The in situ production and separation of surfactin, iturin, and fengycin lipopeptides in an aqueous two-phase system

Aqueous two-phase systems (ATPS) were used to evaluate the in situ production and separation of lipopeptides produced by Bacillus amyloliquefaciens. This method demonstrates the first step towards continuous production of lipopeptides. Growth and lipopeptide production were evaluated at elevated trisodium citrate, sodium potassium tartrate, dipotassium hydrogen phosphate, and sodium sulphate concentrations (from 0.2 to 0.8 M). Citrate concentrations negatively impacted growth, while phosphate, sulphate, and tartrate showed little effect, with cultures reaching a maximum cell dry weight between 3.98 and 4.5 g L⁻¹, in line with the 4.5 g L⁻¹ observed with standard media optimized for lipopeptide production. These results were coupled with the production of 137 mg L⁻¹ lipopeptides at 0.8 M tartrate, 95 mg L⁻¹ at 0.8 M phosphate, and 14 mg L⁻¹ at 0.8 M sulphate. Two-phase forming systems were tested, with the addition of various concentrations of PEG 8000, excluding citrate. In the phosphate ATPS, iturin partitioned primarily to the polymer phase with concentrations up to 267 mg L⁻¹, while the fengycin primarily partitioned to the salt phase with concentrations of 118 mg L⁻¹. Surfactin was found to accumulate in the solid phase under ATPS conditions. This method is simple, scalable, and novel.