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.

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.

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.

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.

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.