Harvey Bolton

Soil microbial biomass and activity of a disturbed and undisturbed shrub-steppe ecosystem

Abstract Disturbance of shrub-steppe soils and alterations in plant cover may affect the distribution, size and activity of soil microorganisms and their ability to biogeochemically cycle essential nutrients. Therefore, the soil microbial biomass and activity and selected soil enzyme activities were determined for two arid ecosystems, an undisturbed perennial shrub-steppe and annual grassland, which was initially shrub-steppe and has been an annual grassland since the disturbance caused by farming ceased in the 1940s. Soils were sampled at 0–5 and 5–15 cm depths beneath sagebrush ( Artemisia tridentata Mutt.), bluebunch wheatgrass [ Elytrigia spicata (Pursh) D.R. Dewey] and cryptogamic soil lichen crust at the perennial site and beneath downy brome ( Bromus tectorum L.) at the annual grassland site. Soils were analyzed for physical properties, inorganic N, microbial biomass C and N, respiration and several enzymes. The soil pH and bulk density usually increased, while inorganic N, total N and total C decreased as a function of soil depth. Soil microbial biomass C and N, soil respiration and soil dehydrogenase activity were 2–15 times higher in the top 5 cm of soil than at the 5–15 cm depth regardless of plant type. Loss of this surface soil would therefore be detrimental to microbially-mediated cycling of nutrients. Surface soil (0–5 cm depth) microbial biomass C and N and soil respiration, dehydrogenase and phosphatase activity were influenced by plant type and decreased in the order B. tectorum A. tridentata = E. spicata soil crust. Spatial distribution of plant species at the shrub-steppe site resulted in “islands” of enhanced microbial biomass and activity underneath the shrubs and grasses when compared to the interplant areas covered with soil crust. When plant cover was used to compute a landscape estimate of soil microbial biomass C and N for the perennial shrub-steppe and the annual grassland, similar values were obtained. This indicates that while the distribution of microorganisms may be more heterogeneous in the shrub-steppe, the average across the landscape is the same as the more homogeneous annual grassland.

Spatial relationships of soil microbial biomass and C and N Mineralization in a semi-arid shrub-steppe ecosystem

Abstract Microbial mineralization of both C and N in the shrub-steppe soils of central Washington influence plant productivity and ecosystem stability. Microbial processes in arid ecosystems are in turn influenced by heterogeneously-spaced plants and abiotic variables. Our objective was to determine the spatial relationships of C and N mineralization and microbial biomass to plant location. We measured inorganic N pools, microbial biomass and C and N mineralization potentials (C o , N o ) from 205 soil samples positioned around five Artemisia tridentata shrub plants. Most variables showed log normal distributions and all were significantly correlated to each other. Microbial biomass had the highest positive correlation with C mineralization and soluble C. The metabolic C quotient ( q CO 2 ) was twice that of other natural forest and grassland ecosystems. In addition the metabolic N quotient ( q N) was lower in the shrub-steppe soil compared to other ecosystems. The coupled metabolic quotients indicate the shrub-steppe soil has low substrate quality with a high N immobilization capacity. Geostatistical analysis of spatial relationships showed that samples were spatially related with each other to a distance of 0.5–1.0 m. At sample locations where microbial biomass was high Co was also high. In contrast. No was low in these areas. Cross-correlation with plants showed that C o was spatially related to shrubs and not to grass plants and that No was not related to any plant location. Cross-correlation analysis revealed that variables that are linearly correlated may not necessarily be spatially correlated. Our study showed that the resource island effect of nutrients and microbial biomass in the shrub-steppe ecosystem is important when estimating microbial processes at the landscape level.

Soil properties and microbial activity across a 500 m elevation gradient in a semi-arid environment

If climate change causes the semi-arid shrub-steppe to become hotter and drier it may affect soil C and N cycling and precipitate changes in soil processes and microbial and plant community structure. This study was conducted, using an elevation gradient as an analog of climate change, to analyze climatic influence on soil microbial activity and soil properties. We collected soil from under cryptogamic crust and bunchgrass plants at 25 sites over a 500 m elevation transect in a shrub-steppe ecosystem located in eastern Washington State of the US. The samples were analyzed for several chemical and microbiological attributes including pH, microbial biomass and nitrification potential and the data grouped into five climate sites for statistical analysis. Soil pH decreased over the transect with higher pH values in the grass soil than the crust. In contrast soil electrical conductivity (EC) increased with increasing elevation as did both ammonium and nitrate. Ammonium and EC were greater in the crust soil than the grass soil but nitrate concentration was the same under both plant covers. Both total C and N amounts increased with elevation as did nitrification potential. Due to high sample spatial variability microbial biomass, respiration and N mineralization showed non-significant trends over the 500 m elevation transect. Using these measured gradient relationships the increase in temperature and decrease in precipitation that is expected in this shrub-steppe ecosystem over the next 100 years would eventually cause the pH to increase and the EC to decrease. Plants would become more sparse, nitrification potential would decrease and ammonium would increase. Total C, N and microbial biomass concentrations would begin decreasing and may shift the controlling factors of the ecosystem to abiotic factors. The changes in the cycling of N and to some extent C due to climate change could alter the microbial and plant community structure and function of this ecosystem and cause it to move in the direction of desertification.

Nitrous oxide flux from a shrub-steppe ecosystem: Sources and regulation

The semi-arid shrub-steppe is the largest grassland-type ecosystem of North America and may make significant contributions to the global atmospheric N2O budget. However, little information is available concerning sources and regulation of N2O flux in this ecosystem. Experiments were made to determine the relative importance of nitrification, denitrification and abiotic sources to total N2O flux and to investigate the factors regulating N2O flux rates from an undisturbed shrub-steppe ecosystem. The contributions to N2O flux by nitrification and denitrification were estimated using acetylene (10 Pa) to selectively inhibit N2O production by nitrifiers. Abiotic sources of N2O were evaluated using sterilized soil. Factors limiting N2O production were evaluated by monitoring N2O flux rates from soil-cores amended with combinations of NO3−-N, NH4+-N, soluble C and water. The effect of wet-dry cycles on N2O flux was determined by wetting field dry soil to field capacity and monitoring N2O flux rates, soil NH4+-N, NO3−-N and water content throughout a drying period. Our results showed that nitrification accounts for 61–98% of the N2O produced from soil at water contents below saturation and that denitrification is the primary N2O source at saturated water contents. No detectable N2O was produced by abiotic sources. In intact soil cores N2O flux rates were found to be most limited by water and N availability. Wetting of dry soil resulted in a pulse of N2O flux due to increased N availability. It is likely that this ecosystem exhibits relatively low N2O flux rates for much of the year due to low soil moisture and inorganic N contents. Since soil moisture content is generally well below field capacity in this ecosystem, nitrification must be the dominant N2O source. These results suggest that conditions favorable for substantial N2O production in shrub-steppe ecosystems probably exist only at times following precipitation events.

Priming effect and C storage in semi-arid no-till spring crop rotations

Adoption of less invasive management practices, such as no-till (NT) and continuous cropping, could reduce CO2 emissions from agricultural soils by retaining soil organic matter (SOM). We hypothesized that C storage increases as cropping intensity increases and tillage decreases. We also hypothesized that pulsed addition of C increases the mineralization of native SOM. We evaluated C storage at the 0- to 5-cm depth in soils from four crop rotations: winter wheat-fallow, spring wheat-chemical fallow, continuous hard red spring wheat, and spring wheat-spring barley on a Ritzville silt loam (Calcidic Haploxeroll). In two incubation studies using 14C-labeled wheat straw, we traced the decomposition of added residue as influenced by (1) cropping frequency, (2) tillage, and (3) pulsed additions of C. Differences in 14C mineralization did not exist among the four rotations at any time throughout the incubations. However, differences in total CO2 production between the continuous wheat rotations and the fallow rotations point to a priming of native SOM, the degree of which appears to be related to the relative contributions of fungi and bacteria to the decomposition of added residue. Addition of non-labeled wheat straw to select samples in the second incubation resulted in a flush of 14C-CO2 not seen in the controls. This priming effect suggests C inputs have a greater effect on mineralization of residual C compared to disturbance and endogenous metabolism appears to be the source of primed C, with priming becoming more pronounced as the fungal:bacterial ratio in the soil increases.

Novel antibiotics as inhibitors for the selective respiratory inhibition method of measuring fungal:bacterial ratios in soil

The use of the selective inhibition (SI) method for measuring fungal:bacterial ratios may be limited due to biocide selectivity and the overlap of antibiotic activity. This study evaluated novel pairs of antibiotics for their specificity in soils of different origins and their potential reduction in inhibition of non-target organisms. Four soils selected for this study were from a semi-arid shrub-steppe, a loblolly pine forest and two grassland sites (restored and farmed prairie plots). Three bactericides were tested: oxytetracycline hydrochloride, streptomycin sulphate, and bronopol. Three fungicides were tested: captan, ketoconazole, and nystatin. The inhibitor additivity ratio and fungal:bacterial ratios were calculated from control and treated soils where inhibition was measured as CO2 respiration reduction with biocides. We were able to minimize non-target inhibition by the antibiotics to <5% and thus calculate reliable fungal:bacterial ratios using captan to inhibit fungi in all four soils, and bronopol to inhibit bacteria in three of the four soils. The most successful bactericide in the restored prairie was oxytetracycline-HCl. Our results demonstrate that application of novel antibiotics is not uniformly successful in soils of different origin and that the SI technique requires more than just optimization of antibiotic concentration; it also requires optimization of antibiotic selection.

Small-scale spatial and temporal variability of N2O flux from a shrub-steppe ecosystem

Abstract Nitrous oxide (N 2 O) is a trace greenhouse gas that catalyses ozone destruction. It is also the major gaseous loss of N from the N-limited shrub-steppe ecosystem. We examined the spatial and temporal flux of N 2 O from small plots in an undisturbed shrub-steppe ecosystem having spatially heterogeneous plant cover. The N 2 O flux from the soil surface and NH 4 + N and NO 3 − N concentrations were measured periodically over 1 y from 44 points in 2.4 m 2 plots centered on individual Artemisia tridentata shrubs. Positive N 2 O flux occurred from March to October, with no detectable flux during the winter months. The spatial (plot) variability of N 2 O flux ranged from 23 to 130%, with lower variability as soil moisture increased. Temporal variability (March to October) was 171%, but decreased to 77% when calculated without the August sampling date. The measured N 2 O flux correlated positively with microbial activity (CO 2 production), with moisture when the soil was fairly wet and usually with NO 3 − N concentrations. After a precipitation event on to dry soil, there was a pulse of N mineralization and N 2 O flux, which strongly correlated with proximity to vegetation. The estimated N 2 O flux occurring within 48 h after warm season precipitation events accounted for 20% of the total annual N 2 O flux from this ecosystem. Thus, small-scale spatial and short-term temporal variations can significantly affect annual estimates of ecosystem N 2 O flux and, thus, gaseous N loss from semi-arid ecosystems.

Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

ABSTRACT EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed inEscherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. TheemoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.

Field calibration of soil-core microcosms: Fate of a genetically altered rhizobacterium

Microcosms containing intact soil-cores are a potential tool for assessing the risks of the release of genetically engineered microorganisms (GEMs) to the environment. Before microcosms become a standard assessment tool, however, they must first be calibrated to ensure that they adequately simulate key parameters in the field. Four systems were compared: intact soil-core microcosms located in the laboratory at ambient temperature and in a growth chamber with temperature fluctuations that simulated average conditions in the field, field lysimeters, and field plots. These four systems were inoculated with rifampicin-resistantPseudomonas sp. and planted to winter wheat. Populations of thePseudomonas sp. in soil decreased more rapidly at ambient temperature, but population size at the three-leaf stage of wheat growth was the same in all four systems. Populations of thePseudomonas sp. on the rhizoplane of wheat were the same at the three-leaf stage in all four systems, and colonization with depth at the final boot stage-sampling was also similar. In general, microcosms incubated at ambient temperature in the laboratory or in the growth chamber were similar to those in the field with respect to survival of and colonization of the rhizoplane by the introducedPseudomonas sp.

Electroelution to remove humic compounds from soil DNA and RNA extracts

The application of nucleic acid techniques to detect, identify, and monitor specific genes or organisms in soils or sediments is often complicated by the inhibitory effects of humic compounds that copurify with nucleic acids. A rapid electroelution technique was developed to separate inhibitory compounds from extracts of soil DNA and RNA. This technique was used in conjunction with PCR to detect nifH, terrestrial ammonia-oxidizer (TAO) 16S rRNA genes and TAO 16S rRNA from a variety of surface soils and contaminated sediments. After electroelution of crude nucleic acid extracts, PCR sensitivity was increased up to a factor of 104 relative to DNA templates that had not been electroeluted. Without electroelution of crude DNA extracts, target genes often remained undetected. Likewise, electroelution of crude RNA extracts increased RT-PCR sensitivity (for TAO 16S rRNA) by a factor of 103 relative to RNA extracts that had not been further purified. The electroelution technique will therefore be useful for rendering environmental nucleic acids extracted from soil and sediment more amenable to PCR methods and nucleic acid analysis.