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T. de Vrije and P.A.M. Claassen 1

Abstract The production of hydrogen is a ubiquitous, natural phenomenon under anoxic or anaerobic condi- tions. A wide variety of , in swamps, sewage, hot springs, the rumen of cattle etc. is able to convert organic matter to hydrogen, CO2 and metabolites like acetic acid, lactate, ethanol and alanine. In general, these bacteria live in the close vicinity of other bacteria which consume these metabolites, including hydrogen, producing their own endproducts like methane and CO2. In this way, a stable ecosystem is formed where potential feedback inhibition of the hydrogen producers by hydrogen, is annulled by the action of the hydrogen consumers. In view of the design of a bioprocess for the production of hydrogen from biomass, extreme thermophi- lic anaerobic bacteria have been selected because of their high yield with respect to hydrogen produc- tion. The yield is reported to be approximately 83-100% of the maximal theoretical value of 4 mol hydrogen/mol , in contrast to the strict anaerobic Clostridia which produce hydrogen with an approximate yield of 2 mol/mol and the facultative anaerobes which show a H2 yield of less than 2. Besides optimal H2 molar yields, high hydrogen production rates are needed. Product formation appeared to be dependent on cell densities. usually grow to low densities and, therefore production rates are expected to be low. High production rates are reported for Clostridia and Enterobacter of maximal 23 and 58 mmol/L.h, respectively. Hydrogen fermentations by co- and mixed cultures showed production rates of approximately 30-50 mmol/L.h.

5.1 Introduction is provided in sections 5.2 and 5.3. In section 5.4 the capacity to utilize various substrates is discus- This Chapter focusses on the microbial produc- sed. The design of a bioprocess for hydrogen from tion of hydrogen from biomass by . biomass and potential process improvements are In anoxic or anaerobic environments, hydrogen is dicussed in section 5.5. This section is followed commonly produced during microbial breakdown by a description of the economical consequences of organic compounds. In case organic com- of this bioprocess in section 5.6. In section 5.7 the pounds are the sole carbon and energy source current (inter)national developments and pro- providing metabolic energy, the process is termed grams are summarized. This Chapter is concluded ‘dark’ hydrogen fermentation. When light is by section 5.8 in which the conclusions and per- required to provide additional energy, the process spectives are presented. belongs to the category of photobiological proces- ses (discussed in Chapter 6). In this Chapter, 5.2 Physiology of dark hydrogen several features of dark hydrogen fermentation are fermentation presented and discussed to provide an insight in the state of the art and the presently recognized Dark hydrogen production is a ubiquitous pheno- bottlenecks and R&D challenges associated with menon under anoxic or anaerobic conditions (i.e. the first design of an envisaged production plant no present as an electron acceptor). A for hydrogen from biomass. wide variety of bacteria use the reduction of pro- An overview of basic hydrogen fermentation pro- tons to hydrogen to dispose of reducing equiva- cesses and hydrogen producing micro-organisms lents which result from primary metabolism. In

1 Corresponding author. See list of contributors. - 103 - other words: when bacteria grow on organic sub- yield of hydrogen during dark fermentation is strates (heterotrophic growth), these substrates severely affected by the partial pressure of the pro- are degraded by oxidation to provide for building duct. At high H2 partial pressures a metabolic shift blocks and metabolic energy for growth. This oxi- to production of more reduced products, like lac- dation generates electrons which need to be dis- tate [3] or alanine [4] occurs, thereby decreasing posed of for maintaining electrical neutrality. In the yield of H2. aerobic environments, oxygen is reduced and Having established that microbial hydrogen pro- water is the product. In anaerobic or anoxic envi- duction is a ubiquitous phenomenon, it must be ronments, other compounds need to act as elec- surprising that no hydrogen bubbles are coming tron acceptor, e.g. protons, which are reduced to out of organic waste piles or the sewer. The molecular hydrogen (H2). Other examples of underlying reason is the fact that, in natural envi- alternative electron acceptors in anaerobic envi- ronments, microbial activity is governed by an ronments are nitrate with nitrogen gas (N2) as the ecological niche. This niche is the resultant of product or sulfate with dihydrogensulfide (H2S) many contributing factors including the presence as the reduced product. Even organic compounds and quality of available organic matter, presence can act as electron acceptors as e.g. in the micro- of minerals, temperature, light, pH, salinity, redox bial production of butanol which is done through potential, synergistic or antagonistic activity of the reduction of butyric acid. The capacity to microbial populations etc. The ecological niche reduce other electron acceptors than oxygen governs the activity of certain microbial popula- requires the presence of a specific enzyme system tions and thus the concentration and variety of in the micro-organisms: hydrogen producing bac- the final products, i.e. CO2, water, hydrogen, teria possess hydrogenase enzymes; nitrate redu- nitrate, CH4 etc. No hydrogen is bubbling out of cing bacteria possess an elaborate set of enzymes the sewer since in nature there are numerous catalyzing the stepwise reduction of nitrate to other bacteria, which readily consume hydrogen nitrogen etc. as a source of reducing power. When the aim is to produce hydrogen from organic matter, a specific Even though many organic compounds enable the environment needs to be created in which hydro- production of hydrogen during dark fermenta- gen producing microorganisms flourish and tion, estimations of potential yields are mostly others perish (Fig. 1). based on hexose conversions. The theoretical yield per mole of glucose is described in the follo- 5.3 Hydrogen producing micro- wing reaction [1, 2]: organisms

C6H12O6 + 4H2O In reviews by Kosaric and Lyng [5] and Nandi and - - + 2CH3COO + 2HCO3 + 4H + 4H2 Sengupta [6] extensive lists of heterotrophic bac- teria known to produce hydrogen have been -1 ∆G’0 = -206 kJ.mol published. An update is listed in this chapter.

5.3.1. Strict anaerobes A maximum of 4 moles of H2 per mole of glucose 5.3.1.1. Clostridia can be produced concurrently with the produc- Many anaerobes produce hydrogen from hexoses tion of energy (206 kJ per mole of glucose) which in acetic acid, butyric acid and acetone-butanol- is sufficient to support microbial growth. The ethanol fermentations. The highest maximal yield remainder of the hydrogen in the hexose is con- of 4 mole H2 from 1 mole of glucose is produced served in the byproduct , and under non- in acetic acid fermentations. The production of ideal circumstances, more reduced products like other, more reduced organic acids and/or alcohols ethanol, lactate or alanine. The complete oxida- lowers the yield of H2. For instance the conver- tion of glucose to H2 and CO2 yields a stoichio- sion of one mole of glucose into butyrate is metry of 12 mole H2 per mole of glucose but in accompanied by the production of only 2 moles of this case no metabolic energy is obtained. The H2. Usually a mixture of products is produced by

- 104 - A

B1

B2 B3 Figure 1. Growth of Caldicellulosiruptor saccharolyticus (A) on in flasks (B1) and under controlled condi- tions in fermentors (B2, B3). Photo of the fermentor (B3) by courtesy of TNO Environment, Energy and Process Innovation, Apeldoorn, The Netherlands.

Clostridia and the available H2 from glucose is 14.6 mmol/g or 2.1 and 2.2 mol/mol) than from determined by the butyrate/acetate ratio. glucose (11.1 mmol/g or 2.0 mol/mol) [9]. These C. butyricum, C. welchii, C. pasteurianum, C. results suggest that both Clostridia spp. can be beijerincki, newly isolated Clostridium spp. and used for the production of H2 from both cellulose mixtures of Clostridia have been used in studies and hemicellulose present in plant biomass. The dedicated to produce high amounts of hydrogen. hydrolysis of biomass for the production of a fer- Taguchi and collegues isolated various new mentable substrate can either be done during fer- Clostridia strains. A growing culture of C. beijerin- mentation, in a simultaneous saccharification and cki AM21B isolated from termites yielded 1.8 to fermentation process, or in a separate process pre- 2.0 mole H2 on glucose [7]. The strain could also ceding fermentation. Using Clostridium sp. strain utilize a large number of other carbohydrates, no.2 it was found that the simultaneous sacchari- such as , , galactose, cellobiose, fication of xylan with a crude xylanase prepara- sucrose, and with efficiencies from 15.7 tion and hydrogen fermentation of the resulting to 19.0 mmol/g of substrate in batch fermenta- hydrolysate by strain no. 2 could proceed in a tions of 24 h [8]. H2 was also produced from single fermentor [10]. However, simultaneous starch with equal efficiencies, but sustained pro- conversion was less efficient than the independent duction was not achieved and production ceased conversion of the hydrolysate. Furthermore, before the exhaustion of carbohydrates in the hydrolysis of a cellulose preparation (Avicel) with medium. Another Clostridium sp., strain no. 2, a commercial cellulase preparation and hydrogen also isolated from termites, produced H2 more fermentation of the hydrolysate by strain no. 2 efficiently from xylose and arabinose (13.7 and could not proceed in one flask because the condi-

- 105 - tions required for the enzyme activity and the other products like acetate, ethanol, formate and growth of strain no. 2 differed significantly. CO2 from carbohydrates. In a continuous culture Another approach is using Clostridia strains which a H2 yield of 2.4 mol/mol glucose was reported by are known to produce cellulase or xylanase activi- Innotti et al. [20]. Since then production of H2 by ty. At present, no strains are available that are able R. albus was not studied further. to hydrolyse both glucans and xylans. Taguchi et al. [11] isolated a novel strain, Clostridium sp. 5.3.1.3. Thermophiles strain X53 from wild termites, which produced The hyperthermophile Pyrococcus furiosus, an xylanase in a batch culture and converted xylan to archaebacterium, produces H2, organic acids and hydrogen. In comparison to xylose, the kinetics of CO2 from carbohydrates [21, 22, 23]. Hydrogen hydrogen production from xylan were not signifi- production efficiencies were not evaluated. From cantly different, but the total yield from xylan was characterization studies of utilized substrates and lower than from xylose. Clostridia have also been produced products many extreme- and hyper- used in continuous hydrogen fermentations on thermophiles are known to produce hydrogen glucose [12, 13, 14, 15, 16]. For Clostridium sp. from carbohydrates (reviewed by [24]). strain no. 2 it has been shown that the maximal Cellulolytic thermophiles and extreme and hyper- H2 production rate and the molar yield were com- thermophilic bacteria producing hydrogen are e.g. parable in batch and continuous fermentations. species of Anaerocellum, Caldicellulosiruptor, Continuous H2 production by fermentation of a Clostridium, Dictyoglomus, Fervidobacterium, continuously produced hydrolysate of cellulose in Spirocheta, and Thermoanaerobacter. an aqueous two-phase system (polyethylene gly- Schröder et al. [25] reported on batch fermenta- col and dextran) has been studied. The H2 pro- tions at 80 ºC with Thermotoga maritima. A H2 duction rate and the H2 yield were higher with yield on glucose of 4 mol/mol was obtained which Avicel hydrolysate compared to glucose. Since the is equal to the maximal theoretical value. H2 yield on Avicel is higher than the theoretical However, glucose consumption was low (1.6 mM) maximum the presence of hydrolyzed dextran in and low cell densities (1.4 x 108 per mL) were the Avicel hydrolysate was suggested [17]. In con- reached. Maximal hydrogen production rates of tinuous fermentations maximal H2 production approximately 10 mmol/L.h were measured. rates of 20.4 and 21.7 mmol/L.h have been meas- Similar stoichiometries as for T. maritima were ured [13, 15] at low yields of 1.4 mole H2 per obtained for two moderate thermophiles, mole glucose. Higher yields, 2.4 mol/mol were Acetothermus paucivorans and Acetomicrobium flavi- accompanied by lower H2 production rates of 7 dum, grown at 60 ºC [26, 27]. Recently, results on mmol/L.h [15]. On xylose similar results were growth and hydrogen production by two other obtained, maximal H2 production rates of 21.0 extreme thermophiles during sugar fermentation mmol/L.h have been measured at yields of 1.7 H2 have been published [28]. In cultures of mol/mol xylose. Hydrogen has also been produ- Caldicellulosiruptor saccharolyticus grown on sucro- ced from N-acetyl-D-glucosamine and chitin se at 70 ºC and Thermotoga elfii grown on glucose waste products. In batch fermentations by the chi- at 65 ºC stoichiometries of 3.3 mole H2 per mole tinolytic bacterium C. paraputrificum M-21 a H2 hexose were obtained which is 83% of the theore- yield of 2.2 mol/mol N-acetyl-D-glucosamine has tical maximum. Maximal hydrogen production been obtained at production rates of 31 mmol/L.h rates of 8.4 and 2.7 mmol/L.h, respectively, were [18, 19]. The ability of Clostridia to produce H2 measured. These results show that higher hydr- looks very promising. Hydrogen yields and pro- ogen yields on hexose can be reached by extreme duction rates can still be improved by optimizing and hyper-themophiles compared to mesophilic process conditions. facultative and strict anaerobes.

5.3.1.2. Rumen bacteria 5.3.1.4. Other strict anaerobic bacteria producing hydr- Methanogens are characterized by the presence of ogen are rumen bacteria. Ruminococcus albus has hydrogenase, which is usually involved in the oxi- long been known to produce H2 together with dation of H2 coupled to CH4 production and CO2

- 106 - reduction. However, under conditions of inhibi- in the production of other metabolites, alcohols tion of CH4 formation Bott et al. [29] reported and organic acids, which normally decreases production of H2 and CO2 in stoichiometric hydrogen production. In an E. aerogenes double amounts from CO and H2O by a strain of mutant which produced lower amounts of ethanol Methanosarcina barkeri, the so-called water-gas and butanediol but comparable amounts of orga- shift reaction. nic acids the hydrogen production and yield were 2 times higher compared to the wild type [35]. 5.3.2. Facultative anaerobes Kumar and Das [36] have isolated E. cloacae IIT- Facultative anaerobes are resistant to oxygen. BT 08 strain from leaf extracts which was capable These bacteria have the advantage of rapidly con- to grow and produce hydrogen by using different suming oxygen thereby restoring anaerobic condi- carbon sources. In batch fermentations the maxi- tions immediately in reactors. Strict anaerobes are mum H2 yield was 2.2 mol/mol glucose and 6.0 very sensitive to oxygen and often do not survive and 5.4 on sucrose and cellobiose, respectively. low oxygen concentrations. The maximum H2 production rate measured was 35 mmol/L.h on sucrose as substrate. The same 5.3.2.1. Enterobacter approach as for E. aerogenes has been used to Enterobacter as well as other members of the develop mutants with enhanced H2 production Enterobacteriaceae can have several beneficial pro- [37]. In batch fermentations a double mutant pro- perties favourable for H2 production. In addition duced less ethanol and butanediol, and lower to high growth rates and utilization of a wide yields of lactate and butyrate were obtained. The range of carbon sources, H2 production by yield of acetate was similar as the wild type strain. Enterobacter is not inhibited by high H2 pressures The block in formation pathways of alcohols and [30]. However, the H2 yield on glucose is normal- organic acids was accompanied by a 1.5 times ly lower compared to that of e.g. Clostridia. increased H2 yield on glucose, i.e. 3.4 mole per Tanisho et al. [31] isolated strain E. aerogenes mole glucose. Continuous fermentations were E.82005 from leaves of Mirabilis jalapa. Under performed with E. aerogenes wild types and a dou- batch cultivation a hydrogen production rate of ble mutant [38]. Due to self-flocculation, cells 21 mmol/L.h was obtained over a period of 23 h. were retained in the reactor even at high dilution The H2 yield was 1.0 mol/mol glucose. In a conti- rates. Maximum production rates of 58 mmol/L.h nuous fermentation hydrogen was produced at a dilution rate of 0.67 h-1 were reached for the during 42 days using the same strain and molas- double mutant, which was nearly 2 times higher ses as the substrate. The average H2 production compared to the wild type. The molar H2 yield on rate was approximately 17 mmol/L.h. The average glucose was maintained at 1.1. In a packed H2 yield on sucrose was 1.5 mole. In contrast to column with spongy material an E. aerogenes wild batch fermentations lactate was the major product type strain produced hydrogen on a starch hydro- and butyrate and acetic acid were produced in lysate with a yield of 1.5 mol/mol glucose at a lower amounts [32]. Although production by dilution rate of 0.1 h-1 [39]. Enterobacter is not inhibited by high H2 pressures flushing the culture medium with argon enhanced 5.3.2.2. E. coli the H2 yield to 1.6 mole per mole glucose. It was E. coli has been shown to be capable of producing suggested that the removal of CO2 was responsi- H2 and CO2 from formate in the absence of oxy- ble for the yield improvement [33]. Yokoi et al. gen [40, 72]. The catalytic activity, called formate [34] isolated an aciduric E. aerogenes, strain HO- hydrogen lyase, was shown to be a membrane- 39, which was able to grow and produce hydro- bound multi-enzyme complex, consisting of a for- gen at low pH of 4.5. In a continuous culture wit- mate dehydrogenase and a hydrogenase [73]. hout pH control hydrogen was produced at a rate Sustained lysis of formate required blocking of of approximately 5 mmol/L.h for 26 days with other anaerobic reductases [74]. Production of a yield of 0.8 on glucose. To enhance H2 hydrogen from carbohydrates was also reported production rates mutants of E. aerogenes and [40]. Inconsistency exists on the pathway leading E. cloacae were developed. The mutants are blocked to H2 production, either via formate or without

- 107 - formate as an intermediate [40, 41]. The molar H2 meal or sludges from anaerobic digesters of muni- yield on glucose by growing E. coli was 0.9 [41, cipal sewage or organic waste and sludge from 42] or 1.2 by immobilized cells. kitchen waste water. These microflora often con- tain unwanted bacteria such as methanogens 5.3.2.3. Citrobacter which consume the produced hydrogen and con- A Citrobacter species, Citrobacter sp. Y19 isolated vert it to methane. Enrichment cultures of the from sludge digesters, has been shown to produce microflora are prepared by forced aeration of the hydrogen from CO and H2O by the water-gas shift sludge or by heat treatment which inhibits the reaction under anaerobic conditions [43, 44]. H2 activity of the hydrogen consumers while the production was observed in serum-bottles and spore forming anaerobic bacteria survive. during continuous operations. In the latter case Additionally, in continuous fermentations higher H2 production rates of approximately 15 dilution rates are used to wash out the slow gro- mmol/L.h have been observed. Equimolar wing methanogens and select for the acid produ- amounts of H2 were produced from the consumed cing bacteria. In industrial applications the use of CO, but the conversion efficiency of CO, which mixed cultures for hydrogen production from was about 20%, was relatively low. This was attri- organic wastes might be more advantageous buted to short retention times of CO. because pure cultures can easily become contami- nated with H2 consuming bacteria. The first 5.3.3. Aerobes reports published on hydrogen production during 5.3.3.1 Alcaligenes wastewater treatment showed inhibited methane A. eutrophus has been shown to grow heterotrop- production but low H2 yields and lack of stability hically on gluconate and fructose, and when [51, 52]. Ueno et al. [53] have found that the exposed to anaerobic conditions produced hydro- anaerobic microflora in sludge compost converted gen [45]. It contains a soluble NAD-reducing cellulose to hydrogen with high efficiency of 2.4 hydrogenase [46]. mol/mol hexose in batch experiments at 60 ºC. Furthermore, stable hydrogen production for 190 5.3.3.2 Bacillus days from industrial wastewater from a sugar fac- A hydrogen-producing B. licheniformis was isola- tory by the same microflora in a chemostat cultu- ted from cattle dung [47]. It produced 0.5 mol re was reported [54]. Hydrogen yield on hexose H2/mol glucose [48]. Immobilized cells had an was similar as in the batch culture and a H2 pro- average H2 yield of 1.5 mole per mole glucose and duction rate of 1.4 mmol/L.h at a HRT of 3 days cells were stable during 60 days. was obtained. The maximal removal efficiency of carbohydrates was approximately 97% and also 5.3.4. Co- and mixed cultures other organic compounds were converted. The Yokoi et al. [49, 50] reported on a co-culture in a hydrogen producers in the thermophilic microflo- continuous fermentation of Clostridium butyricum ra were identified. Sixty-eight strains were isola- and Enterobacter aerogenes in which the higher H2 ted and classified in 9 distinct groups and it was yield of the strict anaerobe and the oxygen con- suggested that hydrogen production from cellulo- sumption by the facultative anaerobe were combi- se is performed by a consortium of several species ned. This resulted in fermentations with no need of microorganisms [55]. Thermoanaerobacterium for an expensive reducing agent since the presence thermosaccharolyticum was the dominant strain in of E. aerogenes was sufficient to rapidly restore the enrichment cultures. A patent has been issued anaerobic conditions in the fermentor upon short [56] claiming a process for stable H2 production at oxygen exposures. A continuous fermentation by high efficiency by anaerobic bacteria from sludge immobilized mixed cells on porous glass beads compost under anaerobic conditions in combina- and starch as the substrate showed a H2 produc- tion with treatment of wastewater. Other recent tion rate of approximately 50 mmol/L.h and a H2 work utilized sewage sludge to convert glucose yield of 2.6 on glucose at dilution rates of 1 h-1. and sucrose into hydrogen in continuous cultures Microflora for mixed cultures have been isolated at 35 ºC [57, 58]. At retention times of 6 to 8 from various sources, such as fermented soybean hours a molar H2 yield of 1.7 and 3.4 on glucose

- 108 - and sucrose, respectively, was obtained at produc- respectively. Hydrogen fermentations by co- and tion rates of approximately 26 to 29 mmol/L.h mixed cultures showed production rates of over a two-week period. Kinetic models were approximately 30-50 mmol/L.h. developed to describe and predict the results and based on this it was suggested that product for- 5.4. Feedstocks for dark hydrogen mation was essentially a linear function of bio- fermentation mass concentration. Results on mixed cultures from digester sludge have been reported by Lay In this section two aspects of the feedstock for this [59, 60]. Conditions were varied according to type of hydrogen production are discussed. The central composite design methodology in order to first concerns the range of organic compounds model and optimize the anaerobic digested sludge which can be utilised. The second concerns the converting starch and cellulose to hydrogen. quality of the feedstocks which can be used for Mizuno et al. [61] improved the H2 yield on glu- dark hydrogen fermentation. cose by mixed cultures isolated from fermented soybean meal through sparging the medium in a With respect to the range of potential substrates continuous stirred-tank reactor with N2. During which can be utilised by the broad range of an 8 week period of continuous operation stable hydrogen producing bacteria it can be stated that, H2 production rates of approximately 8 mmol/L.h at present, it is vast and open for further explora- were obtained. Noike and Mizuno [62] reported tion. From a thermodynamic point of view, the on hydrogen fermentations of organic waste, such conversion of carbohydrates to hydrogen and as bean curd manufacturing waste, rice and wheat organic acids is preferred because it yields the bran by the same mixed culture in batch reactors. highest amount of hydrogen per mole of substra- The H2 yield varied from 1.7 to 2.5 mol/mol hexo- te. These carbohydrates can be monosaccharides se and the carbohydrates were used as the main but may also be polymers such as starch, cellulo- source while soluble protein was hardly degraded. se or xylan, as discussed in section 3. Besides car- bohydrates (Fig. 2) also formate and peptides To summarize, the highest H2 yields on hexose have, until now, been studied as substrates for have been obtained by hydrogen producing extre- dark hydrogen production. me thermophilic anaerobic bacteria (Table 1). The From our own work with extreme thermophilic yields were approximately 83-100% of the maxi- bacteria, it has recently become clear that amino mal theoretical value of 4 mol/mol. More research acids can also be oxidised to hydrogen by certain is needed to confirm these results and to determi- strains. It is not clear whether specific amino ne whether this is a general property of thermop- acids, entering bacterial metabolism at the level of hilic bacteria. The strict anaerobic Clostridia pro- pyruvate are selected or whether this pheno- duce hydrogen with higher yields, approximately menon is more general. Furthermore, we have 2 mol/mol, than facultative anaerobes which show observed that growth of hydrogen producers on a H2 yield of less than 2. However, higher molar certain saccharides is not always associated with yields of more than 3 have been obtained in reduction of protons to hydrogen but limited to mutants of Enterobacter which are blocked in bio- reduction of other electron acceptors which were, synthetic pathways leading to organic acid and allegedly, used for analytical reasons, e.g. like alcohol production. In mixed cultures molar H2 thiosulfate being reduced to the easily detected yields of around 2 are obtained, which reflects the H2S in the study of Ravot [63]. dominant presence of Clostridia in enriched cul- Finally, to add to the confusion, there is the fact tures. Besides optimal H2 molar yields, high that there have not (yet) been many studies on hydrogen production rates are needed. Product dark hydrogen production. This, together with formation appears to be dependent on cell density. the large number of hydrogen producing micro- Thermophiles usually grow to low densities and, bial species, allows the suggestion that most car- therefore production rates are expected to be low. bohydrates are a suitable feedstock for dark High production rates are reported for Clostridia hydrogen fermentation. Proteins, peptides and and Enterobacter of maximal 23 and 58 mmol/L.h, amino acids are probably less suitable for dark

- 109 - production source production 2 yield H 2 mol/mol rate (maximal) mol/mol rate monosaccharide mmol/h.L 2.5 31.0 [18, 19] 1 sugar factory pH ºC T, 1 - , h D HRT, h HRT, culture batch uncontrolled 36 glucose 3.4 batch 6.0 36 xylose 2.1 21.7 continuouscontinuous 1.16batch 0.96batchbatch 6.0 6.0 36 36 uncontrolled 7.4 80 7.0 glucose xylose 65 glucose 1.4 70 1.7 4.0 glucose 20.4 sucrose 21.0 3.3 10 3.3 2.7 [25] 8.4 [28] [28] batch uncontrolled 36 sucrose 3.0 35 continuous 8 6.7 35 sucrose 1.7 26.2 [58] 11 M-21 batch uncontrolled 37 GlcNAc from: HO-39 LMG1213tl continuous 0.222 5.8 36 glucose 1.5 21.7 [13] IFO13949 + continuous 1.0 5.2 36 starch 2.6 53 [49, 50] E.82005 continuous 0.32 6.0 38 molasses 0.7 20 [9] E.82005 batch 6.0 38 glucose 1.0 21 [30, 31] HU-101 wtHU-101 m AY-2 continuous continuous 0.67 0.67 uncontrolled uncontrolled 37 37 glucose glucose 1.1 0.6 58 31 [38] sp. no 2 batch 6.0 36 glucose 2.0 23.9 [9] sp. no 2 continuous 0.18 6.0 36 glucose 2.4 7.1 [15] Hydrogen yields and production rates by microorganisms as reported in the literature. as reported rates by microorganisms yields and production Hydrogen -acetyl-D-glucosamine IIT-BT 08 wtIIT-BT batch uncontrolled 36 glucose 2.2 [36, 37] IIT-BT 08 m DM IIT-BT N E. aerogenes saccharolyticus GlcNAc= Strict anaerobes Clostridia Clostridium C. butyricum Clostridium TABLE 1. TABLE microorganism conditions substrate H 1 E. aerogenes E. aerogenes E. aerogenes Co-culture C. butyricum C. paraputrificum Facultative anaerobes Enterobacter E. aerogenes Thermophiles Thermotoga maritima Thermotoga elfii Caldicellulosiruptor E. cloacae E. cloacae Mixed cultures - sludge compost- sewage sludge- fermented soybean meal continuous continuous 12 continuous 8.5 6 6.8 6.0 5.7 60 35 35 waste water glucose 2.5 glucose 1.4 1.7 8.3 8 29.6 [54] [61] [57]

- 110 - 10

75

7.5

Hydrogen mM

50 Glc mM Acetate mM 5 Xyl mM

OD580, pH OD580 pH 25 2.5 H2, Glc, Xyl, Ac (mM)

0 0 024487696 time (h)

Figure 2. Simultaneous consumption of glucose and xylose during growth of Thermotoga elfii on Miscanthus hydrolysate at 65 ºC. (from: de Vrije et al., 2002 [75])

hydrogen production whereas biopolymers like both by CO removal and H2 production. A tech- lipids will be unsuited. nological challenge is to enhance CO mass trans- This great potential of dark hydrogen fermenta- fer which is the rate limiting step in the process. tion, i.e. the vast range of potential organic sub- strates, has also been recognized by other workers At this point the second aspect of the feedstock, in the field. Noike and Mizuno [62] and Yu et al. i.e. the quality, enters the discussion. Even though [78] refer to several forms of organic waste there have been reports on dark hydrogen fer- streams ranging from solid wastes like rice straw mentation using solid organic waste, this pheno- to waste water from a sugar factory and a rice menon has also been denied. Besides this contrast winery, which have been successfully used for which is probably due to different species being dark hydrogen production. Besides the organic involved, there is the even more basic discussion substrates, CO in syngas has been used as feed- concerning the configuration of the feedstock on stock for biological H2 production. Syngas or fuel the molecular level. As stated above and shown in gas is a mixture of (mainly) CO and H2, which can section 3 several hydrogen producers are able to be produced cheaply and on a large scale by ther- convert biopolymers like starch, cellulose and mochemical gasification of coal or wood. A wide xylan to hydrogen and organic acids (Fig. 3). range of anaerobic micro-organisms are capable of This is very convenient, because pretreatment of CO oxidation with concomitant H2 production in biomass is then only, eventually, needed from the a biological variant of the water-gas shift reaction process technological point of view e.g. for [29, 77]. These organisms could serve as a biolo- improving rheological properties. However, apart gical alternative for chemical catalysts to remove from the more easily degradable feedstocks such CO from H2 rich gases, and produce H2 linked to as starch and cellulose, the main components of CO oxydation. CO uptake has been shown to future feedstocks will, most probably, to a large occur at very high rates. The process could be extent be derived from lignocellulosic raw mate- used for fuel gas conditioning and upgrading, rials. Lignocellulose is a biopolymer consisting of

- 111 - C

AB

E

D

Figure 3. Feedstocks which have been successfully used for hydrogen production by extreme thermophiles (A. Sweet Sorghum, B. Miscanthus, C. paper sludge, D. potato steam peels, E. domestic organic waste). tightly bound lignin, cellulose and hemicellulose. lignocellulose resists mobilisation and b) chemi- Whereas cellulose and hemicellulose can be feed- cally degraded lignin is often inhibitory to micro- stocks for hydrogen fermentation, lignin is not bial growth. degraded under anaerobic conditions. Moreover, These parameters need to be studied in view of lignin strongly hampers the utilisation of cellulo- producing cheap feedstocks for dark hydrogen se and hemicellulose because a) the bonding in fermentation from lignocellulosic biomass residues

Figure 4. Chopped Miscanthus before (Left) and after extrusion in combination with a sodium hydroxide treatment (Right). The pretreatment removed 77% of the lignin.

- 112 - black liquor: hydrolyzed xylan decomposed lignin

Miscanthus pretreated C 5 + C6 biomass biomass sugars extrusion enzymatic C & C + NaOH H O wash 5 6 2 hydrolysis fermentation pretreatment 100 g: 58 g: 37 g: glucan 3 6 glucan 3 5 Glc 27 xylan 24 xylan 1 3 Xyl+ Ara 1 0 lignin 2 4 lignin 6

residue: glucan 1 0 xylan 5 lignin 6

Figure 5. Flowsheet of pretreatment (extrusion combined with sodium hydroxide) and enzymatic hydrolysis of Miscanthus. Pretreatment and enzymatic hydrolysis results in fermentable sugars and (non)fermentable sidestreams. (from: de Vrije et al., 2002 [75]) and/or energy crops. It seems obvious that produ- gen producing bacteria flourish and others perish. cing cheap feedstocks will require the develop- However, as outlined above, dark hydrogen fer- ment of cost effective pretreatment methods with mentation is an incomplete oxidation. This means a low energy demand (Fig. 4). that organic matter is not completely oxidised to CO2 but to intermediate compounds, like acetic This constraint in dark hydrogen fermentation is acid or lactate. Further oxidation of these pro- also studied in our own hydrogen projects which ducts in the dark, to hydrogen and CO2, is ther- focus on the whole chain of events for hydrogen modynamically very unfavourable. Thus, dark production from biomass. The approach for suc- hydrogen fermentation delivers, besides very pure cessful pretreatment is to use a combination of hydrogen, other, reduced carbonaceous products physical and (bio)chemical methods for mobilisa- which need to be utilised for making a sound tion of saccharides for fermentation, while simul- balance when considering energy production taneously preserving the nonfermentable compo- from organic matter. As can be read in the chap- nents for further valorisation, e.g. lignin as a bio- ters dealing with methane production (Chapter 4) fuel (Fig. 5). and photobiological hydrogen production (Chapter 6) these intermediate products can be further metabolised to methane or converted to 5.5. Bioprocesses for hydrogen from hydrogen in the presence of light, respectively. biomass Our approach is to counter one of the physiologi- cal drawbacks of dark hydrogen fermentation, i.e. 5.5.1. Current process development the incomplete oxidation, by coupling the process The physiology of dark hydrogen fermentation to a subsequent fermentation (Fig. 6). explains, thisfar, that hydrogen production is a In this way the chemical energy present in the ini- common microbial asset and that for obtaining tial organic matter remains preserved as much as this hydrogen as the final product, only a specific possible. This also applies to situations where the environment needs to be created in which hydro- yield of hydrogen is lower than theoretically

- 113 - expected. Theoretically, 1 mole of glucose is con- Several approaches can be considered to increase verted to 4 moles of hydrogen, 2 moles of acetic hydrogen yields in the dark fermentation. The acid and 2 moles of CO2. As can be read in section optimisation of methane and photobiological fer- 3 of this chapter, there are several microbial con- mentations, is discussed in Chapters 3 and 5, versions where the yield is less than 4 moles of respectively. hydrogen. Other workers in the field [64] strong- Metabolic engineering of hydrogen producing ly adhere to attaining this theoretical maximum. micro-organisms to minimise production of other Even though our present projects aim at reaching more reduced products by blocking their biosyn- a yield of 4 moles of hydrogen in the dark fer- thetic pathways will provide higher yields [35, 37, mentation, it is our conviction that the whole 38]. Additionally, higher hydrogen yields will chain should be considered here. The substrate most probably be achieved by limiting cell growth range of phototrophic bacteria in a subsequent through nutrient limitations, thereby enhancing fermentation is fairly wide. This means that a catabolic processes. High cell densities are needed lower hydrogen production during dark fermen- to maximize hydrogen production rates. tation is compensated by a higher hydrogen pro- Therefore, major improvements are expected in duction in e.g. a consecutive photobiological fer- systems with biomass retention, e.g. by immobili- mentation. Thus, in the end, the same amount of zed cells [79], under nutrient limitations opera- hydrogen will be produced. When the dark ting in a continuous mode. hydrogen fermentation is followed by a methane As another potential improvement the production fermentation, there may be less room for negotia- of hydrogen at high temperatures by extreme and tion, although acetic acid is amongst the favorite hyper-thermophiles could be considered [65]. At substrates for methane production (Fig. 7). increased temperatures hydrogen production

5.5.2. Process optimisation

Gas separator

BIOMASS

Gas separator CO2

H2 + CO2 H2 + CO2

H2 carbohydrates organic acids LIGHT organic acids

Stage 1 Stage 2

Figure 6. Outline of the bioprocess for production of hydrogen from biomass in a 2 stage fermentation. Stage 1 is for heterotrophic fermentation of carbohydrates to hydrogen, and organic acids. In stage 2 the photohetero- trophic fermentation of organic acids to hydrogen and carbon dioxide takes place.

- 114 - 16 160 80 160

12 120 60 120

8 80 40 80

Glucose/H2 mM 40 4 CH4/acetate mM 20 40 H2/Acetate mM Glucose/Fructose mM

0 0 0 0 0 40 80 120 In mM 0 100 200 300 A Time (h) H2 B Time (h) CH4 Glucose Fructose Acetate

Figure 7. Results from a bioprocess for production of hydrogen and methane from domestic organic waste in a 2 stage bioprocess. In stage 1 hydrogen and acetic acid are produced during growth of Thermotoga elfii on domestic organic waste hydrolysate (A). In stage 2 the methanogenic fermentation of acetic acid to methane takes place (B). (from: Claassen et al., 2002 [76]) becomes more exergonic [66]. Pyrococcus furiosus 5.6. Economics for hydrogen from hydrogenase showed a dramatic increase in H2 biomass evolution activity above 80º C which may be part- ly due to a decreased affinity of the enzyme for H2 Here data are presented which were collected [67]. Therefore, extreme- and hyper-thermophiles during a Kiem-EET study (Nov 1998–Feb 2000) show a better resistance to high hydrogen partial performed by ATO (co-ordinator), WU- pressures [68] which otherwise cause a metabolic Laboratory for Microbiology, WU-Department shift to production of more reduced lactate or ala- Agrotechnology and Food Sciences, TNO-MEP nine instead of acetate. This could be one of the and Paques Biosystems [69] and financially sup- reasons that extreme- and hyper-thermophiles ported by the Dutch Ministries of Economic produce hydrogen with an efficiency of almost the Affairs (EZ), Education, Culture and Science theoretical maximum. Another advantage of fer- (OCenW), and Housing, Spatial Planning and the mentations at extreme temperatures is that the Environment (VROM) via the Economy, Ecology, process is less sensitive to contaminations by e.g. Technology Programme (EET) and the Ministry of hydrogen consumers, thus establishing a specific Agriculture, Nature Management and Fisheries. environment enabling maximum evolution of As stated above, dark hydrogen fermentation is an hydrogen. In spite of the higher tolerance for incomplete oxidation, yielding not only hydrogen hydrogen in thermophiles, this product may still and CO2, but also organic acids like acetic acid. impose feed-back inhibition. Therefore, the For an economically sound process, the reduced design of a highly efficient hydrogen removal step carbonaceous compounds need to be converted is needed to further augment productivity [80, too; either in a photo-bioreactor to H2 and CO2 or 81]. in a methane reactor to CH4 and CO2. If the dark hydrogen fermentation is not followed by further conversion, the H2 yield will not warrant econo- mic feasibility. Therefore, the costs of hydrogen production were estimated from a first design of a complete bioprocess for hydrogen from biomass, consisting of an extruder for preparing fermenta- ble feedstock, a thermo-bioreactor (95 m3) for

- 115 - H2

BIOMASS

H2 SUN LIGHT

NUTRIENTS

NaOH

ORGANIC MATERIAL

EFFLUENT

BIOMASS

Figure 8. First design of a bioprocess for the conversion of biomass to hydrogen in a thermo-bioreactor, followed by a photo-bioreactor (adapted by ATO from the conceptual design by Paques Biosystems B.V.). dark hydrogen fermentation and a photobioreac- produced feedstock. As feedstock a common lig- tor (300 m3) equipped with a sunlight collector, nocellulosic substrate was chosen consisting of for the conversion of acetic acid to hydrogen and 65% (w/w dry matter) (hemi)cellulose of which CO2 (Fig. 8). 65% becomes available for fermentation after pretreatment in the extruder. On this basis it was The estimate is based on the use of extreme ther- calculated that 1000 kg biomass (dry mophilic bacteria for the dark hydrogen fermenta- weight)/hour is required to produce 425 m3 tion since only these have been shown, until now, H2/hour. to achieve the theoretical production of 4 moles of The contribution of the investment costs and hydrogen per mole of glucose consumed. The energy demand of the separate steps to the pro- conversion efficiency in the thermo-bioreactor duction costs of hydrogen from biomass is shown was assumed to be 80%. The same efficiency was in Tables 2 and 3. As the current costs of biomass assumed for the consecutive photo-bioreactor range from low to even negative, being very vola- where acetate from the effluent of the thermo-bio- tile in view of future demands, no value was reactor was converted to hydrogen at a ratio of 4 included for the acquisition of the feedstock. The moles hydrogen per mole of acetate. same accounts for the mobilisation of fermentable Hydrogen produced in the thermo-bioreactor was substrates from the feedstock. This is a potential recovered using gas stripping and this hydrogen cost factor which is dependent on intrinsic feed- was further purified to specifications for fuel cell stock properties and applied pretreatment and application using pressure swing adsorption. hydrolysis schemes and therefore impossible to The size of the plant was set at a production capa- consider under a common denominator. 3 city of 425 Nm H2/hour (39 kg H2/hour), aiming The data shown in Tables 2 and 3 result in an esti- at relatively small scale systems, fed by locally mated overall cost of EURO 2.74/kg H2, equiva-

- 116 - TABLE 2. First estimate of investment costs for equipment required for a hydrogen from lignocellulosic biomass 3 production process for production of 425 m H2 /h or 312 tonne H2 /year, and the contribution to the H2 production costs.

Item Investment costs Annual capital Costs/kg H2 (EURO) costs (EURO) (EURO)

Extruder 1,045,455 156,818 0.50 Bioreactors, pumps etc. 1,295,455 194,318 0.62 Sunlight collector 811,064 121,660 0.39

Equipment for H2 recovery from thermo-bioreactor 403,182 60,509 0.20

Equipment for H2 recovery from photobioreactor 196,803 29,520 0.10

Total: 3,751,959 562,825 1.81

TABLE 3. First estimate of energy consumption and costs for a hydrogen from lignocellulosic biomass production 3 process for production of 425 m H2 /h or 312 tonne H2 /year. Assumed electricity costs 0.068 EURO / kWh.

Item Energy consumption Energy costs/year Costs/kg H2 (GJ/h) (EURO) (EURO)

Extruder 0.547 82,879 0.26 Bioreactors 0.842 127,576 0.41

Recovery of H2 from thermo-bioreactor 0.225 34,030 0.11

Recovery of H2 from photobioreactor 0.281 42,545 0.14

H2 Purification 0.010 1,515 0.01

Total: 1.90 288,545 0.93

3 lent to 0.25 EURO/Nm H2 or 19.2 EURO/GJ derived from biomass, no net emission occurs and (based on upper combustion value). This final thus this process is considered as CO2 neutral. cost estimation is based on acquisition of biomass Besides CO2, formed in a ratio of 1 : 2 (CO2 : H2 at zero value, zero hydrolysis costs and excludes v/v), no other volatile products are expected from personnel costs and costs for civil works, all the dark fermentation. So, besides being CO2 neu- potential cost factors. On the other hand, process tral, another advantage of this bioprocess is the units have been considered separately, thus pre- production of pure product streams. cluding the opportunity to couple technical devi- Besides the final cost of the produced hydrogen, ces and energy requiring and energy yielding pro- the energy balance of this bioprocess has been 3 cess units. For comparative purposes, present considered. The production of 425 m H2/h is production costs for hydrogen produced in small equivalent to an energy production of 5.4 GJ/h, scale production plants based on alternative tech- based on the upper combustion value of 12.74 nologies are presented in Table 4. Except for the MJ/Nm3. The electricity requirement in this technologies where natural gas or conventional design is estimated at 1.9 GJe/h. These observa- electricity is used for the production of hydrogen, tions show the weak points in the first design these production methods are without net CO2 which are now being studied in new projects. emission. First of all the yield in terms of hydrogen is In the bioprocess for hydrogen from biomass, CO2 addressed. The yield is inherently related to the is one of the products. However, since this CO2 is mobilisation of fermentable feedstock which can

- 117 - TABLE 4. Comparison of H2 production costs and net CO2 emissions in small-scale production plants with capacities in 3 the range 100 – 1000 Nm H2 /h.

1) Technology Production costs CO2-emission 3 3 (EURO/Nm H2) (kg/Nm H2)

Steam-reforming of natural gas 0.32 0.8 Electrolysis with conventional electricity 0.23 1.8 2) Electrolysis with CO2-lean electricity 0.27-0.36 0 2-stage bioprocess for hydrogen from biomass (this estimate) 0.25 0 Steam-reforming of bio-methane 0.32 0 Electrolysis with electricity from wind turbines 0.25 0 Electrolysis with electricity from photovoltaic cells 2.95 0

1) Data for alternative technologies were provided by TNO Environment, Energy and Process Innovation (personal communication). 2) Here CO2 is sequestered in e.g. aquifers, rendering this process CO2 neutral.

TABLE 5. Estimated energy potential of a bioprocess for hydrogen from biomass in The Netherlands.

Units

Available biomass for biohydrogen production1) 2,650 ktonne d.w./year 2) Total bio-H2 production (39 kg H2 /tonne biomass) 103 ktonne / year 3) Total energy content of Bio-H2 produced 14.7 PJ 2) Potential number of Bio-H2 plants 330 Total electricity production 4) 7.4 PJe / year

Electricity production per bio-H2 plant 22,294 GJe / year Electricity consumption per household; 3380 kWh/yr5) 12.2 GJe / year

Number of households per Bio-H2 plant 1830

Total number of households with Bio-H2 electricity 600,000

1) 50 % of total available biomass (5.3 Mtonne/a according to Faaij et al. [71]) 2) For a plant producing 39 kg H2 /hour from 1 tonne (d.w.) of biomass. 3) Based on upper combustion value: 142.7 GJ/tonne H2 4) Fuel cell with 50% conversion efficiency 5) Ref: http://www.energie.nl/ 6) Total number of households is 6.86 million Ref: http://www.energie.nl/ be converted to hydrogen. Besides increasing the mass for the production of energy in a non-fer- efficiency of mobilisation, e.g. from 65% to 85%, mentative way. It seems feasible to generate suffi- also other feedstocks besides lignocellulosic feed- cient energy from the residues to prevent the stocks, e.g. energy crops (Sweet Sorghum) or star- addition of external electricity from the grid. chy wastes (potato steam peels) are now under An indication of the energy potential of the bio- consideration. Secondly, the energy requirement process for The Netherlands is provided in Table of the bioprocess is addressed. By a new reactor 5. The estimate is based on data provided by Faaij design the energy demand has become signifi- et al (1997) [71] for the total biomass availability cantly decreased [81]. A new, but certainly neces- (5.3 Mtonne/yr) in The Netherlands. It is assumed sary strategy, is the utilisation of the residual bio- that 50% of the total amount of biomass (2,650

- 118 - ktonne/y) is available for biohydrogen production field is the development of combined two-stage (103 ktonne H2/y) and that the energy requi H2 and CH4 fermentation systems e.g in The rement of the bioprocess is fully covered by ther- Netherlands and Japan. A major technical prere- mochemical conversion of residual non-fermen- quisite for efficient H2 fermentations is the main- ted biomass. It is further assumed that the produ- tenance of low H2 partial pressures through conti- ced hydrogen is converted to electricity in fuel nuous removal of H2 from the fermentation broth. cells at 50% efficiency. As the total number of At the Biohydrogen 2002 conference several households is 6.86 million [2000; ref recent developments were presented including http://www.energie.nl/], the coverage with respect the use of membranes, which indicates that this to electricity demand is 9% of all households. If obstacle can be overcome with continued deve- the biomass availability increases, e.g. due to a lopment [82]. The development of dark hydrogen decreased demand in the animal feed industry, the fermentations also benefits from the rapid coverage increases accordingly. Finally, fuel cells progress in the field of fundamental hydrogenase generate heat besides electricity. This aspect has research that includes the recent elucidation of not yet been introduced in the calculations pre- the structure of the catalytic sites and basic phy- sented here because at present its quantification is siological research. unsure. In order to accelerate the technological develop- ment and to generate critical mass for the deve- 5.7. International status of development lopment of a hydrogen based economy interna- tional knowledge exchange and co-operation are The potential of biological hydrogen production is required. The IEA Hydrogen Program supports recognized worldwide. At the recent international collaborative activities for the advancement of conference Biohydrogen 2002, with 150 partici- hydrogen technologies in various tasks focusing pating researchers from around the world, the sta- on hydrogen production and storage technologies tus and progress in fundamental microbiologi- and infrastructural integration [83]. Work on dark cal/biochemical research and technological R&D hydrogen fermentations is included in Task 15, in the field of both photobiological and dark fer- ‘Photobiological hydrogen production’ that has mentative hydrogen production was reviewed recently been extended. The objectives of the [82]. Biohydrogen 2002 shows that the interna- European COST Action 841 ‘Biological and tional attention and R&D efforts in the field of Biochemical Diversity of Hydrogen Metabolism’ dark fermentative hydrogen production from bio- are to enhance the understanding of the basic wastes and wastewater are rapidly increasing. At molecular and physiological aspects of biological present, The Netherlands is leading in research on hydrogen metabolism as an indispensable basis application of thermophilic bacteria for hydrogen for fundamental and applied research [84]. The production in projects supported by national European Union further supports the European governmental organisations as well as the Thematic Network on Hydrogen: HyNet [85]. European Union. The targeted feedstocks include In the USA the activities for the transition to a biowastes (potato-processing residues, organic hydrogen economy are combined in the fraction of municipal solid wastes, paper sludge) ‘Hydrogen, Fuel Cells & Infrastructure as well as energy crops such as Miscanthus and Technologies Program’ supported by the Sweet Sorghum. In some countries R&D focuses Department of Energy [86]. In November 2002 primarily on mesophilic H2 fermentations. In the ‘National Hydrogen Energy Roadmap’ was Japan H2 fermentation R&D has included feed- presented in which biological hydrogen produc- stocks such as bean manufacturing waste tion is seen as one of the options for renewable (‘okara’), rice bran, wheat bran, apple and potato hydrogen production on the longer term. peels, palm oil mill effluent and tofu waste water. Increasingly, expert groups of various disciplines Additional R&D takes place in China (rice winery throughout Europe, Canada, Asia and, to a smal- wastewater) and in Hungary (in co-operation with ler extent, the USA are focussing on the biological The Netherlands) on H2 fermentation of paper production and application of hydrogen, as well sludge hydrolysate. A relatively new focus in the as the societal impacts of implementation. The

- 119 - insight that hydrogen needs to be produced from technology development. renewable sources in the future is recognised at • The realization of a bioprocess for hydrogen the level of the European Commission and des- requires two consecutive steps for complete cribed in reports like ‘Future Needs and utilisation of the chemical energy in the Challenges for Non-Nuclear Energy Research in substrate. In the first step hydrogen is produced the European Union’ (2002). The future role of by dark hydrogen fermentation. In the second hydrogen as a clean fuel for fuel cells producing step the effluent is converted to either near-zero emissions and as an intermediate energy hydrogen or methane. Solutions for obtaining carrier for storage and transport of renewable high yields and high production rates in the energy is increasingly recognized in Europe. The dark fermentation phase are envisaged. EU will therefore intensify the R&D in the field of • For an economically feasible hydrogen from hydrogen and related technologies. Several biomass production process, a two stage Expression of Interests related to biological hydr- fermentation is required for complete conver- ogen production and supported by more than 30 sion of sugars to hydrogen. workers in the field have been submitted to the A tentative cost estimate for hydrogen from bio- European Commission in June 2002. This may mass in a first design shows a cost for hydrogen form the basis for continuation and expansion of which is favourable as compared to other sustain- bio-hydrogen development in Europe. Finally, the able hydrogen production processes. The energy combination of fermentation processes with the demand of the bioprocess should be covered by use of product gas in fuel cells is particularly rele- utilisation of the non-fermentable residual bio- vant for the future application of bio-hydrogen. mass. This topic is being explored in ‘BFCNet’: ‘Network on Biomass Fermentation Towards Usage in Fuel 5.8.2. R&D challenges and Cells’ [87]. The objectives of BFCNet include joint perspectives research and demonstration and the development The mobilisation of fermentable feedstocks from of standards on EU level. biomass is an important R&D issue for every fer- It is clear from the above that biological hydrogen mentative biofuel process, including the hydrogen production through fermentation of biowastes is from biomass process described here. This tech- receiving increased attention. Furthermore, the nological obstacle can be tackled by further deve- potential of (renewable) hydrogen is increasingly lopment and optimisation of pretreatment techni- recognized internationally, which provides a sti- ques, aiming at an increase in efficiency of the mulus for the further development of biological current 65% to 85%. Improved mobilisation of hydrogen production. fermentable substrate will increase the yield of hydrogen from biomass and hence the overall 5.8. Conclusions and perspectives for energy efficiency of the bioprocess. Here it is further development important to realise that some pretreatment tech- niques are more suitable for pretreatment of a spe- 5.8.1. Brief conclusions cific biomass type than others. When different • Dark hydrogen fermentation is a natural biomass types are considered, e.g. steam explo- phenomenon but is, in natural environments sion or other techniques may be more adequate. often obscured due to rapid consumption of In general, pretreatment is followed by, presently hydrogen by other species. very expensive or environmentally undesirable, •The capacity to ferment organic compounds (bio)chemical hydrolysis. Even though many to hydrogen is widespread amongst micro- hydrogen producing micro-organisms are able to organisms. There are great differences in yields directly convert (hemi)cellulose to hydrogen, this and production rates. conversion may be rate-limiting or impair proper • Dark hydrogen fermentation can be done with process performance. Therefore, this topic has to almost all carbohydrates. The production of be considered too in further optimisation. fermentable feedstocks from lignocellulosic In addition, it is very important that new techni- material requires further investigation and ques are developed for the utilisation of non-fer-

- 120 - mentable biomass fractions, such as lignin, for have been considered separately. It is expected energy production (Supercritical Water that process integration of the separate units will Gasification) or other product (composites) appli- enable substantial reduction of both the invest- cations. For the designed 2-stage bioprocess, the ment costs and the energy consumption. Finally, volumetric H2 productivity in the thermophilic other products from the bioprocess such as new, fermentation step must be increased at least 10- thermostable proteins from the dark fermentation, fold in order to meet the production capacity of new secondary metabolites (including vitamins) 3 425 m H2 /h. This can be done by conventional from the photobiological fermentation, and even methods such as optimisation of culture condi- clean carbon dioxide produced on site, may find tions, biomass retention, or selection of highly their own application and thus contribute to productive species or strains from the broad range making the bioprocess even more economically of available hydrogen producing micro-orga- viable. nisms. The productivity of the photobiological fermenta- 5.9. References tion step should be increased by at least a factor 15 through optimization of sunlight conversion 1. Solomon BO, Zeng A-P, Biebl H, Schlieker H, Posten C, Deckwer WD (1995) Comparison of the energetic efficiencies efficiency. In this case the main improvement has of hydrogen and oxychemicals formation in Klebsiella pneumo- to come from technological improvements of sun- niae and Clostridium butyricum during anaerobic growth on light collection and light transfer systems, and glycerol. 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Kosaric N, Lyng RP (1988) Microbial production of hydrogen, of storing the effluent, methane production might In: Biotechnology, vol 6b, eds, Rehn RJ and Reed G, Springer- act as a substitute. When this option turns out Verlag, Berlin, 100 6. Nandi R, Sengupta S (1998) Microbial production of favourable, the advantages of a partial photofer- hydrogen: an overview. Crit Rev Microbiol 24, 61-84 mentation supplemented with a methane fermen- 7. Taguchi F, Chang JD, Takiguchi S, Morimoto M (1992) tation have to be weighed against a complete Efficient hydrogen production from starch by a bacterium isolated from termites. J Ferment Bioeng 73, 244-245 replacement of the photofermentation step. This 8. Taguchi F, Chang JD, Mizukami N, Saito-Taki T, Hasegawa K, issue needs to be evaluated together with the Morimoto M (1993) Isolation of a hydrogen producing progress in the field of the application of methane bacterium, Clostridium beijerinckii strain AM 21B from termites. 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