Dark fermentation on biohydrogen production: Pure culture

Bioresource Technology 102 (2011) 8393–8402

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Dark fermentation on biohydrogen production: Pure culture Duu-Jong Lee a,b,⇑, Kuan-Yeow Show c, Ay Su d a

Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan c Department of Environmental Engineering, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Jalan University, Bandar Barat, 31900 Kampar, Perak, Malaysia d Fuel Cell Center, Department of Mechanical Engineering, Yuan Ze University, Taoyuan 320, Taiwan b

a r t i c l e

i n f o

Article history: Received 12 January 2011 Received in revised form 14 March 2011 Accepted 16 March 2011 Available online 21 March 2011 Keywords: Biohydrogen Biofuel Pure cultures Dark fermentation

a b s t r a c t Biohydrogen is regarded as an attractive future clean energy carrier due to its high energy content and environmental-friendly conversion. While biohydrogen production is still in the early stage of development, there have been a variety of laboratory- and pilot-scale systems developed with promising potential. This work presents a review of literature reports on the pure hydrogen-producers under anaerobic environment. Challenges and perspective of biohydrogen production with pure cultures are also outlined. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Hydrogen is a promising alternative to conventional fossil fuels because it has the potential to eliminate most of the problems that the fossil fuels create, and hydrogen itself is proposed as the ultimate transport fuel for cars, trucks, and buses (Forsberg, 2007). However, a major doubt on hydrogen as a clean energy alternative is that most of the hydrogen gas at present is generated from fossil fuels by thermochemical processes, such as hydrocarbon reforming, coal gasification and partial oxidation of heavier hydrocarbons. Biohydrogen production, unlike their chemical or electrochemical counterparts, is catalyzed by microorganisms in an environment at ambient temperature and pressure. Microorganisms can recover and concentrate the energy from aqueous organic resources such as, industrial wastewater and sludge in a usable form. Biohydrogen production, in a sense, is an entropy reducing process, which could not be realized by mechanical or chemical systems. Moreover, biological techniques are well suited for decentralized energy production in small-scale installations in locations where biomass or wastes are available, thus avoiding energy expenditure and costs for transport. Studies on biohydrogen production have been focused on biophotolysis of water using algae and cyanobacteria, photo-decompo-

sition of organic compounds by photosynthetic bacteria and dark fermentation from organic compounds with anaerobes. Among these biological processes, anaerobic hydrogen fermentation seems to be more favorable, since hydrogen is yielded at a high rate and various organic waste and wastewater enriched with carbohydrates as the substrate results in low cost for producing hydrogen. Extensive research in the past two decades have reviewed promising prospect of biohydrogen production via dark fermentation. There have been substantial improvement and development in both the yield and volumetric production rates of hydrogen fermentations. For realistic applications that make economic sense, hydrogen yields and production rates must at least surpass considerably the present achievements, if not the metabolic threshold of 4 H2/glucose. Technological breakthrough must be sought after in order to extract most of the hydrogen from the substrate, if not all. Investigation addressing this challenge should be viewed as one of the focuses of future research. Study of microbiological aspects of biohydrogen production is still in the early stage of development. As most of the current research is focusing on pure culture, pure hydrogen producers reported in dark fermentation literature were reviewed and discussed in the present work. 2. Dark fermentation pathway

⇑ Corresponding author at: Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China. Tel.: +886 2 27376545; fax: +886 2 23623040. E-mail address: [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.041

Dark fermentative hydrogen production is a ubiquitous phenomenon under anoxic or anaerobic conditions. Many bacteria use reduction of protons to hydrogen via hydrogenases as a means

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of oxidizing the carriers reduced during fermentation, which is required to allow the carriers to recycle and maintain electrical neutrality so that a continuous supply of adenosine triphosphate (ATP) can be generated by substrate-level phosphorylation (Adams et al., 1980). Molecular hydrogen formation is generally followed two routes in the presence of specific coenzymes, i.e., either by formic acid decomposition pathway or by the re-oxidization of nicotinamide adenine dinucleotide (NADH) pathway [Eqs. (1) and (2)]. 2þ

NADH þ Hþ þ 2Fd þ

þ hydrogenase

2Fd 2H

!

þ

! 2Hþ þ NADþ þ 2Fd 2þ

2Fd

þ H2

ð1Þ ð2Þ

The Embden–Meyerhof or glycolytic pathway is applied to convert glucose into pyruvate associated with the conversion of NADH from NAD+ via anaerobic glycolysis, which could be represented by Eq.(3):

C6 H12 O6 þ 2NADþ ! 2CH3 COCOOH þ 2NADH þ 2Hþ

ð3Þ

Hydrogen is generated through the re-oxidization of NADH by some specific microorganisms under acidogenic conditions in the presence of ferredoxin oxidoreductase and hydrogenase (Tanisho et al., 1998). This metabolic route is present in some species of Clostridium. The obligate anaerobic Clostridia lack a typical cytochrome system and obtain energy by substrate-level phosphorylation during fermentation. Oxidizing carbohydrates generates electrons which need to be disposed of to maintain electrical neutrality. For the saccharolytic Clostridia, hydrogen evolution via a hydrogenase is a major route through which the cells dispose excess electrons produced from the oxidative breakdown of carbohydrates (Chen and Mortenson, 1974). NADH-ferredoxin reductase functions primarily as an electron carrier and is involved in pyruvate oxidation to acetyl-CoA and carbon dioxides as well as proton reduction to molecular hydrogen. Clostridia break down pyruvate to acetyl-CoA to produce 2 mol of NADH and 2 mol of reduced ferredoxin. Four moles of hydrogen per mole of glucose is achieved, which is the theoretical maximum yield of dark hydrogen fermentation if all of the substrate would be converted to acetic acid. If all the substrate would be converted to butyric acid, this value is two moles of hydrogen per more of glucose, since 2 mol of NADH has been consumed during the conversion of intermediate products. The available hydrogen from glucose fermentation is determined by the ratio of butyrate/acetate produced during fermentation. In the cases mentioned above, no NADH is used as a reductant for alcohol production. However, the disposal of electrons via pyruvate-ferredoxin oxidoreductase or NADH-ferredoxin oxidoreductase and hydrogenase might be affected by the corresponding NADH and acetyl-CoA levels as well as environmental conditions. As a result, the oxidation–reduction state has to be balanced through the NADH consumption to form some reduced compounds, i.e., lactate, ethanol and butanol, resulting in a lowered hydrogen yield. In the anaerobic Clostridia formate is not a major intermediate in the breakdown of pyruvate, although such organisms possess formate dehydrogenase (Adams et al., 1980). In contrast, the facultative anaerobes, especially members of the family Enterobacteriaceae, can metabolize pyruvate to formic acid and other products in a process sometimes called the formic acid fermentation. As with the Clostridia these organisms have to dispose of excess reductant produced during fermentation, and this is accomplished by the eventual production of hydrogen. Pyruvate is first converted into to formic acid by pyruvate-formate lyase with the production of acetyl-CoA, and energy is conserved by the formation of ATP via acetyl phosphate. Under anaerobic condition and in the absence of suitable electron acceptors, the formic acid will be further

degraded into hydrogen and carbon dioxides via formic hydrogenlyase [Eqs. (4) and (5)].

CH3 COCOOH þ H CoA ! CH3 COCoA þ HCOOH

ð4Þ

HCOOH ! H2 þ CO2

ð5Þ

It is noted that NADH produced during anaerobic glycolysis is rarely used for hydrogen production by the bacteria of genus Enterobacteriaceae, due to the absence of specific coenzymes such as ferredoxin oxidoreductase. Nevertheless, NADH must still oxidized back to NAD+, otherwise anaerobic glycolysis will cease. Many microorganisms solve this problem by slowing or stopping pyruvate dehydrogenase activity and using pyruvate or one of its derivatives as an electron and hydrogen acceptor for the re-oxidization of NADH in a fermentation process. As a result, the oxidation– reduction state has to be balanced through the NADH consumption to form a large amount of mixed acids and alcohols, most of which are the hydrogen-containing reduced products, are accompanied with the formation of formate. There are two types of formic acid fermentation. Mixed-acid fermentation results in the excretion of ethanol and a complex mixture of acids, particularly acetic, lactic, succinic and formic acids. This pattern is seen in Escherichia, Salmonella, Proteus, and other genera. The second type, butanediol fermentation, is characteristics of Enterobacter, Serratia, Erwinia, and some spices of Bacillus. Pyruvate is converted to acetoin, which is then reduced to 2,3-butanediol with NADH. At the same time, ethanol is also produced, together with smaller amount of the acids found in mixed acid fermentation. Production of these reduced compounds limits hydrogen release in a gas form and results in a lowered hydrogen yield which is generally less than 2 mol-H2/mol-glucose by facultative anaerobes compared with anaerobic Clostridia. The biochemical reactions involved in thermophilic hydrogen production seem different from those mesophiles as mentioned above. Conversion of glucose to pyruvate might be accomplished through either an Embden–Meyerhof pathway (eubacterium Thermotoga maritima) or a modified Embden-Meyerhof pathway (archaeon Thermococcales, pyrococcus furiosus), dependent on microbial population. The fermentation following the latter pathway is not coupled with ATP synthesis; energy is conserved in the course of acetate formation from acetyl-CoA catalyzed by acetyl-CoA synthetase (ADP forming). Based on glucose fermentation in growing cultures of hyperthermophilic Thermotoga maritime (eubacterium), Schröder et al. (1994) proposed a metabolic route of glucose fermentation in such a hyper-thermophilic eubacterium. Acetate is found to produce as the unique soluble metabolite from the pyruvate, whereas hydrogen and carbon dioxide are gaseous products. The enzymes found in T. maritime, which are involved in pyruvate conversion to acetate (pyruvate: ferredoxin oxidoreductase, phosphate acetyltransferase and acetate kinase) and in hydrogen formation (NADH: ferredoxin oxidoreductase and hydrogenase) are typical for anaerobic bacteria, such as Clostridia, indicating that the re-oxidization of NADH pathway is followed to form hydrogen. Such a metabolic pathway of pyruvate conversion to acetate was also reported for Thermococcus kodakaraensis KOD1 (archaeon) (Kanai et al., 2005). Besides acetate, however, a reduced end product of L-alanine is formed in another metabolic branch. Such a acetate–alanine pathway has also been reported in carbohydrate fermentation with other species of Thermococcales (Kanai et al., 2005). Alanine is formed by alanine aminotransferase directly from pyruvate via transamination with glutamate. In P. furiosus and T. litoralis, alanine production was found to increase with increased H2 partial pressure, suggesting that the generation of H2 and alanine are competitive means of disposing intracellular reducing equivalents.

D.-J. Lee et al. / Bioresource Technology 102 (2011) 8393–8402

Preventing alanine formation should contribute to an increase in microbial H2 production. A great deal of bacterial species with vastly different taxonomic and physiological characteristics can produce hydrogen via biochemical reactions. Hydrogen-producing microorganisms use hydrogenase and/or nitrogenase enzyme as hydrogen yielding protein. This enzyme regulates the hydrogen-metabolism of uncountable prokaryotes and some eukaryotic organisms including green algae. The function of nitrogenase and/or hydrogenase enzyme is associated with the use of the products of photosynthetic reactions that generate reactants from water. Combination of the enzymes of the pentose phosphate pathway with hydrogenase has been demonstrated to increase the hydrogen yield from glucose to nearly theoretical yield (Woodward et al., 2002). The instability of the commercially available mesophilic enzymes as well as that of the cofactor NADP+ is an obstacle for practical application of the method to hydrogen production. Improvement of enzyme and cofactor stability was pursued by employment of thermophilic enzymes and encapsulation of the enzymes in a gas-permeable matrix. To obtain thermophilic enzymes, the pentose phosphate pathway enzymes from the thermophile Thermotoga maritime are being cloned and expressed in Escherichia coli. Primers were designed for the cloning of the target genes. The genes encoding the two NADP-dependent dehydrogenases, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were cloned into the vector PCR2.1 and expressed in E. coli. Production of hydrogen by enzymes in cell-free extracts of T. maritima, alone or in combination with Pyrococcus furiosus hydrogenase, was demonstrated. Studies were initiated on a novel method for practical implementation of enzymes for production of hydrogen, the incorporation of enzymes and cofactors into stable liposomes that will function as nanobioreactors. Hydrogenase and glucose dehydrogenase were encapsulated in liposomes and subsequently demonstrated to produce hydrogen (Woodward et al., 2002). 3. Hydrogen-producing microbes According to operating temperature conditions for microbial cultures, hydrogen production studies are conducted in four temperature regimes: ambient (15–30 °C), mesophilic (30–39 °C) and thermophilic (50–64 °C) and hyper-thermophilic (>65 °C). 3.1. Ambient and mesophilic strains Hydrogen is consumed by the consortia as it is produced, mainly by methanogenic archaea, acetogenic bacteria and sulfate-reducing bacteria. Diverse microbes capable of hydrogen production are distributed across a wide variety of bacterial groups. Research studies on anaerobic microbes have been intensively developed in recent years, and some new or efficient bacterial species and strains for dark hydrogen fermentation have been isolated and recognized (Fang et al., 2002). In general, the isolated and identified mesophiles are mainly affiliated with two genera: facultative Enterobactericeae (Kumar and Das, 2000) and strictly anaerobic Clostridiaceae (Collet et al., 2004; Evvyernie et al., 2001; Wang et al., 2003), whereas most thermophiles belong to genus Thermoanaerobacterium (Ahn et al., 2005; Ueno et al., 2001; Zhang et al., 2003). In addition, aerobes such as Bacillus (Kalia et al., 1994; Kumar et al., 1995; Shin et al., 2004), Aeromonons spp., Pseudomonos spp. and Vibrio spp. (Oh et al., 2003b) have been cultivated or isolated, but their hydrogen yields are generally less than 1.2 mol-H2/mol-glucose under anaerobic conditions. Xu et al. (2008) achieved a hydrogen yield of 2.26 mol-H2/mol-glucose using a new strain Ethanoligenens harbinense B49.

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Since facultative anaerobes can be grown more easily than obligate ones, attempts have been made to work with pure cultures of facultative anaerobes of genus Enterobactericeae. Tanisho and Ishiwata (1995) isolated and characterized as E. aerogenes strain E. 82005, and some other bacterial strains such as E. aerogenes strain HO 39, E. aerogenes HU-101 strain AY2 (Rachman et al., 1997) and E. cloacae IIT-BT 08 (Kumar and Das, 2000; Kumar et al., 2001) were also harvested by different researchers. Hydrogen yields of Escherichia species as obtained in a pure culture of E. coli NCIMB 11943 are in a range of 0.2–1.8 mol-H2/mol-hexose while fermenting glucose or starch hydrolysate (Perego et al., 1998), whereas the hydrogen yields demonstrated by the pure cultures of Enterobacter species are much higher, ranging from 1.1 mol-H2/mol-hexose to ca. 3.0 mol-H2/mol-hexose (Fabiano and Perego, 2002; Kumar and Das, 2000; Palazzi et al., 2000; Rachman et al., 1997). In addition, the enhanced hydrogen yields can be achieved through redirection of metabolic pathways of facultative anaerobes such as E. aerogenes and E. cloacae (Kumar et al., 2001; Nath and Das, 2004). The yield was reportedly increased to 3.8 mol/mol-glucose by blocking the pathways of organic acid formation using the proton-suicide technique with NaBr and NaBrO3 on bacterial species of E. cloacae (Kumar et al., 2001). A similar enhancement of hydrogen yield using E. aerogenes HU-101 was reported while blocking the formation of alcoholic and acidic metabolites by both allyl alcohol and the proton suicide technique (Rachman et al., 1997). Hydrogen yield might also be improved through genetic modification of facultative anaerobes. Chittibabu et al. (2006) demonstrated the fermentative hydrogen production by hydA over-expressed recombinant gene (Fe-hydrogenase coded gene from E. cloacae IIT BT 08) in non-hydrogen producing E. coli BL-21. The yield of hydrogen with recombinant E. coli BL-21 is 3.12 mol-H2/mol-glucose, which is much higher than that reported for the wild strain E. cloacae IIT BT 08. The hydrogen production capabilities of pure cultures of microorganisms are tabulated in Table 1. Oh et al. (2002, 2003a,b) have successfully isolated two new microorganisms producing H2 from CO and water, Rhodopseudomonas palustris P4 and Citrobacter sp. Y19, both affiliated with genus Enterobactericeae. Rps. palustris P4, isolated from a sludge digester, is a facultative anaerobe and can grow in a photoautotrophic or chemoheterotrophic manner. Citrobacter sp. Y19 isolated from an anaerobic wastewater sludge digester, is also a facultative anaerobe. The authors pointed out that hydrogen production is observed only under anaerobic conditions for both strains. Rps. palustris P4 and Citrobacter sp. Y1 can produce hydrogen from the sugars under a wide range of pH (5–9) and temperature (25–40 °C), giving the maximum hydrogen yields of 2.8 mmol-H2/mmol-glucose (Oh et al., 2002) and 2.5 mol-H2/mol-glucose (Oh et al., 2003a), respectively. Among the fermentative anaerobes, Clostridia have been well known and studied extensively, not for their hydrogen production capability but for their role in the industrial solvent production from various carbohydrates. The hydrogen yields of the pure cultures belonging to genus Clostridiaceae have been examined, including C. paraputrificum (Evvyernie et al., 2001), C. lentocellum (Ravinder et al., 2000), C. thermosuccinogenes, C. bifermentans (Wang et al., 2003), C. thermolacticum (Collet et al., 2004), C. butyricum (Chen et al., 2005), C. saccharoperbutylacetonicum (Ferchichi et al., 2005b), C. acetobutylicum (Zhang et al., 2006a,b) and C. pasteurianum. The optimum hydrogen yields observed of each species vary between 1.1 mol-H2/mol-hexose and 2.6 molH2/mol-hexose, dependent on the organism per se as well as environmental conditions (Table 1). Clostridia are strict anaerobes and extremely sensitive to oxygen so that the presence of a trace amount of dissolved oxygen can completely stop the hydrogen production. Addition of E. aerogenes,

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Table 1 Microbial yield and production rate by pure cultures of dark fermentative microorganisms. Substrate

Culture operation

Temperature

H2 yield (mol-H2/mol hexose or glucose)

Hydrogen production rate (L/Lh)

References

Facultative anaerobes E. coli NCIMB 11943 E. aerogenes NCIMB 10102 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. cloacae IIT-BT08 E. aerogenes HU-101 AY2 Enterobacter aerogens DM11 Escherichia coli BL-21 Klebsiella oxytoca HP1 Rhodopseudomonas palustris P4 Citrobacter sp. Y19 F.p 01

Starch hydrolysate Starch hydrolysate Glucose Sucrose Cellobiose L-Arabinose Fructose Maltose Potato starch C.M. cellulose Glucose Glucose Glucose Sucrose Glucose Glucose Maltose

Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Continuous Continuous Batch Batch Batch

37 40 36 36 36 36 36 36 36 36 37 36 37 38 37 36 36

1.8 1.1 2.2 3.0 2.7 1.5 1.6 1.4 – – 1.2 3.80 3.12 1.30 2.76 2.49 2.52

0.33 0.24 0.45 0.66 0.65 0.36 0.44 0.18 0.20 0.09 – – 2.18 0.35 – – –

Perego et al. (1998) Perego et al. (1998) Kumar and Das (2000) Kumar and Das (2000) Kumar and Das (2000) Kumar and Das (2000) Kumar and Das, (2000) Kumar and Das (2000) Kumar and Das (2000) Kumar and Das (2000) Rachman et al. (1997) Kumar et al. (2001) Chittibabu et al. (2006)

Strict anaerobes C. amygdalinum C9

Xylan

Batch

37 (pH 7.5)

0.04

–

C. amygdalinum C9

Xylose

Batch

2.2–2.5

–

C. amygdalinum C9

Arabinose

Batch

37 (pH 7.5– 8.5) 37 (pH 8.5)

1.78

–

C. amygdalinum C9

Starch

Batch

37 (7.5)

390 ml/g@

–

C. C. C. C. C. C. C. C. C.

Glucose Glucose Lactose Glucose Starch N-acetyl-v-glucosamine (GlcNAc) Ball-milled Raw shrimp and lobster shells Acid/alkali treated raw shrimp and lobster shells Corn fiber Cellobiose Glucose Wastewater sludge Sucrose Glucose Lactose

Continuous Continuous Continuous Batch Batch Batch Batch Batch Batch

30 36 58 36 36 45 45 45 45

2.3 2.3 1.5 2.0 1.8 2.4a 1.5a 1.2a 1.3a

– 1.15 0.06 0.66 0.41 – 0.21 0.06 0.1

Batch Batch Batch Batch Batch Continuous Batch

45 45 45 35 37 30 30

1.1 1.4 1.1 2.1b 1.39 1.08 1.41

– – – – 0.21 0.22 0.12

Evvyernie et al. (2001) Evvyernie et al. (2001) Evvyernie et al. (2001) Wang et al. (2003) Chen et al. (2005) Zhang et al. (2006) Ferchichi et al. (2005)

Sucrose

Batch

30

1.42

0.20

Ferchichi et al. (2005)

Maltose

Batch

30

1.39

0.12

Ferchichi et al. (2005)

Glucose

Batch

30

1.37

0.16

Ferchichi et al. (2005)

butyricum SC E1 butyricum IFO13949 thermolaiticum DSM 2910 beijirinchi AM21B beijirinchi AM21B paraputrificum M-21 paraputrificum M-21 paraputrificum M-21 paraputrificum M-21

C. paraputrificum M-21 C. paraputrificum M-21 C. paraputrificum M-21 C. bifermentans C. butyricum CGS5 C. acetobutylicum ATCC 824 C. saccharoperbutylacetonicum ATCC 27021 C. saccharoperbutylacetonicum ATCC 27021 C. saccharoperbutylacetonicum ATCC 27021 C. saccharoperbutylacetonicum ATCC 27021

Oh et al. (2002) Oh et al. (2003a) Zhao et al. (2010) Jayasinghearachchi (2010) Jayasinghearachchi (2010) Jayasinghearachchi (2010) Jayasinghearachchi (2010)

et al. et al. et al. et al.

Yokoi et al. (1995) Collet et al. (2004)

Evvyernie Evvyernie Evvyernie Evvyernie

et et et et

al. al. al. al.

(2001) (2001) (2001) (2001)

D.-J. Lee et al. / Bioresource Technology 102 (2011) 8393–8402

Genus classification

Fructose

Batch

30

1.20

0.14

Ferchichi et al. (2005)

Cheese whey

Batch

30

1.35

0.14

Ferchichi et al. (2005)

Cellobiose (with phenol or cresols) Distillery wastewater Sucrose

30 37 (pH 5.0) 37 (pH 7.5)

3.5+ 3.35 5.85#

– – 0.67

(Ho et al. (2010) Kamalaskar et al. (2010) Lo et al. (2010)

Halanaerobium saccharolyticum Halanaerobium senegalensis Ethanoligenens harbinense Escherichia coli (engineered)%

Glycerol Glycerol Glucose Glucose

batch batch Continuous (followed by photo reactor) Batch Batch Batch Batch

37 (pH 7.4) 37 (pH 7.0) 35 (pH 3.5) 37

0.58 1.21 2.26 1.82

– – – –

Kivisto et al. (2010) Kivisto et al. (2010) Xu et al. (2008) Mathews et al. (2010)

Starch Glucose Paper sludge hydrolysate Sucrose Glucose Glucose Hydrolyzed potato steam peels Glucose Hydrolyzed potato steam peels Xylose

Continuous Batch Batch Batch Batch Batch Batch Batch Batch Batch

3.33 4.00 3.84 3.33 3.33 3.4 3.4 2.9 3,3 2.0*

0.21 – 0.12 0.20

Kanai et al. (2005) Schröder et al. (1994)

Bagasse

Continuous

85 80 70 70 65 70 70 80 80 30 50 (pH 4.8) 38

1.60

0.35

van Niel et al. (2002) van Niel et al. (2002) Mars et al. (2010) Mars et al. (2010) Mars et al. (2010) Mars et al. (2010) Long et al. (2010) Long et al. (2005) Wu et al. (2010)

Wheat slurries

Continuous

38–40

0.20

–

Kalia et al. (1994)

(Hyper)thermophiles Thermococcus kodakaraensis KOD1 Thermotoga maritime Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Thermotoga elfi Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Thermotoga neapolitana Thermotoga neapolitana Enterobacter sp. CN1 Klebsiella oxytoca HP1 Klebsiella oxytoca HP1 Aerobes Bacillus licheniformis Jk1 a b @ + # * %

mol/mol-GlcNAc. mmol H2/g-COD. ml-H2/g starch. mol-H2/mol-cellobiose Dark fermentation + photo fermentation. mol-H2/mol xylose. E. Coli with lactate dehydrogenase and fumarate reductase deleted.

– – – – –

D.-J. Lee et al. / Bioresource Technology 102 (2011) 8393–8402

C. saccharoperbutylacetonicum ATCC 27021 C. saccharoperbutylacetonicum ATCC 27021 Clostridium sp. R1 Clostridium sp. DMHC-10 C. butyricum CGS5

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a facultative anaerobe, might accelerate the reactor startup by eliminating the inhibition of oxygen or other factors on Clostridia, which has been validated by Yokoi et al. (1998) in batch and continuous fermentation on starch. The authors found that hydrogen yield and metabolite distribution of the mixed culture are equivalent to those of C. butyricum when culture pH is controlled at 5.0 to 5.5. This is mainly attributed to the facts that E. aerogenes could not degrade starch directly and the cells of C. butyricum are substantially predominated over E. aerogenes in the mixed cultures. As a consequence, only few cells of E. aerogenes might be surviving in the mixed culture by assimilating of polypeptone and a very slight amount of sugars such as glucose and maltose derived from starch decomposed by the amylase produced by C. butyricum. Clostridium may form endospores, which can be considered ‘‘survival mechanisms’’ developed by these organisms when unfavorable environmental conditions are encountered (e.g., high temperature, desiccation, lack of carbon or nitrogen source, and chemical toxicity) (Sung et al., 2002). It should be realized that several highest hydrogen production rates reported so far were achieved in the cell-immobilized (granules or biofilms) systems wherein Clostridia were formed predominant in the immobilized cultures (Lee et al., 2004; Wu et al., 2006; Zhang et al., 2007b). Among them, the culture population with the highest hydrogen production rate was dominated by a presumably excellent hydrogen-producing bacterial species identified as C. pasteurianum (Wu et al., 2006). 3.2. Thermophilies and hyper-thermophilies Karadag and Puhakka (2010) noted from a mixed culture the microbial community shift for hydrogen production from Clostridium species predominating at 37–45 °C to Thermoanaerobacterium species at 60 °C. Hydrogen yields are comparable at mesophilic and thermophilic temperatures, but lower at the ambient temperature (Kanai et al., 2005; van Niel et al., 2002). However, hyperthermophilic cultures seem to posses a superior ability to generate more hydrogen, and the highest hydrogen yields reported in the world are close to the theoretical maximum of 4.0 mol-H2/molglucose which was achieved by using extreme thermophiles (Schröder et al., 1994). Thermoanaerobacterium species are found to be most popular thermophiles in thermophilic hydrogen fermentation (Ahn et al., 2005; Zhang et al., 2003). Ueno et al. (2001) demonstrated that most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria, but the most dominant bands showed a high sequence similarity to T. thermosaccharolyticum in a thermophilic culture fed with cellulose. A similar microbial composition was reported by Ahn et al. (2005), when fermenting synthetic glucose wastewater with a thermophilic culture in the trickling biofilter reactors. Zhang et al. (2003) summarized several species of Thermoanaerobacterium known for their hydrogen producing characteristics, including T. thermosaccharolyticum, T. polysaccharolyticum, T. zeae, T. lactoethylicum and T. aotearoense. Species of Thermoanaerobacterium have been isolated from thermal volcanic spring, hot spring, and a high temperature acidic leachate of a waste pile from a canning factory, and have the optimal growth conditions of 55–70 °C and pH 5.2– 7.8 (Zhang et al., 2003). Besides T. thermosaccharolyticum, in a thermophilic culture fermenting food wastes, it was found that Desulfotomaculum geothermicum was present as one of main hydrogen-producing bacterial strains (Shin et al., 2004). Some thermophilic species of Clostridia are also detected in thermophilic hydrogen-producing cultures. Thermophilic cellulolytic bacteria, C. thermocellum and C. cellulosi are detected by Ueno et al. (2001) with cellulose powder as hydrogen-producing substrate. Working with a culture of C. thermolaiticum fed with lactose, Collet et al. (2004) obtained a hydrogen yield of 1.5 mol-H2/mol-hexose.

A thermophilic hydrogen-producing bacterial strain Klebsiella oxytoca HP1 which belongs to genus Enterobactericeae was isolated from a 65 °C hot spring (Long et al., 2005). Klebsiella oxytoca HP1 is highly resistant to oxygen inhibition (up to 10%), and its hydrogen production activity increases with temperature increasing from 25 °C to 35 °C in batch culture on media with glucose, achieving the maximum hydrogen yield of 1.0 mol-H2/mol-glucose. In continuous hydrogen production from sucrose, a much higher hydrogen yield of 3.6 mol-H2/mol-sucrose is attained at 38 °C and pH 6.5 (Wu et al., 2010). (Interestingly, although the Klebsiella oxytoca HP1 was isolated from a hot spring, the studies were conducted at mesophilic condition.). Van Niel et al. (2002) obtained a hydrogen yield of 3.33 mol-H2/ mol-hexose by using a pure culture of extremophiles either Caldicellulosiruptor saccharolyticus on sucrose (70 °C) or Thermotoga elfi on glucose (65 °C), and the same yield was reported by Kanai et al. (2005) from starch fermentation with hyper-thermophilic archaeon, Thermococcus kodakaraensis KOD1 at 85 °C. Moreover, it is stressed that a yield of 4 mol-H2/mol-hexose, the maximal theoretical value, was reported by Schröder et al. (1994) with a batch eubacterial culture of Thermotoga maritima on glucose at 80 °C. However, the authors pointed out that both microbial growth of the culture and glucose utilization are low. In addition, comparing with mesophilic cultures, hyper-thermophiles exhibit much lower production rates of hydrogen, generally ranging from 0.01 to 0.2 LH2/Lh (Table 1), which is largely attributed to the slow-growing characteristics of hyperthermophiles.

3.3. The role of pure or mixed cultures Comparative study of the role of pure or mixed cultures in the microbial community on hydrogen production is somewhat limited. Fermentative hydrogen production from the same substrate source (sorghum extract) can be achieved either by using mixed acidogenic microbial cultures or a pure culture of a saccharolytic strain Ruminococcus albus (Antonopoulou et al., 2007). The highest hydrogen yield obtained from the sorghum extract fermented with mixed microbial cultures in continuous system was 0.86 mol hydrogen per mol of glucose consumed at the hydraulic retention time (HRT) of 12 h. This corresponded to a hydrogen productivity of 10.4 l hydrogen per kg of sorghum biomass. On the other hand, the hydrogen yield obtained from sorghum extract treated with the pure culture R. albus was as high as 2.1–2.6 mol hydrogen per mol of glucose consumed. It is obvious that use of R. albus could increase hydrogen yield from sorghum extract compared to that obtained from mixed acidogenic culture. However, the drawback of using a pure microbial culture for hydrogen production from sorghum biomass compared to the use of the mixed indigenous acidogenic culture is the necessity for maintaining sterilized conditions with consequent extra energy requirements, unless R. albus can indeed proliferate under nonsterile conditions (Antonopoulou et al., 2007). In another study by Zhang et al. (2006), it was postulated that the HRT was able to reduce the diversity of microbial community associated with an elimination of propionate production without affecting the existence of dominant pure cultures, presumably causing the observed increase in hydrogen yield. On the other hand, steady microbial community and hydrogen yield were observed as the HRT increases, indicating the capability of microbial community to convert carbohydrate into hydrogen gas is not dependent on the HRT. As a result, the hydrogen yield could be considered as a function of the microbial populations in the culture, but the HRT affects the microbial community to a certain extent, and in turn shows an impact on hydrogen yield.

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4. Challenges and prospects Biologically produced hydrogen is currently more expensive than other fuel options (Nath and Das, 2004). The relatively low hydrogen yield and production rate are two common challenges for the biological hydrogen-producing systems, preventing them from becoming a practical means of hydrogen production. Although positive features of anaerobic hydrogen fermentation technology such as high production rate, low energy demand, easy operation and sustainability have been demonstrated in laboratory studies, this technology is yet to compete with those commercial hydrogen production processes from fossil fuels in terms of cost, efficiency and reliability. Economics would strongly favor largescale hydrogen production systems. However, the quantities of hydrogen that can be produced are limited by available biomass and the efficiency of converting that biomass into hydrogen. Up to now, the research activities have been mainly focused on mesophilic and thermophilic dark hydrogen production from organic substance enriched with carbohydrates (starch, cellulose and hemicellulose). The experimental results indicate that dark hydrogen fermentation is a feasible option to produce hydrogen from renewable biomass. Biohydrogen can be produced at a high yield (4 mol-H2/mol-hexose) or a high production rate (15 L/Lh) from carbohydrates-based, but not from proteins- and lipid-rich wastewaters and solid wastes. It should be realized that most studies were conducted in a laboratory scale, only few of pilot-scale studies have been demonstrated so far (Ren et al., 2006). Some technical issues need to be addressed in the laboratory in view of efficient producing hydrogen before the scale-up and commercialization of the dark hydrogen fermentation process.

4.1. Enhancement of hydrogen yield and production rate Enhancing the hydrogen production efficiency is one of the major challenges to dark hydrogen fermentation. To achieve such a purpose, numerous studies have been conducted, and several hundreds of public reports were published during the past decade. In practice, high H2 yields are usually associated with butyrate production, while low yields associated with the production of propionate and reduced end products (e.g., alcohols, lactic acid). There are great differences in yields and production rates. Hyper-thermophiles seem to be capable of gaining much high hydrogen yields, approximately 83–100% of the theoretical maximum of 4 mol-H2/ mol-hexose, while they usually grow to low densities, and hence hydrogen production rates are limited in a range of 0.01–0.2 LH2/Lh. Although mixed cultures of hyper-thermophilic bacteria have been immobilized through forming granular sludge, and biomass retention might be improved to a large extent, this mixed hyper-thermophilic culture are not advantageous, and even inferior compared to some mesophilic mixed cultures with respect to hydrogen production efficiency as the hydrogen yield and production rate of such hyper-thermophilic cultures are about 2.47 molH2/mol-glucose and 0.05 L-H2/Lh, respectively (Kotsopoulos et al., 2006). On the contrary, immobilization of mesophilic cultures substantially improves the biomass retention and hydrogen production rate. The highest rate of hydrogen production reported in the world is 15 L/Lh (i.e., 360 L/Lday or 613.5 mmol/Lh) in a bioreactor with granular sludge predominated with Clostridial species (Wu et al., 2006). However, the yield (3.50 mol-H2/mol-sucrose) of such a mesophilic culture of granular sludge is about 44% of the theoretical maximum. An increase in efficiency of the dark hydrogen production would be expected if biomass retention of hyperthermophiles is improved to a level of the mesophilic granular sludge; alternatively, if the hydrogen yields of mesophilic bacteria

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are enhanced. Therefore, the research direction is indicated here, this is, improving both hydrogen yield and hydrogen production rate simultaneously. In order to yield as much hydrogen as possible, a niche favorable hydrogen evolution has to be created by regulating microbial metabolism away from formation of alcohols and reduced acids (e.g., lactate) and towards production of acetate and butyrate. Metabolic engineering has received increasing attention in improving biohydrogen production. Applying genetic and metabolic engineering techniques to improve the hydrogen yields of those microbial cultures with a higher hydrogen production rate might be an feasible option as few cases indicated (Chittibabu et al., 2006; Kumar et al., 2001; Nath and Das 2004; Rachman et al., 1997). In a recent review, Nath and Das (2004) summarized several genetic approaches to enhance hydrogen productivity, including (1) overexpression of cellulases, hemicellulases, and lignases that can maximize substrate availability; (2) elimination of uptake hydrogenase; (3) overexpression of hydrogen-evolving hydrogenases that have themselves been modified to be hydrogen tolerant. Improvements in hydrogen yields by existing pathways has been attempted by increasing the flux through gene knockouts of competing pathways or increased homologous expression of enzymes involved in the hydrogen-generating pathways (Hallenbeck and Ghosh, 2009). On the downside, its metabolism restricts hydrogen yields to 2 H2/glucose. Mathews et al. (2010) found that deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdBC) of Escherichia coli increased hydrogen yield from 1.37 to 1.82 mol-H2/mol glucose. Wu et al. (2010) noted that an acetaldehyde dehydrogenase (adhE) gene inactivated Klebsiella oxytoca HP1 (DadhE HP1) mutant could enhance biohydrogen yield by reducing ethanol production from bagasse. The vast majority of the hydrogen-producing microbial diversity however, is yet to be discovered. This unexplored biodiversity will be tapped as more research work is engaged in future and with setting up of mechanisms for integrated management and utilization of these microbial resources. The potential and strategies for harnessing microbial resources and their gene resources in dark fermentation could shed light in further improving the yield and production rates of hydrogen fermentations. 4.2. Process performance The inhibition of hydrogen consumers present in the mixed cultures is essential for net hydrogen production and for further scale-up and industrial application. The pure culture is easily contaminated by other bacteria including hydrogen consumers such as methane producing bacteria and sulfate-reducing bacteria (Ueno et al., 2001). Several types of anaerobic bacteria are able to use hydrogen as a source of energy by coupling its oxidation to the reduction of a variety of electron acceptors. This group also includes facultative anaerobes such as E. coli (subsection IIB), which is capable of hydrogen-dependent fumarate reduction, and Paracoccus denitrificans, which, although classified as an aerobic hydrogen bacterium (subsection IID), uses either O2 or nitrate as a terminal electron acceptor during respiratory hydrogen oxidation. A sufficiently high growth rate of the adopted strain allows the reactors to be operated at low hydraulic retention time (HRT) with no severe washout of the functional strains. Alternatively, the hydrogen producers can be immobilized as biofilms or granules for easy retention of biomass in the reactor. The future use of hyper-thermophilic cultures in dark hydrogen fermentation, in particular in a large scale, remains unclear, because of their extremely low cell densities. Cell densities of hyperthermophic T. maritima were maintained in a range of 1.0–1.5  108 cells/mL, corresponding to biomass concentrations of 30–45 mg/L, even

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though the molar growth yields of on glucose (Yglucose) were 43–48 g cell dry mass/mol glucose) (Schröder et al., 1994). Likewise, with hyper-thermophilic C. saccharolyticus and T. elfii maximum cell densities was found to reach the order of 109 cells/mL (van Niel et al., 2002), which is comparable to the concentrations of 0.39–0.48 g dw/L indicated by Thermococcus kodakaraensis KOD1 (Kanai et al., 2005). Mohan et al. (2010) identified microbial community of a biofilm anaerobic biohydrogen reactor operated for wastewater treatment. These authors noted that Clostridium species are dominant in the observed microbial community, effectively producing biohydrogen via acetate-producing pathway. Restated, functional strains immobilized in the forms of biofilms or granules retain biomass in the reactor for achieving high volumetric hydrogen production rate. During the dark fermentation, carbohydrates are converted into hydrogen gas, and VFAs and alcohols which are organic pollutants and energy carriers. For the purpose of energy production and environmental protection, a second stage process is necessary to follow the dark fermentation, recovering the energy residues remaining in the effluent in the form of VFAs and alcohols. Thus the fermentative reactor becomes part of a process wherein the effluent post-treatment process and hydrogen utilization should also be included. The possible second state process is photo-fermentation, anaerobic digestion, or microbial fuel cells, which has been assessed in a recent review (Hawkes et al., 2002). Lo et al. (2010) adopted sequential dark fermentation (with Clostridium butyricum CGS5), photo fermentation (with Rhodopseudomonas palutris WP3-5) and autotrophic microalgae cultivation (with Chlorella vulgaris C-C) to establish a maximum hydrogen yield of 11.61 mol H2/mol sucrose and production rate of 0.67 L-H2/h/L with no carbon dioxide emission. Ho et al. (2010) yielded biohydrogen from cellobiose in phenol and cresol-containing medium using strain Clostridium sp R1. This work revealed the possibility to harvest bio-H2 from toxic wastewaters, whose toxicity prevents the bioenergy content of certain industrial effluents from being recovered. Kivisto et al. (2010) isolated new strain that can produce hydrogen from glycerol in saline environment. Novel hydrogen producers can be cultivated for harvest bioenergy from substrates in harsh environments. 4.3. Economic aspects In all of the biomass systems, a significant fraction of the energy produced is required to grow, harvest and process the biomass to produce hydrogen. Besides hydrogen, carbohydrates-based feedstocks can also be used for the production of other energy carriers, such as methane, ethanol, and acetone–butanol–ethanol (ABE). Claassen et al. (1999) briefly assessed those fermentative processes with respect to process and energy recovery efficiency. Although the authors attempted to compare energy from different resources and different processes on the basis of energy obtained from combustion, they also admitted that such a comparison is not adequate to determine which process is better since it refers to ‘pure’ compounds, and the cost of handling, distributing and storing etc. of the energy carriers remains neglected. Forsberg (2007) proposed that the cost of distributing and storing liquid fuels might be much lower than that of gaseous fuels. Each approach can provide the biofuel for the transport sector without increasing atmospheric greenhouse concentrations. It is still too early to predict which process is the process of choice. Economics and demand will likely determine the preferred option. Most of the hydrogen currently in use is not produced from renewable sources (Lee, 2011). Hydrogen production using biomass or carbohydrate-based substrates represent a promising route to biological hydrogen production (Perera et al., 2010;

Abreu et al., 2010; Kivisto et al., 2010). Biohydrogen can also be produced from biowaste such as municipal solid waste (Ljunggren and Zacchi, 2010; Prakasham et al., 2010; Luo et al., 2010; Geng et al., 2010). The cost of hydrogen production using lignocellulosic feedstock available locally was reported (de Vrije and Claassen, 2003). The plant was operated at a capacity of 10,200 Nm3 hydrogen/day and consisted of a 95 m3 thermobioreactor for hydrogen fermentation followed by a 300 m3 photo bioreactor for conversion of acetic acid to hydrogen and carbon dioxide. An overall cost of €2.74 for each kg hydrogen produced was estimated. The estimate was based on zero feedstock value and zero hydrolysis costs, and personnel costs and costs for civil works were omitted. Exploitation of energy crops as feedstock for biofuels production has been escalating in the recent years. However, there has been a growing resistance against the use of energy crops as feedstocks for biofuels generation. This backlash was centred on the food-vs-fuel debate, with the main arguments that crops that could support human dietary needs are diverted to the production of biofuels thus inflates food prices. As an answer to those issues, the production of second generation biofuels is proposed, i.e., biofuels produced by feedstocks that are not competitive to edible crops such as wastes and residues. Numerous biomass and wastes including the lignocellulosic residues remaining from such crops, together with other agricultural and forestall residues have been tested as potential feedstocks. Their exploitation, not only leads to energy recovery but can also be a cost reducing management method. The problem with the dark hydrogen fermentation is that in the dark, fermentative bacteria produce only relatively small amounts of hydrogen, typically less than 30% stoichiometrically (based on 12 mol-H2/mol-glucose). Economic feasibility will not be sustainable until these yields reach the 60–80% (Benemann, 1996). Further recovery of residue hydrogen is essential. Fang et al. (2006) showed that mixed photofermentative microflora could be used convert acetate and butyrate effectively to hydrogen. If technological and cost effective photobioreactors were available, the two-stage dark and light hydrogen fermentation process would be a promising method as it has a theoretical maximum molar hydrogen yield of 12 mol-H2/mol-hexose converted in the two-stage process (Fang et al., 2005; Hawkes et al., 2002). In addition thermo-chemical synthesis process could be integrated the hydrogen production bioprocess such that the biomass carbohydrate fraction is converted to hydrogen and the lignin-rich residue is gasified and used to produce heat, electricity and/or fuels, thus greatly increasing the overall energy recovery efficiency of biomass. Ruggeri et al. (2010) conducted energy balance for a batch anaerobic bioreactor operated at 16–50 °C for a mesophilic consortium to do dark fermentation on biohydrogen production. These authors noted that only with a minimal-sized reactor operated in summer time with energy recovery steps can the system have a positive net energy gain. Perera et al. (2010) conducted energy gain analysis for dark fermentation at ambient or elevated temperatures. These authors concluded that the energy gain decreases with increasing operating temperature of a dark fermentating reactor. Negative energy gain can be obtained to operate the reactor at high temperature. The biohydrogen production should be followed by methanogenesis reactor or by a MFC to recover energy from organic metabolites in the effluent, with the latter being the better choice than the former in terms of energy gain. In the scale of economy-wide analysis, Lee (2011) and Lee and Lee (2008) noted that with a very strong Governmental support biohydrogen with efficient and reliant hydrogen producers can yield a promising alternative energy sector to other energy sectors up to 2040.

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5. Conclusions Extensive research in the past two decades have reviewed promising prospect of biohydrogen production via dark fermentation. There have been substantial improvement and development in both the yield and volumetric production rates of hydrogen fermentations. A great deal of bacterial species with vastly different taxonomic and physiological characteristics can produce hydrogen via biochemical reactions. This work reviewed the mesophilic and thermophilic hydrogen-producing microorganisms reported in literature. The challenges to get positive energy gain via dark fermentation strains were discussed.

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