Simazine biodegradation and community structures of ammonia-oxidizing microorganisms in bioaugmented soil: impact of ammonia and nitrate nitrogen sources

The objective of the present study was to investigate the impact of ammonia and nitrate nitrogen sources on simazine biodegradation by Arthrobacter sp. strain SD1 and the community structures of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in non-agricultural soil. Soil microcosms with different treatments were constructed for herbicide biodegradation test. The relative abundance of the strain SD1 and the structures of AOA and AOB communities were assessed using quantitative PCR (q-PCR) and terminal restriction fragment length polymorphism (TRFLP), respectively. The co-existence of two inorganic nitrogen sources (ammonia and nitrate) had certain impact on simazine dissipation by the strain SD1. Bioaugmentation could induce a shift in the community structures of both AOA and AOB, but AOA were more responsive. Nitrogen application had significant impacts on AOA and AOB communities in bioaugmented soils. Moreover, in non-bioaugmented soil, the community structure of AOA, instead of AOB, could be quickly recovered after herbicide application. This study could add some new insights towards the impacts of nitrogen sources on s-triazine bioremediation and ammonia-oxidizing microorganisms in soil ecosystem.     

Effects of mercury on the activity and community composition of soil ammonia oxidizers

Purpose  

Experiments were conducted to examine the effects of mercury (Hg) on soil nitrification activities and the microbial communities of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA).     

Influence of the DMPP (3,4-dimethyl pyrazole phosphate) on nitrogen transformation and leaching in multi-layer soil columns

The application of nitrogen fertilizers leads to various ecological problems such as nitrate leaching. The use of nitrification inhibitors as nitrate leaching retardants is a proposal that has been suggested for inclusion in regulations in many countries. In this study, using a multi-layer soil column device, the influence of new nitrification inhibitor DMPP (3,4-dimethyl pyrazole phosphate) was studied for understanding the nitrogen vertical transformation and lowering the nitrate leaching at different soil profile depths. The results indicated that, within 60 d of experiment, the regular urea added 1.0% DMPP can effectively inhibit the ammonium oxidation in the soil, and improve the ammonium concentration in soil solution over the 20cm depths of soil profile, while decline the concentrations of nitrate and nitrite. No obvious difference was found on ammonium concentrations in soil solution collected from deep profile under 20cm depths between regular urea and the urea added 1.0% DMPP. There was also no significant difference for the nitrate, ammonium and nitrite concentrations in the soil solution under 40cm depths of soil profile with the increasing nitrogen application level, among the treatments of urea added 1.0% DMPP within 60 d. It is proposed that DMPP could be used as an effective nitrification inhibitor in some region to control ammonium oxidation and decline the ion-nitrogen leaching, minimizing the shallow groundwater pollution risk and being beneficial for the ecological environment.     

Nitrous oxide emission from polyculture constructed wetlands: effect of plant species

Loss of nitrogen from the soil-plant system has raised environmental concern. This study assessed the fluxes of nitrous oxide (N2O) in the subsurface flow constructed wetlands (CWs). To better understand the mechanism of N2O emission, spatial distribution of ammonia-oxidizing bacteria (AOB) in four kinds of wetlands soil were compared. N2O emission data showed large temporal and spatial variation ranging from -5.5 to 32.7 mg N2O m(-2) d(-1). The highest N2O emission occurred in the cell planted with Phragmites australis and Zizania latifolia. Whereas, the lower emission rate were obtained in the cell planted with P. australis and Typha latifolia. These revealed that Z. latifolia stimulated the N2O emission. Transportation of more organic matter and oxygen for AOB growth may be the reason. The study of AOB also supported this result, indicating that the root structure of Z. latifolia was favored by AOB for N2O formation.     

Evaluation of nitrification inhibitor 3,4-dimethyl pyrazole phosphate on nitrogen leaching in undisturbed soil columns

The application of nitrogen fertilizers leads to various ecological problems such as nitrate leaching. The use of nitrification inhibitors (NI) as nitrate leaching retardants is a proposal that has been suggested for inclusion in regulations in many countries. In this study, the efficacy of the new NI, 3,4-dimethyl pyrazole phosphate (DMPP), was tested under simulated high-risk leaching situations in two types of undisturbed soil columns. The results showed that the accumulative leaching losses of soil nitrate under treatment of urea with 1.0% DMPP, from columns of silt loam soil and heavy clay soil, were 66.8% and 69.5% lower than those soil columns tested with regular urea application within the 60 days observation, respectively. However, the losses of ammonium leaching were reversely increased 9.7% and 6.7% under the former treatment than the latter one. Application of regular urea with 1.0% DMPP addition can reduce about 59.3%-63.1% of total losses of inorganic nitrogen via leaching. The application of DMPP to urea had stimulated the inhibition effects of DMPP on the ammonium nitrification process in the soil up to 60 days. It is proposed that the DMPP could be used as an effective NI to control inorganic N leaching losses, minimizing the risk of nitrate pollution in shallow groundwater.     

Effects of selected pharmaceutically active compounds on the ammonia oxidizing bacterium Nitrosomonas europaea

Pharmaceutically active compounds (PhACs) are commonly found in wastewater influent. However, little research has focused on determining their impact on fundamental processes in wastewater treatment such as nitrogen removal. In this study, focus was placed on 4 commonly occurring PhACs (ketoprofen, naproxen, carbamazepine and gemfibrozil). Their effect was ascertained in the ammonia oxidizing bacterium (AOB), Nitrosomonaseuropaea in terms of membrane integrity and nitrite production. These PhACs were shown to inhibit nitrite production at concentrations of 1 and 10 μM while no effect was observed at 0.1 μM. The maximum observed nitrification inhibition was 25%, 29%, 22% and 26% for ketoprofen, naproxen, carbamazepine and gemfibrozil, respectively. A decrease in the live/dead ratio ranging from 10% to 16% suggests that these PhACs affect membrane integrity in N.europaea. The difference in nitrite production between PhACs treated cells and non PhAC treated controls was still significant following washing suggesting that inhibition is irreversible. Finally, nitrite production when adjusted to the live fraction of cells was also found to decrease suggesting that PhACs inhibited the activity of surviving cells. These results suggest that the presence of PhACs may affect AOB activity and may impact nitrogen removal, a key function in wastewater treatment. Follow up studies with additional AOB and in mixed culture are needed to further confirm these results.     

Seasonal effect on N2O formation in nitrification in constructed wetlands

Constructed wetlands are considered to be important sources of nitrous oxide (N(2)O). In order to investigate the contribution of nitrification in N(2)O formation, some environmental factors, plant species and ammonia-oxidizing bacteria (AOB) in active layers have been compared. Vegetation cells indicated remarkable effect of seasons and different plant species on N(2)O emission and AOB amount. Nitrous oxide data showed large temporal and spatial fluctuations ranging 0-52.8 mg N(2)O m(-2)d(-1). Higher AOB amount and N(2)O flux rate were observed in the Zizania latifolia cell, reflecting high potential of global warming. Roles of plants as ecosystem engineers are summarized with rhizosphere oxygen release and organic matter transportation to affect nitrogen transformation. The Phragmites australis cell contributed to keeping high T-N removal performance and lower N(2)O emission. The distribution of AOB also supported this result. Statistical analysis showed several environmental parameters affecting the strength of observed greenhouse gases emission, such as water temperature, water level, TOC, plant species and plant cover.     

Simazine degradation in bioaugmented soil: urea impact and response of ammonia-oxidizing bacteria and other soil bacterial communities

The objective of this study was to investigate the impact of exogenous urea nitrogen on ammonia-oxidizing bacteria (AOB) and other soil bacterial communities in soil bioaugmented for simazine remediation. The previously isolated simazine-degrading Arthrobacter sp. strain SD1 was used to degrade the herbicide. The effect of urea on the simazine degradation capacity of the soil bioaugmented with Arthrobacter strain SD1 was assessed using quantitative PCR targeting the s-triazine-degrading trzN and atzC genes. Structures of bacterial and AOB communities were characterized using terminal restriction fragment length polymorphism. Urea fertilizer could affect simazine biodegradation and decreased the proportion of its trzN and atzC genes in soil augmented with Arthrobacter strain SD1. Bioaugmentation process could significantly alter the structures of both bacterial and AOB communities, which were strongly affected by urea amendment, depending on the dosage. This study could provide some new insights towards s-triazine bioremediation and microbial ecology in a bioaugmented system. However, further studies are necessary in order to elucidate the impact of different types and levels of nitrogen sources on s-triazine-degraders and bacterial and AOB communities in bioaugmented soil.     

污泥好氧颗粒化过程中氨氧化菌群结构的演替与分析

为了揭示颗粒污泥形成过程中氨氧化菌(AOB)群落结构的演替规律,利用变性梯度凝胶电泳(DGGE)、克隆测序和实时定量聚合酶链式反应(real-time PCR)等分子生物学技术对AOB群落的演替进行了研究。DGGE结果表明,污泥接种驯化期,AOB群结构的变化较为剧烈,在外加选择压的作用下,种群多样性迅速下降;但随着污泥颗粒化的完成而趋于稳定。测序结果表明,接种污泥中的大多数亚硝化单胞菌属因可快速适应工艺的淘洗过程而被保留在系统内,而亚硝化螺菌属逐渐被淘汰。real-time PCR结果表明,在经历了运行初期的淘洗后,AOB含量随着污泥浓度的提高而逐渐增长;但污泥的氨氧化活性随着污泥浓度的增长而降低。     

Effect of biochar on nitrous oxide emission and its potential mechanisms

Extensive use of biochar to mitigate nitrous oxide (N₂O) emission is limited by the lack of understanding on the exact mechanisms altering N₂O emission from biochar-amended soil. Biochars produced from rice straw and dairy manure at 350 and 500 °C by oxygen-limited pyrolysis were used to investigate their influence on N₂O emission. A quadratic effect of biochar levels was observed on the N₂O emissions. The potential mechanisms were explored by terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (qPCR). A lower relative abundance of bacteria, which included ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), was observed at 4% biochar application rate. Reduced copy numbers of the ammonia monooxygenase gene amoA and the nitrite reductase gene nirS coincided with decreased N₂O emissions. Therefore, biochar may potentially alter N₂O emission by affecting ammonia-oxidizing and denitrification bacteria, which is determined by the application rate of biochar in soil.

Implications:Biochar research has received increased interest in recent years because of the potential beneficial effects of biochar on soil properties. Recent research shows that biochar can alter the rates of nitrogen cycling in soil systems by influencing nitrification and denitrification, which are key sources of the greenhouse gas nitrous oxide (N₂O). However, there are still some controversial data. The purpose of this research was to (1) examine how applications of different dose of biochar to soil affect emission of N₂O and (2) improve the understanding of the underlying mechanisms.     

有机负荷对膜-生物反应器硝化性能的影响

采用厌氧动态膜-生物反应器(AnDMBR)组合自养膜-生物反应器(MBR)工艺,研究冬季低温条件下系统的硝化效果以及TP的去除效果,并与单级MBR工艺进行对比。结果表明:(1)AnDMBR对COD的去除率基本保持在50%～60%,AnDMBR组合自养MBR工艺对COD的去除率为80%～85%;单级MBR工艺对COD的去除率为80%左右。(2)总体上,AnDMBR组合自养MBR工艺对NH4+-N的去除率大于95%;单级MBR对NH4+-N的去除效果比AnDMBR组合自养MBR工艺差。(3)AnDMBR组合自养MBR工艺中,出水NO2--N与NO3--N均有积累;单级MBR工艺中,出水NO2--N积累不明显。(4)相对于亚硝酸盐氧化菌(NOB),氨氧化菌(AOB)对有机负荷更敏感,当有机负荷高时,AOB更易受到异养菌活动的抑制;当有机负荷降低、异养菌活性减弱时,AOB活性明显增强,系统的硝化效果得到明显改善。(5)AnDMBR组合自养MBR工艺对TP的去除率高于80%,单级MBR工艺稳定后对TP的去除率仅为20%～30%。(6)从呼吸速率和硝化速率可知,自养MBR的硝化效果优于单级MBR。     

Nitrifying community analysis in a single submerged attached-growth bioreactor for treatment of high-ammonia waste stream.

This study investigated the nitrifying community structure in a single-stage submerged attached-growth bioreactor (SAGB) that successfully achieved stable nitrogen removal over nitrite of a high-strength ammonia wastewater. The reactor was operated with intermittent aeration and external carbon addition (methanol). With influent ammonia and total Kjeldahl nitrogen ranging from 537 to 968 mg/L and 643 to 1510 mg/L, respectively, 85% nitrogen removal was obtained, and effluent was dominated by nitrite (NO2-/NOx > 0.95). Nitrifying community analysis using fluorescence in situ hybridization (FISH), with a hierarchical set of probes targeting known ammonia-oxidizing bacteria (AOB) within beta-proteobacteria, showed that the AOB community of the biofilter consists almost entirely of members of the Nitrosomonas europaea/eutropha and the Nitrosococcus mobilis lineages. Image analysis of FISH pictures was used to quantify the identified AOB, and it was estimated that Nitrosomonas europaea/eutropha-like AOB accounted for 4.3% of the total volume of the biofilm, while Nitrosococcus mobilis-like AOB made up 1.2%; these numbers summed up to a total AOB fraction of 5.5% of the total volume on the biofilm. Nitrite-oxidizing bacteria (NOB) were not detectable in the biofilm samples with probes for either Nitrospira sp. or Nitrobacter sp., which indicated that NOB were either absent from the biofilters or present in numbers below the detection limit of FISH (< 0.1% of the total biofilm). Nitrite oxidizers were likely outcompeted from the system because of the free ammonia inhibition and the possibility that the aeration period (from intermittent aeration) was not sufficiently long for the NOB to be released from the competition for oxygen with heterotrophs and AOB. The nitrogen removal via nitrite in a SAGB reactor described in this study is applicable for high-ammonia-strength wastewater treatment, such as centrate or industrial wastes.     

实时荧光定量PCR对A2/O短程硝化系统内氨氧化菌的定量分析

通过控制好氧区低DO浓度以及缩短好氧实际水力停留时间(actual hydraulic retention time,AHRT),在处理低C/N比实际生活污水的A2/O工艺中,成功启动并维持了短程硝化反硝化;系统亚硝酸盐积累率稳定维持在90%左右,氨氮去除率在95%以上。通过提取富集氨氧化菌(ammonia oxidizing bacteria,AOB)的基因组DNA,经两次常规PCR扩增和琼脂糖凝胶电泳,以纯化回收的DNA扩增片段作为实时荧光定量PCR检测AOB数量的DNA标准品,建立了检测AOB数量的实时荧光定量PCR标准曲线。利用实时荧光定量PCR技术比较了A2/O系统在不同运行条件及亚硝酸盐积累率情况下AOB菌群数量。结果表明,随着系统亚硝酸盐积累率的上升,系统内AOB菌群数量也大幅上升。全程硝化和短程硝化时,系统内的AOB菌群数量分别为5.28×109cells/g MLVSS和3.95×1010cells/g MLVSS。此外,亚硝酸盐积累率的下降相对于AOB菌群数量的下降有一定的滞后性。     

生物质碳源组合型生态浮床系统脱氮效果研究

利用农业废弃物和水生植物,结合水下空间与水面景观,构建集成脱氮技术的新型立体组合式生态浮床系统用以地表水体中氮的脱除以净化水质。立体生态浮床运行2个月之后的结果显示,NH3-N、TN和CODMn的去除率可分别达到96.77%、95.51%和77.75%,本浮床具有较高脱氮能力,水体净化效果明显;同时,整个生态浮床系统的生物多样性较为丰富,原水分别为Ⅴ类和劣Ⅴ类的池2和池5中分别鉴定出6种和8种主要氨氧化菌谱带,位于系统中部的玉米芯和稻草表面微生物多样性丰富程度较高;稻草和底泥中氨氧化菌的谱带种类最多。与池2相比,池5中除了存在共有谱带外,还存在特有谱带h和j带,表明可能有特殊氨氧化菌群的存在。本组合型生态浮床具有同步硝化与反硝化作用,利于沉水植物生长,并能促进水生态系统构建。     

Effects of submerged macrophytes on the abundance and community composition of ammonia-oxidizing prokaryotes in a eutrophic lake

Abundances and community compositions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in unvegetated sediment and the rhizosphere sediments of three submerged macrophytes (Ceratophyllum demersum, Vallisneria spinulosa, and Potamogeton crispus) were investigated in a large, eutrophic freshwater lake, Lake Taihu. Abundances of archaeal ammonia monooxygenase alpha-subunit (amoA) gene (from 6.56?×?10⁶ copies to 1.06?×?10⁷ copies per gram of dry sediment) were higher than those of bacterial amoA (from 6.13?×?10⁵ to 3.21?×?10⁶ copies per gram of dry sediment) in all samples. Submerged macrophytes exhibited no significant effect on the abundance and diversity of archaeal amoA gene. C. demersum and V. spinulosa increased the abundance and diversity of bacterial amoA gene in their rhizosphere sediment. However, the diversity of bacterial amoA gene in the rhizosphere sediments of P. crispus was decreased. The data obtained in this study would be helpful to elucidate the roles of submerged macrophytes involved in the nitrogen cycling of eutrophic lake ecosystems.     

Quantifying the contribution of nitrification and denitrification to the nitrous oxide flux using 15N tracers

Microbial transformations of nitrification and denitrification are the main sources of nitrous oxide (N2O) from soils. Relative contributions of both processes to N2O emissions were estimated on an agricultural soil using 15N isotope tracers (15NH4+ or 15NO3-), for a 10-day batch experiment. Under unsaturated and saturated conditions, both processes were significantly involved in N2O production. Under unsaturated conditions, 60% of N-N2O came from nitrification, while denitrification contributed around 85-90% under saturated conditions. Estimated nitrification rates were not significantly different whatever the soil moisture content, whereas the proportion of nitrified N emitted as N2O changed from 0.13 to 2.32%. In coherence with previous studies, we interpreted this high value as resulting from the decrease in O2 availability through the increase in soil moisture content. It thus appears that, under limiting aeration conditions, some values for N2O emissions through nitrification could be underestimated.     

Feasibility of a membrane-aerated biofilm reactor to achieve single-stage autotrophic nitrogen removal based on Anammox

A laboratory-scale membrane-aerated biofilm bioreactor (MABR) equipped with non-woven fabrics support around the gas-permeable carbon tube was developed for single-stage autotrophic nitrogen removal based on partial nitrification and anaerobic ammonium oxidization. This reactor allowed air to be supplied through the microporous carbon tube wall to the biofilm that was supported by non-woven fabrics. The partial nitrification and consumption of dissolved oxygen occurred in the inner layer and Anammox in the anoxic outer layer of the non-woven fabrics, thus realizing autotrophic nitrogen removal in a single reactor. After 116d of operation, the maximal nitrogen removal of 0.77kgNm(-3)d(-1) at a volumetric ammonium loading rate of 0.87kgNm(-3)d(-1) was achieved. The spatial profiles of the ammonia-oxidizing bacteria and Anammox bacteria were evaluated by fluorescence in situ hybridization. This study demonstrated that MABR was a very suitable experimental set-up for the operation of the single-stage autotrophic nitrogen removal process.     

Unraveling the source of nitric oxide emission during nitrification.

Nitric oxide production was measured during nitrification in a laboratory-scale bioreactor, operated at conditions relevant to municipal nitrifying wastewater treatment plants. This study aims to determine which type of microorganism and which metabolic pathway is responsible for nitric oxide emission during nitrification. Simulation studies were used to identify which pathway is the main source of nitric oxide emission, based on the following three hypothetical pathways for nitric oxide emission: (a) nitrification, (b) denitrification by ammonia-oxidizing bacteria with ammonium as electron donor, and (c) heterotrophic denitrification. The results of the study suggest that, in a nitrifying reactor treating wastewater containing solely ammonium and nutrients, denitrification by ammonia-oxidizing bacteria is the main nitric-oxide-producing pathway. During the experiments, 0.025% of the treated ammonium is emitted as nitric oxide, independent of the aeration rate imposed. Nitrite presence and oxygen limitation were found to increase the nitric oxide emission.     

NaCl盐度对耐盐脱氮污泥硝化功能的影响

利用序批式活性污泥反应器(SBR)研究NaCl盐度对耐盐脱氮污泥硝化功能的影响,在此基础上考察瞬时盐度冲击对氨氧化细菌(AOB)、亚硝酸氮氧化细菌(NOB)的抑制及恢复情况。实验结果表明,当废水中NaCl浓度为0~50g/L时,AOB几乎没有影响,NOB影响较小。当NaCl浓度为60 g/L时,AOB影响较小,NOB受到一定程度的抑制。当NaCl浓度为70 g/L时,AOB、NOB均受到严重的抑制;耐盐脱氮污泥能够适应NaCl浓度50 g/L的瞬时冲击,盐度降低有利于AOB、NOB的恢复。当耐盐脱氮污泥受到NaCl浓度60 g/L的瞬时冲击时,系统发生"中毒"现象,盐度降低至0 g/L时,AOB、NOB均有不同程度恢复。     

分段进水SBR短程生物脱氮过程中N_2O产生及控制

利用SBR,控制曝气量为60 L/h,利用在线pH曲线控制曝气时间,成功实现了短程生物脱氮过程,并考察了不同进水方式下SBR运行性能及N2O产量。结果表明,分段进水能够有效降低短程生物脱氮过程中外加碳源投加量。在原水进水碳氮比较低时,采用递增进水量的进水方式,能够有效降低生物脱氮过程中NO-2积累量,从而降低系统N2O产量。1次进水、2次等量进水和2次递增进水方式下,生物脱氮过程中N2O产量分别为11.1、8.86和5.04 mg/L。硝化过程中NO-2-N的积累是导致系统N2O产生的主要原因。部分氨氧化菌（AOB）在限氧条件下以NH＋4-N作为电子供体,NO-2-N作为电子受体进行反硝化,最终产物是N2O。