The microbial ecology of Acidovorax temperans in activated sludge

Abstract

Acidovorax species have been identified as common constituents of the microbial community in activated sludge wastewater treatment systems and the strain Acidovorax temperans CB2 has been proposed as a model organism for studying microbial processes in AS. To support this, the distribution, abundance and diversity of A. temperans was investigated using a polyphasic approach to determine the functional role of this putatively representative member of the AS community. Three methods targeted toward the 16S rRNA gene were assessed for their utility in monitoring the occurrence and abundance of Acidovorax and the family Comamonadaceae; fluorescence in situ hybridization, real-time PCR and targeted cleavage of 16S hybrids with oligonucleotides and RNase H. These methods gave significantly different results for the abundance of Acidovorax based on a preliminary assessment of six samples (FISH 1.5%±0.4; real-time PCR 2.8%±1.1; oligonucleotides and RNase H 1.0%±0.7) but all indicated that Acidovorax was consistently present in AS WWTPs at levels between 1-5% of the total community. Based on the assessment of methods, FISH was chosen to monitor the distribution and abundance of members of the genus Acidovorax and closely related species within the Comamonadaceae in three full-scale NZ activated sludge wastewater treatment plants. The results from this year-long monitoring study confirm that Acidovorax is a low-abundance (0.5-6% of total bacterial cell biovolume; average 1%±0.5) and active member of the microbial community, occurring primarily as single cells or pairs and occasionally as microcolonies. Acidovorax typically made up a small fraction of the family Comamonadaceae which were found in abundances ranging from 4-45% of biovolume (average 17%±6). A simultaneous 16S rRNA gene clone library survey gave significantly different values for both groups in all samples (0-4.4% Acidovorax based on number of clone sequences; 1-16% Comamonadaceae). While Acidovorax cells were observed primarily on the periphery of floes and in the planktonic phase of AS mixed liquor it was observed that their abundance was higher in return activated sludge and therefore this organism may be retained in AS systems through the recycling of settled solids. A culture collection of 11 A. temperans strains were isolated from 3 AS WWTPs over a three year period. Though all strains shared ^39% identity 16S rRNA gene identity, phenotypic characterisation indicated significant variability in functionally relevant characteristics including capacity for aggregation, biofilm formation, motility and carbon-source utilization, with most strains actively utilizing nitrogenous substrates including amino acids and carboxylic acids rather than carbohydrates . The underlying genetic causes of this diversity were investigated by comparing draft genome sequences of six strains with the previously completed and annotated wastewater strain CB2 Hp and publicly available genome sequences. A total of 1973 genes (representing 44% of the total) were identified in all strains, and putatively represent the core genome of this species. 2484 genes were shared by two or more, but not all, strains while the number of unique genes in each strain ranged from 122 to 827. Comparative genome analysis centred on three functionally relevant sets of genes that code for flagella biosynthesis, type IV pili and nitrogen metabolism. In contrast to the type strain for A. temperans which is motile, the majority of AS strains had lost all the genes for flagella biosynthesis. However all strains contained genes coding for synthesis of type IV pili although some genetic diversity was observed in coding sequence for the major pilin subunit. The functional relevance of this was not determined. All strains also contained conserved genes for denitrification of nitrates and nitroalkanes to N2 gas. Collectively these results confirm that Acidovorax is a consistent member of the AS community where it occurs as small clusters of suspended or loosely attached cells. Though the function of A. temperans in AS remains unclear, carbon utilization patterns indicate that it is well adapted to proteinaceous waste streams, while potential for denitrification may enhance retention in AS systems configured for biological N-removal. The genetic and phenotypic diversity of strains with identical 16S sequences raises the question of whether Acidovorax represents a functionally coherent group within the context of AS. However variation in capacity for aggregation and biofilm support this use of this organism as a model to investigate the mechanisms regulating these functionally relevant characteristics. Further comparison of genome sequences is expected to uncover whether this organism contains novel genes that explain the functional diversity displayed between isolates in aggregation and biofilm formation.