Elsevier

Journal of Biotechnology

Volume 64, Issue 1, 17 September 1998, Pages 53-62
Journal of Biotechnology

Review article
Novel techniques for analysing microbial diversity in natural and perturbed environments

https://doi.org/10.1016/S0168-1656(98)00103-5Get rights and content

Abstract

Molecular techniques were applied for analysing the entire bacterial community, including both the cultivated and non-cultivated part of the community. DNA was extracted from samples of soils and sediments, and a combination of different molecular methods were used to investigate community structure and diversity in these environments. Reassociation of sheared and thermally denatured DNA in solution was used to measure the total genetical diversity. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of rRNA genes gave information about changes in the numerically dominating bacterial populations. Hybridisation with phylogenetic group specific probes, and sequencing provided information about the affiliation of the bacterial populations. Using DNA reassociation analysis we demonstrated that bacterial communities in pristine soil and sediments may contain more than 10 000 different bacterial types. The diversity of the total soil community was at least 200 times higher than the diversity of bacterial isolates from the same soil. This indicates that the culturing conditions select for a distinct subpopulation of the bacteria present in the environment. Molecular methods were applied to monitor the effects of perturbations due to antropogenic activities and pollution on microbial communities. Our investigations show that agricultural management, fish farming and pollution may lead to profound changes in the community structure and a reduction in the bacterial diversity.

Introduction

Biodiversity has been defined as the range of significantly different types of organisms and their relative abundance in an assemblage or community. The diversity has also been defined according to information theory, as the amount and distribution of information in an assemblage or community (Atlas, 1984).

Traditionally, biodiversity is based upon the species as a unit. Species diversity consists of two components, species richness and species evenness (distribution). For prokaryotic organisms the species concept is obscure. This problem has been circumvented by replacing traditional identification and classification with numerical taxonomy. With this method the distances between isolates are calculated and they are then clustered into biotypes. The biotype is an operational taxonomic unit (OTU) which can be used instead of species to characterise and compare populations and communities.

One gram of soil or sediment may contain more than 1010 bacteria as counted in fluorescence microscope after staining with a fluorescent dye (Fægri et al., 1977, Torsvik et al., 1990a). A serious problem in microbial ecology is that the relative proportion of bacteria growing on agar plates (CFU) vary from 0.1 to 1% in pristine forest soils to 10% in environments like arable soil. This implies that investigations based on bacterial isolates may include only a minor part of the total bacterial diversity.

Molecular methods provide tools for analysing the entire bacterial community, covering also those bacteria that have not been cultured in the laboratory. Therefore, such methods are becoming increasingly important in microbial ecology (Pickup, 1991, Stackebrandt et al., 1993, Amann et al., 1995, Holben and Harris, 1995). We have applied DNA analysis at different resolution levels to analyse whole communities, bacterial isolates, and clones of specific genes. Low resolution and broad scale analysis of community DNA, like DNA-reassociation, allow assessment of the total genetical diversity of bacterial communities (Torsvik et al., 1996). PCR-denaturing gradient gel electrophoresis (DGGE) analysis of rRNA genes gives somewhat higher resolution and provides information about changes in the gross community structure (Muyzer et al., 1993). When DGGE analyses of rRNA genes are combined with hybridisation using phylogenetic probes or with sequencing, assessment of the phylogenetic affiliation of the numerically dominating members of a community can be obtained (Øvreås et al., 1997). Fluorescent in situ hybridisation (FISH) of bacterial cells with phylogenetic probes provides information about the overall taxon composition of bacterial communities or assemblies (Hahn et al., 1992, Amann et al., 1995). By cloning PCR-products from rRNA genes in whole community DNA, information about non-cultured bacteria is gained. This approach also allows comparison of the structure of the cultivated fraction of a bacterial community with the total community. To discriminate at bacterial isolate and clone levels, DNA fingerprinting and sequencing have been applied (de Bruijn, 1992, Massol-Deya et al., 1995, Stackebrandt and Rainey, 1995).

An important part of our investigations has been to evaluate the different methods and their feasibility in monitoring changes in microbial communities due to antropogenic impact. We have mainly studied soil systems, but marine sediments have also been included. The soil environments investigated comprise soil under different agricultural management, sewage sludge amended soils with heavy metal contamination, and chemically polluted soils. The perturbed soils have been compared to unpolluted and pristine soils. The sediments investigated were polluted fish farm sediments that were compared to pristine sediments with similar amount of organic matter.

Section snippets

DNA melting-profiles and reassociation analysis

The gross genetical structure and diversity in bacterial communities were assessed by measuring the base composition and complexity of total community DNA. Base composition profiles expressed as mole percent guanine+cytosine (%G+C), were determined by analysing the melting curves of DNA (Torsvik et al., 1995). The melting profiles were converted to %G+C profiles by calculating the first derivative of the melting curve (Ritz et al., 1997).

The sequence complexity of DNA was determined by

Bacterial diversity in natural environments

The genetical diversity index C0t1/2, provides a measure of the total genetical complexity in a community. Like the Shannon Weaver index for species diversity it includes the amount of information in the community and how this information is distributed. The reassociation rates of bacterial community DNA from pristine soil and sediments were very slow, giving C0t1/2 of 4500–9000 (reassociation experiments with such complex DNA takes several weeks). In contrast the C0t1/2 of DNA from a mixture

Bacterial diversity in perturbed and polluted environments

Diversity measurements have been performed in perturbed environments like marine fish farm sediments, in heavy metal polluted soils, and in model experiments with soils supplied with a sole carbon source.

The mole% G+C profile of DNA from a pristine marine sediment with high organic content (Torsvik et al., 1993b) was very broad, with a plateau ranging from 35 to 60 mole% G+C (Fig. 1). This indicates that the bacterial community comprised a wide range of bacterial groups with entirely different

Concluding remarks

We have evaluated the applicability of molecular methods for analysing bacterial populations and communities. Some of the molecular fingerprinting methods (RFLP, ARDRA) were found to be too sensitive, giving too high resolution to provide reliable and robust genotypic characterisation at the community level. These methods were feasible for monitoring specific populations in microbial communities, and for assessing the diversity of bacterial isolates and cloned genes. Total DNA from complex

Acknowledgements

This work was supported by funds provided by the Norwegian Research Council and by the EC Commission (Projects No. BIO2-CT94-3098 and EV5V-CT94-0415).

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