Monitoring the development of anaerobic biofilms using fluorescent in situ hybridization and confocal laser scanning microscopy

Abstract

In this study we investigated the development of anaerobic biofilm using a laboratory reactor. We were especially interested in comparing the organization of anaerobic cells (particularly those that are very common in domestic sewage sludge) in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescent in situ hybridization (FISH) with domain and group specific probes directed against 16S ribosomal RNA were used to quantify microbial composition in the biofilm. FISH and confocal laser scanning microscopy (CLSM) were used to elucidate spatial distribution of microbes in the biofilms. Two experiments were carried out, one with pure methanogenic organisms and the other with a microbial anaerobic consortium. The pure methanogen cultures, Methanobacterium formicicum (DSM 1535); Methanosaeta concilli (DSM 3671) and Methanosarcina barkeri (DSM 800) were used to seed the modified Robbins Device (MRD) to allow the development of biofilms on polypropylene and glass surfaces during the 9-days experiment. The results showed that all the three species were colonizing both surfaces after two and nine days of experimental period. In another experiment, with polypropylene coupons only, MRD was seeded with a microbial anaerobic consortium and biofilm formation was studied during 11 days. At the end of this period, the biofilms generated were of uneven thickness with areas of minimal or no surface coverage and areas where the biofilm attained a thickness of 7.0 to 9.0 μm as revealed by CLSM. The results showed that the modified Robbins Device together with the fluorescent in situ hybridization and confocal laser scanning microscopy are suitable tools to study anaerobic biofilm development in different kinds of support materials.