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). 01/2001; 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.
    Journal of Environmental Engineering 10/2015; DOI:10.1061/(ASCE)EE.1943-7870.0001031 · 1.27 Impact Factor
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    • "V3–V5 16S rRNA gene tag sequencing Variable regions 3 to 5 (V4–V5) of the 16S rRNA gene from Bacteria and Archaea were amplified from a selected number of unamended slurry DNA samples covering a range of temperatures (25, 35, 38, 46, 66 • C) and time points (0, 15, 35, 62 days) using barcoded fusion primers 357F/907R (Muyzer, Dewaal and Uitterlinden 1993; Muyzer et al. 1998 "
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    ABSTRACT: The impact of temperature (0 - 80°C) on anaerobic biogeochemical processes and prokaryotic communities in marine sediments (tidal flat) was investigated in slurries for up to 100 days. Temperature had a non-linear effect on biogeochemistry and prokaryotes with rapid changes over small temperature intervals. Some activities (e.g. methanogenesis) had multiple "windows" within a large temperature range (∼10 - 80°C). Others, including acetate oxidation, had maximum activities within a temperature zone, which varied with electron acceptor (metal oxide [up to ∼34°C] and sulphate [up to ∼50°C]). Substrates for sulphate reduction changed from predominantly acetate below, and H2 above, a 43°C critical temperature; along with changes in activation energies and types of sulphate-reducing Bacteria. Above ∼43°C, methylamine metabolism ceased with changes in methanogen types and increased acetate concentrations (>1mM). Abundances of uncultured Archaea, characteristic of deep marine sediments (e.g. MBGD Euryarchaeota, 'Bathyarchaeota') changed, indicating their possible metabolic activity and temperature range. Bacterial cell numbers were consistently higher than archaeal cells and both decreased above ∼15°C. Substrate addition stimulated activities, widened some activity temperature ranges (methanogenesis) and increased bacterial (x10) more than archaeal cell numbers. Hence, additional organic matter input from climate-related eutrophication may amplify the impact of temperature increases on sedimentary biogeochemistry. © FEMS 2015.
    FEMS Microbiology Ecology 07/2015; 91(8). DOI:10.1093/femsec/fiv084 · 3.57 Impact Factor
    • "The second step used a lower annealing temperature of 52 °C instead of 55 °C. The 18S and 16S rRNA gene amplicons were applied onto a 6 % polyacrylamide gel with a denaturing gradient from 20 to 50 % and 10 to 70 % UF, respectively (Schäfer and Muyzer 2001). Individual bands were excised with a sterile razor and incubated overnight in 40 μL 10 mM tris solution water at 4 °C. "
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    ABSTRACT: Large-scale production of microalgal storage compounds will likely involve marine microalgae. Previously, we described a method to enrich microalgae with a high storage compound productivity from a natural inoculum. Here, this strategy was implemented under marine conditions in a sequencing batch reactor. The influence of the volume exchange ratio and the moment of ammonium addition in the day-night cycle on the storage compound productivity are described. Storage compound productivity was maximal if ammonium was supplied at the start of the dark period rather than the light period, irrespective of the volume exchange ratio. Increasing the volume exchange ratio from 33 to 50 % per cycle induced a decrease in storage compound production if ammonium was supplied in the light whereas the storage compound productivity was comparable when ammonium was supplied in the dark. The latter indicates a shift of cell division processes to the light period at increasing volume exchange ratio, although ammonium uptake completely occurred in the dark period.
    Journal of Applied Phycology 06/2015; 27(3):1121-1126. DOI:10.1007/s10811-014-0430-3 · 2.56 Impact Factor
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