The ABE fermentation is a complicated multistage process with a series of consecutive and parallel reactions influenced by a series of technological factors. The presence or absence of natural constituents or contaminations in the used raw materials has important influence on the productivity and product distribution. The ABE fermentation is controlled by intracellular redox processes which is influenced by a variety of technological conditions.

2.1. General mechanism of ABE fermentation

The ABE fermentation is a two-stage process: first, an acid-producing and then a solvent producing process takes place, but the solvent producing metabolic pathway could be observed only above 20 g/L starting sugar concentration [8]. Key factors in starting of solven — togenesis are the undissociated intracellular butyric acid concentration and the summarized amount of the undissociated butyric and acetic acids within the cells. These are in relationships with the pH and the concentration of butyric and acetic acids in the ferment mash of course, and a boundary condition is that glucose concentration should be above 15 g/L at the moment of the final consumption of butyric acid, because a high glucose flux is required to generate as much amount of ATP as is enough to supply the energy demand of the butyric acid-butanol transformation [9]. Hartmanis et al. studied the pathway for uptake of acids during the solvent formation phase of ABE fermentation by C. acetobutylicum using 13C NMR [10]. Actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phos — photransacetylase, acetate kinase, and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid-forming enzymes. No short-chain acyl-CoA synthetase activity could be detected. Apparently, an acetoacetyl-CoA:acetate (butyrate) CoA — transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase which is induced late in the fermentation and pulls the transfer reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise BuOH fermentation without acetone formation and retention of a good yield of BuOH [10]. Ferredoxin enzymes also play important role in the ABE processes, thus the presence of iron in the appropriate form and concentration is essential factor in the appropriate solvent production. When Clostridium acetobutylicum was grown in batch culture under Fe limitation (0.2 mg/L) at pH 4.8, glucose was fermented to BuOH as the major fermentation end product, and small quantities of HOAc were produced. The final conversion yield of glucose into BuOH could be increased from 20% to 30% by Fe limitation. The BuOH- acetone ratio was changed from 3.7 (control) to 11.8. Hydrogenase specific activity was decreased by 40% and acetoacetate decarboxylase specific activity by 25% under Fe limitation. Thus, Fe limitation affects C and electron flow in addition to hydrogenase [11].

Terracciano and Kashket investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undis­sociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM [12]. C. acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate and butanol which were 13-, 7- and 1.3- fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate, which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration — dependent decrease in the intracellular pH, while butanol had relatively little effect until the external concentration reached 150 mM. Acetone had no effect at concentrations <200 mM. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic
conditions and thus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads to induction of solvent production thereby circumventing a decrease in the intracellular pH great enough to be deleterious to

Harris et al. suggested [14] that butyryl phosphate (BuP) is a regulator of solventogenesis in Clostridium acetobutylicum. Determination of BuP and acetyl phosphate (AcP) levels in various C. acetobutylicum strains (wild(WT), M5, a butyrate kinase (buk) and a phosphotran — sacetylase (pta) mutant) showed that the buk mutant had higher levels of BuP and AcP than the wild strain; the BuP levels were high during the early exponential phase, and there was a peak corresponding to solvent production [15]. Consistently with this, solvent formation was initiated significantly earlier and was much stronger in the buk mutant than in all other strains. For all strains, initiation of butanol formation corresponded to a BuP peak concentration that was more than 60 to 70 pmol/g (dry wt.), and higher and sustained levels corresponded to higher butanol formation fluxes. The BuP levels never exceeded 40 to 50 pmol/g (dry wt.) in strain M5, which produces no solvents. The BuP profiles were bimodal, and there was a second
peak midway through solventogenesis that corresponded to carboxylic acid reutilization. AcP showed a delayed single peak during late solventogenesis corresponding to acetate reutiliza­tion. As expected, in the pta mutant AcP levels were very low, yet this strain exhibited strong butanol prodn. These data suggest that BuP is a regulatory mol. that may act as a phospho — donor of transcriptional factors. DNA array-based transcriptional anal. of the buk and M5 mutants demonstrated that high BuP levels corresponded to downregulation of flagellar genes and upregulation of solvent formation and stress

The toxicity of accumulated butanol and the intermediates is a very important feature of the ABE fermentation. Costa studied [16] the growth rates of Clostridium acetobutylicum in presence of BuOH, EtOH, Me2CO, acetate and butyrate. Acetate and butyrate were the most toxic compounds, with concentrations of 5 and 8.5 g/L, respectively, stopped the cell growth. An EtOH concentration of 51 g/L or 11 g BuOH/L reduced cell growth by
50%. Acetone did not inhibit cell growth at 29 g/L, thus ethanol and acetone were non­toxic at a normal fermentation. Some mutant strains, however, more tolerant towards bu­tanol, for example Lin and Bladchek [17] obtained a derivative of C. acetobutylicum ATCC 824 which grew at concentrations of BuOH that prevented growth of the wild — type strain at a rate which was 66% of the uninhibited control. This strain produced con­sistently higher concentrations of BuOH (5-14%) and lower concentrations of acetone (12.5-40%) than the wild-type strain in 4-20% extruded corn broth. Characterization of the wild-type and the mutant strain demonstrated the superiority of the latter in terms of growth rate, time of onset of BuOH production, carbohydrate utilization, pH resist­ance, and final BuOH concentration in the fermentation broth [17]. Moreira et al. [18] ini­tiated a fundamental study attempting to elucidate the mechanism for BuOH toxicity in the acetone-BuOH fermentation by Clostridium acetobutylicum. Butanol as a hydropho­bic compound inserted into the membrane increases the passive proton flux, forms a "hole" for proton on the membrane. This eliminates hydrogen ions form the cell and the intracellular pH increases. The strains which are able to decrease the membrane fluidity are more resistant towards butanol. The cells have deacidifying mechanism to keep the intracellular pH value at 6 when the pH value of the ferment liquor is located between 4 and 5 can reduce acids into alcohols, which increases their butanol producing ability. Lepage et al [19] studied the changes in membrane lipid composition of C. acetobutyli­cum during ABE fermentation. Large changes were found in phospholipid composition and in fatty acid composition, the latter characterized mainly by a decrease in the unsa- turated/saturated fatty acid (U/S) ratio.

Effects of the addition of alcohols (EtOH, BuOH, hexanol, and octanol) and of acetone were also studied. In all cases, large changes were observed in the U/S ratio, but with differences which were related to the chain length of the alcohols. The effect of solvents appears to account for a large part of changes in lipid composition observed during the fermentation. The pH was also important, a decrease in pH resulting in a decrease in the U/S ratio and in an increase in cyclopropane fatty acids. The effect of increasing temperature was mainly to increase fatty acid chain lengths [19].

3. General conditions of the ABE fermentation

Optimal conditions of ABE fermentation strongly depend on many factors such as the selected raw materials or their composition, and are essentially influenced by the selected strain as well. Furthermore, a series of important factors can decrease or increase the yield and changes the distribution of the ABE solvents even with the same raw material or bacterium strain. Some selected pieces of information are summarized below.