Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones

Lands under riparian and agricultural management differ in soil properties, water content, plant species and nutrient content and are therefore expected to influence denitrifier communities, denitrification and nitrous oxide (N(2) O) emissions. Denitrifier community abundance, denitrifier community structure, denitrification gene expression and activity were quantified on three dates in a maize field and adjacent riparian zone. N(2) O emissions were greater in the agricultural zone, whereas complete denitrification to N(2) was greater in the riparian zone. In general, the targeted denitrifier community abundance did not change between agricultural and riparian zones. However, nosZ gene expression was greater in the riparian zone than the agricultural zone. The community structure of nirS-gene-bearing denitrifiers differed in June only, whereas the nirK-gene-bearing community structure differed significantly between the riparian and the agricultural zones at all dates. The nirK-gene-bearing community structure was correlated with soil pH, while no significant correlations were found between nirS-gene-bearing community structure and soil environmental variables or N(2) O emissions, denitrification or denitrifier enzyme activity. The results suggested for the nirK and nirS-gene-bearing communities different factors control abundance vs. community structure. The nirK-gene-bearing community structure was also more responsive than the nirS-gene-bearing community structure to change between the two ecosystems.     

崇明岛不同土地利用类型河岸带土壤反硝化酶活性特征

以崇明岛河岸带为研究对象,采用乙炔抑制法,研究了不同土地利用类型河岸带(农田河岸带、林地河岸带、草地河岸带)土壤反硝化酶活性及其影响因素.结果表明：河岸带反硝化酶活性在(0.69±0.11)～(134.93±33.72) μg N·kg^-1·h^-1,不同土地类型河岸带土壤反硝化酶活性存在明显差异,整体趋势为林地河岸带>农田河岸带>草地河岸带.河岸带表层土壤(0～10 cm)反硝化酶活性与其他土层(10～30、30～50和50～70 cm)呈显著差异(P<0.05).反硝化酶活性与土壤有机碳、土壤全氮和土壤硝态氮呈极显著正相关关系(P<0.01).土地利用类型的变化主要通过改变河岸带土壤自然结构和理化性质、降低土壤有机质的积累、影响土壤氮素的转化,从而抑制河岸带土壤反硝化作用的发生.     

Plant Trait Diversity Buffers Variability in Denitrification Potential over Changes in Season and Soil Conditions

Background

Denitrification is an important ecosystem service that removes nitrogen (N) from N-polluted watersheds, buffering soil, stream, and river water quality from excess N by returning N to the atmosphere before it reaches lakes or oceans and leads to eutrophication. The denitrification enzyme activity (DEA) assay is widely used for measuring denitrification potential. Because DEA is a function of enzyme levels in soils, most ecologists studying denitrification have assumed that DEA is less sensitive to ambient levels of nitrate (NO₃ ⁻) and soil carbon and thus, less variable over time than field measurements. In addition, plant diversity has been shown to have strong effects on microbial communities and belowground processes and could potentially alter the functional capacity of denitrifiers. Here, we examined three questions: (1) Does DEA vary through the growing season? (2) If so, can we predict DEA variability with environmental variables? (3) Does plant functional diversity affect DEA variability?

Methodology/Principal Findings

The study site is a restored wetland in North Carolina, US with native wetland herbs planted in monocultures or mixes of four or eight species. We found that denitrification potentials for soils collected in July 2006 were significantly greater than for soils collected in May and late August 2006 (p<0.0001). Similarly, microbial biomass standardized DEA rates were significantly greater in July than May and August (p<0.0001). Of the soil variables measured—soil moisture, organic matter, total inorganic nitrogen, and microbial biomass—none consistently explained the pattern observed in DEA through time. There was no significant relationship between DEA and plant species richness or functional diversity. However, the seasonal variance in microbial biomass standardized DEA rates was significantly inversely related to plant species functional diversity (p<0.01).

Conclusions/Significance

These findings suggest that higher plant functional diversity may support a more constant level of DEA through time, buffering the ecosystem from changes in season and soil conditions.     

Denitrification Potential, Root Biomass, and Organic Matter in Degraded and Restored Urban Riparian Zones

Hydrologic changes associated with urbanization often lead to lower water tables and drier, more aerobic soils in riparian zones. These changes reduce the potential for denitrification, an anaerobic microbial process that converts nitrate, a common water pollutant, into nitrogen gas. In addition to oxygen, denitrification is controlled by soil organic matter and nitrate. Geomorphic stream restorations are common in urban areas, but their effects on riparian soil conditions and denitrification have not been evaluated. We measured root biomass, soil organic matter, and denitrification potential (anaerobic slurry assay) at four depths in duplicate degraded, restored, and reference riparian zones in the Baltimore, Maryland, U.S.A., metropolitan area. There were three main findings in this study. First, although reference sites were wet and had high soil organic matter, they had low levels of nitrate relative to degraded and restored sites and therefore there were few differences in denitrification potential among sites. Evaluations of riparian restorations that have nitrate removal by denitrification as a goal should consider the complex controls of this process and how they vary between sites. Second, all variables declined markedly with depth in the soil. Restorations that increase riparian water tables will thus foster interaction of groundwater nitrate with near-surface soils with higher denitrification potential. Third, we observed strong positive relationships between root biomass and soil organic matter and between soil organic matter and denitrification potential, which suggest that establishment of deep-rooted vegetation may be particularly important for increasing the depth of the active denitrification zone in restored riparian zones.     

Dynamics of Soil Denitrifier Populations: Relationships between Enzyme Activity, Most-Probable-Number Counts, and Actual N Gas Loss

To better understand temporal variability in soil denitrification, denitrifying enzyme activity (DEA) and denitrifier populations (as determined by most-probable-number [MPN] counts) were measured in field and laboratory experiments. Measurements of DEA and MPN provided highly contradictory indications of denitrifier dynamics. In laboratory incubations, under conditions favoring active denitrification, the synthesis of new denitrifying enzymes and the actual amount of denitrification were closely related. In other experiments, however, both DEA and MPN counts were poor indicators of actual denitrification. In some cases, we found significant increases in DEA but no significant production of N gas. Except with unnaturally high substrate amendments, changes in DEA were small relative both to the persistently high DEA background and to changes in MPN. As estimated by MPN counts, denitrifier populations increased significantly during denitrification events. It was apparent that only a small fraction of the denitrifiers were included in the MPN counts, but it appeared that this isolatable fraction increased during periods of active denitrifier growth. Use of DEA as an index of biomass of cells which have synthesized denitrifying enzymes suggested that denitrifier populations were persistent, stable, and much larger than indicated by MPN procedures.     

Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in alpha-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.     

Tree species, root decomposition and subsurface denitrification potential in riparian wetlands

Patches of organic matter have been found to be important `hotspots' of denitrification in both surface and subsurface soils, but the factors controlling the formation and maintenance of these patches are not well established. We compared the concentration of patches of organic matter and root biomass in the subsurface (saturated zone) beneath poorly drained riparian wetland soils at four sites in Rhode Island, USA - two dominated by red maple (Acer rubrum) and two dominated by white pine (Pinus strobus). Denitrification enzyme activity (DEA) and carbon (C) content of patch material were compared between sites and between patches with different visual characteristics. Root decomposition was measured in an 8-week ex-situ incubation experiment that compared the effects of water content, root species, and soil matrix origin on CO₂ evolution. We observed significantly greater concentrations of patches at 55 cm at one red maple site than all other sites. DEA and percent C in patches was generally higher in patches than matrix soil and did not vary between sites or by patch type. White pine roots decomposed at a faster rate than red maple roots under unsaturated conditions. Our results suggest that faster root decomposition could result in lower concentrations of patches of organic material in subsurface soils at sites dominated by white pine. Tree species composition and root decomposition may play a significant role in the formation of patches and the creation and maintenance of groundwater denitrification hotspots in the subsurface of riparian wetlands. Abbreviations: DEA – denitrification enzyme activity; DOC – dissolved organic carbon; PD – poorly drained; RM-1 – red maple-1 site; RM-2 – red maple-2 site; WP-1 – white pine-1 site; WP-2 – white pine-2 site.     

Challenges for simultaneous nitrification, denitrification, and phosphorus removal in microbial aggregates: mass transfer limitation and nitrous oxide production

The microbial community composition and activity was investigated in aggregates from a lab-scale bioreactor, in which nitrification, denitrification and phosphorus removal occurred simultaneously. The biomass was highly enriched for polyphosphate accumulating organisms facilitating complete removal of phosphorus from the bulk liquid; however, some inorganic nitrogen still remained at the end of the reactor cycle. This was ascribed to incomplete coupling of nitrification and denitrification causing NO(3)(-) accumulation. After 2 h of aeration, denitrification was dependent on the activity of nitrifying bacteria facilitating the formation of anoxic zones in the aggregates; hence, denitrification could not occur without simultaneous nitrification towards the end of the reactor cycle. Nitrous oxide was identified as a product of denitrification, when based on stored PHA as carbon source. This observation is of critical importance to the outlook of applying PHA-driven denitrification in activated sludge processes.     

Interactions among nitrogen-transforming bacteria and nitrogen-fixingPisum sativum L. in laboratory sand columns

Plants of barley (Hordeum vulgare), ryegrass (Lolium perenne), pea (Pisum sativum) or turnip (Brassica campestris rapifera) were grown in pots of unfertilised soil for 10 weeks together with unplanted control pots. A wide range of soil microbiological parameters was measured on bulk soil samples 2, 4, 7 and 10 weeks after seedlings were transplanted. There was no effect of planting or differential effect of plant species upon respiration rate, microbial biomass N, or biomass of microbial predators, but these parameters all varied significantly over time. Respiration, biomass N and nematode biomass all increased, whilst protozoan biomass decreased. Microbial biomass C showed no significant temporal changes or effect of planting. There was evidence for differential plant effects on potential nitrification and denitrification. Nitrification rates were depressed, compared with the fallow, in all treatments except the pea, at some time in the experiment. Conversely denitrification rates were enhanced in all treatments, except the grass, at specific times. Denitrification rates were greater in the pea treatment than the fallow on all occasions. These results demonstrate that plants do not necessarily influence the gross microbiology of the soil, but may affect physiologically distinct sub-components of the microbial biomass.     

Long-term warming effects on root morphology, root mass distribution, and microbial activity in two dry tundra plant communities in northern Sweden

Effects of warming on root morphology, root mass distribution and microbial activity were studied in organic and mineral soil layers in two alpine ecosystems over>10 yr, using open-top chambers, in Swedish Lapland. Root mass was estimated using soil cores. Washed roots were scanned and sorted into four diameter classes, for which variables including root mass (g dry matter (g DM) m(-2)), root length density (RLD; cm cm(-3) soil), specific root length (SRL; m g DM(-1)), specific root area (SRA; m2 kg DM(-1)), and number of root tips m(-2) were determined. Nitrification (NEA) and denitrification enzyme activity (DEA) in the top 10 cm of soil were measured. Soil warming shifted the rooting zone towards the upper soil organic layer in both plant communities. In the dry heath, warming increased SRL and SRA of the finest roots in both soil layers, whereas the dry meadow was unaffected. Neither NEA nor DEA exhibited differences attributable to warming. Tundra plants may respond to climate change by altering their root morphology and mass while microbial activity may be unaffected. This suggests that carbon may be incorporated in tundra soils partly as a result of increases in the mass of the finer roots if temperatures rise.     

帽儿山地区不同类型河岸带土壤的反硝化效率

以帽儿山地区森林背景下的森林、皆伐、草地河岸带和农田背景下的森林、裸地河岸带土壤为研究对象,采用硝态氮消失法,研究了不同背景下各类型河岸带的反硝化强度及其影响因素．结果表明：各类型河岸带中,农田背景下的森林河岸带土壤反硝化强度最大,其硝态氮消失率的变化范围为46．79％～91．13％,农田背景下的裸地河岸带土壤反硝化强度最小,其硝态氮消失率的变化范围为15．64％～81．84％;森林背景下土壤反硝化强度的大小顺序为皆伐河岸带〉森林河岸带〉草地河岸带,其硝态氮消失率的变化范围依次为42．06％～90．39％、28．24％～85．73％、21．44％～83．11％．研究区河岸带表层土壤的反硝化强度大于底层．河岸带土壤反硝化强度均受可利用碳、硝态氮的限制,各类型河岸带以农田背景下森林河岸带土壤反硝化潜力最大．     

Tree Species Composition Influences Enzyme Activities and Microbial Biomass in the Rhizosphere: A Rhizobox Approach

Monoculture causes nutrient losses and leads to declines in soil fertility and biomass production over successive cultivation. The rhizosphere, a zone of usually high microbial activities and clearly distinct from bulk soil, is defined as the volume of soil around living roots and influenced by root activities. Here we investigated enzyme activities and microbial biomass in the rhizosphere under different tree compositions. Six treatments with poplar, willow, and alder mono- or mixed seedlings were grown in rhizoboxes. Enzyme activities associated with nitrogen cycling and microbial biomass were measured in all rhizosphere and bulk soils. Both enzyme activities and microbial biomass in the rhizosphere differed significantly tree compositions. Microbial biomass contents were more sensitive to the changes of the rhizosphere environment than enzyme activities. Tree species coexistence did not consistently increase tested enzyme activities and microbial biomass, but varied depending on the complementarities of species traits. In general, impacts of tree species and coexistence were more pronounced on microbial composition than total biomass, evidenced by differences in microbial biomass C/N ratios stratified across the rhizosphere soils. Compared to poplar clone monoculture, other tree species addition obviously increased rhizosphere urease activity, but greatly reduced rhizosphere L-asparaginase activity. Poplar growth was enhanced only when coexisted with alder. Our results suggested that a highly productive or keystone plant species in a community had greater influence over soil functions than the contribution of diversity.     

Plant presence and species combination, but not diversity, influence denitrifier activity and the composition of nirK-type denitrifier communities in grassland soil

To explore potential links between plant communities, soil denitrifiers and denitrifier function, the impact of presence, diversity (i.e. species richness) and plant combination on nirK -type denitrifier community composition and on denitrifier activity was studied in artificial grassland plant assemblages over two consecutive years. Mesocosms containing zero, four and eight species and different combinations of two species were set up. Differences in denitrifier community composition were analysed by canonical correspondence analyses following terminal restriction fragment length polymorphism analysis of PCR-amplified nirK gene fragments coding for the copper-containing nitrite reductase. As a measure of denitrifier function, denitrifier enzyme activity (DEA) was determined in the soil samples. The presence as well as the combination of plants and sampling time, but not plant diversity, affected the composition of the nirK -type denitrifier community and DEA. Denitrifier activity significantly increased in the presence of plants, especially when they were growing during summer and autumn. Overall, we found a strong and direct linkage of denitrifier community composition and functioning, but also that plants had additional effects on denitrifier function that could not be solely explained by their effects on nirK -type denitrifier community composition.     

Community Composition and Functioning of Denitrifying Bacteria from Adjacent Meadow and Forest Soils

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in α-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.     

Grass species and soil type effects on microbial biomass and activity

We evaluated plant versus soil type controls on microbial biomass and activity by comparing microbial biomass C, soil respiration, denitrification potential, potential net N mineralization and nitrification in different soils supporting four grass species, and by growing a group of 10 different grass species on the same soil, in two experiments respectively. In the first experiment, none of the microbial variables showed significant variation with grass species while all variables showed significant variation with soil type, likely due to variation in soil texture. In the second experiment, there were few significant differences in microbial biomass C among the 10 grasses but there were significant relationships between variation in microbial biomass C and potential net N mineralization (negative), soil respiration (positive) and denitrification (positive). There was no relationship between microbial biomass C and either plant yield or plant N concentration. The results suggest that 1) soil type is a more important controller of microbial biomass and activity than grass species, 2) that different grass species can create significant, but small and infrequent, differences in microbial biomass and activity in soil, and 3) that plant-induced variation in microbial biomass and activity is caused by variation in labile C input to soil.     

Stimulation of soil nitrification and denitrification by grazing in grasslands: do changes in plant species composition matter?

Stimulation of nitrification and denitrification by long term (from years to decades) grazing has commonly been reported in different grassland ecosystems. However, grazing generally induces important changes in plant species composition, and whether changes in nitrification and denitrification are primarily due to changes in vegetation composition has never been tested. We compared soil nitrification- and denitrification-enzyme activities (NEA and DEA, respectively) between semi-natural grassland sites experiencing intensive (IG) and light (LG) grazing/mowing regimes for 13 years. Mean NEA and DEA (i.e. observed from random soil sampling) were higher in IG than LG sites. The NEA/DEA ratio was higher in IG than LG sites, indicating a higher stimulation of nitrification. Marked changes in plant species composition were observed in response to the grazing/mowing regime. In particular, the specific phytomass volume of Elymus repens was lower in IG than LG sites, whereas the specific volume of Lolium perenne was higher in IG than LG sites. In contrast, the specific volume of Holcus lanatus, Poa trivialis and Arrhenatherum elatius were not significantly different between treatments. Soils sampled beneath grass tussocks of the last three species exhibited higher DEA, NEA and NEA/DEA ratio in IG than LG sites. For a given grazing regime, plant species did not affect significantly soil DEA, NEA and NEA/DEA ratio. The modification of plant species composition is thus not the primary factor driving changes in nitrification and denitrification in semi-natural grassland ecosystems experiencing long term intensive grazing. Factors such as trampling, N returned in animal excreta, and/or modification of N uptake and C exudation by frequently defoliated plants could be responsible for the enhanced microbial activities.     

Greenhouse gas emissions from a Cu-contaminated soil remediated by in situ stabilization and phytomanaged by a mixed stand of poplar,willows, and false indigo-bush

Phytomanagement of trace element-contaminated soils can reduce soil toxicity and restore soil ecological functions, including the soil gas exchange with the atmosphere. We studied the emission rate of the greenhouse gases (GHGs) CO₂, CH₄, and N₂O; the potential CH₄ oxidation; denitrification enzyme activity (DEA), and glucose mineralization of a Cu-contaminated soil amended with dolomitic limestone and compost, alone or in combination, after a 2-year phytomanagement with a mixed stand of Populus nigra, Salix viminalis, S. caprea, and Amorpha fruticosa. Soil microbial biomass and microbial community composition after analysis of the phospholipid fatty acids (PLFA) profile were determined. Phytomanagement significantly reduced Cu availability and soil toxicity, increased soil microbial biomass and glucose mineralization capacity, changed the composition of soil microbial communities, and increased the CO₂ and N₂O emission rates and DEA. Despite such increases, microbial communities were evolving toward less GHG emission per unit of microbial biomass than in untreated soils. Overall, the aided phytostabilization option would allow methanotrophic populations to establish in the remediated soils due to decreased soil toxicity and increased nutrient availability.     

Occurrence of nitric and nitrous oxides in a coastal marine sediment.

The distribution of denitrification activity in a coastal marine sediment was determined by the acetylene inhibition technique and compared to concentration profiles of NO3-, NO2-, NO, and N2O. The bulk of the denitrification activity was associated with the accumulation of NO3- in the oxidized surface zone of the sediment, but a secondary denitrification zone was occasionally found in the deeper layers where oxidized patches had been introduced by the burrowing activity of the macrofauna. Maxima of NO and N2O were not associated with the peak activity of denitrification in the surface zone but were located at the lower edge of the activity profile. Significant accumulation of NO was found at the redox transition zone towards the deeper, sulfide-rich layers.     

Metabolic Status of Bacteria and Fungi in the Rhizosphere of Ponderosa Pine Seedlings

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.     

太湖流域上游竹林河岸带土壤反硝化酶活性及其影响因素

本研究以太湖流域上游竹林河岸带为对象,采用乙炔抑制法分析了夏季河岸带土壤反硝化酶活性(DEA)及其影响因素,以期为竹林河岸带在减少河流氮污染方面的生态功能评估提供数据支持。结果表明: 河岸带土壤DEA为6.32～23.22 μg N·kg^-1·h^-1,平均值为14.65 μg N·kg^-1·h^-1。河岸带土壤有机碳(SOC)、总氮、硝态氮含量、含水量和碳氮水解酶活性共同影响着DEA的垂直分布,使DEA随土壤深度(0～40 cm)的增加而递减;而DEA在水平方向上(同一土壤深度离水距离不同)的变化主要受SOC含量的影响。太湖流域上游竹林河岸带土壤在夏季可能会由于缺乏相对充足的可溶性有机碳而对DEA产生一定的限制。