Review

Developments and constraints in fermentative hydrogen production

Jan Bartacek, Wageningen University, Wageningen, The Netherlands and Institute of Chemical Technology in Prague, Prague, Czech Republic Jana Zabranska, Institute of Chemical Technology in Prague, Prague, Czech Republic Piet N. L. Lens, Wageningen University, Wageningen, The Netherlands

Received May 14, 2007; revised version received July 16, 2007; accepted July 16, 2007 Published online September 21, 2007 in Wiley InterScience (www.interscience.wiley.com); DOI: 10.1002/bbb.17; Biofuels, Bioprod. Bioref. 1:201214 (2007) Abstract: Fermentative hydrogen production is a novel aspect of anaerobic digestion. The main advantage of hydrogen is that it is a clean and renewable energy source/carrier with high specic heat of combustion and no contribution to the Greenhouse effect, and can be used in many industrial applications. This review discusses fermentative hydrogen production from various points of view. First, the theoretical principles of the biological processes taking place in hydrogen production, as well as the organisms responsible for this process, are described. Second, practical aspects of fermentative hydrogen production are overviewed. Suitable conditions for the hydrogen-producers (pH and temperature), suitable substrates for hydrogen production and applicable reactor designs are discussed. Finally, the challenges faced by fermentative hydrogen production are discussed. Current research directions are listed together with the most important problems currently constraining full-scale application. 2007 Society of Chemical Industry and John Wiley & Sons, Ltd Keywords: fermentation; hydrogen production; substrates; reactors

Introduction

ecently, the international community has expressed huge interest in renewable energy sources. Special attention is being focused on research on hydrogen production and utilization.1 The effort directed towards hydrogen production and utilization is worldwide: the initiative of the USA to establish a so called hydrogen economy

is particularly important.2 The European Community is also extremely interested in this.1 Hydrogen can be produced from fossil fuels, water, or biomass. At present, most hydrogen is produced from natural gas via steam reforming.2 However, to be sustainable, hydrogen production technologies need to utilize renewable sources. In this context, the most promising technology is hydrolysis of water to hydrogen and oxygen using

Correspondence to: P. N .L. Lens, Subdepartment of Environmental Technology, Wageningen University, Biotechnion - Bomenweg 2, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. E-mail: piet.lens@wur.nl

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Review: Fermentative hydrogen production

solar power.2 In contrast to these technologies, biological hydrogen production from wastewater or biowaste is a very inexpensive and very simple prospective method.3 Most studies on biological hydrogen production carried out in the last 20 years have focused on the use of photosynthetic bacteria. However, hydrogen production via dark fermentation is now arousing interest as well. Th is process has the advantage that the hydrogen can be produced in a reactor without the requirement for light energy, which is the limiting factor in light-driven processes.4,5 In contrast to the production of bioethanol, fermentative hydrogen production has the advantage that the mixed bacterial cultures can utilize a substantially wider range of substrates than yeasts, which are usually used for ethanol production.6 Why produce hydrogen? The main advantage of hydrogen is that it is a clean and renewable energy carrier, possessing a high specific heat of combustion (122 kJ g1), not directly (during the process of energy generation) contributing to the Greenhouse effect.3, 7 In comparison, the energy yield of methane is 50.1 kJ g1 and of ethanol only 26.5 kJ g1. Moreover, hydrogen can be used directly as a fuel in fuel cells. Although methane, methanol, ethanol, etc. can also be used, hydrogen is at present the fuel used most often.8 This is because of its high reactivity and high energy density. Hydrogen can be used for many industrial applications, including synthesis of ammonia, alcohols and aldehydes as well as hydrogenation of petroleum. Methane, in contrast, is of little commercial value, except as a fuel.9 As hydrogen can be produced from many natural sources, it is expected to have a stable price in the future, independent of the fluctuation in price and availability of single sources. Hydrogen also allows flexibility in balancing centralized and decentralized power supply.1 Biological hydrogen production The proposed biohydrogen processes can be divided into two groups: dark fermentation light-driven processes (direct biophotolysis, indirect biophotolysis, photofermentation).

Dark fermentation Hydrogen production via dark fermentation is a special type of anaerobic digestion comprising only hydrolysis and acidogenesis. It leads to the production of hydrogen, carbon dioxide and some simple organic compounds (volatile fatty acids (VFA) and alcohols). These readily degradable organic compounds can be used for further methane production. Light-driven processes In direct biophotolysis solar energy is used to convert water to oxygen and hydrogen which are produced together. For example, the green algae Chlamydomonas reinhardtii was used for hydrogen production in laboratory experiments.10 The process is highly oxygen sensitive and the overall efficiency is too low for practical application.11 In indirect biophotolysis oxygen evolution and hydrogen evolution are temporally and spatially separated. Cyanobacteria are ideal organisms for use in indirect biophotolysi.12,13 Recently, for example, Synechocystis sp. was used to produce hydrogen.14 During photofermentation, photosynthetic bacteria produce hydrogen through the action of their nitrogenase system. It has been considered that this process is ineffective and it would be simpler and more efficient to extract the hydrogen from organic substrates using a dark fermentation process.11 Recently, Oh et al.15,16 suggested a two-stage system for hydrogen production using fermentative bacteria Rhodopseudomonas palustris P4. In the fi rst step, glucose was fermented in dark conditions to hydrogen and acetate as main products. In the second step, acetate was utilized via photofermentation by the same hydrogen-producing bacterium. The conversion yield of acetate to H2 was estimated to be 2.42.8 mol H2 mol1 acetate, indicating that the overall yield from glucose to H2 by a two-step process (dark- and photo-fermentation) can be increased two-fold compared to that by a dark-only process. However, the low hydrogen volumetric production rate in the second step remains a problem for the economic feasibility of the whole process. Evaluation Light-driven processes are still at the conceptual stage: they are of questionable economics and remain to be demonstrated

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at full-scale. Dark fermentation offers (in comparison with light-driven processes) one important advantage: no problems with light-energy supply have to be solved. This decreases the energy demand and the technology can be simpler.35,11 A promising possibility is the combination of the dark fermentation with a light-dependent step, and a combination of thermophilic fermentation and photofermentation has been described.17 Fundamentals of fermentative hydrogen production All processes of biological hydrogen production are fundamentally dependent upon the presence of a hydrogenproducing enzyme containing complex metallo-clusters as active sites. These hydrogen-producing enzymes catalyze proton reduction to molecular hydrogen:11 2H 2e H2 (1)

4 mol hydrogen mol1 glucose.6,9,19,21,22 Theoretically, a maximum of 33% of the chemical oxygen demand (COD) can be transformed from glucose into hydrogen. The rest of the energy is transformed into acetate. C6H12O6 12H2O 6HCO3 12H2 6H G 0 241 kJ mol1 C6H12O6 4H2O 2CH3COO 2HCO3 4H2 4H G 0 48 kJ mol1 C6H12O6 2H2O CH3CH2CH2COO 2HCO3 2H2 3H G 0 137 kJ mol1 (4)

(2)

(3)

C6H12O6 3H2O CH3CH2OH CH3COO 2HCO3 2H2 3H G 0 97 kJ mol1 (5) In contrast to the former reactions, production of propionate (Equation (6)) decreases the production of hydrogen, 9,21 as was shown experimentally by Shin et al.23 C6H12O6 2H2 2CH3CH2COO 2H2O 2H Undesirable consumption of hydrogen (reacion (7)) or glucose (reaction (8)) can be caused by the activity of homoacetogens such as Clostridium aceticum:24 2HCO3 4H2 H CH3COO 4H2O C6H12O6 2CH3CH2COO 2H (7) (8) (6)

Three enzymes are known to be responsible for hydrogen production: nitrogenase, Fe-hydrogenase and NiFehydrogenase. It has been shown that Fe-hydrogenase is the fastest hydrogen-producing enzyme owing to its highly reactive complex FeS center. This cluster is responsible for the enormous sensitivity of hydrogen producers to oxygen.11 Theoretically, 12 mol of hydrogen can be produced from one mol of glucose (reaction (2)), however, it is generally considered unrealistic.6,11 As seen from the values of standard Gibbs free energy (25C) of reactions (2), (3), (4) and (5) (the G 0 values were calculated based on data from Amend and Shock18), production of 12 mol of hydrogen (reaction (2)) is thermodynamically unfavorable. This is also under hyperthermophilic conditions.19 Transformation of the acetate produced to hydrogen is feasible via photosynthesis at very low hydrogen partial pressure and a temperature higher than 40C.19 There are metabolic pathways in microalgae (e.g. the oxidative pentose phosphate pathway) that can produce a stoichiometric amount (12 mol H2 mol1 glucose) of hydrogen.11 This pathway was demonstrated experimentally by Woodward et al.20 under nearly equilibrium conditions (low rate and very low hydrogen pressure). The summary reactions of possible fermentative hydrogen production from glucose ((3), (4) and (5)) show that the most desirable end-product is acetate, with production rate

In practice, fermentation with butyrate as the major product is considered to be the most effective hydrogenproducing pathway.25,26 Th is type of fermentation is carried out by Clostridium sp., 26 with a maximum hydrogen yield, demonstrated experimentally, of 2.9 mol H2 mol1 glucose.27 Indeed, VFAs are not the only product of acidogenesis: acidogenic bacteria are also very important organisms for industrial solvent production, e.g. ethanol or butanol.28 As shown by Lay, 25 hydrogen is produced mainly in the acid-forming, not in the solvent-production metabolism. The production trend of hydrogen/VFA is opposite to that of alcohols: the higher the hydrogen/VFA production, the lower the alcohol production.

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Practical aspects
Microorganisms employed Much data can be found in the literature regarding hydrogen production using pure cultures or mixed cultures of microorganisms. Although it is possible to achieve a higher hydrogen yield with use of a pure culture, 29 it is less useful for industrial applications, because the pure culture can easily be contaminated by various hydrogen utilizers, such as methanogens or sulfate-reducing bacteria. In addition, a mixed culture is more stable during the process of hydrogen production from biowaste.30,31 Clostridium sp.3234 and Enterobacter sp.35,36 are frequently reported to produce hydrogen from carbohydrates in pure culture. Rhodopseudomonas sp.,16 Citrobacter sp.37 and Bacillus sp.38 have also been used. Caldicellulosiruptor saccharolyticus has been used under hyperthermophilic conditions.39 Regarding mixed cultures, Clostridium sp. and Thermoanaerobacterium sp. are the species most often indicated under mesophilic and thermophilic conditions, respectively. Clostridium sp. and Enterobacter sp. are the most effective fermentative hydrogen producers. The main advantage of Enterobacter sp. is their resistivity to oxygen. In contrast, Clostridium sp. is known for its extreme sensitivity to oxygen, which can cause severe operational problems (via air contamination), especially when using pure cultures.40 However, Clostridia possess a substantially higher hydrogen yield than Enterobacter sp. (more then twice) and, moreover, it is able to form spores in response to unfavorable environmental conditions, such as lack of nutrients or rising temperature.6 Sporulation of Clostridia can be used to select these bacteria from natural environments (e.g. composts or anaerobic digester sludges). Various methods have been used to inhibit methanogens and other hydrogen consumers present in mixed cultures. Boiling the inoculum is a frequently used inhibitory method.4143 When solid materials (soya bean soil, potato soil, compost, etc.) have been used, they were also heattreated (104C for 2 h).44,45 Inhibitory agents (oxygen, 2-bromoethane-sulfonate and acetylene) have also been used,46 but this method is less successful and more expensive. Optimized reactor performance may be a better alternative than the inhibitory procedures. As the optimum pH for growth of Clostridium is in the range 4.55.5,25,42,47

methanogens (optimum pH around 7.0) can be suppressed by keeping the pH low in the reactor and operating with short hydraulic retention times.5,48 Substrates The sustainability of fermentative hydrogen production is very dependent on the substrate used, and it has been shown that the physicalchemical properties of the substrate strongly determine the overall efficiency of the process. Although laboratory studies have generally focused on pure substrates (mostly glucose, sucrose, starch and cellulose), full-scale applications require more complex substrates. For sustainable biohydrogen production, the feedstock has to meet the following criteria:6 high content of carbohydrates require minimum pre-treatment sufficient concentration that fermentative conversion and energy recovery is energetically favorable sustainable resources low cost.

Generally, the biodegradability of waste is related to carbohydrate materials, which are the main source of hydrogen production.43 Lay et al.49 found that the hydrogen-producing potential of carbohydrate-rich waste (rice and potato) was approximately 20 times larger than that of fat-rich waste (fat meat and chicken skin) and of protein-rich waste (egg and lean meat). Types of substrates The substrates usable for fermentative hydrogen production can be divided into four main groups: Pure substrates (e.g. glucose, cellulose, starch) Energy crops (e.g. Miscanthus, amaranth, grass, sugarbeet) Solid waste (e.g. food waste, organic fraction of municipal solid waste) Industrial wastewaters (e.g. wastewaters from tofu or sugar factory, wastewater from pulp and paper industry).

Pure substrates To date, the majority of research has been directed at pure substrates. Glucose and sucrose are the most often used pure

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chemicals. Tables 1 and 2 summarize the results of some of the research focused on these two compounds. These substrates are suitable for basic experimental work because of their simplicity and because the catabolic pathways of these compounds have been described in the past. However, the high price of these substrates prevents their utilization on an industrial scale.6

is cheap, and is produced in large quantities.67 Thus, this material is very suitable as a source of sustainable energy. Food waste is the most important part of the organic fraction of municipal solid waste: an important part of this originates from fruit and vegetables,68,69 and can be considered a lignocellulosic material.

Energy crops There are a huge number plants that are suitable for fermentative hydrogen production. The basic requirements of these plants are (1) high yield per land area, (2) high sugar content, (3) low lignin content and (4) low demand in terms of cultivation. Generally, energy crops may include sugarcontaining (e.g. sweet sorghum and sugar beet), starch based (e.g. corn and wheat), or lignocellulose based (e.g. fodder grass and Miscanthus) crops.6

Industrial solid waste Industrial solid waste suitable for fermentative hydrogen production is commonly of lignocellulosic nature, the most important fraction being the carbohydrates. Noike and Mizuno30 observed that the carbohydrate fraction of the waste was rapidly consumed just after inoculation, while soluble protein was hardly degraded for each substrate (bean curd manufacturing waste, rice bran and wheat bran), indicating that carbohydrate was the main source of hydrogen production. Waste from plants has often been used for hydrogen production; e.g. starch-manufacturing waste, 70 sweet potato starch residue,70 jackfruit peel,71 bean curd manufacturing waste,72 rice bran, wheat bran, 30 rice slurry42 or olive pulp.73

Sugar-containing crops Hussy et al.59 successfully used sugar beet extract as a substrate for continuous fermentative hydrogen production. The reactor fed by this substrate was operated for 45 days. Hydrogen production was very stable and hydrogen yield averaged 0.9 mol mol1 hexose converted from sugar beet water extract. In comparison to lignocellulosic energy crops, sugar beet offers several advantages. It is already commonly present in European crop rotation, and owing to its high water and sucrose content, it is well-established as a substrate for fermentation, e.g. to bioethanol.59

Lignocellulosic biomass Lignocellulosic biomass is widely available as a cheap raw material for biological energy production. However, it is known to be difficult to ferment. Successful biological conversion of lignocellulose to hydrogen strongly depends on its biodegradability by fermentative bacteria. The main focus in ongoing research is on delignification and hydrolysis of lignocellulosic biomass.66

Wastewaters Some industrial wastewaters are a suitable substrate for hydrogen production. Criteria for a suitable wastewater include: (1) high concentration of organic compounds (in terms of COD); (2) high proportion of readily degradable compounds (carbohydrates); and (3) higher temperature (if possible). Examples of suitable wastewaters are shown in Table 3. Yu et al.5 used rice winery wastewater as a substrate. Th is is a promising method because there are over ten thousand rice fermentation plants for the production of rice wine in China alone. Other authors have used sugar factory wastewater,74 noodle manufacturing waste,75 starch-rich wastewater,76 olive pulp wastewater77,78 or tofu wastewater79. Most of these wastewaters originate from the food industry and they meet the criteria for suitable substrates detailed above.

Solid waste Waste of biological origin (i.e. from municipal markets or food-processing industries) has high organic matter content,

Comparison of different substrates Complex substrates (solid as well as liquid) are more suitable for industrial use than pure substrates. The specific

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Table 1. Hydrogen yield and specific hydrogen production rate (HPR) from glucose. Hydrogen yield Seed
Soyabean-meal silo Enterobacter aerogenes Clostridium acetobutylicum soil from tomato plants 104C, 2 h Enterobacter aerogenes Activated and digested sludge from water reclamation plant Mixed anaerobic culture Soyabean-meal silo Waste sludge Mesophilic sewage sludge digester - acclimated for glucose Secondary sedimentation tank WWTP Sludge adapted to sucrose n.a. Citrobacter sp. Y19 Agricultural soil 104 C 2h Clostridium sp. Strain No. 2 n.a. Clostridium sp. Enterobacter sp. 1.85 2.10 2.49 2.80 2.92 Clostridium sp. 1.76 Clostridium sp. 1.44 15.0 18.3 Clostridium sp. 1.43 14.9 n.a. 1.42 14.8 11.9 11.9 12.8 14.7 Clostridium sp. 1.16 12.1 9.7 1.15 12.0 9.6 n.a. 0.98 10.2 8.2 n.a. n.a. 4.34 0.90 9.4 7.5 n.a. 0.90 9.4 7.5 n.a. Clostridium sp. 0.85 8.9 7.1 3.4 53 n.a. 7079 6064 n.a. 59

Temp. (C)

Reactor type

Dominating bacteria

(mol mol1)

(g kg1 COD) (% COD)* H2 in gas (%)

HPR (mmol g1 VSS h1)

Ref.
50 51 34 44 51 52

35

CSTR

37

CSTR - free cells

30

Trickle bed

26

Batch

37

CSTR immobilized cells

37

GAC-AFBR

35

SBR

0.8 7.4 n.a. 19.0

2540 n.a. n.a. 4454

53 50 54 26

35

CSTR - gas sparging

30

CSTR

35

CSTR

36

CSTR

19.2 21.9 25.9 29.2 30.4

15.4 16.4 20.7 23.3 24.3

7.6 8.4 32.3 7.0 20.4

64 64 n.a. 6072 n.a.

55 47 37 45 56

36

CSTR

36

Batch

30

CSTR

2007 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 1:201214 (2007); DOI: 10.1002/bbb

36

Batch

*Hydrogen yield expressed in percentage of glucose COD transformed into hydrogen COD.

n.a. not available.

Review: Fermentative hydrogen production

GAC-AFBR - granular activated carbon anaerobic uidized bed reactor.

Table 2. Hydrogen yield and specific hydrogen production rate (HPR) from sucrose. Hydrogen yield Dominating bacteria
n.a. n.a. n.a. n.a. 2.05 10.7 8.5 1.90 9.9 7.9 n.a. n.a. 1.14 5.9 4.7 2.2 1.03 5.4 4.3 3.6 2535 42 n.a. 6065

Review: Fermentative hydrogen production

Temp. (C)

Reactor type

Seed

(mol mol1)

(g kg1 COD) (% COD)* H2 in gas (%)

HPR (mmol g1 VSS h1)

Ref.
57 58 59 44

35

Fixed-bed reactor

Domestic sewage sludge - pH treatment

35

UASB

Domestic sewage sludge - pH treatment

32

CSTR sparging

Digested sludge

26

Batch

Soil from tomato plants

104C, 2 h Clostridium sp. n.a. C. pasteurianum Clostridium sp. Clostridium butyricum Clostridium sp. C. pasteurianum 4.80 4.26 4.02 21.0 22.4 25.0 3.76 19.6 3.43 17.9 2.88 15.0 12.0 14.3 15.7 16.8 17.9 20.0 2.15 11.2 9.0 0.1 39.5 1.9 1.2 10.1 1.2 5.9 61** 3547 51 63 38 63 55 60 61 62 63 64 63 65

36

Batch

Cow dung composter

35

CSTR

Waste sludge

35

CSTR

Anaerobic sewage sludge

26

CSTR

Secondary sedimentation tank of WWTP granular sludge

35

CIGSB

Sludge from WWTP acidic pretreatment

26

UASB

Sludge from secondary settling tam from WWTP

35

Batch

Anaerobic sewage sludge acclimated with sucrose

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* Hydrogen yield expressed in percentage of sucrose COD transformed into hydrogen COD.

** Maximum value. J Bartacek, J Zabranska, PNL Lens

CIGSB - carrier-induced granular sludge bed.

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Table 3. Characteristics of various wastewater types that have been used as substrate for fermentative hydrogen production. Wastewater type
COD (g L
1

Rice winery
29.535.4 15.018.7

Sugar factory
31.85 n.d. 9.85 n.d. 2.165 n.d. 0.02 9.5 74

Noodle manufacture
n.d. n.d. 5.62 0.32 n.d. n.d. n.d. 4.8 75

) )
1

BOD5 (g L

Carbohydrates (g L Protein (g L TN (g L TP (g L
1 1

n.d. n.d. 0.070.14 0.020.03 0.310.73 4.85.9 5

) )

SS (g L pH

Reference

n.d. not dened, TN total nitrogen, TP total phosphorus, SS suspended solids.

hydrogen yields from these materials are lower than from pure substrates, but the overall economics of the technology is more sustainable. The highest hydrogen yields (other than for pure substrates) were obtained from rice slurry (14.6% of COD utilized),42 food waste (19.3% COD utilized)43 and fi ltrate from waste sludge (24% COD utilized).29 As one of the most important process criteria is economic feasibility, the cheapest substrates are favored. Therefore, any kind of waste (especially food waste with a high sugar content) is suitable. Concentrated wastewaters from the food industry have the additional advantage of a low solids content. Thus, the hydrolysis of the feedstock does not influence the overall kinetics. Moreover, the reactor operation is much simpler with liquid substrates. Therefore, wastewaters from the food industry are an ideal substrate for hydrogen production. Technology Reactor design Systems for fermentative hydrogen production usually consist of two reactors. The first acidogenic reactor produces hydrogen (and carbon dioxide) and readily degradable organic compounds (VFAs, alcohols). The second methanogenic reactor produces methane. Since the substrate originating from the acidogenic stage is readily degradable, some type of high-rate anaerobic reactor can be employed for the second step. As a relatively low amount of substrate COD is transformed into hydrogen (for very good

substrates, a maximum of 25%; normally only 510 %), the second step is necessary to further utilize the remaining organic matter.80 Any of the current anaerobic reactor designs can be used for liquid substrates (wastewater), e.g. UASB (upflow anaerobic sludge blanket58), fluidized bed reactor,81 fi xedbed bioreactor57 or (the most often used at laboratory scale) completely stirred tank reactor (CSTR).50 Recently, the unsaturated trickle-bed reactor was developed by Zhang et al.34 CSTRs are most often used as continuously working reactors (Tables 1 and 2), the reason being their simplicity of building and operation. However, this system does not allow use of high organic loading rates (OLR) and high biomass concentrations. The volumetric hydrogen production range is, therefore, relatively low.24 The highest reported biomass concentration (expressed as volatile suspended solids-VSS) using a CSTR (with sucrose as the substrate) was only 5.7 g VSS L1.82 The typical values (using pure substrates) lie within the range 1.52.5 g VSS L1.50,61,83 To increase the biomass concentration, various separation techniques have been used. Oh et al.83 used membranes to increase the biomass concentration (expressed as mixed liquor suspended solids-MLSS) in a CSTR from 2.2 to 5.8 g MLSS L1. Ren et al.84 used a CSTR with an internal three-phase separator and achieved 10.6 g VSS L1. Similar to methane-producing reactors, biomass immobilization is widely used for hydrogen-producing microorganisms. Theoretically, biomass retention can allow growth of

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slow-growing bacteria, which can consume hydrogen (e.g. methanogens). However, this phenomenon has not been confirmed experimentally.24 Many authors have used reactors with granular sludge (e.g. UASB). The usual biomass concentration in such reactors is 68 g VSS L1, 58,85,86 but Fang and Zhang63 achieved 20 g VSS L1. Immobilization using a support medium is also possible. The best results were obtained by Chang et al.57 with activated carbon (15.8 g VSS L1), Lee et al.87 with activated carbon (26.0 g VSS L1), Oh et al.88 with fibrous polymeric material (24 g VSS L1) and Wu et al.89 with activated carbon powder (32.5 g VSS/L). Tables 1 and 2 illustrate that the specific hydrogen production rates achieved with immobilized biomass reactors is not substantially higher than the rate in a CSTR. Also, the hydrogen yield is similar for both systems. However, owing to the higher biomass concentration, the volumetric hydrogen production rate (mol hydrogen.day1 L1 reactor) can be increased by up to three times. For solid substrates, some type of discontinuously or semicontinuously working reactor must be used. Leaching-bed reactors have often been used.69 A system of four semicontinuous leaching-bed reactors for hydrogen production coupled to a UASB reactor (as the second stage) was designed by Shin et al.90 for solid waste (e.g. food waste) degradation.

Operational parameters pH Hydrogen is produced during the exponential growth phase of Clostridia. When the population reaches the stationary growth phase, the reactions shift from the hydrogen/acids production phase to a solvent production phase. This shift can be enabled by an unsuitable level of pH. Thus, it is important to control the pH within an optimum range to maintain hydrogen production.91 Lay25 found pH 5.05.5 to be optimum (considering the hydrogen production rate) for hydrogen production with Clostridium-rich sludge. Values outside this interval supported solvent production. As mentioned above, other bacteria can dominate the sludge. Then, the pH optimum will be different. Because there are many conflicting recommendations from various authors, several conclusions regarding the optimum pH are summarized in Table 4. Liu et al.92 tested thermophilic (55C) hydrogen production under various pH levels with the dominating bacterium Thermoanaerobacterium sp. The optimum (initial) pH value (considering hydrogen yield as well as hydrogen production rate) was 6.5. Yokoi et al.51 and Fabiano and Perego93 stated that the optimum pH for Enterobacter aerogenes was within the range of 6.06.5. Citrobacter sp., a chemoheterotrophic bacterium, requires a pH of about 7.0.37

Table 4. Optimum pH range for fermentative hydrogen production. Dominating bacteria

n.d. n.d. Clostridium sp. Clostridium sp. Clostridium sp. Clostridium sp. Thermoanaerobacterium sp. Thermoanaerobacterium sp. Enterobacter aerogenes Enterobacter aerogenes Citrobacter sp. Rhodobacter sp. n.d. not dened. * All the optimum values correspond to the maximum hydrogen production rate. ** The values in parentheses correspond to the maximum hydrogen yield.

Optimum pH value*
5.25.5 5.5 4.55.5 4.5 (5.5)** 5.56.0 (4.5)** 5.05.5 6.5 7.0 (6.0)** 6.06.5 6.06.5 7.0 7.5

Reference
75 5 47 42 90 25 93 76 92 51 37 70

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Temperature Most of the work in the area of fermentative hydrogen production has focused on mesophilic temperatures (about 37C), because most of the hydrogen-producing Clostridia prefer these conditions. However, thermophilic bacteria (e.g. Thermoanaerobacterium sp.) can be employed. Lin and Chang 95 studied the process at ambient temperature (around 20C). The thermophilic process has the potential to achieve a greater hydrogen yield and higher hydrogen production rate. Yu et al.5 compared the hydrogen production at 20, 30, 35 and 55C: the highest temperature enabled the highest hydrogen yield. Zhang et al.,76 Shin et al.23 and Cheng et al.96 stated that the thermophilic (55C) hydrogen production rate was substantially higher than the mesophilic (3537C) hydrogen production rate. Shin et al.23 obtained a nine times higher hydrogen production rate under thermophilic conditions (55C) when food waste was used as a substrate, compared to rates at mesophilic (35C) temperature. Lin and Chang 95 studied the process at ambient temperatures (1534C). They obtained an average hydrogen yield of 1.15 mol mol1 glucose, which is rather low compared to that at mesophilic temperature (usually 1.42.9 mol mol1 at 37 C).

with photo-bioreactors as suggested by Claassen and de Vrije17 or Oh et al.16 However, two-step processes are still in the research phase and far from full-scale application. To fi nd a complex solution for biological energy production from organic substrates, fermentative hydrogen production can be included among the so-called biorefi neries.99 Th is concept is based on gradual production of liquid (ethanol) and gaseous (hydrogen, methane) energy carriers. Thus, minimum residues will remain and a maximum amount of substrate will be utilized and recycled. Moreover, the diversity in products can contribute to higher flexibility and sustainability of the technology. Much research must be performed to reach hydrogen yields comparable with the theoretical efficiency maximum. Although a relatively high efficiency has been reached using pure substrates, the low hydrogen yield with complex (real) substrates remains a great challenge. Another limit is the relatively pure substrate variability. Since hydrogen can be formed only from carbohydrates, many available substrates are not usable for hydrogen production. The practical value of the process would dramatically increase if the number of usable substrates could be expanded, e.g. to fats and proteins. For the same reason, a cheap and effective method of substrate pre-treatment should be developed. The optimal reactor design also remains an important issue. Many recent publications have focused on this topic.9,24 However, the majority of the research is still conducted on batch reactors or CSTRs. The main issues in this area are (1) retention of the hydrogen-producing microorganisms and (2) recovery of end-products. As the current methods for hydrogen (or carbon dioxide) release from the reaction systems are far from practical, prevention of endproduct toxicity must be studied intensively. The required quality of the hydrogen gas produced depends on the type of utilization (e.g. various types of fuel cells), and gas treatment (to achieve the required quality) may involve several steps.100 Hydrogen sulfide, siloxanes, water, ammonia and carbon dioxide may be present in the hydrogen-rich fermentation gas.100,101 To date, these compounds have been removed using expensive physicalchemical methods (especially hydrogen sulfide and siloxanes). Further research should be focused on developing less costly biological treatment processes.101,102

Challenges
Despite the huge number of scientific papers published on biological hydrogen production, practical applications of the process remain few. So far, few pilot-scale applications have been reported,97 and no full-scale process has yet been reported. The low efficiency of the hydrogen production process remains the main limiting factor. The maximum theoretical hydrogen production is well below the stoichiometric amount (e.g. only 4 mol of hydrogen can be produced from 1 mol of glucose). This results in relatively low energy utilization only 33% of the COD can be transformed from glucose into hydrogen. The remainder is mainly formed as VFAs (acetate, butyrate). These facts imply that hydrogen production has always to be coupled with a second step methane production98 or another VFA/solvents utilization process. One of the most promising options is a coupling

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Recently, improvements in hydrogen production have been achieved through genetic modification of hydrogenproducing bacteria. The main principles of genetic engineering include: (1) overexpression of cellulases, hemicellulases and lignases to maximize substrate availability, (2) elimination of hydrogen-consuming hydrogenases and (3) overexpression of hydrogen-producing hydrogenases.103 Increase of cellulolytic activity (the first principle) has been achieved for Clostridium beijerinckii.19 Elimination of hydrogen-utilizing hydrogenases (the second principle) has been most often used with cyanobacteria.104,105 Overexpression of formate hydrogen lyase (FHL) (the third principle) has recently been reported with Escherichia coli.106108 The FHL complex consists of formate dehydrogenase, and electron transfer mediators106 present in various microbial genera, including Enterobacter, Methanogenes and photosynthetic bacteria.109 Penfold et al.107 achieved a doubled hydrogen production rate with the modified E. coli strain HD701 in comparison with the parent strain MC4100. Even better results were achieved by Yoshida et al.109, who obtained a 2.8-fold higher hydrogen production rate with a modified E. coli strain SR13. References
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