Summary
Soil is a complex and dynamic biological system, and still in 2003 it is difficult to determine the composition of microbial communities in soil. We are also limited in the determination of microbially mediated reactions because present assays for determining the overall rate of entire metabolic processes (such as respiration) or specific enzyme activities (such as urease, protease and phosphomonoesterase activity) do not allow any identification of the microbial species directly involved in the measured processes. The central problem posed by the link between microbial diversity and soil function is to understand the relations between genetic diversity and community structure and between community structure and function. A better understanding of the relations between microbial diversity and soil functions requires not only the use of more accurate assays for taxonomically and functionally characterizing DNA and RNA extracted from soil, but also high-resolution techniques with which to detect inactive and active microbial cells in the soil matrix.

Soil seems to be characterized by a redundancy of functions; for example, no relationship has been shown to exist between microbial diversity and decomposition of organic matter. Generally, a reduction in any group of species has little effect on overall processes in soil because other microorganisms can take on its function.

The determination of the composition of microbial communities in soil is not necessary for a better quantification of nutrient transformations. The holistic approach, based on the division of the systems in pools and the measurement of fluxes linking these pools, is the most efficient. The determination of microbial C, N, P and S contents by fumigation techniques has allowed a better quantification of nutrient dynamics in soil. However, further advances require determining new pools, such as active microbial biomass, also with molecular techniques. Recently investigators have separated 13C- and 12C-DNA, both extracted from soil treated with a 13C source, by density-gradient centrifugation. This technique should allow us to calculate the active microbial C pool by multiplying the ratio between labelled and total DNA by the microbial biomass C content of soil. In addition, the taxonomic and functional characterization of 13C-DNA allows us to understand more precisely the changes in the composition of microbial communities affected by the C-substrate added to soil.

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Microbial diversity and soil functions

From near to far ,

from here to there ,

funny things are everywhere. 

—Dr. Seuss, One Fish Two Fish Red Fish Blue Fish 

In 1909, H. Joel Conn ([25][1]) expressed the hope that methods would soon be at hand by which the significance of the different bacteria present in any soil could be

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Identifying the Dominant Soil Bacterial Taxa in Libraries of 16S rRNA and 16S rRNA Genes

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

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DNA extraction from soils: old bias for new microbial diversity analysis methods.

Knowledge of microbial numbers and activity in subsoils is essential for understanding the transformation and downward movement of natural and synthetic organics. Soil cores were taken from two soil profiles (surface textures: silty clay loam and loamy sand), and samples extracted from the 0-30 cm (surface), 1.0-1.3 m (mid) and 2.7-3.0 m deep; clay) and 3.9-4.2 m (deep: sand) layers. A variety of soil biotic (microbial numbers, microbial biomass, enzyme activities) and abiotic properties (pH, organic C, texture, CEC) were measured. Bacterial numbers decreased with depth as indicated by viable counts and by calculations based upon biomass carbon and extracted DNA. Direct microscopic counts were the most sensitive method of enumeration and gave bacterial numbers between 37 and 442 X greater than colony formin g units and those calculated from DNA extracted from soil. DNA extracted from soil ranged from 1.23 (sand surface) and 1.34 (clay surface) mug g(-1) d wt soil to 0.02 (sand deep) and 0.01 (clay deep) mug g(-1) d wt soil. Bacterial numbers, estimated from biomass-C measurements, were comparable to direct counts. Large numbers of bacteria were recorded in the subsoils (direct counts: 5.6 X 10(8) sand, 4.5 X 10(8) clay) even though this was equivalent to only 4.7 and 1.7% of those in the surface soils. Fungi were isolated from surface and mid-depth layers of both soils but were absent from the deep soil samples. Enzymatic activities (arylsulphatase, beta-glucosidase, phosphomonoesterases, urease, dehydrogenase, FDA hydrolysis), assayed with or without buffers, also decreased with depth. The exception was urease activity in the clay soil where no difference was seen between mid and deep in non-buffered assays but a 2.9-fold greater activity was exhibited in the mid than in the surface soil when buffered, Strong positive correlations (R > 0.95) were observed between all enzyme activities (except with urease activity in clay soil and non-buffered phosphatase activity in sand soil) and between all methods of estimating bacterial abundance. Strong positive correlations (R > 0.90) were also found between bacterial abundance and enzyme activities and between enzyme activities and organic matter content. (C) 2002 Elsevier Science Ltd. All rights reserved.

Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

Vincent Ramisse

Papers

Quantification of bias related to the extraction of DNA directly from soils.