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1.
国内首次运用FastPrep○R 核酸快速提取系统提取了重金属复合污染农田土壤的DNA ,并对其进行了聚合酶链反应—变性梯度凝胶电泳 (PCR DGGE)分析。结果表明 ,FastPrep○R核酸提取仪与相应的FastD NASPINKitforSoil试剂盒联用时 ,能有效地分离到纯度较高的重金属污染农田土壤的DNA。PCR DGGE电泳图谱表明 ,PCR产物经DGGE检测后得到的电泳条带清晰且分离效果好 ,可以明显反映出重金属复合污染导致了农田土壤微生物在基因上的损伤 ,影响到农田土壤生态系统的细菌丰富度 ,改变了土壤环境的优势菌群 ,从而使农田土壤微生物群落结构多样性发生变化。可见 ,FastPrep○R核酸提取系统同样适用于重金属污染农田土壤环境中微生物基因组DNA的快速分离和纯化 ,得到的DNA可直接用于PCR DGGE分析。  相似文献   

2.
We characterized humic acids (HAs) and glycerol-extractable organic fractions (GEOFs) extracted from four Andisols, taken from comparable soil-climate conditions on the east side of Mount Etna. The soils were formed on old lava (about 9 000 years ago), old tephra (about 8 700 years ago), recent lava (about 2 600 years ago) and recent tephra (about 3 600 years ago). A part of the organic matter of the soils, deprived of HAs and fulvic acids (FAs), was isolated by glycerol extraction. The GEOF can not be extracted with alkaline solutions, probably because it is closely bound to the mineral component of the soil. The characterization of the extracted organic fraction was carried out using elementary and functional group analysis and Fourier transform infrared (FT-IR) spectroscopy. About 20 extractions were necessary to extract the HA and FA from the older soils and about 10 extractions to extract them from the younger soils. Data showed that the GEOFs had a greater ash content and a smaller N content, as well as a greater presence of aliphatic compounds and carboxylic groups as compared to the HA extracted from the same soil. The GEOFs extracted from younger soils also had a lower yield, ash and COOH-group content, and were more aliphatic than the GEOF extracted from older soils. Finally, the GEOFs were more closely bound to the amorphous component of the soil (‘short-range’ minerals) and consequently less subject to biodegradation.  相似文献   

3.
The biodegradation of polyaromatic hydrocarbons (PAHs) has been well documented; however, the biodegradation of PAHs in contaminated soil has proved to be problematic. Sorption of PAHs to soil over time can significantly decrease their availability for extraction much less than for biodegradation. In this study the ability of various organic solvents to extract PAHs from coal tar-contaminated soil obtained from former manufactured gas plant (MGP) sites was investigated. Solvents investigated included acetone/hexane, dichloromethane, ethanol, methanol, toluene, and water. The extraction of MGP soils with solvents was investigated using soxhlet extraction, multiple soxhlet extractions, sonication, and brief agitation at ambient temperature with a range of solvent concentrations. Of particular interest was the documentation of the recalcitrance of PAHs in weathered MGP soils to extraction and to bioremediation, as well as to demonstrate the ease with which PAHs extracted from these soils can be biodegraded. The efficiency of extraction of PAHs from MGP soils was found to be more dependent upon the choice of solvent. The environmentally-benign solvent ethanol, was shown to be equal to if not better than acetone/hexane (the EPA recommended solvent) for the extraction of PAHs from MGP soils, brief contact/agitation times (minutes) using small quantities of ethanol (2 volumes or less) can achieve nearly quantitative extraction of PAHs from MGP soils. Moreover aqueous slurries of an MGP soils experienced less than 10% biodegradation of PAHs in 14 days while in the same period about 95% biodegradation was acieved using PAHs extracted from this soil by ethanol and subsequently added to aqueous bacterial suspensions.  相似文献   

4.
A comparative analysis of five methods of extraction and purification of soil DNA, including a modification of the authors, was performed for the further molecular investigation of various ammonium-oxidizing bacteria and archaea in soils. Experiments using soil samples from natural ecosystems and agroecosystems of the European area of Russia established that the amount of DNA extracted by different methods depended significantly on the type of soil. The subsequent molecular analysis (PCR-DGGE) of ribosomal (16S rRNA) and functional (amoA) genes demonstrated significant differences in the community structure of ammonium oxidizers depending on the method of DNA extraction. The best results were obtained for acidic soil (soddy-podzolic and gray forest soils) when using the method of Griffiths et al. [4] with our own modification. On the other hand, application of commercial DNA extraction kits was most efficient for soils with a high content of humus (black and chestnut soils). According to the results obtained, molecular analysis of soil microbe communities required selection of optimum conditions for DNA extraction, especially for soils with high contents of organic compounds and clay minerals at different pH levels.  相似文献   

5.
Many studies have highlighted the importance of the Amorphous Silica (ASi) pool to the overall mass balance in the biogeosphere. In order to advance our knowledge of measurements and quantification of this pool, it is necessary to compare the ability of different extractants to dissolve ASi in soils and to test methods developed in the aquatic sciences to soils systems. The methods used in this work included three acid extraction techniques (0.2 m NH4‐oxalate, 0.1 m NH4‐citrate and 0.5 m NH4‐acetate) and two alkaline extraction techniques (0.094 m Na2CO3 and 0.5 m NaOH), which are more commonly used for the measurement of ASi in aquatic sediments. Our results indicate that the amount of Si extracted from phytolith samples with the acid methods was two orders of magnitude less than the amount of extracted by alkaline extractions. When applied to natural soil samples, these extractions show that the acid techniques are only able to extract loosely‐bound components such as adsorbed Si and Si bound in amorphous matrices with Al and Fe. While Na2CO3 or NaOH extracted the same amount of ASi in Podzols, Na2CO3 was able to extract only part of the ASi extracted with NaOH in Chernozems. Pre‐treatment of the samples with 0.1 m HCl before the Na2CO3 extraction did not increase amounts of ASi extracted. The present work suggests that alkaline methods used commonly for ASi on aquatic sediment samples can be used on a wide variety of soils.  相似文献   

6.
 We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods, i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples. The profiles generated by all four methods were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community. However, the patterns of clustering also indicated that different populations of bacteria could be detected in the same soil using different soil DNA extraction methods. The application of two dilution levels of DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods. The indirect methods gave clustered profiles at both dilution levels. It is likely that these methods extracted DNA from a major, easily desorbed, bacterial fraction, consisting of low-density populations. PCR-DGGE was found to be a suitable technique with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination of microbial community diversity at the molecular level. Received: 22 June 1999  相似文献   

7.
Batch extraction of zein from dry‐milled whole corn with ethanol was optimum with 70% ethanol in water, an extraction time of 30–40 min, and temperature of 50°C. High yields (60% of the zein in corn) and high zein contents in the extracted solids (50%) were obtained at a solvent‐to‐solids ratio of 8 mL of 70% ethanol/g of corn. However, zein concentration in the extract was higher at lower ratios. Multiple extraction of the same corn with fresh ethanol resulted in a yield of 85% after four extractions, whereas multiple extractions of fresh corn with the same ethanol resulted in high (15 g/L) zein concentration in the extract. Optimum conditions for batch extraction of zein were 45°C, with 68% ethanol at a solvent‐to‐solids ratio of 7.8 mL/g for an extraction time of 55 min. Column extractions were also best at 50°C and 70% ethanol; a solvent ratio of 1 mL/g resulted in high zein concentrations in the extract (17 g/L) but yields were low (20%).  相似文献   

8.
Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996  相似文献   

9.
Abstract

Sequential extraction techniques have been used to make inferences about speciation of phosphorus (P) and to a lesser extent arsenic (As) in soils. However, sequential extraction studies on the less‐abundant group V element, antimony (Sb), are limited. In this work, a widely used P sequential extraction scheme was modified and used to extract P, As, and Sb from two acidic soils from the Macleay River floodplain, NSW, that were enriched with Sb (26.9 and 23.0 mg kg?1). An ammonium oxalate–oxalic acid step was included in the extraction sequence to dissolve the noncrystalline iron (Fe) and aluminium (Al) hydroxide phase. It extracted 30 to 47% of Sb, indicating the importance of this fraction, which may be mobilized in the floodplain by acid sulfate soil processes and periodic waterlogging. The original method overestimated P, As, and Sb in the residual fraction (30–71%). Relative efficiency values of extracts for P, As, and Sb were compared, and inferences about phase distributions were made. The results suggest some potential in using extractions to assess bioavailability of Sb in soil.  相似文献   

10.
In this study, the interrelation between nitrogen availability and prokaryotic diversity are studied using a well-characterised system from a long-term field experiment on a loamy sandy soil. The prokaryotic potential functional diversity and community composition were assessed using community-level physiological profiling (CLPP), and their phylogenetic diversity was analysed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in combination with sequencing analysis. Highest prokaryotic potential functional diversity was measured in the control soil receiving no N fertilisation, indicating an efficient as well as versatile utilisation of the substrates in this soil. Both substrate utilisation richness and substrate utilisation evenness, the two constituents of the functional diversity, were decreased with increasing N supply. Furthermore, distinct prokaryotic community compositions were generated in N-enriched soils compared to unfertilised control soils. These differences suggest a dominance of populations adapted to utilising readily available substrates. We demonstrated that the shift in prokaryotic functional diversity was connected to a shift in the phylogenetic structure of the bacterial and archaeal communities. Taken together, our data clearly show that, for the sandy soil system, prokaryotic diversity and N availability were interrelated.  相似文献   

11.
The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grinding-freezing-thawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel electrophoresis. This indicates the importance of the DNA extraction protocol on data for evaluating soil bacterial diversity. Consistently, the bead-beating procedure gave rise to the highest number of DNA bands, indicating the highest number of bacterial species. Supplementing the bead-beating procedure with additional cell-rupture steps generally did not change the bacterial community profile. The same consistency was not observed when evaluating the efficiency of the different methods on soil DNA yield. This parameter depended on soil type. The DNA size was of highest molecular weight with the sonication and grinding-freezing-thawing procedures (approx. 20 kb). In contrast, the inclusion of bead beating resulted in more sheared DNA (approx. 6-20 kb), and the longer the bead-beating time, the higher the fraction of low-molecular weight DNA. Clearly, the choice of DNA extraction protocol depends on soil type. We found, however, that for the analysis of indigenous soil bacterial communities the bead-beating procedure was appropriate because it is fast, reproducible, and gives very pure DNA of relatively high molecular weight. And very importantly, with this protocol the highest soil bacterial diversity was obtained. We believe that the choice of DNA extraction protocol will influence not only the determined phylogenetic diversity of indigenous microbial communities, but also the obtained functional diversity. This means that the detected presence of a functional gene—and thus the indication of enzyme activity—may depend on the nature of the applied DNA extraction procedure.  相似文献   

12.
Abstract

The chemical characterization of soil phosphorus (P) desorbed by anion and cation exchange membranes is of major importance to better understand which P forms are available to plants in short‐ and long‐term time periods. Two distinct soils, one acidic and one calcareous, were analyzed for P using two extraction procedures with mixed anion and cation exchange membranes. The short‐term (ST) experiment evaluated the effect of increasing the extraction periods up to 24 h, whereas the long‐term (LT) experiment consisted of a sequential extraction procedure using up to seven successive 24‐h extractions. In both experiments, the Chang and Jackson inorganic P fractionation methodology was carried out after each extraction treatment, and each treatment consisted of three replicates. Data were statistically analyzed by ANOVA and nonlinear regressions. In the ST experiment, increasing the extraction time increased the extracted P according to an asymptotic relationship (y=c?ab x ). Extracted P proceeded from the most labile fractions in the acidic soil. In calcareous soils, calcium phosphates may also contribute for extractable P. The LT experiment revealed that a single extraction, regardless of that extraction method, cannot predict the long‐term capacity of soils to supply P to the plants. An exponential relationship (P=a×n b ) was found between extracted P and the extraction number. Desorbed P proceeded from the most labile fractions in the acidic soil. However, in calcareous soils, some precaution is needed when considering the biological meaning of the results, because the occluded Fe phosphates also revealed significant decreases, probably due to the redox conditions in which these long extractions are performed.  相似文献   

13.
The soil accumulation of metals and other elements as a result of human activities is a global concern. This investigation involves the use of commercially available extractions to estimate elemental abundance and availability. The need exists, especially in poor nations, to have cost-effective analytical capabilities to perform an initial screening of a soil resource to determine if a problem exists. Three extraction protocols are proposed: (i) an aqua regia digestion to estimate a baseline geochemistry, (ii) a pyrophosphate extraction to estimate elemental abundances associated with the soil organic fraction, and (iii) a water extraction to estimate the most available fraction. If the soil resource is impacted, then more refined and traditional methods are required to document the extent of impact for possible remediation. The three extraction protocols were applied to soils with little impact to illustrate the assessment potential for selected metals and p-block elements.  相似文献   

14.
One of the most influential factors determining the growth and composition of soil bacterial communities is pH. However, soil pH is often correlated with many other factors, including nutrient availability and plant community, and causality among factors is not easily determined. If soil pH is directly influencing the bacterial community, this must lead to a bacterial community growth optimised for the in situ pH. Using one set of Iberian soils (46 soils covering pH 4.2-7.3) and one set of UK grassland soils (16 soils covering pH 3.3-7.5) we measured the pH-optima for the growth of bacterial communities. Bacterial growth was estimated by the leucine incorporation method. The pH-optima for bacterial growth were positively correlated with soil pH, demonstrating its direct influence on the soil bacterial community. We found that the pH from a water extraction better matched the bacterial growth optimum compared with salt extractions of soil. Furthermore, we also showed a more subtle pattern between bacterial pH growth optima and soil pH. While closely matched at neutral pHs, pH-optima became higher than the in situ pH in more acid soils, resulting in a difference of about one pH-unit at the low-pH end. We propose that an explanation for the pattern is an interaction between increasing overall bacterial growth with higher pHs and the unimodal pH-response for growth of bacterial communities.  相似文献   

15.
We used chemical extraction methods and 31P-nuclear magnetic resonance (NMR) to investigate the effects of vegetation on the amount and structural composition of phosphorous (P) in the sub-alpine soils of central Taiwan. Chemical extraction methods were used to measure inorganic P (Pi) and organic P (Po) in main soil horizons. The soil P composition was assessed by 31P-NMR spectroscopy on alkaline EDTA–NaOH extracts. According to the results of chemical extractions, the forest soil had a higher amount of Pi than the grassland soil, which might be a result of the mineralization of Po. 31P-NMR spectra showed inorganic orthophosphate (up to 67%) and orthophosphate monoesters (up to 75%) as the major forms of P extracted in forest and grassland soils, respectively. Smaller proportions of orthophosphate diesters and trace amounts of phosphonates and pyrophosphate were found. With possible hydrolysis of P compounds during chemical extraction and slight systemic error in the processes of extraction with NMR, the results from NMR analysis are, in general, consistent with those of chemical extraction.  相似文献   

16.
Soil microorganisms are key regulators of the biogeochemical phosphorus (P) cycle. Microbial P limitation in highly weathered tropical soils has been reported, but whether it affects the cellular P content of indigenous soil microorganisms and its biochemical composition is unknown. We investigated the effect of microbial P limitation by measuring respiration, microbial growth, community composition and P content of microbial cells in a Ferralsol with low amounts of available P as affected by amendments with C substrates with ample nitrogen (CN) with and without extra phosphate (P). Microbial biomass and community composition were quantified by phospholipid fatty acid (PLFA) analyses. Cellular P content and P pools (PLFA, DNA and RNA per cell) were determined after extraction of microbial cells from soil by density gradient centrifugation. The apparent microbial growth rate during exponential increase in respiration rates in response to CNP addition was 0.072 h−1, compared to 0.017 h−1 for the CN amendment (no extra P added). This suggests that the microbial growth after a combined C and N addition was retarded by P limitation in the native soil (without added P). The net increase in microbial biomass, however, reached similar levels for both the CN and CNP treatment (measured at the point in time when respiration rates peaked). This outcome was unexpected since maximum respiration rates were about three times higher in the CNP compared to the CN treatment. Total P in extracted cells ranged from 2.1 to 8.9 fg P cell−1 (microscopic counts), with a tendency for lower values for treatments without C amendments. Only 10-25% of the measured total P in extracted cells was accounted for by the measured RNA, DNA and PLFA. This low percentage could partly be due to underestimation of the RNA pool (degradation during extraction). PLFA analyses showed that substrate induced growth, regardless of P addition, led to a change in microbial community composition and was dominated by fungi. The extraction of microbial cells from soil by density gradient centrifugation, however, discriminates against fungi. Accordingly, the extracted cells were not fully representative for the entire soil microbiota regarding the community composition and metabolic state. Nevertheless, for the first time microbial cell P content and P pools are reported for microorganisms that actually grew in soil and not in chemostat or batch cultures.  相似文献   

17.
Abstract

Soil extraction techniques to measure the status of available micronutrients for plants are important in the diagnosis of deficiency or toxicity. Mehlich 3 (M3), EDTA (pH=8.2), DTPA‐TEA, and Soltanpour and Schwab (SS) solutions were confronted for their ability to extract simultaneously copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe). Argentinean soils from different taxonomic orders with widely varying properties were investigated. The values obtained showed that DTPA‐TEA and SS solutions extracted similar amounts of Zn, Fe, and Mn, while EDTA dissolved comparatively higher amounts of Fe and Mn. Mehlich 3 yielded the highest extractions for the four micronutrients. Soil pH not only affected the extraction of Mn by DTPA‐TEA, SS, and EDTA extractions, but also the extraction of Fe by EDTA. The organic carbon affected the determination of Fe and Zn in all cases. The correlations of the different tests for Cu, Zn, Mn, and Fe were significant. The results suggest that for the determination of the bioavailable status of micronutrients, any of the studied tests could be applied using the soil edaphic properties as factors to improve the correlations between them and standardize the methods.  相似文献   

18.
Solubility of the inorganic soil phosphorus and fertilizer phosphate The solubility of the inorganic soil phosphate of different soils was determined in up to 40 consecutive extractions with water and lactate solutions. The graphs showing the relationship between total extracted P, and P concentration in the extract ants (Fig. 1 and 3) were of an exponential type, independent of chemical bonding of the soil phosphorus, and independent of other soil properties. P contents in the first extractions were closely correlated with the sum of the P contents of all extractions with a solubility > 100 μg P/1 (fig. 2). All of the soil phosphate capable of diffusion is plant available. Therefore all phosphate in the soil which originated from fertilizer phosphate will be utilized. This follows from the data of solubility of the soil phosphorus (20–140 μg P/1) after repeated water and lactate (CAL) extractions. The slow adjustment of equilibrium, which is common with phosphate adsorption experiments is due to the influence of diffusion. The relationships between P fertilization, solubility of soil phosphate, and the recommended soil P content for economical P utilization with optimum plant growth (e.g. Tab. 2) is shown.  相似文献   

19.
Extracellular DNA in soil and sediment: fate and ecological relevance   总被引：1，自引：0，他引：1  
The review discusses origin, state and function of extracellular DNA in soils and sediments. Extracellular DNA can be released from prokaryotic and eukaryotic cells and can be protected against nuclease degradation by its adsorption on soil colloids and sand particles. Laboratory experiments have shown that DNA adsorbed by colloids and sand particles can be taken up by prokaryotic competent cells and be involved in natural transformation. Most of these experiments have been carried out under artificial conditions with pure DNA molecules and pure adsorbing matrices, but in soils and sediments, pure surface-reactive colloids are not present and DNA is present with other cellular components (wall debris, proteins, lipids, RNA, etc.) especially if released after cell lysis. The presence of inorganic compounds and organic molecules on both soil particles and DNA molecules can influence the DNA adsorption, degradation and transformation of competent cells. Extracellular DNA can be used as C, N and P sources by heterotrophic microorganisms and plays a significant role in bacterial biofilm formation. The nucleotides and nucleosides originated from the degradation of extracellular DNA can be re-assimilated by soil microorganisms. Extracellular DNA in soil can be leached and moved by water through the soil profile by capillarity. In this way, the extracellular DNA secreted by a cell can reach a competent bacterial cell far from the donor cell. Finally, the characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA.  相似文献   

20.
Extracting and purifying a representative fraction of bacteria from soil is necessary for the application of many techniques of microbial ecology. Here the influence of different soil types on the quantitative and qualitative recovery of bacteria by soil grinding and Nycodenz density gradient centrifugation was investigated. Three soils presenting contrasted physicochemical characteristics were used for this study. For each soil, the total (AODC: acridine orange direct count) and culturable (cfu: colony-forming units) bacterial densities were measured in three distinct fractions: (i) the primary soil, (ii) the soil pellet (soil remaining after centrifugation), and (iii) the extracted cells. The automated–ribosomal intergenic spacer analysis (A-RISA) was used to characterize the community structure directly from the DNA extracted from each fraction. The physicochemical characteristics of soils were found to influence both the efficiency of bacterial cell recovery and the representativeness of the extracted cells in term of community structures between the different fractions. Surprisingly, the most representative extracted cells were obtained from the soil exhibiting the lowest efficiency of cell recovery. Our results demonstrated that quantitative and qualitative cell recovery using Nycodenz density gradient centrifugation are not necessarily related and could be differentially biased according to soil type.  相似文献   

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