June 2011, Volume 2, No.

3 International Journal of Chemical and Environmental Engineering

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

Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective

Table 1. Comparison of energy and emissions of combustible fuels [4] Fuel type Energy Energy Kg of per unit per carbon mass volume release per (MJ/kg) (MJ/l) kg of fuel (approx.) used H2 gas 120 2 0 H2 liquid Coal (anthracite) Coal (subbituminous) Natural gas Petrol Oil Diesel Bio-diesel Ethanol Charcoal Agricultural residue Wood 120 1519 2730 3350 4043 4245 42.8 37 21 30 1017 15 8.5 9 31.5 38 35 33 23 0 0.5 0.7 0.46 0.86 0.84 0.9 0.5 0.5 0.5 0.5 0.5

common energy intensive processes Water splitting by artificial photosynthesis, photobiological methods based on algae, and high temperatures obtained by nuclear or concentrated solar power plants are promising approaches [5]. The H2 economy is an inevitable energy system of the future where the renewable sources will be used to generate H2 and electricity as energy carriers, which are capable of satisfying all the energy needs of human civilization. However nearly all H2 produced today for the industrial sector, is largely by thermal processes with natural gas as the H2 feedstock. Thus the development of alternative and renewable pathways for producing H2 fuels is of utmost importance. The purpose of this paper is to provide a brief summary of significant current and developing biological H2 production technologies. A vision for H2 economy in Pakistan is also discussed.

2. Industrial Applications of Hydrogen

Approximately 49% of hydrogen produced is used for the manufacture of ammonia, 37% for petroleum refining, 8% for methanol production and about 6% for miscellaneous smaller-volume uses [6]. It is also used in the petrochemical manufacturing, glass purification, semiconductor industry and for the hydrogenation of unsaturated fats in vegetable oil [7]. In metallurgical processes, hydrogen mixed with N2, is used for heat treating applications to remove O2 as O2 scavenger. The future widespread use of hydrogen is likely to be in the transportation sector, where it will help reduce pollution. Vehicles can be powered with hydrogen fuel cells, which are three times more efficient than a gasoline-powered engine [8, 9].

H2 has some unique characteristics which make it suitable for H2 economy, namely: H2 is one of the most plentiful elements on Earth and in the Cosmos Combustion of molecular H2 with oxygen produces heat. H2 has the highest energy content per unit weight of any known fuel (142 KJ /g or 61,000 Btu/lb) H2 can be produced from and converted into electricity at a relatively high efficiency. The only byproduct is water, while burning of fossil fuels generates CO2 and a variety of pollutants. H2 may be completely renewable fuel It can be stored as liquid, gas It can be transported over large distances using pipelines, tankers, or rail trucks. It can be converted into other forms of energy in more ways and more efficiently than any other fuel, i.e., in addition to flame combustion (like any other fuel) H2 may be converted through catalytic combustion, electro-chemical conversion, and hydriding. Some vehicle manufacturers have already demonstrated that H2 can be used directly in an internal combustion engine, and fuel cell-powered prototype cars have also been constructed. H2 can be transported for domestic/industrial consumption through conventional means. Production of H2 from petroleum product or natural gas does not offer any advantage over the direct use of such fuels while Production from coal by gasification techniques with capture and sequestration of CO2 could be an interim solution [5]. The key issue to make H2 an attractive alternative fuel particularly for the transportation sector is to optimize the production process from renewable raw materials instead of the more

3. Current Hydrogen Production

Worldwide, H2 is being considered as a fuel for the future. It is an environmentally benign replacement for gasoline, diesel, heating oil, natural gas, and other fuels in both the transportation and non-transportation sectors. Although abundant on earth as an element, H2 combines readily with other elements and is almost always found as part of some other substances, such as water, biomass and hydrocarbons like petroleum and natural Gas. Currently 500 billion cubic meters H2 are produced annually worldwide. Presently, 40 % H2 is produced from natural gas, 30 % from heavy oils and naphtha, 18 % from coal, and 4 % from electrolysis and about 1 % is produced from biomass [8, 10] Currently, the most developed and most used technology is the reforming of natural gas/ hydrocarbon fuels [11]. Each method of H2 production requires a source of energy, i.e., thermal or electrolytic.

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective The merits and demerits of the biomass processes are discussed in Table 2.
Table 2. Advantages and disadvantages of different H2 production processes from biomass [7, 12] Process Advantages Disadvantages Thermochemical (i)Maximum (i)Significant gas gasification conversion can be conditioning is achieved required (ii)Removal of tar Pyrolysis (i)Produces carbonaceous material along with bio-oil, (ii)chemicals and minerals (i)Good H2 yield (i)Chances of catalyst deactivation

Solar gasification

Supercritical conversion

(i)Can process sewage sludge, which is difficult to gasify

(i)Required effective collector plates (i)Selection of supercritical medium

4. Biological H2 production processes

Producing H2 using conventional methods defeats the purpose of using H2 as a clean alternative fuel. The production of H2 from non-fossil fuel sources has becomes central for better transition to H2 economy. Certain microorganisms can produce enzymes that can produce H2 provides an attractive option to produce hydrogen through microbial process. A large number of microbial species, including significantly different taxonomic and physiological types, can produce H2. Diversity in microbial physiology and metabolism means that there are a variety of different ways in which microorganisms can produce H2, each one with seeming advantages, as well as problematic issues [13]. From an engineering perspective, they all potentially offer the advantages of lower cost catalysts (microbial cells) and less energy intensive reactor operation (mesophilic) than the present industrial process for making hydrogen (steam reformation of methane) [14]. The H2 metabolism of green algae was first discovered in the early 1940s by Hans Gaffron. He observed that green algae (under anaerobic conditions) can either use H2 as an electron donor in the CO2-fixation process or evolve H2 in both dark and the light [15-17]. Although the physiological significance of H2 metabolism in algae is still a matter of basic research, the process of photohydrogen production by green algae is of interest because it generates H2 gas from the most plentiful resources, light and water [18-21].

All microbial conversions can be carried out at ambient conditions, however lower rate of H2 production and low yield are chief drawbacks. All processes are controlled by the hydrogen-producing enzymes, such as hydrogenase and nitrogenase. Hydrogenases exist in most of the photosynthetic microorganisms and they can be classified into two categories: (i) uptake hydrogenases and (ii) reversible hydrogenases. Uptake hydrogenases, such as NiFe hydrogenases and NiFeSe hydro genases, act as important catalysts for hydrogen consumption. Reversible hydrogenases, as indicated by its name, have the ability to produce H2 as well as consume hydrogen depending on the reaction condition. The major components of nitrogenase are MoFe protein and Fe protein. Nitrogenase has the ability to use magnesium adenosine triphosphate (MgATP) and electrons to reduce a variety of substrates (including protons). This chemical reaction yields hydrogen production by a nitrogenase-based system where ADP and Pi refer to adenosine diphosphate and inorganic phosphate, respectively 2e- + 2H+ + 4ATP ---->H2 + 4ADP + 4Pi The processes of biological H2 production can be broadly classified into following distinct approaches for include: 1) Direct biophotolysis 2) Indirect biophotolysis 3) Photofermentation 4) Dark fermentation 5) Microbial fuel cell (MFC) (bioelectrohydrogenesis )

4.1. Direct Biophotolysis

The process of photosynthetic H2-production with electrons derived from H2O [18, 22] entails H2Ooxidation and a light-dependent transfer of electrons to the [Fe]-hydrogenase, leading to the synthesis of molecular H2. The concerted action of the two photosystems of plant-type photosynthesis to split water with absorbed photons and generate reduced ferredoxin to drive the reduction of protons to hydrogen, is carried out by some green algae and some cyanobacteria as shown in (Fig. 1). The two photosynthetic systems responsible for photosynthesis process are: (i) photo system I (PSI) which produces reductant for CO2 and (ii) photo system II (PSII) which splits water to evolve O2. The two photons obtained from the splitting of water can either reduce CO2 by PSI or form H2 in the presence of hydrogenase. In plants, due to the lack of hydrogenase, only CO2 reduction takes place. On the contrary, green algae and cyanobacteria (blue-green algae) contain hydrogenase and thus have the ability to produce H2 [23]. In these organisms, electrons are generated when PSII absorbs light energy, which is then transferred to ferredoxin. A reversible hydrogenase accepts electrons directly from the

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective reduced ferredoxin to generate H2 in the presence of hydrogenase. Even though photosynthetic hydrogen production is a 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 the toxic nature of the effluent, or its color/ opaqueness.

Figure-1. Direct Biophotolysis (green algae cyanobacteria) [14]

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 expression. At present, this direct mechanism has limitations as a tool of further research and for practical application, mainly due to the great sensitivity of the [Fe]hydrogenase to O2, which is evolved upon illumination by the water-oxidizing reactions of PSII [25]. Nevertheless, such H2 co-production can be prolonged under conditions designed to actively remove O2 from the reaction mixture.

Figure-2. Photofermentation (Photosynthetic bacteria) [14]

4.3. Dark fermentation

In dark fermentation, H2 production is inherently more stable since it takes place in the absence of oxygen. The oxidation of the substrate by bacteria generates electrons which need to be disposed off in order to maintain the electrical neutrality. Under the aerobic conditions O2

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective 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 can degrade the soluble metabolites from the fermentative step using sunlight to overcome the energy barrier. Dark fermentation reactions can be operated at mesophilic (25 40C), thermophilic (4065C), extreme thermophilic (6580C), or hyperthermophilic (80C) temperatures. Biohydrogen production by dark fermentation is highly dependent on the process conditions such as temperature, pH, mineral medium formulation, type of organic acids produced, hydraulic residence time (HRT), type of substrate and concentration, hydrogen partial pressure, and reactor configuration [31]. Since organic substrates are the ultimate source of hydrogen in photofermentations or indirect biophotolysis processes, it can be argued that it should be simpler and more efficient to extract the hydrogen from such substrates using a dark fermentation process [13].

Figure 3. Dark fermentation (Clostridia, Enterobacteracae) [14]

4.4. Microbial fuel cell (MFC)

It is based on the concept and practice of a microbial fuel cell (MFC). Fact the idea is to add a little electrical potential to that generated by a microbial fuel cell, thus reaching a sufficient force to reduce protons to hydrogen, in a process that can be called bioelectrohydrogenesis. A MEC consists of four parts: first, the anodic chamber with the anode; second, the cathodic chamber with cathode; third, an external electrical power source; and fourth, an electronic separator [32, 33] as shown in Fig. 4.Thus the cell could be called a microbial electrohydrogenesis cell (MEC). Acetate is typically used as the electron donor and it is oxidized according to the following reaction [34]: Acetate - + 4H2O 2HCO3- + 9H+ +9eThe 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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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective 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 (80100%) [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]

Figure-4. Layout of the microbial electrolysis cell. Microorganisms present on the anode catalyze the oxidation of substrate to bicarbonate, protons and electrons. The production of hydrogen on the cathode may be catalyzed by a chemical catalyst or by microorganisms (biocathode) [32]

The performance of a MEC is determined on the one hand by the physiology of the microorganisms, and on the other hand by the physical chemical transport processes involved. There remains a great challenge to reduce the overpotential at both the bioanode and biocathode [32]. A typical application of a MEC would be wastewater treatment, in which the organic compounds in the wastewater serve as electron donors for the bioanode [38, 39]. MEC could also produce hydrogen from agroindustrial residues containing biopolymers like cellulose and starch.

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 convert the two acetates to 8 mol of H2. After this last step the algal biomass would be returned to the ponds, to repeat the cycle. Support systems included the anaerobic digestion (methane fermentation) of any wasted biomass (assumed at 10% for each cycle), an inoculum production system to provide make-up biomass and a gas handling and separation system (to recycle the CO2 from the H2 back to the ponds) [13]. Genetic modifcation of strains to eliminate uptake hydrogenases and increase levels of bidirectional hydrogenase activity may yield signi6cant increases in H2 production.

4.6. Two-stage System

Photosynthetic O2 formation and H2 evolution occur simultaneously in green algae as electrons and protons released from photosynthetic H2O oxidation are used in the hydrogenase catalysed H2 evolution [30, 40]. In this one-stage process, H2 evolution is transient and cannot be sustained due to strong deactivation of hydrogenase activity by O2 (at as low as 2% partial pressure) evolved from photosynthesis [41]. This mutually exclusive nature of the O2 and H2 photoproduction reactions has halted the development of H2 production process by green algae under ambient conditions [41]. To overcome this problem, a two-stage protocol has been developed to evolve H2 from green algae, in which photosynthetic O2 evolution and carbon accumulation

4.5. Indirect Biophotolysis

Indirect biophotolysis processes involve separation of the H2 and O2 evolution reactions into separate stages (Fig. 5), coupled through CO2 fixation/evolution. Indirect biophotolysis, consists of two stages in series: photosynthesis for carbohydrate accumulation, and dark fermentation of the carbon reserve for hydrogen production. In this way, the oxygen and hydrogen evolutions are temporally and/or spatially separated. This separation not only avoids the incompatibility of oxygen and hydrogen evolution (e.g., enzyme deactivation and the explosive property of the gas mixture), but also makes hydrogen purification relatively easy because CO2 can be conveniently removed from the H2/CO2 mixture.

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective (stage 1) are temporally separated from the consumption of cellular metabolites and concomitant H2 production (stage 2) [18, 42]. H2 evolution strongly depended upon the duration of anaerobic incubation, deprivation of sulphur (S) from the medium and the medium pH [43]. It has been reported that inhibition of the hydrogenase by oxygen can be partially overcome by cultivation of algae under sulfur deprivation for 23 days to provide anaerobic conditions under the light [26, 44]. Melis et al. [42] and Ghirardi et al. [25] devised a mechanism to partially inactivate PSII activity to a point where all the O2 evolved by photosynthesis is immediately taken up by the respiratory activity of the culture. This mechanism is based on a two-step process. The steps, growth mode and H2 production mode, are initiated by cycling between sulfur-containing and sulfur-free culture medium. This results in a temporal separation of net O2- and H2evolution activities in the green alga Chlamydomonas reinhardtii. This discovery eliminates the need for a purge gas, but introduces the need for careful sulfate controls in the aqueous medium. The absence of sulfur nutrients from the growth medium of algae acts as a metabolic switch, one that 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 solutions with carbon dioxide and a small amount of oxygen, the cells are able to remain in Reactor 1 indefinitely, obtaining some energy from photosynthesis and some energy through respiration of acetate in solution. Cells from Reactor 1 are transferred to Reactor 2, which is maintained under anaerobic conditions. Cells entering Reactor 2 already have suppressed PS-II systems, so they will not cause Reactor 2 to go aerobic. Any residual oxygen is quickly consumed by the algae in Reactor 2. Finding themselves under anaerobic conditions, the cells will start producing hydrogenase and subsequently, H2. The transition step that consumes the oxygen in solution in the batch system is avoided by having Reactor 2 already anaerobic. At the same time, some cells are continuously removed from Reactor 2. The effect is that the cells are removed from Reactor 2 before they completely stop producing H2. Successful operation has been shown with a dilution rate of 0.5/day, which is equivalent to an average residence time of 2 days for the cells. Because Reactor 2 is a continuously-stirred reactor (like Reactor 1), the average residence time is 2 days, but some individual cells removed from the reactor may have been there longer or shorter times. With an average residence time of 2 days, one would expect a H2 production rate lower than the initial production rate of the batch system, but higher than the production rate at the end of a batch production cycle.

Figure 6. Continuous H2 Production

The merits and demerits of each biological process are discussed in Table 3.

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Table 3. Comparison of important biological H2 production processes [12, 47] Advantages Disadvantages Maximum reported rate (mmol H2 /L h) -Can produce H2 directly from water and sunlight -Solar conversion energy increased by ten folds as compared to trees, crops -Requires high intensity of light -Simultaneous production of O2 and H2. O2 can be dangerous for the system -Hydrogenase (green algae) is highly sensitive to even moderately low concentrations of O2 -Lower photochemical efficiency -Uptake hydrogenase enzymes are to be removed to stop degradation of H2 -About 30% O2 present in gas mixture 0.07

Process

General reaction

Reference

Direct biophotolysis

2 H2O + light 2 H2 + O2

[48, 49]

Indirect biophotolysis

Photofermentation

(a) 6H2O + 6CO2 + light C6H12O6 + 6O2 (b) C6H12O6 + 2H2O 4H2 + 2CH3COOH + 2CO2 (c) 2CH3COOH + 4H2O + light 8 H2 + 4CO2 Overall reaction 12H2O + light 12 H2 + 6O2 CH3COOH + 2H2O + light 4H2 + 2CO2

-Cyanobacteria can produce H2 from water -Has the ability to fix N2 from atmosphere

0.36

[50, 51]

-A wide spectral light energy can be used by these bacteria -Can use different organic wastes -High substrate conversion efficiencies -Degrade a wide range of substrates.

Dark Fermentation

C6H12O6 + 2H2O 2CH3COOH + 4H2 + 2CO2

Microbial fuel cell (MFC)

C6H12O6 + 2H2O 4H2 + 2CO2 + 2CH3COOH Anode: CH3COOH + 2H2O 2CO2 + 8e- + 8H+ (15) Cathode: 8H+ + 8e- 4H2

-Simpler, less expensive, and produce hydrogen at much higher rate -It can produce H2 all day long without light -A variety of carbon sources can be used as substrates -It produces valuable metabolites such as butyric, lactic and acetic acids as by products -It is anaerobic process, so there is no O2 limitation problem -Energy available in waste streams can be directly recovered as electricity (MFC) or hydrogen (MEC). promising future approach to hydrogen generation from wastewater, especially for effluents with low organic content.

-Production rate of H2 is slow -O2 has an inhibitory effect on nitrogenase -Light conversion efficiency is very low, only 15% -Pre-treatment may be needed due to either the toxic nature of the substrate (effluent), or its color/opaqueness. -Large reactor surface areas requirement -Expensive equipment -O2 is a strong inhibitor of hydrogenase -Relatively lower achievable yields of H2 -As yields increase H2 fermentation becomes thermodynamically unfavorable -Product gas mixture contains CO2 which has to be separated

0.16

[52]

75.60 64.50

[53, 54]

-Metabolic pathways involved are not clear -MEC studies have been carried out only with mixed cultures, often using those already enriched and active in microbial fuel cells (MFC). -Power densities at the electrode surface are low, which translates into low volumetric hydrogen production. -Higher yields require increased voltage, adversely affecting energy efficiency. 51.20 47.92 [15, 55]

Two-stage fermentation (dark + photo)

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5. Barriers for biohydrogen production

The diffuse nature of solar energy and the consequent low energy density places severe economic restrictions on potential light-driven processes for biological conversion of solar energy to hydrogen [13]. Major challenges need to be overcome for the smooth transition from the fossil fuel based economy to the H2 energy based economy and may be outlined as follows [56]: The yield of H2 from any of the processes defined above is low for commercial application. The pathways of H2 production have not been identified and the reaction remains energetically unfavorable. There is no clear contender for a robust, industrially capable microorganism that can be metabolically engineered to produce more than 4 mol H2/mol of glucose. Several engineering issues need to be addressed which include the appropriate bioreactor design for H2 production, difficult to sustain steady continuous H2 production rate in the long term, scale-up, preventing interspecies H2 transfer in non sterile conditions and separation/purification of H2. Sensitivity of hydrogenase to O2 and H2 partial pressure severely disrupts the efficiency of the processes and adds to the problems of lower yields. Insufficient knowledge on the metabolism of H2 producing bacteria and the levels of H2 concentration tolerance of these bacteria.

Other barriers to microbial based, large-scale production of H2 include [57] [Maness el al 2009] (a) inherent properties of the microbes that preclude continuity and efficiency of H2 production; (b) underlying limitations of photosynthetic efficiency; and (c) limitations of the hydrogenase catalytic function. Scientific and technical barriers for biohydrogen production have been summarized in Table IV.

6. Immobilization
One of the largest challenges of optimizing molecular H2 production by Chlamydomonas reinhardtii cells is the transfer of the cells from sulfur deficient conditions to sulfur rich conditions (for regenerative purposes) and then back to sulfur deficient conditions (for further H2 production). Recent research in immobilization has provided a new technique to eliminate this challenge. Prior to the development of immobilizations, cells were suspended in aqueous media with either sulfur rich or deficient conditions present. This posed a problem for scientists because the cells had to be filtered out of the media to be transferred to the next media in the cycle of molecular H2 production. The filtration process was very time consuming and so was not feasible on an industrial scale. Another dilemma that plagued the free suspension in liquid media technique was the inability to make the media with cells very concentrated. This restricted the amount of light that could interact with the cells decreasing the overall yield of molecular H2. To avoid 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 Bacteria do not produce more than 4 mol H2/mol glucose naturally Putative Solution Isolate more novel microbes and combinational screen for H2 production rates yields, and durability. Genetic manipulation of established bacteria. Greater understanding of the enzyme regulation and expression. Mutagenic studies. Low H2 partial pressure fermentation. Renewable biomass as feedstock. Co-digestion/use of microbial consortia which can increase the yield Development of industrially viable strain(s)/consortia

Organism Basic science Enzyme (hydrogenase)

Hydrogenase over expression not stable O2 sensitivity H2 feed back inhibition High cost of suitable feedstock (glucose) Low yield using renewable biomass Lack of industrial-suitable strain

Fermentative

Feedstock Strain

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective

Commercially feasible product yield Incomplete substrate utilization Sustainable process Sterilization Hybrid system (photo + dark fermentation) Link fermentation to a second process that makes both economically possible Application and utilization of fermentation tools such as continuous culture Development of low-cost stream sterilization technology/process that can bypass sterilization Incorporation of process engineering concepts to develop a suitable reactor for the defined strain/process, flat panel or hollow tube reactor can be employed Reverse electron transport to drive H2 production past barrier Selection absorption of CO2 /H2S Basic studies on H2 storage

Process

Engineering Reactor

Lack of kinetics/appropriate reactor design for H2 production Light intensity in case of photo-bioreactor Thermodynamic barrier NAD(P)H H2 (+4.62 kJ/mol) H2 purification/separation Storage

Thermodynamic H2

7. Maximum possible yield of H2 by green algae

Even though the catalytic activity of the various enzymes differs enormously, there is no evidence for the quantity of hydrogen-producing enzyme being the limiting factor. Indeed, in many microbial systems, potential catalytic activity far surpasses the amount of hydrogen produced, suggesting that other metabolic factors are limiting [13]. The use of light attenuation devices that transfer sunlight into the depths of a dense algal culture is an approach to overcoming the light saturation effect in light driven processes. The simplest approach is to arrange photobioreactors in vertical arrays to reduce direct sunlight. Of course, this arrangement also proportionally increases the area of required photobioreactors, which is the limiting economic factor in any photobiological fuelproduction process. Another alternative is the use of optical fiber photobioreactors, in which light energy is collected by large concentrating mirrors and piped into small photobioreactors with optical 1bers [13].

Application of the two-stage photosynthesis and H2 production protocol to a green alga mass culture could provide a commercially viable method of renewable H2 generation. Table 5 provides preliminary estimates of maximum possible yield of H2 by green algae, based on the luminosity of the sun and the green algal photosynthesis characteristics. Calculations were based on the integrated luminosity of the sun during a cloudless spring day. In mid-latitudes at springtime, this would entail delivery of approximately 50 mol photons m 2 d 1 (Table 5). It is generally accepted that electron transport by the two photosystems and via the hydrogenase pathway for the production of 1 mol H2 requires the absorption and utilization of a minimum of 5 mol photons in the photosynthetic apparatus (Table 5). On the basis of these optimal assumptions, it can be calculated that green algae could produce a maximum 10 mol (20 g) H2 per m2 culture area per day. If yields of such magnitude could be approached in mass culture, this would constitute a viable and profitable method of renewable H2 production.

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 d 1 (based on a Gaussian solar intensity profile in which the peak solar irradiance reaches 2,200 mol photons m 2 s 1) Theoretical minimum photon requirement for H2 production in green algae: 5 mol photons/mol H2 Theoretical maximum yield of H2 production by green algae: 10 mol H2 m 2 d 1 (20 g H2 m 2 d 1; ~80 kg H2 acre 1 d 1) Daily irradiance can vary significantly depending on season and cloud cover. It can be greater than 50 mol photons m 2 d 1 in the summer and much less than that on cloudy days and in the winter. [29]. Based on the requirement of 10 photons for the oxidation of two water molecules and the release of four electrons and four protons in photosynthesis [30, 31] Assuming that all incoming photosynthetically active radiation will be 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 green algae

Light absorption by the photosynthetic apparatus is essential for the generation of H2 gas. However the optical properties of light absorption by green algae impose a limitation in terms of solar conversion efficiency in the algae chloroplast. This is because wildtype green algae are equipped with a large size lightharvesting chlorophyll antenna to absorb as much sunlight as they can. Under direct and bright sunlight, they could waste up to 60% of the absorbed irradiance [47, 62]. This evolutionary trait may be good for survival of the organism in the wild, where light is often limiting, but it is not good for the photosynthetic productivity of a green algal mass culture. This optical property of the cells could further lower the productivity of a commercial H2 production farm. The analysis up to this point has shown that H2 production can be limited by the photons available or the capacity of algae to process the photons into H2. Another observation is that the number of photons absorbed is much higher than the algaes ability to process the photons. By reducing the number of excess photons absorbed and let them reach deeper into the liquid, it may be possible to produce more H2. By reducing the size of the algaes light collecting antennae, but not affecting the organisms ability to process the photons to produce H2, one gets deeper light penetration for the same cell concentration, which means more photons are available at the lower depths for H2 production. While regular green algae absorb most of the light falling on them, algae engineered to have less chlorophyll let some light left through. In University of California, Berkeley, Melis and his colleagues are designing algae that have less chlorophyll so that they absorb less sunlight [63]. When grown in large, open bioreactors in dense cultures, the chlorophyll-deficient algae will let sunlight penetrate to the deeper algae layers and thereby utilize sunlight more efficiently [64]. The critical enzymatic component of this photosynthetic reaction is the reversible hydrogenase enzyme, which reduces protons with high potential energy electrons to form H2. During normal photosynthesis, algae focus on using the suns energy to convert carbon dioxide and water into glucose, releasing oxygen in the process. Only about 3 to 5 percent of photosynthesis leads to H2. Because hydrogenase is sensitive to oxygen, this H2 production must be carried out in an anaerobic environment

Photosynthetic H2 production by green algae involves water splitting to produce H2 and oxygen. Unfortunately, H2 production by this process is quite ineffective since it simultaneously produces oxygen, which inhibits the hydrogenase enzyme. Thus, during light reaction, H2 evolution ceases due to an accumulation of oxygen. Therefore the prerequisite for photohydrogen production by green algae is that they have to adapt to an anaerobic condition. By exposing the cells to specific conditions scientists are able to modify photosynthesis so that oxygen will not act as the final electron carrier of the electron transport chain; rather H2 will allow the cells to release molecular H2 as opposed to molecular oxygen. Melis [45] estimates that, if the entire capacity of the photosynthesis of the algae could be directed toward H2 production, 80 kilograms of H2 could be produced commercially per acre per day. The yield of H2 production currently achieved in the laboratory corresponds to only 15 to 20% of the measured capacity of the photosynthetic apparatus for electron transport [63]. In a laboratory, Melis [45] worked with low-density cultures and have thin bottles so that light penetrates from all sides. Because of this, the cells use all the light falling on them. But in a commercial bioreactor, where dense algae cultures would be spread out in open ponds under the sun, the top layers of algae absorb all the sunlight but can only use a fraction of it [63]. Further research and development aimed at increasing rates of synthesis and final l yields of H2 are essential. Optimization of bioreactor designs, rapid removal and puri6cation of gases, and genetic modifcation of enzyme pathways that compete with hydrogen producing enzyme systems offer exciting prospects for biohydrogen systems [48]. Increase in the rate of H2 would reduce bioreactor size dramatically to overcome the engineering challenges of scale up, and create new opportunities for practical applications.

9. H2 Economy
A typical energy chain for sustainable H2 comprises the harvesting of sunlight into H2 as energy carrier, the storage and distribution of this energy carrier to the enddevice where it is converted to power. The key market for fuel cells has always assumed to be the automotive industry. The great expectation that hydrogen fuel-cell powered vehicles will displace gasoline and diesel powered vehicles has not materialized for a variety of reasons, but primarily because fuel cell technology has

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective not yet matured and the infrastructure required for hydrogen storage, transportation, and refueling has been slow to develop. Consumer energy applications will require delivery systems that can supply H2 as readily as gasoline and natural gas are supplied today. Higherpressure gaseous storage and non-conventional storage technologies will be used to meet the requirements of transportation applications (storage at 350700 bar compared to the 200 bar storage pressure commonly used in normal merchant gas systems) [65]. Gas purity requirements are important for the H2 energy market. They very much depend on the energy conversion device used, as well as on the storage technology. Combustion systems are much less sensitive to impurity levels, however, fuel cells are very sensitive to CO and sulfur poisoning. The U.S. Department of Energy has developed a multiyear plan with aggressive milestones and targets for the development of H2 infrastructure, fuel cells, and storage technologies. The targeted H2 cost is $24 kg-1 (energy equivalent of 1 gallon of gasoline) delivered [66, 67] A rollout of such a sustainable H2 chain in developed countries could go either gradually via a H2 economy based on fossil fuels or discontinuously in the case of inventions of disruptive technologies. For developing countries the situation may be different. Introduction of such H2 chains for their fast-growing primary energy demands might enable them to skip the stage of conventional, fossil fuel-based technologies and markets and leapfrog directly to a sustainable H2 economy [68]. The salient features of a H2 economy will be as follows [69]: A H2-based energy system will increase the opportunity to use renewable energy in the transport sector. This will increase the diversity of energy sources and reduce overall greenhouse gas emissions. H2 in the transport sector can reduce local pollution, which is a high priority in many large cities. The robustness and flexibility of the energy system will be increased by the introduction of H2 as a strong new energy carrier that can interconnect different parts of the energy system. The targets for reducing vehicle noise may be met by replacing conventional engines with H2powered fuel cells. Fuel cells for battery replacement and backup power systems are niche markets in which price and efficiency are relatively unimportant. Sales in this market will drive the technology forward towards the point at which fuel cells will become economic for the introduction into the energy sector. H2 electrolysers/fuel cells connected directly to wind turbines are a convenient way to balance out local fluctuations in the availability of wind power. The development of fuel cells and a H2 economy will provide new market opportunities and new jobs. Present knowledge indicates that H2 as an energy carrier will involve little environmental risk. All renewable hydrogen production technologies face the common challenge of integration with hydrogen purification and storage [65].

10. Present energy scenario of Pakistan

Pakistan is basically an energy deficient country. Pakistans per capita energy consumption, 3894kWh as against the world average of 17620kWh, gives it a ranking of 100 amongst the nations of the world [70]. The demand for primary energy in Pakistan has increased considerably over the last few decades and the country is facing serious energy shortage problems. The energy supply is not increasing by any means to cope with the rising energy demands. As a result the gap between the energy demand and supply is growing every year. The country is meeting about 86% of oil demand from imports by spending around US$6.65 billion per annum [71]. Pakistans future energy system looks rather uncertain. In recent years, the combination of rising oil consumption and flat oil production in Pakistan has led to rising oil imports from Middle East exporters. The balance recoverable reserves of crude oil in the country as on January 1st 2010 have been estimated at 303.63 million barrels [72]. Natural gas accounts for the largest share of Pakistans energy use, amounting to nearly 43.7 percent of total energy consumption. As on January 1, 2010, the balance recoverable natural gas reserves have been estimated at 28.33 trillion cubic feet. The average production of natural gas during July- March 2009-10 was 4,048.76 million cubic feet per day (mmcfd) [72]. As the demand of natural gas exceeds the supply, country is already facing shortage of natural gas and during the peak demand most of the gas fired generating units are shutdown while duel fuel units are fired by oil. Pakistan is presently facing shortage of around 300-350 MMCDF of natural gas which is likely to go up because of rising needs and slowing down of supplies at home [73]. According to The Energy Security Action Plan of the Planning Commission, Pakistan will be facing a shortfall in gas supplies rising from 1.4 Billion Cubic Feet (BCF) per day in 2012 to 2.7 BCF in 2015 and escalating to 10.3 BCF per day by the year 2025 [74]. It is therefore a matter

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective of economic security to develop alternative H2 resources to avoid mid century energy crises in the country. Natural gas is used in general industry to prepare consumer items, produce cement, fertilizer and generate electricity. At present, the power sector is the largest user of gas accounting for 33.5 percent share followed by the industrial sector (23.8 percent), household (18.1 percent), fertilizer (15.6 percent), transport (5.4 percent) and cement (0.9 percent) [75]. Natural gas is used in the transport sector in the form of CNG. There are about 3,116 established CNG stations in the country and approximately 2 million vehicles are using CNG. Pakistan has become the largest CNG consuming country among Natural Gas Vehicle (NGV) countries. According to Petroleum Policy 1997; the use of CNG in vehicles was encouraged by Government to reduce pressure on petroleum imports, to curb pollution and to improve the environment [75]. Transport sector is one of the major consumers of commercial energy in Pakistan. It accounted for about 28% of the total final commercial energy consumed (33.95 MTOE) and 55.8% of the total petroleum products consumed (15 MTOE) in the country. economy, and will have a positive impact on the environment in which atmospheric pollution is all but alleviated and the so-called greenhouse effect is mitigated. To ensure a sustainable energy future for Pakistan, it is necessary that the energy sector be accorded a high priority. In Pakistan efforts to reduce reliance on fossil fuels through increasing the share of renewable energy in the energy supply systems have met with little success so far. Mirza et al. [77] and Sahir and Qureshi [78] have discussed the barriers to development of renewable energy. Mirza, et al. [77] has broadly classified these barriers as policy and regulatory barriers, institutional barriers, fiscal and financial barriers, market-related barriers, technological barriers and information and social barriers. They have also suggested better coordination among various stakeholders and indigenization of renewable energy technologies to overcome these barriers. Sahir, and Qureshi [78] has suggested an integrated energy planning approach, consistency in government policies and rational policy instruments to deal with the techno-economic and socio-political barriers are the prerequisites for long-term sustainable development of the renewable energy technologies. There is little doubt that power production by renewable energies, energy storage by H2, and electric power transportation and distribution by smart electric grids will play an essential role in phasing out fossil fuels.

11. H2 Production in Pakistan

In Pakistan H2 is largely produced in the fertilizer industry from natural gas, which is used for the production of anhydrous ammonia. All urea plants in the country are based on natural gas as feedstock. On an average, the fertilizer sector consumes 15.6 per cent of natural gas produced in country. The government provides an indirect subsidy to fertilizer manufacturers by selling feedstock gas at rates ranging up to $1.0 against commercial rates of $4.0 per MMBTU. The return on paid-up capital in the fertilizer industry is about 80-100 per cent per annum [73]. The current energy scenario in the country, already discussed above , identifies the transport sector and fertilizer sector as key sectors where the H2 gas can be immediately employed as substitute to fossil fuel. Mirza et al. [76] has presented complete road map to H2 economy in Pakistan. They have concluded that the H2 economy potentially offers the possibility to deliver a range of benefits for the country including reducing dependence on oil imports, environmental sustainability and economic competitiveness. In medium term advent of H2 will bring about technological developments in many fields, including power generation, agriculture, the automotive industry, and other as yet unforeseen applications. It will increase employment, stimulate the

12. Conclusions
Concerns about global warming and environmental pollution due to the use of fossil fuels, combined with projections of potential fossil fuel shortfall toward the middle of the 21st century, make it imperative to develop alternative energy sources that would clean, renewable, and environmentally friendly. It is important to note that hydrogen can be produced from a wide variety of feed stocks available almost anywhere. There are many processes under development which will have a minimal environmental impact. Development of these technologies may decrease the worlds dependence on fuels that come primarily from unstable regions. The in house hydrogen production may increase both national energy and economic security. The ability of hydrogen to be produced from a wide variety of feedstocks and using a wide variety of processes makes it so that every region of the world may be able to produce much of their own energy. It is clear

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Developments in Hydrogen Production through Microbial Processes; Pakistans Prospective that as the technologies develop and mature, hydrogen may prove to be the most ubiquitous fuel available. The vision for a H2 future is one based on clean sustainable renewable energy supply of global proportions that plays a key role in all sectors of the economy. Microbial Processes provides an attractive option to produce H2 at ambient conditions. A large number of microbial species, including significantly different taxonomic and physiological types, can produce H2, Diversity in microbial physiology and metabolism means that there are a variety of different ways in which microorganisms can produce H2, each one with seeming advantages, as well as problematic issues. Lower rate of H2 production and low yield are chief drawbacks. From an engineering perspective, they all potentially offer the advantages of lower cost catalysts (microbial cells) and less energy intensive reactor operation (mesophilic) than the present industrial process for making hydrogen. The recently developed single-organism, two-stage photosynthesis and H2 production protocol with green algae is of interest because significant amounts of H2 gas can be generated essentially from sunlight and water. Further, this method does not entail the generation of any undesirable, harmful, or polluting byproducts and it may even offer the advantage of value-added products as a result of the mass cultivation of green algae. However, several biological and engineering challenges must be overcome before this promising technology becomes a practical reality. Foremost, the cellular metabolism and basic biochemistry that support this process must be well understood and much fundamental research on the mechanism of H2 production by S- deprivation remains to be done. For Pakistan the indigenous H2 production may increase both national energy and economic security. The ability of H2 to be produced from a wide variety of feedstocks and using a wide variety of processes makes it so Pakistan may be able to produce much of her own energy. Fertilizer sector is key area where the H2 gas can be immediately employed as substitute to natural gas. The advent of H2 will bring about technological developments in many fields, including power generation, agriculture, the automotive industry, and other as yet unforeseen applications. It will increase employment, stimulate the economy of all nations on earth, and will have a positive impact on the environment in which atmospheric pollution is all but alleviated and the socalled greenhouse effect is mitigated. Non-incorporation of renewable energy issues in the regulatory policy and lack of awareness among regulators restrict technology penetration. There is a lack of financial resources and proper lending facilities, particularly for small-scale projects in country. In addition, the absence of a central body for overall coordination of energy sector activities results in duplication of R&D activities. Unfortunately private sector especially transports and fertilizer sector has made no contributions to promote research activities to produce H2 from renewable resources. REFERENCES
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