Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.     

Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes

A lab-scale sequencing batch reactor fed with real municipal wastewater was used to study nitrous oxide (N(2)O) emissions from simulated wastewater treatment processes. The experiments were performed under four different controlled conditions as follows: (1) fully aerobic, (2) anoxic-aerobic with high dissolved oxygen (DO) concentration, (3) anoxic-aerobic with low DO concentration, and 4) intermittent aeration. The results indicated that N(2)O production can occur from both incomplete nitrification and incomplete denitrification. N(2)O production from denitrification was observed in both aerobic and anoxic phases. However, N(2)O production from aerobic conditions occurred only when both low DO concentrations and high nitrite concentration existed simultaneously. The magnitude of N(2) O produced via anoxic denitrification was lower than via oxic denitrification and required the presence of nitrite. Changes in DO, ammonium, and nitrite concentrations influenced the magnitude of N(2)O production through denitrification. The results also suggested that N(2)O can be produced from incomplete denitrification and then released to the atmosphere during aeration phase due to air stripping. Therefore, biological nitrogen removal systems should be optimized to promote complete nitrification and denitrification to minimize N(2)O emissions.     

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.     

Respirometric Activities of Heterotrophic and Nitrifying Populations in Aerobic Granules Developed at Different Substrate N/COD Ratios

Aerobic granules were successfully developed at substrate N/COD ratios ranging from 5/100 to 30/100 by weight. By measuring respective respirometric activities of heterotrophic, ammonia-oxidizing, and nitrite-oxidizing bacteria, it was found that the relative abundance of nitrifying bacteria over heterotrophs in aerobic granules was closely related to the substrate N/COD ratios. Results further showed that the populations of both ammonia and nitrite oxidizers were significantly enriched with the increase of the substrate N/COD ratio, while a decreasing trend of heterotrophic population was observed in the aerobic granules. These seem to indicate that high substrate N/COD ratio favors the selection of nitrifying bacteria in the aerobic granules, while the relative activity of nitrifying population against heterotrophic population evolved until a balance between two populations was reached in the aerobic granular sludge community. Moreover, cell elemental composition was correlated with the shift in microbial populations, e.g., the enriched nitrifying population in the aerobic granules resulted in a high cell nitrogen content normalized to cell carbon content. This study provides a good insight into microbial interaction in aerobic granules.     

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.     

Evaluation on factors influencing the heterotrophic growth on the soluble microbial products of autotrophs

In this work, the heterotrophic growth on the microbial products of autotrophs and the effecting factors were evaluated with both experimental and modeling approaches. Fluorescence in situ hybridization (FISH) analysis illustrated that ammonia oxidizers (AOB), nitrite oxidizers (NOB), and heterotrophs accounted for about 65%, 20%, and 15% of the total bacteria, respectively. The mathematical evaluation of experimental data reported in literature indicated that heterotrophic growth in nitrifying biofilm (30–50%) and granules (30%) was significantly higher than that of nitrifying sludge (15%). It was found that low influent ammonium resulted in a lower availability of soluble microbial products (SMP) and a slower heterotrophic growth, but high ammonium (>150 mg N L⁻¹) feeding would lead to purely AOB dominated sludge with high biomass‐associated products contained effluent, although the absolute heterotrophic growth increased. Meanwhile, the total active biomass concentration increased gradually with the increasing solids retention time, whereas the factions of active AOB, NOB, and heterotrophs varied a lot at different solids retention times. This work could be useful for better understanding of the autotrophic wastewater treatment systems. Biotechnol. Bioeng. 2011; 108:804–812. © 2010 Wiley Periodicals, Inc.     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。     

Aerobic denitrification by novel isolated strain using NO-₂-N as nitrogen source

Biological denitrification reaction can be achieved under aerobic environment. Few aerobic denitrifiers using nitrite as sole nitrogen source were identified. Using nitrite as the sole nitrogen source, this work assessed the denitrification activity of yy7, an aerobic heterotrophic denitrifier identified as Pseudomonas sp. (94% similarity) by 16S rRNA sequencing analysis. The logistic equation describes the cell growth curve, yielding a generation time of 2.9h at an initial 18 mg l(-1)NO(-)?-N. Reduction of NO(-)?-N was primarily achieved during its logarithmic growth phase, and was accompanied by an increase in suspension pH and near complete consumption of dissolved oxygen. Three genes relating to nirK, norB, and nosZ were noted to involve in isolate strain. Isolate yy7 can survive and remove up to 40 mg l(-1)NO(-)?-N and, hence, can be applied as an effective aerobic denitrifier during simultaneous nitrification and denitrification via nitrite processes.     

Shortcut biological nitrogen removal in hybrid biofilm/suspended growth reactors

A shortcut biological nitrogen removal (SBNR) process converts ammonium directly through nitrite to nitrogen gas, thus requiring less aeration and carbon. We evaluated a hybrid SBNR (HSBNR) reactor containing an anoxic tank followed by an aerobic tank and a settling tank. The aerobic tank was filled with polyvinyl alcohol sponge media (20%, v/v) to attach and retain ammonium oxidizers. Two configurations of the HSBNR reactor were tested for treating a wastewater with high strength ammonium and organic electron donor. The HSNBR reactors accumulated nitrite stably for 1.5 years and maintained a high free ammonia (FA) concentration (20–25 mg/L) and a low dissolved oxygen (DO) concentration (<1 mg/L) in the aerobic tank. Apparently, the biofilm carriers increased the solids retention time (SRT) for ammonium oxidizers, while high FA and low DO selected against nitrite oxidizers and promoted direct denitrification of nitrite in the aerobic tank. The significant amount of chemical oxygen demand (COD) was removed by shortcut denitrification of nitrite in the anoxic tank.     

Simultaneous Nitrification and Denitrification in Aerobic Chemostat Cultures of Thiosphaera pantotropha

Thiosphaera pantotropha is capable of simultaneous heterotrophic nitrification and aerobic denitrification. Consequently, its nitrification potential could not be judged from nitrite accumulation, but was estimated from complete nitrogen balances. The maximum rate of nitrification obtained during these experiments was 93.9 nmol min⁻¹ mg of protein⁻¹. The nitrification rate could be reduced by the provision of nitrate, nitrite, or thiosulfate to the culture medium. Both nitrification and denitrification increased as the dissolved oxygen concentration fell, until a critical level was reached at approximately 25% of air saturation. At this point, the rate of (aerobic) denitrification was equivalent to the anaerobic rate. At this dissolved oxygen concentration, the combined nitrification and denitrification was such that cultures receiving ammonium as their sole source of nitrogen appeared to become oxygen limited and the nitrification rate fell. It appeared that, under carbon-and energy-limited conditions, a high nitrification rate was correlated with a reduced biomass yield. To facilitate experimental design, a working hypothesis for the mechanism behind nitrification and denitrification by T. pantotropha was formulated. This involved the basic assumption that this species has a “bottleneck” in its cytochrome chain to oxygen and that denitrification and nitrification are used to overcome this. The nitrification potential of other heterotrophic nitrifiers has been reconsidered. Several species considered to be “poor” nitrifiers also simultaneously nitrify and denitrify, thus giving a falsely low nitrification potential.     

Modelling of biological processes during aerobic treatment of piggery wastewater aiming at process optimisation

A dynamic mathematical model was developed for the simulation of the aerobic treatment of piggery wastewater. This model includes the carbon oxidation, the nitrification and the denitrification. According to the experimental results obtained during this study, a modified version of the activated sludge model No. 1 has been developed. The model includes (1) nitrite as intermediate of nitrification and denitrification, (2) the distinction between the anoxic heterotrophic yield and the aerobic heterotrophic yield, respectively equal to 0.53 and 0.6 and (3) the first-order hydrolysis of the slowly biodegradable fraction. The calibration and the validation of the model was performed using experimental data from three experiments with two piggery wastewaters. A set of kinetic and stoichiometric parameters emerged from these tests. Except the kinetic of hydrolysis of the slowly biodegradable organic matter varying from 6 to 25 gCOD(gCODday)(-1), all other parameters were similar for all experiments. The dissolved oxygen concentration was identified as the main variable influencing the nitrite accumulation during nitrification. In the calibrated model, the oxygen half-saturation coefficient of the ammonium oxidisers (0.3g O(2)m(-3)) was lower than for the nitrite oxidisers (1.1 gO(2)m(-3)), leading to nitrite accumulation when the dissolved oxygen concentration was low. Simulations with the proposed model could be very useful for improved design and management of biological treatment of piggery wastewaters, particularly in case of partial nitrification to nitrite directly followed by denitrification.     

细菌好氧反硝化研究进展

阐述了好氧反硝化细菌的种类及有关性质 ,并从电子理论、氧的浓度、反硝化酶系等方面对细菌好氧反硝化的作用机制进行了探讨 ,对细菌好氧反硝化的研究概况、进展及其研究意义和目前存在的问题作了较为详细的介绍。     

湖泊氮素氧化及脱氮过程研究进展

自然界中氮的生物地球化学循环主要由微生物驱动,由固氮作用、硝化作用、反硝化作用和氨化作用来完成。过去数十年间,随着异养硝化、厌氧氨氧化和古菌氨氧化作用的发现,人们对环境中氮素循环认识逐步深入,提出了多种脱氮途径新假说。对湖泊生态系统中氮素的输入、输出及其在水体、沉积物和水土界面的迁移转化过程进行了概括,对湖泊生态系统中反硝化和厌氧氨氧化脱氮机理及脱氮效率的最新研究进展进行了探讨,并对以后的氮素循环研究进行了展望。     

Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification

A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification. A mixture of NaHCO₃ and CH₃COONa was selected as the appropriate carbon source for cell growth and nitrogen removal, the concentrations of carbon and nitrogen sources were also examined. Ammonia could be oxidized aerobically to nitrite by the mixed culture, and the intermediate nitrite was then reduced to dinitrogen gas. No nitrite was detected during the process. 0.212 g of ammonia/l could be removed in 30 h and nitrate could not be utilized aerobically by the mixed culture. Nitrite could be degraded aerobically as well as anaerobically. Very little ammonia was degraded anaerobically, but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42 h.     

Microbial niche differentiation explains nitrite oxidation in marine oxygen minimum zones

Nitrite is a pivotal component of the marine nitrogen cycle. The fate of nitrite determines the loss or retention of fixed nitrogen, an essential nutrient for all organisms. Loss occurs via anaerobic nitrite reduction to gases during denitrification and anammox, while retention occurs via nitrite oxidation to nitrate. Nitrite oxidation is usually represented in biogeochemical models by one kinetic parameter and one oxygen threshold, below which nitrite oxidation is set to zero. Here we find that the responses of nitrite oxidation to nitrite and oxygen concentrations vary along a redox gradient in a Pacific Ocean oxygen minimum zone, indicating niche differentiation of nitrite-oxidizing assemblages. Notably, we observe the full inhibition of nitrite oxidation by oxygen addition and nitrite oxidation coupled with nitrogen loss in the absence of oxygen consumption in samples collected from anoxic waters. Nitrite-oxidizing bacteria, including novel clades with high relative abundance in anoxic depths, were also detected in the same samples. Mechanisms corresponding to niche differentiation of nitrite-oxidizing bacteria across the redox gradient are considered. Implementing these mechanisms in biogeochemical models has a significant effect on the estimated fixed nitrogen budget.Subject terms: Biogeochemistry, Water microbiology, Microbial ecology     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

Simultaneous nitrification,denitrification, and phosphorus removal in a lab-scale sequencing batch reactor

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic-anoxic-enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the energy and COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab-scale sequencing batch reactor (SBR) was operated in alternating anaerobic-aerobic mode with a low dissolved oxygen (DO) concentration (0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHAs), accompanied by phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to <0.5 mg/L by the end of the cycle. Ammonia was also oxidized during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off-gas analysis showed that the final denitrification product was mainly nitrous oxide (N(2)O), not N(2). Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen-accumulating organisms (DGAOs), rather than denitrifying polyphosphate-accumulating organisms (DPAOs), were responsible for the denitrification activity.     

Sulfide persistence in oil field waters amended with nitrate and acetate

Nitrate amendment is normally an effective method for sulfide control in oil field-produced waters. However, this approach has occasionally failed to prevent sulfide accumulation, despite the presence of active nitrate-reducing bacterial populations. Here, we report our study of bulk chemical transformations in microcosms of oil field waters containing nitrate-reducing, sulfide-oxidizing bacteria, but lacking denitrifying heterotrophs. Amendment with combinations of nitrate, acetate, and phosphate altered the microbial sulfur and nitrogen transformations. Elemental sulfur produced by chemotrophic nitrate-reducing bacteria was re-reduced heterotrophically to sulfide. Ammonification, rather than denitrification, was the predominant pathway for nitrate reduction. The application of nitrite led to transient sulfide depletion, possibly due to higher rates of nitrite reduction. The addition of molybdate suppressed both the accumulation of sulfide and the heterotrophic reduction of nitrate. Therefore, sulfidogenesis was likely due to elemental sulfur-reducing heterotrophic bacteria, and the nitrate-reducing microbial community consisted mainly of facultatively chemotrophic microbes. This study describes one set of conditions for continued sulfidogenesis during nitrate reduction, with important implications for nitrate control of sulfide production in oil fields.     

Role of heterotrophic nitrifiers and aerobic denitrifiers in simultaneous nitrification and denitrification process: a nonconventional nitrogen removal pathway in wastewater treatment

Bacterial species capable of performing both nitrification and denitrification in a single vessel under similar conditions have gained significance in the wastewater treatment scenario considering their unique character of performing the above reactions under heterotrophic and aerobic conditions respectively. Such a novel strategy often referred to as simultaneous nitrification and denitrification (SND) has a tremendous potential in dealing with various wastewaters having low C : N content, considering that the process needs very little or no external carbon source and oxygen supply thus adding to its cost-effective and environmentally friendly nature. Though like other micro-organisms, heterotrophic nitrifiers and aerobic denitrifiers convert inorganic or organic nitrogen-containing substances into harmless dinitrogen gas in the wastewater, their ecophysiological role in the global nitrogen cycle is still not fully understood. Attempts to highlight the role played by the heterotrophic nitrifiers and aerobic denitrifiers in dealing with nitrogen pollution under various environmental operating conditions will help in developing a mechanistic understanding of the SND process to address the issues faced by the traditional methods of aerobic autotrophic nitrification–anaerobic heterotrophic denitrification.