In situ identification of polyphosphate‐ and polyhydroxyalkanoate‐accumulating traits for microbial populations in a biological phosphorus removal process

Abstract

Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

“…The research described in this paper was motivated by several reported probing inconsistencies where many Competibacter (a member of the Gammaproteobacteria) cells did not bind GAM42a (for Gammaproteobacteria) (Crocetti et al, 2002;Kong et al, 2002;Liu et al, 2001;Nielsen et al, 1999) and many Competibacter cells did bind BET42a (for Betaproteobacteria) (Crocetti et al, 2002;Kong et al, 2002 It is known that G-T and G-A hybrids are stronger than many other mismatches such as A-A, T-T, C-T or C-A (Lathe, 1990). The addition of an unlabelled competitor probe is required to differentiate the relatively weak A-A or T-T mismatch at position 1033 between GAM42a-Betaproteobacteria cells and BET42a-Gammaproteobacteria cells, respectively.…”

“…So therefore, the absence of a competitor probe in FISH experiments studying organisms like Competibacter with the previously unknown G at position 1033 in their 23S rRNA, could readily lead to hybridization between them and GAM42a (G-A) or BET42a (G-T). The Competibacter cells with the perfect GAM42a probe target would bind GAM42a, but the Competibacter cells with G at position 1033 were likely to be responsible for the reported probing inconsistencies (Crocetti et al, 2002;Kong et al, 2002;Liu et al, 2001;Nielsen et al, 1999).…”

“…The taxon 'Candidatus Competibacter phosphatis' (henceforth called Competibacter) was recently identified and linked to the in situ GAO phenotype (Crocetti et al, 2002) and numerous PGS probes targeting Competibacter have been reported (Crocetti et al, 2002;Kong et al, 2002;Nielsen et al, 1999). Several researchers have noted that some cells hybridizing these Competibacter-specific FISH probes did not bind GAM42a, despite the phylogenetic placement of Competibacter in the Gammaproteobacteria subphylum (Crocetti et al, 2002;Kong et al, 2002;Liu et al, 2001;Nielsen et al, 1999). Nielsen et al (1999) suggested this was related to the specificity of GAM42a which was originally designed based on a limited number of 23S rRNA sequences.…”

Anaerobic and aerobic metabolism of glycogen-accumulating organisms selected with propionate as the sole carbon source

“…Fluorescence in situ hybridization (FISH) has shown Accumulibacter to be an abundant organism in laboratory-scale EBPR cultures with various carbon sources (Levantesi et al, 2002;Liu et al, 2001;Oehmen et al, 2004Oehmen et al, , 2005bOnda et al, 2002;Pijuan et al, 2004;Zeng et al, 2003a). Accumulibacter has also been reported in full-scale EBPR systems (Saunders et al, 2003;Zilles et al, 2002).…”

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

“…The shortage of information is due, in part, to the lack of reliable techniques for analyzing microbial community structure and diversities in environmental samples. Recently, 16S rRNA/DNA-based molecular methods with high degrees of precision and specificity have been widely used for environmental microbial studies (Wagner et al, 1994;Bond et al, 1995;Liu et al, 2001). The full-length 16S rDNA sequence contains variable and conserved regions, which accurately reflect the phylogenetic position of the corresponding 16S rDNA sequences and the total degree of diversity (Lane et al, 1985).…”