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Dominance of sphingomonads in a copper-exposed biofilm community for groundwater treatment

R. Vílchez^1,

¹ Grupo de Microbiología Ambiental (Environmental Microbiology Research Group), Instituto del Agua, Escuela Superior de Ingenieros de Caminos, Canales y Puertos, Universidad de Granada, 18071 Granada, Spain
² Grupo de Microbiología Ambiental (Environmental Microbiology Research Group), Departamento de Microbiología, Facultad de Farmacia, Escuela Superior de Ingenieros de Caminos, Canales y Puertos, Universidad de Granada, 18071 Granada, Spain
³ Grupo de Microbiología Ambiental (Environmental Microbiology Research Group), Departamento de Ingeniería Civil, Escuela Superior de Ingenieros de Caminos, Canales y Puertos, Universidad de Granada, 18071 Granada, Spain

Correspondence
J. González-López
jgl{at}ugr.es

The structure, biological activity and microbial biodiversity of a biofilm used for the removal of copper from groundwater were studied and compared with those of a biofilm grown under copper-free conditions. A laboratory-scale submerged fixed biofilter was fed with groundwater (2.3 l h^–1) artificially polluted with Cu(II) (15 mg l^–1) and amended with sucrose (150 mg l^–1) as carbon source. Between 73 and 90 % of the Cu(II) was removed from water during long-term operation (over 200 days). The biofilm was a complex ecosystem, consisting of eukaryotic and prokaryotic micro-organisms. Scanning electron microscopy revealed marked structural changes in the biofilm induced by Cu(II), compared to the biofilm grown in absence of the heavy metal. Analysis of cell-bound extracellular polymeric substances (EPS) demonstrated a significant modification of the composition of cell envelopes in response to Cu(II). Transmission electron microscopy and energy-dispersive X-ray microanalysis (EDX) showed that copper bioaccumulated in the EPS matrix by becoming bound to phosphates and/or silicates, whereas copper accumulated only intracytoplasmically in cells of eukaryotic microbes. Cu(II) also decreased sucrose consumption, ATP content and alkaline phosphatase activity of the biofilm. A detailed study of the bacterial community composition was conducted by 16S rRNA-based temperature gradient gel electrophoresis (TGGE) profiling, which showed spatial and temporal stability of the species diversity of copper-exposed biofilms during biofilter operation. PCR reamplification and sequencing of 14 TGGE bands showed the prevalence of alphaproteobacteria, with most sequences (78 %) affiliated to the Sphingomonadaceae. The major cultivable colony type in plate counts of the copper-exposed biofilm was also identified as that of Sphingomonas sp. These data confirm a major role of these organisms in the composition of the Cu(II)-removing community.

Present address: Helmholtz Center for Infection Research, Department of Cell Biology and Immunology, Inhoffenstraße 7, D-38124 Braunschweig, Germany.

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