Flow cytometric quantification of viruses in activated sludge.

Viruses may play a critical role in the microbial dynamics of activated sludge systems; however the difficulty of their quantification makes long term and large scale studies costly, timely and challenging. Thus a flow cytometric protocol was optimised and employed to determine virus abundance in activated sludge samples. The best flow cytometry signature and highest virus count was obtained by separating the indigenous floc-associated viruses using Tween 80 and sodium pyrophosphate, diluting the sample with Tris-EDTA and staining with SYBR Green II. Using the optimised protocol viral concentrations from 25 activated sludge plants were determined, with average concentrations of 2.35 × 109 mL⁻¹ observed. Direct counts by transmission electron microscopy were highly correlated with flow cytometric counts (p = <0.05 and r² = 0.77), with concentrations from both quantification methods comparable at the order of magnitude level. The high counting efficiency, ease of preparation and rapidity and reproducibility of analysis makes flow cytometric quantification of viruses in activated sludge ideal for routine investigation and thus invaluable in unravelling the complexity of phage host interactions in such systems.

Characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (T-RFLP).

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community "genome" had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences.

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the alpha-subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the alpha-subunit of methanol dehydrogenase (MDH) present in all gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.

Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities.

Abstract In this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.

Novel techniques for analysing microbial diversity in natural and perturbed environments.

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.

Microbial community changes in a perturbed agricultural soil investigated by molecular and physiological approaches.

Changes in soil microbial activity and diversity after incubation either with nitrogen or with a mixture of methane and air were examined. The perturbation by methane and air were characterized in detail and led to reduced diversity and enrichment of methanotrophs which were identified by denaturing gradient gel electrophoresis and 16S rRNA sequencing.

Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

The community structure of bacterioplankton in meromictic Lake Saelenvannet was examined by PCR amplification of the V3 region of 16S rRNA from microbial communities recovered from various depths in the water column. Two different primer sets were used, one for amplification of DNA from the domain Bacteria and another specific for DNA from the domain Archaea. Amplified DNA fragments were resolved by denaturing gradient gel electrophoresis (DGGE), and the resulting profiles were reproducible and specific for the communities from different depths. Bacterial diversity estimated from the number and intensity of specific fragments in DGGE profiles decreased with depth. The reverse was true for the Archaea, with the diversity increasing with depth. Hybridization of DGGE profiles with oligonucleotide probes specific for phylogenetic groups of microorganisms showed the presence of both sulfate-reducing bacteria and methanogens throughout the water column, but they appeared to be most abundant below the chemocline. Several dominant fragments in the DGGE profiles were excised and sequenced. Among the dominant populations were representatives related to Chlorobium phaeovibrioides, chloroplasts from eukaryotic algae, and unidentified Archaea.