Denitrifier Communities Research Articles

The Functional Significance of Denitrifier Community Composition in a Terrestrial Ecosystem

We tested the hypothesis that soil microbial diversity affects ecosystem function by evaluating the effect of denitrifier community composition on nitrous oxide (N2O) production. Denitrification is a major source of atmospheric N2O, an important greenhouse gas and a natural catalyst of stratospheric ozone decay. The major environmental controls on denitrification rate and the mole ratio of N2O produced during denitrification have been incorporated into mechanistic models, but these models are, in general, poor predictors of in situ N2O flux rates. We sampled two geomorphically similar soils from fields in southwest Michigan that differed in plant community composition and disturbance regime: a conventionally tilled agricultural field and a never-tilled successional field. We tested whether denitrifier community composition influences denitrification rate and the relative rate of N2O production [△N2O/△(N2O + N2)], or rN2O, using a soil enzyme assay designed to evaluate the effect of oxygen concentration and pH on the activity of denitrification enzymes responsible for the production and consumption of N2O. By controlling, or providing in nonlimiting amounts, all known environmental regulators of denitrifier N2O production and consumption, we created conditions in which the only variable contributing to differences in denitrification rate and rN2O in the two soils was denitrifier community composition. We found that both denitrification rate and rN2O differed for the two soils under controlled incubation conditions. Oxygen inhibited the activity of enzymes involved in N2O production (nitrate reductase, Nar; nitrite reductase, Nir; and nitric oxide reductase, Nor) to a greater extent in the denitrifying community from the agricultural field than in the community from the successional field. The Nar, Nir, and Nor enzymes of the denitrifying community from the successional field, on the other hand, were more sensitive to pH than were those in the denitrifying community from the agricultural field. Moreover, the denitrifying community in the soil from the successional field had relatively more active nitrous oxide reductase (Nos) enzymes, which reduce N2O to N2, than the denitrifying community in the agricultural field. Also, the shape of the rN2O curve with increasing oxygen was different for each denitrifying community. Each of these differences suggests that the denitrifying communities in these two soils are different and that they do not respond to environmental regulators in the same manner. We thus conclude that native microbial community composition regulates an important ecosystem function in these soils.

THE FUNCTIONAL SIGNIFICANCE OF DENITRIFIER COMMUNITY COMPOSITION IN A TERRESTRIAL ECOSYSTEM

We tested the hypothesis that soil microbial diversity affects ecosystem func- tion by evaluating the effect of denitrifier community composition on nitrous oxide (N20) production. Denitrification is a major source of atmospheric N20, an important greenhouse gas and a natural catalyst of stratospheric ozone decay. The major environmental controls on denitrification rate and the mole ratio of N20 produced during denitrification have been incorporated into mechanistic models, but these models are, in general, poor predictors of in situ N20 flux rates. We sampled two geomorphically similar soils from fields in southwest Michigan that differed in plant community composition and disturbance regime: a con- ventionally tilled agricultural field and a never-tilled successional field. We tested whether denitrifier community composition influences denitrification rate and the relative rate of N20 production (AN20/A(N20 + N2)), or rN20, using a soil enzyme assay designed to evaluate the effect of oxygen concentration and pH on the activity of denitrification enzymes responsible for the production and consumption of N20. By controlling, or providing in nonlimiting amounts, all known environmental regulators of denitrifier N20 production and consumption, we created conditions in which the only variable contributing to differences in denitrification rate and rN2O in the two soils was denitrifier community composition. We found that both denitrification rate and rN2O differed for the two soils under controlled incubation conditions. Oxygen inhibited the activity of enzymes involved in N20 production (nitrate reductase, Nar; nitrite reductase, Nir; and nitric oxide reductase, Nor) to a greater extent in the denitrifying community from the agricultural field than in the community from the successional field. The Nar, Nir, and Nor enzymes of the denitrifying community from the successional field, on the other hand, were more sensitive to pH than were those in the denitrifying community from the agricultural field. Moreover, the denitrifying community in the soil from the successional field had relatively more active nitrous oxide reductase (Nos) enzymes, which reduce N20 to N2, than the denitrifying community in the agricultural field. Also, the shape of the rN20 curve with increasing oxygen was different for each denitrifying community. Each of these differences suggests that the denitrifying commu- nities in these two soils are different and that they do not respond to environmental regulators in the same manner. We thus conclude that native microbial community composition reg- ulates an important ecosystem function in these soils.

Comparison of denitrifying communities in organic soils: kinetics of NO −3 and N 2O reduction

Our aim was to determine whether intrinsic differences in the denitrifier communities existed in farmed organic soils from three different sites (Germany, Sweden and Finland) on which field fluxes of N 2O had been measured continuously over 2 y. To estimate enzyme kinetic parameters (i.e. V max and K m) for N 2O reductase, NO − 3 was first removed by a combination of soil washing and anaerobic incubation. Then the samples were incubated anaerobically (as slurries) with or without added NO − 3, N 2O and C 2H 2. The estimated half saturation constants for N 2O reductase were similar for all soils, and very low ( K m=0.1–0.4 μM) compared to other investigations. In response to a prolonged anaerobic conditioning incubation (48 h), the K m values increased significantly and similarly for all soils, suggesting a shift in the dominant members of the denitrifier communities. The ratio between the estimated V max values for NO − 3 reduction-to-N 2O and N 2O reduction-to-N 2 was much lower for the Swedish than for the other two, indicating that the community of the Swedish soil would be more efficient in reducing NO − 3 all the way to N 2. This difference is congruent with differences in annual field fluxes. The results thus suggest that intrinsic differences in community composition of soils exist, with consequences for the emission of N 2O. Prolonged anaerobic incubation (48 h at 20°C) resulted in a convergence of the communities towards similar ratios between the two V max values, suggesting that the apparent intrinsic differences may disappear in response to such severe treatment. Thus, the persistence of such patterns may depend on the drainage capacity of the soils. The results indicate that qualities of the denitrifying community must be taken into account when trying to understand and to model field fluxes of N 2O from soils. They also illustrate how global trace gas emissions can be affected by changes in the community compositions of soils.

Denitrification in arable soils in relation to their physico-chemical properties and fertilization practice

Increased use of N-fertilizers in the Czech Republic in recent decades has been accompanied by increased amounts of N compounds in the general environment, and also by shifts in the rates of biological N-processes in agricultural soils. Knowledge of these changes, including rates, distribution and variability of denitrification in soils in the Czech Republic is, however, very limited. In the period 1993–1995, potential denitrification (i.e. denitrifying enzyme activity (DEA) and denitrification potential (DP)), was determined at 13 different sites, in soils with differing physico-chemical and biological properties, but which were typical of large areas in the Czech Republic. In 1994–1995, more detailed research on four differently fertilized plots of a sandy-loam cambisol was conducted on nine occasions in order to analyze seasonal effects and to establish if more than 20 yr of fertilization and selected environmental factors had influenced denitrification. This research was completed in 1996, when 12 differently fertilized plots of the same soil were examined. When a range of soils was investigated, DEA reached values between 15 and 680 ng N 2O-N g −1 h −1, corresponding roughly to 1.3–16 mg N kg −1 d −1, and DP reached values between 6.6–52 mg N kg −1 d −1. No significant relationship was found between DEA and DP, indicating the independence of the existing metabolic activity of the denitrifier community (DEA) and its potential for rapid development if the environmental conditions change in its favor (DP). While DEA did not correlate with any of the measured soil properties, DP correlated highly significantly with soil pH (H 2O) and organic carbon content. Detailed research on a sandy loam cambisol showed (i) a large temporal variability of DEA and DP, (ii) an influence of long-term fertilization on denitrification characteristics; DEA and DP responded differently: while DEA increased with all fertilization rates, DP increased in moderately fertilized soils, but decreased significantly in a highly fertilized soil and (iii) a positive correlation of DP with many soil properties, including pH, microbial biomass and dehydrogenase activity.

Regulation of potential denitrification by soil pH in long-term fertilized arable soils

The denitrifying enzyme activity (DEA), denitrification potential (DP) and anaerobic respiration (RESP) together with chemical characteristics were measured in three contrasting soils collected from experimental arable plots that had been subjected to long-term (21–23 years) fertilizer treatments. The plots sampled were either unfertilized or had received either annual inorganic NPK, manure and lime, or inorganic NPK and manure treatments. Addition of inorganic NPK, manure and lime led to large increases in the DEA for two of the three soils, but in the absence of lime, inorganic NPK and manure caused only small increases in DEA compared to unfertilized soils. Both DP and RESP were increased by the addition of inorganic NPK, manure and lime, but were substantially decreased by fertilizer treatments without lime. In most cases there was a simple relationship between soil pH and either DEA and DP, with those treatments that reduced soil pH also leading to reduced denitrification and vice versa. The effects of artificially increasing the pH to a value close to the pH in unfertilized soils (6.3) by addition of NaOH to the soils that had received inorganic NPK, and which had the lowest soil pH values, were to increase substantially DEA, DP and RESP. In soil from one of the sites that had been stored for 5 weeks, the DP values responded differently between the fertilizer treatments. The DP value was lowest in the soil that had inorganic NPK and manure, higher in the soil that received inorganic NPK, manure and lime and it was the highest in unfertilized (control) soil. The soil pH values for these treatments were 4.47, 5.79 and 6.58, respectively. However, when the soil pHs were adjusted by addition of either H2SO4 or NaOH to give a range between pH 2 and 12, the DP values from all three fertilizer treatments showed almost identical responses. The optimum pH value for DP was between 7 and 8 for all three fertilizer treatments. Substrate-induced respiration values from all fertilizer treatments showed a similar trend to DP when the soil pHs were modified. The results show that soil pH was an important factor which in the studied soils controls the microbial community in general and the community of denitrifiers in particular. However, denitrifiers showed a high pH resilience leading to no marked change of the pH optimum for potential denitrification.

Denitrification in the subsoil of the Broadbalk Continuous Wheat Experiment

Denitrification in subsoils may be a natural process able to decrease NO 3 contamination of groundwater. We studied this process and how it was affected by long-term fertilization practices. We collected soil to a depth of 2 m from four treatments in the Broadbalk Continuous Wheat Experiment. One treatment (FYM) had received manure applications annually since 1843, while the other treatments were N0 (0 kg N ha −1 since 1843), N4 (196 kg N ha −1 since 1968) and N6 (288 kg N ha −1 since 1984). Using the acetylene inhibition method, we measured N 2O production from anaerobic slurries of either unamended or amended (plus C and NO 3) samples of surface soil and three subsoil depth layers. These results will not equate to actual field denitrification rates, but the amended treatments (i.e. plus C and NO 3) will indicate the relative denitrification capacities of the soils. The unamended treatment will more accurately indicate the relative field denitrification rates. In the surface soils, denitrification rates were approximately proportional to total organic C (TOC) and they increased in the following order: N0, N4, N6, FYM. In the subsoil, denitrification declined with depth, and at 1.2 m was only a small fraction of that at the surface. There was no relationship between surface soil fertilization practices and either subsoil denitrification capacity, TOC, dissolved organic carbon (DOC) or total N. Indeed subsoil (60–200 cm) denitrification capacity was not significantly different between treatments. Denitrification capacity in unamended subsoils was not related to DOC. However, there was a significant relationship ( R 2=0.41) between TOC and denitrification rate. When amended soils were conditioned aerobically for 5 days, N 2O production increased, indicating the presence of a small community of denitrifiers which multiplied when given a C source. We conclude that subsoil denitrification has the potential to decrease NO 3 concentrations of percolating waters but is in reality limited by biologically available C. On our Broadbalk site, surface soil management had no effect upon the amount of `available' C in the subsoil.

Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments.

Diversity of the nitrous oxide reductase (nosZ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the nosZ gene were amplified via PCR, using nosZ gene-specific primers. Thirty-seven unique copies of the nosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% +/- 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (CuZ) of the mature protein. Phylogenetic analysis demonstrated three major clusters of nosZ genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.