Denitrification: ecological niches,competition and survival

Organisms with the denitrification capacity are widely distributed and in high density in nature. It is not well understood why they are so successful. A survey of denitrifying enzyme content of various habitats is presented which indicates a role of carbon and oxygen, but not nitrate, in affecting denitrifier populations. It is suggested that organic carbon is more important than oxygen status in determining denitrifying enzyme content of habitats. In low oxygen environments, denitrifiers compete with organisms that dissimilate nitrate to ammonium, a process which conserves nitrogen. The energetic and kinetic parameters that affect this competition are evaluated. The latter is examined using Michaelis-Menten theoretical models by varying V_max, K_m, and S_o (substrate concentration) for the two competing populations. The outcome predicted by these models is presented and discussed in relation to previous data on population densities and K_m values for representatives of these competing groups. These models suggest the conditions required to achieve changes in partitioning between the two fates of nitrate. These considerations are important if one is to be able to evaluate and successfully “manage” the fate of nitrate in any habitat.     

Exploration of Inorganic C and N Assimilation by Soil Microbes with Time-of-Flight Secondary Ion Mass Spectrometry

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect ¹³C and ¹⁵N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (¹²C¹⁴N⁻) and mass 27 (¹³C¹⁴N⁻ and ¹²C¹⁵N⁻) ions sputtered with a Ga⁺ probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative ¹⁵N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with ¹⁵NO₃⁻. We observed that the ¹⁵N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the ¹⁵N content of cells that have assimilated ¹⁵N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.     

Long-term effects of elevated nitrogen on forest soil organic matter stability

Nitrogen addition may alter the decomposition rate for different organic-matter pools in contrasting ways. Using a paired-plot design, we sought to determine the effects of long-term elevated N on the stability of five organic-matter pools: organic horizons (Oe+a), whole mineral soil (WS), mineral soil fractions including the light fraction (LF), heavy fraction (HF), and a physically recombined fraction (RF). These substrates were incubated for 300 days, and respiration, mineralized N, and active microbial biomass were measured. Samples with elevated N gave 15% lower cumulative respiration for all five substrates. Over the 300-day incubation, the Oe+a gave twice the cumulative respiration (gCkg^–1 initial C) as the LF, which gave slightly higher respiration than the HF. Respiration was 35% higher for the WS than for the RF. Mineralized N was similar between N treatments and between the LF and HF. Net N mineralized by the LF over the course of the 300-day incubation decreased with higher C:N ratio, due presumably to N immobilization to meet metabolic demands. The pattern was opposite for HF, however, which could be explained by a release of N in excess of metabolic demands due to recalcitrance of the HF organic matter. Mineralized N increased with respiration for the HF but showed no pattern, or perhaps even decreased, for the LF. WS and RF showed decreasing active microbial biomass near the end of the incubation, which corresponded with decreasing respiration and increasing nitrate. Our results show that long-term elevated N stabilized organic matter in whole soil and soil fractions.     

Recovery of ectomycorrhiza after 'nitrogen saturation' of a conifer forest

Trees reduce their carbon (C) allocation to roots and mycorrhizal fungi in response to high nitrogen (N) additions, which should reduce the N retention capacity of forests. The time needed for recovery of mycorrhizas after termination of N loading remains unknown. Here, we report the long-term impact of N loading and the recovery of ectomycorrhiza after high N loading on a Pinus sylvestris forest. We analysed the N% and abundance of the stable isotope (15) N in tree needles and soil, soil microbial fatty acid biomarkers and fungal DNA. Needles in N-loaded plots became enriched in (15) N, reflecting decreased N retention by mycorrhizal fungi and isotopic discrimination against (15) N during loss of N. Meanwhile, needles in N-limited (control) plots became depleted in (15) N, reflecting high retention of (15) N by mycorrhizal fungi. N loading was terminated after 20yr. The δ(15) N and N% of the needles decreased 6yr after N loading had been terminated, and approached values in control plots after 15yr. This decrease, and the larger contributions compared with N-loaded plots of a fungal fatty acid biomarker and ectomycorrhizal sequences, suggest recovery of ectomycorrhiza. High N loading rapidly decreased the functional role of ectomycorrhiza in the forest N cycle, but significant recovery occurred within 6-15yr after termination of N loading.     

Alder and lupine enhance nitrogen cycling in a degraded forest soil in Northern Sweden

Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N₂ fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine (Lupinus nootkatensis Donn) and gray alder (Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH⁺ ₄ and NO^– ₃ as collected on ion exchange membranes. We also estimated N₂ fixation activity of the N₂-fixing plants by the natural abundance of ¹⁵N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N₂-fixing plants was almost totally derived from N₂ fixation. Plots containing N₂-fixing species generally had significantly higher soil N and N availability than a control plot without N₂-fixing plants. Taken together, all measurements indicated that N₂-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden.     

Ammonia-Oxidizing Bacteria along Meadow-to-Forest Transects in the Oregon Cascade Mountains

Although nitrification has been well studied in coniferous forests of Western North America, communities of NH₃-oxidizing bacteria in these forests have not been characterized. Studies were conducted along meadow-to-forest transects at two sites (Lookout and Carpenter) in the H. J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon. Soil samples taken at 10- or 20-m intervals along the transects showed that several soil properties, including net nitrogen mineralization and nitrification potential rates changed significantly between vegetation zones. Nonetheless, terminal restriction fragment length polymorphism (T-RFLP) analysis of the PCR-amplified NH₃ monooxygenase subunit A gene (amoA) showed the same DNA fragments (TaqI [283 bp], CfoI [66 bp], and AluI [392 bp]) to dominate ≥45 of 47 soil samples recovered from both sites. Two fragments (491-bp AluI [AluI491] and CfoI135) were found more frequently in meadow and transition zone soil samples than in forest samples at both sites. At the Lookout site the combination AluI491-CfoI135 was found primarily in meadow samples expressing the highest N mineralization rates. Four unique amoA sequences were identified among 15 isolates recovered into pure culture from various transect locations. Six isolates possessed the most common T-RFLP amoA fingerprint of the soil samples (TaqI283-AluI392-CfoI66), and their amoA sequences shared 99.8% similarity with a cultured species, Nitrosospira sp. strain Ka4 (cluster 4). The other three amoA sequences were most similar to sequences of Nitrosospira sp. strain Nsp1 and Nitrosospira briensis (cluster 3). 16S ribosomal DNA sequence analysis confirmed the affiliation of these isolates with Nitrosospira clusters 3 and 4. Two amoA clone sequences matched T-RFLP fingerprints found in soil, but they were not found among the isolates.     

Microbial Community Dynamics Associated with Rhizosphere Carbon Flow

Root-deposited photosynthate (rhizodeposition) is an important source of readily available carbon (C) for microbes in the vicinity of growing roots. Plant nutrient availability is controlled, to a large extent, by the cycling of this and other organic materials through the soil microbial community. Currently, our understanding of microbial community dynamics associated with rhizodeposition is limited. We used a ¹³C pulse-chase labeling procedure to examine the incorporation of rhizodeposition into individual phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of greenhouse-grown annual ryegrass (Lolium multiflorum Lam. var. Gulf). Labeling took place during a growth stage in transition between active root growth and rapid shoot growth on one set of plants (labeling period 1) and 9 days later during the rapid shoot growth stage on another set of plants (labeling period 2). Temporal differences in microbial community composition were more apparent than spatial differences, with a greater relative abundance of PLFAs from gram-positive organisms (i15:0 and a15:0) in the second labeling period. Although more abundant, gram-positive organisms appeared to be less actively utilizing rhizodeposited C in labeling period 2 than in labeling period 1. Gram-negative bacteria associated with the 16:1ω5 PLFA were more active in utilizing ¹³C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:2ω6,9 was the most highly labeled. These results demonstrate the effectiveness of using ¹³C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.     

Geographic distribution and genetic diversity of Ceanothus-infective Frankia strains

Little is known about Ceanothus-infective Frankia strains because no Frankia strains that can reinfect the host plants have been isolated from Ceonothus spp. Therefore, we studied the diversity of the Ceonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infect Ceanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3' end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among the Ceanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, the Frankia strains present were related to the sample collection locales.     

Microbial community dynamics associated with rhizosphere carbon flow

Root-deposited photosynthate (rhizodeposition) is an important source of readily available carbon (C) for microbes in the vicinity of growing roots. Plant nutrient availability is controlled, to a large extent, by the cycling of this and other organic materials through the soil microbial community. Currently, our understanding of microbial community dynamics associated with rhizodeposition is limited. We used a (13)C pulse-chase labeling procedure to examine the incorporation of rhizodeposition into individual phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of greenhouse-grown annual ryegrass (Lolium multiflorum Lam. var. Gulf). Labeling took place during a growth stage in transition between active root growth and rapid shoot growth on one set of plants (labeling period 1) and 9 days later during the rapid shoot growth stage on another set of plants (labeling period 2). Temporal differences in microbial community composition were more apparent than spatial differences, with a greater relative abundance of PLFAs from gram-positive organisms (i15:0 and a15:0) in the second labeling period. Although more abundant, gram-positive organisms appeared to be less actively utilizing rhizodeposited C in labeling period 2 than in labeling period 1. Gram-negative bacteria associated with the 16:1omega5 PLFA were more active in utilizing (13)C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:2omega6,9 was the most highly labeled. These results demonstrate the effectiveness of using (13)C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.