Gordonia pseudamarae sp. nov., a home for novel actinobacteria isolated from stable foams on activated sludge wastewater treatment plants.

The taxonomic status of two Gordonia strains, designated BEN371 and CON9T, isolated from stable foams on activated sludge plants was the subject of a polyphasic study which also included the type strains of Gordonia species and three authenticated Gordonia amarae strains recovered from such foams. Phylogenetic analyses of 16S rRNA gene sequences showed that these isolates formed a compact cluster suggesting a well-supported lineage together with a second branch containing the G. amarae strains. A phylogenomic tree based on sequences of 92 core genes extracted from whole genome sequences of the isolates, the G. amarae strains and Gordonia type strains confirmed the assignment of the isolates and the G. amarae strains to separate but closely associated lineages. Average nucleotide index (ANI) and digital DNA-DNA hybridisation (dDDH) similarities showed that BEN371 and CON9T belonged to the same species and had chemotaxonomic and morphological features consistent with their assignment to the genus Gordonia. The isolates and the G. amarae strains were distinguished using a range of phenotypic features and by low ANI and dDDH values of 84.2 and 27.0 %, respectively. These data supplemented with associated genome characteristics show that BEN371 and CON9T represent a novel species of the genus Gordonia. The name proposed for members of this taxon is Gordonia pseudamarae sp. nov. with isolate CON9T (=DSM 43602T=JCM 35249T) as the type strain.

The phylogeny, ecology and ecophysiology of the glycogen accumulating organism (GAO) Defluviicoccus in wastewater treatment plants.

This comprehensive review looks critically what is known about members of the genus Defluviicoccus, an example of a glycogen accumulating organism (GAO), in wastewater treatment plants, but found also in other habitats. It considers the operating conditions thought to affect its performance in activated sludge plants designed to remove phosphorus microbiologically, including the still controversial view that it competes with the polyphosphate accumulating bacterium Ca. Accumulibacter for readily biodegradable substrates in the anaerobic zone receiving the influent raw sewage. It looks at its present phylogeny and what is known about it's physiology and biochemistry under the highly selective conditions of these plants, where the biomass is recycled continuously through alternative anaerobic (feed); aerobic (famine) conditions encountered there. The impact of whole genome sequence data, which have revealed considerable intra- and interclade genotypic diversity, on our understanding of its in situ behaviour is also addressed. Particular attention is paid to the problems in much of the literature data based on clone library and next generation DNA sequencing data, where Defluviicoccus identification is restricted to genus level only. Equally problematic, in many publications no attempt has been made to distinguish between Defluviicoccus and the other known GAO, especially Ca. Competibacter, which, as shown here, has a very different ecophysiology. The impact this has had and continues to have on our understanding of members of this genus is discussed, as is the present controversy over its taxonomy. It also suggests where research should be directed to answer some of the important research questions raised in this review.

Effects of different carbon sources on enhanced biological phosphorus removal and "Candidatus Accumulibacter" community composition under continuous aerobic condition.

Previous studies have shown that enhanced biological phosphorus removal (EBPR) performance under continuous aerobic conditions always eventually deteriorates; however, the speed at which this happens depends on the carbon source supplied. The published data suggest that propionate is a better carbon source than acetate is for maintaining operational stability, although it is not clear why. A lab-scale sequencing batch reactor was run initially under conventional anaerobic/aerobic conditions with either acetate or propionate as the carbon source. Chemical and microbiological analyses revealed that both sources performed as expected for such systems. When continuous aerobic conditions were imposed on both these established communities, marked shifts of the "Candidatus Accumulibacter" clades were recorded for both carbon sources. Here, we discuss whether this shift could explain the prolonged EBPR stability observed with propionate.

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal.

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

Influence of temperature, pH and dissolved oxygen concentration on enhanced biological phosphorus removal under strictly aerobic conditions.

Previous research has suggested that enhanced biological phosphorus removal (EBPR) from wastewater can be achieved under continuous aerobic conditions over the short term. However, little is known how environmental conditions might affect aerobic EBPR performance. Consequently we have investigated the impact of temperature, pH and dissolved oxygen (DO) concentrations on EBPR performance under strictly aerobic conditions. A sequencing batch reactor (SBR) was operated for 108 days on a six-hour cycle (four cycles a day). The SBR ran under alternating anaerobic-aerobic conditions as standard and then operated under strictly aerobic conditions for one cycle every three or four days. SBR operational temperature (10, 15, 20, 25 and 30°C), pH (6, 7, 8 and 9) and DO concentration (0.5, 2.0 and 3.5mg/L) were changed consecutively during the aerobic cycle. Recorded increases in mixed liquor phosphorus (P) concentrations during aerobic carbon source uptake (P release) were affected by the biomass P content rather than the imposed changes in the operational conditions. Thus, P release levels increased with biomass P content. By contrast, subsequent aerobic P assimilation (P uptake) levels were both affected by changes in operational temperature and pH, and peaked at 20-25°C and pH 7-8. Highest P uptake detected under these SBR operating conditions was 15.4 mg Pg-MLSS(-1) (at 25°C, pH 7 and DO 2.0mg/L). The ability of the community for linked aerobic P release and P uptake required the presence of acetate in the medium, a finding which differs from previous data, where these are reported to occur in the absence of any exogenous carbon source. Fluorescence in situ hybridization was performed on samples collected from the SBR, and Candidatus 'Accumulibacter phosphatis' cells were detected with PAOmix probes through the operational periods. Thus, Candidatus 'Accumulibacter phosphatis' seemed to perform P removal in the SBR as shown in previous studies on P removal under strictly aerobic conditions.

The microbiology of phosphorus removal in activated sludge processes-the current state of play.

This review discusses critically what we know and would like to know about the microbiology of phosphorus (P) removal in activated sludge systems. In particular, the description of the genome sequences of two strains of the polyphosphate accumulating organism found in these processes, Candidatus 'Accumulibacter phosphatis', allows us to address many of the previously unanswered questions relating to how these processes behave, and to raise new questions about the microbiology of P removal. This article attempts to be deliberately speculative, and inevitably subjective, but hopefully at the same time useful to those who have an active interest in these environmentally very important processes.

FACS enrichment and identification of floc-associated alphaproteobacterial tetrad-forming organisms in an activated sludge community.

A precise phylogenetic identity of the Defluviicoccus-related glycogen-accumulating organisms (GAO) observed after FISH probing in a novel activated sludge process removing phosphorus was sought with the aim of exploring the phylogenetic diversity of this important group. These organisms, whose sequences were not revealed in previously generated community wide 16S rRNA gene clone libraries, were identified using flow cytometry cell sorting of FISH-positive cells. Sequencing of a 16S rRNA gene clone library created from this sorted population identified the Defluviicoccus-related GAO as being highly related to previous identified GAO from enhanced biological phosphorus removal systems, despite a marked environmental difference between the two systems.

Ecophysiology of the Actinobacteria in activated sludge systems.

This review considers what is known about the Actinobacteria in activated sludge systems, their abundance and their functional roles there. Participation in processes leading to the microbiological removal of phosphate and in the operational problems of bulking and foaming are discussed in terms of their ecophysiological traits. We consider critically whether elucidation of their nutritional requirements and other physiological properties allow us to understand better what might affect their survival capabilities in these highly competitive systems. Furthermore, how this information might allow us to improve how these processes work is discussed.

In situ identification and characterization of the microbial community structure of full-scale enhanced biological phosphorous removal plants in Japan.

Fluorescent in situ hybridization (FISH) and polyphosphate (polyP) staining methods were used to characterize the microbial community structure of 13 activated sludge samples taken from nine different Japanese wastewater treatment plants with and without enhanced biological phosphorous removal (EBPR) activities. FISH with published rRNA-targeted oligonucleotide probes for important bacterial groups involving in the EBPR process revealed that Rhodocyclus-related polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms from a gammaproteobacterial lineage GB were the predominant populations detected, representing 4-18% and 10-31% of EUBmix-stained cells, respectively, in those samples. However, a considerable proportion of Rhodocyclus-related PAO cells were observed with no polyP granules accumulated based on polyP staining. This was further supported by a poor correlation between Rhodocyclus-related PAO population and sludge total phosphorous (TP) contents. In contrast, high correlations between polyP-stained cells and sludge TP contents were observed. In particular, among those polyP-stained cells in samples Ariake_A2O and Nakano_AO, more than 85% of them could not be targeted by probe PAOmix. These non-Rhodocyclus-related PAOs included populations from other bacterial divisions and members of the Betaproteobacteria other than those in Rhodocyclus-related group.

Are some putative glycogen accumulating organisms (GAO) in anaerobic : aerobic activated sludge systems members of the alpha-Proteobacteria?

Activated sludge plants designed to remove phosphorus microbiologically often perform unreliably. One suggestion is that the polyphosphate-accumulating organisms (PAO) are out-competed for substrates by another group of bacteria, the glycogen-accumulating organisms (GAO) in the anaerobic zones of these processes. This study used fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) to analyse the communities from laboratory-scale anaerobic : aerobic sequencing batch reactors. Members of the genus Sphingomonas in the alpha-Proteobacteria were present in large numbers in communities with poor phosphorus removal capacity where the biomass had a high glycogen content. Their ability to store poly-beta-hydroxyalkanoates anaerobically, but not aerobically, and not accumulate polyphosphate aerobically is consistent with these organisms behaving as GAO there. No evidence was found to support an important role for the gamma-Proteobacteria as possible GAO in these communities, although these bacterial populations have been considered in other studies to act as possible competitors for the PAO.

The microbiology of biological phosphorus removal in activated sludge systems.

Activated sludge systems are designed and operated globally to remove phosphorus microbiologically, a process called enhanced biological phosphorus removal (EBPR). Yet little is still known about the ecology of EBPR processes, the microbes involved, their functions there and the possible reasons why they often perform unreliably. The application of rRNA-based methods to analyze EBPR community structure has changed dramatically our understanding of the microbial populations responsible for EBPR, but many substantial gaps in our knowledge of the population dynamics of EBPR and its underlying mechanisms remain. This review critically examines what we once thought we knew about the microbial ecology of EBPR, what we think we now know, and what still needs to be elucidated before these processes can be operated and controlled more reliably than is currently possible. It looks at the history of EBPR, the currently available biochemical models, the structure of the microbial communities found in EBPR systems, possible identities of the bacteria responsible, and the evidence why these systems might operate suboptimally. The review stresses the need to extend what have been predominantly laboratory-based studies to full-scale operating plants. It aims to encourage microbiologists and process engineers to collaborate more closely and to bring an interdisciplinary approach to bear on this complex ecosystem.

Functional analysis of microbial communities in aerobic-anaerobic sequencing batch reactors fed with different phosphorus/carbon (P/C) ratios.

Fluorescence in situ hybridization (FISH) was used to analyse the community composition of a sequencing batch reactor (SBR) operating with aerobic-anaerobic cycling and fed acetate as its sole carbon source. Phosphorus was removed from the SBR microbiologically. Marked shifts in the community structure occurred as the phosphorus/carbon (P/C) ratio in the feed was changed. When the P/C ratio was shifted from 1:10 to 1:50, FISH analysis showed that the percentage of beta-Proteobacteria fell from ca 77% of the total bacteria to ca 38%. This decrease in the beta-Proteobacteria coincided with a reduction in both the proportions of the beta-proteobacterial Rhodocyclus-related phosphorus-accumulating bacteria and the biomass phosphorus content. FISH/microautoradiography and FISH/poly beta-hydroxyalkanoate (PHA) staining showed that the Rhodocyclus-related bacteria assimilated acetate and synthesized PHAs anaerobically, and that they accumulated phosphorus aerobically. No Acinetobacter spp. could be detected in any of the communities, casting further doubt on their role in phosphorus-removing activated sludge systems. As the feed P/C ratio decreased there was a corresponding increase in the proportion of alpha-Proteobacteria and, to a smaller extent, in the proportion of gamma-Proteobacteria; both the alpha- and gamma-Proteobacteria consisted mostly of tetrad-forming cocci, fitting the description of the so-called 'G-bacteria' morphotype. The change in the proportions of Proteobacteria present paralleled increases in the biomass glycogen content. Both the alpha- and beta-proteobacterial 'G-bacterial' populations assimilated acetate and synthesized PHA anaerobically. The alpha-Proteobacteria are considered responsible for glycogen production in these SBR systems.