 nm.
Figure 7-8 ESEM photograph of Chlorella
93
Figure 7-8 and Figure 7-9 show the ESEM photograph of C. pyrenoidosa and S. platensis.
It is clearly shown that C. pyrenoidosa is a microalgal species with a single round cell. However,
the shape of a single S. platensis is spiral. Since the C. pyrenoidosa was purchased from a food
store as a nutrient supplement, the photograph also shows the cell of C. pyrenoidosa was broken.
From these two figures, it is also known that a single cell of S. platensis is much larger than that
of C. pyrenoidosa.
Figure 7-9 ESEM photograph of Spirulina
The catalyst specimen was recovered after the HTL process by using toluene to extract the
raw oil product. Therefore unreacted algal biomass, ash content in the algal biomass and
inorganic compounds could mix with the catalysts. In fact, the sepration of catalysts from the
solid residue and ash content is extremely difficult, because during the HTL experiment,
catalysts were completely mixed with the feedstock.
Figure 7-10 shows the ESEM photograph of the Pt/C catalyst before and after HTL with C.
pyrenoidosa. The EADX spectra represent the elemental compositions of the specific spots on
the photograph. At 240°C, it was found the pores of the activated carbon were clogged by the
unreacted algal biomass and ash content in the feedstock. Through the EADX spectra (Figure
7-10 (d)), it was also found the elemental composition on the surface of activated carbon was not
significantly changed compared with the EADX spectra for the original Pt/C catalyst. With the
temperature increasing, the unreacted algal biomass materials in the porous disappeared and only
the inorganic compounds remained (Figure 7-10 (c) and (e)). The results indicate that during the
HTL process, some of the algal biomass could temporarily clog the pore of activated carbon at
low temperature; those biomass materials would be converted into HTL products as temperature
increased. Finally, only the ash and inorganic compounds would remain in the porous structure
of activated carbon.
94
(a)
(b) (c)
C C
(d) (e)
O Pt
Mg
O Au K
Pt Pd Ca
Energy (Kev) Energy (Kev)
Figure 7-10 ESEM photograph of catalyst sample with EDAX spectra: (a) Pt/C without HTL process; (b)
Chlorella with Pt/C at 240°C with 30 min; (c) Chlorella with Pt/C at 280°C with 30 min; (d) EDAX spectra
of representative spot on (b); (e) EDAX spectra of representative spot on (c)
Although the HTL process is an intensive chemical conversion process, it does not change
the porous structure of activated carbon. Meanwhile, it was found the carbon content in the solid
residue product was about 70 wt% (Yu et al., 2011b), therefore, it is very difficult to find
whether there was carbon deposition occurring on the activated carbon using the EDAX spectra.
95
A previous study suggested that Pt/C was a better catalyst to promote fatty acids
decarboxylation compared with activated carbon alone since the activation energy was about 50%
lower (Fu et al., 2011b). From this aspect, if carbon deposition could occur on the surface of
catalyst, it could influence its activity.
(a) (b)
Al Al
(c) (d)
P
O
Mg
Au
Si
P C Na
K
C Si
Mg Au Pd Ca
Na Pd K Fe
S
Figure 7-11 ESEM photograph of catalyst sample with EDAX spectra: (a) Spirulina with Pd/Al2O3 at
240°C with 30 min; (b) Spirulina with Pd/Al2O3 at 280°C with 30 min; (c) EDAX spectra of representative
spot on (a); (d) EDAX spectra of representative spot on (b)
Figure 7-11 shows the ESEM photograph and the elemental composition of Pd/Al2O3 after
HTL with S. platensis at 240°C and 280°C with 30 minutes retention time. The EDAX spectra
showed the semi-quantitative elemental composition on the surface of the catalyst. The peak area
of different elements represents its relative weight fraction. It was clearly found under the two
different temperature levels, carbon deposition occurred on the surface of the catalyst. From
Figure 7-11 (a), it was found the surface of alumina was no longer smooth and there were
inorganic compounds and carbon depositing on it.
96
Table 7-2 summarizes the elemental composition on the surface of Pd/Al2O3 after HTL
with S. platensis at 240°C and 280°C with 30 minutes retention time. It presents the quantitative
data for Figure 7-11 (c) and (d). There is Au presenting in Table 7-2 because it was used as the
coating materials to increase the conductivity of the specimen.
Table 7-2 Elemental composition on the surface of Pd/Al2O3 after HTL of Spirulina
HTL conditions Element Wt% a At% b

C 24.16 37.32
O 32.33 37.50
Na 0.78 0.63
Mg 1.62 1.23
Al 25.10 17.26
Si 2.49 1.65
240°C, 30 min
P 4.37 2.62
Au 3.92 0.37
S 0.40 0.23
Pd 3.68 0.64
K 1.16 0.55
Total 100.00 100.00
C 19.61 30.86
O 34.92 41.26
Na 2.61 2.15
Mg 5.00 3.89
Al 15.72 11.01
Si 3.12 2.10
280°C, 30 min P 8.99 5.48
Au 1.49 0.14
S 2.10 0.37
Pd 2.36 1.14
K 1.56 0.74
Fe 2.52 0.85
Total 100.00 100.00
a
weight percentage; b atom percentage
It was found with increasing reaction temperature, less carbon remained on the surface of
the catalyst. As mentioned above, at 240°C some of the algal biomass was unreacted and
remained mixed with the catalyst. When the temperature increased to 280°C, most of the algal
biomass had been converted into the HTL products. Other than carbon deposition, the other
materials on the surface of the catalyst after the HTL process were inorganic compounds and ash
content. From Table 7-2, it was found that the fractions of O, Na, Mg, P, S, and K contents,
which represent the inorganic compounds and ash content in the feedstock, are all higher at
280°C compared with those at 240°C.
97
Figure 7-12 shows the ESEM photograph of Raney nickel before and after HTL with S.
platensis. From the EDAX spectra and the photos it is clearly shown that after the HTL process,
a lot of carbon and inorganic compounds deposited on the surface of Raney nickel. Carbon
contributes about 59% of total weight for the materials on the surface of Raney nickel (Figure
7-12 (d)). At the same time, other inorganic elements such as O, Na, Mg, P, K, Ca and Fe
contribute about 30% of the total weight.
(a) (b)
Al
C
(c) (d)
Ni O
Si
Ni Mg
Al Au
Na
Ni K Ca Fe Ni
Figure 7-12 ESEM photograph of catalyst sample with EDAX spectra: (a) Raney Ni without HTL process;
(b) Spirulina with Raney Ni at 280°C with 30 min; (c) EDAX spectra of (a); (d) EDAX spectra of (b)
Under the operating conditions of this study, it was shown from the ESEM photograph that
the structures and shapes of catalysts were stable during the HTL process. Carbon from the solid
residue and inorganic compounds in the algal biomass materials mixed and deposited on the
surface of the catalysts after HTL. This phenomenon implies that the ash and inorganic contents
in the biomass materials could decrease the contact area between feedstock and the noble metal
atoms; therefore, the activity of the catalysts was decreased. On the other hand, a previous study
98
suggested that thermal degradation (sintering) could happen on metal catalysts, such as Ni
(Elliott et al., 1993). Some supporting materials of metal catalysts, such as Al2O3, could be
transformed into AlO(OH) under hydrothermal conditions (Yu and Savage, 2001).
Obviously, there is a lot of unknown information about how catalysts can affect the HTL
process and how the HTL process can deactivate the metal catalysts. For example, a previous
study reports significant loss of surface areas of MnO2, TiO2, and CuO/Al2O3 under
hydrothermal conditions (Yu and Savage, 2001). Although it was found the shape of activated
carbon was not changed much during the HTL, how the surface area of this porous material is
changed during HTL is still unknown. Through present study, it was found carbon and inorganic
compounds deposition occurred during HTL of microalgae, but how it affects the lifespan of
catalyst and whether some elements can lead to catalysts poison are still unknown. To
summarize, in this study we provide the preliminary information about how catalysts could
influence the HTL process. In the future, more efforts are needed to study the effects of catalysts
on the HTL process and finally develop specific catalysts with long lifespans to improve the
conversion efficiency and quality of bio-crude oil produced from low-lipid microalgae.
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CHAPTER 8. ELEMENTAL AND ENERGY BALANCE OF HTL OF
LOW-LIPID MICROALGAE
Since microalgae usually have faster growth rates, shorter growing cycles and higher
photosynthetic efficiencies than terrestrial lignocellulosic biomass (Pirt, 1986), they are viewed
as suitable feedstocks for producing next generation biofuel (Chisti, 2007; Chisti, 2008; Luque et
al., 2008; Luque et al., 2010). In addition, algae production can occur on marginal lands and
different bodies of water, resulting in much less impact on current land-use for food production.
Microalgae are usually composed of lipids, proteins, nucleic acids, and non-cellulosic
carbohydrates. Conventional algae-to-biodiesel technology requires drying of the algal biomass
followed by solvent and/or mechanical extraction. The energy consumed in the drying process is
more than 75% of the total energy consumption (Lardon et al., 2009). Additionally, this
conventional approach leads to a focus on growing relatively pure cultures of high-lipid algae,
which usually have lower biomass productivities compared to low-lipid algal strains (Mata et al.,
2010; Rodolfi et al., 2009; Williams and Laurens, 2010). In other words, focusing on lipid
accumulation in microalgae under a stressed condition such as nitrogen depletion, sacrifices
biomass productivity, reduces the net energy yield, and makes the process very sensitive to
contamination, therefore, it could cause a net reduction in lipid productivity (Sheehan et al.,
1998). In contrast, it would be preferable to have an algal biofuel technology designed to work
with wet biomass, low-lipid algae and potential co-growth of non-target species.
Compared with pyrolysis, HTL requires lower temperatures for conversion; and most
importantly does not require energy intensive drying of the feedstock (Demirba, 2001; Huber et
al., 2006). Although fast pyrolysis has the advantages of shorter residence times and lower
capital costs because of lower operating pressures, the oil products from pyrolysis usually have
higher moisture and oxygen content compared to oils from HTL (Bridgwater et al., 1999;
Peterson et al., 2008). Therefore, HTL oils typically have more desirable quantities than
pyrolysis oils and can be produced with higher energetic efficiency (Peterson et al., 2008),
especially for wet biomass feedstocks such as animal waste, human waste, food-processing waste
and algae. As mentioned before, in a typical HTL process feedstock is converted into bio-crude
oil, solid residue, gaseous product, and aqueous product, in which the aqueous product contains
water soluble constituents produced from HTL. Bio-crude oil from HTL needs further upgrading
100
to be used as transportation fuels because of higher oxygen and nitrogen contents than
conventional petroleum (He et al., 2000a; Minowa et al., 1995a). Previous studies have reported
that, during the HTL process, only about 40% of the carbon and 35% of the hydrogen in algal
feedstocks were converted into oil, which means most of the organic compounds in the feedstock
were converted into water soluble or gaseous products (Yang et al., 2004). Most of the nitrogen
in the feedstock was converted into water soluble products though the HTL process (Dote et al.,
1996); and homogeneous catalysts could affect nitrogen distribution in HTL products. For
instance, the use of organic acids could result in increase of nitrogen in the bio-crude oil (Ross et
al., 2010). However, the majority of previous studies have focused on high-lipid content algae
(Brown et al., 2010; Dote et al., 1994; Duan and Savage, 2011b; Minowa et al., 1995a; Zou et al.,
2010c), and there is little known about the effect of operating conditions, such as reaction
temperature and retention time, on the distribution of nutrients and energy recovery when fast-
growing, low-lipid content algae are used as feedstocks in an HTL process.
Carbon and nitrogen remaining in the HTL aqueous product are generally not suitable to be
discharged to the environment without additional treatment. Management strategies for the
nutrients in the HTL products streams can significantly affect the viability of an HTL process.
One approach to integrate bio-crude oil production via HTL and wastewater treatment through
growing algae is an integrated process we refer to as Environment-Enhancing-Energy (E2-
Energy) (Zhang and Schideman, 2010), which is illustrated in Figure 4-6. Nutrient distribution in
HTL products is one of the key research questions in the E2-Energy paradigm because the
amount of nutrients and energy remaining in the aqueous products and gas stream affects the
wastewater treatment by algae growing. The recycling of nutrients to the algae growing ponds
allows more algae to be grown, and thus, increases the amount of biofuel created. In this
approach, growing algae is used as a wastewater treatment method to uptake nutrients and
capture CO2 from HTL products. Subsequently, the resulting algal biomass will be further
converted into bio-crude oil via HTL. Since nitrogen and carbon contents of the HTL aqueous
stream are high, the algal species that grow in the post-HTL water are expected to be low-lipid
content microalgae (Zhou, 2010).
In this chapter, the effects of reaction temperature, holding time (retention time) and
catalysts on the carbon and nitrogen distributions in the products from HTL of low-lipid
microalgae were investigated. The elemental distribution under different operating conditions
101
observed in this study can be used to better understand the reaction mechanism of HTL and
provide useful information to optimize the HTL process. This information can also be utilized in
the life-cycle assessment to evaluate the viability of the entire E2-Energy system.
8.1 ELEMENTAL DISTRIBUTION OF HTL OF CHLORELLA
In this study, carbon, hydrogen and nitrogen content of both feedstock and HTL products
were analyzed using a CHN analyzer; and the oxygen content was calculated by difference. Thus,
the oxygen content is slightly larger than the true value since it includes the other minor elements.
The elemental compositions of raw oil and solid residue were analyzed first, and then the
elemental composition of the bio-crude oil product was calculated by difference between these
two fractions. The recovery of carbon or nitrogen is defined as the ratio of carbon or nitrogen in
the HTL product to the carbon or nitrogen in the original algal feedstock. The calculation
equations are shown below. The carbon and nitrogen recoveries for the aqueous product were
calculated by difference once the other three components were determined.
Carbon Recovery (CR):
( )
Nitrogen Recovery (NR):
( )
8.1.1 Effects of reaction temperature on C and N recovery of HTL products from

Chlorella
Figure 8-1 illustrates the effect of reaction temperature on the carbon recoveries in
different phases after HTL of C. pyrenoidosa, including oil/solid phase (raw oil), gas phase and
aqueous phase (aqueous product). The tests in Figure 8-1 were conducted at reaction
temperatures ranging from 100°C to 300°C with a constant retention time of 30 minutes and 0.69
MPa initial pressure of nitrogen.
102
Figure 8-1 CR in different phases vs. reaction temperatures with 30 min retention time (Chlorella)
The raw oil is the combination of bio-crude oil and the solid residue, which are considered
together in some of the data below, because these components are often mixed together in the
reaction products such as the char and bitumen-like products, and can be used as the substitute of
asphalt binder (Minarick et al., 2011).
Although the physical properties such as the viscosity and pour point of raw oil were not
discussed in this study, the appearance of the raw oil product was changed substantially by
increasing reaction temperature, which is summarized in Table 8-1.
Table 8-1 Appearance of raw oil products from different temperatures (Chlorella)
Reaction Temperature Raw oil product appearance Stage
<180°C Green algal cake Stage 1
180-200°C Black solid, looking like a bio-char product Stage 2
200-240°C High viscosity asphalt/bitumen-like product Stage 2

Self-separating, flowable oil phase on the
>240°C Stage 3
top of the aqueous phase
103
The trend of CR in the raw oil with increasing temperature can be divided into three stages
as shown in Figure 8-1. First, when the temperature was lower than 160°C, the CR values of raw
oil were all higher than 90%, which may be due in part to unreacted solids given the green
appearance of the reaction products. Secondly, it decreased from 92.0% to 52.5% when the
reaction temperature increased from 160°C to 260°C. Finally, after the CR in the raw oil reached
the minimum value at 260°C, its values were slightly increased from 52.5% to 58.2%.
In contrast, the CR of the aqueous product initially increased with reaction temperature; but
the value reached at peak of 39.7% at 260°C, after which it started to decrease slightly. Figure
8-1 shows that more than one-third of the total carbon in the original materials was converted
into water soluble organic matters at temperatures above 220°C. This was likely due to
hydrolysis of proteins in the algal biomass and the resulting formation of hydrophilic amino
acids, organic acids, and their derivatives.
The bio-crude oil was defined as the toluene soluble fraction of the raw oil and it is the
most important fraction for determining the potential of HTL to produce petroleum-like products.
The bio-crude oil yield, based on the dry matter of feedstock, increased from 0.4% to 35.4%,
when the reaction temperature increased from 100°C to 300°C. Figure 8-2 shows the CR of bio-
crude oil and the solid residue vs. reaction temperature. The changes of CR for the bio-crude oil
and solid residue can also be divided into three stages, as was noted above for the raw oil product.
For Stage 1, when the reaction temperature was lower than 160°C, most of the carbon in the raw
oil was in solid residue fraction and the CR of the bio-crude oil was less than 5%. Considering
that the toluene soluble fraction of the original C. pyrenoidosa material was less than 0.1%, it
indicates bio-crude oil formation did not occur when the temperature was lower than 160°C. For
Stage 2, as the temperature increased from 180°C to 240°C, CR in the bio-crude oil increased
from 3.1% to 43.2%. On the other hand, CR in the solid residue rapidly decreased from 73.9% to
11.8%. Thus some of the solid residue was converted into the aqueous fraction at the same time
when oil formation was occurring. At Stage 3, when the temperature increased from 240°C to
300°C, the change of CR for both bio-crude oil and the solid residue slowed down and tended to
plateau. The highest CR for the bio-crude oil was 55.3% at 300°C. Compared with other
previous studies that reported CR values for HTL oil in the range of 20% to 40%, the carbon
recovery of the bio-crude oil in present work was higher (Heilmann et al., 2010; Ross et al., 2010;
Yang et al., 2004). This is probably due to the differences in algal species, operating conditions
104
and solvent extraction methods. For reaction temperatures higher than 200°C, the carbon content
of bio-crude oil was consistently around 74% based on the total mass, and did not change much
with increasing temperature. Therefore, the increase in CR for the bio-crude oil above 200°C was
mainly due to the increase of bio-crude oil yield.
Figure 8-2 CR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention time
(Chlorella)
It can be concluded that the minimum reaction temperature for bio-crude oil formation
should be in the range of 180°C to 240°C for C. pyrenoidosa. However, further temperature
increase was also important for changing the physical property of the product because raw oil
could be turned from an asphalt/bitumen-like product into a self-separating, flowable oil product
at higher temperatures. The mechanism of HTL can be briefly explained as large molecular
compounds being decomposed into fragments of small molecules, while at the same time these
unstable and reactive fragments can repolymerize into oily compounds (Demirbaş, 2001). The
repolymerization of these reactive fragments is influenced by the temperature, which
subsequently affects the physical properties of the oil product. The details of the reaction
mechanism are discussed in CHAPTER 9.
105
Figure 8-3 shows the classification of C. pyrenoidosa feedstock, raw oil and bio-crude oil
from different reaction temperatures based on a Van Krevelen diagram. Since bio-crude oil yield
was lower than 5% at 180°C, it is expected that formation of bio-crude oil would mainly occur at
temperature higher than 180°C. At the same time Figure 8-3 also shows the H/C and O/C ratios
of bio-crude oil produced in the temperature ranges from 200°C to 300°C. When the reaction
temperature was lower than 180°C, the atomic H/C and O/C ratios of raw oil were similar to the
algal feedstock. Then both H/C and O/C ratios of the raw oil decreased substantially when the
reaction temperatures were 200°C and 220°C. Since amino acids and organic acids could be
produced through protein hydrolysis, decarboxylation and deamination could be the main
pathways of amino acids decomposition under hydrothermal conditions (Sato et al., 2004).
Under this assumption, amine and carboxyl groups were removed from larger protein molecules
at the relatively low temperatures.
Figure 8-3 Van Krevelen diagram of raw oil and bio-crude oil (Chlorella)
The deamination could remove nitrogen and hydrogen from the amino acids and produce
hydroxyacid. For example, the glycine could be converted into glycolic acid through
deamination (Klingler et al., 2007). The deoxygenation could occur through decarboxylation
106
(Demirbaş, 2000b) as the carbon was removed as CO2. When the temperature was increased
from 220°C to 300°C, the O/C ratio still decreased while the H/C ratio started to increase. In this
temperature range, the H/C ratios of the raw oil were around 1.5 and very similar to the original
C. pyrenoidosa. On the other hand, the O/C ratios of the raw oil were much lower compared to
the algal feedstock. This finding implies that the oxygen was continuously removed via HTL as
the reaction temperature increased from 100°C to 300°C, while the hydrogen was removed from
the feedstock initially at temperatures below 220°C, and then the addition of hydrogen atoms
occurred at higher temperatures. The H/C and O/C ratios of bio-crude oil produced from
temperatures higher than 220°C were similar to those found in another study (Ross et al., 2010).
Increasing the temperature tended to decrease O/C ratio and increase H/C ratio of the bio-crude
oil (colored data points enclosed by dashed line in Figure 8-3).
The change of the atomic H/C and O/C ratios of raw oil with temperature in this study was
different from biochars produced from pistachio shell biomass via slow pyrolysis, in which both
the H/C and O/C ratios of biochars produced at high temperature were much lower than the raw
material and the biochars produced from low temperature (Apaydin-Varol et al., 2007).
Additionally the H/C ratios of raw oil product across the reaction temperature range were all
higher than the chars from pyrolysis of lignocellulosic biomass (Apaydin-Varol et al., 2007;
Mahinpey et al., 2009). Furthermore, when the reaction temperature was higher than 220°C, O/C
ratios of bio-crude oil were all smaller than O/C ratios of bio-oil produced from microalgae via a
fast pyrolysis process (Miao et al., 2004). These observations imply that bio-crude oils produced
from HTL usually have lower oxygen contents than the biofuels produced from pyrolysis
processes, which is advantageous for biofuel applications because of higher energy density
(Huber et al., 2006; Peterson et al., 2008).
Carbon monoxide, carbon dioxide and methane were three carbon-source gaseous products
analyzed in this study, and carbon dioxide was the predominant gas. The CR of gaseous product
increased from 0% to 8.7% as the reaction temperature increased. Another study indicated the
CR from the gas and oil could be equal at 500°C, and the gaseous product would contain more
carbon at even modestly higher temperatures (>500°C) (Brown et al., 2010). Carbon dioxide was
usually the predominant gaseous product at relatively low temperatures (<350°C) in a HTL
process (Brown et al., 2010; Heilmann et al., 2010; Yang et al., 2004). Although there were some
molecules with short carbon chain existing in the gaseous product, the fractions of these gases
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were small and even negligible when the reaction temperature was lower than 300°C (Brown et
al., 2010). In the present study, when the reaction temperature was lower than 220°C, CO2 was
the only detected carbon-source gas. After the reaction temperature was increased above 220°C,
small amounts of CO and CH4 were detected. CR contributed by CO2 in the gaseous products
increased from 0% to 8.61% when the temperature increased from 100°C to 300°C, which
indicated more than 98% of the carbon in the gaseous product was attributed to CO2. This result
indicates that the idea of E2-Energy to utilize HTL gaseous product with high CO2 concentration
as carbon source for algae growing is a desirable approach (Figure 4-6). On the other hand, CR
of CO reached a peak value at 220°C and then decreased quickly with the increase of reaction
temperature. This finding suggested that CO was produced during the HTL process and then
consumed. At the same time, from 240°C to 300°C the CR of CH4 increased with increasing
reaction temperature. The relationship of CR of CO and CH4 with reaction temperature is shown
in Figure 8-4.
Figure 8-4 CR of CO and CH4 from Chlorella
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Another study also found the concentration of CO in the HTL gaseous product was
negligible and speculated that the consumption of CO and formation of CH4 in the HTL process
could be involved in a water-gas shift reaction and a methanation reaction (Brown et al., 2010).
However, the water-gas shift reaction is usually favorable in supercritical water, therefore, more
experimental evidences are needed to study whether it could occur in the subcritical water.
Figure 8-5 pH of post-HTL water (Chlorella)
Figure 8-5 shows the change of pH value of the post-HTL water with reaction temperature.
The pH value of the liquid first decreased when the reaction temperature increased from 100°C
to 180°C, and then started to increase with reaction temperature. This trend indicated some acids
were produced first in the HTL process and then decomposed; some free hydrogen ions were
produced and then consumed. This is consistent with the results shown in Figure 8-3, in which
the addition of hydrogen to the oil product occurred at high temperatures. Since the protein
content of the raw algal material was high, it was reasonable to assume that the peptide bonds in
the protein molecules were broken down under relatively low temperature so that amino acids or
other short carbon chain organic acids were produced. These organic acids could be further
broken down to produce carbon monoxide and hydrogen ions, therefore, enabling other
109
recombination reactions as the temperature continued to increase. Some previous studies found
that the “Fischer-Tropsch” (FT) reaction could occur in a hydrothermal system, with water or
other short carbon chain organic acids acting as hydrogen sources, where CO apparently reacted
with H2 to produce alkanes under milder temperatures (150-250°C) in a stainless steel reactor
with 2-3 days retention time (McCollom et al., 1999; Rushdi and Simoneit, 2001). Since the
reaction intermediates produced from the HTL of microalgae are complex, more research efforts
are required to study the reactions between the intermediates produced from protein, lipid and
carbohydrates in the HTL system.
Deamination is usually accompanied by liberation of ammonia from the amino acids
molecules (Sato et al., 2004). Although ammonium could be one of the sources of nitrogen in the
HTL aqueous product when alkaline catalysts were used (Ross et al., 2010) and at a higher
temperature (350°C) than this study (Minowa and Sawayama, 1999), there was substantial
nitrogen recovered by organic compounds containing nitrogen with the presence of organic acids
and amino acids under the hydrothermal condition at a temperature of 300°C (Dote et al., 1998a).
This could explain why, in many algae HTL studies, including the present work, there was no
ammonia gas detected in the gaseous products (Brown et al., 2010; Elliott et al., 1988; Yang et
al., 2004). The author, therefore, assume that all of the nitrogen in the algal feedstock is
recovered in the aqueous and raw oil products. The relationship of the NR in the raw oil and the
aqueous product to reaction temperature is shown in Figure 8-6. NR of the raw oil product kept
decreasing with the increase of reaction temperature, while the nitrogen recovery in the aqueous
product increased from 1.1% to 73.5%. This result implies that the majority of the nitrogen in the
original material would be converted into water soluble products if the reaction temperature
increased. It also was evidence for the deamination of the amino acids, and for the susceptibility
of amino acid degradation under hydrothermal conditions corresponding to the decomposition
temperature (Sato et al., 2004). The rate of change of the nitrogen recovery for both raw oil and
the aqueous product decreased at the high reaction temperature; the NR of those two products
reached respective plateaus when the reaction temperature increased above 240°C.
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Figure 8-6 NR of raw oil and aqueous product vs. reaction temperature with 30 min retention time
(Chlorella)
To further understand the distribution of nitrogen content in the raw oil, Figure 8-7 presents
the relationship of NR of the bio-crude oil and the solid residue to reaction temperature. The NR
of bio-crude oil increased from 0.4% to 23.4% when the reaction temperature increased from
100°C to 300°C, and the NR of solid residue decreased substantially from 98.6% to 3.1%.
Therefore, even though the total nitrogen recovery of the raw oil decreased as the temperature
increased, more nitrogen was recovered by the bio-crude oil instead of the solid residue. In
general, oils from algae via thermochemical conversion contain a higher proportion of nitrogen
compared to oils from lignocellulosic biomass because of the high protein content in algae.
The undesirable high nitrogen content indicates upgrading of the HTL bio-crude oil should
include nitrogen removal. Zou et al. (2009) reported the nitrogen content in the oil product could
be significantly decreased to less than 1% if the liquefaction was performed in ethylene glycol
medium with sulphuric acid as a catalyst (Zou et al., 2009). While the nitrogen result is
promising, in this case, economic constraints would suggest the need to re-use the solvent media
and catalyst, which is not easily accomplished. Thus, additional work on the removal of nitrogen
from HTL oil products is an important topic for the future work.
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Figure 8-7 NR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention time
(Chlorella)
8.1.2 Effects of retention time on C and N recovery of HTL products from Chlorella
For investigating the retention time effects on CR and NR values of HTL products, HTL
tests were conducted under 200°C, 220°C, 240°C, 260°C and 280°C with 0, 10, 30, 60 and 120
minutes of retention time using the same 0.69 MPa initial pressure of nitrogen. Zero minute
retention time means as soon as the temperature reached the designated reaction temperature, the
cooling water was introduced into the cooling coil inside the reactor to cool down the reactor to
room temperature. The bio-crude oil yield increased from 3.2% to 22.6%, from 16.7% to 30.5%,
from 26.0% to 33.4%, from 25.8% to 35.9%, and from 29.6% to 39.4% when the retention time
increased from 0 to 120 minutes at 200°C, 220°C, 240°C, 260°C and 280°C, respectively. Thus,
increasing retention time had the most impact at lower temperatures (200°C and 220°C). It was
observed that at any given retention time, higher oil yields were achieved with higher reaction
temperature.
Figure 8-8 shows how the retention time affected the CR of bio-crude oil and solid residue.
At five reaction temperature levels, CR of bio-crude oil all increased with the increasing of
retention time, at the same time the solid residue CR values all decreased. Prolonging the
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retention time helps to convert toluene insoluble organic carbon into toluene soluble organic
carbon. It was also found that the effects of retention time on CR and NR values at high
temperatures were less substantial than that at low temperatures. For instance, at 280°C CR of
bio-crude oil increased from 39.0% to 55.3%, while it increased from 4.0% to 31.9%% at 200°C.
Figure 8-8 CR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Chlorella)
Meanwhile, CR of raw oil decreased from 62.9% to 52.5% at 240°C, and increased from
49.9% to 56.9% at 280°C when the retention time increased from 0 minute to 120 minutes.
However, when the temperature was 260°C, CR of raw oil appeared to be stable around 52%
regardless of how long the retention time was. The result indicates that 260°C could be a
threshold value or turning point of reaction temperature, above which the relationships between
raw oil carbon recovery and the reaction temperature were opposite to each other. This
conclusion is consistent with the results in Figure 8-1, which showed that when the reaction
temperature was lower than 260°C, the CR of the raw oil decreased with increasing of reaction
temperature but when the reaction temperature was higher than 260°C, the CR of the raw oil
started to increase. The carbon in the feedstock was removed as CO2 through decarboxylation;
thus, the carbon recovery of raw oil first decreased with increasing of reaction temperature. Since
deamination could introduce hydroxyl groups by removing nitrogen from amino acids, it is
possible that amino acids containing hydroxyl groups were produced. On the other hand, amino
acids with aldehyde groups can also be produced through deamination. These organic molecules
with hydroxyl, aldehyde and carboxyl groups may react with each other through aldol
condensation or esterification. These recombination reactions could produce water insoluble
113
organic molecules; therefore, the carbon recovery of raw oil could increase as the temperature
was further increased. Testing of this hypothetical mechanism could be accomplished by analysis
of the organic molecules and functional groups in the aqueous and oil phases (CHAPTER 9).
The retention time effects on the nitrogen recoveries of bio-crude oil and solid residue are
shown in Figure 8-9. The NR of the bio-crude oil increased with retention time, while the NR of
solid residue decreased substantially. At all reaction temperatures, the nitrogen recoveries for
raw oil decreased as reaction temperatures increased (from 47.5% to 29.7%. from 41.0% to
26.4%, from 42.7% to 23.1%, from 31.3% to 23.5% and from 29.1% to 23.7% when the
retention time increased from 0 minutes to 120 minutes at 200°C, 220°C, 240°C, 260°C and
280°C reaction temperature, respectively). Thus, nitrogen generally accumulated in the bio-crude
oil with longer retention time. Note that at 200°C and 220°C, the nitrogen content in bio-crude
oil increased from 1.6% to 5.9% and from 1.8% to 6.5% respectively, when the retention time
increased from 0 minutes to 120 minutes. However, at higher temperatures, nitrogen content in
bio-crude oil did not change much with retention time. These results implied that at low
temperature, some of the toluene insoluble nitrogen in organic molecules was converted into
toluene soluble molecules when the retention time increased; but increased NR of bio-crude oil
at high temperature were mainly due to the increasing of its yield.
Figure 8-9 NR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Chlorella)
Combined with the results from Section 8.1.1, it is evident that neither increasing reaction
temperature nor prolonging retention time helps to decrease the NR of the bio-crude oil. Even so,
at the five reaction temperature levels, most of the nitrogen in the original material was
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converted into aqueous product. The nitrogen recoveries of the aqueous product all increased
with retention time, and could be expected to approach more than 75% with a sufficiently long
retention time. Unfortunately, there is a strong correlation between operating conditions (reaction
temperature/retention time) that increase CR in the bio-crude oil and those that increase NR in
the bio-crude oil. Thus, other approaches, such as catalysts, would appear to be more promising
for finding ways to reduce the nitrogen content of the HTL oil. Otherwise additional upgrading
to remove the nitrogen from bio-crude oil will be needed to produce transportation grade fuel
from high protein feedstocks (Duan and Savage, 2011b).
8.1.3 Effects of catalysts on C and N recovery of HTL products from Chlorella

Figure 8-10 shows the CR of HTL products produced at 240°C with different catalysts. CR
values of gas product in all HTL tests ranged from 1.9% to 2.7%, which was a small fraction of
the total carbon in the algal biomass. Applying different catalysts at 240°C did not change the
CR of bio-crude oil substantially. The highest CR of bio-crude oil was obtained in the
experiment with the addition of 5% NaOH.
However, the carbon content of the bio-crude oil with 5% NaOH addition was 68.3%
(weight percentage), which was lower than that obtained without catalyst (75.7%). Therefore the
highest CR was mainly due to its highest bio-crude oil yield (Figure 7-1). It should be noted that
with the presences of alkaline catalysts, the CR of the aqueous product also increased. Especially
when 5% of Na2CO3 was applied in the HTL test, there was about 52.1% of carbon was
recovered in the aqueous product, which was much higher than the value obtained without a
catalyst (32.0%). At the same time, the carbon contents in the solid residue were lowered with
the addition of alkaline catalysts. For instance, the carbon content of solid residue with 5%
NaOH and Na2CO3 were 34.0% and 34.9%, respectively, which were much lower than those
obtained without a catalyst (58.0%) and with metal catalysts (ranged from 58.9% to 73.7%). At
the same time, it was found that the solid residue yields with the addition of alkaline catalysts
were lower than those achieved with metal catalysts at 240°C. It indicates that an alkaline
catalyst could benefit the liquefaction of biomass materials into water soluble product. Previous
studies also demonstrated that the solid residue or char yield from HTL of lignocellulose and
protein could be lowered with the addition of Na2CO3 (Minowa et al., 1998; Wang, 2011).
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Na2CO3
NaOH
Raney Ni
Pt/C Bio-crude oil

Solid residue
Pt/Al2O3 Aqueous product
Gas
Pd/C
Pd/Al2O3
No Catalyst
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Figure 8-10 CR of HTL product at 240°C with different catalysts (Chlorella)
Figure 8-11 shows the CR of HTL products with different catalysts at 280°C. Compared
with Figure 8-10, it was found the CR of bio-crude oil was greatly increased and CR of solid
residue and aqueous product were both decreased. Compared with uncatalyzed test at 280°C, CR
of bio-crude oil was increased with the addition of different catalysts. At the same time, the CR
of solid residue was largely decreased with the addition of different catalysts. Especially, when
alkaline catalysts were applied, the CR of solid residue was lower than 2%. This again confirms
the addition of alkaline catalysts could decrease solid residue or char formation under
hydrothermal conditions.
Meanwhile, since gas yield increased with the increase of reaction temperature, the CR of
gas product was higher than that at 240°C. Meanwhile, the CR of gas product at 280°C was still
less than 5%, which indicates most of carbon was recovered by bio-crude oil. Another study
implied that with the addition of alkaline catalysts, the CR of gas product could be substantially
increased when the reaction temperature was higher than 300°C (Ross et al., 2010).
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Na2CO3
NaOH
Raney Ni
Pt/C Bio-crude oil

Solid residue
Pt/Al2O3 Aqueous product
Gas
Pd/C
Pd/Al2O3
No Catalyst
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Figure 8-11 CR of HTL product at 280°C with different catalysts (Chlorella)
Figure 8-12 reflects the NR of HTL products with different catalysts at 240°C. It was found
that the majority of nitrogen in the original biomass material was recovered by the aqueous
product. In other words, more than 60% of nitrogen was converted into water soluble products.
Compared with the additions of metal catalysts, the addition of alkaline catalysts substantially
lowered the NR of solid residue and increased the NR of bio-crude oil. At the same time,
nitrogen contents of bio-crude oil produced at 240°C with different catalysts were similar to each
other, so the high NR of bio-crude oil with alkaline catalysts was due to the higher bio-crude oil
yields.
The nitrogen content of the solid residue was lowered with the addition of alkaline catalysts.
For example, under uncatalyzed conditions the nitrogen content of solid residue was 9.5% and it
decreased to 5.8% and 5.6% with the addition of NaOH and Na2CO3, respectively.
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Na2CO3
NaOH
Raney Ni
Pt/C Bio-crude oil
Solid residue
Pt/Al2O3
Aqueous product
Pd/C
Pd/Al2O3
No Catalyst
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Figure 8-12 NR of HTL product at 240°C with different catalysts (Chlorella)
Figure 8-13 shows the NR of HTL products with different catalysts at 280°C. Compared
with Figure 8-12, the NR of the aqueous product did not change much with increased
temperature, but the NR in the solid/oil phase was greatly changed. Most of the nitrogen in the
solid residue was converted into nitrogen in the bio-crude oil. With catalysts applied, NR values
of solid residue at 280°C were substantially decreased because the yield of solid residue
decreased from 15.6% (without catalysts) to around 5% (with catalysts). It was also found that
except for Pt/Al2O3 and Raney nickel, the nitrogen content of the solid residue deceased with the
addition of different catalysts. Especially, when alkaline catalysts were applied the nitrogen
content of the solid residue decreased from 7.4% (without catalyst) to 5.0% (with NaOH) and 2.5%
(with Na2CO3). Compared with the nitrogen content of solid residue obtained at a lower
temperature (240°C) with the different catalysts, increasing temperature could lower the nitrogen
content of the solid residue.
A previous study showed that a significant fraction of nitrogen was recovered by the gas
product (Ross et al., 2010). However, in that study only nitrogen in the bio-crude oil, solid
residue and ammonia concentration in the aqueous product were analyzed, and then the NR of
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the gas product was calculated by difference. In other words, organic nitrogen in the aqueous
phase was not considered. In fact, since a lot of organic compounds with nitrogen atom were
detected in the aqueous phase (CHAPTER 9), the NR of aqueous product should be higher than
the literature value.
Na2CO3
NaOH
Raney Ni
Pt/C Bio-crude oil
Solid residue
Pt/Al2O3
Aqueous product
Pd/C
Pd/Al2O3
No Catalyst
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Figure 8-13 NR of HTL product at 280°C with different catalysts (Chlorella)
Our results also implies that majority of nitrogen was recovered by aqueous product when
the temperature was lower than 300°C (Yu et al., 2011b). At same time, including present study,
there was no ammonia gas detected in the HTL gaseous products (Brown et al., 2010; Elliott et
al., 1988; Yang et al., 2004). Therefore the NR of gas product should be negligible under
subcritical conditions.
8.2 ELEMENTAL DISTRIBUTION OF HTL OF SPIRULINA
8.2.1 Effects of reaction temperature on C and N recovery of HTL products from

Spirulina
Figure 8-14 shows the CR of different HTL products produced from S. platensis. The trend
of CR in the raw oil with increasing temperature can be divided into three stages as shown in
Figure 8-14. First, when the temperature was lower than 200°C, the CR values of raw oil were
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all higher than 90%, which was partially due to unreacted algal feedstock given the green
appearance of the reaction products. Secondly, it decreased from 94.1% to 33.3% when the
reaction temperature increased from 200°C to 220°C. Finally, after the CR in the raw oil reached
the minimum at 220°C, its values were increased from 40.2% to 59.2% when the temperature
increased from 240°C to 300°C. Compared with the results from C. pyrenoidosa (Figure 8-1), it
was found that the highest CR of the aqueous product was obtained at 220°C for S. platensis,
while it was 260°C for C. pyrenoidosa. This is because the highest aqueous yield of S. platensis
was achieved at 220°C and then the yield of aqueous product decreased with the further increase
of reaction temperature.
Figure 8-14 CR in different phases vs. reaction temperatures with 30 min retention time (Spirulina)
Figure 8-15 shows the CR of bio-crude oil and solid residue produced from S. platensis. It
was found that with the increase of reaction temperature, the CR of solid residue decreased. At
the same time the CR of bio-crude oil increased. It suggests that more carbon accumulated in the
bio-crude oil phase, even the CR of raw oil product decreased with temperature increased from
160°C to 220°C, and then started to increase when the temperature increased from 220°C to
300°C. From Figure 8-15, the temperature at which the CR of the bio-crude oil and solid residue
was equal to each other, was between 220°C to 240°C.
120
At 300°C with 30 minutes retention time, only 2.9% of the total carbon in the original
biomass remained in the solid residue product. Therefore, HTL is an effective process to convert
organic carbon in biomass materials into the bio-crude oil, which can be dissolved into solvents.
Even CR of the aqueous product decreased with temperature when temperature was higher than
220°C; there was about 34.7% of carbon recovered by the water soluble product at 300°C. Those
carbons in the aqueous phase were primarily organic compounds with short carbon chains.
Therefore, how to treat and reuse those carbons in the E2-Energy system will be a critical issue
because some of the organic compounds may be toxic for algae growing.
Figure 8-15 CR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention time
(Spirulina)
The relationship of the NR in the raw oil and the aqueous product with reaction temperature
is shown in Figure 8-16. The NR of raw oil decreased substantially from 93.3% to 24.2% when
the temperature increased from 200°C to 220°C. After that, the NR of raw oil remained relatively
stable around 25%. NR of the aqueous product increased from 6.7% to 75.8% when the
temperature increased from 200°C to 220°C. Figure 8-16 shows that the majority of the nitrogen
content was converted into water soluble products via the HTL process, which was consistent
with the results from C. pyrenoidosa.
121
Some of the nitrogen was converted into inorganic form, such as ammonia via deamination.
On the other hand, some organic nitrogen compounds were also produced via deamination and
dissolved into water due to the amino acid degradation under hydrothermal conditions
corresponding to the decomposition temperature (Sato et al., 2004) . The organic form of the
nitrogen is often contained in the amine group (CHAPTER 9). Since a lot of aromatic amines are
usually toxic to the aqueous species, such as Chlorella vulgaris (Netzeva et al., 2004), the
presence of large amounts of amines in the aqueous phase could influence the reuse of post-HTL
water to grow algae.
Figure 8-16 NR of raw oil and aqueous product vs. reaction temperature with 30 min retention time
(Spirulina)
To investigate the NR in the bio-crude oil and solid residue and how the nitrogen content
distributed in the raw oil product, Figure 8-17 shows the relationship of NR of bio-crude oil and
solid residue with the reaction temperature. It was found that by increasing temperature, the NR
of the bio-crude oil increased. At 300°C, about 23.3% of total nitrogen in the original feedstock
was recovered by the bio-crude oil. The nitrogen content (weight percentage) in the bio-crude oil
decreased with the increase of temperature. For instance, at 220°C, the nitrogen content in the
bio-crude oil was around 10.9%. When the temperature reached 300°C, the nitrogen content of
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the bio-crude oil was 6.9%. Since the yield of bio-crude oil increased from 9.7% to 37.3% as
temperature increased from 220°C to 300°C, the NR of bio-crude oil increased with the increase
of temperature. This result was consistent with the data in literature and is much higher than the
nitrogen content in conventional crude oil, therefore, additional upgrading of the bio-crude oil
was necessary to remove the nitrogen content (Duan and Savage, 2011a).
As discussed before, using solvent as a reaction medium could be helpful in lowering the
nitrogen content of the bio-crude oil. At the same time, using solvent as reaction medium could
also increase the bio-crude oil yield because some of the water soluble organic compounds can
be extracted by the solvents. However, reuse of the organic solvents, which is not easy to achieve,
would be a critical issue to demonstrate the economically feasibility of this approach.
Figure 8-17 NR of bio-crude oil & solid residue vs. reaction temperature with 30 min retention time
(Spirulina)
8.2.2 Effects of retention time on C and N recovery of HTL products from Spirulina
Figure 8-18 shows the CR for the bio-crude oil and solid residue produced from S. platensis
with 30 minutes retention time at different temperature levels. At all temperature levels, the CR
of bio-crude oil increased with the increase of retention time. At the same time, the CR of solid
residue decreased with prolonged retention time. These findings are similar to the results from
123
HTL of C. pyrenoidosa. At 280°C with 120 minutes retention time, there was about 68.8% of
total carbon in the original algal biomass remaining in the bio-crude oil phase. At this operating
condition, solid residue only contained 4.9% of the total carbon in the algal feedstock. Compared
with the results obtained from C. pyrenoidosa at this condition, less carbon was recovered by the
aqueous phase, implying that more carbon can be retained in the bio-crude oil phase for HTL of
S. platensis.
(a) (b)
Figure 8-18 CR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Spirulina)
Figure 8-19 shows the relationship of NR for bio-crude oil and solid residue vs. retention
time at five reaction temperature levels. The NR of solid residue decreased substantially by
prolonging retention time at all temperature levels. At the same time, the NR of bio-crude oil
increased with the increase of retention time. It should be noted that the nitrogen content in the
bio-crude oil (weight percentage) decreased with increased retention time at 220°C, 240°C,
260°C and 280°C. For instance, the nitrogen content of bio-crude oil decreased from 11.4% to
6.3% when the retention time increased from 0 minute to 120 minutes at 280°C. Therefore, the
NR of bio-crude oil increased with retention time was mainly due to the increase of its yield.
As the retention time increased, the nitrogen recovered by the aqueous phase also increased.
At 280°C, NR of the aqueous phase increased from 58.3% to 74.6% when the retention time
increased from 0 to 120 minutes.
124
(a) (b)
Figure 8-19 NR vs. retention time: (a) for bio-crude oil; and (b) for solid residue (Spirulina)
8.2.3 Effects of catalysts on C and N recovery of HTL products from Spirulina

CR of HTL products produced from S. platensis with the presence of different catalysts at
240°C and 280°C are illustrated in Figure 8-20. Except for Pt/C, CR in the solid/oil phase (raw
oil) increased as temperature increased with the addition of the same catalysts. At the same time,
CR of the aqueous product decreased as temperature increased from 240°C to 280°C. Therefore,
higher operating temperatures could be helpful in converting carbon from the aqueous phase into
the bio-crude oil phase. CR of the gaseous product also increased due to the increase of its yield.
(a) (b)
Na2CO3 Na2CO3
NaOH NaOH
Raney Ni Raney Ni
Pt/C Pt/C Bio-crude oil

Solid residue
Pt/Al2O3 Pt/Al2O3 Aqueous product
Gas
Pd/C Pd/C
Pd/Al2O3 Pd/Al2O3
No Catalyst No Catalyst
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Figure 8-20 CR of HTL product with different catalysts (Spirulina): (a) 240°C; and (b) 280°C
125
When Pt/C was applied into the HTL process at 240°C and 280°C, the bio-crude oil yields
were 31.3% and 32.8%, respectively. Although the carbon content of the bio-crude oil increased
from 64.6% to 70.8% as temperature increased, carbon content in the solid residue was
substantially lowered from 45.2% to 24.0%. At the same time, solid residue yield also decreased
from 16.4% to 7.8% as temperature increased from 240°C to 280°C. Therefore, the small
increase of CR in the aqueous product (from 41.7% to 45.5%) as temperature increased from
240°C to 280°C was primarily due to the substantial decrease of CR in the solid residue.
(a) (b)
Na2CO3 Na2CO3
NaOH NaOH
Raney Ni Raney Ni
Pt/C Pt/C Bio-crude oil
Solid residue
Pt/Al2O3 Pt/Al2O3
Aqueous product
Pd/C Pd/C
Pd/Al2O3 Pd/Al2O3
No Catalyst No Catalyst
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Figure 8-21 NR of HTL product with different catalysts (Spirulina): (a) 240°C; and (b) 280°C
Figure 8-21 shows the NR of HTL products from S. platensis with different catalysts.
Although NR of solid residue decreased as temperature increased from 240°C to 280°C, NR of
bio-crude oil did not change much. The nitrogen content of the bio-crude oil slightly decreased
with the increase of temperature. However, the NRs of bio-crude oil were close to each other due
to the increase of bio-crude oil yield as reaction temperature increased.
It was found at the two temperature levels, the addition of metal catalysts could decrease
NR of the aqueous product. It implied more nitrogen would remain in the bio-crude oil phase
with the addition of metal catalysts. This was mainly due to the increase of bio-crude oil yield
because the nitrogen contents of bio-crude oil at the two temperatures were close to each other.
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Nitrogen content of bio-crude oil can significantly influence its quality and potential
application; therefore, nitrogen removal is always important for upgrading bio-crude oil. Based
on the results obtained from the present study, the nitrogen content in the bio-crude oil will not
be significantly lowered with the increase in temperature, prolonging retention time or directly
applying catalysts into the HTL process.
Upgrading bio-crude oil from algae with metal catalysts in supercritical water could lower
the nitrogen content of the bio-crude oil to about 2% (Duan and Savage, 2011a), but the nitrogen
content was still much higher than that in conventional petroleum. Therefore, many more studies
and efforts are needed to explore an effective way to remove nitrogen from bio-crude oil in the
future.
8.3 ENERGY BALANCE OF HTL OF MICROALGAE
8.3.1 Energy consumption ratio of HTL of microalgae

The energy balance of HTL of microalgae is evaluated using two parameters: energy
recovery (ER) and energy consumption ratio (ECR). Energy recovery was defined as how much
higher heating value (HHV) of the original algal biomass can be recovered by bio-crude oil,
which can be calcultaed as:
( )
HHV values in this study were calculated using the Dulong formula:
( ) ( )
where C, H, and O are the mass percentages of carbon, hydrogen and oxygen, respectively.
ECR was defined to estimate the energy required for HTL of microalgae against the total
energy that can be recovered by combusting the bio-crude oil. An ECR value above 1 means the
energy balance is negative. ECR values less than 1 indicate the energy balance for the conversion
is positive. The ECR values were calculated according to the equation below, which was adapted
and modified from the methods in literature (Biller and Ross, 2011; Sawayama et al., 1999;
Vardon et al., 2012).
( ) ( ) ( )
( )
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where wi is the moisture content of the initial feedstock before HTL, Cpw is the specific heat of
water (4.18 kJ∙kg-1∙K-1), Cps is the specific heat of dry algae (1.25 kJ∙kg-1∙K-1), T is the designated
reaction temperature, Tini is the initial temperature before conversion which is assumed to be
25°C, Ybio is the bio-crude oil yield, HHVbio is the higher heating value of bio-crude oil, rh is the
efficiency of heat recovery assumed to be 0.5, rc is the efficiency of available combustion energy
assumed to be 0.6.
Table 8-2 summarizes the element composition and calculated HHV values of bio-crude oil
produced from C. pyrenoidosa. It was found that reaction temperature has substantial effects on
the heating value of bio-crude oil. At 240°C reaction temperature with 0 minutes reaction time,
HHV of bio-crude oil was about 59% higher than that of C. pyrenoidosa feedstock, indicating
that HTL has already occurred and formation of bio-crude oil has already started under the
operating conditions.
Table 8-2 Element analysis and HHV of Chlorella and bio-crude oil
Reaction Temperature Retention Time HHV
C (%) H (%) N (%) O (%) a
(°C) (min) (MJ∙kg-1)
C. pyrenoidosa -- 51.38 6.62 11.13 30.87 21.31
160 30 40.82 6.18 11.58 41.42 15.25
180 30 40.90 5.92 17.04 36.14 15.86
200 0 63.59 9.25 1.58 25.58 30.24
200 10 64.83 9.36 3.06 22.75 31.33
200 30 72.40 4.38 6.85 16.36 27.87
200 60 71.76 10.06 4.97 13.21 36.40
200 120 72.50 9.80 5.86 11.84 36.52
220 0 64.78 9.58 1.80 20.47 32.03
220 10 73.78 10.87 4.55 10.79 38.69
220 30 74.03 4.47 7.00 14.51 28.87
220 60 73.98 9.95 6.12 9.95 37.58
220 120 71.36 9.62 6.46 12.56 35.74
240 0 67.29 9.78 5.82 17.11 33.79
240 10 70.49 9.61 5.84 14.07 35.16
240 30 75.60 9.39 7.38 7.63 37.74
240 60 71.13 9.98 7.17 11.73 36.34
240 120 71.27 8.99 6.61 13.13 34.71
260 0 67.91 9.90 6.14 16.05 34.35
260 10 72.36 10.83 6.84 11.6 38.00
260 30 73.26 9.32 7.56 9.86 36.45
260 60 72.82 9.66 7.43 10.10 36.74
260 120 71.27 8.99 6.61 13.13 34.71
280 0 67.66 9.76 7.57 15.01 34.25
280 10 71.22 8.47 7.46 12.85 33.99
280 30 74.49 10.13 7.17 8.22 38.32
280 60 72.83 8.93 6.72 11.51 35.45
280 120 72.21 9.15 6.43 12.21 35.42
300 30 75.42 9.95 7.35 7.28 38.54
a
Calculated by difference
128
The Dulong formula was widely used to estimate the HHVs of bio-crude oil in other studies
(Vardon et al., 2012; Xu and Lad, 2007; Zhou et al., 2010a). The HHVs of raw oil produced at
280°C and 300°C reaction temperatures with 30 minutes reaction time were also analyzed using
a bomb calorimeter (Model 1281, Parr Instrument Co., Moline, IL), and the HHV of the bio-
crude oil was calculated using the percentage of solid residue in the raw oil and their HHVs. In
comparison with the experimental results, the Dulong formula gave overestimations of only 6.25%
and 1.83% at reaction temperatures of 280°C and 300°C, respectively. Therefore, the HHVs
estimated by Dulong formula were acceptable.
Figure 8-22 is a contour plot of the energy recovery of bio-crude oil at different reaction
temperatures and retention times. The figure indicates that, with the temperature ranging from
100°C to 300°C and retention time ranging from 0 to 120 minutes, energy recovery of bio-crude
oil increased with reaction temperature and retention time. The highest energy recovery of bio-
crude oil was 65.4%, achieved at 280°C reaction temperature and 120 minutes retention time.
This result is comparable with one study using a higher reaction temperature (350°C) and high-
lipid content microalgae (Nannochloropsis) as HTL feedstock (Biller and Ross, 2011). The
results also indicated that about two thirds of the total energy in the algal feedstock was
contained in the HTL oil product. Finding an economical way, such as the concept of E2-Energy,
to recover the fraction of energy contained in other HTL products would enhance the viability of
the HTL process.
129
Figure 8-22 Energy recovery (%) of bio-crude oil produced from Chlorella
Energy consumption ratio (ECR) of HTL can reflect whether this conversion process has
positive energy balance or not. In this study, the moisture contents of microalgal feedstock were
both 80% for C. pyrenoidosa and S. platensis when the effect of temperature on HTL of
microalgae was investigated. Figure 8-23 shows the ECR values of C. pyrenoidosa and S.
platensis at different temperature levels.
(a) (b)
Figure 8-23 ECR of HTL at different temperature: (a) Chlorella; (b) Spirulina
130
As temperature increased from 220°C to 240°C, ECR values decreased from 0.41 to 0.31
(with 50% heat recovery) for HTL of C. pyrenoidosa. Although the ECR still decreased with
temperature further increasing from 240°C to 300°C, the rate of ECR decrease was slow.
Compared with C. pyrenoidosa, the ECR values for HTL of S. platensis were higher at the same
temperature. Especially, when the temperature was lower than 260°C, ECR values exceeded
unity for HTL of S. platensis if there is no heat recovery.
Based on the equation to calculate the ECR value, it is known that efficiency of heat
recovery can significantly influence the energy balance of the HTL process. As shown in Figure
8-23, ECR values were lowered by 50% if 50% of heat used for HTL can be recovered. As a
result, although increasing reaction temperature means more energy was consumed during the
HTL process, the decrease of ECR values indicates the energy balance of the HTL process was
improved at high temperatures. In other words, HTL became more energetically favorable at
higher reaction temperature.
Initial moisture content of the HTL feedstock not only impacts the energy balance of the
conversion process (ECR value), but also determines how much energy is required to pre-
dewater the feedstock. Unlike lignocellulosic biomass, dewatering is often needed for microalgae
harvesting. For algae-to-biodiesel, energy consumed in the dewatering process contributed
around 75% of total energy consumption (Lardon et al., 2009). In the present study, the initial
moisture content of the microalgal feedstock was adjusted to 80% by weight. However, the solid
content of microalgae in an open pond or photobioreactor system is usually lower than 5%,
which means the moisture content is higher than 95%. Therefore, it is necessary to investigate
how the initial moisture content of feedstock can influence the energy balance of the HTL
process.
131
Figure 8-24 ECR of HTL vs. initial moisture content of feedstock (Chlorella)
Figure 8-24 shows how the initial moisture content of feedstock affects the energy balance
of an HTL process. It was assumed that the bio-crude oil yield and the HHV of bio-crude oil
remain constant at the same temperature with different initial moisture content of the feedstock.
It was found the moisture content of the feedstock can substantially affect the energy balance of
the HTL process. Theoretically, lower moisture content is preferred because of the resulting
lower ECR value. However, achieving lower moisture content implies more energy will be
consumed in the pre-dewatering process. On the other hand, water serves as the reaction medium
of the HTL process and sufficient water can guarantee the feedstock slurry is pumpable in the
continuous HTL process.
It was found that at the three temperature levels in Figure 8-24, when the moisture content
of the feedstock exceeds 93%, the ECR value is larger than unity and changes the HTL process
from energy positive to negative. This result suggests that HTL may not be an energetically
favorable process to convert high-diluted fresh microalgae into bio-crude oil. However, the ECR
value decreases substantially with the decrease of the moisture content of feedstock. For example,
with 80% moisture content the ECR value is lowered to 0.30 at reaction temperature of 300°C.
132
From this aspect, HTL still has many advantages compared with traditional algae-to-biodiesel
technology and pyrolysis process, which often require completely drying of algal feedstock.
Furthermore, in Figure 8-24 the efficiency of heat recovery rh and the combustion of bio-crude
oil rc were assumed to be 0.5 and 0.6, respectively. If both of the efficiencies can be increased,
HTL could still be energetically favorable with higher initial moisture content of the feedstock.
For instance, if heat recovery efficiency and the efficiency of bio-crude oil combustion are both
increased to 0.8, HTL is still an energetically favorable process even the initial moisture content
of feedstock is as high as 97%. Under the circumstances, the energy required for pre-dewatering
microalgae feedstock can be greatly decreased.
Obviously, moisture content of the feedstock will not only influence the energy balance of
the HTL process but also determine how much energy is needed to pre-dewater the algal
feedstock. Therefore in the future a comprehensive study on the energy balance of the entire
process, which includes algae cultivation, harvesting, dewatering and conversion, is needed to
investigate the economical and energetic favorability of utilizing HTL to produce biofuel from
microalgae.
8.3.2 Comparison of energy balances of different biofuel productions

Although ECR value is efficient to demonstrate that HTL of microalgae is an energetically
favorable method to produce bio-crude oil, it is necessary to compare the energy balance of bio-
crude oil produced from HTL with other biofuel production methods in order to demonstrate the
advantages of producing biofuel from microalgae via HTL. In this study, an energy balance
index, fossil energy balance (FEB) is used to compare the energy balances of different biofuel
products produced from different methods. The FEB is defined as:
( ) ( )
From the definition, a positive FEB value indicates the energy balance is energetically
favorable and a negative FEB value indicates the conversion process is energetically unfavorable.
Figure 8-25 shows the comparison of FEB values for different biofuels. The FEB values of
petroleum gasoline, corn ethanol, cellulosic ethanol and algae biodiesel were calculated based on
the data in the literature (Lardon et al., 2009; Wang et al., 2011). It should be noted that any
energy products based on fossil energy conversion would result in a negative FEB because the
energy in fossil-based products is part of the energy in fossil feedstock (Wang et al., 2011). It
133
was found that bio-ethanol produced from corn and lignocelluloses all have positive FEB values.
On the other hand, for the algae-biodiesel, only the wet extraction on algae grown in low
nitrogen conditions result in positive FEB values, which again confirms that water evaporation is
the most energy intensive process for producing algae-biodiesel.
1.5
This Study
1
0.5
MJ per MJ biofuel produced
0
1 2 3 4 5 6 7 8 9 10 11
-0.5
1. Petroleum gasoline
2. Corn ethanol
-1
3. Corn Stover ethanol
4. Switch Grass ethanol
-1.5 5. Forest Residue ethanol
6. Algae-biodiesel (LowN-Wet)
-2 7. Algae-biodiesel (LowN-Dry)
8. Algae-biodiesel (Normal-Wet)
9. Algae-biodiesel (Normal-Dry)
-2.5
10. Algae-bio-crude oil (Chlorella)
11. Algae-bio-crude oil (Spirulina)
-3
Figure 8-25 Fossil energy balance of different biofuels
The FEB values for bio-crude oil produced from HTL of Chlorella and Spirulina were
calculated based on the assumption of 50% of heat recovery. It should be noted that only the
energy used to produce algae bio-crude oil via HTL was taken into account in the FEB
calculations. The FEB values of bio-crude oils are both positive and higher than the FEB values
of petroleum gasoline, corn ethanol, all of algae-biodiesel, and ethanol produced from forest
residue. Only the ethanol produced from corn stover and switch grass have higher FEB values
than bio-crude oil produced from HTL. Since the higher heat recovery may be obtained, the FEB
value of bio-crude oil could be increased in the future.
Bio-crude oil produced from HTL of algae can be upgraded into a better quality biofuel
with lower nitrogen, oxygen, and sulfur content through a catalytic hydrotreatment under
supercritical conditions (Duan and Savage, 2011a). Another application of bio-crude oil and raw
134
oil produced from HTL is being used as an asphalt binder. The raw oil may do a better job of
approximating the amount of asphalt binder production since asphalt binder is not a highly
refined product (Minarick et al., 2011). Therefore, the economic balance of HTL of microalgae
could be largely increased since the raw oil yield is much higher than the bio-crude oil yield.
Moreover, the economic outlook of bio-crude oil from HTL of microalgae could be largely
improved if the benefits that may be obtained from recycling nutrients and wastewater treatment
through the E2-Energy design are both considered.
135
CHAPTER 9. CHARACTERIZATION OF HTL PRODUCTS AND
EXPLORATION OF REACTION KINETICS AND MECHANISMS
The characteristics of HTL products provide key information for understanding the reaction
pathway of HTL of microalgae. Although there are an abundance of studies in literature to
explore the reaction mechanisms of different components in biomass materials under
hydrothermal conditions, such as lipid, protein, cellulose, and lignin, reaction mechanisms of real
biomass materials are still largely unknown due to the complexity of their compositions. And to
some extent, the possible reactions among the intermediates derived from different components
under hydrothermal conditions largely increase the difficulty to finally understand the reaction
pathways of real biomass materials. Synergistic and antagonistic effects may coexist due to the
instability of those reaction intermediates. Therefore, although a lot of research has been
conducted on studying reaction pathways of those relatively simple components using model
compounds, investigating what reactions really occur during HTL of microalgae is still very
important and can provide essential information to understand the interactions among reaction
intermediates produced from individual components in biomass materials. The ultimate goal of
understanding the reaction pathway and kinetics of HTL of microalgae can only be achieved
after the information about the complex series of reactions under hydrothermal conditions, such
as decomposition, degradation, depolymerization, recombination and repolymerization, are all
well known.
The gaseous product and solid residue produced from HTL were already characterized in
CHAPTER 8; therefore, in this chapter the emphasis is to investigate the characterization of bio-
crude oil and the aqueous product produced from HTL of microalgae.
9.1 EFFECT OF REACTION TEMPERATURE
9.1.1 Bio-crude oil produced at different temperatures

The composition of bio-crude oil produced from algae via HTL is much more complicated
than conventional fuel (Duan and Savage, 2011b; Jena and Das, 2011; Ross et al., 2010; Vardon
et al., 2012; Zou et al., 2010a). GC-MS is often used to identify the volatile organic molecules in
bio-crude oil samples. In a typical ion chromatogram, hundreds of peaks could be detected.
However, not all the peaks can be identified using a popular database, such as NIST Mass
136
Spectral Database (NIST08) and W8N08 library. Typically, the peak areas of the identified
compounds contribute about 60%-80% of the total ion current chromatogram (TIC) (Duan and
Savage, 2011b; Jena and Das, 2011; Ross et al., 2010; Vardon et al., 2012; Wang, 2011; Xiu et
al., 2011; Zou et al., 2010a).
Figure 9-1 Total ion chromatogram of bio-crude oil (Chlorella, 200°C, 30 min)
For example, Figure 9-1 shows the total ion chromatogram of bio-crude oil produced from
C. pyrenoidosa at 200°C with 30 minutes retention time. It was found under this condition the
bio-crude oil mainly consists of cyclic nitrogenates (peak 1 and 2), straight and branched
hydrocarbon (peak 3 and 4), branched oxygenates (peak 5, 6 and 8), and fatty acids (peak 7, 9
and 10). Fatty acid is often produced through hydrolysis of lipid content. Although the crude fat,
which represents the lipid content in C. pyrenoidosa was 0.1% based on the dry weight of the
feedstock, it was found the percentage of fatty acids peak area was more than 50% of the total
peak area in Figure 9-1. It should be pointed that this finding cannot simply lead to the
conclusion that the lipid content in C. pyrenoidosa is high because the bio-crude oil yield was
relatively low under this condition (22.4%). Additionally, the percentage of peak area does not
represent the actual concentration of the compounds although there is a positively correlated
137
relationship between these two parameters. However, this finding implies that crude fat, which is
analyzed using the Soxhlet extraction, could lead to underestimation of lipid content in
microalgae. The Folch extraction was also conducted to analyze lipid content of C. pyrenoidosa
and S. platensis. The comparison of these two different methods is summarized in Table 9-1. The
uncertainties represent standard deviations of triplicate results.
Table 9-1 Comparisons of different methods to analyze lipid content in algae
Crude fat (%) a Lipid (%) b

C. pyrenoidosa 0.40.3 14.31.8
S. platensis 4.80.3 12.31.6
a b
analyzed using Soxhlet extraction; analyzed using Folch extraction.
It was found the Folch extraction resulted a much higher lipid content compared with the
Soxhlet extraction method. However, Folch extraction could lead to overestimation of lipid
content because other components such as pigment in microalgae, can also be extracted at the
same time. Since the method for analyzing algae lipid content has not been standardized yet, we
can only estimate that the true value of lipid content should be between the results produced from
these two different methods. However, no matter which method is applied, the two algal species,
C. pyrenoidosa and S. platensis, still belong to low-lipid content microalgae and are not suitable
for biodiesel production.
Figure 9-2 Total ion chromatogram of bio-crude oil (Chlorella, 300°C, 30 min)
138
Figure 9-2 shows the TIC of bio-crude oil produced from C. pyrenoidosa at 300°C with 30
minutes retention time. Compared with Figure 9-1, much more compounds were detected at
higher reaction temperature, which indicates the compositions of bio-crude oil became more
complicated at a higher reaction temperature. The major compounds and their chemical
structures in Figure 9-2 are summarized in Table 9-2.
Table 9-2 Major compounds in bio-crude oil produced from Chlorella (300°C, 30 min)
Retention
No. Compound Area (%) Structure
time (min)
1 8.32 Pyrazine, 2,5-dimethyl- 0.55
2 10.45 Benzaldehyde 0.57
3 11.50 Phenol 1.35
4 11.65 Pyrazine, 2-ethyl-6-methyl- 0.77
5 14.19 Pyrazine, 3-ethyl-2,5-dimethyl- 0.99
6 16.02 N-(3-Methylbutyl) acetamide 1.17
7 20.35 Indole 1.76
N-[2-Hydroxyethyl]
8 22.45 1.84
succinimide
9 22.69 1H-Indole, 4-methyl- 0.97
139
Table 9-2(cont.)
Retention
No. Compound Area (%) Structure
time (min)
10 24.95 Benzonitrile, 2,4,6-trimethyl- 0.96
11 25.63 Acetamide, N-(2-phenylethyl)- 1.34
12 29.67 n-Octacosane 4.02

2-Hexadecene, 3,7,11,15-
13 32.88 8.78
tetramethyl-, [R-[R*,R*-(E)]]-
3,7,11,15-Tetramethyl-2-
14 33.29 1.91
hexadecen-1-ol
15 35.18 n-Hexadecanoic acid 15.51
16 38.26 cis-Vaccenic acid 2.84
17 39.74 Hexadecanamide 1.80
9,12-Octadecadienoic acid
18 41.52 6.74
(Z,Z)-, methyl ester
19 42.00 9-Octadecenamide, (Z)- 6.14
20 43.74 N-Decanoylmorpholine 10.21
Hexadecanoic acid,
21 44.79 4.45
pyrrolidide
Pyrrolidine, 1-(1-oxo-9,12-
22 47.24 2.37
octadecadienyl)-
Total 77.04
It was found that the fraction of N-heterocyclic compounds increased as temperature

increased. On the other hand, the fraction of organic acids decreased to 21.8% of total peak area,
it is probably due to their reactions with ammonia and organic compounds with the amine group
140
and more compounds being produced and detected under the higher temperature. This
assumption can be supported by the evidence that a lot of amide and amide derivatives were
produced at 300°C. From Table 9-2 it was found large amounts of molecules with N-heterocyclic
structure were produced. This also confirms that the organic acid or fatty acid could react with
cyclic or branched amine at high temperatures. It should be pointed out these N-heterocyclic
compounds are usually stable under subcritical hydrothermal conditions. Jena and Das (2011)
found at 350°C the N-heterocyclic compounds, still largely presented in bio-oil produced from
Spirulina (Jena and Das, 2011).
Figure 9-3 Temperature effect on relative percentages of major compound classes in bio-crude oil
Classifying different organic compounds into different groups can benefit the study of
reaction mechanism because it can group the compounds with a similar reaction pathway under
hydrothermal conditions. Effects of temperature on the relative peak area percentages of four
major compound classes in bio-crude oil produced from C. pyrenoidosa are illustrated in Figure
9-3.
The hydrocarbon includes straight and branched hydrocarbon. Organic acid includes fatty
acid and other organic acids. Cyclic oxygenates include phenols, phenol derivatives and fused
ring compounds. Straight and branched amides are produced from reaction of organic acids and
141
ammonia or amines. Nitrogen and oxygen heterocyclic compounds are produced via
recombination or repolymerization of protein derivatives under hydrothermal condition, such as
pyrazine, pyrolle, indole, piperazinedione, and derivatives of pyrrolidine.
It was found fractions of hydrocarbon increased from 3.3% to 15.3% as temperature
increased from 200°C to 300°C. On the other hand, at 200°C, organic acids represent more than
50% of the total detected organic compounds and its fraction decreased to 15.3% at 280°C and
then slightly increased to 21.8% at 300°C. The hydrocarbon was partially produced from organic
acids via decarboxylation. Fractions of cyclic oxygenate and heterocyclic compounds increased
with temperature, which indicates the decomposition of protein and repolymerization of reaction
intermediates were both promoted at high reaction temperatures. The fraction of straight and
branched amides increased from 2.1% to 21.7% when the temperature increased from 200°C to
280°C, then decreased to 14.8% when temperature increased from 280°C to 300°C. It was found
that under hydrothermal conditions amide with short carbon chain, for example N-
methylacetamide, can be hydrolyzed to yield acetic acid and methylamine via a reversible
reaction (Duan et al., 2010). Since the straight and branched amides detected in bio-crude oil
usually have a long carbon chain, such as hexadecanamide and 9-Octadecenamide, (Z)-, more
studies are needed to investigate their pathways under hydrothermal conditions.
9.1.2 Aqueous product produced at different temperatures

The characteristics of the aqueous product are directly related to the nutrients recycle and
algae growth and as a result greatly affect the feasibility of the entire E2-Energy system. It is
necessary to analyze its composition, which represents the water soluble organic compounds
produced via HTL of microalgae.
Figure 9-4 shows the TIC of aqueous product produced from C. pyrenoidosa at different
temperatures with 30 minutes retention time. The molecule structures associated to the peaks in
Figure 9-4 were summarized in Table 9-3. It was found the compositions of the aqueous product
were changed substantially as temperature increased. Note that some compounds were co-eluted
from the GC column using the current analytical method and it is extremely difficult to separate
them perfectly due to their similarities in structure after derivatization. Although quantitatively
analyzing the concentration of each molecule in the aqueous product seems to be difficult, the
TIC still reveals important information to predict the reaction pathways of protein, lipid and
carbohydrates in algae under hydrothermal conditions.
142
8
8 11
100 C 200 C
18
11 14
12 10 17 19
23 9
4 13 15 19
11
89 240 C 11 300 C
8
3 17 17
10
5
14
3
7 6 16
2
Figure 9-4 Total ion chromatogram of aqueous product at different temperature (Chlorella)
The peak No. 8 was detected in the aqueous product at different temperatures, which
represents both phosphorus acid and glycerol since they were co-eluted. However, at 100°C,
peak No. 8 was only identified as phosphorus acid and its peak area was more than 75% of the
total peak area. At temperatures of 200°C, 240°C and 300°C, both phosphoric acid and glycerol
were identified. The pH value of post-HTL water was lower than 7.0 when reaction temperature
was below 220°C. When the reaction temperature was higher than 240°C, the pH value of post-
HTL water was above 7.0 (Figure 8-5). Therefore, it is possible that phosphoric acid can exist at
temperature of 100°C and 200°C since it is a strong inorganic acid (pKa1: 2.12, pKa2: 7.21,
pKa3: 12.67). However, at temperatures of 240°C and 300°C abundant phosphoric acid cannot
exist in the alkaline environment. Before the aqueous samples were analyzed using GC-MS,
derivatization (trimethylsilylation) was applied, during which the pH value was adjusted by
methoxyamine hydrochloride (Section 4.3.6). In this pretreatment process, phosphoric acid could
143
be generated from the phosphate group in post-HTL water. The phosphate group was probably
produced from hydrolysis of phosphate ester. Therefore, the identification of phosphoric acid in
aqueous product suggests notable amounts of phosphorus in the original algal feedstock were
converted into water soluble products after the HTL process.
It should be noted that the phosphorus content in the post-HTL water is largely affected by
the metal composition in the feedstock. One explanation for this conclusion is some metal
elements could bond with phosphorus to form precipitates so that the phosphorus will be
recovered by solid residue. For instance, the calcium and magnesium could bond with
phosphorus to form calcium phosphate and magnesium phosphate which are both insoluble in
water, respectively. The phosphorus content in the algal feedstock is 1.68%, the calcium and
magnesium content are 0.23% and 0.27% (dry weight basis), respectively. Therefore, even all of
the calcium and magnesium can bond with phosphorus to form precipitates during HTL and
finally form solid residue, only 0.33% of the phosphorus in the algal feedstock will be recovered
by solid residue. In other words, more than 80% of phosphorus in the algal feedstock was
recovered in the aqueous phase. From this aspect, the phosphorus recovery in HTL products is
largely affected by the metal compositions of the feedstock. For instance, the calcium and
magnesium content in swine manure is 2.02% and 0.79%, respectively. Since the phosphorus
content in swine manure is 1.87%, almost all of the phosphorus could be bonded with calcium
and magnesium to form solid residue after the HTL process.
Three major compounds in the aqueous product produced at 200°C and 240°C, were not
detected at 300°C, including glycine (No. 9), pyrimidine (No. 10) and adenine (No.14). Those
compounds are intermediates produced from protein hydrolysis and their stabilities under
hydrothermal conditions were quite different. For example, glycine was found unstable under
hydrothermal conditions and degraded quickly to produce glycolic acid via deamination
(Klingler et al., 2007) or to yield methylamine via decarboxylation (Snider and Wolfenden,
2000). A previous study also reported that the glycine degradation under hydrothermal
conditions followed simultaneous decarboxylation and deamination pathways (Sato et al., 2004).
However, another study reported glycine preferentially undergo dimerization and subsequent
cyclization when heated in an inert reactor under hydrothermal conditions (Cox and Seward,
2007). Another possible reaction pathway of glycine degradation is the Maillard reaction,
through which the amino acids and carbohydrates or derivatives from amino acids and
144
carbohydrates may react to each other. In the Maillard reaction, the amine group in amino acids
will react with the carbonyl group presenting in carbohydrates to generate complicated amine
and water (Peterson et al., 2010). In fact, the glycine peak was first identified in the aqueous
product at a reaction temperature of 160°C and its relative concentration (% area) increased
when the temperature increased from 160°C to 260°C, then greatly decreased when the
temperature increased from 260°C to 300°C.
On the other hand, with presences of N2, CO2 and H2, under hydrothermal conditions at
300°C the adenine decreased rapidly during the first 24 hours and kept decreasing slowly
afterwards so that adenine was still detected in the hydrothermal solution after 200 hours
(Franiatte et al., 2008).
These findings in literature imply that even the intermediates produced from one source
component in microalgae, such as protein, have different reaction rates, degradation pathways
under hydrothermal conditions and could interact with other possible intermediates produced
from other components, such as lipids and carbohydrates in microalgae. Therefore, the reaction
mechanisms of the real biomass materials are expected to be extremely complicated.
Glycerol, which is the main product from lipid hydrolysis under hydrothermal conditions,
could also degrade to yield acrolein and acetaldehyde in subcritical conditions, and acrolein and
allyl alcohol in supercritical conditions (Qadariyah et al., 2011). Therefore, glycerol could also
react with other intermediates under hydrothermal conditions. It was interesting to find that a
large fraction of 2-O-Glycerol-α-d-galactopyranoside (floridoside) (8.3% of the total peak area),
which is represented by peak No. 18, was only detected at a reaction temperature of 200°C in the
aqueous product. Meanwhile, a small fraction of floridoside (less than 1% of total peak area) was
also detected between 120°C to 240°C. Although floridoside is a natural glycerol galactoside
widely found in red algae (Weïwer et al., 2008), it was reasonable to speculate that it could be
produced from the reaction between glycerol and carbohydrate derivatives in this study. Lactic
acid (peak No. 1) could also be generated from glycerol degradation under hydrothermal
conditions (Ram rez-L pez et al., 2010) and the highest fraction of lactic acid was also detected
at 200°C. Therefore, it was evident that glycerol degradation could occur and its derivative could
react with other reaction intermediates under hydrothermal conditions. However, some pathways
could only be preferable at certain temperatures due to the instabilities and further reactions of
the products.
145
Table 9-3 Major compounds in aqueous product produced from Chlorella
Retention
No. Compound Formula Structure
time (min)
1 15.46 Lactic acid C3H6O3
2 15.91 Acetic acid C2H4O2
3 16.83 Alanine C3H7NO2
4 18.00 Carboxy-clycine C3H5NO4
5 18.43 Cadaverine C5H14N2
6 19.24 1,4-Butanediamine C4H12N2
7 20.25 Valine C5H11NO2
8 21.99* Phosphoric acid H3PO4
8 21.99* Glycerol C3H8O3
9 22.91 Glycine C2H5NO2
10 23.66 Pyrimidine C4H4N2
11 28.44* Proline C5H9NO2
11 28.44* Pyroglutamic acid C5H7NO3
146
Table 9-3(cont.)
Retention
No. Compound Formula Structure
time (min)
12 32.11 Ribose C5H10O5
13 33.17 Ribitol C5H12O5
14 35.92 Adenine C5H5N5
15 36.94 Lysine C6H14N2O2
16 36.80 Tyramine C8H11NO
17 40.51 Inositol C6H12O6
2-O-Glycerol-α-d-
18 44.34 C9H18O8
galactopyranoside
19 46.33 Uridine C9H12N2O6
*: Compounds are eluted simultaneously

To summarize the effect of reaction temperature on HTL product characteristics,
temperature greatly affects the compositions of bio-crude oil and the aqueous product. Increase
in temperature led to further degradation and repolymerization of initial intermediates produced
from protein, lipid and carbohydrates in microalgae. The instabilities, or in other words,
reactivities of the reaction intermediates make the reaction pathways of microalgae under
hydrothermal conditions extremely complicated.
147
9.2 EFFECT OF RETENTION TIME
The effect of retention time on bio-crude oil characteristics is shown in Figure 9-5 and
Figure 9-6. The relative concentrations of different major compound classes are represented by
the percentage of their peak areas to the total peak area.
Figure 9-5 Effect of retention time on bio-crude oil characteristics (Chlorella, 240°C)
At the two temperature levels, fraction of organic acids decreased with the increase of
retention time, while fraction of amides and N&O heterocyclic compounds increased with
retention time increasing. On the other hand, effect of retention time on fraction of cyclic
oxygenates was not pronounced. As defined previously, cyclic oxygenates include phenols,
phenol derivatives and fused ring compounds. It was already found that the fraction of cyclic
oxygenates was increasing with reaction temperature (Figure 9-3). Therefore, the relative
concentrations of the cyclic oxygenates were less influence by retention time compared to
reaction temperature.
148
Figure 9-6 Effect of retention time on bio-crude oil characteristics (Chlorella, 280°C)
The N&O heterocyclic compounds, such as pyrazine and derivatives of pyrrolidine are
produced from the recombination or repolymerization of the small molecules produced from
degradation of protein, lipid and carbohydrates. Under the two temperature levels, the fraction of
pyrazine, which represents the product produced from recombination of amine and straight
oxygenates with a short carbon chain; and the fraction of derivatives of pyrrolidine, which
represents the product produced from recombination of pyrrolidine and fatty acid with a long
carbon chain, were both increased. Meanwhile, higher relative concentrations of complicated
compounds produced from recombination also implies that more small compounds were
produced via degradation of protein, lipid and carbohydrates. For example, the scission of C-C
bond in amino acids and the recombination of amino acid with organic acids could happen
simultaneously. Degradation pathways of amino acids, including decarboxylation and
deamination, could occur simultaneously under hydrothermal conditions with elevated
temperature and prolonged retention time. Additionally, the hydrolysis of amino acids under
hydrothermal conditions may result in interconversion among amino acids (Sato et al., 2004).
149
240 C, 0min 2 240 C, 10min
3 5
5 4
3 4
1
2
1
2 2 240 C, 60min
240 C, 30min
1
1 5 5
3 3
4
4
2
240 C, 120min
1
5
3
4
Figure 9-7 Effect of retention time on TIC of aqueous product at 240°C (Chlorella)
Figure 9-7 shows the effects of retention time on the aqueous product produced at 240°C
with different retention times. For the two detected major peaks, which eluted time are 21.99
minute and 28.44 minute, respectively, there are two molecules associated with each peak. The
150
peak with 21.99 minute eluted time represented both phosphoric acid and glycerol; and the peak
with 28.44 minute eluted time represented both proline and pyroglutamic acid. Other than the
two major peaks, it was found the relative concentrations of five major compounds (Peak No.1-5
in Figure 9-7) changed with prolonged retention time. The No.1-5 peaks represent alanine,
glycine, pyrimidine, adenine and inositol, respectively. Their structures and compound formulas
were already summarized in Table 9-3. Figure 9-8 shows the changes of relative concentrations
of alanine, glycine, pyrimidine, adenine and inositol with increase of retention time at 240°C.
Figure 9-8 Effect of retention time on relative concentrations of 5 compounds in aqueous product
(Chlorella, 240°C)
It was found the relative concentrations of alanine and glycine first increased with retention
time, and then their fractions decreased with further increase of retention time. The highest
fraction of glycine (5.51%) and alanine (3.74%) was obtained at 10 and 60 minutes retention
time, respectively. Alanine and glycine are two of the simplest amino acids produced from
protein hydrolysis. The results in Figure 9-8 indicate the increase of retention time could benefit
protein hydrolysis to produce amino acids first, but increasing additional retention time could
lead to amino acid degradation or reacting with other intermediates.
151
Fractions of pyrimidine and adenine were both decreased with retention time. It implies
both of the two compounds either degraded or reacted with other intermediates by prolonging
retention time. The instability of adenine under hydrothermal conditions with increasing
retention time found in this study is consistent with results in the literature (Franiatte et al., 2008).
Inositol (peak No. 5 in Figure 9-7), which is not a common sugar, belongs to carbohydrates. Its
fraction decreased first with retention time and then started to increase with the further increase
of retention time. Since the carbohydrates contribute about 22.0% of the total weight of C.
pyrenoidosa (Table 5-1), it could be released from hydrolysis of carbohydrates under
hydrothermal conditions. The decrease of the inositol fraction could confirm the Maillard
reaction occurrence when microalgae are converted under hydrothermal conditions.
Figure 9-9 shows the effect of retention time on the aqueous product TIC at 280°C. The
relative concentrations of five major compounds (Peak No.1-5 in Figure 9-9) changed with
prolonged retention time. The No.1-5 peaks represent alanine, (2Z)-(Hydroxyimino) acetic acid,
glycine, adenine, and inositol, respectively. Their structures and compound formulas were
summarized in Table 9-3. The fraction of pyrimidine detected at 240°C was negligible at 280°C.
The trend of the alanine fraction change was found similar to that at 240°C: it first
increased with retention time, and then started to decrease after its relative concentration reached
the maximum. However, the fraction of glycine decreased with the increase of retention time all
the time. At 280°C with 120 minutes retention time, the relative concentrations of alanine and
glycine were both negligible, which indicates both of the two amino acids have degraded or
reacted with other intermediates. This result is consistent with that obtained at 240°C but it might
be concluded that the production of glycine was already completed even with 0 minute retention
time at 280°C. (2Z)-(Hydroxyimino) acetic acid is a derivative probably produced from acetic
acid and ammonia. The increasing of its fraction implies some interactions between reaction
intermediates and ammonia could be promoted by prolonging retention time. Since the ammonia
is the inorganic nitrogen in the aqueous product produced from deamination of amino acids, this
finding suggests that the inorganic nitrogen in the aqueous product could be converted into
organic nitrogen under certain hydrothermal conditions.
152
280 C, 0min 280 C, 10min
3
5
5
1
4 3
1
2 2 4
5
280 C, 60min
280 C, 30min
5
1
1
3 2
2
4
3
280 C, 120min
5
1
2
Figure 9-9 Effect of retention time on TIC of aqueous product at 280°C (Chlorella)
Figure 9-10 shows the changes of relative concentrations of alanine, (2Z)-(Hydroxyimino)

acetic acid, glycine, adenine, and inositol with increase of retention time at 280°C. The fraction
of adenine decreased with the increase of retention time at 280°C. When the retention time was
153
120 minutes, the relative concentration of adenine in the aqueous product was negligible. At the
same time, the trend for the inositol fraction change was different from that at 240°C. The
fraction of inositol decreased first with prolonging of retention time and then started to increase
at 240°C, probably due to the production rate of inositol was faster than the consumption rate at
240°C. However, the consumption rate could exceed the production rate at 280°C due to the
more intensive reaction conditions. No matter under which assumption, as soon as the fractions
of carbohydrates and amino acids decreased, it might be concluded the interaction between them
could happen, which confirms the Maillard reaction pathway is highly possible under
experimental conditions.
Figure 9-10 Effect of retention time on relative concentrations of 5 compounds in aqueous product
(Chlorella, 280°C)
9.3 EFFECT OF CATALYSTS
The effects of catalysts on bio-crude oil compositions at 240°C and 280°C were
summarized in Figure 9-11 and Figure 9-12. The major compounds here were classified as
hydrocarbon, organic acids (including fatty acids), amides and N&O heterocyclic compounds.
154
At 240°C, additions of heterogeneous catalysts increased the fraction of hydrocarbons and
at the same time the fraction of organic acids was decreased. This implies heterogeneous
catalysts promoted the decarboxylation of organic acid; therefore, the organic acids were
converted into hydrocarbons. Meanwhile, fractions of amides and N&O heterocyclic compounds
were not greatly changed when heterogeneous catalysts were applied at 240°C.
On the other hand, when alkaline catalysts were applied, the fraction of organic acids was
substantially decreased. However, the fraction of hydrocarbons did not change much compared
with experiments without the addition of catalysts. At the same time, it was found the fraction of
N&O heterocyclic compounds was largely increased. Therefore, although the addition of
alkaline catalysts could lower the fraction of organic acids, the main pathway of organic acids
decreased might not be decarboxylation. Alkaline catalysts might enhance the dissociation of
hydrogen ion in fatty acids and then promote the recombination or repolymerization of the
intermediates.
Figure 9-11 Effects of catalysts on relative concentration of major compound classes in bio-crude oil
(Chlorella, 240°C)
155
At 280°C, the catalysts effects on the relative concentrations of major compounds in bio-
crude oil were less substantial than at 240°C. It was found the addition of Pd/Al2O3 decreased the
fraction of N&O heterocyclic compounds from 36.1% (no catalyst) to 22.7%, and increased the
fraction of amides from 21.7% (no catalyst) to 31.2%.
On the other hand, a large increase of N&O-heterocyclic compounds was found when
alkaline catalysts were applied, which suggests the addition of alkaline catalysts could increase
the possibilities of intermediates recombination or repolymerization. Production of large N&O-
heterocyclic compounds may decrease the quality of the bio-crude oil due to the increase of NR
of bio-crude oil. Moreover, the increase of N&O heterocyclic compounds may also shift the BP
distribution of bio-crude oil to the high temperature range because heterocyclic compounds
generally have high boiling points. This result keeps consistencies with the BP distribution
results illustrated in Figure 7-7 and Figure 8-13.
Figure 9-12 Effects of catalysts on relative concentration of major compound classes in bio-crude oil
(Chlorella, 280°C)
The effects of catalysts on major compounds in the aqueous product produced from C.
pyrenoidosa at 240°C are illustrated in Figure 9-13. From comparisons, it was found the TICs of
the aqueous product with heterogeneous catalysts additions were very similar to that obtained
156
without the addition of a catalyst. However, with the addition of alkaline catalysts, peak areas of
some major compounds decreased substantially. For instance, peak No. 1 and No. 2 represent
glycine and inositol, respectively. With the addition of NaOH, both of the peak areas were
greatly decreased, which implies the relative concentrations of those two compounds were
largely decreased so that the addition of NaOH could decrease the concentration of glycine in the
aqueous product. With the addition of another alkaline catalyst, Na2CO3, only the peak of
inositol was largely decreased, which suggests the addition of alkaline catalysts could lower the
concentration of inositol in aqueous product at 240°C.
On the other hand, with the addition of alkaline catalysts the relative concentration of
floridoside (peak No. 3 in Figure 9-13) was increased. Since floridoside was expected to be
produced from the reaction between glycerol and carbohydrate derivative, it suggests that
alkaline catalysts could promote the reaction between the carbohydrate derivatives and lipid
hydrolysis products. One of the carbohydrates or sugar derivatives was also detected when the
alkaline catalysts were applied in HTL (peak No. 4 in Figure 9-13). However, in current
available databases the structures of this sugar derivative could not be identified. It was only
classified as a sugar derivative because signals at m/z 191 and m/z 361, which are the typical
sugar signals, presented in its spectrum. But this already provides the information that the
addition of alkaline during HTL of microalgae can promote the hydrolysis of carbohydrates
(Zhao et al., 2008).
Figure 9-14 shows the effects of catalysts on the relative concentrations of the major
compounds in the aqueous products produced from HTL of C. pyrenoidosa at 280°C. Compared
with Figure 9-13, TICs of the aqueous product produced with the addition of different catalysts
were much more similar to each other at the higher temperature. Heterogeneous catalysts seem to
have negligible effects on the major compounds in the aqueous product. On the other hand, the
main difference between the aqueous product produced with the addition of alkaline catalysts
and other experiments includes three peaks (peak No. 1-3 in Figure 9-14). Compared with the
results from other experiments, fraction of N-carboxyglycine (peak No. 1 in Figure 9-14)
increased from about 0.5% to 2.2% when 5% NaOH was applied in HTL. At the same time, with
addition of NaOH and Na2CO3, some organic acids, such as the pipecolic acid (peak No. 2 in
Figure 9-14) were largely removed. Since the relative concentration of pipecolic acid was low
even without the addition of alkaline catalysts (usually less than 1% of the total peak area), and it
157
is a strong acid with pKa value of 2.28, it is reasonable to speculate that it could be neutralized
by additions of alkaline catalysts. Finally, the fraction of inositol (peak No. 3 in Figure 9-14)
decreased from 4%-6% of the total peak area to 2.3% and 2.6% of the total peak area when
NaOH and Na2CO3 was applied in HTL respectively. This finding is similar with that obtained at
240°C.
158
Figure 9-13 Effect of catalysts on TIC of aqueous product at 240°C (Chlorella)
159
Figure 9-14 Effect of catalysts on TIC of aqueous product at 280°C (Chlorella)
160
9.4 EXPLORATION OF HTL KINETICS AND MECHANISMS
9.4.1 Kinetic model for HTL of microalgae

As discussed in this chapter, the composition of bio-crude oil and the aqueous product are
both very complicated. Tens of organic compounds were detected in bio-crude oil and the
aqueous products (Table 9-2, Table 9-3). Most importantly, since no single dominant product (%
area larger than 50%) was detected in the bio-crude oil or in the aqueous product, it is very
difficult to quantitatively analyze the concentration of each organic compound in the bio-crude
oil and aqueous product. For these reasons, the detailed kinetic modeling of HTL of microalgae
becomes highly challenging. Based on the author’s knowledge, there was no similar study
available in literature.
Meanwhile, kinetic models on model compounds such as protein, lipid and carbohydrate
under hydrothermal conditions were well documented in the literature (Khuwijitjaru et al., 2004;
Rogalinski et al., 2005; Zhang et al., 2008; Zhu et al., 2008). Since proteins and carbohydrates
are the main components in the two microalgal species used in this study, we attempted to
develop a similar kinetic model based on the information derived from pure compounds and data
obtained from this study.
The reaction of HTL of microalgae can be simplified as a solid state decomposition reaction
as shown below:
Since the reaction order for protein, lipid and carbohydrate hydrolysis were widely assumed as
first-order reaction, the hydrothermal liquefaction of microalgae can be treated as a pseudo-first-
order decomposition reaction as well. Therefore, the rate of microalgal biomass conversion via
HTL, dα/dt, can be described as equation (9-1):
( ) (9-1)
Where k is the reaction rate constant at different temperature, f(α) is the function of the reaction
model, and α is the conversion fraction. It is worth pointing out, since the heating period has a
great effect on the HTL product yields (Figure 6-8), it is not reasonable to combine the heating
period and iso-thermal period together to establish the kinetic model. From this consideration,
the conversion fraction α is defined as:
161
( )
( )
Where mt is the sum of the carbon recoveries of bio-crude oil, aqueous product, and gaseous
product at a given retention time t, mini is the sum of carbon recoveries of bio-crude oil, aqueous
product and gaseous product as soon as the temperature reached to the designated terminal
reaction temperature. From this definition, it is clear that the carbon in solid residue is defined as
the un-converted material; therefore, there is a relationship between m and carbon recovery of
solid residue:
where CRsolid represents the carbon recovery of solid residue.

At the same time, the reaction rate constant k can be described by the Arrhenius law:
( ) (9-2)
where Ea is the activation energy of the reaction, A is the pre-exponential factor, R is the ideal
gas constant (8.314 Jmol-1K-1), and T is the Kelvin temperature.
Combining equation (9-1) and (9-2) gives:
( ) ( ) (9-3)
For the pseudo-first order reaction, there is a relationship between conversion fraction α and
reaction rate constant k (Coats and Redfern, 1964; Luo et al., 2011; Zou et al., 2010b):
[ ] (9-4)
Therefore, the plot of -ln(1-α) against t (retention time) should result in a straight line with a
slope of reaction rate constant k at different temperatures. The k values of HTL of C.
pyrenoidosa at different temperatures are summarized in Table 9-4.
Table 9-4 Reaction rate constants at different temperatures (Chlorella)
T(°C) k (s-1) R2
200 0.0000798552 0.98
220 0.0001274990 0.93
240 0.0001552200 0.89
260 0.0001580119 0.96
280 0.0002831621 0.92
162
Integrate equation (9-2), the relationship between k and temperature can be derived:
(9-5)
From equation (9-5), the plot of lnk against 1/T should result in a straight line with a slope of –
Ea/R and intersection of lnA. Based on the data in Table 9-4, the activation energy for HTL of C.
pyrenoidosa can be calculated. The calculation of Ea and A is illustrated in Figure 9-15.
-8.1
-8.3
-8.5
y = -3586.5x - 1.8065
R² = 0.9059
-8.7
lnk
-8.9
Ea=29.82 kJ/mol
-9.1 A=0.1642 s-1
-9.3
-9.5
0.001750 0.001800 0.001850 0.001900 0.001950 0.002000 0.002050 0.002100 0.002150
1/T (K-1)
Figure 9-15 Determinations of Arrhenius kinetic parameters
The same method can be used to derive the apparent activation energy and pre-exponential
factor for HTL of S. platensis. The results of the kinetic study are summarized in Table 9-5. It
was found the activation energy for HTL of C. pyrenoidosa and S. platensis was 29.82 kJ∙mol-1
and 24.02 kJ∙mol-1, respectively.
Table 9-5 Apparent kinetic parameters for HTL of microalgae
Algal species Ea (kJ/mol) A (s-1) R2

C. pyrenoidosa 29.82 0.1642 0.91
S. platensis 24.02 0.0699 0.83
163
Compared with kinetic parameters derived for hydrolysis of the pure compounds, the
kinetic model set-up in this study was relatively simple due to the complexity of the HTL
products. Although only an apparent decomposition first-order reaction kinetic model was
derived based on existing information, it shows that the organic carbon in algal feedstock quickly
decomposed and was converted into bio-crude oil, aqueous product and gaseous product under
hydrothermal conditions. The lower activation energy indicates energy needed for microalgae
decomposition is less than producing certain amounts of reaction intermediates. In fact, with
similar assumption, activation energy for decompositions of other biomass components was
derived in literature. For example, Zhang et al. (2008) used a 75 mL Parr batch reactor to study
the kinetics of hydrothermal treatment of lignin. With a high heating rate of 140°C∙min-1, the
activation energy for hydrothermal treatment of lignin was as low as 37 kJ∙mol-1 (Zhang et al.,
2008). Kanetake et al. (2007) used guaiacol as the representative of lignin and obtained the
activation energy for its decomposition under hydrothermal condition was 39.35 kJ∙mol-1
(Kanetake et al., 2007).
It was also found the activation energies of HTL of microalgae were lower than that of
protein, lipid and carbohydrate individual hydrolysis (Khuwijitjaru et al., 2004; Rogalinski et al.,
2005; Zhang et al., 2008; Zhu et al., 2008). Note that most of the literature data were obtained
through directly measuring the concentrations of certain products. For instance, the amino acids
concentrations were directly measured to study the kinetics of protein hydrolysis (Rogalinski et
al., 2005; Zhu et al., 2008). On the other hand, some literature data was obtained under
circumstances that either a tubular plug-flow reactor was available or direct samplings were
conducted during regular intervals without interrupting or quenching the experiments. Therefore,
the effects of heating period on product yields were minimized. Due to the limitations of heat
transfer for the batch reactor used in this study, certain fractions of biomass materials had already
been degraded during the heating period especially when the final reaction temperature was high.
Thus, in the iso-thermal period, some fraction of algal biomass were already converted into the
reaction intermediates for bio-crude oil formation so that the reaction rate constant was
constrained largely by the interactions among reaction intermediates. The apparent reaction rate
constant was not greatly enhanced with increasing temperature, which leads to the relatively low
activation energy derived from the Arrhenius law.
164
9.4.2 Reaction mechanism for HTL of microalgae
As previously discussed, the major components in microalgal species include protein, lipid
and carbohydrates. However, the reaction mechanism of HTL of microalgae is much more
complicated than simply combining the reaction pathways of those components under
hydrothermal conditions due to the occurrences of a large amount of interactions among the
reaction intermediates produced from different components. Based on the data obtained from this
study and the literature, the reaction pathways of HTL microalgae under hydrothermal conditions
can be simplified as:
First step, hydrolysis of protein, carbohydrate and lipid:
(1) Protein is hydrolyzed to produce amino acids.
(2) Carbohydrate is hydrolyzed to produce sugars.
(3) Lipid is hydrolyzed to produce fatty acids.
Second step, decomposition of amino acids, fatty acids and sugars:
(4) Oxygen is removed from carboxyl group in amino acids to form carbon dioxide via
decarboxylation.
(5) Nitrogen is removed from amine group in amino acids to form ammonia via deamination.
(6) Alkanes and alkenes are produced from fatty acids and organic acids via decarboxylation.
(7) Sugars are degraded to produced cyclic oxygenates.
Third step, recombination and decomposition of reaction intermediates:
(8) Carboxyl group in amino acid or fatty acid react with amine to produce N-heterocyclic
compounds, such as pyrazine, pyrrole, etc.
(9) Amino acids react with reducing sugars to produce N&O-heterocyclic compounds via
Maillard reaction.
(10) Further recombination and decomposition of the reaction intermediates produced from
the previous steps.
A simplified scheme of the reaction pathways for HTL of microalgae is illustrated in Figure
9-16. The positions of the compounds on the associated temperature axis represent the lowest
temperature under which the compounds were produced in bio-crude oil or aqueous product. It
should be pointed out, after the cyclic oxygenates and cyclic nitrogenates were produced, further
recombination or degradation of these reaction intermediates could occur at higher temperatures
(not shown in Figure 9-16). From this proposed reaction pathway, it is assumed that reaction
165
intermediates produced from protein hydrolysis can actively react with products from lipid and
carbohydrate hydrolysis. Due to the high reactivities of the products from protein hydrolysis, the
reactions among the intermediates are very complicated. Many more efforts are needed to study
the interactions among reaction intermediates, and then it would be possible to illustrate the
comprehensive reaction pathways for HTL of microalgae.
166
hydrolysis
+ Fatty acids
Glycerol
Decarboxylation Alkanes
Lipid
Carbon dioxide Alkenes
+
Pyrrolidine derivatives of fatty acid
Decarboxylation
Amine
Amide
Amino acids
hydrolysis Alcohol
Pyrazine
Protein
Deamination Pyrrolidine Indole
+
Organic acid Ammonia
Maillard Reaction Degradation

Melanoidin
hydrolysis
Reducing Sugar
Carbohydrate Degradation
+
Cyclic oxygenates
Sugar
0 50 100 150 200 250 300

Temperature ( C)
Figure 9-16 Simplified reaction pathway scheme for HTL of microalgae
167
CHAPTER 10. SUMMARY AND RECOMMENDATIONS
10.1 SUMMARY AND FINDINGS
Microalgae are treated as suitable feedstocks for next-generation biofuel production

because they have faster growth rates, shorter growing cycles and higher photosynthetic
efficiencies than lignocellulosic plants. Additionally, microalgae growing can occur on marginal
land, and therefore, has less impact on current land use for food production systems. High-lipid
microalgae were widely studied in literature for the feasibilities of lipid-to-biodiesel conversion.
However, lipid accumulation in microalgae under a stressed condition such as nitrogen depletion,
sacrifices biomass productivity, reduces the net energy yield and makes the process very
sensitive to contamination. Therefore, high-lipid microalgae usually have lower biomass
productivities than low-lipid microalgae.
Hydrothermal liquefaction (HTL) was approved to be an effective way to convert wet
biomass materials into bio-crude oil. In current work, two low-lipid microalgal species,
Chlorella pyrenoidosa and Spirulina platensis, have been successfully converted into bio-crude
oil via HTL. The main findings of this study are summarized as follows:
(1) Algae were converted into bio-crude oil, aqueous product, gaseous product and solid
residue via HTL. The sum of bio-crude oil and solid residue was defined as raw oil product.
More than 10 algal species were converted via HTL. Solubility of raw oil reflects the quality of
oil product. There was a positive relationship between the protein content of algae and the
toluene solubility of raw oil product. On the other hand, ash content in algae had a negative
effect on the oil quality. The average bio-crude oil yield from microalgae was higher than that
from macroalgae under the same operating conditions. Based on the results from screening tests
and the availability of feedstock, two low-lipid and fast-growing microalgae, Chlorella
pyrenoidosa and Spirulina platensis were selected to study the feasibility of producing bio-crude
oil from microalgae via HTL.
(2) Increasing reaction temperature could increase bio-crude oil yield. For Chlorella, bio-
crude oil yield increased from 0.4% to 39.8% when the reaction temperature increased from
100°C to 320°C. When the temperature increased from 320°C to 340°C, the bio-crude oil yield
slightly decreased to 35.0%. For Spirulina, as the reaction temperature increased from 200°C to
168
300°C, the bio-crude oil yield increased from 9.7% to 37.3%. As temperature increased, the yield
of aqueous product, which represents the water-soluble fraction of HTL products, first increased
and then slightly decreased. At 300°C, the aqueous product yield was 44.0% and 48.1% for
Chlorella and Spirulina, respectively. For both microalgal species, the gaseous product yield was
negligible when the temperature was below 160°C. Gas yield was also increased with
temperature, but the increase was slow from 240°C to 300°C. Solid residue yield substantially
decreased as reaction temperature increased. At 300°C with 30 minutes retention time, the solid
residue yield was 4.3% and 3.2% for Chlorella and Spirulina, respectively. The toluene
solubility of raw oil increased with temperature, meaning that the quality of oil could be
improved by increasing operating temperature.
(3) At five temperature levels (200°C, 220°C, 240°C, 260°C, and 280°C), bio-crude oil
yield increased with prolonging of retention time. The effect of retention time at a lower
temperature was more pronounced than at higher temperature. Solid residue yield decreased
substantially with the increase of retention time. At 280°C with 120 minutes retention time, both
highest bio-crude oil yield and lowest solid residue yield were obtained for the two microalgal
species. The highest bio-crude oil yield was 39.4% for Chlorella and 50.1% for Spirulina. One
reason that Spirulina had a higher bio-crude oil yield than Chlorella was that the experiment for
converting Spirulina was conducted in a smaller batch reactor (100 mL vs. 2000 mL), and thus,
fewer products were lost during the recovery process. Typically, the bio-crude oil yield obtained
with 100 mL reactor was about 4% higher than that obtained with the 2 L reactor under the same
operating conditions. The toluene-soluble fraction of raw oil increased with retention time. At
280°C with 120 minutes retention time, more than 97% and 93% of raw oil could be dissolved in
toluene for Chlorella and Spirulina, respectively. Increasing initial pressure can increase the
maximum system pressure during the HTL. However, its effects on HTL products yields were
not pronounced in the range of 0-0.69 MPa (gauge pressure). Therefore the effect of nitrogen
was mainly to remove the residue air from the reactor and the initial pressure was introduced to
prevent the extensive boiling of water during HTL.
(4) Five metal catalysts with different supporting materials and two alkaline catalysts (5%
based on the dry matter of feedstock) were applied in HTL to investigate their effects on bio-
crude oil yield and quality. Catalysts had more substantial effects on bio-crude oil yield at 280°C
than at 240°C. The highest increase of bio-crude oil yield with the addition of catalysts compared
169
with uncatalyzed experiments was about 10%. At 240°C, the addition of different catalysts could
slightly improve the bio-crude oil boiling point (BP) distribution. At 280°C, heterogeneous
catalysts had little effect on bio-crude oil BP distribution, while the addition of alkaline catalysts
made the bio-crude oil BP distribution curve shift to the high temperature range, indicating more
complicated compounds with higher BPs were produced probably through recombination of
small compounds. The morphology change of catalysts was found not substantial after
hydrothermal treatment. However, carbon deposition and mineral mixing were found on the
surface of catalysts after hydrothermal treatment. The carbon deposition might decrease the
contact area between feedstock and the noble metal atoms, and therefore decrease the activity of
the catalysts. After the HTL process, minerals and ash content in microalgal feedstock mixed
with the granular catalysts, which may hinder the catalyst recovery.
(5) Carbon recovery (CR) and nitrogen recovery (NR) of HTL products were affected by
reaction temperature, retention time and different catalysts. As reaction temperature and
retention time increased, CR and NR of bio-crude oil increased and those of solid residue
decreased. From 100°C to 300°C with 30 minutes retention time, CR of bio-crude oil produced
from Chlorella increased from 0.1% to 55.3% and NR increased from 0.4% to 23.4%. CR of bio-
crude oil produced from Spirulina increased from 0.5% to 56.3% when the temperature
increased from 200°C to 300°C, and NR of bio-crude oil also increased from 0.5% to 23.3%.
After the HTL process, approximately 40% carbon and 75% nitrogen in the original algal
feedstock remained in the aqueous phase. CR and NR of bio-crude oil did not change much with
the addition of catalysts at 240°C. At 280°C, both CR and NR of bio-crude oil increased with the
addition of catalysts mainly due to the increase of bio-crude oil yield, so that NR of aqueous
product decreased. A novel treatment process, Environment-Enhancing Energy (E2-Energy), was
designed to recycle/reuse the nutrients in the post-HTL water and the carbon in the HTL gaseous
product to grow algae, while at the same time to treat wastewater and capture carbon.
(6) As temperature increased from 100°C to 300°C and retention time increased from 0 to
120 minutes, energy recovery (ER) of bio-crude oil increased. At 280°C with 120 minutes
retention time, the highest ER of bio-crude oil (65.4%) was obtained for Chlorella. Energy
consumption ratio (ECR) decreased with the temperature, indicating the HTL becomes more
energetically favorable at higher temperatures. Moisture content of feedstock substantially
affects the energy balance of the HTL process; it was found that if the moisture content of algal
170
feedstock was higher than 93%, with 50% of heat recovery and 60% combustion efficiency, the
ECR value of the HTL process could exceed 1.0, which makes the conversion energetically
unfavorable. The fossil energy balance (FEB) value of producing algae bio-crude oil via HTL is
higher than that of corn ethanol, algae-biodiesel and petroleum gasoline. The energy balance of
algae bio-crude oil is comparable with lignocellulosic ethanol in terms of similar FEB values.
(7) With increase of reaction temperature, fractions of hydrocarbon, cyclic oxygenates
and heterocyclic compounds in bio-crude oil increased. This indicates the decomposition of
protein and repolymerization of reaction intermediates were both promoted by increasing
temperature. At the same time, the fraction of organic acids decreased. Some intermediates
produced from protein hydrolysis, which can be dissolved in water, degraded with temperature
due to their instabilities under hydrothermal conditions. For instance, the glycine peak was first
identified in aqueous product at the reaction temperature of 160°C and its relative concentration
increased when the temperature increased from 160°C to 260°C, then greatly decreased as
temperature increased from 260°C to 300°C.
(8) As retention time increased, fraction of amides and N&O heterocyclic compounds in
bio-crude oil increased, and the fraction of organic acids decreased. The impact of retention time
on fraction of cyclic oxygenates was not pronounced. The fractions of some amino acids in
aqueous product, such as alanine and glycine, first increased with retention time and then
decreased. This confirms that the reaction intermediates produced from protein are not stable and
may react with intermediates produced from other components in microalgae. For example, the
Maillard reaction could occur between amino acids and reducing sugar from carbohydrates.
(9) Additions of heterogeneous catalysts increased the fraction of hydrocarbons and at the
same time decreased the fraction of organic acids. The fraction of N&O heterocyclic compounds
was largely increased with additions of alkaline catalysts. The total ion current chromatograms
(TIC) of aqueous product with different catalysts were similar to each other but some organic
acids peaks in TIC disappeared with presence of alkaline catalysts.
171
(10) The kinetic model was established for microalgae decomposition under hydrothermal
conditions. The decomposition model of HTL of microalgae was assumed as a first-order
reaction, and the activation energy for Chlorella and Spirulina decomposition was 29.8 kJ∙mol-1
and 24.0 kJ∙mol-1, respectively. The reaction mechanisms were expected to involve three steps:
hydrolysis of protein, lipid and carbohydrates; decomposition of amino acids, fatty acids and
sugars; and further degradation and recombination/repolymerization of the reaction intermediates.
10.2 RECOMMENDATIONS AND FUTURE WORK
Utilizing low-lipid microalgae to produce bio-crude oil via HTL was systematically
investigated in this work. Although the effects of operating conditions including temperature,
retention time and initial pressure; and the effects of different catalysts on HTL product yields
and qualities were investigated, and a preliminary reaction kinetic and mechanism were
developed based on the characterizations of HTL products, there are still many unexplored areas
under this research topic. More studies are needed in the future in order to improve the
conversion efficiency, bio-crude oil quality and finally, to understand the detailed reaction
mechanisms.
10.2.1 Upgrading and refinery of bio-crude oil

The bio-crude oil produced from algae has higher oxygen and nitrogen content than
conventional crude oil. This characteristic could greatly influence the application of bio-crude oil
in the future. Although operating conditions and direct additions of catalysts could affect the
nitrogen content and nitrogen distribution of HTL products, a way has not been found through
which the oxygen and nitrogen content in bio-crude oil can be substantially reduced to the levels
in petroleum. Therefore bio-crude oil upgrading and refining is necessary before it can be used as
alternative fuel to transportation fuels. Upgrading bio-crude oil at a supercritical condition with
the addition of catalysts may be helpful. However, how to improve the contacting area between
catalysts and bio-crude oil as well as increase the catalysts lifespan are two important issues in
this approach. In literature, adding granular or powdered catalysts directly in a traditional batch
reactor or continuous stirred-tank reactor is widely used for upgrading bio-crude oil. However, in
this kind of experiment set-ups, the catalysts are very difficult to recover after conversion or
upgrading because they are completely mixed with ash, minerals and chars produced at the same
172
time. Therefore, it is important to design new reactors in order to improve the contacting area
between catalysts and bio-crude oil and recover the catalysts after bio-crude oil upgrading. For
example, the pellet catalyst could be held in the catalyst basket and then the basket could be put
in the reactor. Through this design, the catalyst can be easily recovered after the experiments.
In this study, the metal catalysts were used as received to investigate whether the
commercially available catalysts could benefit the HTL process. Although there is concern that
the pre-reduction is not necessary because the catalysts will be exposed to the oxidizing
hydrothermal environment, it is worth conducting pre-reduction of the catalyst by adding H2 in
the HTL process to study whether the hydrogenation could occur. This is necessary for bio-crude
oil upgrading because in this case, the addition of hydrogen to remove oxygen and nitrogen via
hydrotreating is the main goal.
10.2.2 Characterization of HTL products

The bio-crude oil and aqueous product samples were analyzed using GC-MS in this study.
It was found the compositions of the products were very complicated. In each analysis, more
than 100 compounds could be separated but not all of them could be identified with the current
database or library. Some compounds were co-eluted so that their peaks overlapped each other in
the ion chromatogram. Under the circumstances, it is difficult and challenging to quantitatively
analyze the concentration of each compound in bio-crude oil or aqueous product. A pretreatment
of HTL products is then necessary and useful to obtain better analytic results. For example, the
bio-crude oil samples can be separated as polar fractions, neutral fractions and non-polar
fractions, or can be separated by the molecule weight. With this kind of pretreatments before the
samples are injected into GC-MS, the composition of each fraction becomes much simpler than
the original samples. Therefore, they could be better separated in the GC column to avoid peak
overlapping. Most importantly, fewer compounds in each fraction can make the quantitative
analysis possible and benefit the study of reaction kinetics and mechanisms of HTL.
10.2.3 Challenges for series HTL experimental design

Although the proposed reaction mechanism for HTL of microalgae is simplified, it is still
very complicated due to the interactions among the intermediates. To avoid or decrease the
interactions among reaction intermediates, conducting hydrothermal treatment for each
component of microalgae in separated series reactors could be helpful. However, the hydrolysis
of protein, which is the major composition in low-lipid microalgae, gradually happens in a wide
173
temperature range under hydrothermal conditions. Compared to lipid and carbohydrates, the
reaction pathways of amino acids, which are products from protein hydrolysis, are more
complicated. Moreover, it was found that the temperatures ranges, in which different
components can be hydrolyzed, could overlap each other (Figure 9-16). Therefore, the idea of
hydrothermal treatment of different components in series batch reactors is great, but the goal is
not easy to achieve. Maybe applying different catalysts for different components hydrolysis
could enhance the hydrolysis rates and narrow hydrolysis temperature ranges, but more studies
on the behavior of individual components in microalgae under hydrothermal conditions are
needed first.
10.2.4 Kinetic study of HTL

In this study, a kinetic model was established to describe the decomposition of microalgae
under hydrothermal conditions. This model is simplified to describe the apparent kinetics of HTL
of microalgae. A more detailed intrinsic kinetic model should be developed in the future. For
current batch reactors, it takes 20-50 minutes to reach the designated temperature due to the
limitation of heater and heat transfer efficiency. The effect of the heating period on HTL of
microalgae was investigated in this work and the long heating period was found to have a
pronounced effect on bio-crude oil yield (Figure 6-8). The reaction rate constants were found to
be low and the increase of reaction rate constant with temperature was small. In order to conduct
an accurate study on the intrinsic kinetics of HTL in the future, a tubular, plug-flow reactor
should be used because it can minimize the effect of the heating period. Conducting sampling
during batch HTL tests and then analyzing the product concentrations is another approach. In the
latter case, conducting small amounts of sampling and recovering products from the pipes
connecting the sampler to the reactor are both important; otherwise the mass balance could be
influenced.
10.2.5 Life cycle assessment (LCA)

Although microalgae are treated as one of the most popular feedstocks for producing next
generation biofuel, how to economically and energetically produce large amounts of algal
biomass for biofuel production has not been achieved yet. Therefore, no matter what
technologies are selected as the conversion process, a comprehensive life cycle assessment (LCA)
is essential to evaluate the feasibility of the system. In this study, energy recovery (ER) and
energy consumption ratio (ECR) were used to study the energy balance of HTL of microalgae.
174
The results provided preliminary evaluations on the HTL process, but the upstream processes,
including algae growing, harvesting and dewatering, and the downstream processes, including
the bio-crude oil upgrading or refining were not considered. In other words, the boundary of the
LCA must be expanded to cover the range from algae growing to biofuel delivery at least, to
evaluate whether this approach is economically and energetically favorable.
175
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©2012 by Guo Yu. All rights reserved.

HYDROTHERMAL LIQUEFACTION OF LOW-LIPID MICROALGAE TO

Submitted in partial fulfillment of the requirements

Professor Yuanhui Zhang, Chair

For their love, support and understanding

To my wife, YAN-JU CHEN

Who loves me, and whom I dearly love.

LIST OF TABLES ..................................................................................................................... xvi

LIST OF ABBREVIATIONS AND ACRONYMS ............................................................... xviii

1.1 BACKGROUND ............................................................................................................................. 1

1.1.1 Feedstocks for biofuel production ........................................................................................... 3

1.1.2 Bio-crude oil produced from thermochemical conversion ...................................................... 5

1.2 ALGAE AS FEEDSTOCK FOR BIO-CRUDE OIL PRODUCTION ............................................. 7

1.2.1 Classification of algae ............................................................................................................. 8

1.2.2 Bottlenecks for producing biodiesel from microalgae ............................................................ 9

CHAPTER 2. LITERATURE REVIEW .............................................................................. 12

2.1 WATER PROPERTIES AT HIGH TEMPERATURE .................................................................. 12

2.2 DEVELOPMENT OF HTL ........................................................................................................... 14

2.3 HTL OF DIFFERENT BIOMASS INTO BIO-CRUDE OIL ......................................................... 16

2.3.1 Lignocellulosic biomass and biowaste .................................................................................. 16

2.3.2 Algae as feedstock for bio-crude oil production ................................................................... 20

2.4 REACTION MECHANISMS AND KINETICS OF HTL ............................................................. 24

2.4.1 Lipids/Fats ............................................................................................................................. 24

2.4.2 Proteins/Amino acids ............................................................................................................ 25

2.4.3 Carbohydrates and lignocellulose ......................................................................................... 27

2.5 MIMICKING MOTHER NATURE’S OIL FORMATION VIA HTL .......................................... 32

2.5.1 Formation of conventional petroleum ................................................................................... 32

2.5.2 Similarities between HTL and natural geochemical processes ............................................. 35

CHAPTER 3. OBJECTIVES ................................................................................................. 37

CHAPTER 4. EXPERIMENTAL DESIGNS AND PROCEDURES ................................. 39

4.2 HYDROTHERMAL LIQUEFACTION PROCEDURE ............................................................... 40

4.2.1 Reactor types and experiment procedure .............................................................................. 40

4.2.2 Recovery procedures of HTL products ................................................................................. 42

4.3 EXPERIMENTAL DESIGN ......................................................................................................... 44

4.3.1 Effect of temperature............................................................................................................. 44

4.3.2 Effect of retention time ......................................................................................................... 47

4.3.3 Effect of initial pressure ........................................................................................................ 48

4.3.4 Effects of catalysts/additives on HTL process ...................................................................... 48

4.3.5 Elemental balance of HTL process ....................................................................................... 52

4.3.6 Characterization of HTL products......................................................................................... 54

4.3.7 Study reaction kinetics and mechanism of HTL process ...................................................... 57

CHAPTER 5. SCREENING ALGAL SPECIES FOR HTL EXPERIMENTS................. 59

5.1 CHARACTERISTICS OF DIFFERENT ALGAL SPECIES ........................................................ 59

5.2 HTL RESULTS OF DIFFERENT ALGAE SPECIES ................................................................... 61

5.2.1 Bio-crude oil yield of different algae species ........................................................................ 61

5.2.2 Relationship between oil quality and feedstock characteristics ............................................ 62

CHAPTER 6. EFFECTS OF OPERATING CONDITIONS ON PRODUCT YIELDS

6.1 HTL OF CHLORELLA PYRENOIDOSA .................................................................................... 64

6.1.1 Replicates of experiments ..................................................................................................... 64

6.1.2 Effect of reaction temperature ............................................................................................... 65

6.1.3 Effect of retention time ......................................................................................................... 67

6.1.4 Effect of initial pressure ........................................................................................................ 71

6.1.5 Effect of heating period ......................................................................................................... 74

6.2 HTL OF SPIRULINA PLATENSIS .............................................................................................. 76

6.2.2 Effect of retention time ......................................................................................................... 78

CHAPTER 7. EFFECTS OF CATALYSTS ON HTL PRODUCT YIELDS .................... 84

7.1 CATALYST EFFECTS ON BIO-CRUDE OIL YIELD ................................................................ 85

7.1.1 Effects of catalysts on bio-crude oil yield of Chlorella ......................................................... 86

7.1.2 Effects of catalysts on bio-crude oil yield of Spirulina ......................................................... 88