Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing, denitrifying and nitrifying bacterial communities in grassland soils

Management by combined grazing and mowing events is commonly used in grasslands, which influences the activity and composition of soil bacterial communities. Whether observed effects are mediated by management-induced disturbances, or indirectly by changes in the identity of major plant species, is still unknown. To address this issue, we quantified substrate-induced respiration (SIR), and the nitrification, denitrification and free-living N(2)-fixation enzyme activities below grass tufts of three major plant species (Holcus lanatus, Arrhenatherum elatius and Dactylis glomerata) in extensively or intensively managed grasslands. The genetic structures of eubacterial, ammonia oxidizing, nitrate reducing, and free-living N(2)-fixing communities were also characterized by ribosomal intergenic spacer analysis, and denaturing gradient gel electrophoresis (DGGE) or restriction fragment length polymorphism (RFLP) targeting group-specific genes. SIR was not influenced by management and plant species, whereas denitrification enzyme activity was influenced only by plant species, and management-plant species interactions were observed for fixation and nitrification enzyme activities. Changes in nitrification enzyme activity were likely largely explained by the observed changes in ammonium concentration, whereas N availability was not a major factor explaining changes in denitrification and fixation enzyme activities. The structures of eubacterial and free-living N(2)-fixing communities were essentially controlled by management, whereas the diversity of nitrate reducers and ammonia oxidizers depended on both management and plant species. For each functional group, changes in enzyme activity were not correlated or were weakly correlated to overall changes in genetic structure, but around 60% of activity variance was correlated to changes in five RFLP or DGGE bands. Although our conclusions should be tested for other ecosystems and seasons, these results show that predicting microbial changes induced by management in grasslands requires consideration of management-plant species interactions.     

Microbial succession of nitrate-reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central Alps

Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (>2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community.     

Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO3)2, (NH4)2SO4, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH4)2SO4 and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO3)2 or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.     

Denitrifying genes in bacterial and Archaeal genomes

Denitrification, the reduction of nitrate or nitrite to nitrous oxide or dinitrogen, is the major mechanism by which fixed nitrogen returns to the atmosphere from soil and water. Although the denitrifying ability has been found in microorganisms belonging to numerous groups of bacteria and Archaea, the genes encoding the denitrifying reductases have been studied in only few species. Recent investigations have led to the identification of new classes of denitrifying reductases, indicating a more complex genetic basis of this process than previously recognized. The increasing number of genome sequencing projects has opened a new way to study the genetics of the denitrifying process in bacteria and Archaea. In this review, we summarized the current knowledge on denitrifying genes and compared their genetic organizations by using new sequences resulting from the analysis of finished and unfinished microbial genomes with a special attention paid to the clustering of genes encoding different classes of reductases. In addition, some evolutionary relationships between the structural genes are presented.     

DNA extraction from soils: old bias for new microbial diversity analysis methods

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.     

Determinants of the distribution of nitrogen-cycling microbial communities at the landscape scale

Little information is available regarding the landscape-scale distribution of microbial communities and its environmental determinants. However, a landscape perspective is needed to understand the relative importance of local and regional factors and land management for the microbial communities and the ecosystem services they provide. In the most comprehensive analysis of spatial patterns of microbial communities to date, we investigated the distribution of functional microbial communities involved in N-cycling and of the total bacterial and crenarchaeal communities over 107 sites in Burgundy, a 31 500 km² region of France, using a 16 × 16 km² sampling grid. At each sampling site, the abundance of total bacteria, crenarchaea, nitrate reducers, denitrifiers- and ammonia oxidizers were estimated by quantitative PCR and 42 soil physico-chemical properties were measured. The relative contributions of land use, spatial distance, climatic conditions, time, and soil physico-chemical properties to the spatial distribution of the different communities were analyzed by canonical variation partitioning. Our results indicate that 43–85% of the spatial variation in community abundances could be explained by the measured environmental parameters, with soil chemical properties (mostly pH) being the main driver. We found spatial autocorrelation up to 739 km and used geostatistical modelling to generate predictive maps of the distribution of microbial communities at the landscape scale. The present study highlights the potential of a spatially explicit approach for microbial ecology to identify the overarching factors driving the spatial heterogeneity of microbial communities even at the landscape scale.     

Atmospheric hydrogen peroxide and Eoarchean iron formations

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean–Early Paleoproterozoic (2.7–2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H₂O₂), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H₂O₂ that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO₂, O₂, and CH₄ mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H₂O₂ rainout was calculated to be <10⁶ molecules cm^?2 s^?1. Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H₂O₂ was insufficient by several orders of magnitude to account for IF deposition (requiring ~10¹¹ H₂O₂ molecules cm^?2 s^?1). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)‐oxidizing micro‐organisms the most likely mechanism responsible for Earth's oldest IF.     

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.     

Fluid-deposited graphite and its geobiological implications in early Archean gneiss from Akilia, Greenland

Graphite, interpreted as altered bioorganic matter in an early Archean, ca. 3.83‐Ga‐old quartz‐amphibole‐pyroxene gneiss on Akilia Island, Greenland, has previously been claimed to be the earliest trace of life on Earth. Our petrographic and Raman spectroscopy data from this gneiss reveal the occurrence of graphitic material with the structure of nano‐crystalline to crystalline graphite in trails and clusters of CO₂, CH₄ and H₂O bearing fluid inclusions. Irregular particles of graphitic material without a fluid phase, representing decrepitated fluid inclusions are common in such trails too, but occur also as dispersed individual or clustered particles. The occurrence of graphitic material associated with carbonic fluid inclusions is consistent with an abiologic, fluid deposited origin during a poly‐metamorphic history. The evidence for fluid‐deposited graphitic material greatly complicates any claim about remnants of early life in the Akilia rock.