ANAMMOX process start up and stabilization with an anaerobic seed in Anaerobic Membrane Bioreactor (AnMBR)

ANaerobic AMMonium OXidation (ANAMMOX) process, an advanced biological nitrogen removal alternative to traditional nitrification--denitrification removes ammonia using nitrite as the electron acceptor without oxygen. The feasibility of enriching anammox bacteria from anaerobic seed culture to start up an Anaerobic Membrane Bioreactor (AnMBR) for N-removal is reported in this paper. The Anammox activity was established in the AnMBR with anaerobic digester seed culture from a Sewage Treatment Plant in batch mode with recirculation followed by semi continuous process and continuous modes of operation. The AnMBR performance under varying Nitrogen Loading Rates (NLR) and HRTs is reported for a year, in terms of nitrogen transformations to ammoniacal nitrogen, nitrite and nitrate along with hydrazine and hydroxylamine. Interestingly ANAMMOX process was evident from simultaneous Amm-N and nitrite reduction, consistent nitrate production, hydrazine and hydroxylamine presence, notable organic load reduction and bicarbonate consumption.     

Reactivation of biocatalysts after long-term storage and startup of the DEAMOX process

Studies of our laboratory have led to elaboration of the DEAMOX (DEnitrifying AMmonia OXidation) technology intended for removal of nitrogen contaminants from wastewater. The DEAMOX process comprises two anaerobic stages implemented by the same sludge biocatalyst, namely, denitratation (conversion of nitrate to nitrite) and anammox reaction (ANaerobic AMmonium nitrogen OXidation by nitrite). The results of reactivation of biocatalysts after their long-term storage (5 and 16 months) and successful startup of the DEAMOX process in two modifications (S- and O-) are described. An S-DEAMOX process was launched using a sludge biocatalyst with restored anammox activity of 20.1 mg N/g VSS/day; this process provided removal of 78% of nitrogen in reactor over 20 days. The launched O-DEAMOX process with the sludge biocatalyst with anammox activity of 6.1 mg N/g VSS/day provided for 87% removal of the total nitrogen compounds over 30 days. Two different electron donors were used at the stage of nitrate conversion to nitrite, namely, an inorganic donor, sulfide (S-DEAMOX), and an organic one, acetate (O-DEAMOX).     

Nitrogen removal from wastewaters at low C/N ratios with ammonium and acetate as electron donors

A denitrifying upflow anaerobic sludge blanket (UASB) reactor was operated at different nitrate loading rates at a C/N ratio of 1.2, with acetate as an electron donor. This resulted in an increase in the accumulation of nitrite. After this, the UASB reactor was supplemented with 100 mg NH4+-Nl(-1) d(-1), while acetate was gradually limited in the medium. This prevented nitrite accumulation at a C/N ratio of 0.6 due to an enhanced nitrite reduction rate achieved in the reactor. An increasing amount of ammonium was consumed when the C/N ratio was lowered in the medium. This suggested that ammonium was used as an alternative electron donor during denitrification, which is supported by nitrogen balances. Nitrite was shown to be toxic for the nitrogen removal process at 200-400 mg NO2--N(l(-1) when the C/N ratio was decreased to 0.4 leading to formation of ammonium. The present study showed that addition of ammonium as an alternative electron donor for denitrification achieved a nitrogen removal process with negligible accumulation of undesirable intermediates.     

亚硝酸盐对污水生物除磷影响的研究进展

亚硝酸盐作为生物硝化和反硝化的中间产物, 存在于污水生物脱氮除磷系统中。对于生物强化除磷工艺亚硝酸盐既是电子受体用于反硝化除磷, 同时又是抑制剂影响生物除磷过程。本文综述了聚磷菌在厌氧、好氧和缺氧环境中的代谢机理, 在此基础上分别从好氧除磷和反硝化除磷两方面介绍了亚硝酸盐对污水生物除磷影响的研究, 同时概述了亚硝酸盐对生物除磷的抑制机理, 并对该领域的研究提出了个人见解。     

Evaluation of Anammox and denitrification during anaerobic digestion of poultry manure

Two approaches based on ne w process development and biological nitrogen transformation were investigated in a bench study for removing nitrogen as N2 gas from poultry waste while stabilizing the wastes. The process, known as "Anammox", was explored in batch anaerobic culture using serum bottles. The Anammox process involves the use of nitrite as an electron acceptor in the bacterially mediated oxidation of ammonia to yield N2. Studies are described wherein nitrite was added to poultry waste and the effects on ammonium levels were monitored. About 13-22% ammonium removal was observed with the inoculation of returned activated sludge, and the total ammonium reduction was not proportional to the reduction of nitrite, thereby suggesting that Anammox was less competitive under the conditions in our studies. The addition of nitrite and nitrate was not inhibitory to the process based on gas generation and COD reduction. The classical nitrogen removal process of nitrification followed with denitrification offers a more reliable basis for nitrogen removal from poultry wastes.     

Batch culture enrichment of ANAMMOX populations from anaerobic and aerobic seed cultures

Discharge of nitrate and ammonia rich wastewaters into the natural waters encourage eutrophication, and contribute to aquatic toxicity. Anaerobic ammonium oxidation process (ANAMMOX) is a novel biological nitrogen removal alternative to nitrification-denitrification, that removes ammonia using nitrite as the electron acceptor. The feasibility of enriching the ANAMMOX bacteria from the anaerobic digester sludge of a biomethanation plant treating vegetable waste and aerobic sludge from an activated sludge process treating domestic sewage is reported in this paper. ANAMMOX bacterial activity was monitored and established in terms of nitrogen transformations to ammonia, nitrite and nitrate along with formation of hydrazine and hydroxylamine.     

Utilization of nitrate byBeggiatoa alba

Beggiatoa alba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 mM.B. alba coupledin vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction.B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction byB. alba was ammonia; N₂ or N₂O were not produced. The nitrate reductase activity inB. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. TheB. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of theB. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus,B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.Non-standard abbreviations MV Methyl viologen - DT dithionite - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase - PPO 2-diphenyloxazole - POPOP 1,4-(bis)-[2-(5-phenyloxazolyl)] benzene - TCA trichloroacetic acid - CCCP carbonylcyanidem-chlorophenylhydrazone - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - TTFA thenoyltrifluoroacetone - PHEN 1,10-phenanthroline - HOQNO 2-heptyl 4-hydroxyquinoline-n-oxide - 8HQ 8-hydroxyquinoline     

Graphite electrodes as electron donors for anaerobic respiration

It has been demonstrated previously that Geobacter species can transfer electrons directly to electrodes. In order to determine whether electrodes could serve as electron donors for microbial respiration, enrichment cultures were established from a sediment inoculum with a potentiostat-poised graphite electrode as the sole electron donor and nitrate as the electron acceptor. Nitrate was reduced to nitrite with the consumption of electrical current. The stoichiometry of electron and nitrate consumption and nitrite accumulation were consistent with the electrode serving as the sole electron donor for nitrate reduction. Analysis of 16 rRNA gene sequences demonstrated that the electrodes supplied with current were specifically enriched in microorganisms with sequences most closely related to the sequences of known Geobacter species. A pure culture of Geobacter metallireducens was shown to reduce nitrate to nitrite with the electrode as the sole electron donor with the expected stoichiometry of electron consumption. Cells attached to the electrode appeared to be responsible for the nitrate reduction. Attached cells of Geobacter sulfurreducens reduced fumarate to succinate with the electrode as an electron donor. These results demonstrate for the first time that electrodes may serve as a direct electron donor for anaerobic respiration. This finding has implications for the harvesting of electricity from anaerobic sediments and the bioremediation of oxidized contaminants.     

Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway

Abstract: A total of 28 nitrate-reducing bacteria were isolated from marine sediment (Mediterranean coast of France) in which dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. ¹⁵N ammonium production from ¹⁵N nitrate by an Enterobacter sp. and a Vibrio sp., the predominant bacteria involved in nitrate ammonification in marine sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (1 mM) produced ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in the presence of nitrate, ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium. These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate formation, instead of ethanol formation which was favoured in the absence of nitrate.     

Oxygen exposure promotes fuel diversity for Shewanella oneidensis microbial fuel cells

Miniature microbial fuel cells (mini-MFCs) were used to monitor the current generated by Shewanella oneidensis DSP10 under both anaerobic and aerobic conditions when exposed to glucose as a potential electron donor. In addition to glucose, other carbon fuels including fructose, sucrose, acetate, and ascorbic acid were also tested. When the anolyte containing S. oneidensis was grown in the presence of oxygen, power densities of 270+/-10, 350+/-20, and 120+/-10 W/m(3) were recorded from the mini-MFC for glucose, fructose, and ascorbic acid electron donors, respectively, while sucrose and acetate produced no response. The power produced from glucose decreased considerably (相似文献   

以N-甲基吡咯烷酮(NMP)为电子供体进行反硝化的可行性及实验研究

【目的】利用N-甲基吡咯烷酮(N-methylpyrrolidone, NMP)作为电子供体进行反硝化实验,以实现废水资源化。【方法】分别将NMP废水和葡萄糖作为电子供体加入到模拟的城市污水处理尾水中进行反硝化,比较2种电子供体去除硝酸盐的规律。同时考查NMP在反硝化过程中的氮素释放规律,并对所释放的氮素进行后续处理。最后再对它们作为电子供体时的反硝化污泥采用高通量测序,从微生物群落的角度分析NMP作为电子供体时其作用机理是否相同。【结果】当以NMP为电子供体时,硝酸盐氮的去除速率比葡萄糖为电子供体时要快67%。在8 h的反硝化结束后,剩余的硝酸盐氮、累积的亚硝酸盐氮和NMP本身所释放氨氮之和的总氮,与葡萄糖为电子供体时相近。【结论】NMP废水可以作为电子供体用于城镇污水处理厂的深度脱氮。对2种碳源所驯化的反硝化污泥样品进行高通量分析表明,NMP与葡萄糖作为电子供体用于反硝化反应时,相关的作用机理是不同的。该项研究结果对利用含氮杂环化合物作为电子供体进行反硝化具有重要的理论指导意义。     

利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation,DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程,对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor,SBR)内接种混合物,分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d,定期检测反应器基质浓度变化、计算转化速率;利用16S rRNA基因系统发育分析研究功能微生物的多样性,利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌,未检测到DAMO古菌;以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物;3个反应器的脱氮速率经过初始低速期、快速提升期,最终达到稳定,但2号快速提升期开始时间比1、3号晚了80 d左右,达到稳定的时间更长,稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响;以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存,DAMO古菌可能是协同体系中脱氮速率的限制性因素。     

Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor

Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate.     

The effect of propionic to acetic acid ratio on anaerobic-aerobic (low dissolved oxygen) biological phosphorus and nitrogen removal

In this paper, three lab-scale sequencing batch reactors (SBR-A, B, and C) operated with anaerobic/aerobic (low dissolved oxygen, 0.15-0.45 mg L(-1)) configuration were long-term cultured, respectively with single acetic acid and propionic/acetic acid of 1/1 and 2/1 (carbon molar ratio), and the comparisons of anaerobic and aerobic transformations of phosphorus and nitrogen among them were made. With the increase of propionic/acetic acid, lower anaerobic phosphorus release and higher phosphorus release to short-chain fatty acids uptake ratio were observed, and less anaerobic and aerobic transformations of glycogen and poly-3-hydroxybutyrate as well as total polyhydroxyalkanoates occurred, but the transformations of poly-3-hydroxyvalerate and poly-3-hydroxy-2-methyvalerate increased. The phosphorus removal efficiency was respectively 81, 94 and 97% in SBR-A, B and C. Almost all ammonium was removed and no significant nitrite was accumulated at different propionic/acetic acid ratios. However, the nitrate accumulation and total nitrogen removal were observed to be affected by propionic/acetic acid ratio. The total nitrogen removal efficiency was 61, 68 and 82%, and the aerobic end nitrate concentration was 8.05, 6.40 and 3.54 mg L(-1) in three SBRs, respectively. All the above studies indicated that the sole acetic acid caused more nitrate accumulation than propionic and acetic acids mixture, and a pertinent increase of wastewater propionic/acetic acid ratio was of benefit to both nitrogen and phosphorus removal in an anaerobic/aerobic (low dissolved oxygen) biological wastewater treatment process.     

Coupling between anammox and autotrophic denitrification for simultaneous removal of ammonium and sulfide by enriched marine sediments

In the present study, the capacity of enrichments derived from marine sediments collected from different sites of the Mexican littoral to perform anaerobic ammonium oxidation (anammox) coupled to sulfide-dependent denitrification for simultaneous removal of ammonium and sulfide linked to nitrite reduction was evaluated. Sulfide-dependent denitrification out-competed anammox during the simultaneous oxidation of sulfide and ammonium. Significant accumulation of elemental sulfur (ca. 14–30 % of added sulfide) occurred during the coupling between the two respiratory processes, while ammonium was partly oxidized (31–47 %) due to nitrite limitation imposed in sediment incubations. Nevertheless, mass balances revealed up to 38 % more oxidation of the electron donors available (ammonium and sulfide) than that expected from stoichiometry. Recycling of nitrite, from nitrate produced through anammox, is proposed to contribute to extra oxidation of sulfide, while additional ammonium oxidation is suggested by sulfate-reducing anammox (SR-anammox). The complex interaction between nitrogenous and sulfurous compounds occurring through the concomitant presence of autotrophic denitrification, conventional anammox and SR-anammox may significantly drive the nitrogen and sulfur fluxes in marine environments.     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998     

Carbohydrate oxidation coupled to Fe(III) reduction, a novel form of anaerobic metabolism

An isolate, designated GC-29, that could incompletely oxidize glucose to acetate and carbon dioxide with Fe(III) serving as the electron acceptor was recovered from freshwater sediments of the Potomac River, Maryland. This metabolism yielded energy to support cell growth. Strain GC-29 is a facultatively anaerobic, gram-negative motile rod which, in addition to glucose, also used sucrose, lactate, pyruvate, yeast extract, casamino acids or H2 as alternative electron donors for Fe(III) reduction. Stain GC-29 could reduce NO3(-), Mn(IV), U(VI), fumarate, malate, S2O3(2-), and colloidal S0 as well as the humics analog, 2,6-anthraquinone disulfonate. Analysis of the almost complete 16S rRNA sequence indicated that strain GC-29 belongs in the Shewanella genus in the epsilon subdivision of the Proteobacteria. The name Shewanella saccharophilia is proposed. Shewanella saccharophilia differs from previously described fermentative microorganisms that metabolize glucose with the reduction of Fe(III) because it transfers significantly more electron equivalents to Fe(III); acetate and carbon dioxide are the only products of glucose metabolism; energy is conserved from Fe(III) reduction; and glucose is not metabolized in the absence of Fe(III). The metabolism of organisms like S. saccharophilia may account for the fact that glucose is metabolized primarily to acetate and carbon dioxide in a variety of sediments in which Fe(III) reduction is the terminal electron accepting process.     

Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15 degrees C and a maximum temperature of 37 degrees C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K(m) of nitrite consumption was less than 3 microM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp. (Lupinus)

Induction, energy gain, effect on growth, and interaction of nitrate and nitrite reduction of Bradyrhizobium sp. (Lupinus) USDA 3045 were characterized. Both nitrate and nitrite were reduced in air, although nitrite reduction was insensitive to ammonium inhibition. Anaerobic reduction of both ions was shown to be linked with energy conservation. A dissimilatory ammonification process was detected, which has not been reported in rhizobia so far. Nevertheless, anaerobic conversion of nitrate to ammonium was lower than 40%, which suggests the presence of an additional, nitrite reductase of denitrifying type. Nitrite toxicity caused a non-linear relationship between biomass produced and >2 mM concentrations of each N oxyanion consumed. At > or =5 mM initial concentrations of nitrate, a stoichiometric nitrite accumulation occurred and nitrite remained in the medium. This suggests an inhibition of nitrite reductase activity by nitrate, presumably due to competition with nitrate reductase for electron donors. Lowering of growth temperature almost completely diminished nitrite accumulation and enabled consumption as high as 10 mM nitrate, which confirms such a conclusion.     

Relationship of hydrogen bioavailability to chromate reduction in aquifer sediments

Biological Cr(VI) reduction was studied in anaerobic sediments from an aquifer in Norman, Okla. Microcosms containing sediment and mineral medium were amended with various electron donors to determine those most important for biological Cr(VI) reduction. Cr(VI) (about 340 microM) was reduced with endogenous substrates (no donor), or acetate was added. The addition of formate, hydrogen, and glucose stimulated Cr(VI) reduction compared with reduction in unamended controls. From these sediments, an anaerobic Cr(VI)-utilizing enrichment was obtained that was dependent upon hydrogen for both growth and Cr(VI) reduction. No methane was produced by the enrichment, which reduced about 750 microM Cr(VI) in less than six days. The dissolved hydrogen concentration was used as an indicator of the terminal electron accepting process occurring in the sediments. Microcosms with sediments, groundwater, and chromate metabolized hydrogen to a concentration below the detection limits of the mercury vapor gas chromatograph. In microcosms without chromate, the hydrogen concentration was about 8 nM, a concentration comparable to that under methanogenic conditions. When these microcosms were amended with 500 microM Cr(VI), the dissolved hydrogen concentration quickly fell below the detection limits. These results showed that the hydrogen concentration under chromate-reducing conditions became very low, as low as that reported under nitrate- and manganese-reducing conditions, a result consistent with the free energy changes for these reactions. The utilization of formate, lactate, hydrogen, and glucose as electron donors for Cr(VI) reduction indicates that increasing the availability of hydrogen results in a greater capacity for Cr(VI) reduction. This conclusion is supported by the existence of an enrichment dependent upon hydrogen for growth and Cr(VI) reduction.