氮磷添加对草甸草原土壤氮磷转化功能基因丰度的影响

氮添加是提高退化草地生产力的主要养分管理措施,而过量的氮输入会导致土壤酸化、增加硝酸盐淋溶损失和温室气体排放。旨在明确草原割草利用下土壤氮、磷转化功能基因丰度对氮磷添加的响应规律,为定向调控打草场土壤氮、磷转化过程,提高养分利用效率,减少温室气体N₂O排放提供科学依据。2018—2020年在呼伦贝尔草甸草原打草场设置了5个施氮水平(0、1.55、4.65、13.95、27.9 g N m^-2 a^-1)和3个磷水平(0、5.24、10.48 g P m^-2 a^-1),裂区试验设计,在植物不同生长时期测定土壤氨氧化(amoA-AOA和amoA-AOB)、反硝化(narG、nirK、nirS和nosZ)和磷转化(phoD)基因丰度。结果表明,土壤氮转化基因丰度受到氮、磷添加的调控,而氮、磷添加对土壤磷转化功能基因丰度无显著影响(P>0.05)。氮添加可提高amoA-AOB基因丰度,增加氨氧化细菌调控土壤总硝化速率的相对重要性,因此能增加硝酸盐淋溶损失潜势。高氮处理下添加磷可降低...     

Abundance of Microbes Involved in Nitrogen Transformation in the Rhizosphere of Leucanthemopsis alpina (L.) Heywood Grown in Soils from Different Sites of the Damma Glacier Forefield

Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant–microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Especially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) Heywood as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the soils as well as to the nitrogen and carbon status of the plants. The experiment was performed in a greenhouse simulating the climatic conditions of the glacier forefield. Samples were taken after 7 and 13 weeks of plant growth. Highest nifH gene abundance in connection with lowest nitrogen content of L. alpina was observed in the 10-year soil after 7 weeks of plant growth, demonstrating the important role of associative nitrogen fixation for plant development in this soil. In contrast, in the 120-year soil copy numbers of genes involved in denitrification, mainly nosZ were increased after 13 weeks of plant growth, indicating an overall increased microbial activity status as well as higher concentrations of nitrate in this soil.     

Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ _I and nosZ _II) in lake sediments. Summertime N₂O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS, nirK, nosZ _I, and nosZ _II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O reductase genes, i.e. the (nirS+nirK)/nosZ _I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ _I at the inter-lake scale and (nirS+nirK)/nosZ _I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.     

Nitrification,denitrification, and related functional genes under elevated CO2: A meta-analysis in terrestrial ecosystems

Global change may have profound effects on soil nitrogen (N) cycling that can induce positive feedback to climate change through increased nitrous oxide (N₂O) emissions mediated by nitrification and denitrification. We conducted a meta-analysis of the effects of elevated CO₂ on nitrification and denitrification based on 879 observations from 58 publications and 46 independent elevated CO₂ experiments in terrestrial ecosystems. We investigated the effects of elevated CO₂ alone or combined with elevated temperature, increased precipitation, drought, and N addition. We assessed the response to elevated CO₂ of gross and potential nitrification, potential denitrification, and abundances of related functional genes (archaeal amoA, bacterial amoA, nirK, nirS, and nosZ). Elevated CO₂ increased potential nitrification (+28%) and the abundance of bacterial amoA functional gene (+62%) in cropland ecosystems. Elevated CO₂ increased potential denitrification when combined with N addition and higher precipitation (+116%). Elevated CO₂ also increased the abundance of nirK (+25%) and nirS (+27%) functional genes in terrestrial ecosystems and of nosZ (+32%) functional gene in cropland ecosystems. The increase in the abundance of nosZ under elevated CO₂ was larger at elevated temperature and high N (+62%). Four out of 14 two-way interactions tested between elevated CO₂ and elevated temperature, elevated CO₂ and increased precipitation, and elevated CO₂ and N addition were marginally significant and mostly synergistic. The effects of elevated CO₂ on potential nitrification and abundances of bacterial amoA and nirS functional genes increased with mean annual temperature and mean annual precipitation. Our meta-analysis thus suggests that warming and increased precipitation in large areas of the world could reinforce positive responses of nitrification and denitrification to elevated CO₂ and urges the need for more investigations in the tropical zone and on interactive effects among multiple global change factors, as we may largely underestimate the effects of global change on soil N₂O emissions.     

不同土地利用方式土壤氨氧化微生物和反硝化微生物时空分布特征

研究不同土地利用方式下氮循环相关微生物在不同土壤剖面的分布,可为认识和理解土壤氮转化过程提供科学依据。土壤氨氧化微生物和反硝化微生物在调节氮肥利用率、硝态氮淋溶和氧化亚氮(N₂O)排放等方面有着重要作用。以北京郊区农田和林地两种土地利用方式为研究对象,分析土壤氨氧化潜势和亚硝酸盐氧化潜势在0—100 cm土壤剖面上的季节分布(春季和秋季),并通过实时荧光定量PCR方法表征土壤氨氧化和反硝化微生物的时空分布特征。结果表明,农田土壤氨氧化潜势、亚硝酸盐氧化潜势、氨氧化微生物和反硝化微生物丰度均显著高于林地土壤,且随土壤深度增加而显著降低。除氨氧化古菌amoA基因丰度在不同季节间无显著差异外,春季土壤氨氧化细菌(amoA基因)、反硝化微生物nirS、nirK和典型nosZ I基因的丰度均显著高于秋季。土壤有机质、总氮、NH~+₄-N、NO~-₃-N含量与氨氧化微生物和反硝化微生物的功能基因丰度显著相关。综上,不同土地利用方式下土壤氮循环相关微生物的丰度与土壤氮素的可利用性和转化过程紧密相关,研究结果对土壤氮素利用和养分管理提供...     

The Different Potential of Sponge Bacterial Symbionts in N2 Release Indicated by the Phylogenetic Diversity and Abundance Analyses of Denitrification Genes,nirK and nosZ

Nitrogen cycle is a critical biogeochemical process of the oceans. The nitrogen fixation by sponge cyanobacteria was early observed. Until recently, sponges were found to be able to release nitrogen gas. However the gene-level evidence for the role of bacterial symbionts from different species sponges in nitrogen gas release is limited. And meanwhile, the quanitative analysis of nitrogen cycle-related genes of sponge microbial symbionts is relatively lacking. The nirK gene encoding nitrite reductase which catalyzes soluble nitrite into gas NO and nosZ gene encoding nitrous oxide reductase which catalyzes N₂O into N₂ are two key functional genes in the complete denitrification pathway. In this study, using nirK and nosZ genes as markers, the potential of bacterial symbionts in six species of sponges in the release of N₂ was investigated by phylogenetic analysis and real-time qPCR. As a result, totally, 2 OTUs of nirK and 5 OTUs of nosZ genes were detected by gene library-based saturated sequencing. Difference phylogenetic diversity of nirK and nosZ genes were observed at OTU level in sponges. Meanwhile, real-time qPCR analysis showed that Xestospongia testudinaria had the highest abundance of nosZ gene, while Cinachyrella sp. had the greatest abundance of nirK gene. Phylogenetic analysis showed that the nirK and nosZ genes were probably of Alpha-, Beta-, and Gammaproteobacteria origin. The results from this study suggest that the denitrification potential of bacteria varies among sponges because of the different phylogenetic diversity and relative abundance of nosZ and nirK genes in sponges. Totally, both the qualitative and quantitative analyses of nirK and nosZ genes indicated the different potential of sponge bacterial symbionts in the release of nitrogen gas.     

Molecular analysis of the nitrogen cycle in deep-sea microorganisms from the Nankai Trough: genes for nitrification and denitrification from deep-sea environmental DNA

Nitrification and denitrification are bacterial functions, which are important for the global nitrogen cycle. Thus, it is important to study the diversity and distribution of bacteria in the environment, which are involved in the nitrogen cycle on the earth. Ammonia monooxygenase encoded by the amoA gene and nitrite reductase encoded by nirK or nirS are essential enzymes for nitrificaton and denitrification, respectively. These genes can be used as markers for the identification of organisms in the nitrogen cycle. In this study, we identified amoA (42 clones) and nirS (98 clones) genes in parallel from samples recovered from the deep-sea of the Nankai Trough. Genes for nirK could not be amplified from these samples. The obtained amoA sequences were not so closely related to those of amoA genes from previously isolated environmental organisms and those of genes from environmental DNAs. On the other hand, the nirS genes sequenced showed some relationship to some extent with the latter genes. However, some of the newly sequenced genes formed clusters, which contained no previously identified genes on a phylogenetic tree. These are likely present in specific denitrifiers from the deep-sea. The results of this study further suggest that nitrifiers and denitrifiers live in the same area of the Nankai Trough and the nitrogen cycle exists even in the deep-sea.     

生物炭对褐土旱地玉米季氮转化功能基因、丛枝菌根真菌及N2O释放的影响

以豫西旱地玉米农田为研究对象,设置不同生物炭施用量处理（T0：不施用生物炭;T1：施用生物炭20 t/hm²;T2：施用生物炭40 t/hm²）,采用密闭式静态箱法测定N₂O排放通量和荧光定量PCR法分析丛枝菌根（arbuscular mycorrhizal,AM）真菌、氨单加氧酶（amoA）、亚硝酸盐还原酶（nirS、nirK）以及氧化亚氮还原酶（nosZ）的基因丰度,同时测定土壤理化性状的变化。研究结果表明,随着生物炭施用量的增加,土壤pH和含水量呈增加趋势,土壤有机碳、全氮和铵态氮含量显著提高,土壤容重和硝态氮含量显著降低。T1和T2处理土壤有机碳含量分别较T0显著提高38.44%和71.01%;T1和T2处理土壤铵态氮含量分别较T0显著增加15.89%和30.46%;T2处理土壤全氮含量较T0处理显著提高14.87%;T1和T2处理土壤硝态氮含量分别较T0减少10.57%和21.40%。随着生物炭施用量的增加,AM真菌侵染率显著增加,T1和T2处理分别较T0处理提高71.88%和115.88%;AOA、AOB、nirK和nirS基因丰度显著降低;nosZ基因丰度增加。施加生物炭处理的N₂O排放通量和累积排放量均低于不施生物炭处理,具体表现为：T0 > T1 > T2。相关分析表明,生物炭施用量与AM真菌基因丰度呈显著正相关;与nosZ基因丰度呈正相关;与AOA、AOB、nirK、nirS基因丰度呈极显著负相关。N₂O排放通量与AOA、nirK、nirS基因丰度呈极显著正相关;与土壤含水量和土壤硝态氮含量呈显著正相关;与AM真菌、nosZ基因丰度、易提取球囊霉素含量、铵态氮含量呈极显著负相关。集成推进树（ABT）分析表明,AOA对N₂O排放的影响最大,其次是AM真菌和nirS。总之,生物炭处理改善土壤理化性质、提高土壤AM真菌侵染率、调节硝化、反硝化相关功能基因的丰度,减少N₂O气体排放,为旱地农田合理施用生物炭减少N₂O气体排放提供理论依据。     

Abundance and Diversity of Bacterial Nitrifiers and Denitrifiers and Their Functional Genes in Tannery Wastewater Treatment Plants Revealed by High-Throughput Sequencing

Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.     

不同放牧强度下土壤氨氧化和反硝化微生物的变化特征

土壤硝化及反硝化功能微生物在氮素可利用性、硝酸盐淋溶和氧化亚氮温室气体排放等方面起着关键作用,在指示不同放牧强度对生态系统的影响及预测草地生态系统退化状况等方面具有重要意义。以内蒙古干旱半干旱草原不同放牧强度(轻度、中度和重度)的长期试验样地为对象,应用定量PCR和限制性末端片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)的方法,研究土壤氨氧化古菌(ammonia-oxidizing archaea,AOA)、氨氧化细菌(ammonia-oxidizing bacteria,AOB)和反硝化细菌的丰度、群落结构和多样性对不同放牧强度的响应。结果表明,土壤p H和铵态氮含量分别在7.90—8.18和6.37—35.92 mg/kg之间,中度放牧处理显著增高了土壤pH(P=0.03),而铵态氮含量在重度放牧处理中最高(P=0.02)。不同放牧强度下土壤异养呼吸相比未放牧处理均显著降低(P=0.02)。土壤AOA-amoA和AOB-amoA基因丰度范围分别为每克干土(4.94—7.60)×10~9个拷贝数和(0.68—3.75)×10~6个拷贝数,放牧处理对AOA-amoA基因丰度无显著影响,中度放牧处理显著降低了AOB-amoA基因丰度(P=0.04);反硝化微生物nosZ基因丰度随在轻度放牧处理中最低(P=0.03)。土壤铵态氮含量是影响AOA-amoA和AOB-amoA基因丰度的主要因子,而nosZ基因丰度主要受反硝化底物含量及土壤通气状况的影响。冗余分析表明由放牧所引起的可利用性氮含量的变化是导致氨氧化和反硝化微生物群落结构显著变化的主要因素。     

Ectomycorrhizal fungi inoculation alleviates simulated acid rain effects on soil ammonia oxidizers and denitrifiers in Masson pine forest

Acid rain can cause severe effects on soil biota and nutrient biogeochemical cycles in the forest ecosystem, but how plant-symbiotic ectomycorrhizal fungi will modulate the effects remains unknown. Here, we conducted a full factorial field experiment in a Masson pine forest by simultaneously controlling the acidity of the simulated rain (pH 5.6 vs. pH 3.5) and the ectomycorrhizal fungi Pisolithus tinctorius inoculation (non-inoculation vs. inoculation), to investigate the effects on ammonia oxidizers and denitrifiers. After 10 months, compared with the control (rain pH 5.6, and non-inoculation), simulated acid rain (pH 3.5) reduced soil nutrient content, decreased archaeal amoA gene abundance and inhibited denitrification enzyme activity. Also, simulated acid rain altered the community compositions of all the examined functional genes (archaeal amoA, bacterial amoA, nirK, nirS and nosZ). However, inoculation with ectomycorrhizal fungi under acid rain stress recovered soil nutrient content, archaeal amoA gene abundance and denitrification enzyme activity to levels comparable to the control, suggesting that ectomycorrhizal fungi inoculation ameliorates simulated acid rain effects. Taken together, ectomycorrhizal fungi inoculation – potentially through improving soil substrate availability – could alleviate the deleterious effects of acid rain on nitrogen cycling microbes in forest soils.     

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.     

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).     

Elevated temperature shifts soil N cycling from microbial immobilization to enhanced mineralization,nitrification and denitrification across global terrestrial ecosystems

We assessed the response of soil microbial nitrogen (N) cycling and associated functional genes to elevated temperature at the global scale. A meta‐analysis of 1,270 observations from 134 publications indicated that elevated temperature decreased soil microbial biomass N and increased N mineralization rates, both in the presence and absence of plants. These findings infer that elevated temperature drives microbially mediated N cycling processes from dominance by anabolic to catabolic reaction processes. Elevated temperature increased soil nitrification and denitrification rates, leading to an increase in N₂O emissions of up to 227%, whether plants were present or not. Rates of N mineralization, denitrification and N₂O emission demonstrated significant positive relationships with rates of CO₂ emissions under elevated temperatures, suggesting that microbial N cycling processes were associated with enhanced microbial carbon (C) metabolism due to soil warming. The response in the abundance of relevant genes to elevated temperature was not always consistent with changes in N cycling processes. While elevated temperature increased the abundances of the nirS gene with plants and nosZ genes without plants, there was no effect on the abundances of the ammonia‐oxidizing archaea amoA gene, ammonia‐oxidizing bacteria amoA and nirK genes. This study provides the first global‐scale assessment demonstrating that elevated temperature shifts N cycling from microbial immobilization to enhanced mineralization, nitrification and denitrification in terrestrial ecosystems. These findings infer that elevated temperatures have a profound impact on global N cycling processes with implications of a positive feedback to global climate and emphasize the close linkage between soil microbial C and N cycling.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Oligonucleotide Microarray for the Study of Functional Gene Diversity in the Nitrogen Cycle in the Environment

The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 10⁷ copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications.     

Can differences in microbial abundances help explain enhanced N2O emissions in a permanent grassland under elevated atmospheric CO2?

Long‐term effects of elevated atmospheric CO₂ on the ammonia‐oxidizing and denitrifying bacteria in a grassland soil were investigated to test whether a shift in abundance of these N‐cycling microorganisms was responsible for enhanced N₂O emissions under elevated atmospheric CO₂. Soil samples (7.5 cm increments to 45 cm depth) were collected in 2008 from the University of Giessen Free Air Carbon dioxide Enrichment (GiFACE), a permanent grassland exposed to moderately elevated atmospheric CO₂ (+20%) since 1998. GiFACE plots lay on a soil moisture gradient because of gradually changing depth to the underlying water table and labeled as the DRY block (furthest from water table), MED block (intermediate to water table), and WET block (nearest to water table). Mean N₂O emissions measured since 1998 have been significantly higher under elevated CO₂. This study sought to identify microbial and biochemical parameters that might explain higher N₂O emissions under elevated CO₂. Soil biochemical parameters [extractable organic carbon (EOC), dissolved organic nitrogen (DON), NH₄⁺, NO₃^?], and abundances of genes encoding the key enzymes involved in ammonia oxidation (amoA) and denitrification (nirK, nirS, nosZ) depended more on soil depth and block (underlying soil moisture gradient) than on elevated CO₂. Ammonia oxidation and denitrification gene abundances, relative abundances (ratios) of nirS to nirK, of nosZ to both nirS and to nirK, and of the measured soil biochemical properties DON and NO₃^? tended to be lower in elevated CO₂ plots as compared with ambient plots in the MED and WET blocks while the DRY block exhibited an opposite trend. High N₂O emissions under elevated CO₂ in the MED and WET blocks correlated with lower nosZ to nirK ratios, suggesting that increased N₂O emissions under elevated CO₂ might be caused by a higher proportion of N₂O‐producing rather than N₂O consuming (N₂ producing) denitrifiers.     

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.     

Impacts of Long-Term Irrigation of Domestic Treated Wastewater on Soil Biogeochemistry and Bacterial Community Structure

Freshwater scarcity and regulations on wastewater disposal have necessitated the reuse of treated wastewater (TWW) for soil irrigation, which has several environmental and economic benefits. However, TWW irrigation can cause nutrient loading to the receiving environments. We assessed bacterial community structure and associated biogeochemical changes in soil plots irrigated with nitrate-rich TWW (referred to as pivots) for periods ranging from 13 to 30 years. Soil cores (0 to 40 cm) were collected in summer and winter from five irrigated pivots and three adjacently located nonirrigated plots. Total bacterial and denitrifier gene abundances were estimated by quantitative PCR (qPCR), and community structure was assessed by 454 massively parallel tag sequencing (MPTS) of small-subunit (SSU) rRNA genes along with terminal restriction fragment length polymorphism (T-RFLP) analysis of nirK, nirS, and nosZ functional genes responsible for denitrification of the TWW-associated nitrate. Soil physicochemical analyses showed that, regardless of the seasons, pH and moisture contents (MC) were higher in the irrigated (IR) pivots than in the nonirrigated (NIR) plots; organic matter (OM) and microbial biomass carbon (MBC) were higher as a function of season but not of irrigation treatment. MPTS analysis showed that TWW loading resulted in the following: (i) an increase in the relative abundance of Proteobacteria, especially Betaproteobacteria and Gammaproteobacteria; (ii) a decrease in the relative abundance of Actinobacteria; (iii) shifts in the communities of acidobacterial groups, along with a shift in the nirK and nirS denitrifier guilds as shown by T-RFLP analysis. Additionally, bacterial biomass estimated by genus/group-specific real-time qPCR analyses revealed that higher numbers of total bacteria, Acidobacteria, Actinobacteria, Alphaproteobacteria, and the nirS denitrifier guilds were present in the IR pivots than in the NIR plots. Identification of the nirK-containing microbiota as a proxy for the denitrifier community indicated that bacteria belonged to alphaproteobacteria from the Rhizobiaceae family within the agroecosystem studied. Multivariate statistical analyses further confirmed some of the above soil physicochemical and bacterial community structure changes as a function of long-term TWW application within this agroecosystem.