海洋氮循环中细菌的厌氧氨氧化

细菌厌氧氨氧化过程是在一类特殊细菌的厌氧氨氧化体内完成的以氨作为电子供体硝酸盐作为电子受体的一种新型脱氮反应.厌氧氨氧化菌的发现,改变人们对传统氮的生物地球化学循环的认识:反硝化细菌并不是大气中氮气产生的唯一生物类群.而且越来越多的证据表明,细菌厌氧氨氧化与全球的氮物质循环密切相关,估计海洋细菌的厌氧氨氧化过程占到全球海洋氮气产生的一半左右.由于氮与碳的循环密切相关,因此可以推测,细菌的厌氧氨氧化会影响大气中的二氧化碳浓度,从而对全球气候变化产生重要影响.另外,由于细菌厌氧氨氧化菌实现了氨氮的短程转化,缩短了氮素的转化过程,因此为开发更节约能源、更符合可持续发展要求的废水脱氮新技术提供了生物学基础.     

Influence of Plants and Organic Matter on the Nitrogen Removal in Laboratory‐Scale Model Subsurface Flow Constructed Wetlands Inoculated with Anaerobic Ammonium Oxidizing Bacteria

Anaerobic oxidation of ammonium has become an alternative for the treatment of wastewater with high ammonium loads, and it was also suggested to be involved in the nitrogen removal process in constructed wetlands. Nonetheless, its role has not been well evaluated as yet. In this paper, results of a lab‐scale study are presented focusing on the evaluation of the role of Anammox bacteria, plants, applied ammonia, nitrite nitrogen loads, and the presence of organic matter in nitrogen transformation processes in subsurface‐flow constructed wetlands. The inoculation of the experimental model wetlands with active Anammox biomass increased the total nitrogen and ammonium removal rates to values up to 5.7 g N/m² d, which is almost 10 times higher than those values reported for subsurface flow constructed wetlands. Although the presence of plants caused a higher removal rate, the role of the plants became less important with high nitrite influent concentration. Because the unplanted experimental system without the addition of any organic carbon source showed also high nitrogen removal rates, it can be concluded that beside the potential for “conventional” denitrification in the planted systems the main mechanism for explaining the high nitrogen removal rates obtained during the experiments was the anaerobic ammonia oxidation. The assay of the formation of hydrazine from hydroxylamine and the findings of the molecular biology tests fitted with the positive results for potential Anammox activity obtained in the bottle test. The addition of organic carbon, specifically acetate, apparently had no great influence on Anammox activity, which is in agreement with the findings reported by other authors. Nevertheless, the addition influenced the redox potential. Some questions are still left open, which are mainly associated with the scaling up of these results and the inoculation of Anammox biomass in full‐scale systems.     

厌氧氨氧化菌的中心代谢研究进展

摘要: 厌氧氨氧化是以NH +4为电子供体,以NO－2为电子受体产生N2的生物反应。厌氧氨氧化菌是厌氧氨氧化过程的执行者,在废水生物脱氮和地球氮素循环中扮演着重要角色。研究厌氧氨氧化菌的代谢特性,将有助于理解厌氧氨氧化过程,开发厌氧氨氧化工艺。厌氧氨氧化菌是化能自养型细菌,以CO2或HCO－3为碳源,并通过偶联NH+4氧化和NO －2还原的生物反应获得能量。在NH+4/NO－2的生物氧化还原反应过程中,检出了中间产物N2H4,但未检出其他中间产物(如NH2OH、NO)。此外,由基因组信息推断,厌氧氨氧化菌     

Anammox-zeolite system acting as buffer to achieve stable effluent nitrogen values

For a successful nitrogen removal, Anammox process needs to be established in line with a stable partial nitritation pretreatment unit since wastewater influent is mostly unsuitable for direct treatment by Anammox. Partial nitritation is, however, a critical bottleneck for the nitrogen removal since it is often difficult to maintain the right proportions of NO₂-N and NH₄-N during long periods of time for Anammox process. This study investigated the potential of Anammox-zeolite biofilter to buffer inequalities in nitrite and ammonium nitrogen in the influent feed. Anammox-zeolite biofilter combines the ion-exchange property of zeolite with the biological removal by Anammox process. Continuous-flow biofilter was operated for 570 days to test the response of Anammox-zeolite system for irregular ammonium and nitrite nitrogen entries. The reactor demonstrated stable and high nitrogen removal efficiencies (approximately 95 %) even when the influent NO₂-N to NH₄-N ratios were far from the stoichiometric ratio for Anammox reaction (i.e. NO₂-N to NH₄-N ranging from 0 to infinity). This is achieved by the sorption of surplus NH₄-N by zeolite particles in case ammonium rich influent came in excess with respect to Anammox stoichiometry. Similarly, when ammonium-poor influent is fed to the reactor, ammonium desorption took place due to shifts in ion-exchange equilibrium and deficient amount were supplied by previously sorbed NH₄-N. Here, zeolite acted as a preserving reservoir of ammonium where both sorption and desorption took place when needed and this caused the Anammox-zeolite system to act as a buffer system to generate a stable effluent.     

Removal of multiple nitrogenous wastes by Aspergillus niger in a continuous fixed-slab reactor

A biofilter reactor, to which is attached a large variety of microorganisms, can be employed to treat circulating water in an intensive aquaculture system. Some nitrogen-containing wastes, such as ammonium and nitrite, are toxic to the aquatic organisms. The removal rates of the nitrogenous wastes are regarded as indices for the efficiency of treatment by biofilters. In this study, a fungus that was characterized as being able to remediate multiple nitrogenous wastes was identified as Aspergillus niger NBG5. In a continuous fixed-slab reactor, the heterotrophic fungus utilized ammonium, nitrite, protein, and glucose simultaneously. The fungus assimilated ammonium, nitrite and protein at rates of 0.247, 0.07 and 0.096 g-N/g-cell/day, respectively, at 22 degrees C. The remediation rates of ammonium nitrogenous wastes decreased by a factor of eight at 35 degrees C, while the specific growth rates slightly increased. For nitrogenous wastes, ammonium was a preferred substrate but its rate of consumption declined significantly as temperature increased. The nitrogen consumption rates were inconsistent with the cell yields at high temperature. Further analysis of consumption ratios of C/N revealed that cells grew predominantly from the carbon at high temperature. The A. niger NBG5 consumed glucose rapidly at specific rates of 2-2.5 g-C/g-cell/day at 35 degrees C in the presence of ammonium and nitrite; while sluggish consumption of glucose was observed in the protein substrate. The protein could serve as an alternative carbon source. Further ANOVA statistical analysis with P < 0.05 revealed no significant effects of temperature on the specific growth rates of A. niger on the SG-NH4 and milk-protein substrates, whereas significant effects on the C/N ratio at culture temperatures higher than 25 degrees C were observed. These findings indicated that the carbon utilization rate increased with high temperature, whereas nitrogen utilization increased as temperature declined. A suitable operational temperature was suggested, depending upon the amount of waste contents of C/N. A high temperature stimulates the use of carbon waste, while a low temperature favors remediation of all nitrogenous wastes.     

Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium

Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite and produce N(2). They reside in many natural ecosystems and contribute significantly to the cycling of marine nitrogen. Anammox bacteria generally live under ammonium limitation, and it was assumed that in nature anammox bacteria depend on other biochemical processes for ammonium. In this study we investigated the possibility of dissimilatory nitrate reduction to ammonium by anammox bacteria. Physically purified Kuenenia stuttgartiensis cells reduced (15)NO(3) (-) to (15)NH(4) (+) via (15)NO(2) (-) as the intermediate. This was followed by the anaerobic oxidation of the produced ammonium and nitrite. The overall end-product of this metabolism of anammox bacteria was (15)N(15)N dinitrogen gas. The nitrate reduction to nitrite proceeds at a rate of 0.3 +/- 0.02 fmol cell(-1) day(-1) (10% of the 'normal' anammox rate). A calcium-dependent cytochrome c protein with a high (305 mumol min(-1) mg protein(-1)) rate of nitrite reduction to ammonium was partially purified. We present evidence that dissimilatory nitrate reduction to ammonium occurs in Benguela upwelling system at the same site where anammox bacteria were previously detected. This indicates that anammox bacteria could be mediating dissimilatory nitrate reduction to ammonium in natural ecosystems.     

厌氧铵氧化过程中关键酶及相关分子标记在生态学研究中的应用进展

厌氧铵氧化是由微生物介导的氮素循环过程中的重要途径之一。近20年来,通过对厌氧铵氧化细菌生态学、基因组学和生理代谢特性的探索,人们对其微生物学机制已经有了较多的认识:厌氧铵氧化细菌通过亚硝酸盐还原酶将亚硝酸根离子还原为一氧化氮,进而与铵离子结合在联氨合成酶的作用下生成联氨,最后通过联氨氧化酶的催化产生终产物氮气。同时,对参与这些过程的关键酶及其功能基因的认识有助于选择新的分子标记,从而为研究厌氧铵氧化细菌的多样性和分子生态学特征提供新的工具,以弥补16S rRNA基因特异性相对较低且难以与生态功能关联等方面的不足。对目前已知的参与厌氧铵氧化过程的3种关键酶的研究历程和现状进行了评述,并总结了利用3种功能基因进行厌氧铵氧化细菌生态学研究的最新进展。     

Anaerobic ammonium oxidation in marine environments: contribution to biogeochemical cycles and biotechnological developments for wastewater treatment

Microbial processes are responsible for most reactions involved in the nitrogen cycle in the oceans, which determine the fluxes of this crucial nutrient in these environments. The present review provides an overview of the contribution of anaerobic ammonium oxidation (Anammox) to marine biogeochemical processes. Besides the conventional Anammox process, anaerobic ammonium oxidation coupled to the microbial reduction of alternative electron acceptors, such as sulfate (Sulfammox), ferric iron (Feammox), and natural organic matter (NOM-dependent Anammox) is also described in the context of global marine biogeochemical cycles. Also, the complex interactions among the oceanic biogeochemical cycles of N, S and Fe are discussed at the light of the new findings available in the literature. The review also underlines the important role of the microbial processes performing the Anammox reaction in the development of wastewater treatment systems for the removal of nitrogen from saline effluents. Strategies to enrich and immobilize Anammox bacteria in different reactor configurations for the treatment of saline wastewaters are also described as well as future directions for novel biotechnological developments based on Anammox.

     

流加菌种对厌氧氨氧化工艺的影响

厌氧氨氧化工艺具有很高的容积氮去除速率,现已成功应用于污泥压滤液等含氨废水的脱氮处理,容积氮去除速率高达9.5 kg/(m3·d)。但由于厌氧氨氧化菌为自养型细菌,生长缓慢,对环境条件敏感,致使厌氧氨氧化工艺启动时间过长,运行容易失稳,并且不适合处理有机含氨废水和毒性含氨废水,极大地限制了该工艺的进一步推广应用。为了克服厌氧氨氧化工艺实际应用中存在的问题,结合发酵工业中常用的菌种流加技术,提出了一种新型的菌种流加式厌氧氨氧化工艺,研究了该新型工艺在厌氧氨氧化工艺的启动过程、稳定运行以及处理有机含氨废水和毒性含氨废水等方面的应用情况。结果表明,通过向反应器内补加优质厌氧氨氧化菌种,可提高厌氧氨氧化菌数量及其在菌群中的比例,强化厌氧氨氧化功能。据此研发的菌种流加式厌氧氨氧化工艺不仅可以实现快速启动,而且可以稳定运行,并突破了有机物和毒物所致的运行障碍,拓展了厌氧氨氧化工艺的应用范围。     

Anaerobic oxidation of ammonium is a biologically mediated process.

A newly discovered process by which ammonium is converted to dinitrogen gas under anaerobic conditions (the Anammox process) has now been examined in detail. In order to confirm the biological nature of this process, anaerobic batch culture experiments were used. All of the ammonium provided in the medium was oxidized within 9 days. In control experiments with autoclaved or raw wastewater, without added sludge or with added sterilized (either autoclaved or gamma irradiated) sludge, no changes in the ammonium and nitrate concentrations were observed. Chemical reactions could therefore not be responsible for the ammonium conversion. The addition of chloramphenicol, ampicillin, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and mercuric chloride (HgIICl2) completely inhibited the activity of the ammonium-oxidizing sludge. Furthermore, the rate of ammonium oxidation was proportional to the initial amount of sludge used. It was therefore concluded that anaerobic ammonium oxidation was a microbiological process. As the experiments were carried out in an oxygen-free atmosphere, the conversion of ammonium to dinitrogen gas did not even require a trace of O2. That the end product of the reaction was nitrogen gas has been confirmed by using 15NH4+ and 14NO3-. The dominant product was 14-15N2. Only 1.7% of the total labelled nitrogen gas produced was 15-15N2. It is therefore proposed that the N2 produced by the Anammox process is formed from equimolar amounts of NH4+ and NO3-.     

Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene.

15N tracer methods and gas chromatography coupled to an electron capture detector were used to investigate dissimilatory reduction of nitrate and nitrite by the rumen microbiota of a fistulated cow. Ammonium was the only 15N-labeled end product of quantitative significance. Only traces of nitrous oxide were detected as a product of nitrate reduction; but in experiments with nitrite, up to 0.3% of the added nitrogen accumulated as nitrous oxide, but it was not further reduced. Furthermore, when 13NO3- was incubated with rumen microbiota virtually no [13N]N2 was produced. Acetylene partially inhibited the reduction of nitrite to ammonium as well as the formation of nitrous oxide. It is suggested that in the rumen ecosystem nitrous oxide is a byproduct of dissimilatory nitrite reduction to ammonium rather than a product of denitrification and that the latter process is absent from the rumen habitat.     

高含氮稻田深层土壤的氨氧化古菌和厌氧氨氧化菌共存及对氮循环影响

随着海洋生态系统中的厌氧氨氧化反应和氨氧化古菌的发现,自然生态系统的氮循环过程被重新认识,但是目前尚无在陆地深层的相关报道。结合同位素示踪与分子生物学技术探索了稻田深层土壤中anammox与AOA的存在及特性。结果表明,在沼渣处理废水浇灌的高含氮稻田深层土壤中,anammox与AOA共存。通过构建克隆文库发现,此土壤中厌氧氨氧化菌的生物多样性相对较低,35个克隆序列只分为4个独立操作单元(OTU),代表序列与Genebank数据库中已探明的厌氧氨氧化菌Candidatus 'Kuenenia stuttgartiensis’的同源性超过95%;对氨氧化古菌的分析发现,20个克隆子共得到5个OTU,其与基因库中土壤/沉积物进化分支关系最近,序列的同源性部分超过98%。同位素示踪的初步结果表明,anammox产生的氮气占此土壤总氮气生成量的24.1%-29.8%。AOA与anammox的共存为anammox反应的广泛存在与发生提供了新思路。     

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.     

Long-term stability of partial nitritation of swine wastewater digester liquor and its subsequent treatment by Anammox

Partial nitritation using inhibition of free ammonia and free nitric acid is an effective technique for the treatment of high concentrations of ammonium in wastewaters. This technique was applied to the digester liquor of swine wastewater and the stability of its long-term operation was investigated. Partial nitritation was successfully maintained at a nitrogen loading rate (NLR) of 1.0 kg N m(-3)d(-1) for 120 days without acclimatization of nitrite oxidizing bacteria (NOB) to the inhibitory compounds (free ammonia and free nitric acid). The conversion efficiencies of NH(4)-N to NO(2)-N and to NO(3)-N were determined to be around 58% and <5%, respectively. After the establishment of partial nitritation, the influence of swine wastewater on the Anammox reaction was examined using continuous flow treatment experiments. Consistent nitrogen removal was achieved for 70 days at a nitrogen removal rate (NRR) of 0.22 kg N m(-3)d(-1) and the color of Anammox bacteria changed from red to greyish black. The NO(2)-N consumption and the NO(3)-N production increased concurrently and the Anammox reaction ratio was estimated to be 1:1.67:0.53, which is different from that reported previously (1:1.32:0.26).     

New pathways for ammonia conversion in soil and aquatic systems

Ammonia conversion processes are essential for most soil and aquatic systems. Under natural conditions, the many possible reactions are difficult to analyze. For example, nitrification and denitrification have long been regarded as separate phenomena performed by different groups of bacteria in segregated areas of soils, sediments or aquatic systems sequentially in time. It has now been established that strict segregation in place and time of the two processes is not necessary and that both denitrifiers and nitrifiers have versatile metabolisms. However, the rates described for aerobic denitrifiers are very low compared to the rates observed under anoxic conditions. Also the rates of nitrifier denitrification are quite low, indicating that these conversions may not play an important role under natural conditions. In addition, these processes often result in the emission of quite large amounts of undesirable products, NO and N₂O. Heterotrophic nitrification might be of relevance for systems, that contain a high carbon to nitrogen ratio. Recently, a novel process (Anammox) has been discovered in which ammonium serves as the electron donor for denitrification of nitrite into dinitrogen gas. ¹⁵N labeling studies showed that hydrazine and hydroxylamine were important intermediates in this process. Enrichment cultures on ammonium, nitrite and bicarbonate resulted in the dominance of one morphotypical microorganism. The growth rate of the cultures is extremely low (doubling time 11 days), but the affinity for ammonium and nitrite and the conversion rates (9.2 10^–4 mol kg^–1 s^–1) are quite high. Some of the reported high nitrogen losses in soil and aquatic systems might be attributed to anaerobic ammonium oxidation. In addition, this conversion offers new opportunities for nitrogen removal, when it is combined with recently developed processes for partial nitrification.     

Heterotrophic nitrogen removal by <Emphasis Type="Italic">Providencia rettgeri</Emphasis> strain YL

Providencia rettgeri strain YL was found to be efficient in heterotrophic nitrogen removal under aerobic conditions. Maximum removal of NH₄ ⁺–N occurred under the conditions of pH 7 and supplemented with glucose as the carbon source. Inorganic ions such as Mg²⁺, Mn²⁺, and Zn²⁺ largely influenced the growth and nitrogen removal efficiency. A quantitative detection of nitrogen gas by gas chromatography was conducted to evaluate the nitrogen removal by strain YL. From the nitrogen balance during heterotrophic growth with 180 mg/l of NH₄ ⁺–N, 44.5% of NH₄ ⁺–N was in the form of N₂ and 49.7% was found in biomass, with only a trace amount of either nitrite or nitrate. The utilization of nitrite and nitrate during the ammonium removal process demonstrated that the nitrogen removal pathway by strain YL was heterotrophic nitrification-aerobic denitrification. A further enzyme assay of nitrate reductase and nitrite reductase activity under the aerobic condition confirmed this nitrogen removal pathway.     

Effects of mechanical stress on Anammox granules in a sequencing batch reactor (SBR)

The effect of shear stress on Anammox process was studied in a sequencing batch reactor (SBR). The reactor was operated during 218 days under different stirring speeds (60-250 rpm) in order to expose the system to different shear conditions and to study the stability of the Anammox granules referred to their biological activity and size. The nitrogen loading rate (NLR) fed to the SBR was kept around 0.3g N(L day)(-1). The nitrite (limiting substrate) removal percentage was 98% during most of the operational period. The specific Anammox activity of the biomass was practically constant and around 0.4 g N(g VSSday)(-1) and the average feret diameter of the formed granules was 0.64 mm. Obtained results indicated that stirring speeds up to 180 rpm have no negative effect on the performance of the Anammox process, whereas Anammox activity decreased to 40% when a rotating speed of 250 rpm was tested and the average diameter decreased in 45%, the concentration of solids in the effluent increased to 0.2g TSSL(-1) and nitrite was accumulated in the reactor up to 60 mg NL(-1).     

Combined process of urea nitrogen removal in anaerobic Anammox co-culture reactor

In this study, the feasibility of biological urea nitrogen removal in anaerobic Anammox co-culture was investigated. After 100 days of operation, complete urea nitrogen removal of 0.35 g (NH(2))(2)CO-N L(-1) d(-1) was achieved. The pure Anammox bacteria were obtained by percoll density-gradient centrifugation and found to be of incapable to hydrolyze urea. The ureolytic bacteria were isolated from the Anammox co-culture by the spread plate and streak. Comparative analysis of partial 16S rDNA sequence presented it belongs to Bacillus sp., and so named as Bacillus sp. LST-1. Fluorescence in situ hybridization was applied to identify the ratio of Bacillus sp. and Anammox in the reactor and the value was approximately 1:4. Urea nitrogen removal was realized in this autotrophic, anoxic reactor via the combined process of urea hydrolysis by Bacillus sp. LST-1 and ammonium oxidizing by Anammox. The investigation of this combined process might have an actual significance in engineering application for its low operational cost.     

Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost‐effective and environment‐friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere.     

Sensitivity analysis of a biofilm model describing a one-stage completely autotrophic nitrogen removal (CANON) process.

A mathematical model for nitrification and anaerobic ammonium oxidation (ANAMMOX) processes in a single biofilm reactor (CANON) was developed. This model describes completely autotrophic conversion of ammonium to dinitrogen gas. Aerobic ammonium and nitrite oxidation were modeled together with ANAMMOX. The sensitivity of kinetic constants and biofilm and process parameters to the process performance was evaluated, and the total effluent concentrations were, in general, found to be insensitive to affinity constants. Increasing the amount of biomass by either increasing biofilm thickness and density or decreasing porosity had no significant influence on the total effluent concentrations, provided that a minimum total biomass was present in the reactor. The ANAMMOX process always occurred in the depth of the biofilm provided that the oxygen concentration was limiting. The optimal dissolved oxygen concentration level at which the maximum nitrogen removal occurred is related to a certain ammonium surface load on the biofilm. An ammonium surface load of 2 g N/m2. d, associated with a dissolved oxygen concentration level of 1.3 g O2/m3 in the bulk liquid and with a minimum biofilm depth of 1 mm seems a proper design condition for the one-stage ammonium removal process. Under this condition, the ammonium removal efficiency is 94% (82% for the total nitrogen removal efficiency) (30 degrees C). Better ammonium removal could be achieved with an increase in the dissolved oxygen concentration level, but this would strongly limit the ANAMMOX process and decrease total nitrogen removal. It can be concluded that a one-stage process is probably not optimal if a good nitrogen effluent is required. A two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.