European Journal of Biotechnology and Bioscience 2013; 1 (2): 84-93
Review of Research on Production Methods
ISSN 2321-9122
EJBB 2013; 1 (2): 84-93
© 2013 AkiNik Publications
Received 16-8-2013
Accepted: 23-9-2013
Dr. R. Hema Krishna
Department of Chemistry, University
of Toronto, Ontario, Canada. M5S
3H6, Canada.
West
Correspondence:
Dr. R. Hema Krishna
Department of Chemistry,
University of Toronto, Ontario,
Canada. M5S 3H6, Canada.
E-mail: hkravuri32@gmail.com
Tel: 001-6475026323
of Hydrogen: Future Fuel
Dr. R. Hema Krishna
ABSTRACT
Hydrogen is considered to be the most viable energy carrier for the future. Hydrogen is high in
energy content as it contains 120.7 kilojoules/gram. This is the highest energy content per unit
mass among known fuels. This paper provides a comprehensive review of the various Physico-
Chemical Production methods(Fossil Fuels ,Steam Reforming of Natural Gas, Thermal Cracking
of Natural Gas, Partial Oxidation of Heavier Hydrocarbons,Coal Gassification, Water,
Electrolysis, Photolysis, Thermochemical Process, Thermolysis) & biological approaches (Bio-
Photolysis of water, Photodecomposition of organic compounds, Fermentative Hydrogen
Production from Organic Compounds, Hybrid System) that are being studied for biohydrogen
production. A succinct discussion of production techniques for enhancing hydrogen production
from Physical-Chemical Production has also been provided where Steam reforming of natural gas
is currently the least expensive method of producing hydrogen, and used for about half of the
world's production of hydrogen, where as in biological approaches Hybrid System have been
found to be among the best production technologies for enhanced biohydrogen production.
Keywords: Hydrogen, Future fuel, Thermochemical Process, biological approaches, Hybrid System
1. Introduction
The energy demand of the world has been continually and increasingly dependent on earth’s
fossil fuels reserves particularly oil, coal and natural gas. Over 90% of world’s energy demands
are made by fossil fuels as they are readily available and convenient to use. However the non-
renewability of these resources and their uneven distribution around the globe are the limiting
factors. The rate at which we consume these resources will for exceed the rate at which they
are being recycled. This will lead to the eventual depletion such as readily accessible reserves
according to [1-3] they may well get exhausted by 2030. Although conventional energy sources
have heralded technological progress and are holding a unique position because of their high
energy intensity, they have got serious drawbacks such as having end products that are
extremely harmful to human health, and our the major contributors of serious environmental
pollution. The combustion of fossil fuels leads to air pollution, acid rain, increasing levels of
tropospheric ozone, depletion of stratospheric ozone, greenhouse effect and there by global
warming. The rising costs of fossil fuels pollution and the finite nature of conventional fuels
have forced the world to look for an alternative energy source. The desire for change in the
present pattern of energy use stems from the helplessness to curb power consumption and the
need for clean energy to eliminate growing environmental threat. The desirable characteristics
of such a system are abundant availabilities, low cost, conveniently usable, renewable nature,
economically transportable and socially compatible. Though alternate energy sources such as
solar, wind, tidal, wave, ocean, thermal, geothermal etc. are available in plenty and
environmental compatible, there are still in the experimental stages and are having one (or) the
other drawbacks such as productive cost and unfeasible technological Know-How. By contrast
Hydrogen is consider as the only conceivable energy source by many researches as an ideal
fuel of the future because of its non-polluting (The end product of combustion being water) and
renewable nature [4-5]. This paper provides a comprehensive review of the various Physico-
Chemical Production methods & biological approaches that are being studied for biohydrogen
production.
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 European Journal of Biotechnology and Bioscience
2. Profile of Research in Hydrogen Production
The popularity of Hydrogen as a fuel source followed from a crisis
that resulted on greater usage of non-renewable fuels. During the
Energy crisis of the 1970’s, Hydrogen was touted as the “fuel of
the future” and a great deal of time and money were put on
research on its possible sources and applications. The biological
hydrogen production was first seriously considered as a practical
capability through a series of sponsored meetings conducted by
National Science Foundation, Washington D.C [6]. The focus of
these meetings were chiefly on hydrogen production through
photosynthetic processes. The initial processes provided
encouraging results demonstrating that hydrogen could be
produced from water by illuminating a mixture of spinach
chloroplast membranes and two bacterial proteins, a hydrogenase
for production of hydrogen and a ferredoxin for shuttling the
electrons from the photosynthetic membranes to the hydrogenase
[7]
. Experiments with Anabaena cylindria, a nitrogen fixing
cyanobacteria (blue-green algae), demonstrated the in-vivo
hydrogen production. With the promise of practical photobiological
hydrogen production on the horizon, a great deal of research was
conducted in this area until early 1980’s [8-9] when the energy crisis
was declared as over. After the oil prices dropped, the international
agenda slowed down its research on procurement of hydrogen and
other alternative energy technologies.
The interest in hydrogen resurfaced in the 1990’s [10-11]. when it
became apparent that the atmospheric pollution by fossil fuels was
considered not only unhealthy but also as a major cause for global
climatic changes. At the moment, the US Department of energy,
Fig 1: Sources and Methods of Hydrogen Production
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Washington DC, is currently spending about 1 million $ per year in
Biohydrogen research while Japan is spending five times as much
in that area. These sums are far below to what both the long-term
promises and near-term opportunities of their practical applications
can ensure. Given the economic uncertainties and environmental
hazards of fossil fuels, working out the technical and economic
feasibility of hydrogen has gained major importance as we entered
the 21st century [12]. At present, the research divests on splitting of
water into hydrogen and oxygen by photosynthetic systems. This
approach, if successful, would virtually allow unlimited production
of hydrogen from earth’s most plentiful resource- water and
sunlight. There are, however, tremendous biological and
engineering challenges to overcome for realizing this goal.
Additional technical challenges are presented in engineering low-
cost photobioreactors that can simultaneously provide an
environment conducive to the efficient microbial catalysis of
hydrogen production from water and sunlight as well as capture of
hydrogen.
3. Hydrogen Production Methods
Increase in industrial processes has been resulting in enormous
amount of effluents generation for which selection of appropriate
treatment methodology is extremely challenging. At present,
hydrogen is mainly produced from fossil fuels, biomass and water.
Among these methods, steam-reforming process alone produces
about 90% of hydrogen [13]. The various methods adopted to
produce hydrogen are given in the (figure .1)
 European Journal of Biotechnology and Bioscience
4. From Fossil Fuels
Over 90% of the world's hydrogen of around 45 million metric tons
are produced from fossil fuels. The largest producers of hydrogen
are the artificial fertilizer and petroleum industries, with
respectively 47% and 37%. Sales of hydrogen have increased by
6% annually in the last five years, which is closely related to the
increased use of hydrogen in refineries as a result of stricter
standards for fuel quality. This development is expected to
increase. In addition, it is expected that the next five years will see
a dramatic increase in local small-scale production of hydrogen as
fuel cell technology is introduced on the mass market. Removing
the environmentally dangerous chemicals from fuel in central
plants will spare the local environment, and it is also easier to
handle a few central discharge stations than several smaller ones.
Hydrogen can be produced from several different hydrocarbon
compounds by varying techniques. Different hydrocarbon
compounds have different amounts of hydrogen. Natural gas has a
ratio of hydrogen to carbon four times higher than in coal. The
several techniques used are:
4.1 Steam Reforming Process
Steam reforming of natural gas is currently the least expensive
method of producing hydrogen, and used for about half of the
world's production of hydrogen. Steam, at a temperature of 700-
1,100 °C is mixed with methane gas in a reactor with a catalyser at
3-25 bar pressure. Thirty percent more natural gas is required for
this process, but new processes are constantly being developed to
increase the rate of production. It is possible to increase the
efficiency to over 85% with an economic profit at higher thermal
integration. A large steam reformer which produces 100,000 tons
of hydrogen a year can supply roughly one million fuel cell cars
with an annual average driving range of 16,000 km. There are two
types of steam reformers for small-scale hydrogen production:
Conventional reduced-scale reformers and specially designed
reformers for fuel cells. The latter operates under lower pressure
and temperatures than conventional reformers and is more
compact. The formula for this process is:
CH4 + H2 O => CO + 3H2
It is usually followed by the shift reaction:
CO + H2O => CO2 + H2
1 mol methane => 4 mol hydrogen
The percentage of hydrogen to water is 50%. In steam reforming of
natural gas, 7.05 kg CO2 are produced per kilogram hydrogen
4.2 Partial Oxidation of Heavier Hydrocarbons
Heavy hydrocarbon's low volatility and often high sulfur content
prevents using steam reforming. Instead, the oil is treated
autothermically in a combustion reaction and fed with steam and
oxygen at 1300-1500oC. The oxygen to steam ratio is controlled so
that the gasification continues without the use of additional energy.
The formula for this process is:
CH1,4 + 0,3 H20 + 0,4 02 => 0,9 CO + 0,1 CO2 + H2
This is followed by the shift reaction:
CO + H20 => CO2 + H2
1 mol CH 1,4 => 1,9 mol hydrogen
4.3 Thermal Cracking Process
Burning hydrocarbons with reduced access to oxygen is called
partial oxidation. Thermal Cracking is a mixture of partial
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oxidation and steam reforming. The name refers to the heat
exchange between the endothermic steam reforming process and
the exothermic partial oxidation. Hydrocarbons react with a
mixture of oxygen and steam in a "thermos reactor" with a
catalyzer.
4.4 Coal Gasification
Gasification of coal is the oldest method for production of
hydrogen. Generally, coal is heated up to 900o C with a catalyser
and without air. There are also more complex ways of gasifying
coal, such as the Lurgi, Winkler and Koppers-Totzek methods.
These techniques are similar in that by using steam and oxygen at
temperatures over 1400o, they change carbon into H2, CO and CO2,
and in addition create some sulfur and nitrogen, which like CO and
CO2, must be treated in the same way, environmentally speaking
[14]
. It is almost twice as expensive to produce hydrogen from coal
as from natural gas. This has to do with the relation between
hydrogen and carbon, which in natural gas is 4:1 and in carbon
0.8:1. Germany, South Africa and the USA presently have large
gasification plants, and technology for gasification of coal in
thermal power plants is the subject of much R&D by the coal
industry.
4.5 Pyrolysis
Hydrogen can also be produced by thermal gasification of organic
compounds like heavier hydrocarbons at very high temperatures in
absence of air. Under high temperatures, the organic compounds
break down to gas. The gas consists mainly of H2, CO and CH4
(methane). Steam is then introduced to reform CH4 to H2 and CO.
CO is then put through the shift process to attain a higher level of
hydrogen. The by-product from this process is CO2. The mixed gas
can also be used in fuel cells for electricity production. Compared
to conventional processes for production of electric energy from
biomass or waste, integrated gasification fuel cell systems are
preferable.
4.6 From Water
Water is the most inexpensive and a highly renewable source of
hydrogen production taking into account that over three-fourths of
our world is filled with water. Thermal and Electrochemical
decomposition of water shows greater hydrogen yields and is also
less energy intensive. However, the temperature and pressure
conditions required to be maintained for splitting of water to
hydrogen and oxygen is very high in the range of 2000-30000C.
This appeared to be the major disadvantage of using water as a
source of hydrogen production but further research is being carried
out to prove energy-cost savings. The various processes studied for
hydrogen production using water source are:
4.7 Electrolysis
Water electrolysis is splitting water into hydrogen and oxygen. An
electrolyser is a device for electrolysis. Water is subjected to
electrical power and the result is hydrogen and oxygen.
2H2O + Energy => 2H2 + O2
4.8 Photolysis
Instead of first converting sunlight to electricity and then using an
electrolyser to produce hydrogen from water, it is possible to
combine these two steps. The photovoltaic cell combines with a
catalyst, which acts as an electrolyser and splits hydrogen and
oxygen directly from the surface of the cell. This can quite
 European Journal of Biotechnology and Bioscience

realistically be a commercially viable means of producing
hydrogen. The advantage with these systems is that they eliminate
the cost of electrolysers and increase the systems’ efficiency. Tests
performed outdoors with silicon based cells have shown an
efficiency of 7.8% in natural sunlight. Research is being done to
increase the efficiency factor and the life span for such cells.
4.9 Thermochemical Process
Thermochemical hydrogen production technologies use heat and
chemical reactions to convert hydrocarbon feedstocks to hydrogen.
This process involves use of a high-temperature (800-1000°C) and
low-pressure endothermic (heat absorbing) decomposition of water
to yield hydrogen. An overall efficiency of greater than 50% was
projected.
4.9.1 Thermolysis or Direct Thermal Decomposition of water
In a thermal solar power plant with a central collector, the
temperatures can reach over 3,000ºC. Heating water to over 2,000
ºC can break it down into hydrogen and oxygen. This is considered
to be an interesting and inexpensive method of producing hydrogen
directly from solar energy. Research is also being done on the use
of catalysts to reduce the temperature for dissociation. One central
problem is the separation of gases at high temperatures to avoid
recombining. The efficiency factor is uncertain.
5. Biological Hydrogen Production processes
At present hydrogen gas is mainly produced from fossil fuels
which will soon bee depleted. In addition hydrogen production
from fossil fuels normally requires energy intensive processes such
as thermal and chemical process [13], which are typically expensive
and often cause pollution to the environment. Therefore there is an
urgent need to develop more environmental friendly and cost
effective alternatives to hydrogen production and concerns about
global warming increased the interest in hydrogen as a fuel.
Biohydrogen production played an important role in making this
interest feasible. This production process makes use of bacterial
microorganisms that have the tendency of fermenting natural
wastes and organic compounds to hydrogen and any other non-
polluting gas. Different microorganisms participate in biological
hydrogen generation system as shown in Table 1 given below.

Table 1: Microorganisms for hydrogen generation

Broad Classification Name of the microorganisms

Scenedesmus obliquus
Chlamydomonas reinhardii
Green Algae
C.moewusii
Anabaena azollae
Anabaena CA
A.variabilis
A.cylindrical
Nostoc muscorum
Cyanobacteria Heterocystous
N.spongiaeforme
Westiellopsis prolifica
Plectonema boryanum
Oscillotoria Miami BG7
O.limnetica
Synechococcus sp.
Aphanothece halophytico
Cyanobacteria Nonheterocysous
Mastidocladus laminosus
Phormidium valderianum
Rhodobacter sphaeroides
R. capsulatus
R. sulidophilus
Rhodopsuedomonas
Sphaeroides
R. palustris
R. capsulata
Photosynthetic Bacteria
Rhodospirillium rubnum
Chromatium sp.
Miami PSB 1071
Enterobacter aerogenes
E. cloacae
Clostridium butyricum
C. pasteurianum
Desulfovibrio vulgaris
Fermentative Bacteria Magashaera elsdenii
Citrobacter intermedius
Escherichia Coli

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 European Journal of Biotechnology and Bioscience

5.1 Bio-Photolysis of water using algae and cyanobacteria hydrogen producers, utilizing organic acids such as lactic, succinic
This method uses the same processes found in plants and algal and butyric acids, or alcohols as electron donors. Since light energy
photosynthesis, but adapts them for generation of hydrogen gas is not required for water oxidation, the efficiency of light energy
instead of the carbon containing biomass. conversion to hydrogen gas by photosynthetic bacteria is in
Microalgae are primitive microscopic plants living in aqueous principle much higher than that by cyanobacteria. Hydrogen
environments. Cyanobacteria, formerly known as blue-green algae, production by photosynthetic bacteria is mediated by nitrogenase
are now recognized as bacteria since the anatomical characteristics activity, although hydrogenases may be active for both hydrogen
of their cells are prokaryotic (bacterial type). Miroalgae and production and hydrogen uptake under some conditions.The major
Cyanobacteria along with higher plants, are capable of oxygenic benefits of photosynthetic bacterium as a microbial system for
photosynthesis according to the following reaction: biological hydrogen production are as follows: (a)High theoretical
conversion yields, (b)Lack of oxygen evolving activity, which
CO2 + H2O =>HCHO + O2.
causes problem of oxygen interactivation of different biological
Photosynthesis consists of two processes: light energy conversion systems,(c)Ability to use a wide spectrum of light,(d)Ability to
to biochemical energy by a photochemical reaction, and CO2 consume organic substrates derivable from the wastes and their
reduction to organic compounds such as sugar phosphates, through potential to be used in association with waste water treatment.The
the use of this biochemical energy by Calvin-cycle enzymes. Under overall bio-chemical pathways for photo fermentation process can
certain conditions, however, instead of reducing CO2, a few groups be expressed as follows: -
of microalgae and Cyanobacteria consume biochemical energy to
produce molecular hydrogen. Hydrogenase and nitrogenase
enzymes are both capable of hydrogen production.
Gaffron and Rubin reported that a green alga, Scenedesmus,
CO can also be used for the production of hydrogen using
produced molecular hydrogen under light conditions after being
kept under anaerobic and dark conditions [15-16] studied the effect of microbial shift reaction by the photosynthetic bacteria as follows:-
oxygen on the water splitting reaction. They established that in a
direct biophotolysis reaction, electron flow from water through the CO +H2O CO2 + H2
photosystems of plant photosynthesis, to the hydrogen evolving
enzyme hydrogenase via a ferrodoxin electron carrier. Greenbaum
5.3 Fermentative Hydrogen Production from Organic
and co-workers reported very high (10 to 20%) efficiencies of light
Compounds
conversion to hydrogen, based on PAR (photosynthetically active
radiation which includes light energy of 400-700nm in Hydrogen evolution by fermentation has been shown lesser
attention whereas the hydrogen evolution by photosynthetic
wavelength). These authors recently reported what may represent a
microorganisms has been extensively studied. The evolution of
"short circuit" of photosynthesis, whereby hydrogen production and
hydrogen by fermentation has, however, several advantages over
CO2 fixation occurred by a single photosystem (photosystem II
industrial production processes such as 1.Fermentative bacteria
only) of a Chlamydomonas mutant [17] demonstrated that a
have a very high evolution rate of hydrogen 2.Fermentative
nitrogen-fixing cyanobacterium, Anabaena cylindrica, produced
bacteria can produce hydrogen constantly throughout day and night
hydrogen and oxygen gas simultaneously in an argon atmosphere
for several hours. Nitrogenase is responsible for nitrogen fixation from organic substrates.3.These bacteria have a good growth rate
for supply of microorganisms to the production systems. Therefore,
and is distributed mainly among prokaryotes, including
the fermentative evolution is more advantageous than
cyanobacteria, but does not occur in eukaryotes, under which
photochemical evolution for the mass production of hydrogen by
microalgae are classified. Molecular nitrogen is reduced to
microorganisms. Fermentative hydrogen production can be
ammonium with consumption of reducing power (e' mediated by
maximized through the effective coupling of the factors like an
ferredoxin) and ATP.However, the rate of hydrogen production
accessible and a rich source of electron and biochemical electron
was lower than typical rate for CO2 reduction. One of the major
pump and an active hydrogenase.
obstacles in obtaining sustained hydrogen photo production is the
photosynthetically generated oxygen, which irreversibly inactivates
5.4 Hybrid System using photosynthetic and fermentative
hydrogen producing systems and supports oxygen dependent
bacteria
hydrogen uptake activity. Many microalgeae in particular species
Anaerobic bacteria metabolize sugars to produce hydrogen gas and
classified as “green algae” produced hydrogen after a period of
organic acids, but are incapable of further breaking down the
anaerobic condition in dark, during which the hydrogenase enzyme
organic acids formed [18] proposed the combined use of
is activated and synthesized and small amounts of hydrogen
photosynthetic and anaerobic bacteria for the conversion of organic
production are observed, when such “anaerobically adopted” algae
acids to hydrogen. Theoretically, one mole of glucose can be
are returned to light (but still under anaerobic condition) Hydrogen
converted to 12 moles of hydrogen through the use of
production rates after increases dramatically, but cease once normal
photosynthetic bacteria capable of capturing light energy in such a
photosynthesis (oxygen evolution, CO2 fixation is reestablished.
combined system. From a practical point of view, organic wastes
frequently contain sugar or sugar polymers. It is not however easy
5.2 Photodecomposition of Organic compounds by
to obtain organic wastes containing organic acids as the main
Photosynthetic bacteria
components. The combined use of photosynthetic and anaerobic
Photosynthetic bacteria undergo anoxygenic photosynthesis with
bacteria should potentially increase the likelihood of their
organic compounds or reduced sulfur compounds as electron
application in photobiological hydrogen production.
donors. Some non-sulfur photosynthetic bacteria are potent

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 European Journal of Biotechnology and Bioscience

The first type are normally catalyzed by intra or extracellular

enzymes and act on the pool of biologically available organic
material disintegration composites (such as dead biomass) to
particulate constituents and the subsequent enzymatic hydrolysis of
these to their soluble monomers are extracellular. Organisms
mediate degradation of soluble materials intracellularly, resulting
in biomass growth and subsequent decay. Second type is not
biologically mediated and encompasses ion association/
dissociation and gas liquid transfer [21]. A simplified schematic of
overall mechanism of anaerobic digestion, [22] which involves a
multitude of microbial species, is:

Fig 2: A schematic pathway for conversion of renewables to

hydrogen via fermentation
5.4.1 Dark Fermentation Processes
Fermentation is a metabolic process aiming at regeneration of ATP, In anaerobic digestion, organic matter is completely degraded to
the metabolic energy currency, and at supplying metabolites for methane and carbon dioxide in discrete steps by concerted action of
biosynthetic processes without involvement of oxygen. Dark several different metabolic groups of microorganisms shown in
hydrogen fermentation is a ubiquitous phenomenon under anoxic (Figure. 3) [23]
or anaerobic conditions (i.e., no oxygen present as an electron
acceptor). When bacteria grow on organic substrates (heterotrophic
growth), these substrates are degraded by oxidation to provide
building blocks and metabolic energy for growth. This oxidation
process generates electrons, which need to be disposed of to
maintain neutrality. In aerobic or oxic environments, oxygen is
reduced and water is the product. In anaerobic or anoxic
environments, other compounds e.g., protons, which are reduced to
molecular hydrogen; need to act as an electron acceptor. [19] (Figure
.2) illustrates the biochemical pathway for the conversion of
renewable biomass in to hydrogen via fermentation. In the
fermentation process of glucose to hydrogen, pyruvate, the product
of glucose metabolism, is oxidized to acetyl-CoA, which can be
converted to acetyl phosphate and results in generation of ATP and
the excretion of acetate. Pyruvate oxidation to acetyl-CoA requires
ferredoxin (Fd) reduction. Reduced Fd is oxidized by hydrogenase,
which generates Fd and releases electrons as molecular hydrogen
[20]
.
Pyruvate + CoA + 2Fd (ox)  Acetyl-CoA + 2Fd (red) + CO2
2Fd (red)  2Fd (ox) + H2
Anaerobic fermentation enables the mass production of hydrogen
via relatively simple processes from a wide spectrum of potentially
utilizable substrate, including refuse and waste products. Before,
going deeply into hydrogen production from fermentation, it is
necessary to first analyze the process used for the fermentation,
namely anaerobic wastewater treatment.

5.4.2 Mechanism of Anaerobic Treatment

Anaerobic process is a complex process mediated by various
anaerobic microorganisms in a series of biochemical reactions
specifically designed for a particular purpose. The understanding of
anaerobic process is one of the important aspects and the system
can be divided into two main types:
Fig 3: schematic representations of the different metabolic steps
involved in the complete degradation of organic matter to methane
and carbon dioxide [23].

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 European Journal of Biotechnology and Bioscience
In the first step, large solid sludge material is solubilized or
dispersed by extracellular enzymes synthesized by a broad
spectrum of bacteria. Among the enzymes found in anaerobic
digesters are proteolytic, lipolytic, and several cellulolytic
enzymes. Since solids don’t build up in anaerobic digester, these
solubilization reactions apparently proceed fast enough to prevent
this step from limiting the rate of overall reaction sequence. This
step involving degradation of polymeric materials such as
polysaccharides and proteins to monomers is commonly termed as
‘Hydrolysis’. Experimental Studies of the next portion of digestion
reaction, namely bacterial synthesis of short chain fatty and volatile
acids from soluble organic material, reveal that these steps also
occur at a relatively rapid rate. The organisms responsible for these
transformations called acid formers, for obvious reasons, are
facultative anaerobic heterotrophs which function best in the range
of pH from 4 to 6.5. The acid formers are relatively tolerant to
changes in pH and temperature and grow at a more rapid rate than
methane producing bacteria. These facultative organisms can use
molecular oxygen (dissolved oxygen) during metabolism.
Therefore, these microbes protect the strictly anaerobic bacteria
from small amounts of free dissolved oxygen that maybe
introduced into digestion system with the feed sludge. These
organisms responsible for fermentation and hydrolysis (to a small
extent) include Clostridium spp., Peptococcus anaerobus,
Bifidobacterium spp., Lactobacillus, Actinomyces, Staphylococcus
and Escherichia Coli. Other physiological groups present include
those producing proteolytic, lipolytic, ureolytioc or cellulolytic
enzymes. This process by which soluble organic compounds are
fermented by acid producing facultative bacteria to volatile acids,
carbon dioxide and hydrogen gas is usually termed as
‘Acidogenesis’. During this acid production phase there is almost
no change in quantity of organic material in the system. There is,
however, redistribution among various types of simpler organic
compounds and a release of CO 2, H2 and H2S [24]. While Acetic
acid is the major product, Butyric acid and Propionic acids are also
formed. Acetic acid is the most important substrate for final
reaction of the sequence resulting in about 70% of total methane
production. Other acids commonly formed during this phase are
Formic acid, Valeric acid, Iso-Valeric acid and Caproic acid. The
propionate and butyrate are further fermented to produce H2, CO2
and acetates. These act as precursors of methane formation. The
energy change associated with the conversion of propionate and
butyrate to acetate and hydrogen requires that hydrogen is at a low
concentration in the system (almost < 10-4 atm), or the reaction will
not proceed further [21]. In the final and the most important step, the
acids formed react with hydrogen in presence of a class of bacteria
called methanogenic organisms to produce methane and carbon
dioxide. This rate-limiting step in anaerobic digestion is known as
‘Methanogenesis’. A narrower range of pH, from 7 to 7.8, is
optimal for these organisms, which, although difficult to isolate in
pure culture, thrive in a mixed culture in a properly operated
digester [22]. These microorganisms responsible for methane
production, classified as archaea, are strict obligate anaerobes.
Many of methanogenic organisms identified in anaerobic digesters
are similar to those found in stomachs of ruminant animals and in
organic sediments taken from lakes and rivers. The principal genre
of microorganisms that have been identified at mesophilic
conditions include rods (Methanobacterium, Methnaobacillus) and
spheres (Methanococcus, Methanothrix and Methanosarcina).
These methanogenic organisms are classified into two groups. One
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group, termed as aceticlastic methanogens, split the acetate into
methane and CO2. The second group, termed Hydrogen-utilizing
methanogens, use hydrogen as electron donor and CO2 as the
electron acceptor to produce methane. These organisms including
species of Methanobacterium have shown abundance at
temperatures of 60 0C. Bacteria within anaerobic process, termed
acetogens, are also able to use CO2 to oxidize hydrogen and form
acetic acid. However, acetic acid will be converted to methane, so
the impact of the reaction is minor. About 72 % of methane
produced in anaerobic digestion are from acetate formation.
Methanosarcina and Methanothrix (also termed as Methyanosaeta)
are the only organisms able to use acetate to produce methane and
CO2. The acetate-utilizing methanogens were also observed in
thermophilic reactors. Some species of Methanosarcina were
inhibited at temperature of 65 0C, while others like species of
Methanothrix did not show any inhibition .The methanogens and
acidogens form a syntrophic (mutually beneficial) relationship in
which the methanogens convert the fermentation end products such
as hydrogen, formate and acetate to produce methane and CO2.
Since the methanogens are able to maintain an extremely low
pressure of hydrogen, the equilibrium of fermentation reactions is
shifted towards the formation of more oxidized end products (e.g.
formate and acetate). The utilization of hydrogen produced by
acidogens and other anaerobes by methanogens is termed
Interspecies hydrogen transfer. In effect, methanogenic organisms
serve as a hydrogen sink that allows fermentation reactions to
proceed. If process upsets occur and the methanogenic organisms
do not utilize the hydrogen produced fast enough, the propionate
and butyrate fermentation will be slowed with accumulation of
volatile fatty acids in the anaerobic reactor and a possible reduction
of pH [21]. Once the lower pH-tolerance level of methane former is
reached methane production ceases and pH can be lowered to toxic
level of acid formers unless the system is rectified quickly. The
buffering capacity of the digester is therefore very important.
Fortunately, alkalinity of digesting sludge is naturally high because
of solubilization of CO2 production by biological processes and its
subsequent conversion to bicarbonates. A sudden reduction in
alkalinity heralds a pH drop, and more alkalinity usually in form of
lime, must be added to maintain buffering capacity.
(Tchobanoglous, Peavy and Rowe)Product gases from anaerobic
digestion typically contain 65-70 % methane, 25-30 % CO2 and
trace amounts of other gases. (Tchobanoglous, Peavy and Rowe)
.In addition, anaerobic process can break down a variety of
aromatic compounds by a process called anaerobic breakdown of
the benzene nucleus by two different pathways, photometabolism
and methanogenic formation. It has been reported that benzoate,
phenylacetage, phenylpropionate and animate are completely
degraded to CO2 and CH4. It is also reported that phenol, cresol and
resorcinol are completely converted to CH4 and CO2.
Biodegradation of refractory/inhibitory organic by microbes will
not yield any energy. If a co-substrate (such as sugar, methanol and
lactate) is added along with a refractory organic such as
chloroform, the biomass is able to metabolize. Numerous
investigators have shown that a variety of pesticides including
lindane and isomers of benzene hexachloride degrade under
anaerobic conditions [25] .Comparing the 3 different fermentation
processes, it has been observed that a combination of dark/photo
fermentation gives a much higher hydrogen yield as compared to
standalone dark or photo fermentation process.
 European Journal of Biotechnology and Bioscience

Table 2: Feed stocks For Biohydrogen Production

Feedstock Technology Key Findings References
Employed
[26]
Water and Algae Direct biophotolysis 2.152 mL of H2 for a 10-mL culture of alga
Water and Carbon [27]
Indirect biophotolysis Hydrogen production rate: 0.18 mL/mg/day
Dioxide
Water and Carbon Biological water-gas Volumetric hydrogen production: 41 mmol H2 L- [28]
Monoxide shift reaction 1h−1
Anaerobic
[29]
Dairy Manures fermentation and acid Hydrogen yield: 31.5 ml/g-total volatile solids
pretreatment
Palm Oil Mill Effluent Hydrogen yield: 3195 ml H2/L-medium Volumetric [30]
Dark fermentation
(POME) hydrogen production rate: 1034 ml H2/L-medium/h
Hydrogen yield: 1.73 mol H2/mol total sugar [31]
Sugarcane Bagasse Dark fermentation
Hydrogen production rate: 1611 ml H2/L/day
Anaerobic [32]
Corn Stover Hydrogen yield: 2.84 molar
fermentation
[33]
Sludge Dark fermentation Hydrogen yield: 2.28 mol H2/mol glucose
[34]
Glucose Dark fermentation Hydrogen yield: 1.0 mol/mol glucose
Wastewater (WW):
Hydrogen yield: 0.01 L H2/L WW Hydrogen yield: [35]
Domestic Potato Dark fermentation
1.0 L H2/L WW
Processing
Anaerobic [36]
Food Wastewater Hydrogen production rate: 97.5 mmol H2/L/d
fermentation
Hydrogen production: 178 ml Hydrogen yield: 1.23
[37]
Wheat Starch Photo fermentation mol H2 mol−1 glucose Specific hydrogen rate: 46
ml H2 g−1 biomass h−1
Dark fermentation
[38]
Cassava Starch and photo Hydrogen yield: 840 ml H2/g starch
fermentation
Dark fermentation Hydrogen yield (DF): 120.3 ± 5.2 mL H2/g-corncob
[39]
Corncob and photo Hydrogen rate (DF): 150 mL H2/(L h) Hydrogen
fermentation yield (PF): 713.6 ± 44.1 mL H2/g-COD
[40]
Dark fermentation
Olive Mill Wastewater
and photo Hydrogen yield: 29 l H2/lOMW
(OMW)
fermentation
Hydrogenyield:2.95(mmol/day)
Pharmaceutical Anaerobic
5.90 Organic loading rate (OLR)(Kg COD/cum- [41]
Wastewater fermentation
day)

6. Acknowledgement
The author gratefully acknowledge the co-faculty members of the
Department of chemistry, University of Toronto, for their moral
support to complete this manuscript.
7. Conclusion
1. The scientists have estimated that the potential effects on
climate from hydrogen-based energy systems would be much
lower than those from fossil fuel-based energy systems.
However, such impacts will depend on the rate of hydrogen
leakage during its synthesis, storage and use. The researchers
have calculated that a global hydrogen economy with a
leakage rate of 1% of the produced would produce a climate
impact of 0.6% of the fossil fuel system it replaces. If the
leakage rate was 10%, then the climate impact would be 6% of
that of the fossil fuel system. The current study suggests that a
future hydrogen-based economy would not be free from
climate disturbance, although this may be considerably less
pronounced that that caused by the current fossil fuel energy
systems. Careful attention would have to be paid to reducing
hydrogen leakage to a minimum if the potential climate
benefits of a future global hydrogen economy are to be
realised.
2. The promise of a clean and sustainable energy future lies in
technologies that capture natural energy flows as well as in
technologies that store and distribute that energy effectively.
Hydrogen pathways that are coupled with low carbon energy
sources remain among the most promising of long-term
options for providing sustainable energy with low fuel-cycle
emissions of greenhouse gases and air pollutants. Hydrogen,
fuel cell, and renewable energy technologies have developed
rapidly in recent years, and the introduction of commercial
systems is accelerating. Technological challenges remain,
particularly for hydrogen in transportation markets, but
growing needs for improved energy efficiency, greater energy
security, and reduced emissions are continuing to highlight the

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 European Journal of Biotechnology and Bioscience
need to explore promising longer-term options for these goals.
These farsighted energy R&D efforts should be complemented
with other near-term strategies to achieve more modest but
immediate steps toward societal goals. biohydrogen production
from Physico-Chemical Production has also been provided
where Steam reforming of natural gas is currently the least
expensive method of producing hydrogen, where as in
biological approaches Hybrid System have been found to be
among the best production technologies for enhanced
biohydrogen production. As for other treatment methods such
as microwave and ultrasound irradiation, these have not been
extensively studied and as such, represent potential areas of
future research.
8. Reference:
1. Kovacs KL, Kaman . Recent advances in biohydrogen
research Europe physiol 2000;439 [suppl] : r 81-r83 .
2. Khanal SK, Chen WH, Li L. and Sung S. Biological
hydrogen production: effects of pH and intermediate
products. Int. J. Hydrogen Energy2004:29: 1123-1131.
3. Bicelli LP. Hydrogen A Clean Energy Sources. Fast
Journal of hydrogen energy 1986;11:555-562.
4. Aullffe MC. The Hydrogen Economy Chem. Britain
1973; 9: 559-561
5. Gregory DP. The Hydrogen Economy. Scientific
American. 1973; 228:13-21.
6. Gibbs M Hollaender A, Kok B, Krampitz, LO, and
SanPietro A. "Proceedings of the workshop on Bio Solar
Hydrogen Conversion." September 5-6 1973, Bethesda,
MD.
7. Benemann JR, Berenson JA, Kaplan NO and Kamen MD.
" Hydrogen evolution by a chloroplast-ferredoxin-
hydrogenase sustem." Proc. Nat. Acad. Sci. USA 1973;70:
2317-2320.
8. Winter CJ and Nitsch J. Hydrogen as an Energy Carrier.
Springer-Verlag. (Applied Sciences Library, STRO) 1988.
9. Benemann JR, Miyamoto K, and Hallenbeck PC.
"Bioengineering aspects of biophotolysis "Enzyme
Microb. Technol 1980 ;2: 103-111.
10. Boichenko VA. and Hoffman P. "Photosynthetic
hydrogen-production in prokaryotes and eukaryotes:
occurrence, mechanism, and functions" Photosynthetica
1994;30: 527-552.
11. Markov SA, Bazin M, and Hall DO. The potential of
using cyanobacteria in photobioreactors for hydrogen
production Adv. Biochem. Eng./Biotechnol. 1995;52: 61-
86.
12. Benemann JR. " Hydrogen biotechnology: progress and
prospects" Nature Biotechnol. 1996;14: 1101-1103.
13. Das D,and Veziroglu TN. Hydrogen production by
biological processes:a survey of literature. Int J Hydrogen
Energy2001; 26:13 –28.
14. Winter, C..J. and Nitsch J. (1988). "Hydrogen as an
Energy Carrier." Springer-Verlag. (Applied Sciences
Library, STRO).
15. Gaffron, H. and Rubin, J. Fermentation and
Photochemical production of hydrogen in algae", J. Gen.
Physiol. 1942;26: 219-240
16. Benemann, J.R., Berenson, J.A., Kaplan, N.O.and Kamen,
M.D. 1973. ; Hydrogen evolution by a chloroplast-
~ 92 ~
ferredoxin-hydrogenase sustem.; Proc. Nat. Acad. Sci.
USA 70: 2317-2320
17. Benemann JR, Weare NM , Hydrogen evolution by
nitrogen-fixing Anabaena cylindrica cultures. Science
1974;184: 174-175 .
18. Markwell .A and Vogrinetz. Int. J. Hydrogen Energy.
1983;8: 281
19. Nandi R and Sengupta S. Microbial Production of
hydrogen: an overview. Crit Rev Microbiol 1998;24:61-
84.
20. Hallenbeck PC, Benemann JR. Biological hydrogen
production: fundamentals and limiting processes. Int. J
Hydrogen Energy 2002; 27: 1185-1193
21. Metcalf & Eddy Inc.. Wastewater Engineering: Treatment
and Reuse. 4th ed. New York: McGraw-Hill 2003.
22. Bailey JE and Ollis DF. Biochemical Engineering
Fundamentals. 2nd ed. New York. Mc-Graw-Hill 1986.
23. McInerney MJ and Bryant MP. Fuel Gas Production from
Biomass. CRC Press, Boca Raton 1981;1: 19-46.
24. Malina JF and Poland FG. Design of anaerobic processes
for treatment of industrial and municipal wastes.
Technomic Publishing Co Inc, Lanchester 1992.
25. Krishna RH, Swamy AVVS and Mohan SV.Evaluation
of kinetic parameters for hydrogen production by
anaerobic suspended growth reactor using synthetic feed
and complex Feed " International Journal of Chemistry
2011:3(2):124-139.
26. Jo JH, Lee DS, Park LM. Modeling and Optimization of
Photosynthetic Hydrogen Gas Production by Green Alga
Chlamydomonas reinhardtii in Sulfur-Deprived
Circumstance, Biotechnology Progress 2006; 22: 431-437.
27. Huesemann MH, Hausmann TS, Carter BM, Gerschler JJ,
Benemann JR. Hydrogen generation through indirect
biophotolysis in batch cultures of the nonheterocystous
nitrogen-fixing Cyanobacterium plectonema boryanum,
Applied Biochemistry and Biotechnology, 2010; 162:208-
220.
28. Oh YK, Kim YJ, Park JY, Lee TH, Kim MS, and Park S.
“Biohydrogen production from carbon monoxide and
water using Rhodopseudomonas palustris P4,
Biotechnology and Bioprocess Engineering, 2005; 10:
270-274.
29. Xing Y, Li Z, Fan Y, Hou H. Biohydrogen production
from dairy manures with acidification pre-treatment by
anaerobic fermentation, Environmental Science and
Pollution Research 2010 ; 17: 392-399.
30. Chong ML, Rahim RA, Shirai Y . MA Hassan,
Biohydrogen production by Clostridium butyricum EB6
from palm oil mill effluent,” International Journal of
Hydrogen Energy 2009 ; 34: 764-771.
31. Pattra S. Sangyoka S, Boonmee M, Reungsang A, Bio-
hydrogen production from the fermentation of sugarcane
bagasse hydrolysate by Clostridium butyricum
International Journal of Hydrogen Energy 2008 ; 33:
5256-5265.
32. Datar R, Huang J, Maness PC, Mohagheghi A, Czernik S,
and Chornet E. Hydrogen production from the
fermentation of corn stover biomass pretreated with a
steam-explosion process, International Journal of
Hydrogen Energy, vol. 2007; 32 : 932-939.
European Journal of Biotechnology and Bioscience

33. Kotay SM, Das D. Microbial hydrogen production with

Bacillus coagulans IIT-BT S1 isolated from anaerobic
sewage sludge. Bioresource Technology 2007: 98; 1183-
1190,
34. Minnan L, Jinli H, Xiaobin W, Huijuan X, Jinzao C ,
Chuannan L, Fengzhang Z, and Liangshu X, Isolation and
characterization of a high H2 producing strain Klebsiella
oxytoca HP1 from a hot spring, Research in Microbiology
2005;156:76-81.
35. Van Ginkel SW, Oh SE, Logan BE, Biohydrogen gas
production from food processing anddomestic
Wastewaters International Journal of Hydrogen
Energy2010 ; 30: 1535- 1542.
36. Wu JH, Lin CY. Biohydrogen production by mesophilic
fermentation of food wastewater, Water Science and
Technology 2004 ; 49: 223-228.
37. Kapdan IK, Kargi F, Oztekin R, Argun H. Bio-hydrogen
production from acid hydrolyzed wheat starch by photo-
fermentation using Rhodobacter sp, International Journal
of Hydrogen Energy2009; 34:2201-2207.
38. Cheng J, Su H, Zhou J, Song W, Cen K. Hydrogen
production by mixed bacteria through dark and photo
fermentation, International Journal of Hydrogen
Energy2011 ; 36: 450-457.
39. Yang H, Guo L, Lie F. Enhanced bio-hydrogen production
from corncob by a two-step process: Dark- and photo-
fermentation, Bioresource Technology, 2010; 101: 2049-
2052.
40. Eroğlu E, Eroğlu I, Gündüz U, Türker L, Yücel M.
Biological hydrogen production from olive mill
wastewater with two-stage processes, International Journal
of Hydrogen Energy, 2006; 31: 1527-1535.
41. Krishna RH, Swamy AVVS and Mohan SV. Bio
Hydrogen Production from Pharmaceutical Waste Water
Treatment by a Suspended Growth Reactor Using
Environmental Anaerobic Technology, American
Chemical Science Journal 2013; 3 (2): 80-97.

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