Microbial population analysis of nutrient removal-related organisms in membrane bioreactors.

Membrane bioreactors (MBR) are an important and increasingly implemented wastewater treatment technology, which are operated at low food to microorganism ratios (F/M) and retain slow-growing organisms. Enhanced biological phosphorus removal (EBPR)-related organisms grow slower than ordinary heterotrophs, but have never been studied in detail in MBRs. This study presents a comprehensive analysis of the microorganisms involved in EBPR in pilot- and full-scale MBRs, using fluorescence in situ hybridization (FISH), as well as an overall assessment of other relevant microbial groups. The results showed that polyphosphate accumulating organisms (PAOs) were present at similar levels in all studied MBRs (10% ± 6%), even those without a defined anaerobic zone. Glycogen accumulating organisms were also detected, although rarely. The FISH results correlated well with the observed P removal performance of each plant. The results from this study suggest that a defined anaerobic zone is not necessarily required for putative PAO growth in MBRs, since polyphosphate storage may provide a selective advantage in fulfilling cell maintenance requirements in substrate-limited conditions (low F/M).

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting.

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13- PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome's sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria.

Microbial characterization of mercury-reducing mixed cultures enriched with different carbon sources.

The use of mixed microbial cultures enriched for biological mercury removal is explored in this paper, focusing on the ecological shifts occurring throughout acclimatization to mercury and on the long-term stability of four microbial enrichments. The 16S rRNA genetic profiles obtained by denaturing gradient gel electrophoresis (DGGE) revealed that the glucose and ethanol cultures had similar profiles, whereas the acetate cultures diverged into a totally dissimilar cluster. Quantification of the merA gene copies in each enrichment showed higher values for the glucose culture, followed by the ethanol and then the acetate cultures, which was consistent with the mercury removal performance throughout the study. Isolates were obtained from the four cultures and analyzed with respect to their genetic (16S rRNA) and functional (merA) phylogenies in order to identify mercury-resistant species enriched with different carbon sources. All mercury-resistant isolates obtained from the glucose and ethanol cultures belonged to the Gammaproteobacteria, whereas acetate cultures also contained members of other phyla, with differences in merA sequences. Higher phylogenetic than functional diversity of the isolates, together with increasing merA copies even after culture stabilisation, highlight the role of horizontal gene transfer in the acclimatization process.