Commensal interactions in a dual-species biofilm exposed to mixed organic compounds

Abstract

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.

FIG. 1

Growth and inhibition kinetics. (A and C) P. putida DMP1. (B and D) Pseudomonas sp. strain GJ1. (A) P. putida DMP1 growth on p-cresol. (B) Pseudomonas sp. strain GJ1 growth on 2-CE. (C) Inhibition of P. putida DMP1 growth with p-cresol as the carbon source and 2-CE as the inhibitor. (D) Inhibition of Pseudomonas sp. strain GJ1 growth with: 2-CE as the carbon source and p-cresol as the inhibitor.

FIG. 2

Vertical sections of biofilms. (A to C) Pseudomonas sp. strain GJ1 (green) monoculture biofilms. (D to F) Pseudomonas sp. strain GJ1 (yellow) and P. putida DMP1 (red) coculture biofilms. (G to I) Pseudomonas sp. strain GJ1 (yellow) and E. coli ATCC 33456 (red) coculture biofilms. (A, D, and G) Cells were grown on succinate. (B, E, and H) Cells were grown on 20 mM 2-CE and 0.7 mM p-cresol. (C, F, and I) Cells were grown on 20 mM 2-CE and 1.9 mM p-cresol. Magnification, ×200.

FIG. 3

Horizontal and vertical sections of Pseudomonas sp. strain GJ1 (yellow) and P. putida DMP1 (red) coculture biofilms with exopolysaccharides (blue). (A to C) Cells grown in succinate. (D to F) Cells grown on 20 mM 2-CE and 0.7 mM p-cresol. (G to I) Cells grown on 20 mM 2-CE and 1.9 mM p-cresol. Magnification, ×200.