Characterization of bacterial communities from seawater samples: the study case of Kandalaksha Bay, Russia
Author(s)
Pesciaroli, Chiara
Date Issued
July 18, 2011
Type
Doctoral Thesis
Abstract
In this work we studied the bacterial community obtained from water samples collected in Kandalaksha Bay (White Sea), Russia in order to obtain information both at the ecological level and for possible future application in biotechnology. The study has been carried out both on cultivable strains, isolated by traditional methods, and on the total bacterial (Eubacteria) community by molecular methods.
The isolates were preliminary investigated in order to understand their temperature preferences then their metabolic competences, including the production of extracellular enzyme activities, were tested for possible further biotechnological applications. Furthermore, a detailed taxonomical study was carried out both on cultivable and total bacterial population
Strain isolation and preliminary tests
Sea water was sampled in various areas of Kandalaksha Bay. The majority of samples were collected, at minimum tide level using sterile containers, in an intertidal zone pool and from the adjacent water surface. Others, from different offshore locations and depths (0.5, 2.5, 15, 70 m), were taken by scuba divers or boats using Niskin bottles. Water was filtered on membranes in order to obtain both bacteria pure cultures and DNA for molecular to studies. Pure cultures of isolates (ca. 500) were obtained by plate streak method. To discharge evident replicates of same isolates, preliminary tests were carried out considering strain morphological characteristics (shape, color and dimensions), Gram reaction and simple biochemical tests (catalase and oxidase production). This preliminary selection permitted to remove the majority of replicates and to keep 52 isolates.
Taxonomical identification of isolates by 16S rDNA
2
Bacterial genomic DNA was extracted from pure cultures and used for amplification of 16S rDNA, to allows taxonomical identification of each isolate.
Only 20 strains out of 52 (ca. 38%), showing high identity (99-100%) with a single known microorganism, were identified at species level. All other isolates were identified at the genus level only: for most of them, affiliation was not possible because 98-99% of identity was recorded with various species of the same genus. Moreover 3 strains showed a very low percentage of identity (96-97%).
The majority of sequences were phylogenetically related to the genera Pseudomonas and Serratia that are well worldwide distributed. However, only a typical bacteria of cold marine environment Shewanella baltica (KB30) have been revealed.
When Blastn analyses supplied uncertain identification, sequences were aligned with highly similar 16S in the Genbank and phylogenetic analysis was performed. To get a confident branch-length, three different trees, Pseudomonas, Serratia and a group containing all the other genera, were inferred. Basing on the results some of the strains were identified with certainty at species level while some others, due to the branch length or the external position, probably belonged to new species. For all other strains identification was possible at genus level only. In many cases this could be due to the scarce informative power of the gene target used to discern the relations below the genus level.
Temperature growth profiles
The optimal temperatures for growth of the various strains were tested in the range 0-45°C on PCA plates (steps of 5 °C). Most of the strains (42% ca.) showed the optimum at 30°C. The lowest optimum was recorded at 15°C for one strain only (KB75), while the highest was at 40°C (KB22 and KB49). Most of the strains (56%) were able to growth at 0°C while only 25% grew at 45°C.In addition, the majority were able to grow in a rather broad range of temperature. The majority of the isolates (42%) seems to be psychrotrophics while no psychrophiles were detected. Almost all the strains could be considered as eurythermics,
3
indicating adaptation to frequent and wide temperature variations such as those of Kandalaksha Bay. Moreover, it was evident that many KB strains had wider ranges of growth if compared with same species described in literature; optima were also different.
Extracellular enzyme activity
The strains where submitted to plate screening for the production of various extracellular enzyme activities (amylase, cellulase, chitinase, pectinase, phosphatase, protease, urease, lipases), at their optimal temperature, in order to obtain metabolic information and to find new microorganisms for possible applications.
Lipases, phosphatase and protease were common (ca. 54% and 44% of the isolates, respectively). Pectinase and amylase were present in about 32 % of the strains, while chitinase and urease were detected in a limited number of isolates (17% both); cellulase was not detected. Organisms producing large halos of activity were considered as possible high producers and could be further investigated for biotechnological application. However, none of the isolates produced all the tested activities and a rather large number of strains (17%) produced no activity at all. The results of the screening could be useful also at ecological level. In fact, the isolates producing a limited number of enzymes could be considered as specialized, while those with more diversified enzymatic competence, showing a higher eco-nutritional versatility, are probably advantaged in the harsh White Sea environment.
Metabolic competences by BIOLOG system
The BIOLOG system was used to test the metabolic competences of the strains by their ability to use different compounds as carbon sources. The system is able to detect the oxidation of 95 compounds (including sugars, fatty, organic amino and acids), used by microorganisms as sole carbon sources.In our case, amino acids were generally the preferred compounds: the most utilized carbon sources were L-Glutamic Acid and L-asparagine (96% of strains). Also use of sugars was, as expected, rather common, being α-D-Glucose oxidized by ca. 92%of strains. The information obtained by BIOLOG could be considered as an index
4
regarding the strain metabolic complexity. A small number of strain apparently showed a rather simple metabolism being using a limited number of carbon sources and only few showed a very diversified metabolic competence. However, the majority of the bacteria tested used about 30-50 compounds showing medium-high competence. In general, the strains able to use a wide array of carbon sources were also able to produce diversified extracellular enzyme patterns confirming their high eco-nutritional versatility.
Study on the total bacterial (Eubacteria) community
In order to have a complete overview of the bacterial community structure, by a cultivation-independent approach, total DNA was extracted from the filter-membranes. The bacteria biodiversity was studied by PCR-TGGE fingerprinting of partial 16S-rRNA gene amplicons.
TGGE band patterns were normalized, compared and clustered. showed The community structure was revealed by the cluster analysis of the fingerprints. Samples collected both from the intertidal zone and the nearby sea surface, grouped together (80% similarity). Samples from open sea clearly clustered away. This was particularly evident for the sample collected at -70 m, which branched away at only 40% similarity. TGGE gel images allow the analysis of band patterns generated from the environmental samples representing the various species present in the community. A single species is identified by a single band and the relative abundance of a single species is determined by the band intensity. Among the KB samples, a total of 70 different banding positions (band classes) were detected. The average number of bands per sample was 26 with a maximum of 30 in the samples from open sea. Simpson's Diversity Index calculated for all the samples showed very high level of diversity. Based on the total number of bands in each TGGE pattern and the relative temperature gradient, range-weighted richness indexes (Rr) were calculated and indicated a very high community biodiversity. Relative bands intensities were also calculated and expressed as percentages of the total band intensity in each TGGE lane. To render a graphic representation of the bacterial communities evenness, Pareto-Lorenz distribution curves were drawn based on the intensities.
5
The functional community redundancy (response to perturbing environmental conditions) is evaluated by the Functional organization index (Fo) considering curves slope. Results showed a balanced community with medium Fo and a medium evenness. In other words, due to the elevated concentration of some species and the availability of many others, the community can potentially deal with environmental conditions changes thus preserving its functionality.
Prominent TGGE bands were excised from the gel, re-amplified and sequenced, to obtain the identities of the community predominant populations. Sequences phylogenetic analysis, showed a great presence of α-proteobacteria (16 sequences out of 27) with some γ-proteobacteria and some actinobacteria too. Some cyanobacteria were revealed also. Among α -proteobacteria strains can be affiliated mainly to the genus Roseobacter and one sequence was related to Ruegeria. All γ-proteobacteria showed highly similarity with the species of Cobetia marina, while all the cyanobacteria were unknown.
Taxonomic results obtained by the total community study were quite different from those gained by the pure cultures: the pure culture strains were completely different (genera and species) from those by 16S rDNA analysis. This could be explained by the limits of TGGE technique. In fact, due to the bias introduced by the PCR reaction, this methodology can only detect bacteria representing at least 1% of the total community. It is possible that some species, even if present in a very low percentage, had prevailed due to favorable culture conditions of the isolation procedures. However, it is known that a large number of marine bacteria are uncultivable.
Another important information was that some bands showed very low sequence identity if compared with sequences present in the database, suggesting the possible presence of unknown bacteria.
Study on Pseudomonas species present in the community
In the TGGE gel relative to the total bacterial community, no Pseudomonas species were revealed. By contrast, the majority (ca. 45%) of the cultivable KB strains were affiliated to
6
this genus. In order to find possible explications to this apparent incongruity, further TGGE analyses have been carried out focusing on Pseudomonas. Thus, total DNA was used for PCR-TGGE fingerprinting of partial 16S-rRNA gene amplicons using specific primers for the amplification of this Genus and different temperature gradients.
Again, prominent TGGE bands, excised from the gel, were re-amplified and sequenced. The phylogenetic analysis showed that only 6 sequences out of 22 can be affiliated to Pseudomonas. Although the primers used were specific for this genus, possible amplification of other similar 16S rDNA (in particular γ-proteobacteria) could occur. In fact, a big cluster, comprising the majority of the sequences, showed very low similarity with Pseudomonas but high similarity with clones of marine invertebrates symbiotic γ-proteobacteria. It is possible that some new genera and/or species were present in our samples.
TGGE patterns were normalized, compared and clustered as reported for the total bacteria community. Cluster analysis of the fingerprints was similar to that obtained for total community. However, in this case sample collected at -70 m, was even more clearly separated from all the others samples: similarity was only 20%.
Analysis of bands number allowed to detect a total of 26 different banding positions
All the statistical indexes, discussed for total bacteria and related to community diversity and organization, in this case are meaningless because they would refer only to a limited portion of the entire population.
To the best of our knowledge, this work represents the first and extensive study carried out on Kandalaksha Bay bacterial community.
Additional information
Dottorato di ricerca in Scienze ambientali
File(s)![Thumbnail Image]()
Name
cpesciaroli_tesid.pdf
Size
3.24 MB
Format
Adobe PDF
Checksum (MD5)
aaab1f276b30fecfc51b3cd78ff4ad79