Fatty acid methyl ester (FAME) profiles as a tool to investigate community structure of two agricultural soils

Soil microbiological parameters may be the earliest predictors of soil quality changes. Recently, molecular techniques such as fatty acid methyl ester (FAME) profiles have been used to characterize soil microbial communities. Fatty acid methyl ester (FAME) from whole soil may be derived from live cells, dead cells, humic materials, as well as plant and root exudates. Our objective was to verify differences in FAME profiles from two agricultural soils with different plants. Soil samples were collected from Ritzville and Palouse silt loams for fatty acid analysis. Soil samples from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), jointed goatgrass ( Aegilops cylindrica L.) and downy brome (Bromus tectorum L.) rhizospheres were also collected for fatty acid analysis. Principal component analysis (PCA) of the two soils explained 42% of the variance on PC1, which accounted for Palouse soil. Ritzville soil accounted for 19% of the variance on PC2. Factor analysis showed that rhizosphere microbial communities from various plant species may differ depending on the plant species. Presence of Gram-positive bacteria as identified by a15:0, i15:0, a17:0 and i17:0 peaks were similar between rhizosphere and nonrhizosphere soils. Gram-negative bacteria characterized by short chain hydroxy acids (10:03OH and 12:03OH) as well as cyclopropane acids (cy17:0) were higher in rhizosphere soil than nonrhizosphere. This indicates a possible shift in the bacterial community to more Gram-negative bacteria and fewer Gram-positive bacteria in the rhizospheres of the plants species studied.     

Microbial community in the rhizosphere of young maize seedlings is susceptible to the impact of introduced pseudomonads as indicated by FAME analysis

Two species of Pseudomonas (i.e. P. chlororaphis or P. putida) derived from a maize rhizosphere were studied for their impact on the structure of the microbial community in the rhizosphere of young maize seedlings after inoculation. The culturable bacteria and total microbial communities were analyzed based on profiles of whole-cell fatty acid methyl esters (MIDI-FAME). The introduction of Pseudomonas species resulted in the shift from the Gram-positive dominated culturable community in the rhizosphere of uninoculated maize to more Gram-negative populations in the rhizospheres of the inoculated plants. For the total rhizosphere communities, 43, 47 and 42 FAMEs were detected in the uninoculated maize and the samples inoculated with P. chlororaphis or P. putida, respectively. In contrast to the culturable communities, low concentrations of marker FAMEs for Gram-positives (i15:0, a15:0, i16:0) were found in the profiles of the total rhizosphere communities. The maize inoculations resulted in an enrichment of some Gram-negative isolates; however, Gram-positive bacteria, Cytophaga/Flavobacterium and saprophytic fungi were found in the uninoculated rhizosphere.     

Microbial indices of soil fertility

AIMS: To find the new microbial parameters explaining the soil fertility from the microbial community viewpoint. METHODS AND RESULTS: Fatty acid methyl ester (FAME) analysis and terminal-restriction fragment length polymorphism (T-RFLP) analysis were carried out using 16 differently treated plots from the same field that had been kept under different fertilizer management systems since 1984. It was found that organic fertilizer application had small impact, whereas chemical fertilizer application, especially ammonium-nitrogen fertilizer, had strong impact on microbial community structures. Principal component analysis was conducted based on soil chemical and physical parameters, crop yields, FAMEs and terminal-restriction fragments (T-RFs) to provide 10 FAMEs and 10 T-RFs showing strong relation with soil fertility. CONCLUSION: We defined these 10 FAMEs and 10 T-RFs as 'keystone' biological parameters explaining soil fertility in the soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the FAMEs and T-RFs related to soil fertility. Both analyses are rapid, inexpensive and reproducible means. As field assessment needs precise and rapid analysis, FAME and T-RFLP analyses and these new parameters are very useful to analyse soil fertility at biological viewpoint.     

Use of the MIDI-FAME technique to characterize groundwater communities

Fatty acid methyl ester (FAME) profiles were identified directly from groundwater microbial communities concentrated on and extracted with polycarbonate filters. The sensitivity of this direct extraction method was determined using pure cultures of Acinetobacter junii, Pseudomonas putida and Stenotrophomonas maltophilia. A minimum concentration of 107 cells filter-1 was required to identify the predominant fatty acids from each culture. However, at least 3.7 x 109 cells filter-1 were required to obtain fatty acid profiles that matched the signature profiles for pure cultures in a commercial database. While several saturated fatty acids (i.e. 14 : 0, 16 : 0, 18 : 0) were extracted from the polycarbonate filters, they were readily subtracted from microbial fatty acid profiles and did not interfere with the characterization of pure cultures or environmental samples. For the environmental samples, 3 l of groundwater from the Savannah River Site, Aiken, SC, (USA) contained sufficient biomass for direct extraction. A comparative analysis of FAME groundwater profiles demonstrated a qualitative difference among communities sampled from spatially discrete locations, while a groundwater well that was sampled at two time points showed strong similarities over time. Concentration of microbial biomass on polycarbonate filters coupled with the MIDI-FAME extraction of both biomass and filter was a useful technique to characterize microbial communities from groundwater.     

Community Structure Comparison Using FAME Analysis of Desert Varnish and Soil,Mojave Desert,California

While a number of studies have shown that a close association exists between microorganisms and varnished rocks, there is little hard evidence to support the existence of either specific desert varnish communities, or any role these microbes might play in the genesis of the varnish layers. To this end, we analyzed fatty acid methyl esters (FAMEs) of samples collected from the Mojave desert of southern California to compare the microbial community structure of desert varnish with the adjacent desert soil. These analyses indicated prokaryotic and fungal communities in both desert varnish and soil samples. FAMEs specific to gram-positive bacteria were found more often, and in greater abundance in varnish samples than in adjacent soils. This may represent a higher preservation potential of gram-positive bacteria fatty acids in varnish, a source area of varnish microorganisms dominated by gram-positive bacteria, or a varnish community dominated by gram-positive microorganisms. Heterogeneity in fatty acids was documented between varnished rocks and soils from different localities, as well as between samples collected from the same locality. This heterogeneity suggests that there are significant differences in the community structure of the microbial fauna found in varnish samples compared to the adjacent soil, and that desert varnish in the Mojave desert is not characterized by a unique and ubiquitous microbial community. These results suggest that the varnish is not a homogeneous and unique environment for biota, and provide no support for the hypothesis that the varnish layers are biogenic in origin.     

Evaluating the effect of fertilizer application on soil microbial community structure in rice based cropping system using fatty acid methyl esters (FAME) analysis

The present study was undertaken to observe the effects of chemical fertilizers and compost amendments on microbial community structure in rice based cropping system under long term fertilizer application experiment. Changes in microbial community structure assessed by the relative abundance of fatty acid methyl ester (FAME) profiles revealed that compost amendments led to promoting the abundance of Gram-positive bacterial FAMEs. Multivariate analysis of FAME profiles separated the treatments primarily on the basis of the amount of added compost. A link between different fertilizer application treatment and soil microbial components was tentatively established, but it needs to be verified in further studies.     

Accuracy, Reproducibility, and Interpretation of Fatty Acid Methyl Ester Profiles of Model Bacterial Communities

We determined the accuracy and reproducibility of whole-community fatty acid methyl ester (FAME) analysis with two model bacterial communities differing in composition by using the Microbial ID, Inc. (MIDI), system. The biomass, taxonomic structure, and expected MIDI-FAME profiles under a variety of environmental conditions were known for these model communities a priori. Not all members of each community could be detected in the composite profile because of lack of fatty acid “signatures” in some isolates or because of variations (approximately fivefold) in fatty acid yield across taxa. MIDI-FAME profiles of replicate subsamples of a given community were similar in terms of fatty acid yield per unit of community dry weight and relative proportions of specific fatty acids. Principal-components analysis (PCA) of MIDI-FAME profiles resulted in a clear separation of the two different communities and a clustering of replicates of each community from two separate experiments on the first PCA axis. The first PCA axis accounted for 57.1% of the variance in the data and was correlated with fatty acids that varied significantly between communities and reflected the underlying community taxonomic structure. On the basis of our data, community fatty acid profiles can be used to assess the relative similarities and differences of microbial communities that differ in taxonomic composition. However, detailed interpretation of community fatty acid profiles in terms of biomass or community taxonomic composition must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

Microbial community succession and bacterial diversity in soils during 77,000 years of ecosystem development

The origins of the biological complexity and the factors that regulate the development of community composition, diversity and richness in soil remain largely unknown. To gain a better understanding of how bacterial communities change during soil ecosystem development, their composition and diversity in soils that developed over c. 77 000 years of intermittent aeolian deposition were studied. 16S rRNA gene clone libraries and fatty acid methyl ester (FAME) analyses were used to assess the diversity and composition of the communities. The bacterial community composition changed with soil age, and the overall diversity, richness and evenness of the communities increased as the soil habitat matured. When analysed using a multivariate Bray-Curtis ordination technique, the distribution of ribotypes showed an orderly pattern of bacterial community development that was clearly associated with soil and ecosystem development. Similarly, changes in the composition of the FAMEs across the chronosequence were associated with biomarkers for fungi, actinomycetes and Gram-positive bacteria. The development of the soil ecosystem promoted the development of distinctive microbial communities that were reminiscent of successional processes often evoked to describe change during the development of plant communities in terrestrial ecosystems.     

Comparison of microbial and meiofaunal community analyses for determining impact of heavy metal contamination

The impact of long-term heavy metal contamination on soil communities was assessed by a number of methods. These included plate counts of culturable bacteria, community level physiological profiling (CLPP) by analysis of the utilization of multiple carbon sources in BIOLOG plates, community fatty acid methyl ester (C-FAME) profiling and dehydrogenase enzyme activity measurements. These approaches were complemented with microscopic assessments of the diversity of the nematode community. Samples from two sites with different histories of heavy-metal input were assessed. Major differences in microbial and meiofaunal parameters were observed both between and within the sites. There was a large degree of congruence between each of the microbiological approaches. In particular, one sample appeared to be distinguished by a reduction in culturable bacteria (especially pseudomonads), limited response to carbon sources in CLPP, and major differences in extracted fatty acid profiles. The use of multivariate analysis to examine the relationship between microbial and physicochemical measurements revealed that CLPP and plate counts were useful for determining the gross effect of metals on soil microbial communities, whereas proportions of metal-resistant bacteria and dehydrogenase activity differentiated between the two sites. Copper and zinc concentrations and pH all showed significant correlation with the microbial parameters. Nematode community structure was affected to a greater extent by soil pH than by metal content, but the within-site rankings were the same as those achieved for microbiological analyses. The use of these methods for field evaluation of the impact of industrial pollution may be possible provided care is taken when interpreting the data.     

Characterization of the rhizosphere microbial community from different Arabidopsis thaliana genotypes using phospholipid fatty acids (PLFA) analysis

Total and culturable rhizosphere microbial communities structure from three different genotypes of Arabidopsis thaliana growing on three different substrates was studied with phospholipid fatty acid analysis (PLFA) and multivariate statistical analyses: correspondence analysis (CA) and distance based redundancy analyses (db-RDA). In addition, microbial biomass from different groups (total bacteria, Gram+, Gram? and fungi) was calculated from biomarkers PLFA peak area, both from total and culturable microbial community. db-RDA analysis showed significant differences between soils but not between plant genotypes for culturable microbial community structure. Total microbial community was significantly different between soils, and also between plant lines in each soil. Biomass of different bacterial groups showed significant higher values in soil two rhizosphere irrespective of the plant line. In addition, significant differences between plant lines were also found for microbial biomass of different bacterial groups both in total and culturable microbial community. Throughout the work we have demonstrated that PLFA analysis has been able to show a different behaviour of total microbial community with regard to the culturable fraction analyzed in this work under the influence of plant roots. Microbial biomass of different microbial groups calculated with PLFA biomarkers was a suitable tool to detect differences between soils irrespective of the plant line, and differences in the same soil between plant lines. According to this data, a previous study should be carried out before GMPs are used in field conditions to evaluate the potential alterations that may take place on rhizosphere microbial communities structure which may further affect soil productivity. In conclusion, based on data presented in this work, GMPs alter rhizosphere microbial communities structure and this effect is different depending on the soil. Furthermore, total microbial community is affected to a greater extent than the culturable fraction analyzed.     

脂肪酸甲酯法检测空心莲子草入侵影响土壤微生物群落结构的初步研究

空心莲子草是我国2003年公布的第一批外来入侵物种。为了进一步了解该植物的入侵机制,采集湖北咸宁、仙桃和武汉三地的土样,采用土壤脂肪酸甲酯谱图分析的方法探讨该植物入侵对土壤微生物的影响。结果显示：空心莲子草入侵后土壤可培养细菌、真菌的数量显著增加,而放线菌的数量显著下降。脂肪酸分析表明土壤微生物群落结构发生一定程度的改变,但其变化因土壤的不同而有差异。     

Fatty acid analysis as a chemotaxonomic tool for taxonomic and epidemiological characterization of four fish pathogenic Tenacibaculum species

Aims: In this work, fatty acid content and profiles were analysed in order to differentiate the species Tenacibaculum maritimum, Tenacibaculum gallaicum, Tenacibaculum discolor and Tenacibaculum ovolyticum that are pathogenic for cultured marine fish and to assess the potential of fatty acid profiles as a tool for epizootiological typing. Methods and Results: The fatty acid methylesters (FAMEs) were extracted from cells grown on marine agar for 48 h at 25°C and were prepared and analysed according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System. The cellular fatty acid profiles of Tenacibaculum strains tested were characterized by the presence of large amounts of branched (36·1–40·2%) and hydroxylated (29·6–31·7%) fatty acids. The FAME products from the four species significantly (P < 0·05) differed in the content of iso‐C_15:03‐OH, iso‐C_16:03‐OH, iso‐C_15:1G, summed feature 3 (a component that contains C_16:1ω7c and/or iso‐C_15:0 2‐OH), iso‐C_16:0, C_17:1ω6c, C_15:03‐OH, iso‐C_17:03‐OH. Conclusions: Results of present study demonstrated the existence of differences in the fatty acids content between the T. maritimum isolates from different marine fish/geographical origin and between strains of T. maritimum, T. discolor, T. gallaicum and T. ovolyticum. Significance and Impact of the Study: Profiling of fatty acids may be a useful tool to distinguish T. maritimum from other Tenacibaculum species pathogenic for fish as well as for epizootiological differentiation of T. maritimum isolates.     

The use of phospholipid fatty acids (PL-FA) in the determination of rhizosphere specific microbial communities (RSMC) of two wheat cultivars

To determine differences in microbial community structure, phospholipid fatty acids (PL-FA) from rhizosphere bacteria of two different wheat cultivars Triticum aestivum L. (cv. Bohouth-6 and cv. Salamouni) were extracted and analyzed by gas chromatography. This approach was used to overcome the methodological underestimation of microbial densities obtained with isolation, culture techniques and microscopic observations. Our objective was to verify differences in PL-FA profiles from two wheat cultivars grown under controlled environmental conditions. Principal component analysis (PCA) and cluster analysis were used to detect dissimilarities between rhizosphere microbial communities of the two wheat cultivars and signature fatty acids (FA) were used to determine specific differences in the community structures. PCA of the two cultivars explained 79.18% of the variance on principal component 1 (PC1), which accounted for Bohouth-6 rhizosphere soil. The rhizosphere soil of Salamouni accounted for 11.66% of the variance on principal component 2 (PC2). The results demonstrated repeatedly the clustering of the samples into two distinct groups; each group belonging specifically to one of the two wheat cultivars. Profiles of Bohouth-6 showed higher amounts of cyclopropane acid 19:0cy and Sif 7 (Sum in feature 7) than Salamouni. Those FA are known as signature molecules for Gram-negative bacteria. This was also reflected by the higher bacterial counts (cfu g^–1 fresh root weight) of Gram-negative bacteria from the rhizosphere of the former than the latter. The results indicated that under controlled environmental conditions, wheat cultivars of different genotypes exhibit distinct microbial colonization in their rhizosphere.     

Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.

The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.     

德兴铜矿尾矿重金属污染对土壤中微生物多样性的影响

【目的】为更好地了解重金属污染与微生物多样性之间的相互作用关系,以江西德兴铜矿4#尾砂库为研究对象,采集野外实地样品共16件进行分析(包括尾砂样品以及周围农田和菜地土壤样品)。【方法】一方面对样品中可培养异养细菌进行平板计数,一方面采用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)对样品中可培养和不可培养微生物分子生态多样性进行研究;同时采用PCA(Principle component analysis)方法分析样品理化性质、重金属及主要元素与可培养细菌数量及微生物多样性之间的相互关系。【结果】元素分析结果表明该尾矿区样品受到不同程度重金属Cu、Cd、Zn、Ni、Pb和Cr的污染;可培养异养细菌在尾砂样品中数量最少,在菜地和农田土壤样品中有明显增加;多样性指数(Shannon-Weaver index H)计算结果发现H最大值出现在距离尾矿中等距离、重金属浓度在中等程度的样品中。PCA分析结果表明可培养异养菌数量与理化性质如有机碳、有机质、含水率等相关性较大,重金属影响不明显;而多样性指数H除与上述理化性质相关性较大外,还受到重金属Ag、Zn、As、Pb、Ni、Cr等的影响,而在样品中含量普遍比较高的重金属如Cu、Cd等并不成为影响微生物多样性的主要因素。【结论】从这些长期受重金属污染的野外实地样品来看,以上结果说明不同重金属浓度对微生物多样性的影响可能并不是实验室研究的简单的线性关系。     

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.     

Broad-scale approaches to the determination of soil microbial community structure: Application of the community DNA hybridization technique

Broad-scale approaches seek to integrate information on whole microbial communities. It is widely recognized that culture techniques are too selective and unrepresentative to allow a realistic assessment of the overall structure of microbial communities. Techniques based on fatty acid or metabolic profiles determine the phenotypic composition of the community. Complementary information about the genotypic structure of soil microbial communities necessitates analysis of community DNA. To determine broad-scale differences in soil microbial community structure (i.e., differences at the whole community level, rather than specific differences in species composition), we have applied a community hybridization technique to determine the similarity and relative diversity of two samples by cross hybridization. In previous studies this assay failed with whole-soil community DNA. Usable hybridization signals were obtained using whole-soil DNA, in this study, by digesting the DNA with restriction enzymes before the labeling with a random-primer reaction. The community hybridization technique was tested using a graded series of microbial fractions, increasing in complexity, all isolated from the same soil sample. This demonstrated that single bacterial species and a mixture of cultivable bacteria were less complex and only 5% similar to whole-community DNA or bacteria directly extracted from the soil. Extracted bacterial and whole-community DNA were 75% similar to each other and equally complex. When DNA was extracted from four different agricultural soils, their similarities ranged from 35 to 75%. The potential usefulness of community hybridization applied to soil microbial communities is discussed.