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. 2020 Oct 29:8:572892.
doi: 10.3389/fbioe.2020.572892. eCollection 2020.

Genetic Cell-Surface Modification for Optimized Foam Fractionation

Christian C Blesken  1 , Isabel Bator  1   2 , Christian Eberlein  3 , Hermann J Heipieper  3 , Till Tiso  1   2 , Lars M Blank  1   2
Affiliations

Genetic Cell-Surface Modification for Optimized Foam Fractionation

Christian C Blesken et al. Front Bioeng Biotechnol. .

Abstract

Rhamnolipids are among the glycolipids that have been investigated intensively in the last decades, mostly produced by the facultative pathogen Pseudomonas aeruginosa using plant oils as carbon source and antifoam agent. Simplification of downstream processing is envisaged using hydrophilic carbon sources, such as glucose, employing recombinant non-pathogenic Pseudomonas putida KT2440 for rhamnolipid or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., rhamnolipid precursors) production. However, during scale-up of the cultivation from shake flask to bioreactor, excessive foam formation hinders the use of standard fermentation protocols. In this study, the foam was guided from the reactor to a foam fractionation column to separate biosurfactants from medium and bacterial cells. Applying this integrated unit operation, the space-time yield (STY) for rhamnolipid synthesis could be increased by a factor of 2.8 (STY = 0.17 gRL/L·h) compared to the production in shake flasks. The accumulation of bacteria at the gas-liquid interface of the foam resulted in removal of whole-cell biocatalyst from the reactor with the strong consequence of reduced rhamnolipid production. To diminish the accumulation of bacteria at the gas-liquid interface, we deleted genes encoding cell-surface structures, focusing on hydrophobic proteins present on P. putida KT2440. Strains lacking, e.g., the flagellum, fimbriae, exopolysaccharides, and specific surface proteins, were tested for cell surface hydrophobicity and foam adsorption. Without flagellum or the large adhesion protein F (LapF), foam enrichment of these modified P. putida KT2440 was reduced by 23 and 51%, respectively. In a bioreactor cultivation of the non-motile strain with integrated rhamnolipid production genes, biomass enrichment in the foam was reduced by 46% compared to the reference strain. The intensification of rhamnolipid production from hydrophilic carbon sources presented here is an example for integrated strain and process engineering. This approach will become routine in the development of whole-cell catalysts for the envisaged bioeconomy. The results are discussed in the context of the importance of interacting strain and process engineering early in the development of bioprocesses.

Keywords: 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA); cell surface hydrophobicity; flagellum; foam fractionation; integrated product recovery; large adhesion protein; metabolic engineering; rhamnolipid.

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Figures

Figure 1
Figure 1
Foam fractionation setup to determine cell agglomeration in foam. The culture broth was centrifuged, and the pellet was washed (A) before it was resuspended in an isotonic solution containing rhamnolipids (B). The cell suspension was filled into the fractionation column, and the gas flow was turned on (C). The foam leaving the upper opening was collapsed by transfer through a thin tube and collected as foamate in a vacuum bottle (D).
Figure 2
Figure 2
Fermentation setup with three stages in the growth phase and the harvest phase. Growth phase: 1st stage: no gassing into the stirred bioreactor (A). 2nd stage: activated aeration; discharging foam through the exhaust directly into the foam centrifuge (C) (bypass in red); foamate reflux into reactor (blue). 3rd stage: bypass cut-off and integration of a foam fractionation column (B) between reactor and centrifuge. From the column, a pump returns the drained liquid while the fractionated foam is guided through an upper outlet. Harvest phase: product harvest by stopping the foamate reflux and collecting the foamate in a bottle. Regular refill of fresh medium to control working volume in the reactor. Sampling points are marked as ➀ (reactor) and ➁ (foamate outlet).
Figure 3
Figure 3
Congener composition of produced mono-rhamnolipids (black) and HAAs (gray) with P. putida KT2440 SK4 (left) and P. putida KT2440 KS3 (right) on minimal medium with glucose as sole carbon source. The error bars indicate the deviation from the mean of two biological replicates.
Figure 4
Figure 4
Shake flask cultivation of P. putida KT2440 SK4 (blue) and P. putida KT2440 KS3 (black) on minimal medium with 9 g/L glucose. (A) Biomass concentration vs. time (squares) and surfactant concentration vs. time (empty squares); (B) surfactant yield from biomass vs. time (YP/X); (C) biomass yield from glucose vs. time (YX/S, points) and biomass per ammonium vs. time (YX/NH4+, empty points). The error bars indicate the deviation from the mean of two biological replicates.
Figure 5
Figure 5
Cultivation of P. putida KT2440 SK4 (A–C) and P. putida KT2440 KS3 (D–F) in a bioreactor. Growth phase (gray background) and a subsequent continuous product separation during the harvest phase (t = 10.6 h to t = 20.6 h). (A,D) Biomass concentration in the reactor (blue squares) and in the foamate (black squares) and (B,E) Surfactant concentrations were measured in the fermentation broth of the reactor (blue points) and in the foamate after fractionation (black points). The biomass and surfactant enrichment factors (Ebiomass & Esurfactant) are depicted in violet. (C,F) The total volume of separated foamate was measured continuously.
Figure 6
Figure 6
Biomass enrichment factor EOD600·V (n = 2) in fractionated foam vs. water contact angle (n = 9) for Pseudomonas wild types (black) and P. putida KT2440 knock-out strains. P. putida KT2440 knock-out strains used for rhlA and rhlAB integration in the following are marked by filled squares. Vertical error bars indicate the deviation from the mean for n = 2 and horizontal error bars the standard deviation of the mean for n = 9.
Figure 7
Figure 7
Comparison of non-modified production hosts (black) with knock-out strains negative for the synthesis of surface structures (colored). Harvest phase at the (A–E) rhamnolipid and (F–J) HAA production and separation as foamate in the designed bioreactor setup. (A,F) Biomass concentration in the reactor vs. time, (B,G) biosurfactant concentration in the foamate at the inlet of the collection bottle vs. time, (C,D,H,I) biomass and surfactant enrichment in the foamate (Ebiomass & Esurfactant) vs. time, and (E, J) the volume of the collected foamate vs. time.
Figure 8
Figure 8
Abiotic influence factors on biosurfactant production in the applied aerated bioreactor system with product separation via integrated foam fractionation.
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