Bio Hydrogen Production From Biomass and Wastes Via Dark Fermentation A Review

Waste Biomass Valor (2010) 1:2139 DOI 10.
1007/s12649-009-9001-2
Biohydrogen Production from Biomass and Wastes via Dark Fermentation: A Review
I. Ntaikou G. Antonopoulou G. Lyberatos
Received: 15 November 2009 / Accepted: 14 December 2009 / Published online: 4 February 2010 Springer Science+Business Media B.V. 2010
Abstract The present review article aims to summarize the microbiological and technological background of the dark fermentation processes for hydrogen generation, emphasising on the exploitation of biomass and wastes as potential feedstocks. The basic principles, the microbiology and the current technology of the processes are outlined. Subsequently, the use of different types of biomass and wastes that have so far been tested as feedstocks is analysed focusing on the advantages, possible limitations and future prospects of their exploitation. Moreover, different types of so far suggested pretreatment methods for better utilisation of the feedstocks are presented, pointing out the advantages and disadvantages of each method. Finally, methods for possible further utilisation of the generated by-products are laid out as well as the present status of the real scale applications. Keywords Biohydrogen Energy Biomass Wastes Wastewater
Introduction Woody biomass has been the major source of energy for mankind throughout the ages. During the st steps of man on earth the combustion of wood was more than enough to meet his energy requirements, which actually were limited
I. Ntaikou G. Antonopoulou G. Lyberatos (&) Department of Chemical Engineering, University of Patras, Karatheodori 1 st., 265 00 Patras, Greece e-mail: lyberatos@chemeng.upatras.gr I. Ntaikou G. Antonopoulou G. Lyberatos Institute of Chemical Engineering and High Temperature Chemical Processes, 265 04 Patras, Greece
to cooking, warming and protection. The idea of using waste as a source of energy is not new either. As a matter of fact the use of animal manure as fuel via combustion is a quite ancient practice, too. However as the world population started to grow, mankind started searching for more effective means of energy production, turning thus initially towards the use of coal, and later onto oil, an even more energy efcient fossil fuel. Nowadays, the overexploitation of fossil fuels has brought mankind to a dead end from both an environmental and energy resource standpoint. It is indeed true that the fossil fuel reserves are not endless. Consequently, their heedless use in order to cover the continuously increasing energy demands [1] has resulted to the excessive accumulation of carbon dioxide in the atmosphere on one hand [2], while bringing humanity to an imminent world energy crisis on the other [1, 3]. As a result of those facts, an exigent need for identifying and exploiting alternative energy sources has emerged; energy sources that would be renewable and environmental friendly. Hydrogen, although not being readily available in its molecular form, is quite abundant in nature and compared to other fuels, it has the highest specic energy and energetic density. Moreover it can be produced via a number of processes from renewable sources (it is called in such cases biohydrogen), and it generates zero emissions when burned for energy recovery [4, 5]. Those characteristics make hydrogen an appealing candidate for a future energy system based on sustainability. Biological hydrogen production methods include biophotolysis of water, photofermentation and dark fermentation of organic matter [6]. Among them, dark fermentative hydrogen production comprises the simplest technology, resulting at the same time to highest yields. For the scaling up of such a process, the raw material cost is considered to be among the major limitations. As raw materials some carbohydrate- and/or, starch-rich wastes/
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wastewaters as well as cellulose-rich biomass are considered the most suitable feedstocks. Main Principles of Hydrogen Production via Dark Fermentation The generation of biohydrogen via dark fermentation is achieved mainly by strictly anaerobic or facultative anaerobic bacteria under anaerobic conditions [7]. Although different organic substances, such as carbohydrates, sugars, proteins and lipids can in principle be used as substrates, for the estimation of the theoretical yields of fermentative hydrogen the reaction of glucose biotransformation towards acetate is widely accepted as reference. According to that reaction (1) [8, 9] the maximum theoretical yield of biohydrogen from glucose fermentation is 4 mol H2 per mol of consumed glucose: C6 H12 O6 4H2 O ! 2CH3 COO 2HCO 4H 4H2 3 DGo 206:3 kJ mol1 1
that will actually dene whether the maximum theoretical hydrogen yield will be 4 or 2 mol H2 per mol of consumed sugar, but also whether the observed nal yield will reach its maximum value. Depending on the enzymatic system that a microorganism has, acetylCoA can be generated either via reactions (5) or (6). pyruvate CoA Fdox $ acetylCoA CO2 Fdred DGo 19:2 kJ mol1 pyruvate CoA $ acetylCoA formate DGo 16:3 kJ mol1 6 5
The maximum hydrogen theoretical yield of 4 mol H2 per mol of consumed glucose can also be achieved in two steps via the fermentation of glucose towards acetate and formate according to reactions (2a) and (2b) [10, 11]: C6 H12 O6 2H2 O ! 2CH3 COO 2HCOO 4H 2H2 DGo 209:1 kJ mol1 2HCOOH ! 2CO2 2H2 DGo 6 kJ mol1 2b 2a
In the case that instead of acetate, butyrate is the sole organic byproduct, the maximum hydrogen theoretical yield becomes 2 H2 per mol of consumed glucose according to reaction (3) [8]: C6 H12 O6 2H2 O ! CH3 CH2 CH2 COO 2HCO 3H 2H2 3 DGo 254:8 kJ mol1 3
The basic step in all the above reactions is the metabolism of glucose towards pyruvate as shown in reaction 4, through which 2 mol of hydrogen can theoretically be generated during the subsequent regeneration of the produced NADH (NADH ? H? ? NAD? ? H2). In most bacteria, the metabolism of glucose to pyruvate is usually carried out via the EMP metabolic path. C6 H12 O6 2NAD ! 2CH3 COCOO 4H 2NADH DGo 112:1 kJ mol1 4
Nevertheless the key substance for hydrogen production from organic matter is acetylCoA, which is further produced by pyruvate. The subsequent fate of acetylCoA is the factor
Reaction (5) is catalyzed via the enzyme pyruvateferredoxin oxidoreductase, where ferredoxin is the coenzyme that acts as electron receiver [12]. This enzyme has been found in many strictly anaerobic [13] as well as in facultative anaerobic bacteria [14] but also in cyanobacteria [15]. AcetylCoA can further be metabolized either to acetate (Fig. 1a), or to butyrate (Fig. 1b), and in both cases from the re-oxidation of each mole of ferredoxin one mole of hydrogen will be generated via a hydrogenase enzyme. In the case that acetate is the nal product, one extra mole of hydrogen will also occur from the reduction of each mole of NADH, that was produced during glycolysis, to NAD?, leading thus to a total hydrogen yield of 4 moles/mol of consumed glucose. In the case that butyrate is the nal product, the produced from glycolysis NADH is actually used for the oxidation of acetoacetylCoA to butyrate, and thus the total nal hydrogen yield is 2 mol/mol of consumed glucose. Depending on the culture conditions as well as the type of microorganism, simultaneous generation of acetate and butyrate can often be observed, leading thus to a hydrogen yield with values between 2 and 4. Such reactions are quite typical for some clostridia such as Clostridium pasteurianum [16] and C. butyricum [17]. The second way of acetylCoA generation, as described by reaction (6), leads to the simultaneous production of formate [18]. This reaction, as shown in Fig. 2, is catalysed by the enzyme pyruvate-formate lyase [19], and is typical of the metabolism of enterobacteria, such as Enterobacter aerogenes and Escherichia coli [20] under anaerobic conditions. The function of pyruvate-formate lyase in enterobecteria is regulated at the transcription level based on the oxygen level in the environment [21]. Besides enterobacteria, the presence of pyruvate-formate lyase has also been reported for some clostridia [22]. It is nevertheless true that glucose fermentation can also lead to the generation of other products besides acetate, butyrate and formate. Such products are propionate, succinate, lactate and 2,3 butanediol that emerge directly from pyruvate, as well as ethanol, butanol and isopropanol that emerge through a further step from acetylCoA metabolism
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Waste Biomass Valor (2010) 1:2139 Fig. 1 Generation of hydrogen with simultaneous production of acetate (a) and butyrate (b) via glycolysis. Extra molecular hydrogen can be generated in the case of acetate production by NADH that is generated during glycolysis
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undesired byproducts, the generation of which leads to lower overall hydrogen yield. Those products can often emerge when fermentations are carried out via mixed metabolism bacteria or via mixed cultures. The distribution of metabolites seems to be highly dependent on the prevailing culture conditions. It is reported that for E. coli, for example, for pH values below 7 the generation of lactate is favored [23], leading thus to reduced hydrogen yields. In such cases, a strict selection of proper culture conditions has to be made so as to lead the metabolism towards hydrogen generation and/or towards the dominance of hydrogen producing microorganisms when mixed cultures are used.
Microorganisms Fermentative hydrogen production can be carried out via a wide range of microorganisms, with quite diverse requirements in terms of substrate preference, pH and temperatures [24]. Those parameters do not only determine the growth of the microorganisms, but also have a crucial role on the metabolic path that the microorganisms will follow, affecting thus severely the nal observed hydrogen yield from the whole process. Hydrogen production can be achieved either through mixed acidogenic microbial
Fig. 2 Generation of hydrogen with simultaneous production of acetate, ethanol, lactate and formate (mixed acid fermentation) via glycolysis. Extra molecular hydrogen can be generated by NADH that is generated during glycolysis
[21]. The generation of all abovementioned metabolites is not accompanied by hydrogen generation, and thus during dark fermentative production, these are characterized as
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cultures, derived from natural environments such as soil, wastewater sludge, and compost, or through pure cultures of selected hydrogen producing bacteria. Such bacteria can be mesophilic (2540C), thermophilic (4065C), extreme thermophilic (6580C), or even hyperthermophilic ([80C). In any case, the nal selection of the type of culture to be used (mixed, pure, co-culture) as well as the specic microorganism, has to be based on the specic requirements of each possess. Mixed Cultures In general, for a full-scale application the selection of mixed cultures is considered to be favourable, at least from an engineering standpoint. This is due to the fact that the control and operation of the process is facilitated when no medium sterilisation is required, reducing thus the overall cost, whereas it also allows for a broader choice of feedstocks selection [25]. The mixed consortia can be derived from a variety of different natural sources, such as sewage sludge [2628], anaerobically digested sludge [2932], acclimated sludge [33], compost [3437], animal manure [35] and soil [38, 39] or even from the indigenous microorganisms found in certain wastes [4043]. Despite the advantages of mixed cultures in terms of the economical viability of a process, their use always lurks the possible predominance of non-hydrogen producing species such as methanogens, homoacetogens and lactic acid bacteria; a case which could eventually lead to the dramatic failure of the viability of process. The strategy followed in order to minimise such a possibility includes on the one hand an initial pretreatment of the seed so as to remove to the highest possible degree the undesired microorganisms, and on the other, the maintenance of such environmental and operational conditions that favour the predominance of the desired hydrogen producing species. In terms of the seed pretreatment, heat treatment is generally the most common practice [40, 44]. By subjecting seed cultures to high temperatures, only the spore-forming acidogenic microorganisms survive the thermal shock, whereas the methanogenic non-spore-forming bacteria die. The use of temperatures reported are 75C [45], 100C [4649] or even 121C [42] whereas the duration of the process has been reported to vary between 15 min and 2 h [42, 4549]. Alternatively to heat treatment, an acid/base treatment of the seed has also been suggested as a possible way for ensuring the dominance of hydrogen producing bacteria [26, 50]. The principle of this method is to maintain the seed microorganisms for a prolonged period of time at very acidic or very basic conditions, which would eventually lead to the removal of methanogens that cannot survive such extreme pH values.
Pure Cultures of Wild Strains Alternatively to mixed cultures, many researchers have focused on the use of pure cultures of selected hydrogen producing species. The main arguments for their advantageous use are, the selectivity of substrates, the easiest manipulation of the metabolism by altering growth conditions, the higher observed hydrogen yields as an effect of the reduction of undesired by-products as well as the repeatability of the process. On the other side of the coin, pure cultures can be quite sensitive to contaminations and thus their use demands, in most cases, the presence of aseptic conditions, which signicantly increases the overall cost of the process. There is a wide range of microorganisms that are capable of producing hydrogen via dark fermentation. This includes strict anaerobes (Clostridia, methylotrophs, rumen bacteria, methanogenic bacteria, archaea), facultative anaerobes (E. coli, Enterobacter, Citrobacter), and even aerobes (Alcaligenes, Bacillus). Among the hydrogenproducing bacteria, Clostridium sp. and Enterobacter, are the most widely studied. Species of genus Clostridium such as C. butyricum [51], C. acetobutyricum and C. beijerinckii [52], C. thermolacticum [53], C. tyrobutyricum [54], C. thermocellum [55] and C. paraputricum [56] are examples of strict anaerobic and spore forming microorganisms, generating hydrogen gas during the exponential growth phase. In parallel, facultative anaerobes such as E. coli and species of genus Enterobacter, such as E. aerogenes [57, 58] and E. cloacae [59, 60] have also been used for hydrogen production. In the recent years, extensive research has also been carried out in hydrogen production at high temperature, using thermophilic or hyperthermophilic bacteria, since the increase of temperature in principle improves the reaction kinetics. The thermophiles that have been studied include Caldicellulosiruptor saccharolyticus [61], Thermoanaerobacterium sp. such as T. thermosaccharolyticum [62] and Thermotoga sp. such as T. maritima [63] and T. eli [64]. Among them, the use of hyperthermophilic bacteria has lately attracted increasing interest due to their many outstanding properties. Since, they can grow successfully at very high temperatures (reaching even 110C) hyperthermophiles make hydrogen fermentation less sensitive to contaminations by undesirable intruders. Moreover, hyperthermophiles show a better resistance to high hydrogen partial pressures [65], one of most inhibitory factors in fermentative hydrogen production processes. Genetically Modied Microorganisms Fermentative hydrogen production processes, either conducted via mixed or pure cultures, still have certain
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limitations that could be crucial for scaling up. Inefcient substrate conversion, formation of by-products, low resistance to high H2 partial pressures and oxygen are only some of them. On top of all those, comes the limiting upper value of hydrogen yields which is 4 mol of H2/mol hexose consumed, a limit that could be theoretically highly increased taking into account that one mol of hexose contains 12 gram-atoms of hydrogen. As an answer to the abovementioned limitations several researchers have proposed the use of genetically modied microorganisms with advanced selected properties, constructed either via mutagenesis or through genetic engineering. The main areas of modication can be summarized as follows: (a) introduction or manipulation of genes responsible for the overexpression of enzymes with cellulolytic activities such as cellulases, hemicellulases and lignases, or enzymes for uptake of different types of sugars, both aiming at the maximization of substrate availability and conversion, (b) elimination of hydrogen-consuming hydrogenases and (c) overexpression of hydrogen-producing hydrogenases, that have also been modied so as to be hydrogen tolerant [66]. E. coli, one of mostly studied facultative anaerobes for hydrogen generation has also been widely used as the carrier of different genes through mutagenesis and genetic engineering. So far, the construction of many different genetically modied strains has been reported including strains capable of consuming pentoses (case a) [67], strains with suppressed lactate and succinate dehydrogenases activities (case b) [68, 69] and strains showing overexpression of formate hydrogenlyase [7072]. Increase of cellulolytic activity (case a) for enhancing hydrogen production from lignocellulosic biomass has been achieved for Clostridium beijerinckii [73].
The major criteria that have to be met for the selection of substrates suitable for fermentative bio-hydrogen production are availability, cost, carbohydrate content and biodegradability [74]. Simple sugars such as glucose, sucrose and lactose are readily biodegradable and thus preferred as model substrates for hydrogen production [7578]. However, pure carbohydrate sources are expensive raw materials for real scale hydrogen production, which can only be viable when based on renewable and low cost sources [79, 80]. Different types of classications have been reported so far for the description of biomass and wastes that have been used for fermentative hydrogen productions. Most often, the classication criteria used are either the chemical composition of the major present substrate [6, 51] and/or the origin of the biomass/waste [6, 8183]. The most frequently exploited types of biomass/wastes used as feedstocks for hydrogen production are analysed below. Energy Crops The idea of exploiting whole crops for energy production had already emerged by the early 1980s [84, 85]. By definition energy crops refer to certain plants that are cultivated solely for the further exploitation of their biomass (either whole or part of it) as feedstock for energy production, which can be directly exploited for its energy content via combustion or be biotransformed to biofuels. In general, the sustainability of such processes can only be assured if (a) the crops are produced at low cost, thus with minimum nutrient and water requirements, (b) they are resistant to environmental stresses, and (c) they are highly biomass yielding [74]. Furthermore, such plans in order to be suitable for hydrogen production via dark fermentation should also have high sugar and/or carbohydrates content and low lignin content [74]. According to their chemical composition, energy crops used for fermentative hydrogen production can be divided in sugar based crops (e.g. sweet sorghum, sugar cane and sugar beet), starch based crops (e.g. corn and wheat), and lignocellulose based crops including herbaceous (e.g. switch grass and fodder grass) and woody (e.g. Miscanthus and poplar). The results from studies conducted for hydrogen production via dark fermentation with different types of energy crops are summarised in Table 1. As shown, energy crops can be quite sufcient for hydrogen generation, especially when the latter is based on cultures of pure microorganisms. Lately however, the continuously rising food prices, the sustainability doubts and the energy-equation challenges have led to a backlash against the use of energy crops as feedstocks for biofuels generation. This backlash was centred on the food-vs.-fuel debate [95, 96], since in many countries huge agricultural areas have been turned into
Types of Feedstocks Theoretically any organic substrate rich in carbohydrates, fats and proteins could be considered as possible substrate for biohydrogen production. However, as reported by numerous studies, carbohydrates are the main source of hydrogen during fermentative processes and therefore wastes and biomass rich in sugars and/or complex carbohydrates turn out to be most suitable feedstocks for biohydrogen generation [6]. According to a comparative study by Lay et al. [35], using substrates of different chemical composition treated with the same mixed consortium, it was shown that the hydrogen-producing potential of carbohydrate-rich waste (rice and potato) was approximately 20 times higher than that of fat-rich waste (fat meat and chicken skin) and of protein-rich waste (egg and lean meat).
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26 Table 1 Fermentative hydrogen production from energy crops Crop Microorganism Operation mode Batch Continuous Continuous Continuous Continuous Batch Batch Continuous Batch Batch Batch Batch Batch Batch Maximum H2 production rate (LH2/l/day) 3 2.2b 2.57 8.52 6 13.1d 12.6d
Maximum H2 yield (mol H2/mol cons. hexose) 1.1a 1.26 1.9 0.51 0.86 3.15 (59 l/kg wet biomass) 2.61 82c 1.75 (30.17 l/kg dry biomass) 3.2 3.4
References
Miscanthus (pretreatment: mechanical and NaOH) Wheat starch Sugarbeet juice Corn starch Sweet sorghum extract Sweet sorghum stalks Sweet sorghum extract Ryegrass Sweet sorghum Sugar beet extract Barley grains Corn grains Miscanthus (pretreatment: NaOH, Ca(OH)2) Miscanthus (pretreatment: NaOH, Ca(OH)2)
a b c d
Thermotoga eli Mixed mesophilic cultures Mixed mesophilic cultures Mixed mesophilic cultures Indigenous microbial mesophilic culture Rumicococcus albus Rumicococcus albus Mixed mesophilic cultures Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Thermotoga neapolitana Caldicellulosiruptor saccharolyticus
[64] [86] [87] [88] [89] [90] [90] [91] [92] [93] [93] [93] [94] [94]
mol H2/mol consumed sugars ml/min l ml H2/g dry mass mmol H2/l h
feedstock industries for the production of biofuels. The main arguments against the use of energy plants is that crops that could support human dietary needseither directly or indirectly through farmed animalsare diverted to the production of biofuels. Even in the case of non-food crops cultivation, the sustainability issue is getting under question. 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. Wastes and Residues Lignocellulosic Residues Different types of lignocellulosic residues have been studied as potential renewable feedstocks for fermentative biohydrogen production. These include agricultural residues such as sugar cane and sweet sorghum bagasse, corn stalks and stover, fodder maize, wheat straw, etc. and forestry residues such as wood trimmings. Among them agricultural residues seem to be the most abundant as estimated by the Food and Agriculture Organization.
Around 2.9 9 103 million tons from cereal crops and 1.6 9 102 million tons from pulse crops, 1.4 9 10 million tons from oil seed crops and 5.4 9 102 million tons from plantation crops are produced annually worldwide [97], being thus a voluminous source of lignocellulose that could be potentially utilised via bioconversions. Compared to the use of energy crops, the exploitation of residues, remaining after the harvesting and processing of the starch or sugar crops that cannot be further exploited in the food industry chain, is more likely to yield a solution with far better overall prospects for economic and environmental sustainability [98]. Hydrogen generated from such feedstocks can be characterised as 2nd generation hydrogen since its production is not competitive to food. Although being an abundant and almost zero cost feedstock, agricultural and forestry residues do not contain easily fermentable free sugars, but complex carbohydrate polymers, i.e. cellulose and hemicellulose, which are tightly bonded to lignin [99, 100]. Thus, their biotransformation to hydrogen is not an easy task in most cases. Even in the case that cellulolytic microorganisms are used for fermentative hydrogen production, residues have to be subjected to some kind of pretreatment, so that their
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Waste Biomass Valor (2010) 1:2139 Table 2 Fermentative hydrogen production from lignocellulosic residues Lignocellulosic residue Wood bers Corn stover Sugarcane bagasse hydrolysate Pretreatment Microorganism Operation mode Batch Continuous Batch H2 production rate 10.56 mmol/h 1.611 l/l/day Maximum H2 yield (mol/mol cons. hexose) 1.47 3 1.73b
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References
Mechanical Steam explosion (90220C, 35 min) Acid-thermal hydrolysis H2SO4 0.277(v/v), ?121C, 60 min
Clostridium thermocellum Mixed mesophilic cultures Clostridium butyricum
[55] [101] [102]
Fobber maize juice Sweet sorghum residues Wheat straw Maize leaves Barley straw Corn stalks Bagasse
Mechanical Mechanical Mechanical Mechanical Mild acid 1.8% H2SO4w/w Mild acid 1.8% H2SO4w/w Alkali-thermal 0.24 g/l NaOH, 100C, 2 h
Mixed mesophilic cultures Rumicococcus albus Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Caldicellulosiruptor saccharolyticus Mixed thermophilic cultures Thermoanaerobacterium thermosaccharolyticum
Continuous Batch Batch Batch Batch Batch Batch
0.28 mmol/h/g TVS 3.305 l/day
69.4c 2.59 3.8 (44.7 l/kg dry biomass) 3.6 (81.5 l/kg dry biomass) 13.39d
[91] [90] [92] [92] [93] [93] [103]
Corn stover
Acid-thermal hydrolysis H2SO4 0.254(v/v), ?121C, 30180 min
Batch
2.24
[104]
a b c d
l/kg TVS mol/mol total sugar ml H2/g dry mass mmol H2/g TVS
delignication and the subsequent loosening of the structure of cellulose and hemicellulose can be achieved, facilitating thus the subsequent liberation and uptake of sugars [93]. In Table 2 different types of residues used as feedstocks for hydrogen production are presented, as along with the achieved hydrogen yields and rates. Wastes and Wastewaters The biotransformation of wastes and wastewater towards hydrogen can be considered quite appealing from both the environmental (pollution control, renewable energy) and the economical (resources recovery, low total cost waste management) standpoint. The criteria according to which a waste/wastewater would be characterised as efcient feedstock for hydrogen generation are a high concentration of degradable organic compounds, high proportion of readily fermentable compounds such as sugars and
carbohydrates and low concentration of inhibitory to microbiological activity compounds. Many types of food processing industrial wastewaters can be considered suitable feedstocks for hydrogen production via dark fermentation. Rice winery, noodle, sugar, and molasses manufacturing, olive mill wastewater, olive pulp and cheese whey are some of the wastewaters that have been successfully tested for hydrogen production at laboratory scale (Table 3). Coming from food industry, such wastewaters theoretically meet all three abovementioned criteria, which can make them ideal candidates for hydrogen production via microbial processes. Indeed, quite high yields of hydrogen have been achieved from different food industry wastes without any pretreatment. However, it has to be mentioned that in most cases dilution of the raw waste has to be performed so as to lower the organic loading which otherwise can prove to be inhibitory for the success of the process [32, 43, 49, 114, 117]. Moreover,
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28 Table 3 Fermentative hydrogen production from different types of waste and wastewaters Type of waste/ wastewater Sugar factory wastewater OFMSW Rice winery wastewater Microorganism Mixed thermophilic culture Mixed mesophilic culture Mixed culture Operation mode H2 production rate
Maximum H2 yield 2.6 mol/mol hexose 0.15 l/g OFMSW 2.14 mol/mol hexose 122.9 ml/g COD carbohydrate 1.8 mol/mol hexose 7.89 mmol/g lactose 2.8 l/l wastewater 10 mM/g COD
References [105] [40] [106] [107] [108] [109] [110] [111] [112] [113] [114] [43] [115] [49]
Continuous 4.4 l/l/day Batch 0.4 l/g VSS/day
Continuous 9.33 l/g VSS/day 3.81 l/l/day Batch Batch Batch Batch 2.67 l/g VSS/day 0.288 l/g VSS/day 28.3 ml/h
Food wastesewage Mixed mesophilic culture sludge Food waste Cheese whey Potato processing wastewater Cheese whey Dairy wastewater Molasses Cheese whey Cheese whey Olive pulp Olive oil mill wastewater Wastepaper Mixed thermophilic culture Mixed mesophilic culture Mixed mesophilic culture Mixed mesophilic culture Mixed mesophilic culture Mixed mesophilic culture Mixed mesophilic indigenous microbial culture Mixed mesophilic culture Mixed mesophilic culture Ruminococcus albus
Clostridium saccharoperbutylacetonicum Batch
Continuous 1.59 mmol H2/l/day Continuous 4.8 l/l/day Batch 8.1 mmol/l/h 5.9 mol/mol lactose 0.9 mol/mol hexose 0.19 mol/kg TS 196.2 ml/g hexose Continuous 2.51 l/l/day Continuous 0.26 l/l/day Continuous 201.6 ml/day Batch
2.29 mol/mol hexose [116] (282.76 l/kg dry biomass)
such wastes can have a quite complex chemical composition, including different organic and inorganic substances with varying concentrations that may limit the reproducibility of a proposed process. Consequently, dening the exact conditions for the efcient treatment of a certain waste is not always possible. For example, in the case of cheese whey, a rich in readily fermentable sugars wastewater, Davila-Vazquez et al. [114] suggest that a pH between 6 and 7 is optimum for obtaining the highest hydrogen yield and production rate, respectively, whereas Yang et al. [118] suggest that a pH in the range 45 is the best in terms of both hydrogen yield and rate. In both cases mixed mesophilic cultures where used. Complex solid wastes, such as wastes from kitchen, food processing, mixed wastes, and municipal wastes have also been tested as feedstocks for fermentative hydrogen production. Such wastes apart from carbohydrates usually have quite high contents of proteins and fats, and thus their conversion efciencies to hydrogen are comparatively lower than those obtained from carbohydrate based wastewaters. Indeed, according to previous studies, the hydrogen production potential of carbohydrate-based wastes, was 20 times higher than that of fat-based and protein-based wastes [35]. This observation was partially
attributed to the consumption of hydrogen towards ammonium using nitrogen generated from protein biodegradation. Among different types of wastes, the organic fraction of municipal solid waste (OFMSW) could be considered quite promising as a potential feedstock for hydrogen production, since it can represent up to 70% of the total MSW produced, consisting of paper (up to 40%), garden residues, food wastes and wood [99]. It has to be noted though that such exploitation would require an initial selection/separation of the suitable substrates, which will lead to an additional cost in the overall process. A special type of waste that is attracting increased attention lately is crude glycerol, coming from the biodiesel production industry. Glycerol is a valuable chemical that is widely used in the cosmetics industry. The recent increase of the biodiesel production from vegetable oils and fats nowadays has lead to the generation of large quantities that have to be disposed somehow. In addition, the potential of glycerol utilisation would help improve the economics of biodiesel production. Although it is well established that the maximum theoretical hydrogen yield that can be obtained from carbohydrates is 4 [7], the maximum possible theoretical hydrogen yield from glycerol is still under question. Akutsu et al. [119] based on the study of Thauer et al. [8] on microbial metabolism, assume
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that the maximum theoretical yield is 3 mol H2/mol glycerol, according to the reaction: C3 H8 O3 2H2 O ! CH3 COO HCO 2H 3H2 3 7 In the study of Akutsu et al. [119] a mixed mesophilic consortium was used as inoculum, in which it proven that clostridia were the predominant species. The maximum obtained yield was 38.1 ml H2/g-CODglycerol, with 1,3 propanediol being the main by-product, followed by acetate, whereas no butyrate (the main by-product generated from Clostridia during carbohydrates fermentation) was detected. In contrast to the assumption of Akatsu et al., in the studies of Ito et al. [120] and Sakai and Yagishita [121] the maximum theoretical hydrogen yield from glycerol is assumed to have the value of 1 mol H2/mol glycerol, based on the reaction of glycerol conversion to H2, CO2, and ethanol as described by the reaction: C3 H8 O3 ! H2 CO2 C2 H5 OH 8
Pretreatment of Lignocellulose-Based Biomass and Wastes Although starchy and sugar based biomass and wastes are readily fermentable by microorganisms for hydrogen generation, lignocellulosic biomass needs to be pretreated so as to be exploitable. Pretreatment is commonly accepted to be an essential prerequisite to make lignocellulosic biomass accessible to enzymatic attack (either by microorganisms or enzymes), by breaking the lignin seal, removing hemicellulose, or disrupting the crystalline structure of cellulose [123]. The limiting factors that affect enzymatic hydrolysis of biomass have been traditionally divided into two groups: biomass structural features and enzyme mechanism. Pretreatment is responsible for altering the structural features of biomass which are classied as physical or chemical. The chemical structural features involve the compositions of cellulose, hemicellulose, lignin, and acetyl groups bound to hemicellulose. The physical structural features include the accessible surface area, the crystallinity, the physical distribution of lignin in the biomass matrix, the degree of polymerization, the pore volume, and the biomass particle size [124]. The correlation between structural features and digestibility is reported to be of varying importance, depending on the type of biomass to be treated [125, 126]. Consequently, because of the heterogeneity of lignocellulosic biomass, the application of the same pretreatment method can affect to a different extent each of the above parameters, whereas different pretreatment methods have a different effect on each of the above parameters. In general, pretreatment methods of lignocellulosic biomass can be divided into three main types, according to the means used for altering its structural features: mechanical, physicochemical and biological. Mechanical pretreatment is almost always applied before any other kind of pretreatment, and actually refers to milling, though which reduction of particle size and crystallinity of biomass is achieved. The reduction in particle size leads to an increase of available specic surface and a reduction of the degree of polymerization [127]. Physicochemical pretreatment was the rst to be applied successfully in lignocellulosic biomass for subsequent ethanol production [128131] and this knowledge has already been transferred to hydrogen production eld. However, though research on biological pretreatment of lignocellulosic biomass for ethanol production has also been performed [132, 133], the application of biological pretreatment methods to the fermentative production eld has not been so far studied. The main principles of different pretreatment methods are analysed below.
Both studies were conducted by pure cultures of the bacterium Enterobacter aerogenes which is known to follow the enterobacteriaceae pattern for hydrogen production from carbohydrates [21] The study of Ito et al. [120], has shown that crude glycerol waste has to be diluted so as to lower the organic loading and the salt concentrations, both of which seemed to be inhibitory above a certain limit. It was shown that batch cultures with suspended microbial cells could lead to a hydrogen production rate of 30 mmol/ l/h, which though was doubled (63 mmol/l/h) when using porous ceramics as a support material to x cells in the reactor. Another interesting nding from that study was that the addition of nutrients, such as yeast extract and peptone could enhance highly the hydrogen producing capacity of the microorganism from the particular waste, something though that could be expected to some degree, since the waste is by denition poor in nitrogen, an element which is necessary for microbial growth. The maximum observed hydrogen yield observed by Sakai and Yagishita [121] was 0.77 mol H2/mol glycerol with ethanol being the dominant by-product, whereas no propanediol was detected. On the other hand, 1,3 propanediol was the main by-product during fermentative hydrogen production by pure cultures of Klebsiella pneumoniae [122]. The maximum obtained hydrogen yield from that study was reported to be 0.53 mol H2/mol glycerol, whereas the maximum hydrogen production rate was 17.8 mmol/l/h. Based on the ndings from those studies, it can be assumed that the exploitation of crude glycerol towards hydrogen can be more efcient when based on enterobacteria fermentations. It has to be noted, however, that this is a rather new eld that needs to be further optimised.
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Physicochemical Pretreatment During physicochemical pretreatment, lignocellulosic biomass is exposed to acidic, alkaline or oxidative conditions, at ambient or elevated temperature. The use of high temperatures without the addition of some chemical agent can also be effectuated, leading to a process simply called thermal pretreatment. In any of the above cases, the result is that lignocellulosic biomass is fractionated since the bonds that hold together cellulose, hemicellulose and lignin are loosened and changes to the chemical structure of all three polymers occur to a different degree, according to the severity of each method. Among celluloses, hemicelluloses and lignin, hemicelluloses are the most sensitive when thermal-chemically treated and thus are the rst to be degraded [134]. Combinations of the methods have been examined, combining two or more physical and chemical pretreatment elements. For acid pretreatment of lignocellulosic biomass, either dilute or concentrated acids, such as H2SO4 and HCl, can be used. The main reaction that occurs during acid pretreatment is the hydrolysis of hemicellulose, especially xylan, since glucomannan is more stable. Under such conditions furfural (FF) and hydroxymethylfurfural (HMF) generation can occur because of dehydration of xylose and galactose, mannose and glucose respectively, whereas further production of formic and levulinic acids can also be observed [99]. Lignin is hardly dissolved in most cases, but it is disrupted to a high degree, leading to increased cellulose susceptibility to enzymes [135]. Alkaline pretreatment refers to the addition of dilute bases on the biomass leading to an increase of internal surface by swelling, a decrease of polymerization degree and crystallinity, destruction of links between lignin and other polymers, and lignin breakdown. The effectiveness of this method depends on the lignin content of the biomass [136]. Lignin is primarily affected by alkaline pretreatment methods, causing depolymerisation and cleavage of lignin-carbohydrate linkages [137]. Hemicellulose solubilisation into its oligomers also takes place whereas cellulose structure is affected to a less degree. When thermally pretreated the lignocellulosic biomass is actually heated at temperatures between 150 and 220C. When treated at temperatures above 160 180C, part of the lignocellulosic biomass, rst the hemicelluloses and lignin shortly after, solubilise [138]. During thermal processes a part of the hemicellulose is hydrolyzed, forming acids, which subsequently act as catalysts for the further hydrolysis of hemicellulose. However, it is concluded that other so far unknown factors, apart from the formed acids, can play a role in the solubilisation of hemicellulose [128, 139]. Thermal pretreatment with temperatures of 160C also lead to the solubilisation of lignin. The produced compounds
are almost always phenolic compounds and have in many cases an inhibitory or toxic effect on bacteria, yeast and methanogens/archae [140]. Temperatures of 250C and higher should be avoided during pretreatment, as unwanted pyrolysis reactions start to take place. The application of different physicochemical pretreatment methods on lignocellulosic biomass aiming at facilitating hydrogen production, are summarized in Tables 1 and 2. Biological Pretreatment Biological pretreatment of lignocellulosic biomass aims at the removal of lignin so that the complex carbohydrates cellulose and hemicellulose are liberated, becoming thus accessible to enzymatic attack for their depolymerization (saccharication). It can be performed either by whole cell microorganisms or enzymes capable of degrading lignin by oxidation [141]. Lignin is a polymeric substance of coniferyl alcohol and sinapyl alcohol, with functional groups such as hydroxyl, methoxyl and carbonyl. The amorphous heteropolymer is non-water soluble and optically inactive, making thus its degradation very tough [99]. Consequently very few species are capable of performing biological break down of lignin. Among them bacteria symbiotic to termites [142] and white rot fungi are included. The enzymatic degradation of lignin proceeds through the concerted action of specic enzymes, i.e. lignin peroxidase, manganese peroxidase, H2O2-generating enzymes and laccase, which produce strong oxidants and thus combust, the lignin framework [143]. On comparing the efciency of whole cell and enzymatic delignication, a possible drawback of the rst could be the degradation and consumption of carbohydrates, resulting in lower yields during the subsequent bioconversion to hydrogen. However, white-rot fungi can perform selective or non-selective delignication of wood. In selective delignication, lignin is removed without any marked loss of cellulose, whereas in non-selective delignication all the major cell wall components are degraded [144]. Among the best studied white-rot fungi are Phanerochaete chrysosporium and Phlebia radiata which degrade lignin selectively, and Pleurotus sp. which degrades lignin and hemicelluloses selectively. The nal delignication efciency is affected by several parameters such as the oxygen and moisture level, the C:N ratio and concentration of specic ions like Cu2?. The reactor system choice and development, solid state or submerged fermentation system, is of crucial importance as well. In overall, biological pretreatment, using microorganisms and/or their enzyme systems to breakdown the lignin present in lignocellulosic biomass, is an environmentally friendly approach, and according to some researchers with signicant advantages over the
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physicochemical pretreatment technologies, such as reduced energy and material costs and simplied processes and equipment [145, 146]. Research results are very encouraging so far; however, the development and application of a fungi-based pretreatment step for efcient and cost-effective lignocellulose break-down has a long way to go. Formation of Inhibitory Compounds due to Pretreatment Upon either physicochemical or biological pretreatment of lignocellulosic biomass, various organics substances are generated that are considered to be undesirable bioproducts since they may be inhibitory to the microbial metabolism as has been demonstrated by several researchers [102, 146149]. The most commonly generated, being also the best studied are furfural, hydroxyfurfural and phenolics. Furfural (FF, C5H4O2) and 5-hydroxymethylfurfural (HMF, C6H6O3) are aldehyde and furan compounds, which are formed during the thermal decomposition of sugars and carbohydrates, whereas phenolics are chemical compounds containing a hydroxyl group (OH) bonded directly to an aromatic hydrocarbon group and are produced during the degradation of lignin. During thermochemical pretreatment, all three inhibitory compounds are formed, whereas during biological pretreatment only phenolics emerge. The severity of the inhibitory effect of these compounds depends on their concentrations and thus, in order to achieve efcient fermentations, either mild pretreatment methods should be selected, or the inhibitory compounds should be removed prior to feeding the fermentation bioreactors [102, 147149]. Such removal is indeed possible and can be achieved by several methods, such as extraction [150], ion exchange [151], activated carbon [152], overliming [153] or laccase and peroxidase treatment [154, 155]. Although the inhibitory effect of FF, FHM and phenolic compounds on hydrogen producing bacteria is not yet well dened, it is expected that the results would be similar to those observed during fermentations for ethanol or methane production. Summarizing, an ideal effective pretreatment process would produce reactive bre, yield pentoses in nondegraded form, exhibit no signicant inhibition of fermentation, require little or no feedstock size reduction, entail reactors of reasonable size (high solids loading), built of materials with moderate cost, not produce solid residues, and have a high degree of simplicity [155]. A process meeting all those criteria at the same time unfortunately has not yet been developed. Each type of pretreatment has certain advantages in comparison to the others and certain
limitations too. One has to select the most efcient one depending on the specic circumstances (feedstock type and fermentation type to follow).
Types of Reactors Reactor conguration is considered to be crucial for the overall performance of fermentative hydrogen production. It inuences the reactor microenvironment, the prevailing microbial population, the established hydrodynamic behaviour, the contact between substrate and consortia, etc. [156]. The most commonly used reactor type is the mixed (continuously stirred) suspended growth reactor. In general, mixed reactors for fermentative hydrogen production can operate in either batch or continuous mode. Batch mode fermentative hydrogen production has been shown to be more suitable for research purposes [26, 157], but any industrially feasible process would most likely have to be performed on a continuous or at least semicontinuous (fed or sequencing batch) basis. The CSTR (Continuously Stirred Tank Reactor) is the most commonly used continuous reactor system, offering simple construction, ease of operation and effective homogenous mixing as well as temperature and pH control. In a conventional CSTR, biomass is well suspended in the mixed liquor, which has the same composition as the efuent. The production of hydrogen in a CSTR may be inuenced by several operating parameters. The most important parameter is the HRT (Hydraulic Retention Time). A small enough SRT (Solids Retention Time) in a mixed culture CSTR secures that no methanogenesis takes place, since methanogens are slowly growing species. Typically a HRT of 1236 h, depending on the substrate, provides complete conversion of carbohydrates and highest hydrogen yields, avoiding the generation of methane [32, 49]. The temperature and the pH are also important operating parameters as outlined in section Microorganisms. Finally, the partial pressure of hydrogen seems to be a very important parameter. High partial pressures of hydrogen have been shown to inhibit hydrogen production. One way to avoid high partial pressures is to sparge an inert gas through the reactor [158, 159]. In some studies it has been found that batch hydrogen production gives higher hydrogen yields than continuous operation [32, 89, 90]. This could be attributed to the substantially different microenvironment of the microbes in a batch reactor (time-varying and consequently higher substrate concentrations in the early part of the batch) leading to a different metabolic ux than in the constant low-substrate microenvironment in a CSTR. A batch bioreactor has the disadvantage of requiring long down-times
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and startup procedures. An interesting alternative operation of a mixed bioreactor is the sequencing-batch operation: In a cycle, we distinguish feed, react, settle, decant phases, so that the bioreactor is in essence operating in a semi-continuous mode. The optimisation of a mixed bioreactor operation requires appropriate modelling. Relatively few studies have been presented so far on modelling fermentative hydrogen production [160, 161]. The most promising framework is the ADM 1 (anaerobic digestion model 1) framework of Batstone et al. [162], which needs to be appropriately modied to properly describe fermentative hydrogen production [52, 163165]. In a CSTR, biomass has the same retention time (SRT) as the HRT, and thus, its concentration in the mixed liquor is limited and this limits the rate of hydrogen production. Another category of reactors are systems characterized by physical retention of the microbial biomass, which offer several advantages compared to the conventional CSTRs. In these systems, the SRT is independent of HRT due to physical retention of the microbial biomass inside the reactor, allowing high cell concentrations and thus high hydrogen volumetric production rates with relatively small reactor volumes. Physical retention of microbial biomass could be accomplished by several different means, including the use of naturally forming ocs or granules of self immobilized microbes, microbial immobilization on inert materials, microbial-based biolms or retentive membranes [166]. It has been recently found that hydrogen producing biomass in a CSTR could be self-granulated or occulated under proper conditions [167]. Another approach is to immobilize biomass on biolms or articial granules made of various support materials such as cuprammonium rayon [168], polyvinyl alcohol, polyacrylamide and anionic silica sol [125, 169]. A potential problem with attached growth bioreactors is the loss of hydrogen through the formation of methane due to extended retention of the biomass inside the reactor permitting the establishment of slow-growing methanogenic populations. Among the types of reactor used for continuous hydrogen production, are the CSTR [32, 89, 90, 117, 170] and several variations of hybrid and attached growth reactors, such as the Upow Anaerobic Sludge Blanket reactor, the UASB [171], the Packed Bed Reactor, PBR [172], Anaerobic Sequencing Batch Reactor, ASBR [173], the Fixed Bed Bioreactor with Activated Carbon, the FBBAC [27], the Anaerobic Fluidized Bed Reactor, the AFBR [174, 175], the Carrier-Induced Granular Sludge Bed, the CIGSB [176], Membrane Bioreactor, the MBR [50] and the Rhomboidal reactor [60]. A direct comparison of the performance of the different types of reactor congurations that have been studied in terms of hydrogen productivity is not possible, since the operational parameters (feedstocks
and operating conditions) along with the reactor conguration, in all these studies, are quite different.
Exploitation of By-Products Hydrogen yields of fermentative hydrogen production processes are restricted by the existing metabolic pathways to 2 or 4 mol H2/mol glucose consumed, for butyrate or acetate fermentation, respectively. Moreover, regardless the nal hydrogen yield achieved, fermentative hydrogen production processes are not generally expected to be effective in terms of Chemical Oxygen Demand (COD) removal. In contrast to the anaerobic digestion processes during which organic carbon is nally converted to methane, thus diminishing the organic load, in the acidication processes carbohydrates are transformed into more oxidized soluble compounds, thus remaining in the liquid phase in the form of various volatile fatty acids (VFAs) and solvents. Consequently acidication is accompanied by limited removal of COD [21]. Apart from the carbon that remains trapped in the generated oxidative products, a remarkable amount of hydrogen also remains trapped in these compounds, resulting thus in nal hydrogen yields in the range of about 12 mol H2/mol of hexose. Even under optimum conditions of 4 mol H2/mol glucose, about 6070% of the organic matter of the feed remains in solution [156]. Further utilization of the organic matter contained in the efuent of a fermentative hydrogen producing bioreactor, could increase the overall energy output of the process and/or more products recovery. The development of a two-stage process so far, involves the fermentation of the substrate to hydrogen and organic acids in the rst stage and in a second stage either the additional energy extraction or the generation of high added value products by exploiting the efuent of the rst stage reactor. Biomethane One approach in order to further utilize/reuse the remaining organic matter is to produce a second useable form for energy (an energy carrier) such as CH4 in a second stage. Integration of an acidogenic process with a subsequent methanogenic process for combined hydrogen and methane generation, offers several advantages such as a higher performance of the process in terms of waste stabilization efciency and net energy recovery [177]. Such a two-stage system has been proposed so far for organic solid wastes, rich in carbohydrates such as food wastes [31], cheese whey [43, 117], olive mill wastewaters [115], household solid waste [178], a mixture of pulverized garbage and shredded paper wastes [179] and wastewater sludge [180]. A combined hydrogen and methane generation process has
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already been scaled up to the pilot plant stage, for organic solid wastes [181]. The hydrogen and methane production rates were 5.4 m3/m3/day and 6.1 m3/m3/day, respectively while the process COD removal efciency was 80%. The overall efciency of this combined process is demonstrated by the fact that methane yields were twofold higher than a comparable single-stage process [181]. Photoheterotrophic Hydrogen Production A different approach for increasing the overall energy extraction is to couple the fermentative hydrogen production with photofermentation aiming to the recovery of additional hydrogen. In such a two-stage process, the rich in organic acids efuent produced in the rst stage by fermentative bacteria could be converted to hydrogen in the second step by photosynthetic bacteria which capture and use light energy. Light as energy source is necessary for the conduction of such reactions since their Gibbs energy is positive (e.g. Eq. 1) and thus they are not thermodynamically favoured. CH3 COOH 2H2 O hv ! 2CO2 4H2 DGo 75:2 kJ mol1 9
This combination of both kinds of bacteria in a two stage system not only reduces the light energy demand of the photosynthetic bacteria but also enhances the hydrogen yield as well [79]. Intensive research has been carried out in this area [182, 183] in the recent years. However, there are important factors limiting the practical application of such a process. One of them is that the involved hydrogen enzyme, nitrogenase, is potentially sensitive to the nitrogen content of the medium/substrate, since nitrogen, especially in the form of NH4?, not only inhibits the enzymatic activity, but also represses the synthesis of the enzyme [184, 185]. This limitation can be potentially overcome either by genetic manipulation [186] or selection [187] to remove nitrogenase regulation. In addition, one of the most severe constraints is that photosynthetic efciencies are very low, since at even moderate light intensities, the main part of captured light is dissipated as heat [188]. This means that there will be a demand of large surface areas for the production of hydrogen contributing to the total cost and render the development of a two-stage process of fermentationphotofermentation, far from practical application. Production of Polyhydroxyalkanoates In both methods analysed above, the rich in acids efuent from dark fermentation hydrogen producing systems is used for extra energy generation. An alternative approach is their exploitation for the production of other high added
value products such as polyhydroxyalkaonates (PHAs), via selected microorganisms. The current sharp focus of attention on the environmental pollution caused by discarded petrochemical plastics, has given added impetus to research on the production of plastics that can be fully biodegraded in the natural environment, such as PHAs. PHAs are biodegradable polyesters that can be produced by bacteria [189], which they accumulate as intracellular storage reserves of carbon and energy under stress conditions in the form of inclusion bodies [190]. There are several types of biopolyesters but poly(3-hydroxybutyrate) and poly(3-hydroxyvalerate) are the most well-known since they bear similar properties to conventional plastics such as polypropylene and polyethylene [191]. The copolymers of PHAs are less penetrable to oxygen than conventional plastics, being thus ideal as packaging material in food industry, but also in many other applications of the medical sector [192]. The production of PHAs from acidied wastewaters had been previously investigated in both lab [193] and pilot [194] scale, showing very promising results. Combining dark hydrogen and PHAs production in a two stage system has been proposed by Ntaikou et al. [49] using three phase olive mill wastewater as feedstock. Both processes were continuous; hydrogen production was conducted anaerobically in a CSTR and the efuent, consisting mainly of acetate, butyrate, propionate and ethanol, was subsequently fed to an SBR where PHAs were produced aerobically. Lately, the scale up of the process has also been investigated [195], conducting the two stage PHAs production at semi-pilot scale.
Real Scale Applications Up to now, a continuous scaled-up process for sustainable fermentative H2 production has not been reported in the literature. Only very few studies on the fermentation of sugars to hydrogen, at pilot scale, are available so far. Ren et al. [196] performed a pilot scale study in a continuous ow anaerobic fermentative reactor with an active volume of 1.48 m3 using molasses as feedstock. The reactor operated at organic loading rates of 3.1185.57 kg COD/m3 reactor/day and produced 5.57 m3 H2/m3 reactor/ day or 8240 l H2/day with a hydrogen yield of 26.13 mol/ kg COD removed. The efuent which was produced, contained primarily acetate and ethanol and was as high as 3,000 l/day. This, rich in acetate, efuent could be further exploited for hydrogen production through a subsequent photoheterotrophic stage, which could increase hydrogen production by 317%. Vatsala et al. [197] evaluated the feasibility of hydrogen production from a sugar cane distillery efuent using co-cultures of Citrobacter freundii 01, Enterobacter
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aerogenes E10 and Rhodopseudomonas palustris P2, at 100 m3 scale. The reactor operated in batch mode for 40 h, and the hydrogen production was 21.38 kg with an average yield of 2.76 mol H2/mol glucose and a rate of 0.53 kg/ 100 m3/h. The results showed that distillery efuent could be used as a source of hydrogen providing insights into treatment for industrial exploitation. Since data for real applications are not available so far, designing of such a process will have to be based on the respective lab scale experiments. The problem is that the hydrogen productivity and yields depend signicantly on the prevailing conditions, the feedstocks as well as the inoculum used. However, from laboratory-scale work on continuous processes, it could be suggested that such a process may operate at a mesophilic temperature, at a pH around 5.5 and an HRT approximately 812 h, for simple substrates. Higher HRTs are indicative for complex carbohydrate-rich feedstocks. Finally, such a process may use as microbial inoculum, heat treated sludge form aerobic or anaerobic process or the indigenous microbial species available in the feedstock/waste, which has often proved to work optimally [43, 89].
major limitation of hydrogen production from biomass and wastes, however, remains the relatively low rates and yields of hydrogen generation. A possible solution to that could be combining the fermentative hydrogen producing systems with a second stage process, via which the further use of the emerging by-products would be feasible for extra energy and/or materials recovery either via hydrogen, biogas or other valuable products in a biorenery framework. Future progress leading to more efcient fermentative hydrogen production will depend on research efforts to (a) identify the most suitable feedstocks, (b) develop more efcient pretreatment methods for saccharication of lignocellulosics, (c) isolate and/or develop through genetic engineering high hydrogen producing strains, (d) develop optimal reactor congurations and operating strategies through modelling and optimisation.
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Conclusions and Outlook It is widely believed that the future energy economy will be based on hydrogen; this is due to the fact that hydrogen is a clean and efcient energy carrier, with zero emissions when burned, that can be produced by renewable sources such as biomass and wastes. Fermentative hydrogen production from biomass and wastes refers to the exploitation of the latter via microorganisms which are capable of converting organic matter to acids and alcohols with simultaneous liberation of molecular hydrogen. Different types of biomass and wastes, in terms of both chemical composition and origin, have been tested as potential feedstocks, leading in many cases to very promising results. Among them the use of energy crops, although being very effective, has lately been put under question due the arising food versus fuel debate. On the other hand the lignocellulosic residues remaining from such crops, together with other agricultural and forestal residues remain an abundant source of biomass, on the use of which, 2nd generation hydrogen production can be based. Among the limiting steps for the efcient exploitation of such biomass is identifying effective economical viable and environmental friendly solutions for the liberation of carbohydrates via pretreatment. On the other hand readily fermentative carbohydrates are included to a high degree in sugar and starch based wastes and wastewaters. Their exploitation, not only leads to energy recovery but can also be a cost reducing management method. The
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Bio Hydrogen Production From Biomass and Wastes Via Dark Fermentation A Review

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Waste Biomass Valor (2010) 1:2139 DOI 10.

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Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

Clostridium thermocellum Mixed mesophilic cultures Clostridium butyricum

[55] [101] [102]

Continuous Batch Batch Batch Batch Batch Batch

0.28 mmol/h/g TVS 3.305 l/day

[91] [90] [92] [92] [93] [93] [103]

Acid-thermal hydrolysis H2SO4 0.254(v/v), ?121C, 30180 min

Waste Biomass Valor (2010) 1:2139

Continuous 4.4 l/l/day Batch 0.4 l/g VSS/day

Clostridium saccharoperbutylacetonicum Batch

2.29 mol/mol hexose [116] (282.76 l/kg dry biomass)

Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

Waste Biomass Valor (2010) 1:2139

95. 96. 97.

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