Formation of fermentation products and extracellular protease during anaerobic growth of Bacillus licheniformis in chemostat and batch-culture

Abstract

For a relaxed (rel ⁻), protease producing (A-type) and a stringent (rel ⁺), not-protease producing (B-type) variant of Bacillus licheniformis we determined fermentation patterns and products, growth parameters and alkaline protease-production (if any) in anaerobic, glucose-grown chemostats and batch-cultures. Glucose is dissimilated via glycolysis and oxidative pentose phosphate pathway simultaneously; the relative share of these two routes depends on growth phase (in batch) and specific growth rate (in chemostat). Predominant products are lactate, glycerol and acetaldehyde for A-type batches and acetaldehyde, ethanol, acetate and lactate for B-type batches. Both types show a considerable acetaldehyde production. In chemostat cultures, the fermentation products resemble those in batch-culture.

From the anaerobic batches and chemostats, we conclude that the A-type (with low ATP-yield) will have a Y_ATP ^max of probably 12.9 g/mol and the B-type (with high ATP-yield) a Y_ATP ^max of about 10.1 g/mol. For batch-cultures, both types have about the same, high Y_glucose (12 g/mol). So, the slow-growing A-type has a relatively high efficiency of anaerobic growth (i.e. an efficient use of ATP) and the fast-growing B-type a relatively low efficiency of anaerobic growth. In aerobic batch-cultures, we found 48, respectively 41% glucose-carbon conversion into mainly glycerol and pyruvate, respectively acetate as overflow metabolites in the A- and B-type.

In both aerobic and anaerobic batch-cultures of the A-type, protease is produced predominantly in the logarithmic and early stationary phase, while a low but steady production is maintained in the stationary phase. Protease production occurs via de novo synthesis; up to 10% of the total protease in a culture is present in a cell-associated form. Although anaerobic protease production (expressed as protease per amount of biomass) is much higher than for aerobic conditions, specific rates of production are in the same range as for aerobic conditions while, most important, the substrate costs of anaerobic production are very much higher than for aerobic conditions.

Abbreviations and symbols: C-rec = %-age carbon-recovery; C-rec-Fp = agreement between glucose dissimilated and fermentation products (including CO₂) found; DW = dry weight of biomass (g/L); E ₄₄₀ = light-extinction at 440 nm; Fp = fermentation products; γ = reduction degree (no dimension); γ(ferm) = average reduction degree of all ‘products’ i.e. fermentation products + biomass + cxocellular protein); L = liter; m = maintenance requirement (mol/g DW X h); M _b = ‘molar weight’ of bacteria (147.6 g/mol) with the general elementary cell composition COOHlIIR030Nu); µ = specific growth-rate (h^-1); O/R = average oxidationlreduction quotient (of all fermentation products); q = specific rate of consumption or production (mol/g DW x h); rel ⁺, rel ^- = stringent. relaxed genotype; RI = refraction index; rpm = rotations per minute; UV = ultraviolet; X (or x) = biomass (g/L); Y = molar growth yield (g DW/mol); sub-Isuper-scripts: b = biomass, corr = corrected for exocellular protein, pr = exocellular protein, s = substrate, t = time.