9.5.1 ABE Fermentation Processes
An ABE batch fermentation usually starts with a heatshock of the spore stocks in order to induce expression of heat shock proteins that enhance butanol toler- ance and production and avoid possible culture degeneration (Ezeji et al., 2007c).
Various carbon sources and different media have been tested for ABE produc- tion. Nevertheless, besides the carbon source, proper buffer, essential minerals, and vitamins usually compose the synthetic medium. An acetone:butanol:ethanol ratio of 3:6:1 with small variations is usually obtained from the fermentation pro- cess with solventogenic clostridia. ABE fermentation is a very complex process and is influenced by many factors. A number of studies have been carried out to investigate the effects of different factors on butanol production, including pH, tem- perature, agitation, and so on (Nishio et al., 1983; Welsh and Veliky, 1984; Soni et al., 1992; Geng and Park, 1993; Salleh et al., 2008). Wang and Blaschek (2011) investigated the individual and interactive effects of pH, initial substrate concen- tration, and agitation rate on ABE fermentation using a multifactorial experimental design approach and determined the optimum conditions for butanol production when mixed sugars from tropical maize stalk juice were used as the carbon source.
Notwithstanding, the effects of various operational parameters on ABE fermenta- tion vary significantly depending on different strains, carbon sources, and media compositions.
Batch fermentation is the most often used operation mode in many industries due to its high efficiency and easy operation. However, fed-batch fermentation is advan- tageous in cases where there is a potential substrate inhibition at the beginning of fermentation (Ezeji et al., 2004). Fed-batch fermentation is an industrial technique where the reactor is started with a relatively low substrate concentration, and as the substrate is consumed, more concentrated substrate solution is added at a low rate.
Through the dilution effect, the toxicity from the end products could be meanwhile alleviated, while the substrate concentration in the reactor is kept below the toxic level. Although it requires continuous and long-term manual work for the operation, continuous fermentation has various advantages over the batch and fed-batch fer- mentation processes. First, it only needs one inoculum for a long time of operation;
in addition, it saves time for sterilization and re-inoculation and thus enhances the overall productivity (Kumar and Gayen, 2011). The cultivation ofC. beijerinckii
BA101 free cells in degermed corn-based P2 medium for continuous butanol pro- duction has been investigated (Ezeji et al., 2007b). Results indicated that long-term continuous cultivation ofC. beijerinckiiBA101 in a degermed corn-based medium was not successful because of the “retrogradation” of the gelatinized degermed corn starch; however, continuous fermentation of saccharified degermed corn with normal and half P2 medium nutrients were successful, and an ABE concentration of up to 14.28 g/L was obtained during 504 hours of operation, after which the experiment was stopped intentionally. Cell immobilization is often employed in continuous fer- mentation in order to maintain high cell density in the reactor during the fermentation process. In a study for the continuous production of isopropanol and butanol with C. beijerinckiiDSM 6423 (C. beijerinckiiNRRL B593), both single- and two-stage continuous fermentation modes were investigated, where wood pulp was employed for holding the cell. While the single-stage continuous fermentation demonstrated much higher solvent productivity (5.52 g/L⋅h in single stage vs. 0.84 g/L⋅h in two stage), the two-stage fermentation generated higher solvent yield (0.32 g/g) and final solvent titer (7.51 g/L) (Survase et al., 2011). Li et al. (2011) compared the per- formance of batch, fed-batch, and continuous ABE fermentation processes under pH-controlled conditions (Li et al., 2011). They reported that while the batch mode provides the highest overall solvent yield, the continuous fermentation worked better in terms of butanol yield and productivity. Interestingly, they also concluded that the fed-batch mode is not suggested for solvent production due to the long time needed for passing from acidogenesis to solventogenesis.
9.5.2 Butanol Toxicity and Butanol-Tolerant Strains
During ABE fermentation, solvents in the fermentation broth become very toxic when the level is beyond 2%. Butanol is the most toxic end product for solventogenic clostridia, and rare butanol-producing strains can tolerate more than 2% butanol (Lin and Blaschek, 1983). Butanol toxicity has been attributed to the chaotropic effects on the cell membrane and the inhibitory effects on sugar uptake, nutrient transport, membrane-bound ATPase activity, disruption of the proton motive force (or intracellular pH), etc. (Bowles and Ellefson, 1985; Ounine et al., 1985). It was reported that 20–30% increase in membrane fluidity was observed whenC. acetobutylicumwas exposed to 1% butanol (Vollherbstschneck et al., 1984). The incompetence of butanol tolerance of the fermentative microorganism thus leads to a very low final solvent titer in the ABE fermentation, which results in a high energy cost for the following solvent recovery process. It was estimated that if the final butanol concentration in the fermentation broth is increased from 1% to 2%, the energy requirement for recovering butanol by distillation would be lowered by 62% (Phillips and Humphrey, 1983). The poor tolerance (and thus the low final solvent titer) in the traditional ABE fermentation process is a major limitation for scaling up the butanol fermentation to an economic viable industrial scale.
Great efforts have been made to improve the butanol tolerance of the solvento- genic clostridia stains. A butanol-tolerant derivative (called SA-1) ofC. beijerinckii
NCIMB 8052 was obtained by serial enrichment with butanol challenge (Lin and Blaschek, 1983). The mutant was superior over other mutants in terms of growth rate, butanol tolerance, butanol production, and butanol/acetone ratio. Later, another butanol-tolerant mutantC. beijerinckiiSA-2 was obtained through similar procedure by the same group (Baer et al., 1987). The consequences of butanol challenge on cell membrane of SA-2 mutant were investigated; results demonstrated that SA-2 strain developed a mechanism for maintaining a more stable membrane environ- ment by synthesizing an increased amount of saturated fatty acids, which might be closely related to the enhanced butanol tolerance. Recently, inC. acetobutylicum DSM 1731, a functionally unknown protein with a hypothetical alcohol-interacting domain was identified, and its disruption led to an increased butanol tolerance; thus it can be considered as a potential target for engineering alcohol tolerance (Jia et al., 2012).
9.5.3 Fermentation Products Recovery
Numerous endeavors have been implemented in fermentation process development in order to alleviate the solvent-induced inhibitory effects and thus improve the fer- mentation productivity. Usually, the selective removal of fermentation products was integrated simultaneously into the fermentation process in order to maintain a low solvent concentration in the fermentation broth. Many online butanol removal tech- niques have been reported with various advantages and efficiencies, including liquid–
liquid extraction, perstraction, gas stripping, pervaporation, adsorption, reverse osmosis, etc.
For the liquid–liquid extraction technique, a water-insoluble organic extractant is mixed with the fermentation broth. Since butanol is more soluble in the organic extractant than in the fermentation broth, high-concentrated solvents can be obtained from the organic extractant phase, which is immiscible and separated from the fer- mentation broth. Thus, the solvents can then be recovered by distillation with a relatively low energy cost. Oleyl alcohol, which is relatively nontoxic to the fermen- tative microorganism, has been proved to be a good extractant for this purpose (Ezeji et al., 2007c). Recently, nonionic surfactants have also been reported to effectively separate butanol from the fermentation broth and thus significantly enhanced the butanol production in an ABE batch fermentation withC. pasteurianum(Dhamole et al., 2012).
However, liquid–liquid extraction can form emulsions, and the extractant is usu- ally expensive and sometimes toxic to the cell culture (D¨urre, 1998). To solve these problems, the perstraction technique was developed based on the liquid–liquid extrac- tion concept. In the perstraction separation process, the fermentation broth and the extractant are separated by a membrane, where two immiscible phases can exchange butanol. In this process, butanol can diffuse preferentially across the membrane, while other components (substrates, cell culture, and other fermentation products) are retained in the fermentation broth. This strategy effectively avoids the poten- tial problems in the liquid–liquid extraction system, but it needs to point out that
the membrane can decrease the butanol extraction rate as a physical barrier (Ezeji et al., 2007c).
Gas stripping is another attractive method for integrated product recovery in the ABE fermentation process. In this system, the gas is directly sparged through the fermentation system, and thus the evaporable solvents can be easily recovered and other essential components for the fermentation and cell culture can be retained in the broth. Inert gas, like nitrogen, could be employed for this purpose, and the mixed gases (carbon dioxide and hydrogen) generated during the fermentation could also be collected and used in order to save the cost for the exogenous nitrogen (Qureshi et al., 2008). Gas stripping is very simple to operate and usually leads to a low chance of clogging or fouling for the fermentation and thus became very attractive recently (Shen et al., 2011; Lee et al., 2012). Actually, till now, the gas stripping process has been proved to be the most effective process for online recovery of butanol (Zheng et al., 2009).
Recently, a novel process with simultaneous ABE fermentation andin situproduct recovery with vacuum was reported (Mariano et al., 2011). Results indicated that fer- mentation coupled within situvacuum recovery led to complete substrate utilization, greater solvent productivity, and improved cell growth.