In this study, the microbial community structure and carbon source utilisation profile of activated sludge samples collected from full-scale municipal wastewater treatment plants (WWTPs) operated under different conditions were characterised and compared, respectively, using terminal-restriction fragment length polymorphism (T-RFLP) analysis and Biolog assay. Samples were collected from each biological treatment tank of six conventional activated sludge, two anaerobic-oxic, two anaerobic-anoxic-oxic, and one step-aeration processes in eight full-scale WWTPs in Osaka, Japan. Results of the T-RFLP analysis of eubacterial 16S rDNA showed that microbial communities of activated sludge differed greatly among samples, and that they were affected by process-based operational conditions. In contrast, the carbon source utilisation profiles of activated sludge samples were mutually similar, but appeared to be influenced slightly by aerated conditions at each reaction tank. Similar carbon source utilisation profiles among all samples suggest that the activated sludge community possesses functions that are necessary for wastewater treatment even if the phylogenetic composition is different. Different results from the T-RFLP analysis and Biolog assay suggest that the phylogenetic composition of microbial community might not necessarily reflect the wastewater treatment functions of the activated sludge.

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Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.

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