Application of fluorescent in-situ hybridization in the evaluation of the biocides used in the oil industry

Abstract

Microbiologically influenced corrosion (MIC) and souring of oilfield reservoirs as result of the presence of sulfate-reducingbacteria (SRB) are of great concern in the oil industry. Considering the limitations of conventional culture-based methods forstudying SRB, techniques involving the direct analysis of the microbial population from their genetic material are getting moreattention. One of them is Fluorescent in situ Hybridization (FISH), which uses fluorescently labeled oligonucleotide probes thathybridizes specifically to its complementary 16S rRNA target sequence within the intact cell. Using FISH, the abundance of thedetected microorganisms can be determined by counting the cells stained with a general DNA-binding dye and the cellshybridized with a specific probe. To control bacterial populations, biocides are commonly applied to injection waters andproduction facilities. The aim of this work was to evaluate the possibility of using FISH to help in the selection of an appropriatebiocide for the water treatment plant of an oil secondary recovery plant (OSRP). Three commercial biocides based on THPS andquaternary ammonium salts were used in the assay in a concentration of 200mg/l. Three replicates of each biocide in eachconcentration were done. The following probes (5P end-labeled with Cy3) were used: Eub338; Non338 and SRB385. Thefluorescence was detected with a Leica microscope, analyzing 15 to 20 images per sample. The inhibition effect of the biocideswas tested in OSRP water filtered inoculated with a microbial culture in PostagteB medium, obtained from the same water. Theinhibitory effect was determined by counting the cells hybridized with the Eub338 and SRB385 probes. The percentage ofhybridized vs DAPI-stained cells, RS%, was calculated. The RS% values for the Eub338 probe obtained after 4h of incubationat60ºC(water treatment plant condition) in the presence of biocides B1 and B3 were higher than those obtained with the control(without biocide). This behavior suggested a metabolic activation, associated with a high content of RNA in the cells. It could berelated with the mode of action of the chemicals or the application of a sub lethal dose. The RS% values obtained with B2 wereno significantly different from those of the control, associated with no inhibitory effect. The RS% values obtained after 4h ofincubation for SRB population showed that none of the tested biocides produced metabolic activation. B1 and B3 biocidal effectswere not different from that observed in the control. Instead, the lower RS% values obtained with B2 were associated with asignificant inhibitory effect on SRB population, showing that the SRB population was more sensitive to B2 effect under the testedcondition. These results suggested that FISH could be helpful in the screening of biocides, being a responsive and suitable testto detected inhibitory concentrations.