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Denaturing Gradient Gel Electrophoresis in Marine Microbial Ecology

Koninklijk Nederlands Instituut voor Onderzoek der Zee - NIOZ, Burg, North Holland, Netherlands
Methods in Microbiology (Impact Factor: 0.08). 12/2001; 30:425-468. DOI: 10.1016/S0580-9517(01)30057-0
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    • "The resulting DNA samples were then subsequently stored at −20°C until further analysis. PCR Amplification of the 16 S rRNA V3 Region from the Biofilm Microbes From the extracted biofilm DNA, the V3 region of the 16S bacterial rRNA gene was amplified through polymerase chain reaction (PCR) according to previously-described conditions (Schafer and Muyzer 2001). Briefly, 40-μL reactions were prepared consisting of 1 × iTaq amplification buffer [Bio-Rad Laboratories (Canada), Mississauga, Ontario], 200 nM of each dNTP, 500 nM of each primer, 2 mg ml −1 BSA, and 2U iTaq polymerase [Bio-Rad Laboratories (Canada) Mississauga, Ontario]. "
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    ABSTRACT: Phosphate, a key nutrient for bacterial growth, is also a key component of many corrosion-control programs to manage lead and copper corrosion in premise plumbing. Bench-scale stagnant water galvanic macrocells with lead and copper components were fed with drinking water containing three levels of zinc orthophosphate [0 (control), 1, and 3 mg l −1-PO 4 ]. Suspended polycarbonate coupons, representing benign downstream fixtures, were placed in the macrocells, thus enabling biofilm formation on this material. Community profiling using denaturing gradient gel electrophoresis (16S rDNA PCR-DGGE) revealed that phosphate dose (primarily) and metal type (to a lesser extent) influenced biofilm community diversity. Generally, community diversity increased with increasing heterotrophic plate counts that in turn rose in response to elevated phosphate. Partial 16s rDNA sequences obtained from DGGE gel bands identified the dominant bacterial taxa as the phyla Verrumicrobia, Firmicutes, Bacteroidetes, and α-Proteobacteria. The increase in size and diversity of biofilm communities as a result of phosphate treatment further highlights the challenges of a phosphate corrosion-control program.
    Full-text · Article · Feb 2016 · Journal of Environmental Engineering
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    • "The extracted DNA was used as for PCR amplifica - tion of the 16S rRNA gene . The set of primers used is the 341F ( containing a 40 - bp GC clamp ) and 907R ( Schäfer and Muyzer , 2001 ) . The used PCR thermal profile started with a pre - cooling phase at 4 °C for 1 min , followed by initial denaturation at 95 °C for 5 min , 32 cycles of 95 °C for 30 s , 55 °C for 40 s , 72 °C for 40 s , followed by an additional extension step at 72 °C for 30 min . "

    Full-text · Article · Dec 2015 · The ISME Journal
    • "The extracted DNA was used as for PCR amplification of the 16S rRNA gene. The set of primers used is the 341F (containing a 40-bp GC clamp) and 907R (Schäfer and Muyzer, 2001). The used PCR thermal profile started with a pre-cooling phase at 4 °C for 1 min, followed by initial denaturation at 95 °C for 5 min, 32 cycles of 95 °C for 30 s, 55 °C for 40 s, 72 °C for 40 s, followed by an additional extension step at 72 °C for 30 min. "
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    ABSTRACT: Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are competing microbial nitrate-reduction processes. The occurrence of DNRA has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in a laboratory reactor, yet no successful DNRA enrichment culture has been described. We showed that a stable DNRA-dominated enrichment culture can be obtained in a chemostat system. The enrichment was based on the hypothesis that nitrate limitation is the dominant factor in selecting for DNRA. First, a conventional denitrifying culture was enriched from activated sludge, with acetate and nitrate as substrates. Next, the acetate concentration in the medium was increased to obtain nitrate-limiting conditions. As a result, conversions shifted from denitrification to DNRA. In this selection of a DNRA culture, two important factors were the nitrate limitation and a relatively low dilution rate (0.026 h(-1)). The culture was a highly enriched population of Deltaproteobacteria most closely related to Geobacter lovleyi, based on 16S rRNA gene sequencing (97% similarity). We established a stable and reproducible cultivation method for the enrichment of DNRA bacteria in a continuously operated reactor system. This enrichment method allows to further investigate the DNRA process and address the factors for competition between DNRA and denitrification, or other N-conversion pathways.
    No preview · Article · Dec 2015 · The ISME Journal
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