Selection and dominance mechanisms of denitrifying phosphate-accumulating organisms in biological phosphate removal process

A sequencing batch reactor under different electron acceptor conditions was operated serially to investigate the selection and dominance mechanisms of denitrifying phosphate-accumulating organisms (DNPAOs) in a biological nutrient removal process. The presence of a small amount of NO^– ₃ at the start of the anaerobic phase stimulated the selection of DNPAOs in an anaerobic/aerobic system, and switching O₂ to NO^– ₃ as an electron acceptor enhanced the activity of anoxic phosphate uptake.     

Effects of acetate and nitrite addition on fraction of denitrifying phosphate-accumulating organisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying phosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.     

Fluctuation of microbial activities after influent load variations in a full-scale SBR: recovery of the biomass after starvation

Due to variations in the production levels, a full-scale sequencing batch reactor (SBR) for post-treatment of tannery wastewater was exposed to low and high ammonia load periods. In order to study how these changes affected the N-removal capacity, the microbiology of the reactor was studied by a diverse set of techniques including molecular tools, activity tests, and microbial counts in samples taken along 3 years. The recover capacity of the biomass was also studied in a lab-scale reactor operated with intermittent aeration without feeding for 36 days. The results showed that changes in the feeding negatively affected the nitrifying community, but the nitrogen removal efficiencies could be restored after the concentration stress. Species substitution was observed within the nitrifying bacteria, Nitrosomonas europaea and Nitrobacter predominated initially, and after an ammonia overload period, Nitrosomonas nitrosa and Nitrospira became dominant. Some denitrifiers, with nirS related to Alicycliphilus, Azospirillum, and Marinobacter nirS, persisted during long-term reactor operation, but the community fluctuated both in composition and in abundance. This fluctuating community may better resist the continuous changes in the feeding regime. Our results showed that a nitrifying–denitrifying SBR could be operated with low loads or even without feeding during production shut down periods.     

Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge

Aerobic granular sludge technology offers a possibility to design compact wastewater treatment plants based on simultaneous chemical oxygen demand (COD), nitrogen and phosphate removal in one sequencing batch reactor. In earlier studies, it was shown that aerobic granules, cultivated with an aerobic pulse-feeding pattern, were not stable at low dissolved oxygen concentrations. Selection for slow-growing organisms such as phosphate-accumulating organisms (PAO) was shown to be a measure for improved granule stability, particularly at low oxygen concentrations. Moreover, this allows long feeding periods needed for economically feasible full-scale applications. Simultaneous nutrient removal was possible, because of heterotrophic growth inside the granules (denitrifying PAO). At low oxygen saturation (20%) high removal efficiencies were obtained; 100% COD removal, 94% phosphate (P-) removal and 94% total nitrogen (N-) removal (with 100% ammonium removal). Experimental results strongly suggest that P-removal occurs partly by (biologically induced) precipitation. Monitoring the laboratory scale reactors for a long period showed that N-removal efficiency highly depends on the diameter of the granules.     

Effect of anaerobic reaction time on denitrifying phosphorus removal and N2O production

Nitrous oxide (N₂O) is a highly potent greenhouse gas; however, the characteristics of N₂O production during denitrification using poly-β-hydroxyalkanoates (PHA) as a carbon source are not well understood. In this study, effects of anaerobic reaction time (AnRT) on PHA formation, denitrifying phosphorus removal and N₂O production were investigated using a laboratory-scale anaerobic/anoxic/oxic sequencing batch reactor (An/A/O SBR). The results showed that operation of the An/A/O SBR for 0.78 SRT (47 cycles) after the AnRT was shortened from 90 min to 60 min resulted in anaerobically synthesized PHA improving by 1.8 times. This improvement was accompanied by increased phosphorus removal efficiency and denitrification. Accordingly, the N₂O-N production was reduced by 6.7 times. Parallel batch experiments were also conducted with AnRTs of 60, 90 and 120 min. All results indicated that in addition to the amount of anaerobically synthesized PHA, the kinetics of PHA degradation also regulated denitrifying phosphorus removal and N₂O production.     

传统厌氧/好氧生物除磷与厌氧/缺氧反硝化除磷效能的比较

采用序批式反应器(SBR),对比厌氧/好氧(A/O)和厌氧/缺氧(A/A)2种运行模式对模拟生活和工业混合污水同时脱氮除磷的效能。结果表明:反硝化聚磷菌完全可以在厌氧/缺氧交替运行条件下得到富集,稳定运行的2种模式对有机物和P的去除率分别保持在90%和85%以上,且A/A SBR具有更强的释磷能力,其释磷量比A/O SBR高出1.2倍。进一步试验表明:磷的释放在有无硝酸盐的情况下效果是不同的。2个系统内污泥均有反硝化除磷能力,A/A SBR中所含反硝化聚磷菌(DPAO)的比例是A/O SBR的4.56倍。2种模式出水水质都能取得较好的效果,且能实现同步除磷脱氮,而反硝化除磷在生物除磷方面更具优势。     

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of ¹³C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived ¹³C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real‐time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors. Biotechnol. Bioeng. 2009;102: 1527–1536. © 2008 Wiley Periodicals, Inc.     

Removal of phosphate in a sequencing batch reactor by Staphylococcus auricularis

A sequencing batch reactor (SBR) was used to remove phosphate in biological wastewater treatment as an alternative to the activated sludge process, in order to improve the low removal efficiency of phosphate and the operational instability. After a cycle of 2 h anaerobic and 4 h aerobic conditions, phosphate removal was optimized. The removal efficiencies of 5 and 50 mg phosphate l^–1 by Staphylococcus auricularis under repeated anaerobic and aerobic conditions were above 90%. These results showed that a long adaptation time, one of the major problems in biological phosphate removal process, was overcome by SBR.     

Analysis of denitrifier community in a bioaugmented sequencing batch reactor for the treatment of coking wastewater containing pyridine and quinoline

The denitrifier community and associated nitrate and nitrite reduction in the bioaugmented and general sequencing batch reactors (SBRs) during the treatment of coking wastewater containing pyridine and quinoline were investigated. The efficiency and stability of nitrate and nitrite reduction in SBR was considerably improved after inoculation with four pyridine- or quinoline-degrading bacterial strains (including three denitrifying strains). Terminal restriction fragment length polymorphism (T-RFLP) based on the nosZ gene revealed that the structures of the denitrifier communities in bioaugmented and non-bioaugmented reactors were distinct and varied during the course of the experiment. Bioaugmentation protected indigenous denitrifiers from disruptions caused by pyridine and quinoline. Clone library analysis showed that one of the added denitrifiers comprised approximately 6% of the denitrifier population in the bioaugmented sludge.     

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO₃⁻-N mg of mixed-liquor volatile suspended solids (MLVSS)⁻¹ h⁻¹ to a steady-state value of 0.06 mg of NO₃⁻-N mg of MLVSS⁻¹ h⁻¹ over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [¹³C]methanol to biomark the DNA of the denitrifiers. The extracted [¹³C]DNA and [¹²C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [¹³C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [¹²C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [¹⁴C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part I. Optimal condition for lactic acid fermentation using a microaerobic sequencing batch reactor (microaerobic SBR) with immobilized Lactobacillus plantarum TISTR 2265 and microbial communities

Cheese whey contains a high organic content and causes serious problems if it is released into the environment when untreated. This study aimed to investigate the optimum condition of lactic acid production using the microaerobic sequencing batch reactor (microaerobic SBR) in a nonsterile system. The high production of lactic acid was achieved by immobilized Lactobacillus plantarum TISTR 2265 to generate an acidic pH condition below 4.5 and then to support single-cell protein (SCP) production in the second aerobic sequencing batch reactor (aerobic SBR). A hydraulic retention time (HRT) of 4 days and a whey concentration of 80% feeding gave a high lactic acid yield of 12.58 g/L, chemical oxygen demand (COD) removal of 62.38%, and lactose utilization of 61.54%. The microbial communities in the nonsterile system were dominated by members of lactic acid bacteria, and it was shown that the inoculum remained in the system up to 330 days.     

A screen-printed biosensor using pyruvate oxidase for rapid determination of phosphate in synthetic wastewater

A screen-printed phosphate biosensor based on immobilized pyruvate oxidase (PyOD, E.C. 1.2.3.3) has been developed for monitoring phosphate concentrations in a sequencing batch reactor (SBR) system. The enzyme was immobilized by a nafion matrix and covered a poly(carbamoyl) sulfonate (PCS) hydrogel on a screen-printed electrode. PyOD consumes phosphate in the presence of pyruvate and oxygen and generates hydrogen peroxide (H₂O₂), carbon dioxide and acetylphosphate. The electroactive H₂O₂, monitored at +420 mV vs Ag/AgCl, is generated in proportion to the concentration of phosphate. The sensor has a fast response time (2 s) and a short recovery period (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 M to 625 M phosphate with a detection limit of 3.6 M. There was good agreement (R²=0.9848) between the commercial phosphate testing kit and the phosphate sensor in measurements of synthetic wastewater in a SBR system. This sensor maintained a high working stability (>85%) after 12 h of operation and involved a simple operation procedure. It therefore serves as a useful tool for rapid and accurate phosphate measurements in the SBR system and probably for process control.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10⁸ copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

     

Biodegradation of methyl <Emphasis Type="Italic">t</Emphasis>-butyl ether by aerobic granules under a cosubstrate condition

Aerobic granules efficient at degrading methyl tert-butyl ether (MTBE) with ethanol as a cosubstrate were successfully developed in a well-mixed sequencing batch reactor (SBR). Aerobic granules were first observed about 100 days after reactor startup. Treatment efficiency of MTBE in the reactor during stable operation exceeded 99.9%, and effluent MTBE was in the range of 15–50 μg/L. The specific MTBE degradation rate was observed to increase with increasing MTBE initial concentration from 25 to 500 mg/L, which peaked at 22.7 mg MTBE/g (volatile suspended solids)·h and declined with further increases in MTBE concentration as substrate inhibition effects became significant. Microbial-community deoxyribonucleic acid profiling was carried out using denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal ribonucleic acid. The reactor was found to be inhabited by several diverse bacterial species, most notably microorganisms related to the genera Sphingomonas, Methylobacterium, and Hyphomicrobium vulgare. These organisms were previously reported to be associated with MTBE biodegradation. A majority of the bands in the reactor represented a group of organisms belonging to the Flavobacteria–Proteobacteria–Actinobacteridae class of bacteria. This study demonstrates that MTBE can be effectively degraded by aerobic granules under a cosubstrate condition and gives insight into the microorganisms potentially involved in the process.     

Isolation and Characterization of Pseudoxanthomonas sp. Strain YP1 Capable of Denitrifying Phosphorus Removal (DPR)

A bacterial strain (designated as YP1) was isolated from an aerobic granular sequence batch reactor (SBR) performing simultaneous nitrogen and phosphorus removal. Based on the morphological, biochemical characteristics, and phylogenetic analysis of 16S rRNA gene sequence, YP1 was identified as Pseudoxanthomonas sp. strain. Strain YP1 was confirmed to have the ability to conduct denitrifying phosphorus removal (DPR). The optimal conditions for YP1 were pH 8.0, phosphorus (PO₄^3?-P) concentration of 8.0 mg/L, sodium citrate as carbon source, and nitrate nitrogen (NO₃^?-N) concentration of 30 mg/L. The functional genes including ppk and ppx, narG and narA, nirS and nirK were amplified for understanding the DPR pathways. The results provide more information about denitrifying polyphosphate-accumulating organisms (DPAOs) in aerobic granular sludge (AGS) and lay the foundations for full-scale DPR.     

Effect of aspartate and glutamate on the fate of enhanced biological phosphorus removal process and microbial community structure

This study investigated the fate of enhanced biological phosphorus removal (EBPR) and changes in microbial speciation in a sequencing batch reactor (SBR) fed with aspartate and glutamate. It involved SBR operation for 288 days, batch tests for observation of metabolic functions together with microscopic and phylogenetic analyses. Polyphosphate accumulating organisms (PAOs) were observed in abundance with complete removal of phosphorus. Fluorescence in situ hybridization (FISH) combined with 4′,6-dia-midino-2-phenylindole (DAPI) staining confirmed the accumulation of polyphosphate by Rhodocyclus-related and Actinobacterial PAOs. Aspartate seemed to favor the competitive growth of Rhodocyclus-related PAOs since EBPR population used the common biochemical pathways followed by Rhodocyclus-related PAOs in the aspartate fed batch tests. In the glutamate fed batch reactors, however, Actinobacterial PAOs appeared to be competitively selected which explains the lower levels of PHA generation. Even though operational conditions did not change, effective EBPR could not be maintained during the latter part of the study.     

Free nitrous acid selectively inhibits and eliminates nitrite oxidizers from nitrifying sequencing batch reactor

In a complete nitrification sequencing batch reactor (CNSBR), where ammonium containing wastewater (200–1,000 mg N/L) is completely oxidized to nitrate up to 2.4 kg NH₄ ⁺–N/m³ d, both ammonia oxidizers and nitrite oxidizers were enriched in the sludge granules. Quantitative fluorescence in situ hybridization analyses of the sludge granules of the CNSBR showed that ammonia oxidizers and nitrite oxidizers occupied 31 and 4.2% of total bacteria, respectively. Most of the nitrite oxidizers were Nitrobacter species (95% of the nitrite oxidizers) and the remainder was Nitrospira species. The population of nitrite oxidizers was significantly higher than that of partial nitrification SBR (PNSBR) where most of the ammonium was oxidized to nitrite. The PNSBR had 37% (ammonia oxidizers) and 0.4% (nitrite oxidizers) of total bacteria. Comparative study with CNSBR and PNSBR revealed that free nitrous acid, rather than free ammonia, played a critical inhibition role to wash out nitrite oxidizers from the reactor. The concentrations of free ammonia and nitrite as well as free nitrous acid in the CNSBR selected Nitrobacter as the dominant nitrite oxidizers rather than Nitrospira.     

Competition and coexistence of aerobic ammonium- and nitrite-oxidizing bacteria at low oxygen concentrations

In natural and man-made ecosystems nitrifying bacteria experience frequent exposure to oxygen-limited conditions and thus have to compete for oxygen. In several reactor systems (retentostat, chemostat and sequencing batch reactors) it was possible to establish co-cultures of aerobic ammonium- and nitrite-oxidizing bacteria at very low oxygen concentrations (2–8 μM) provided that ammonium was the limiting N compound. When ammonia was in excess of oxygen, the nitrite-oxidizing bacteria were washed out of the reactors, and ammonium was converted to mainly nitrite, nitric oxide and nitrous oxide by Nitrosomonas-related bacteria. The situation could be rapidly reversed by adjusting the oxygen to ammonium ratio in the reactor. In batch and continuous tests, no inhibitory effect of ammonium, nitric oxide or nitrous oxide on nitrite-oxidizing bacteria could be detected in our studies. The recently developed oxygen microsensors may be helpful to determine the kinetic parameters of the nitrifying bacteria, which are needed to make predictive kinetic models of their competition.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.