Temporal Dynamics and Performance Association of the Tetrasphaera-Enriched Microbiome for Enhanced Biological Phosphorus Removal

Tetrasphaera have been recently identified based on the 16S ribosomal RNA(rRNA)gene as among the most abundant polyphosphate-accumulating organisms(PAOs)in global full-scale wastewater treatment plants(WWTPs)with enhanced biological phosphorus removal(EBPR).However,it is unclear how Tetrasphaera PAOs are selectively enriched in the context of the EBPR microbiome.In this study,an EBPR microbiome enriched with Tetrasphaera(accounting for 40%of 16S sequences on day 113)was built using a top-down design approach featuring multicarbon sources and a low dosage of allylthiourea.The microbiome showed enhanced nutrient removal(phosphorus removal~85%and nitrogen removal~80%)and increased phosphorus recovery(up to 23.2 times)compared with the seeding activated sludge from a local full-scale WWTP.The supply of 1 mg·L^(-1)allylthiourea promoted the coselection of Tetrasphaera PAOs and Microlunatus PAOs and sharply reduced the relative abundance of both ammonia oxidizer Nitrosomonas and putative competitors Brevundimonas and Paracoccus,facilitating the establishment of the EBPR microbiome.Based on 16S rRNA gene analysis,a putative novel PAO species,EBPR-ASV0001,was identified with Tetrasphaera japonica as its closest relative.This study provides new knowledge on the establishment of a Tetrasphaera-enriched microbiome facilitated by allylthiourea,which can be further exploited to guide future process upgrading and optimization to achieve and/or enhance simultaneous biological phosphorus and nitrogen removal from high-strength wastewater.

Enhanced Biological Phosphorus Removal with Pseudomonas putida GM6 from Activated Sludge

The enhanced biological phosphorus removal （EBPR） method is widely adopted for phosphorus removal from wastewater, yet little is known about its microbiological and molecular mechanisms. Therefore, it is difficult to predict and control the deterioration of the EBPR process in a large-scale municipal sewage treatment plant. This study used a novel strain isolated in the laboratory, Pseudomonas putida GM6, which had a high phosphate accumulating ability and could recover rapidly from the deteriorated system and enhance the capability of phosphorus removal in activated sludge. Strain GM6 marked with gfp gene, which was called GMTR, was delivered into a bench-scale sequencing batch reactor （SBR） of low efficiency, to investigate the colonization of GMTR and removal of phosphorus. After 21 days, the proportion of GMTR in the total bacteria of the sludge reached 9.2%, whereas the phosphorus removal rate was 96%, with an effluent concentration of about 0.2 mg L^-1. In the reactor with the addition of GMTR, phosphorus was removed quickly, in 1 h under anaerobic conditions, and in 2 h under aerobic conditions. These evidences were characteristic of EBPR processes. Field testing was conducted at a hospital sewage treatment facility with low phosphorus removal capability. Twentyone days after Pseudomonas putida GM6 was added, effluent phosphorus concentration remained around 0.3 mg L^-1, corresponding to a removal rate of 96.8%. It was therefore demonstrated that Pseudomonas putida GM6 could be used for a quick startup and enhancement of wastewater biological phosphorus removal, which provided a scientific basis for potential large-scale engineering application.

Effects of COD to Phosphorus Ratios on the Metabolism of PAOs in Enhanced Biological Phosphorus Removal with Different Carbon Sources

To elucidate the phosphorus removal and metabolism under various COD / P ratio,a sludge highly enriched in PAOs was used to investigate the impacts of COD / P in batch tests under different carbon supply conditions. Acetate,propionate and a mixture of acetate and propionate at a ratio of 3 ∶ 1( COD basis) was used as carbon sources with the COD / P of 20,15,10 and 5. 0 g COD /gP,respectively. The minimum COD / P ratios for complete P removal were found to be 8. 24 g COD /gP for acetate,11. 40 g COD /gP for propionate and9. 10 g COD /gP for the 3 ∶ 1 mixture of acetate and propionate. Converted to a mass basis,all three cases had a very similar ratio of 7. 7 g VFA /gP,which represented a useful guide for operation of EBPR plants to identify possible shortages in VFAs. The trend in PHV accumulation during the anaerobic period along with the decrease of COD / P ratios suggested that,PAOs may use the TCA pathway for anaerobic VFA uptake to maintain the required NADH production with reduced glycogen degradation. During the aerobic phase,the glycogen pool was reduced but remained enough compared to the requirement for anaerobic VFA uptake,and the synthesis and degradation of glycogen was not the inhibition factor of PAOs.

Enhanced Biological Phosphorus Removal in SBR Using Glucose as a Single Organic Substrate

Enhanced biological phosphorus removal(EBPR) was investigated in an anaerobic/aerobic sequencing batch reactor(SBR) supplied with glucose as a single organic substrate.The results illustrated that EBPR process could also occur successfully with glucose other than short chain fatty acids(SCFAs).High phosphorus release and poly-hydroxyalkanoate(PHA) accumulation in the anaerobic phase was found vital for the removal of phosphorus during the aerobic phase.The measurement of intracellular reserves revealed that glycogen had a higher chance to replace the energy role of poly-P under anaerobic conditions.Moreover,glycogen was also utilized as the carbon source for PHA synthesis,as well as a reducing power as reported earlier.The accumulated PHA in this system was mainly in the form of poly-hydroxyvalerate(PHV) instead of poly-hydroxybutyrate(PHB),and was inferred to be caused by the excess reducing power contained in glucose.Lactate as a fermentation product was also found released into the bulk solution.Applying fundamental biochemistry knowledge to the experimental results,a conceptual biochemical model was developed to explain the metabolism of the glucose-induced EBPR.

Nitrous oxide emission by denitrifying phosphorus removal culture using polyhydroxyalkanoates as carbon source

Nitrous oxide （N2O） emission has been reported to be enhanced during denitrification when internally-stored compounds are used as carbon sources. However, negligible N2O emissions have been detected in the few studies where polyhydroxyalkanoates （PHA） were specifically used. This study investigated and compared the potential enhancement of N2O production, based on utilization of an internally-stored polymer and external carbon （acetate） by a denitrifying phosphorus removal culture. Results indicated that at relatively low chemical oxygen demand-to-nitrogen （COD/N） ratios, more nitrite was reduced to N2O in the presence of an external carbon source as compared to an internal carbon source （PHA）. At relatively higher COD/N ratios, similar N2O reduction rates were obtained in all cases regardless of the type of carbon source available. N2O reduction rates were, however, generally higher in the presence of an internal carbon source. Results from the study imply that when the presence of an external carbon source is not sufficient to support denitrification, it is likely competitively utilized by different metabolic pathways of denitrifying polyphosphate accumulating organisms （DPAOs） and other ordinary denitfifiers. This study also reveals that the consumption of PHA is potentially the rate-limiting step for N2O reduction during denitrification.

Effects of different external carbon sources and electron acceptors on interactions between denitrification and phosphorus removal in biological nutrient removal processes

The effects of two different external carbon sources （acetate and ethanol） and electron acceptors （dissolved oxygen, nitrate, and nitrite） were investigated under aerobic and anoxic conditions with non-acclimated process bi- omass from a full-scale biological nutrient removal-activated sludge system. When acetate was added as an external carbon source, phosphate release was observed even in the presence of electron acceptors. The release rates were 1.7, 7.8, and 3.5 mg P/（g MLVSS.h） （MLVSS： mixed liquor volatile suspended solids）, respectively, for dissolved oxygen, nitrate, and nitrite. In the case of ethanol, no phosphate release was observed in the presence of electron acceptors. Results of the experiments with nitrite showed that approximately 25 mg NO2-N/L of nitrite inhibited anoxic phosphorus uptake regardless of the concentration of the tested external carbon sources. Furthermore, higher deni- trification rates were obtained with acetate （1.4 and 0.8 mg N/（g MLVSS.h）） compared to ethanol （1.1 and 0.7 mg N/ （g MLVSS.h）） for both anoxic electron acceptors （nitrate and nitrite）.

Hypothesis for metabolites of denitrifying phosphate accumulating organisms by the restriction of denitrifying bacteria in anoxic phase

The objective of this work is to verify a hypothesis that nitrite accumulation comes from the metabolites of denitrification phosphate accumulating organisms (DPAOs),not denitrifying bacteria.On the precondition of the restriction of denitrifying bacteria in anoxic phase,static experimental test was designed using NO3-as electron acceptor,effluent was removed after sedimentation in anaerobic phase,and the same concentration solution of PO43--P was returned,so that TOC was excluded and denitrification was inhibited in the next phases.A parallel experiment was carried out simultaneously with the normal anaerobic-anoxic progress.The results showed that,in static test,by keeping the normal growth of DPAO and inhibiting denitrification of denitrifying bacteria,P-release in anaerobic and P-uptake in anoxic phase proceeded normally.DPAO had obvious effect on P-removal and the P-removal efficiency was 69%.The effluent concentration of NO3--N and NO2--N was 7.62 mg/L and 6.05 mg/L respectively,compared with parallel experiments,and nitrogen removal rate was lower.No nitrite residue was found in parallel test.Therefore,it can confirm the hypothesis that the metabolites of DPAO are both nitrogen and nitrite when nitrate is taken as electron acceptor,and nitrite is subsequently converted to nitrogen by denitrifying bacteria.

Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection

Currently,the most cost-effective and efficient method for phosphorus(P)removal from wastewater is enhanced biological P removal(EPBR)via polyphosphate-accumulating organisms(PAOs).This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment.The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment.Notably,Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates,possess specific enzymes for ATP production from polyphosphate,and have electrochemical transporters for acetate and C4-dicarboxylates.In contrast,Tetrasphaera,Candidatus Phosphoribacter,Knoellia,and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids.Additionally,this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants.Notably,FISH-Raman spectroscopy emerges as one of the most advanced detection techniques.Overall,this review provides critical insights into PAO research,underscoring the need for enhanced strategies in biological phosphorus removal.

Denitrification and phosphorus uptake by DPAOs using nitrite as an electron acceptor by step-feed strategies

Denitrifying phosphorus accumulating organ- isms （DPAOs） using nitrite as an electron acceptor can reduce more energy. However, nitrite has been reported to have an inhibition on denitrifying phosphorus removal. In this study, the step-feed strategy was proposed to achieve low nitrite concentration, which can avoid or relieve nitrite inhibition. The results showed that denitrification rate, phosphorus uptake rate and the ratio of the phosphorus uptaken to nitrite denitrified （anoxic P/N ratio） increased when the nitrite concentration was 15 rag. L-1 after step- feeding nitrite. The maximum denitrification rate and phosphorus uptake rate was 12.73 mg NO2-N.g MLSS- 1· h- 1 and 18.75 mg PO34--P- g MLSS- 1. h- 1, respec- tively. These rates were higher than that using nitrate （15 mg. L-l） as an electron acceptor. The maximum anoxic P/N ratio was 1.55 mg PO43- -Pmg NO2-N-1. When the nitrite concentration increased from 15 to 20 mg NO2 -N ~ L-I after addition of nitrite, the anoxic phosphorus uptake was inhibited by 64.85%, and the denitrification by DPAOs was inhibited by 61.25%. Denitrification rate by DPAOs decreased gradually when nitrite （about 20 mg · L-1） was added in the step-feed SBR. These results indicated that the step-feed strategy can be used to achieve denitrifying phosphorus removal using nitrite as an electron acceptor, and nitrite concentration should be maintained at low level （ 〈 15 mg. L-1 in this study）.

Cassava stillage and its anaerobic fermentation liquid as external carbon sources in biological nutrient removal

The aim of this study was to investigate the effects of one kind of food industry effluent, cassava stillage and its anaerobic fermentation liquid, on biological nutrient removal （BNR） from municipal wastewater in anaerobic- anoxic-aerobic sequencing batch reactors （SBRs）. Experiments were carried out with cassava stillage supernatant and its anaerobic fermentation liquid, and one pure compound （sodium acetate） served as an external carbon source. Cyclic studies indicated that the cassava by-products not only affected the transformation of nitrogen, phosphorus, poly-13-hydroxyalkanoates （PHAs）, and glycogen in the BNR process, but also resulted in higher removal efficiencies for phosphorus and nitrogen compared with sodium acetate. Furthermore, assays for phosphorus accumulating or- ganisms （PAOs） and denitrifying phosphorus accumulating organisms （DPAOs） demonstrated that the proportion of DPAOs to PAOs reached 62.6% （Day 86） and 61.8% （Day 65） when using cassava stillage and its anaerobic fer- mentation liquid, respectively, as the external carbon source. In addition, the nitrate utilization rates （NURs） of the cassava by-products were in the range of 5.49-5.99 g N/（kg MLVSS.h） （MLVSS is mixed liquor volatile suspended solids） and 6.63-6.81 g N/（kg MLVSS.h）, respectively. The improvement in BNR performance and the reduction in the amount of cassava stillage to be treated in-situ make cassava stillage and its anaerobic fermentation liquid attractive alternatives to sodium acetate as external carbon sources for BNR processes.

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℃;6-13 d,29±1℃;14-34 d,14±1℃).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℃ to 29±1℃ and then to 14±1℃,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℃ 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.