Soil biodiversity and DNA barcodes: opportunities and challenges

Soils encompass a huge diversity of organisms which mostly remains to be characterized due to a number of methodological and logistical issues. Nonetheless, remarkable progress has been made in recent years toward developing strategies to characterize and describe soil biodiversity, especially thanks to the development of molecular approaches relying on direct DNA extraction from the soil matrix.Metabarcoding can be applied to DNA from any environment or organism, and is gaining increasing prominence in biodiversity studies. This approach is already commonly used to characterize soil microbial communities and its application is now being extended to other soil organisms, i.e. meso- and macro-fauna.These developments offer unprecedented scientific and operational opportunities in order to better understand soil biodiversity distribution and dynamics, and to propose tools and strategies for biodiversity diagnosis. However, these opportunities also come with challenges that the scientific community must face. Such challenges are related to i) clarification of terminology, (ii) standardisation of methods and further methodological development for additional taxonomic groups, (iii) development of a common database, and (iv) ways to avoid waste of information and data derived from metabarcoding. In order to facilitate common application of metabarcoding in soil biodiversity assessment, we discuss these opportunities and challenges and propose solutions towards a more homogeneous framework.     

Long-term effects of devegetation on composition and activities (including transcription) of fungal communities of a semi-arid soil

A stable plant cover is essential in the semi-arid soils of the Mediterranean area to maintain their fertility and functionality. In a semi-arid area, we have studied abundance, structure, and presence of active species of fungal communities of a devegetated soil (disturbed soil) and vegetated soil (undisturbed soil). Disturbed soil was covered by small spontaneous vegetation (5–10%) compared to undisturbed soils (70%), and this decreased the content of the total organic C, microbial biomass, microbial activity (adenosine triphosphate), and fungal counts. The composition and activities of fungal communities were also investigated by direct extraction of DNA and RNA from soil. Denaturing gradient gel electrophoresis analysis of 18S ribosomal DNA and 18S ribosomal RNA profiles indicated that total and active fungal communities were changed after vegetation removal.     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

Plant residues do not have an immediate impact on soil bacterial community composition and abundance

Plant residues are often used as soil amendments in laboratory experiments, but they can reportedly release compounds interfering with soil DNA extraction and subsequent molecular biological analyses. Theoretically, for accurate comparison of microbial community composition in soils with and without added plant residues after a period of incubation, no significant difference at the beginning of the experiment is required between the amended and unamended control soils. We mixed plant residue into soil and immediately (within 10 min) commenced DNA extraction, and then performed 16S rRNA gene sequencing and quantitative PCR (qPCR) to determine bacterial community composition and abundance. Soil without plant residue addition served as a control. Five commonly used DNA extraction kits, 16S rRNA gene primer pairs, and soils, and two types (rice straw and alfalfa shoots) and three addition rates (2%, 4%, and 6%; w/w) of plant residue, were tested. In all cases, we found no significant difference in measured bacterial community composition or abundance between the treatments with and without added plant residue.     

稻秸对土壤细菌群落分子多态性的影响

模拟稻秸原位还田条件,分别在水稻土和红壤中添加水稻秸秆培养70d ,第0、5、2 5、4 5、70天采集土样。采用非机械破壁法直接提取水稻土和红壤细菌总DNA ,水稻土细菌总DNA经过二次纯化;红壤细菌总DNA经过一次纯化后,PCR扩增其16SrDNAV3可变区,均可获得清晰的目的条带,对扩增产物进行DGGE分析,结果显示:水稻土和红壤样品的DGGE条带增加,说明稻秸能够增加土壤细菌群落分子多态性的丰富度,随着培养期的延长,施有稻秸的处理中土壤细菌群落多态性的变化远远复杂于空白对照土壤中的细菌群落变化;同时发现在稻秸刺激下不同土壤细菌群落多态性高峰期出现时间不同     

土壤微生物多样性研究的新方法

传统的分离培养和鉴定土壤微生物方法所具有的困难性和局限性 ,是造成难以深入了解土壤微生物生态学特性和多样性组成方面的主要障碍。本文运用分子生物学技术 ,以澳大利亚两种主要森林类型的土壤微生物多样性研究为实例 ,介绍了从土壤中直接提取土壤微生物DNA的方法以及末端限制性酶切片段长度多态性 (T RFLP)分析的基本原理和方法。作者认为 ,用该方法提取的土壤真菌DNA的纯度高 ,完全适合PCR扩增和T RFLP分析的要求。T RFLP已成为国外深入研究土壤微生物多样性的理想方法之一     

Bacterial pH-optima for growth track soil pH, but are higher than expected at low pH

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.     

The influence of soil properties on the structure of bacterial and fungal communities across land-use types

Land-use change can have significant impacts on soil conditions and microbial communities are likely to respond to these changes. However, such responses are poorly characterized as few studies have examined how specific changes in edaphic characteristics do, or do not, influence the composition of soil bacterial and fungal communities across land-use types. Soil samples were collected from four replicated (n = 3) land-use types (hardwood and pine forests, cultivated and livestock pasture lands) in the southeastern US to assess the effects of land-use change on microbial community structure and distribution. We used quantitative PCR to estimate bacterial–fungal ratios and clone libraries targeting small-subunit rRNA genes to independently characterize the bacterial and fungal communities. Although some soil properties (soil texture and nutrient status) did significantly differ across land-use types, other edaphic factors (e.g., pH) did not vary consistently with land-use. Bacterial–fungal ratios were not significantly different across the land-uses and distinct land-use types did not necessarily harbor distinct soil fungal or bacterial communities. Rather, the composition of bacterial and fungal communities was most strongly correlated with specific soil properties. Soil pH was the best predictor of bacterial community composition across this landscape while fungal community composition was most closely associated with changes in soil nutrient status. Together these results suggest that specific changes in edaphic properties, not necessarily land-use type itself, may best predict shifts in microbial community composition across a given landscape. In addition, our results demonstrate the utility of using sequence-based approaches to concurrently analyze bacterial and fungal communities as such analyses provide detailed phylogenetic information on individual communities and permit the robust assessment of the biogeographical patterns exhibited by soil microbial communities.     

Assessing the relationship between patch type and soil mites: A molecular approach

An urgent need exists for indicators of soil health and patch functionality in extensive rangelands that can be measured efficiently and at low cost. Soil mites are candidate indicators, but their identification and handling is so specialised and time-consuming that their inclusion in routine monitoring is unlikely. The aim of this study was to measure the relationship between patch type and mite assemblages using a conventional approach. An additional aim was to determine if a molecular approach traditionally used for soil microbes could be adapted for soil mites to overcome some of the bottlenecks associated with soil fauna diversity assessment. Soil mite species abundance and diversity were measured using conventional ecological methods in soil from patches with perennial grass and litter cover (PGL), and compared to soil from bare patches with annual grasses and/or litter cover (BAL). Soil mite assemblages were also assessed using a molecular method called terminal-restriction fragment length polymorphism (T-RFLP) analysis. The conventional data showed a relationship between patch type and mite assemblage. The Prostigmata and Oribatida were well represented in the PGL sites, particularly the Aphelacaridae (Oribatida). For T-RFLP analysis, the mite community was represented by a series of DNA fragment lengths that reflected mite sequence diversity. The T-RFLP data showed a distinct difference in the mite assemblage between the patch types. Where possible, T-RFLP peaks were matched to mite families using a reference 18S rDNA database, and the Aphelacaridae prevalent in the conventional samples at PGL sites were identified, as were prostigmatids and oribatids. We identified limits to the T-RFLP approach and this included an inability to distinguish some species whose DNA sequences were similar. Despite these limitations, the data still showed a clear difference between sites, and the molecular taxonomic inferences also compared well with the conventional ecological data. The results from this study indicated that the T-RFLP approach was effective in measuring mite assemblages in this system. The power of this technique lies in the fact that species diversity and abundance data can be obtained quickly because of the time taken to process hundreds of samples, from soil DNA extraction to data output on the gene analyser, can be as little as 4 days.     

Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods

Several biochemical and molecular methods are used to investigate the microbial diversity and changes in microbial community structure in rhizospheres and bulk soils resulting from changes in management. We have compared the effects of plants on the microbial community, using several methods, in three different types of soils. Pots containing soil from three contrasting sites were planted with Lolium perenne (rye grass). Physiological (Biolog), biochemical (PLFA) and molecular (DGGE and TRFLP) fingerprinting methods were employed to study the change in soil microbial communities caused by the growth of rye grass. Different methods of DNA extraction and nested PCR on TRFLP profiles were examined to investigate whether they gave different views of community structure. Molecular methods were used for both fungal and bacterial diversity. Principal component analysis of Biolog data suggested a significant effect of the plants on the microbial community structure. We found significant effects of both soil type and plants on microbial communities in PLFA data. Data from TRFLP of soil bacterial communities showed large effects of soil type and smaller but significant effects of plants. Effects of plant growth on soil fungal communities were measured by TRFLP and DGGE. Multiple Procrustes analysis suggested that both methods gave similar results, with only soil types having a significant effect on fungal communities. However, TRFLP was more discriminatory as it generated more ribotype fragments for each sample than the number of bands detected by DGGE. Neither methods of DNA extraction nor the nested PCR had any effect on the evaluation of soil microbial community structure. In conclusion, the different methods of microbial fingerprinting gave qualitatively similar results when samples were processed consistently and compatible statistical methods used. However, the molecular methods were more discriminatory than the physiological and biochemical approaches. We believe results obtained from this experiment will have a major impact on soil microbial ecology in general and rhizosphere–microbial interaction studies in particular, as we showed that the different fingerprinting methods for microbial communities gave qualitatively similar results.     

Experimental warming effects on the microbial community of a temperate mountain forest soil

Soil microbial communities mediate the decomposition of soil organic matter (SOM). The amount of carbon (C) that is respired leaves the soil as CO₂ (soil respiration) and causes one of the greatest fluxes in the global carbon cycle. How soil microbial communities will respond to global warming, however, is not well understood. To elucidate the effect of warming on the microbial community we analyzed soil from the soil warming experiment Achenkirch, Austria. Soil of a mature spruce forest was warmed by 4 °C during snow-free seasons since 2004. Repeated soil sampling from control and warmed plots took place from 2008 until 2010. We monitored microbial biomass C and nitrogen (N). Microbial community composition was assessed by phospholipid fatty acid analysis (PLFA) and by quantitative real time polymerase chain reaction (qPCR) of ribosomal RNA genes. Microbial metabolic activity was estimated by soil respiration to biomass ratios and RNA to DNA ratios. Soil warming did not affect microbial biomass, nor did warming affect the abundances of most microbial groups. Warming significantly enhanced microbial metabolic activity in terms of soil respiration per amount of microbial biomass C. Microbial stress biomarkers were elevated in warmed plots. In summary, the 4 °C increase in soil temperature during the snow-free season had no influence on microbial community composition and biomass but strongly increased microbial metabolic activity and hence reduced carbon use efficiency.     

Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres

Lumbricus terrestris is a deep-burrowing anecic earthworm that builds permanent, vertical burrows with linings (e.g., drilosphere) that are stable and long-lived microhabitats for bacteria, fungi, micro- and mesofauna. We conducted the first non-culture based field study to assess simultaneously the drilosphere (here sampled as 0–2 mm burrow lining) composition of microbial and micro/mesofaunal communities relative to bulk soil. Our study also included a treatment of surface-applied ¹³C- and ¹⁵N-labeled plant residue to trace the short-term (40 d) translocation of residue C and N into the drilosphere, and potentially the assimilation of residue C into drilosphere microbial phospholipid fatty acids (PLFAs). Total C concentration was 23%, microbial PLFA biomass was 58%, and PLFAs associated with protozoa, nematodes, Collembola and other fauna were 200-to-300% greater in the drilosphere than in nearby bulk soil. Principal components analysis of community PLFAs revealed that distributions of Gram-negative bacteria and actinomycetes and other Gram-positive bacteria were highly variable among drilosphere samples, and that drilosphere communities were distinct from bulk soil communities due to the atypical distribution of PLFA biomarkers for micro- and mesofauna. The degree of microbial PLFA ¹³C enrichment in drilosphere soils receiving ¹³C-labeled residue was highly variable, and only one PLFA, 18:1ω9c, was significantly enriched. In contrast, 11 PLFAs from diverse microbial groups where enriched in response to residue amendment in bulk soil 0–5 cm deep. Among control soils, however, a significant δ¹³C shift between drilosphere and bulk soil at the same depth (5–15 cm) revealed the importance of L. terrestris for translocating perennial ryegrass-derived C into the soil at depth, where we estimated the contribution of the recent grass C (8 years) to be at least 26% of the drilosphere soil C. We conclude that L. terrestris facilitates the translocation of plant C into soil at depth and promotes the maintenance of distinct soil microbial and faunal communities that are unlike those found in the bulk soil.     

Linking species richness, biodiversity and ecosystem function in soil systems

Soils are the central organizing entities in terrestrial ecosystems and possess extremely diverse prokaryotic and eukaryotic biota. They are physically and chemically complex, with micro- and macro-aggregates embedded within a solid, liquid and gaseous matrix that is continually changing in response to natural and human-induced perturbations. Recent advances in molecular techniques in systematics have provided opportunities for the study of biodiversity and biocomplexity of soil biota. A symposium and workshop on soil biogeochemistry and biodiversity International Symposium on Impacts of Soil Biodiversity on Biogeochemical Processes in Ecosystems, Taiwan Forestry Research Institute, Taipei, Taiwan April 18-24, 2004. Convened an international array of participants working in biomes on virtually every continent on the planet (ranging from polar to tropical regions). This special issue reports on the theoretical bases and applications of molecular methods for the measurement of soil biodiversity.

Themes addressed include a melding of classical taxonomic investigations with biochemical fingerprinting and molecular probing of organism identities. Several papers highlight new advances in identifications of prokaryotic and eukaryotic organisms. Examples include new developments in “fingerprinting” of microbes active in “mycorrhizospheres” using immunocapture and other innovative techniques. Developments in the study of impacts of invasive plant and animal species on ecosystem function and subsequent microbial community composition and function have been very great in the last 2-3 years. Soils are major repositories of legacies, including fine and coarse woody debris and other organic products, which have feedbacks on soil diversity. The ways in which species diversity and function of microbial and faunal communities interact and their importance to ecosystem function are examined in biological and biochemical detail. This paper provides an overview of soil biodiversity and its feedbacks on soil biogeochemical processes in ecosystems.     

Molecular profiling of soil animal diversity in natural ecosystems: Incongruence of molecular and morphological results

A major problem facing ecologists is obtaining a complete picture of the highly complex soil community. While DNA-based methods are routinely used to assess prokaryote community structure and diversity in soil, approaches for measuring the total faunal community are not yet available. This is due to difficulties such as designing primers specific to a range of soil animals while excluding other eukaryotes. Instead, scientists use laborious and specialized taxonomic methods for extracting and identifying soil fauna. We examined this problem using DNA sequencing to profile soil animal diversity across two Alaskan ecosystems and compare the results with morphological analyses. Of 5267 sequences, representing 549 operational taxonomic units (OTU), only 18 OTUs were common to both sites. Representatives included 8 phyla, dominated by arthropods and nematodes. This is the most comprehensive molecular analysis of soil fauna to date, and provides a tool to rapidly assess a missing component of soil biodiversity.     

Soil microarthropod community testing: A new approach to increase the ecological relevance of effect data for pesticide risk assessment

In the present study, a new complementary approach combining the use of the natural soil microarthropod community and conventional test methods was used. The effects of soil contamination with the insecticide carbofuran on two geographically distinct microarthropod communities (Mediterranean and Tropical) were evaluated in their soils of origin under controlled laboratory conditions.After contamination of two agricultural soils from Portugal and Brazil, a gradient of concentrations was prepared. Soil cores were taken from the respective uncontaminated surrounding areas and the mesofauna of three cores was extracted directly to the test soil. After extracting the microarthropod communities to the test soil, these were incubated under laboratory conditions for 4 weeks, after which the mesofauna was extracted again. The organisms were assorted into higher taxonomic groups and Acari and Collembola were respectively assorted into order/sub-order/cohort and family. Collembolans were still classified according to morphological traits and used as a case-study of trait based risk assessment (TERA; Baird et al., 2008) of pesticides.The exposure to insecticide contamination caused the impoverishment of the taxonomic diversity in both communities. Significant shifts in the microarthropod community structure in the different carbofuran treatments were found for both soils, although effects were more pronounced in the assay performed with the soil from Brazil. Collembolans were the most affected group with a strong decline in their abundance. A dose–response relationship was observed, showing a consistent decline on the relative abundance of Isotomidae, closely followed by an increase of Entomobryidae. Contrastingly, Acari (especially Oribatida) tended to increase their numbers with higher concentrations.Trait based analysis of Collembola data suggested that a shift in the functional composition of the communities occurred due to carbofuran soil contamination and that species adapted to deeper soil layers were more vulnerable to insecticide toxicity.     

Shifts in bacterial community structure associated with inputs of low molecular weight carbon compounds to soil

Low molecular weight carbon (C) substrates are major drivers of bacterial activity and diversity in the soil environment. However, it is not well understood how specific low molecular weight C compounds, which are frequently found in root exudates and litter leachates, influence bacterial community structure or if there are specific groups of soil bacteria that preferentially respond to these C inputs. To address these knowledge gaps, we added three simple C substrates representative of common root exudate compounds (glucose, glycine, and citric acid) to microcosms containing three distinct soils from a grassland, hardwood forest, and coniferous forest. CO₂ production was assessed over a 24 h incubation period and, at the end of the incubation, DNA was extracted from the samples for assessment of bacterial community structure via bar-coded pyrosequencing of the 16S rRNA gene. All three C substrates significantly increased CO₂ production in all soils; however, there was no relationship between the magnitude of the increase in CO₂ production and the shift in bacterial community composition. All three substrates had significant effects on overall community structure with the changes primarily driven by relative increases in β-Proteobacteria, γ-Proteobacteria, and Actinobacteria. Citric acid additions had a particularly strong influence on bacterial communities, producing a 2-5-fold increase in the relative abundance of the β-Proteobacteria subphylum. These results suggest that although community-level responses to substrate additions vary depending on the substrate and soil in question, there are specific bacterial taxa that preferentially respond to the substrate additions across soil types.     

Determination of the optimal soil sample size to accurately characterise nematode communities in soil

Analysis of nematode communities is a potential biological proxy to monitor soil health. Traditionally, nematodes are extracted from 200 g of soil and then identified based on morphology. However, the optimal soil sample size to accurately characterise nematode communities using molecular methods is unknown. Using a combined relative real-time PCR and T-RFLP approach we analysed nematode communities extracted from triplicate samples ranging from 1 to 200 g soil. Our data indicated that for molecular based analyses, soil sample sizes <200 g of soil do not accurately represent the abundance of nematodes and <100 g samples are less likely to reflect the true community composition. Thus characterisation of nematode communities from low sample sizes may not be robust.     

Bacterial phylogenetic diversity and a novel candidate division of two humid region, sandy surface soils

The extent of microbial community diversity in two similar sandy surface soils from Virginia and Delaware (USA) was analysed with a culture-independent small subunit ribosomal RNA (SSU rRNA) gene-based cloning approach with about 400-700 SSU rDNA clones obtained from each sample. While there were no operational taxonomic units (OTUs) having more than three individuals, about 96-99% of the OTUs had only a single individual. The clones showing less than 85% similarity to the sequences in the current databases were fully sequenced. The majority of the clones (55%) had sequences that were more than 20% different from those in the current databases. About 37% of the clones differed by 15-20% in sequence from the database, 16% of the clones differed by 10-15%, and 5% of the clones differed by only 1-10%. Phylogenetic analysis indicated that these sequences fell into 10 of the 35-40 known phylogenetic divisions. Many of the clones were affiliated with Acidobacterium (35%). While a substantial portion of the clones belong to alpha (24%) and beta (12%) Proteobacteria, a few of them were affiliated with delta (6%) and gamma (3%) Proteobacteria. About 6% of the clones belong to Planctomycetes, and 4% of the clones were related to gram-positive bacteria. About 4% of clones were related to other bacterial divisions, including Cytophaga, Green sulfur bacteria, Nitrospira, OP10, and Verrucomicrobia. Eight sequences had no specific association with any of the known divisions or candidate divisions and were phylogenetically divided into three novel division level groups, named AD1, AD2 and AD3. Candidate division AD1 represented by six clones (4%) was found in both sites and consisted of two subdivisions. The community structures were similar between these two widely separated, sandy, oligotrophic, surface soils under grass vegetation in a temperate, humid climate but somewhat dissimilar to community structures revealed in similar studies in other types of soil habitats.     

Urbanization alters the functional composition,but not taxonomic diversity,of the soil nematode community

We evaluated the response of riparian forest soil nematode community structure to the physico-chemical environment associated with urban land use. Soils were sampled seasonally between December 2000 and October 2002 along an urban—rural transect in Asheville, North Carolina. We characterized the taxonomic (to genus) and functional composition (trophic groups) of the nematode community of forest soils, as well as several nematode ecological indicators (maturity index, channel index, weighted faunal index). The diversity of nematode genera was not affected by urban land use. However, there tended to be functional differences in the nematode communities along the land use gradient. The urban soils tended to have lower abundances of predatory and omnivorous nematodes. Differences in channel index scores indicated that there was less fungal dominance in the soil food webs of the urban soils. Our results indicate that the functional composition of the soil food web is an important component of soil biodiversity that can be affected by land use practices. This study was conducted in a relatively small city; hence the influence of pollutants on the soil environment was not as great as in larger cities. Correspondingly, the impact on the soil nematode community was not very severe. The utilization of the nematode community assemblage as an indicator of soil conditions should be further explored in urban places of differing magnitudes of environmental effects.     

Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis

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.