scholarly journals Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies

2020 ◽  
Vol 8 (8) ◽  
pp. 1099
Author(s):  
Lucilla Iacumin ◽  
Francesca Cecchini ◽  
Marco Vendrame ◽  
Giuseppe Comi
Keyword(s):  
Microbial Population ◽  
Population Studies ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Conventional Pcr ◽  
Dgge Analysis ◽  
Emulsion Pcr ◽  
Gradient Gel Electrophoresis ◽  
Preferential Amplification ◽  
Polymerase Chain ◽  
Higher Sensitivity

To the authors’ knowledge, this is the first report of the use of emulsion-Polymerase chain reaction (e-PCR) coupled with denaturing gradient gel electrophoresis (DGGE) analysis. In the present work the effectiveness of ePCR in improving the power of the DGGE technique for microbial population studies was tested. Our results indicated that ePCR results in uniform amplification of several DNA molecules, overcoming the major limitations of conventional PCR, such as preferential amplification and DNA concentration dependence. Moreover, ePCR-DGGE resulted in higher sensitivity when compared to conventional PCR-DGGE methods used for studying microbial populations in a complex matrix. In fact, compared to conventional PCR, the DGGE profiles of ePCR products permitted the detection of a higher number of the species that were present in the tested sample.

scholarly journals PCR-based DGGE fingerprinting and identification of the microbial population in South African red grape must and wine

OENO One ◽  
2013 ◽  
Vol 47 (1) ◽  
pp. 47 ◽  
Author(s):  
Michelle Cameron ◽  
Leoni Siebrits ◽  
Maret Du Toit ◽  
Corli Witthuhn
Keyword(s):  
South African ◽  
Alcoholic Fermentation ◽  
Microbial Population ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Grape Must ◽  
Microbiological Methods ◽  
Spontaneous Fermentations ◽  
Gradient Gel Electrophoresis ◽  
Polymerase Chain ◽  
Red Grape

<p style="text-align: justify;"><strong>Aim</strong>: The aim of this study was to evaluate the microbial population present in red grape must and wine using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE).</p><p style="text-align: justify;"><strong>Methods and results</strong>: Red wine from the cultivars Pinotage and Merlot were produced and samples taken throughout alcoholic and malolactic fermentation (MLF). PCR fragments were resolved by DGGE and unique fingerprints were obtained for the bacteria and yeasts present in the wines. <em>Lactobacillus plantarum</em>, <em>Enterobacter</em> <em>sakazakii</em> (<em>Cronobacter</em> sp.) and <em>Pantoea agglomerans</em> were present in the Pinotage during both alcoholic and MLF, and in both inoculated and spontaneous fermentations. <em>E. sakazakii</em> (<em>Cronobacter</em> sp.) and <em>P. agglomerans</em> were also observed in the Merlot wines during alcoholic fermentation as well as MLF. <em>Saccharomyces cerevisiae</em> was the most dominant yeast observed in Pinotage, and was the only yeast observed in Merlot. This yeast was observed until the end of MLF.</p><p style="text-align: justify;"><strong>Conclusions</strong>: Results showed that the microbial flora that participates in the winemaking process is more diverse than commonly thought.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This method may serve as an alternative to conventional microbiological methods for the identification of the microbial species in red grape must and wine.</p>

scholarly journals Investigations on the Effects of Dietary Essential Oils and Different Husbandry Conditions on the Gut Ecology in Piglets after Weaning

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
P. Janczyk ◽  
R. Pieper ◽  
V. Urubschurov ◽  
K. R. Wendler ◽  
W. B. Souffrant
Keyword(s):  
Essential Oils ◽  
Dry Matter ◽  
Distal Part ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Control Diet ◽  
Short Chain Fatty Acids ◽  
Growth Promoters ◽  
Gradient Gel Electrophoresis ◽  
Antibiotic Growth Promoters ◽  
Polymerase Chain

Essential oils (EO) are being considered as possible alternatives to in-feed antibiotic growth promoters in pig nutrition. The effects of an EO mixture consisting of limonene, eugenol and pinene (10.0, 2.0, and 4.8 mg/kg diet, resp.) on gut physiology and ecology were studied in piglets. The experiment was conducted at low (commercial farm) and high hygienic conditions (experimental farm), to elucidate interactions between EO supplementation and husbandry methods. Piglets were weaned at 28 days of age, when they were offered either a control diet (C) or C with EO. Four piglets were sacrificed in each group on day 29, 30, 33 and 39. Digesta from the third distal part of the small intestine and from the colon were sampled and analysed for pH, dry matter, lactic acid, short chain fatty acids and ammonia concentrations. Enterobacteria, enterococci, lactobacilli and yeast counts were obtained by plating. Genomic DNA was extracted from digesta and polymerase chain reaction—denaturing gradient gel electrophoresis was performed. Individual microbial communities were identified at each farm. Age affected the intestinal parameters. No effects of the EO with exception for a significant reduction in colon bacterial diversity at 39 days of age could be recorded at experimental farm.

The assessment of the coke wastewater treatment efficacy in rotating biological contractor

2015 ◽  
Vol 73 (5) ◽  
pp. 1202-1210 ◽  
Author(s):  
G. Cema ◽  
S. Żabczyński ◽  
A. Ziembińska-Buczyńska
Keyword(s):  
Biological Treatment ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rotating Biological Contactor ◽  
Coke Wastewater ◽  
Promising Tool ◽  
Start Up ◽  
Gradient Gel Electrophoresis ◽  
Real Wastewater ◽  
Polymerase Chain ◽  
Anammox Process

Coke wastewater is known to be relatively difficult for biological treatment. Nonetheless, biofilm-based systems seem to be promising tool for such treatment. That is why a rotating biological contactor (RBC) system focused on the Anammox process was used in this study. The experiment was divided into two parts with synthetic and then real wastewater. It was proven that it is possible to treat coke wastewater with RBC but such a procedure requires a very long start-up period for the nitritation (190 days), as well as for the Anammox process, where stable nitrogen removal over 70% was achieved after 400 days of experiment. Interestingly, it was possible at a relatively low (20.2 ± 2.2 °C) temperature. The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) based monitoring of the bacterial community showed that its biodiversity decreased when the real wastewater was treated and it was composed mainly of GC-rich genotypes, probably because of the modeling influence of this wastewater and the genotypes specialization.

PCR-DGGE analysis of denitrifying bacteria in a metallurgic wastewater treatment process

2002 ◽  
Vol 46 (1-2) ◽  
pp. 333-336 ◽  
Author(s):  
N. Noda ◽  
S. Yoshie ◽  
T. Miyano ◽  
S. Tsuneda ◽  
A. Hirata ◽  
...  
Keyword(s):  
Fluidized Bed ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Precious Metals ◽  
Packed Bed ◽  
Population Diversity ◽  
Operating Conditions ◽  
Industrial Wastes ◽  
Wastewater Treatment Process ◽  
Dgge Analysis ◽  
Gradient Gel Electrophoresis

The wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of acids such as nitric acid and of salts. Biological nitrogen removal from this wastewater was attempted by using a circulating bioreactor system equipped with an anoxic packed bed or an anoxic fluidized bed and an aerobic three-phase fluidized bed. The system was found to effectively remove nitrogen from the diluted wastewater (T-N; 1,000–4,000 mg litre−1). The microbial population structure of activated sludge in an anoxic reactor was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. DGGE analysis under different operating conditions demonstrated the presence of some distinguishable bands in the separation pattern, which were most likely derived from many different species constituting the microbial communities. Furthermore, the population diversity varied in accordance with the nitrate-loading rate, water temperature and reactor condition. Some major DGGE bands were excised, reamplified and directly sequenced. It was revealed that the dominant population in the anoxic reactor were affiliated with the β subclass of the class Proteobacteria.

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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