ADVANCES IN BIOLOGICAL HYDROGEN PRODUCTION PROCESSES Текст научной статьи по специальности «Промышленные биотехнологии»

ЭНЕРГИЯ БИОМАССЫ

ENERGY OF BIOMASS

ADVANCES IN BIOLOGICAL HYDROGEN PRODUCTION

PROCESSES

Debabrata Das1,2, T. Nejat Veziroglu2^

d^T Honorable Editor-in-Chief

1 Department of Biotechnology, Indian Institute of Technology Kharagpur 721302, INDIA E-mail: [email protected]

2 Clean Energy research Institute, College of Engineering, University of Miami Coral Gables, Florida 33124-0622, USA E-mail: [email protected]

Biological hydrogen production processes offer a technique through which renewable energy sources like biomass can be utilized for the generation of cleanest energy carrier for the use of mankind, hydrogen. Intensive research work has already been carried out on the advancement of these processes, such as the development of genetically modified microorganism, improvement of the reactor designs, use of different solid matrices for the immobilization of whole cells, development of two-stage processes, etc. higher H2 production rates. Maximum H2 yield is found to be 7.1 molH2/mol glucose. However, major bottlenecks for the commercialization of these processes are lower H2 yield and rate of H2 production. Competant microbial cultures are required to handle waste materials efficiently, which are usually complex in nature. This will serve dual purposes: clean energy generation and bioremediation. Scale-up studies on fermentative H2 production processes have been done successfully. Pilot plant trials of the photo-fermentation processes require more attention. Use of cheaper raw materials and efficient biological hydrogen production processes will surely make them more competitive with the conventional H2 gerneration processes in near future.

Nomenclature

ADP adenosine diphosphate

ATP adenosine triphosphate

BEAMR bioelectrochemically assisted

microbial reactor CEM cation exchange membrane

CoA coenzyme A (P-Mercaptoethylamine +

+ Pantothenic acid + ADP with 3'-phosphate group) CSTR continuous stirred tank reactor

Fd(ox) ferredoxin (oxidised form) Fd(red) ferredoxin (reduced form) LP Leudeking-Piret

NAD+ necotinamide adenine dinucleotide

MFC microbial fuel cell

MLVSS mixed liquor volatile suspended solids

MSW municipal solid wastes

NADH necotinamide adenine dinucleotide

(reduced form) NADP necotinamide adenine dinucleotide phosphate

NADPH necotinamide adenine dinucleotide

phosphate (reduced form) OLR organic loading rate

PBR packed bed reactor

PNS purple non-sulfur

PSI photosystem-I

PSII photosystem-II

UASB upflow anaerobic sludge blanket

VSS volatile suspended solids

Статья поступила в редакцию 01.07.2007 г. Ред. per. № 104. The article has entered in publishing office 01.07.2007. Ed. reg. No. 104.

International Scientific Journal for Alternative Energy and Ecology № 7(51) 2007

© 2007 Scientific Technical Centre «TATA»

1. Introduction

Hydrogen is considered as a 'dream fuel' for the future mainly due its non-polluting nature as compared to other fuels. It has the highest energy content per unit weight of any known fuel (143 GJ/tonne) [1]. It is the only common fuel that is not chemically bound to carbon. Therefore, major environmental damage, such as global warming, ozone layer depletion and acid rains, may be avoided, if hydrogen is utilized as fuel. Presently, hydrogen is produced 40 % from natural gas, 30 % from heavy oils and Naphtha, 18 % from coal, and 4 % from electrolysis [2-3]. Advantages and disadvantages of different H2 production processes are shown in Table 1. Biological hydrogen production processes are becoming important mainly due to two reasons: (i) they can utilize renewable energy resources, and (ii) they are usually operated at ambient temperature and atmospheric pressure. These processes are usually controlled by different anaerobic bacteria and/or algae. The characteristics of these microorganisms widely differ from each other with respect to substrates and process conditions. The merits and demerits of the processes have already been discussed by different authors [4, 5]. The key question is from which process hydrogen can be produced in a sustainable manner, in large quantities, and at an acceptable cost.

Present paper deals with the review of the advances in biological hydrogen production processes and also discusses major bottlenecks to commercialization.

2. Basic concepts

Biological hydrogen production processes can be classified as follows:

♦ Biophotolysis of water using green algae and blue-green algae (cyanobacteria)

- Direct biophotolysis

- Indirect biophotolysis

♦ Photodecomposition of organic compounds by photosynthetic bacteria;

♦ Dark fermentation of organic compounds;

and

♦ Hybrid systems

- Using fermentative and photosynthetic bacteria,

- Using bioelectrochemical assisted bioreactor.

2.1. Biophotolysis of water using green algae and blue-green algae (cyanobacteria)

Green algae and blue-green algae split water molecules to hydrogen ion and oxygen via direct and indirect biophotolysis.

2.1.1. Direct biophotolysis

The conversion of water to hydrogen by green algae may be represented by the following general reaction:

2H2O + Light energy ^ 2H2 + O2. (1)

Well known H2-producing green algae, Chla-mydomonas reinhardtii, under anaerobic conditions, can either generate H2 or use H2 as an electron donor [6]. The generated hydrogen ions are converted into hydrogen gas in the medium with electrons (donated by reduced ferredoxin) by hydrogenase enzyme present in the cells. Light energy absorbed by photosystem II (PS II) generates electrons which transferred to ferredoxin using light energy absorbed by photosytem I (PS I). A reversible hydrogenase accepts electrons directly from the reduced ferredoxin to generate H2 in presence of hydrogenase enzyme, as follws:

H2O ^ PSII ^ PSI ^ Fd ^ Hydrogenase ^ H2

+ (2) O2.

This enzyme is very sensitive to O2. Hydroge-nase activity has also been observed in other green algae like Scenedesmus obliquus [6], Chlorococ-cum littorale [5], Playtmonas subcordiformis [5] and Chlorella fusca [6]. On the other hand, there are several green algae types that do not have hydrogenase activity such as Duneliella salina and C. vulgaris [5].

2.1.2. Indirect biophotolysis General reaction for hydrogen formation from water by cyanobacteria can be represented by following reactions:

-C6H12O6 + 6O2 (3)

12H2O + 6CO2 + light energy -

and

C6H12O6 + 12H2O + light energy ^ 12H2O6 + 6CO2. (4)

Cyanobacteria is also known as blue-green algae, cyanophyceae or cyanophytes. It has a large and diverse group of photoautotrophic microorganism. Cyanobacteria contain photosynthetic pigments, such as chl a, carotenoids and phycobilipro-teins, and can perform oxygenic photosynthesis. Morphologically these organisms fall into a diverse group, that includes unicellular, filamentous and colonial species. Hydrogen is produced both by hydrogenase and nitrogenase enzymes. Within the filamentous cyanobacteria, vegetative cell may develop into structurally modified and functionally specialized cells. The nutritional requirements of cyanobacteria are simple: air (N2 and O2), water, mineral salts and light.

Hydrogen producing cyanobacteria may be either nitrogen fixing or non-nitrogen fixing. The examples of nitrogen fixing organisms are nonmarine Anabaena sp., marine cyanobacter Calothrix sp., Oscillatoria sp., and non-nitrogen fixing organisms such as Synechococcus sp., Gloebacter sp. and Anabaena sp. They are found suitable for higher hydrogen evolution as compared to other cyanobacter species [5-8]. Heterocystous filamen-

tous Anabaena cylindrica is a well-known hydrogen producing cyanobacter [5]. But, A. variabilis has received more attention in recent years, because of higher hydrogen yield [9].

Hydrogen production by vegetative cells can take two routes [9]:

A. Heterocystous nitrogen fixing bacteria:

► Ferredoxin ^ Nitrogenase

H2O ^ Photosystems ^ [CH2O]2 -(vegetative cells)

1 t INADPH I (5)

02 CO2 Recycle CO2 H2

B. Nonheterocystous nitrogen fixing bacteria:

H2O ^ Photosystems ^ [CH2O]2 ^ Ferredoxin ^ Nitrogenase (vegetative cells)

1 t I NADPH or Hydrogenase

02 CO2 Recycle CO2 t I

PS-I ^ ATP H2

(6)

The growth conditions for Anabaena is simple which include nitrogen free media, illumination, CO2 and N2. Nitrogenase plays important role for the hydrogen generation. Activity of the nitrogenase enzyme is inhibited by oxygen. Hydrogen production takes place under anaerobic conditions. Some cultures require CO2 during hydrogen evolution phase, although CO2 is reported to give some inhibition effects on photo-production of H2. Lower CO2 concentrations (4-18 % w/v) have been reported to increase cell density during growth phase, resulting in higher hydrogen evolution in the later stage. Simple sugars have been found suitable for hydrogen production. Recently more stress has been given to increase hydrogenase activity and bi-directional hydrogenase deficient mutant of Anabaena sp. in order to increase the rate of hydrogen production. However, at the present time the rate of hydrogen production by Anabaena sp. is considerably lower than that obtained by dark or photo-fermentations [5-9].

With molecule N2

N2 + 8H+ + 8ë + 16ATP ^ 2NH3 + H2 + 16ADP + 16P; (7) or, without molecular N2

8H+ + 8e + 16ATP-

■ 4H2 + 16ADP + 16P/.

(8)

use is in the versatile metabolic capabilities of these organisms and the lack of Photosystem II (PSII), which automatically eliminates the difficulties associated with O2 inhibition of H2 production. Pho-totrophic bacteria require organic or inorganic electron source to drive their photosynthesis. They can utilize a wide range of cheap compounds. These photoheterotrophic bacteria have been found suitable to convert light energy into H2 using organic wastes as substrate [10-12] in batch processes [13], continuous cultures [14], or immobilized whole cell system using different solid matrices like car-rageenan [15], agar gel [16], porous glass [12], and polyurethane foam [11].

The overall biochemical pathways for the photo fermentation process can be expressed as follows:

NADPH

(CH^O) ^ Ferredoxin ^ Nitrogenase ^ H2 (10)

t ATP

t ATP

Certain photoheterotrophic bacteria within the superfamily Rhodospirillaceae can grow in the dark using CO as the sole carbon source to generate ATP with the simultaneous release of H2 and CO2 [6]. The oxidation of CO to CO2 with the release of H2 occurs via a water gas shift reaction as shown below:

CO + H2O-

CO2 + H

2-

(11)

2.2. Photo-decomposition of organic compounds by photosynthetic bacteria

H2 production by purple non-sulfur bacteria is mainly due to the presence of nitrogenase under nitrogen-deficient conditions using light energy and reduced compounds (organic acids). The reaction is as follows:

C6H12O6 + 12H2O + light energy ^ 12H2 + 6CO2. (9) Photosynthetic bacteria have long been studied for their capacity to produce significant amounts of hydrogen [10]. The advantage of their

2.3. Fermentative hydrogen production from organic compounds

Dark hydrogen fermentation is a ubiquitous phenomenon under anoxic or anaerobic conditions (i.e., no oxygen present as an electron acceptor). When bacteria grow on organic substrates (heterotrophic growth), these substrates are degraded by oxidation to provide building blocks and metabolic energy for growth. This oxidation generates electrons which need to be disposed of to maintain electrical neutrality. In aerobic or oxic environments, oxygen is reduced and water is the product. In anaerobic or anoxic environments, other compounds, e.g., protons, which are reduced to molecular hydrogen (H2), need to act as electron acceptor [4, 7]. In the hydrogen fermentation process, glucose is initially converted to pyruvate by the glycolytic pathways. This is oxidized to acetyl-CoA, which can be converted to acetyl phosphate and results in the generation of ATP and the excretion of acetate. Pyruvate oxidation to acetyl-CoA requires ferredoxin (Fd) reduction. Reduced Fd is oxidized by hydrogenase which generates Fd and releases electrons as molecular hydrogen [17, 18]. The overall reaction of the processes can be described as follows:

Pyruvate + CoA + 2Fd(ox) ^ • Acetyl - CoA + 2Fd(red) + CO2

(12)

International Scientific Journal for Alternative Energy and Ecology № 7(51) 2007

© 2007 Scientific Technical Centre «TATA»

2H+ + Fd(red) ^ H2 + Fd(ox).

(l3)

Anaerobic fermentation enables the mass production of hydrogen via relatively simple processes from a wide spectrum of potentially utilizable substrates, including refuse and waste products. Moreover, fermentative hydrogen production generally proceeds at a higher rate and does not rely on the availability of light sources. Carbohydrates, mainly glucose, are the preferred carbon sources for fermentation process, which predominantly give rise to acetic and butyric acids together with hydrogen gas [18], as follows:

ОД^ + 2H2O-

■ 2CH3COOH + 2CO2 -

■4H2

(l4)

C6H12O6 + 2H2O ^ CH3CH2COOH + 2CO2 + 2H2. (15) The end-products of glucose fermentation by anaerobic and facultative anaerobic chemohetero-trophs, e.g., clostridia and enteric bacteria, are

produced through pyruvate. Facultative anaerobic bacteria give 2 mol of hydrogen per mol of glucose, whereas strictly anaerobic bacteria give four. Facultative anaerobes are less sensitive to oxygen, and are sometimes able to recover hydrogen production activity after accidental oxygen damage to them by rapidly depleting oxygen present in the broth. As a consequence, a facultative anaerobe is considered a better microorganism than a strict anaerobe to carry out fermentative hydrogen production process [19]. One of the main constraints of fermentative biohydrogenation process is the lower yield of hydrogen, maximally 4 mol/mol glucose, compared with other processes (Table 1). A yield of 2 mol H2/mol glucose was reported for butyrate fermentation [20]. However, by a modification of fermentation pathways only a maximum of 4 mol H2/mol glucose can be expected from ideal acetate fermentation. This yield is too

Table l

Advantages and disadvantages of different hydrogen production processes

from biomass

Process Advantages Disadvantages

Thermochemical gasification Maximum conversion can be achieved Significant gas conditioning is required Removal of tar

Pyrolysis Produces carbonaceous material along with bio-oil, chemicals and minerals Chances of catalyst deactivation

Solar gasification Good hydrogen yield Required effective collector plates

Supercritical conversion Can process sewage sludge, which is difficult to gasify Selection of supercritical medium

Microbial conversion: Can be operated at ambient temperature and atmospheric pressure Lower rate of hydrogen production and yield

• Direct biophotolysis 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 O2 can be dangerous for the system Lower photochemical efficiency

• Indirect biophotolysis Cyanobacteria can produce H2 from water Has the ability to fix N2 from atmosphere Uptake hydrogenase enzymes are to be removed to stop degradation of H2 About 30 % O2 present in gas mixture O2 has an inhibitory effect on nitrogenase

• Photofermentation A wide spectral light energy can be used by these bacteria Can use different organic wastes Light conversion efficiency is very low, only 1-5 % O2 is a strong inhibitor of hydrogenase

• Dark fermentation 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 Relatively lower achievable yields of H2 As yields increase H2 fermentation becomes thermodynamically unfavorable Product gas mixture contains CO2 which has to be separated

low to be economically viable as an alternative to existing chemical or electrochemical processes of hydrogen generation [21]. Therefore, the ultimate goal, and challenge, for fermentative hydrogen research and development focuses essentially on attaining higher yields of hydrogen. The present review provides a critical discussion of the various practical and theoretical approaches towards improvement of overall yield of hydrogen in fermentative process.

2.3.1. Hybrid system using fermentative and photosynthetic bacteria

The microbial production of hydrogen by fermentation can be broadly classified into two main categories. One category, light independent bacteria could provide an integrated system for maximizing the hydrogen yield [22]. In such a system, the anaerobic fermentation of carbohydrate (or organic wastes) produces intermediates, such as low-molecular-weight organic acids, which are then converted into hydrogen by photosynthetic bacteria in the second step in a photo-bioreactor. The overall reactions of the process can be represented as:

(1) Stage I. Dark fermentation (facultative anaerobes):

C6H12O6 + 2H2O-

■2CH3CQOH + 2CO2 -

4H2

producing current. At the cathode oxygen reacts with the electrons and protons to form a reduced compound, such as water. In a bioelectrochemical-ly assisted microbial reactor (BEAMR), hydrogen is evolved at the cathode according to Geobacter or Shewanella sps. or Rhodoferax ferrireducens

C6H12O6 + 2H2O-Anode:

■ 4H2 + 2CO2 + 2CH3COOH. (18)

2

CH3COOH + 2H2O

Cathode:

8H+ + 8e -

2CO2 + 8e- + 8H +

• 4H2

(19)

(16)

(2) Stage II. Photo-fermentation (photosyn-thetic bacteria):

2CH3COOH + 4H2O ^ 8H2 + 4CO2. (17)

So, theoretically it is evident that using glucose as the sole substrate in dark anaerobic fermentation, where acetic acid is the predominant metabolite product, a total of 12 mol hydrogen could be expected in a combined process from one mol of glucose. Lee et al. (2002) studied the combination of purple nonsulfur (PNS) photosynthetic bacteria and anaerobic bacteria for the efficient conversion of wastewater into hydrogen [23]. In this study, effluents from three carbohydrate-fed reactors (CSTR, UASB) have been used for hydrogen production. In another study, Kim et al. (2001) combined dark fermentation with photo-fermentation to improve hydrogen productivity from food processing wastewater and sewage sludge [24]. Similar studies have been reported by Nath et al. (2005) using glucose as a substrate in the dark fermentation process, and the spent medium from this process has been used as a substrate for anoxygenic phototrophic PNS bacteria for hydrogen production in photo-fermentation [18].

2.3.2. Hybrid system using bioelectrochemical assisted bioreactor

Microbial fuel cell (MFC) produces protons and electrons due to the oxidation of organic matter by the bacteria [25, 26]. Protons diffuse through the electrolyte towards the cathode. The electrons travel around a circuit to the cathode,

,2 (20)

by eliminating oxygen at the cathode, and adding a small voltage to the circuit [27]. The potential at pH = 7 that is required to produce hydrogen is theoretically -0.61 V (VCat; versus Ag/AgCl). The anode potential produced by the oxidation of the organic matter by the bacteria is approximately -0.50 V (V ), so that the minimum theoretical applied voltage is 0.11V (Vapp = Van - VCat). In practice, the minimum applied voltage to produce hydrogen from the bioelectrolysis of acetate has been found to be more than ~0.25 V due to ohmic resistance and electrode over potentials [27] which is is still substantially less than the 1.8-2.0 V needed for hydrogen production via water electrolysis (alkaline conditions). The BEAMR and MFC systems share many similar characteristics, and therefore many findings for improving electricity generation in MFCs should be applicable for increasing hydrogen production in the BEAMR system. BEAMR process differs from MFC with respect to loss of hydrogen due to its diffusion from the cathode chamber through the cation exchange membrane (CEM) into the anode chamber. In addition, in the BEAMR process there is no potential for loss of substrate resulting from aerobic growth of bacteria due to oxygen diffusion into the anode chamber from the cathode chamber. This could allow higher Coulombic efficiencies (CEs) in the BEAMR than in the MFC, but it could also affect redox conditions in the anode chamber (and the development of the bacterial community), and therefore the performance of the system.

3. Major progress in biological hydrogen production processes

Research work in biological hydrogen production has been carried out in different areas which are broadly classified as follows:

♦ Development of microbial consortium,

♦ Genetic modification of the microorganisms,

♦ Metabolic engineering,

♦ Effect on physico-chemical parameters,

♦ Performances of different bioreactors, and

♦ Use of cheaper raw material as substrate.

International Scientific Journal for Alternative Energy and Ecology № 7(51) 2007

© 2007 Scientific Technical Centre «TATA»

3.1. Development of microbial consortium

Different microorganisms participate in the biological hydrogen generation system such as green algae, cyanobacteria (or blue-green algae), photo-synthetic bacteria and fermentative bacteria [4, 5]. Intensive research work has been done to find out the suitability of different microorganisms for the hydrogen generation (Table 2). The hydrogen production by green algae could be considered as an economical and sustainable method in terms of water utilization as a renewable resource and recycling CO2, a greenhouse gas. However, strong inhibition effect of generated oxygen on hydrogenase enzyme is the major bottleneck for the process. It has been reported that inhibition of the hydrogenase enzyme by oxygen can be partially overcome by cultivation of algae under sulfur deprivation for 2-3 days to provide anaerobic conditions under the light [5, 8]. Major drawbacks of this process are low hydrogen production potential and inability to use organic wastes. The hydrogenase activity of the C. reindhartii [200 nmol/(gChl a.h)] is higher than Scenedesmus sp. [150 nmol/(g Chl a.h)] [6]. Rates of hydrogen production by photoheterotrophic bacteria are higher in case of immobilized cells than

that of the suspended cells. Continuous cultures of Rhodobacter spheroides and Rhodopseudomonas capsulata have been reported to produce H2 at rates 80 ml to 100 ml H2/(l of culture.h) and 40 to 50 ml H2/(lof culture. H) respectively [7]. Major drawbacks of the photo-biological hydrogen production processes are:

1. Presence of H2-uptake enzyme, Ni-Fe-hy-drogenase,

2. O2 toxicity to Fe-hydrogenase and nitroge-nase enzymes,

3. Requirement of light source, and

4. Scaling up problems.

Different strategies have been taken to improve upon these organisms. Photosynthetic bacteria can use different organic matter as substrate. So, this can not only be used for the hydrogen generation, but also for the bioremediation of the wastewater containg organic matters. Fermentative microorganism such as Enterobacter cloacae, E. aerogenes, Clostridium sp., Bacillus sp. etc. are found very effective for the hydrogen generation within short time as compared to photo-biological processes [34-39]. E. cloacae can tolerate little oxygen concentration because O2 favours the

Table 2

Reported hydrogen yield by different microorganisms

Maximum

rate of H2 production (ml/l. h) Maximum H2 yield

Microorganisms Raw material used References

Green algae Scenedesmus obliquus TAP-S medium 3.6* 6

Chlmydomonas reinhardii A. moewusii -do-do- 4.5* 10.0* — 6 6

Cyanobacteria Heterocystous Anabaena variabilis BG-11 medium 20 9

A. cylindrica Nutrient medium 8 — 28

Nonhetrocystous Oscillotoria Miami BG7 Medium-A except NH4Cl 90 0.3 29

Photosynthetic bacteria Rhodobacter sphaeroides R. capsulatus R. palustris Rhodospirillum rubnum Minimal medium -do- Rhodospirillaceae medium CO & H2O 5 25 24.9 358** — 30 31 32 33

Fermentative bacteria

Enterobacter aerogenes Glucose 390 — 34

E. cloacae IIT-BT08 Sucrose 660 6.0 35

Clostridium butyricum Glucose 205 — 36

Citrobacter spY19 Glucose — 2.49 37

Bacillus coagulans Glucose 2.28 — 38

Clostridium

acetobutlricum ATCC 824 Glucose 220 — 39

* ml /pg Chl a.l, ** ml/g cell. H

growth of the cells, but not the hydrogen production. This pH of the fermentation broth in case of aerobic growth will increae after 10 h of fermentation, but in case of anaerobic growth it will mentain the decline profile as shown in Fig. 1. This organism has been successfully used for the continuous production of hydrogen.

3.2. Genetic modification of the microoganisms

Knowledge on molecular fundamentals of hydrogen production and utilization in biological system is very much important for the basic and applied research. A tentative mechanism has been proposed for the high H2-yielding microbial strain, Enterobacter cloacae IIT-BT 08, for the transfer of electron donor to H-cluster without the mediation of the F-cluster present in hydrogenase coded gene [40-42]. Several research work has been reported on the use of different genetically mofied microorganisms. Two groups of enzymes participate in biological hydrogen metabolism: nitroge-nases and hydrogenases. Nitrogenase produces H2 as the byproduct of N2 fixation In order to maintain the N2-fixation reaction in the desired direction, nitrogenase produces H2 in excess, which then diffuses out of the cell. Hydrogenase is the key enzyme in the biological hydrogen production processes. Major genetically modified research work is concentrated in two major areas: one is development of H2-uptake Ni-Fe hydrogenase negative mutant and second is to produce oxygen tolerant enzyme [6, 43].

3.3. Metabolic engineering

Much progress has been made in the elucidation of gene expression, structure and regulation of nitrogenase and hydrogenase. No practical and economically competitive process for the continuous production of biological H2 has been available on the market. One of the difficulties is due to the fact that H2 output represents an energy loss for the cell, and that microbial metabolic network has evolved for rationalization of energy use and optimization of specific growth rate. The study of the physiology of genetically modified photosyn-thetic microorganisms has shown that electron flux could be redirected to the bidirectional hydroge-nase in a ndhB mutant of Synechocystis, and that a change in carbon metabolism in mutants of Rho-dobacter capsulatus unable to grow photoautotroph-ically could affect the flow of reducing equivalent from organic substrates to nitrogenase. Increasing the flux through an existing pathway or redirecting enzyme-catalyzed reactions is an approach referred to as metabolic engineering. Various «naturally engineered» organisms such as Dehalococ-coides ethenogenes, which is the only bacterium known to reductively dechlorinate the ground water pollutants tetrachloroethene and trichloethene to ethene. D. ethenogenes exhibits an unusual met-

0 2 4 6 8 10 12 14 16

Time(h)

Fig. 1. pH profiles of the medium of E. cloacae IIT-BT 08 grown in aerobic and anaerobic conditions

abolic specialization; it uses only H2 as an electron donor and chlorinated compounds as electron acceptors to support growth. The sequence of its genome has revealed the presence of five hy-drogenase complexes [44].

3.4. Effect of physico-chemical parameters

Temperature, pH and medium composition play very important role for the H2 production. The pH of the fermentative bacteria usually produces hydrogen under acidic condition pH 4.5-6.5), but photosynthetic bacteria works well at pH more than 7 [6, 9, 28-33]. In case PNS photosynthetic bacteria the energy conversion efficiency is inversely proportional to the intensity of light [18]. Malate and glutamate play important role in this fermentation process. The initial acetic acid concentration present in the spent medium of dark fermentation process has a profound effect on hydrogen production. Acetate concentration up to 55 mM is found non-toxic to the photo-fermentation of hydrogen [18]. Effect of several physico-chemical parameters on the photo-biological processes have already been reported [4]. Substrate concentration affects the fermentative H2 production processes to a great extent. Glucose concentration of 1 % w/v is found suitable [35]. Partial pressure of H2 effects the efficiency of the process to a great extends [45]. H2 production is found to increase through redirection of metabolic pathways by blocking formation of alcohol and some organic acids in E. cloacae [46]. The fermentative H2 production is carried out at different temperatures 20, 30, 35 and 55 °C [47]. The higher temperatures improve the H2 yield.

3.5. Performances of different bioreactors

Different types of bioreactors are reported for the different biological H2 production process, e.g. multi-layer bioreactor, UASB, rhomboidal reactor, etc. (Table 3). Different solid matrices are used for the immobilization of whole cells. Major problem lies on the gas-hold up, which decreases the working volume of the reactor to a great extend.

78

International Scientific Journal for Alternative Energy and Ecology № 7(51) 2007

© 2007 Scientific Technical Centre «TATA»

IotJ

Table 3

Performances of modified bioreactor and solid matrices for the H2 production

Maximum rate of H2 production (ml Hi/l.h) Maximum H2

Microorganisms Raw materials Type of modification yield (mol H2/ References

/mol glucose)

Rhodobacter sphaeroides RV Basal medium with lactate and glutamate Multi-layered photobioreactor (MLPR) 2000* — 48

Rhodopseudomonas palustris WP3-5 Acetate Internal optical fiber illumination 28.5 2.97** 49

Sewage sludge Sucrose Fixed bed bioreactor with activated carbon 1210 — 50

Activated sludge and digested sludge Glucose Anaerobic fluidized bed reactor 2360 — 51

Sludge from the treatment plant Sucrose Up-flow anaerobic sludge blanket reactor 252.4 — 52

Sludge from domestic wastewater Molasses CSTR 201.4 — 53

Anaerobic sludge Sucrose Polymethy methacrylate(PMMA) immobilized cells 1800 2.25*** 54

Sludge from wastewater treatment plant Sucrose Carrier-induced granular sludge bed (CIGSB) 9310 -4.02 55

Sludge from wastewater Sucrose Fluisized bed reactor (FBR) 1321.6 56

treatment plant

Enterobacter cloacae IIT-BT 08 Sucrose Rhomboidal bioreactor 77 3**** — 57

* ml/m2.h; ** mol H2/mol acetate, *** mol H2/mol sucrose; **** 77.3 mmol/l.h

This can be patially overcome by using rhomboi-dal bioreactor [57] (Fig. 2). Light distribution in the photo-biological reaction can be improved by using multi-layer photo-bioreactor [48]. The conversion efficiencies of a BEAMR can be as high as 92 ± 6.3 % with acetate, while in MFCs this varies from 10 % to 78 % using mixed cultures and acetate. It cannot be predicted based on MFC tests what the minimum applied voltage will be for hydrogen generation in a BEAMR process for different substrates, or how current density might be affected by the applied voltage in these systems [27].

Fig. 2. Continuous hydrogen production by immobilized whole cell reactor using rhomboidal bioreactor

3.6. Use of cheaper raw materials as substrate

Cost of the raw materials play very important role for the overall economy of the hydrogen generation process. Different waste materials have been successfully used in different processes for the hydrogen generation (Table 4). Starch based wastewater has great potentiality for the H2 production [47, 59]. Major problem of using industrial wastewater is the presence of different components in the reaction mixture. Recently, author observed that sewage sludge in combination with molasses improves the hydrogen yield of the process to a great extend (data not published yet).

3.7. Performance of two-stage processes

H2-production could significantly improve by using two-stage processes (Table 5). Hydrogen production with glucose by using co-immobilized cultures of a lactic acid bacterium, Lactobacillus del-brueckii NBRC13953, and a photosynthetic bacterium, Rhodobacter sphaeroides RV, in agar gels have been studied. The maximum yield of the two stage process is 7.1 mol H2/mol glucose at a maximum under illuminated conditions [67]. In an another study, the hydrogen yield has been found to be equal to 5.3 mol H2/mol glucose in a two-stage process using E. cloacae IIT-BT 08 and Rho-dobacter sphaeroides [18].

Table 4

Use of different waste materials for the biohydrogen production processes

Name of the wastes Organism used Process Maximum rate of H2 production (ml H2 /l.h) Maximum H2 yield (mol H2/ /mol glucose) References

Dairy wastewater Anaerobic mixed consortia UASB — 0.122* 58

Starch manufacturing wastes Closdridium butyricum & Enterobacter aerogenes HO-39 and Rhodobacter sp. M-19 Repeated batch culture — 7.2 59

Sewage biosolids Mixed culture Batch — 0 7** 60

Rice winery wastewater Sludge from wastewater treatment plant Upflow reactor — 2.14 47

Potato processing wastewater Sludge from wastewater treatment plant Batch — 6* 47

MSW Mixed culture PBR — 99** * 61

Food wastes Sewage sludge Batch 112 2**** 5.48* 62

Food wastes Mesophilic and thermophilic cultures Batch — 1.8 63

Jackfruit peel Cow dung Anaerobic contact filter — 18***** 64

Olive mill wastewater Activated sludge and Rhodobacte rsphaeroides O.U.001 Two-stage process 11 — 65

* mmol H2/g COD; ** mol H2/g COD; *** ml H2/g VS removed; **** ml H2/g VSS. H; ***** mmol H2/VS destroyed

Table 5

Comparison of different two-stage processes for hydrogen production

Maximum Maximum Rate of H2

Organic acid concentration (mg/l) H2 yield (mol H2/mol glucose)

Microorganisms used Raw materials production References

(ml H2/l.h)

Activated sludge and Rhodobacter sphaeroides Ü.U.001 Olive mill wastewater — 11 66

Lactobacillus delbrueckii 16 mM acetic

and Rhodobacter Glucose acid and 10 7.1 — 67

sphaeroides RV mM lactic acid

Escherichia coli HD701 20 mM acetate, 15 mM lactate, 3 mM succinate

and Rhodobacter Glucose — 5.2 68

sphaeroides Ü.U.001

Clostridium butyricum NCIB 9576 and Makkoli 16 69

Rhodobacter sphaeroides E15-1 wastewater

Rhodobacter sphaeroides Ü.U.001 and Basal medium with lactate and 27 70

Halobacterium salinarum glutamate

Enterobacter cloacae 2,500 mg/l VFA

DM11 and Rhodobacter Glucose 5.3 — 18

sphaeroides Ü.U.001

Anaerobic mesophilic and thermophilic acidogenesis Food waste 1,337 mg/l VFA 1.8 — 72

Closdridium butyricum & Enterobacter aerogenes HÜ-39 and Rhodobacter Starch manufacturing wastes — 7.2 — 59

sp. M-19

International Scientific Journal for Alternative Energy and Ecology № 7(51) 2007

© 2007 Scientific Technical Centre «TATA»

4. Pilot plant studies

Very little information is available on the pilot plant plant studies for the biological H2 production. A pilot-plant of 1.48 m3 capacity has been studied continuously for 200 days. The hydrogen bio-producing reactor (HBR) system has been operated under the organic loading rates (OLR) of 3.11-85.57 kg COD/m3 d with molasses as the substrate. Both biogas and hydrogen yields increase with OLR at the range of 3.11-68.21 kg COD/m3d, but decrease at high OLR (68.2185.57 kg COD/m3 reactor.d). The biogas is main-

Fig. 3. Pilot plant studies at Indian Institute of Technology Kharagpur, India using immobilized Enterobacter cloacae IIT-BT 08 for hydrogen production from cane molasses

ly composed of CO2 and H2 with the percentage of H2 ranging from 40 % to 52 % in biogas. A maximum hydrogen production rate of 5.57 m3 H2/m3 reactor.d, with a specific hydrogen production rate of 0.75 m3 H2/kg MLVSS.d, has been obtained in the reactor. The hydrogen yield is 26.13 mol/kg COD removed within OLR range of 35-55 kg COD/ m3d. In addition, it has been reported that the hydrogen yield is affected by the presence of etha-nol and acetate in the liquid phase, and the maximum hydrogen production rate occurred while the ratio of ethanol to acetate was close to 1. Ethanol-type fermentation was favorable for hydrogen production [75]. One pilot plant of capacity 800 L is in operation at the Indian Institute of Technology Kharagpur using immobilized whole cell (E. cloacae IIT-BT 08) system (Fig. 3). The rate of H2 production of the reactor is comparable with that of the bench scale data (77.3 mmol H2/l. h) [57].

5. Conclusion

Biohydrogen production has been established as a prospective alternative and integral component of green sustainable energy. A challenging problem in establishing biohydrogen as a source of energy is the renewable and environmentally friendly generation of large quantities of hydrogen gas. However, two major aspects need indispensable optimization, viz., a suitable renewable biomass / wastewater and ideal microbial consortia that can convert this biomass efficiently to hydrogen. E. cloacae IIT-BT 08 has greater potentiality for the hydrogen production. Comparative studies on the availables processes indicate

Table 6

Comparison of biological hydrogen production processes with that of

conventional processes

Energy Unit cost of

Name of the process Raw materials conversion content of the fuel References

efficiency

Photobiological hydrogen H2O and organic acids 10 10 72

Fermentative hydrogen Molasses 28.34 30 73

Fast pyrolysis for hydrogen production Coal, biomass — 4 74

H2 from advanced electrolysis H2O — 10 74

H2 from thermal

decomposition of H2O — 13 74

steam

H2 from photochemical Organic acids — 21 74

Fermentative ethanol Molasses — 31.5 75

Gasoline Crude petroleum — 6 74

that biohydrogen production requires greater improvement on the process mainly with respect to hydrogen yield from the cheaper raw materials (Table 6).

6. Future goal

Significant progress on biological hydrogen production processes has already been carried out by several research groups, but still the economy of the process is not attractive as compared to the conventional H2 production processes. The following points require immediate attention:

I. Improvement of H2 yield of the processes using cheaper raw materials,

II. Development of mixed microbial consortium or metagenomic approach may be used to develop efficient microbial strain for the better utilization of industrial wastewater, which has different carbon content,

III. In two stage processes, major bottleneck lies on the photo-fermentation process. Improvement of these processes surely will improve overall hydrogen yield as well as economy of the process.

Acknowledgement

The financial support of the National Science Foundation, USA, and Department of Science & Technology, Government of India, to carry out this project is gratefully acknowledged.

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