Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting

The purpose of this study was to investigate the diversity of denitrifier community during agricultural waste composting. The diversity and dynamics of the denitrifying genes (nirK and nirS) were determined using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE). Relationships between physico-chemical parameters and denitrifying genes structures were simultaneously evaluated by redundancy analysis (RDA). Phylogenetic analysis indicated that nirK clones grouped into six clusters and nirS clones into two major clusters, respectively. The results showed a very high diversity of nir gene sequences within composting samples. RDA showed that the nirK and nirS gene structures were significantly related to pH and pile temperature (P?<?0.05). Significant amounts of the variation (49.2 and 38.3 % for nirK and nirS genes, respectively) were explained by pH and pile temperature, suggesting that those two parameters were the most likely ones to influence, or be influenced by the denitrifiers harboring nirK and nirS genes.     

Impact of Long-Term Fertilization on the Composition of Denitrifier Communities Based on Nitrite Reductase Analyses in a Paddy Soil

The effect of long-term fertilization on soil-denitrifying communities was determined by measuring the abundance and diversity of the nitrite reductase genes nirK and nirS. Soil samples were collected from plots of a long-term fertilization experiment started in 1990, located in Taoyuan (110°72″ E, 28°52″ N), China. The treatments were no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). The abundance, diversity, and composition of the soil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nirK and nirS genes. There was a pronounced difference in the community composition and diversity of nirK-containing denitrifiers responding to the long-term fertilization regimes; however, less variation was observed in communities of nirS-containing denitrifiers, indicating that denitrifiers possessing nirK were more sensitive to the fertilization practices than those with nirS. In contrast, fertilization regimes had similar effects on the copy numbers of nirK and nirS genes. The BMR treatment had the highest copy numbers of nirK and nirS, followed by the two mineral fertilization regimes (UR and BM), and the lowest was in the NF treatment. Of the measured soil parameters, the differences in the community composition of nirK and the abundance of nir denitrifiers were highly correlated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on the community structure of nirK populations only, and total organic carbon was the dominant factor in relation to the variations of nir community sizes.     

Molecular Characterization of Diazotrophic and Denitrifying Bacteria Associated with Mangrove Roots

An analysis of the molecular diversity of N₂ fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of nifH (N₂ fixation) and nirS and nirK (denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for nifH was higher than that for nirK or nirS at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest nifH diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For nirS, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for nifH. OTU defined on the basis of nirK were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of nifH, nirK, and nirS showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.     

牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。     

Genetic and Functional Variation in Denitrifier Populations along a Short-Term Restoration Chronosequence

Complete removal of plants and soil to exposed bedrock, in order to eradicate the Hole-in-the-Donut (HID) region of the Everglades National Park, FL, of exotic invasive plants, presented the opportunity to monitor the redevelopment of soil and the associated microbial communities along a short-term restoration chronosequence. Sampling plots were established for sites restored in 1989, 1997, 2000, 2001, and 2003. The goal of this study was to characterize the activity and diversity of denitrifying bacterial populations in developing HID soils in an effort to understand changes in nitrogen (N) cycling during short-term primary succession. Denitrifying enzyme activity (DEA) was detected in soils from all sites, indicating a potential for N loss via denitrification. However, no correlation between DEA and time since disturbance was observed. Diversity of bacterial denitrifiers in soils was characterized by sequence analysis of nitrite reductase genes (nirK and nirS) in DNA extracts from soils ranging in nitrate concentrations from 1.8 to 7.8 mg kg⁻¹. High levels of diversity were observed in both nirK and nirS clone libraries. Statistical analyses of clone libraries suggest a different response of nirS- and nirK-type denitrifiers to factors associated with soil redevelopment. nirS populations demonstrated a linear pattern of succession, with individual lineages represented at each site, while multiple levels of analysis suggest nirK populations respond in a grouped pattern. These findings suggest that nirK communities are more sensitive than nirS communities to environmental gradients in these soils.     

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2–6 times more abundant than those revealed with the conventional primers. RNA- and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.     

Effect of Sulfadiazine on Abundance and Diversity of Denitrifying Bacteria by Determining nirK and nirS Genes in Two Arable Soils

Sulfadiazine (SDZ) is an antibiotic frequently used in agricultural husbandry. Via manuring of excrements of medicated animals, the drug reaches the soil and might impair important biochemical transformation processes performed by microbes, e.g., the nitrogen turnover. We studied the effect of pig manure and SDZ-spiked pig manure on denitrifying bacteria by quantifying nirK and nirS nitrite reductase genes in two arable soils. Addition of manure entailed mainly an increase of nirK-harboring denitrifiers in both soils, whereas in the SDZ-amended treatments, primarily the nirS denitrifiers increased in abundance after the bioavailable SDZ had declined. However, the community composition of nirS nitrite reducers investigated by denaturing gradient gel electrophoresis did not change despite the observed alterations in abundance.     

Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

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.     

Nitrite Reductase Genes (nirK and nirS) as Functional Markers To Investigate Diversity of Denitrifying Bacteria in Pacific Northwest Marine Sediment Communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

N<Subscript>2</Subscript>O production by denitrification in an urban river: evidence from isotopes,functional genes,and dissolved organic matter

Rivers are important sources of N₂O emissions into the atmosphere. Nevertheless, N₂O production processes in rivers are not well identified. We measured concentrations and isotopic ratios of N₂O, NH₄ ⁺, NO₂ ^?, and NO₃ ^? in surface water to identify the microbial processes of N₂O production along the Tama River in Japan. We also measured the functional gene abundance of nitrifiers and denitrifiers (amoA-bacteria, nirK, nirS, nosZ clade I, nosZ clade II) together with concentrations of dissolved organic carbon (DOC) and fluorescence intensities of protein and humic components of dissolved organic matter (DOM) to support the elucidation of N₂O production processes. The observed nitrogen (δ¹⁵N) and oxygen (δ¹⁸O) of N₂O were within the expected isotopic range of N₂O produced by nitrate reduction, indicating that N₂O was dominantly produced by denitrification. The positive significant correlation between N₂O_Net concentration and nirK gene abundance implied that nitrifiers and denitrifiers are contributors to N₂O production. Fluorescence intensities of protein and humic components of DOM and concentrations of DOC did not show significant correlations with N₂O concentrations, which suggests that DOC and abundance of DOM components do not control dissolved N₂O. Measurement of isotope ratios of N₂O and its substrates was found to be a useful tool to obtain evidence of denitrification as the main source of N₂O production along the Tama River.     

Potential effects of leaf litter on water quality in urban watersheds

Leaf litter plays a critical role in regulating ecological functions in headwater forest streams, whereas the effects of leaves on water quality in urbanized streams are not fully understood. This study examined the potential importance of leaf litter for the release and transformations of organic carbon and nutrients in urban streams, and compared the effects with other types of natural organic substrates (periphyton and stream sediment). Nutrients and organic carbon were leached from senescent leaves of 6 tree species in the laboratory with deionized water, and maximal releases, leaching rate constants, composition and bioavailability of the leached dissolved organic carbon (DOC) were determined. Stream substrates (leaf debris, rocks with periphyton, and sediment) were seasonally collected from urban and forest reference streams of the NSF Baltimore Long-term Ecological Research Site and incubated with overlying stream water to estimate areal fluxes of DOC and nitrogen. Leaf litter leaching showed large ranges in maximal releases of DOC (7.0–131 mg g^?1), dissolved organic nitrogen (DON; 0.07–1.39 mg g^?1) and total dissolved phosphorus (TDP; 0.14–0.70 mg g^?1) among tree species. DOC leaching rate constants, carbon to nitrogen ratios, and DOC bioavailability were all correlated with organic matter quality indicated by fluorescence spectroscopy. Results from substrate incubation experiments showed far higher DOC and DON release and nitrate retention with leaf debris than with sediment, or rocks with periphyton. DOC release from leaf debris was positively correlated with stream nitrate retention at residential and urban sites, with the highest values observed during the fall and lowest during the summer. This study suggests the potential importance of leaf litter quantity and quality on fostering DOC and nutrient release and transformations in urban streams. It also suggests that species-specific impacts of leaves should be considered in riparian buffer and stream restoration strategies.     

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。     

The effects of heating,rhizosphere, and depth on root litter decomposition are mediated by soil moisture

The breakdown and decomposition of plant inputs are critical for nutrient cycling, soil development, and climate-ecosystem feedbacks, but uncertainties persist in how the rates and products of litter decomposition are affected by soil temperature, rhizosphere, and depth of input. We investigated the effects of soil warming (+ 4 °C), rhizosphere, and depth of litter placement on the decomposition of Avena fatua (wild oat grass) root litter in a Mediterranean grassland ecosystem. Field lysimeters were subjected to three environmental treatments (heating, control, and plant removal) and three ¹³C-labeled root litter addition treatments (to A horizon, to B horizon, and no-addition disturbance control) for each of two harvest time points. We buried root litter in February 2014 and measured loss of ¹³C in CO₂ from the soil surface and in leachate as dissolved organic carbon (DOC) over two growing seasons. At the end of each growing season we recovered the ¹³C remaining in the soil. Loss of root litter C occurred almost entirely via heterotrophic respiration, with an estimated < 2% lost as DOC during the initial decay period. The added roots were broken down and incorporated into bulk soil material very quickly; only ~ 30% of added root was visible after 6 months. In the first growing season, decomposition occurred faster in the B than in the A horizon, the latter having greater moisture limitation. Subsequently, there was almost no further decomposition in the B horizon. After two growing seasons, less than 20% of the added root litter C remained in the A or B horizons of all environmental treatments. Heating did not stimulate decomposition, likely because it exacerbated the moisture limitation. However, while plots without plants dried down more slowly than plots with plants, their decomposition rate was not significantly greater, possibly due to the lack of rhizosphere processes such as priming. We conclude that in this Mediterranean grassland ecosystem, soil moisture, which is affected by season, depth, heating, and rhizosphere, plays a dominant role in mediating the effect of those factors on root litter decomposition, which after two seasons did not differ by depth or by treatment.     

Hydrologic Drivers and Seasonality of Dissolved Organic Carbon Concentration, Nitrogen Content, Bioavailability, and Export in a Forested New England Stream

We present the results of a full year of high-resolution monitoring of hydrologic event-driven export of stream dissolved organic matter (DOM) from the forested Bigelow Brook watershed in Harvard Forest, Massachusetts, USA. A combination of in situ fluorescent dissolved organic matter (FDOM) measurement, grab samples, and bioassays was utilized. FDOM was identified as a strong indicator of concentration for dissolved organic carbon (DOC, r ² = 0.96), dissolved organic nitrogen (DON, r ² = 0.81), and bioavailable DOC (BDOC, r ² = 0.81). Relationships between FDOM and concentration were utilized to improve characterization of patterns of hydrological event-driven export and the quantification of annual export. This characterization was possible because DOM composition remained relatively consistent seasonally; however, a subtle shift to increased fluorescence per unit absorbance was observed for summer and fall seasons and percent BDOC did increase slightly with increasing concentrations. The majority of export occurred during pulsed hydrological events, so the greatest impact of bioavailable exports may be on downstream aquatic ecosystems. Export from individual events was highly seasonal in nature with the highest flow weighted mean concentrations (DOC_FW) being observed in late summer and fall months, but the highest total export being observed for larger winter storms. Seasonal trends in DOC export coincide with weather driven changes in surface and subsurface flow paths, potential for depletion and rebuilding of a flushable soil organic matter pool, and the availability of terrestrial carbon sources such as leaf litter. Our approach and findings demonstrate the utility of high frequency FDOM measurement to improve estimates of intra-annual temporal trends of DOM export.     

Quantification of Nitrogen Reductase and Nitrite Reductase Genes in Soil of Thinned and Clear-Cut Douglas-Fir Stands by Using Real-Time PCR

The abundance of nifH, nirS, and nirK gene fragments involved in nitrogen (N) fixation and denitrification in thinned second-growth Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest soil was investigated by using quantitative real-time PCR. Prokaryotic N cycling is an important aspect of N availability in forest soil. The abundance of universal nifH, Azotobacter sp.-specific nifH (nifH-g1), nirS, and nirK gene fragments in unthinned control and 30, 90, and 100% thinning treatments were compared at two long-term research sites on Vancouver Island, Canada. The soil was analyzed for organic matter (OM), total carbon (C), total N, NH₄-N, NO₃-N, and phosphorus (P). The soil horizon accounted for the greatest variation in nutrient status, followed by the site location. The 30% thinning treatment was associated with significantly greater nifH-g1 abundance than the control treatment in one site; at the same site, nirS in the mineral soil horizon was significantly reduced by thinning. The abundance of nirS genes significantly correlated with the abundance of nirK genes. In addition, significant correlations were observed between nifH-g1 abundance and C and N in the organic horizon and between nirS and nirK and N in the mineral horizon. Overall, no clear influence of tree thinning on nifH, nirS, and nirK was observed. However, soil OM, C, and N were found to significantly influence N-cycling gene abundance.Nitrogen (N) is a limiting nutrient in most Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest ecosystems. Understanding the links between forest management and forest ecosystem function, including the cycling of N, is of paramount importance to researchers and forest managers. Management practices such as thinning and clear-cutting can alter the soil microbial community, potentially altering the rate and amount of net N addition or loss to the forest floor. Clear-cutting alters the functional diversity of soil microorganisms and alters soil characteristics (temperature, pH, moisture, and nutrient status). Thinning and clear-cutting can increase nitrification, denitrification, and leaching of N in soil, all of which can reduce the available N (2, 13, 22, 41, 47). Clear-cutting in Douglas-fir forests can also remove associated gene pools of diazotrophic microorganisms (46). It is not yet well understood how clear-cutting or thinning affects the abundance of N-cycling microorganisms. We focus on two populations of N-cycling microorganisms: diazotrophs, which biologically fix N₂ gas to ammonia, and denitrifiers, which reduce N oxides and result in the release of N-containing gasses.Fixation of N by diazotrophic microorganisms is the primary source of N addition to undisturbed, unfertilized forest soil ecosystems (9, 39). The diazotrophic community is most often studied in situ using the marker gene for nitrogenase reductase (nifH); the diversity and abundance of diazotrophic microorganisms as determined by nifH characterization may be used as an indicator of overall soil ecological health. Diazotrophs can be symbiotic, associated (e.g., with a specific plant or fungal biomass), or free-living in the soil. Endophytic diazotrophs fix ∼100 times more N than free-living strains (9). Free-living diazotrophs such as Azotobacter vinelandii and A. chroococcum may fix between 0 and 60 kg of N ha⁻¹ year⁻¹ (9) and, because of a relative dearth of endophytic interactions in coniferous forests, free-living diazotrophs can be an important source of N in these soils. Cultural studies have shown that free-living diazotrophs improve the establishment of mycorrhizae and conifer seedlings, with relative activity fluctuating according to season, site aspect, and moisture conditions (11). Fixed-N inputs act as a catalyst for interlinked N-cycling events, e.g., fungal decomposition of woody debris and organic material (28). Nitrogen fixation in temperate forest soil is directly related to the amounts of soil organic matter (17). However, it is unclear how nifH gene abundance relates to the amount of total carbon (C) and organic matter (OM) and N in forest soil. It is also unknown how common silvicultural practices (e.g., clear-cutting and thinning) affect diazotrophic abundance or how diazotrophic abundance may in turn affect cycling of soil nutrients.The reduction of inorganic N oxides by denitrifying microorganisms can cause N loss from forest soil ecosystems, as well as the release of greenhouse gases into the atmosphere. The loss of N from temperate forest soil as N₂O has been reported as ranging from 0.2 to 7.0 kg ha⁻¹ year⁻¹, depending largely on soil nitrogen status, soil moisture, and temperature (57). Robertson and Tiedje (44) state that soil N loss in coniferous ecosystems due to denitrification is regulated by nitrification potential (e.g., nitrate levels) in the soil, and while not considered a major N loss component following clear-cutting, this loss is generally of the same magnitude as the N loss due to leaching. Denitrification is primarily studied using molecular approaches by monitoring several genes in the denitrification pathway: cytochrome cd₁-containing nitrite reductase (nirS), Cu-containing nitrite reductase (nirK), nitrous oxide reductase (nosZ), and membrane-bound nitrate reductase A (narG). The nirS and nirK genes were the denitrification genes used in the present study. Studies demonstrating (i) that the nirS gene is more diverse than nirK in soil and (ii) the domination of the nirK population by a relatively reduced number of clones have been published (42, 45). However, recent meta-analysis of studies involving nirK and nirS has shown that both communities tend to be phylogenetically clustered in undisturbed soils (23).To compare the effects of silvicultural practices on the abundance of diazotrophs and denitrifiers, we used quantitative real-time PCR (qPCR) assays to quantify nifH, nirS, and nirK genes in soil. This method can be used to quantify target sequences in environmental samples. Several qPCR protocols for the analysis of functional gene abundance in soil have been developed for N-cycling genes, including nifH, ammonia monooxygenase (amoA), nirK, nirS, nosZ, and narG (21, 24, 31, 38, 43, 54, 55). The objectives of the present study were (i) to quantify nifH, nirS, and nirK; (ii) to compare the effects of thinning and clear-cutting in Douglas-fir stands on the abundance of total diazotrophs, free-living diazotrophs, and denitrifiers; and (iii) to elucidate the relationships between N-cycling genes and nutrient abundance in forest soils.     

Biodegradability of Vegetation-Derived Dissolved Organic Carbon in a Cool Temperate Ombrotrophic Bog

Dissolved organic carbon (DOC) plays a key role in the peatland carbon balance and serves numerous ecological and chemical functions including acting as a microbial substrate. In this study, we quantify the concentration, biodegradability, and intrinsic properties of DOC obtained from peat, fresh material, and litter from nine species of ombrotrophic bog vegetation. Potential biodegradability was assessed by incubating vegetation extracts for 28 days in the dark and measuring percent DOC loss as the fraction of biodegradable DOC (%BDOC) while DOC properties were characterized using UV–Vis absorbance and fluorescence measurements. The mean initial DOC concentration extracted differed significantly among species (P < 0.05) and was significantly higher in fresh material, 217 ± 259 mg DOC l^?1, than either litter or peat extracts with mean concentrations of 82.1 ± 117 mg DOC l^?1 and 12.7 ± 1.0 mg DOC l^?1, respectively (P < 0.05). %BDOC also differed significantly among species (P < 0.05) and ranged from 52 to 73% in fresh cuttings with the greatest fraction observed in S. magellanicum; 22–46% in litter; and 24% in peat. The majority of variability (82.5%) in BDOC was explained by initial absorbance at 254 nm and total dissolved nitrogen concentration which was further resolved into significant non-linear relationships between %BDOC and both humic-like and protein-like DOC fractions (P < 0.05). Our results highlight the extremely heterogeneous nature of the surface vegetation-derived DOC input in peatlands and stress the importance of vegetation species in peatland ecosystem function.     

Experimental summer drought reduces soil CO2 effluxes and DOC leaching in Swiss grassland soils along an elevational gradient

Soil moisture affects belowground activity in grasslands, but the effects of summer drought on different soil C fluxes is uncertain. Soil respiration (SR), dissolved organic carbon (DOC) leaching and their components may all respond differently and drought effects will interact with other factors such as temperature, making a priori predictions of soil C balances difficult. In this study, we used rain shelters to simulate summer droughts by reducing annual precipitation by around 30 % in three managed grassland sites at 400, 1,000 and 2,000 m a.s.l. in Switzerland covering a gradient in mean annual temperatures of 7.5 °C. During the growing season, we quantified the impacts of drought on SR, DOC leaching, litter decomposition and the contribution of ¹³C-depleted litter to DOC fluxes. Along the elevational gradient, SR rates did not decrease with increasing altitude. Thus, SR was higher at a given temperature at higher altitudes, which probably reflects more labile soil C and hence greater substrate availability in a colder climate. Fluxes of DOC at 5 cm depth were a magnitude smaller than SR and did not show a pattern with elevation. At all altitudes, the experimental summer drought significantly reduced SR rates by 25–57 % and DOC leaching by 80–100 %, with a declining contribution of ¹³C-depleted litter-DOC. The remaining litter mass after drought was two to seven times larger as compared to the control. We did not observe a strong C release upon rewetting and hence, there was no compensation for the reduced soil C fluxes during drought. The more sensitive drought response in the litter layer than in the deeper soil and the declining DOC fluxes indicate an altered soil C balance with a C preservation in the topsoil, but ongoing losses of probably ‘older’ C in subsoils under drought.     

Distribution of denitrifying bacterial communities in the stratified water column and sediment–water interface in two freshwater lakes and the Baltic Sea

We have studied the distribution and community composition of denitrifying bacteria in the stratified water column and at the sediment–water interface in lakes Plußsee and Schöhsee, and a near-shore site in the Baltic Sea in Germany. Although environmental changes induced by the stratification of the water column in marine environments are known to affect specific populations of denitrifying bacteria, little information is available for stratified freshwater lakes and brackish water. The aim of the present study was to fill this gap and to demonstrate specific distribution patterns of denitrifying bacteria in specific aquatic habitats using two functional markers for the nitrite reductase (nirK and nirS genes) as a proxy for the communities. The leading question to be answered was whether communities containing the genes nirK and nirS have similar, identical, or different distribution patterns, and occupy the same or different ecological niches. The genes nirK and nirS were analyzed by PCR amplification with specific primers followed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequence analysis. Overall, nirS-denitrifiers were more diverse than nirK-denitrifiers. Denitrifying communities in sediments were clearly different from those in the water column in all aquatic systems, regardless of the gene analyzed. A differential distribution of denitrifying assemblages was observed for each particular site. In the Baltic Sea and Lake Plußsee, nirK-denitrifiers were more diverse throughout the water column, while nirS-denitrifiers were more diverse in the sediment. In Lake Schöhsee, nirS-denitrifiers showed high diversity across the whole water body. Habitat-specific clusters of nirS sequences were observed for the freshwater lakes, while nirK sequences from both freshwater lakes and the Baltic Sea were found in common phylogenetic clusters. These results demonstrated differences in the distribution of bacteria containing nirS and those containing nirK indicating that both types of denitrifiers apparently occupy different ecological niches.     

Intergenomic Comparisons Highlight Modularity of the Denitrification Pathway and Underpin the Importance of Community Structure for N2O Emissions

Nitrous oxide (N₂O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N₂O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N₂O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N₂O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N₂O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N₂O emissions than nirK type denitrifiers under favorable environmental conditions_. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N₂O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N₂O emissions.