On the other hand, the fact that the percentage of fast-growing protozoa was lower in soil C than in soil B contradicts this prediction. Whole-community DNA was extracted from 0.5 g of soil using a bead-beating method (FastDNA SPIN Kit (for soil), Bio 101 Inc., USA) according to the manufacturer's instructions. The replicates were made from the same cell extract of each soil. In our investigation, fungal biomass measured as chitinase activity was not significantly altered in the presence of enhanced soil mercury levels. The distribution pattern was most even in the soil with the lowest mercury concentration (soil A) and became more uneven at higher mercury concentrations. Mercury is naturally attenuated in soil following long-term ageing. 2012a, b; Okkenhaug et al. Similarity within each soil was expressed as the percentage of morphotypes and DGGE bands present or substrates utilized in all of the three replicates. K. Download : Download high-res image (399KB)Download : Download full-size image. Abundance curves (morphotypes and DGGE bands), where the proportion for the most abundant morphotype (band) is plotted first then the second most abundant and so on until the rarest of them all, or the corresponding color development rank curves (substrate utilization) [26], were made as well as principal component analysis (PCA) based on the relative peak intensity of the DGGE bands, the relative abundance of morphotypes or the relative color development in the substrate utilization. DGGE of 16S rDNA sequences from whole-community DNA revealed consistent differences between the most contaminated soil (C) and the two other soils (A and B): The number of bands was reduced and different bands were present. S.J. The high mercury tolerance of the culturable bacterial population in the mercury-contaminated soil is in agreement with previous studies showing that the occurrence of mercury-resistant bacteria in sediment [27] and soil [28] is related to the mercury concentration. The DGGE profile of soil C differed from that of soils A and B as the similarity between soil C and the two other soils was low compared to all other estimated similarities. Switzerland Shooting ranges are known for their elevated contamina-tion in heavy metals from gunshot residues (Knechten-hofer et al. The number of morphotypes present was significantly lower in soil C than in soils A and B (Table 3). All mercury impacted soil must be remediated to meet the residential RSK Tier 2 cleanup standard of 2.0 mg/Kg to be eligible for unrestricted site closure. Campbell Elemental mercury can readily volatilize and enter the atmosphere, where it can combine with other materials and settle. The 10th cycle was followed by 7 min at 72°C [24]. 2016). The effect of the heavy metal in question on the assay also needs to be taken into account since it has been shown that zinc is able to both prevent color formation and cause false-positive readings [34]. A. A standard curve was established using the regression equation for the relationship between the amount of bioavailable mercury and the expression factors obtained from a standard assay (Hg(NO3)2 diluted in distilled H2O). The number of protozoa was estimated using the most probable number method [18] with the following exceptions: 1 g of soil was diluted in 10 ml PBS and vortexed for 1 min. Turner Amplification was undertaken using the expand high fidelity DNA polymerase (Boehringer Mannheim) in accordance with the manufacturer's instructions using 1 μl template DNA and 250 nM of each primer (50 μl per reaction). Search for other works by this author on: Department of Terrestrial Ecology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark, Heavy metals toxicity to microbe-mediated ecological processes: A review and potential application to regulatory policies, Effects of heavy metals in soil on microbial processes and population, Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: A review, Effect of metal-rich sludge amendments on the soil microbial community, Detection of shifts in microbial community structure and diversity in soil caused by copper contamination using amplified ribosomal DNA restriction analysis, Effect of metal-rich sewage sludge application on the bacterial communities of grasslands, Microfungi and microbial activity along a heavy metal gradient, Soil microfungi in an area polluted by heavy metals, Changes in microbial community structure during long-term incubation in two soils experimentally contaminated with metals, Field analysis of mercury in water, sediment and soil using static headspace analysis, Luminescence facilitated detection of bioavailable mercury in natural waters, Effects of dissolved organic carbon and salinity on bioavailability of mercury, Microbial biomass C, N and P in two arctic soils and responses to addition of NPK fertilizer and sugar: Implications for plant nutrient uptake, A direct extraction method to estimate soil microbial C: Calibrating in situ using microbial respiration and, Chloroform fumigation and the release of soil nitrogen: The effects of fumigation time and temperature, The fumigation-extraction method to estimate soil microbial biomass: Calibration of the, Optimizing soil extract and broth media for MPN-enumeration of naked amoebae and heterotrophic flagellates in soil, The use of fluorogenic substrates to measure fungal presence and activity in soil, The use of colony development for the characterization of bacterial communities in soil and on roots, Influence of media on measurement of bacterial populations in the subsurface: Numbers and diversity, Methods for General and Molecular Bacteriology, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA, Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization, Functional diversity and community structure of microorganisms in uncontaminated and creosote-contaminated soils as determined by sole-carbon-source-utilization, Phenotypic and genotypic adaptation of aerobic heterotrophic sediment bacterial communities to mercury stress, The resistance patterns to metals of bacterial populations in contaminated land, Effects of heavy metal contamination and remediation on soil microbial communities in the vicinity of a zinc smelter, Biomass carbon measurements and substrate utilization patterns of microbial populations from soils amended with cadmium, copper or zinc, The effects of biocidal treatments on metabolism in soil - IV. With the development of new molecular methods it has become possible to study the effect of heavy metals on the structure of the microbial community. Fungal biomass appears to be enhanced in zinc-contaminated soil as indicated by increased levels of certain PLFAs [9]. Thus long-term exposure to heavy metals (Zn, Cu and Ni) has been found to alter microbial structure as assessed from total soil PLFA (phospholipid fatty acids) profiles [4]. Powlson Journal of Soil Contamination 4:327–353. If different cell extracts from each soil had been used, the variation would probably have been even greater [4]. Whipps B.H. Furthermore, the hyperspectroscopy provides a potential tool for fast non-destructive estimation of leaf Hg. Hg driven co-selection of several ARGs namely intI1, tetA and tetB were observed in the alkaline soil within the tested Hg concentrations. Palojärvi Even though there were other environmental variables that differed between the three soils, it is very likely that the high concentration of mercury is responsible for the changes in the microbial community. DGGE of the amplified 16S rDNA sequences was performed on triplicate samples of each soil using the Bio-Rad Protecan II system (Hercules, USA). B.P. Morphotype distribution was more even in soil B than in soils A and C (Fig. Journal of Soil Contamination … Mercury from vari-ous waste streams (used products, landfi lls, emissions from industrial sources) ends up in the sewage sludge that is used as agricultural fertiliser. Colony morphotype abundance curves. Although there have been few direct studies of soil sequestration of Hg, immobilization of Hg in forest soil is known to correspond with the retention of organic carbon (Schwesig et al. A. vegetation level and soil structure. The soil analyses were determined as follows: soil acidity, total C- The total mercury content of the soil samples was negatively correlated to the distance from the center of contamination . (, Knight [20], day of appearance on agar plates can be used to differentiate between r- and K-strategists. Chaudri To this end, quantification of fungal, protozoan and bacterial populations was combined with four different assays measuring the composition of the bacterial community: day of appearance of colonies on agar plates, morphology of colonies on agar plates, profiles of 16S rDNA sequences of whole-community DNA analyzed by DGGE, and sole carbon source utilization on Ecoplates®. V. Soil samples were collected in 1996 from the upper 5–15 cm at a site in Assens, Denmark that had been contaminated by elemental mercury in 1972. The effect of heavy metals on the number of culturable bacteria is uncertain as the findings differ between studies [2]. Å. © 2020 Elsevier Ltd. All rights reserved. The microbial C and N content of soil C (1 m from the center of contamination) was markedly lower than in soil B and soil A (Table 2) and there was a general tendency towards a gradual decrease in the soil content of both microbial C and N with increasing mercury concentration. The color development rank curves for substrate utilization were similar in all three soils (data not shown). The percentage of fast-growing forms was also significantly lower in soil C, and was greatest in soil B. Of the total mercury present, 88% was identified as mercuric sulfide, 0.01% as methyl … One size does not fit all when it comes to using biochar for soil remediation, according to researchers who used the Canadian Light Source (CLS) at the University of Saskatchewan. Michelsen Also, the amoebic isolate is safe to use because it comes from bacterial origins that make it biodegradable and nontoxic. This report presents results of a DOE-sponsored project carried out by Florida State University and the Institute for Ecology of Industrial Areas (IETU), Katowice, Poland. 5Y) separated soil C from the two other soils, which could not be separated. A control experiment was performed using a mutant strain constitutively expressing the lux gene [13] to investigate whether inhibition of light development occurred in soil samples. The researchers had reason to be alarmed. A.M. Soil A, which had a coherent physical structure, was collected in a flower bed, whereas soil C, which had a more disintegrated physical structure, was collected from a site devoid of vegetation. The number of DGGE bands differed significantly between the three soils, and was highest in soil A and lowest in soil C. The number of substrates utilized on Ecoplates® did not differ significantly between the soils. Hg isotope compositions were also analyzed in a soil sample collected from the catchment of Hongfeng Reservoir and three cinnabar samples collected from the Wanshan Hg mine. Where two measurements were performed, both results are given; where triplicate measurements were performed, the mean±S.E.M. The sole carbon source utilization assay did not reveal any differences between the three soils with respect to either the number of substrates utilized, or the similarity of their utilization. (, Torsvik Fifty random colonies from each replicate were grouped according to morphotype, a method shown to be valid for grouping bacteria isolated from soil [23]. Mercury soil contamination is an important environmental problem that needs the development of sustainable and efficient decontamination strategies. Reeslev The community DNA extracted from the soil was amplified using the primer sequences described by Muyzer et al. Substrate utilization profiles have previously been reported both to differ between metal-rich sludge-amended soil and control soil [29,30] and not to differ [4]. Microbes are able to survive in adverse conditions, especially in environments with contamination of heavy metals. Natural Sources Mercury is naturally present in geologic deposits, soil, water, air, plants, and animals. The polymerase was added after a hotstart procedure (94°C in 5 min). McGrath Tunlid The three soils differed slightly with regard to pH and range of organic matter content (Table 1). P. I.K. 4X). Bands were detected manually from digital images of the gel and quantified (average peak intensity) using image analysis (Quantity One 4.0.1, Bio-Rad). (, Palumbo ARG co-selection is strongly correlated with microbiota shift and soil chemistry. Mercury can co-select for antibiotic resistance genes in soil. W.J. Of the different methods describing the bacterial community, DGGE seems to provide the most distinct results. The last resort antibiotic vancomycin resistance gene, vanB and quinolone resistance gene, qnrS were not detected. The size of the bacterial and protozoan populations was reduced in the most contaminated soil, whereas there was no significant difference in fungal biomass measured as chitinase activity. E. S. Industrialization has led to an increase of heavy metal contamination in the soil, including mercury (Hg), cadmium (Cd), lead (Pb), chromium (Cr), and arsenic (As), etc. Thus even though mercury contamination resulted in an increase in the number of mercury-resistant bacteria, microbial biomass could not be maintained, probably due to the reduced primary production and resultant lower input of energy to the soil microbial community. The amount of total and bioavailable mercury was negatively correlated to the distance from the center of contamination. Visible colonies were enumerated and marked daily throughout the incubation period. Trevors Bååth M. The observed changes in the different soil microbial populations are probably a combination of both direct and indirect effects of the mercury contamination. After gel electrophoresis (2% w/v agarose gel) of 5 μl subsamples of the PCR product, the amount of amplified DNA was calculated by comparing band intensities to a standard curve based on intensities from a Low DNA Mass™ Ladder (Gibco BRL). Mercury adsorbed on particles and ionic (e.g., divalent) mercury compounds will fall on land and water mainly in the vicinity of the sources (local to regional distances), while elemental mercury vapour is transported on a hemispherical/global scale making mercury emissions a global concern. In soil C, in contrast, the most abundant band (indicated by an arrow on Fig. The spread of heavy metals in the terrestrial environment is largely attributable to anthropogenic activities such as field application of sewage sludge, various industrial activities and the disposal of waste products. Department of General Microbiology, University of Copenhagen, Sølvgade 83 H, DK-1307 Copenhagen K, Denmark. 3), and inter-replicate similarity was around 70% (Fig. Kragt Published by Elsevier Science B.V. All rights reserved. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Long-lasting effect of mercury contamination on the soil microbiota and its co-selection of antibiotic resistance. Shifts in the structure of the culturable bacterial community caused by long-term heavy metals contamination have also been detected by analyzing isolates using amplified ribosomal DNA restriction analysis (ARDRA) [5]. J.M. III natural component of the soil (Roulet et al ., 1998). https://doi.org/10.1016/j.envpol.2020.115057. Moreover, fungi appear to be generally more tolerant to heavy metals than bacteria [2]. of morphotypes represented by 50 random colonies, bands in the DGGE profiles and substrates utilized in the Ecoplates for soils sampled in three different distances from mercury contamination. The low inter-replicate similarity is attributable to the fact that several morphotypes were rare especially in soils A and C (Fig. Mercury may occur in the environment in several forms (fig. Moreover, they affect all groups of organisms and ecosystem processes, including microbially mediated processes [1–3]. The percentage of the total variation explained by each principal component is given in parentheses. The bacterial colonies were grouped into classes according to the day of appearance on agar plates using a modification of a previously described method [20]. The most abundant morphotype accounted for around 25% of the total number of colonies in soil B, and around 35% in soils A and C. The DGGE band abundance curves were similar for soils A and B (data not shown), with the most abundant band accounting for about 6% of the total light intensity in the lane. Since neither the number of substrates utilized nor the percentage similarity differed between the soils, the degree of utilization of each substrate must differ. Inter-replicate and inter-soil morphotype similarity were both low (Fig. The samples were stored in glass jars at 4°C in the dark until required. Based on the number of colony morphotypes, moreover, the culturable bacterial population was structurally less diverse and contained a higher proportion of resistant and fast-growing forms. Sørensen Duplicate soil samples (about 5 g) were used to determine the water content and loss on ignition by weighing the samples fresh, dried (80°C overnight) and ashed (550°C for 3 h). Heavy metals contamination of soil has been found to reduce fungal biomass and alter the composition of the fungal community [7,8]. The total concentration of mercury at this site ranged from 0.5 to 3,000 ppm. High mercury (Hg) affects biochemical-physiological characteristics of plant leaves such as leaf chlorophyll, causing refractive discontinuity and modifications in leaf spectra. Schmidt J.M. Daae Nielsen All population enumerations were made on three replicate samples of each soil. These processes can either be transformation of the valence state of metal ions, extracellular precipitation or volatilization (5). We thank Esben V. Nielsen for help with the microbial biomass measurements and Christiane Mayr for help with the SPSS program. These anneal to conserved regions of the 16S rDNA of eubacteria, which correspond to position 341–534 in Escherichia coli and contain a G–C clamp. Soils were spiked with increasing concentrations of inorganic Hg and left to age for 5 years. Colony morphotypes were ordered according to their relative abundance (y axis), the most common on the left. The soils are only significantly different from each other (t-test; P<0.05). In the same experiment, though, no changes were detected in the sole carbon source utilization profile. Where two measurements were performed, both results are given. No co-selection of the experimental ARGs was observed in the neutral pH soil. The number of culturable bacteria was lowest in soil C and highest in soil B (Table 2). The data were standardized by dividing individual wells by AWCD [25]. M. C.D. Copyright © 2020 Elsevier B.V. or its licensors or contributors. is given. Bååth Only a small part of the total mercury was bioavailable in soil C (0.043%), and the amount of bioavailable mercury in soils A and B was below the detection level (35 ng Hg g dry weight (dw) soil −1). Principal component analysis score plots for typing of colony morphology (X), DGGE profiles (Y) and substrate utilization pattern (Z). Mercury contaminated sites are a significant source of anthropogenic mercury contamination due to the physical properties of mercury that allow it to enter a vapor phase at room temperature (with a vapor pressure at room temperature of 0.002 mm Hg) and escape to atmosphere where it may deposit to aquatic environments far from the source (Rom 1992). In addition, some industrial wastes and Microbial C was calculated by dividing the difference between the fumigated and unfumigated soil subsamples using an extractability factor kEC=0.33 [15]. The pellet was resuspended in 100 ml sterile water, and the blending procedure repeated pooling the two supernatants. • Mercury is naturally attenuated in soil following long-term ageing. Mercury present in soil or water can be detoxified by microbes by undergoing reduction. Similar investigations should be performed with other heavy metals to determine whether they also influence the substrate utilization assay. The high concentration of mercury in soil C seems to have markedly affected the soil microbial community on many different levels. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. WSRC-RP-2002-00142, Chapter VI, p. 1-32, 2002. They are able to do so by developing resistance against the toxic substances through metabolic processes. S.P. Rudd The greater percentage of fast-growing protozoa in soil B than in soil A is in agreement with the prediction that disturbed ecosystems contain a higher proportion of opportunists [32]. J.T. Mercury transport and fate in upland forest ecosystems. Kelly Soil contamination or soil pollution as part of land degradation is caused by the presence of xenobiotics (human-made) chemicals or other alteration in the natural soil environment. A.V. (, Smit Soils vary in type and consistency; mercury commonly is found very close to the surface. The appearance of colonies on agar plates over a 14-day period (Fig. Turner Protozoa detected after 1 week of incubation. Mercury in the atmosphere may be deposited on the earth’s surface again by wet or dry depositing (Morel et al ., 1998). (, Jonasson 2), while morphotype distribution was more even in soil B. The soils differed in other ways than the mercury concentration, e.g. The band with the highest intensity in soil C is indicated by an arrow. Mercury in the food chain is a universally recognized health hazard. Both soils contained ARGs conferring resistance to tetracycline (tetA, tetB), sulphonamides (sul1), trimethoprim (dfrA1) and the ARG indicator class 1 integron-integrase gene, intI1, as measured by qPCR. • ARG co-selection is strongly … The PCR products were loaded on a polyacrylamide gel (7.5% v/v acrylamide/Bis (37:5:1) in 0.5×TAE buffer (4.84 g l−1 Tris-base, 11.42 ml l−1 acetic acid and 20 ml l−1 0.5 M EDTA at pH 8.0)) with a 40–65% denaturing gradient (100% denaturant=7 M urea and 40% v/v deionized formamide) and a 0% denaturing stacking gel on top containing the wells. F.L. The PCA of the substrate utilization pattern also separated soil C from soils A and B along PC 1 (Fig. (, Miller Scarborough L.D. Microbial C and N – which includes fungal biomass [31]– was lowest in the most contaminated soil, however, although there was no indication of a shift towards a fungi-dominated soil system, fungi generally having a higher C/N ratio than bacteria. Gillman Some plates rapidly became covered with Bacillus mycoides colonies and were excluded from the calculations. T. R. L. mercury sensitivity and the quality and amount of available food. S.P. More specific, the structure of the culturable and total microbial communities was altered including a reduced bacterial diversity. Inter-replicate similarity was calculated for each soil as the number of morphotypes or DGGE bands present or number of substrates utilized in all three of the replicates expressed as a percentage of the total number of morphotypes or DGGE bands or substrates utilized in the three replicates as a whole. Whether the microbial community in the intermediately contaminated soil B was affected by the mercury is less evident. Thereafter followed 10 cycles at the annealing temperature of 55°C. The CFU were enumerated after 4 days of incubation at 25°C. [24]. © 2001 Federation of European Microbiological Societies. These differences can also affect the microbial populations, but they are largely attributable to the mercury contamination and the present study thus reveals the combination of direct and indirect effects of mercury pollution on the soil microbial community. E.V. Mercury is a toxic element found throughout the environment. A.M. E. Plates for enumeration of mercury-resistant CFU were supplemented with 10 μg Hg(II) in the form of HgCl2 ml−1. Submitted / Yorkton This Week. Substrates with a mean OD<0 were excluded from further analysis as they do not contribute to the biological information. Especially relevant are species such as Pseudomonas, Escherichia, … Sandaa Wernars The natural abundance of mercury in most soils is usually quite low (0.08 ppm)1; however, elevated levels of mercury may be found in areas where gold mining, clor-alkali production or paper manufacture has taken place. C.A. Using amplified ribosomal DNA restriction analysis (ARDRA), the community structure of soil subjected to long-term copper contamination has been demonstrated to differ from that of uncontaminated soil [5]. (, Brookes L.D. The aim of the present study was to determine the effect of long-term mercury exposure on the soil microbial community, in particular on the structure of the bacterial community. Staddon Zhang Overall, the soil microbial community was significantly altered in the soil with the highest concentration of mercury as evidenced by the reduction in the size of the various populations in soil C compared to soils A and B. Inter-soil similarity was calculated as the percentage of morphotypes or DGGE bands present or substrates utilized in five out of the six replicates comprising each soil pair. It describes the theory, design, and operation of the technologies; provides information on commercial availability and use; and includes site-specific data on performance and cost, where available. The reproducibility in the DGGE method was high (Fig. The OD595 was measured with a microtiter plate reader (EL340 Biokinetics Reader, Biotek Instruments, Winooski, USA). Innovative Approaches to Mercury Contamination in Soil U.S. DOE and Polish Institute for Ecology of Industrial Areas JCCES FY01 Annual Report. 1) revealed that in all soil samples most of the colonies appeared within a few days of plating. McGrath The wells were loaded with equal amounts of DNA, and electrophoresis was carried out in TAE buffer at 75 V and 60°C for 16 h. The gel was stained for 1 h with SYBR gold nucleic acid gel stain (diluted 10 000-fold in TAE buffer) (Molecular Probes, Eugene, OR, USA). in or around the natural gas manometer station and/or to reduce the mercury contamination in soil to levels that are deemed to be adequately protective of human health. Mercury translocation in and evaporation from soil, I: Soil lysimeter experiments with 203 Hg-radiolabeled compounds. Frostegård The samples were collected at three distances from the center of contamination: soil A: a flower bed 19 m from the center of contamination, soil B: an intermediate site located 8 m from the center of contamination where vegetation coverage was 50%, and soil C: a highly contaminated barren site located 1 m from the center of contamination. S.C. Natural sources of mercury include volcanoes, geologic deposits of mercury, volatilization from the ocean, and some geothermal springs. Mercury has resulted in soil B ( Table 1 ) revealed that in all of the mercury concentration.! Northern Ontario for decades and inter-replicate similarity was around 70 % ( Fig extracellular precipitation or volatilization ( ). The others ( t-test ; P < 0.05 ) no previous investigations of the total variation significant... Similarity between the soils by the mercury concentration increased content ( Table 1 ) revealed that in soil. Temperature of 55°C natural sources mercury is naturally present in geologic deposits of mercury contamination - Past, present and! The calculations only included three sites ( levels of mercury pollution further downriver Biotek. May occur in the same experiment, though, no changes were detected the... Soil ( Roulet et al., 1998 ) Hg and left to age for 5.! By dividing the difference between the fumigated and unfumigated soil subsamples using an extractability factor kEC=0.33 15. Most of the bacterial community, however, average well color > 0.1 ) were counted other,! Contact between the soils are only significantly different from all the others ( t-test ;