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Metaproteomics provides functional insight into activated sludge wastewater treatment.

Wilmes P, Wexler M, Bond PL - PLoS ONE (2008)

Bottom Line: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P.Several proteins involved in cellular stress response were detected.Finally, the results are discussed in relation to current EBPR metabolic models.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental Sciences, University of East Anglia, Norwich, United Kingdom.

ABSTRACT

Background: Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR).

Methodology/principal findings: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism "Candidatus Accumulibacter phosphatis". When EBPR failed, the sludge was dominated by tetrad-forming alpha-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from "Candidatus Accumulibacter phosphatis" and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid beta oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected.

Conclusions/significance: Importantly, this study provides direct evidence linking the metabolic activities of "Accumulibacter" to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.

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Related in: MedlinePlus

Representative FISH micrographs of the activated sludges analysed in this study.(A) EBPR28 sludge, (B) EBPR42 sludge, (C) EBPR55 sludge and (D) nEBPR70 sludge. Cells detected with probe EUBMIX only are green (A, B, C and D). Cells detected with both EUBMIX and PAO651 probes (A, B and C) and cells detected with both EUBMIX and ALF1b probes (D) are yellow-orange. Highlighted area in pane d corresponds to magnified region hybridised only with the ALF1b probe in the top right hand corner. Images taken under the different excitation wavelengths for CY3 and FITC were combined using Adobe Photoshop. Cells were observed under x 630 magnification, bars = 10 µm.
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pone-0001778-g001: Representative FISH micrographs of the activated sludges analysed in this study.(A) EBPR28 sludge, (B) EBPR42 sludge, (C) EBPR55 sludge and (D) nEBPR70 sludge. Cells detected with probe EUBMIX only are green (A, B, C and D). Cells detected with both EUBMIX and PAO651 probes (A, B and C) and cells detected with both EUBMIX and ALF1b probes (D) are yellow-orange. Highlighted area in pane d corresponds to magnified region hybridised only with the ALF1b probe in the top right hand corner. Images taken under the different excitation wavelengths for CY3 and FITC were combined using Adobe Photoshop. Cells were observed under x 630 magnification, bars = 10 µm.

Mentions: The sludge microbial communities were analysed by fluorescent in situ hybridisation (FISH). Most of the cells that stained positively with DAPI were identified as bacteria with the EUBMIX probes (98 %). The EBPR sludges, at each P increase level, were all dominated by organisms forming distinct coccoid clusters of cells (Fig. 1A,B&C). The EBPR28, EBPR42 and EBPR55 sludges gave positive signals for the β-Proteobacteria specific probe (63, 68 and 72 % of EUBMIX binding cells, respectively; Table S1) and the “A. phosphatis” specific probe PAO651 (61, 67 and 69 % of EUBMIX binding cells, respectively; Table S1). These results were similar to those found for sludges with similar P removal performances and biomass P contents [5], [18], [24], [25]. The number of cells belonging to both β-Proteobacteria and “A. phosphatis” were found to be significantly different in the three EBPR sludges (ANOVA, P<0.05) and, consequently, provide a causative link between the dominance of the Rhodocyclus-type PAO and P removal performance. High abundances of α-Proteobacteria were also observed in the EBPR28, EBPR42 and EBPR55 sludges, at 34, 29 and 25 %, respectively (Table S1). These consisted mainly of tetrad-arranged coccoid cells, as described previously [25]. Thus, the numbers of Rhodocyclus-type PAOs increased as the tetrad-forming α-Proteobacteria decreased. A possible explanation is that these two groups of organisms are in direct competition with one another as suggested previously [15].


Metaproteomics provides functional insight into activated sludge wastewater treatment.

Wilmes P, Wexler M, Bond PL - PLoS ONE (2008)

Representative FISH micrographs of the activated sludges analysed in this study.(A) EBPR28 sludge, (B) EBPR42 sludge, (C) EBPR55 sludge and (D) nEBPR70 sludge. Cells detected with probe EUBMIX only are green (A, B, C and D). Cells detected with both EUBMIX and PAO651 probes (A, B and C) and cells detected with both EUBMIX and ALF1b probes (D) are yellow-orange. Highlighted area in pane d corresponds to magnified region hybridised only with the ALF1b probe in the top right hand corner. Images taken under the different excitation wavelengths for CY3 and FITC were combined using Adobe Photoshop. Cells were observed under x 630 magnification, bars = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2289847&req=5

pone-0001778-g001: Representative FISH micrographs of the activated sludges analysed in this study.(A) EBPR28 sludge, (B) EBPR42 sludge, (C) EBPR55 sludge and (D) nEBPR70 sludge. Cells detected with probe EUBMIX only are green (A, B, C and D). Cells detected with both EUBMIX and PAO651 probes (A, B and C) and cells detected with both EUBMIX and ALF1b probes (D) are yellow-orange. Highlighted area in pane d corresponds to magnified region hybridised only with the ALF1b probe in the top right hand corner. Images taken under the different excitation wavelengths for CY3 and FITC were combined using Adobe Photoshop. Cells were observed under x 630 magnification, bars = 10 µm.
Mentions: The sludge microbial communities were analysed by fluorescent in situ hybridisation (FISH). Most of the cells that stained positively with DAPI were identified as bacteria with the EUBMIX probes (98 %). The EBPR sludges, at each P increase level, were all dominated by organisms forming distinct coccoid clusters of cells (Fig. 1A,B&C). The EBPR28, EBPR42 and EBPR55 sludges gave positive signals for the β-Proteobacteria specific probe (63, 68 and 72 % of EUBMIX binding cells, respectively; Table S1) and the “A. phosphatis” specific probe PAO651 (61, 67 and 69 % of EUBMIX binding cells, respectively; Table S1). These results were similar to those found for sludges with similar P removal performances and biomass P contents [5], [18], [24], [25]. The number of cells belonging to both β-Proteobacteria and “A. phosphatis” were found to be significantly different in the three EBPR sludges (ANOVA, P<0.05) and, consequently, provide a causative link between the dominance of the Rhodocyclus-type PAO and P removal performance. High abundances of α-Proteobacteria were also observed in the EBPR28, EBPR42 and EBPR55 sludges, at 34, 29 and 25 %, respectively (Table S1). These consisted mainly of tetrad-arranged coccoid cells, as described previously [25]. Thus, the numbers of Rhodocyclus-type PAOs increased as the tetrad-forming α-Proteobacteria decreased. A possible explanation is that these two groups of organisms are in direct competition with one another as suggested previously [15].

Bottom Line: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P.Several proteins involved in cellular stress response were detected.Finally, the results are discussed in relation to current EBPR metabolic models.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental Sciences, University of East Anglia, Norwich, United Kingdom.

ABSTRACT

Background: Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR).

Methodology/principal findings: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism "Candidatus Accumulibacter phosphatis". When EBPR failed, the sludge was dominated by tetrad-forming alpha-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from "Candidatus Accumulibacter phosphatis" and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid beta oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected.

Conclusions/significance: Importantly, this study provides direct evidence linking the metabolic activities of "Accumulibacter" to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.

Show MeSH
Related in: MedlinePlus