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1 Advanced Wastewater Management Centre, The University of Queensland, St Lucia, QLD 4072, Australia
2 Section of Environmental Engineering, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark
Correspondence
Per H. Nielsen
phn{at}bio.aau.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; van Loosdrecht et al., 1997). However, the anaerobic–aerobic and feast–famine selective pressures used in EBPR plants may also favour growth of a different phenotypic group of organisms known as glycogen-accumulating organisms (GAOs) (Liu et al., 1996; Satoh et al., 1992). GAOs also take up and store organic carbon under anaerobic conditions but do not cycle Pi (anaerobic release and aerobic uptake), and their selective enrichment over PAOs via competition for sparingly available VFAs in EBPR plants leads to excessive Pi in plant effluents (Liu et al., 1996; Mino et al., 1995). Lab-scale (Bond et al., 1999; Oehmen et al., 2005) and full-scale (Kong et al., 2004, 2006; Saunders et al., 2003) EBPR studies showed this PAO–GAO competition for VFAs. Pure cultures that display the PAO or GAO phenotype as described above have not yet been acquired (Blackall et al., 2002; Seviour et al., 2003).
While numerous investigations of putative GAOs have been reported (Seviour et al., 2000, 2003), only ‘Candidatus Competibacter phosphatis' (hereafter called Competibacter spp.) has been demonstrated to be important in full-scale plants (Crocetti et al., 2002; Kong et al., 2006; Saunders et al., 2003). However, some lab-scale investigations have suggested that uncultured organisms belonging to the Alphaproteobacteria also play a role in breakdown of stable EBPR. These organisms have been phylogenetically identified as closely related to the pure culture Defluviicoccus vanus (Meyer et al., 2006; Wong et al., 2004) and Sphingomonas spp. (Beer et al., 2004). Due to the nature of the lab-scale studies carried out by enrichment of micro-organisms on sole organic carbon substrates, key aspects of their ecophysiology in full-scale plants are not known.
The aim of this investigation was to determine the role, if any, of the putative GAOs Defluviicoccus spp. and Sphingomonas spp. in full-scale EBPR processes. Traits of their ecophysiology including substrate uptake patterns, fate of assimilated substrates, anaerobic activity of particular central metabolic pathways and capability to denitrify were investigated. Factors of relevance to the competition between Defluviicoccus spp., PAOs and other putative GAOs in EBPR systems are discussed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and in these two, the abundance of other important EBPR micro-organisms (i.e. Accumulibacter spp. and Competibacter spp.) was determined. Ecophysiological investigations of Defluviicoccus spp. using FISH-microautoradiography (FISH-MAR) and post-FISH chemical staining techniques were only carried out in plants that contained a relatively high abundance of Defluviicoccus spp. Two plants (Aalborg West and Helsingborg) were chosen to study Cluster 1 Defluviicoccus. Four plants (Aalborg West, Skagen, Bjerringbro and Helsingborg) were chosen to study Cluster 2 Defluviicoccus. For each ecophysiological investigation, fresh biomass was sampled from the aerobic zone and transferred to the laboratory within 2–4 h.
FISH.
FISH was done according to the method of Amann (1995). FISH probes used in this study were NON338 for non-specific binding (Wallner et al., 1993), EUBMIX for Bacteria (Amann et al., 1990; Daims et al., 1999), DF1MIX (TFO_DF218 plus TFO_DF618) for the Alphaproteobacteria from Cluster 1 Defluviicoccus spp. (Wong et al., 2004), DF2MIX (DF988, DF1020 plus helper probes H966 and H1038) for Cluster 2 Defluviicoccus spp. (Meyer et al., 2006), SBR9-1a for Alphaproteobacteria Sphingomonas spp. (Beer et al., 2004), PAOMIX (Crocetti et al., 2000) for the Betaproteobacteria Accumulibacter spp., and GAOMIX (equal amounts of GAOQ989 and GB_G2 (Crocetti et al., 2002; Kong et al., 2002) for the Gammaproteobacteria Competibacter spp. All microscopic examinations were carried out using a Zeiss LSM510 Meta confocal laser scanning microscope (CLSM).
Quantitative FISH.
FISH quantification was performed by digital image analysis of FISH images using ImageJ V1.35k (http://rsb.info.nih.gov/ij/). FISH images were captured using a Zeiss LSM510 Meta CLSM. Samples were hybridized with Cy3-labelled specific probes and FLUOS-labelled EUBMIX probes. A total of 45 images were taken from three separate hybridizations for quantification of a specific FISH-probe-defined organism in each sample. The pixel area of the cells positive for the Cy3-labelled specific probe was quantified as a percentage of the pixel area for cells positive with the FLUOS-labelled EUBMIX probe. The percentage abundance reported for a specific organism was expressed as the mean of three separate measurements, with each measurement derived from 15 images. A standard deviation for each mean measurement was calculated as the mean of the squared differences between each value and the mean value.
FISH-microautoradiography (FISH-MAR) and post-FISH chemical staining.
FISH-MAR experiments were carried out using previously described methods (Kong et al., 2004; Lee et al., 1999) and different radiolabelled substrates (Kong et al., 2004, 2005, 2006). The manipulations and incubation conditions of biomass samples for FISH-MAR batch tests were fully described by Kong et al. (2004). Briefly, biomass samples were incubated with a radiolabelled substrate under different electron acceptor conditions for various time periods before fixation with paraformaldehyde (4 %) in phosphate-buffered saline at 4 °C.
To investigate the fate of acetate taken up by Defluviicoccus spp. under different electron acceptor conditions, pre-incubations with unlabelled acetate were employed. All incubations were carried out on a shaker (Kikalabortechnik, Denmark) at 250 r.p.m. and kept at 20±1 °C.
At least 100 MAR-positive and/or MAR-negative Defluviicoccus cells were counted. Uptake of a radiolabelled substrate was reported as positive (+) if >90 % and negative (–) if 0 % of cells were visualized by silver grain formation. Typical exposure time was 4 days; however, cells incubated with radiolabelled propionate required up to 12 days' exposure time. Generally a clear positive or negative result was demonstrated. However, we always found that 25–90 % of Defluviicoccus cells took up glucose for all incubation times and for substrate uptake experiments with Defluviicoccus cells incubated in the absence of an electron acceptor pre-incubated with unlabelled acetate for 3 h. This was reported as a variable result (+/–). Negative MAR results (0 % of cells with silver grain formation) remained negative after extension of exposure time to 12 days, except in the case of radiolabelled propionate incubations, during which with extension of exposure time to 12 days, Defluviicoccus cells went from variable (25–90 % of cells with silver grain formation) to MAR-positive (>90 % of cells with silver grain formation).
FISH and post-FISH chemical staining was carried out using methods fully described by Crocetti et al. (2000), with intracellular PHA and polyphosphate (polyP) determined by staining with Nile blue A (Ostle & Holt, 1982) and Neisser (Lindrea et al., 1999), respectively. Briefly, FISH was carried out and images were captured. Then cells on the slide were stained for PHA or polyP, the fields of view captured in FISH were relocated and images from chemical staining were taken. Prior to carrying out FISH and chemical staining, biomass samples were subjected to the different incubation conditions described by Kong et al. (2004) with unlabelled acetate (final concentration of either 1.5 mM or 0.5 mM).
For visualization of PHA accumulation, biomass samples were incubated under anaerobic conditions (nitrogen gassing, no external electron acceptor provided) with unlabelled acetate at a final concentration of 1.5 mM for 2 h. To visualize PHA exhaustion (PAO and GAO properties) and polyP accumulation (an exclusive PAO property), biomass samples were first incubated under anaerobic conditions with unlabelled acetate at a final concentration of 0.5 mM for 2 h. A further 2 h aerobic incubation without added acetate followed the anaerobic incubation. A lower concentration of acetate was used for the PHA exhaustion and polyP accumulation experiments than for the PHA accumulation experiments to ensure that complete uptake of acetate had occurred prior to aerobic incubation. Accumulation of polyP was also determined in biomass sampled directly from the aerobic zone of full-scale plants.
Metabolic inhibitors.
Some experiments used FISH-MAR or post-FISH chemical staining in combination with metabolic inhibitors. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was inhibited using sodium iodoacetate (Bickis & Quastel, 1965) at final concentrations of 0.5, 1.0 or 2.0 mM, and aconitase, which catalyses conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, was inhibited using sodium fluoroacetate (Lotspeich et al., 1952) at final concentrations of 0.5, 1.0 or 2.0 mM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cluster 1 and Cluster 2 Defluviicoccus cells (targeted by DF1MIX and DF2MIX, respectively) were all targeted by EUBMIX. The NON338 probe did not bind DF1MIX- or DF2MIX-positive cells. Cluster 2 Defluviicoccus (cocci in tetrads or clumps) were present in 7 of the 10 plants surveyed, with their abundance varying from <1 % to 9 % of Bacteria. Cluster 1 Defluviicoccus (also cocci in tetrads or clumps) were present in 3 of the 10 plants; however; they were always <1 % of all Bacteria. Sphingomonas spp. (targeted by SBR9-1a) were not observed in any of the 10 plants surveyed. Biomass from plants Bjerringbro and Skagen had the highest abundance of Cluster 2 Defluviicoccus (3 % and 9 % of Bacteria, respectively) and also contained ‘Candidatus Accumulibacter phosphatis’, hereafter called Accumulibacter spp. (2 % and 6 % of Bacteria, respectively), and Competibacter spp. (1 % and 6 % of Bacteria, respectively).
Substrate uptake patterns of Defluviicoccus spp.
All the experiments carried out with either radiolabelled or unlabelled substrates were repeated at least twice (total of three separate trials) in four different plants for Cluster 2 Defluviicoccus or two plants for Cluster 1 Defluviicoccus. Under separate anaerobic or aerobic incubation conditions both Cluster 1 and Cluster 2 Defluviicoccus exhibited identical substrate uptake behaviour, utilizing acetate, propionate, pyruvate and glucose. Cluster 2 Defluviicoccus could also take up an amino acid mixture. Other substrates, including formate, butyrate, ethanol, mannose, oleic acid, aspartic acid, glutamic acid, leucine, glycine and thymidine were not taken up by Defluviicoccus cells (Table 2).
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). Propionate, leucine and thymidine were separately taken up in the presence of acetate under anaerobic incubation conditions (i.e. no external electron acceptor). Butyrate and oleic acid were not taken up as sole substrates and neither could be taken up in the presence of acetate. The nitrogen-containing substrates tested, leucine and thymidine, were unable to be taken up as sole substrates but they were taken up in the presence of acetate.
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). The results of the separate test incubations with four sludges under the various electron acceptor conditions (no electron acceptor, oxygen, nitrate or nitrite) and different durations of pre-incubation with unlabelled substrate (3, 6 or 8 h) are reported in Table 4. A 3 h anaerobic (no electron acceptor) pre-incubation with unlabelled acetate (1C in Table 4) resulted in only a fraction of Cluster 2 Defluviicoccus (25–90 % cells) being able to take up radiolabelled acetate anaerobically. However, when a 6 h anaerobic pre-incubation with unlabelled acetate was employed, no Cluster 2 Defluviicoccus cells were capable of radiolabelled acetate uptake (1D in Table 4). Under prolonged aerobic pre-incubation conditions (2A–2C in Table 4), Cluster 2 Defluviicoccus spp. in all four sludges were positive (>90 % cells) for radiolabelled acetate uptake. Cluster 2 Defluviicoccus cells could not take up radiolabelled acetate under anoxic conditions (nitrate or nitrite provided as the only external electron acceptor) after a 6 h anoxic (nitrate or nitrite) pre-incubation with unlabelled acetate (3A and 3B, Table 4).
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Anaerobic incubation of biomass with acetate and the glycolysis inhibitor sodium iodoacetate (at 0.5, 1.0 or 2.0 mM) resulted in no PHA formation and no acetate uptake in Cluster 2 Defluviicoccus cells. However, PHA formation and acetate uptake were observed in Cluster 2 Defluviicoccus cells when biomass was incubated anaerobically with acetate and sodium fluoroacetate (at 0.5, 1.0 or 2.0 mM), an inhibitor of the TCA cycle enzyme aconitase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Wong et al., 2004) and Sphingomonas spp. (Beer et al., 2004). We showed that Defluviicoccus spp. are present in low abundance in most full-scale nutrient removal plants (9 of the 10 were EBPR plants). However, in one EBPR plant receiving mainly industrial wastewater, Cluster 2 Defluviicoccus constituted 9 % of all Bacteria. A further investigation of the two plants with the highest abundance of Cluster 2 Defluviicoccus (Bjerringbro and Skagen) revealed that these bacteria co-existed with both Accumulibacter spp. and Competibacter spp. and indeed were present in higher abundance than either of the latter two. Meyer et al. (2006) observed Cluster 2 Defluviicoccus in two Australian full-scale plants and concluded that they might be industrially relevant. Our findings are in agreement, since we demonstrated that Cluster 2 Defluviicoccus are relatively abundant members of the microbial community in two full-scale EBPR plants. In our investigation they were more abundant in plants receiving mainly industrial sources of wastewater. The Bjerringbro plant recently stopped receiving wastewater from slaughterhouses and treats mainly municipal wastewater. Interestingly, Cluster 2 Defluviicoccus constituted 3 % of Bacteria in this plant when it received both municipal and slaughterhouse wastewater, but <1 % when the plant received only municipal wastewater (Table 1). Some components of the slaughterhouse wastewater might be responsible for the selection of Cluster 2 Defluviicoccus, leading to its relatively higher abundance (3 % of all Bacteria) in this plant. Cluster 1 Defluviicoccus always constituted <1 % of Bacteria in plants surveyed here. Previous surveys of full-scale plants from several countries (Wong et al., 2004, 2005) also demonstrated low numbers of Cluster 1 Defluviicoccus. Micro-organisms related to Sphingomonas spp. were not observed in any of the 10 plants, suggesting that they are unimportant. Previously, Competibacter spp. (putative GAOs) were found to be more widely distributed and typically in higher abundances than Cluster 1 or Cluster 2 Defluviicoccus (Kong et al., 2002, 2006; Saunders et al., 2003).
The physiology of Defluviicoccus spp. is relatively poorly understood and if they are GAOs, their detailed ecophysiology in full-scale EBPR plants is critical in understanding how the EBPR process deteriorates. It has been proposed that GAOs are capable of utilizing VFAs, carboxylic acids, sugars and amino acids (Mino et al., 1998; Seviour et al., 2003). However, only one study has investigated the capability of putative GAOs to take up this range of substrates (Kong et al., 2006). Here we demonstrated that Defluviicoccus spp. take up acetate, propionate, pyruvate, glucose and a mixture of amino acids under either anaerobic or aerobic conditions, but not formate, butyrate, ethanol and several other organic substrates. Defluviicoccus spp. clearly have many similarities to Accumulibacter spp. and Competibacter spp. in substrate uptake. A notable difference was the direct uptake of glucose by Defluviicoccus spp., a capability not shared by Accumulibacter spp. (Kong et al., 2004) or Competibacter spp. (Kong et al., 2006). Uptake of glucose and assimilation as PHA has previously been reported for the only extant pure culture of Defluviicoccus, Defluviicoccus vanus strain Ben114 (Maszenan et al., 2005).
Cluster 2 Defluviicoccus were capable of simultaneous uptake of two different organic carbon substrates. The nitrogen-containing compounds leucine and thymidine, which were not taken up as sole substrates, were taken up in the presence of acetate. Accumulibacter spp. (Kong et al., 2004) and Competibacter spp. (Kong et al., 2006) have been reported to have similar capabilities. Substrate uptake similarities between these three organisms relevant to EBPR (carbon uptake and simultaneous uptake of two different organic carbon substrates) make it difficult to design strategies that specifically select for the EBPR desired organism, Accumulibacter.
The currently accepted biochemical model for GAOs hypothesizes that acetate is taken up anaerobically and stored as PHA and that there is a finite amount of PHA that can be formed (Filipe et al., 2001; Zeng et al., 2003b). The stored PHA is metabolized aerobically as a carbon and energy source. Under anaerobic pre-incubation conditions, Cluster 2 Defluviicoccus progressively lost their ability to take up radiolabelled acetate (1A–1C, Table 4). We concluded that Cluster 2 Defluviicoccus saturated their PHA storage capacity during the pre-incubation, thus preventing them from further uptake of radiolabelled acetate. However, under aerobic conditions (2A–2C, Table 4), radiolabelled acetate was continuously taken up. We concluded that Cluster 2 Defluviicoccus are aerobically growing on acetate. FISH and post-FISH chemical staining revealed that acetate was anaerobically stored as PHA. By the same methods, aerobic PHA consumption occurred but polyP was not accumulated. The type of organic carbon cycling and absence of P transformations in these micro-organisms is in accordance with the GAO biochemical model (Filipe et al., 2001; Zeng et al., 2003b).
Some GAOs have the capacity to utilize nitrate/nitrite as an electron acceptor (instead of oxygen) in the phase following the anaerobic zone (Zeng et al., 2003c). Our experiments support the notion that Cluster 2 Defluviicoccus are unlikely to denitrify with acetate as a carbon source (3A and 3B, Table 4). However, we did not explore anaerobic–anoxic conditions in which the organisms could have anaerobically stored acetate as PHA that could have been used as a carbon and energy source anoxically. This latter phenotype has been demonstrated for enrichment cultures of Accumulibacter spp. (Zeng et al., 2003a) and enrichments of Competibacter spp. (Zeng et al., 2003c).
According to the GAO biochemical model (Filipe et al., 2001; Zeng et al., 2003b), glycolysis and the TCA cycle provide energy and reducing equivalents essential for anaerobic acetate assimilation and reduction to PHA. Incubation of Cluster 2 Defluviicoccus with iodoacetate (a glycolysis inhibitor) essentially stopped acetate uptake and PHA formation. However, inhibition of the TCA cycle had undetectable effects on acetate uptake and PHA formation. Thus under anaerobic conditions, Cluster 2 Defluviicoccus can take up acetate and reduce it to PHA even when the TCA cycle is limited. We conclude that the energy and reducing equivalents required for anaerobic acetate uptake and PHA formation can be generated solely via glycolysis in Cluster 2 Defluviicoccus. However, we cannot conclude that this organism does not use the TCA cycle when it is not restricted.
A comparison of the ecophysiological traits of Cluster 2 Defluviicoccus with Accumulibacter spp. (Kong et al., 2004) and Competibacter spp. (Kong et al., 2006) reveals a similar, shared physiology. To survive and proliferate under the anaerobic–aerobic cycling conditions applied in EBPR systems, it is critical for micro-organisms to take up and store carbon under carbon-rich, anaerobic conditions. The internally stored carbon is then utilized for growth in the subsequent aerobic, external-carbon-poor conditions. Accumulibacter spp., Competibacter spp. and Defluviicoccus spp. take up identical organic carbon compounds (typically VFAs such as acetate and propionate) and therefore, in full-scale EBPR plants, they likely compete for sparingly available VFAs. It could be that physiological factors, such as substrate uptake rates or substrate affinities for different organic carbon compounds, differ between Accumulibacter spp., Competibacter spp. and Defluviicoccus spp., facilitating selection for different microbial communities. Future research using techniques based on quantitative MAR in combination with FISH, as described by Nielsen et al. (2003), could be used to address these hypothetical physiological variations between these organisms.
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ACKNOWLEDGEMENTS |
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Edited by: W. Liesack
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 7 August 2006;
revised 28 September 2006;
accepted 9 October 2006.
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  Y. Kong, Y. Xia, J. L. Nielsen, and P. H. Nielsen Structure and function of the microbial community in a full-scale enhanced biological phosphorus removal plant Microbiology, December 1, 2007; 153(12): 4061 - 4073. [Abstract] [Full Text] [PDF]
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