Accumulation and degradation of diclofop methyl by cultured biofilm communities

Traditionally, bacteria are viewed as unicellular organisms and are studied as isolated cell lines. However, in natural systems they rarely exist as pure cultures, and thus there is a need to study them as communities under defined laboratory conditions. In this study, a degradative microbial community consisting of nine bacterial and one algal species was isolated from soil using the herbicide diclofop methyl as sole carbon source. The presence of the algae, or addition of an exogenous carbon source, significantly increased diclofop mineralization in continuous flow (36% increase) and batch (11% increase) cultures. Pure cultures isolated from this bacterial consortium could not mineralize diclofop. However, when supplied with an additional carbon source, two strains could degrade more than 25% of it to CO2. Biofilm formation also resulted in more efficient degradation (36% increase). These observations indicated that interspecies interactions and spatial organization within biofilms enhance degradative efficiency. Subsequent analysis of the degradative biofilms, using scanning confocal laser microscopy, revealed distinctive spatial relationships among members of the consortium when grown on diclofop. These relationships, such as the formation of cell clusters within biofilms and an irregular surface topography, were absent when more labile substrates were provided as the carbon source. Stable biofilms having these distinctive features, normally developed in 14 to 21 days. Their development was accompanied by an increase in autofluorescence, indicating accumulation of diclofop and its aromatic breakdown products. Probe mass spectroscopy confirmed the presence of these compounds in the biofilm matrix. Most accumulation occurred in cell capsules and exopolymeric materials within specific regions of the biofilm matrix. Additional evidence for spatial organization in the degradative biofilms was obtained using a set of FITC-conjugated probes. These revealed that regions of the exopolymer matrix which accumulated diclofop had a unique chemical composition and charge. These regions were not present in biofilms grown on a more labile growth medium. Fluorescence extinction and radioisotopic techniques demonstrated that the adsorbed diclofop was utilized during periods of carbon deprivation, indicating that exopolymers can store adsorbed carbon for subsequent utilization