Clade IIC Research Articles

Metabolic response and clade variation in Accumulibacter under phosphorus deprivation stress

Two enhanced biological phosphorus removal systems (R1 and R2) fed with different carbon sources (acetic acid and propionic acid) were used to investigate the metabolic response of nitrite-type denitrifying phosphorus removal functional bacteria Accumulibacter to different phosphorus concentrations and the changes of its clade community structure. The results indicated that the systems almost lost the ability to release phosphorus after 15 d phosphorus deprivation, the contents of poly-P in the sludge of R1 and R2 decreased from 0.63 to 0.02 mgP/mgVSS and from 1.23 to 0.05 mgP/mgVSS, respectively. According to the analysis of stoichiometric parameters, the functional bacteria in both systems performed glycogen accumulating organism metabolism to survive under phosphorus-poor condition. Besides, the two systems exhibited a stronger EBPR ability even after 42 d phosphorus deprivation, and the maximum anoxic phosphorus uptake rate was 1.92 times and 2.29 times of those under phosphorus-rich conditions. Real-time quantitative Polymerase Chain Reaction detection indicated that the proportion of Accumulibacter in the two systems decreased from 16.45 % to 8.78 % and from 31.00 % to 8.43 %, respectively, as phosphorus was deprived. Clade IIC maintained its dominance in both systems, while clade IID was gradually washed out from propionic acid system, suggesting that clade IIC had a more flexible metabolic response to phosphorus deprivation stress.

Two new clades recovered at high temperatures provide novel phylogenetic and genomic insights into Candidatus Accumulibacter.

Candidatus Accumulibacter, a key genus of polyphosphate-accumulating organisms, plays key roles in lab- and full-scale enhanced biological phosphorus removal (EBPR) systems. A total of 10 high-quality Ca. Accumulibacter genomes were recovered from EBPR systems operated at high temperatures, providing significantly updated phylogenetic and genomic insights into the Ca. Accumulibacter lineage. Among these genomes, clade IIF members SCELSE-3, SCELSE-4, and SCELSE-6 represent the to-date known genomes encoding a complete denitrification pathway, suggesting that Ca. Accumulibacter alone could achieve complete denitrification. Clade IIC members SSA1, SCUT-1, SCELCE-2, and SCELSE-8 lack the entire set of denitrifying genes, representing to-date known non-denitrifying Ca. Accumulibacter. A pan-genomic analysis with other Ca. Accumulibacter members suggested that all Ca. Accumulibacter likely has the potential to use dicarboxylic amino acids. Ca. Accumulibacter aalborgensis AALB and Ca. Accumulibacter affinis BAT3C720 seemed to be the only two members capable of using glucose for EBPR. A heat shock protein Hsp20 encoding gene was found exclusively in genomes recovered at high temperatures, which was absent in clades IA, IC, IG, IIA, IIB, IID, IIG, and II-I members. High transcription of this gene in clade IIC members SCUT-2 and SCUT-3 suggested its role in surviving high temperatures for Ca. Accumulibacter. Ambiguous clade identity was observed for newly recovered genomes (SCELSE-9 and SCELSE-10). Five machine learning models were developed using orthogroups as input features. Prediction results suggested that they belong to a new clade (IIK). The phylogeny of Ca. Accumulibacter was re-evaluated based on the laterally derived polyphosphokinase 2 gene, showing improved resolution in differentiating different clades.

Candidatus Accumulibacter use fermentation products for enhanced biological phosphorus removal

Previous research suggested that two major groups of polyphosphate-accumulating organisms (PAOs), i.e., Ca. Accumulibacter and Tetrasphaera, play cooperative roles in enhanced biological phosphorus removal (EBPR). The fermentation of complex organic compounds by Tetrasphaera provides carbon sources for Ca. Accumulibacter. However, the viability of the fermentation products (e.g., lactate, succinate, alanine) as carbon sources for Ca. Accumulibacter and their potential effects on the metabolism of Ca. Accumulibacter were largely unknown. This work for the first time investigated the capability and metabolic details of Ca. Accumulibacter cognatus clade IIC strain SCUT-2 (enriched in a lab-scale reactor with a relative abundance of 42.8%) in using these fermentation products for EBPR. The enrichment culture was able to assimilate lactate and succinate with the anaerobic P release to carbon uptake ratios of 0.28 and 0.36 P mol/C mol, respectively. In the co-presence of acetate, the uptake of lactate was strongly inhibited, since two substrates shared the same transporter as suggested by the carbon uptake bioenergetic analysis. When acetate and succinate were fed at the same time, Ca. Accumulibacter assimilated two carbon sources simultaneously. Proton motive force (PMF) was the key driving force (up to 90%) for the uptake of lactate and succinate by Ca. Accumulibacter. Apart from the efflux of proton in symport with phosphate via the inorganic phosphate transport system, translocation of proton via the activity of fumarate reductase contributed to the generation of PMF, which agreed with the fact that PHV was a major component of PHA when lactate and succinate were used as carbon sources, involving the succinate-propionate pathway. Metabolic models for the usage of lactate and succinate by Ca. Accumulibacter for EBPR were built based on the combined physiological, biochemical, metagenomic, and metatranscriptomic analyses. Alanine was shown as an invalid carbon source for Ca. Accumulibacter. Instead, it significantly and adversely affected Ca. Accumulibacter-mediated EBPR. Phosphate release was observed without alanine uptake. Significant inhibitions on the aerobic phosphate uptake was also evident. Overall, this study suggested that there might not be a simply synergic relationship between Ca. Accumulibacter and Tetrasphaera. Their interactions would largely be determined by the kind of fermentation products released by the latter.

Effect of different types of volatile fatty acids on the performance and bacterial population in a batch reactor performing biological nutrient removal

Different ratios of four volatile fatty acids (VFAs) were used as the primary feed to a laboratory scale biological nutrient reactor during four operational stages. The reactor performed efficiently over 500 days of operation with over 90% dissolved phosphorus and over 98% ammonium-nitrogen (NH4+-N) removal. Through in the first experimental phase, acetate and propionate were present in a significant proportion as carbon sources, the relative abundance of Candidatus Accumulibacter, a potential polyphosphate accumulating organism, increased from 10% to 57% and the Defluviicoccus genus, a known glycogen accumulating organism (GAO), decreased from 41% to 5%. Further tests indicated the presence of denitrifying phosphorus accumulating organisms (DPAO) belonging to Clade IIC, that could use nitrite as the electron acceptor during P-uptake. In general, VFAs favored the increase of the genus Defluviicoccus and Candidatus Accumulibacter. High relative abundance of Defluviicoccus did not affect the stability and the performance of the BNR process.

Trebonskia zoosporica, gen. et sp. nov., a new member of the Goniochloridales (Eustigmatophyceae, Stramenopiles) with an unusual mode of reproduction

ABSTRACT A new freshwater microalgal isolate, CCALA 1135, was characterized using a polyphasic approach (morphology, ultrastructure, fatty acid composition, 18S rRNA gene and rbcL analyses). Non-motile vegetative cells were spherical with thick, smooth cell walls lacking ornamentation, with multiple overlapping chloroplasts and sometimes more than one red globule in the cytoplasm. The nuclear and chloroplast envelopes formed a continuous membrane and there was no pyrenoid. These morphological and ultrastructural features indicated assignment to the class Eustigmatophyceae. The strain was, however, unique in its mode of reproduction: only zoospores were formed, but no autospores were observed (i.e. obligate zoospory, which is rare in the Eustigmatophyceae). The zoospores were relatively fragile and their morphology was similar to those of other members of the clade Goniochloridales (a clade name under the PhyloCode) that have been studied, being uniflagellate and lacking an eyespot. A very high proportion of polyunsaturated fatty acids (up to 57% of total fatty acids) was found, dominated by nutritionally valuable arachidonic and eicosapentaenoic acids, the latter being the most abundant FA (up to 35%) in the total profile. Phylogenetic analysis based on 18S rDNA and rbcL sequences confirmed that CCALA 1135 represents a new member of Goniochloridales clade IIc. Based on cellular morphology, ultrastructure and molecular data we propose this strain as a new species and new genus of the Eustigmatophyceae, Trebonskia zoosporica gen. et sp. nov. Our work is a step toward clarifying the taxonomy of a group of the Eustigmatophyceae with many unnamed strains awaiting characterization.

Biological phosphorus removal and its microbial community in a modified full-scale activated sludge system under dry and wet weather dynamics

Enhanced biological phosphorus removal (EBPR) performance and microbial community dynamics during dry and wet-weather conditions of a full-scale treatment plant was evaluated by converting a section of activated sludge basins using low-cost operational modifications into an anoxic/anaerobic zone to promote EBPR. Two trains of the activated sludge system at the Des Moines, Iowa Metropolitan Wastewater Reclamation Facility were used for the study with one train modified for EBPR, and the other remained as nitrification-only for comparison.In addition to measuring the modification effectiveness for phosphorus removal, performance was compared during dry and wet weather conditions over the course of two summer seasons to improve understanding of wet and dry weather dynamics for EBPR. DNA sequencing and qPCR tests were conducted to develop an understanding of microbial population changes between control and modified basins and wet and dry weather conditions.Basin hydraulic retention times varied from 2.6 to 12.7 hours with an average of 8.9 hours. EBPR activity was successfully established in the modified basins with average phosphorus content of the return activated sludge 0.032 ± 0.002 compared to 0.016 ± 0.001 mg TP/mg TSS (95% confidence) in the control basins. Phosphorus removal was significantly decreased by prolonged wet weather conditions, particularly in year two of the study, however the modified basin maximum removal of 96% and average of 43.7 ± 5.3% remained significantly higher than the maximum of 46% and average 12.6 ± 2.4% removal in the control basins.DNA sequencing showed a significant increase in relative abundance of phyla Chloroflexi, Nitrospirae, and Verrucomicrobia in the modified basins, but no correlation to EBPR performance. qPCR indicated significant increase in relative quantity of Accumulibacter, but not for Actinetobacter-like phosphorus accumulating organisms (PAOs), which includes the PAO Tetrasphaera. Significant abundance of some Accumulibacter clades found within the modified basins was contrary to previous literature which focused on small-scale and batch studies. A higher than expected dominance of clade I and increased relative quantities of clades IIB and IIC during extended wet weather was observed which may have contributed to rapid recovery of phosphorus removal when dry weather resumed. The abundance of PAOs did not significantly correlate with changes in phosphorous removal performance, contrary to reports from previous small-scale and batch studies.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.

P.080 A translational Homer 1a-based network approach: imaging how haloperidol modulates glutamate system functional connectivity

The dynamic response mechanism of Candidatus Accumulibacter clades to environmental factors in enhanced biological phosphorus removal (EBPR) was unclear. This study investigated the relationship between the transcriptional responses of Candidatus Accumulibacter clades and environmental dynamics. Results suggested that Candidatus Accumulibacter clade IIA only responded in initial 20 and 30 min of P-release and P-uptake stage, respectively, and was also the first clade to stop responding among the six Candidatus Accumulibacter clades. Clade IIC and IID responded at rising stage of P-release and P-uptake rate. Clade IA and IIB responded at decreasing stage of P-release and P-uptake rate. The transcriptional response duration of clade IIF was the longest, which constantly responded throughout anaerobic, anoxic and oxic phase. The transcriptional responses of Candidatus Accumulibacter clades to environmental dynamics revealed the microorganisms actually working in P-release and P-uptake, and gave a new insight into the transcriptional responses related to the EBPR performance.

The saprotrophic Pleurotus ostreatus species complex: late Eocene origin in East Asia, multiple dispersal, and complex speciation

The Pleurotus ostreatus species complex is saprotrophic and of significant economic and ecological importance. However, species delimitation has long been problematic because of phenotypic plasticity and morphological stasis. In addition, the evolutionary history is poorly understood due to limited sampling and insufficient gene fragments employed for phylogenetic analyses. Comprehensive sampling from Asia, Europe, North and South America and Africa was used to run phylogenetic analyses of the P. ostreatus species complex based on 40 nuclear single-copy orthologous genes using maximum likelihood and Bayesian inference analyses. Here, we present a robust phylogeny of the P. ostreatus species complex, fully resolved from the deepest nodes to species level. The P. ostreatus species complex was strongly supported as monophyletic, and 20 phylogenetic species were recognized, with seven putatively new species. Data from our molecular clock analyses suggested that divergence of the genus Pleurotus probably occurred in the late Jurassic, while the most recent common ancestor of the P. ostreatus species complex diversified about 39 Ma in East Asia. Species of the P. ostreatus complex might migrate from the East Asia into North America across the North Atlantic Land Bridge or the Bering Land Bridge at different times during the late Oligocene, late Miocene and late Pliocene, and then diversified in the Old and New Worlds simultaneously through multiple dispersal and vicariance events. The dispersal from East Asia to South America in the middle Oligocene was probably achieved by a long-distance dispersal event. Intensification of aridity and climate cooling events in the late Miocene and Quaternary glacial cycling probably had a significant influence on diversification patterns of the complex. The disjunctions among East Asia, Europe, North America and Africa within Clade IIc are hypothesized to be a result of allopatric speciation. Substrate transitions to Apiaceae probably occurred no earlier than 6 Ma. Biogeographic analyses suggested that the global cooling of the late Eocene, intensification of aridity caused by rapid uplift of the QTP and retreat of the Tethys Sea in the late Miocene, climate cooling events in Quaternary glacial cycling, and substrate transitions have contributed jointly to diversification of the species complex.

Transcriptional responses of Candidatus Accumulibacter clades to environmental dynamics in enhanced biological phosphorus removal

The dynamic response mechanism of Candidatus Accumulibacter clades to environmental factors in enhanced biological phosphorus removal (EBPR) was unclear. This study investigated the relationship between the transcriptional responses of Candidatus Accumulibacter clades and environmental dynamics. Results suggested that Candidatus Accumulibacter clade IIA only responded in initial 20 and 30 min of P-release and P-uptake stage, respectively, and was also the first clade to stop responding among the six Candidatus Accumulibacter clades. Clade IIC and IID responded at rising stage of P-release and P-uptake rate. Clade IA and IIB responded at decreasing stage of P-release and P-uptake rate. The transcriptional response duration of clade IIF was the longest, which constantly responded throughout anaerobic, anoxic and oxic phase. The transcriptional responses of Candidatus Accumulibacter clades to environmental dynamics revealed the microorganisms actually working in P-release and P-uptake, and gave a new insight into the transcriptional responses related to the EBPR performance.

New insights in the competition of polyphosphate-accumulating organisms and glycogen-accumulating organisms under glycogen accumulating metabolism with trace Poly-P using flow cytometry

In enhanced biological phosphorus removal (EBPR) from wastewater, the metabolism pathway of polyphosphate accumulating organisms (PAOs) converted from polyphosphate accumulating metabolism (PAM) to glycogen accumulating metabolism (GAM) under a high ratio of carbon to phosphorus (C/P). This study investigated the competition mechanism between PAOs and glycogen accumulating organisms (GAOs) under GAM pathway. Low acetic acid (HAC) concentration made the GAM of PAOs outcompete GAOs in high C/P ratio (100:1), and the effect of influent acetate concentration on GAOs was stronger than PAOs. “Candidatus Accumulibacter” clades IIC and IID were the main dominant PAOs, and Thiothrix caldifontis (potential PAOs) was discovered and significantly increased from 0.53% to 5.75%. Flow cytometry results indicated that PAOs had higher specific surface area and lower nucleic acid content than GAOs, and high C/P promoted GAM rather than GAOs growth, which maybe important reasons causing PAOs dominant growth. PAOs dominance under GAM ensured stable performance of EBPR when C/P ratio in real wastewater increased.

Profiling population-level diversity and dynamics of Accumulibacter via high throughput sequencing of ppk1.

As the key organism for enhanced biological phosphorus removal, Accumulibacter has shown high intragenus diversity based on the phylogeny of polyphosphate kinase1 gene (ppk1) and many clade-specific features related to performance of wastewater treatment. However, the widely used molecular approaches are deficient or cost-inefficient in providing a comprehensive and quantitative population-level profile for Accumulibacter in complex community. In this study, we introduced a pipeline to analyze the population-level diversity and dynamics of Accumulibacter via high throughput sequencing (HTS) of ppk1 and 16S rRNA gene simultaneously. The HTS approach was assessed by testing primer coverage, performing sample replication, and comparing with a traditional clone library. Based on survey on full-scale activated sludge samples, unexpected high microdiversity in Accumulibacter and a tendency of exclusivity between two phylogenetic types were discovered. Moreover, the pipeline facilitated monitoring the population-level dynamics and co-occurrence pattern under various laboratory enriching conditions. The results revealed previously uncharacterized intraclade dynamics during enrichment, little effect of denitrifying process on the Accumulibacter diversity, and the niche adaption of Clade IIC on propionate as sole carbon source. Co-occurrence of Accumulibacter populations further partially supported the exclusivity of two types. A few bacterial taxa, including Cytophagaceae-, Prosthecobacter-, and Compteibacter-related taxa, showed co-occurrence with many Accumulibacter populations, suggesting their niche co-selection or potential metabolic interactions with Accumulibacter. The present pipeline is transplantable for studying microdiversity and niche differentiation of other functional microorganisms in complex microbial systems.

Evolutionary Analyses Reveal Diverged Patterns of SQUAMOSA Promoter Binding Protein-Like (SPL) Gene Family in Oryza Genus.

The SPL (SQUAMOSA promoter binding protein-like) gene family is one of the plant-specific transcription factor families and controls a considerable number of biological functions, including floral development, phytohormone signaling, and toxin resistance. However, the evolutionary patterns and driving forces of SPL genes in the Oryza genus are still not well-characterized. In this study, we investigated a total of 105 SPL genes from six AA genome Oryza representative species (O. barthii, O. glumipatula, O. nivara, O. rufipogon, O. glaberrima, and O. sativa). Phylogenetic and motif analyses indicated that SPL proteins could be divided into two distinct lineages (I and II), and further studies showed lineage II consisted of three clades (IIA, IIB, and IIC). We found that clade I had comparable structural features with clade IIA, whereas genes in clade IIC displayed intrinsic differences, such as lower exon numbers and the presence of miR156 regulation elements. Nineteen orthologous groups of OsSPLs in Oryza were also identified, and most exons within those genes maintained constant length, whereas length of intron changed relatively. All groups were constrained by stronger purifying selection and diversified continually including alterative gene number, intron length, and miR156 regulation. Subsequently, cis-acting element analyses revealed the potential role of SPLs in wild rice, which might participate in light-responsive, phytohormone response, and plant growth and development. Our results shed light on that different evolutionary rates and duplication events might result in divergent evolutionary patterns in each lineage of SPL genes, providing a guide in exploring diverse function in the rice gene family among six closely related Oryza species.

Population Structure and Morphotype Analysis of "Candidatus Accumulibacter" Using Fluorescence In Situ Hybridization-Staining-Flow Cytometry.

"Candidatus Accumulibacter" is the dominant polyphosphate-accumulating organism (PAO) in denitrifying phosphorus removal (DPR) systems. In order to investigate the community structure and clade morphotypes of "Candidatus Accumulibacter" in DPR systems through flow cytometry (FCM), denitrifying phosphorus removal of almost 100% using nitrite and nitrate as the electron acceptor was achieved in sequencing batch reactors (SBRs). An optimal method of flow cytometry combined with fluorescence in situ hybridization and SYBR green I staining (FISH-staining-flow cytometry) was developed to quantify PAOs in DPR systems. By setting the width value of FCM, bacterial cells in a sludge sample were divided into three groups in different morphotypes, namely, coccus, coccobacillus, and bacillus. Average percentages that the three different PAO populations accounted for among total bacteria from SBR1 (SBR2) were 42% (45%), 14% (13%), and 4% (2%). FCM showed that the ratios of PAOs to total bacteria in the two reactors were 61% and 59%, and the quantitative PCR (qPCR) results indicated that IIC was the dominant "Candidatus Accumulibacter" clade in both denitrifying phosphorus removal systems, reaching 50% of the total "Candidatus Accumulibacter" bacteria. The subdominant clade in the reactor with nitrite as the electron acceptor was IID, accounting for 31% of the total "Candidatus Accumulibacter" bacteria. The FCM and qPCR results suggested that clades IIC and IID were both coccus, clade IIF was coccobacillus, and clade IA was bacillus. FISH analysis also indicated that PAOs were major cocci in the systems. An equivalence test of FCM-based quantification confirmed the accuracy of FISH-staining-flow cytometry, which can meet the quantitative requirements for PAOs in complex activated sludge samples.IMPORTANCE As one group of the most important functional phosphorus removal organisms, "Candidatus Accumulibacter," affiliated with the Rhodocyclus group of the Betaproteobacteria, is a widely recognized and studied PAO in the field of biological wastewater treatment. The morphotypes and population structure of clade-level "Candidatus Accumulibacter" were studied through novel FISH-staining-flow cytometry, which involved denitrifying phosphorus removal (DPR) achieving carbon and energy savings and simultaneous removal of N and P, thus inferring the different denitrifying phosphorus removal abilities of these clades. Additionally, based on this method, in situ quantification for specific polyphosphate-accumulating organisms (PAOs) enables a more efficient process and more accurate result. The establishment of FISH-staining-flow cytometry makes cell sorting of clade-level noncultivated organisms available.

Community structures and population dynamics of “Candidatus Accumulibacter” in activated sludges of wastewater treatment plants using ppk1 as phylogenetic marker

Candidatus Accumulibacter has been identified as dominant polyphosphate-accumulating organisms (PAOs) in enhanced biological phosphorus (P) removal (EBPR) from wastewater. This study revealed the relevance of community structure, abundance and seasonal population dynamics of Candidatus Accumulibacter to process operation of wastewater treatment plants (WWTPs) in China using ppk1 gene as phylogenetic marker. All sludge samples had properties of denitrifying P removal using nitrate as an electron acceptor. Accumulibacter abundance in the anaerobic-anoxic-oxic (A2O) process was the highest (26% of total bacteria), and higher in winter than in summer with a better EBPR performance. Type-II was the dominant Accumulibacter in all processes, and type-I accounted for a small proportion of total Accumulibacter. The abundance of Clade-IIC as the most dominant clade reached 2.59×109 cells/g MLSS and accounted for 87.3% of total Accumulibacter. Clade IIC mainly contributed to denitrifying P removal. Clades IIA, IIC and IID were found in all processes, while clade-IIF was only found in oxidation ditch process through phylogenetic analysis. High proportion of clade IID to total Accumulibacter led to poor performance of aerobic P-uptake in inverted A2O process. Therefore, Accumulibacter clades in WWTPs were diverse, and EBPR performance was closely related to the clade-level community structures and abundances of Accumulibacter.

Interaction of "Candidatus Accumulibacter" and nitrifying bacteria to achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage.

To achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage, interaction of "Candidatus Accumulibacter" and nitrifying bacteria was investigated in a continuous-flow process. When nitrite in returned sludge of secondary settler was above 13mg/L, nitrite inhibition on anaerobic P-release of poly-phosphate organisms (PAOs) occurred. Clades IIC and IID were dominant, reaching 3.1%-11.9% of total bacteria. Clade IIC was sensitive to nitrite. Under low concentration of nitrite (<8mg/L), clade IIC primarily contributed to anoxic P-uptake. Clade IID had a strong tolerance to nitrite exposure. At high nitrite level (above 16mg/L), anoxic P-uptake was mainly performed by clade IID due to its strong tolerance to nitrite exposure. Ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and Accumulibacter interacted through variations of nitrite accumulation. High AOB abundance coupled with inhibition of NOB favored denitrifying phosphorus removal by clade IID. All Accumulibacter lineages were sorted into four clades of Type II. The most dominant ppk1 gene homologs were affiliated with clade IID, accounting for 69% of ppk1 clone library, and thus played an important role in denitrifying phosphorus removal via nitrite pathway.

Population dynamics of “Candidatus Accumulibacter phosphatis” under the modes of complete nitrification and partial nitrification (nitritation) in domestic wastewater treatment system

In order to understand the enhanced biological phosphorus removal in a continuous-flow domestic wastewater treatment system, population dynamics of “Candidatus Accumulibacter phosphatis” using polyphosphate kinase (ppk1) gene as genetic marker was investigated under the modes of complete nitrification and partial nitrification (nitritation). Nitritation was achieved at hydraulic retention time (HRT) of 6.49h and dissolved oxygen (DO) of 0.5mg O2/L. Denitrifying phosphate removal via nitrite pathway caused low PHAs consumption. Regardless of change of nitrifying modes, phosphorus removal always maintained at a high level of about 100%. Abundance of total Accumulibacter increased along with rising of carbon to nitrogen ratios (C/N). Under good performance of complete nitrification and nitritation, IIC and IID clade affiliated with type II (including clades II A-I) of Accumulibacter lineage was dominant (70%–80%), respectively. Clade IID exhibited high tolerance to starving, oxygen-limiting and high nitrite accumulation, and thus became dominant clade under insufficient carbon sources, low DO and nitritation mode. Clade IIC had a positive correlation with C/N ratios, and IIF had a positive correlation with temperature (p<0.05). Shannon index under nitrification and nitritation mode was 3.54 and 2.38, respectively. That indicated the change of Accumulibacter diversities under different operational conditions.

Complete Nutrient Removal Coupled to Nitrous Oxide Production as a Bioenergy Source by Denitrifying Polyphosphate-Accumulating Organisms.

Coupled aerobic-anoxic nitrous decomposition operation (CANDO) is a promising emerging bioprocess for wastewater treatment that enables direct energy recovery from nitrogen (N) in three steps: (1) ammonium oxidation to nitrite; (2) denitrification of nitrite to nitrous oxide (N2O); and (3) N2O conversion to N2 with energy generation. However, CANDO does not currently target phosphorus (P) removal. Here, we demonstrate that denitrifying polyphosphate-accumulating organism (PAO) enrichment cultures are capable of catalyzing simultaneous biological N and P removal coupled to N2O generation in a second generation CANDO process, CANDO+P. Over 7 months (>300 cycles) of operation of a prototype lab-scale CANDO+P sequencing batch reactor treating synthetic municipal wastewater, we observed stable and near-complete N removal accompanied by sustained high-rate, high-yield N2O production with partial P removal. A substantial increase in abundance of the PAO Candidatus Accumulibacter phosphatis was observed, increasing from 5% of the total bacterial community in the inoculum to over 50% after 4 months. PAO enrichment was accompanied by a strong shift in the dominant Accumulibacter population from clade IIC to clade IA, based on qPCR monitoring of polyphosphate kinase 1 (ppk1) gene variants. Our work demonstrates the feasibility of combining high-rate, high-yield N2O production for bioenergy production with combined N and P removal from wastewater, and it further suggests a putative denitrifying PAO niche for Accumulibacter clade IA.

Multi-cycle operation of enhanced biological phosphorus removal (EBPR) with different carbon sources under high temperature

Many studies reported that it is challenging to apply enhanced biological phosphorus removal (EBPR) process at high temperature. Glycogen accumulating organisms (GAOs) could easily gain their dominance over poly-phosphate accumulating organisms (PAOs) when the operating temperature was in the range of 25 °C–30 °C. However, a few successful EBPR processes operated at high temperature have been reported recently. This study aimed to have an in-depth understanding on the impact of feeding strategy and carbon source types on EBPR performance in tropical climate. P-removal performance of two EBPR systems was monitored through tracking effluent quality and cyclic studies. The results confirmed that EBPR was successfully obtained and maintained at high temperature with a multi-cycle strategy. More stable performance was observed with acetate as the sole carbon source compared to propionate. Stoichiometric ratios of phosphorus and carbon transformation during both anaerobic and aerobic phases were higher at high temperature than low temperature (20±1 °C) except anaerobic PHA/C ratios within most of the sub-cycles. Furthermore, the fractions of PHA and glycogen in biomass were lower compared with one-cycle pulse feed operation. The microbial community structure was more stable in acetate-fed sequencing batch reactor (C2-SBR) than that in propionate-fed reactor (C3-SBR). Accumulibacter Clade IIC was found to be highly abundant in both reactors.

Metabolic Response of "Candidatus Accumulibacter Phosphatis" Clade II C to Changes in Influent P/C Ratio.

The objective of this study was to investigate the ability of a culture highly enriched with the polyphosphate-accumulating organism, “Candidatus Accumulibacter phosphatis” clade IIC, to adjust their metabolism to different phosphate availabilities. For this purpose the biomass was cultivated in a sequencing batch reactor with acetate and exposed to different phosphate/carbon influent ratios during six experimental phases. Activity tests were conducted to determine the anaerobic kinetic and stoichiometric parameters as well as the composition of the microbial community. Increasing influent phosphate concentrations led to increased poly-phosphate content and decreased glycogen content of the biomass. In response to higher biomass poly-phosphate content, the biomass showed higher specific phosphate release rates. Together with the phosphate release rates, acetate uptake rates also increased up to an optimal poly-phosphate/glycogen ratio of 0.3 P-mol/C-mol. At higher poly-phosphate/glycogen ratios (obtained at influent P/C ratios above 0.051 P-mol/C-mol), the acetate uptake rates started to decrease. The stoichiometry of the anaerobic conversions clearly demonstrated a metabolic shift from a glycogen dominated to a poly-phosphate dominated metabolism as the biomass poly-phosphate content increased. FISH and DGGE analyses confirmed that no significant changes occurred in the microbial community, suggesting that the changes in the biomass activity were due to different metabolic behavior, allowing the organisms to proliferate under conditions with fluctuating phosphate levels.