不同季节艾比湖湿地盐角草根际nirS-型与nirK-型反硝化细菌群落组成分析

旨在了解艾比湖湿地盐生植物盐角草根际与非根际中不同类型反硝化细菌的分布及其随季节变化情况,为温带干旱地区荒漠盐化生态系统的代表-艾比湖湿地在生态植被恢复过程中,由微生物推动的土壤氮素循环过程提供数据支撑。采集了艾比湖湿地夏、秋、春三个季节的盐角草根际和非根际土壤样本,通过高通量测序技术,比较分析了nirS-型和nirK-型两种类型的反硝化细菌的多样性和群落结构特点;利用RDA (redundancy analysis)探究了土壤理化因素对反硝化细菌多样性及群落结构的影响。艾比湖湿地盐角草根际与非根际中,nirS-型和nirK-型反硝化细菌多样性最高的为秋季根际土壤样本;各土壤样本中的反硝化细菌多样性均呈现根际>非根际。盐角草各土壤样本中的nirS-型反硝化细菌在门分类水平上隶属于变形菌门(Proteobacteria),厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),而nirK-型反硝化细菌在门水平上分类仅包括了Proteobacteria和Firmicutes。Proteobacteria在各土壤样本中的占比均较高;其中Gamma-Proteobacteria的盐单胞菌属(Halomonas)和假单胞菌属(Pseudomonas)是各土壤样本所共有的nirS-型反硝化菌的优势菌属,但它们在每个土壤样本中的相对丰度各有差异。Alpha-Proteobacteria的根瘤菌属(Rhizobium)是盐角草各土壤样本中较为广泛存在的nirK-型反硝化细菌。艾比湖湿地盐角草各土壤样本中的反硝化细菌群落结构存在着一定的差异。RDA结果显示含水量、有机质、全氮和铵态氮等对各土壤样本中的nirS-型反硝化细菌的多样性影响较大,含水量、有机质、全氮、碱解氮等是nirK-型反硝化细菌多样性的主要影响因素。土壤电导率、全磷、全钾、全氮和碱解氮协同影响nirS-型反硝化细菌的群落结构,有机质、速效钾、速效磷、pH和硝态氮是nirK-型反硝化细菌群落结构组成的主要影响因素。艾比湖湿地反硝化细菌呈现季节性变化,nirS-型和nirK-型反硝化细菌以不同的主要菌属,共同推进湿地反硝化作用。而对于湿地生态系统的保护,则需要进行长期而广泛的土壤状态评估和土壤反硝化微生物菌群的动态监测。     

Long-term fertilization and tillage regimes have limited effects on structuring bacterial and denitrifier communities in a sandy loam UK soil

Denitrification causes loss of available nitrogen from soil systems, thereby reducing crop productivity and increasing reliance on agrochemicals. The dynamics of denitrification and denitrifying communities are thought to be altered by land management practices, which affect the physicochemical properties of the soil. In this study, we look at the effects of long-term tillage and fertilization regimes on arable soils following 16 years of treatment in a factorial field trial. By studying the bacterial community composition based on 16S rRNA amplicons, absolute bacterial abundance and diversity of denitrification functional genes (nirK, nirS and nosZ), under conditions of minimum/conventional tillage and organic/synthetic mineral fertilizer, we tested how specific land management histories affect the diversity and distribution of both bacteria and denitrification genes. Bacterial and denitrifier communities were largely unaffected by land management history and clustered predominantly by spatial location, indicating that the variability in bacterial community composition in these arable soils is governed by innate environmental differences and Euclidean distance rather than agricultural management intervention.     

深松与包膜尿素对玉米田土壤氮素转化及利用的影响

耕作方式和氮肥施用是影响土壤中氮肥转化、利用效率和作物产量的重要因素。通过夏玉米田的2a(2011—2012)定位试验,研究了两种耕作方式(深松、旋耕)配合不同尿素类型(包膜尿素、普通尿素)的施用对玉米田土壤硝态氮和铵态氮含量、脲酶活性、硝化细菌和反硝化细菌数量、玉米产量以及氮肥农学效率的影响。研究结果表明:相同耕作方式下,包膜尿素处理土壤中脲酶活性较稳定,且增加了旱田土壤亚硝酸细菌数量而降低了反硝化细菌数量,有利于土壤硝态氮含量的提高,尤其是作物生长的中后期;包膜尿素处理的产量比普通尿素提高7.25%—10.82%,同时提高氮肥农学效率。深松处理增加了土壤中的反硝化细菌数量,配合施用包膜尿素进一步提高了土壤脲酶活性,增加了亚硝酸细菌数量;旋耕与包膜尿素配合施用在一段时期内能显著增加土壤硝态氮含量,减少反硝化细菌数量。深松配合包膜尿素处理能够显著的增加玉米产量,2a分别比旋耕配合包膜尿素增加1.41%和10.62%。因此,深松措施配合施用包膜尿素能够增强土壤脲酶活性,增加亚硝酸细菌数量,提高氮素转化速率,增加作物产量和氮肥农学效率,其稳产效果在干旱年份尤为显著。     

Diversity,abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface sediments of the South China Sea

In marine ecosystems, both nitrite‐reducing bacteria and anaerobic ammonium‐oxidizing (anammox) bacteria, containing different types of NO‐forming nitrite reductase–encoding genes, contribute to the nitrogen cycle. The objectives of study were to reveal the diversity, abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface environments. Results showed that higher diversity and abundance of nirS gene than nirK and Scalindua‐nirS genes were evident in the sediments of the South China Sea (SCS), indicating bacteria containing nirS gene dominated the NO‐forming nitrite‐reducing microbial community in this ecosystem. Similar diversity and abundance distribution patterns of both nirS and Scalindua‐nirS genes were detected in this study sites, but different from nirK gene. Further statistical analyses also showed both nirS and Scalindua‐nirS genes respond similarly to environmental factors, but differed from nirK gene. These results suggest that bacteria containing nirS and Scalindua‐nirS genes share similar niche in deep‐sea subsurface sediments of the SCS, but differed from those containing nirK gene, indicating that community structures of nitrite‐reducing bacteria are segregated by the functional modules (NirS vs. NirK) rather than the competing processes (anammox vs. classical denitrification).     

Persistence of Denitrifying Enzyme Activity in Dried Soils

The effects of air drying soil on denitrifying enzyme activity, denitrifier numbers, and rates of N gas loss from soil cores were measured. Only 29 and 16% of the initial denitrifying enzyme activity in fresh, near field capacity samples of Maury and Donerail soils, respectively, were lost after 7 days of air drying. The denitrifying activity of bacteria added to soil and activity recently formed in situ were not stable during drying. When dried and moist soil cores were irrigated, evolution of N gas began, and it maximized sooner in the dried cores. This suggests that the persistence of denitrifying enzymes permits accelerated denitrification when dried soils are remoistened. Enzyme activity increased significantly in these waterlogged cores, but fluctuations in enzyme activity were small compared with fluctuations in actual denitrification rate, and enzyme activities were always greater than denitrification rates. Apparent numbers of isolatable denitrifiers (most-probable-number counts) decreased more than enzyme activity as the soils were dried, but after the soils were rewetted, the extent of apparent growth was not consistently related to the magnitude of N loss. We hypothesize that activation-inactivation of existing enzymes by soil O₂ is of greater significance in transient denitrification events than is growth of denitrifiers or synthesis of new enzymes.     

Metagenomic analysis reveals distinct patterns of denitrification gene abundance across soil moisture,nitrate gradients

This study coupled a landscape-scale metagenomic survey of denitrification gene abundance in soils with in situ denitrification measurements to show how environmental factors shape distinct denitrification communities that exhibit varying denitrification activity. Across a hydrologic gradient, the distribution of total denitrification genes (nap/nar + nirK/nirS + cNor/qNor + nosZ) inferred from metagenomic read abundance exhibited no consistent patterns. However, when genes were considered independently, nirS, cNor and nosZ read abundance was positively associated with areas of higher soil moisture, higher nitrate and higher annual denitrification rates, whereas nirK and qNor read abundance was negatively associated with these factors. These results suggest that environmental conditions, in particular soil moisture and nitrate, select for distinct denitrification communities that are characterized by differential abundance of genes encoding apparently functionally redundant proteins. In contrast, taxonomic analysis did not identify notable variability in denitrifying community composition across sites. While the capacity to denitrify was ubiquitous across sites, denitrification genes with higher energetic costs, such as nirS and cNor, appear to confer a selective advantage in microbial communities experiencing more frequent soil saturation and greater nitrate inputs. This study suggests metagenomics can help identify denitrification hotspots that could be protected or enhanced to treat non-point source nitrogen pollution.     

The denitrification potential and density of denitrifying bacteria in the bottom sediments of Matupi Harbor (Papua New Guinea)

The denitrification potential and density of denitrifying bacteria were determined in suboxidized sediments of Matupi Harbor (Papua New Guinea). The sediments were characterized by low denitrification potentials, not exceeding 0.8 nM/(g h). The maximum density of denitrifying bacteria was recorded in the upper sediment horizons and was 10⁴ cells/g of wet ground.     

NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand

The genetic heterogeneity of the nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in a non-agricultural forest soil in Thailand was investigated using soil samples from the Plant Germplasm-Royal Initiation Project area in Kanchanaburi Province, Thailand. Soil bacteria were screened for denitrification activity and 13 (from 211) positive isolates were obtained and further evaluated for their ability to reduce nitrate and to accumulate or reduce nitrite. Three species with potentially previously unreported denitrifying activities were recorded. Analysis of the partial nirK and nirS sequences of these 13 strains revealed a diverse sequence heterogeneity in these two genes within the same environment and even potentially within the same host species, the potential existence of lateral gene transfer and the first record of both nirK and nirS homologues in one bacterial species. Finally, isolates of two species of bacteria (Corynebacterium propinquum and Micrococcus lylae) are recorded as denitrifiers for the first time.     

Urban Stormwater Runoff Drives Denitrifying Community Composition Through Changes in Sediment Texture and Carbon Content

The export of nitrogen from urban catchments is a global problem, and denitrifying bacteria in stream ecosystems are critical for reducing in-stream N. However, the environmental factors that control the composition of denitrifying communities in streams are not well understood. We determined whether denitrifying community composition in sediments of nine streams on the eastern fringe of Melbourne, Australia was correlated with two measures of catchment urban impact: effective imperviousness (EI, the proportion of a catchment covered by impervious surfaces with direct connection to streams) or septic tank density (which affects stream water chemistry, particularly stream N concentrations). Denitrifying community structure was examined by comparing terminal restriction fragment length polymorphisms of nosZ genes in the sediments, as the nosZ gene codes for nitrous oxide reductase, the last step in the denitrification pathway. We also determined the chemical and physical characteristics of the streams that were best correlated with denitrifying community composition. EI was strongly correlated with community composition and sediment physical and chemical properties, while septic tank density was not. Sites with high EI were sandier, with less fine sediment and lower organic carbon content, higher sediment cations (calcium, sodium and magnesium) and water filterable reactive phosphorus concentrations. These were also the best small-scale environmental variables that explained denitrifying community composition. Among our study streams, which differed in the degree of urban stormwater impact, sediment grain size and carbon content are the most likely drivers of change in community composition. Denitrifying community composition is another in a long list of ecological indicators that suggest the profound degradation of streams is caused by urban stormwater runoff. While the relationships between denitrifying community composition and denitrification rates are yet to be unequivocally established, landscape-scale indices of environmental impact such as EI may prove to be useful indicators of change in microbial communities.     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO₃) from agricultural runoff through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N₂O) emissions from riparian forest soils exposed to prolonged nutrient runoff from plant nurseries and compares these to similar forest soils not exposed to nutrient runoff. Nursery runoff also contains high levels of phosphate (PO₄). Since there are conflicting reports on the impact of PO₄ on the activity of denitrifying microbes, the impact of PO₄ on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N₂O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO₃-N g⁻¹ soil, respectively. Net N₂O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO₃-N g⁻¹ soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO₃-N g⁻¹ in soil slurries. The addition of PO₄ (5 μg PO₄-P g⁻¹) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N₂O emissions compared to emission rates from non-exposed forest soils.     

Perspectives on measurement of denitrification in the field including recommended protocols for acetylene based methods

Of the biogeochemical processes, denitrification has perhaps been the most difficult to study in the field because of the inability to measure the product of the process. The last decade of research, however, has provided both acetylene and¹⁵N based methods as well as undisturbed soil core andin situ soil cover sampling approaches to implementing these methods. All of these methods, if used appropriately, give comparable results. Thus, we now have several methods, each with advantages for particular sites or objectives, that accurately measure denitrification in nature. Because of the general usefulness of the acetylene methods, updated protocols for the following three methods are given: gas-phase recirculation soil cores; static soil cores; and the denitrifying enzyme assay also known as the phase 1 assay. Despite the availability of these and other methods, denitrification budgets remain difficult to accurately establish in most environments because of the high spatial and temporal variability inherent in denitrification. Appropriate analysis of those data includes a distribution analysis of the data, and if highly skewed as is typically the case, the most accurate method to estimate the mean and the population variance is the UMVUE method (uniformly minimum variance unbiased estimator). Geostatistical methods have also been employed to improve spatial and temporal estimates of denitrification. These have occasionally been successful for spatial analysis but in the attempt described here for temporal analysis the approach was not useful.Discussions of the importance of denitrification have always focused on quantifying the process and whether particular measured quantities are judged to be a significant amount of nitrogen. A second line of evidence discussed here is the extant genetic record that results from natural selection. These analysis lead to the conclusion that strong selection for denitrification must currently be occurring, which implies that the process is of general significance in soils.     

Metabolic Profiles and Genetic Diversity of Denitrifying Communities in Activated Sludge after Addition of Methanol or Ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

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。     

Plant, soil microbial and soil inorganic nitrogen responses to elevated CO2: a study in microcosms of Holcus lanatus

The impact of elevated atmospheric CO₂ concentrations on the nitrogen cycle was evaluated in a 2-month experiment in monospecific grassland microcosms (Holcus lanatus L.) grown on reconstituted grassland soil. The responses of the N pools in the plants, soil, and soil microbes were studied. The impact of high CO₂ on key stages of the N cycle, especially nitrification and denitrification processes, were also measured. Our study showed a strong plant response to high CO₂: total biomass increased by 76% (P < 0.001) and root length density increased by 77% (P = 0.010). However, total plant N was not significantly modified by high CO₂, because the percent N in the plant decreased by 40% (P < 0.001). We observed a large decrease in soil NO₃^– concentration under elevated CO₂ (–50%; P = 0.002). Soil ammonium concentrations were much less affected by CO₂ enrichment, and only in resin bags (–8%, P = 0.019). Soil nitrifying enzyme activity (NEA) had a tendency to increase (+17%; P = 0.061) and denitrifying enzyme activity (DEA) decreased (-12%; P = 0.013). We found evidence of increased microbial N sink (microbial N increased by 17%, P = 0.004). This and other studies suggest that rising CO₂ often reduces soil nitrate concentrations, which may lead to decreased nitrate leaching. Elevated CO₂ led to environmental conditions that were less favourable for denitrification in our study.     

Comparative Denitrification of Selected Microorganisms in a Culture Medium and in Autoclaved Soil

The denitrifying behavior of selected soil bacteria was compared in a culture solution and in soil that was sterilized by autoclaving. The essential characteristics concerning nitrate reduction and the formation of nitrogenous gases did not change significantly for most bacteria in the two environments. Bacteria whose denitrification product was nitrous oxide evolved the same gas both in soil and in a liquid system, whereas other bacteria formed only nitrogen gas. The validity of laboratory observations in relation to field studies in the domain of denitrification is discussed and evaluated.     

Nitrogen dynamics in seasonally flooded soils in the Amazon floodplain

Large areas of the Amazon are subject to seasonal flooding due to water level changes of the river. This flood pulse causes rapidly changing conditions for microorganisms living in the soils which affects the cycling of nitrogen in the ecosystem. An understanding of the nitrogen dynamics in the seasonally flooded soils is essential for the development of productive and sustainable management concepts. We measured nitrogen concentrations, denitrifier enzyme activity (DEA), cell numbers of nitrifying and denitrifying bacteria, respiration, pH and total carbon in the seasonally flooded soils over one entire annual hydrological cycle. By comparing three sites with different vegetation (forest, aquatic macrophyte stand and bare sediment with annual herbs) we assessed the effect of vegetation on soil nitrogen dynamics. Inorganic nitrogen was always dominated by ammonium indicating reduced conditions in the soil even during the terrestrial phase. Although conditions were generally poor for nitrification we observed high numbers of nitrifying bacteria between 10⁴ and 10⁷cells g^–1. Pulses of ammonium as well as high DEA were observed during the transition periods between aquatic and terrestrial phase. Thus the alternation between aquatic and terrestrial phase promotes nitrogen mineralization and denitrification in the soils. There were no plausible correlations between microbial activities and numbers with soil physical or chemical parameters except a relation between the numbers of nitrate reducing bacteria and soil moisture (R² = 0.81) and ammonium (R² = 0.92) at one site. This shows the complex regulation patterns in this habitat. Different vegetation did not alter the general patterns of nitrogen dynamics but the absolute extend of fluctuations. We conclude that both the soil physical and chemical changes directly caused by the flood pulse and the vegetation have a great impact on microbial nitrogen turnover in the soils. The effects of the flood pulse can be buffered by a fine soil texture or a litter layer which prevents desiccation of the soil during the terrestrial phase.     

Denitrification in Lake Kinneret in the presence of oxygen*

SUMMARY. Denitrification experiments under anaerobic and aerated conditions were carried out in the laboratory with Lake Kinneret water and with pure cultures of the denitrifying bacteria Pseudomonas aeruginosa 2 Kin isolated from the lake. Although losses of nitrogen in Lake Kinneret due to denitrification have been found to occur during periods when dissolved oxygen exceeded 5 mg l^?1 it was found that under aerated conditions glucose as a carbon source must be added in order to get denitrification in the laboratory. Disappearance of nitrogen during the experiments was due to denitrification as shown by the nitrogen balance calculated for each sampling. The ATP content showed that no proliferation of cells took place during the experiment. The rate of denitrification was strongly influenced by and was directly proportional to nitrate concentrations. Temperature has a very slight effect on the denitrification rate. Q₁₀ for the range 15–30°C was 1.35. The role of denitrification in the nitrogen balance of Lake Kinneret is discussed.     

Effects of photoperiod on growth of and denitrification by Pseudomonas chlororaphis in the root zone of Glyceria maxima,studied in a gnotobiotic microcosm

The emergent macrophyte Glyceria maxima was subjected to different photoperiods and grown with ammonium or nitrate as nitrogen source in presterilized microcosms with spatially separated root and non-root compartments. The microcosms were inoculated with the denitrifying bacterium Pseudomonas chlororaphis. The effect of the plant and the photoperiod on growth and denitrification by P. chlororaphis was assessed. The plant had a strong positive effect on the growth of the bacteria. The bacterial numbers in the root compartment of the planted microcosms were 19-32 times higher than found in the non-root sediment of the unplanted systems. Lengthening the photoperiod resulted in elevated bacterial numbers due to the higher carbon exudation of the plant. This effect was greater still with the nitrate-fed plants, where additional P. chlororaphis growth could proceed via denitrification, indicating oxygen-limiting conditions in the microcosms. Higher porewater N₂O concentrations in the root compartments as compared to the non-root compartments, which were highest for the long photoperiod, were also indicative of a plant-induced stimulation of denitrification. An effect of a diurnal oxygen release pattern of G. maxima on denitrification could not be detected. The gnotobiotic microcosm used in this study represents a potential system for the study of the behaviour and interactions of important bacterial groups, such as nitrifying and denitrifying bacteria where plant roots drive bacterial activity.     

Identification of Denitrifying Bacteria Diversity in an Activated Sludge System by using Nitrite Reductase Genes

Nitrite reduction is the key step in the denitrification reaction with two predominant types of nitrite reductase genes: nirS and nirK. The diversity of denitrifying bacteria in a municipal wastewater treatment plant is described by using both these genes. Of the cultured colonies, 22.5% contained the NirS gene and 12.5% the nirK gene. These nitrite reductase-containing colonies could be further divided into five different types by using both restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis. Phylogenetic analysis showed that these five types of denitrifying bacteria were phylogenetically diverse. Finally, one nirS gene was obtained and compared with the published sequences.