Tetrad-forming Organisms Research Articles

Nitrification and aerobic denitrification in anoxic–aerobic sequencing batch reactor

The aim of this study was to evaluate the feasibility of achieving nitrogen (N) removal using a lab-scale sequencing batch reactor (SBR) exposed to anoxic/aerobic (AN/OX) phases, focusing to achieve aerobic denitrification. This process will minimize emissions of N2O greenhouse gas. The effects of different operating parameters on the reactor performance were studied: cycle duration, AN/OX ratio, pH, dissolved oxygen concentration (DOC), and organic load. The highest inorganic N removal (NiR), close to 70%, was obtained at pH=7.5, low organic load (440mgCOD/(Lday)) and high aeration given by 12h cycle, AN/OX ratio=0.5:1.0 and DOC higher than 4.0mgO2/L. Nitrification followed by high-rate aerobic denitrification took place during the aerobic phase. Aerobic denitrification could be attributed to Tetrad-forming organisms (TFOs) with phenotype of glycogen accumulating organisms using polyhydroxyalkanoate and/or glycogen storage. The proposed AN/OX system constitutes an eco-friendly N removal process providing N2 as the end product.

Denitrifying capability and community dynamics of glycogen accumulating organisms during sludge granulation in an anaerobic-aerobic sequencing batch reactor.

Denitrifying capability of glycogen accumulating organisms (GAOs) has received great attention in environmental science and microbial ecology. Combining this ability with granule processes would be an interesting attempt. Here, a laboratory-scale sequencing batch reactor (SBR) was operated to enrich GAOs and enable sludge granulation. The results showed that the GAO granules were cultivated successfully and the granules had denitrifying capability. The batch experiments demonstrated that all NO3−-N could be removed or reduced, some amount of NO2−-N were accumulated in the reactor, and N2 was the main gaseous product. SEM analysis suggested that the granules were tightly packed with a large amount of tetrad-forming organisms (TFOs); filamentous bacteria served as the supporting structures for the granules. The microbial community structure of GAO granules was differed substantially from the inoculant conventional activated sludge. Most of the bacteria in the seed sludge grouped with members of Proteobacterium. FISH analysis confirmed that GAOs were the predominant members in the granules and were distributed evenly throughout the granular space. In contrast, PAOs were severely inhibited. Overall, cultivation of the GAO granules and utilizing their denitrifying capability can provide us with a new approach of nitrogen removal and saving more energy.

Metagenomic characterization of ‘Candidatus Defluviicoccus tetraformis strain TFO71’, a tetrad‐forming organism, predominant in an anaerobic–aerobic membrane bioreactor with deteriorated biological phosphorus removal

In an acetate-fed anaerobic-aerobic membrane bioreactor with deteriorated enhanced biological phosphorus removal (EBPR), Defluviicoccus-related tetrad-forming organisms (DTFO) were observed to predominate in the microbial community. Using metagenomics, a partial genome of the predominant DTFO, 'Candidatus Defluviicoccus tetraformis strain TFO71', was successfully constructed and characterized. Examining the genome confirmed the presence of genes related to the synthesis and degradation of glycogen and polyhydroxyalkanoate (PHA), which function as energy and carbon storage compounds. TFO71 and 'Candidatus Accumulibacter phosphatis' (CAP) UW-1 and CAP UW-2, representative polyphosphate-accumulating organisms (PAO), have PHA metabolism-related genes with high homology, but TFO71 has unique genes for PHA synthesis, gene regulation and granule management. We further discovered genes encoding DTFO polyphosphate (polyP) synthesis, suggesting that TFO71 may synthesize polyP under untested conditions. However, TFO71 may not activate these genes under EBPR conditions because the retrieved genome does not contain all inorganic phosphate transporters that are characteristic of PAOs (CAP UW-1, CAP UW-2, Microlunatus phosphovorus NM-1 and Tetrasphaera species). As a first step in characterizing EBPR-associated DTFO metabolism, this study identifies important differences between DTFO and PAO that may contribute to EBPR community competition and deterioration.

In Situ Profiling of Microbial Communities in Full-Scale Aerobic Sequencing Batch Reactors Treating Winery Waste in Australia

On-site aerobic sequencing batch reactor (SBR) treatment plants are implemented in many Australian wineries to treat the large volumes of associated wastewater they generate. Yet very little is known about their microbiology. This paper represents the first attempt to analyze the communities of three such systems sampled during both vintage and nonvintage operational periods using molecular methods. Alphaproteobacterial tetrad forming organisms (TFO) related to members of the genus Defluviicoccus and Amaricoccus dominated all three systems in both operational periods. Candidatus 'Alysiosphaera europaea' and Zoogloea were codominant in two communities. Production of high levels of exocellular capsular material by Zoogloea and Amaricoccus is thought to explain the poor settleability of solids in one of these plants. The dominance of these organisms is thought to result from the high COD to N/P ratios that characterize winery wastes, and it is suggested that manipulating this ratio with nutrient dosing may help control the problems they cause.

Short-term effect of temperature variation on the competition between PAOs and GAOs during acclimation period of an EBPR system

Sequencing batch reactor (SBR) for enhanced biological phosphorus removal (EBPR) processes was used to investigate the impact of the temperature shock on the competition between phosphorus-accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in start-up stage. During the 34 days operation, SBR was set with temperature variation(0–5 d, 22±1°C; 6–13 d, 29±1°C; 14–34 d, 14±1°C). PAOs and GAOs were analyzed by fluorescent in situ hybridization (FISH), and intracellular polyphosphate granules were stained by Neisser-stain. The results showed that the influence of temperature shock on PAOs’ abundance was more serious than that on GAOs in the enriching process. Under sudden and substantially temperature variation, from 22±1°C to 29±1°C and then to 14±1°C, the domination of PAOs was deteriorated. After temperature shock, PAOs’ competitive advantages at low temperature that concluded in other study did not appear in our study. As mesophilic, GAOs (indicated by Alphaproteobacteria and Gammaproteobacteria) were more temperature adaptive and better grew and took the domination at 14±1°C in the end. In the competition process, organisms of tetrad forming organisms (TFOs)-like shape which were considered as typical GAOs, were observed. With the evidence of poly-P granules containing by Neisser-straining and result of FISH, these organisms of TFOs-like shape were better to be assumed as adaption state or a special self-protecting shape of PAOs.

Elucidating further phylogenetic diversity among the Defluviicoccus‐related glycogen‐accumulating organisms in activated sludge

Glycogen-accumulating organisms (GAO) are thought to out-compete the polyphosphate-accumulating organisms (PAO) in activated sludge communities removing phosphate (P). Two GAO groups are currently recognized, the gammaproteobacterial Candidatus'Competibacter phosphatis', and the alphaproteobacterial Defluviicoccus vanus-related tetrad forming organisms (TFOs). Both are phylogenetically diverse based on their 16S rRNA sequences, with the latter currently considered to contain members falling into three distinct clusters. This paper identifies members of an additional fourth Defluviicoccus cluster from 16S rRNA gene clone library data obtained from a laboratory-scale activated sludge plant community removing P, and details FISH probes designed against them. Probe DF181A was designed to target a single sequence and DF181B designed against the remaining sequences in the cluster. Cells hybridizing with these probes in the biomass samples tested always appeared as either TFOs or in large clusters of small cocci. Members of the Defluviicoccus-related organisms were commonly found in full-scale wastewater treatments plants, sometimes as a dominant population.

Functionally Relevant Microorganisms to Enhanced Biological Phosphorus Removal Performance at Full‐Scale Wastewater Treatment Plants in the United States

The abundance and relevance ofAccumulibacter phosphatis (presumed to be polyphosphate-accumulating organisms [PAOs]), Competibacter phosphatis (presumed to be glycogen-accumulating organisms [GAOs]), and tetrad-forming organisms (TFOs) to phosphorus removal performance at six full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants were investigated. Coexistence of various levels of candidate PAOs and GAOs were found at these facilities. Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The TFO abundance varied from nondetectable to dominant. Anaerobic phosphorus (P) release to acetate uptake ratios (P(rel)/HAc(up)) obtained from bench tests were correlated positively with the abundance ratio of Accumulibacter/(Competibacter +TFOs) and negatively with the abundance of (Competibacter +TFOs) for all plants except one, suggesting the relevance of these candidate organisms to EBPR processes. However, effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system were not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. The plant that had the lowest average effluent phosphorus and highest stability rating had the lowest P(rel)/HAc(up) and the most TFOs. Evaluation of full-scale EBPR performance data indicated that low effluent phosphorus concentration and high process stability are positively correlated with the influent readily biodegradable chemical oxygen demand-to-phosphorus ratio. A system-level carbon-distribution-based conceptual model is proposed for capturing the dynamic competition between PAOs and GAOs and their effect on an EBPR process, and the results from this study seem to support the model hypothesis.

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 Defluviicoccus‐related tetrad‐forming organisms in an anaerobic–aerobic activated sludge process

A group of uncultured tetrad-forming organisms (TFOs) was enriched in an acetate-fed anaerobic-aerobic sequencing membrane bioreactor showing deteriorated enhanced biological phosphorus removal capacity. Based on 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses, these TFOs were identified as novel members of the Defluviicoccus cluster in the Alphaproteobacteria, accounting for 90 +/- 5% of the EUBmix FISH-detectable bacterial cell area in the reactor biomass. Microautoradiography in combination with FISH and polyhydroxyalkanoate (PHA) staining revealed that these Defluviicoccus-related TFOs could take up and transform acetate, lactate, propionate and pyruvate, but not aspartic acid and glucose, into PHA under anaerobic conditions. In contrast, under continuous anaerobic-aerobic cultivation, Defluviicoccus vanus, the only cultured strain from the cluster, was able to take up glucose with concurrent glycogen consumption and PHA production under anaerobic conditions. Under subsequent aerobic conditions, the accumulated PHA was utilized and the biomass glycogen levels were restored. These findings not only re-confirmed these Defluviicoccus-related TFOs as glycogen-accumulating organisms, but also revealed unexpected levels of physiological, phylogenetic and morphological diversity among members of the Defluviicoccus cluster.

Ecology of the Microbial Community Removing Phosphate from Wastewater under Continuously Aerobic Conditions in a Sequencing Batch Reactor

All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter(-1) to less than 0.1 mg P liter(-1) (when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly-beta-hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that "Candidatus Accumulibacter phosphatis" bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial Dechloromonas and alphaproteobacterial tetrad-forming organisms related to Defluviicoccus in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.

Microbial succession of glycogen accumulating organisms in an anaerobic-aerobic membrane bioreactor with no phosphorus removal

The succession of glycogen accumulating organisms (GAOs) has been observed in an acetate-fed, anaerobic-aerobic sequencing membrane bioreactor (MBR) operated for 260 days without enhanced biological phosphorus removal (EBPR) activity. Semi-quantitative fluorescence in situ hybridization results showed that a gammaproteobacterial lineage GB frequently observed in EBPR processes was initially the numerically dominant species (50-66% of total cells) of the GAO in the MBR from day 1 to day 38. During this period, succession of two different subgroups of group GB was also observed. On day 85 onward, a population shift from GB group to 'Defluvicoccus'-related tetrad-forming organisms (TFO) occurred. This microbial succession was suspected to be related to the applied operating conditions (long hydraulic retention time and long solid residence time) which favored the proliferation of 'Defluvicoccus'-related TFO rather than the GB group. Application of terminal restriction fragment length polymorphism on selected samples further revealed that the microbial diversity of the seeding sludge as determined by the number of terminal restriction fragments was higher than that of sludge samples taken after day 85.

Proliferation of glycogen accumulating organisms induced by Fe(III) dosing in a domestic wastewater treatment plant

To meet the effluent requirements given for the sensitive receiving body, the Southpest Wastewater Treatment Plant of Budapest, Hungary uses a combined activated sludge-biofilter system with chemical precipitation for P removal. Causes of the proliferation of glycogen accumulating organisms (GAOs) observed in the unaerated/oxic activated sludge unit of this system were investigated both in full-scale and in lab-scale experiments combined with a detailed analysis of the microbial communities. Concentration profile measurements throughout the 8-stage activated sludge unit indicated anaerobic conditions in the first two unaerated reactors and low orthophosphate level (< 1 mg l(-1)) in all of the stages that could not be attributed to the influent quality, but to Fe (III)-dosing to the returned activated sludge. Microbiological analysis revealed the presence of GAOs from the GB group in the Gammaproteobacteria and occasionally tetrad-forming organisms from Actinobacteria, and the absence of Rhodocyclus-related polyphosphate accumulating organisms (PAOs) in the activated sludge samples. Comparative lab-scale studies carried out in two identically arranged UCT-systems with staged anoxic reactors also confirmed that Fe (III)-dosing may result in phosphorus deficiency of the microbial niche, leading to the suppression of growth and EBPR activity of PAOs and to the proliferation of GAOs.

Quadrisphaera granulorum gen. nov., sp. nov., a Gram-positive polyphosphate-accumulating coccus in tetrads or aggregates isolated from aerobic granules

A Gram-positive bacterium, designated strain AG019(T), was isolated by micromanipulation from aerobic granules obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci clustered predominantly in tetrads, and was morphologically similar to the dominant organisms observed in the biomass. The morphology also resembled that of the tetrad-forming organisms commonly seen in activated sludge samples. Strain AG019(T) was found to be an oxidase-negative, catalase-positive, non-motile aerobe that does not reduce nitrate and grows at temperatures between 15 and 40 degrees C, with an optimum at 37 degrees C. The pH range for growth was 5.0-9.0, with an optimum at pH 7.5. Strain AG019(T) contained a peptidoglycan with directly cross-linked meso-diaminopimelic acid (type A1gamma) and lacked mycolic acids. The G+C content of the DNA was 75 mol%. Menaquinone MK-8(H(2)) was the major isoprenoid quinone. The bacterium stained positively for intracellular polyphosphate granules but not for poly-beta-hydroxyalkanoates. It produced capsular material and showed autoaggregation ability. Phenotypic and 16S rRNA gene analyses showed that the bacterium differed sufficiently from its closest phylogenetic relatives, namely members of the suborder Frankineae, which includes the genera Geodermatophilus, Blastococcus, Frankia, Sporichthya, Acidothermus and Microsphaera, that it is proposed that it be placed in a novel genus, Quadrisphaera, as Quadrisphaera granulorum gen. nov., sp. nov. The type strain is AG019(T) (=ATCC BAA-1104(T)=DSM 44889(T)).

Identification and occurrence of tetrad-forming Alphaproteobacteria in anaerobic–aerobic activated sludge processes

In an acetate-fed anaerobic-aerobic membrane bioreactor, a deteriorated enhanced biological phosphorus removal (EBPR) community was developed (as determined based on the chemical profiles of organic substrate, soluble phosphate, and intracellular carbohydrate and polyhydroxyalkanote (PHA) concentrations). Microscopic observations revealed the dominance of tetrad-forming organisms (TFOs), of which the majority stained positively for PHA under anaerobic conditions. Fluorescence in situ hybridization (FISH) confirmed that the Alphaproteobacteria (85.0+/-7.0% of total cells) were the most dominant group. A 16S rRNA gene clone library specific for the Alphaproteobacteria indicated that most 16S rRNA gene clones (61% of total clones) were closely affiliated with 'Defluvicoccus vanus', forming a cluster within subgroup 1 of the Alphaproteobacteria. Combined PHA staining and FISH with specific probes designed for the members of the 'Defluvicoccus' cluster suggested diversity within this TFO cluster, and that these TFOs were newly identified glycogen-accumulating organisms in EBPR systems. However, these 'Defluvicoccus'-related TFOs were only seen in low abundance in 12 different EBPR and non-EBPR systems, suggesting that they were not the key populations responsible for the deterioration of full-scale EBPR processes.

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor.

A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67 +/- 13.86 mg P l-1 was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04 +/- 1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 micro m) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 micro m) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria, but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.

Phylogenetic and physiological diversity of tetrad-forming organisms in deteriorated biological phosphorus removal systems

Polyhydroxyalkanoate (PHA)- and polyphosphate-accumulating traits of different taxonomic tetrad-forming organisms (TFOs) in two anaerobic-aerobic sequential batch reactors (SBRs) were characterized by the simultaneous use of fluorescence in-situ hybridization, PHB stain and DAPI stain. The two SBRs with glucose as the main carbon source were operated under different P:total organic carbon feeding ratios for more than 300 days, but both exhibited no enhanced biological phosphorus removal (EBPR) activity. Microscopic observations on sludge samples taken at various times from those two SBRs revealed that TFOs consistently accounted for more than 50% of total cells, and were mostly affiliated with the beta- and gamma-subclasses of Proteobacteria and the high G+C phylum of gram-positive bacteria (HGC). Those TFOs from the beta-Proteobcateria exhibited PHB stain positive and DAPI stain negative, indicating that they could utilize compounds other than polyphosphate (i.e. glycogen) as reducing power for PHA synthesis from glucose. In contrast, two types of TFOs within the HGC group showed negative PHB stain and positive DAPI stain, indicating their capacity to accumulate polyphosphate without the synthesis and degradation of PHA. This metabolic trait was different from the widely accepted biochemical model of EBPR and non-EBPR metabolisms. Other TFOs within the HGC group and gamma-Proteobacteria showed negative responses to both PHA and DAPI stains, and their function in the deteriorated EBPR system need to be further clarified. Overall findings suggested that the phylogenic and physiological heterogeneity of TFOs in anaerobic-aerobic activated sludge systems were diverse and greatly exceeded the current understanding.