Lars R. Bakken

Viability of Soil Bacteria: Optimization of Plate-Counting Technique and Comparison Between Total Counts and Plate Counts Within Different Size Groups

Viable counts of heterotropic soil bacteria were 3–5 times higher on low-nutrient agar media compared with a series of conventional agar media. Substantial amounts of monosaccharides and amino acids were present in solid media made from distilled water and agar powder, and a salt-solution agar medium (without organic substrates added) gave practically the same colony counts as the low nutrient soil extract agar medium. MPN values were comparable to or lower than plate counts. A search for slow-growing cells in the negative MPN tubes by fluorescence microscopical examination after 3 months incubation was negative.The viable counts were 2–4% of the total microscopical counts in different soils. Assuming that the colony-forming cells did not derive from the numerous “dwarf” cells present in soil, a calculated percent viability of the larger cells was about 10%. The ecological significance of the plate-counting technique is discussed.

Denitrification gene pools, transcription and kinetics of NO, N2O and N2 production as affected by soil pH

The N(2)O : N(2) product ratio of denitrification is negatively correlated with soil pH, but the mechanisms involved are not clear. We compared soils from field experiments where the pH had been maintained at different levels (pH 4.0-8.0) by liming (> or = 20 years), and quantified functional gene pools (nirS, nirK and nosZ), their transcription and gas kinetics (NO, N(2)O and N(2)) of denitrification as induced by anoxic incubation with and without a carbon substrate (glutamate). Denitrification in unamended soil appeared to be based largely on the activation of a pre-existing denitrification proteome, because constant rates of N(2) and N(2)O production were observed, and the transcription of functional genes was below the detection level. In contrast, glutamate-amended soils showed sharp peaks in the transcripts of nirS and nosZ, increasing the rates of denitrification and pH-dependent transient accumulation of N(2)O. The results indicate that the high N(2)O : N(2) product ratio at low pH is a post-transcriptional phenomenon, because the transcription rate of nosZ relative to that of nirS was higher at pH 6.1 than at pH 8.0. The most plausible explanation is that the translation/assembly of N(2)O reductase is more sensitive to low pH than that of the other reductases involved in denitrification.

CH4 uptake by temperate forest soil: Effect of N input and soil acidification

N fertilization and soil acidification effects on the uptake of atmospheric CH4 and soil CH4 concentrations were studied in lysimeter soil from a 100-y-old Scots pine forest in Norway. N fertilization with NH4NO3 significantly reduced the uptake of atmospheric CH4. Medium N application (30 kg N ha−1 y−1) resulted in a CH4 uptake rate which was 85 ± 3% of that in the control, while the uptake rate with high N (90 kg N ha−1 y−1) was 62 ± 2% of the control. The most acidic irrigation (pH = 3) increased CH4 uptake compared to pH 5.5 and 4 irrigations. This effect was the same at all N treatments (no interaction). The positive effect of acidification indicates that autotrophic NH4 oxidation is of minor importance for CH4 oxidation in soil. Increased soil moisture from 32 to 42% (v/v) significantly reduced CH4 uptake. CH4 uptake was observed during winter measurements, when the mean soil temperature was <1°C.

Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils

Denitrifying prokaryotes use NOx as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N2O and N2, depending on the relative activity of the enzymes catalysing the stepwise reduction of NO3− to N2O and finally to N2. Cultured denitrifying prokaryotes show characteristic transient accumulation of NO2−, NO and N2O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N2O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N2O produced (N2O/(N2+N2O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N2O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N2O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level.

The Relationship Between Cell Size and Viability of Soil Bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (“dwarf”) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4μm, about 10% for cells with diameters between 0.4 and 0.6μm, and 30–40% for cells with diameters above 0.6μm. Only 10–20% of the viable cells with diameters <0.4μm increased their diameter to >0.4μm prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.

Denitrification Response Patterns during the Transition to Anoxic Respiration and Posttranscriptional Effects of Suboptimal pH on Nitrogen Oxide Reductase in Paracoccus denitrificans

ABSTRACT Denitrification in soil is a major source of atmospheric N2O. Soil pH appears to exert a strong control on the N2O/N2 product ratio (high ratios at low pH), but the reasons for this are not well understood. To explore the possible mechanisms involved, we conducted an in-depth investigation of the regulation of denitrification in the model organism Paracoccus denitrificans during transition to anoxia both at pH 7 and when challenged with pHs ranging from 6 to 7.5. The kinetics of gas transformations (O2, NO, N2O, and N2) were monitored using a robotic incubation system. Combined with quantification of gene transcription, this yields high-resolution data for direct response patterns to single factors. P. denitrificans demonstrated robustly balanced transitions from O2 to nitric oxide-based respiration, with NO concentrations in the low nanomolar range and marginal N2O production at an optimal pH of 7. Transcription of nosZ (encoding N2O reductase) preceded that of nirS and norB (encoding nitrite and NO reductase, respectively) by 5 to 7 h, which was confirmed by observed reduction of externally supplied N2O. Reduction of N2O was severely inhibited by suboptimal pH. The relative transcription rates of nosZ versus nirS and norB were unaffected by pH, and low pH had a moderate effect on the N2O reductase activity in cells with a denitrification proteome assembled at pH 7. We thus concluded that the inhibition occurred during protein synthesis/assembly rather than transcription. The study shed new light on the regulation of the environmentally essential N2O reductase and the important role of pH in N2O emission.

Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2− and O2 concentrations

The oxygen control of denitrification and its emission of NO/N2O/N2 was investigated by incubation of Nycodenz-extracted soil bacteria in an incubation robot which monitors O2, NO, N2O and N2 concentrations (in He+O2 atmosphere). Two consecutive incubations were undertaken to determine (1) the regulation of denitrification by O2 and NO2(-) during respiratory O2 depletion and (2) the effects of re-exposure to O2 of cultures with fully expressed denitrification proteome. Early denitrification was only detected (as NO and N2O) at <or=80 microM O2 in treatments with NO2(-), and the rates were three orders of magnitude lower than the rates observed after oxygen depletion (with N2 as the primary product). When re-exposed to O2, the cultures continued to denitrify (8-55% of the rates during the foregoing anoxic phase), but its main product was N2O. The N2O reductase activity recovered as oxygen was being depleted. The results suggest that expression of the denitrifying proteome may result in significant subsequent aerobic denitrification, and this has profound implications for the understanding and modelling of denitrification and N2O emission. Short anoxic spells caused by transient flooding during rainfall, could lead to subsequent unbalanced aerobic denitrification, in which N2O is a major end product.

Nitrous oxide release from spruce forest soil: Relationships with nitrification, methane uptake, temperature, moisture and fertilization

Abstract The spatial variability in a homogeneous 63 yr old spruce forest stand was investigated with respect to potential release of N2O in relation to nitrification rate, N mineralization rate, CH4 uptake, organic C, total N and pH in soil under standardized laboratory incubation conditions. Soil samples were taken at random within a 100 m2 forest stand, and gas fluxes at three temperatures (3, 10 and 15°C) and two soil moisture contents (35 and 45% v/v) were measured. In addition, a number of other relevant factors was measured in each soil sample. There was large spatial variation in N2O release [coefficient of variation (CV)=up to 152%], nitrification rate (CV=65%) and N-mineralization rate (CV = 137%). Comparatively low spatial variations in organic C (CV=31%), total N (CV =29%), CO2 evolution (CV = 38%), CH4 uptake (CV = 37%) and pH (CV = 4.4%) were observed. The rate of N2O release was positively correlated with nitrification rate and negatively correlated with pH. The rate of CH4 uptake was negatively correlated (r = − 0.61) with nitrification rate. No significant correlation was observed between the N-mineralization rate and any of the variables measured. N2O fluxes measured at 10 and 3°C were 76% (±14%) and 17% (±4%) of that measured at 15°C, respectively. We found a significant increase in N2O accumulation by increasing the moisture content from 35 to 45%. Ammonium sulphate additions stimulated N2O release but not the nitrification. The investigation demonstrated large spatial variation in the rates of nitrification, N2O release, but it was difficult to identify regulating factors for this spatial variability.

COMPETITION FOR NITROGEN DURING DECOMPOSITION OF PLANT RESIDUES IN SOIL: EFFECT OF SPATIAL PLACEMENT OF N-RICH AND N-POOR PLANT RESIDUES

Abstract The distance between “hot spots” for N-mineralization (N-rich clover residues) and for N-immobilization (high C-to-N straw) in soil was experimentally manipulated to investigate its effect on the competition between plant roots and microorganisms for mineralized N. The experiment demonstrated that plant roots were reasonably competitive, resulting in deprivation of the N-supply to the microorganisms growing on the straw material, but this was totally dependent on the distance between the N-rich and the N-poor sites in the soil. The critical distance was somewhere between 3 and 6 mm, above which plant roots outcompeted the microorganisms more or less completely. Our study illustrates an important mechanism by which plant roots can interfere with microbial N-transformations in soil. It may be the mechanism responsible for an often alleged “stimulation” of N-mineralization by plant roots. The mineralization rate of clover- and straw-C was measured in planted as well as unplanted soil. The presence of plant roots retarded the straw-C mineralization significantly, but not clover C.

N-fertilization and soil acidification effects on N2O and CO2 emission from temperate pine forest soil

The fluxes and soil atmospheric concentrations of N2O were studied in field lysimeters containing reestablished soil profiles from 100-y-old Scots pine (Pinus sylvestris) forest of Norway. The experiment was designed as a full factorial with 3 N-fertilization rates [0 (Control), 30 kg (Medium) and 90 kg (High) N ha−1 y−1] with NH4NO3 and 3 pH values of soil acidification (acid irrigation with pH 3, 4 and 5.5). The most acidic treatment (pH 3) significantly reduced both N2O fluxes and soil N2O concentrations. The highest N2O fluxes were observed in the intermediate pH treatment (pH 4). There was a significant increase in N2O release due to input of N. The average fertilizer-derived N2O-N emission during the summer period was 94 and 93 mg kg−1 of added NH4NO3-N d−1 for Medium N and High N, respectively. Surprisingly, despite a strong effect of acidification on the N2O flux, we could not find any significant interaction between acidification and N-fertilization. This means that N-deposition effects on the N2O fluxes will be more or less unaffected by previous acidification due to deposition of sulphuric acid. Soil acidification with the most acidic “rain” (pH 3) resulted in decreased CO2 fluxes and concentrations in soil.