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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

Proposed metabolic model for the (A) anaerobic and (B) aerobic phase of EBPR inferred from the proteomic data.Identified proteins catalysing individual reactions are highlighted in green [best MASCOT metagenomic sequence match located on a scaffold source binned as “A. phosphatis”, i.e. strong association with the “A. phosphatis” composite genome], orange [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a strong BLAST hit (>90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. medium strong association with the “A. phosphatis” composite genome], and red [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a weak BLAST hit (<90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. weak association with the “A. phosphatis” composite genome]. Not all intermediate metabolites are shown. Abbreviations: ACC, acetyl-CoA carboxylase; ACD, acyl-CoA dehydrogenase; ACS, acyl-CoA synthetase; AGP, ADP-glucose pyrophosphorylase; ATPsyn, F0F1-type ATP synthase; CSY, citrate synthase; Fba, fructose bisphosphate aldolase; HpI, hydroxypyruvate isomerase; Ily, isocitrate lyase; Mdh, malate dehydrogenase; MalS, malate synthase; NADH, uncharacterised NAD(FAD)-dependent dehydrogenase; PhaA, acetyl-CoA acetyltransferase; PhaC, poly(3-hydroxyalkanoate) synthetase; PhaJ, enoyl-CoA hydratase; PpS, phosphoenolpyruvate synthase; Pst, ABC-type phosphate transport system; SCFA, short chain fatty acids; SuccDH, succinate dehydrogenase; TpI, triosephosphate isomerase.
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pone-0001778-g004: Proposed metabolic model for the (A) anaerobic and (B) aerobic phase of EBPR inferred from the proteomic data.Identified proteins catalysing individual reactions are highlighted in green [best MASCOT metagenomic sequence match located on a scaffold source binned as “A. phosphatis”, i.e. strong association with the “A. phosphatis” composite genome], orange [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a strong BLAST hit (>90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. medium strong association with the “A. phosphatis” composite genome], and red [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a weak BLAST hit (<90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. weak association with the “A. phosphatis” composite genome]. Not all intermediate metabolites are shown. Abbreviations: ACC, acetyl-CoA carboxylase; ACD, acyl-CoA dehydrogenase; ACS, acyl-CoA synthetase; AGP, ADP-glucose pyrophosphorylase; ATPsyn, F0F1-type ATP synthase; CSY, citrate synthase; Fba, fructose bisphosphate aldolase; HpI, hydroxypyruvate isomerase; Ily, isocitrate lyase; Mdh, malate dehydrogenase; MalS, malate synthase; NADH, uncharacterised NAD(FAD)-dependent dehydrogenase; PhaA, acetyl-CoA acetyltransferase; PhaC, poly(3-hydroxyalkanoate) synthetase; PhaJ, enoyl-CoA hydratase; PpS, phosphoenolpyruvate synthase; Pst, ABC-type phosphate transport system; SCFA, short chain fatty acids; SuccDH, succinate dehydrogenase; TpI, triosephosphate isomerase.

Mentions: The detected proteins along with possible functions are listed in Table 2. Several proteins putatively involved in PHA synthesis and fatty acid oxidation were highly expressed. These included acetyl-CoA acetyltransferase (PhaA; spot 41), which is involved in the formation of acetoacetyl-CoA, the first step of PHA synthesis, and poly (3-hydroxyalkanoate) synthetase (PhaC; spots 2, 5, 9, 27 and 28). The activity of PhaC links (R)-3-hydroxyacyl-CoA to an existing PHA molecule, the last step in the formation of PHA (Fig. 4a). These transformations would be an integral part of anaerobic EBPR metabolism.


Metaproteomics provides functional insight into activated sludge wastewater treatment.

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

Proposed metabolic model for the (A) anaerobic and (B) aerobic phase of EBPR inferred from the proteomic data.Identified proteins catalysing individual reactions are highlighted in green [best MASCOT metagenomic sequence match located on a scaffold source binned as “A. phosphatis”, i.e. strong association with the “A. phosphatis” composite genome], orange [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a strong BLAST hit (>90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. medium strong association with the “A. phosphatis” composite genome], and red [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a weak BLAST hit (<90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. weak association with the “A. phosphatis” composite genome]. Not all intermediate metabolites are shown. Abbreviations: ACC, acetyl-CoA carboxylase; ACD, acyl-CoA dehydrogenase; ACS, acyl-CoA synthetase; AGP, ADP-glucose pyrophosphorylase; ATPsyn, F0F1-type ATP synthase; CSY, citrate synthase; Fba, fructose bisphosphate aldolase; HpI, hydroxypyruvate isomerase; Ily, isocitrate lyase; Mdh, malate dehydrogenase; MalS, malate synthase; NADH, uncharacterised NAD(FAD)-dependent dehydrogenase; PhaA, acetyl-CoA acetyltransferase; PhaC, poly(3-hydroxyalkanoate) synthetase; PhaJ, enoyl-CoA hydratase; PpS, phosphoenolpyruvate synthase; Pst, ABC-type phosphate transport system; SCFA, short chain fatty acids; SuccDH, succinate dehydrogenase; TpI, triosephosphate isomerase.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2289847&req=5

pone-0001778-g004: Proposed metabolic model for the (A) anaerobic and (B) aerobic phase of EBPR inferred from the proteomic data.Identified proteins catalysing individual reactions are highlighted in green [best MASCOT metagenomic sequence match located on a scaffold source binned as “A. phosphatis”, i.e. strong association with the “A. phosphatis” composite genome], orange [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a strong BLAST hit (>90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. medium strong association with the “A. phosphatis” composite genome], and red [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a weak BLAST hit (<90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. weak association with the “A. phosphatis” composite genome]. Not all intermediate metabolites are shown. Abbreviations: ACC, acetyl-CoA carboxylase; ACD, acyl-CoA dehydrogenase; ACS, acyl-CoA synthetase; AGP, ADP-glucose pyrophosphorylase; ATPsyn, F0F1-type ATP synthase; CSY, citrate synthase; Fba, fructose bisphosphate aldolase; HpI, hydroxypyruvate isomerase; Ily, isocitrate lyase; Mdh, malate dehydrogenase; MalS, malate synthase; NADH, uncharacterised NAD(FAD)-dependent dehydrogenase; PhaA, acetyl-CoA acetyltransferase; PhaC, poly(3-hydroxyalkanoate) synthetase; PhaJ, enoyl-CoA hydratase; PpS, phosphoenolpyruvate synthase; Pst, ABC-type phosphate transport system; SCFA, short chain fatty acids; SuccDH, succinate dehydrogenase; TpI, triosephosphate isomerase.
Mentions: The detected proteins along with possible functions are listed in Table 2. Several proteins putatively involved in PHA synthesis and fatty acid oxidation were highly expressed. These included acetyl-CoA acetyltransferase (PhaA; spot 41), which is involved in the formation of acetoacetyl-CoA, the first step of PHA synthesis, and poly (3-hydroxyalkanoate) synthetase (PhaC; spots 2, 5, 9, 27 and 28). The activity of PhaC links (R)-3-hydroxyacyl-CoA to an existing PHA molecule, the last step in the formation of PHA (Fig. 4a). These transformations would be an integral part of anaerobic EBPR metabolism.

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