Cultivation and molecular monitoring of halophilic microorganisms inhabiting an extreme environment presented by a salt-attacked monument

2009 ◽  
Vol 9 (1) ◽  
pp. 59-72 ◽  
Author(s):  
Jörg Ettenauer ◽  
Katja Sterflinger ◽  
Guadalupe Piñar
Keyword(s):  
Microbial Community ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Random Amplified Polymorphic Dna ◽  
Extreme Environment ◽  
Salt Crystallization ◽  
Porous Space ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Halophilic Microorganisms ◽  
Culture Dependent

AbstractIn the last few years several investigations, based on culture-dependent and -independent techniques, have shown that salt-attacked stone surfaces present a habitat for extremely salt tolerant and moderate halophilic microorganisms. The inner walls of the Chapel of St. Virgil in Vienna (Austria) are an example of this phenomenon. Salt crusts cover most of the wall surfaces and salt crystallization in the porous space of the stone is causing decohesion of material and destruction of the original medieval paintings. The salt, together with the oligotrophic conditions, creates a very special and extreme habitat for halotolerant and halophilic microorganisms.In this study we investigate and monitor the cultivable and non-cultivable members of the microbial community present on the stonework of the medieval Chapel of St. Virgil after several severe disturbances of the microbial environment caused by desalination and disinfection treatments. With this finality, a combination of culture-dependent and -independent techniques was selected. The genetic diversity of a total of 104 bacterial strains isolated from the stone samples was analysed by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD) analysis and 16S rRNA gene sequencing. Strains were distributed over 29 groups on the basis of their RAPD patterns. Only 19 groups were differentiated by DGGE. Comparative sequence analyses showed that the isolated strains belong to related species of the generaHalobacillus(47.1%),Bacillus(35.6%),Acinetobacter(4.8%),Halomonas(3.9%),Nesterenkonia(2.9%),Paucisalibacillus(2.9%),Paenibacillus(1%),Staphylococcus(1%) andExiguobacterium(1%).In addition, polymerase chain reaction DGGE fingerprints, in combination with the creation of clone libraries and sequencing analyses, were used to monitor and identifyArchaea, the non-cultivable fraction of the microbial community. The detected archaeal sequences were closely related to different uncultured archaeons as well as to the cultured generaHalococcusandHalalkalicoccusandHalobacterium.Cultivation and molecular analyses revealed the presence of highly specialized microorganisms that were able to thrive and survive after several desalination and disinfection treatments in the extreme environment presented by the salt-attacked Chapel of St. Virgil.

scholarly journals Microbial Ecology of Greek Wheat Sourdoughs, Identified by a Culture-Dependent and a Culture-Independent Approach

Foods ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1603
Author(s):  
Maria K. Syrokou ◽  
Christina Themeli ◽  
Spiros Paramithiotis ◽  
Marios Mataragas ◽  
Loulouda Bosnea ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Microbial Ecology ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Random Amplified Polymorphic Dna ◽  
Titratable Acidity ◽  
Rrna Gene ◽  
Pichia Membranifaciens ◽  
Of Greece ◽  
Culture Dependent

The aim of the present study was to assess the microecosystem of 13 homemade spontaneously fermented wheat sourdoughs from different regions of Greece, through the combined use of culture-dependent (classical approach; clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and identification by PCR species-specific for Lactiplantibacillus plantarum, and sequencing of the 16S-rRNA and 26S-rRNA gene, for Lactic Acid Bacteria (LAB) and yeasts, respectively) and independent approaches [DNA- and RNA-based PCR-Denaturing Gradient Gel Electrophoresis (DGGE)]. The pH and Total Titratable Acidity (TTA) values ranged from 3.64–5.05 and from 0.50–1.59% lactic acid, respectively. Yeast and lactic acid bacteria populations ranged within 4.60–6.32 and 6.28–9.20 log CFU/g, respectively. The yeast: LAB ratio varied from 1:23–1:10,000. A total of 207 bacterial and 195 yeast isolates were obtained and a culture-dependent assessment of their taxonomic affiliation revealed dominance of Lb. plantarum in three sourdoughs, Levilactobacillus brevis in four sourdoughs and co-dominance of these species in two sourdoughs. In addition, Companilactobacillusparalimentarius dominated in two sourdoughs and Fructilactobacillussanfranciscensis and Latilactobacillus sakei in one sourdough each. Lactococcus lactis, Lb. curvatus, Leuconostoc citreum, Ln. mesenteroides and Lb. zymae were also recovered from some samples. Regarding the yeast microbiota, it was dominated by Saccharomyces cerevisiae in 11 sourdoughs and Pichia membranifaciens and P. fermentans in one sourdough each. Wickerhamomyces anomalus and Kazachstania humilis were also recovered from one sample. RNA-based PCR-DGGE provided with nearly identical results with DNA-based one; in only one sample the latter provided an additional band. In general, the limitations of this approach, namely co-migration of amplicons from different species to the same electrophoretic position and multiband profile of specific isolates, greatly reduced resolution capacity, which resulted in only partial verification of the microbial ecology detected by culture-dependent approach in the majority of sourdough samples. Our knowledge regarding the microecosystem of spontaneously fermented Greek wheat-based sourdoughs was expanded, through the study of sourdoughs originating from regions of Greece that were not previously assessed.

Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene

2001 ◽  
Vol 43 (1) ◽  
pp. 77-82 ◽  
Author(s):  
O.-C. Chan ◽  
W.-T. Liu ◽  
H. H. Fang
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Granular Sludge ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Rrna Gene ◽  
Related Sequence ◽  
Gradient Gel Electrophoresis

The microbial community structure of granular sludge from an upflow anaerobic sludge blanket (UASB) reactor treating brewery effluent was studied by denaturing gradient gel electrophoresis (DGGE). Twelve major bands were observed in the DGGE fingerprint for the Bacteria domain and four bands for the Archaea domain. Of the bacterial bands observed, six were successfully purified and sequenced. Among them, three were related to the gram-positive low G+C group, one to the Delta subclass of the Proteobacteria, one to the Gamma subclass, and one to the Cytophaga group with no close related sequence. The 16S rRNA sequences of the four archaeal bands were closely associated with Methanosaeta concilii and Methanobacterium formicum.

scholarly journals Tracking of Intentionally Inoculated Lactic Acid Bacteria Strains in Yogurt and Probiotic Powder

2019 ◽  
Vol 8 (1) ◽  
pp. 5 ◽  
Author(s):  
Anshul Sharma ◽  
Jasmine Kaur ◽  
Sulhee Lee ◽  
Young-Seo Park
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Gene Sequence ◽  
Random Amplified Polymorphic Dna ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Sequence Alignments ◽  
Gene Sequence Analysis ◽  
Rrna Gene Sequencing

The present work aimed at tracking intentionally inoculated lactic acid bacteria (LAB) strains in yogurt and probiotic powder. Leuconostoc (Leu.) mesenteroides (11251), Lactobacillus (L.) brevis (B151), and Lactobacillus plantarum (LB41K) strains were tracked in yogurt, and L. plantarum (LB41P) was tracked in a commercial probiotic powder. The yogurt was intentionally inoculated with the selected bacterial strains. Two types of yogurt with known and unknown bacterial pools were utilized. The standard 16S rRNA gene sequencing was used to evaluate the initial screening. The molecular typing tools, random amplified polymorphic DNA (RAPD), repetitive element palindromic PCR (rep-PCR), and comparative gene sequence analysis of selected housekeeping loci were used to track the inoculated dubious strains. Out of 30 random selections for each inoculation, the developed method identified seven (11251), nine (B151), and five (LB41K) colonies in the yogurt. The validation was performed by identifying 7 colonies (LB41P) out of 30 in the probiotic powder. The DNA banding profiles and the gene sequence alignments led to the identification of the correct inoculated strains. Overall, the study summarizes the use of molecular tools to identify the deliberately inoculated LAB strains. In conclusion, the proposed polyphasic approach effectively tracked the intentionally inoculated strains: Leu. mesenteroides, L. brevis, and L. plantarum (LB41K) in yogurt and L. plantarum (LB41P) in probiotic powder. The study demonstrates how to track industrially relevant misused LAB strains in marketable food products.

scholarly journals Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter

2003 ◽  
Vol 69 (6) ◽  
pp. 3607-3616 ◽  
Author(s):  
Veljo Kisand ◽  
Johan Wikner
Keyword(s):  
Organic Matter ◽  
Dissolved Organic Matter ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Experimental System ◽  
Community Diversity ◽  
Rrna Gene ◽  
Solid Media ◽  
Gradient Gel Electrophoresis ◽  
Culture Dependent

ABSTRACT Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated β-proteobacteria to be RDOM consumers.

scholarly journals Enrichment, Isolation, and Phylogenetic Identification of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria from Elizabeth River Sediments†

2007 ◽  
Vol 74 (4) ◽  
pp. 1176-1182 ◽  
Author(s):  
Edward J. Hilyard ◽  
Joanne M. Jones-Meehan ◽  
Barry J. Spargo ◽  
Russell T. Hill
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
River Sediments ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Molecular Analyses ◽  
Selective Enrichment ◽  
Elizabeth River ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic ◽  
Gradient Gel Electrophoresis

ABSTRACT The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of 14C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.

Comparative Analysis of Bacterial Diversity for Cutlassfish (Trichiurus haumela) during Cold Storage by the Culture-Dependent and Culture-Independent Methods

2012 ◽  
Vol 535-537 ◽  
pp. 1046-1053 ◽  
Author(s):  
Wei Qing Lan ◽  
Jing Xie
Keyword(s):  
16S Rrna ◽  
Pseudomonas Fluorescens ◽  
Cold Storage ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Region Gene ◽  
Gradient Gel Electrophoresis ◽  
Dominant Bacteria ◽  
V3 Region ◽  
Culture Dependent

The methods of culture-dependent and denaturing gradient gel electrophoresis (DGGE) based on the sequence of 16S rRNA V3 region gene were described to comparatively characterize the microbial population and community structure of cutlassfish (Trichiurus haumela) under the cold storage. The results showed that 13 kinds of bacteria were identified by the traditional culture-dependent methods, the dominant bacteria belonged to Shewanella putrefaciens and Pseudomonas fluorescens. To determine the community profiles of the samples on variable V3 region, the bacteria of 16S rRNA gene were amplified by PCR and 11 distinct PCR products were separated by DGGE fingerprinting technology. From the sequence analysis, Psychrobacter sp. was found to be the predominant bacteria in the initial stage of the storage. The proportion of Shewanella sp., Pseudomonas sp. increased gradually with the extension of storage time, and they took the place of Psychrobacter sp. to be the dominant bacteria. Thereinto, both Pseudomonas fluorescens and Vibrio sp. took high proportions in the process of storage due to the deterioration of cutlassfish (Trichiurus haumela).

scholarly journals Molecular Analysis of the Diversity of Sulfate-Reducing and Sulfur-Oxidizing Prokaryotes in the Environment, Using aprA as Functional Marker Gene

2007 ◽  
Vol 73 (23) ◽  
pp. 7664-7679 ◽  
Author(s):  
Birte Meyer ◽  
Jan Kuever
Keyword(s):  
Microbial Community ◽  
Microbial Diversity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Marker Gene ◽  
Sulfur Cycle ◽  
Estuarine Sediments ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Manganese Crust ◽  
Sulfur Oxidizing

ABSTRACT The dissimilatory adenosine-5′-phosposulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The aprA gene-based approach revealed putative sulfur-oxidizing Alphaproteobacteria of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the Betaproteobacteria and Gammaproteobacteria.

Culture-dependent and -independent approaches establish the complexity of a PAH-degrading microbial consortium

2005 ◽  
Vol 51 (11) ◽  
pp. 897-909 ◽  
Author(s):  
Marc Viñas ◽  
Jordi Sabaté ◽  
Caterina Guasp ◽  
Jorge Lalucat ◽  
Anna M Solanas
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Consortium ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Sole Source ◽  
Rrna Gene ◽  
Dominant Group ◽  
Clone Libraries ◽  
Variable Regions ◽  
Culture Dependent

A microbial consortium (AM) obtained by sequential enrichment in liquid culture with a polycyclic aromatic hydrocarbon (PAH) mixture of three- and four-ringed PAHs as a sole source of carbon and energy was examined using a triple-approach method based on various cultivation strategies, denaturing gradient gel electrophoresis (DGGE), and the screening of 16S and 18S rRNA gene clone libraries. Eleven different sequences by culture-dependent techniques and seven by both DGGE and clone libraries were obtained. The comparison of three variable regions (V3–V5) of the 16S rRNA gene between the sequences obtained yielded 19 different microbial components. Proteobacteria were the dominant group, representing 83% of the total, while the Cytophaga–Flexibacter–Bacteroides group (CFB) was 11% and the Ascomycota fungi 6%. β-Proteobacteria were predominant in the DGGE and clone library methods, whereas they were a minority in culturable strains. The highest diversity and number of noncoincident sequences were achieved by the cultivation method that showed members of the α-, β-, and γ-Proteobacteria; CFB bacterial group; and Asco mycota fungi. Only six of the 11 strains isolated showed PAH-degrading capability. The bacterial strain (AMS7) and the fungal strain (AMF1), which were similar to Sphingomonas sp. and Fusarium sp., respectively, achieved the greatest PAH depletion. The results indicate that polyphasic assessment is necessary for a proper understanding of the composition of a microbial consortium.Key words: microbial consortium, microbial diversity, PAHs, DGGE, 16S rRNA gene.

scholarly journals Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

2012 ◽  
Vol 78 (6) ◽  
pp. 1890-1898 ◽  
Author(s):  
Ángel Alegría ◽  
Pawel Szczesny ◽  
Baltasar Mayo ◽  
Jacek Bardowski ◽  
Magdalena Kowalczyk
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Content Type ◽  
Protected Designation Of Origin ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups ◽  
The Tatra Mountains ◽  
Culture Dependent

ABSTRACTOscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g.,Lactococcus,Lactobacillus,Leuconostoc,Streptococcus, andEnterococcus, identified by all three methods, other, subdominant bacteria belonging to the familiesBifidobacteriaceaeandMoraxellaceae(mostlyEnhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.

scholarly journals Heterotrophic and Autotrophic Microbial Populations in Cold Perennial Springs of the High Arctic

2008 ◽  
Vol 74 (22) ◽  
pp. 6898-6907 ◽  
Author(s):  
Nancy N. Perreault ◽  
Charles W. Greer ◽  
Dale T. Andersen ◽  
Stefanie Tille ◽  
Georges Lacrampe-Couloume ◽  
...  
Keyword(s):  
Microbial Community ◽  
Heterotrophic Bacteria ◽  
Continuous Light ◽  
Sulfur Oxidation ◽  
Extreme Environment ◽  
High Arctic ◽  
The Arctic ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Moderate Salinity

ABSTRACT The saline springs of Gypsum Hill in the Canadian high Arctic are a rare example of cold springs originating from deep groundwater and rising to the surface through thick permafrost. The heterotrophic bacteria and autotrophic sulfur-oxidizing bacteria (up to 40% of the total microbial community) isolated from the spring waters and sediments were classified into four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) based on 16S rRNA gene analysis; heterotrophic isolates were primarily psychrotolerant, salt-tolerant, facultative anaerobes. Some of the isolates contained genes for thiosulfate oxidation (soxB) and anoxygenic photosynthesis (pufM), possibly enabling the strains to better compete in these sulfur-rich environments subject to long periods of illumination in the Arctic summer. Although leucine uptake by the spring water microbial community was low, CO2 uptake was relatively high under dark incubation, reinforcing the idea that primary production by chemoautotrophs is an important process in the springs. The small amounts of hydrocarbons in gases exsolving from the springs (0.38 to 0.51% CH4) were compositionally and isotopically consistent with microbial methanogenesis and possible methanotrophy. Anaerobic heterotrophic sulfur oxidation and aerobic autotrophic sulfur oxidation activities were demonstrated in sediment slurries. Overall, our results describe an active microbial community capable of sustainability in an extreme environment that experiences prolonged periods of continuous light or darkness, low temperatures, and moderate salinity, where life seems to rely on chemolithoautotrophy.

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