Biotechnology Reports 15 (2017) 63–69

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Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre

Review

Hydrogen from algal biomass: A review of production process MARK

⁎
Archita Sharma, Shailendra Kumar Arya
Department of Biotechnology, University Institute of Engineering and Technology, Panjab University, Chandigarh, India

A R T I C L E I N F O A B S T R A C T

Keywords: Multifariousness of biofuel sources has marked an edge to an imperative energy issue. Production of hydrogen
Hydrogen from microalgae has been gathering much contemplation right away. But, mercantile production of microalgae
Algae biofuels considering bio-hydrogen is still not practicable because of low biomass concentration and costly down
Biofuels streaming processes. This review has taken up the hydrogen production by microalgae. Biofuels are the up and
Immobilization
coming alternative to exhaustible, environmentally and unsafe fossil fuels. Algal biomass has been considered as
Bioreactors
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.

1. Introduction fertilizer and petroleum industries with approximately 50% and 37%
respectively [6]. Hydrogen production has been determined only at the
Forthwith, the world is bearing the challenges of high energy de- laboratory scale with yield still low for commercial application and so,
mands as well as escalating fuel prices because of breakneck growth of the optimization of design and operating parameters for maximum
world population and hasty industrialization. So there is a need of an hydrogen production is a must step while addressing the subject of
hour to cope up with such challenges and for this researchers are now hydrogen production rate. The optimization basically counts on the
paying much appreciable attention by recommending sustainable and microalgae strain along with the available growth conditions [7]. For
cost-effective methods for energy production [1,2]. Fossil fuels are as- biofuels to be broadly authorized in the energy merchandise, spotlight
sociated with the environmental pollution and thus, more efforts have must be on acclimatizing and improving photosynthetic organisms for
been evolved in renewable energy sources being economic and en- biofuel production [8].
vironmental friendly [2]. Governments are now proactive in addressing By the time mentioned, the sucrose and starch crops, for instance,
secured supply of raw materials with limiting climate change and many sugarcane and corn as well as lignocellulosic materials like rice straw
potential candidate fuels have been studied in the energy area, subse- and switchgrass are being used as biofuel feedstock’s. But, high cost in
quently [3,4]. Biomass is one of the most encouraging renewable re- the hydrolysis of lignocellulosic materials is a matter of concern. Sugars
sources being used to bring about different types of biofuels, serving as come in several forms, containing approximately four calories per
biodiesel, bioethanol, biogas and biohydrogen. Energy from biomass gram. Simple sugars like monosaccharides like glucose, fructose and
would contribute to a stable energy supply and to local society due to galactose. Biohydrogen production offers a sustainable alternate and by
an increase in commercial activities [5]. Biomass can be derived from utilizing renewable carbon sources can be considered as carbon dioxide
cultivation of dedicated energy crops; by harvesting forestry and other offset. This can utilize various carbon sources including wastewater.
plant residues; and from biomass wastes [6]. Hydrogen is extensively Glucose, sucrose are readily degradable and hence preferred as model
being seen as a clean fuel, environmentally safe, renewable energy re- substrates for hydrogen production. Because of complex composition
source and an excellent substitute of fossil fuels and a potential can- and polymeric structure complex carbon must released or converted to
didate with highest energy density with many of the technical, socio- simple sugars. Complex polymer consists of tightly bound lignin, cel-
economic and environmental benefits to its credence among all other lulose and hemicelluloses. Cellulose and hemicelluloses can be de-
known fuels (143 GJ per tonne) and is the only acknowledged fuel that graded under same conditions and add up the cost factor which is a
does not produce carbon dioxide as a by-product when used in fuel cells matter of concern [2,9]. A lot of microorganisms are involved in the
for electricity generation [2,6]. The bulkiest users of hydrogen are the production of biofuels like hydrogen, but most accepted are

⁎
Corresponding author.
E-mail address: skarya_kr@yahoo.co.in (S.K. Arya).

http://dx.doi.org/10.1016/j.btre.2017.06.001
Received 15 February 2017; Received in revised form 20 May 2017; Accepted 1 June 2017
Available online 14 June 2017
2215-017X/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
A. Sharma, S.K. Arya Biotechnology Reports 15 (2017) 63–69

cyanobacteria and green microalgae freshly considered as third gen- conversion efficiencies are certain practical hindrances need to over-
eration feedstock’s being more efficient at converting sunlight into the come for the successful hydrogen production.
chemical energy and require a smaller footprint and less water for
cultivation [7,10,11]. 2.2.1. Direct photolysis
Diverse pretreatment methods and physic-chemical pretreatments There is a dissociation of water into hydrogen and oxygen in the
have been revised for the hydrogen production. It is necessary step to presence of light, that is, H2O → H2 + ½O2 [16,17]. Green microalgae
breach the algal cell wall along with the complex carbohydrate to re- can use light to carry out photosynthesis as they possess chlorophyll a
lease simple sugars. Pretreatment methods such as physical (sonication, and the photosynthetic systems: Photosystem (PS) II and Photosystem
milling, grinding, pyrolysis), chemical (acid, alkali, thermal) and bio- (PS) I, respectively [18,19]. Disadvantages are the enzyme hydrogenase
logical methods (enzymatic) is being employed to break algal cell wall, is very sensitive to oxygen so when a certain amount of oxygen is
to hydrolyze the complex carbohydrates and to release fermentable present, will inhibit hydrogenase activity and will stop it from produ-
sugars [9]. cing hydrogen. Also, it requires high intensity of light. The advantages
An immobilized cell means a cell by natural or artificial paths is include tenfold more solar conversion in green microalgae.
being prevented independent movement from its neighbouring en-
vironment to all parts of the system which is under consideration [12]. 2.2.2. Indirect photolysis
Basically, there are six different types of cell immobilization methods. There is two step processes, firstly there is a splitting of water mo-
They are covalent coupling, affinity immobilization, adsorption, con- lecules in the presence of sunlight and protons and oxygen is formed.
finement in the liquid–liquid emulsion, capture behind semi-permeable Secondly carbon dioxide fixation occurs storage carbohydrate is being
membrane and entrapment. Utilization of immobilization technique produced, followed by the production of hydrogen gas by hydrogenase
contributes more resilience while designing a reactor comparing con- [20].
ventional suspension systems. Furthermore, increase in cell density,
12H2O + 6CO2 + light energy → C6H12O6 + 6O2
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 ad- C6H12O6 + 12H2O + light energy → 12H2 + 6CO2
hesive attachment of cells onto surfaces of solid support are usually
more common. Important criteria for successful entrapment are to Blue-green algae (cyanobacteria) are promising microorganisms for
set algal cells from within their partition, while pores inside gel matrix this. Advantages are hydrogen evolution is separated from oxygen
allow diffusion of substrates and metabolic products towards and from evolution. It can also produce relatively higher hydrogen yields.
cells [13]. Furthermore, by-products can be efficiently converted to hydrogen.
In these, bioreactors which are being considered for hydrogen Disadvantages are like significant adenosine triphosphate (ATP) re-
production from algal biomass are matter of concern. Biohydrogen quirement of nitrogenase. Also, this requires continuous light source
production by microorganisms has attracted our increasing worldwide which is difficult for large scale processes [21–24].
attention, having its potential for inexhaustible, low-cost and a re-
newable source of energy. They are categorically pre-requisite for large 2.2.3. Dark fermentation
scale hydrogen production by microorganisms. Certain microorganisms Hydrogen production in a dark environment without the presence of
have been evolved for hydrogen production either from organic mate- sunlight, water and oxygen. Fermentative microorganisms hydrolyze
rials like sugar or biomass. Bioreactors are closed systems that have complex organic polymers to monomers that are further converted to a
varied size from the small (5 mL–10 mL) to the larger scale or more mixture of lower molecular weight organic acids and alcohols by ne-
than 500,000 L industrial scale. Photobioreactors are made up of an cessary hydrogen producing bacteria [24–26]. Advantages consist of
array of tubes, tanks bags, where photosynthetic microorganisms in- use of a variety of carbon sources and production of hydrogen without
cluding algae are being cultivated and later monitored as light is the light. It produces valuable by-products like butyric acid, lactic acid and
essential component for growing photosynthetic microorganisms. acetic acid etc. Disadvantages are relatively lower hydrogen yields. Also
Bioreactors that have been mentioned later in this are photobioreactors, the product gas mixture contains carbon dioxide which has to be se-
continuous stirred tank reactor (CSTR), fixed bed bioreactors, mem- parated [3]. Fig. 2 explains different types of microorganisms capable
brane bioreactors, multi-stage bioreactors and hybrid bioreactors [69]. of dark fermentation with examples.

2. Hydrogen production 2.2.4. Photo-fermentation

It is a fermentative conversion of organic substrates into hydrogen
2.1. Principle and carbon dioxide by use of sunlight as an energy source.

CH3COOH + 2H2O + light → 4H2 + 2CO2

Hydrogen makes up about three quarters of all matter and thus the
most plentiful element of universe. Compelling hydrogen sources in- Using light as the energy source, the organic acid substrates are
cludes fossil fuels (95–99%) and water [14]. Dealing with future hy- oxidized using the tricarboxylic acid cycle (TCA), producing electrons,
drogen demands independent of fossil fuels, it is necessary to appreciate protons and carbon dioxide. Example includes purple non sulfur bac-
all available renewable resources [15]. teria (PNS) [11,17]. Advantages in removal of environmental pollu-
The classical methods for hydrogen production consists of steam tants, use of industrial waste and use of organic acids produced from
reforming of natural gases, coal gasification and electrolysis of water dark fermentation. Disadvantages are need to nitrogen limit condition
are energy intensive processes that requires high temperatures and pretreatment of industrial effluent as it may be toxic [23].
(> 840 °C) and are not environmental friendly as such. Electrolysis of
water being the cleanest technology for hydrogen production, can only 3. Hydrogen production from algal biomass
used in sectors with cheap electricity as adds up to 80% of the operating
cost [3]. Fig. 1 explains hydrogen production processes with examples. The renewable energy sources play crucial role in decreasing the
greenhouse effect but also provides an alternative approach regarding
2.2. Mechanism increasing global energy demands resulting into depletion of energy
reserves [27]. Many algal species show potential to produce hydrogen
With the noteworthy merits, the low production rates, low substrate under certain conditions [28]. Nonetheless, certain technical barriers

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A. Sharma, S.K. Arya Biotechnology Reports 15 (2017) 63–69

Fig. 1. Hydrogen production methods.

like developing low-energy methods to harvest microalgal cells, diffi- Over 40,000 species of algae have been described, and this is likely
culties in continuously producing biomass at a large scale, the presence only a small fraction of the total number of available species. The U.S.
of invasive species in large-scale ponds, low light penetrance in dense Department of Energy’s Aquatic Species Program analyzed approxi-
microalgal cultures, and the lack of cost-effective bioenergy carrier mately 3000 different microalgae for their potential to produce biofuels
extraction techniques, are required to overcome before using micro- [30]. Table 1 explains numerous microorganisms involved in hydrogen
algae as an economically viable biofuel feedstock [29]. production from algal biomass with their processes.

3.1.1. Culture conditions

3.1. Microrganisms
Inescapably, accomplishments of hydrogen-producing bioreactor
systems are governed by various factors like:
Algae are the association of organisms similar in their morpholo-
gical and physiological features, that is, ability of photoautotrophic
metabolic pathways, exists predominantly in water etc. Microalgae can 3.1.2. pH
fix carbon dioxide from the atmosphere and with this fact; they are pH has been sanctioned as one of the most important environmental
positively characterized in terms of biomass cultivation convenience factors that affects metabolic pathways and yields hydrogen.
[29]. Comparative studies have shown that the ideal pH range should be

Fig. 2. Dark fermentation with different type of microorganisms.

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Table 1 nitrogen, phosphate and other inorganic trace minerals are imperative
Microorganisms used in hydrogen production. supplements for carbohydrate based feedstocks. Organic nitrogen is
more favourable compared to inorganic one. Phosphate is required for
Microrganism Mode of Operation References
optimal hydrogen production. The elements like Mg, Na, Zn and Fe are
C. reinhardtii Phaeodactylum Genetic engineering using [30] crucial supplements and have suggested an optimum nutrient for-
tricornutum expressed sequence tags mulation. Iron is of utmost importance in the enzymatic activity of
Thalassiosira pseudonana (ESTs)
hydrogen production that is [NiFe]-hydrogenases, [FeFe]-hydro-
Cyanidioschyzon merolae
Ostreococcus lucimarinus genases, and [Fe]-hydrogenase [47–54].
Ostreococcus tauri The apical level production from sweet potato starch residue by a
Micromonas pusilla repeated batch culture containing C. butyricum IFO13949 when 1.0%
Fragilariopsis cylindrus polypepton was added as nitrogen source was reported. In contra-
Pseudo-nitzschia
diction, addition of urea (NH4)2SO4 or NH4Cl resulted in the absence of
Thalassiosira rotula
Chlorella vulgaris hydrogen evolution by the same culture [47].
Dunaliella salina
Micromonas pusilla 3.1.6. Temperature
Galdieria sulphuraria
Microbes are competent in a temperature range of 15–85 °C but may
Porphyra purpurea
Volvox carteri vary under temperatures from 15 to 34 °C for mixed cultures [6,27].
Aureococcus anophageferrens It has been considered that the hydrogen production capacity of a
Chlorella pyrenoidosa Lipid Biosynthesis [31] mixed culture under varying temperatures from 15 to 34 °C and found
Chlamydomonasmoewusii Anaerobic Fermentation [32] that hydrogen yield and specific hydrogen production rate increased
Scenedesmus oblique Anaerobic Fermentation [33]
with temperature, achieving respective maximum values of
Anabaena variabilis Photo-Fermentation [34]
Rhodobactersphaeroides Transcriptional analysis [29] 359 mmol l−1 d−1 and 1.42 mol H2 mol−1 glucose at 30–34 °C and
Enterobacter aerogenes Batch Fermentation [35] 28–32 °C respectively [6].
Clostridium butyricum Anaerobic Fermentation [36]
Bacillus coagulans Anaerobic Fermentation [10]
3.1.7. Substrate concentration
Clostridium acetobutylicum ATCC Anaerobic Fermentation [37]
824
This factor, moreover, has been a point of debate. Recent studies
Laminaria japonica Anaerobic Fermentation [38] have found that hydrogen yield to increase with increasing glucose
Gelidium amansii Dark Fermentation [39] concentration [55]. Along with substrate concentration other operating
conditions like hydraulic retention time, composition of microbial
cultures also affects the same [56,57].
around 5.2 and 6.0 [39,40]. The effect of pH in a CSTR must be in the It was found that hydrogen yield to increase with increasing glucose
range of 4.0–7.0, optimum at a pH of 5.5 [41]. concentration from 10 to 35 g l−1at a hydraulic retention time of 12 h
It has been observed hydrogen production from glucose using con- [58] when in fact it was investigated that there is continuous hydrogen
tinuous cultures under identical conditions except pH values, and production at 12 h hydraulic retention time on 10–50 g l−1 sucrose and
concluded that higher hydrogen yield and production rate are attained found that the hydrogen yield decreased from 1.7 ± 0.2 mol H2 mol−1
at a pH of 5.7 compared with pH 6.4 [6]. More emphasis was on the hexose added at 10 g l−1 sucrose to 0.8 ± 0.1 mol H2 mol l−1 hexose
maximum hydrogen yield which was achieved only when microbial added at 50 g l−1 [58]. These considerations indicated that besides
reactions followed an ethanol fermentation type that occurred at a pH substrate concentration conditions as in hydraulic retention time and
of about 4.5 [42]. composition of microbial cultures also influences hydrogen production.

3.1.3. Hydraulic retention time (HRT) 3.1.8. Seed culture

Hydraulic retention time is required to prefer microbial populations Clostridium and Enterobacter are extensively used as inoculums for
with growth rates being able to catch up with mechanical dilution set fermentative hydrogen production [59]. A lot of studies have shown
up by continuous volumetric flow. It was found that shortening the that using pure cultures of bacteria for fermentative hydrogen pro-
hydraulic retention time from 8 h to 6 h would reduce microbial di- duction were conducted in batch mode and used glucose as substrate
versity leading to an increase in the hydrogen yield [43]. [60,61]. Mixed bacterial cultures from anaerobic sludge, compost and
It was found that the methane concentration ranged between soil is used as inoculums for fermentative hydrogen production.
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 3.1.9. Feedstock
(D > 0.075 h−1), indicating negligible methanogenic activity at high Simple sugars such as glucose, sucrose and lactose being biode-
dilution rates. This means that hydraulic retention time is capable of gradable are preferred as model substrates for hydrogen production.
inhibiting or terminating methanogenesis in hydrogen production via But, the costs for pure carbohydrate sources are high on practical-scale
anaerobic fermentation [44]. production that is feasible on renewable and low cost sources [62–65].
Proclaimed by plentiful studies of hydrogen fermentative processes,
3.1.4. Hydrogen partial pressure carbohydrates are the essential source of hydrogen. Along the lines,
The dissolved hydrogen concentration adding to the hydrogen wastes and biomass rich in sugars and complex carbohydrates turn out
partial pressure is the key factors affecting microbial pathways. to be the most apt feedstocks for biohydrogen generation [66].
Hydrogen production is less favourable as the hydrogen partial pressure
rises. Thus it is mandatory to remove excess hydrogen from the system 3.2. Immobilization
for maintaining hydrogen production [43,45].
It was found an increase in the hydrogen yield from 0.85 to The usefulness of microalgae in biotechnology has been heightened
1.43 mol mol−1 hexose when the reactor is sparged with nitrogen at in fresh years. These organisms being involved in food, cosmetic,
fifteen times hydrogen produced rate [46]. aquaculture, pharmaceutical industries and various other industries but
small size poses an obstacle in the biotechnological applications. Cell
3.1.5. Nutrients immobilization techniques have been refined to solve above mentioned
For accessing optimal cell cultivation and hydrogen production complications [4]. Immobilized algae have been used for biomass

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Table 2
Immobilization methods.

Method Matrix Remarks Refrences

Encapsulation Alginate beads Initial cell concentration of 100 × 106 cells ml−1 of alginate and 1 × 106 cells ml−1 could be entrapped. [69]
Encapsulation Calcium alginate With apt light intensity and pH of the medium for optimal values for the suspension culture the immobilized culture was [70]
beads evolving hydrogen for approximately three weeks of S depletion.
Entrapment Alginate films It has been observed that there were higher cell densities and a specific hydrogen production rates after the immobilization [71–75]
process.
Binding Glass beads Bound cells are more easily cycled between growth mode and hydrogen production mode. [76]
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.

obtain and macronutrient removal. The extremely high accumulation contact and mass transfer can be absolutely accomplished while on the
capacity of some of these organisms for potentially dangerous sub- other hand, the continuous stirred tank reactor is not be able to
stances has been also exploited for bioremediation techniques applied maintain high levels of fermentative biomass because of the rapid
on polluted waters especially involving metals [67]. mixing operating pattern. Biomass washout is possible to occur at short
Environmental benefits of immobilized algal cells are not confined hydraulic retention times (HRTs), and thus the hydrogen production
to pollutant removal level only. These techniques have been currently rates are noticeably restricted [66,74,78,79].
being used in the area of toxicity measurement experiments. Utmost,
the immobilization techniques being devised for microorganisms in 3.3.3. Fixed-bed bioreactor
general can also be applied for microalgae, with the check of light This is negotiated with support carriers packed within the tank. The
transmission if living cells are intended to be immobilized [68]. Table 2 hydraulic mixing regime is less turbulent comparing with the con-
give examples of immobilization method and their matrix uses. tinuous stirred tank reactor with a consequence of higher mass transfer
resistance along with lowered rates of substrate conversion and hy-
3.3. Bioreactor drogen production. High hydrogen yield could not be cultivated per-
sistently in a fixed-bed reactor as pH gradient distribution along the
Traditional industrial methods are quite costly. It is imaginable to reactor column will cause a heterogeneous distribution of microbial
design bioreactors on large scale using microorganisms as bioreactors activity. So, to overcome this, recirculation flow was recommended
are categorically a pre-requisite for large-scale hydrogen production by [43,82].
microorganisms [77,78].
3.3.4. Membrane bioreactor
3.3.1. Photo-bioreactors Membrane bioreactor (MBR) is basically used to control biomass
Design depends on microbiological processes linked with micro- concentration. MBR did not exhibit superiority other high-rate hy-
algae or cyanobacteria. With differing photochemical efficiency, ab- drogen production systems. Membrane fouling and high operating cost
sorption coefficient and size, the light regime including light and dark also limit the use of membrane bioreactor process in bio hydrogen
cycles is hypothetically is much more determining than biological fac- fermentation [68].
tors [71]. Therefore, productivity of a photo-bioreactor is light-depen-
dent with large surface to volume ratio as a pre-requisite. Photo-bior- 3.3.5. Multi-stage bioreactors
eactors have been designed in such a way to access an economical, Multi-stage bioreactors consisting of three four stages have been
rapid multiplication and high density of the microalgae culture prospective to maximize the production from the substrate [66,75].
[66,80,81]. Sunlight is first percolated via the first stage called direct photolysis
reactor where the visible light will be exploited by blue green algae
3.3.2. Continuous stirred tank reactor (CSTR) whereas the unfiltered infrared light is used by photo-synthetic mi-
Continuous stirred tank reactors are frequently being used for hy- crobes in the second stage called photo-fermentative reactor. The ef-
drogen production. In a continuous stirred tank reactor, hydrogen- fluent from the second stage along with feedstock is stuffed into a third
producing microbes are thoroughly mixed and suspended in the reactor stage called dark fermentation reactor where the bacteria convert the
liquid from the mixing pattern. Under this, a good substrate-microbes substrate to hydrogen and organic acids. The fourth stage is the use of a

Fig. 3. Multi-stage bioreactor system.

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