scholarly journals Functional stabilisation and partner selection during repeated co-culivation in a methanotrophic interactome

2016 ◽  
Author(s):  
Frederiek - Maarten Kerckhof ◽  
Charlotte De Rudder ◽  
Varvara Tsilia ◽  
Ruben Props ◽  
Adrian Ho ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Genome Mining ◽  
Partner Selection ◽  
Sugar Metabolism ◽  
Rrna Gene ◽  
Metabolic Potential ◽  
Oxidation Of Methane ◽  
Genes Encoding ◽  
Gradient Gel Electrophoresis ◽  
Ecological Sorting

Background Biological oxidation of methane (CH4) is an essential ecosystem function. Accumulating evidence indicated that this function is mediated by associations of methanotrophic bacteria (MOB) with non-methanotrophic partners; together referred to as a methanotrophic interactome. Given the potency of CH4 as a greenhouse gas, a thorough understanding of how these interactomes exert an effect on methane oxidation is of special interest. Furthermore, MOB - non-MOB associations could be exploited for sustainable biotechnological applications in light of the renewed interest in MOB as natural and cost-efficient biocatalysts. The selectivity of MOB for non-MOB partners, as well as the stimulation of MOB activity (CH4 oxidation rate, MOR) with increasing non-MOB richness have both been recently described for a single batch incubation period. Therefore, we hypothesized that during repeated co-cultivation of MOB with non-MOB, ecological sorting would guide the methanotrophic interactome towards its optimal composition, which could additionally boost functionality (MOR). Methods Co-cultures of 8 non-MOB partners with a single alpha- or a single gammaproteobacterial MOB were repeatedly sub-cultivated. In every cycle, the headspace CH4 concentration was measured to over time to determine the MOR, while headspace CO2 concentrations and total protein in the culture were determined to track the fate of CH4-derived carbon (catabolism and assimilation respectively). Finally, the relative abundance of each co-culture partner was assessed using a 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Results and Discussion While no significant improvement of functionality was observed, the biological variability of MOR was stabilized by co-cultivation with non-MOB partners. Overall, higher biomass yields were obtained when MOB were co-cultivated with non-MOB partners and the alphaproteobacterial MOB appeared to be able to support more non-MOB biomass than the gammaproteobacterial MOB, which could be linked to the proposed life-strategies of these clades. A clear partner selection was observed as only 4 out of 8 initial partners were found to persist during repeated cycles of co-cultivation. While 2 of the persisting partners could coexist with either MOB type, the other two were more restricted to a specific MOB. Differential metabolic potential of non-MOB was resolved by genome mining publicly available genomes; our attempt to find clues for the partner selectivity did not reveal a clear link with the potential for C1-compound metabolism. However, genes for sugar metabolism (fructose, mannose, sucrose) were restricted to the persisting partners while genes encoding an ATP-dependent vitamin B12 importer were restricted to the non-persisting partners, underlining the importance of metabolic exchange in the methanotrophic interactome.

scholarly journals Functional stabilisation and partner selection during repeated co-culivation in a methanotrophic interactome

2016 ◽  
Author(s):  
Frederiek - Maarten Kerckhof ◽  
Charlotte De Rudder ◽  
Varvara Tsilia ◽  
Ruben Props ◽  
Adrian Ho ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Genome Mining ◽  
Partner Selection ◽  
Sugar Metabolism ◽  
Rrna Gene ◽  
Metabolic Potential ◽  
Oxidation Of Methane ◽  
Genes Encoding ◽  
Gradient Gel Electrophoresis ◽  
Ecological Sorting

Background Biological oxidation of methane (CH4) is an essential ecosystem function. Accumulating evidence indicated that this function is mediated by associations of methanotrophic bacteria (MOB) with non-methanotrophic partners; together referred to as a methanotrophic interactome. Given the potency of CH4 as a greenhouse gas, a thorough understanding of how these interactomes exert an effect on methane oxidation is of special interest. Furthermore, MOB - non-MOB associations could be exploited for sustainable biotechnological applications in light of the renewed interest in MOB as natural and cost-efficient biocatalysts. The selectivity of MOB for non-MOB partners, as well as the stimulation of MOB activity (CH4 oxidation rate, MOR) with increasing non-MOB richness have both been recently described for a single batch incubation period. Therefore, we hypothesized that during repeated co-cultivation of MOB with non-MOB, ecological sorting would guide the methanotrophic interactome towards its optimal composition, which could additionally boost functionality (MOR). Methods Co-cultures of 8 non-MOB partners with a single alpha- or a single gammaproteobacterial MOB were repeatedly sub-cultivated. In every cycle, the headspace CH4 concentration was measured to over time to determine the MOR, while headspace CO2 concentrations and total protein in the culture were determined to track the fate of CH4-derived carbon (catabolism and assimilation respectively). Finally, the relative abundance of each co-culture partner was assessed using a 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Results and Discussion While no significant improvement of functionality was observed, the biological variability of MOR was stabilized by co-cultivation with non-MOB partners. Overall, higher biomass yields were obtained when MOB were co-cultivated with non-MOB partners and the alphaproteobacterial MOB appeared to be able to support more non-MOB biomass than the gammaproteobacterial MOB, which could be linked to the proposed life-strategies of these clades. A clear partner selection was observed as only 4 out of 8 initial partners were found to persist during repeated cycles of co-cultivation. While 2 of the persisting partners could coexist with either MOB type, the other two were more restricted to a specific MOB. Differential metabolic potential of non-MOB was resolved by genome mining publicly available genomes; our attempt to find clues for the partner selectivity did not reveal a clear link with the potential for C1-compound metabolism. However, genes for sugar metabolism (fructose, mannose, sucrose) were restricted to the persisting partners while genes encoding an ATP-dependent vitamin B12 importer were restricted to the non-persisting partners, underlining the importance of metabolic exchange in the methanotrophic interactome.

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 Denaturing gradient gel electrophoresis as a fingerprinting method for the analysis of soil microbial communities

2009 ◽  
Vol 55 (No. 10) ◽  
pp. 413-423 ◽  
Author(s):  
V. Valášková ◽  
P. Baldrian
Keyword(s):  
Microbial Communities ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soil Microbial Communities ◽  
Soil Microbial ◽  
Genes Encoding ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Experimental Treatments ◽  
Multiple Samples

In soil microbial ecology, the effects of environmental factors and their gradients, temporal changes or the response to specific experimental treatments of microbial communities can only be effectively analyzed using methods that address the structural differences among whole communities. Fingerprinting methods are the most appropriate technique for this task when multiple samples must be analyzed. Among the methods currently used to compare microbial communities based on nucleic acid sequences, the techniques based on differences in the melting properties of double-stranded molecules, denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE), are the most widely used. Their main advantage is that they provide the possibility to further analyze whole sequences contained in fingerprints using molecular methods. In addition to the analysis of microbial communities based on DNA extracted from soils, DGGE/TGGE can also be used for the assessment of the active part of the community based on the analysis of RNA-derived sequences or for the analysis of sequences of functional genes encoding for proteins involved in important soil processes.

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.

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