Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples

An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment sizeof approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in-gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18SrDNA groups under standard polymerase chain reaction conditions.     

Method for evaluating the kinetics of dynamic gas extraction

Summary New methods have been developed for the study of the kinetics of the dynamic gas extraction process based on instrumental recording of the extraction curve and division into half-lives whose temporal values can be employed for the calculation of the distribution coefficient. The dependence of this process on the thermodynamical system as well as on instrumental factors for the testing of field equipment was determined using toluene dissolved in water, by means of which in connection with sorption process isolation, enrichment and preservation of volatile organic compounds in samples were achieved.

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Auswertung der Kinetik der dynamischen Gasextraktion

     

Ultrasound-assisted sequential extraction method for the evaluation of mobility of toxic elements in contaminated soils

A method for the fast sequential extraction of toxic elements in contaminated soil samples using an ultrasonic water bath, followed by determination with inductively coupled plasma optical emission spectrometry (ICP-OES), was developed and compared with other methods introduced in the literature. The five-step sequential extraction (Tessier scheme) was shortened using ultrasound-assisted sequential extraction (UASE). The optimization of the five-step sequential extraction was based on the analysis of SRM 2710 using Tessiers’s method as a reference. Several extracting solutions with different sonication times and temperatures were tested in the optimization procedure. Concentrations of arsenic, cadmium, copper, lead, and zinc were determined in SRM 2710, SRM 2711, and contaminated soil samples with high accuracy and precision. The certified acid-leachable concentrations of the SRM 2710 were obtained for all elements investigated by using an optimized UASE method. Tessiers’s method yielded total element concentrations that were too high. The determination of zinc in SRM 2711 yielded concentrations that were too low, whereas arsenic determination yielded concentrations that were too high by the UASE method and analysis by ICP-OES. The analysis of the SRMs showed that the UASE method is highly comparable with the other methods used for such purposes. The major advantages of the UASE method are the high treatment rate (40 samples simultaneously with a sonication time of 54 min) and a low sample and reagent usage.     

Selectivity assessment of an arsenic sequential extraction procedure for evaluating mobility in mine wastes

An optimized sequential extraction (SE) scheme for mine waste materials has been developed and tested for As partitioning over a range of pure As-bearing mineral phases, their model mixtures, and natural mine waste materials. This optimized SE procedure employs five extraction steps: (1) nitrogen-purged deionized water, 10 h; (2) 0.01 M NH₄H₂PO₄, 16 h; (3) 0.2 M NH₄-oxalate in the dark, pH3, 2 h; (4) 0.2 M NH₄-oxalate, pH3/80 °C, 4 h; (5) KClO₃/HCl/HNO₃ digestion. Selectivity and specificity tests on natural mine wastes and major pure As-bearing mineral phases showed that these As fractions appear to be primarily associated with: (1) readily soluble; (2) adsorbed; (3) amorphous and poorly-crystalline arsenates, oxides and hydroxosulfates of Fe; (4) well-crystalline arsenates, oxides, and hydroxosulfates of Fe; as well as (5) sulfides and arsenides. The specificity and selectivity of extractants, and the reproducibility of the optimized SE procedure were further verified by artificial model mineral mixtures and different natural mine waste materials. Partitioning data for extraction steps 3, 4, and 5 showed good agreement with those calculated in the model mineral mixtures (<15% difference), as well as that expected in different natural mine waste materials. The sum of the As recovered in the different extractant pools was not significantly different (89–112%) than the results for acid digestion. This suggests that the optimized SE scheme can reliably be employed for As partitioning in mine waste materials.     

熏蒸提取法对比测定不同土种中的微生物碳含量

土种是土壤发生分类中的最基础分类单元,基于土种的微生物量调查是土壤生物调查中的重要工作,其中微生物量以微生物碳含量表征。采用氯仿熏蒸提取法-碳光谱分析法和重铬酸钾氧化法对比测定土壤中的微生物碳含量,对于提取液中的有机碳含量,碳光谱分析法的检出限为0.11 mg/L,重铬酸钾氧化法的检出限为1.55 mg/L。在精密度试验中,对于熏蒸后的土样,碳光谱分析法的RSD为8.8%~9.9%,重铬酸钾氧化法的RSD为7.0%~17.6%。对于未熏蒸处理的土样,两种方法的RSD分别为5.3%~9.3%和9.3%~12.0%。微生物碳测试结果与土壤的水分含量相关性显著（p<0.05）。对比两种方法的测定结果,发现熏蒸土样的相对偏差范围为0.1%~49.4%,未熏蒸土样的相对偏差范围为0.0%~60.9%,低浓度样品的偏差较大。对于不同土种中微生物碳含量的测定,碳光谱分析法测得结果范围为146 μg/g dry soil~414 μg/g dry soil,重铬酸钾氧化法的范围为131 μg/g dry soil~407 μg/g dry soil,两种方法测定结果间无显著差异。结果表明,低浓度样品测定时采用重铬酸钾氧化法造成的误差较大,宜采用碳光谱分析法,两种方法均能有效测定不同土种中的微生物碳含量。     

On-line coupling of dynamic microwave-assisted extraction to solid-phase extraction for the determination of sulfonamide antibiotics in soil

A rapid technique based on dynamic microwave-assisted extraction (DMAE) coupled on-line with solid-phase extraction (SPE) was developed for the determination of sulfonamides (SAs) including sulfadiazine, sulfameter, sulfamonomethoxine and sulfaquinoxaline in soil. The SAs were first extracted with acetonitrile under the action of microwave energy, and then directly introduced into the SPE column which was packed with neutral alumina for preconcentration of analytes and clean-up of sample matrix. Subsequently, the SAs trapped on the alumina were eluted with 0.3% acetic acid aqueous solution and determined by liquid chromatography-tandem mass spectrometry. The DMAE parameters were optimized by the Box-Behnken design. Maximum extraction efficiency was achieved using 320 W of microwave power; 12 mL of extraction solvent and 0.8 mL min⁻¹ of extraction solvent flow rate. The limits of detection and quantification obtained are in the range of 1.4-4.8 ng g⁻¹ and 4.6-16.0 ng g⁻¹ for the SAs, respectively. The mean values of relative standard deviation of intra- and inter-day ranging from 2.7% to 5.3% and from 5.6% to 6.7% are obtained, respectively. The recoveries of SAs obtained by analyzing four spiked soil samples at three fortified levels (20 ng g⁻¹, 100 ng g⁻¹ and 500 ng g⁻¹) were from 82.6 ± 6.0% to 93.7 ± 5.5%. The effect of standing time of spiked soil sample on the SAs recoveries was examined. The recoveries of SAs decreased from (86.3-101.9)% to (37.6-47.5)% when the standing time changed from one day to four weeks.     

Comparison of different methods of extraction for the determination of polychlorinated dibenzo-p-dioxins in soil

Summary The application of five different extraction methods including the extraction with liquid and supercritical carbon dioxide were tested on various soil samples with regard to their extraction efficiency. The carbon dioxide liquid extraction has proven to be not suitable for these applications.

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Vergleich verschiedener Extraktionsverfahren zur Bestimmung von polychlorierten Dibenzo-p-dioxinen in Böden

     

Comparison of different extraction methods for the determination of polycyclic aromatic hydrocarbons in soil

Two different extraction methods for the determination of polycyclic aromatic hydrocarbons in soil are compared: the extraction in combination with ultrasonic treatment, and the Soxhlet extraction method according to DIN-draft 38414 Part 21. Different types of real soil were extracted and analysed by HPLC with diode-array detector and fluorescence detection. The results show that the efficiency of the ultrasonic method is comparable to the Soxhlet method.     

Comparison of the TEVA-Spec resin and liquid-liquid extraction methods for the separation of technetium in soil samples

When ICP-MS is used for⁹⁹Tc measurements, elements which interfere with⁹⁹Tc counting should be removed. In this study, a separation method using a novel chromatographic resin (TEVA·Spec^® resin) was compared with a liquid-liquid extraction (LLX) method for separation and concentration of⁹⁹Tc which was trapped in a solution after combustion of environmental soil samples. The results showed that Tc could be separated from Ru at high recoveries with both separation methods. Molybdenum, however, was not removed by LLX, while more than 99% of Mo in soil samples were removed by TEVA·Spec resin.     

Comparative study of pertechnetate reduction by thiourea using spectrophotometric,solvent extraction and ion-exchange methods

The results of a comparative study of pertechnetate reduction by thiourea in mineral acid (HCl, HNO₃, HClO₄, H₂SO₄) media using spectrophotometric, solvent extraction and ion-exchange chromatographic methods are presented. Changes of spectral characteristics of redox systems and distribution characteristics of reduced technetium (cationic) forms on extraction with bis(1,2-dicarbollide)cobaltic acid and on ion exchange on Dowex 50WX8 were established as a function of time and either aqueous or organic phase composition. Thiourea was tested as a reagent for reduction back-extraction of technetium from organic phases containing the amines Aliquat 336 and trilaurylmethylammonium as convenient extraction reagents for pertechnetate.     

The use of polarographic methods for the evaluation of the stability of element species in food extracts

Summary A new approach to the evaluation of binding stabilities and capacities of metals in their intrinsic matrices is presented. The applicability of polarography without previous matrix separation is demonstrated by the example of copper and nickel in food extracts. In such native solutions the interdependence of the metals and their potential binding partners is examined. The results show that copper is very specifically bound to sulfhydryl groups, whereas nickel is bound more or less unspecifically. Distribution patterns for binding stabilities and capacities are given for soybean flour and mate tea extract. Generally, copper has higher stabilities and capacities than nickel. As a special feature the displacement of nickel by copper is demonstrated.     

Comparative study of solvent extraction and thermal desorption methods for determining a wide range of volatile organic compounds in ambient air

This paper compares two analytical methods for determining levels of 90 volatile organic compounds (VOCs) commonly found in industrial and urban atmospheres. Both methods are based on two official methods for determining benzene levels and involve collecting samples by active adsorptive enrichment on solid sorbents. The first method involves solvent extraction and uses activated charcoal as a sorbent. After sampling, the sorbent is extracted with 1 mL of carbon disulfide and then 1 μL of the extract is analysed in a GC-MS. The second method involves thermal desorption (TD) and uses Tenax TA and Carbograph 1TD as sorbents, which allows the whole sample to be analysed. In general, the thermal desorption method showed the best repetitivity and recovery and the lowest limit of detection and quantification for all target compounds. Because of its lower sensitivity, the solvent extraction method needs the preconcentration of large sample volumes of air (720 L vs. 2.64 L for the thermal desorption method) to yield similar limits of detection.The performance of both methods in real samples was tested in a location near to a petrochemical complex. The results of the 24-h samples for the solvent extraction method were compared with the average of 12 2-h samples for the TD method. In some cases, both methods found differences in the VOC concentrations, especially in those compounds whose concentrations fluctuate significantly during the day.     

Pilot plant study of the microbial flora in a diesel fuel contaminated soil

This study focused on the impact of a recent soil pollution of diesel fuel on a site and its indigenous microbial flora. A pilot plant (0.5 m3) was set up and filled with a soil (about 700 kg), artificially and uniformly polluted with diesel fuel (7 g/kg). This plant was then chemically and biologically monitored during the whole experiment (about two years). During the monitoring, a morphological change of the microbial colonies was observed. This was probably due to the acclimation phenomena to the pollution. With batch kinetic studies (10 ml) and increasing the selective pressure of the pollutant, it was possible to select and isolate a microbial consortium and a single strain that developed the ability to use different diesel fuel fractions as carbon sources. GC-MS analytical techniques were used. Results showed that different fractions were degraded at different times. In the batch system, in 7 days, the microbial consortium degraded some aromatic hydrocarbons. The isolate strain, in 20 days, degraded linear hydrocarbons. After a two years acclimation, it was possible to obtain, from a pilot plant, a microbial consortium and a strain able to degrade diesel fuel, for a future bioremediation in situ process.     

Study of partitioning and dynamics of metals in contaminated soil using modified four-step BCR sequential extraction procedure

The modified four-step BCR sequential extraction procedure (exchangeable and weak acid available species, reducible, oxidisable and residual fractions) was used to examine the distribution of As, Cd, Cr, Cu, Pb, and Zn with soil depth in an area (Baia Mare — Bozanta, Romania) with both high natural level of elements considered as toxic and historical pollution resulting from nonferrous metallurgy. The BCR approach proved a high metal input of anthropogenic origin down to 40 cm, while at lower depths the naturally elevated metal content must be considered. Results of the partitioning study and XRD analysis of solid matrix showed the greatest potential for chemical remobilisation of Cd, Zn, and Cu in weak acidic medium as well as their affinity for the oxidisable fraction (organic matter/sulphide). The tendency of Cr, Pb, and As to be immobilised as residual or reducible species on Fe-Mn oxides was evident. Although the partitioning of As in chemically inactive forms such as scorodite (FeAsO₄ · 2H₂O) soluble under reducible conditions and beudantite (PbFe₃(AsO₄)(SO₄)(OH)₂)), a residual species soluble in acid media, chemical mobilisation from soil in groundwater was confirmed. Dynamic processes of metal retention in soil under different conditions, namely acidic, reducing or oxidisable, were predicted from the Pearsonșs correlation analysis of element species with soil characteristics and components such as Fe, Mn, organic matter content, pH, and total element content, respectively. At the moment of the study, soil and groundwater in the area were found to be polluted with As, Cd, Cu, Pb and Zn. Presented at the 35th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 26–30 May 2008.     

Evaluation of extraction methods for arsenic speciation in polluted soil and rotten ore by HPLC-HG-AFS analysis

Three extraction systems including shaking, ultrasonic and microwave-assisted extraction were evaluated. Water and phosphate buffer were tested for the extraction of arsenic compounds in polluted soil, describing the water-soluble or plant-available fraction. The stabilities and recoveries of various arsenic species indicated that no obvious changes of species occurred during the extraction process. The raw extracts were cleaned up by C₁₈ cartridge before analysis. Having optimized the extraction conditions, the arsenic species in polluted soil and ore from the different pollution sources were extracted by microwave-assisted extraction with 0.5 M phosphate buffer as extractant. Arsenic species were quantitatively determined by high performance liquid chromatography on-line coupled with hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS). As(III) and As(V) were the major arsenic species in the polluted soil samples resulting from irrigation by waste water. As^V was the only form found in the rotten ore sampled in mining area. During the extraction process, the recoveries of spiked As(III), As(V), DMA(V) and MMA(V) were 85.4 ± 7.2%, 80.2 ± 6.7%, 101.6 ± 6.7% and 98.8 ± 9.1%, respectively, showing that most water-soluble arsenic could be measured.     

Sprayed liquid-gas extraction in combination with ion mobility spectrometry: a novel approach for the fast determination of semi-volatile compounds in air and from contaminated surfaces

We developed a fast, simple and highly-efficient enrichment procedure for trace levels of semi volatile organic compounds from air and surfaces and combined it with ion mobility spectrometry as field-deployable and rapid analytical technique. Our new technique, the sprayed liquid-gas extraction, was developed and optimized to allow the enrichment of semi volatile organic compounds. The air sample is pumped through a flow blurring nebulizer together with water. The sprayed liquid is collected and the organic compounds are transferred from the water phase to n-hexane via a miniscale liquid-liquid extraction. 50 μL of the n-hexane extract is applied to a fiber tape. After the n-hexane has evaporated, the fiber tape is transferred to the thermodesorber unit of a GDA-X ion mobility spectrometer (Airsense, Schwerin, Germany). The whole sampling and the sample preparation procedure takes no longer than 15 min and only requires 2.5 mL organic solvent. The method was optimized for Malathion, a widely used organophosphate insecticide and an accepted simulant for the nerve-agent, VX. Malathion provides defined ion mobility spectra in both, the positive and negative mode. The positive spectra show one major peak with a reduced mobility of 1.197 cm² Vs^?1 and an additional peak at 1.449 cm² Vs^?1 with lower intensity. A major product ion peak of 1.720 cm² Vs^?1 can be detected in negative mode together with an additional peak of low intensity at 1.403 cm² Vs^?1. The detection limit of the ion mobility spectrometer is approximately 20 ng absolute.     

Fluidized-bed column method for automatic dynamic extraction and determination of trace element bioaccessibility in highly heterogeneous solid wastes

Dynamic flow-through extraction/fractionation methods have recently drawn much attention as appealing alternatives to the batchwise steady-state counterparts for the evaluation of environmentally available pools of potentially hazardous trace elements in solid matrices. The most critical weakness of flow-based column approaches lies in the small amount of solid that can be handled, whereby their applicability has been merely limited to date to the extraction of trace elements in highly homogeneous solid substrates; otherwise the representativeness of the test portion might not be assured.To tackle this limitation, we have devised an automated flow-through system incorporating a specially designed extraction column with a large volume capacity, wherein up to 2 g of solid sample could be handled without undue backpressure. The assembled flow setup was exploited for fast screening of potentially hazardous trace elements (namely, Cd, Cr, Cu, Pb, and Zn) in highly inhomogeneous municipal solid waste incineration (MSWI) bottom ashes. The pools of readily mobilizable metal forms were ascertained using the Toxicity Characteristic Leaching Procedure (TCLP) based on the usage of 0.1 mol L⁻¹ CH₃COOH as leachant and analysis of extracts by inductively coupled optical emission spectrometry. The application of a two-level full factorial (screening) design revealed that the effect of sample fluidization primarily but other experimental factors such as the solid to liquid ratio and extractant flow rate significantly influenced the leachability of given elements in raw bottom ashes at the 0.05 significance level.The analytical performance of the novel flow-based method capitalized on fluidized-bed extraction was evaluated in terms of accuracy, through the use of mass balance validation, reproducibility and operational time as compared to batchwise extraction and earlier flow injection/sequential injection microcolum-based leaching tests.