scholarly journals Rapid Mineralization of Benzo[a]pyrene by a Microbial Consortium Growing on Diesel Fuel

2000 ◽  
Vol 66 (10) ◽  
pp. 4205-4211 ◽  
Author(s):  
Robert A. Kanaly ◽  
Richard Bartha ◽  
Kazuya Watanabe ◽  
Shigeaki Harayama
Keyword(s):  
Incubation Period ◽  
Diesel Fuel ◽  
Microbial Consortium ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Cell Protein ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Balance Analysis ◽  
Pyrene Concentration ◽  
Sequence Types

ABSTRACT A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-14C]benzo[a]pyrene to14CO2 when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations. Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter−1 and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter−1. A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds. The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the classProteobacteria but that the consortium also included members which were related to the genera Mycobacterium andSphingobacterium.

scholarly journals Characterization of a Highly Enriched Microbial Consortium Reductively Dechlorinating 2,3-Dichlorophenol and 2,4,6-Trichlorophenol and the Corresponding cprA Genes from River Sediment

2016 ◽  
Vol 65 (3) ◽  
pp. 341-352 ◽  
Author(s):  
Wael S. El-Sayed
Keyword(s):  
Electron Donor ◽  
Reductive Dechlorination ◽  
River Sediment ◽  
Microbial Consortium ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Dominant Bacteria

Anaerobic reductive dechlorination of 2,3-dichlorophenol (2,3DCP) and 2,4,6-trichlorophenol (2,4,6TCP) was investigated in microcosms from River Nile sediment. A stable sediment-free anaerobic microbial consortium reductively dechlorinating 2,3DCP and 2,4,6TCP was established. Defined sediment-free cultures showing stable dechlorination were restricted to ortho chlorine when enriched with hydrogen as the electron donor, acetate as the carbon source, and either 2,3-DCP or 2,4,6-TCP as electron acceptors. When acetate, formate, or pyruvate were used as electron donors, dechlorination activity was lost. Only lactate can replace dihydrogen as an electron donor. However, the dechlorination potential was decreased after successive transfers. To reveal chlororespiring species, the microbial community structure of chlorophenol-reductive dechlorinating enrichment cultures was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Eight dominant bacteria were detected in the dechlorinating microcosms including members of the genera Citrobacter, Geobacter, Pseudomonas, Desulfitobacterium, Desulfovibrio, and Clostridium. Highly enriched dechlorinating cultures were dominated by four bacterial species belonging to the genera Pseudomonas, Desulfitobacterium, and Clostridium. Desulfitobacterium represented the major fraction in DGGE profiles indicating its importance in dechlorination activity, which was further confirmed by its absence resulting in complete loss of dechlorination. Reductive dechlorination was confirmed by the stoichiometric dechlorination of 2,3DCP and 2,4,6TCP to metabolites with less chloride groups and by the detection of chlorophenol RD cprA gene fragments in dechlorinating cultures. PCR amplified cprA gene fragments were cloned and sequenced and found to cluster with the cprA/pceA type genes of Dehalobacter restrictus.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

scholarly journals Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

2006 ◽  
Vol 72 (10) ◽  
pp. 6452-6460 ◽  
Author(s):  
Paul J. Hunter ◽  
Geoff M. Petch ◽  
Leo A. Calvo-Bado ◽  
Tim R. Pettitt ◽  
Nick R. Parsons ◽  
...  
Keyword(s):  
Microbial Activity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Small Subunit ◽  
Disease Suppression ◽  
Rrna Gene ◽  
Damping Off ◽  
Small Subunit Rrna ◽  
Pythium Sylvaticum ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

scholarly journals MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR-DGGE, A TRADITIONAL MEXICANBEVERAGE

2015 ◽  
Vol 3 (3) ◽  
pp. 1-15
Author(s):  
Marcial-Quino J. ◽  
Garcia-Ocón B. ◽  
Mendoza-Espinoza J.A. ◽  
Gómez-Manzo S. ◽  
Sierra-Palacios E
Keyword(s):  
Gel Electrophoresis ◽  
Fermentation Process ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Beverage Samples ◽  
D2 Domain ◽  
Gradient Gel Electrophoresis ◽  
26S Rrna

Currently it is well known that yeasts play an essential role in the production of different beverages. In this paper, were identified some of the yeasts involved in the fermentation process of the pulque, a Mexican traditional beverage. Samples were collected from different regions of Mexico and yeasts were detected directly from samples without cultivation. Identifying the yeasts was obtained using amplification the D1/D2 domain of the 26S rRNA gene and Denaturing Gradient Gel Electrophoresis (DGGE). The results of DGGE showed different profiles of bands in each of the analyzed samples, indicating the presence of several species of yeast, which was also confirmed by sequencing of the bands corresponding to the domain D1/D2, succeeded in identifying five species of yeasts. The results obtained in this work demonstrated that the technique used for identification of yeasts of pulque was efficient. Besides, the optimization of this method could also allow rapid identification of yeasts and help understand the role of these in the fermentation process of this beverage, as well as the isolation of strains of interest for biotechnological purposes such as production of ethanol or metabolites with nutraceutical activity.

scholarly journals Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

2001 ◽  
Vol 67 (11) ◽  
pp. 5113-5121 ◽  
Author(s):  
Luca Cocolin ◽  
Marisa Manzano ◽  
Carlo Cantoni ◽  
Giuseppe Comi
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Dynamic Changes ◽  
Good Tool ◽  
Reverse Transcription Pcr ◽  
Dna And Rna ◽  
Fermented Sausages ◽  
Brochothrix Thermosphacta ◽  
Gradient Gel Electrophoresis

ABSTRACT In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta andEnterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

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 Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil

2000 ◽  
Vol 66 (7) ◽  
pp. 2959-2964 ◽  
Author(s):  
Gregory M. Colores ◽  
Richard E. Macur ◽  
David M. Ward ◽  
William P. Inskeep
Keyword(s):  
Gel Electrophoresis ◽  
Microbial Population ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Populations ◽  
Alcohol Ethoxylate ◽  
Rrna Gene ◽  
Mineralization Kinetics ◽  
Gradient Gel Electrophoresis ◽  
The Impact ◽  
Parallel Cultivation

ABSTRACT We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g−1). Addition of the surfactant at a concentration below the CMC′ (2 mg g−1) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g−1), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g−1) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulatedRhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenespopulations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas andAlcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.

scholarly journals Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

2008 ◽  
Vol 2008 ◽  
pp. 1-26 ◽  
Author(s):  
Geert Huys ◽  
Tom Vanhoutte ◽  
Peter Vandamme
Keyword(s):  
Population Dynamics ◽  
Intestinal Microbiota ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Therapeutic Interventions ◽  
Rrna Gene ◽  
Sequence Dependent ◽  
Human Intestinal Microbiota ◽  
Gradient Gel Electrophoresis ◽  
Community Fingerprinting

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated.

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