The influence of dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

Abstract

The antibiotic, difficidin, and its hydroxylated derivative, oxydifficidin, were synthesized by cultures of Bacillus subtilis grown on a complex medium. Maximum titers of about 200 and 130 mg/L, respectively, were obtained. In fermentations where the dissolved oxygen tension (DOT) was controlled, the maximum specific growth rate was only reduced below 5% air saturation. DOT had little effect on the volumetric rate of synthesis of oxydifficidin but greatly influenced the rate for difficidin, which was reduced at DOT values below 40% air saturation. Key words: difficidin oxydifficidin Bacillus subtilis dissolved oxygen tension Dissolved oxygen tension (DOT) is known to affect the microbial synthesis of many antibiotics. Early reports' 9 3 described the influence of aeration and agitation, but later it was possible to correlate product formation directly with DOT measured using electrodes. Various workers have demonstrated that penicillin synthesis is suppressed at DOT values below about 10% air saturation8'16 and only reaches a maximum rate at higher DOT values. Critical DOT values below which synthesis is reduced have been reported for other antibiotics, such as cephalosporins,6 cephamycin C,'5,2' and candicidin. l 2In large-scale fermentations, due to inefficient bulk mixing and changes in hydrostatic pressure, microorganisms are exposed to a continually changing environment as they pass through different regions in a fermenter. Dissolved oxygen concentration gradients have been observed for an unspecified 19 m3 fermentati~n'~ and for chlorotetracycline production by strains of Streptomyces aureofaciens in a 1 12-m3 fermenter." Previously we reported16 that the specific penicillin production rate (q,,) of Penicillium chrysogenum strain P1 fell at DOT values below 35% air saturation. When the conditions in a large fermenter were simulated by cycling the DOT (23-37%) in a small fermenter, the qpen was lower than for fermentations run at a constant * To whom all correspondence should be addressed. 30% air saturation. Yegneswaran et al.,' reported that cycling of DOT reduced biosynthesis of cephamycin C by Streptomyces cluvuligerus. Using an alternative approach where the microorganisms were cycled between two vessels maintained at different DOT levels, Larsson and Enfors' observed changes in the respiration rate of Penicillium chrysogenum under certain conditions.With mycelial organisms, any change in the morphology can affect the response to DOT. l7 We have selected, therefore, for further studies on the effect of DOT on antibiotic production, a nonfilamentous bacterium, Bacillus subtilis, which synthesizes difficidin and its hydroxylated derivative, o~ydifficidin'~ ( Fig.

The effect of cycling dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

“…In each case the titers rose almost linearly over these 9 h periods allowing volumetric production rates to be calculated (Table 3). There was no significant trend in the volumetric production rates for oxydifficidin with the values (0.9-1.3 mg/lh) in the same range as reported for constant DOT fermentations [8]. However, it was clear that the volumetric difficidin production rate in the fermentation with cycling at 40% DOT increased with fermentation time up to 70 h. Whereas the difficidin production rates rose substantially between the initial constant DOT period and the subsequent cycling period for fermentations at 20 and 40% DOT there was only a small increase in the fermentation at 15% DOT indicating a deleterious effect of cycling about that DOT value.…”

“…In a previous paper [8] we showed that the rate of difficidin synthesis was affected by the level of DOT and that a dramatic change was observed at approximately 20% air saturation. We have, therefore, carried out fermentations cycling the DOT (i) below this value, about 15% air saturation; (ii) at this critical value, about 20% air saturation and (iii) above this apparent critical value, about 40% air saturation.…”

“…The examination of cycling DOT about 20% and 15% saturation would determine the effect of changing DOT on antibiotic production rates and cycling the DOT around 40% should illustrate whether or not the DOT cycling itself is a factor. The cycling DOT experiments were conducted during the growth phase (between 28 to 70 h of cultivation) when antibiotic production was almost linear during fermentations at constant DOT [8]. Each experiment was divided into three parts: (i) fermentation with controlled constant DOT; (ii) fermentation with cycling DOT and (iii) fermentation with controlled constant DOT.…”

“…The volumetric production rates reported previously [8] were based in each case on seven measurements of antibiotic titers between 23 and 71 h of the fermentations. In the cycling experiments about thirty measurements were made between 28 and 71 h allowing the profiles to be examined in more detail.…”

“…Critical DOT values below which synthesis is reduced have been reported for other antibiotics such as cephalosporins [5], cephamycin C [6] and candicidin [7]. We have previously reported [8] the effect of DOT on antibiotic production in a non-filamentous bacterium, Bacillus subtilis, which synthesises difficidin and its hydroxylated derivative, oxydifficidin [9] and a brief description of the work was reported earlier [10]. In fermentations where the DOT was controlled, the maximum specific growth rate was only reduced below 5% air saturation.…”