Ronald F. Turco

The use of carbon substrate utilization patterns in environmental and ecological microbiology

A bstractCarbon substrate utilization patterns have found increasing use in environmental and ecological microbiology over the past five years. Ninety six-well microtiter plates containing various carbon substrates permit these patterns to be determined quickly, economically, and effectively. The use of these patterns to characterize and differentiate strains isolated from the environment has been very effective in providing information on the culturable fraction of the microbial community. Another approach involves the direct inoculation of natural samples into these microtiter plates; this approach has several fundamental problems. The inoculation of low cell densities into the wells means that the technique is a culture-based method in which the biases of enrichment culture may render the results unrepresentative of the native microbiota. The physiological state of the inoculated microbes may affect the kinetics and pattern of substrate utilization. As a measure of the functional diversity of microbial communities, this approach suffers because the tested substrates do not accurately represent the types of substrates present in ecosystems, and the metabolic redundancy of species implies that changes in the response may only crudely represent the actual microbial population dynamics. Therefore, although this approach can be used to determine whether environmental samples differ in their response patterns, it is unclear how it can be used to provide fundamental information on questions of microbial diversity.

Surface and subsurface microbial biomass, community structure and metabolic activity as a function of soil depth and season

Microbial biomass, size and community structure along with an estimate of microbial activity and soil chemical parameters were determined at three depths in two soils (e.g. sandy loam Ultic Hapludalf and silt loam Mollic Hapludalf ) replicated three times under one winter and summer season. Microbial biomass and community structure were estimated from phospholipid-PO4 content and fatty acid methyl ester (FAME) measurements. Microbial activity and assimilative capacity were estimated using a 3 H-acetate incorporation into phospholipids and by incubating the soil samples at the average winter and summer temperatures, 3 and 20 ◦ C, respectively. We found that the size of the microbial biomass in both the surface and the subsurface soils was not significantly affected by the seasonal variation but activity increased by as much as 83% at the summer temperatures in the surface soil. We demonstrated using FAME analysis that for both soils seasonal changes in the subsurface microbial community occurred. These findings suggest that winter conditions will shift the population activity level in both the surface and subsurface systems and the biochemical structure of the community in the subsurface. In all cases, the inorganic chemical properties of the soil, as a function of season, remained constant. The greatly increased activity of microbial population at the higher temperature will favor the capacity of the system to utilize nutrients or organic materials that may enter soil. During low temperature seasons the capacity of either surface or subsurface soils to assimilate materials is generally diminished but the reduction reflects changes in metabolism and not a reduced biomass size.

The impact of chlorothalonil application on soil bacterial and fungal populations as assessed by denaturing gradient gel electrophoresis

Abstract The impact of the fungicide chlorothalonil on dominant bacterial and fungal populations following application to turfgrass, forest, and agricultural soils was investigated. Chlorothalonil was applied to each soil at three rates, representing 0.2, 1 and 5 times the recommended label rate for turfgrass, and incubated for a 2-week period. Changes to the microbial community caused by the chlorothalonil application were assessed following DNA extraction, PCR-amplification using both bacteria domain- and fungal-specific primers, then separation with denaturing gradient gel electrophoresis (DGGE). Digitized DGGE images were used to determine two parameters: the number of bands per lane and the Shannon–Wiener index of diversity ( H ′), both of which were used only for comparison of the different treatments, and not as true diversity measurements. Bands appearing to be either enhanced or inhibited as a result of the chlorothalonil treatment were excised and sequenced. Increased rates of chlorothalonil impacted eight bacterial populations (two inhibitions, four enhancements, and two non-specific responses) and four fungal populations (all inhibitions). Band number and H ′ indicated an altered but not significantly different ( P Cytophaga–Flavobacterium–Bacteroides , α-, β-, γ-, and δ-proteobacteria) and two fungal taxa (zygomycota and ascomycota). Although changes to the overall community structure of dominant species were non-significant, we conclude that following a single chlorothalonil application and a short incubation period, community changes including both enhancement and inhibition of a variety of dominant organisms can occur.

Association of Microbial Community Composition and Activity with Lead, Chromium, and Hydrocarbon Contamination

ABSTRACT Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil−1, 3 to 3,300 mg of total Cr kg of soil−1, and 1 to 17,100 mg of Pb kg of soil−1. Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [3H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC50 values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO42− and 0.01 mM for Pb2+. Much higher levels of Pb were required to inhibit [14C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.

Microbial community analysis of soils contaminated with lead, chromium and petroleum hydrocarbons.

The impact on the microbial community of long-term environmental exposure to metal and organic contamination was investigated. Twenty-four soil samples were collected along a transect dug in soils contaminated with road paint and paint solvents, mainly toluene. Chemical analysis along the transect revealed a range from high to low concentrations of metals (lead and chromium) and organic solvent compounds. Principal components analysis of microbial community structure based on denaturing gradient gel electrophoresis of the V3 region of the 16S rRNA gene and fatty acid methyl esters derived from phospholipids (phospholipid fatty acid analysis) showing samples with similar fingerprints also had similar contaminant concentrations. There was also a weak positive correlation between microbial biomass and the organic carbon concentration. Results indicated that microbial populations are present despite some extreme contaminant levels in this mixed-waste contaminated site. Nucleotide sequence determination of the 16S rRNA gene indicated the presence of phylogenetically diverse bacteria belonging to the α-, β-, γ-, and δ-Proteobacteria, the high and low G + C Gram-positive bacteria, green nonsulfur, OP8, and others that did not group within a described division. This indicates that soils contaminated with both heavy metals and hydrocarbons for several decades have undergone changes in community composition, but still contain a phylogenetically diverse group of bacteria (including novel phylotypes) that warrant further investigation.

Accelerated biodegradation of atrazine by a microbial consortium is possible in culture and soil

A mixed enrichment culture of microorganisms capable of accelerated mineralization of atrazine was isolated from soil treated with successive applications of the herbicide. Liquid cultures of this consortium, in the presence of simple carbon sources, mineralized 96% of the applied atrazine (0.56 mM) within 7 days. Atrazine mineralization in culture is initiated with the formation of the metabolite hydroxyatrazine. In soil treated with atrazine at a concentration of 0.14 mM (concentration is based on total soil mass), and then inoculated with the microbial consortium, the parent compound was completely transformed in 25 days. After 30 days of incubation, 60% of the applied atrazine was accounted for as14CO2. As was found with the liquid cultures, hydroxyatrazine was the major metabolite. After 145 days, soil extractable hydroxyatrazine declined to zero and 86% of the applied atrazine was accounted for as14CO2. No metabolites, other than hydroxyatrazine, were recovered from either the liquid culture or soil inoculated with the consortium. The use of the mixed microbial culture enhanced mineralization more than 20 fold as compared to uninoculated soil.

Long-term effects of chromium and lead upon the activity of soil microbial communities

The inhibitory effects of heavy metals upon microbial populations and the factors limiting microbial activity and growth were determined in soils that had been contaminated with lead (Pb) or chromium (Cr) for more than 40 years. Total Cr and Pb concentrations were 260,000 and 10,000 mg kg −1 soil in Cr- and Pb-contaminated soils, respectively. The tolerance of bacteria extracted from soil particles to Pb or Cr was assayed by measuring the incorporation of 3 H -leucine into macromolecules at a series of CrO4 2− or Pb 2+ concentrations. IC50 (heavy-metal concentration giving 50% reduction of microbial 3 H -leucine incorporation compared to the control) for the two metals was similar in the two soils, despite their differences in metal contamination; the IC50 values were 4 and 0.02 mM for CrO4 2− and Pb 2+ , respectively. Stimulation of microbial activity and biomass by organic C was measured in microcosms over 56 days. The adverse effects of Cr and Pb were characterized in terms of the ratio of microbial biomass C to soil organic C, basal respiration per unit microbial biomass (qCO2), and the ratio of substrate-responsive respiration to microbial biomass C (substrate-responsive qCO2). Ratios of microbial biomass C to soil organic C were small: 0.42% in Cr-contaminated soil and 0.36% in Pb-contaminated soil. Values of qCO2 and substrate-responsive qCO2 were significantly higher in Pb- than in Cr-contaminated soils ( P< 0.01). The microcosm results indicate that Cr and Pb decreased microbial activities and led to the accumulation of soil organic C, and that Pb posed greater stress to soil microbes than Cr.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Contribution of soil-borne bacteria to the rotation effect in corn

Few efforts have been directed at understanding how the rhizosphere microbiology of continuous corn may effect crop yields. This relationship may explain, in part, the decreases in yield associated with continuous corn as compared to the corn in rotation with a second crop. This study was conducted to determine the importance of soil-borne microorganisms to yield declines in long term continuous corn. Continuous corn (Zea mays L.) or rotated corn-soybean (Glycine max L.) field plots, established in 1975, under either fall plowing or no-till tillage treatments were used. Treatments consisted of methyl bromide applied at 48.8 g m−2 3 days prior to planting in all four combinations. Total plant samples from both the fumigated and non-fumigated areas were collected 14 days after planting. Rhizosphere bacteria were recovered and tested for their ability to impact plant growth. Bacterial assessments were made in a test tube bioassay where germinated corn was transported in to agar containing a bacterial isolate. In the first year of the study a highly significant interaction of fumigation and rotation was indicated. With fumigation continuous corn yields were similar to that of rotated corn-bean. Rotated corn yields were less affected by fumigation. In the second year, the effects were similar but less significant. Over 130 bacterial isolates were tested for their effect on plant growth. Approximately 22% were able to inhibit plant growth. Of these, 72% were from the continuous corn system. Clearly, the interaction of rotation and yield is at a microbiological level. The suggestion that microorganisms similar to those isolated are responsible for controlling early plant growth in the continuous corn system is indicated.

Response of Soil Microorganisms to As-Produced and Functionalized Single-Wall Carbon Nanotubes (SWNTs)

The use of single-wall carbon nanotubes (SWNTs) in manufacturing and biomedical applications is increasing at a rapid rate; however data on the effects of a potential environmental release of the materials remain sparse. In this study, soils with either low or high organic matter contents as well as pure cultures of E. coli are challenged with either raw as-produced SWNTs (AP-SWNTs) or SWNTs functionalized with either polyethyleneglycol (PEG-SWNTs) or m-polyaminobenzene sulfonic acid (PABS-SWNTs). To mimic chronic exposure, the soil systems were challenged weekly for six weeks; microbial activities and community structures for both the prokaryote and eukaryote community were evaluated. Results show that repeated applications of AP-SWNTs can affect microbial community structures and induce minor changes in soil metabolic activity in the low organic matter systems. Toxicity of the three types of SWNTs was also assessed in liquid cultures using a bioluminescent E. coli-O157:H7 strain. Although decreases in light were detected in all treated samples, low light recovery following glucose addition in AP-SWNTs treatment and light absorption property of SWNTs particles suggest that AP-SWNTs suppressed metabolic activity of the E. coli, whereas the two functionalized SWNTs are less toxic. The metals released from the raw forms of SWNTs would not play a role in the effects seen in soil or the pure culture. We suggest that sorption to soil organic matter plays a controlling role in the soil microbiological responses to these nanomaterials.