Oxygen transfer rate determines molecular weight and production of poly(γ‐glutamic acid) as well as carbon utilization by Bacillus velezensis 83

Abstract

BACKGROUNDThe objective of the study was to evaluate the influence of the oxygen transfer rate (OTR) on poly(γ‐glutamic acid) (γ‐PGA) production and molecular weight and the carbon source utilization in cultures of Bacillus velezensis 83 developed in shake flasks.RESULTSThe cellular metabolism of B. velezensis 83 was affected by the OTR conditions. At maximum oxygen transfer rates (OTRmax) of 12, 17 and 19 mmol L−1 h−1, glucose was mainly used for cell growth and γ‐PGA production. In contrast, under oxygen‐limiting conditions (2.5 and 5.7 mmol L−1 h−1), the carbon source was also directed to acetoin, 2,3‐butanediol and CO2 production. Changes in OTRmax significantly impacted γ‐PGA production, its maximum mean molecular weight (MMWmax) and its depolymerization kinetics. In cultures developed at an OTRmax of 2.5 mmol L−1 h−1, the bacteria synthesized a polymer with a MMWmax of 2400 kDa, whereas at OTR greater than 5.7 mmol L−1 h−1 a polymer with a MMWmax of 2850 kDa was obtained and the γ‐PGA concentration increased up to 2.8‐fold. In addition, γ‐PGA depolymerization, occurring even in the presence of glucose, increased at non‐limiting OTR conditions.CONCLUSIONSThe production, yield and molecular weight of γ‐PGA are determined by OTR conditions. Our results showed, for the first time, that there is a direct relationship between OTRmax and the γ‐PGA depolymerization rate in B. velezensis 83 cultures. © 2020 Society of Chemical Industry

Online measurement of the viscosity in shake flasks enables monitoring of γ‐PGA production in depolymerase knockout mutants of Bacillus subtilis with the phosphate‐starvation inducible promoter Ppst

“…This may be due to an increased γ‐PGA production under oxygen‐limited cultivation conditions 30 . Possibly, it also may depend on the higher molecular weight of the produced γ‐PGA induced by the changed cultivation conditions 1,30,44 …”

Oxygen transfer and bubble sizes occurring in a pilot‐scale cultivation of Bacillus velezensis 83 for the production of poly(γ‐glutamic acid) under two schemes of power drawn

“…Glucose in supernatants was determined using a biochemistry analyzer (YSI, Yellow Springs Instruments, Ohio, USA). PGA concentration and molecular weight were measured using gel permeation chromatography with a serial set of ultrahydrogel columns (UG 500 and Linear, Waters), using a highperformance liquid chromatography system (Waters 2414) equipped with a differential refractometer detector (Waters 410) as reported by Flores et al 30 Surfactin and bacillomycin in the culture broth were determined according to Balderas-Ruíz et al 35 Surface tension was measured in the culture broth previously centrifuged (10 000 rpm, 10 min), using a Wilhelmy plate tensiometer (ST-PLUS, Tantech Inc., USA) as previously described by Brito-Bazán et al, 13 at the temperature at which the fermentations were carried out (30 °C). The rheological properties of the broth were measured using a stress-controlled DHR-3 (TA Instruments) rheometer, using a concentric cylinder with double gap, at a shear rate of 100 s −1 and at 30 °C.…”

“…CP034203) was provided by Agro&Biotecnia S de RL de CV. The strain was kept at −18 °C in 80% (w/w) glycerol–water before use and recovered in a Petri dish with YPG solid medium containing (in g L −1 ): yeast extract, 10; peptone, 10; glucose, 10; agar, 15 30 …”

“…The culture medium used in the 100 L bioreactor was previously reported by Flores et al 30 . During the culture time, three nutrient pulses (glucose and ammonium sulfate) were added to these cultures at different times of cultivation (16, 22 and 28 h), to avoid glucose and ammonium limitation and in such a way that the glucose concentration was 15 g L −1 after the addition and 6 g L −1 in the case of ammonium sulfate.…”

“…Glucose in supernatants was determined using a biochemistry analyzer (YSI, Yellow Springs Instruments, Ohio, USA). PGA concentration and molecular weight were measured using gel permeation chromatography with a serial set of ultrahydrogel columns (UG 500 and Linear, Waters), using a high‐performance liquid chromatography system (Waters 2414) equipped with a differential refractometer detector (Waters 410) as reported by Flores et al 30 . Surfactin and bacillomycin in the culture broth were determined according to Balderas‐Ruíz et al 35 .…”