Progress in liquid biofuel and biohydrogen from agro-industrial wastes by clostridia

Progress in liquid biofuel and biohydrogen from agro-industrial wastes by clostridia Mohamed Hemida Abd-Alla*, Ahmed Abdel-salam Issa, Fatthy Mohamed Morsy and Magdy Khalil Bagy Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt *Corresponding author: E-mail: mhabdalla2002@yahoo.com The increase in prices of petroleum based fuels, future depletion of worldwide petroleum reserves and environmental policies to reduce CO 2 emissions have stimulated research into the development of biotechnology to produce chemicals and fuels from renewable resources. The most commonly used metabolically derived biofuels are hydrogen, acetone, butanol and ethanol. Biofuel is produced biologically from renewable biomass by Clostridium spp. under strictly anaerobic condition. Substrate costs can make up to about 63% of the total cost of biofuel production. This is not because of the expense of the substrate itself, but mainly because of the low efficiency of Clostridium to convert substrate into biofuel, i.e. the yield of biofuel is often low, and this together with the formation of by-products leads to a high cost for butanol recovery. In addition, the maintenance of strict anaerobic conditions for Clostridium requires special conditions e.g. addition of costly reducing agents, and flushing with N 2 gas, which increase the cost of the fermentation process. Hence, substrates such as agricultural residues, including wheat straw, barley straw, maize stover, wood hydrolysate, and switchgrass as well as dairy industry waste offer potential alternatives. To reduce the costs of producing hydrogen and ABE from fermentation, include using a low cost fermentation substrate and/or optimizing the fermentation conditions to improve the efficiency of converting substrate to biofuel. The facultative anaerobes are able to consume O 2 in a medium and so a steady anaerobic condition in a fermentor was attained without addition of any reducing agent. Significant progress has been made towards genetically engineering clostridia to utilize a variety of substrates, and to reduce the need for pretreatment processes as well as reduce the application of reducing agents for creation anaerobic conditions. Among the cheap and readily available substrates for biohydrogen and liquid biofuel production, agro-industrial wastes are possibly one of the better choices. The possibility of using cheaper resources, such as lignocelluloses, whey cheese or any agro-industrial and domestic organic wastes, as the alternative substrates for biofuel production over more expensive substrates. Selection of cellulolytic clostridia in applying biotechnology to acetone-butanol fermentation revived interest in research on solvent production by fermentation. Keywords: Biofuel, Biohydrogen, clostridia, agro-industrial wastes 1. Hydrogen production by clostridia Many anaerobic organisms can produce hydrogen from carbohydrate containing organic wastes. The organisms belonging to genus Clostridium such as C. buytricum [1], C. thermolacticum [2], C. pasteurianum [3,4], C. paraputrificum M-21 [5] and C. bifermentants [6] are obligate anaerobes and spore forming organisms. Clostridia species produce hydrogen gas during the exponential growth phase. In batch growth of clostridia the metabolism shifts from a hydrogen/acid production phase to a solvent production phase, when the population reaches to the stationary growth phase. 1.1 Hydrogenase Clostridia can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Hydrogen is produced in clostridia by the (FeFe)-hydrogenase catalyzing the reversible reduction of protons. The [FeFe]-hydrogenase is of particular interest for bioenergy applications because of its high catalytic rates of proton reduction. Owing to their highly reactive and complex metallocenters, hydrogenases are regarded as the most efficient with turnover rates 1000 times higher than for nitrogenases [7]. The structures of the iron hydrogenases from Clostridium pasteurianum has been investigated by x-ray crystallography [8]. The structure of the enzyme was also investigated in other bacteria such as Desulfovibrio desulfuricans [9]. The proteins consist of one or two subunits and have a remarkable iron cofactor (H cluster) in the catalytic site. The H cluster contains an unusual supercluster comprising a [4Fe4S] subcluster and a [2Fe] center, which are bridged together by single cysteinyl sulfur [10]. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species [11]. The properties of nitrogenase and hydrogenases are summarized in table 1 as described by [12, 13]. 340 FORMATEX 2013

Table 1. Comparison between nitrogenase and hydrogenases Property Nitrogenase Hydrogenase Substrates ATP, H, N 2, electrons H +, H 2 products H 2, NH 4 ATP,H +, H 2, electrons Number of proteins Two (Mo-Fe and Fe) one Metal components Mo, Fe Ni, Fe, S inhibitors NO3, NH 4, O 2 CO, EDTA, O 2 1.2 Methods for hydrogen production by clostridia Clostridium sp. is extremely sensitive to O 2 and their H 2 -producing abilities are inhibited by a slight amount of O 2 in a fermentor, so the addition of a reducing agent such as L-cysteine in a medium is indispensable for its H 2 production. However, such reducing agents are expensive and so the procedure for H 2 production by Clostridium sp. without the reducing agent is desired [14]. The facultative anaerobes such as E. coli EGY are able to consume O 2 in a medium and so a steady anaerobic condition in a fermentor was attained without addition of any reducing agent [14]. A mixed continuous culture of Clostridium butyricum and Enterobacter aerogenes removed O 2 in a fermentor and produced H 2 from starch with yield of more than 2 mol H 2 mol -1 glucose without any reducing agents in the medium [15]. The use of facultative anaerobes to remove oxygen from the medium was effective for H 2 and acetone-butanol production by Clostridium [16]. Moreover, aerobic Bacillus may function as an oxygen consumer in the mixed culture to create an anaerobic environment for Clostridium [17, 18]. The application of N 2 sparging and addition of a reducing agent (Lcysteine) produced similar amounts of hydrogen as compared with anaerobiosis generated by E. coli. [14]. 1.2.1 Enrichment of hydrogen yield by a second stage photofermentation Although as many as 12 mol hydrogen can theoretically be derived from glucose, there is no known natural metabolic pathway that could provide this yield, due to the presence of other products [19, 20]. The theoretical maximum hydrogen yield in dark fermentation of glucose is 4 mol H 2 /mol glucose when acetic acid is the only product. With butyric acid only 2 mol hydrogen are produced. Acetic acid fermentation: C6H12O6 + 2 H 2 O------> 2 CH 3 COOH + 4H2 + 2CO2 Butyric acid fermentation: C6H12O6 + 2 H 2 O------>CH 3 CH2CH2COOH + 2H2 + 2CO2 Since photosynthetic bacteria utilize organic acids, by products produced by fermentative bacteria in H 2 production, a microbial H 2 production by a mixed culture of the fermentative bacteria and the photosynthetic bacteria has become a subject of considerable interest [1]. Dark fermentation with mainly acidogenic bacteria (Clostridium sp. and Enterobacter sp.) has the ability to produce H 2 while converting organic substrates into volatile fatty acids and alcohols [21, 22]. These soluble metabolites (e.g., acetic acid, butyric acid) can be further utilized via photofermentation (with photosynthetic bacteria, such as purple non-sulfur bacteria) resulting in more H 2 production at the expense of light energy [23-28]. 2. Acetone-butanol-ethanol (ABE) fermentation Acetone-butanol-ethanol (ABE) fermentation was an important industrial process during the early 1900s, and was first reported for butanol production by Louis Pasteur in 1861 [29, 30]. In 1945 66% of the total butanol and 10% of the total acetone production were obtained by ABE fermentation, making it the largest scale bioindustry ever run second to ethanol fermentation [31]. However, butanol production by ABE fermentation declined rapidly during the 1950 s due to the rise of cheaper petrochemical synthesis and increased cost of fermentation raw materials [32,33]. 2.1 Return to ABE fermentation Society in the early 21st century appears to be undergoing an unprecedented transition with respect to the fundamental source of its materials and energy. Petroleum, the fuel that has been driving modern society for one century, is showing signs of scarcity [34, 35]. With the growing concerns of environmental issues, depleting fossil resources and increasing crude oil price, renewed interest has returned to fermentative butanol production, not only as a chemical but also as an alternative biofuel [33, 36-38]. To overcome the limitations of conventional ABE fermentation such as low titer and high substrate cost, areas under research and development include utilization of renewable and low-cost feedstocks, development in novel fermentation processes, alternative product recovery technologies, and metabolic engineering of solvent-producing microorganisms [39-43]. FORMATEX 2013 341

2.2 Enhancing ABE resistance in solventogenic clostridia In recent years, butanol has been attracting research attention as an alternative biofuel to bioethanol. Compared to ethanol, butanol is considered as the next generation biofuel due to many advantages it offers, such as higher energy content and lower volatility [32, 38, 44, 45]. Butanol can be used directly or blended with gasoline and diesel as fuel additives in the current automobile engine without any modification or substitution. In addition, butanol is compatible with the current transportation pipeline for gasoline [32, 44, 38]. In the traditional and historic batch ABE process, C. acetobutylicum produces some hydrogen, carbon dioxide, acetate, and butyrate during the initial growth phase, resulting in decreasing ph. Clostridium species secrete enzymes that facilitate the breakdown of polymeric carbohydrates such as starch into monomers that can be transported into the cells using the phosphoenolpyruvate-dependent phosphotransferase system for glucose and non-pts mechanism for galactose. As the batch culture enters the stationary phase, a metabolic shift to solventogenesis occurs with the assimilation of the acids and concomitant release of n-butanol, acetone and ethanol. The biochemical pathways followed in clostridia are fairly well described [46]. However, the multiple metabolic pathways and two-stage nature of ABE fermentation still prevent a clear and conclusive calculation of maximum theoretical yield. C. acetobutylicum has been successfully adopted for the production of acetone and butanol. Under batch conditions the fermentation process of solvent-producing Clostridium strains proceed with the production of cells, hydrogen, carbon dioxide, acetic acid and butyric acid during the initial growth phase (acidogenesis). As the acid concentrations increase (ph decrease), the metabolism of cells shifts to solvent production (solventogenesis) and acidogenic cells able to reproduce themselves - shift to the solventogenesis state with a morphological change. During solventogenesis the active cells become endospores unable to reproduce themselves. The solventogenic clostridia have historically been the most studied acetone-butanol-producing organisms. Two Genome sequenced solventogenic microorganisms - C. acetobutylicum824 and C. beijerinckii NCIMB 8052 have been the focus of significant analysis and adaptive engineering in an effort to increase resistance to solvents (particularly butanol) in the fermentation broth. While significant advancements have been made, low solvent concentration remains a hurdle for commercialization of biologically produced butanol and acetone. Typically, the concentration of total solvents in the bioreactor during the ABE fermentation rarely exceeds 20 g/l, with butanol concentrations rarely exceeding 13 g/l [47]. Efforts in strain improvement have increased butanol concentration to as much as 19 g/l, but concentrations approaching 40 g/l could significantly reduce the energy used for butanol recovery [48]. Borden and Papoutsak are expanded the search for genes to target C. acetobutylicum's ability to withstand greater solvent concentrations using a genomic library. Plasmids were inserted into wild type C. acetobutylicum cells via electroporation, and the cells were challenged with various amounts of butanol [49]. Sixteen genes were identified as contributing to the cells ability to withstand greater concentrations of butanol; pcac1869 in particular showed a 45% increase in tolerance. Similarly, pcac0003 was found to have a 24%increase in butanol tolerance. CAC1869 is suspected to be a transcriptional regulator (KEGG) and was found to have maximal transcription preceding induction of the solventogenic genes aad, ctfa, and ctfb. This gene is actively transcribed throughout the transitional phase. 2.3 Recent advances in ABE fermentation from agro-industrial wastes In the past, ABE fermentation was done employing easily fermentable carbohydrates in mashes derived from maize, grains, beets or potatoes. These starchy substrates could be converted by clostridia to ABE without prior pretreatments [50]. Challenging the biofuel development, the cost of substrate is the most important economic factor in ABE production (14, 38, 47, 51, 52]. The ABE fermentation is receiving renewed interest as a way to upgrade renewable resources into valuable base chemicals and liquid fuels [53]. Major challenges with ABE fermentation concern low solvent concentrations, yields and productivities due to butanol toxicity to microbial cells [54]. Recent developments of molecular techniques applied to solventogenic microorganisms in combination with advances in fermentation technology and downstream processing have contributed to improve feasibility and competitiveness of the ABE fermentation process. Biological conversion of lignocellulose to biofuels usually pretreatment/hydrolysis of the substrate, followed by fermentative production of biofuels [55]. Among those steps, hydrolysis that yields fermentable reducing sugars is often the rate-limiting step of the overall cellulosic biofuels production process [56]. In general practice, physical and chemical treatments are usually applied for pretreatment and partial hydrolysis of the lignocellulosic feedstock, while enzymatic hydrolysis is effective in obtaining simple reducing sugars or monosaccharides [57]. Recently, improved technologies have been developed for the biobutanol fermentation process: higher butanol concentrations and productivities are achieved during fermentation, and separation and purification techniques are less energy intensive. This may result in an economically viable process when compared to the petrochemical pathway for butanol production. Improved fermentation strains currently available are not sufficient to attain a profitable process design without implementation of advanced processing techniques. Among the novel technologies for fermentation and downstream processing, fed-batch fermentation with in situ product recovery by gas- 342 FORMATEX 2013

stripping, followed by either liquid-liquid extraction or adsorption, appears to be the most promising techniques for current industrial application. The ABE concentration and productivity in typical batch fermentation by solventogenic Clostridium species can be increased to values between 13 to 18 g/l and 0.2 to 0.3 g/l/h, respectively [58]. Modest increases in ABE titer and productivity can be achieved by supplementing acetate [59] or butanol [60, 61] to the fermentation medium. Fermentation substrate is an important factor influencing the cost of butanol production [33, 62, 63]. Lignocellulose is the most abundant renewable resource on the planet and it has great potential as a substrate for fermentation because of the un-competitiveness with food resources. Despite the advantages in sustainability and availability, commercial use of lignocelluloses is still problematic. Due to the complexity of lignocellulosic materials, hydrolysis of hemicellulose and cellulose into five- and six-carbon sugars has to be carried out prior to, or concurrently with, the fermentation [64]. 2.4 Selection of clostridial isolates for maximizing ABE production Some reports on the corresponding ability of clostridia strains are available in the scientific literature, but no systematic investigation has been carried out. Present contribution regards the characterization of the ABE fermentation by different clostridia adopting sugars representative for hydrolysis products of lignocellulosic biomass: hexoses (glucose and mannose) and pentoses (arabinose and xylose). Batch tests were characterized in terms of butanol and solvent yield and maximum solvent concentration. Ten clostridial isolates belonging to five species were evaluated for their ability to utilize non cellulosic (non salty cheese whey, sugarcane molasses and beet molasses) and lignocelluosic (wheat straw, rice straw, beet residues and corn husks) substrates as source of carbohydrate for production of acetone, butanol and ethanol. Unpublished data presented in Table 2 indicated that in case of fermentation of cheese whey, C. bejerinkii isolate STDF 1(6,8 g/l ABE), C. butyricum isolate STDF 2 (5.07 g/l ABE) and C. acetobutylicium isolate STDF 55 (4.95 g/l ABE) were the best producers of total acetone, butanol and ethanol. Application of sugarcane molasses as substrate indicated that the superior producers of total acetone, butanol and ethanol (32.97 g/l ABE) were C. pasteurianum isolate STDF12 followed by C. saccharobutylicum isolate STDF 36 (29.23 g/l ABE) and C. acetobutylicium isolate 55 (27.74 g/l ABE). Similar to sugarcane molases, fermentation of beet molasses indicated that C. pasteurianum STDF 12 (16.85 g/l ABE), C. saccharobutylicum isolate STDF 36 (13.12 g/l ABE) and C. acetobutylicium isolate STDF 55 were the best. Table 2. Production of acetone (A), butanol (B) and ethanol (E) (g/l) from cheese whey, sugar cane molasses and beet molasses (49.5 g/l reducing sugars) after 5 days of fermentation by different clostridial isolates. STDF Chesses whey Sugarcane molasses beet molasses Isolate * A B E ABE A B E ABE A B E ABE 1 2.16 2.64 2.00 6.80 4.88 8.97 0.55 14.41 2.18 5.98 0.43 8.59 5 2.52 1.55 1.00 5.07 1.97 2.07 0.37 4.40 0.97 1.56 0.23 2.76 10 2.47 2.30 0.06 4.83 1.76 2.72 0.48 4.95 1.06 1.8 0.28 3.14 11 1.12 0.20 0.02 1.34 1.23 1.48 1.23 3.94 0.94 1.41 0.23 2.58 12 1.01 0.60 0.10 1.72 10.2 19.77 2.81 32.78 4.28 10.9 1.72 16.85 36 0.10 0.18 0.11 0.39 8.43 19.88 0.92 29.23 2.48 9.65 0.99 13.12 47 1.78 1.65 0.17 3.60 1.26 3.55 0.20 5.01 1.35 2.67 0.89 4.91 55 0.23 3.07 1.66 4.95 9.67 16.87 1.20 27.74 2.31 7.97 0.78 11.06 58 0.53 2.66 1.08 4.27 1.72 1.40 0.14 3.27 1.16 1.56 0.23 2.95 59 0.10 1.50 0.54 2.14 1.50 2.86 1.50 5.87 1.26 3.25 0.26 4.77 * C. bejerinkii STDF 1, 10; C. butyricum STDF 5, C. acetobutylicium isolates STDF 11,47,55,58 and 59; C. pasteurianum STDF 12 and C. saccharobutylicum STDF 36. The annual lignocellulosic biomass generated by the primary agricultural sector has been evaluated at approximately 200 billion tons worldwide [65]. In Egypt, around 32.81 Mt of agricultural residues are produced every year [66]. Some effective lignocellulose pretreatment and hydrolysis procedures have been developed and applied to the ABE fermentation in Europe, Russia, and USA [37, 63, 67, 68]. In Egypt, ABE entrepreneurs are increasingly interested in the utilization of lignocellulosic biomass, while research progress has remained in a preliminary stage. Data presented in table 3 showed that the best isolates for ABE production were C. acetobutylicium isolate STDF55 and C. saccharobutylicum isolate STDF36. It is also noted that the best substrate for ABE production was recorded in the following order: Corn husk > Beet residue > Rice straw > Wheat straw. FORMATEX 2013 343

Table 3. Production of acetone (A), butanol (B) and ethanol (E) (g/l) from hydrolysate of wheat straw, rice straw, beet residues and corn husk (37.5 g/l reducing sugars) after 5 days of fermentation by different clostridial isolates STDF Wheat straw Rice straw Beet residues Corn husk Isolate * A B E ABE A B E ABE A B E ABE A B E ABE 1 1.54 2.23 0.95 4.72 1.63 2.56 1.16 5.35 1.34 2.56 1.67 5.57 1.53 2.75 1.97 6.25 5 2.33 1.65 0.64 4.62 2.11 1.86 1.25 5.22 2.92 1.95 0.90 5.77 3.11 2.11 1.1 6.32 10 0.95 1.33 0.11 2.39 1.15 1.55 0.55 3.25 1.11 2.24 0.10 3.45 1.96 3.11 0.42 5.49 11 1.13 1.13 0.18 2.44 1.09 1.43 0.42 2.94 1.43 1.48 0.21 3.12 1.89 2.11 0.43 4.43 12 0.65 0.34 0.14 1.13 0.99 1.03 0.34 2.36 0.85 0.32 0.11 1.28 1.16 0.47 0.73 2.36 36 3.11 6.39 1.11 10.61 3.99 7.67 2.12 13.78 5.48 11.28 1.51 18.27 5.27 13.27 2.11 20.65 47 1.36 2.25 0.19 3.80 1.99 3.71 0.54 6.24 2.16 4.55 0.25 6.96 3.01 5.16 0.65 8.82 55 4.56 6.89 1.21 12.66 4.97 7.69 2.72 15.38 7.78 9.68 2.21 19.67 8.92 11.89 3.12 23.93 58 1.89 2.87 0.66 5.42 2.69 3.87 1.56 8.12 3.29 3.78 0.75 7.82 4.11 4.15 0.75 9.01 59 1.78 3.48 0.77 6.03 2.23 3.98 0.94 7.15 2.46 5.85 0.68 8.99 3.25 6.58 1.13 10.96 * C. bejerinkii STDF 1, 10; C. butyricum STDF 5, C. acetobutylicium isolates STDF 11,47,55,58 and 59; C. pasteurianum STDF 12 and C. saccharobutylicum STDF 36. The conversion process was also interpreted on a metabolic level by comparison with glucose fermentations. The choice of strain used in a production facility depends on a number of factors, and must be well-suited to the locally available substrate and production conditions. The production of biohydrogen and liquid biofuel from crop waste biomass is limited by the hydrolytic activity of the microorganisms involved in the biological attack of the heterogeneous and microcrystalline structure of lignocellulosic component, and in the decomposition of cellulose-like compounds to soluble sugars [69]. Appropriate pretreatment steps for the raw material are often required in order to favor hydrolysis. The main pretreatments are based on mechanical, physical, chemical and biological techniques [70]. A mechanical shredding step is essential to reduce particle size and increase the surface area of the organic waste prior to fermentation. The two primary solventogenic Clostridium organisms that have been investigated for the production of n-butanol are C. acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB805212.The hyper-butanogenic C. beijerinckii BA101 strain was generated by chemical mutagenesis from C. beijerinckii NCIMB 8052 [37]. C. beijerinckii BA101 has enhanced capability to utilize starch and tolerates 0.017 0.021 kg n-butanol per liter of fermentation broth [36]. Various agricultural residues, such as corn stover, corn fiber and fiber-rich distillers dried grains and soluble as substrates have been reported as substrate for this strain [36]. Though pentoses and hexoses were used concurrently for n-butanol production, the highest concentration of n-butanol was produced when cellobiose was used, whereas the least amount of n-butanol was produced using galactose [36]. Fermentation inhibitors such as furfural, hydroxymethyl furfural (HMF), acetic, ferulic, glucuronic and phenolic compounds are generally formed during pretreatment of fiberrich cellulosic biomass. Of these, furfural and HMF are not inhibitory to C. beijerinckii BA101, however, even 300 g of r-coumaric and ferulic acids per m 3 fermentation broth reduced n-butanol production significantly [36]. The current biobutanol production using the existing Clostridium species suffer compared to yeast-based bio-ethanol from low final n- butanol titer, low yield, and low productivity (longer fermentation times). Recombinant DNA technology along with traditional mutagenesis and selection has been employed to modify targeted metabolic pathways in the solventogenic Clostridium species. [37]. For example, [71] used antisense RNA to downregulate the enzymes in the acetone formation pathway. Even though lower levels of acetone formation were achieved there was no redirection of carbon flux towards n-butanol synthesis. The solvent tolerance was similar to ABE fermentation and this is perhaps not surprising due to the physical impact of the solvent butanol on organisms. Butanol will dissolve cell membranes and the low saturation concentration of n-butanol in water (about 8 wt %) leads to high and lethal thermodynamic activity already at butanol concentrations that are modest compared to concentrations in ethanol fermentation. The strains for industrial ABE fermentation are mainly Clostridium strains, including C. acetobutylicum, Clostridium beijerinkii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum [72]. Strains of C. acetobutylicum and C. beijerinkii are suitable for acetone butanol fermentation from corn, while C. saccharobutylicum and C. saccharoperbutylacetonicum preferably utilize molasses as a substrate for ABE production [32, 73]. A variety of biomass resources can be used to convert to energy by clostridia (Table 4). In the past 20 years, research and development efforts have focused on various aspects of the ABE process. Molecular biology research has achieved major breakthroughs in strain/mutant development that dramatically improved microbial tolerance to butanol toxicity, which resulted in a significant increase in ABE solvent production yield [74]. Biobutanol production is biphasic fermentation where acetic and butyric acids are produced during the acidogenic phase followed by their conversion into acetone and butanol (solventogenic phase). At the end of the fermentation, cell mass and other suspended solids are removed by centrifugation and can be sold as cattle feed [79, 80]. 344 FORMATEX 2013

Table 4. Comparison of fermentation processes based on substrate, and total ABE production and ABE productivity. Substrate Strain Total ABE Yield References (g g -1 ) Synthetic medium C. Saccharoperbutylacetonicum N1 4 0.42 75 with butyric acid Corn stover C. beijerinckii P260 0.21 76 and switchgrass Switchgrass C. beijerinckii P260 0.09 76 Barley straw C. beijerinckii P260 0.39 74 Degermed Corn + P2 medium C. beijerinckii BA101 0.42 77 Cassava chip hydrolysate C. saccharoperbutylacetonicum N1-4 0.34 78 Corn fiber C. beijerinckii BA101 0.10 79 Sago starch C. saccharobutylicum DSM 0.29 51 13864 Whey permeate C. acetobutylicum P262 0.35 81 Corn C. acetobutylicum ATCC 55025 0.42 82 Defidered-sweetpotatoslurry C. acetobutylicum P262 0.2 83 Synthetic medium C. beijerinckii BA101 0.36 84 Sludge with sago Clostridium saccharoperbutylacetonicum 0.62 85 starch N1-4 (ATCC 13564) Corn straw C. acetobutylicum 0.41 86 Spoilage date fruit C. acetobutylicum ATCC 824 & 0.39 63 Bacillus subtilis DSM 4451 Rice straw Clostridium spp 0.67 87 In several recent approaches, agricultural waste such as packing peanuts, orchard waste, corn fiber, wheat straw, barley straw, grass, etc. have been used as substrates [37, 76, 79, 88-90]. Huang et al. [91] reported an experimental process that uses continuous immobilized cultures of C. tyrobutyricum and C. acetobutylicum to maximize the production of hydrogen and butyric acid and convert butyric acid to butanol separately in two steps. Extensive research has been performed on the use of alternative fermentation and product recovery techniques for biobutanol production. These techniques have involved the use of immobilized and cell recycle continuous bioreactors and alternative product recovery technique, for example adsorption, gas stripping, ionic liquids, liquideliquid extraction, pervaporation, aqueous two-phase separation, supercritical extraction, and perstraction,etc. [79]. 3. Recent advances in Hydrogen production from agro-industrial wastes Results of fermentation of sugarcane molasses presented in table 5 (unpublished data) indicated that maximum hydrogen production (2 L) was produced by C. pasteurianum 12 in 40 hour with hydrogen production rate of 50 ml h -1. C. saccharobutylicum STDF36 and C.acetobutylicium STDF 55 produced comparable amount of hydrogen with the same flow rate of hydrogen (45 ml h -1 ), the same trend was achieved in beet molasses, but the amount of hydrogen was slightly low. While in case of cheese whey (table 5), the maximum hydrogen production peaked at 870 ml in 24 hour fermentation and hydrogen production rate at 36.2 ml h -1 were achieved by C. bejerinkii isolate STDF 1 followed by C.butyricum STDF 2 and C.acetobutylicium STDF 55. In case of lignocellulosic substrate,c. acetobutylicium 55 shows higher production of hydrogen than C. saccharobutylicum isolate 36 whatever hydrolysate used. Corn husk was the best hydrolysate to produce hydrogen (1629 ml) followed by beet residues (1340 ml H 2, 21.64), rice straw (1176 ml H 2 ) and wheat straw (1080 ml H 2 ).The highest production of hydrogen from hydrolysate of corn husk may be attributed to high concentration of glactose and fructose that utilized by C. acetobutylicium 55. FORMATEX 2013 345

Table 5. Production of hydrogen from agro-industrial wastes by different clostridial isolates. substrate Isolate* Hydrogen (ml L -1 ) Cheese whey STDF1 STDF2 STDF55 Sugarcane molasses STDF 12 STDF 36 STDF 55 Beet molasses STDF 12 STDF 36 STDF 55 Wheat straw STDF55 hydrolysate STDF36 870 790 756 2000 1810 1800 1300 950 1100 1080 898 Hydrogen rate (ml h -1 ) 36.2 32.2 31.50 50 45.25 45 32.5 23.75 24.45 45 37.4 Rice straw hydrolysate STDF55 STDF36 1176 978 49 40.75 Beet residues hydrolysate STDF55 STDF36 1340 1078 56 50 Corn husk hydrolysate STDF55 STDF36 1629 1359 C. bejerinkii STDF 1, 10; C. butyricum STDF 2, C. acetobutylicium isolates STDF 1,47, 55,58 and 59; C. pasteurianum STDF 12 and C. saccharobutylicum STDF 36. Hydrogen yields from different agriculture substrates, as recorded in the literature, are summarized in Table 6. The origins of the organic substrates are quite similar; nevertheless, untreated raw material presents generally lower yields, ranging from 0.5 to 16 ml H 2 g -1 sugar. Under mesophilic conditions the lowest yield was reported from the conversion of wheat straw to hydrogen in a batch reactor [92], while the highest was obtained using cornstalks [93]. The yield of fermentative hydrogen from crop residues in thermophilic conditions at 70 0 C was higher than that in mesophilic conditions indicating that temperature favors hydrolysis [94]. Indeed, the cornstalks category shows variable hydrogen yields, likely because of the varied composition of the carbohydrates, which include cellulose, hemicellulose and lignin [93, 94]. Moreover, as reported in anaerobic digesters producing methane from agricultural waste, the crop species, the harvesting time and the variable silage period must all be considered as main factors impacting on biogas fermentation [95]. A recent review of the literature summarized the composition of different crops residues, e.g. wheat straw, corn stover and rice straw as containing cellulose, hemicelluloses and lignin in a range of approx. 32-47%, 19-27% and 5-24%, respectively [96]. Although no trend was observed in the reported data, a reasonable hypothesis is that biohydrogen yields may be inversely correlated to the cellulose and lignin contents of the waste, as observed by [97] for methane production. 67.8 56.6 346 FORMATEX 2013

Table 6. Comparison of hydrogen yield from different substrates by sequential dark and photo fermentation. Substrate Dark fermentation Photofermentation Total H 2 yield References Microorganism(s) Microorganism(s) Potato starch Mixed culture Rhodobacter capsulatus 5.6 mol H 2 mol -1 hexose [98] Potato steam peel Caldicellulusiruptor saccharolyticus Rhodobacter capsulatus 5.81 mol H 2 mol -1 hexose [99] Beet molasses Caldicellulusiruptor saccharolyticus Rhodobacter capsulatus 13.7 mol H 2 mol -1 sucrose [100] Cheese whey Mixed culture Rhodobacter palustris 10 mol H 2 mol -1 lactose [99] Sweet potato starch Mixed culture; Clostridium butyricum, E. aerogenes Rhodobacter sp. 7 mol H 2 mol -1 hexose [1] corncob Mixed culture Rhodobacter sphaeroides 714 ml H 2 g -1 COD Cassava starch Activated sludge Rhodobacter palustris 503 ml H 2 g -1 starch Cassava starch Mixed culture Rhodobacter palustris 840 ml H 2 g -1 starch [101] [102, 20] [103] Glucose Enterobacter cloacae DM11 Rhodobacter sphaeroides 6.61-6.75 mol H 2 mol -1 hexose [104] Glucose Clostridium butyricum Rhodopseudomonas faecalis RLD-53 Glucose Clostridium butyricum Rhodopseudomonas faecalis RLD-53 4.13 mol H 2 mol -1 hexose 5.31 mol H 2 mol -1 hexose [105] [106] Sucrose Clostridium butyricum CGS5 Rhodopseudomonas palustris WP3-5 5.99 mol H 2 mol -1 hexose [107] Rotten date fruits Clostridium acetobutylicum + E. coli Rhodobacter capsulatus 7.8 mol H 2 mol -1 sucrose [14] Water hyacinth Mushroom waste mixed microflora. 0.42 mol H 2 g-1 substarte 0.86 42 mol H 2 g-1 substarte [108] [108] Straw Mixed culture 0.29 mol H 2 /mol glucose [109] FORMATEX 2013 347

4. Conclusion This chapter has tried to summarize the recent discoveries and developments in basic scientific aspects of the historically important clostridial solvent fermentation. Most of the respective metabolic reactions and their regulation and provided the basis for strain and process improvement by genetic manipulation and substrate choice. Liquid biofuel and biohydrogen can be produced from various simple sugars, molasses, cheese whey, starch (starchy crops) and lignocellulosic biomass. Biomass is potentially a reliable energy resource for hydrogen production. It is renewable, abundant and easy to use. The cellulosic biomass that has been used for this fermentation includes maize fiber, wheat straw, barley straw, maize stover, corn husk, beet residues and rice straw. Selection of agriculture waste with superior clostridial strain for fermentation process plays a major role in economizing the process of liquid biofuel and biohydrogen production by fermentation. 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