Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Use of the IUL Countermat Automatic Colony Counter for Spiral Plated Total Viable Counts

An IUL Countermat automatic colony counter was used to enumerate colonies on spiral total viable count plates made with a wide variety of foods. The counter results exhibited a correlation with manual counting results similar to the reproducibility obtained with manually counted spiral plates. Use of this machine results in a large time saving compared with the conventional counting method and is recommended as a generally suitable method for counting spiral total viable count plates.     

Comparison of Several Algorithms to Estimate Activity Counts with Smartphones as an Indication of Physical Activity Level

Background

Dedicated devices like GT3X+, Actical or ActivPal have been widely used to measure physical activity (PA) levels by using cut-points on activity counts. However, the calculation of activity counts relies on proprietary software. Since smartphones incorporate accelerometers they are suitable candidates to determine PA levels in a wider population.

Variation in Heterotrophic and Autotrophic Nitrifier Populations in Relation to Nitrification in Organic Soils

The occurrence of heterotrophic and autotrophic nitrifiers in Pahokee muck and the role of these organisms in the ecosystem were assessed by surveying their population densities under different field conditions and by observing the relationship of these populations with aerobic bacteria and soil moisture. Heterotrophic nitrifier populations varied from 2.0 × 10⁵ to 3.8 × 10⁶ bacteria per cm³ of muck in surface fallow (bare) Pahokee muck during the annual cycle. This population decreased 40-fold between the surface and the 60- to 70-cm depths of soil. Similar variations were noted with autotrophic nitrifier populations. Significant correlations were found between heterotrophic nitrifiers and both soil moisture and aerobic bacteria. These relationships did not exist for the autotrophic nitrifiers. In soil that had been heated to kill the autotrophic nitrifiers, while preserving a population of the heterotrophs, and then amended with sodium acetate or ammonium sulfate or both, no nitrate or nitrite accumulated, although significant increases in heterotrophic nitrifiers were detected. In unheated control soil, nitrate plus nitrite-N increased from 14.3 to 181 μg/g of wet soil, and 48 μg of nitrite-N per g was produced. These data suggest that the autotrophic nitrifiers were the sole population responsible for nitrification in Pahokee muck.     

Specific Inhibition of Nitrite Oxidation by Chlorate and Its Use in Assessing Nitrification in Soils and Sediments

A method was developed to determine the ammonium oxidation rate (potential) of unenriched natural samples by measuring the nitrite produced in shaken slurries. Addition of chlorate to the samples prevented nitrite from being oxidized to nitrate. The effectiveness and specificity of chlorate were tested with pure cultures of nitrite and ammonium oxidizers, as well as in soil and sediment slurries. It was concluded that chlorate had relatively little inhibitory effect on ammonium oxidation. However, under some conditions chlorate was not completely effective in blocking nitrite oxidation, and the causes of this were investigated. The technique was designed to check for incomplete blockage.     

Determination of Microbial Extracellular Enzyme Activity in Waters,Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.     

Invertebrate Biodiversity in Antarctic Dry Valley Soils and Sediments

We studied invertebrate communities across a transition zone between soils and stream sediments in the cold desert landscape of Taylor Valley, Antarctica. We hypothesized that hydrological and biogeochemical linkages in the functionally important transition zone between streams and surrounding soils should be important in structuring invertebrate communities. We compared invertebrate communities along transects beginning in the saturated sediments under flowing stream water and extending laterally through the hyporheic zone to the dry soils that characterize most of the dry valley landscape. Nematodes, rotifers, and tardigrades assembled into different communities in soils and sediments, but there was no relationship between the total abundance of invertebrates and moisture. Community diversity was, however, influenced by the moisture and salinity gradients created with distance from flowing waters. The wet, low-salinity sediments in the center of the stream contained the most invertebrates and had the highest taxonomic diversity. Adjacent to the stream, communities in the hyporheic zone were influenced strongly by salt deposition. Abundance of invertebrates was low in the hyporheic zone, but this area contained the most co-occurring nematode species (three species). In dry soils, communities were composed almost entirely of a single species of nematode, Scottnema lindsayae, an organism not found in the stream center. These results suggest spatially-partitioned niches for invertebrates in soils and sediments in the dry valley landscape based on proximity to sources of moisture and the interactive effects of salinity. Received 22 September 1998; accepted 16 April 1999.     

Trichloroethene Biodegradation Potential in Wetland Soils and Paleowetland Sediments

Trichloroethene (TCE) plumes extend north-northeast toward the Ohio River from the Paducah Gaseous Diffusion Plant (PGDP), a Superfund site in the Gulf Coastal Plain of western Kentucky. Wetlands in the floodplain are in the paths of these plumes, and on-site contamination has migrated downward from the Regional Gravel Aquifer (RGA) into the upper McNairy Formation, which overlies a bedrock aquifer. Intrinsic biodegradation in these two environments at the margins of the RGA could limit further contaminant migration and ecosystem or water-quality degradation. To assess cometabolic biodegradation potential in these uncontaminated environments, we attempted to culture and enumerate methanogens, sulfate- and Fe(III)-reducers, and methanotrophs, which have been implicated elsewhere as TCE degraders. Soil samples were collected at three wetland sites in the floodplain. McNairy sediments were collected beneath one of the suspected source areas at PGDP. Methanogens, sulfate reducers, and methanotrophs were abundant in wetland soils, with populations generally decreasing with depth. Methanogens were the only group cultured from McNairy sediments, and they showed little activity compared with wetland methanogen cultures. TCE loss in methanogenic batch cultures by chemoautotrophic and acetoclastic methanogens was monitored, but no significant degradation was observed.     

Use of Carbon Isotope Composition for Characterization of Microbial Activity in Arable Soils

The effect of glucose on microbial mineralization of soil organic matter (SOM) was studied in arable soil specimens. The fluxes of carbon dioxide generated during this degradation were deduced from differences in the carbon isotope abundance ratios of glucose δ¹³C = –11.4 per mil) and SOM δ¹³C = –27.01 per mil). The priming effect of glucose and respiratory quotient (RQ) were taken as indices of activation of SOM-consuming microbiota. The data on microbial mineralization of organic matter in soil obtained in this study show that the addition of a readily consumable substance (glucose) to soil favors SOM degradation and increases the release of carbon dioxide from soil to atmosphere.     

Prediction of Petroleum Hydrocarbon Bioavailability in Contaminated Soils and Sediments

Recently, several laboratory methods have been developed for the prediction of contaminant bioavailability. So far, none of these methods has been extensively tested for petroleum hydrocarbons. In the present study we investigated solid-phase extraction and persulfate oxidation for the prediction of total petroleum hydrocarbon (TPH) bioavailability. One sediment and two soil samples were subjected to solid-phase extraction, persulfate oxidation, and biodegradation, after which hydrocarbon removal was compared. It was demonstrated that a short solid-phase extraction (168?h) provided a good method for the prediction of the extent of TPH degradation in an optimized slurry reactor (84?d). Solid-phase extraction slightly underestimated the degradation of readily biodegradable hydrocarbons, whereas it slightly overestimated the degradation of poorly biodegradable hydrocarbons. Persulfate oxidation appeared to be unfit for the prediction of TPH bioavailability as persulfate was unable to oxidize hydrocarbons with a high ionization potential. Hydrocarbons that were affected were likely to be transformed rather than completely oxidized. Nevertheless, persulfate oxidation provided a good method for the prediction of polycyclic aromatic hydrocarbon (PAH) bioavailability.     

Forest Productivity and Efficiency of Resource Use Across a Chronosequence of Tropical Montane Soils

We tested the hypothesis that plants adjust to nutrient availability by altering carbon allocation patterns and nutrient-use efficiency (NUE = net primary production [NPP] per unit nutrient uptake), but are constrained by a trade-off between NUE and light-use efficiency () = NPP per unit intercepted light). NPP, NUE and ) were measured in montane Metrosideros polymorpha forest across a 4.1 x 10⁶ yr space for time substitution chronosequence in which available soil N and P pools change with site age. Although the range of N and P availability across sites was broad, there was little difference in NPP between sites, and in contrast to theories of carbon allocation relative to limiting resources, we found no consistent relationships in production allocation to leaves, fine roots or wood. However, canopy nutrient pools and fluxes were correlated with the mass of fine roots per unit soil volume and there was a weak but positive correlation of NPP with LAI. Patterns of ) and NUE across the soil developmental sequence were opposite to each other. ) increased as nutrient availability and nutrient turnover increased, while NUE decreased in response to the same influences but reached its highest values where either N or P availability and turnover of both N and P were low. A negative correlation between ) and NUE supports the hypothesis that a trade-off exists between ) and leaf characteristics affecting NUE.     

Lignin Degradation and Humus Formation in Alluvial Soils and Sediments

The contribution of lignin to the formation of humic compounds was examined in different environments of the terrestrial-aquatic interface in the Garonne River valley in southwestern France. Alluvial soils and submerged or nonsubmerged river and pond sediments containing alder, poplar, or willow [¹⁴C-lignin]ligno-celluloses were incubated. After a 49-day incubation period, 10 to 15% of labeled lignins in alluvial soils was recovered as evolved ¹⁴CO₂. In nonsubmerged sediments, 10% of the applied activity was released as ¹⁴CO₂, and in submerged sediments, only 5% was released after 60 days of incubation. In the different alluvial soils and sediments, the bulk of residual activity (70 to 85%) remained in the two coarsest-grain fractions (2,000 to 100 and 100 to 50 μm). Only 2 to 6% of the residual activity of these two coarse fractions was recovered as humic and fulvic acids, except in the case of alder [¹⁴C-lignin]lignocellulose, which had decomposed in a soil collected beneath alders. In this one 55% of the residual activity was extracted as humic substances from the 2,000- to 100-μm fraction. Humic and fulvic acids represented from 6 to 50% of the residual activity in the finest-grain fractions (50 to 20 and 20 to 0 μm). The highest percentages were obtained in soil collected beneath alders and in submerged pond sediment. The contribution of different groups of microorganisms, as well as nutrients and clay content, may influence humic-substance formation in such environments. Physical stability also may be an important factor for complex microbial activity involved in this process.     

The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil

Our objective was to determine if 4-methylumbelliferyl-labelled enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF) N-acetyl-β-d-glucosaminide and MUF β-d-lactoside were used for the detection and quantification of β-N-acetylglucosaminidase (EC 3.2.1.30) (NAGase) and endo 1,4-β-glucanase (EC 3.2.1.4)/cellobiohydrolase (EC 3.2.1.91) (CELase), respectively. Culture screenings on solid media showed a widespread ability to produce NAGase among a taxonomically diverse selection of fungi on media with and without added chitin. NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of two fungus-specific indicator molecules, 18:2ω6 phospholipid fatty acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil, but no correlation was found with ergosterol-based estimates of fungal biomass.The determination of enzyme activities is a simple approach to the study of microbially mediated processes within the soil environment. Thus, soil enzyme activities have been interpreted as indirect measures of microbial biomass, rhizosphere effects, soil productivity, and mineralization potential of naturally occurring substrates or xenobiotics (4). However, few studies have attempted to correlate soil enzyme activities with the presence and activities of specific components of the microbial community. The ability of soil-inhabiting fungi to produce a range of enzymes capable of degrading complex litter substances could make the use of an enzymatic approach to study soil fungal populations possible. These enzymes must be specific for fungal presence and activity. In one study of chitinase in soil (24), chitinase activity and the number of fungal propagules in chitin-amended soils were strongly correlated. The same correlation was not found for actinomycetes or bacteria. Thus, chitinase activity appears to be a suitable indicator of actively growing fungi in the soil. The hydrolysis of cellulose requires the interaction of a number of hydrolases produced by cellulolytic microorganisms. A major role is played by the cellulase system, which consists of several distinct enzymes that are produced by a large number of microorganisms, including fungi, actinomycetes, and bacteria. However, fungi have been suggested as the predominant source of β-d-glucosidase (EC 3.2.1.21) (16, 17) and endo 1,4-β-glucanase (EC 3.2.1.4) (23) activity in soils.Fluorogenic 4-methylumbelliferyl (MUF)-labelled enzyme substrates have been introduced for process-oriented studies in aquatic systems (3, 18) and, more recently, in peatlands (11). MUF substrates have been used to assay cell-bound activities in pure cultures of fungi, as the soluble substrate can enter the cell wall, making periplasmic enzyme activity detectable (15). These substrates have been used to detect fungal chitinolytic activities (17a) and cellulases (6) in vitro. The substrates may be added to environmental samples and, when hydrolyzed, release 4-methyl-umbelliferone (4-MU), which fluoresces and can be quantified in nanomolar concentrations (3).A variety of methods to quantify fungi in soil have been described. The techniques include direct microscopic observation and extraction of fungus-specific indicator molecules such as glucosamine or ergosterol (9). More recently, the phospholipid fatty acid (PLFA) 18:2ω6 has been proposed as an indicator of fungal biomass (7, 12). Our objectives in the present study were to determine if (i) components of chitinase and cellulase activities could be used as indicators of the presence and activity of fungal biomass and (ii) enzyme activities detected with specific MUF substrates in soil samples were correlated with the content of the fungus-specific indicator molecules 18:2ω6 PLFA and ergosterol.     

Influence of Biochar on Nitrogen Use Efficiency and Root Morphology of Rice-Seedling in Two Contrasting Paddy Soils

Biochar may affect the root morphology and nitrogen (N) use efficiency (NUE) of rice at seedling stage, which has not been clearly verified until now. To clarify it, we conducted a pot experiment regarding to two soil types (Hydragric Anthrosol and Haplic Acrisol), two biochar application rates (0.5 wt% and 1.5 wt %) and two rice varieties (common rice var. Xiushui134 and hybrid super rice var. Zhongkejiayou12-6) meanwhile. Seedling NUE of common rice Xiuhui134 was significantly increased (p < 0.05) by 78.2% in Hydragric Anthrosol and by 91.4% in Haplic Acrisol following biochar addition with 1.5 wt%. However, biochar addition exerted no influence on seedling NUE of super rice Zhongkejiayou12-6 in both soils. Overall, 0.09–0.10 units higher soil pH and 105– 116% higher soil NH₄⁺ -N were observed in Xiushui134 growing two soils with 1.5 wt% biochar. In addition, improved root morphology (including longer root length, larger root surface area, bigger root volume, and more root tips) contributed to the higher seedling NUE of Xiushui134 in two soils. The soil pH and NH₄⁺ -N content, also the root morphology were influenced by biochar, which though could not thoroughly explained the NUE of Zhongkejiayou12-6. In conclusion, biochar application to paddy soil changed soil pH and NH₄⁺ -N content, root growth, and the consequent seedling NUE of rice, which effects are relative with rice cultivar, biochar addition rate, and soil type.     

Microcosm Enrichment of Biphenyl-Degrading Microbial Communities from Soils and Sediments

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 × 10⁹ and 2 × 10¹¹ CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The R. opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R. opacus, independent of the pollution history of the original sample. R. opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.