An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

Aims: To evaluate six commercial DNA extraction kits for their ability to isolate PCR‐quality DNA from Bacillus spores in various soil samples. Methods and Results: Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real‐time PCR assay that included an internal positive control. The FastDNA^® SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.^® Soil DNA and PowerSoil^® DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. Conclusions: The results of this study suggest that commercially available extraction kits can be used to extract PCR‐quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. Significance and Impact of the Study: The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real‐time PCR detection of a pathogen in soil.     

Influx and efflux of arsenic in cotton fields irrigated with arsenic-contaminated groundwater

Abstract

Samples of rhizosphere soil, groundwater used for drip irrigation, and mature cotton plants were collected from farms at Kuitun, Xinjiang and analyzed for their arsenic (As) levels to account for the influx and efflux of arsenic in the field. The pH value and the soil-grain-size distribution were found to correlate with the different levels of arsenic accumulated by cotton plants and arable soil layers. The principal component analysis (PCA) indicates that the arsenic level in soil is influenced by the silt content and the arsenic level in groundwater, while the arsenic level in plant roots is affected mainly by the silt content and the arsenic level in soil. As estimated, about 64.6?±?2.7?μg of arsenic was extracted from soil by one cotton plant, and more than 44% of it was stored in husks. About 0.8-2.6 mg As/(m² yr) was extracted by plants from soil according to the average dry weight of the plant and the field planting density, while 2.4–28.0?mg As/(m² yr) entered the soil based on the irrigated groundwater volume and the arsenic level in groundwater. Taking account of the cotton residues left after harvest, up to 85.8% of arsenic in the plants returned to the soil. After calculation, about 0.7–2.2?mg As/(m² yr) returned to the soil, and thus the actual net arsenic input to the soil could reach 2.3–27.6?mg As/(m² yr). Because of an obvious rise of the soil arsenic level in Kuitun, Xinjiang, and certain preventive measures should be taken to prevent the propagation of arsenic in the agricultural field.     

Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10³ to 1.8 × 10³ CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

A real‐time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil

Aim: To develop a rapid real‐time PCR method for the specific detection and quantification of Bacillus thuringiensis var. israelensis (Bti) spores present in the environment. Methods and Results: Seven soil samples as well as one sediment sample obtained from various regions of Switzerland and characterized by different granulometry, pH values, organic matter and carbonate content were artificially inoculated with known amounts of Bti spores. After DNA extraction, DNA templates were amplified using TaqMan real‐time PCR targeting the cry4Aa and cry4Ba plasmid genes encoding two insecticidal toxins (δ‐endotoxins), and quantitative standard curves were created for each sample. Physicochemical characteristics of the samples tested did not influence DNA extraction efficiency. Real‐time PCR inhibition because of the presence of co‐extracted humic substances from the soil was observed only for undiluted DNA extracts from samples with very high organic matter content (68%). The developed real‐time PCR system proved to be sensitive, detecting down to 1 × 10³ Bti spores per g soil. One‐way analysis of variance confirmed the accuracy of the method. Conclusions: Direct extraction of DNA from environmental samples without culturing, followed by a specific real‐time PCR allowed for a fast and reliable identification and quantification of Bti spores in soil and sediment. Significance and Impact of the Study: The developed real‐time PCR system can be used as a tool for ecological surveys of areas where treatments with Bti are carried out.     

Determination of trace cadmium in saliva samples using spray assisted droplet formation-liquid phase microextraction prior to the measurement by slotted quartz tube-flame atomic absorption spectrophotometry

BackgroundAn effective, green and rapid analytical strategy namely the simultaneous spray assisted droplet formation-liquid phase microextraction (S-SADF-LPME) method was developed for the determination of trace quantity of cadmium in saliva samples by using the slotted quartz tube-flame atomic absorption spectrophotometry (SQT-FAAS). By the developed method, external dispersive solvent usage for droplet formation was reduced to obtain a more environmental-friendly method.MethodsMethod consists of a simultaneous complexing and extraction step, which was based on spraying an extraction solvent containing a solid ligand into the aqueous sample solution, forming fine droplets without the use of dispersive solvent. The procedure was implemented using a customized, cost effective and portable spray apparatus to minimize the consumption of reagent, analysis time and operation steps. Thus, this methodology ensures better repeatability and accuracy while minimizing the relative errors caused by the experimental steps. Parameters including the buffer amount, extractant/ligand concentration, extraction solvent type, extraction/ligand solution volume, spraying number and vortex period were systemically optimized to lower the detection limit.ResultsUnder the optimal extraction conditions, 96.9-folds enhancement in the detection power of the traditional FAAS was achieved. The limit of detection and limit of quantification values of presented method were calculated to be 0.65 and 2.17 ng mL⁻¹, respectively. Accuracy and applicability of the optimized method was investigated by collecting saliva samples from smokers. Satisfactory percent recovery values wereachieved for cadmium with a low standard deviation in the acceptable range of 84.9–109.6 %.ConclusionThe developed dispersive solvent-free S-SADF-LPME technique presents a fast, simple, cost-effective and eco-friendly microextraction method based on the use of an easily accessible and functional spray apparatus.     

Arsenic in Agricultural Soils of a Historically Mined and Industrial Region of Southern Serbia and Northern Kosovo: Bioavailability and Uptake by Plants Species Zea mays L. and Solanum tuberosum L.

This article reports the results of a study focused on the presence and bioavailability of arsenic in agricultural soil in the mining and industrial regions of northern Kosovo and southern Serbia, as well as uptake and bioaccumulation of arsenic in two commonly cultivated plant species (Zea mays L. and Solanum tuberosum L.). This area was one of the most important mining districts in Europe. The collected soil samples were subjected to a modified BCR three-step sequential extraction procedure in order to investigate the chemical partitioning of arsenic in the soils. The general distribution of arsenic in various fractions was: exchangeable < reducible < oxidizable fractions. Highest concentrations of total arsenic in soil were found close to industrial facilities and tailing ponds. In addition, fluvisols were significantly more enriched with arsenic than soils at a distance from the river flows. The edible parts of the plant specimen showed different As contents, suggesting that these plant species have different propensities for the uptake and bioaccumulation of arsenic from soil.     

Trace-Level Determination of Oxolinic Acid and Flumequine in Soil,River Bed Sediment,and River Water Using Microwave-Assisted Extraction and High-Performance Liquid Chromatography with Fluorimetric Detection

This work presents the optimization of analytical procedures for the determination of two antibiotics, oxolinic acid (OA) and flumequine (FL), in bed sediment, river water, and soil samples. Three extraction methods (microwave-assisted extraction (MAE), ultrasonication, and reflux) were tested, and the highest recoveries were obtained with MAE (94 ± 3% and 95 ± 3% for OA and FL, respectively). A solid-phase extraction (SPE) clean-up step was optimized by comparing two polymeric sorbents: Oasis HLB and Oasis MAX. The final extracts were analyzed by liquid chromatography with fluorimetric detection. Limits of detection (LOD) obtained for OA and FL in soil and sediment ranged from 0.3 to 0.5 µg kg^?1. Meanwhile, a novel SPE procedure was also implemented for OA and FL determination in river water samples. It also relied on the use of Oasis MAX, and recovery rates were in the range 90–94%; LODs were 2 ng L^?1 for both OA and FL. These methods were applied for the analysis of samples taken in the Seine River basin (France). The obtained results demonstrated the widespread occurrence of OA and FL, at ng L^?1 and µg kg^?1 levels in water and sediment/soil, respectively, and their persistence in the environment.     

Microwave-Assisted Process (MAPTM) for the Extraction of Contaminants from Soil

Laboratory-scale tests were performed to evaluate the use of Environment Canada's patented Microwave-Assisted Process (MAP_TM) for the extraction of petroleum hydrocarbons from contaminated soil. The purpose of these tests was to determine the potential for using the process for large-scale processing of contaminated soil. Tests were performed using three soil types: a certified sediment and certified soil, both contaminated with polycyclic aromatic hydrocarbons (PAHs), and spiked peat soil contaminated with long-chain petroleum hydrocarbons. The test methods used were based on existing MAP techniques that have been proven for the sample preparation of contaminated soils for analytical purposes. The parameters evaluated concentrated on those that are amenable to a continuous large-scale process running at atmospheric pressures. This meant using solvents that are inexpensive and readily available in large volumes, low solvent to material ratios, and optimized energy inputs. In general, it was found that microwaves could be used to enhance the solvent extraction of the contaminants from the soil and that the properties of the soil greatly affected the extent to which the contaminants were removed.     

Characterization of an ecotype of brake-fern, Pteris vittata, for arsenic tolerance and accumulation in plant biomass

An ecotype of brake fern (Pteris vittata) was assessed for arsenic tolerance and accumulation in its biomass under in vivo and in vitro condition; using soil, and agar-gelled Murashige and Skoog (MS) medium supplemented with different concentrations of arsenic. The plants were raised in soil amended with 100–1000 mg arsenic kg⁻¹ soil, and MS medium was supplemented with 10–300 mg arsenic 1⁻¹ medium using Na₂HAsO₄ · 7H₂O. The spores and haploid gametophytic-prothalli were raised in vitro on MS medium supplemented with arsenic. The field plants showed normal growth and biomass formation in arsenic amended soil, and accumulated 1908–4700 mg arsenic kg⁻¹ dry aerial biomass after 10 weeks of growth. Arsenic toxicity was observed above >200 mg arsenic kg⁻¹ soil. The concentrations of arsenic accumulated in the plant biomass were statistically significant (p < 0.05). Normal plants were developed from spores and gametophyte prothalli on the MS media supplemented with 50–200 mg arsenic 1⁻¹ medium. The in vitro raised plants were tolerant to 300 mg arsenic kg⁻¹ of soil and accumulated up to 3232 mg arsenic kg⁻¹ dry aerial biomass that showed better growth performance, biomass generation and arsenic accumulation in comparison to the field plants. The text was submitted by the authors in English.     

Distinguishing the effects of vegetation restoration on runoff and sediment generation on simulated rainfall on the hillslopes of the loess plateau of China

Aims

Since the 1970s, extensive croplands were converted to forest and pasture lands to control severe soil erosion on the Loess Plateau of China. We quantify the direct and indirect effects of vegetation restoration on runoff and sediment yield on hillslopes in the field to improve environmental governance.

Methods

An artificial rainfall experiment at a rainfall intensity of 120 mm h⁻¹ and a slope gradient of 22° were used to distinguish the effects of vegetation restoration on runoff and sediment yield.

Results

Compared to the farmland slopes, vegetation restoration directly prolonged the time-to-runoff by 140%, reduced the runoff rate by 20%, and increased the soil infiltration capacity by 15%. Vegetation restoration indirectly delayed the time-to-runoff by 120%, reduced the runoff rate and sediment yield rate by 50% and 94%, respectively, and increased the soil infiltration capacity by 58% on the hillslopes with vegetation restoration.

Conclusions

The direct effects of vegetation restoration on runoff and sediment yield were lower than its indirect impacts. Vegetation cover, decreases in soil bulk density, and increases in belowground root biomasses and > 0.25 mm aggregate stability were the primary causes of runoff and sediment yield reduction on the slopes with vegetation restoration.

     

The contents of risk elements,arsenic speciation,and possible interactions of elements and betalains in beetroot (<Emphasis Type="Italic">Beta vulgaris</Emphasis>, L.) growing in contaminated soil

The effect of enhanced soil risk element contents on the uptake of As, Cd, Pb, and Zn was determined in two pot experiments. Simultaneously, transformation of arsenic and its compounds in beetroot (Beta vulgaris L.) plants was investigated. The mobile fractions of elements were determined in 0.05 mol L⁻¹ (NH₄)₂SO₄ extracts and did not exceed 2% of total soil arsenic, 9% of total cadmium, 3% of total lead, and 8% of total zinc, respectively. Although the soils were extremely contaminated the mobile portions of the elements represented only a small fragment of the total element content. Arsenic contents in beet plants reached up to 25 mg As kg⁻¹ in roots and 48 mg As kg⁻¹ in leaves in the soil characterized by the highest mobile arsenic portion. Arsenic portions extractable with water and phosphate buffer from the beetroot samples did not show significant differences between the extraction agents but the extractability was affected by the arsenic concentration. Arsenic was almost quantitatively extractable from the samples with the lowest total arsenic concentration, whereas in the samples with the highest total arsenic concentration less than 25% was extractable. Arsenate was the dominant arsenic compound in the extracts (70% in phosphate buffer, 50% in water extracts). A small portion of dimethylarsinic acid, not exceeding 0.5%, was detected only in the sample growing in the soil with the highest arsenic concentration. The role of betalains (betanin, isobetanin, vulgaxanthin I and vulgaxanthin II) in transformation/detoxification of arsenic in plants was not confirmed in this experiment because the plants were able to grow in the contaminated soil without any symptoms of arsenic toxicity.     

Speciation of trace metals in chromated-copper-arsenate contaminated sediment of Ogba River

Abstract

The impact of storm water run-off from a location of intensive application of CCA in wood treatment on the quality of sediment of the receiving water body was examined. Sediment samples were collected along Ogba River. Physico-chemical characteristics as well as total levels of As, Cr and Cu in sediment samples were determined. Physico-chemical analysis of composite sediment samples upstream and downstream showed pH of sediments to be neutral (6.95 upstream and 6.90 downstream). The sand content for downstream sediment was 90.52 and 87.52% for upstream sediment sample. Total levels of 3.3–14.9 mg kg^?1 As, 3.1–116.6 mg kg^?1 Cr, and 13.7–16.4mg kg^?1 Cu were obtained for downstream sediment samples and 2.2–2.3mg kg^?1 As, 8.1–13.1 mg kg^?1 Cr, and 6.0–7.4mg kg^?1 Cu were obtained for upstream samples. Sequential extraction procedure involving six steps was used to evaluate the partitioning of As, Cr and Cu in the sediments. The results showed that the bioavailable fraction of sediment 20 m downstream was 10%, 100% and 20% of the total levels of As, Cr and Cu respectively in the sediment.     

Equilibrium solution composition and exchange properties of disturbed and undisturbed soil samples from an acid forest soil

Microscalic heterogeneity of soil chemical properties caused by soil structure has been reported for several soils. We investigated exchange properties and soil solution composition of disturbed and undisturbed samples of an acid forest soil lacking visible structure. Cation concentrations in the soil solution resulting from two extraction procedures and two analytical methods were compared. The effective cation exchange capacity (CEC_e) of the undisturbed sample represented 56–69% of the bulk soil CEC_e. Base saturation of undisturbed samples equalled that of disturbed samples for EA, Bhs, and Bsh horizons, and was higher for the Bw horizon. Contradicting the results of other authors, soil pore solution obtained by percolating soil cores under conditions of low water tension offered more favourable conditions for plant roots when compared to the equilibrium soil solution of the bulk soil sample in all except the Bsh horizon. Ca²⁺/Al³⁺ molar ratios were higher and fractions of H⁺ + Al³⁺ on total cationic charge were lower in the soil pore solution. These results were obtained employing soil: solution ratios of about 1:0.5 during the extraction of soil pore solution, and by determination of free cations. Other authors used a water extraction with soil:solution ratios up to 1:2 and took total metal for ion concentrations. The combination of the latter extraction and analytical method in our study, too, led to unfavourable Ca²⁺/Al³⁺ ratios and high tractions of H⁺ + Al³⁺. The choice of analytical and extraction method are thus decisive for the valuation of the soil solution composition in view of plant nutrition.     

Phosphorus bioavailability of fluvial sediments determined by algal assays

Eroding bank soils and riverine suspended sediments from the Flathead River-Lake ecosystem, Montana, USA, were cultured with the alga Selenastrum capricornutum Printz in PAAP medium with the sediments as the sole source of phosphorus. Extraction of phosphorus by NaOH and nitrilotriacetic acid (NTA) solutions were compared to results from algal bioassays. The fine sediment particles transported into Flathead Lake during spring runoff had the highest availability (i.e. 6% of total phosphorus). Bank soils which contained the greatest percentage of fine clays exhibited similar (i.e. 4% of total phosphorus) availability. Bank soils containing predominantly organic phosphorus had the lowest availability. Spearman's rank correlation indicated significance at the 5% test level between algal assay estimates of available phosphorus and both chemical extraction techniques.     

Validation of chemical speciation model of inorganic arsenic in river waters with mobilisation processes

Abstract

The thermodynamic model of inorganic arsenic (acid–base, complexation, precipitation and redox equilibria) was validated with results obtained in arsenic mobilisation processes taking place in several river sediment samples of the Basque Country (North of Spain). Natural waters in contact with the sediment were collected at each sampling point, together with the sediment samples. These waters were analysed and then used as the extractant in the mobilisation studies. Considering pH, redox potential and total concentration values of the main cations and anions, the stoichiometric equilibrium constant were computed for each river water situation. The activity coefficient values of the inorganic arsenic species were estimated by means of the Modified Bromley Methodology (MBM). Both water and sediment for each sampling site were used in the mobility experiments, and the results were compared with the theoretical predictions of the thermodynamic model extrapolated to the chemical conditions of the sampling point. Moreover, a correlation analysis was also performed taking into account all the data and sampling sites; these results were also discussed and compared with the theoretical speciation obtained from the basic model of inorganic arsenic.     

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1 × 10⁶ cells/cm³. This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.     

Uptake of arsenic by mushrooms from soil

ABSTRACT

Graphite furnace atomic absorption spectrometry (standard addition method) was used to determine the total arsenic in wild-growing mushrooms after digestion with nitric acid, then with perchloric acid and in associated soils after digestion with mixtures of nitric and hydrofluoric acids in a microwave system. Among 83 species of mushrooms the highest concentrations of arsenic on a dry mass basis were found in Laccaria amethystea (26–125 mg kg^?1), Laccaria laccata (11–33 mg kg^?1), Thelephora terrestris (38 mg kg^?1), Boletus cavipes (11.6 mg kg^?1) and Ramaria botrytis (10 mg kg^?1). Mushroom caps of L. laccata, L. amethystea, and B. cavipes had approximately double the arsenic concentrations found in stems. The arsenic concentrations in caps of L. amethystea and L. laccata were directly proportional to the concentrations in the soils. The concentrations of arsenic in the soils were in the range 6.5–65 mg kg^?1. Among the 19 mushroom caps with arsenic concentrations above the method detection limit of 0.2 mg As/kg dry mass, only L. amethystea and L. laccata had arsenic concentration ratios ‘cap/soil’ higher than 1 (between 1.1 and 1.9). Thelephora terrestris had a ratio of 2.37.     

Arsenopyrite Weathering and Leaching of Arsenic in an Austrian Soil

The extent of arsenopyrite weathering in relation to co-existing minerals in an Austrian soil and the leaching of arsenic from the soil has been investigated. Soil and underlying bedrock samples were collected and characterized by chemical and mineralogical analyses. The solubility of the soil arsenic under anaerobic conditions was studied by incubating the soil sample in distilled water for different periods of time using a customized lycimeter. The solubility of arsenic from pure arsenopyrite mineral and mixtures of arsenopyrite with chalcopyrite or pyrite was studied by incubating the pulverized minerals. Speciation of arsenic in the incubated and non-incubated soil samples was carried out by sequential leaching, solvent-extraction, and ion exchange chromatographic techniques.

Results of SEM analysis indicated that arsenopyite (FeAsS), the most common mineral in the area, occurs in paragenesis with pyrite (FeS₂) and chalcopyrite (CuFeS₂). The existence of these minerals with arsenopyrite was found to enhance its solubilization. From the speciation study it was found that nearly all (92%) of the arsenic in the soil exists in the inorganic form. Out of the total inorganic arsenic, the trivalent inorganic species accounted for only 3% and the remaining 89% was found to be the pentavalent form. The low solubility of As in the Graz soil is attributed to the prevalence of this pentavalent inorganic species.     

Irrigation of Three Wetland Species and a Hyperaccumlating Fern with Arsenic-Laden Solutions: Observations of Growth,Arsenic Uptake,Nutrient Status,and Chlorophyll Content

Engineered wetlands can be an integral part of a treatment strategy for remediating arsenic-contaminated wastewater, wherein, As is removed by adsorption to soil particles, chemical transformation, precipitation, or accumulation by plants. The remediation process could be optimized by choosing plant species that take up As throughout the seasonal growing period. This report details experiments that utilize wetland plant species native to Ohio (Carex stricta, Pycnanthemum virginianum, and Spartina pectinata) that exhibit seasonally related maximal growth rates, plus one hyperaccumulating fern (Pteris vittata) that was used to compare arsenic tolerance. All plants were irrigated with control or As-laden nutrient solutions (either 0, 1.5, or 25 mg As L^?1) for 52 d. Biomass, nutrient content, and chlorophyll content were compared between plants treated and control plants (n = 5). At the higher concentration of arsenic (25 mg L^?1), plant biomass, leaf area, and total chlorophyll were all lower than values in control plants. A tolerance index, based on total plant biomass at the end of the experiment, indicated C. stricta (0.99) and S. pectinata (0.84) were more tolerant than the other plant species when irrigated with 1.5 mg As L^?1. These plant species can be considered as candidates for engineered wetlands.