# US 10,767,159 B2 · Automated method for selecting microbial strains which can degrade or emulsify oil > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,159 B2 - Document ID: US10767159B2 - Status: Granted - Kind: B2 - Application number: 15/546,771 - Filed: 2016-01-27 - Published: 2018-01-18 - Granted: 2020-09-08 - Earliest claimed: 2015-01-28 - Assignees: Equinor Energy AS - Applicants: Equinor Energy AS - Inventors: Ane Kjølhamar; Anita Skarstad; Hans-Kristian Kotlar - Web page: https://patentgrep.com/patent/US10767159B2 ## CPC - C12N 1/26: Processes using, or culture media containing, hydrocarbons (refining of hydrocarbon oils by using microorganisms C10G32/00) - B01F 23/40: Mixing liquids with liquids; Emulsifying - C09K 8/582: characterised by the use of bacteria - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06) - C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE ## Abstract The invention provides an automated method for selecting a microbial strain from within a microbial strain library which can degrade or emulsify a target oil substrate. An apparatus, device or system adapted to perform the method of the invention is further provided. The use of the method of the invention in a method for preparing a microbial inoculum suitable for use to degrade or emulsify a target oil substrate; in a method of treating an oil reservoir or environmental remediation; and in a method for producing a biosurfactant-like substance is also provided. ## Claims ### Claim 1 An automated method for selecting a microbial strain from within a microbial strain library which can degrade or emulsify a target oil substrate, said method comprising in no particular order, unless specified: (a) providing a plurality of receptacles adapted to receive a liquid microbial cell culture as part of a multi-well culture plate wherein the internal surface(s) of the receptacles are coated, at least in part, with a layer of the target oil substrate; (b) applying to each of said oil-coated receptacle a sample of one of the members of the strain library, wherein a plurality of members of the strain library are applied to the plate; (c) culturing said samples in a liquid cell culture medium and monitoring said oil layer coating at least part of the internal surface(s) of said receptacles for a change in its appearance, wherein a change in the appearance of said oil layer coating at least part of the internal surface(s) of said receptacles is indicative of degradation or emulsification of said target oil substrate; (d) selecting those samples from step (c) which caused degradation or emulsification of the oil substrate; (e) separately culturing either (i) all the members of the library applied in step (b) or (ii) the library members selected in step (d) in the presence of an n-alkane and monitoring for degradation of said n-alkane; (f) separately culturing either (i) all the members of the library applied in step (b) or (ii) the library members selected in step (d) in the presence of a polycyclic aromatic hydrocarbon (PAH) and monitoring for degradation of said PAH; (g) subjecting either (i) all the members of the library applied in step (b) or (ii) the library members selected in step (d) to environmental tolerance testing, wherein said testing comprises culturing the library members under varying environmental conditions and determining the limits of these conditions at which colony viability ceases, wherein said environmental conditions tested comprising temperature, pH and ionic concentration, and optionally further comprising O2 concentration or pressure; (h) optionally repeating steps (a) to (c) with the library members selected at step (d); and (i) selecting a microbial strain on the basis of its performance in steps (c), (e), (f), (g) and optionally (h). ### Claim 2 (depends on 1) The automated method of claim 1 wherein the microbial strain selected on the basis of its performance in steps (c), (e), (f), (g) and optionally (h) is a microbial strain which causes biodegradation or emulsification of the oil substrate, degradation of said n-alkane or said PAH and which tolerates environmental conditions selected from temperature, pH and ionic concentration, and optionally O2 concentration or pressure that expected to be encountered during its use. ### Claim 3 (depends on 2) The automated method of claim 2 wherein each microbial strain of the library is scored in order of relative performance during steps (c), (e), (f), (g) and optionally (h) and selection is based on said scores. ### Claim 4 (depends on 3) The automated method of claim 3, wherein greater weight is given to the score for steps (c) and optionally (h) than steps (e), (f) or (g). ### Claim 5 (depends on 3) The automated method of claim 3 wherein the microbial strains of the library are ranked based on said scores and selection is of the portion of strains at the top of the rankings. ### Claim 6 (depends on 1) The automated method of claim 1, wherein said multi-well plate is a microplate. ### Claim 7 (depends on 1) The automated method of claim 1, wherein said oil is crude oil. ### Claim 8 (depends on 1) The automated method of claim 1, wherein said culturing in any one of steps (c), (e) or (f) is in a minimal medium. ### Claim 9 (depends on 1) The automated method of claim 1, wherein the n-alkane is a C16 or a C32 alkane. ### Claim 10 (depends on 1) The automated method of claim 1, wherein the PAH is dibenzothiophene. ### Claim 11 (depends on 1) A method for preparing a microbial inoculum suitable for use to degrade or emulsify a target oil substrate, said method comprising (i) performing the automated method as defined in claim 1, (ii) culturing a sample of said selected strain to increase the numbers of cells in the culture. ### Claim 12 (depends on 1) A method of treating an oil reservoir or environmental remediation, said method comprising (i) performing the automated method as defined in claim 1, and (ii) introducing a sample of said selected microbial strain or said microbial inoculum to an oil reservoir or contacting a site or material undergoing environmental remediation with a sample of said selected microbial strain or said microbial inoculum. ### Claim 13 (depends on 1) A method for producing a biosurfactant-like substance (BLS), said method comprising (i) performing the automated method as defined in claim 1 and selecting a strain which can emulsify the target oil substrate, (ii) culturing a sample of said selected strain under conditions which result in BLS production. ### Claim 14 (depends on 11) A method of treating an oil reservoir or environmental remediation, said method comprising (i) the method for preparing a microbial inoculum as defined in claim 11, and (ii) introducing a sample of said selected microbial strain or said microbial inoculum to an oil reservoir or contacting a site or material undergoing environmental remediation with a sample of said selected microbial strain or said microbial inoculum.