Real-time solvent tolerance analysis of pseudomonas sp. strain VLB120{Delta}C catalytic biofilms

Abstract

Biofilms are ubiquitous surface-associated microbial communities embedded in an extracellular polymeric (EPS) matrix, which gives the biofilm structural integrity and strength. It is often reported that biofilm-grown cells exhibit enhanced tolerance toward adverse environmental stress conditions, and thus there has been a growing interest in recent years to use biofilms for biotechnological applications. We present a time- and locus-resolved, noninvasive, quantitative approach to study biofilm development and its response to the toxic solvent styrene. Pseudomonas sp. strain VLB120ΔC-BT-gfp1 was grown in modified flow-cell reactors and exposed to the solvent styrene. Biofilm-grown cells displayed stable catalytic activity, producing (S)-styrene oxide continuously during the experimental period. The pillar-like structure and growth rate of the biofilm was not influenced by the presence of the solvent. However, the cells experience severe membrane damage during styrene treatment, although they obviously are able to adapt to the solvent, as the amount of permeabilized cells decreased from 75 to 80% down to 40% in 48 h. Concomitantly, the fraction of concanavalin A (ConA)-stainable EPS increased, substantiating the assumption that those polysaccharides play a major role in structural integrity and enhanced biofilm tolerance toward toxic environments. Compared to control experiments with planktonic grown cells, the Pseudomonas biofilm adapted much better to toxic concentrations of styrene, as nearly 65% of biofilm cells were not permeabilized (viable), compared to only 7% in analogous planktonic cultures. These findings underline the robustness of biofilms under stress conditions and its potential for fine chemical syntheses.

FIG. 1.

Confocal micrographs showing biofilm development stages under standard growth conditions (no solvents) and in the styrene environment. (A to C) gfp-expressing intact and PI-stained biofilms under normal growth conditions after 24 (A), 48 (B), and 72 (C). (D to F) gfp-expressing intact and PI-stained biofilms in the styrene environment after 24 (D), 48 (E), and 72 h (F). (G to I) ConA-stained biofilm under normal growth conditions after 24 (G), 48 (H), and 72 h (I). (J to L) ConA-stained biofilm in the styrene environment after 24 (J), 48 (K), and 72 h (L). (M) Top view of 48-h-old ConA-stained biofilm with water channels as indicated by arrows. Green color represents the intact gfp-expressing cells, red color represents PI-stained dead or permeabilized cells, and violet color (ConA) represents polysaccharides in the EPS matrix. Representative IMARIS-treated and 3D-reconstructed images from three independent experiments are shown. Scale bar, 20 μm.

FIG. 2.

Planktonic cell response to styrene. The graph shows the planktonic live and dead/permeabilized cells (A), as well as respective cell growth (B), from 0 to 96 h. □, percent live cells of styrene culture; ▪, percent dead cells of styrene culture; ○, percent dead cells as a control (without styrene); ▵, cell dry weight of control cultures; ▴, cell dry weight of cultures grown in the styrene environment. Data are mean values from three independent growth experiments.

FIG. 3.

Graphical representation of fractional dynamics of live and dead (permeabilized) cells (A) and the (S)-styrene oxide production of biofilms growing in a styrene environment (B). Biofilm-forming cells were inoculated into the styrene-saturated flow-cell environment as described in Materials and Methods. ▴, percent intact gfp-expressing live cells; ▪, percent live cells as controls (without styrene); •, total styrene oxide produced in aqueous and organic phase. Live and dead (permeabilized) cells were determined by surface area covered compared to the total area. The aqueous- and organic-phase samples were collected every 24 h, and the concentration of styrene oxide was determined by GC. The organic-phase volume was 70 μl and was exchanged every 24 h. Data presented here are mean values from three independent growth experiments.

FIG. 4.

Polysaccharide amount in biofilms grown in the presence of styrene and under standard/nonsolvent conditions. (A) Graph shows microbial polysaccharide production (given in volume covered) as analyzed during a period of 96 h. Biofilms were allowed to grow for 24 h under standard conditions before styrene was added. Data presented here are mean values from three independent growth experiments. (B) Increase in biofilm biovolume over time in standard and in styrene environments. ▵, biovolume of the biofilm grown in styrene environment; ▴, biovolume of the biofilm grown in a nonsolvent environment.

FIG. 5.

Confocal micrographs (sectional views) showing the distribution of live and dead (permeabilized) cells in the layers of biofilm at different time frames. (A) Early stage (24 h); (B) intermediate stage (51 h); (C and D) matured biofilms (72 and 96 h, respectively). Arrows indicate the intact cells that are located nearly exclusively inside the red (permeabilized) cell layers. Scale bar, 30 μm.