Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Accelerating effect of hydroxylamine and hydrazine on nitrogen removal rate in moving bed biofilm reactor

In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N?H? and NH?OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L?1 of each NH?OH and N?H?, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO??. Various combinations of N?H?, NH?OH, NH??, and NO?? were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N?H? concentration (4.38 mg N L?1) present in these batches was 5.43 mg N g?1 TSS h?1, whereas equimolar concentrations of N?H? and NH?OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.     

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m³/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m³/day at a nitrogen loading rate of 1.0 kg-N/m³/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m³/day at a nitrogen loading rate of 0.93 kg-N/m³/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5–6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.     

Potential coupling effects of ammonia-oxidizing and anaerobic ammonium-oxidizing bacteria on completely autotrophic nitrogen removal over nitrite biofilm formation induced by the second messenger cyclic diguanylate

The objective of this study was to investigate the influence of extracellular polymeric substance (EPS) on the coupling effects between ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria for the completely autotrophic nitrogen removal over nitrite (CANON) biofilm formation in a moving bed biofilm reactor (MBBR). Analysis of the quantity of EPS and cyclic diguanylate (c-di-GMP) confirmed that the contents of polysaccharides and c-di-GMP were correlated in the AOB sludge, anammox sludge, and CANON biofilm. The anammox sludge secreted more EPS (especially polysaccharides) than AOB with a markedly higher c-di-GMP content, which could be used by the bacteria to regulate the synthesis of exopolysaccharides that are ultimately used as a fixation matrix, for the adhesion of biomass. Indeed, increased intracellular c-di-GMP concentrations in the anammox sludge enhanced the regulation of polysaccharides to promote the adhesion of AOB and formation of the CANON biofilm. Overall, the results of this study provide new comprehensive information regarding the coupling effects of AOB and anammox bacteria for the nitrogen removal process.

     

Innovative treatment system for digester liquor using anammox process

This study demonstrated that partial nitritation using nitrifying activated sludge entrapped in a polyethylene glycol (PEG) gel carrier, as a pretreatment to anammox process, could be successfully applied to digester liquor of biogas plant at a nitrogen loading rate of 3.0 kg-N/m³/d. The nitritation process produced an effluent with a NO₂–N/NH₄–N ratio between 1.0 and 1.4, which was found to be suitable for the subsequent anammox process. A high SS concentration (2000–3000 mg/l) in the digester liquor did not affect partial nitritation treatment performances. Effluent from this partial nitritation reactor was successfully treated in the anammox reactor using anammox sludge entrapped in the PEG gel carrier with T-N removal rates of greater than 4.0 kg-N/m³/d. Influent BOD and SS contents did not inhibit anammox activity of the anammox gel carrier. The combination of partial nitritation and anammox reactors using PEG entrapped nitrifying and anammox bacteria was shown to be effective for the removal of high concentration ammonium in the digester liquor of a biogas plant.     

Importance of the operating pH in maintaining the stability of anoxic ammonium oxidation (anammox) activity in moving bed biofilm reactors

Two bench-scale parallel moving bed biofilm reactors (MBBR) were operated to assess pH-associated anammox activity changes during long term treatment of anaerobically digested sludge centrate pre-treated in a suspended growth partial nitrification reactor. The pH was maintained at 6.5 in reactor R1, while it was allowed to vary naturally between 7.5 and 8.1 in reactor R2. At high nitrogen loads reactor R2 had a 61% lower volumetric specific nitrogen removal rate than reactor R1. The low pH and the associated low free ammonia (FA) concentrations were found to be critical to stable anammox activity in the MBBR. Nitrite enhanced the nitrogen removal rate in the conditions of low pH, all the way up to the investigated level of 50 mg NO₂-N/L. At low FA levels nitrite concentrations up to 250 mg NO₂-N/L did not cause inactivation of anammox consortia over a 2-days exposure time.     

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation–anammox process

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m³/day under aerobic nitrogen loading rate of 3.8 kg-N/m³/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH₃/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m³/day under anaerobic nitrogen loading rate of 4.1 kg-N/m³/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation–anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.     

Development of a fixed-bed anammox reactor with high treatment potential

A plug-flow type anaerobic ammonium oxidation (anammox) reactor was developed using malt ceramics (MC) produced from carbonized spent grains as the biomass carriers for anammox sludge. Partial nitrified effluent of the filtrate from the sludge dehydrator of a brewery company was used as influent to a 20 L anammox reactor using MC. An average volumetric nitrogen removal rate (VNR) of 8.78 kg-N/m³/day was maintained stably for 76 days with 1 h of HRT. In a larger anammox reactor (400 L), an average VNR of 4.84 kg-N/m³/day could be maintained for 86 days during the treatment of low strength synthetic inorganic wastewater. As a result of bacterial community analysis for the 20 L anammox reactor, Asahi BRW1, probably originating from the wastewater collected at Asahi Breweries, was detected as the dominant anammox bacterium. These anammox reactors were characterized by a high NH₄-N removal capacity for low strength wastewater with a short hydraulic retention time.     

Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor

Anammox process has attracted considerable attention in the recent years as an alternative to conventional nitrogen removal technologies. In this study, a column type reactor using a novel net type acrylic fiber (Biofix) support material was used for anammox treatment. The Biofix reactor was operated at a temperature of 25°C (peak summer temperature, 31.5°C). During more than 340 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m³/d with TN removal efficiencies reaching 81.3%. When the reactor was used for raw anaerobic sludge digester liquor treatment, an average TN removal efficiency of 72% was obtained with highest removal efficiency of 81.6% at a nitrogen loading rate of 2.2 kg-N/m³/d. Results of extracellular polymeric substances (EPS) quantification revealed that protein was the most abundant component in the granular sludge and was found to be almost twice than that in the sludge attached to the biomass carriers. The anammox granules in the Biofix reactor illustrated a dense morphology substantiated by scanning electron microscopy and EPS results. The results of DNA analyses indicated that the anammox strain KSU-1 might prefer relatively low nutrient levels, while the anammox strain KU2 strain might be better suited at high nutrient concentration. Other types of bacteria were also identified with the potential of consuming dissolved oxygen in the influent and facilitating survival of anammox bacteria under aerobic conditions.     

Simultaneous nitrite-dependent anaerobic methane and ammonium oxidation processes

Nitrite-dependent anaerobic oxidation of methane (n-damo) and ammonium (anammox) are two recently discovered processes in the nitrogen cycle that are catalyzed by n-damo bacteria, including "Candidatus Methylomirabilis oxyfera," and anammox bacteria, respectively. The feasibility of coculturing anammox and n-damo bacteria is important for implementation in wastewater treatment systems that contain substantial amounts of both methane and ammonium. Here we tested this possible coexistence experimentally. To obtain such a coculture, ammonium was fed to a stable enrichment culture of n-damo bacteria that still contained some residual anammox bacteria. The ammonium supplied to the reactor was consumed rapidly and could be gradually increased from 1 to 20 mM/day. The enriched coculture was monitored by fluorescence in situ hybridization and 16S rRNA and pmoA gene clone libraries and activity measurements. After 161 days, a coculture with about equal amounts of n-damo and anammox bacteria was established that converted nitrite at a rate of 0.1 kg-N/m(3)/day (17.2 mmol day(-1)). This indicated that the application of such a coculture for nitrogen removal may be feasible in the near future.     

厌氧氨氧化在污水处理中的研究进展

厌氧氨氧化(ANAMMOX)是指厌氧氨氧化细菌在厌氧条件下以亚硝酸盐为电子受体将氨氮氧化为氮气的过程。由于在节能降耗和环境友好上的独特优点,基于厌氧氨氧化原理的脱氮技术被公认是目前最具应用前景的生物脱氮技术,因此自发现以来一直是国内外研究的热点。综述近年有关厌氧氨氧化细菌、厌氧氨氧化机理、反应的影响因素及其在污水处理应用方面的研究进展,并展望厌氧氨氧化在污水处理领域的发展方向。     

Nitrogen removal from animal waste treatment water by anammox enrichment

The aim of this work was to examine the applicability of the anaerobic ammonium oxidation (anammox) process to three kinds of low BOD/N ratio wastewaters from animal waste treatment processes in batch mode. A rapid decrease of NO(2)(-) and NH(4)(+) was observed during incubation with wastewaters from AS and UASB/trickling filter and their corresponding control artificial wastewaters. This nitrogen removal resulted from the anammox reaction, because the ratio of removed NO(2)(-) and NH(4)(+) was close to the theoretical ratio of the anammox reaction. Comparison of the inorganic nitrogen removal rate of the actual wastewater and that of control artificial wastewater showed that these two kinds of wastewater were very suitable for anammox treatment. Incubation with wastewater from RW did not show a clear anammox reaction; however, diluting it by half enabled the reaction, suggesting the presence of an inhibitory factor. This study showed that the three kinds of wastewater from animal waste treatment processes were suitable for anammox treatment.     

基于好氧反硝化及反硝化聚磷菌强化的低温低碳氮比生活污水生物处理中试研究

【背景】低碳氮比生活污水很难达标处理,多级A/O工艺、生物强化技术及生物膜技术的有机结合可有效解决这一问题。【目的】开发出一种泥膜共生多级A/O工艺并进行中试研究,驯化出高效脱氮除磷菌剂并对系统进行生物强化。【方法】通过测定中试设备出水及污水处理厂出水化学需氧量(Chemical oxygen demand,COD)、氨氮(NH_4~+-N)、硝氮(NO_3~--N)、总氮(Total nitrogen,TN)、总磷(Total phosphorus,TP)对比分析两种工艺的污染物去除效能,利用高通量测序技术对比生物强化技术对系统微生物群落结构的影响。【结果】中试设备对COD、NH_4~+-N、NO_3~--N、TN、TP的去除效果均优于污水处理厂的处理工艺;驯化的低温好氧反硝化菌TN去除率最大值可达84.21%,驯化的低温反硝化聚磷菌群对磷的去除率最高可达85.75%;利用驯化菌群对中试设备进行生物强化后较好地改善了系统NH_4~+-N、NO_3~--N、TN、TP的去除效果;经生物强化后,具有好氧反硝化和反硝化聚磷功能的Pseudomonas菌群明显增多。【结论】泥膜共生多级A/O工艺对于低碳氮比生活污水的处理具有很好的效果,利用生物强化技术可有效提高低温条件下系统污染物去除效能。     

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Anammox start-up in sequencing batch biofilm reactors using different inoculating sludge

In this study, anammox bacteria were rapidly enriched in sequencing batch biofilm reactors (SBBRs) with different inoculations. The activated sludge taken from a sequencing batch reactor was used and inoculated to SBBR1, while SBBR2 was seeded with stored anaerobic sludge from an upflow anaerobic fixed bed (2-year stored at 5–15 °C). Nitrogen removal performance, anammox activity, biofilm characteristics and variation of the microbial community were evaluated. The maximum total nitrogen loading rate (NLR) of SBBR1 gradually reached to 1.62 kg?N/(m³/day) with a removal efficiency higher than 88 % and the NLR of SBBR2 reached to 1.43 kg?N/(m³/day) with a removal efficiency of 86 %. SBBR2 was more stable compared to SBBR1. These results, combined with molecular techniques such as scanning electron microscope, fluorescence in situ hybridization, and terminal restriction fragment length polymorphism, indicated that different genera of anammox bacteria became dominant. This research also demonstrates that SBBR is a promising bioreactor for starting up and enriching anammox bacteria.     

Characterization of microbial community in an aerobic moving bed biofilm reactor applied for simultaneous nitrification and denitrification

A continuous-flow moving bed biofilm reactor (MBBR) under aerobic conditions was established for simultaneous nitrification and denitrification (SND), and microbial communities were investigated by a combination of denaturing gel gradient electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). DGGE analysis has revealed more similar microbial community structures formed in the biofilms with more similar carbon nitrogen (C/N) ratios. FISH analysis shows that the dominance of both Betaproteobacteria ammonia-oxidizing bacteria and Nitrospira-like nitrite-oxidizing bacteria were negatively correlated to C/N ratios. Sequence analysis of DGGE bands has indicated the presence of anoxic denitrifying bacteria Agrobacterium tumefaciens and Rhizobium sp., suggesting that the oxygen gradient inside the biofilm may be responsible for the mechanism of SND in aerobic MBBRs. The study confirms that appropriate control of microbial community structure resulting from optimal C/N ratio is beneficial in improving SND, thus optimizing nitrogen removal in aerobic MBBR. The established SND-based MBBR can save operation space and time in comparison to the traditional nitrogen removal process, and might be very attractive for future practical applications.     

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor

Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (± 0.19) kg-N m⁻³ d⁻¹ than the reactor initiated as the partial nitrifying reactor (0.23 (± 0.16) kg-N m⁻³ d⁻¹). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor.     

Simultaneous ammonium-nitrogen and copper removal, and copper recovery using nitrifying biofilm from the ultra-compact biofilm reactor

Simultaneous ammonium-nitrogen (NH(4)(+)-N) and copper removal, and copper recovery in synthetic wastewater using nitrifying biofilm from an ultra-compact biofilm reactor (UCBR) was demonstrated in batch studies, which consisted of three phases: Phase 1 for NH(4)(+)-N and copper removals, Phase 2 for copper recovery, and Phase 3 for NH(4)(+)-N removal. The results showed that more than 96.3% of copper was removed within 60min, while 60.1% of the adsorbed copper was recovered through rinsing the biofilms with 0.1mM of ethylenediaminetetraacetic acid (EDTA). The nitrifying biofilm was able to adsorb 0.245mg of copper/g of biofilms. After recovery treatment, 29.4% of copper remained bound within the nitrifying biofilms. No significant inhibitory effects towards NH(4)(+)-N removal in the presence of 0.92mg copper/L was noted in Phase 1 compared with the control test. However, lower initial pH condition in the recovery process and the accumulation of copper on the biofilm led to 50% inhibition on NH(4)(+)-N removal efficiency in the subsequent phase.     

PVA-alginate immobilized cells for anaerobic ammonium oxidation (anammox) process

The feasibility of an anaerobic ammonium oxidation (anammox) process combined with a cell-immobilization technique for autotrophic nitrogen removal was investigated. Anammox biomass was cultivated from local activated sludge and achieved significant anammox activity in 6 months. The development of a mature anammox biomass was confirmed by fluorescence in situ hybridization (FISH) analysis and off-line activity measurements. The abundance fraction of the anammox bacteria determined by FISH analysis was estimated by software. The anaerobic ammonia oxidizers occupied almost half of the total cells. Additionally, the anammox biomass was granulated as spherical gel beads of 3-4 mm in diameter by using a cell-immobilization technique. The nitrogen removal activity was proved to be successfully retained in the beads, with about 80% of nitrogenous compounds (NH(4) (+), NO(2) (- )and total nitrogen) removed after 48 h. These results offer a promising technique for the preservation of anammox microorganisms, the protection of them against the unfavorable surroundings, and the prevention of biomass washout towards the implementation of sustainable nitrogen elimination biotechnology. This is the first report on the immobilization of anammox biomass as gel beads.     

焦化废水工业处理装置和实验室装置硝化菌群的分子比较

反应器的群落结构分析有助于对工业装置的故障原因进行诊断。为了解决某焦化废水处理装置硝化功能低下的故障,构建了一套相似的实验室装置作为参照系统,该装置的硝化功能良好。通过工业装置和实验室装置好氧池生物膜16SrDNA克隆文库的比较,分析了它们之间硝化菌群的组成差异。实验室装置克隆文库的构成说明Nitrosomonas europaea-Nitrosoccus mobilis类群和Nitrospira属Ⅰ亚区系分别是该工艺条件下优势的氨氧化菌和亚硝酸氧化菌,但工业装置的克隆文库中却没有找到任何与硝化菌序列相近的克隆,这说明工业装置中硝化菌的多度较低。进一步使用Taqman荧光探针实时定量PCR测定了样品中Nitrospira属的多度,实验室装置中Nitrospira属16S rDNA的拷贝数达到3.4×106个/微克基因组DNA,而工业装置的测定值不到实验室装置的1/300。这些试验结果都表明工业装置好氧池微生物群落中缺少适当的硝化菌群是造成其硝化能力低下的重要原因。提高菌群中Nitrosomonas属和Nitrospira属的多度是解决工业装置硝化能力低下的关键。