Challenges of butanol production

Production of biobutanol by clostridia is not straightforward process and 1-butanol is neither a typical primary metabolite, the formation of which is connected with cells growth, nor a typical secondary metabolite like antibiotics or pigments. The metabolic switch from acido — to solventogenesis, regulation of which is usually connected with sporulation initiation, does not need to happen necessarily during the fermentation. Actually, when cells are well nourished and their growth rate approaches its maximum then cells reproduce and form only acids; this state has been many times observed in continuous cultivations (Ezeji et al., 2005) but sometimes it can occur even in batch cultivation as so-called "acid crash" (Maddox et al., 2000; Rychtera et al., 2010) which was generally ascribed to fast acetic and butyric acids formation. The proposed acid crash prevention was careful pH control or metabolism slowdown by lowering cultivation temperature (Maddox et al., 2000). However, very recently the novel possible explanation of this phenomenon has been revealed in intracellular accumulation of formic acid by C. acetobutylicum DSM 1731 (Wang et al., 2011). If acid crash is the phenomenon that usually happens at random in the particular fermentation, so-called strain degeneration is a more serious problem when the production culture loses either transiently or permanently its ability to undergo the metabolic shift and to produce solvents. The reliable prevention of the degeneration is maintaining the culture in the form of spore suspension (Kashket & Cao, 1995). A cause of degeneration was investigated in many laboratories using various clostridial strains and therefore also with different results. The degeneration of C. acetobutylicum ATCC 824 is probably caused by loss of its mega plasmid containing genes for both sporulation and solvents production (Cornillot et al., 1997) but mechanism and reason of this degeneration were not offered by this study. Actually the authors (Cornillot et al., 1997) compared wild-type strain C. acetobutylicum ATCC 824 with isolated degenerated mutants. It is questionable how often or under which conditions the degeneration of C. acetobutylicum ATCC 824 happens because in the past, it was reported 218 passages of vegetative C. acetobutylicum ATCC 824 cells did not almost influence their solvents formation (Hartmanis et al., 1986). The cells of C. saccharoperbutylacetonicum N1-4 degenerated when quorum sensing mechanism in the

population was impaired (Kosaka et al., 2007). The very detailed study of C. beijerinckii NCIMB 8052 degeneration disclosed two different degeneration causes: involvement of global regulatory gene and defect in NADH generation (Kashket & Cao, 1995). It seems probable that degeneration has no single reason and if other strains were studied different reasons would be found.

ABE industrial fermentation was probably the first process that had to cope with bacteriophage infection of producing microorganism. The first severe bacteriophage attack was reported from Terre Haute plant in the U. S.A. in 1923 and the problems occurred at fermentation of corn by Clostridium acetobutylicum (the solvents yield was decreased by half for a year). From that time, Clostridium strains used for either starch or saccharose fermentations were attacked by various both lysogenic and lytic bacteriophages what was documented in the literature. The ABE plant in Germiston in South Africa faced to confirmed bacteriophage infection 4- times in its 46-year history (plus two unconfirmed cases). Till now, the best solution in battle against Clostridium bacteriophages seems to be the prevention i. e. good process hygiene, sterilization, decontamination and disinfection (Jones et al., 2000).

Lactic acid bacteria represent the most common type of contamination having very similar requests for cultivation conditions (temperature, pH, anaerobiosis, composition of cultivation media) as clostridia and grow faster. These bacteria can cause not only losses in solvents yield but also can hamper the metabolic switch of clostridia because formed lactic acid over-acidifies the medium and poisons the clostridia in higher concentration. Other contaminants like Bacillus bacteria or yeast are encountered only scarcely (Beesch, 1953).

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