Jean-Claude Germon

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

ABSTRACT 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.

Nitrous oxide emissions under different soil and land management conditions

Abstract Nitrous oxide (N2O) emissions of three different soils – a rendzina on cryoturbed soil, a hydromorphic leached brown soil and a superficial soil on a calcareous plateau – were measured using the chamber method. Each site included four types of land management: bare soil, seeded unfertilized soil, a suboptimally fertilized rapeseed crop and an overfertilized rapeseed crop. Fluxes varied from –1g to 100g N2O-nitrogen ha–1 day–1. The highest rates of N2O emissions were measured during spring on the hydromorphic leached brown soil which had been fertilized with nitrogen (N); the total emissions during a 5-month period exceeded 3500gNha–1. Significant fluxes were also observed during the summer. Very marked effects of soil type and management were observed. Two factors – the soil hydraulic behaviour and the ability of the microbial population to reduce N2O – appear to be essential in determining emissions of N2O by soils. In fact, the hydromorphic leached brown soil showed the highest emissions, despite having the lowest denitrification potential because of its water-filled pore space and low N2O reductase activity. Soil management also appears to affect both soil nitrate content and N2O emissions.

Characterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor and nos genes.

In this study, we report the cloning and characterization of denitrifying gene clusters of Pseudomonas fluorescens C7R12 containing the narXLDKGHJI, nirPOQSM, norCB and nosRZDFYL genes. While consensus sequences for Fnr-like protein binding sites were identified in the promoter regions of the nar, nir, nor and nos genes, consensus sequences corresponding to the NarL binding sites were identified only upstream the nar genes. Monitoring by mRNA analysis the expression of the narG, nirS, norB and nosZ structural genes suggests a sequential induction of the denitrification system in P. fluorescens.

N2O and NO emissions by agricultural soils with low hydraulic potentials

N2O and NO production were studied on five agricultural soils with low hydraulic potentials. All experiments were performed in a laboratory under standard incubation conditions to limit any intrinsic soil heterogeneity. The mechanisms involved in NO and N2O production was investigated using the inhibitory properties of acetylene on nitrification and N2O reduction. This work confirmed that N2O and NO could be produced by soils under aerobic conditions. Nitrification seemed to be the only process involved in NO production and the main process involved in N2O production by the five studied soils when the water content was low. Nevertheless, aerobic denitrification with N2O release was observed in one soil. The proportion of N emitted as NO and N2O through nitrification varied considerably from soil to soil and, in some soils, also varied with soil hydraulic potential, ranging from 0 to 2.5%, and from 0.03 to 1%, respectively. This study clearly shows that both NO emission and the gaseous N emitted in aerated conditions should be taken into account in determining the N-budget on cultivated soils.

Influence of different agricultural practices (type of crop, form of N-fertilizer) on soil nitrous oxide emissions

Abstract N2O emissions were periodically measured using the static chamber method over a 1-year period in a cultivated field subjected to different agricultural practices including the type of N fertilizer (NH4NO3, (NH4)2SO4, CO(NH2)2 or KNO3 and the type of crop (rapeseed and winter wheat). N2O emissions exhibited the same seasonal pattern whatever the treatment, with emissions between 1.5 and 15 g N ha–1 day–1 during the autumn, 16–56 g N ha–1 day–1 in winter after a lengthy period of freezing, 0.5–70 g N ha–1 day–1 during the spring and lower emissions during the summer. The type of crop had little impact on the level of N2O emission. These emissions were a little higher under wheat during the autumn in relation to an higher soil NO3– content, but the level of emissions was similar over a 7-month period (2163 and 2093 g N ha–1 for rape and wheat, respectively). The form of N fertilizer affected N2O emissions during the month following fertilizer application, with higher emissions in the case of NH4NO3 and (NH4)2SO4, and a different temporal pattern of emissions after CO(NH2)2 application. The proportion of applied N lost as N2O varied from 0.42% to 0.55% with the form of N applied, suggesting that controlling this agricultural factor would not be an efficient way of limiting N2O emissions under certain climatic and pedological situations.

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.

Inhibitory capacities of acetylene on nitrification in two agricultural soils

Abstract Acetylene is currently used to distinguish between the relative contribution of nitrification and denitrification to soil emissions of the greenhouse gas, N 2 O. The basis of this method is that acetylene at low partial pressures inhibits nitrification without affecting N 2 O reduction. This paper reports experiments where low acetylene partial pressures were insufficient to totally inhibit nitrification in an hypercalcareous rendosol at water potentials higher than −3.5 MPa while they were always sufficient in a redoxic luvisol.

Effets des techniques culturales sans labour sur le stockage de carbone dans le sol en contexte climatique tempéré

En regions de climat tempere, les terres cultivees ont un potentiel de stockage de carbone que l’on peut tenter d’utiliser pour reduire d’autant les emissions de CO2 atmospherique par des pratiques culturales adaptees et notamment par les techniques culturales sans labour (TCSL). Cette capacite de stockage de carbone dans le sol a ete evaluee sur l’essai de longue duree de Boigneville (Bassin de Paris, France) et a ete comparee aux donnees de la litterature internationale. Le suivi du stock de carbone du sol sous une rotation mais-ble indique une tendance a un stockage modere tant en systeme laboure (0,10 t C ha-1 an-1 sur 28 ans) qu’en TCSL (respectivement 0,21 t et 0,19 t C ha-1 an-1 pour le travail superficiel et le semis direct sur cette meme periode). Avec une absence de difference significative entre le semis direct et le travail superficiel, l’effet specifique moyen des TCSL evalue a 0,10 t C ha-1 an-1 sur 28 ans apparait sensiblement plus faible que celui mesure sur les 20 premieres annees et eval...

Estimation of N2O fluxes under rape for biological fuel production, bare soil and grass fallow

Nitrous oxide emission from agricultural soils is an important environmental concern as this gas contributes to the partial destruction of the stratospheric ozone layer and to global warming. European farmers are being encouraged to develop crops for producing biofuel. However, the excessive use of nitrogenous fertilizers on these crops could substantially increase agricultural emissions of N2O and hence eliminate the beneficial effects of recycling CO2. N2O fluxes were estimated from soil under four treatments: (1) bare soil, (2) unfertilized grassland fallow, (3) soil cropped to rapeseed and fertilized at rate of 180 kg N ha−1 yr−1 and (4) as for (3) but fertilized at 280 kg N ha−1 yr−1. Measurements were made using the static chamber method throughout spring and summer 1994. No particularly beneficial effect on N2O production was observed under reasonable fertilized rapeseed. Fluxes in the order of 1 kg N ha−1 yr −1 were measured under rape fertilized at 180 kg N ha−1 yr−1. The amount of fertilizer applied to rape has a very definite effect on the amount of N2O released. Bare soils constitute a risk in terms of N2O release as a ‘hot spot’ was observed after the harvest. Grass fallow seemed to be best adapted to limiting N2O release.