Real-Time Solvent Tolerance Analysis ofPseudomonassp. Strain VLB120ΔC 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 VLB120⌬C-BT-gfp1 was grown in modified flow-cell reactors and exposed to the solvent styrene. Biofilmgrown 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.

Catalytic Pseudomonas taiwanensis VLB120ΔC Biofilms Thrive in a Continuous Pure Styrene Generated by Multiphasic Segmented Flow in a Capillary Microreactor

“…During this time period, a flat biofilm was developed (Figure 1), which fully adapted to the fluidic and hydrophobic stresses and recovered its structure ( Figure 1J). Our previous findings revealed that addition of solvents such as styrene stimulates and increases the EPS production in biofilms [16]. Similar findings of alteration and enhancement of EPS production was observed after chromium exposure in membrane cultivated Pseudomonas putida biofilms [17].…”

“…Cells in the biofilm experienced severe membrane damage upon styrene exposure through the silicone membrane but were able to adapt to the solvent over 48 h in minimal medium containing glucose as sole carbon and energy source [16]. This work here focuses on understanding the cellular response, growth, and biocatalytic performance when exposed to a pure styrene environment in a continuous-flow capillary microreactor.…”

“…The physiological effect of the substrate styrene on the biofilm catalyst was investigated in our previous work using confocal † † microscopy [16]. Cells in the biofilm experienced severe membrane damage upon styrene exposure through the silicone membrane but were able to adapt to the solvent over 48 h in minimal medium containing glucose as sole carbon and energy source [16].…”

“…Carl-Zeiss LSM5 Pascal confocal laser-scanning microscope (CLSM; Carl Zeiss, Jena, Germany) equipped with an argon and helium-neon laser was used to acquire biofilm images in situ noninvasively in real time at regular time intervals. Details of image acquisition and data treatment were described in Halan et al [16] and Karande et al [5].…”

“…Pseudomonas taiwanensis VLB120∆C and a variant synthesizing the green fluorescent protein (GFP) as fluorescent marker were used as a model biofilm-forming strain [16]. For precultures, the strains were grown overnight at 30°C (200 rpm, Multitron, Infors HT, Bottmingen, Switzerland) in lysogeny broth (LB) medium supplemented with streptomycin (100 μg/mL).…”