SCIENCE, ENGINEERING AND ECONOMICS
2.4 Substrates for ABE Fermentation
BIOBUTANOL:SCIENCE,ENGINEERING AND ECONOMICS
This species was initially known as an acid producer that fermented carbohydrates to butyrate, acetate, CO2 and H2 [84]. However, Harris et al. [85] reported that the species could also produce significant quantities of acetone, butanol, and ethanol, when grown in media of high glucose content. Nakas et al. [86] have reported conversion of algal biomass supported with 4%w/v glycerol to butanol, 1,3–propanediol and ethanol. In another interesting study, Heyndrickx et al. [87] compared the fermentation characteristics of C. butyricum (LMG 1212t2) and C. pasteurianum (LMG 3285) with glycerol and a glycerol-acetae mixture as the carbon source. C. butyricum converted more than half of glycerol to n–butanol. Addition of acetate to glycerol resulted in less 1,3 propanediol and H2 from C. butyricum, while fermentation products of C. pasteurianum remained unaffected. More recently, a few papers have appeared in the literature investigating conversion of crude glycerol resulting from transesterification process for biodiesel production by C. pasteurianum species [88, 89].
Dabrock et al. [90] have reported product ratios of 38 mol:18 mol:18 mol for ethanol:butanol:1,3 propanediol per 100 mol of glycerol fermented under phosphate limitation. Biebl [91] has also reported butanol (with concentration up to 17 g/L) as a major product of glycerol fermentation using C. pasteurianum. Taconi et al. [92] have reported butanol yields of 30 g/g on crude glycerol and 38 g/g with pure glycerol. Although these studies have shown high potential for industrial use of C. pasteurianum, especially for improvement of the economics of the biodiesel industry, significant research is yet to be done on process engineering aspect of C. pasteurianum based fermentation.
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were necessary [14, 18, 62, 93-95]. Molasses had many distinct advantages over corn mash such as a lower operational temperature and a higher solvent yield. As a result, many plants utilizing corn mash as substrate were switched over to using molasses, along with other substrates such as grains and corn cob hydrolyzates [19-20, 96-97].
After World War II, most of the conventional substrates were increasingly utilized as cattle feed, and the prices become unaffordable to the distiller industry. As the cost of substrate is a major factor determining the overall economy of ABE fermentation industry, significant research has been done on alternative cheap substrates. Initial research focused mainly on alternate sources of starch/carbohydrate. These include potatoes, rice, jawari, bajra, apple pomace, cheese whey, and Jerusalem artichokes [86, 95, 98-108]. Jerusalem artichokes require hydrolysis (acid/enzyme) prior to fermentation, while cheese whey requires the removal of casein by precipitation. Solvent production from Jerusalem artichokes was similar to that from molasses (23–24 g/L) [109-115], while cheese whey gave much lower productivity (5–15 g/L solvents). It was, however, observed that the relative yield of butanol (or the distribution of ABE products) was much higher as compared to when glucose was used. Voget et al. [106] investigated use of apple pomace as a substrate for butanol fermentation and reported a yield of 2% w/w. Nakas et al. [86] has reported algal biomass (sp. Dunaliella) as a substrate for fermentation with C. pasteurianum. Algal biomass supplemented with 4% glycerol produced a mixture (16 g/L) of butanol and 1,3 propanediol.
Another alternative substrate for fermentation is lignocellulosic biomass. Being most abundant and renewable, this source offers great promise for improvement of the economy of the ABE fermentation. However, effective hydrolysis of the hemicellulosic and cellulosic fractions of biomass is a major factor. Hydrolyzates of cellulosic fractions are mostly comprised of hexose sugars (glucose, fructose, mannose, sucrose), which are completely consumed; while hydrolyzates of hemicellulose are made up of pentose sugars (xylose,
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BIOBUTANOL:SCIENCE,ENGINEERING AND ECONOMICS
galactose, arabinose and raffinose), which are only partially consumed. Extensive literature has been published since the 1920‘s that presents a comparative evaluation of fermentation of different sugars by clostridial cultures [20,116-121]. Fond et al. [122,123] have studied the kinetics of utilization of glucose, xylose, and a mixture of these in a fermentation employing C. acetobutylicum in batch and fed–batch mode. The principal findings of this study were as follows: (1) the fastest initial growth and transition from acidogenesis to solventogenesis occurs at a glucose concentration of 62 g/L. Relatively slower initial growth rate and transition from acidogenesis and solventogenesis is seen with xylose, which results in higher cellular concentration. (2) For the mixture of these two sugars in batch fermentation, glucose is fermented more rapidly. The highest fermented sugar concentration was 68 g/L for the mixture of glucose and xylose. (3) For the fed batch mode, the fermentation process is limited by the low sugar concentration feed rate. This has several consequences such as slower cellular growth, slower metabolic transitions, and higher accumulation of acids. At higher feed rates, results comparable with batch fermentation were obtained. Solvent production was triggered at a total acid concentration of 4.5 g/L, while final inhibition of fermentation occurred at a solvent concentration of 20 g/L. (4) For low glucose concentrations in the feed, both sugars are consumed at same rate, while at higher glucose levels, xylose utilization is inhibited as catabolic flux of glucose can alone satisfy metabolic activities of cell. (5) The acid concentration for the switchover from acidogenesis to solventogenesis, and final inhibition concentration of butanol was same for fed-batch mode as for the batch mode.
Hydrolyzates of lignocellulosic biomass are increasingly being used as substrates for the ABE fermentation. The hydrolyzates were prepared using either acid or enzyme hydrolysis. Lee et al. [124] have reviewed technological aspects and economic factors of acid hydrolysis of lignocellulosic biomass. Prospects of utilization of hydolyzates for ABE fermentation have been investigated for more than half a century. Earliest studies in this area
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include those of Waksman and Kirsh [125], Underkofler et al. [126], Sjolander et al. [127], Leonard et al. [129], Langlykke et al. [130], Nakhmanovich and Shcheblykina [20].
Acid treated hydrolyzates are reported to contain inhibitory compounds such as salts, furfurals, glucuronic, p–coumaric acid, phenolics [130] etc. These need to be removed by techniques such as adsorption or solvent wash etc. On the other hand, no inhibitory compounds content has been reported in the hydrolyzates produced by enzymatic treatments.
Another pretreatment for separation of lignocellulose biomass into cellulose, hemicellulose and lignin is steam cracking [131-134]. After this treatment, the hemicellulosic fraction is present in water (in solubilized form), while the cellulosic fraction is present in the insoluble substrate. Both of these fractions could be hydrolyzed (by acid/enzyme) to allow for further release of sugars. Although this pretreatment showed positive results for wood chips during a pilot scale process [135], Marchal et al. [136] reported that hydrolyzates obtained by enzymatic saccharification of wheat straw or corn stover, pretreated with steam explosion were non-fermentable. Heating of hydrozylates with alkaline compounds such as Ca(OH)2 or MgCO3 to restore neutral pH was necessary to make the hydrolyzates fermentable.
2.4.1 Cocultures
Utilization of cellulosic, hemicellulosic or lignocellulosic biomass as substrates for fermentation requires a hydrolysis step prior to fermentation. Enzymatic hydrolysis is relatively expensive, while acid hydrolysis yields hydrolyzates containing inhibitory compounds. Purification of hydrolyzates prior to fermentation adds additional cost component. A viable solution to this has been explored by some research groups in terms of examination of co-cultures of Clostridium acetobutylicum with microorganisms having enzymes capable of simultaneous hydrolyzing cellulose and hemicellulose, as the fermentation proceeds. Double et.al has briefly stidued the production cost of liquid fuels from varios biomass through fermentative and thermochemical route [137]. Literature in this
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area, however, is limited. We give below a brief summary of some important publications in this area:
Soni et al. [138] used mixed cultures of cellulolytic fungi T. reesei and A. wentii to obtain hydrolyzates of agricultural wastes such as bagasse, rice straw and wheat straw. These hydrolyzates fermented either with cultures of C. saccharoperbutylacetonicum or C.
acetobutylicum yielded 16 g/L and 17.3 g/L of butanol, respectively. Petitdemange et al.
[139, 140] and Fond et al. [141] attempted use of mesophilic cocultures of cellulolytic (C.
cellulolyticum H10) and glycolytic (C. acetobutylicum) clostridial strains for direct fermentation of cellulose. However, the major fermentation product was butyric acid, with a small amount of acetone, ethanol and butanol. Due to the relatively slow hydrolysis of cellulose by C. cellulolyticum, the level of glucose in the medium was low, which resulted in low solvent production. Yu et al. [142] attempted conversion of solka floc and aspenwood xylan (basically, a lignocellulosic substrate) with co-cultures of C. acetobutylicum (glycolytic bacteria) and C. thermocellum (cellulolytic bacteria). This co culture resulted in a 1.7–2.6 fold increase in total fermentation products with effective utilization of all hydrolysis products. However, the majority of the fermentation products were acids and not solvents.
This result was similar to the earlier studies of Fond et al.[141] and Petitdemange et al. [139, 140], and was attributed to a rather slow rate of hydrolysis. These results suggest the importance of the development of mutant strains of cellulolytic microbes, which would give relatively faster rate of hydrolysis (at par with fermentation rates of glycolytic species). Two- stage fermentation process [143] with co culture C. butyricum and C. pasteurianum in the first stage, and C. beijerincki and C. pasteurianum in the second stage yielded 20% more butanol. This was a consequence of high level of butyric acid production in the first stage, which was effectively reduced to butanol in the second stage. A new process reported by Ramey [144] employs continuous immobilized cultures of C. tyrobutyricum and C.
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acetobutylicuim with corn as substrate, at a productivity of 4.64 g/L–h and yield of 42%. C.
tyrobutyricum maximizes production of hydrogen and butyric acid, while C. acetobutylicum effectively converts butyric acid to butanol. This new route has several advantages such as elimination of products such as acetic, lactic and propionic acids
Table 2.4 presents a summary of several papers published since 1985 that investigate viability of alternate substrates for ABE fermentation when using different cultures of clostridia.