Architecture and spatial organization in a triple-species bacterial biofilm synergistically degrading the phenylurea herbicide linuron

RESEARCH ARTICLE Architecture and spatial organization in a triple-species bacterial bio¢lm synergistically degrading the phenylurea herbicide linuron Philip Breugelmans1, Kim Bundvig Barken2, Tim Tolker-Nielsen2, Johan Hofkens3, Winnie Dejonghe4 & Dirk Springael1 1 Division of Soil and Water Management, Catholic University of Leuven, Kasteelpark Arenberg, Leuven, Belgium; 2BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark; 3Division of Molecular and Nanomaterials, Catholic University of Leuven, Leuven, Belgium; and 4 Environmental and Process Technology, Flemish Institute for Technological Research (VITO), Boeretang, Belgium Received 20 November 2007; revised 23 January 2008; accepted 25 January 2008. First published online 28 March 2008. DOI:10.1111/j.1574-6941.2008.00470.x Editor: Max Häggblom Keywords biofilm; consortium; linuron; metabolic interactions. Abstract Members of a triple-species 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-mineralizing consortium, i.e. the linuron- and 3,4-dichloroanilinedegrading Variovorax sp. WDL1, the 3,4-dichloroaniline-degrading Comamonas testosteroni WDL7 and the N,O-dimethylhydroxylamine-degrading Hyphomicrobium sulfonivorans WDL6, were cultivated as mono- or multi-species biofilms in flow cells irrigated with selective or nonselective media, and examined with confocal laser scanning microscopy. In contrast to mono-species biofilms of Variovorax sp. WDL1, the triple-species consortium biofilm degraded linuron completely through apparent synergistic interactions. The triple-species linuronfed consortium biofilm displayed a heterogeneous structure with an irregular surface topography that most resembled the topography of linuron-fed monospecies WDL1 biofilms, indicating that WDL1 had a dominating influence on the triple-species biofilm architecture. This architecture was dependent on the carbon source supplied, as the biofilm architecture of WDL1 growing on alternative carbon sources was different from that observed under linuron-fed conditions. Linuron-fed triple-species consortium biofilms consisted of mounds composed of closely associated WDL1, WDL7 and WDL6 cells, while this association was lost when the consortium was grown on a nonselective carbon source. In addition, under linuron-fed conditions, microcolonies displaying associated growth developed rapidly after inoculation. These observations indicate that the spatial organization in the linuron-fed consortium biofilm reflected the metabolic interactions within the consortium. Introduction Herbicides mainly enter the environment as nonpoint contamination from agricultural sources. Following spraying practices, specific environmental conditions such as heavy precipitation can result in the migration of these pollutants into water bodies (Gooddy et al., 2002). Phenylurea herbicides such as linuron are commonly used for pre- and postemergence control of annual grasses and broad-leafed weeds and are frequently detected in groundwater and surface water worldwide (e.g., Caux et al., 1998; Carabias-Martı́nez et al., 2003). The potential negative impact of 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea FEMS Microbiol Ecol 64 (2008) 271–282 (linuron) and its metabolites on ecosystem and human health (Kegley et al., 2007) has stimulated research to study its natural attenuation in the environment. Microbial degradation is the primary mechanism by which linuron is removed from the environment, and the activity of linurontransforming organisms in the topsoil is considered to determine the amount of herbicide residues that leach and run off to water bodies. Although strains that mineralize linuron individually have been reported (Sørensen et al., 2005), linuron-mineralizing bacterial cultures enriched from treated agricultural soils often consist of multiple-strain consortia, indicating that linuron in agricultural soils is degraded by co-operative 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c Correspondence: Dirk Springael, Division of Soil and Water Management, Catholic University of Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium. Tel.: 132 0 16321604; fax: 132 0 16321997; e-mail: 272 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c Appropriate mono-, dual- and triple-species biofilms were grown in flow cells under nutritionally different conditions (commensal and noncommensal), and effluent concentrations of linuron and 3,4-DCA were monitored. The spatial organization of the biofilms was monitored using CLSM, and the relative abundance of each strain in the multispecies biofilms was evaluated using the image analysis program DAIME (Daims & Wagner, 2005). Materials and methods Bacteria and culture conditions The linuron-mineralizing consortium used in this study was described previously by Dejonghe et al. (2003) and consists of the linuron- and 3,4-DCA-degrading strain Variovorax sp. WDL1, the 3,4-DCA-degrading strain C. testosteroni WDL7 and the N,O-DMHA-degrading strain H. sulfonivorans WDL6. Tagged derivatives of C. testosteroni WDL7 and H. sulfonivorans WDL6 were used. A red fluorescent derivative of C. testosteroni WDL7, i.e. strain WDL7-Rfp, was constructed by introduction of the miniTn5(Km)PA1/04/03rfp gene cassette (Tolker-Nielsen et al., 2000) into the chromosome by tri-parental conjugation. Hyphomicrobium sulfonivorans WDL6 was chromosomally tagged with the miniTn7(Km,Sm)PA1/04/03-eyfp-a gene cassette (Lambertsen et al., 2004) by four-parental conjugation, resulting in a yellow fluorescent derivative of WDL6 designated as WDL6-Yfp. No significant difference in 3,4-DCA degradation kinetics between wild type and tagged derivatives of WDL7, and growth rate on N,O-DMHA between wild type and tagged derivatives of WDL6 were observed. To visualize Variovorax sp. WDL1, the unspecific cyanine nucleic acid stain Syto 62 (Molecular Probes, Invitrogen) was applied. Using two specific labels and one unspecific stain, the three consortium members could be visualized and distinguished from each other using CLSM. As observed previously with DNA stains in combination with green fluorescent protein-expressing strains (Wuertz et al., 2001), Syto 62 did not penetrate red fluorescent protein (RFP)- or yellow fluorescent protein (YFP)-labeled cells. Although the reason for this phenomenon is not clear (Nancharaiah et al., 2005), it facilitated discrimination between the three community members. To obtain high-density cultures for inoculation, each strain was individually precultured in a medium, which ensured optimal growth conditions for that particular strain. Variovorax sp. WDL1, C. testosteroni WDL7-Rfp and H. sulfonivorans WDL6-Yfp were grown in 25 mL of, respectively, R2A (0.5 g tryptone, 0.5 g yeast extract, 0.5 g casein hydrolysate, 0.5 g glucose D1, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0 (...truncated)