APPLICATION OF FLOURESCENT IN-SITU HYBRIDIZATION IN THE EVALUATION OF THE BIOCIDES USED IN THE OIL INDUSTRY.

C. TERADA; DEL PANNO M.T; VIERA M.

Congreso; XI Congreso Argentino de Microbiología General. SAMIGE; 2015

Microbiologically influencedcorrosion (MIC) and souring of oilfield reservoirs as a result of the presenceof sulfate-reducing bacteria (SRB) are of great concern in the oil industry.Considering the limitations of conventional culture-based methods for studyingSRB, techniques involving the direct analysis of the microbial population fromtheir genetic material are getting more attention. One of them is Fluorescent in situ Hybridization (FISH), which usesfluorescently labeled oligonucleotide probes that hybridizes specifically toits complementary 16S rRNA target sequence within the intact cell. Using FISH, theabundance of the detected microorganisms can be determined by counting thecells stained with a general DNA-binding dye and the cells hybridized with aspecific probe. To control bacterial populations, biocidesare commonly applied to injection waters and production facilities. The aim ofthis work was to evaluate the possibility of using FISH to help in theselection of an appropriate biocide to be used in the water treatment plant ofan oil secondary recovery plant (OSRP). Three available commercial biocidesbased on THPS and quaternary ammonium salts were used in the assay. Threereplicates of each biocide in each concentration were done. The followingprobes (5P end-labeled with Cy3) were used Eub338; Non338 and SRB385. The fluorescencewas detected with a Leica microscope. Among 15 to 20 images were analyzed foreach sample.The inhibition effect of thebiocides was tested in OSRP water filtered inoculated with a microbial culturein PGB medium, obtained from the same water. One concentration of 200mg/l ofthe three biocides was assayed. The inhibitory effect was determined by countof the cells hybridized with the Eub338 and SRB385 probes. The percentage relationship of hybridizedversus DAPI stain cells, RS%, was calculated. The RS% values obtained after 4hs of the incubation at 60ºC (water treatment plantcondition) for bacterial population with B1 and B3 biocides were higher thanthose obtained with the control (without biocides). These behavior suggested ametabolic activation, associated with a high content of RNA from the cells. Itcould be produced by the mode of action of the chemicals or the application ofa sub lethal dose. The RS% values obtained with B2 biocide were nosignificantly different from those with the control, associated with no inhibitoryeffect. The RS% values obtained after 4hs ofthe incubation for SRB population showed that none tested biocides produced metabolicactivation. B1 and B3 biocide effect were no different from that observed inthe control. Instead, the lower RS% values obtained with B2 were associatedwith a significant inhibitory effect on SRB population. The results evidencedthat the SRB population was more sensible to B2 biocide effect under the testedcondition.  As an initial approach for the useof the FISH technique to evaluate the efficiency of biocides in water treatmentplant, the results demonstrated that it could be useful inscreening of biocides, resulting a responsive and suitable test to detectedinhibitory agent concentrations in a relative short time.