Fermentation patterns of gramicidin S formation by Bacillus brevis ATCC 9999 in high‐yielding synthetic medium

Abstract

Addition of the gramicidin S (GS)‐constituent amino acids, other than the limiting precursor L‐phenylalanine, to the high‐yielding chemically defined F3/6 and G3/6 media, enhanced growth and volumetric GS‐production by Bacillus brevis ATCC 9999 considerably, but did not yield a higher specific GS‐production level. L‐Leucine alone could duplicate this stimulatory effect in G3/6 medium. Replacing the fructose component of F3/6 medium by these four amino acids yielded a high specific GS‐production level, but resulted in poor growth and low volumetric antibiotic production. Nutrient‐utilisation patterns in F3/6 medium revealed that B. brevis initially grew at the expense of L‐glutamine and L‐arginine. After a diauxic lag period, D‐fructose was consumed together with L‐histidine. L‐Proline was mainly used during the stationary phase. L‐Methionine was broken down gradually throughout the whole fermentation cycle. L‐Phenylalanine was metabolised only after GS formation started, and its disappearance was proportional to the amount of GS produced. Lowering the aeration rate caused an acidification of the medium, resulting in a slower and incomplete, although similar, nutrient‐utilisation pattern. At a controlled pH of 7.3, under lowered aeration, utilisation patterns were again comparable with those of a fully aerated fermentation, but GS levels were enhanced considerably (0.220 mg of GS mg−1 dry cell wt). Depending on environmental culture conditions, B. brevis also excreted different amino acids (L‐lysine, L‐alanine, L‐valine, L‐serine), which were in turn metabolised during late growth and differentiation stages. The onset of GS synthesis occurred on depletion of L‐glutamine and L‐arginine. Soluble GS synthetase 1 and 2 peaks coincided with ‘diauxic’ lag phases; this supported the idea that a high growth rate is incompatible with GS synthetase formation.