Abstract: Atmospheric nitrogen that is fixed by associative cyanobacteria can be released into the surrounding soil environment providing a key source of N for arctic ecosystems. Yet, little is known about nitrogen fixation by Biological Soil Crusts (BSCs) within hummock-hollow complexes that are typical of many arctic environments. In this study, we examined spatial and temporal patterns in N-2-fixation, dinitrogenase reductase (nifH) gene abundance and release of N in a low arctic hummock-hollow ecosystem. The impacts of cyanobacteria on N status in soil were evaluated by assessing soil nitrogen in relation to the cyanobacterial associations found on Hummock and Hollow BSCs. In addition, potential P limitation of N-2-fixation by cyanobacteria was assessed for Hummock and Hollow BSCs. The tops of hummocks and the bottoms of hollows were areas of high N-2-fixation, whereas minimal N-2-fixation occurred on the sides of hummock-hollow complexes. Compared with Hummock BSCs, Hollow BSCs had a higher mean growing season N-2-fixation rate, a higher mean growing season nifH abundance, a higher mean total %N and delta N-15 values closer to that of atmospheric N-2. Soil N status was linked to rates of N-2-fixation by BSCs indicating that these N-2-fixing associations act as important point sources of soil N in this low arctic ecosystem. Over the course of a growing season temporal variation in N-2-fixation and nifH abundance were weakly linked suggesting that N-2-fixation was carried out by complex communities of diazotrophic microorganisms and that factors such as nutrient availability may limit N-2-fixation to a greater extent than nifH abundance. (C) 2010 Elsevier Ltd. All rights reserved.
Abstract: Nitrogen inputs via biological N-2-fixation are important in arctic environments where N often limits plant productivity. An understanding of the direct and indirect theoretical causal relationships between key intercorrelated variables that drive the process of N-2-fixation is essential to understanding N input. An exploratory multi-group Structural Equation Modeling (SEM) approach was used to examine the direct and indirect effects of soil moisture, plant community functional composition, and bryophyte and lichen abundance on rates of nitrogen fixation at a low arctic ecosystem, two high arctic oases and a high arctic polar desert in the Canadian Arctic. Increasing soil moisture was strongly associated with an increasing presence of bryophytes and increasing bryophyte abundance was a major factor determining higher N-2-fixation rates at all sites. Shrubs had a negative effect on bryophyte abundance at all sites with the exception of the polar desert site at Alexandra Fjord highland. The importance of competition from vascular plants appears to be greater in more productive sites and may increase at lower latitudes. Moisture availability may have an indirect effect on ecosystem development by affecting N input into the system with bryophyte-cyanobacterial associations playing an important intermediary role in the process.
Abstract: Understanding the links between plant diversity and soil communities is critical to disentangling the mechanisms by which plant communities modulate ecosystem function. Experimental plant communities varying in species richness, evenness, and density were established using a response surface design and soil community properties including bacterial and archaeal abundance, richness, and evenness were measured. The potential to perform a representative soil ecosystem function, oxidation of ammonium to nitrite, was measured via archaeal and bacterial amoA genes. Structural equation modeling was used to explore the direct and indirect effects of the plant community on soil diversity and potential function. Plant communities influenced archaea and bacteria via different pathways. Species richness and evenness had significant direct effects on soil microbial community structure, but the mechanisms driving these effects did not include either root biomass or the pools of carbon and nitrogen available to the soil microbial community. Species richness had direct positive effects on archaeal amoA prevalence, but only indirect impacts on bacterial communities through modulation of plant evenness. Increased plant evenness increased bacterial abundance which in turn increased bacterial amoA abundance. These results suggest that plant community evenness may have a strong impact on some aspects of soil ecosystem function. We show that a more even plant community increased bacterial abundance, which then increased the potential for bacterial nitrification. A more even plant community also increased total dissolved nitrogen in the soil, which decreased the potential for archaeal nitrification. The role of plant evenness in structuring the soil community suggests mechanisms including complementarity in root exudate profiles or root foraging patterns.
Abstract: Site-specific risk assessments often incorporate the concepts of bioaccessibility (i.e., contaminant fraction released into gastrointestinal fluids) or bioavailability (i.e., contaminant fraction absorbed into systemic circulation) into the calculation of ingestion exposure. We evaluated total and bioaccessible metal concentrations for 19 soil samples under simulated stomach and duodenal conditions using an in vitro gastrointestinal model. We demonstrated that the median bioaccessibility of 23 metals ranged between <1 and 41% under simulated stomach conditions and <1 and 63% under simulated duodenal conditions. Notably, these large differences in metal bioaccessibility were independent of equilibrium solubility and stability constants. Instead, the relationship (stomach phase R = 0.927; duodenum phase R = 0.891) between bioaccessibility and water exchange rates of metal cations (kappa(H2O)) indicated that desorption kinetics may influence if not control metal bioaccessibility.
Abstract: New technologies in trace gas detection are revolutionizing our ability to study soil microbiological ecosystems. Field-deployable infrared-spectroscopy detectors capable of rapidly measuring multiple analyte gases simultaneously allow estimates of soil:atmosphere gas exchange and below-ground gas concentrations, and production dynamics across divergent ecosystems, creating opportunities to study interactions between microorganisms, soils, atmospheres, and global cycling, as well as interactions between different gases. The greenhouse gases CO2, CH4, and N2O can be measured in the field and compared to each other to uncover links between the biochemical pathways responsible for the production and consumption of these gases. We have developed techniques using a nondestructive, Fourier-transform infrared detector under remote field conditions in three campaigns in the Canadian High Arctic to measure highly variable gas processes in soils.
Abstract: In vitro digestors can be used to provide bioaccessibility values to help assess the risk from incidental human ingestion of contaminated soils. It has been suggested that these digestors may need to include a lipid sink to mimic human uptake processes. We compare the correspondence between in vivo polycydic aromatic hydrocarbon (PAR) uptake for eight different PAH contaminated soils with PAR release in in vitro digestors in the presence and absence of a lipid sink. Lipid sinks were essential to the success of the in vitro digestors in predicting juvenile swine PAH uptake, In the presence of the lipid sink, results of the In Vitro Digestion model (IVD) closely corresponded with a slope of 0.85 (r(2) = 0.45, P < 0.07) to the in vivo results. The Relative Bioaccessibility Leaching Procedure (RBALP) results did not correspond to the in vivo study but did tightly reflect total soil PAR concentration. We conclude that the basis of this difference between digestors is that the RBALP used an aggressive extraction technique that maximized PAR release from soil. Systemic uptake in juvenile swine was not linked to soil PAH concentration but rather to the thermodynamic properties of the soil.
Abstract: The health risk associated with exposure to urban brownfields is often driven by the incidental ingestion of soil by humans. Recent evidence found that humans likely ingest the fraction of soil that passes a 45-mu m sieve, which is the particle size adhered to the hands. We evaluated if PAH concentrations were enriched in this soil fraction compared to the bulk soil and if this enrichment lead to an increase in bio-accessability and thus an increase in incremental lifetime cancer risk for exposed persons. Soils (n = 18) with PAH concentrations below the current Canadian soil quality guidelines for human health were collected from an Arctic urban site and were sieved to pass a 45-mu m sieve. Soil PAH profiles were measured and bioaccessibility was assessed using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). PAHs were significantly enriched in the <45 mu m size fraction (3.7-fold) and this enrichment could be predicted according to the fugacity capacity of soil (Enrichment = 2.18-0.055Z(soil), r(2) = 0.65, p < 0.001). PAH release in the stomach and small intestine compartments of the SHIME was low (8%) and could not be predicted by PAH concentrations in 45-mu m sieved soil. In fact, PAH release in the SHIME was lower from the <45 mu m size fraction despite the fact that this fraction had higher levels of PAHs than the bulk soil. We postulate that this occurs because PAHs adsorbed to soil did not reach equilibrium with the small intestinal fluid. In contrast, PAH release in the colonic compartment of the SHIME reached equilibrium and was linked to soil concentration. Bioaccessibility in the SHIME colon could be predicted by the ratio of fugacity capacity of soil to water for a PAH (Bioaccessibilty = 0.15e((-6.4 x 10E-7) (Z-soil/Zwater)), r(2) = 0.53, p < 0.01). The estimated incremental lifetime cancer risk was significantly greater for the <45 mu m soil fraction compared to the bulk fraction: however, when bioaccessible PAH concentrations in a simulated small intestine were used in the risk assessment calculations, cancer risk was slightly lower in the <45 mu m soil fraction for these soils. Our results highlight the importance of using a small soil size fraction, e.g. 45 mu m, for contaminated site human health risk assessment. However, further work is needed to estimate the bioavailability of this size fraction in an in vivo model and to assess the correlation between in vitro and in vivo gastrointestinal models. (C) 2010 Elsevier Ltd. All rights reserved.
Abstract: Few soil invertebrate species are available for the toxic assessment of soils from boreal or other northern ecozones, yet these soils cover the majority of Canada's landmass as well as significant portions of Eurasia. Oppia Miens (C.L. Koch) is an herbivorous and fungivorous oribatid mite found in soil throughout Holarctic regions, including Canada. Soil tests using O. nitens were performed using 15 different forest soil types and horizons to investigate test variability in adult survival and reproduction. Adult survival (86.1 +/- 1.1%) was consistent across soil types, with a coefficient of variation (CV) of 15%. However, reproduction varied significantly, ranging from 2.9 (+/-1.1) to 86.2 (+/-11.7) individuals, with a corresponding CV of 118 and 30%, respectively. Of the soil factors assessed (NH3, NO3, pH, phosphorus [P], organic matter content (OM), carbon:nitrogen (C:N), sand, silt, clay, and sodium adsorption ratio), soil organic matter (OM) explained 68% of the variation observed for reproduction. Increasing the OM using Sphagnum sp. peat moss resulted in optimal reproduction at 7% OM (8% peat content) with the lowest variability (CV of 20%). When assessing the toxicity of a reference chemical, boric acid, the effect of peat amendment reduced lethality to adults with no observable difference on reproduction. The use an age-synchronized culture reduced the test variability for reproduction relative to the use of unsynchronized cultures. Oppia linens is a good candidate species for a standardized test design, with adult survival easily assessed in a relatively simple design. A long-term reproduction test with O. nitens will require the use of a synchronized population and, on occasion, OM amendment when testing soils with low organic matter content. Environ. Toxicol. Chem. 2010;29:971-979. (C) 2009 SETAC
Abstract: This study examined the effect of water filled pore space (WFPS) on gross N fluxes and community structure and abundance of ammonia oxidizing archaea and bacteria in a semi-arid soil. Different WFPS altered the community structure of both AOA and AOB. Ammonia oxidizer communities (for both archaea and bacteria) from 'wet' soils (95, 85 and 75% WFPS) and 'dry' soils (25, 45 and 55% WFPS) were distinctly different from one another. Additionally there was a significant relationship between community structure and gross rates of nitrification. There was also a significant relationship between WFPS and bacterial amoA abundance but not archaeal amoA abundance suggesting that bacterial ammonia oxidizers are more responsive to changes in soil water availability. These results are in agreement with other studies suggesting that both groups of ammonia oxidizers have distinct physiological characteristics and ecological niches with consequences for nitrification in response to WFPS. Overall findings from this study indicate that nitrification, both in terms of process rates and populations responsible for nitrification activity, is highly responsive to soil water availability. (C) 2010 Elsevier Ltd. All rights reserved.
Abstract: Percent arsenic bioaccessibility is occasionally dependent upon arsenic concentration; however, the mechanism(s) of this relationship has not yet been defined. To evaluate the mechanism of this relationship, the arsenic bioaccessibility from freshly synthesized poorly crystalline scorodite was measured in the stomach, small intestine, and colon stages of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The shape of the arsenic dissolution isotherms were different between stages (stomach: linear; small intestine: exponential rise to maxima; colon: sigmoidal). These results indicate that arsenic bioaccessibility may be limited by either in vitro GI fluid saturation or in vitro GI model residence time, depending upon the chemical/microbiological conditions of the model. Gastrointestinal microorganisms increased arsenic bioaccessibility of scorodite up to two-fold in the SHIME colon; however, this was dependent upon the sample arsenic concentration. Up to 40% of the bioaccessible arsenic was reduced to arsenite; however this process was neither mediated by GI microorganisms nor associated with increased arsenic bioaccessibility.
Abstract: Fuel spills in Antarctica typically occur in rare ice-free oases along the coast, which are areas of extreme seasonal freezing. Spills often occur at subzero temperatures, but little is known of ecosystem sensitivity to pollutants, in particular the influence that soil liquid water and low temperature have on toxicity of petroleum hydrocarbons (PHC) in Antarctic soil. To evaluate PHC toxicity, 32 locations at an aged diesel spill site in Antarctica were sampled nine times to encompass frozen, thaw, and refreeze periods. Toxicity was assessed using potential activities of substrate-induced respiration, basal respiration, nitrification, denitrification, and metabolic quotient as well as microbial community composition and bacterial biomass. The most sensitive indicator was community composition with a PHC concentration effecting 25% of the population (EC25) of 800 mg/kg, followed by nitrification (2,000 mg/kg), microbial biomass (2,400 mg/kg), and soil respiration (3,500 mg/kg). Despite changes in potential microbial activities and composition over the frozen, thaw, and refreeze period, the sensitivity of these endpoints to PHC did not change with liquid water or temperature. However, the variability associated with ecotoxicity data increased at low liquid water contents. As a consequence of this variability, highly replicated (n = 50) experiments are needed to quantify a 25% ecological impairment by PHCs in Antarctic soils at a 95% level of significance. Increases in biomass and respiration associated with changes in community composition suggest that PHC contamination in Antarctic soils may have irrevocable effects on the ecosystem.
Abstract: Mercury has been found in polar bears and other top predators in the Arctic at concentrations that pose a risk to the indigenous population, however, the means by which this occurs is uncertain. There has been extensive research on the atmospheric cycling of mercury but little is known about mercury cycling in Arctic terrestrial ecosystems. The objective of this study was to determine whether wet sedge meadow soils within the Truelove Lowlands, Devon Island, NT, Canada (75 degrees 33'N, 84 degrees 40'N) were acting as sources or sinks for methylmercury (MeHg). Over the course of an Arctic summer, MeHg concentrations and other biophysical characteristics were measured at four wet sedge meadows over a 19 day study period that commenced approximately 1 month after snowmelt. Soil MeHg concentrations declined during the study period, indicating a net loss of MeHg over the summer. The dominant ligand in solution appeared to be dissolved organic matter, little sulfide was detected, and it would seem that most of the mercury was unavailable for methylation during the summer sampling period. In soil microcosms, spiked with 5.0 nmol g(-1) (1 mu g g(-1)) HgCl2, the soil did methylate mercury suggesting that there is the potential for mercury methylation. We also noted significant spatial variability in MeHg concentrations between catenas that could not be explained by other biophysical parameters, which are known to affect methylation. Given our data and previous geochemical data collected from suprapermafrost groundwater during snowmelt, it seems likely that methylation may occur during the spring melt period in the arctic. Furthermore the geochemical variability of the melt water may lead to the spatial variability observed in MeHg concentrations in this study. (C) 2008 Elsevier B.V. All rights reserved.
Abstract: Soil physicochemical characteristics and contamination levels alter the bioavailability of metals to terrestrial invertebrates. Current laboratory-derived benchmark concentrations used to estimate risk do not take into account site-specific conditions, such as contaminant sequestration, and site-specific risk assessment requires a battery of time-consuming and costly toxicity tests. The development of an in vitro simulator for earthworm bioaccessibility would significantly shorten analytical time and enable site managers to focus on areas of greatest concern. The simulated earthworm gut (SEG) was developed to measure the bioaccessibility of metals in soil to earthworms by mimicking the gastrointestinal fluid composition of earthworms. Three formulations of the SEG (enzymes, microbial culture, enzymes and microbial culture) were developed and used to digest field soils from a former industrial site with varying physicochemical characteristics and contamination levels. Formulations containing enzymes released between two to 10 times more arsenic, copper, and zinc from contaminated soils compared with control and 0.01 M CaCl2 extractions. Metal concentrations in extracts from SEG formulation with microbial culture alone were not different from values for chemical extractions. The mechanism for greater bioaccessible metal concentrations from enzyme-treated soils is uncertain, but it is postulated that enzymatic digestion of soil organic matter might release sequestered metal. The relevance of these SEG results will need validation through further comparison and correlation with bioaccumulation tests, alternative chemical extraction tests, and a battery of chronic toxicity tests with invertebrates and plants.
Abstract: Arctic soils emit nitrous oxide, which is a potent greenhouse gas and also represents an important loss of nitrogen to oligotrophic Arctic ecosystems. However, little is known about the temperature sensitivity of nitrous oxide release in Arctic soils or the organisms mainly responsible for it. We investigated controls on nitrous oxide emissions in an Arctic soil across a typical temperature range (between 4 and 13 degrees C) on Truelove Lowland, Devon Island, Canada (75 degrees 40'N 84 degrees 35'W) at two different moisture contents. When fertilized with ammonia or nitrate, nitrous oxide emissions and temperature dependence of nitrous oxide emissions were insensitive to soil moisture content but linked to nitrification rates. Stable isotope analysis revealed that nitrous oxide was predominantly released by nitrifiers. However, nitrous oxide emissions were not linked to nitrifier prevalence with an insignificant (P < 0.219) increase in amoA genes and a (P < 0.01) decrease in archaeal nitrifiers. In contrast, denitrifier nosZ prevalence was 10,000 times greater than that of nitrifiers and was related to nitrous oxide emission potential when soils were fertilized with nitrate. Manipulating water-filled pore space should have changed the pattern of N2O emissions. We used selective inhibitors to further explore why denitrification did not occur under field conditions when we manipulated water-filled pore space or when we used N-15 analysis. When fungi were inhibited in the soil, nitrous oxide emissions from denitrifiers increased with no change in nitrous oxide released by nitrifiers. When fungi were active in the soil, there was little available nitrate but when fungi were inhibited, available soil nitrate increased over the incubation period. The dominance of nitrifiers in nitrous oxide emissions from Arctic soils under field conditions is linked to the competition for nitrate between fungi and denitrifiers. (C) 2009 Elsevier Ltd. All rights reserved.
Abstract: Human exposure to contaminated soils drives clean up criteria at many urban brownfields, Current risk assessment guidelines assume that humans ingest some fraction of soil smaller than 4 mm but have no estimates of what fraction of soil is ingested by humans. Here, we evaluated soil adherence to human hands for 13 agricultural soils from Saskatchewan, Canada and 17 different soils from a brownfield located in Iqaluit, Nunavut, Canada. In addition, we estimated average particle size adhering to human hands for residents of a northern urban setting. Further, we estimated how metal concentrations differed between the adhered and bulk (<4 mm) fraction of soil. The average particle size for adhered agricultural soils was 34 mu m, adhered brownfield soils was 105 mu m, and particles adhered to human residents was 36 mu m. Metals were significantly enriched in these adhered fractions with an average enrichment [(adheredbulk)/bulk] in metal concentration of 184% (113% median) for 24 different elements. Enrichment was greater for key toxicological elements of concern such as chromium (140%), copper (140%), nickel (130%), lead (110%), and zinc (130%) and was highest for silver (810%), mercury (630%), selenium (500%), and arsenic (420%). Enrichment were positively correlated with carbonate complexation constants (but not bulk solubility products) and suggests that the dominant mechanism controlling metal enrichment in these samples is a precipitation of carbonate surfaces that subsequently adsorb metals. Our results suggest that metals of toxicological concern are selectively enriched in the fraction of soil that humans incidentally ingest. Investigators should likely process soil samples through a 45 mu m sieve before estimating the risk associated with contaminated soils to humans. The chemical mechanisms resulting in metal enrichment likely differ between sites but at our site were linked to surface complexation with carbonates.
Abstract: In vitro gastrointestinal models, used to measure the metal(loid) bioaccessibility for site specific risk assessment are typically operated under fasted conditions. We evaluated the hypothesis that fed conditions increase arsenic bioaccessibility on three reference soils (NIST 2711, NIST 2709, and BGS 102) and the bulk and <38 mu m size fractions of a mine tailing. The three nutritional states included a fed state with a carbohydrate mixture, a second fed state with homogenized crowberries (Empetrum nigrum), and a fasted state. The carbohydrate mixture increased arsenic bioaccessibility from four of five samples in the simulator of the human intestinal microbial ecosystem (SHIME) stomach but only three of five samples in the SHIME small intestine and colon. In contrast crowberries increased arsenic bioaccessibility from four of five samples in the SHIME small intestine but had variable affects in the SHIME stomach and colon. The effect of nutritional status on arsenic bioaccessibility was potentially mediated via ligand-promoted dissolution in the SHIME stomach and small intestne. The displacement of arsenic with phosphate was potentially present in the SHIME small intestine but not the SHIME stomach. Microbial activity increased arsenic bioaccessibility relative to sterile conditions from four of five samples under fasted conditions and three of the five samples under fed conditions, which may suggest that in vitro gastrointestinal (GI)models operated underfed conditions and with microbes provide a more conservative estimate of in vitro bioaccessibility. However, for some samples, the arsenic bioaccessibility in the SHIME colon (with microbial activity) was equivalent to values observed in a separate physiologically based extraction test under small intestinal conditions (without microbial activity). These results suggest that the incorporation of microbial activity into in vitro GI models does not necessarily make estimates of arsenic bioaccessibility more protective than those generated using in vitro models that do not include microbial activity.
Abstract: Human health risk assessment of dietary mercury (Hg) exposure in Canada assumes that all Hg from fish consumption is in the form of methylmercury (MeHg), the more bioavailable and hazardous form of Hg. In contrast, the risk assessment of dietary Hg to Inuit in northern Canada assumes that no more than two-thirds of dietary Hg is MeHg since mammal organs consumed by Inuit contain substantial concentrations of inorganic Hg. In vitro gastrointestinal models (e.g., the Simulator of the Human Intestinal Microbial Ecosystem) are often used for the evaluation of soil contaminant bioaccessibility, i.e., the fraction solubilized into gastrointestinal fluids, for use in site-specific human health risk assessment. In this research, we digested northern country foods using the SHIME for the measurement of Hg bioaccessibility, a novel approach for the assessment of dietary Hg bioavailability. We demonstrated that small intestinal Hg bioaccessibility from 16 fish. wild game, and marine mammal samples consumed by Inuit in northern Canada ranged between 1 and 93% and was independent of food HgT (MeHg + Hg-II) concentration. Additionally, we demonstrated that gastrointestinal microbes may affect Hg bioaccessibility of the 16 country foods, either increasing or decreasing bioaccessibility depending upon the type of food. These results indicate that gastrointestinal absorption of Hg is not likely limited by the concentration of Hg in the food, which is in agreement with in vivo Hg bioavailability studies. Furthermore, these in vitro results support the hypothesis that the gastrointestinal absorption of Hg from Inuit country foods is dependent upon food type. (C) 2009 Elsevier B.V. All rights reserved.
Abstract: Industrial and human activities in the Arctic regions may pose a risk to terrestrial Arctic ecosystem functions. One of the most common terrestrial toxicological end points, primary productivity, typically is assessed using a plant phytotoxicity test. Because of cryoturbation, a soil mixing process common in polar regions, we hypothesized that phytotoxicity test results in Arctic soils would be highly variable compared to other terrestrial ecosystems. The variability associated with phytotoxicity tests was evaluated using Environment Canada's standardized plant toxicity test in three cryoturbated soils from Canada's Arctic exposed to a reference toxicant, boric acid. Northern wheatgrass (Elymus lanceolatus) not only was more sensitive to toxicants in Arctic soils, its response to toxicants was more variable compared to that in temperate soils. The phytotoxicity of boric acid in cryosols was much greater than commonly reported in other soils, with a boric acid concentration of less than 150 mu g/g soil needed to inhibit root and shoot growth by 20%. Large variability also was found in the phytotoxicity test results, with coefficients of variation for 10 samples ranging from 160 to 79%. The increased toxicity of boric acid in cryosols and variability in test response was not explained by soil properties. Based on our admittedly limited data set of three different Arctic soils, we recommend that more than 30 samples be taken from each control and potentially impacted area to accurately assess contaminant effects at sites in northern Canada. Such intensive sampling will insure that false-negative results for toxicant impacts in Arctic soils are minimized.
Abstract: Amnionium nitrate (NH4NO3) is a common water pollutant associated with many industrial and municipal activities. One solution to reduce exposure of sensitive aquatic systems to nitrogenous compounds is to atomize (atmospherically disperse in fine particles) contaminated water over the Arctic tundra, which will reduce nitrogen loading to surface water. The toxicity of arturionium nitrate to Arctic soils, however, is poorly understood. In the present study, we characterized the biogeochemical toxicity and phytotoxicity of ammonium nitrate solutions in four different Arctic soils and in a temperate soil. Soil was exposed to a range of ammonium nitrate concentrations over a 90-d period. Dose responses of carbon mineralization, nitrification, and phytotoxicity endpoints were estimated. In addition to direct toxicity, the effect of ammonium nitrate on ecosystem resilience was investigated by dosing nitrogen-impacted soils with boric acid. Ammonium nitrate had no effect on carbon mineralization activity and only affected nitrification in one soil, a polar desert soil from Cornwallis Island, Northwest Territories, Canada. In contrast, ammonium nitrate applications (43 mmol N/L soil water) significantly impaired seedling emergence, root length, and shoot length of northern wheatgrass (Elymus lanceolatus). Concentrations of ammonium nitrate in soil water that inhibited plant parameters by 20% varied between 43 and 280 mmol N/L soil water, which corresponds to 2,100 to 15,801 mg/L of ammonium nitrate in the application water. Arctic soils were more resistant to ammonium nitrate toxicity compared with the temperate soil under these study conditions. It is not clear, however, if this represents a general trend for all polar soils, and because nitrogen is an essential macronutrient, nitrogenous toxicity likely should be considered as a special case for soil toxicity.
Abstract: The link between differences in the community composition of nitrifiers and denitrifiers to differences in the emission of nitrous oxide (N2O) from soils remains unclear. Nitrifier and denitrifier community composition, abundance and N2O emission activity were determined for two common landscapes characteristic of the North American "prairie pothole region": cultivated wetlands (CW) vs. uncultivated wetlands (UW). The hypotheses of this study were: (1) landscape selects for different nitrifier and denitrifier communities, (2) denitrification was the dominant N2O emitting process, and (3) a relationship exists between nitrifier and denitrifier community composition, their abundance, and N2O emission. Comparisons were made among soils from three CW and three UW at the St. Denis National Wildlife Area. Denaturing gradient gel electrophoresis was used to compare community composition, and quantitative polymerase chain reaction was used to estimate community size. Incubation experiments on re-packed soil cores with N-15-labeled nitrate were performed to assess the relative contributions of nitrification and denitrification to total N2O emission. Results indicate: (1) nitrification was the primary source of N2O emission, (2) cultivation increased nitrifier abundance but decreased nitrifier richness, (3) denitrifier abundance was not affected by cultivation but richness was increased by cultivation, and (4) differences in nitrifier and denitrifier communities composition and abundance between kind-use and landform did not correspond to differences in N2O emission. (C) 2007 Elsevier Ltd. All rights reserved.
Abstract: Fungal activity is a major driver in the global nitrogen cycle, and mounting evidence suggests that fungal denitrification activity contributes significantly to soil emissions of the greenhouse gas nitrous oxide (N2O). The metabolic pathway and oxygen requirement for fungal denitrification are different from those for bacterial denitrification. We hypothesized that the soil N2O emission from fungi is formate and O-2 dependent and that land use and landforms could influence the proportion of N2O coming from fungi. Using substrate-induced respiration inhibition under anaerobic and aerobic conditions in combination with N-15 gas analysis, we found that formate and hypoxia (versus anaerobiosis) were essential for the fungal reduction of N-15-labeled nitrate to (N2O)-N-15. As much as 65% of soil-emitted N2O was attributable to fungi; however, this was found only in soils from water-accumulating landforms. From these results, we hypothesize that plant root exudates could affect N2O production from fungi via the proposed formate-dependent pathway.
Abstract: We do not yet understand why fuel spills can cause greater damage in polar soils than in temperate soils, The role of water in the freezing environment may partly be responsible for why polar soils are more sensitive to pollution. We hypothesized that hydrocarbons alter the liquid water in frozen soil, and we evaluated this hypothesis by conducting laboratory and field experiments at Casey Station, Antarctica. Liquid water content in frozen Soils (theta(liquid)) was estimated by time domain reflectometry in laboratory, field collected soils, and in situ field measurements. Our results demonstrate an increase in liquid water associated with hydrocarbon contamination in frozen soils. The dependence of theta(liquid) on aged fuel and spiked fuel were almost identical, with a slope of 2.6 x 10(-6) mg TPH (total vertical bar petroleum hydrocarbons) kg(-1) for aged fuel and 3.1 x 10(-6) mg TPH kg(-1) for spiked fuel. In situ measurements found theta(liquid) depends, r(2) = 0.75, on fuel for silt loam Soils (theta(liquid) = 0.094 + 7.8 x 10(-6) mg TPH kg(-1)) but not on fuel for silt clay loam soils. In our study, theta(liquid) doubled infield soils and quadrupled in laboratory soils contaminated with diesel which may have profound implications on frost heave models in contaminated soils.
Abstract: Sub-Antarctic islands have been subjected to petroleum hydrocarbon spills, yet no information is available regarding the toxicity of petroleum hydrocarbons to these subpolar soils. The purpose of the present study was to identify soil biogeochemical toxicity end points for petroleum hydrocarbon contamination in sub-Antarctic soil. Soil from Macquarie Island, a sub-Antarctic island south of Australia, was collected and exposed to 10 concentrations of Special Antarctic Blend (SAB) diesel fuel, ranging from 0 to 50,000 mg fuel/kg soil, for a 21-d period. The sensitivity of nitrification, denitrification, carbohydrate utilization, and total soil respiration to SAB fuel was assessed. Potential nitrification activity was the most sensitive indicator of SAB contamination assessed for nitrogen cycling, with an IC20 (concentration that results in a 20% change from the control response) of 190 mg fuel/kg soil. Potential denitrification activity was not as sensitive to SAB contamination, with an IC20 of 950 mg fuel/kg soil for nitrous oxide production. Nitrous oxide consumption was unaffected by SAB contamination. Carbohydrate utilization (respiration caused by sucrose) was a more sensitive indicator (IC20, 16 mg fuel/kg soil) of SAB contamination than total respiration (IC20, 220 mg fuel/kg soil). However, total soil respiration was a more responsive measurement end point, increasing soil respiration over a 72-h period by 17 mg of CO2, compared to a change of only 2.1 mg of CO2 for carbohydrate utilization. Our results indicate that IC20s varied between 16 to 950 mg fuel/kg soil for Macquarie Island soil spiked with SAB diesel fuel. These results indicate that current cleanup levels derived from temperate zones may be too liberal for soil contamination in sub-Antarctic islands.
Abstract: Through their diet, herbivores inhabiting contaminated sites may be chronically exposed to a variety of aryl hydrocarbons (e.g., dioxins and polycyclic aromatic hydrocarbons [PAHs]). However, little is known about how differences in morphology and physiology among plant species alter the environmental accumulation of aryl hydrocarbons or their release and subsequent activity in the gastrointestinal tract of herbivores after ingestion. In the present study, the activity of aryl hydrocarbons during digestion was examined using six Arctic plant species growing in impacted and reference sites near Inuvik, Northwest Territories, Canada. The plant species studied were black spruce (Picea mariana), labrador tea (Ledum groenlandicum), bog birch (Betula glandulosa), green alder (Alnus crispa), water sedge (Carex aquatilis), and little-tree willow (Salix arbusculoides). Plants were digested using a simulator of the upper digestive tract, and aryl hydrocarbon release was evaluated using an aryl hydrocarbon-receptor assay. Bioaccessible aryl hydrocarbon activity varied among the plant species tested. The species with the greatest activity was green alder, and the species with the least activity was black spruce. Further investigation revealed that digested plant extracts may antagonize the aryl hydrocarbon receptor and prevent bioactivation of the aryl compound benzo[a]pyrene. Thus, PAH risk from the ingestion of vegetation varies among plant species and may depend on antagonists present in the vegetation.
Abstract: The contribution of nitrifiers (ammonia-oxidizing bacteria (AOB)) and denitrifiers to nitrous oxide (N2O) emission from arctic soils remains inconclusive. Based on preliminary experiments, we hypothesized that AOB are the primary producers of N2O in a high arctic lowland ecosystem on Devon Island, Nunavut, Canada. In part I of the Study, flux chambers were installed in a catena to determine in situ fluxes of gases (N2O and carbon dioxide (CO2)) from 16 June to 13 July 2004. Although fluxes were low, N2O production occurred in the wettest area of the landscape when ammonium levels were high. As ammonium, but not nitrate, levels declined in the wet sedge meadow, N2O emissions correspondingly decreased. In part 2, the contribution of nitrification and denitrification to N2O production was assessed by Acetylene Inhibition Assay and N-15 isotopically enriched incubations. Ammonium fertilization stimulated N2O emissions to a greater extent than nitrate, and acetylene had a greater inipact on N2O emissions in ammoniurn-fertilized soils than in nitrate-amended soils. Stable isotope analysis indicated that at 50-55% water filled pore space, nitrification was the dominant (> 80%) N2O emitting process. In part 3, molecular analyses of the two N2O producing groups indicated the both nitrifiers and denitrifiers did not differ between landforms. Our results suggest nitrifier denitrification is the dominant process occurring in these arctic soils and that the role of denitrifiers in N2O release from arctic soils needs to be re-evaluated. (c) 2007 Elsevier Ltd. All rights reserved.
Abstract: It is widely accepted that the use of total metal concentrations in soil overestimates metal risk from human ingestion of contaminated soils. In vitro simulators have been used to estimate the fraction of arsenic present in soil that is bioaccessible in the human digestive track. These approaches assume that the bioaccessible fraction remains constant across soil total metal concentrations and that intestinal microbiota do not contribute to arsenic release. Here, we evaluate both of these assumptions in two size fractions (bulk and < 38 mu m) of arsenic-rich mine tailings from the Goldenville, Lower Seal Harbour, and Montague Gold Districts, Nova Scotia. These samples were evaluated using an in vitro gastrointestinal model, the Simulator of the Human Intestinal Ecosystem (SHIME). Arsenic bioaccessibility, which ranged between 2 and 20% in the small intestine and 4 and 70% in the colon, was inversely related to total arsenic concentration in the mine tailings. Additionally, arsenic bioaccessibility was greater in the bulk fraction than in the < 38 mu m fraction in the small intestine and colon while colon microbes increased the bioaccessibility of arsenic in mine tailings. These results suggest that the practice of using a constant percent arsenic bioaccessibility across all metal concentrations in risk assessment should be revisited.
Abstract: Differentiation of DNA derived from viable or non-viable microorganisms within mixed microbial communities continues to be one of the Greatest challenges in molecular studies of environmental samples. A novel method developed for microbial food pathogens is tested here on environmental samples. This technique involves the use of ethidium monoazide bromide (EMA) for the distinction of live/dead cells. In nonviable cells E.,MA intercalates into the DNA which prevents amplification by PCR. We adapted and evaluated the EMA technique for soil, elemental sulfur and river biofilm samples. Quantitative PCR determined that EMA suppressed 99.99% of E. coli LKI gfp+ signal in non-viable cultures and 100.00% when the cultures were added to soil samples. The same technique was also successful at suppressing DNA amplification from spiked non-viable cells in elemental sulfur samples by 100.00%, but not in three Saskatchewan River biofilms. In sub Antarctic soil, EMAQ-PCR was used to detect the prevalence of a functional gene, amoA, and this was closely correlated to nitrification activity measurements. The ability of EMA to differentiate between viable and non-viable populations in soil was confirmed by the similarity of the 16S rRNA denaturing-gradient-gel electrophoresis DNA fingerprint of EMA treated soil and the 16S rRNA cDNA fingerprint of non-EMA treated soil. The EMA technique effectively suppressed amplification of non-viable spiked controls, closely mirrored activity assays and yielded community composition profiles similar to rRNA techniques. The use of EMA in soil effectively suppressed amplification of non-viable organism DNA, however it was not effective in biofilm samples and EMA partially inhibited amplification of viable organism DNA in elemental sulfur samples. (c) 2007 Elsevier B.V. All rights reserved.
Abstract: The usefulness of quantitative polymerase chain reaction (QPCR) to measure nosZ gene prevalence in a multitemplate reaction was assessed by comparing 19 nosZ template DNA samples and 91 model Communities. Efficiencies of the QPCR varied but were not significantly different among nosZ genotypes and were not linked to genetic distance from Ralstonia eutropha. nosZ genotype QPCR efficiencies obtained from isolated denitrifiers were higher (84.8%) than those obtained from excised denaturing gradient gel electrophoresis bands or clones of PCR products from total community DNA (ca. 60%). Analysis of the model communities indicated that QPCR accurately predicts gene prevalence in communities composed of up to six templates.
Abstract: Porewater samples were obtained on five occasions during spring, summer and fall by in situ dialysis from three sites of a large freshwater wetland situated along the St. Lawrence River. These samples were analysed for total dissolved mercury ([Hg](T)) and methylmercury ([MeHg]) concentrations and for complementary variables including dissolved sulfate, sulfide and elemental sulfur concentrations. Sediment cores were obtained on three occasions from one of these sites for the determination of total mercury ({Hg)(T)) and methylmercury ((MeHg)) concentration as well as mercury methyltransferase (HgMT) activity profiles. 1 MeHg and HgMT activity varied with time and sediment depth. The porewater [Hg](T) and [MeHg] depth profiles varied with time and among sites. Modeling the porewater [MeHg] profiles with a one-dimensional reactiontransport equation allowed identification of the sediment depths where MeHg is produced or consumed, as well as an estimate of the net in situ MeHg production rates in the sediments. The model-predicted depths of MeHg production, as well as the sulfate concentration and the HgMT activity depth distributions are all consistent with the involvement of sulfate reducing bacteria in the production of MeHg. (C) 2007 Elsevier Ltd. All rights reserved.
Abstract: Previous published measurements of mercury photoreduction are for net-photoreduction, since photooxidation processes occur simultaneously. In this research we combine continuous dissolved-gaseous mercury (DGM) analysis with a photoreactor and a quartz sparger in order to derive mercury gross photoreduction rate constants for UVB and UVA irradiations. The DGM concentration in each filter-sterilized freshwater was measured at 5 min intervals over a period of 23 h. Photoreduction proceeded for the initial 200 min, after which, reducible mercury was depleted in the sample. Substantial losses in DOC fluorescence were observed during the incubations for UVA radiation but not for UVB; therefore, UVB photoreduction dynamics are not linked to a loss in DOC fluorescence. Pseudo first-order reaction kinetics fit the data well (r(2) > 0.87). The rate constants appear divided between lakes and rivers with the mean lake UVB rate constant (k(UVB) = 8.91 X 10(-5) s(-1)), significantly less than the mean rate constant (k(UVB) = 1.81 x 10(-4) s(-1)) for the river samples. However, while there were differences for the UVB rates between lakes and rivers, the mean and median rate constants for UVA in lakes (k(UVA) = 7.76 x 10(-5) s(-1)) did not differ significantly from the mean rate constant for the river sites (k(UVA) = 1.78 x 10(-4) s(-1)). Here, we propose a model for mercury photoredox dynamics for both temperate lake and river systems. The lake model was validated using principal axis analysis to compare observed and predicted DGM data (n = 279) from a variety of lake sites in Nova Scotia and Central Quebec. Principal axis analysis found a linear fit (correlation = 0.81; slope = 2.13) between predicted and observed environmental DGM values when log-normalized. The constant bias on the predicted values was attributed to estimates of available reducible mercury and the effect of DGM volatilization on observed data.
Abstract: Human activities in the Antarctic have resulted in hydrocarbon contamination of these fragile polar soils. Bioremediation is one of the options for remediation of these sites. However, little is known about anaerobic hydrocarbon degradation in polar soils and the influence of bioremediation practices on these processes. Using a field trial at Old Casey Station, Antarctica, we assessed the influence of fertilization on the anaerobic degradation of a 20-year old fuel spill. Fertilization increased hydrocarbon degradation in both anaerobic and aerobic soils when compared to controls, but was of most benefit for anaerobic soils where evaporation was negligible. This increased biodegradation in the anaerobic soils corresponded with a shift in the denitrifier community composition and an increased abundance of denitrifiers and benzoyl-CoA reductase. A microcosm study using toluene and hexadecane confirmed the degradative capacity within these soils under anaerobic conditions. It was observed that fertilized anaerobic soil degraded more of this hydrocarbon spike when incubated anaerobically than when incubated aerobically. We conclude that denitrifiers are actively involved in hydrocarbon degradation in Antarctic soils and that fertilization is an effective means of stimulating their activity. Further, when communities stimulated to degrade hydrocarbons under anaerobic conditions are exposed to oxygen, hydrocarbon degradation is suppressed. The commonly accepted belief that remediation of polar soils requires aeration needs to be reevaluated in light of this new data.
Abstract: It is well known that dissolved organic matter (DOM) increases in lakes associated with forestry activity but characterization of the DOM structure is incomplete. Twenty-three lakes with a wide range of forestry activities located in central Quebec, Canada were sampled and analyzed for dissolved organic carbon (DOC) concentration, DOC fluorescence, and ultra violet-visible (UV-VIS) absorption spectra. The results show that DOC increases (as does the associated DOC fluorescence) with increased logging (slope=0.122, r(2)=0.581, p < 0.001; and slope=0.283, r(2)=0.308, p < 0.01, respectively) in the 23 lakes sampled however, the aromaticity of the DOM does not change with changes in logging (as found by UV-VIS ratios, absorbance slope in the UV region, and DOC normalized fluorescence (slope=1.42 x 10(-2) r(2)=0.331, p < 0.01). The DOM from four of these lakes was concentrated using reverse osmosis (RO) followed by freeze-drying. The structures of the concentrated dissolved organic matter (DOM) samples were analyzed using X-ray analysis of near edge structures (XANES), X-ray diffraction (XRD), and C-13 solid-state nuclear magnetic resonance (C-13 NMR) analysis. XANES analysis of functional groups in the four concentrated samples shows that there are significant differences in reduced sulphur between the samples, however there was no clear relationship with forestry activity in the associated catchment. XRD data showed the presence of amorphous sulphide minerals associated with the DOM concentrate that may be important sites for mercury binding. The C-13 NMR spectra of these samples show that the percentage of carbon present in carboxylic functional groups increases with increasing logging. Such structures are important for binding photo-reducible mercury and their presence may limit mercury photo-reduction and volatilization. We propose a mechanism by which increased logging leads to increased carboxylic groups in DOM and thereby increased weak binding of photo-reducible mercury. These results, in part, explain the decrease in dissolved gaseous mercury (DGM) production rates with increased logging found in our previous work. (c) 2005 Elsevier B.V. All rights reserved.
Abstract: In this study, we report the isolation of denitrifiers from hydrocarbon-contaminated Antarctic soils. Seventy-two isolates were obtained from soils that had received a fertilizer treatment to stimulate hydrocarbon degradation. All isolates, except one, belonged to the genus Pseudomonas. The one exception was a member of the Microbacteriaceae, which was also, coincidentally, the only isolate negative for the nirS gene. The diversity of the 16S rRNA and nosZ genes was assessed by denaturing gradient gel electrophoresis and sequencing. There was a slight correlation between the 16S rRNA and nosZ operational taxonomic units. Surprisingly, many isolates contained nosZ on plasmids and, to the best of our knowledge, this is the first report of nosZ being extra-chromosomally present in Pseudomonas spp.
Abstract: The use of agrochemicals, such as mineral fertilizers and herbicides in agricultural systems, may affect the potential of soils to act as a sink for methane. Typically, the effect of each agrochemical on soil methane oxidation is investigated separately whereas in the field these agrochemicals are used together to form one comprehensive land management system. Here we report the results of field experiments that assessed the combined effect of multiple fertilizer and herbicide (nicosulfuron, dimethenamide and atrazine) applications on the soil methanotrophic community. Soils treated with organic fertilizer had three times higher methane oxidation rates compared to soils receiving mineral fertilizers. These higher oxidation rates were positively reflected in a significantly enhanced abundance of methanotrophs for the organic fertilized soils. In contrast, herbicide application did not alter significantly the soil methane oxidation rate or the methane-oxidizing population abundance. Subsequently, the methanotrophic community structure was analyzed with group-specific DGGE of 16S rRNA genes. Cluster analysis of the methanotrophic patterns clearly separated the mineral from organically fertilized soils. Less pronounced clustering differentiated between chemical and manual weed control. Furthermore, cluster analysis of the methanotrophic community revealed that soil type was the primary determinant of the community structure. Our results indicate that fertilizer type had the greatest influence on methane oxidizer activity and abundance. Soil type had the most pronounced effect on the microbial community structure. (C) 2004 Elsevier Ltd. All rights reserved.
Abstract: The human-specific HF183 Bacteriodes 16S rRNA genetic marker can be used to detect human faecal pollution in water environments. However, there is currently no method to quantify the prevalence of this marker in environmental samples. We developed a real-time polymerase chain reaction (PCR) assay using SYBR Green I detection to quantify this marker in faecal and environmental samples. To decrease the amplicon length to a suitable size for real-time PCR detection, a new reverse primer was designed and validated on human and animal faecal samples. The use of the newly developed reverse primer in combination with the human-specific HF183 primer did not decrease the specificity of the real-time PCR assay but a melting curve analysis must always be included. This new assay was more sensitive than conventional PCR and highly reproducible with a coefficient of variation of less than 1% within an assay and 3% between assays. As the Bacteroides species that carries this human-specific marker has never been isolated, a bacteria real-time assay was used to determine the detection efficiency. The estimated detection efficiency in freshwater ranged from 78% to 91% of the true value with an average detection efficiency of 83 +/- 4% of the true value. Using a simple filtration method, the limit of quantification was 4.7 +/- 0.3 x 10(5) human-specific Bacteroides markers per litre of freshwater. The aerobic incubation of the human-specific Bacteroides marker in freshwater for up to 24 days at 4 and 12degreesC, and up to 8 days at 28degreesC, indicated that the marker persisted up to the end of the incubation period for all incubation temperatures.
Abstract: Arbuscular mycorrhizal (AM) fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Molecular techniques are used to investigate fungal community composition in uncultivated, disturbed, or contaminated soils, but this approach to community analysis of AMF in agricultural soils has not been reported. In this study, a polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) procedure for the detection of fungal 18S ribosomal RNA gene was developed with reference cultures of seven isolates (representing five AMF species). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in the province of Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these cultures. The effectiveness of the procedure was tested by its application to soil samples from 38 farms. Prominent bands from the PCR-DGGE profiles of these samples were excised for sequence analysis. The total number of species recovered was low in comparison to other AMF community surveys of temperate climate locations. The majority of the sequences recovered were Glomus species. Scutellospora calospora, a previously undetected AM fungus in Saskatchewan was found. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods. (c) 2005 Elsevier Ltd. All rights reserved.
Abstract: Methylmercury [MeHg(I)] in the aerobic surface water of lakes is thought to be rapidly degraded, but contrary to expectations, we show that MeHg(I) concentrations often increase during sunlight hours or remain relatively constant. We hypothesized that there wore water column processes that generated MeHg(I) and that these processes were linked to dissolved organic matter (DOM) and solar radiation. A 2-day diurnal pattern of MeHg(I) in surface water with corresponding bottled controls was assessed for two contrasting lakes in Kejimikujik, Nova Scotia, Canada. Following this study, a tangential ultrafiltrator was used to size-fractionate and generate a concentration gradient of DOM from four different lakes located near Lac Berthelot, Quebec, Canada. The watersheds of two of these lakes were not substantially logged whereas the other two had been extensively logged. Different size fractions of DOM as well as different concentrations of DOM were exposed to sunlight for varying periods of time. We observed that, in Kejimikujik, the concentration of MeHg(I) in surface waters peaked in the early,afternoon. Furthermore, this also occurred in bottled water for one of the lakes, Puzzle, eliminating the possibility that in-lake mixing played a role in this pattern. The formation of MeHg(I) was found to be dependent on the size fraction and amount of DOM present in the water. Specifically, DOM less than 5 kDa or between 30 and 300 kDa generated MeHg(I) when exposed to sunlight, but larger fractions did not. Furthermore, although data are limited, we found that water from lakes with logged watersheds generated MeHg(I) when exposed to sunlight, whereas water from lakes with low levels of logging in the undisturbed watersheds did not. Our results demonstrate that MeHg(I) can be formed in freshwaters of certain lakes in response to solar radiation. This photoproduction of MeHg(l) is dependent on DOM concentrations and type, with the importance of water chemistry not yet clear. The significance of this process to freshwater lakes and the mechanism responsible for MeHg(I) photoproduction is still unclear, but a correction in the conventional wisdom that MeHg(I) is rapidly photodegraded is timely.
Abstract: Mercury in humans and other top predators living in the Arctic is present at elevated levels. Since only methylmercury (MeHg) bioaccumulates in food chains, sources of MeHg need to be identified. Recently, wetlands in the High Arctic were found to produce MeHg, and this was confirmed in laboratory soil incubations. In the present study both wetlands and snowmelt water were evaluated as sources of MeHg to Arctic ecosystems in Nunavut. Three substudies took place on Cornwallis Island, and one took place on Ellesmere Island. First, the effect of wetland presence in lake watersheds was evaluated by comparing four lakes with wetlands present to four lakes without wetlands present. Next,two individual wetlands were spatially and temporally investigated. Finally, three basin tributaries were evaluated for snowmelt MeHg sources. Catchments on Cornwallis Island with wetlands did not have an observable effect on MeHg levels in downstream lake water, but the wetland on Ellesmere Island contributed significant MeHg. In contrast, calculated yields of MeHg in tributaries draining snowmelt on Cornwallis Island were higher (ca. 1.5 mg km(-2) day(-1)) than those measured in temperate catchments characterized by wetlands. Methylmercury and total Hg concentrations in lakes, wetlands, and basin tributaries showed a strong temporal trend that corresponded to inputs from snowmelt water in late spring. This study revealed that wetland export of MeHg to downstream Arctic lakes is site dependent, and snowmelt water was the most significant source of MeHg to Arctic ecosystems located on Cornwallis Island.
Abstract: Mercury is present at elevated levels in the top predators living in High Arctic ecosystems. Because only methylmercury (MeHg) bioaccumulates in food chains, the sources need to be identified. In temperate environments, wetlands are considered to be the principal sources of MeHg, with sulfate-reducing bacteria (SRB) thought to be responsible. The present study investigated whether High Arctic wetlands produced MeHg and whether SRB were involved in MeHg formation. Frozen soil was collected from 18 High Arctic wetlands before ground thaw, and when analyzed for MeHg, values were low, averaging 0.065 ng/g. When soils were incubated for 30 and 60 d at typical summer Arctic soil temperatures (4degreesC and 8degreesC), MeHg increased up to 100-fold. These laboratory observations were consistent with field measurements of wetland surface water, where MeHg concentrations increased from near detection limits (0.02 ng/L) at the inflow to an average of 1.21 ng/L at the outflow. Both laboratory and field data showed MeHg production in High Arctic wetlands. The prevalence of SRB in soil was low, however, and DNA analysis of the dissimilatory sulfate-reductase gene specific to SRB was positive at only one site. The present study showed that wetlands in the High Arctic can produce MeHg but that SRB may not the dominant mercury methylators.
Abstract: Described is a liquid chromatography-mass spectrometry (LC-MS) procedure for the determination of hydroxylated biotransformation products of polycyclic aromatic hydrocarbons (PAH) in the human gastrointestinal tract. The formation of hydroxylated PAHs was monitored upon incubation of PAHs with colon microbiota from the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The analytical method consisted of a biomass removal step followed by a solid phase extraction (SPE) step using C18 packed columns to remove non-digested food compounds and microbial metabolites that interfere with the detection of the target compounds. For quantification, 9-hydroxyphenanthrene C-13(6) was used as the internal standard. The detection limits of the hydroxylated PAHs were generally in the range 0.36-14.09 mug l(-1), based on a signal/noise ratio of 3:1. The recovery of hydroxylated PAHs in intestinal suspension was variable ranging from 45 to 107%, with relative standard deviation (R.S.D.) between 5 and 17%. The analytical procedure was used to show the microbial production of I-hydroxypyrene and 7-hydroxybenzo(a)pyrene, metabolites that may give colon incubated PAHs bioactive properties. (C) 2004 Elsevier B.V. All rights reserved.
Abstract: Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type 11 methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.
Abstract: The production of dissolved gaseous mercury (DGM) in freshwater lakes is induced by solar radiation and is also thought to be linked to processes mediated by dissolved organic carbon (DOC). Studies investigating these processes using comparisons between lakes are often confounded by differences in DOC content and structure. In this study, we investigated the link between DOC concentrations and DGM production by using tangential ultrafiltration to manipulate DOC concentrations in water samples taken from a given lake. In this way, a range of samples with different DOC concentrations was produced without substantial changes to DOC structure or dissolved ions. This was repeated for four lakes in central Quebec: two with highly logged drainage basins and two with minimally logged drainage basins. On two separate days for each lake, water samples (filtered to remove >99% of microorganisms) with varying DOC concentrations were incubated in clear and dark Teflon bottles on the lake surface DGM concentrations were measured at 3.5-h intervals over the course of 10.5 h. Levels of DGM concentrations increased with increasing cumulative irradiation for all lakes until approximately 4000 kJ m(-2) (400-750 nm, photosynthetically active radiation (PAR)), when DGM concentrations reached a plateau (between 20 and 200 pg L-1). When we assumed that DGM production was limited by the amount of photoreducible mercury, reversible first-order reaction kinetics fitted the observed data well (r(2) ranging between 0.59 and 0.98, p < 0.05 with the exception of N70 100% DOC, 0% DOC, and K2 0% DOC with p = 0.06, 0.10, and 0.11, respectively). The DGM plateaus were independent of DOC concentrations but differed between lakes. In contrast, photoproduction efficiency (DGM(prod)) (i.e., the amount of DGM produced per unit radiation (fg L-1 (kJ/m(2))(-1)) below 4000 kJ m(-2) PAR) was linearly proportional to DOC concentration for both logged lakes (r(2) = 0.97, p < 0.01) and nonlogged lakes (r(2) = 0.52, p = 0.018) studied. Furthermore, logged lakes had a lower DGM(prod) per unit DOC (p < 0.01) than the nonlogged lakes. In these four lakes, the rate of DGM production per unit PAR was dependent on the concentration of DOC. The DGM plateau was independent of DOC concentration; however, there was a significant difference in DGM plateaus between lakes presumably due to different DOC structures and dissolved ions. This research demonstrates an important mechanism by which logging may exacerbate mercury levels in biota.
Abstract: Soil ingestion is an important exposure route by which immobile soil contaminants enter the human body. We assessed polycyclic aromatic hydrocarbon (PAH) release from a contaminated soil, containing 49 mg PAR kg(-1), using a SHIME (Simulator of the Human Intestinal Microbial Ecosystem) reactor comprising the stomach, duodenal, and colon compartments. Polycyclic aromatic hydrocarbon release was defined as that fraction remaining in the digest supernatant after centrifugation for 5 min at 1500 X g. The PAR release in the stomach digest was only 0.44% of the total PAR present in soil, resulting in PAR concentrations of 23 mug PAR L-1 chyme. The lower PAR releases in duodenum (0.13%) and colon (0.30%) digests, compared with the stomach digest, were thought to be attributed to combined complexation and precipitation with bile salts, dissolved organic matter, or colon microbiota. We studied these complexation processes in an intestinal suspension more in depth by preparing mixtures of 9-anthracenepropionic acid, a Bacillus subtilis culture, and cholin as model compounds for PAHs, organic matter, and bile salts, respectively. Bile salts or organic matter in the aqueous phase initially enhance PAR desorption from soil. However, desorbed PAHs may form large aggregates with bile and organic matter, lowering the freely dissolved PAR fraction in the supernatant. Using the model compounds, mathematical equations were developed and validated to predict PAR complexation processes in the gastrointestinal tract. Contaminant release and subsequent complexation in the gut is an important prerequisite to intestinal absorption and thus bioavailability of that contaminant. The data from this research may help in understanding the processes to which PAHs are subjected in the gastrointestinal tract, before intestinal absorption.
Abstract: The survival and activity of microbial degradative inoculants in bioreactors is critical to obtain successful biodegradation of non- or slowly degradable pollutants. Achieving this in industrial wastewater reactors is technically challenging. We evaluated a strategy to obtain complete and stable bioaugmentation of activated sludge, used to treat a 3-chloroaniline (3-CA) contaminated wastewater in a lab-scale Semi-Continuous Activated Sludge system. A 3-CA metabolizing bacterium, Comamonas testosteroni strain 12, was mixed with molten agar and encapsulated in open ended silicon tubes. The tubes containing the immobilized bacteria represented about 1% of the volume of the mixed liquor. The bioaugmentation activity of a reactor containing the immobilized cells was compared with a reactor with suspended I2gfp cells. From day 25-30 after inoculation, the reactor with only suspended cells failed to degrade 3-CA completely, due to a decrease in specific metabolic activity. In the reactors with immobilized cells however, 3-CA continued to be removed. A mass balance indicated that ca. 10% of the degradation activity was due to the immobilized cells. Slow release of the growing embedded cells from the agar into the activated sludge medium, resulting in a higher number of active 3-CA degrading 12 cells, was responsible for ca. 90% of the degradation. Our results demonstrate that this simple immobilization procedure was effective to maintain a 3-CA degrading population within the activated sludge community.
Abstract: Bioaugmentation of bioreactors generally focuses on the removal of specific xenobiotics. Often, less attention is hereby paid to the recovery of other reactor functions such as ammonium-nitrogen removal. This work evaluated the effects on activated sludge reactor functions of a 3-chloroaniline (3-CA) pulse and the protective bioaugmentation by inoculation with the 3-CA degrading strain Comamonas testosteroni 12gfp. Changes in functions such as nitrification, removal and sludge compaction were monitored with the sludge community structure, in particular the nitrifying populations. Molecular techniques characterized and enumerated the ammonia oxidizing microbial community immediately after a 3-CA shock load. Two days after the 3-CA shock, ammonium accumulated and the nitrification activity did not recover over a 12-day period in the non-bioaugmented reactors. In contrast, nitrification in the bioaugmented reactor started to recover from day 4 on. The molecular analysis showed that the ammonia oxidizing microbial community of the bioaugmented reactor recovered both in structure, activity and abundance, while in the non-bioaugmented reactor the number of ribosomes of the ammonia oxidizers decreased drastically and the community composition changed and did not recover. This study demonstrates that bioaugmentation of wastewater reactors to accelerate the degradation of toxic chlorinated organics such as 3-chloroaniline protected the nitrifying bacterial community, thereby allowing faster recovery from toxic shocks.
Abstract: Microbial fuel cells hold great promise as a sustainable biotechnological solution to future energy needs. Current efforts to improve the efficiency of such fuel cells are limited by the lack of knowledge about the microbial ecology of these systems. The purposes of this study were (i) to elucidate whether a bacterial community, either suspended or attached to an electrode, can evolve in a microbial fuel cell to bring about higher power output, and (ii) to identify species responsible for the electricity generation. Enrichment by repeated transfer of a bacterial consortium harvested from the anode compartment of a biofuel cell in which glucose was used increased the output from an initial level of 0.6 W m(-2) of electrode surface to a maximal level of 4.31 W m(-2) (664 mV, 30.9 mA) when plain graphite electrodes were used. This result was obtained with an average loading rate of I g of glucose liter(-1) day(-1) and corresponded to 81% efficiency for electron transfer from glucose to electricity. Cyclic voltammetry indicated that the enhanced microbial consortium had either membrane-bound or excreted redox components that were not initially detected in the community. Dominant species of the enhanced culture were identified by denaturing gradient gel electrophoresis and culturing. The community consisted mainly of facultative anaerobic bacteria, such as Alcaligenes faecalis and Enterococcus gallinarum, which are capable of hydrogen production. Pseudomonas aeruginosa and other Pseudomonas species were also isolated. For several isolates, electrochemical activity was mainly due to excreted redox mediators, and one of these mediators, pyocyanin produced by P. aeruginosa, could be characterized. Overall, the enrichment procedure, irrespective of whether only attached or suspended bacteria were examined, selected for organisms capable of mediating the electron transfer either by direct bacterial transfer or by excretion of redox components.
Abstract: Diurnal patterns for dissolved gaseous mercury (DGM) concentration, mercury flux, several water variables (pH, oxidation reduction potential (ORP), water temperature), and meteorological variables (wind speed, air temperature, % relative humidity, solar radiation) were measured in two lakes with contrasting dissolved organic carbon (DOC) concentrations in Kejimkujik Park, Nova Scotia. A continuous analysis system made it possible to measure quick changes in DGM over time. Consistently higher DGM concentrations were found in the high DOC lake as compared to the low DOC lake. An examination of current mercury flux models using this quantitative data indicated some good correlations between the data and predicted flux (r ranging from 0.27 to 0.83) but generally poor fit (standard deviation of residuals ranging from 0.97 to 3.38). Cross-correlation analysis indicated that DGM dynamics changed in response to solar radiation with lag-times of 65 and 90 min. This relationship with solar radiation was used to develop new predictive models of DGM and mercury flux dynamics for each lake. We suggest that a generalized approach using time-shifted solar radiation data to predict DGM can be incorporated into existing mercury flux models. It is clear from the work presented that DOC and wind speed may also play important roles in DGM and mercury flux dynamics, and these roles have not been adequately accounted for in current predictive models.
Abstract: High concentrations of soluble calcium in industrial wastewater present problems due to the calcification of downstream processing. The current trend towards circuit closure and increased water re-use will escalate this problem. We investigated ureolytic microbial carbonate precipitation as a novel process for removing excess calcium from industrial effluents. Two laboratory-scale reactors, both with a hydraulic retention time of 8 h, were fed with 1.8 dm(3) of anaerobic effluent (about 11 mm Ca2+) from a paper recycling plant. Both reactors were inoculated with pre-cultivated calcareous sludge and the treatment reactor was additionally dosed with urea to a final concentration of S. 3 mm. Even though the anaerobic wastewater was saturated as such with respect to CaCO3, urea addition and hydrolysis was shown to be a pre-requisite for precipitation. Almost all (85-90% w/v) of the soluble calcium was precipitated as CaCO3 and removed through sedimentation in the treatment reactor. This bio-catalytic process presents an uncomplicated and efficient method for the removal of calcium from industrial wastewater. (C) 2003 Society of Chemical Industry.
Abstract: Methylmercury (MeHg) is photochemically degraded at the surface of freshwater lakes at around 18% day(-1). Since MeHg is not thought to be formed in the water column, concentrations of MeHg at the lake surface should display a strong diurnal pattern due to the photodegradation of MeHg. However, previous investigators have found that MeHg concentrations peak at noon and microbial inhibitors do not prevent mercury methylation. These reports suggest that mercury methylation may mitigate the effect of photodegradation on surface MeHg concentrations. Lake water was fractionated by a tangential ultrafiltrator and exposed in Teflon bottles to sunlight for different periods of time. Concentrations of MeHg initially decreased by 50% but then increased once again. Since, this is a sealed system, we assume that dissolved organic carbon between 300 and 30 kDa was responsible for the abiotic photochemical production of MeHg. Using rate data collected on site, photomethylation rates are estimated at 20 pg L-1 (kW m(-2))(-1) which corresponds to 35% of MeHg inputs in freshwater lakes. The observed dependence of photomethylation on dissolved organic carbon may provide a mechanistic explanation for why clear cutting which increases dissolved organic carbon, increases MeHg in biota.
Abstract: The concentration of dissolved gaseous mercury (DGM) in freshwaters changes more quickly than the 40-min processing time of current analytical methods. A new method for continuous field analysis of DGM was developed using a Tekran 2537A to achieve a DGM analysis time of 5 min. Samples were concurrently analyzed for temperature, oxygen, conductivity, pH, and oxidation-reduction potential using a Hydrolab. The detection limit for DGM ranged between 5 and 20 fmol L-1 with 99% removal efficiency. Control experiments showed that there was no interference due to methyl mercury, which is present in similar concentrations to DGM. Controlled experiments comparing continuous DGM analysis with discrete DGM analysis showed that the results are not significantly affected by typical variations in water temperature. (4-30 degreesC), oxidation-reduction potential (135-355 mV), dissolved organic carbon (4.5-10.5 mg L-1), or pH (3.5-7.8). The continuous analysis was within 4.5% of the discrete analysis when compared across 12 samples analyzed in triplicate. The field performance of this method was tested over two 48-h periods in two lakes in Kejimkujik Park, Nova Scotia where over 1000 data points were collected. (C) 2002 Elsevier Science B.V. All rights reserved.
Abstract: The purpose of this study was to investigate the mechanism by which phytoremediation systems promote hydrocarbon degradation in soil. The composition and degradation capacity of the bulk soil microbial community during the phytoremediation of soil contaminated with aged hydrocarbons was assessed. In the bulk soil, the level of catabolic genes involved in hydrocarbon degradation (ndoB, alkB, and xylE) as well as the mineralization of hexadecane and phenanthrene was higher in planted treatment cells than in treatment cells with no plants. There was no detectable shift in the 16S ribosomal DNA (rDNA) composition of the bulk soil community between treatments, but there were plant-specific and -selective effects on specific catabolic gene prevalence. Tall Fescue (Festuca arundinacea) increased the prevalence of ndoB, alkB, and xylE as well as naphthalene mineralization in rhizosphere soil compared to that in bulk soil. In contrast, Rose Clover (Trifolium hirtum) decreased catabolic gene prevalence and naphthalene mineralization in rhizosphere soil. The results demonstrated that phytoremediation systems increase the catabolic potential of rhizosphere soil by altering the functional composition of the microbial community. This change in composition was not detectable by 16S rDNA but was linked to specific functional genotypes with relevance to petroleum hydrocarbon degradation.
Abstract: Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28degreesC for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.
Abstract: The importance of microbial processes for the regulation of dissolved gaseous mercury (DGM) in deep freshwaters has not been previously investigated. In this study, we evaluated microbial mercury reductase and oxidase activities in depth profiles from Jack's Lake. In addition, detailed DGM depth profiles were determined for four sampling stations on Lake Ontario. Our results illustrate that microbial processes are an important factor regulating DGM in the hypolimnion. Levels of DGM in William's Bay in Jack's Lake were 6 times higher than that observed at Brooke's Bay. This was accompanied by a 10 fold reduction in mercury oxidase activity in William's Bay. When DGM concentrations are expressed on an aerial basis, DGM concentrations above the thermocline in Lake Ontario average 1.5 ng DGM m(-2) and in small freshwater lakes it ranged between 0.1 and 0.8 ng DGM m(-2). Further, it was demonstrated that the majority of DGM in large freshwater lakes such as Lake Ontario exists below the thermocline where photochemical oxidation and reduction processes cannot occur. The importance of this DGM to atmospheric flux rates is discussed.
Abstract: Bioaugmentation of bioreactors focuses on the removal of xenobiotics, with little attention typically paid to the recovery of disrupted reactor functions such as ammonium-nitrogen removal. Chloroanilines are widely used in industry as a precursor to a variety of products and are occasionally released into wastewater streams. This work evaluated the effects on activated-sludge reactor functions of a 3-chloroaniline (3-CA) pulse and bioaugmentation by inoculation with the 3-CA-degrading strain Comamonas testosteroni I2 gfp. Changes in functions such as nitrification, carbon removal, and sludge compaction were studied in relation to the sludge community structure, in particular the nitrifying populations. Denaturing gradient gel electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization (FISH) were used to characterize and enumerate the ammonia-oxidizing microbial community immediately after a 3-CA shock load. Two days after the 3-CA shock, ammonium accumulated, and the nitrification activity did not recover over a 12-day period in the nonbioaugmented reactors. In contrast, nitrification in the bioaugmented reactor started to recover on day 4. The DGGE patterns and the FISH and real-time PCR data showed that the ammonia-oxidizing microbial community of the bioaugmented reactor recovered in structure, activity, and abundance, while the number of ribosomes of the ammonia oxidizers in the nonbioaugmented reactor decreased drastically and the community composition changed and did not recover. The settleability of the activated sludge was negatively influenced by the 3-CA addition, with the sludge volume index increasing by a factor of 2.3. Two days after the 3-CA shock in the nonbioaugmented reactor, chemical oxygen demand (COD) removal efficiency decreased by 36% but recovered fully by day 4. In contrast, in the bioaugmented reactor, no decrease of the COD removal efficiency was observed. This study demonstrates that bioaugmentation of wastewater reactors to accelerate the degradation of toxic chlorinated organics such as 3-CA protected the nitrifying bacterial community, thereby allowing faster recovery from toxic shocks.
Abstract: A microbial fuel cell containing a mixed bacterial culture utilizing glucose as carbon source was enriched to investigate power output in relation to glucose dosage. Electron recovery in terms of electricity up to 89% occurred for glucose feeding rates in the range 0.5-3 g l(-1) d(-1), at powers up to 3.6 W m(-2) of electrode surface, a five fold higher power output than reported thus far. This research indicates that microbial electricity generation offers perspectives for optimization.
Abstract: There is an increasing interest in agricultural systems in which the use of herbicides is forbidden. Therefore, soils treated with herbicides atrazine and metolachlor for the last 20 years were compared with soil samples from the same field that had never been treated (control soil). We determined the pollution induced community tolerance (PICT) by evaluating the methane oxidation capacity of soil samples after adding increasing amounts of a methane oxidation inhibitor, 2,4-dichlorophenoxyacetic acid (2,4-D). Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes assessed whether the soil methanotrophic community differed between the two treatments. Addition of 60 mug 2,4-D per g soil clearly inhibited methane oxidation in both soils but increased the time needed to oxidize 5% methane in the headspace by 250% for the control soil compared with 175% for the herbicide-treated soil. This indicates that the soil with a long-term herbicide history had a greater tolerance to the methane oxidation inhibitor than did the control soil. The DGGE of 16S rRNA genes amplified directly from soil community DNA could also distinguish the two treatments. The banding patterns of the Type I methanotrophs contained fewer bands in the herbicide-treated soil. It seems that both the PICT approach and DGGE analysis are effective assays to distinguish a long-term herbicide-treated soil from an untreated soil.
Abstract: In the relatively pristine ecosystem in Kejimkujik Park, Nova Scotia, methylmercury (MeHg) concentrations in loons, Gavia immer, are among the highest recorded anywhere in the world. This study investigated the influence of bedrock lithology on MeHg concentrations in wetlands. Twenty-five different wetland field sites were sampled over four different bedrock lithologies; Kejimkujik monzogranite, black sulfidic slate, gray slate, and greywacke. Soil samples were analyzed for ethylmercury (EtHg), MeHg, total Hg, acid-volatile sulfides (AVS), organic matter, and water content as well as the biological parameters, mercury methyltransferase (HgMT) activity, sulfate reduction rates, fatty acid methyl ester (FAME) composition, and acidity. Methylmercury concentrations in the wetlands were highly dependent (P < 0.08) on lithology with no significant difference between bogs, fens, and swamps. Methylmercury concentrations in wetland soils developed on Kejimkujik monzogranite averaged 900 ng kg(-1) compared with only 300 ng kg(-1) in wetland soils developed on black sulfidic slate. Fatty acid methyl ester composition was also lithologically dependent (P < 0.001) with biomarkers for Desulfobulbus spp. discriminating between sites containing high and low MeHg concentrations. Levels of MeHg in wetlands were predicted mainly (41% of the sum of squares) by HgMT activity that differed (P < 0.009) between wetlands, with activity in bogs almost three times that present in swamps. Wetland MeHg concentrations are highly dependent on the lithology on which they have developed for largely biological reasons.
Abstract: In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH4+ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% +/- 5% of the influent N at the highest surface load of approximately 8.3 g of N m(-2) day(-1), with N-2 as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 +/- 7.6 and 76.5 +/- 6.4 mg of NH4+-N g of volatile suspended solids [VSS](-1) day(-1), respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS-1 day(-1)). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH4+ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.
Abstract: During a study of ureolytic microbial calcium carbonate (CaCO3) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO3 crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected., Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K-m) and maximum hydrolysis rates (V-max) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.
Abstract: Little is known about the chronic effect of herbicides on the soil microbial community, with most studies focusing on acute impacts. In this study, we investigated the effect of 20 years of atrazine and metolachlor application on the community structure, abundance and function of bacterial groups in the bulk soil of a maize monoculture. Group-specific PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) of 16S rRNA genes was used to characterize the composition of the microbial community. Primers specific for the entire bacterial group, as well as for the Acidobacterium group, the actinomycetes, the ammonium oxidizers, and the Type I and II methanotrophs were used in the PCR-DGGE analysis. Cluster analysis of the DGGE patterns obtained with the different primer sets differentiated between the herbicide-treated and the non-treated control soil. In particular the methanotrophic community differed, with three bands (phylotypes) being absent (or showing a lower intensity) in the DGGE patterns of the herbicide-treated soils compared to the patterns of the control soil. The differences in the methanotrophic community structure were not reflected in the methane oxidation capacity, which was similar for the two treatments. The prevalence of methanotrophs as evaluated with real-time PCR analysis also did not differ between the herbicide-treated and non-treated soil. Our results indicate that the long-term use of the herbicides atrazine and metolachlor resulted in an altered soil community structure, in particular for the methanotrophic bacteria. These observed changes did not cause a decreased community function (methane oxidation), probably because the total abundance of the methanotrophs in the soil system was preserved. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Abstract: In the absence of oxygen, a protective H-2 film is formed around an Fe(0) surface, inhibiting the electron flow from this surface. Our study of anoxic corrosion of Fe(0) beads revealed that, in the presence of Shewanella oneidensis MR-1, H-2 removal and precipitation of Fe mineral particles on the cell surface are determining processes for corrosion. These two biologically mediated processes were governed by cell density. H-2 removal by Shewanella oneidensis was detected at cell concentrations of 1.0 x 10(6) live cells ml(-1) and higher and H-2 was electron donor for denitrification of NO3-. The removal of the protective H-2 layer from Fe(0) beads by Shewanella oneidensis, resulted in an increase of Fe release out of the Fe(0) beads from 153 +/- 25 mg l(-1) to 196 +/- 7 mg l(-1) after 20 h. When the cell concentration exceeded 1.0 x 10(8) live cells ml(-1), precipitation of iron minerals on the cell surface was characteristic for the greatest percentage of MR-1 cells, whereas micrometre-scale iron precipitates not associated with culturable cell biomass significantly decreased in number. Addition of supernatant of a corrosion assay with high cell concentration induced metabolic activity in a corrosion assay with low cell concentration, resulting in increased H-2 consumption and Fe release from Fe(0) beads. Homoserine lactone-like molecules were detected in the supernatant by a bio-assay, suggesting the involvement of a quorum-sensing regulatory mechanism.
Abstract: Bio-catalytic calcification (BCC) reactors utilise microbial urea hydrolysis by autochthonous bacteria for the precipitation-removal of calcium, as calcite, from industrial wastewater. Due to the limited knowledge available concerning natural ureolytic microbial calcium carbonate (CaCO3) precipitation, the microbial ecology of BCC reactors has remained a black box to date. This paper characterises BCC reactor evolution from initialisation to optimisation over a 6-week period. Three key parameters were studied: (1) microbial evolution, (2) the (bio)chemical CaCO3 precipitation pathway, and (3) crystal nucleation site development. Six weeks were required to establish optimal reactor performance, which coincided with an increase in urease activity from an initial 7 mg urea l(-1) reactor h(-1) to about 100 mg urea l(-1) reactor h-1. Urease activity in the optimal period was directly proportional to Ca2+ removal, but urease gene diversity was seemingly limited to a single gene. Denaturing gradient gel electrophoresis of 16S rRNA genes revealed the dynamic evolution of the microbial community structure of the calcareous sludge, which was eventually dominated by a few species including Porphyromonas sp., Arcobacter sp. and Bacteroides sp. Epifluorescence and scanning electron microscopy showed that the calcareous sludge was colonised with living bacteria, as well as the calcified remains of organisms. It appears that the precipitation event is localised in a micro-environment, due to colonisation of crystal nucleation sites (calcareous sludge) by the precipitating organisms.
Abstract: Agricultural practices, such as mineral nitrogen fertilization, have an impact on the soil's ability to oxidize methane, but little is known about the shifts in the methanotrophic community composition associated with these practices. Therefore, the long-term effect of both mineral (NH4NO3) and organic (manure and GFT-compost) fertilizer applications on the soil methanotrophic community activity and structure were investigated. Both high and low affinity methane oxidation rates were lower in the soil treated with mineral fertilizer compared to the other soils. An enhanced nitrate concentration was observed in the mineral fertilized soil but nitrate did not show a direct affect on the high affinity methane oxidation. In contrast, the low affinity methane oxidation was slowed down by increased nitrate concentrations, which suggests a direct effect of nitrate on low affinity methane oxidation. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments specific for methanotrophs revealed a distinct community between the mineral and organic fertilized soils as extra Type I methanotrophic bands (phylotypes) became visible in the organic fertilized soils. These phylotypes were not visible in the patterns of the added organic fertilizers suggesting an indirect effect of the organic fertilizers on the methanotrophic community. Additionally, a molecular analysis was performed after the low affinity methane oxidation test. The enhanced methane concentrations used in the test enriched certain low affinity methanotrophs in the organic fertilized soils but not in the mineral fertilized soil. Supporting the molecular and functional observations, fatty acids characteristic for methanotrophs were less abundant in the soil treated with mineral fertilizer compared to the soil treated with compost. In conclusion, the function and molecular and chemical composition of the methanotrophic community are all altered in soil fertilized with mineral fertilizer.
Abstract: Methylmercury (MeHg) is a highly toxic form of mercury that bioaccumulates in aquatic food chains. However, methods to reliably identify sites of MeHg formation or to quantify MeHg production require the use of isotopic tracers, which limits their use. In this paper, a method is presented to quantify the methylation of mercury by a methyltransferase pathway. This methyltransferase pathway is one of the biochemical pathways responsible for biological mercury methylation. Protein is extracted front environmental samples, and mercury methyltransferase (HgMT) activity of soil extracts is calculated by assessing increases in methyltransferase activity induced by Hg addition. In enzyme extracts from pure cultures or soil sampled,, HgMT activity correlated with net MeHg production and Hg consumption, suggesting that HgMT activity can be used to estimate MeHg production in field samples. Over the course of a three-month period in a freshwater wetland, HgMT activity correlated with not MeHg concentrations (r(2) = 0.55; p < 0.057). Furthermore, HgMT activity predicted (r(2) = 0,80; p < 0.01) gross MeHg formation in freshwater wetlands as well as in laboratory microcosms calculated using previously published rate constants. Our results show that a methyltransferase assay, in combination with demethylation estimates, accurately predicts MeHg formation under field and laboratory conditions. This assay does not require the use of mercury added to field samples to estimate activity but rather estimates the biological activity present in the soil by quantifying the amount of enzyme present in the soil. Such an assay is well suited for use in field surveillance programs assessing MeHg formation in a variety of environments.
Abstract: The fate and environmental threat posed by mercury in aquatic systems is controlled, in part, by the transport of Hg-(II) from oxic to anoxic zones in lakes and its subsequent transformation to organic mercury. The transport of Hg-(II) in aquatic systems can be affected by its partitioning between the dissolved and particulate phases. In this study, batch experiments were performed to quantify Hg(II) adsorption to Bacillus subtilis as bacteria-to-metal ratio, pH, chloride concentration, growth phase, and reaction time were independently varied. The laboratory data were well described by a surface complexation model (SCM) considering the adsorption of neutral Hg(II) hydroxide and chloride complexes by specific functional groups on the bacterial surface. To evaluate its applicability to complex aquatic systems, the SCM was used to predict the distributions of Hg(II) in 36 shallow acidic lakes and wetlands in Kejimkujik National Park, Nova Scotia, Canada. The lab-derived SCM provided a statistically accurate (r(2) = 0.615, P < 0.01) fit to the field data when it was expanded to consider Hg(II) complexation by dissolved organic matter. Inclusion of Hg(II)-mineral adsorption reactions did not improve the fit of the model. The quality of fit provided by the expanded SCM suggested that the major assumptions implicit in applying a lab-derived model to the field were justifiable. Our study has demonstrated that SCMs are powerful tools for dynamic prediction of the sorption of environmental contaminants to biocolloids at the regional scale.
Abstract: The purpose-of our paper is to suggest a novel, hypothetical and yet untested use of the pollution-induced community tolerance concept (PICT). Historically, PICT has been applied to determine whether toxicants are deleterious in microfaunal aquatic and terrestrial communities. We hypothesize that it may be possible to apply PICT to megafaunal organisms (e.g., vertebrates). In doing so, researchers could (1) identify which toxicant, in a complex mixture of toxicants, is harmful since only those contaminants that exert selection pressure are biologically relevant and will result in changes in community structure and (2) determine the transfer of toxicants across trophic levels found within that community. We suggest that community tolerance could be measured in megafaunal communities by measuring biomarkers of exposure and effect in either a number of individuals that make up different populations of animals comprising a community, or in the community as a whole. In this article we discuss the theoretical suitability of our megafaunal PICT approach to the assessment of contaminated sites and some of the potential pitfalls associated with its use. Our intention is that this paper will generate debate and commentary surrounding PICT and its potential uses in the future. Whether this potential approach is feasible remains to be determined.
Abstract: The evasion of elemental mercury represents a significant pathway for reducing the level of this potentially toxic material in aquatic ecosystems, The evasion rate is controlled by the concentration of dissolved gaseous mercury (DGM) across the air-water interface, water, and air temperature as well as wind speed. Here we investigate the role of microbial mercury oxidation and reduction in regulating DGM diet patterns in two freshwater lakes, Jack's Lake and Lake Ontario. Three replicate diurnal cycles of DGM in Brookes Bay, Jack's Lake peaked at 313 fM between 9:00 to 10:30 and decreased to 79.6 fM by 16:00. Microbial mercury reductase activity (converts Hg2+ to Hg-0) increased with DGM concentrations and mercury oxidase activity (converts Hg-0 to Hg2+) increased as DGM concentrations decreased in the mid-afternoon. This illustrates that mercury oxidase activity was linked to hydrogen peroxide (H2O2) diurnal patterns. Thirty minutes after spiking Lake Ontario water with H2O2, mercury oxidase activity increased by 250% and by 60 min, DGM decreased to 28% of its initial value. Two hours after the H2O2 spike, mercury oxidase activity had declined, but mercury reductase activity and DGM both increased. Four hours after the spike, mercury reductase and DGM levels had returned to original levels. Our results are consistent with the following sequence, of events. In the morning, microbial activity produces DGM (in addition to an DGM formed through photoreduction of Hg2+). As photochemically produced H2O2 increases in concentration it induces the biologically mediated decrease in DGM concentrations throughout the afternoon. To oredict concentration of DGM in surface waters and flux rates to the atmosphere, the contribution of photoreduction and photooxidation must be placed in context with reduction and oxidation rates due to microbial activity.
Abstract: The survival and activity of microbial degradative inoculants in bioreactors is critical to obtain successful biodegradation of non- or slowly degradable pollutants. Achieving this in industrial wastewater reactors is technically challenging. We evaluated a strategy to obtain complete and stable bioaugmentation of activated sludge, which is used to treat a 3-chloroaniline (3-CA) contaminated wastewater in a lab-scale semi-continuous activated sludge system. A 3-CA metabolizing bacterium, Comamonas testosteroni strain 12, was mixed with molten agar and encapsulated in 4 mm diameter open-ended silicone tubes of 3 cm long. The tubes containing the immobilized bacteria represented about 1% of the volume of the mixed liquor. The bioaugmentation activity of a reactor containing the immobilized cells was compared with a reactor with suspended I2gfp cells. From day 25-30 after inoculation, the reactor with only suspended cells failed to completely degrade 3-CA because of a decrease in metabolic activity. In the reactors with immobilized cells, however, MA continued to be removed. A mass balance indicated that ca. 10% of the degradation activity was due to the immobilized cells. Slow release of the growing embedded cells from the agar into the activated sludge medium, resulting in a higher number of active 3-CA-degrading 12 cells, was responsible for ca. 90% of the degradation. Our results demonstrate that this simple immobilization procedure was effective to maintain a 3-CA-degrading population within the activated sludge community.
Abstract: This study attempted to investigate if the tolerance of soil bacterial communities in general, and autotrophic ammonia-oxidizing bacteria (AOB) in particular, evolved as a result of prolonged exposure to metals, and could be used as an indigenous bioindicator for soil metal pollution. A soil contaminated with copper, chromium, and arsenic (CCA) was mixed with an uncontaminated garden soil (GS3) to make five test soils with different metal concentrations. A modified potential ammonium oxidation assay was used to determine the metal tolerance of the AOB community. Tolerance to Cr, Cu, and As was tested at the beginning and after up to 13 months of incubation. Compared with the reference GS3 soil, the five CCA soils showed significantly higher tolerance to Cr no matter which form of Cr (Cr3+, CrO42-, or Cr2O72-) Was tested, and the Cr tolerance correlated with the total soil Cr concentration. However, the tolerance to Cu2+, As3+, and As5+ did not differ significantly between the GS3 soil and the five CCA soils. Community level physiological profiles using Biolog microtiter plates were also used to examine the chromate tolerance of the bacterial communities extracted after six months of exposure. Our results showed that the bacterial community tolerance was altered and increased as the soil Cr concentration was increased, indicating that the culturable microbial community and the AOB community responded in a similar manner.
Abstract: Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear, The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants, At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants, Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.
Abstract: Breeding programs for crop plants are designed to improve agronomic characteristics such as yield, fertilizer use efficiency and disease resistance. These programs do not typically consider interactions between plants and soil microflora. This study assessed the bacterial communities associated with roots of various spring wheat (Triticum spp.) cultivars of related Lineage. Bacteria (n=ca. 1900) were isolated from the rhizosphere and root interior of Triticum moncoccum PI 167549 (an ancient land race that originated in Turkey), T. aestivum cv. Red Fife (historical spring wheat cultivar released in Canada ca. 1845) and I aestivum cv. CDC Teal (modem cultivar registered in Canada in 1991) grown at two different field sites. Bacteria were identified by gas chromatography-MIDI (microbial identification software) fatty acid methyl ester analysis. Twenty-eight bacterial genera were identified as being associated with the three wheat cultivars, but only Aureobacter species differed significantly between cultivars with 16 isolates identified from the root interior of PI 167549 compared to one isolate from Red Fife and two from CDC Teal. In contrast, the bacterial endophytic community of the more modern cultivars was more diverse than that seen for the ancient land race. Increases in diversity were not limited to a single genus and some species were selected against. For example, pseudomonads were more numerous and diverse in the root interior (11 species identified in 117 isolates) compared to the rhizosphere (eight species identified in 94 isolates), but Pseudomonas fluorescens abundance decreased in the root interior compared to the rhizosphere. The fact that the roots of newer wheat cultivars were aggressively colonized by endophytic pseudomonads suggests that these bacteria might be exploited as plant growth-promoting rhizosphere bacteria or as a means to establish specific catabolic activities in these plants.
Abstract: The explosive 2,4,6-trinitrotoluene (TNT) is a contaminant of concern at abandoned manufacturing and military sites because of its mobility and toxicity. Phytoremediation may play a role in natural attenuation scenarios by reducing TNT levels at point sources. The purpose of this study was to develop a phytoremediation system suitable for use in soils contaminated with high TNT levels. Sixteen grasses were screened for their tolerance to 41 g TNT kg(-1) soil. Meadow bromegrass (Bromus erectus Huds.), perennial ryegrass (Lolium perenne L.) and sweet vernalgrass (Anthoxanthum odoratum L.) grew in this soil. Inoculating these grasses with Pseudomonas sp. Strain Ii, capable of transforming TNT into mono- and di-amino metabolites, increased the growth of meadow bromegrass but was lethal to perennial ryegrass and sweet vernalgrass. Meadow bromegrass inoculated with Strain I4 reduced TNT levels by 30% compared with the control soil and had 50% more plant biomass than noninoculated plants. Meadow bromegrass;combined with Strain Ii, increased the percentage of the culturable soil heterotrophic population containing the genes involved in 2-nitrotoluene (ntdAa) metabolism 3-fold, as well as the population containing the genes involved in 4-nitrotoluene (ntnM) metabolism 14-fold. Strain I4 inoculation of meadow bromegrass altered the portion of the rhizosphere community involved in nitroaromatic metabolism and Led to a reduction in soil TNT levels.
Abstract: Terrestrial sites contaminated with 2,4,6-trinitrotoluene (TNT) are a widespread and persistent problem and often contain non-vegetated areas with TNT concentrations well in excess of 1000 mg kg(-1). In this study, we examined the effect of TNT on denitrification activity in field soils, and compared the sensitivity of denitrifying: enzymes to TNT. DNA probes assessed the prevalence of nirS, nirK and nosZ (encoding cd(1) or copper nitrite reductase and nitrous oxide reductase, respectively), denitrifying genotypes in the culturable and total microbial community. The nitrate (NaR), nitrite (NiR) and nitrous oxide (N2OR) reductase activities in field soil and in isolates were assessed by gas chromatography. The relative occurrence of the nirK, nirS or nosZ genotypes increased in the cultured community and in total uncultured community DNA as nitroaromatic concentrations increased. However, denitrifying activity decreased in response to increasing TNT concentrations! with an IC50 for NaR+NiR+nitric oxide reductase (NOR) of 400 mg TNT kg(-1) soil and for N2OR of 26 mg TNT kg(-1) soil. The denitrifying activity of four soil isolates also decreased in response to TNT, with N2OR activity being three times more sensitive to TNT than NaR+NiR+NOR activity. Interestingly, there were 118 times more nirK isolates than nirS isolates in uncontaminated soil but only 1.5 times more in soil containing 17400 mg kg(-1) TNT. The results from this study indicated that TNT reduced denitrification activity in field soils, and N2OR was much more sensitive to TNT than NaR+NiR+hTOR. Crown copyright (C) 2000 Published by Elsevier Science B.V. All rights reserved.
Abstract: Determining the toxicity of contaminants to soil organisms under field conditions is hampered by site-specific and temporal factors that modulate contaminant availability. Assessing the pollution-induced community tolerance (PICT) of indigenous microbial communities integrates these complex environmental factors. The purpose of this study was to determine if the PICT response was proportional to 2,3,6-trinitrotoluene (TNT) concentrations in soil, if changes detected by PICT were also evident in soil microbial community composition, and if the PICT response correlated with phytotoxicity assays. Microorganisms extracted from TNT-contaminated field soils were mixed with a solution containing six different concentrations of TNT and inoculated into Biolog ECO plates. The utilization rate of substrates was determined over a 7-d period. Denaturing gradient gel electrophoretic analysis of a portion of the gene encoding 16S rDNA described the structure of the soil microbial community. Phytoindicators (Poa compressa and P. palustris L.) of TNT pollution were identified and used to assess TNT phytotoxicity in soil samples. The TNT (in Biolog wells) greatly inhibited microbial communities from locations with low in situ TNT exposure. The inhibition of microbial use of L-asparagine, L-phenylalanine. and D-glucosaminic acid by TNT (in Biolog wells) increased as TNT concentration in soil decreased. Locations differing in ECO-PICT response also differed in their microbial community composition and TNT phytotoxicity. Decreased phytotoxicity of field soils corresponded to decreases in PICT. The results from this study indicated that ECO-PICT is an effective assay to rapidly detect TNT exposure and toxicity in soil microbial communities.
Abstract: We assessed the effects of the fungicides captan and thiram on the survival and phenotypic characteristics of Rhizobium leguminosarum by. viceae, strain C1. Fungicide was applied to pea seed at rates of 0.25-2 g a.i. kg(-1) seed, and treated seed was inoculated with strain C1. After 24 h, rhizobia were extracted from treated seed and viable numbers determined by plating onto yeast extract mannitol agar. Phenotypic characteristics were assessed using FAME (fatty acid methyl ester) profiles and Biolog substrate utilization patterns. Captan and thiram significantly reduced the numbers of rhizobia recovered from seed and altered the FAME and Biolog profiles of recovered rhizobia. However, only the highest concentrations of captan affected nodulation and plant growth. Contact with some seed-applied fungicides can significantly alter phenotypic characteristics of rhizobia, but these changes might be offset by the presence of host plants.
Abstract: The composition and diversity of the bacterial community associated with plant roots is influenced by a variety of plant factors such as root density and exudation. In turn, these factors are influenced by plant breeding programs. This study assessed the diversity of root-endophytic and rhizosphere bacterial communities associated with three canola cultivars (Parkland, Brassica rapa; Excel, B. napus; and Quest, B. napus) grown at two field sites. Quest, a derivative of Excel developed by the Alberta Wheat Pool, has been genetically engineered to tolerate the herbicide glyphosate. Approximately 2300 bacteria were isolated from roots of plants and identified based on fatty acid methyl ester (FAME) profiles. One third of the isolates were positively identified by FAME analysis (i.e. SIM index greater than or equal to 0.3) with another third assigned tentative identifications (SIM index <0.3). Fewer Bacillus, Micrococcus and Variovorax isolates, and more Flavobacterium and Pseudomonas isolates were found in the root interior of Quest compared to Excel or Parkland. Furthermore, fewer Arthrobacter and Bacillus isolates were recovered from the rhizosphere of Quest compared to Excel or Parkland. The bacterial root-endophytic community of the transgenic cultivar, Quest, was separated by principal component analysis from the other cultivars, and exhibited a lower diversity compared to Excel or Parkland. The rhizosphere of all cultivars yielded more Arthrobacter, Aureobacterium, and Bacillus isolates, but fewer Micrococcus, Variovorax and Xanthomonas isolates compared to the root interior. The results from this study indicate that the composition of the root-endophytic bacterial community of canola differs between cultivars. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Abstract: The use of plants to detoxify contaminated soil sites has the potential to be a cost-effective alternative to traditional remediation technologies. However, plant-bacteria interactions in contaminated soils are not well understood. In this study we investigated the effect of bacterial seed inoculants on the rhizosphere community during the reduction of 2-chlorobenzoic acid (2CBA) concentrations by Dahurian wild rye (Elymus dauricus). Soil was amended with 450 mg 2CBA kg(-1) and aged for 2 yr, at which time the detectable amount of 2CBA was 61 mg kg(-1) Dahurian wild rye inoculated with either Pseudomonas aeruginosa strain R75, P. savastanoi strain CB35 or a 1:1 mixture of these bacteria was grown in aged contaminated soil for 56 d in a growth chamber. The potential of rhizosphere soil to degrade 3-chlorobenzoic acid (3CBA), a contaminant with a similar degradation pathway to 2CBA, versus 2,3-dichlorobenzoic acid (23diCBA) or 2,5-dichlorobenzoic acid (25diCBA), contaminants with pathways dissimilar to 2CBA, was also assessed. Inoculating Dahurian wild rye with the mixed inoculum had decreased the extractable 2CBA from 61 to 29 mg kg-l, 56 d after planting but had no effect on plant growth. Inoculating Dahurian wild rye with a mixture of strains R75 and CB35 increased the potential of rhizosphere microorganisms to reduce 3CBA concentrations by 17% but had no effect on amounts of 23diCBA or 25diCBA. In a sterile hydroponic plant growth system, inoculation of Dahurian wild rye had no effect on 2CBA concentrations; although, the inoculum became established and grew in the hydroponic solution indicating that inoculants required an unknown soil factor to degrade 2CBA. Bacterial seed inoculants selectively enhanced the potential of the rhizosphere community to degrade certain compounds without affecting heterotrophic bacterial communities. (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: The use of soil microorganisms in ecological risk assessment is hampered by an unclear dose-response relationship for most contaminants. Establishing dose-response curves for soil microbial communities requires that one have a clear estimate of exposure at the site of toxic action and a response free of confounding environmental factors. It is not clear what methods can estimate toxicant dose at the site of toxic action or determine microbial response to a toxicant. Pollution-induced community tolerance (PICT) is one possible estimate of microbial toxicant exposure. The PICT hypothesis is that the tolerance of a microbial community is proportional to the in situ dose. This method automatically corrects for differences due to differences in soil physical-chemical variables between samples. Various components of the soil nitrogen cycle can act as microbial bioindicators of toxicant impacts. Estimating denitrification activity presents a number of advantages over other components of the nitrogen cycle. Denitrifying bacteria come fi-om a diversity of habitats, can be autotrophic or heterotrophic, and denitrification is a well-defined enzymatic system, which allows the use of molecular tools. Determining denitrification may be a good estimate of effects of toxicants on microbial communities. However, given the state of our ignorance regarding soil microbial community structure and function, redundant estimates of exposure and effect are necessary to adequately characterize the response of microbial communities to toxicants.
Abstract: The response of potential nitrification activity (PNA), nitrogen-fixation activity (NFA), and dehydrogenase activity (DHA) in soil to 2,4,6-trinitrotoluene (TNT) was assessed. Two garden soils of contrasting texture (sandy loam vs clay loam) were spiked with TNT (25-1,000 mg TNT/kg). Soil microbial activities and TNT residues were analyzed 1 week later. The estimated IC50 (concentration causing 50% inhibition) ranged from 39 to 533 mg/kg of the acetonitrile-extractable (AE) TNT (1 week after spiking), depending on indicators and soils. The lowest LOEC (lowest-observed-effect concentration) was 1 mg AE TNT/kg. Field soil (0-15 cm) was collected from three known contaminated sites in an abandoned TNT manufacturing facility. Microbial toxicity significantly correlated to TNT levels in these soils. The LOEC and NOEC (no-observed-effect concentration) values were sire and indicator specific, with the lowest LOEC bring 1 mg AE TNT/kg and the lowest NOEC being 0.4 mg AE TNT/kg. The IC50 of the pooled held samples was 51 mg AE TNT/kg for PNA or 157 mg AE TNT/kg for DHA. These results indicate that microbial responses were consistent and comparable between the laboratory and the field and that TNT could significantly inhibit soil microbial activities at very low levels. Both AE TNT and deionized water-extractable (DW) TNT concentrations correlated well with microbial toxicity, but AE TNT provided a better evaluation of TNT bioavailability than did DW TNT.
Abstract: Phytoremediation technologies must tolerate and degrade mixtures of contaminants, as most contaminated sites contain mixtures of compounds. This study assessed the ability of plant-bacteria associations to degrade mixtures of mono-and dichlorinated benzoic acids. Sixteen forage grasses and combinations of these grasses with several bacterial inoculants were screened for growth in soil contaminated with various concentrations of mono-or dichlorinated benzoic acids. Dahurian wild rye (Elymus dauricus) inoculated with a combination of Pseudomonas aeruginosa strain R75 and Pseudomonas savastanoi strain CB35 reduced 3-chlorobenzoic acid (3CBA) levels in soil by 74% (i.e., 583 to 149 mg/kg). Meadow brome (Bromus biebersteinii) inoculated with Alcaligenes sp. strain BR60 reduced 2,3-dichlorobenzoic acid (23diCBA) levels in soil by 56% (i.e., 125 to 55 mg/kg). Altai wild rye (Elymus angitus) inoculated with strains R75 and CB35 reduced 2,5-dichlorobenzoic acid (25diCBA) levels in soil by 46% (i.e., 211 to 113 mg/kg). Two plant-bacteria associations and uninoculated Dahurian wild rye also degraded mixtures of 3CBA, 23diCBA, or 25diCBA. When 25diCBA was mixed with 23diCBA, uninoculated Dahurian wild rye reduced levels of 25diCBA in soil by 31% and reduced the levels of both 25diCBA and 3CBA by up to 64% when these two contaminants were present in a mixture. Similarly, meadow brome inoculated with BR60 reduced 23diCBA and 3CBA levels by up to 50%. Levels of all three chlorinated benzoic acids were reduced by 53 to 63% by Altai wild rye inoculated with strains R75 and CB35. Our results indicate that plant-bacteria associations can tolerate and degrade mixtures of contaminants in soil but that predictions about phytoremediation of mixed contaminants may not be straightforward.
Abstract: Little is known about the composition and diversity of the bacterial community associated with plant roots. The purpose of this study was to investigate the diversity of bacteria associated with the roots of canola plants grown at three field locations in Saskatchewan, Canada. Over 300 rhizoplane and 220 endophytic bacteria were randomly selected from agar-solidified trypticase soy broth, and identified using fatty acid methyl ester (FAME) profiles. Based on FAME profiles, 18 bacterial genera were identified with a similarity index > 0.3, but 73% of the identified isolates belonged to four genera: Bacillus (29%), Flavobacterium (12%), Micrococcus (20%) and Rathayibacter (12%). The endophytic community had a lower Shannon-Weaver diversity index (1.35) compared to the rhizoplane (2.15), and a higher proportion of Bacillus, Flavobacterium, Micrococcus and Rathayibacter genera compared to rhizoplane populations. Genera identified in the endophytic isolates were also found in the rhizoplane isolates. Furthermore, principal component analysis indicated three clusters of bacteria regardless of their site of origin, i.e., rhizoplane or endophytic. In addition, the rhizoplane communities of canola and wheat grown at the same site differed significantly. These results indicate that diverse groups of bacteria are associated with field-grown plants and that endophytes are a subset of the rhizoplane community. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Abstract: Inoculating Dahurian wild rye (Elymus dauricus) or meadow brome (Bromus biebersteinii) with a combination of Pseudomonas aeruginosa strain R75 and P. savastanoi strain CB35 increases degradation of 2-chlorobenzoic acid (2CBA) in soil. In this study the effect of these inoculants on the composition and activity of the root surface microbial community of these plants was investigated. The diversity of substrates utilized by the root-associated microbial community was assessed using Biolog GN and GP plates. The communities were also characterized by extracting fatty acid methyl esters (FAME) from roots of the plant-bacteria associations grown in soil. The capacity of the root surface and rhizosphere soil to degrade 2CBA also was assessed. Inoculating Dahurian wild rye increased the potential of rhizosphere soil to degrade 2CBA by 30%, bur had no effect on Biolog substrate utilization patterns or on root FAME profiles. In contrast, inoculating meadow brome increased the potential of the root surface and associated microorganisms to degrade 2CBA 3.5-fold, and also increased the utilization of amine, amide and polymer Biolog substrates. A cluster analysis of FAME profiles indicated that inoculation had a greater effect on root-associated microbial communities of meadow brome compared to Dahurian wild rye. The combination of strains R75 and CB35 increased the potential of Dahurian wild rye and meadow brome in different manners. Inoculating Dahurian wild rye had little effect on the root surface microbial community but increased the rhizosphere soil's potential to degrade 2CBA. Conversely, inoculating meadow brome altered the root surface microbial community and increased the potential of the root surface and associated microorganisms to degrade 2CBA. This suggests that the mechanism by which bacterial inoculants promote phytoremediation differs between plants. (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: Plant characteristics are known to alter endophytic and rhizosphere microbial communities; however, the effect of crop breeding programs on the microbial endophytic and rhizosphere communities is not clear. The purpose of this study was to determine if root-associated microbial communities differed between three cultivars of canola (Brassica spp.) and wheat (Triticum spp.). Biolog(TM) analysis was used to characterize the microbial communities associated with the root interior and rhizosphere: soil of field grown canola (Parkland, Excel, and Quest cultivars) as well as wheat (PI 167549, Red Fife,and CDC Teal cultivars). Fatty acid methyl ester (FAME) profiles of roots and rhizosphere soil of the cultivars were also compared. These crop cultivars represent a continuum from older to the most recent crop cultivars, with Quest being a transgenic canola variety tolerant of the herbicide glyphosate. To the best of our knowledge, Quest is not directly related to Parkland or Excel. The endophytic community of Quest used the Biolog(TM) polymer, carbohydrate, amino acid, and miscellaneous functional guilds at a slower rate than the endophytic community of Excel or Parkland, Furthermore, there were lower levels of the microbial FAMEs, 18:0, 18:3 w6c (6,9,12), 16:0 2OH, and 15:0 2OH in the roots of Quest compared with Excel or Parkland. In contrast, there were no differences in the utilization rate of Biolog(TM) functional guilds or the microbial FAMEs in the roots of the three wheat cultivars studied. The correlation between the ability of endophytic and rhizosphere communities to utilize Biolog(TM) substrates was lower in Quest and CDC Teal compared with earlier crop cultivars. Our results indicate that endophytic and rhizosphere microbial communities of the transgenic cultivar Quest were different from nontransgenic cultivars grown at the same field site.
Abstract: The ability of bacteria to alter phenotypic characteristics during their survival and persistence in soil complicates the risk assessment of bacterial inoculants, especially those genetically engineered. The purpose of this study was to investigate changes in the phenotype of Pseudomonas aureofaciens 3732 RN-LII (lacZY) introduced into and recovered from wheat roots or soil obtained from the field or laboratory microcosms. Fatty acid methyl ester (FAME) and Biolog(TM) analyses were used to characterize the phenotype of 88 isolates obtained from soil collected over a 2-year period. There was variation in the relative proportions of fatty acids found in isolates over the growing season, with FAME profiles of isolates obtained at 14, 28 and 70 days after planting different from isolates obtained at day 0 and day 140. However, these changes were not sufficient to alter the species designation. Similarly, carbon substrate utilization by isolates varied only slightly over the growing season. In addition, there were few phenotypic differences found between isolates obtained from rhizosphere or bulk soil, and no observed differences between isolates recovered from field soil or microcosms. Our results indicate that there was little phenotypic drift of this genetically engineered bacterium during its survival in field and laboratory microcosm soils. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Abstract: The purpose of this study was to assess the potential of prairie grasses as bioindicators of toxicants in soil and to investigate the effect different soil types and organisms have upon the germination of plant bioindicators. As a model compound, we used 2-chlorobenzoic acid (2CBA), a hydrophilic, polar compound present in the degradation pathways of halogenated aromatics. The germination response of prairie grasses to Aroclor(R) 1260-contaminated soil, with concentrations ranging from 13 to 133 mu g kg(-1) total polychlorinated biphenyls, was also investigated. The grasses responded to a wide range of contamination levels, with a 12- and 10-fold difference in the sensitivity of grass species to 2CBA and Aroclor, respectively. Canada blue grass (Pea compressa) and slender wheatgrass (Agropyron trachycaulum) were selected for further study of the effects of soil type and biological treatments on bioindicator response to 2CBA. Canada blue grass response in three of four soils was characterized by Y=110-26xIn(X), while in the fourth soil it was Y=94-1.6xX, where Y equals percent germination and X the 2CBA concentration. Slender wheatgrass response was Y=140-23xIn(X), with no significant difference between soil types. Previous biological treatments of soil significantly affected the response of slender wheatgrass as a bioindicator. Growing plants or plants inoculated with bacteria in noncontaminated soil before slender wheatgrass was planted inhibited emergence, changing the logarithmic relationship between germination and 2CBA concentrations to Y=100-0.28xX. Prairie grasses are potentially useful bioindicators of chlorinated aromatics in soil, but biological interactions may alter the bioindicator response.
Abstract: Biological remediation of contaminated soil is an effective method of reducing risk to human and ecosystem health. Bacteria and plants might be used to enhance remediation of soil pollutants in situ. This study assessed the potential of bacteria (12 isolates), plants (16 forage grasses), and plant-bacteria associations (selected pairings) to remediate 2-chlorobenzoic acid(2CBA)-contaminated soil. Initially, grass viability was assessed in 2CBA-contaminated soil. Soil was contaminated with 2CBA, forage grasses were grown under growth chamber conditions for 42 or 60 d, and the 2CBA concentration in soil was determined by gas chromatography. Only five of 16 forage grasses grew in 2CBA-treated (816 mg/kg) soil. Growth of Bromus inermis had no effect on 2CBA concentration, whereas Agropyron intermedium, B. biebersteinii, A. riparum, and Elymus dauricus decreased 2CBA relative to nonplanted control soil by 32 to 42%. The 12 bacteria isolates were screened for their ability to promote the germination of the five grasses in 2CBA-contaminated soil. Inoculation of A. riparum with Pseudomonas aeruginosa strain R75, a proven plant growth-promoting rhizobacterium, increased seed germination by 80% and disappearance of 2CBA by 20% relative to noninoculated plants. Inoculation of E. dauricus with a mixture of P. savastanoi strain CB35. a 2CBA-degrading bacterium. and P. aeruginosa strain R75 increased disappearance of 2CBA by 112% relative to noninoculated plants. No clear relationship between enhanced 2CBA disappearance and increased plant biomass was found. These results suggest that specific plant-microbial systems can be developed to enhance remediation of pollutants in soil.
