Effects of Stirring and Hydrogen on Fermentation Products of Clostridium thermocellum.

Clostridium thermocellum produces ethanol, acetate, H(2), and CO(2) as major fermentation products from cellulose and cellobiose. The performance of three strains of this microorganism was studied to assess the potential use in producing ethanol directly from cellulosic fiber. Depending on the bacterial strain, an ethanol/acetate product ratio from 1 to as high as 3 was observed in unstirred cultures. Vigorous stirring during growth resulted in a threefold decrease in the ethanol/acetate ratio. The H(2) content in the unstirred culture broth was three times greater than that in the stirred one. Addition of exogenous H(2) to the gas phase during growth increased the ethanol/acetate ratio much more in the stirred than in the unstirred fermentations. The addition of sufficient H(2) to the gas phase almost relieved the effect of stirring, and the ethanol/acetate ratio approached that in the unstirred condition. Addition of tritium to the gas phase of the culture resulted in the formation of tritiated water (H(2)O), which indicates that C. thermocellum possesses hydrogenase(s) that catalyzes the reverse reaction. The rate of H(2)O formation was about three times higher in the stirred culture than in the unstirred culture. These results demonstrate that the H(2) concentration in the broth plays an important role in the product formation. The H(2) supersaturation present in the unstirred cultures is responsible for the observed effect of stirring. A hydrogen feedback control mechanism regulating the relative concentrations of reduced and oxidized electron carriers is proposed to account for the effect of hydrogen on the metabolite distribution.

Novel NADP-linked alcohol--aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria.

An NADP-specific alcohol--aldehyde/ketone oxidoreductase was detected in cell extracts of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum, but not in Thermobacteroides acetoethylicus or Clostridium thermocellum. The enzyme was purified from Ta. brockii by differential procedures that included heat treatment and an affinity-chromatography step on Blue Dextran--Sepharose. The 44-fold-purified enzyme displayed one band (mol.wt. approx. 40000) after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme had a broad substrate specificity that included linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones, and acetaldehyde. The NADP-specific alcohol--aldehyde/ketone oxidoreductase was considerably more active towards secondary alcohols than towards other substrates. The enzyme had remarkable stability to heating at 86 degrees C for 70 min, but was rapidly denatured on boiling. Secondary-alcohol dehydrogenase activity displayed a noticeable inflexion point at 50 degrees C in Arrhenius plots and a high Q10 value (greater than 2.0). The enzyme was inactivated by the thiol-blocking reagent p-chloromercuribenzoate, but was not significantly inhibited by common metal-ion-binding agents. The NADP-linked alcohol--aldehyde/ketone oxidoreductase of Ta. brockii appears to have properties distinct from those of previously described primary- and secondary-alcohol dehydrogenases.

Thermophilic ethanol fermentations.

Thermophilic ethanol fermentations are of interest to industrial alcohol production because both the pentose and hexose fraction of biomass can be directly fermented in high yield (i.e., mol ethanol/mol substrate consumed), and because of potential novel process features associated with high temperature operation. As a net result, the co-culture cellulose fermentations described here may have the potential to convert more substrate to alcohol than some other bioconversion systems described [see Figure 11, (2)]. However, considerably more fundamental and applied research is required before realistic economic assessments can be made. Detailed analysis of the data presented above suggests key control parameters for thermophilic ethanol production (see Table IX). Understanding in detail the physiological and biochemical features that control rate limitation, yield limitation and concentration limitation appears to me as trends for future applied and fundamental studies on thermophilic ethanologenic bacteria. It is worth noting from the data reviewed here that understanding control of any one of these 3 major limitations is complex and multi-faceted. Indeed, improvement of ethanol tolerance (i.e. the ability to produce greater than 1% ethanol at high rates) in these bacteria appears to involve challenges by all three limitations. Furthermore, the biochemical basis for alcohol tolerance in thermophilic ethanologens appears to vary in different species. For example, the ethanol dehydrogenase of C. thermocellum is inhibited by physiological concentrations of alcohol (i.e. 1%) whereas, the reversible activity of T. brockii or C. thermohydrosulfuricum enzyme is increased by higher solvent concentration (greater than 5%).