No abstract

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.

Natural or engineered microbial populations often show variations over time. These variations may be due to environmental fluctuations or intrinsic factors. Thus, studying the dynamics of microbial diversity for different communities living in a spatially homogeneous landscape is of interest. As a model ecosystem, nitrifying biofilm communities were grown in a two litre inverse turbulent bed reactor (ITBR) containing an estimated 200 million small particles (about 150 μm in diameter). Each particulate biofilm is considered as a distinct community growing in the neighborhood of other similar particles, in a homogeneous and well-controlled environmental context. A molecular approach was adopted to test how microbial community structures might evolve: either in synchrony, converging or diverging. The shape of biofilm was observed by microscopy for each particle. The biomass content was evaluated by quantitative PCR and showed similar values for each particle. The microbial community structure was evaluated by Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP) fingerprinting and showed extraordinary homogeneity between particles, even though transitory community structures were observed when reactor operating conditions were modified. This homogeneity was observed for the Bacteria primer set but, more interestingly, was also observed when minor non-nitrifying bacteria making up the biofilm, representing about 5% and 10% of total cells, were targeted.