Structure and functional analysis of the microbial community in an aerobic: anaerobic sequencing batch reactor (SBR) with no Phosphorus removal

La Trobe University Faculty of Science, Technology and Engineering Murray Darling Freshwater Research Centre

MDFRC item.

The bacterial community of an aerobic:anaerobic non-P removing SBR biomass fed a mixture of acetateand glucose was analysed using several 16S rRNA based methods. Populations responsible for anaerobicglucose and acetate assimilation were determined with fluorescent in situ hybridization (FISH) in combinationwith microautoradiography (FISH/MAR). At ‘steady state’ this community consisted of α-Proteobacteria(26%) and γ-Proteobacteria (14%), mainly appearing as large cocci in tetrads (i.e. typical ‘GBacteria’).Large numbers of low G+C bacteria (22%), and high G+C Gram-positive bacteria (29%) seenas small cocci in clusters or in sheets were also detected after FISH. DGGE fingerprinting of PCR amplified16S rDNA fragments and subsequent cloning and sequencing of several of the major bands led tothe identification of some of these populations. They included an organism 98% similar in its 16S rRNAsequence to Micropruina glycogenica, and ca. 76% of the high G+C bacteria responded to a probe MIC184, designed against it. The rest responded to the KSB 531 probe designed against a high G+C clone sequence,sbr-gs28 reported in other similar systems. FISH analyses showed that both these high G+C populationswere almost totally dominated by small clustered cocci. Only ca. 2% of cells were β-Proteobacteria.None of the α- and γ-Proteobacterial ‘G-bacteria’ responded to FISH probes designed for the ‘GBacteria’Amaricoccus spp. or Defluvicoccus vanus. FISH/MAR revealed that not all the α-Proteobacterial‘G-Bacteria’ could take up acetate or glucose anaerobically. Almost all of the γ-Proteobacterial ‘G-Bacteria’assimilated acetate anaerobically but not glucose, the low G+C clustered cocci only took up glucose,whereas the high G+C bacteria including M. glycogenica and the sbr-gs28 clone assimilated both acetateand glucose. All bacteria other than the low G+C small cocci and a few of the α-Proteobacteria accumulatedPHB. The low G+C bacteria showing anaerobic glucose assimilation ability were considered responsiblefor the lactic acid produced anaerobically by this SBR biomass, and M. glycogenica for its highglycogen content.