Characterization of nitrous oxide reduction by Azospira sp. HJ23 isolated from advanced wastewater treatment sludge.

A new nitrous oxide (N2O)-reducing bacterium was isolated from a consortium that was enriched using advanced wastewater treatment sludge as an inoculum and N2O as the sole nitrogen source. The isolated facultative anaerobe was identified as Azospira sp. HJ23. Azospira sp. HJ23 exhibited optimum N2O-reducing activity with a C/N ratio of 62 at pH 6 in the temperature range of 37 °C to 40 °C. The optimum carbon source for N2O reduction was a mixture of glucose and acetate. The maximum rate of N2O reduction by Azospira sp. HJ23 was 4.8 mmol·g-dry cell-1·h-1, and its N2O-reducing activity was higher than other known N2O reducers. Azospira sp. HJ23 possessed several functional genes for denitrification. These included narG (NO3- reductase), nirK (NO2- reductase), norB (NO reductase), and nosZ (N2O reductase) genes. These results suggest that Azospira sp. HJ23 can be applied in the denitrification process to minimalize N2O emission.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1.

Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2ß2γ2 composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αßγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCE The known substrate spectrum of A. aromaticum EbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

Towards the Response Threshold for p-Hydroxyacetophenone in the Denitrifying Bacterium "Aromatoleum aromaticum" EbN1.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 regulates the capacity to anaerobically degrade p-ethylphenol (via p-hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ54-dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with p-hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of A. aromaticum EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which p-hydroxyacetophenone was added at various concentrations (from 500 µM down to 0.1 nM). Depletion profiles of this aromatic substrate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration of p-hydroxyacetophenone, depletion commenced after less than 5 min and suggested a response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degradation and efflux relevant genes (e.g., pchF, encoding a subunit of predicted p-ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM. The most pronounced transcriptional response was observed, as expected, for genes located at the beginning of the two operon-like structures, related to catabolism (i.e., acsA) and potential efflux (i.e., ebA335).IMPORTANCE Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their in situ concentrations and their bioavailabilities varying over several orders of magnitude. Even though degradation pathways and underlying regulatory systems have long been studied with aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little is known about the effector concentration-dependent responsiveness. A. aromaticum EbN1 is a model organism for the anaerobic degradation of aromatic compounds with the architecture of the catabolic network and its substrate-specific regulation having been intensively studied by means of differential proteogenomics. The present study aims at unraveling the minimal concentration of an aromatic growth substrate (p-hydroxyacetophenone here) required to initiate gene expression for its degradation pathway and to learn in principle about the lower limit of catabolic responsiveness of an anaerobic degradation specialist.

Genetic dissection of chlorate respiration in Pseudomonas stutzeri†PDA reveals syntrophic (per)chlorate reduction.

Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.

The predicted σ(54)-dependent regulator EtpR is essential for expression of genes for anaerobic p-ethylphenol and p-hydroxyacetophenone degradation in "Aromatoleum aromaticum" EbN1.

BACKGROUND: The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 anaerobically utilizes a multitude of aromatic compounds via specific peripheral degradation routes. Compound-specific formation of these catabolic modules is assumed to be mediated by specific transcriptional activators. In case of the recently elucidated p-ethylphenol/p-hydroxyacetophenone pathway, the highly substrate-specific regulation was implicated to involve the predicted σ(54)-dependent, NtrC-type regulator EbA324. The latter was suggested to control the expression of the two neighboring gene clusters encoding the catabolic enzymes as well as a corresponding putative solvent efflux system. In the present study, a molecular genetic approach was used to study the predicted function of EbA324. RESULTS: An unmarked in frame ΔebA324 (here renamed as ΔetpR; p-ethylphenol regulator) deletion mutation was generated. The ΔetpR mutant was unable to grow anaerobically with either p-ethylphenol or p-hydroxyacetophenone. Growth similar to the wild type was restored in the ΔetpR mutant background by in trans expression of plasmid-born etpR. Furthermore, expression of the "p-ethylphenol" gene clusters as well as corresponding protein formation was shown to depend on the presence of both, EtpR and either p-ethylphenol or p-hydroxyacetophenone. In the wild type, the etpR gene appears to be constitutively expressed and its expression level not to be modulated upon effector presence. Comparison with the regulatory domains of known phenol- and alkylbenzene-responsive NtrC-type regulators of Pseudomonas spp. and Thauera aromatica allowed identifying >60 amino acid residues in the regulatory domain (in particular positions 149 to 192 of EtpR) that may contribute to the effector specificity viz. presumptively restricted effector spectrum of EtpR. CONCLUSIONS: This study provides experimental evidence for the genome predicted σ(54)-dependent regulator EtpR (formerly EbA324) of "A. aromaticum" EbN1 to be responsive to p-ethylphenol, as well as its degradation intermediate p-hydroxyacetophenone, and to control the expression of genes involved in the anaerobic degradation of these two aromatic growth substrates. Overall, the presented results advance our understanding on the regulation of anaerobic aromatic compound catabolism, foremost based on the sensory discrimination of structurally similar substrates.

Towards habitat-oriented systems biology of "Aromatoleum aromaticum" EbN1: chemical sensing, catabolic network modulation and growth control in anaerobic aromatic compound degradation.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 is a well-studied model organism for anaerobic degradation of aromatic compounds. Following publication of its genome in 2005, comprehensive physiological-proteomic studies were conducted to deduce functional understanding from the genomic blueprint. A catabolic network (85 predicted, 65 identified proteins) for anaerobic degradation of 24 aromatic growth substrates (including 11 newly recognized) was established. Newly elucidated pathways include those for 4-ethylphenol and plant-derived 3-phenylpropanoids, involving functional assignment of several paralogous genes. The substrate-specific regulation of individual peripheral degradation pathways is probably initiated by highly specific chemical sensing via dedicated sensory/regulatory proteins, e.g. three different σ54-dependent one-component sensory/regulatory proteins are predicted to discriminate between three phenolic substrates (phenol, p-cresol and 4-ethylphenol) and two different two-component systems are assumed to differentiate between two alkylbenzenes (toluene, ethylbenzene). Investigations under in situ relevant growth conditions revealed (a) preferred utilization of benzoate from a mixture with succinate results from repressed synthesis of a C4-dicarboxylate TRAP transporter; (b) response to alkylbenzene-induced solvent stress comprises metabolic re-routing of acetyl-CoA and reducing equivalents to poly(3-hydroxybutyrate) synthesis, alteration of cellular membrane composition and formation of putative solvent efflux systems; and (c) multifaceted adaptation to slow growth includes adjustment of energy demand for maintenance and preparedness for future nutritional opportunities, i.e. provision of uptake systems and catabolic enzymes for multiple aromatic substrates despite their absence. This broad knowledge base taken together with the recent development of a genetic system will facilitate future functional, biotechnological (stereospecific dehydrogenases) and habitat re-enacting ("eco-"systems biology) studies with "A. aromaticum" EbN1.

Comparative proteomic analysis reveals insights into anoxic growth of Methyloversatilis universalis FAM5 on methanol and ethanol.

Methyloversatilis universalis FAM5 is a facultative methylotrophic bacterium that has been found in a variety of natural and engineered ecosystems. The goal of this study was to investigate M. universalis FAM5 responses to different electron/carbon donors, e.g. methanol or ethanol, during anoxic growth in chemostats with nitrate as the electron acceptor. During steady-state anoxic growth on either methanol or ethanol, over 90% of the influent nitrate was reduced primarily to nitrite. The cell yield on methanol was lower, possibly due to high energy requirements for C(1) assimilation. Label-free proteomics further revealed that methanol-grown cells displayed elevated concentrations of the enzymes involved in C(1) metabolism (H(4)MPT/H(4) F pathways, formate oxidation and serine cycle). In contrast, C(2) metabolism (glyoxylate shunt and tri-carboxylic acid cycle) and polyhydroxy-ß-butyrate (PHB) synthesis related proteins were overrepresented during subsequent growth on ethanol. Notably, the expression of respiratory nitrate reductase was not affected by the carbon sources applied. Furthermore, the changes in the proteome upon switching back to methanol were mostly reversible. Therefore, M. universalis displays wide-ranging responses to adapt between growth on methanol and ethanol. Such metabolic versatility could be particularly useful in wastewater treatment systems, which need to switch between different electron donors, while still reliably meeting effluent nitrogen discharge goals.

Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors.

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.

Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans.

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1S(T). The second enzyme of the proposed pathway, cholest-4-en-3-one-Delta1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins.

The denitrifying "Aromatoleum aromaticum" strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure ( approximately 15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol- and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2 O respiring bacteria.

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by µmax /Ks , in organisms with nosZ clade II.

Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from 'Aromatoleum aromaticum' EbN1.

The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium 'Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the 'classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD(+)-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe(187)), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr(93)) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93].

Uranium removal and microbial community in a H2-based membrane biofilm reactor.

We evaluated a hydrogen-based membrane biofilm reactor (MBfR) for its capacity to reduce and remove hexavalent uranium [U(VI)] from water. After a startup period that allowed slow-growing U(VI) reducers to form biofilms, the MBfR successfully achieved and maintained 94-95% U(VI) removal over 8 months when the U surface loading was 6-11 e(-) mEq/m(2)-day. The MBfR biofilm was capable of self-recovery after a disturbance due to oxygen exposure. Nanocrystalline UO2 aggregates and amorphous U precipitates were associated with vegetative cells and apparently mature spores that accumulated in the biofilm matrix. Despite inoculation with a concentrated suspension of Desulfovibrio vulgaris, this bacterium was not present in the U(VI)-reducing biofilm. Instead, the most abundant group in the biofilm community contained U(VI) reducers in the Rhodocyclaceae family when U(VI) was the only electron acceptor. When sulfate was present, the community dramatically shifted to the Clostridiaceae family, which included spores that were potentially involved in U(VI) reduction.

[Rapid enrichment and cultivation of denitrifying phosphate-removal bacteria and its identification by fluorescence in situ hybridization technology].

The present work focused on a rapid enrichment and cultivation of denitrifying phosphate-removal bacteria (DPB) in a membrane bio-reactor(MBR) by using A2/O anaerobic sludge from a wastewater treatment plant as seed, as well as providing an identification method. In the experiments, sodium acetate was used as the carbon source and a certain amount of nitrate was added to the MBR in the anoxic stage. Results showed that, with the efficient trap of the hollow-fiber membrane module, the proportion of DPB in all the phosphate-accumulating organisms (PAOs) increased from 24% to 93% within 35 days after two-stage's cultivation including anaerobic/aerobic and anaerobic/anoxic, during which the removal efficiency of nitrogen and phosphorus reached more than 90%. The activated sludge was identified by combining a regular method and the fluorescence in situ hybridization (FISH) technique, which demonstrated that Pseudomonas sp. and Rhodocyclus sp. were the dominant bacteria in the used bioreactor.

Progress toward understanding the distribution of Accumulibacter among full-scale enhanced biological phosphorus removal systems.

This study investigated the role of Accumulibacter-related bacterial populations and factors influencing their distribution in enhanced biological phosphorus removal (EBPR) systems in the USA. For this purpose, five full-scale wastewater treatment facilities performing EBPR were surveyed. The facilities had different configurations but were all treating primarily domestic wastewater. Two facilities had history of poor EBPR performance. Batch-scale acetate uptake and inorganic phosphate (P(i)) release and uptake experiments were conducted to evaluate the EBPR activity of each sludge. Typical P(i) and acetate profiles were observed, and EBPR activity was found to be positively correlated to polyphosphate (polyP)-accumulating organism (PAO) abundance, as determined by staining intracellular polyP. The abundance of Accumulibacter-related organisms was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all full-scale EBPR facilities, at levels ranging from 9 to 24% of total cells. More than 80% of Accumulibacter-related organisms were estimated to have high polyP content, confirming their involvement in EBPR in these five facilities. However, Accumulibacter-related PAOs were only a fraction (40-69%) of the total PAO population. The variation of Accumulibacter-related PAO abundance among these EBPR systems suggests that multiple interacting factors such as wastewater characteristics and operational conditions are structuring PAO communities.

Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures.

It is still a challenge to link specific metabolic activities to certain species in a microbial community because of methodological limitations. We developed a method to analyze the specific metabolic activity of a single bacterial species within a consortium making use of [(13)C(7)]-toluene for metabolic labelling of proteins. Labelled proteins were subsequently analyzed by 2D gel electrophoresis (2-DE) and mass spectrometry (MS) to characterize their identity as well as their (13)C content as an indicator for function and activity of the host organism. To establish this method, we analyzed the metabolic incorporation of (13)C carbon atoms into proteins of Aromatoleum aromaticum strain EbN1. This strain is capable of metabolizing toluene under nitrate-reducing conditions and was grown in either pure culture or in a mixed consortium with a gluconate-consuming enrichment culture. First, strain EbN1 was grown with non-labelled toluene or labelled [(13)C(7)]-toluene as carbon sources, respectively, and their proteins were subjected to 2-DE. In total, 60 unique proteins were identified by MALDI-MS/MS. From 38 proteins, the levels of (13)C incorporation were determined as 92.3+/-0.8%. Subsequently, we mixed strain EbN1 and the enrichment culture UFZ-1, which does not grow on toluene but on gluconate, and added non-labelled toluene, [(13)C(7)]-toluene and/or non-labelled gluconate as carbon sources. The isotope labelling of proteins was analyzed after 2-DE by MS as a quantitative indicator for metabolic transformation of isotopic-labelled toluene by the active species of the consortium. Incorporation of (13)C was exclusively found in proteins from strain EbN1 at a content of 82.6+/-2.3%, as an average calculated from 19 proteins, demonstrating the suitability of the method used to identify metabolic active species with specific properties within a mixed culture.

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants.

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.