Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h−1, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research.

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

The species concept is a recurrent controversial issue that preoccupies philosophers as well as biologists of all disciplines. Prokaryotic species concept has its own history and results from a series of empirical improvements parallel to the development of the techniques of analysis. Among the microbial taxonomists, there is general agreement that the species concept currently in use is useful, pragmatic and universally applicable within the prokaryotic world. However, this empirically designed concept is not encompassed by any of the, at least, 22 concepts described for eukaryotes. The species could be described as ‘a monophyletic and genomically coherent cluster of individual organisms that show a high degree of overall similarity in many independent characteristics, and is diagnosable by a discriminative phenotypic property’. We suggest to refer it as a phylo-phenetic species concept. Here, we discuss the validity of the concept in use which we believe is more pragmatic in comparison with those concepts described for eukaryotes.

The species concept for prokaryotes.

The microbial community structure of anaerobic biological reactors was evaluated by using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens. Phylogenetically defined groups of methanogens were quantified and visualized, respectively, by hybridization of 32P- and fluorescent-dye-labeled probes to the 16S rRNAs from samples taken from laboratory acetate-fed chemostats, laboratory municipal solid waste digestors, and full-scale sewage sludge digestors. Methanosarcina species, members of the order Methanobacteriales, and Methanosaeta species were the most abundant methanogens present in the chemostats, the solid-waste digestors, and the sewage sludge digestors, respectively.

Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridization.

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

http://link.springer.com/content/pdf/10.1007/978-1-4757-0322-1_7.pdf

Molecular Ecology of Gastrointestinal Ecosystems

Use of ribosomal RNA-based molecular probes for characterization of complex microbial communities in anaerobic biofilms

https://www.engr.colostate.edu/~apruden/classes/ce742/Readings/Articles/ED%20SR%20Probe1993.pdf

Monitoring theEnrichment andIsolation ofSulfate-Reducing Bacteria byUsingOligonucleotide Hybridization Probes Designed fromEnvironmentally Derived 16SrRNASequences

Lars K. Poulsen

Papers

Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridization.

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

Molecular Ecology of Gastrointestinal Ecosystems

Use of ribosomal RNA-based molecular probes for characterization of complex microbial communities in anaerobic biofilms

Monitoring theEnrichment andIsolation ofSulfate-Reducing Bacteria byUsingOligonucleotide Hybridization Probes Designed fromEnvironmentally Derived 16SrRNASequences