We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny, Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE fingerprinting, based on cluster analysis of soil bacterial and fungal community fingerprints, was made in terms of the resulting information about microbial community structures and their response to different degrees of cattle impact. Although the comparative DGGE analysis with different DGGE systems provided similar clustering of microbial community structures in correlation with the degree of cattle impact, our results suggest the DGGE system to be a factor influencing DGGE analysis. To our knowledge this is the first attempt to investigate the hypothetical impact of the DGGE system due to different technical characteristics, recommending the use of one and the same DGGE apparatus throughout an experiment, if the monitoring of microbial community structures requires multiple gel-to-gel analysis.

The soil DNA pool consists of an intracellular (iDNA) and extracellular fraction (eDNA). Challenging to improve the extraction efficiency of soil DNA, and to quantitatively and qualitatively characterize both DNA fractions, we set up a molecular approach consisting of sequential and comparative DNA extraction and microbial community fingerprinting. eDNA was extracted by alkaline soil washings (ASW); iDNA by mechanical chemical cell lysis (MCCL) of the residual soil pellet after the extraction of eDNA (ASW–MCCL). The molecular approach was compared in terms of quantity (fluorometer) and quality (agarose gel electrophoresis; small subunit rRNA-denaturing gradient gel electrophoresis) to directly extracted soil DNA comprising both eDNA and iDNA (tDNA; FastDNA Spin Kit for Soil, BIO101). The proposed method appeared to be a potential tool to separately extract and analyze eDNA (6.07μgg−1soil) and iDNA (11.46μgg−1soil) and to obtain a greater amount of DNA from soil with broader genetic information about eubacterial and fungal communities with respect to directly extracted tDNA (8.79μgg−1soil). Our results revealed the extracellular fraction to be quantitatively and qualitatively important of the soil metagenome. As the sequential DNA extraction method not only increased the total amount of extractable soil DNA (17.53μgg−1soil) but also that of iDNA, it is suggested to be suitable for extracting the soil metagenome.

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.