Accepted Manuscript

Title: Hydrogen from Algal Biomass: A review of Production

process

Authors: Archita Sharma, Shailendra Kumar Arya

PII: S2215-017X(17)30041-3
DOI: http://dx.doi.org/doi:10.1016/j.btre.2017.06.001
Reference: BTRE 202

To appear in:

Received date: 15-2-2017

Revised date: 20-5-2017
Accepted date: 1-6-2017

Please cite this article as: Archita Sharma, Shailendra Kumar Arya, Hydrogen
from Algal Biomass: A review of Production process, Biotechnology
Reportshttp://dx.doi.org/10.1016/j.btre.2017.06.001

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Hydrogen from Algal Biomass: A review of Production process

Archita Sharma, Shailendra Kumar Arya1

Department of Biotechnology, University Institute of Engineering and Technology, Panjab

University, Chandigarh, INDIA
1
skarya_kr@yahoo.co.in

Highlights:

 Biofuel alternatives.
 Algal biomass: Importance and need.
 Biohydrogen Production Processes.
 Microorganisms involved in biohydrogen production processes.
 Immobilization methods.
 Bioreactors suitable for biohydrogen production process.

ABSTRACT:
Multifariousness of biofuel sources has marked an edge to an imperative energy issue.
Production of hydrogen from microalgae has been gathering much contemplation right away.
But, mercantile production of microalgae biofuels considering bio-hydrogen is still not
practicable because of low biomass concentration and costly down streaming processes. This
review has taken up the hydrogen production by microalgae. Biofuels are the up and coming
alternative to exhaustible, environmentally and unsafe fossil fuels. Algal biomass has been
considered as an enticing raw material for biofuel production, these days photobioreactors and
open-air systems are being used for hydrogen production from algal biomass. The formers allow
the careful cultivation control whereas the latter ones are cheaper and simpler. A contemporary,
encouraging optimization access has been included called algal cell immobilization on various
matrixes which has resulted in marked increase in the productivity per volume of a reactor and
addition of the hydrogen-production phase.

Keywords: Hydrogen; algae; biofuels; immobilization; bioreactors

INTRODUCTION:
Forthwith, the world is bearing the challenges of high energy demands as well as
escalating fuel prices because of breakneck growth of world population and hasty
industrialization. So there is a need of an hour to cope up with such challenges and for this
researchers are now paying much appreciable attention by recommending sustainable and cost-
effective methods for energy production [1, 2]. Fossil fuels are associated with the environmental
pollution and thus, more efforts have been evolved in renewable energy sources being economic
and environmental friendly [2]. Governments are now proactive in addressing secured supply of
raw materials with limiting climate change and many potential candidate fuels have been studied
in the energy area, subsequently [3,4]. Biomass is one of the most encouraging renewable
resources being used to bring about different types of biofuels, serving as biodiesel, bioethanol,
biogas and biohydrogen. Energy from biomass would contribute to a stable energy supply and to
local society due to an increase in commercial activities [5]. Biomass can be derived from
cultivation of dedicated energy crops; by harvesting forestry and other plant residues; and from
biomass wastes [6]. Hydrogen is extensively being seen as a clean fuel, environmentally safe,
renewable energy resource and an excellent substitute of fossil fuels and a potential candidate
with highest energy density with many of the technical, socio-economic and environmental
benefits to its credence among all other known fuels (143 GJ per tonne) and is the only
acknowledged fuel that does not produce carbon dioxide as a by-product when used in fuel cells
for electricity generation [2, 6]. The bulkiest users of hydrogen are the fertilizer and petroleum
industries with approximately 50% and 37% respectively [6]. Hydrogen production has been
determined only at the laboratory scale with yield still low for commercial application and so, the
optimization of design and operating parameters for maximum hydrogen production is a must
step while addressing the subject of hydrogen production rate. The optimization basically counts
on the microalgae strain along with the available growth conditions [7]. For biofuels to be
broadly authorized in the energy merchandise, spotlight must be on acclimatizing and improving
photosynthetic organisms for biofuel production [8].
By the time mentioned, the sucrose and starch crops, for instance, sugarcane and corn as
well as lignocellulosic materials like rice straw and switchgrass are being used as biofuel
feedstock’s. But, high cost in the hydrolysis of lignocellulosic materials is a matter of concern.
Sugars come in several forms, containing approximately four calories per gram. Simple sugars
like monosaccharides like glucose, fructose and galactose. Biohydrogen production offers a
sustainable alternate and by utilizing renewable carbon sources can be considered as carbon
dioxide offset. This can utilize various carbon sources including wastewater. Glucose, sucrose
are readily degradable and hence preferred as model substrates for hydrogen production. Because
of complex composition and polymeric structure complex carbon must released or converted to
simple sugars. Complex polymer consists of tightly bound lignin, cellulose and hemicelluloses.
Cellulose and hemicelluloses can be degraded under same conditions and add up the cost factor
which is a matter of concern [2, 9]. A lot of microorganisms are involved in the production of
biofuels like hydrogen, but most accepted are cyanobacteria and green microalgae freshly
considered as third generation feedstock’s being more efficient at converting sunlight into the
chemical energy and require a smaller footprint and less water for cultivation [7, 10, 11].
Diverse pretreatment methods and physic-chemical pretreatments have been revised for
the hydrogen production. It is necessary step to breach the algal cell wall along with the complex
carbohydrate to release simple sugars. Pretreatment methods such as physical (sonication,
milling, grinding, pyrolysis), chemical (acid, alkali, thermal) and biological methods (enzymatic)
is being employed to break algal cell wall, to hydrolyze the complex carbohydrates and to release
fermentable sugars [9].
 An immobilized cell means a cell by natural or artificial paths is being prevented
independent movement from its neighbouring environment to all parts of the system which is
under consideration [12]. Basically, there are six different types of cell immobilization methods.
They are covalent coupling, affinity immobilization, adsorption, confinement in the liquid-liquid
emulsion, capture behind semi-permeable membrane and entrapment. Utilization of
immobilization technique contributes more resilience while designing a reactor comparing
conventional suspension systems. Furthermore, increase in cell density, increase in cell wall
permeability, no washout of cells and better system stability are certain additional merits of cell
immobilization technique. Out of all, entrapment of cell within polymeric matrices and self
adhesive attachment of cells onto surfaces of solid support are usually more common. Important
criteria for successful entrapment are to set algal cells from within their partition, while pores
inside gel matrix allow diffusion of substrates and metabolic products towards and from cells
[13].
In these, bioreactors which are being considered for hydrogen production from algal
biomass are matter of concern. Biohydrogen production by microorganisms has attracted our
increasing worldwide attention, having its potential for inexhaustible, low-cost and a renewable
source of energy. They are categorically pre-requisite for large scale hydrogen production by
microorganisms. Certain microorganisms have been evolved for hydrogen production either
from organic materials like sugar or biomass. Bioreactors are closed systems that have varied
size from the small (5 mL to 10 mL) to the larger scale or more than 500,000 L industrial scale.
Photobioreactors are made up of an array of tubes, tanks bags, where photosynthetic
microorganisms including algae are being cultivated and later monitored as light is the essential
component for growing photosynthetic microorganisms. Bioreactors that have been mentioned
later in this are photobioreactors, continuous stirred tank reactor (CSTR), fixed bed bioreactors,
membrane bioreactors, multi-stage bioreactors and hybrid bioreactors [69].

HYDROGEN PRODUCTION:
PRINCIPLE:

Hydrogen makes up about three quarters of all matter and thus the most plentiful element
of universe. Compelling hydrogen sources includes fossil fuels (95-99%) and water [14].
Dealing with future hydrogen demands independent of fossil fuels, it is necessary to appreciate
all available renewable resources [15].
The classical methods for hydrogen production consists of steam reforming of natural
gases, coal gasification and electrolysis of water are energy intensive processes that requires
high temperatures (>840 degree celsius) and are not environmental friendly as such. Electrolysis
of water being the cleanest technology for hydrogen production, can only used in sectors with
cheap electricity as adds up to 80% of the operating cost [3]. Figure 1 explains hydrogen
production processes with examples.
MECHANISM:
With the noteworthy merits, the low production rates, low substrate conversion
efficiencies are certain practical hindrances need to overcome for the successful hydrogen
production.

Direct photolysis:

There is a dissociation of water into hydrogen and oxygen in the presence of light, that is,
H2O → H2 + ½O2 [16, 17]. Green microalgae can use light to carry out photosynthesis as they
possess chlorophyll a and the photosynthetic systems: Photosystem (PS) II and Photosystem (PS)
I, respectively [18, 19]. Disadvantages are the enzyme hydrogenase is very sensitive to oxygen
so when a certain amount of oxygen is present, will inhibit hydrogenase activity and will stop it
from producing hydrogen. Also, it requires high intensity of light. The advantages include
tenfold more solar conversion in green microalgae.

Indirect Photolysis:

There is two step processes, firstly there is a splitting of water molecules in the presence
of sunlight and protons and oxygen is formed. Secondly carbon dioxide fixation occurs storage
carbohydrate is being produced, followed by the production of hydrogen gas by hydrogenase
[20].

12H2O + 6CO2 + light energy → C6H12O6 + 6O2

C6H12O6 + 12H2O + light energy → 12H2 + 6CO2
Blue-green algae (cyanobacteria) are promising microorganisms for this. Advantages are
hydrogen evolution is separated from oxygen evolution. It can also produce relatively higher
hydrogen yields. Furthermore, by-products can be efficiently converted to hydrogen.
Disadvantages are like significant adenosine triphosphate (ATP) requirement of nitrogenase.
Also, this requires continuous light source which is difficult for large scale processes [21- 24].

Dark Fermentation:
Hydrogen production in a dark environment without the presence of sunlight, water and
oxygen. Fermentative microorganisms hydrolyze complex organic polymers to monomers that
are further converted to a mixture of lower molecular weight organic acids and alcohols by
necessary hydrogen producing bacteria [24 - 26]. Advantages consist of use of a variety of
carbon sources and production of hydrogen without light. It produces valuable by-products like
butyric acid, lactic acid and acetic acid etc. Disadvantages are relatively lower hydrogen yields.
Also the product gas mixture contains carbon dioxide which has to be separated [3]. Figure 2
explains different types of microorganisms capable of dark fermentation with examples.

Photo-fermentation:
It is a fermentative conversion of organic substrates into hydrogen and carbon dioxide by use
of sunlight as an energy source.
CH3COOH + 2H2O + light → 4H2 + 2CO2
 Using light as the energy source, the organic acid substrates are oxidized using the
tricarboxylic acid cycle (TCA), producing electrons, protons and carbon dioxide. Example
includes purple non sulfur bacteria (PNS) [11, 17]. Advantages in removal of environmental
pollutants, use of industrial waste and use of organic acids produced from dark fermentation.
Disadvantages are need to nitrogen limit condition and pretreatment of industrial effluent as it
may be toxic [23].

HYDROGEN PRODUCTION FROM ALGAL BIOMASS:

The renewable energy sources play crucial role in decreasing the greenhouse effect but
also provides an alternative approach regarding increasing global energy demands resulting into
depletion of energy reserves [27]. Many algal species show potential to produce hydrogen under
certain conditions [28]. Nonetheless, certain technical barriers like developing low-energy
methods to harvest microalgal cells, difficulties in continuously producing biomass at a large
scale, the presence of invasive species in large-scale ponds, low light penetrance in dense
microalgal cultures, and the lack of cost-effective bioenergy carrier extraction techniques, are
required to overcome before using microalgae as an economically viable biofuel feedstock [29].

MICRORGANISMS:
Algae are the association of organisms similar in their morphological and physiological
features, that is, ability of photoautotrophic metabolic pathways, exists predominantly in water
etc. Microalgae can fix carbon dioxide from the atmosphere and with this fact; they are
positively characterized in terms of biomass cultivation convenience [29].
Over 40,000 species of algae have been described, and this is likely only a small fraction
of the total number of available species. The U.S. Department of Energy’s Aquatic Species
Program analyzed approximately 3,000 different microalgae for their potential to produce
biofuels [30]. Table 1 explains numerous microorganisms involved in hydrogen production from
algal biomass with their processes.
Culture conditions:
Inescapably, accomplishments of hydrogen-producing bioreactor systems are governed
by various factors like:
pH:
pH has been sanctioned as one of the most important environmental factors that affects
metabolic pathways and yields hydrogen. Comparative studies have shown that the ideal pH
range should be around 5.2 and 6.0 [39, 40]. The effect of pH in a CSTR must be in the range of
4.0–7.0, optimum at a pH of 5.5 [41].
It has been observed hydrogen production from glucose using continuous cultures under
identical conditions except pH values, and concluded that higher hydrogen yield and production
rate are attained at a pH of 5.7 compared with pH 6.4 [6]. More emphasis was on the maximum
hydrogen yield which was achieved only when microbial reactions followed an ethanol
fermentation type that occurred at a pH of about 4.5 [42].
Hydraulic retention time (HRT):
Hydraulic retention time is required to prefer microbial populations with growth rates
being able to catch up with mechanical dilution set up by continuous volumetric flow. It was
found that shortening the hydraulic retention time from 8 hours to 6 hours would reduce
microbial diversity leading to an increase in the hydrogen yield. [43].

It was found that the methane concentration ranged between 0.0011 and 0.0058 mol l-1 at
low dilution rates (D = 0.002 – 0.0167 h-1) was hardly measureable at higher dilutions (D >
0.075 h-1), indicating negligible methanogenic activity at high dilution rates. This means that
hydraulic retention time is capable of inhibiting or terminating methanogenesis in hydrogen
production via anaerobic fermentation [44].

Hydrogen partial pressure:

The dissolved hydrogen concentration adding to the hydrogen partial pressure is the key
factors affecting microbial pathways. Hydrogen production is less favourable as the hydrogen
partial pressure rises. Thus it is mandatory to remove excess hydrogen from the system for
maintaining hydrogen production [43, 45].
It was found an increase in the hydrogen yield from 0.85 to 1.43 mol mol-1 hexose when
the reactor is sparged with nitrogen at fifteen times hydrogen produced rate [46].

Nutrients:
For accessing optimal cell cultivation and hydrogen production nitrogen, phosphate and
other inorganic trace minerals are imperative supplements for carbohydrate based feedstocks.
Organic nitrogen is more favourable compared to inorganic one. Phosphate is required for
optimal hydrogen production. The elements like Mg, Na, Zn and Fe are crucial supplements and
have suggested an optimum nutrient formulation. Iron is of utmost importance in the enzymatic
activity of hydrogen production that is [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-
hydrogenase [47- 54].
The apical level production from sweet potato starch residue by a repeated batch culture
containing C. butyricum IFO13949 when 1.0% polypepton was added as nitrogen source was
reported. In contradiction, addition of urea (NH4)2SO4 or NH4Cl resulted in the absence of
hydrogen evolution by the same culture [47].

Temperature:
Microbes are competent in a temperature range of 15–85 degree celsius but may vary
under temperatures from 15 to 34 degree celsius for mixed cultures [6, 27].

It has been considered that the hydrogen production capacity of a mixed culture under
varying temperatures from 15 to 34 degree celsius and found that hydrogen yield and specific
hydrogen production rate increased with temperature, achieving respective maximum values of
359 mmol l-1d-1 and 1.42 mol H2 mol-1 glucose at 30–34 degree celsius and 28–32 degree celsius
respectively [6].
Substrate concentration
This factor, moreover, has been a point of debate. Recent studies have found that
hydrogen yield to increase with increasing glucose concentration [55]. Along with substrate
concentration other operating conditions like hydraulic retention time, composition of microbial
cultures also affects the same [56, 57].

It was found that hydrogen yield to increase with increasing glucose concentration from
10 to 35 g l-1at a hydraulic retention time of 12 hours [58] when in fact it was investigated that
there is continuous hydrogen production at 12 hours hydraulic retention time on 10–50 g l-1
sucrose and found that the hydrogen yield decreased from 1.7 ± 0.2 mol H2 mol-1 hexose added
at 10 g l-1 sucrose to 0.8 ± 0.1 mol H2 mol l-1 hexose added at 50 g l-1 [58]. These considerations
indicated that besides substrate concentration conditions as in hydraulic retention time and
composition of microbial cultures also influences hydrogen production.

Seed culture:
Clostridium and Enterobacter are extensively used as inoculums for fermentative
hydrogen production [59]. A lot of studies have shown that using pure cultures of bacteria for
fermentative hydrogen production were conducted in batch mode and used glucose as substrate
[60, 61]. Mixed bacterial cultures from anaerobic sludge, compost and soil is used as inoculums
for fermentative hydrogen production.

Feedstock:
Simple sugars such as glucose, sucrose and lactose being biodegradable are preferred as
model substrates for hydrogen production. But, the costs for pure carbohydrate sources are high
on practical-scale production that is feasible on renewable and low cost sources [62 - 65].

Proclaimed by plentiful studies of hydrogen fermentative processes, carbohydrates are

the essential source of hydrogen. Along the lines, wastes and biomass rich in sugars and complex
carbohydrates turn out to be the most apt feedstocks for biohydrogen generation [66].

IMMOBILIZATION:
The usefulness of microalgae in biotechnology has been heightened in fresh years. These
organisms being involved in food, cosmetic, aquaculture, pharmaceutical industries and various
other industries but small size poses an obstacle in the biotechnological applications. Cell
immobilization techniques have been refined to solve above mentioned complications [4].
Immobilized algae have been used for biomass obtain and macronutrient removal. The extremely
high accumulation capacity of some of these organisms for potentially dangerous substances has
been also exploited for bioremediation techniques applied on polluted waters (especially
involving metals [67].

Environmental benefits of immobilized algal cells are not confined to pollutant removal
level only. These techniques have been currently being used in the area of toxicity measurement
experiments. Utmost, the immobilization techniques being devised for microorganisms in
general can also be applied for microalgae, with the check of light transmission if living cells are
intended to be immobilized [68]. Table 2 give examples of immobilization method and their
matrix uses.

BIOREACTOR:
Traditional industrial methods are quite costly. It is imaginable to design bioreactors on
large scale using microorganisms as bioreactors are categorically a pre-requisite for large-scale
hydrogen production by microorganisms [78].
Photo-bioreactors:
Design depends on microbiological processes linked with microalgae or cyanobacteria.
With differing photochemical efficiency, absorption coefficient and size, the light regime
including light and dark cycles is hypothetically is much more determining than biological
factors [71]. Therefore, productivity of a photo-bioreactor is light-dependent with large surface
to volume ratio as a pre-requisite. Photo-bioreactors have been designed in such a way to access
an economical, rapid multiplication and high density of the microalgae culture [66, 80, 81].

Continuous stirred tank reactor (CSTR):

Continuous stirred tank reactors are frequently being used for hydrogen production. In a
continuous stirred tank reactor, hydrogen-producing microbes are thoroughly mixed and
suspended in the reactor liquid from the mixing pattern. Under this, a good substrate-microbes
contact and mass transfer can be absolutely accomplished while on the other hand, the
continuous stirred tank reactor is not be able to maintain high levels of fermentative biomass
because of the rapid mixing operating pattern. Biomass washout is possible to occur at short
hydraulic retention times (HRTs), and thus the hydrogen production rates are noticeably
restricted [66, 74, 78, 79].

Fixed-bed bioreactor:
This is negotiated with support carriers packed within the tank. The hydraulic mixing
regime is less turbulent comparing with the continuous stirred tank reactor with a consequence of
higher mass transfer resistance along with lowered rates of substrate conversion and hydrogen
production. High hydrogen yield could not be cultivated persistently in a fixed-bed reactor as pH
gradient distribution along the reactor column will cause a heterogeneous distribution of
microbial activity. So, to overcome this, recirculation flow was recommended [43, 82].

Membrane bioreactor:
Membrane bioreactor (MBR) is basically used to control biomass concentration. MBR
did not exhibit superiority other high-rate hydrogen production systems. Membrane fouling and
high operating cost also limit the use of membrane bioreactor process in bio hydrogen
fermentation [68].

Multi-stage bioreactors:
 Multi-stage bioreactors consisting of three four stages have been prospective to maximize
the production from the substrate [66, 75]. Sunlight is first percolated via the first stage called
direct photolysis reactor where the visible light will be exploited by blue green algae whereas the
unfiltered infrared light is used by photo-synthetic microbes in the second stage called photo-
fermentative reactor. The effluent from the second stage along with feedstock is stuffed into a
third stage called dark fermentation reactor where the bacteria convert the substrate to hydrogen
and organic acids. The fourth stage is the use of a MEC which uses organic acids being
generated from the dark fermentation under light-independent process. So it can be regulated
during night or in low light condition [76]. Figure 3 explains the setup of multi-stage bioreactor
system.

Hybrid bioreactors:
Principle of the hybrid two-stage bioreactors is bio fermentation of substrate to hydrogen
and organic acids that takes place in a conventional reactor in a primary stage of process, and
other gaseous energy in the mode of methane or hydrogen that is being extricated from the
second stage. While optimizing gas production, a different reactor for the second stage is being
operated under different conditions like higher pH and longer hydraulic retention times and is
later is applied. Non-sulphur photosynthetic microbes are capable of converting organic acids to
hydrogen. Combination of dark and photo-fermentation in a hybrid two-stage system can be
helpful in improving the overall hydrogen yield. The synergism lies in the maximal pursuit of the
substrate due to improved thermodynamics. In the first stage, the biomass is fermented to
acetate, carbon dioxide and hydrogen in a thermophilic dark fermentation and in the second stage
photobioreactor, acetate is converted into hydrogen and carbon dioxide [69, 83].

CONCLUSION
The fossil fuel holds back the dearth towards the 21st century due to increase in energy
demand and increase in greenhouse gas emission makes it important to develop alternative
energy carriers that are renewable, clean and environmentally friendly. Hydrogen holds a
promising role as a future fuel and renewable energy source whereas the current classical
methods of producing hydrogen are energy intensive, costly and are not environmentally
friendly. Major technical objection in accomplishing practical applications of bio-hydrogen
includes lowering the cost of production, delivery, storage, conversion and practical applications.
Other objection of the bio-hydrogen production is unstable hydrogen production that possibly
attributes towards the metabolic shift of hydrogen producing organisms. The optimization of key
experimental factors, genetic modification and metabolic engineering of microalgae are the
eventual approaches that make hydrogen production cost-effective and sustainable. The marine
algae are considered an important biomass source; however, their utilization as an energy source
is still quite low. Large number of diverse works is carried out in this area which indicates that
currently algal energy is intensively developing in all directions: increase in growth rate,
improving of harvesting methods, the genetic engineering of crops, optimization of chemical and
thermal methods for producing biofuels due to the urgency of the energy problem: the
environmental risks are associated with the use of fossil fuels and disadvantages of first and
second generation biofuels. But a lot of work on improving of algae cultivation and processing
mechanisms is being done to safeguard commercialization of microalgal biofuels. Jeopardy of
undesirable mutations is mitigated with cell immobilization. Genetic alterations of immobilized
microalgae could also help in improving hydrogen generation processes. The leakage
complication is of utmost concern in cell immobilization as it forestalls the fundamental
objective of demarcating viable cells in a circumscribed matrix. Research efforts have been
concentrated around the design of photobioreactors with the incorporation green algae and
cyanobacteria. The traditional industrial methods of hydrogen production are quite costly and the
problem is to find a cheaper and easiest way for production of hydrogen.

Conflict of Interest

The authors with listed names are immediately certify that they have no affiliations with or
involvement in any organization or entity with any financial interest or non- financial interest in
the subject matter mentioned above.
REFRENCES:
1) N. Mallick, Biotechnological potential of immobilized algae for wastewater N, P and
metal removal: A review, BioMetals 15(2002) 377–390.
2) M.Y. Azwar, M.A. Hussain and A.K. Abdul-Wahab, Development of biohydrogen
production by photobiological, fermentation and electrochemical processes: A review.
Renew. Sust. Energ. Rev: 31 (2014) 158-173.
3) N. Saifuddin and P. Priatharsini, Developments in Bio-hydrogen Production from Algae:
A Review, Research Journal of Applied Sciences, Engineering and Technology 12(2016)
968-982.
4) D. Chaumont, Biotechnology of algal biomass production: a review of systems for
outdoor mass culture, Journal of Applied Phycology, 5 (1993) 593-604.

5) S.A. Sherrif, F.A. Barbir, T. N.Veziroglu, Principles of hydrogen energy production,

storage and utilization, Journal of scientific and industrial research, January – Feburary
62 (2003) 46-63.
6) F.Y.Chang, C.Y. Lin. Biohydrogen production using an up-flow anaerobic sludge blanket
reactor. Int. J. Hydrogen Energy 29 (2004) 33–39.
7) I. Moreno-Garrido, Microalgae immobilization: Current techniques and uses, Bioresource
Technology 99 (2008) 3949–3964.
8) I. Dincer, Green methods for hydrogen production, International journal of hydrogen
energy 37 (2) (2012)1954-1971.
9) S. Behera, R. Singh, R. Arora, N. K. Sharma, M. Shukla and S. Kumar, Scope of algae as
third generation biofuels, Frontiers in bioengineering and biotechnology Marine
Biotechnology, (2) 90 (2015) 1-13.
10) S.M. Kotay and D. Das, Microbial hydrogen production with Bacillus coagulans IIT-BT
S1 isolated from anaerobic sewage sludge. Bioresource Technol., 98(6) (2007)1183-
1190.
11) S Manish. and R. Banerjee, 2008. Comparison of biohydrogen production processes. Int.
J. Hydrogen Energ., 33(1) 279-286.
12) J. Kaparapu, M. N. R. Geddada, Applications of immobilized algae, J. Algal Biomass
Utilization, 2016, 7 (2): 122- 128.
13) M. Brouers, D.J. Shi, D.O. Hall, Immobilization methods for cyanobacteria in solid
matrices, Methods in Enzymology, 167, (1988), 629-636.
14) J. Wang, W. Wan, Factors influencing fermentative hydrogen production: a review. Int. J.
Hydrogen Energy 34 (2009)799–811.
15) R.A. Rozendala, H. V.M. Hamelersa, G. J.W. Euverinkb, S. J. Metzb, C. J.N. Buismana,
Principle and perspectives of hydrogen production through biocatalyzed electrolysis,
International Journal of Hydrogen Energy 31 (2006) 1632 – 1640.
16) J., B., M.C. Mayo and A. Khare. Hydrogen: The energy source for the 21st century.
Technovation, 25(2005) 569-585.
17) I. Akkerman, M. Janssen, J. Rocha and R.H. Wijffels, Photobiological hydrogen
production: photochemical efficiency and bioreactor design. Int. J. Hydrogen Energy,
27(2002) 1195-1208.
18) M.L Ghirardi., Zhang, L., Lee, J.W., Flynn, T., Seibert, M., Greenbaum, E., Melis, A.,
Microalgae: a green source of renewable hydrogen. Trends Biotechnol. 18(2000) 506–
511.
19) D. Das, T. N. Veziroglu, Advances in biological hydrogen production processes,
International Journal of Hydrogen Energy, 33 (2008) 6046–6057.
20) R.C. Prince and H.S. Kheshgi, The photobiological production of hydrogen: Potential
efficiency and effectiveness as a renewable fuel. Crit. Rev. Microbiol., 31(2005) 19-31.
21) A. Melis , M. R. Melnicki, Integrated biological hydrogen production, International
Journal of Hydrogen Energy, 31 (2006), 1563–1573.
22) M. Momirlan and T.N. Veziroglu, The properties of hydrogen as fuel tomorrow in
sustainable energy system for a cleaner, International Journal of Hydrogen Energy, 30
(2005) 681-808.
23) J.Mathews, and G. Wang, Metabolic pathway engineering for enhanced biohydrogen
production. International Journal of Hydrogen Energy, 34 (2009) 7404-7416.
24) D. Das, and T.N. Veziroglu, Advances in biological hydrogen production processes. Int.
J. Hydrogen Energy, 33 (2008) 6046-6057.
25) C.Y. Lin and C.H. Jo, Hydrogen production from sucrose using an anaerobic sequencing
batch reactor process. J. Chem. Technol. Biot., 78 (2003) 678-684.
26) V.Schara, G. T. Maeda and T.K. Wood, Metabolically engineered bacteria for
producing hydrogen via fermentation. Microb. Biotechnol., 1 (2) (2008) 107-125.
27) T Kanai, H Imanaka, A. Nakajima, K. Uwamori, Y.Omori, T.Fukui, , H. Atomi, T.
Imanaka, Continuous hydrogen production by the hyperthermophilic archaeon,
Thermococcus kodakaraensis KOD1. J. Biotechnol. 116 (2005), 271–282.
28) C.L.Li, H.H.P. Fang, Fermentative hydrogen production from wastewater and solid
wastes by mixed cultures. Crit. Rev. Environ. Sci. Technol. 37 (2007) 1–39.
29) R. Radakovits, R. E. Jinkerson, A. Darzins, and M. C. Posewitz, Genetic Engineering of
Algae for Enhanced Biofuel Production, Eukaryotic cell, 9 (2010) 486–501.
30) G. Kars, U. Gündüz , M. Yücel, L. Türker and I. Eroglu, Hydrogen production and
transcriptional analysis of Nifd, Nifk and hups genes in Rhodobacter sphaeroides
O.U.001 grown in media with different concentrations of molybdenum and iron. Int. J.
Hydrogen Energy., 31(2006) 1536-1544.
31) R. A. Voloshin, M. V. Rodionova, S. K. Zharmukhamedov, T. N. Veziroglu , S. I.
Allakhverdiev, International journal of hydrogen energy 41 (2016 ) 17257-17273.
32) H. C. Greenwell, L. M. L. Laurens, R. J. Shields, R. W. Lovitt and K. J. Flynn, Placing
microalgae on the biofuels priority list: a review of the technological challenges J. R.
Soc. Interface 7 (2010) 703–726.
33) M.Winkler, A. Hemschemeier, C. Gotor, A. Melis and T. Happe, [Fe]-hydrogenases sin
green algae: Photo-fermentation and hydrogen evolution under sulfur deprivation. Int. J.
Hydrogen Energy, 27 (2002) 1431-1439.
34) J .Liu, V.E. Bukatin and A.A. Tsygankov, Light energy conversion into H2 by Anabaena
variabilis mutant PK84 dense cultures exposed to nitrogen limitations. Int. J. Hydrogen
Energy, 31(2006)1591-1596.
35) B. Fabiano, and P. Perego Thermodynamic study and optimization of hydrogen
production by Enterobacter aerogenes. Int. J. Hydrogen Energy, 27(2002) 149-156.
36) H.H.P Fang, H. Zhu and T. Zhang, 2006. Phototrophic hydrogen production from glucose
by pure and co-cultures of Clostridium butyricum and Rhodobactersphaeroides. Int. J.
Hydrogen Energy, 31(2006): 2223-2230.
37) H. Zhang, M.A, Bruns and B.E. Logan, Biological hydrogen production by Clostridium
acetobutylicum in an unsaturated flow reactor, Water Res., 40 (2006) 728-734.
38) X. Shi, K. W. Jung, D.H. Kim , Y. T. Ahn and H. S. Shin, , Direct fermentation of
Laminaria japonica for biohydrogen production by anaerobic mixed cultures. Int .J.
Hydrogen Energy 36(2006)5857–5864.
39) J.H. Park, J.J Yoon, H.D. Park, Y.J. Kim, D.J. Lim, and S.H Kim, Feasibility of
biohydrogen production from Gelidium amansii. Int. J. Hydrogen Energy 36(2006)
13997–14003.
40) Y.K Oh, S.H Kim, M.S. Kim, S. Park, Thermophilic biohydrogen production from
glucose with trickling biofilter. Biotechnol. Bioeng. 88 (2006) 690–698.
41) Z.P Zhang, S.S. Adav, K.Y. Show, J.H. Tay, D.T. Liang, D.J. Lee, Characteristics of
rapidly formed hydrogen-producing granules and biofilms. Biotechnol. Bioeng. 101
(2008) 926–936.
42) Z.P Zhang, K.Y. Show, J.H.Tay, T.D.Liang, D.J. Lee, Enhanced continuous biohydrogen
production by immobilized anaerobic microflora. Energy Fuels 22 (2008b) 87–92.
43) H.H.P Fang and H. Liu, Effect of pH on hydrogen production from glucose by a mixed
culture. Bio resource. Technology. 82 (2002) 87–93.
44) R. N. Wang, A. G.Cao, J Xu, L. Gao, Bioconversion of lignocellulosic biomass to
hydrogen: potential and challenges. Biotechnol. Adv. 27 (2009), 1051–1060.
45) Z.P Zhang, K.Y. Show, J.H. Tay, D.T. Liang, D.J. Lee, W.J. Jiang, Effect of hydraulic
retention time on biohydrogen production and anaerobic microbial community. Process
Biochem. 41 (2006) 2118–2123.
46) Y.C Lo, Y.C. Su, C.Y. Chen, W.M. Chen, K.S. Lee, J.S Chang, Biohydrogen production
from cellulosic hydrolysate produced via temperature-shift enhanced bacterial cellulose
hydrolysis. Bioresour. Technol. 100 (2009) 5802– 5807.
47) M.K Tiwari, S. Guha, C.S. Harendranath, S.Tripathi, Influence of extrinsic factors on
granulation in UASB reactor. Appl. Microbiol. Biotechnol. 71 (2006)145– 154.
48) O Mizuno, T. Ohara, M. Shinya, T. Noike, Characteristics of hydrogen production from
bean curd manufacturing waste by anaerobic microflora. Water Sci. Technol. 42 (2000)
345–350.
49) H.Yokoi, A. Saitsu, H.Uchida, J .Hirose, S.Hayashi, Y. Takasaki, Microbial hydrogen
production from sweet potato starch residue. J. Biosci. Bioeng. 91 (2001), 58–63.
50) C.Y., Lin, C.H. Lay, Effects of carbonate and phosphate concentrations on hydrogen
production using anaerobic sewage sludge microflora. Int. J. Hydrogen Energy 29
(2004a), 275–281.
51) C.Y Lin, C.H. Lay, A nutrient formulation for fermentative hydrogen production using
anaerobic sewage sludge microflora. Int. J. Hydrogen Energy 30 (2004)285–292.
52) S.Shima, O. Pilak, S.Vogt, , M. Schick, M.S Stagni, W. M. Klaucke, E. Warkentin,
R.K. Thauer, U. Ermler, The crystal structure of [Fe]-hydrogenase reveals the geometry
of the active site. Science 321 (2008)572–575.
53) B. Bleijlevens, T. Buhrke, E.V.D. Linden, B. Friedrich, S.P. Albracht, The auxiliary
protein HypX provides oxygen tolerance to the soluble [NiFe]- hydrogenase of Ralstonia
eutropha H16 by way of a cyanide ligand to nickel. J. Biol. Chem. 279 (2004), 46686–
46691
54) M. Frey, Hydrogenases: hydrogen-activating enzymes. Chem Biochem 3(2002) 153–160.
55) M.W. Adams, D.O. Hall, Purification of the membrane-bound hydrogenase of
Escherichia coli. J. Biol. Chem. 183 (1979)11–22.
56) H. Nishihara, Y. Miyashita, K. Aoyama, T. Kodama, Y. Igarashi, Y.Takamura,
Characterization of an extremely thermophilic and oxygen-stable membrane bound
hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogeno vibrio marinus.
Biochem. Biophy. Res. Comm. 232(1997)766–770.
57) S.H Kim, Han, S.K., Shin, H.S., Effect of substrate concentration on hydrogen production
and 16S rDNA-based analysis of the microbial community in a continuous fermenter.
Process Biochem. 41 (2006)199–207.
58) S. V. Ginkel, B.E. Logan, Inhibition of biohydrogen production by un-dissociated acetic
and butyric acids. Environ. Sci. Technol. 39 (2005)9351–9356.
59) S. W.V. Ginkel, S.E. Oh, B.E. Logan, Biohydrogen gas production from food processing
and domestic wastewaters. Int. J. Hydrogen Energy 30 (2005)1535– 1542.
60) G. Kyazze, N.M.Perez, R. Dinsdale, G.C. Premier, F.R. Hawkes, A.J. Guwy, D.L.
Hawkes, Influence of substrate concentration on the stability and yield of continuous
biohydrogen production. Biotechnol. Bioeng. 93 (2006)971–979.
61) J. Wang, W.Wan, Factors influencing fermentative hydrogen production: a review. Int. J.
Hydrogen Energy 34 (2009)799–811.
62) H.S. Zhang, M.A.Bruns, B.E. Logan,. Biological hydrogen production by Clostridium
acetobutylicum in an unsaturated flow reactor. Water Res. 40 (2006) 728–734.
63) Z.P. Zhang, K.Y Show, J.H.Tay, T.D. Liang, D.J. Lee, Enhanced continuous
biohydrogen production by immobilized anaerobic microflora. Energy Fuels 22 (2008)
87–92.
64) Y.C. Lo, M.D. Bai, W.M. Chen, J.S. Chang, Cellulosic hydrogen production with a
sequencing bacterial hydrolysis and dark fermentation strategy. Bioresour. Technol. 99
(2008) 8299–8303.
65) Y.C Lo, Y.C. Su, C.Y. Chen, W.M. Chen, K.S Lee, J.S Chang, Biohydrogen production
from cellulosic hydrolysate produced via temperature-shift enhanced bacterial cellulose
hydrolysis. Bioresour. Technol. 100 (2009) 5802– 5807.
66) G. Luo, F. Talebnia, D.Karakashev, L.Xie, Q. Zhou, I.Angelidaki, Enhanced bioenergy
recovery from rapeseed plant in a biorefinery concept. Bioresour. Technol. 102 (2011)
1433–1439.
67) K. B. Nayak, S. Roy, D. Das, Biohydrogen production from algal biomass (Anabaena sp.
PCC 7120) cultivated in airlift Photobioreactor International journal of hydrogen energy
39 (2014)7553-7560.
68) I. Ntaikou, G.Antonopoulou, G. Lyberatos, Biohydrogen production from biomass and
wastes via dark fermentation: A review, Waste Biomass Valoriz. (2010) 1: 21–39.
69) K.Y. Show, D. J. Lee, J. S. Chang, Bioreactor and process design for biohydrogen
production, Bioresource Technology 102 (2011) 8524–8533.
70) J. J. Hahn, M. L. Ghirardi, W. A. Jacoby, Immobilized algal cells used for hydrogen
production, Biochemical Engineering Journal 37 (2007) 75–79.
71) T. V. Laurinavichene, S. N. Kosourov, M. L. Ghirardi, Michael Seibert, Anatoly A.
Prolongation of H2 photoproduction by immobilized, sulfur-limited Chlamydomonas
reinhardtii cultures, Journal of Biotechnology 134 (2008) 275–277
72) A. Melis, L. Zhang, M. Forestier, M. Ghirardi, M. Seibert, Sustained photobiological
hydrogen gas production upon reversible inactivation of oxygen evolution in the green
alga Chlamydomonas reinhardtii, Plant Physiol. 122 (2000) 127–136.
73) T.K. Antal, G. P. Kukarskikh, A. A. Volgusheva, T. E. Krendeleva, E. Tyystjärvi, A. B.
Rubina, Hydrogen photoproduction by immobilized S-deprived Chlamydomonas
reinhardtii: Effect of light intensity and spectrum, and initial medium pH, Algal Research
17 (2016) 38–45.
74) S.N. Kosourov, M. Seibert, Hydrogen photoproduction by nutrient-deprived
Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic
and anaerobic conditions, Biotechnol. Bioeng. 102 (2009) 50–58.
75) S.N. Kosourov, K. Batyrova, E. Petushkova, A. Tsygankov, M. Ghirardi, M. Seibert,
Maximizing the hydrogen photoproduction yields in Chlamydomonas reinhardtii
cultures: he effect of the H2 partial pressure, Int. J. Hydrog. Energy 37 (2012) 8850–
8858.
76) S.M Moreira, M.M. Santos, L. Guilhermino, R. Ribeiro, Immobilization of the marine
microalga Phaeodactylum tricornutum in alginate for in situ experiments: bead stability
and suitability. Enzyme Microb. Technol. 38 (2006) 135–141.
77) I. Akkerman, M. Janssen, J. Rocha, R. H. Wijffels, Potobiological hydrogen production:
photochemical efficiency and bioreactor design. International Journal of Hydrogen
Energy 27 (2002) 1195-1208.
78) H. Younesi, G. Najafpour, K.S.K. Ismail, A.R. Mohamed, A.H. Kamaruddin,
Biohydrogen production in a continuous stirred tank bioreactor from synthesis gas by
anaerobic photosynthetic bacterium: Rhodopirillum rubrum. Bioresour. Technol. 99 (7)
(2008) 2612–2619.
79) J. Ding, X.Wang, X.F. Zhou, N.Q. Ren, W.Q. Guo, 2010. CFD optimization of
continuous stirred-tank (CSTR) reactor for biohydrogen production. Bioresour. Technol.
101 (18) (2010) 7005–7013.
80) T.J. Evens, D.J. Chapman, R.A. Robbins, E.A.D’Asaro, An analytical pat-plate
photobioreactor with a spectrally attenuated light source for the incubation of
phytoplankton under dynamic light regimes, Hydrobiologia 434 (2000) 55–62.
81) W.Z Doenitz, E. E. Dietrich, and R. Streicher, Electrochemical High Technology for
Hydrogen Production or Direct Electricity Generation, International Journal of Hydrogen
Energy, 13(1988) 283-287.

82) A Wang, D. Sun, G.Cao, H.Wang, N.Q. Ren, W.M. Wu, B.E. Logan, Integrated
hydrogen production process from cellulose by combining dark fermentation, microbial
fuel cells, and a microbial electrolysis cell. Bioresour. Technol. 102 (2011) 4137–4143.
83) S.A.Markov, Hydrogen production in bioreactors: Current trends, Energy Procedia 29
(2012) 394 – 400.
Fig.1: Hydrogen production methods

ORGANIC FEEDSTOCK -> HYDROGEN

(DARK FERMENTATION)

MESOPHILLIC THERMOPHILLIC
MICRORGANSMS MICRORGANISM

CLOSTRIDIUM THERMOTOGA
BUTYLICIUM RAEOPLANTIA

Fig. 2: Dark fermentation with different type of microorganisms

Fig. 3: Multi-stage bioreactor system
 Table 1: Microorganisms used in hydrogen production

MICRORGANISM Mode of Operation REFRENCES

C. reinhardtii Genetic engineering using [30]
Phaeodactylum tricornutum expressed sequence tags
Thalassiosira pseudonana (ESTs)
Cyanidioschyzon merolae
Ostreococcus lucimarinus
Ostreococcus tauri
Micromonas pusilla
Fragilariopsis cylindrus
Pseudo-nitzschia
Thalassiosira rotula
Chlorella vulgaris
Dunaliella salina
Micromonas pusilla
Galdieria sulphuraria
Porphyra purpurea
Volvox carteri
Aureococcus anophageferrens
Chlorella pyrenoidosa Lipid Biosynthesis [31]
Chlamydomonasmoewusii Anaerobic Fermentation [32]
Scenedesmus oblique Anaerobic Fermentation [33]
Anabaena variabilis Photo-Fermentation [34]
Rhodobactersphaeroides Transcriptional analysis [29]
Enterobacter aerogenes Batch Fermentation [35]
Clostridium butyricum Anaerobic Fermentation [36]
Bacillus coagulans Anaerobic Fermentation [10]
Clostridium acetobutylicum Anaerobic Fermentation [37]
ATCC 824
Laminaria japonica Anaerobic Fermentation [38]
Gelidium amansii Dark Fermentation [39]
Table 2: Immobilization methods

METHOD MATRIX REMARKS REFRENCES

Encapsulation Alginate beads Initial cell concentration of 100 x106 [69]
cells ml-1 of alginate and 1x106 cells
ml-1 could be entrapped
Encapsulation Calcium With apt light intensity and pH of the [70]
alginate beads medium for optimal values for the
suspension culture the immobilized
culture was evolving hydrogen for
approximately three weeks of S
depletion.
Entrapment Alginate films It has been observed that there were [71-75]
higher cell densities and a specific
hydrogen production rates after the
immobilization process.
Binding Glass beads Bound cells are more easily cycled [76]
between growth mode and hydrogen
production mode.

Fumed silica is an appropriate solid

support for the cells.

Neither growth nor hydrogen

production is inhibited by the presence
of the silica, and the cells are shown to
bind to the particles.