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Anaerobic central metabolic pathways active during polyhydroxyalkanoate production in uncultured cluster 1 Defluviicoccus enriched in activated sludge communities

Luke C. Burow , Amanda N. Mabbett , Luis Borrás , Linda L. Blackall
DOI: http://dx.doi.org/10.1111/j.1574-6968.2009.01695.x 79-84 First published online: 1 September 2009


A glycogen nonpolyphosphate-accumulating organism (GAO) enrichment culture dominated by the Alphaproteobacteria cluster 1 Defluviicoccus was investigated to determine the metabolic pathways involved in the anaerobic formation of polyhydroxyalkanoates, carbon storage polymers important for the proliferation of microorganisms in enhanced biological phosphorus removal processes. FISH–microautoradiography and post-FISH fluorescent chemical staining confirmed acetate assimilation as polyhydroxyalkanoates in cluster 1 Defluviicoccus under anaerobic conditions. Chemical inhibition of glycolysis using iodoacetate, and of isocitrate lyase by 3-nitropropionate and itaconate, indicated that carbon is likely to be channelled through both glycolysis and the glyoxylate cycle in cluster 1 Defluviicoccus. The effect of metabolic inhibitors of aconitase (monofluoroacetate) and succinate dehydrogenase (malonate) suggested that aconitase, but not succinate dehydrogenase, was active, providing further support for the role of the glyoxylate cycle in these GAOs. Metabolic inhibition of fumarate reductase using oxantel decreased polyhydroxyalkanoate production. This indicated reduction of fumarate to succinate and the operation of the reductive branch of the tricarboxylic acid cycle, which is possibly important in the production of the polyhydroxyvalerate component of polyhydroxyalkanoates observed in cluster 1 Defluviicoccus enrichment cultures. These findings were integrated with previous metabolic models for GAOs and enabled an anaerobic central metabolic pathway model for polyhydroxyalkanoate formation in cluster 1 Defluviicoccus to be proposed.

  • wastewater
  • FISH
  • microautoradiography
  • enhanced biological phosphorus removal
  • glycogen (nonpolyphosphate)-accumulating organisms


Microorganisms capable of uptake and assimilation of volatile fatty acids (VFA) as polyhydroxyalkanoates in the absence of external electron acceptors (anaerobic conditions) dominate in the anaerobic : aerobic enhanced biological phosphorus removal (EBPR) wastewater treatment process. Failure of the EBPR process has been attributed to different factors including overloading of phosphorus (Pi), insufficient VFA and the overgrowth of the microbial community by glycogen nonpolyphosphate-accumulating organisms (GAOs) (Seviour et al., 2003; Oehmen et al., 2007). GAOs relevant to EBPR are not available in pure culture and so metabolic models that predict their physiology are based on enrichment cultures (Oehmen et al., 2007). Hypothesized pathways of anaerobic metabolism in GAOs have all inferred that glycogen catabolism provides the reducing power for polyhydroxyalkanoate formation. However, the possibility of reducing power generation through the tricarboxylic acid (TCA) or glyoxylate cycles has been excluded (Filipe et al., 2001) or included (Schuler & Jenkins, 2003) in different stoichiometric models of anaerobic GAO metabolism and their exact contribution is currently unclear (Oehmen et al., 2007).

A combination of in situ techniques were used to characterize the microbial community structure of a lab-scale bioreactor, demonstrating the GAO phenotype and to determine carbon transformations in the enriched GAOs, which were Alphaproteobacteria cluster 1 Defluviicoccus. Determinations of the effects of inhibitors on acetate uptake and polyhydroxyalkanoate formation were carried out to obtain experimental physiological data to identify the operation of pathways of anaerobic polyhydroxyalkanoate formation in cluster 1 Defluviicoccus hypothesized from different metabolic models previously proposed for GAOs.

Materials and methods

Enrichment of GAOs

GAOs were enriched from a seed sludge obtained from a local full-scale biological nutrient removal wastewater treatment plant (Qld, Australia). An 8-L bioreactor was operated to enrich for GAOs by limiting the amount of Pi added under anaerobic conditions. Acetate (200 mg COD L−1 or 3.15 mM) was the only VFA added, which was taken up within 45 min under anaerobic conditions. The anaerobic period was 120 min, the aerobic period was 150 min and the operating pH was 7.0 (±0.2). Further details of the bioreactor setup and operation can be found elsewhere (Dai et al., 2007).

Acetate uptake assays

Harvested biomass samples taken from the bioreactor at the end of the aerobic phase were resuspended in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered salts medium (pH 7.5) at a final concentration of 250 μg mL−1 of total protein. Protein concentration was determined using the BCA protein assay kit using bovine serum albumin as a standard (Pierce Biotechnology). The salts medium and anaerobic acetate uptake assays used in this study are as described previously (Burow et al., 2008a, b). Briefly, acetate uptake assays were performed at pH 7.5 (±0.1) under strictly anaerobic conditions maintained with either oxygen-free helium or oxygen-free nitrogen gas sparging and different inhibitors (Table 1). Batch tests were performed by the addition of inhibitors before adding 2 mM acetate to the HEPES-buffered salts medium, and samples were obtained and uptake rates were calculated and subjected to statistical analysis using Student's t-test. All batch tests were performed in triplicate.

View this table:

Effect of inhibitors on anaerobic acetate uptake and polyhydroxyalkanoate production in cluster 1 Defluviicoccus

Chemical analyses

Biomass samples obtained at T=1, 10, 20 and 30 min during anaerobic uptake assays were filtered using a sterile 0.22-μm filter (Millipore) and stored at 4 or −20 °C for subsequent chemical analyses. Acetate was analysed by HPLC (Shimadzu) and Pi was determined using a flow injection analyser (Lachat Instruments). Glycogen was measured by lyophilization and digestion of biomass with HCl and the resulting supernatant liquid obtained after HCl digestion was analysed for glucose by HPLC (Bond et al., 1999). Polyhydroxyalkanoate was determined in the lyophilized biomass, acidified in a methanol solution using GC (Perkin Elmer Autosystem) (Oehmen et al., 2005).

FISH and quantitative FISH (qFISH)

FISH was carried out on fixed samples of biomass as described by Amann (1995). FISH probes used in this study were EUBMIX for Bacteria (Amann et al., 1990; Daims et al., 1999), PAOMIX (Crocetti et al., 2000) for the Betaproteobacteria PAO ‘Candidatus Accumulibacter phosphatis’ (from now called Accumulibacter), Actino-221 (Actino-221, c1Actino-221 and c2Actino-221) for the Actinobacteria Tetrasphaera-related tetrads (Kong et al., 2005), Actino-658 (Actino-658, c1Actino-658 and c2Actino-658) for the Actinobacteria Tetrasphaera-related rods (Kong et al., 2005), DF1MIX (TFO_DF218 plus TFO_DF618) for the Alphaproteobacteria cluster 1 Defluviicoccus GAOs (Wong et al., 2004), DF2MIX (DF988, DF1020 plus helper probes H966 and H1038) for cluster 2 Defluviicoccus GAOs (Meyer et al., 2006) and GAOMIX (equal amounts of GAOQ989 and GB_G2; Crocetti et al., 2002; Kong et al., 2002) for the GAO Competibacter. FISH quantification was carried out by digital image analysis (imagej V1.35k, http://rsb.info.nih.gov/ij/) of the biomass hybridized with the Cy3-labelled specific probes and Cy5-labelled EUBMIX probes using qFISH methods described previously (Burow et al., 2007).

FISH–microautoradiography (FISH–MAR) and post-FISH chemical staining

Combined FISH and MAR was carried out as described by Lee et al. (1999) on biomass samples enriched in cluster 1 Defluviicoccus using FISH probes (DF1MIX and EUBMIX) and monolabelled [14C]-acetate to determine the uptake of acetate in these microorganisms in the absence of external electron acceptors (anaerobic conditions). A mixture of acetate (final concentration, 2 mM), including radiolabelled acetate (20 μCi mL−1) and unlabelled acetate, was incubated with the biomass for 2 h under anaerobic conditions before FISH–MAR analysis. Reporting of acetate uptake in Defluviicoccus cells was carried out according to the criteria described in Burow et al. (2007). Briefly, at least 100 microautoradiography-positive or microautoradiography-negative cells were counted. Uptake was reported as positive if >90% of cells were visualized with silver grain formation (indicating radioactive labelling). Assimilation of acetate into polyhydroxyalkanoates was determined in cluster 1 Defluviicoccus by FISH (with DF1MIX and EUBMIX) and post-FISH chemical polyhydroxyalkanoate staining (Crocetti et al., 2000) using Nile Blue A (Ostle & Holt, 1982).

Results and discussion

GAO bioreactor

A bioreactor was operated under EBPR conditions, but with limited Pi, so that the biomass demonstrated the GAO phenotype (Dai et al., 2007). This was called the GAO bioreactor, and each time the biomass was harvested for uptake assays (n=12), the microbial community structure was determined using qFISH. The biomass was dominated by cluster 1 Defluviicoccus (targeted by DF1MIX) that represented between 79(±6)% and 83(±3)% of all Bacteria (Figs 1 and 2). This range is statistically insignificant (Student's t-test; P=0.13), supporting the conclusion that the dominant population was similar for each uptake assay. This microbial community was termed the cluster 1 Defluviicoccus enrichment. Competibacter were typically up to 6% of the Bacteria and cluster 2 Defluviicoccus were always ≤1% of the Bacteria in the GAO bioreactor. Accumulibacter and the putative polyphosphate-accumulating organisms (PAOs) in the Actinobacteria (Tetrasphaera-related spp.) were below the detection limit.


FISH–MAR analysis of cluster 1 Defluviicoccus enriched biomass. (a) Micrograph of cluster 1 Defluviicoccus cells [hybridized with both EUBMIX probes (blue) and DF1MIX probes (red)] in magenta. (b) Micrograph of cluster 1 Defluviicoccus taking up radiolabelled acetate (microautoradiography positive) under anaerobic conditions. Scale bar=10 μm.


Post-FISH Nile Blue A staining of cluster 1 Defluviicoccus enriched biomass. (a, c) Magenta cells are cluster 1 Defluviicoccus [hybridized with both EUBMIX probes (blue) and DF1MIX probes (red)]. (b, d) Overlays of phase-contrast (black and white) images and Nile Blue A-stained polyhydroxyalkanoates in red. The same fields of view are shown in the left and right panels. (a, b) Biomass sampled at the end of the aerobic period before anaerobic incubation with acetate. (c, d) Biomass samples after 60 min anaerobic incubation with 2 mM acetate. Scale bar=10 μm.

Although the biomass sampled from the GAO bioreactor consisted of a mixed microbial community, conclusions from the use of metabolic inhibitors were made with regard to cluster 1 Defluviicoccus central metabolism. This was possible as they were by far the dominant microorganisms within the culture and thus these microorganisms are likely to be responsible for the majority of carbon transformations observed in the GAO bioreactor. FISH–MAR and post-FISH chemical staining showed that comparatively very few other microorganisms (<5%) assimilated acetate and/or produced polyhydroxyalkanoates.

Anaerobic acetate uptake and polyhydroxyalkanoate production in the cluster 1 Defluviicoccus enrichment

Radiolabelled acetate was taken up by cluster 1 Defluviicoccus (Fig. 1) and assimilated as polyhydroxyalkanoates (Fig. 2) under anaerobic conditions. These organisms had an average uptake rate of 106±13 nmol acetate min−1 mg−1 protein, which was linear over the initial 30 min. Polyhydroxyalkanoate was produced in the hydroxybutyrate : hydroxyvalerate ratio of 78 : 22 as determined by GC. No change in the Pi concentration under anaerobic conditions was observed (data not shown).

Anaerobic carbohydrate metabolism in the cluster 1 Defluviicoccus enrichment

Glycogen decreased in the cluster 1 Defluviicoccus enrichment from 4.9±0.01 carbon millimole (C-mmol) glucose g−1 dry cell weight before the addition of acetate to 1.8±0.09 C-mmol glucose g−1 dry cell weight after 60 min anaerobic incubation with acetate (2 mM). Under the same incubation conditions and in the presence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor iodoacetate (1 mM) (Even et al., 1999), glycogen did not decrease, but remained at 5.0±0.30 C-mmol glucose g−1 dry cell weight. Anaerobic consumption of glycogen (stored glucose) during acetate assimilation to polyhydroxyalkanoates and the cessation of glycogen consumption in the presence of an inhibitor of GAPDH indicate that cluster 1 Defluviicoccus catabolize glycogen anaerobically via glycolysis, which is consistent with the predicted stoichiometric models for GAOs (Filipe et al., 2001; Oehmen et al., 2007).

Anaerobic assimilation of acetate as polyhydroxyalkanoates and TCA pathway metabolism in the cluster 1 Defluviicoccus enrichment

Uptake of acetate and polyhydroxyalkanoate assimilation in the cluster 1 Defluviicoccus enrichment was investigated in the presence of the aconitase inhibitor, monofluoroacetate (Lauble et al., 1996), which effectively inhibits carbon flux through the TCA and glyoxylate cycles. The acetate uptake rate of the cluster 1 Defluviicoccus enrichment was strongly reduced in the presence of monofluoroacetate compared with control (uninhibited) incubations (Table 1). Incubation with 1 mM monofluoroacetate also reduced polyhydroxyalkanoate production compared with control incubations (Table 1). The isocitrate lyase inhibitors, 3-nitropropionate (Munoz-Elias & McKinney, 2005) and itaconate (Hillier & Charnetzky, 1981), significantly inhibited acetate uptake (Table 1) in cluster 1 Defluviicoccus. Additionally, polyhydroxyalkanoate production was significantly reduced in these organisms in the presence of itaconate compared with control incubations (Table 1).

The glyoxylate cycle activity can be repressed in enteric microorganisms that coutilize acetate and glucose (Clark & Cronan, 1996), whereas in coryneforms its activity is essential for optimal utilization of these substrates (Wendisch et al., 2000). Anaerobic activity of the glyoxylate cycle in cluster 1 Defluviicoccus (Fig. 3) is inferred from the significant reduction in the acetate uptake rate and polyhydroxyalkanoate production in the presence of inhibitors of aconitase (monofluoroacetate) and isocitrate lyase (3-nitropropionate and itaconate). The anaerobic glyoxylate cycle activity in bacteria is known (Blasco et al., 1989) to be an important pathway involved in the generation of carbon storage polymers (Loken & Sirevag, 1982). Therefore, it is not surprising that this cycle would be operating in cluster 1 Defluviicoccus, a microorganism that produces large amounts of the storage polymer polyhydroxyalkanoates under anaerobic conditions.


Hypothetical anaerobic central metabolic pathways in cluster 1 Defluviicoccus. Solid lines indicate probable carbon flux, whereas broken lines represent possible carbon flux not investigated in this study. Filled arrows Embedded Image represent reduced electron carriers (NADH2, NADPH2 or FADH2) and unfilled arrows Embedded Image represent oxidized electron carriers (NAD+, NADP+ or FAD+). Green arrows (Embedded Image, Embedded Image) depict reactions that reduce electron carriers/generate reducing power. Reactions that oxidize electron carriers/consume reducing power are depicted by red arrows (Embedded Image, Embedded Image).

Fumarate reductase activity in cluster 1 Defluviicoccus was previously inferred from reduced rates of acetate uptake in the presence of the fumarate reductase inhibitor oxantel (Mendz et al., 1995; Burow et al., 2008a, b). In the current experiments, cluster 1 Defluviicoccus enrichment produced 6.5±0.27 C-mmol g−1 dry cell weight polyhydroxyalkanoates after incubation with 2 mM acetate (control incubation), whereas in the presence of oxantel, polyhydroxyalkanoate production was significantly lower at 4.2±0.20 C-mmol g−1 dry cell weight. On the other hand, the presence of the succinate dehydrogenase inhibitor malonate (Sumegi et al., 1990) did not affect acetate uptake rates or polyhydroxyalkanoate production significantly (Table 1). These results provide evidence that substantial carbon flux occurs through the reductive branch of the TCA cycle via fumarate reductase and not oxidative reactions that require succinate dehydrogenase activity.

These results are similar to observations made in other GAO enrichments dominated by Competibacter (Lemos et al., 2007). Fumarate reductase activity is likely an important mechanism for balancing reducing equivalents generated by glycogen catabolism and the glyoxylate cycle in cluster 1 Defluviccocus. Flux of pyruvate through the reductive branch of the TCA cycle mediated by fumarate reductase consumes reducing power and is capable of producing hydroxyvalerate monomers following degradation to propionyl-CoA and condensation with acetyl-CoA. Propionyl-CoA is likely formed by succinate decarboxylation via a methylmalonyl-CoA intermediate (Galivan & Allen, 1968) rather than the acrylate pathway (Cardon & Barker, 1947) due to the observed activity of fumarate reductase in cluster 1 Defluviicoccus. Succinate produced by the glyoxylate cycle may also be converted to propionyl-CoA for polyhydroxyvalerate formation (Fig. 3). In contrast to GAO enrichment cultures, PAO (organisms that facilitate phosphorus removal in EBPR sludges) enrichment cultures dominated by Accumulibacter do not seem to possess fumarate reductase activity, at least when utilizing acetate as a carbon source (Burow et al., 2008a). However, they do have the potential to reduce fumarate to succinate (Garcia-Martin et al., 2006) and fumarate reductase activity is hypothesized to be important when PAOs utilize carbon sources (e.g. lactate) that substantially increase the amount of hydroxyvalerate monomers in polyhydroxyalkanoates (Mino & Satoh, 2006).

Cluster 1 Defluviicoccus utilize acetate and glycogen to generate polyhydroxyalkanoates, an essential storage polymer for subsequent aerobic growth under carbon-depleted conditions found in EBPR processes. Anaerobic production of polyhydroxyalkanoates requires these microorganisms to balance the reducing power generated by glycolysis and the glyoxylate cycle. The reducing power would be effectively balanced by the carbon flux through the reducing power-consuming reductive branch of the TCA cycle. Coordinated operation of these pathways is essential to provide cluster 1 Defluviicoccus with the metabolic flexibility to form polyhydroxyalkanoate storage polymers for survival under the feast–famine conditions applied in the EBPR process.


The authors would like to acknowledge the funding support of the Environmental Biotechnology Cooperative Research Centre, an initiative of the Federal Government of Australia and the Queensland State Government – Growing the Smart State Initiative. Thoughtful discussions from Alastair McEwan and Phil Bond are greatly appreciated. We thank Dai Yu and Xialon Wang for providing the cluster 1 Defluviicoccus enrichment and Beatrice Keller-Lehmann for technical support and additional analyses.


  • Editor: Wilfrid Mitchell


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