BR112020001297B1 - METHOD TO DISTRIBUTE EXISTING BIOFILM - Google Patents

Abstract

The present invention relates to a method for distributing biofilm formed by sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfovibrio vulgaris, placing said biofilm in contact with a compound selected from the mannose group, 2-deoxy-D- glucose, methyl-α-D-mannopyranoside and methyl-α-D-glucopyranoside.

Description

[001] The present invention relates to a method to distribute biofilm with the use of mannose and its analogues.

[002] Sulfate Reducing Prokaryotes (SRPs) are known for their detrimental effects on the vast infrastructure needed to produce and transport oil and gas. These organisms, known to cultivate in both planktonic and sessile communities, are some of the major contributors to reservoir acidification, microbially influenced corrosion (MIC) of equipment and other mild steel components, and biofilm-induced plugging and flow restriction. The negative impact of SRPs on both oil production costs and product quality has made these organisms attractive targets for advanced microbial control strategies.

[003] In SRPs, bacteria in the genus Desulfovibrio have been shown to be prevalent in oil and gas applications. In particular, Desulfovibrio vulgaris (D. vulgaris) forms robust biofilms that are capable of inducing pit corrosion in mild steel. U.S. Document No. 7,060,486 discloses that forming biofilms of non-native aerobic bacteria that secrete antimicrobial agents in a system containing SRB can inhibit SRB growth in aqueous systems. Although biocide efficacy and inhibition have been positively demonstrated against SRBs, there is a demand for biocide alternatives that are less toxic, more sustainable and demonstrate the ability to distribute existing biofilms.

[004] Document No. US2016/0000680A1 demonstrates that 2-deoxy-D-glucose has the ability to inhibit biofilm formation in aerobic and facultative bacteria, such as Escherichia coli and oral bacteria; however, there is no teaching of inhibition of biofilm formation in obligate anaerobic SRBs. Additionally, no biofilm distribution of formed biofilms was addressed. Because biofilm inhibition and delivery involve two different processes and mechanisms, there remains a need for an effective method to deliver existing biofilm.

[005] The present invention seeks to solve the problems of the art by providing a method for delivering existing biofilm comprising providing a biofilm; and contacting the biofilm with a compound selected from the group consisting of mannose, 2-deoxy-D-glucose (2DG), methyl α-D-mannopyranoside (αMM), methyl α-D-glycopyranoside (αMG) , and mixtures thereof to distribute the biofilm.

[006] As used herein, the term "biofilm" is defined as a multicellular bacterial community composed of surface-associated microbial cells that are held together by a self-evolving matrix of extracellular polymeric substance.

[007] As used herein, the term “biofilm distribution” is defined as the displacement of all or nearly all sessile cells of the biofilm. Biofilm distribution is the final stage of the biofilm life cycle and could involve several signaling and regulatory processes.

[008] Biofilms of the present invention are found in aqueous systems, such as, for example, industrial wastewater systems and waters resulting from oil and gas operations.

[009] Biofilms of the present invention comprise prokaryotes. Suitable prokaryotes are bacteria, preferably anaerobic bacteria. The biofilms of the present invention may comprise sulfate-reducing prokaryotes (SRPs), suitably sulfate-reducing bacteria. Such sulfate reducing bacteria may be of the genus Desulfovibrio and in particular Desulfovibrio vulgaris (ATCC 29579), Desulfovibrio desulfuricans (DSM 12129) or mixtures thereof.

[0010] To distribute the biofilms, the biofilms are contacted with a compound selected from the group consisting of mannose, 2-deoxy-D-glucose (2DG), α-methyl-D-mannopyranoside (αMM), α- Methyl D-glycopyranoside (αMG) and mixtures thereof. Useful concentrations of mannose and its derivatives are in the range 1 to 500 mM, alternatively 5 to 500 mM, alternatively 30 to 500 mM and alternatively 100 to 500 mM.

[0011] The following examples are provided for illustrative purposes only and should in no way limit the scope of the present invention beyond the scope mentioned above in the specification.

EXAMPLES

[0012] Cultures of Desulfovibrio vulgaris (ATCC 29579) and Desulfovibrio desulfuricans (DSM 12129) were prepared in modified Baar medium at 30 °C under anaerobic conditions. Bacterial cultures were then used to prepare bacterial suspensions in fresh modified Baar medium at a cell density of 0.1 (Tables 2 to 6) or 0.05 (Table 1) at 600 nm. The appropriate bacterial suspension was used to fill 96-well plates and the plates were incubated in a tight chamber with anaerobic gloves for 24 h (Tables 2 to 6) or 48 h (Table 1) at 30 °C to develop biofilms. D-Mannose Reserves (2.85 M, Alpha Aesar, Cat# A10842), 2DG (1.22 M, Alpha Aesar, Cat# AAAL07338-06), αMM (1.22 M, Acros Organics, Cat# C229251000) and αMG (1.22 M, Alfa Aesar, Cat# AAA12484-22) were prepared in sterile distilled water and filtered through a 0.22 µm filter. For biofilm distribution assay, planktonic cells were removed, and plates were washed with 150 µl of 1X phosphate buffered saline (pH 7.4); D-mannose, 2DG, αMM and αMG were added, the volume was adjusted with 1X PBS, pH 7.4 in 150 μl, and the plates were incubated in the airtight chamber with anaerobic gloves for 2 h (Table 1, 2, 4 , 5, 6) or 14 h (Table 3) for biofilm distribution studies. After treatment, supernatants were discarded; the wells were washed three times with deionized water (DW) by dipping the plates in a 1 L solution of DW, and the plates were dried through a piece of paper towel with covers. 300 μl of 0.1% gentian violet was added to each well, the plates were incubated for 20 minutes at room temperature (25 °C), and the staining solution was discarded. The plates were washed three times with DW by immersing the plates in a 1 L DW solution, then 300 µl of 95% ethanol was added to each well, and the plates were soaked for 5 min to dissolve the DW violet. gentian. Total biofilm was measured by spectrophotometry at 540 nm using a Sunrise microplate reader (Tecan, Austria Gesellschaft, Salzburg, Austria). The total biofilm remaining after treatments are summarized in Tables 1 to 6. Table 1. Remaining D. vulgaris biofilm after 2 h of mannose treatment Table 2. Remaining biofilm of D. vulgaris after 2 h of treatment with 2DG Table 3. Remaining biofilm of D. vulgaris after 14 h of treatment with 2DG Table 4. Remaining biofilm of D. vulgaris after 2 h of treatment with αMM Table 5. Remaining D. vulgaris biofilm after 2 h of αMG treatment Table 6. Remaining biofilm of D. desulfuricans after 2 h of treatment with mannose and its analogues

Claims (5)

1. Method for distributing existing biofilm, characterized in that it comprises: i. providing a biofilm, wherein the biofilm comprises sulfate-reducing prokaryotes; and ii. contacting the biofilm with a compound selected from the group consisting of mannose, 2-deoxy-D-glucose (2DG), methyl α-D-mannopyranoside (αMM), methyl α-D-glucopyranoside (αMG), and mixtures thereof to distribute the biofilm. 2. Method according to claim 1, characterized in that the biofilm comprises anaerobic bacteria. 3. Method according to claim 1, characterized in that the biofilm comprises sulfate-reducing bacteria. 4. Method according to claim 2, characterized in that the biofilm comprises Desulfovibrio vulgaris (ATCC 29579). 5. Method according to claim 2, characterized in that the biofilm comprises Desulfovibrio desulfuricans (DSM 12129).