Bioaugmentation of microbial communities in laboratory and pilot scale sequencing batch biofilm reactors using the TOL plasmid.

The aim of this study was to investigate the effectiveness of bioaugmentation and transfer of plasmid pWWO (TOL plasmid) to mixed microbial populations in pilot and laboratory scale sequencing batch biofilm reactors (SBBRs) treating synthetic wastewater containing benzyl alcohol (BA) as a model xenobiotic. The plasmid donor was a Pseudomonas putida strain chromosomally tagged with the gene for the red fluorescent protein carrying a green fluorescent protein labeled TOL plasmid, which confers degradation capacity for several compounds including toluene and BA. In the pilot scale SBBR donor cells were disappeared 84 h after inoculation while transconjugants were not detected at all. In contrast, both donor and transconjugant cells were detected in the laboratory scale reactor where the ratio of transconjugants to donors fluctuated between 1.9 x 10(-1) and 8.9 x 10(-1) during an experimental period of 32 days. BA degradation rate was enhanced after donor inoculation from 0.98 mg BA/min prior to inoculation to 1.9 mg BA/min on the seventeenth day of operation. Survival of a bioaugmented strain, conjugative plasmid transfer and enhanced BA degradation was demonstrated in the laboratory scale SBBR but not in the pilot scale SBBR.

Population changes in a biofilm reactor for phosphorus removal as evidenced by the use of FISH.

Induction of denitrification was investigated for a lab-scale phosphate removing biofilm reactor where oxygen was replaced with nitrate as the electron acceptor. Acetate was used as the carbon source. The original biofilm (acclimatised with oxygen) was taken from a well-established large-scale reactor. During the first run, a decrease in the denitrifying bio-P activity was observed after 1 month following a change in the anaerobic phase length. This was initially interpreted as a shift in the microbial population caused by the changed operation. In the second run, biomass samples were regularly collected and analysed by fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). Concurrently, samples were taken from the original reactor with oxygen as electron acceptor in order to investigate natural microbial fluctuations. A similar decrease in the activity as in the first run was seen after one month, although the phase lengths had not been varied. Hence, the decrease after 1 month in the first and second run should be seen as a start-up phenomenon. FISH could detect a noticeable shift in the microbial population mainly within the first 2 weeks of operation. Almost all bacteria belonging to the alpha subclass disappeared and characteristic clusters of the beta and gamma subclasses were lost. Small clusters of gram-positive bacteria with a high DNA G + C content (GPBHGC) were gradually replaced by filamentous GPBHGC. Most of the bacteria in the denitrifying, phosphate removing biofilm belonged to the beta subclass of Proteobacteria. The applied set of gene probes had been selected based on existing literature on biological phosphate removing organisms and included a recently published probe for a Rhodocyclus-like clone. However, none of the specific probes hybridised to the dominant bacterial groups in the reactors investigated. No noticeable changes were detected in the aerobic bench-scale reactor during this period, indicating that the observed changes in the lab-scale reactor were caused by the changed environment.