D. Chèneby

Insights into the Effect of Soil pH on N2O and N2 Emissions and Denitrifier Community Size and Activity

ABSTRACT The objective of this study was to investigate how changes in soil pH affect the N2O and N2 emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H2SO4 solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N2O and N2 emissions using the 15N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N2O/(N2O + N2) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N2O production were observed. Denitrification activity and N2O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between nirS, napA, and narG gene copy numbers and the N2O/(N2O + N2) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.

Mapping field‐scale spatial patterns of size and activity of the denitrifier community

There is ample evidence that microbial processes can exhibit large variations in activity on a field scale. However, very little is known about the spatial distribution of the microbial communities mediating these processes. Here we used geostatistical modelling to explore spatial patterns of size and activity of the denitrifying community, a functional guild involved in N-cycling, in a grassland field subjected to different cattle grazing regimes. We observed a non-random distribution pattern of the size of the denitrifier community estimated by quantification of the denitrification genes copy numbers with a macro-scale spatial dependence (6-16 m) and mapped the distribution of this functional guild in the field. The spatial patterns of soil properties, which were strongly affected by presence of cattle, imposed significant control on potential denitrification activity, potential N(2)O production and relative abundance of some denitrification genes but not on the size of the denitrifier community. Absolute abundance of most denitrification genes was not correlated with the distribution patterns of potential denitrification activity or potential N(2)O production. However, the relative abundance of bacteria possessing the nosZ gene encoding the N(2)O reductase in the total bacterial community was a strong predictor of the N(2)O/(N(2) + N(2)O) ratio, which provides evidence for a relationship between bacterial community composition based on the relative abundance of denitrifiers in the total bacterial community and ecosystem processes. More generally, the presented geostatistical approach allows integrated mapping of microbial communities, and hence can facilitate our understanding of relationships between the ecology of microbial communities and microbial processes along environmental gradients.

Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: insight into the role of root exudates

To determine to which extent root-derived carbon contributes to the effects of plants on nitrate reducers and denitrifiers, four solutions containing different proportions of sugar, organic acids and amino acids mimicking maize root exudates were added daily to soil microcosms at a concentration of 150 microg C g(-1) of soil. Water-amended soils were used as controls. After 1 month, the size and structure of the nitrate reducer and denitrifier communities were analysed using the narG and napA, and the nirK, nirS and nosZ genes as molecular markers respectively. Addition of artificial root exudates (ARE) did not strongly affect the structure or the density of nitrate reducer and denitrifier communities whereas potential nitrate reductase and denitrification activities were stimulated by the addition of root exudates. An effect of ARE composition was also observed on N(2)O production with an N(2)O:(N(2)O + N(2)) ratio of 0.3 in microcosms amended with ARE containing 80% of sugar and of 1 in microcosms amended with ARE containing 40% of sugar. Our study indicated that ARE stimulated nitrate reduction or denitrification activity with increases in the range of those observed with the whole plant. Furthermore, we demonstrated that the composition of the ARE affected the nature of the end-product of denitrification and could thus have a putative impact on greenhouse gas emissions.

Carbon and nitrogen cycling through soil microbial biomass at various temperatures

Abstract C and N cycling were examined in a soil incubated at 4, 12, 20 or 28°C for 140 days. Before incubation the soil was amended with K 15 NO 3 , and either glucose- 14 C or holocellulose- 14 C. The kinetics of tracer and non-tracer C and N from the biomass, mineralized-C and inorganic-N were measured. C and N behaviours in soil were influenced by temperature, substrate and substrate-temperature interactions. Labelled-C mineralization rates after 140 days ranged from 41 to 58% for glucose and from 34 to 65% for holocellulose. Maximal immobilization was 21.8–31.6 mgN kg −1 soil for holocellulose and 24.3–33.5 mg N kg −1 soil for glucose. Re-mineralization began earlier with glucose and at higher temperatures: 6–23% of immobilized-N were re-mineralized for glucose and 0–19% for holocellulose. More labelled C and N were incorporated into the microbial biomass from both carbon sources at lower temperatures. The biomass turnover was highly influenced by temperature: 40–60% of labelled C or N incorporated in the biomass remained in this compartment at 20–28°C, while corresponding values at 4–12°C were only 0–40%. Organic- 14 C mineralization and immobilization rate constants were influenced by temperature, the different trends depending on the carbon source. Thus an overall temperature coefficient (Q 10 ) could not be determined for these complex transformations. Variations in the rate constant with temperature were described using polynomial regressions.

Diversity of denitrifying microflora and ability to reduce N2O in two soils

Abstract The ozone-depleting gas N2O is an intermediate in denitrification, the biological reduction of NO3– to the gaseous products N2O and N2 gas. The molar ratio of N2O produced (N2O/N2O+N2) varies temporally and spatially, and in some soils N2O may be the dominant end product of denitrification. The fraction of NO3–-N emitted as N2O may be due at least in part to the abundance and activity of denitrifying bacteria which possess N2O reductase. In this study, we enumerated NO3–-reducing and denitrifying bacteria, and compared and contrasted collections of denitrifying bacteria isolated from two agricultural soils, one (Auxonne, soil A) with N2O as the dominant product of denitrification, the other (Châlons, soil C) with N2 gas as the dominant product. Isolates were tested for the ability to reduce N2O, and the presence of the N2O reductase (nosZ)-like gene was evaluated by polymerase chain reaction (PCR) using specific primers coupled with DNA hybridization using a specific probe. The diversity and phylogenetic relationships of members of the collections were established by PCR/restriction fragment length polymorphism of 16s rDNA. The two soils had similar numbers of bacteria which used NO3– as a terminal electron acceptor anaerobically. However, the soil A had many more denitrifiers which reduced NO3– to gaseous products (N2O or N2) than did soil C. Collections of 258 and 281 bacteria able to grow anaerobically in the presence of NO3– were isolated from soil A and soil C, respectively. These two collections contained 66 and 12 denitrifying isolates, respectively, the others reducing NO3– only as far as NO2–. The presence of nosZ sequences was generally a poor predictor of N2O reducing ability: there was agreement between the occurrence of nosZ sequences and the N2O reducing ability for only 42% of the isolates; 35% of the isolates (found exclusively in soil A) without detectable nosZ sequences reduced N2O whereas 21% of the isolates carrying nosZ sequences did not reduce this gas under our assay conditions. Twenty-eight different 16S rDNA restriction patterns (using two restriction endonucleases) were distinguished among the 78 denitrifying isolates. Two types of patterns appeared to be common to both soils. Twenty-three and three types of patterns were found exclusively among bacteria isolated from soils A and C, respectively. The specific composition of denitrifying communities appeared to be different between the two soils studied. This may partly explain the differences in the behaviour of the soils concerning N2O reduction during denitrification.

Genetic Characterization of the Nitrate Reducing Community Based on narG Nucleotide Sequence Analysis

The ability of facultative anerobes to respire nitrate has been ascribed mainly to the activity of a membrane-bound nitrate reductase encoded by the narGHJI operon. Respiratory nitrate reduction is the first step of the denitrification pathway, which is considered as an important soil process since it contributes to the global cycling of nitrogen. In this study, we employed direct PCR, cloning, and sequencing of narG gene fragments to determine the diversity of nitrate-reducing bacteria occurring in soil and in the maize rhizosphere. Libraries containing 727 clones in total were screened by restriction fragment analysis. Phylogenetic analysis of 128 narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria. Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch. All together, our results revealed a more complex nitrate-reducing community than did previous culture-based studies. A significant and consistent shift in the relative abundance of the nitrate-reducing groups within this functional community was detected in the maize rhizosphere. Thus a substantially higher abundance of the dominant clone family and a lower diversity index were observed in the rhizosphere compared to the unplanted soil, suggesting that a bacterial group has been specifically selected within the nitrate-reducing community. Furthermore, restriction fragment length polymorphism analysis of cloned narG gene fragments proved to be a powerful tool in evaluating the structure and the diversity of the nitrate-reducing community and community shifts therein.

Effect of primary mild stresses on resilience and resistance of the nitrate reducer community to a subsequent severe stress.

The factors regulating soil microbial stability (e.g. resistance and resilience) are poorly understood, even though microorganisms are essential for ecosystem functioning. In this study, we tested whether a functional microbial community subjected to different primary mild stresses was equally resistant or resilient to a subsequent severe stress. The nitrate reducers were selected as model community and analysed in terms of nitrate reduction rates and genetic structure by narG PCR-restriction fragment length polymorphism fingerprinting. Heat, copper and atrazine were used as primary stresses and mercury at a high concentration as a severe stress. None of the primary stresses had any significant impact on the nitrate reducer community. Although primary stress with heat, copper or atrazine had no effect on the resilience of the nitrate reducer activity to mercury stress, pre-exposure to copper, another heavy metal, resulted in increased resilience. In contrast, the resistance of both structure and activity of the nitrate reducer community to severe mercury stress was not affected by any of the primary stresses tested. Our experiment suggests that the hypothetical effect of an initial stress on the response of a microbial community to an additional stress is complex and may depend on the relatedness of the two consecutive stresses and the development of positive cotolerance.

Differential responses of nitrate reducer community size, structure, and activity to tillage systems.

ABSTRACT The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients.

Genetic structure and activity of the nitrate-reducers community in the rhizosphere of different cultivars of maize

In this study, the structure and activity of the nitrate-reducers community were analysed in bulk and rhizospheric soils from three different non-isogenic transgenic cultivars of maize (two Bacillus thuringiensis maize and one glyphosate-resistant maize) in a long-term field experiment. DNA was extracted from both rhizospheric and non-rhizospheric soil sampled at three different development stages of the plants and amplified using primers targeting the genes encoding the␣membrane-bound nitrate reductase (narG). Nitrate-reducers community structure was analysed by generating fingerprints and sequencing of narG clone libraries. The season seems to be the most important factor controlling the genetic structure of the nitrate-reducers community. Smaller differences in the narG fingerprints were also observed between bulk and rhizospheric soils suggesting that presence of maize roots was the second important factor affecting the structure of this functional community. Similarly, a rhizosphere effect was observed on the nitrate reductase activity with a 2–3-fold increased in the rhizospheric soil compared to the non-rhizospheric soil. However, for both structure and activity of the nitrate-reducers community, no effect of the maize cultivar was observed. This study suggests that the effect of the cultivar and/or of the agricultural practices associated with the cultivation of transgenic maize is not significant compared to the effect of other environmental factors.

Role of Plant Residues in Determining Temporal Patterns of the Activity, Size, and Structure of Nitrate Reducer Communities in Soil

ABSTRACT The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.