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. 2020 Nov 23:8:565619.
doi: 10.3389/fbioe.2020.565619. eCollection 2020.

Development of a Bioprocess for the Production of Cyclic Lipopeptides Pseudofactins With Efficient Purification From Collected Foam

Piotr Biniarz  1   2 , Marius Henkel  3 , Rudolf Hausmann  3 , Marcin Łukaszewicz  2
Affiliations

Development of a Bioprocess for the Production of Cyclic Lipopeptides Pseudofactins With Efficient Purification From Collected Foam

Piotr Biniarz et al. Front Bioeng Biotechnol. .

Abstract

Microbial surfactants (biosurfactants) have gained interest as promising substitutes of synthetic surface-active compounds. However, their production and purification are still challenging, with significant room for efficiency and costs optimization. In this work, we introduce a method for the enhanced production and purification of cyclic lipopeptides pseudofactins (PFs) from Pseudomonas fluorescens BD5 cultures. The method is directly applicable in a technical scale with the possibility of further upscaling. Comparing to the original protocol for production of PFs (cultures in mineral salt medium in shaken flasks followed by solvent-solvent extraction of PFs), our process offers not only ∼24-fold increased productivity, but also easier and more efficient purification. The new process combines high yield of PFs (∼7.2 grams of PFs per 30 L of working volume), with recovery levels of 80-90% and purity of raw PFs up to 60-70%. These were achieved with an innovative, single-step thermal co-precipitation and extraction of PFs directly from collected foam, as a large amount of PF-enriched foam was produced during the bioprocess. Besides we present a protocol for the selective production of PF structural analogs and their separation with high-performance liquid chromatography. Our approach can be potentially utilized in the efficient production and purification of other lipopeptides of Pseudomonas and Bacillus origin.

Keywords: Pseudomonas fluorescens; bioreactor; biosurfactant production; cyclic lipopeptides (CLPs); foam fractionation; lipopeptide production; lipopeptide purification.

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Figures

FIGURE 1
FIGURE 1
Schematic view of the bioreactor tank set-up used for the production of PFs.
FIGURE 2
FIGURE 2
The schematic representation of PFs purification from foam.
FIGURE 3
FIGURE 3
Batch cultures of P. fluorescens BD5 in 2.5-L (panels A and C) and 30-L (panels B and D) working volumes. Microbial growth (OD, black and gray squares), PFs production (mg/L, black and gray circles) are shown in panels (A,B) for the 2.5- and 30-L cultures, respectively. Alongside, respective models of microbial growth (solid black lines), PFs production (dashed black lines) and PFs specific production (mg/OD, dotted black lines) are shown in panels (A,B). In panels (C,D) data for pO2 (black lines) and pH (gray lines) in cultivation medium is shown.
FIGURE 4
FIGURE 4
Time courses of the cultivation parameters of PFs production in bioreactors with foam overflow in the 2.5-L (panels A,C,E) and 30-L (panels B,D,F) cultures. In panels (A,B) measured OD in culture medium (black squares) and in overflowing foam (gray squares) are shown for the 2.5- and 30-L cultures. PFs concentration in overflowing foam (mg/L, gray circles) is shown together with a mass of collected foam (% of the initial mass of medium in bioreactor, dotted black lines) in panels (B,D) for the 2.5- and 30-L cultures. In panels (E,F) data for pO2 (black lines) and pH (gray lines) in cultivation medium is shown.
FIGURE 5
FIGURE 5
Effect of heating on the clarified foam supernatants. (A) Clarified foam supernatant (SUP) obtained after foam centrifugation and removal of cell pellets. (B) Sample boiling resulted in a formation of pellets in the samples, (C) which can be separated with centrifugation. (D) Relative amounts of PFs in supernatants after boiling. The initial concentration of PFs in the unboiled samples (0 min) was 607.7 ± 2.0 mg/L (100%).
FIGURE 6
FIGURE 6
HPLC analysis of the PF structural analogs purification. (A) methanolic extract of SUP pellet – analytical HPLC chromatogram. (B) methanolic extract of SUP pellet – semi-preparative HPLC chromatogram. Collected PF1 and PF2 fractions are indicated with black lines. (C) Purified PF1, Rf = 3.8 min (D) Purified PF2, Rf = 4.2 min – analytical HPLC chromatograms. PF1 and PF2 peaks are indicated.
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