Diversity and molecular identification of polyhydroxyalkanoate synthases in a mixed culture performing enhanced biological phosphorus removal

Enhanced biological phosphorus removal is a modified activated sludge process commonly used to remove inorganic phosphates from wastewaters. Although EBPR is an operationally well-established process, many full-scale EBPR plants fail periodically. Knowledge of the types of microorganisms responsible for EBPR and the mechanisms involved should provide ways of increasing the reliability of EBPR in practice. Therefore, the overall goal of this research was to improve the understanding of the microbial ecology and ecophysiology of EBPR. The research was performed to bridge the gap between microbial community composition and function and process stability through studying the key genes, namely polyhydroxyalkanoates (PHA) synthases, putatively involved in the carbon transformations during EBPR.; The diversity of putative PHA synthase (phaC) genes in an EBPR community highly enriched for polyphosphate accumulating organisms (PAOs) was investigated using a culture-independent PCR based clone library technique. A library of phaC fragments was generated by PCR on the EBPR metagenomic DNA with degenerate primers targeting phaC s from beta-Proteobacteria. Since the dominant PAO in EBPR systems was found to be closely related to R. tenuis, an internal fragment of R. tenuis phaC was also amplified, cloned, and sequenced. Nine distinct sequence types (EBPR1-9) were obtained. Eight of the sequences were homologous to PHA synthases from beta-Proteobacteria, and one was homologous to the phaC1 of Class II PHA synthases from Pseudomonas. One of the sequence types (EBPR9) clustered closely to the PhaC from R. tenuis on a PhaC phylogenetic tree suggesting that this sequence might belong to a Rhodocyclus related PAO.; Employing inverse PCR to obtain flanking regions of the phaC genes retrieved, a 4.77 kb genomic DNA fragment containing all three of PHA biosynthetic genes from the organism bearing EBPR4 type phaC was obtained. This genomic DNA fragment from an uncultured microorganism in EBPR contained a beta-ketothiolase homolog (phaA ) and an acetoacetyl-CoA reductase (phaB) homolog immediately downstream and a gene of unknown function homologous to yfiH upstream of the phaC on the same strand. The EBPR4 PHA operon was subsequently cloned into an expression vector and phaCABEBPR4 genes were constitutively expressed in the heterologous host E. coli. Using viable-colony staining with Nile Red, it was confirmed that all the genes in EBPR4 PHA operon were functionally active since they conferred non-PHA accumulating E. coli with the ability to accumulate PHAs. Furthermore, to determine the substrate specificity of the synthase, this operon was cloned into a broad host range vector, and expressed in the PHA negative mutant of W. eutropha. It was found that phaCEBPR4 had broader substrate range and could incorporate C6-hydroxy fatty acids (hydroxyhexanoate) into the polymer in addition to C3-C5 monomeric units.; The PHA operon of Rhodocyclus tenuis, an anoxygenic phototrophic non-sulfur bacterium, was also revealed using PCR based molecular techniques. A 6.7-kb genomic DNA fragment harboring phaC also contained a transmembrane protein upstream and an acetoacetyl-CoA reductase (phaB), a PHA regulator protein (phaR), a radical SAM protein (MiaB) and a FAD/FMN containing dehydrogenase downstream. Another PHA synthase homolog was identified on R. tenuis genome in the vicinity of enzymes putatively involved in amino acid catabolism such as branched amino acids (leucine, isoleucine, and valine) and phenylalanine.