环境中的反硝化微生物种群结构和功能研究进展

反硝化作用是生态系统氮循环的重要组成部分,与地下水硝酸盐累积和温室气体排放密切相关。种类繁多的细菌、真菌和古菌参与反硝化过程,并在调控反硝化速率和反硝化气体产物比例等方面发挥重要作用。反硝化微生物的种群结构是一系列环境影响因素长期作用的结果,反硝化微生物种群对温度、水分、pH、O2含量、资源可利用性和植被类型等因素产生不同的响应。环境因素通过对反硝化微生物的影响来调控反硝化速率和反硝化酶的生成。分子技术的应用为自然环境中反硝化微生物的研究开辟了广阔前景,并为进一步认识反硝化微生物种群结构和功能的相互关系提供了新的发展方向。本文总结了关于环境中反硝化微生物种群的研究结果,并为进一步研究反硝化微生物种群结构和功能的联系提供了总体框架。     

华北平原农田厚包气带及含水层反硝化细菌的分离鉴定及作用机制研究

华北平原农田由于长期过量施用氮肥,造成了土壤硝酸盐累积,导致地下水硝酸盐污染日趋严重。微生物的反硝化作用可将土壤中累积的硝酸盐或亚硝酸盐还原为气态产物,是消减厚包气带土壤累积的硝酸盐的重要途径。因此筛选高效反硝化微生物资源,对人工强化厚包气带土壤反硝化脱氮,阻控地下水硝酸盐污染具有重要作用。基于此,本研究采集位于华北平原的中国科学院栾城农业生态系统试验站长期施氮[施氮量为600kg(N)·hm~(-2)·a~(-1)]定位试验0～150m农田厚包气带及含水层土壤样品,从中筛选到62株细菌。16SrRNA基因序列分析表明这62株菌株与变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)中的9个属具有较高的同源性。根据系统发育树的结果,挑选7株亲缘关系较远的菌株进行反硝化潜势试验,结果表明,菌株L71、L13和L103具备反硝化产气能力。电镜观察结果表明,这3株菌均为无鞭毛的杆状细菌,其长度分别为1.0μm、1.5μm和1.5μm,只有L103具有运动能力。此外,菌株L103具有完全反硝化能力,且脱氮能力受到pH的影响,在本试验条件下,菌株L103的反硝化速率高达1.62～2.36g(KNO_3)·d~(-1)·L~(-1),具备实际应用潜力。本研究表明华北平原厚包气带土壤中存在完全反硝化微生物,并可为人工强化治理厚包气带土壤硝酸盐污染提供菌种资源和理论依据。     

一株具有多途径氮代谢功能的荧光假单胞菌

为研究全自养脱氮过程，采用斯凯尔曼亚硝化单胞杆菌培养基，从南京某城市污水处理厂序批式处理系统（SBR）中分离得到一株具有多途径氮代谢功能的荧光假单胞菌Pseudomonas fluorescens dN13。该菌株在有氧条件下，无有机碳源存在，能进行氧化NH3-N产能代谢生长；有有机碳源存在且NO2^- -N和NO3^- -N的浓度较高时，它能将NO2^- -N和NO3^- -N还原为NH3-N。在厌氧条件下，只有乙酸钠作碳源时，该菌株仍能进行硝酸亚硝酸还原（反硝化）作用。在这个具有多途径氮代谢功能的菌株细胞内，硝化反硝化过程耦合成为可能。     

中国北方农田氮磷淋溶损失污染与防控机制

突破厚包气带农田根层氮磷淋溶与地下水污染复杂定量关系和阻控机理是国际研究难点。本文系统梳理了重点研发专项"农田氮磷淋溶损失污染与防控机制"项目取得的主要进展,项目包括以下4方面研究内容:1)北方主要农区农田根层氮磷淋溶时空规律;2)根层—深层包气带氮磷淋溶机制和主控因子;3)黑土、潮土和褐土氮磷淋溶阻控机制及其效果; 4)典型农区氮磷淋溶风险与区域消减途径。主要科学发现包括:1)受土地利用类型、地下水埋深、包气带岩性、水文地质条件等综合因素的影响,黑土区、潮土区和褐土区根层氮磷淋溶规律与地下水硝酸盐超标率体现出空间不一致和较大差异性。黑土区虽然根层淋溶较小,然而受地形地貌影响,地下水水质对淋溶响应更强烈,应该进一步研究黑土区地下水水质对淋溶的响应机制。华北潮土区和褐土区厚包气带具有明显氮阻控能力,应该进一步加强厚包气带对氮磷淋溶减排机理与途径研究。2)基于长期施肥定位试验和12 m深观测井对包气带农田土壤氮盈余累积特征和淋失规律的研究发现,华北平原区的环境安全施氮量约为200kg(N)·hm~(-2)·a~(-1),超过环境安全阈值的多投入氮肥中有51%淋失到1m根层以下,不合理灌溉、强降水、大孔隙和裂隙是造成土壤硝酸盐淋溶的主要因素,对包气带累积硝态氮的淋失作用可影响至6m以下土层。3)利用深层取样和生物学方法结合,对厚包气带0～10.5m原位土壤微生物的反硝化活性和微生物区系组成的研究结果表明,表层土壤是微生物进行反硝化的主要场所,深层土壤中反硝化作用显著减弱,"碳饥饿"是限制底层土壤反硝化微生物丰度与活性的关键因素;室内培养试验证实添加碳源可有效激活土壤微生物的反硝化活性,为"根层截氮包气带脱氮"的淋溶阻控机理找到了突破口。4)利用黑土、潮土和褐土区氮磷淋溶阻控试验、全国农业面源污染国控监测网、北方农区地下水硝酸盐监测网和NUFER (NUtrient flows in Food chains, Environment and Resourcesuse)模型,提出了养分损失脆弱区区划和区域氮磷污染削减草案,可为农业绿色发展和面源污染阻控提供科学依据。     

应重视硝态氮同化过程在降低土壤硝酸盐浓度中的作用

在保证生产力条件下,采取合理的氮肥管理措施降低土壤硝态氮浓度对降低氮污染至关重要。当前,应用硝化抑制剂能够有效延缓铵态氮的硝化速率,进而降低土壤硝态氮淋溶损失和氮氧化物排放,但是其缺点显而易见:促进氨挥发并引起硝化抑制剂污染。好氧条件下,土壤硝态氮净变化量取决于产生(硝化)和消耗(硝态氮同化)的量。但是,一直以来,受微生物优先利用铵态氮这一传统观点的影响,人们普遍认为农田土壤微生物较少利用硝态氮,很大程度上忽视了对硝态氮同化过程的研究。该过程独具优势,它将硝态氮转变为微生物生物量氮进行短期储存并发生再矿化,具有保氮功能且环境友好。加入特定的碳源可以提高硝态氮同化这已是不争的事实,未来应加强硝态氮同化降低土壤硝酸盐累积方面的研究:(1)外源碳影响硝态氮同化的微生物驱动机制是什么?(2)怎样才能操控硝态氮同化和再矿化过程,使得作物氮需求和土壤氮供应相匹配,进而降低氮损失?(3)在碳源充足的条件下,反硝化作用亦会增强,如何才能做到在提高硝态氮同化的同时避免反硝化氮损失?     

反硝化技术对模拟养殖池塘修复的研究

浙江奉化市池塘的底泥经过反复培养、驯化,从中筛选、分离出反硝化细菌,在模拟实验条件下,研究其对不同浓度的硝酸盐氮和亚硝酸盐氮的去除情况,讨论反硝化菌种的生长情况。结果表明,在初始浓度为1、25、50mg·L^-1的硝酸盐氮和亚硝酸盐氮模拟池塘中,随着实验的进行,对污染物的去除效果逐渐提高。其中在1mg·L^-1的浓度组中,3d内硝酸盐氮和亚硝酸盐氮去除率就分别达到了95．8％和90．2％;在25mg·L^-1的浓度组中,第6d硝酸盐氮和亚硝酸盐的去除率分别为93．8％和87．8％;在50mg·L^-1的浓度组中,第6d硝酸盐氮和亚硝酸盐的去除率分别为89．7％和78．7％。此外,反硝化菌对硝酸盐氮的去除效果略优于亚硝酸盐氮,而且硝酸盐氮和亚硝酸盐氮的浓度越低,对其去除效果越好,达到稳定状态的时间越短。在模拟池塘中,菌种的生长情况与硝酸盐氮和亚硝酸盐氮的浓度呈负相关,即污染物的浓度越高反硝化菌的生长情况越差。对反硝化菌的生态影响因子研究表明,其反硝化最适宜的pH值为6～7,温度为25～35℃;而且在同一pH值和温度条件下,硝酸盐氮和亚硝酸盐氮浓度越低,反硝化菌对其去除效果越好。     

土壤氮气排放研究进展

自20世纪初人类发明并掌握工业合成氨的技术以来,氮肥施用量迅速增长。在一部分国家或地区,氮肥的施入量已经超过作物对氮素的需求,导致大量氮素损失到环境中,造成氨挥发、氧化亚氮排放、地下水硝酸盐污染等环境问题。土壤在微生物的作用下可以通过反硝化、厌氧氨氧化等过程将活性氮素转化为惰性氮气,达到清除过多活性氮的目的。由于大气中氮气背景浓度太高,因此很难直接准确测定土壤的氮气排放速率,导致土壤氮气排放通量、过程与调控机制研究远远落后于土壤氮循环的其他方面。本文综述了土壤氮气排放主要途径(反硝化、厌氧氨氧化与共反硝化)及其对土壤氮气排放的贡献;测定土壤氮气排放速率的方法(乙炔抑制法、氮同位素示踪法、N2/Ar比率-膜进样质谱法、氦环境法与N2O同位素自然丰度法)及其优缺点;调控土壤氮气排放通量的主要因素(氧气、可溶性有机碳、硝酸盐、微生物群落结构与功能基因表达等)及其相关作用机制。最后指出研发新的测定原位无扰动土壤氮气通量的方法是推进本领域相关研究的关键;定量典型生态系统(如旱地农田、稻田、森林、草地与湿地)土壤氮气排放通量,阐明其中的微生物学机制,模拟并预测土壤氮气排放对全球变化的响应规律是本领域的研究热点与发展方向。     

脱氮功能菌去除市政废水中氮素的研究

为了探讨实验室筛选获得的氨氧化细菌CM-NR014和反硝化细菌CM-NRD3联合去除市政废水中氮素的应用价值,采用了两级A／O工艺进行菌株去除废水中氮素的小试实验,最后将菌株用于废水脱氮工程中。结果表明,脱氮功能菌实现了短程硝化-反硝化,氨氮去除率在98％以上,总氮去除率在75％以上,COD（化学需氧量）去除率大于90％,出水各项指标均低于城镇污水处理厂污染物排放一级（A）标准。脱氮功能菌在去除市政废水中氮素方面有很高的应用价值,可用于城镇污水处理厂提标改造等。     

长江三角洲农田地下水反硝化对硝酸盐的去除作用

长江三角洲(简称"长三角")农田氮素投入量高,但是否像其他高氮投入农田一样在土壤剖面累积了大量硝酸盐尚不清楚。通过连续两年的野外观测结合室内培养实验,发现长三角地区3种不同类型的高氮投入农田1～4 m地下水硝态氮(NO_3~--N)剖面分布特征存在明显差异,水稻田地下水NO_3~--N浓度始终很低(1mg·L~(-1)),不同深度之间无差异。蔬菜地和葡萄园1 m处地下水NO_3~--N年平均浓度分别为5.6和17.5 mg·L~(-1),但是地下水NO_3~--N浓度随着深度增加急剧下降,至4m处,NO_3~--N浓度降至小于1 mg·L~(-1),与水稻田无差异。蔬菜地和葡萄园地下水高浓度NO_3~--N仅出现在施肥期间,非施肥期地下水NO_3~--N浓度较低,这表明长三角农田不存在明显的NO_3~--N累积。原状土柱培养实验结果表明,0～4 m土壤均存在较强的反硝化活性。通过对地下水中反硝化产物N2及N2O的直接定量测定,发现反硝化对地下水NO_3~--N的去除效率随着深度而增加,至4m处,反硝化对地下水NO_3~--N的去除效率分别为86%(水稻田)、93%(蔬菜地)和89%(葡萄园)。这表明反硝化能有效去除地下水NO_3~--N,是长三角地区农田土壤剖面未产生NO_3~--N累积的重要原因。反硝化产生的溶解性气态氮主要通过地下水流入临近水域,对于蔬菜地和葡萄园而言,溶解性气态氮流失量与NO_3~--N淋溶损失量相当,是一个重要的氮素去向,值得关注。     

耐冷菌强化去除农田径流污染水体中氮磷的模拟研究

通过室内模拟实验研究1株土著反硝化菌(Acinetobacter johnsonii DBP-3)对农田径流污染水体中氮磷的低温生物强化去除特征。结果表明:10℃下避光好氧培养时菌株对水样中的硝酸盐氮和溶解性正磷酸盐具有较强的去除能力,培养8d后灭菌水样和原水样中硝酸盐氮的浓度分别下降了78.5%和70.2%,溶解性正磷酸盐的浓度分别下降了82.4%和74.6%,与未投加菌的对照组相比差异显著。菌株在低温模拟系统中具有较强的适应能力,实验周期内能够保持数量上的优势。与10℃相比,培养温度为25℃时,菌株的脱氮除磷能力明显增强,5℃时菌株的氮磷代谢能力明显降低,但与对照相比,菌株对氮磷仍然保持一定的代谢活性。菌株在模拟系统中对盐度具有较强的抗性,当盐度为10%时,其氮磷代谢能力才受到明显的抑制。多菌灵和毒死蜱的浓度分别为80.0,60.0mg/L时才对菌株的生长代谢产生明显的抑制作用,表明菌株对这2种农药的耐性较强。研究结果说明,实验用菌株在低温条件下不仅具有明显的脱氮除磷能力,而且对盐度和常见的农药具有较强的抗性,在面源污染的治理方面具有广阔的应用前景。     

Biological Denitrification of Groundwater

Nitrate concentrations in groundwater have increased in many areas of the world. This causes serious concerns because of the link found between nitrate and the blue-baby syndrome, and of the possible formation of carcinogenic compounds in the digestive tract. Biological denitrification, bacteria-mediated reduction of nitrate to nitrogen gas, is a method used in the treatment of nitrate contaminated groundwater. The denitrifying microorganisms require carbon and energy substrates which may be organic or inorganic compounds. Treatment can take place in the aquifer (in situ treatment) or in above ground reactors. Numerous biological denitrification processes have been reported; this paper reviews some of this work and studies in progress in the author's laboratory. The choice of a biological denitrification system has to be considered on an individual basis. Although preventive measures are curbing the problem in some developed countries, nitrate pollution is still on the rise in many other countries. Innovative, low-cost biological denitrification processes are specially needed in developing countries.     

基于碳酸氢钠为碳源的氢自养反硝化去除地下水中硝酸盐研究

氢自养反硝化修复地下水中的硝酸盐污染以其清洁、环保又经济而受到广泛重视。利用全自动恒温振荡仪,以NaHCO3为碳源驯化氢自养反硝化细菌,并对影响氢自养反硝化速率的因素进行了研究。结果表明,以NaHCO3作为唯一的无机碳源,不仅可以高效驯化氢自养反硝化细菌,而且可以控制体系的pH值,效果优于单独以CO2或以CO2和NaHCO3共同为碳源的系统;当单独以NaHCO3为碳源时,其浓度为2g·L-1时可以满足氢自养反硝化细菌的生长,并使体系pH保持在8.5±0.2;当初始NO3--N浓度〈135.6mg·L-1时,反硝化速率随着NO3--N浓度的升高而增大,当NO3--N浓度过高时（〉135.6mg·L-1）,会抑制氢自养反硝化的进行;当pH在6.0～9.0时,氢自养反硝化可以进行,但其最适pH为7.0～8.0,而当pH〈6.0或pH〉9.0时,反硝化基本停滞;温度为35℃时反硝化速率最大,为2.83mg·L-·1h-1,当温度为15℃时,有明显的亚硝酸盐积累。     

Nitrogen removal and metabolic profiling of a cold-adaptive and biofilm producing paddy soil bacterium Cupriavidus sp. PDN31

To assess the physiology and low temperature adaptability of the key players of nitrification and denitrification, denitrifying bacteria were isolated and characterized from the selected paddy fields. Bacterial strains belonging to Cupriavidus and Ochrobactrum sp. were explored through the selective screening of heterotrophic nitrifying and aerobic denitrifying bacteria. The direct implication of nitrate removal in the natural sample was estimated by taking the nitrate supplemented soil as well as the enriched culture. A more prominent cold-adaptive bacterium was identified as Cupriavidus sp. PDN31. The utilization of ammonium, nitrate, and nitrite and the presence of nitrous oxide reductase (nosZ) gene, catalyses the first step of the denitrification conferred its heterotrophic nitrification and aerobic denitrification ability. The ammonium, nitrate, and nitrite removal efficiency of PDN31 was found to be 92.1%, 93.5%, and 99.8%, respectively. The functional traits, evaluated from metabolizing various nitrogen substrates (Biolog) suggested its ability to utilize some sources as L-arginine, L-asparagine, L-cysteine, L-glutamic Acid, L-glutamine, L-histidine, L-citrulline and N-acetyl-L glutamic acid. The adaptive behaviour of PDN31 with its ability to remove nitrogen and induced biofilm production under low temperature regime makes it a suitable candidate among the plethora of microorganism resided in any agriculture environment.     

Denitrification enzyme activity is limited by soil aeration in a wastewater-irrigated forest soil

 In land-based wastewater treatment systems (LTS), denitrification is an important nitrogen removal process. We investigated the factors limiting the denitrifying population in a forested LTS, by studying the individual and combined effects of soil aeration, water content, nitrate and carbon on denitrification enzyme activity (DEA). The size of the soil denitrifying population in the LTS appeared to be limited by soil aeration, and limiting oxygen availability increased the denitrifying population above that observed in the field. Furthermore, we found that wastewater irrigation altered the short-term response of denitrifiers to anaerobic soil conditions. Under low oxygen conditions, denitrifiers in the wastewater-irrigated soils produced enzymes sooner and at a greater rate than soils without a history of wastewater irrigation. We propose that the size of the denitrifying population cannot be expected to be large in free-draining, coarsely textured soils even when provided with additional nitrogen and water inputs. Received: 11 October 1999     

Nitrate removal, denitrification and nitrous oxide production in the riparian zone of an ephemeral stream

Riparian zones are important features of the landscape that can buffer waterways from non-point sources of nitrogen pollution. Studies of perennial streams have identified denitrification as one of the dominant mechanisms by which this can occur. This study aimed to assess nitrate removal within the riparian zone of an ephemeral stream and characterise the processes responsible, particularly denitrification, using both in-situ and laboratory techniques. To quantify rates of groundwater nitrate removal and denitrification in-situ, nitrate was added to two separate injection-capture well networks in a perched riparian aquifer of a low order ephemeral stream in South East Queensland, Australia. Both networks also received bromide as a conservative tracer and one received acetylene to inhibit the last step of denitrification. An average of 77 ± 2% and 98 ± 1% of the added nitrate was removed within a distance of 40 cm from the injection wells (networks with acetylene and without, respectively). Based on rates of N₂O production in the network with added acetylene, denitrification was not a major mechanism of nitrate loss, accounting for only 3% of removal. Reduction of nitrate to ammonium was also not a major pathway in either network, contributing <4%. Relatively high concentrations of oxygen in the aquifer following recent filling by stream water may have reduced the importance of these two anaerobic pathways. Alternatively, denitrification may have been underestimated using the in-situ acetylene block technique. In the laboratory, soils taken from two depths at each well network were incubated with four nitrate-N treatments (ranging from ambient concentration to an addition of 15 mg N l⁻¹), with and without added acetylene. Potential rates of denitrification, N₂O production and N₂O:N₂ ratios increased with nitrate additions, particularly in shallow soils. Potential rates of denitrification observed in the laboratory were equivalent in magnitude to nitrate removal measured in the field (mean 0.26 ± 0.12 mg N kg of dry soil⁻¹ d⁻¹), but were two orders of magnitude greater than denitrification measured in the field with added acetylene. The relative importance of assimilatory vs. dissimilatory processes of nitrate removal depends on environmental conditions in the aquifer, particularly hydrology and its effects on dissolved oxygen concentrations. Depending on seasonal conditions, aquifers of ephemeral streams like the study site are likely to fluctuate between oxic and anoxic conditions; nevertheless they may still function as effective buffers. While denitrification to N₂ is a desirable outcome from a management perspective, assimilation into biomass can provide a rapid sink for nitrate, thus helping to reduce short-term delivery of nitrate downstream. Longer-term studies are needed to determine the overall effectiveness of riparian buffers associated with ephemeral streams in mitigating nitrate loads reaching downstream ecosystems.     

Column Experiment on Nitrate Purification with Polylactic Acid and Iron Powder

Groundwater contamination by nitrates is a worldwide problem, but to date there have been no effective measures to purify contaminated groundwater. If a denitrifying system can be constructed in aquifers, it will be able to purify groundwater in situ. This purifying system should also be maintenance free. The purpose of our work is to construct a maintenance-free denitrification system for in situ groundwater purification. This paper presents the result of a basic study on a maintenance-free denitrification system using biodegradable plastic and iron powder. A biodegradable plastic (polylactic acid, PLA) and iron powder were used as detergents, and the nitrate-contaminated groundwater was injected from the bottom of a packed silica sand column filled with a mixture of the detergents. Laboratory experiments were carried out, varying factors such as molecular weight of the biodegradable plastic and mixing ratio of the detergents. Water that permeated through the purification column was sampled to determine the decrease in nitrate concentration. The experimental results suggest that a combination of PLA and iron powder seems effective for denitrification, and that the rate of the PLA degradation is closely related to the molecular weight of the PLA and iron powder content. In this case iron powder acts as accelerator in denitrification. It is concluded that a maintenance-free denitrification system can be built, using PLA and iron powder as detergents. The proposed system may also be used in a wide range of nitrate contamination conditions, adjusting the molecular weight of the PLA and the mixing ratio of the detergents.     

Factors limiting denitrification in a Mediterranean riparian forest

In situ denitrification (DNT) and denitrification enzyme activity (DEA) were measured in a Mediterranean riparian forest soil during two periods under contrasting soil moisture conditions in order to investigate the factors that affect denitrification through the year. Results showed that in summer, soil moisture limited denitrification throughout the entire soil profile, whereas in winter, anaerobic conditions in the soil were more favourable for denitrifiers. The potential for denitrification was larger at shallow depths (<30 cm), and neither nitrate nor organic carbon limited denitrification significantly. Some denitrification was measured during winter at depths below 30 cm, suggesting that a reduction of groundwater nitrate could occur in some areas of this riparian forest during the wet period. In summer, low denitrification, together with high mineralization rates, brought about an increase of soil N, which could be leached to the stream channel during rainfall events. This study suggests that Mediterranean riparian soils act as sources or sinks of dissolved nitrogen depending on the period of the year.     

人工湿地高效好氧反硝化菌的分离鉴定及反硝化特性研究

从增氧型复合垂直流人工湿地中采集样品,利用间歇曝气法富集好氧反硝化菌,并进行分离纯化,共得到10株好氧反硝化菌。其中编号为B13的菌株在初始硝态氮含量为277.23mg·L-1、碳氮比为5的条件下,24h的硝态氮去除率达92.80%,亚硝态氮积累只有12.57mg·L-1,脱氮速率达到20.58mg·L-·1h-1。16S rDNA序列分析表明,该菌与Pseudomonas stutzeri同源性达100%。选用四因素三水平L（934）正交试验表设计实验,通过测定对硝态氮去除能力和亚硝态氮的积累量,研究碳源、碳氮比（C/N）、pH以及溶解氧含量（DO）4种不同因素对B13号菌株好氧反硝化性能的影响。结果表明,该菌株对硝态氮的去除率最大可达99.88%,几乎没有亚硝态氮积累。对硝态氮去除率影响最大的因素为碳氮比,其次为pH,溶解氧含量和碳源。对应的最优条件是碳源为葡萄糖,碳氮比为10,pH为9,溶解氧含量为1.84～3.57mg·L-1。     

Characterization of denitrification activity in zones of groundwater exfiltration within a riparian wetland ecosystem

Movement of agricultural nitrogen (N) into riparian buffers often occurs within discrete seepage or upwelling zones which can limit the ability of the ecosystem to process the nutrient delivered by exfiltrating groundwater. Characterization of the biogeochemical processing of N within these zones is important in assessing the effectiveness of riparian buffers for mitigating nutrient loading of surface waters. The biogeochemical potential for denitrification in zones of exfiltration within a riparian buffer wetland dominated by high-carbon mucky soils was found to be highly stratified by profile depth with substantially higher activity in the surface layer of soil. The denitrification enzyme activity (DEA) within these zones was partly related to the population size of denitrifying microorganisms as measured by the most probable number (MPN) as well as the general microbial population as measured by substrate-induced respiration. The addition of glucose to the DEA assay stimulated enzyme activity indicating that carbon substrate was limiting activity. The stratification patterns of microbial populations and DEA are consistent with new carbon inputs to the ecosystem being most important driver of biogeochemical reactions such as denitrification in this high-carbon environment. A survey of carbon inputs to the ecosystem under study identified two major sources that contribute most of the annual biomass carbon inputs to the wetland: skunk cabbage in early summer and tree leaf litter in the fall. Tests of the ability of annually deposited wetland plant residues to stimulate denitrification and microbial respiration indicated that the degree of stimulation was inversely related to the C/N ratio of these carbon sources.     

Influence of Different Substrates in Wetland Soils on Denitrification

Different substrates were evaluated to investigate their effect on nitrate removal and denitrifying bacterial community in soils obtained from wetland. Serial batch kinetic tests were conducted on soils obtained from wetland mixed with glucose and sawdust using KNO₃ solution. Column tests were also conducted on soils obtained from wetland mixed with three different substrates (glucose, sawdust, and scoria coated with zero-valent iron) using KNO₃ solution. For the batch tests, the nitrate removal efficiency for soil mixed with glucose was comparable to that for soil mixed with sawdust, but the nitrate removal rate for soil mixed with glucose (23.3 NO ₃ ^? -N mg/L-d) was approximately eight times higher than that for soil mixed with sawdust (2.8 NO ₃ ^? -N mg/L-d). For column tests among soil samples, nitrate removal efficiency was highest in soil mixed with glucose, which is an easily biodegradable carbon source. Removal efficiency increased with increasing incubation time for both soil samples with glucose and sawdust. A phylogenetic analysis based on nitrate reductase gene demonstrated that the different carbon sources affected both the diversity and compositions of the denitrifying bacterial in soil samples.