Depending on the composition and properties of raw materials, the selection and conditioning of the appropriate bacterium strain are essential. In order to improve the economic efficacy of ABE fermentation, the butanol ratio is to be increased by eliminating the production of other byproducts such as acetone and specific mutants are to be developed which show high butanol tolerance, high productivity or other advantageous properties.

Harada [28, 29] isolated a new strain of Clostridium (Cl. Madisonii) which produced BuOH amounting to 28.7% of the initial total sugar and the fermented broth included 1.38% BuOH. The age of the culture also plays important role in the productivity. By using older inoculated bacteria, the production of acetone increased and the ratio of BuOH to Me2CO decreased from 2.24 to 1.88 [30]. Harada [31] concluded that the seed culture at the last stage of the acid- decreasing phase gave the best yield as inoculum in the main fermentation. Butanol-resistant mutants have been isolated by Hermann from soil which produced significantly higher solvent concentrations (about 30%) than the wild-type strain [32]. The sporulation-deficient (spo) early-sporulation Clostridium acetobutylicum P262 mutants produced higher solvent yields than did the spoB mutant which was a late-sporulation one. In conventional batch fermenta­tion, the wild-type strain produced 15.44 g L-1 of solvents after 50 h at a productivity of 7.41 g L-1 d-1 of solvents. The spoA2 mutant produced 15.42 g L-1 of solvents at a productivity of 72.4 g L-1 d-1 of solvents with a retention time of 2.4 h in a continuous immobilized cell system employing a fluidized bed reactor [33].

Using two different types of Clostridia to improve the productivity of each (acidogenic and solventogenic) phase is also known. Bergstroem and Foutch [34] improved the BuOH pro­duction from sugars by combining two cultures of Clostridium: one that produces butyric acid, and another that converts butyrate to BuOH. Thus, C. butylicum NRRL B592 and C. pasteur — ianum NRRL B598 were cultured together in thioglycolate medium containing 2.5% added glucose and a CaCO3 chip to maintain pH, at 37 °C under anaerobic conditions. The yield of BuOH was 20 % more as compared to the value when C. butylicum was cultured alone.

Initiation of gene-structure changes by destructive methods such as irradiations or chemicals followed by selection is a well known method in the production of highly effective Cl. Acetobutylicum strains. Yasuda [35] heated ABE producing microorganisms at 100 °C to destroy all vegetative forms except spores which were kept at -10 °C, then treated with electric discharge in vacuum by using 50,000 V and 0.002 A DC for stimulation. High-yield butanol producing Clostridium strain was prepared through irradiation of the wild strain with 60Co y-rays at an irradiation dosage of 100-1,000 Gy and a dosage rate of 3-5 Gy/min [36].

Chemical mutation with N-methyl-N’-nitrosoguanidine is one of the most frequently used method to produce excellent ABE fermenting strains. Hermann et al [37] prepared a strain of C. acetobutylicum that hyperproduces acetone and BuOH by mutation of C. acetobutylicum IFP903. A new mutant (CA101) of C. pasteurianum prepared in this way could produce 2.1 g BuOH/L in 2 days. By using the parental strain, the production of BuOH was only 0.6 g/L [38]. The C. acetobutylicum strain 77 was isolated from the parent strain ATCC 824 with the abovementioned method in the presence of butanol. The mutant grew more rapidly (|j = 0 69 h-1) than the parent strain (p = 0 27 h-1) and, at the stationary phase, the cell dry weight of mutant strain was about 50% higher than that of the parent strain. Strain 77 metabolised glucose faster than wild strain and solvent production started earlier with higher specific production rates than the parent strain. From 65 g of glucose, 20 g L-1 of solvents (butanol, 14 5 g; acetone, 3 5 g; ethanol, 2 g) were formed by the wild strain in 53 h, whereas the mutant used 75 g of glucose and excreted nearly 24 g L-1 of solvents (butanol, 15.6 g; acetone, 4.5 g; ethanol, 3.7 g) in 44 h [39]. A frequently used chemical to inititate mutation in C. Acetobuty — licum strains is methanesulfonic acid ethyl ester (EMS). EMS is effective in inducing mutants resistant to ampicillin, erythromycin, and butanol (15 g/l). Optimal mutagenesis occurs at 85­90% kill corresponding to a 15 minute exposure to 1.0% (v/v) EMS at 35 C. At optimal condi­tions, the frequency of resistant mutant CFU/ total CFU plated increases 100-200 fold [40].

Genetical engineering opened unlimited perspectives in the preparation of ABE ferment­ing microorganisms. Genetically modified C. Acetobutylicum, E. Coli and S. Cereviase and other microorganisms play important role in the future production of ABE solvents under more convenient conditions than in classical ABE fermentation. The acetoacetate decarboxylase gene (adc) in the hyperbutanol-producing industrial strain Clostridium acetobutylicum EA 2018 was disrupted when the butanol ratio was increased from 70 to 80.05%, while acetone production decreased to approx. 0.21 g/L in the adc-disrupted mu­tant (2018adc). Regulation of the electron flow by addition of methylviologen altered the carbon flux from acetic acid production to butanol production in strain 2018adc, which resulted in an increased butanol ratio of 82% and a corresponding improvement in the overall yield of butanol from 57 to 70.8% [41].

Larossa and Smulski found genes involved in a complex that is a three-component proton motive force-dependent multidrug efflux system to be involved in E. coli cell response to butanol by screening of transposon random insertion mutants. Reduced production of the AcrA and/or AcrB proteins of the complex confers increased butanol tolerance [42]. Green and Bennett subcloned the genes coding for enzymes involved in butanol or butyrate formation into a novel Escherichia coli-Clostridium acetobutylicum shuttle vector constructed from pIMPI and a chloramphenicol acetyl transferase gene [43]. The resulting replicative plasmids, referred to as pTHAAD (aldehyde/alcohol dehydrogenase) and pTHBUT (butyrate operon), were used to complement C. acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had been inactivated by recombination with Emr constructs. Complementation of strain PJC4BK (buk mutant) with pTHBUT restored butyrate kinase activity and butyrate production during exponential growth. Complementation of strain PJC4AAD (aad mutant) with pTHAAD restored NAD(H)- dependent butanol dehydrogenase activity, NAD(H)-dependent butyraldehyde dehydrogen­ase activity and butanol production during solventogenic growth [43]. Shen and Liao constructed an Escherichia coli strain that produces 1-butanol and 1-propanol from glucose [44]. First, the strain converts glucose to 2-ketobutyrate, a common keto-acid intermediate for isoleucine biosynthesis. Then, 2-ketobutyrate is converted to 1-butanol via chemicals involved in the synthesis of the unnatural amino acid norvaline. The synthesis of 1-butanol is improved through deregulation of amino-acid biosynthesis and elimination of competing pathways. The final strain demonstrated a production titre of 2 g/L with nearly 1:1 ratio of butanol and propanol [44]. Green et al [45] made recombinant thermophilic bacteria of the family Bacilla — ceae which have been engineered to produce butanol and/or butyrate. The Bacillaceae is preferably of the genus Geobacillus or Ureibacillus [45]. Young et al described a method of modifying prokaryotic and eukaryotic hosts for the fermentation production of aliphatic alcohols. Elements of the gene for a CAAX proteinase (prenylated protein-processing C- terminal proteinase) are used to increase alcohol tolerance. This can be used in combination with other changes to increase alcohol tolerance [46]. Fermenting with modified eukaryotic cells in a suitable fermentation broth, wherein butanol and ethanol are produced at a ratio between 1:2 to 1:100, is described by Dijk et al. [47]. Since fermentations with yeasts do not require sterile environment, genetically modified yeasts are very prosperous microrganisms in ABE fermentation. Yeast cells capable of producing butanol and comprising a nucleotide sequence encoding a butyryl-CoA dehydrogenase and at least one nucleotide sequence encoding an electron transfer flavoprotein were described by Mueller et al. [48].