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Concerted action in Biotechnology: exploration of microbial diversity

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The objectives of the Concerted Action were, as defined in the workprogramme, the following ones:
(1) - organize an extensive consortium of experts into a research network
(2) - exchange environmental samples, microbial strains and researchers
(3) - training in classical and molecular techniques in the various expert groups
(4) - develop a scientific strategy to assess global microbial diversity
Objectives (1) and (2): Establishment of a European network of experts
A consortium of 25 participants (from 9 EU member countries) was formed to concert research efforts in microbial diversity. Networking within the Concerted Action was scientifically most fruitful at the interface of molecular taxonomy and ecology with classical microbiology, e.g. one participant who had isolated novel microorganisms cooperated with another participant specialized in molecular and phylogenetic analysis. In general, the exchange of microbial strains, environmental samples and molecular probes (antibodies, rRNA targeted oligonucleotides) was facilitated substantially. Furthermore, not only hardware was exchanged but also the relevant information went along with it to enable joint publications on the topic of exploitation of microbial diversity. The collaboration with the Concerted Action research network was very substantial during the second half of the project as indicated by about 50 bilateral collaborations. More than half of the participants were major cooperative links, i.e. they had more than two cooperations with other participants. In addition to that the participating culture collections (RNAAS, DSM, LMG) provided reference strains for a great variety of participants.
Objective (3): Training in classical and molecular microbial taxonomy
During the funding period three practical workshops were held and an international symposium on the ecological basis and biotechnological utility of microbial diversity was organized to disseminate technical skills as well as theoretical knowledge for the biotechnological exploitation of microbial diversity in Europe. The practical workshops primarily provided practical training in classical and molecular taxonomy of microorganisms for over 50 scientists from all participating countries. All practical workshops were hands-on training courses at the most advanced scientific and technical level to disseminate solidly the technical skills and the knowledge base for the exploitation of microbial diversity in various fields of application. In detail the following biotechnological techniques and areas are concerted:
The first practical workshop of the Concerted Action was held at the Institute of Freshwater Ecology (IFE), Windermere, UK from the 25 to 30 June 1994. It was organized by B. Finlay (IFE) and T.M. Embley on the topic `Microbial Diversity in Freshwater'. The workshop was attended by 12 researchers from 8 participating laboratories. This practical workshop was centred around the analysis of the microflora in a natural lake (Priest Pot). Depth profiles from the water column as well as sediment samples were analyzed with special reference to the oxic- anoxic boundary layers. The samples were investigated by modern microscopic and molecular methods to estimate the morphological and phylogenetic diversity of the present microbiota. The main techniques exercised were the following: i) rapid techniques for identifying free-living protozoa from the morphological phenotype using epifluorescence microscopy, scanning electron microscopy and transmission electron microscopy, ii) use of 18S rDNA sequences to explore the genetic diversity of the microbial eukaryote community, iii) use of fluorescent oligonucleotide probes for determinative microscopy of in situ hybridized single cells, and iv) cultivation of aerobic and anaerobic protists from pelagic and sediment samples.
The second practical workshop was held at the University of Gent, Belgium, on the 12 to 16 of September 1994. It was organized by K. Kersters and J. Swings on `Fingerprinting techniques for classification and identification of bacteria'. In this hands-on workshop 17 scientists from the participating laboratories were trained in the biochemical and physiological analysis of bacteria for the purpose of identification and classification. The reference bacteria selected were specifically of interest to food industries (lactic acid bacteria) and of agricultural relevance (xanthomonads and pseudomonads). At this workshop basic concepts of taxonomy and identification of bacteria were explained as well as the individual biochemical and physiological fingerprinting techniques. The major techniques exercised were: i) fatty acid methyl ester analysis (FAME), ii) sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins (SDS-PAGE), iii) HPLC analysis of polyamines and iv) phenotypic identification systems: BIOLOG and API systems.
The third practical workshop was held at the National Center for Biotechnology (GBF) Braunschweig, Germany from the 27th of November to the 1st of December 1995. It was organized by M. Höfle, J. Van der Plas and E. Stackebrandt on the topic `Nucleic acid fingerprinting techniques for genotyping of microorganisms'. The workshop was attended by 16 researchers from 10 participating laboratories. This practical workshop focused on the performance of all major nucleic acid fingerprinting techniques that are currently applied to microorganisms for their genotypic characterisation. This molecular techniques were performed with the two major groups of microorganisms that are of biotechnological relevance: bacteria and fungi. The following techniques were exercised: i) low-molecular-weight RNA fingerprinting, ii) pulsed-field analysis of microbial chromosomes, iii) randomly amplified PCR driven DNA fingerprinting (RAPD), iv) selectively amplified PCR driven DNA fingerprinting (ERIC, ARDRA) and v) statistical treatment of fingerprint data and data base handling.
Objective (4): Development of a scientific strategy for the assessment of microbial diversity
An `International Symposium on the Exploration of Microbial Diversity' was held in Goslar from the 12 - 15 of June 1995 as part of the Concerted Action. The focus of the symposium was the evolutionary basis, the underlying ecological principles and the biotechnological utility of microbial diversity. Biodiversity of the whole range of microorganisms was addressed, i.e. bacteria, fungi, protozoa and microalgae. Key areas for the symposium were: i) isolation and cultivation of novel microorganisms, ii) molecular taxonomy and phylogeny, iii) analysis of microbial communities and iv) biotechnological exploitation of microbial diversity. The purpose of the symposium was to evaluate the present knowledge of microbial diversity with state-of- the-art methods and to elucidate its ecological relevance and utility for future biotechnological applications. The current knowledge on microbial diversity was presented in 50 oral and 80 poster communications by European and international scientists. The symposium was attended by about 200 scientists from 22 countries including all EU countries (except Iceland and Luxembourg) and with a strong international participation from the USA and Japan. The barriers for the biotechnological exploitation of microbial diversity were critically discussed with a panel of European and international experts. These discussions and presentations helped formulating a scientific strategy for the assessment of microbial diversity as a whole, i.e. of all major microbial groups (bacteria, fungi, protozoa, microalgae) in the world's major environments and its biotechnological application.
The scientific strategy for the assessment of microbial diversity has to be an integrated and multidisciplinary approach of specialists in microbial ecology, physiology, taxonomy and genetics. The research efforts should concentrate on the following key areas: i) direct analysis of microbial communities, ii) molecular taxonomy, iii) isolation and cultivation, and iv) conservation of microorganisms.
The direct analysis, i.e. analysis without cultivation, of natural microbial communities using molecular methods, such as 5S/16S rRNA profiling and sequencing, is the key to the assessment of microbial diversity because this direct analysis can reveal all the hidden diversity not trackable by culture dependend analysis. In this area more studis are needed that compare different direct assays, e. g. rRNA sequences and lipid biomarkers, in terms of precision and resolution. For this area technologies should be developped that enable direct identification of single microbial cells in natural environments, for e.g. by in situ PCR, and are capable of detecting the functions these cells are carrying out in their environment. The area of molecular taxonomy estabilishes the theoretical framework of the assessment of microbial diversity. Its future research should focus on the development of automated, high resolution genetic fingerprinting techniques for microorganisms that enable detection of single clones. The most relevant task in this area is the development of a generally accessible taxonomic data base for microorganisms that integrates phenotypic and genotypic information on all microbial taxa. Isolation and cultivation of the relevant microorganisms is essential for the biotechnological exploitation of microbial diversity. In this area research efforts should concentrate on the combination of classical enrichment cultures under finely controlled growth conditions and novel isolation techniques for single cells such as laser based cell-sorters and optical tweezers. This area should greatly profit from the methodological progress made in molecular taxonomy and direct analysis of microbial communities that will allow to monitor the success of the isolation procedure and by the combination of genotypic and phenotypic data to elucidate the physiology of the microbes to be isolated. Conservation of the isolated novel microorganisms will finally guarantee the long-lasting success of the efforts in exploiting microbial diversity by generating the genetic resources available to the scientitic community. Bottelnecks in this area are more of organizational character than of scientific character and can be overcome by providing long term funding for culture collections and data bases that contain strain specific taxonomic information. In conclusion, the assessment of global microbial diversity will provide the genetic resources as well as the knowledge base for the full biotechnological exploitation of microorganisms by European industries over the next decades.

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