Developments in Hydrogen Production through Microbial Processes

June 2011, Volume 2, No.3
International Journal of Chemical and Environmental Engineering

Developments in Hydrogen Production through
Microbial Processes; Pakistan’s Prospective
Abdul Waheed Bhutto1, †, *, Aqeel Ahmed Bazmi2,3, Muhammad Nadeem Kardar2 and Muhammad Yaseen2,
Gholamreza Zahedi3 and Sadia Karim1Department of Chemical Engineering, Dawood College of Engineering
and Technology, M.A.Jinnah Road, Karachi-Pakistan
2
Biomass Conversion Research Centre (BCRC), Department of Chemical Engineering,COMSATS Institute of
Information Technology, Defence Road, Off Raiwind Road, Lahore-Pakistan.
3
Process Systems Engineering Centre (PROSPECT), Chemical Engineering Department, Faculty of Chemical
Engineering, Universiti Teknologi Malaysia, Skudai 81310, Johor Bahru (JB), Malaysia.
†
Affiliated member BCRC
*
Corresponding Author Email: abdulwaheed27@hotmail.com

Abstract
Currently, hydrogen (H2) is primarily used in the chemical industry as a reactant, but it is being proposed as future fuel. H2 has great
potential as an environmentally clean energy fuel and as a way to reduce reliance on imported energy sources. A combination of the
need to cut carbon dioxide emissions, the prospect of increasingly expensive oil and the estimated growth in the world's vehicle fleet
indicates that only H2 can plug the gap. There are many processes for H2 production. The key issue to make H2 an attractive
alternative fuel is to optimize its production from renewable raw materials instead of the more common energy intensive processes
such as natural gas reforming or electrolysis of water. With such vision, this paper reviews developments in microbial processes for H2
production followed by a road map to H2 economy in Pakistan. The H2 economy potentially offers the possibility to deliver a range of
benefits for the country; however, significant challenges exist and these are unlikely to be overcome without serious efforts.
Keywords: At least five

1. Introduction

At the start of the 21st century, we face significant being used worldwide. Electricity is a convenient form of
energy challenges. The concept of sustainable energy, which can be produced from various sources and
development is evolved for a livable future where human transported over large distances. Hydrogen is another
needs are met while keeping the balance with nature. clean energy source as well as energy carrier. H2
Driving the global energy system into a sustainable path economy has often been proposed by researchers as
is progressively becoming a major concern and policy another clean, efficient and versatile renewable energy
objective. sources as well as energy carrier [1-3], but the
At the present, world’s energy requirement is by large transformation from the present fossil fuel economy to a
being fulfilled by fossil fuels which serve as a primary H2 economy will need the solution of numerous complex
energy source. Fossil fuel has delivered energy and scientific and technological issues. The provision of cost
convenience, in our homes, for transport and industry. competitive hydrogen in sufficient quantity and quality is
However, the overwhelming scientific evidence is that the the groundwork of a hydrogen energy economy. Presently
unfettered use of fossil fuels is causing the world’s H2 is not an alternative fuel but only an energy carrier
climate to change, with potential disastrous effect on our produced from H2-rich compounds. H2 holds the promise
planet. The dramatic increase in the price of petroleum as a dream fuel of the future with many social, economic
are also forcing for the search for new energy sources and and environmental benefits to its credit. It has the long-
alternative ways. World is in search of convenient, clean, term potential to reduce the dependence on foreign oil and
safe, efficient and versatile energy source as well as lower the carbon and criteria emissions from the
energy carrier that can be delivered to the end user. transportation sector as depicted in Table 1.
Electricity is one of the energy carriers which is already

Developments in Hydrogen Production through Microbial Processes; Pakistan’s Prospective

Table 1. Comparison of energy and emissions of combustible common energy intensive processes Water splitting by
fuels [4]
artificial photosynthesis, photobiological methods based
Fuel type Energy Energy Kg of
per unit per carbon on algae, and high temperatures obtained by nuclear or
mass volume release per concentrated solar power plants are promising approaches
(MJ/kg) (MJ/l) kg of fuel
(approx.) used [5]. The H2 economy is an inevitable energy system of the
H2 gas 120 2 0 future where the renewable sources will be used to
H2 liquid 120 8.5 0 generate H2 and electricity as energy carriers, which are
Coal 15–19
—
0.5 capable of satisfying all the energy needs of human
(anthracite)
Coal (sub- 27–30 0.7
civilization. However nearly all H2 produced today for the
—
bituminous) industrial sector, is largely by thermal processes with
Natural gas 33–50 9 0.46 natural gas as the H2 feedstock. Thus the development of
Petrol 40–43 31.5 0.86
Oil 42–45 38 0.84
alternative and renewable pathways for producing H2
Diesel 42.8 35 0.9 fuels is of utmost importance. The purpose of this paper is
Bio-diesel 37 33 0.5 to provide a brief summary of significant current and
Ethanol 21 23 0.5 developing biological H2 production technologies. A
Charcoal 30 — 0.5
Agricultural vision for H2 economy in Pakistan is also discussed.
10–17 — 0.5
residue
Wood 15 — 0.5 2. Industrial Applications of Hydrogen
Approximately 49% of hydrogen produced is used for
H2 has some unique characteristics which make it the manufacture of ammonia, 37% for petroleum refining,
suitable for H2 economy, namely: H2 is one of the most 8% for methanol production and about 6% for
plentiful elements on Earth and in the Cosmos miscellaneous smaller-volume uses [6]. It is also used in
Combustion of molecular H2 with oxygen produces heat. the petrochemical manufacturing, glass purification,
H2 has the highest energy content per unit weight of any semiconductor industry and for the hydrogenation of
known fuel (142 KJ /g or 61,000 Btu/lb) H2 can be unsaturated fats in vegetable oil [7]. In metallurgical
produced from and converted into electricity at a processes, hydrogen mixed with N2, is used for heat
relatively high efficiency. The only byproduct is water, treating applications to remove O2 as O2 scavenger. The
while burning of fossil fuels generates CO2 and a variety future widespread use of hydrogen is likely to be in the
of pollutants. H2 may be completely renewable fuel It can transportation sector, where it will help reduce pollution.
be stored as liquid, gas It can be transported over large Vehicles can be powered with hydrogen fuel cells, which
distances using pipelines, tankers, or rail trucks. It can be are three times more efficient than a gasoline-powered
converted into other forms of energy in more ways and engine [8, 9].
more efficiently than any other fuel, i.e., in addition to
3. Current Hydrogen Production
flame combustion (like any other fuel) H2 may be
converted through catalytic combustion, electro-chemical Worldwide, H2 is being considered as a fuel for the
conversion, and hydriding. future. It is an environmentally benign replacement for
Some vehicle manufacturers have already gasoline, diesel, heating oil, natural gas, and other fuels in
demonstrated that H2 can be used directly in an internal both the transportation and non-transportation sectors.
combustion engine, and fuel cell-powered prototype cars Although abundant on earth as an element, H2 combines
have also been constructed. H2 can be transported for readily with other elements and is almost always found as
domestic/industrial consumption through conventional part of some other substances, such as water, biomass and
means. hydrocarbons like petroleum and natural Gas. Currently
Production of H2 from petroleum product or natural 500 billion cubic meters H2 are produced annually
gas does not offer any advantage over the direct use of worldwide. Presently, 40 % H2 is produced from natural
such fuels while Production from coal by gasification gas, 30 % from heavy oils and naphtha, 18 % from coal,
techniques with capture and sequestration of CO2 could and 4 % from electrolysis and about 1 % is produced from
be an interim solution [5]. The key issue to make H2 an biomass [8, 10] Currently, the most developed and most
attractive alternative fuel particularly for the used technology is the reforming of natural gas/
transportation sector is to optimize the production process hydrocarbon fuels [11]. Each method of H2 production
from renewable raw materials instead of the more requires a source of energy, i.e., thermal or electrolytic.

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The merits and demerits of the biomass processes are All microbial conversions can be carried out at ambient
discussed in Table 2. conditions, however lower rate of H2 production and low
yield are chief drawbacks. All processes are controlled by
Table 2. Advantages and disadvantages of different H2 the hydrogen-producing enzymes, such as hydrogenase
production processes from biomass [7, 12]
and nitrogenase. Hydrogenases exist in most of the
Process Advantages Disadvantages
photosynthetic microorganisms and they can be classified
Thermochemical (i)Maximum (i)Significant gas
gasification conversion can be conditioning is into two categories: (i) uptake hydrogenases and (ii)
achieved required reversible hydrogenases. Uptake hydrogenases, such as
(ii)Removal of tar
NiFe hydrogenases and NiFeSe hydro genases, act as
Pyrolysis (i)Produces (i)Chances of
important catalysts for hydrogen consumption. Reversible
carbonaceous catalyst deactivation
material along hydrogenases, as indicated by its name, have the ability to
with bio-oil, produce H2 as well as consume hydrogen depending on
(ii)chemicals and the reaction condition. The major components of
minerals
nitrogenase are MoFe protein and Fe protein. Nitrogenase
Solar gasification (i)Good H2 yield (i)Required
effective collector
has the ability to use magnesium adenosine triphosphate
plates (MgATP) and electrons to reduce a variety of substrates
Supercritical (i)Can process (i)Selection of (including protons). This chemical reaction yields
conversion sewage sludge, supercritical hydrogen production by a nitrogenase-based system
which is difficult medium
to gasify
where ADP and Pi refer to adenosine diphosphate and
inorganic phosphate, respectively
2e- + 2H+ + 4ATP ---->H2 + 4ADP + 4Pi
4. Biological H2 production processes The processes of biological H2 production can be
Producing H2 using conventional methods defeats the broadly classified into following distinct approaches for
purpose of using H2 as a clean alternative fuel. The include: 1) Direct biophotolysis 2) Indirect biophotolysis
production of H2 from non-fossil fuel sources has 3) Photofermentation 4) Dark fermentation 5) Microbial
becomes central for better transition to H2 economy. fuel cell (MFC) (bioelectrohydrogenesis )
Certain microorganisms can produce enzymes that can 4.1. Direct Biophotolysis
produce H2 provides an attractive option to produce
hydrogen through microbial process. A large number of The process of photosynthetic H2-production with
microbial species, including significantly different electrons derived from H2O [18, 22] entails H2O-
taxonomic and physiological types, can produce H2. oxidation and a light-dependent transfer of electrons to
Diversity in microbial physiology and metabolism means the [Fe]-hydrogenase, leading to the synthesis of
that there are a variety of different ways in which molecular H2. The concerted action of the two
microorganisms can produce H2, each one with seeming photosystems of plant-type photosynthesis to split water
advantages, as well as problematic issues [13]. From an with absorbed photons and generate reduced ferredoxin to
engineering perspective, they all potentially offer the drive the reduction of protons to hydrogen, is carried out
advantages of lower cost catalysts (microbial cells) and by some green algae and some cyanobacteria as shown in
less energy intensive reactor operation (mesophilic) than (Fig. 1). The two photosynthetic systems responsible for
the present industrial process for making hydrogen (steam photosynthesis process are: (i) photo system I (PSI) which
reformation of methane) [14]. produces reductant for CO2 and (ii) photo system II (PSII)
The H2 metabolism of green algae was first discovered which splits water to evolve O2. The two photons
in the early 1940s by Hans Gaffron. He observed that obtained from the splitting of water can either reduce CO2
green algae (under anaerobic conditions) can either use by PSI or form H2 in the presence of hydrogenase. In
H2 as an electron donor in the CO2-fixation process or plants, due to the lack of hydrogenase, only CO2
evolve H2 in both dark and the light [15-17]. Although the reduction takes place. On the contrary, green algae and
physiological significance of H2 metabolism in algae is cyanobacteria (blue-green algae) contain hydrogenase and
still a matter of basic research, the process of thus have the ability to produce H2 [23]. In these
photohydrogen production by green algae is of interest organisms, electrons are generated when PSII absorbs
because it generates H2 gas from the most plentiful light energy, which is then transferred to ferredoxin. A
resources, light and water [18-21]. reversible hydrogenase accepts electrons directly from the

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reduced ferredoxin to generate H2 in the presence of Even though photosynthetic hydrogen production is a
hydrogenase. theoretically perfect process with transforming solar
energy into hydrogen by photosynthetic bacteria, applying
it to practice is difficult due to the low utilization
efficiency of light and difficulties in designing the
reactors for hydrogen production [26-28].

4.2. Photofermentation
Photofermentation also requires input of light energy
for hydrogen production from various substrates, in
particular organic acids, by photosynthetic bacteria (Fig
.2). Photosynthetic bacteria have long been studied for
their capacity to produce hydrogen through the action of
their nitrogenase system. Fermentative hydrogen
production has the advantages of rapid hydrogen
production rate and simple operation. Photosynthetic
bacteria have long been studied for their capacity to
produce significant amounts of hydrogen due to their high
substrate conversion efficiencies and ability to degrade a
wide range of substrates.
The photosynthetic bacteria have been shown to
produce hydrogen from various organic acids and food
processing and agricultural wastes [13]. Although pure
substrates have usually been used in model studies, some
success in using industrial wastewater as substrate has
been shown [29, 30]. In general, rates of hydrogen
production by photoheterotrophic bacteria are higher
when the cells are immobilized in or on a solid matrix,
than when the cell is free-living.
However, pre-treatment may be needed prior to
photosynthetic biohydrogen gas production due to either
Figure-1. Direct Biophotolysis (green algae – cyanobacteria) [14] the toxic nature of the effluent, or its color/ opaqueness.

Since hydrogenase is sensitive to oxygen, it is
necessary to maintain the oxygen content at a low level
(under 0.1 %) so that the hydrogen production can be
sustained [13]. This process results in the simultaneous
production of O2 and H2 with a H2: O2 = 2:1 ratio [24].
This mechanism holds the promise of generating
hydrogen continuously and efficiently through the solar
conversion ability of the photosynthetic apparatus. In the
absence of provision for the active removal of oxygen,
this mechanism can operate only transiently, as molecular
oxygen is a powerful inhibitor of the enzymatic reaction
and a positive suppressor of [Fe]-hydrogenase gene Figure-2. Photofermentation (Photosynthetic bacteria) [14]
expression. At present, this direct mechanism has
4.3. Dark fermentation
limitations as a tool of further research and for practical
application, mainly due to the great sensitivity of the [Fe]- In dark fermentation, H2 production is inherently more
hydrogenase to O2, which is evolved upon illumination by stable since it takes place in the absence of oxygen. The
the water-oxidizing reactions of PSII [25]. Nevertheless, oxidation of the substrate by bacteria generates electrons
such H2 co-production can be prolonged under conditions which need to be disposed off in order to maintain the
designed to actively remove O2 from the reaction mixture. electrical neutrality. Under the aerobic conditions O2
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serves as the electron acceptor while under the anaerobic
or anoxic conditions other compounds, such as protons,
act as the electron acceptor and are reduced to molecular
H2.
Hydrogen can be produced by anaerobic bacteria,
grown in the dark on carbohydrate-rich substrates.
While direct and indirect photolysis systems produce
pure H2, dark-fermentation processes produce a mixed
biogas containing primarily H2 and carbon dioxide (CO2),
but which may also contain lesser amounts of methane
(CH4), CO, and/or hydrogen sulfide (H2S). The gas
composition presents technical challenges with respect to
using the biogas in fuel cells. In order for hydrogen
production by dark fermentation to be economically
feasible and sustainable, a two-step/hybrid biological
hydrogen production process would be necessary.
Higher overall substrate conversion efficiency is
possible by combining the anaerobic and photosynthetic
steps, as shown in Fig. 3. The photosynthetic microbes
Figure 3. Dark fermentation (Clostridia, Enterobacteracae) [14]
can degrade the soluble metabolites from the fermentative
step using sunlight to overcome the energy barrier.
Dark fermentation reactions can be operated at 4.4. Microbial fuel cell (MFC)
mesophilic (25 –40°C), thermophilic (40–65°C), extreme
thermophilic (65–80°C), or hyperthermophilic (80°C) It is based on the concept and practice of a microbial
temperatures. fuel cell (MFC). Fact the idea is to add a little electrical
Biohydrogen production by dark fermentation is potential to that generated by a microbial fuel cell, thus
highly dependent on the process conditions such as reaching a sufficient force to reduce protons to hydrogen,
temperature, pH, mineral medium formulation, type of in a process that can be called bioelectrohydrogenesis. A
organic acids produced, hydraulic residence time (HRT), MEC consists of four parts: first, the anodic chamber with
type of substrate and concentration, hydrogen partial the anode; second, the cathodic chamber with cathode;
pressure, and reactor configuration [31]. third, an external electrical power source; and fourth, an
electronic separator [32, 33] as shown in Fig. 4.Thus the
Since organic substrates are the ultimate source of cell could be called a microbial electrohydrogenesis cell
hydrogen in photofermentations or indirect biophotolysis (MEC). Acetate is typically used as the electron donor
processes, it can be argued that it should be simpler and and it is oxidized according to the following reaction [34]:
more efficient to extract the hydrogen from such Acetate - + 4H2O  2HCO3- + 9H+ +9e-
substrates using a dark fermentation process [13]. The pH at the anode surface has a strong tendency to
decrease, as one proton is produced per electro transferred
[35, 36]. At the cathode the hydrogen evolution reaction
takes place, in which protons and electrons are combined
to form hydrogen:
2H2 + 2e  H2
The reaction can be catalyzed by microorganisms or
by a chemical catalyst like platinum or nickel. When
microorganisms are used as catalyst these reactions are
essentially anaerobic respirations where the external
electron acceptor is an electrode instead of the more usual
oxidized compound (nitrate, TMAO, fumurate, etc.). Thus
bioelectrohydrogenesis utilizes electrochemically active
micro-organisms which, with a small to moderate voltage

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input, convert dissolved organic matter into hydrogen
inside an electrochemical cell/microbial fuel cell via
coupled anode-cathode reactions. Expressed per amount
of organic matter, the MEC can achieve much higher
hydrogen yields (80–100%) [37] compared to
fermentative hydrogen production (<33%). This is
because the MEC uses electricity to overcome the
energetic barrier for acetate oxidation.

Figure 5. Indirect biophotolysis [13]

One elaboration of this concept [37] involved four distinct
steps:
1. Production in open ponds at 10% solar efficiency of a
biomass high in storage carbohydrates.
2. Concentration of the biomass from the ponds in a
settling pond.
3. Anaerobic dark fermentation to yield 4H2 /glucose
stored in the algal cells, plus 2 acetates.
4. A photobioreactor in which the algal cells would
Figure-4. Layout of the microbial electrolysis cell.
convert the two acetates to 8 mol of H2.
Microorganisms present on the anode catalyze the oxidation of
substrate to bicarbonate, protons and electrons. The production of After this last step the algal biomass would be returned to
hydrogen on the cathode may be catalyzed by a chemical catalyst or the ponds, to repeat the cycle. Support systems
by microorganisms (biocathode) [32] included the anaerobic digestion (methane
fermentation) of any wasted biomass (assumed at 10%
The performance of a MEC is determined on the one for each cycle), an inoculum production system to
hand by the physiology of the microorganisms, and on the
provide make-up biomass and a gas handling and
other hand by the physical chemical transport processes
involved. There remains a great challenge to reduce the separation system (to recycle the CO2 from the H2
overpotential at both the bioanode and biocathode [32]. A back to the ponds) [13].
typical application of a MEC would be wastewater Genetic modifcation of strains to eliminate uptake
treatment, in which the organic compounds in the hydrogenases and increase levels of bidirectional
wastewater serve as electron donors for the bioanode [38, hydrogenase activity may yield signi6cant increases in
39]. MEC could also produce hydrogen from H2 production.
agroindustrial residues containing biopolymers like
cellulose and starch. 4.6. Two-stage System
4.5. Indirect Biophotolysis Photosynthetic O2 formation and H2 evolution occur
Indirect biophotolysis processes involve separation of simultaneously in green algae as electrons and protons
the H2 and O2 evolution reactions into separate stages released from photosynthetic H2O oxidation are used in
(Fig. 5), coupled through CO2 fixation/evolution. Indirect the hydrogenase catalysed H2 evolution [30, 40]. In this
biophotolysis, consists of two stages in series: one-stage process, H2 evolution is transient and cannot be
photosynthesis for carbohydrate accumulation, and dark sustained due to strong deactivation of hydrogenase
fermentation of the carbon reserve for hydrogen activity by O2 (at as low as 2% partial pressure) evolved
production. In this way, the oxygen and hydrogen from photosynthesis [41]. This mutually exclusive nature
evolutions are temporally and/or spatially separated. This of the O2 and H2 photoproduction reactions has halted the
separation not only avoids the incompatibility of oxygen development of H2 production process by green algae
and hydrogen evolution (e.g., enzyme deactivation and under ambient conditions [41].
the explosive property of the gas mixture), but also makes To overcome this problem, a two-stage protocol has
hydrogen purification relatively easy because CO2 can be been developed to evolve H2 from green algae, in which
conveniently removed from the H2/CO2 mixture. photosynthetic O2 evolution and carbon accumulation
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(stage 1) are temporally separated from the consumption Cells from Reactor 1 are transferred to Reactor 2,
of cellular metabolites and concomitant H2 production which is maintained under anaerobic conditions. Cells
(stage 2) [18, 42]. H2 evolution strongly depended upon entering Reactor 2 already have suppressed PS-II
the duration of anaerobic incubation, deprivation of systems, so they will not cause Reactor 2 to go aerobic.
sulphur (S) from the medium and the medium pH [43]. Any residual oxygen is quickly consumed by the algae in
It has been reported that inhibition of the hydrogenase Reactor 2. Finding themselves under anaerobic
by oxygen can be partially overcome by cultivation of conditions, the cells will start producing hydrogenase and
algae under sulfur deprivation for 2–3 days to provide subsequently, H2. The transition step that consumes the
anaerobic conditions under the light [26, 44]. Melis et al. oxygen in solution in the batch system is avoided by
[42] and Ghirardi et al. [25] devised a mechanism to having Reactor 2 already anaerobic. At the same time,
partially inactivate PSII activity to a point where all the some cells are continuously removed from Reactor 2. The
O2 evolved by photosynthesis is immediately taken up by effect is that the cells are removed from Reactor 2 before
the respiratory activity of the culture. This mechanism is they completely stop producing H2. Successful operation
based on a two-step process. The steps, growth mode and has been shown with a dilution rate of 0.5/day, which is
H2 production mode, are initiated by cycling between equivalent to an average residence time of 2 days for the
sulfur-containing and sulfur-free culture medium. This cells. Because Reactor 2 is a continuously-stirred reactor
results in a temporal separation of net O2- and H2- (like Reactor 1), the average residence time is 2 days, but
evolution activities in the green alga Chlamydomonas some individual cells removed from the reactor may have
reinhardtii. This discovery eliminates the need for a purge been there longer or shorter times. With an average
gas, but introduces the need for careful sulfate controls in residence time of 2 days, one would expect a H2
the aqueous medium. production rate lower than the initial production rate of
The absence of sulfur nutrients from the growth the batch system, but higher than the production rate at
medium of algae acts as a metabolic switch, one that the end of a batch production cycle.
selectively and reversibly inhibits photosynthetic O2
production. Thus, in the presence of S, green algae do
normal photosynthesis (H2O-oxidation, O2-evolution and
biomass accumulation) [45].
In 2002, NREL researchers developed a system using
two continuous-flow reactors for producing H2
continuously for periods of up to several weeks [46]. The
continuous H2 production process involves using two
continuously-stirred tanks. Fig.6 shows the tank
configuration. In Reactor 1, cells are cultured in media
containing minimal levels of sulfur. PS-II is slowed and
oxygen production remains lower than oxygen
consumption for cellular respiration, but by bubbling the Figure 6. Continuous H2 Production

solutions with carbon dioxide and a small amount of
oxygen, the cells are able to remain in Reactor 1 The merits and demerits of each biological process are
indefinitely, obtaining some energy from photosynthesis discussed in Table 3.
and some energy through respiration of acetate in
solution.

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Table 3. Comparison of important biological H2 production processes [12, 47]
Process General reaction Advantages Disadvantages Maximum Reference
reported rate
(mmol H2 /L h)

Direct 2 H2O + light → 2 H2 + O2 -Can produce H2 directly -Requires high intensity of light 0.07 [48, 49]
biophotolysis from water and sunlight -Simultaneous production of O2
-Solar conversion energy and H2. O2 can be dangerous for
increased by ten folds as the system
compared to trees, crops -Hydrogenase (green algae) is
highly sensitive to even
moderately low concentrations of
O2
-Lower photochemical efficiency
Indirect (a) 6H2O + 6CO2 + light → -Cyanobacteria can -Uptake hydrogenase enzymes are 0.36 [50, 51]
biophotolysis C6H12O6 + 6O2 produce H2 from water to be removed to stop degradation
(b) C6H12O6 + 2H2O→ 4H2 + -Has the ability to fix N2 of H2
2CH3COOH + 2CO2 from atmosphere -About 30% O2 present in gas
(c) 2CH3COOH + 4H2O + mixture
light → 8 H2 + 4CO2
Overall reaction
12H2O + light → 12 H2 + 6O2
Photo- CH3COOH + 2H2O + light → -A wide spectral light -Production rate of H2 0.16 [52]
fermentation 4H2 + 2CO2 energy can be used by is slow
these bacteria -O2 has an inhibitory effect on
-Can use different organic nitrogenase
wastes -Light conversion efficiency is
-High substrate very low, only 1–5%
conversion efficiencies -Pre-treatment may be needed due
-Degrade a wide range of to either the toxic nature of the
substrates. substrate (effluent), or its
color/opaqueness.
-Large reactor surface areas
requirement -Expensive equipment
Dark C6H12O6 + 2H2O → -Simpler, less expensive, -O2 is a strong inhibitor of 75.60 [53, 54]
Fermentation 2CH3COOH + 4H2 + 2CO2 and produce hydrogen at hydrogenase
much higher rate -Relatively lower achievable yields 64.50
-It can produce H2 all day of H2
long without light -As yields increase H2
-A variety of carbon fermentation becomes
sources can be used as thermodynamically unfavorable
substrates -Product gas mixture contains CO2
-It produces valuable which has to be separated
metabolites such as
butyric, lactic and acetic
acids as by products
-It is anaerobic process,
so there is no O2
limitation problem
Microbial C6H12O6 + 2H2O → 4H2 + -Energy available in -Metabolic pathways involved are
fuel cell 2CO2 + 2CH3COOH waste streams can be not clear
(MFC) directly recovered as -MEC studies have been carried
Anode: CH3COOH + 2H2O → electricity (MFC) or out only with mixed cultures, often
2CO2 + 8e- + 8H+ (15) hydrogen (MEC). using those already enriched and
Cathode: 8H+ + 8e- → 4H2 promising future active in microbial fuel cells
approach to hydrogen (MFC).
generation from -Power densities at the electrode
wastewater, especially for surface are low, which translates
effluents with low organic into low volumetric hydrogen
content. production.
-Higher yields require increased
voltage, adversely affecting energy
efficiency.
Two-stage 51.20 [15, 55]
fermentation
(dark + 47.92
photo)

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Other barriers to microbial based, large-scale
5. Barriers for biohydrogen production
production of H2 include [57] [Maness el al 2009] (a)
The diffuse nature of solar energy and the consequent inherent properties of the microbes that preclude
low energy density places severe economic restrictions on continuity and efficiency of H2 production; (b) underlying
potential light-driven processes for biological conversion limitations of photosynthetic efficiency; and (c)
of solar energy to hydrogen [13]. limitations of the hydrogenase catalytic function.
Major challenges need to be overcome for the smooth Scientific and technical barriers for biohydrogen
transition from the fossil fuel based economy to the H2 production have been summarized in Table IV.
energy based economy and may be outlined as follows
[56]: 6. Immobilization
 The yield of H2 from any of the processes defined One of the largest challenges of optimizing molecular
above is low for commercial application. H2 production by Chlamydomonas reinhardtii cells is the
 The pathways of H2 production have not been transfer of the cells from sulfur deficient conditions to
identified and the reaction remains energetically sulfur rich conditions (for regenerative purposes) and then
unfavorable. back to sulfur deficient conditions (for further H2
 There is no clear contender for a robust, industrially production). Recent research in immobilization has
capable microorganism that can be metabolically provided a new technique to eliminate this challenge.
engineered to produce more than 4 mol H2/mol of Prior to the development of immobilizations, cells were
glucose. suspended in aqueous media with either sulfur rich or
Several engineering issues need to be addressed which deficient conditions present. This posed a problem for
include the appropriate bioreactor design for H2 scientists because the cells had to be filtered out of the
production, difficult to sustain steady continuous H2 media to be transferred to the next media in the cycle of
production rate in the long term, scale-up, preventing molecular H2 production. The filtration process was very
interspecies H2 transfer in non sterile conditions and time consuming and so was not feasible on an industrial
separation/purification of H2. scale. Another dilemma that plagued the free suspension
Sensitivity of hydrogenase to O2 and H2 partial pressure in liquid media technique was the inability to make the
severely disrupts the efficiency of the processes and adds media with cells very concentrated. This restricted the
to the problems of lower yields. Insufficient knowledge amount of light that could interact with the cells
on the metabolism of H2 producing bacteria and the levels decreasing the overall yield of molecular H2. To avoid
of H2 concentration tolerance of these bacteria. difficulties with media transition or cellular concentration
immobilization techniques were developed [58].

Table 4. Scientific and technical barriers for biohydrogen production [7]
Type of barrier Barrier Putative Solution
Bacteria do not produce more than 4 Isolate more novel microbes and combinational
mol H2/mol glucose naturally screen for H2 production rates yields, and
Organism durability. Genetic manipulation of established
Basic science bacteria.
Hydrogenase over expression not Greater understanding of the enzyme regulation
Enzyme stable and expression.
(hydrogenase) O2 sensitivity Mutagenic studies.
H2 feed back inhibition Low H2 partial pressure fermentation.
High cost of suitable feedstock Renewable biomass as feedstock.
Feedstock (glucose) Co-digestion/use of microbial consortia which can
Fermentative Low yield using renewable biomass increase the yield
Lack of industrial-suitable strain Development of industrially viable
Strain strain(s)/consortia

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Commercially feasible product yield Hybrid system (photo + dark fermentation)
Incomplete substrate utilization Link fermentation to a second process that makes
Sustainable process both economically possible
Process Sterilization Application and utilization of fermentation tools
such as continuous culture
Development of low-cost stream sterilization
technology/process that can bypass sterilization

Engineering Lack of kinetics/appropriate reactor Incorporation of process engineering concepts to
design for H2 production Light develop a suitable reactor for the defined
Reactor
intensity in case of photo-bioreactor strain/process, flat panel or hollow tube reactor
can be employed
Thermodynamic barrier Reverse electron transport to drive H2 production
Thermodynamic NAD(P)H → H2 (+4.62 kJ/mol) past barrier
Selection absorption of CO2 /H2S
H2 H2 purification/separation Storage
Basic studies on H2 storage

7. Maximum possible yield of H2 by green algae Application of the two-stage photosynthesis and H2
production protocol to a green alga mass culture could
Even though the catalytic activity of the various provide a commercially viable method of renewable H2
enzymes differs enormously, there is no evidence for the generation.
quantity of hydrogen-producing enzyme being the Table 5 provides preliminary estimates of maximum
limiting factor. Indeed, in many microbial systems, possible yield of H2 by green algae, based on the
potential catalytic activity far surpasses the amount of luminosity of the sun and the green algal photosynthesis
hydrogen produced, suggesting that other metabolic characteristics. Calculations were based on the integrated
factors are limiting [13]. luminosity of the sun during a cloudless spring day. In
The use of light attenuation devices that transfer mid-latitudes at springtime, this would entail delivery of
sunlight into the depths of a dense algal culture is an approximately 50 mol photons m 2 d 1 (Table 5). It is
approach to overcoming the light saturation effect in light generally accepted that electron transport by the two
driven processes. The simplest approach is to arrange photosystems and via the hydrogenase pathway for the
photobioreactors in vertical arrays to reduce direct production of 1 mol H2 requires the absorption and
sunlight. Of course, this arrangement also proportionally utilization of a minimum of 5 mol photons in the
increases the area of required photobioreactors, which is photosynthetic apparatus (Table 5). On the basis of these
the limiting economic factor in any photobiological fuel- “optimal” assumptions, it can be calculated that green
production process. algae could produce a maximum 10 mol (20 g) H2 per m2
Another alternative is the use of optical fiber culture area per day. If yields of such magnitude could be
photobioreactors, in which light energy is collected by approached in mass culture, this would constitute a viable
large concentrating mirrors and piped into small and profitable method of renewable H2 production.
photobioreactors with optical 1bers [13].

Table 5. Yield of H2 photoproduction by green algae (Estimates are based on maximum possible daily integrated irradiance and algal
photosynthesis characteristics.) [20, 59-61]
Photoproduction Characteristics Comments on Assumptions Made

Maximum photosynthetically active radiation, 50 mol photons m 2 Daily irradiance can vary significantly depending on season and cloud
d 1 (based on a Gaussian solar intensity profile in which the peak cover. It can be greater than 50 mol photons m 2 d 1 in the summer and
solar irradiance reaches 2,200 µmol photons m 2 s 1) much less than that on cloudy days and in the winter. [29].
Theoretical minimum photon requirement for H2 production in green Based on the requirement of 10 photons for the oxidation of two water
algae: 5 mol photons/mol H2 molecules and the release of four electrons and four protons in
photosynthesis [30, 31]

Theoretical maximum yield of H2 production by green algae: 10 mol Assuming that all incoming photosynthetically active radiation will be
H2 m 2 d 1 (20 g H2 m 2 d 1; ~80 kg H2 acre 1 d 1) absorbed by the green algae in the culture and that it will be converted into
stable charge separation.

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8. Optical properties of light absorption by Photosynthetic H2 production by green algae involves
green algae water splitting to produce H2 and oxygen. Unfortunately,
Light absorption by the photosynthetic apparatus is H2 production by this process is quite ineffective since it
essential for the generation of H2 gas. However the simultaneously produces oxygen, which inhibits the
optical properties of light absorption by green algae hydrogenase enzyme. Thus, during light reaction, H2
impose a limitation in terms of solar conversion evolution ceases due to an accumulation of oxygen.
efficiency in the algae chloroplast. This is because wild- Therefore the prerequisite for photohydrogen production
type green algae are equipped with a large size light- by green algae is that they have to adapt to an anaerobic
harvesting chlorophyll antenna to absorb as much sunlight condition.
as they can. Under direct and bright sunlight, they could By exposing the cells to specific conditions scientists
waste up to 60% of the absorbed irradiance [47, 62]. This are able to modify photosynthesis so that oxygen will not
evolutionary trait may be good for survival of the act as the final electron carrier of the electron transport
organism in the wild, where light is often limiting, but it chain; rather H2 will allow the cells to release molecular
is not good for the photosynthetic productivity of a green H2 as opposed to molecular oxygen.
algal mass culture. This optical property of the cells could Melis [45] estimates that, if the entire capacity of the
further lower the productivity of a commercial H2 photosynthesis of the algae could be directed toward H2
production farm. production, 80 kilograms of H2 could be produced
The analysis up to this point has shown that H2 commercially per acre per day. The yield of H2
production can be limited by the photons available or the production currently achieved in the laboratory
capacity of algae to process the photons into H2. Another corresponds to only 15 to 20% of the measured capacity
observation is that the number of photons absorbed is of the photosynthetic apparatus for electron transport
much higher than the algae’s ability to process the [63].
photons. By reducing the number of excess photons In a laboratory, Melis [45] worked with low-density
absorbed and let them reach deeper into the liquid, it may cultures and have thin bottles so that light penetrates from
be possible to produce more H2. By reducing the size of all sides. Because of this, the cells use all the light falling
the algae’s light collecting antennae, but not affecting the on them. But in a commercial bioreactor, where dense
organism’s ability to process the photons to produce H2, algae cultures would be spread out in open ponds under
one gets deeper light penetration for the same cell the sun, the top layers of algae absorb all the sunlight but
concentration, which means more photons are available at can only use a fraction of it [63].
the lower depths for H2 production. Further research and development aimed at increasing
While regular green algae absorb most of the light rates of synthesis and final l yields of H2 are essential.
falling on them, algae engineered to have less chlorophyll Optimization of bioreactor designs, rapid removal and
let some light left through. In University of California, puri6cation of gases, and genetic modifcation of enzyme
Berkeley, Melis and his colleagues are designing algae pathways that compete with hydrogen producing enzyme
that have less chlorophyll so that they absorb less sunlight systems offer exciting prospects for biohydrogen systems
[63]. When grown in large, open bioreactors in dense [48]. Increase in the rate of H2 would reduce bioreactor
cultures, the chlorophyll-deficient algae will let sunlight size dramatically to overcome the engineering challenges
penetrate to the deeper algae layers and thereby utilize of scale up, and create new opportunities for practical
sunlight more efficiently [64]. applications.
The critical enzymatic component of this 9. H2 Economy
photosynthetic reaction is the reversible hydrogenase
A typical energy chain for sustainable H2 comprises
enzyme, which reduces protons with high potential
the harvesting of sunlight into H2 as energy carrier, the
energy electrons to form H2. During normal
storage and distribution of this energy carrier to the end-
photosynthesis, algae focus on using the sun’s energy to
device where it is converted to power. The key market for
convert carbon dioxide and water into glucose, releasing
fuel cells has always assumed to be the automotive
oxygen in the process. Only about 3 to 5 percent of
industry. The great expectation that hydrogen fuel-cell
photosynthesis leads to H2. Because hydrogenase is
powered vehicles will displace gasoline and diesel
sensitive to oxygen, this H2 production must be carried
powered vehicles has not materialized for a variety of
out in an anaerobic environment
reasons, but primarily because fuel cell technology has

200


not yet matured and the infrastructure required for cells connected directly to wind turbines are a convenient
hydrogen storage, transportation, and refueling has been way to balance out local fluctuations in the availability of
slow to develop. Consumer energy applications will wind power. The development of fuel cells and a H2
require delivery systems that can supply H2 as readily as economy will provide new market opportunities and new
gasoline and natural gas are supplied today. Higher- jobs. Present knowledge indicates that H2 as an energy
pressure gaseous storage and non-conventional storage carrier will involve little environmental risk. All
technologies will be used to meet the requirements of renewable hydrogen production technologies face the
transportation applications (storage at 350–700 bar common challenge of integration with hydrogen
compared to the 200 bar storage pressure commonly used purification and storage [65].
in normal merchant gas systems) [65].
10. Present energy scenario of Pakistan
Gas purity requirements are important for the H2
energy market. They very much depend on the energy Pakistan is basically an energy deficient country.
conversion device used, as well as on the storage Pakistan’s per capita energy consumption, 3894kWh as
technology. Combustion systems are much less sensitive against the world average of 17620kWh, gives it a
to impurity levels, however, fuel cells are very sensitive ranking of 100 amongst the nations of the world [70]. The
to CO and sulfur poisoning. demand for primary energy in Pakistan has increased
The U.S. Department of Energy has developed a considerably over the last few decades and the country is
multiyear plan with aggressive milestones and targets for facing serious energy shortage problems. The energy
the development of H2 infrastructure, fuel cells, and supply is not increasing by any means to cope with the
storage technologies. The targeted H2 cost is $2–4 kg-1 rising energy demands. As a result the gap between the
(energy equivalent of 1 gallon of gasoline) delivered [66, energy demand and supply is growing every year. The
67] country is meeting about 86% of oil demand from imports
A rollout of such a sustainable H2 chain in developed by spending around US$6.65 billion per annum [71].
countries could go either gradually via a H2 economy Pakistan’s future energy system looks rather
based on fossil fuels or discontinuously in the case of uncertain. In recent years, the combination of rising oil
inventions of disruptive technologies. For developing consumption and flat oil production in Pakistan has led to
countries the situation may be different. Introduction of rising oil imports from Middle East exporters. The
such H2 chains for their fast-growing primary energy balance recoverable reserves of crude oil in the country as
demands might enable them to skip the stage of on January 1st 2010 have been estimated at 303.63
conventional, fossil fuel-based technologies and markets million barrels [72].
and leapfrog directly to a sustainable H2 economy [68]. Natural gas accounts for the largest share of
The salient features of a H2 economy will be as follows Pakistan’s energy use, amounting to nearly 43.7 percent
[69]: of total energy consumption. As on January 1, 2010, the
A H2-based energy system will increase the balance recoverable natural gas reserves have been
opportunity to use renewable energy in the transport estimated at 28.33 trillion cubic feet. The average
sector. This will increase the diversity of energy sources production of natural gas during July- March 2009-10
and reduce overall greenhouse gas emissions. H2 in the was 4,048.76 million cubic feet per day (mmcfd) [72]. As
transport sector can reduce local pollution, which is a the demand of natural gas exceeds the supply, country is
high priority in many large cities. already facing shortage of natural gas and during the peak
The robustness and flexibility of the energy system demand most of the gas fired generating units are
will be increased by the introduction of H2 as a strong shutdown while duel fuel units are fired by oil. Pakistan is
new energy carrier that can interconnect different parts of presently facing shortage of around 300-350 MMCDF of
the energy system. The targets for reducing vehicle noise natural gas which is likely to go up because of rising
may be met by replacing conventional engines with H2- needs and slowing down of supplies at home [73].
powered fuel cells. Fuel cells for battery replacement and According to The Energy Security Action Plan of the
backup power systems are niche markets in which price Planning Commission, Pakistan will be facing a shortfall
and efficiency are relatively unimportant. Sales in this in gas supplies rising from 1.4 Billion Cubic Feet (BCF)
market will drive the technology forward towards the per day in 2012 to 2.7 BCF in 2015 and escalating to 10.3
point at which fuel cells will become economic for the BCF per day by the year 2025 [74]. It is therefore a matter
introduction into the energy sector. H2 electrolysers/fuel

201


of economic security to develop alternative H2 resources economy, and will have a positive impact on the
to avoid mid century energy crises in the country. environment in which atmospheric pollution is all but
Natural gas is used in general industry to prepare alleviated and the so-called greenhouse effect is
consumer items, produce cement, fertilizer and generate mitigated.
electricity. At present, the power sector is the largest user To ensure a sustainable energy future for Pakistan, it
of gas accounting for 33.5 percent share followed by the is necessary that the energy sector be accorded a high
industrial sector (23.8 percent), household (18.1 percent), priority. In Pakistan efforts to reduce reliance on fossil
fertilizer (15.6 percent), transport (5.4 percent) and fuels through increasing the share of renewable energy in
cement (0.9 percent) [75]. Natural gas is used in the the energy supply systems have met with little success so
transport sector in the form of CNG. There are about far. Mirza et al. [77] and Sahir and Qureshi [78] have
3,116 established CNG stations in the country and discussed the barriers to development of renewable
approximately 2 million vehicles are using CNG. Pakistan energy. Mirza, et al. [77] has broadly classified these
has become the largest CNG consuming country among barriers as policy and regulatory barriers, institutional
Natural Gas Vehicle (NGV) countries. According to barriers, fiscal and financial barriers, market-related
Petroleum Policy 1997; the use of CNG in vehicles was barriers, technological barriers and information and social
encouraged by Government to reduce pressure on barriers. They have also suggested better coordination
petroleum imports, to curb pollution and to improve the among various stakeholders and indigenization of
environment [75]. renewable energy technologies to overcome these
Transport sector is one of the major consumers of barriers.
commercial energy in Pakistan. It accounted for about Sahir, and Qureshi [78] has suggested an integrated
28% of the total final commercial energy consumed energy planning approach, consistency in government
(33.95 MTOE) and 55.8% of the total petroleum products policies and rational policy instruments to deal with the
consumed (15 MTOE) in the country. techno-economic and socio-political barriers are the pre-
requisites for long-term sustainable development of the
11. H2 Production in Pakistan
renewable energy technologies.
In Pakistan H2 is largely produced in the fertilizer There is little doubt that power production by
industry from natural gas, which is used for the renewable energies, energy storage by H2, and electric
production of anhydrous ammonia. All urea plants in the power transportation and distribution by smart electric
country are based on natural gas as feedstock. On an grids will play an essential role in phasing out fossil fuels.
average, the fertilizer sector consumes 15.6 per cent of
natural gas produced in country. The government 12. Conclusions
provides an indirect subsidy to fertilizer manufacturers by Concerns about global warming and environmental
selling feedstock gas at rates ranging up to $1.0 against pollution due to the use of fossil fuels, combined with
commercial rates of $4.0 per MMBTU. The return on projections of potential fossil fuel shortfall toward the
paid-up capital in the fertilizer industry is about 80-100 middle of the 21st century, make it imperative to develop
per cent per annum [73]. The current energy scenario in alternative energy sources that would clean, renewable,
the country, already discussed above , identifies the and environmentally friendly.
transport sector and fertilizer sector as key sectors where It is important to note that hydrogen can be produced
the H2 gas can be immediately employed as substitute to from a wide variety of feed stocks available almost
fossil fuel. anywhere. There are many processes under development
Mirza et al. [76] has presented complete road map to which will have a minimal environmental impact.
H2 economy in Pakistan. They have concluded that the H2 Development of these technologies may decrease the
economy potentially offers the possibility to deliver a world’s dependence on fuels that come primarily from
range of benefits for the country including reducing unstable regions. The ‘‘in house’’ hydrogen production
dependence on oil imports, environmental sustainability may increase both national energy and economic security.
and economic competitiveness. In medium term advent of The ability of hydrogen to be produced from a wide
H2 will bring about technological developments in many variety of feedstocks and using a wide variety of
fields, including power generation, agriculture, the processes makes it so that every region of the world may
automotive industry, and other as yet unforeseen be able to produce much of their own energy. It is clear
applications. It will increase employment, stimulate the

202


that as the technologies develop and mature, hydrogen Non-incorporation of renewable energy issues in the
may prove to be the most ubiquitous fuel available. regulatory policy and lack of awareness among regulators
The vision for a H2 future is one based on clean restrict technology penetration. There is a lack of
sustainable renewable energy supply of global financial resources and proper lending facilities,
proportions that plays a key role in all sectors of the particularly for small-scale projects in country. In
economy. addition, the absence of a central body for overall
Microbial Processes provides an attractive option to coordination of energy sector activities results in
produce H2 at ambient conditions. A large number of duplication of R&D activities. Unfortunately private
microbial species, including significantly different sector especially transports and fertilizer sector has made
taxonomic and physiological types, can produce H2, no contributions to promote research activities to produce
Diversity in microbial physiology and metabolism means H2 from renewable resources.
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