# US 9,518,304 B2 · Nicotine resistant microorganisms > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,518,304 B2 - Document ID: US9518304B2 - Status: Granted - Kind: B2 - Application number: 14/723,397 - Filed: 2015-05-27 - Published: 2015-12-03 - Granted: 2016-12-13 - Earliest claimed: 2014-05-28 - Assignees: Tyton Biosciences, LLC - Applicants: Tyton Biosciences, LLC - Inventors: Hsi-Chuan Chen; Peter Majeranowski; Igor Kostenyuk; Iulian Bobe - Web page: https://patentgrep.com/patent/US9518304B2 ## CPC - C12N 1/185: Saccharomyces isolates - C12N 1/16: Yeasts; Culture media therefor - C12N 1/36: Adaptation or attenuation of cells - C12P 7/10: substrate containing cellulosic material - C12R 2001/865: Saccharomyces cerevisiae - Y02E 50/10: Biofuels, e.g. bio-diesel - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract Compositions and methods are provided for enhanced production of ethanol from fermentation of tobacco biomass. Nicotine resistant microorganisms are provided, as well as methods for making these nicotine resistant microorganisms. A biologically pure culture is provided of a nicotine resistant Saccharomyces cerevisiae strain or a mutant thereof having all the identifying characteristics thereof. Methods are provided for producing ethanol from fermentation of tobacco biomass in which the nicotine resistant microorganisms are used in the fermentation of the tobacco biomass, wherein a higher amount of ethanol can be produced from the fermentation. The nicotine resistant yeast strains of the present disclosure can improve ethanol production in tobacco biomass extract fermentations and shorten the fermentation time. ## Claims ### Claim 1 A method for generating a nicotine resistant microorganism, the method comprising: (a) culturing a microorganism in a medium conducive to growth, wherein the medium comprises a first concentration of nicotine that retards but does not inhibit growth of the microorganism; (b) selecting a single colony of the microorganism and inoculating the single colony into a fresh culture of the medium conducive to growth, wherein the fresh medium comprises a second concentration of nicotine that is incrementally higher than the first concentration and that retards but does not inhibit growth of the microorganism; and (c) repeating step (b) until the desired concentration of nicotine is achieved or until growth of the microorganism is completely inhibited, wherein the microorganism produced is resistant to the desired concentration of nicotine. ### Claim 2 (depends on 1) The method of claim 1, wherein the microorganism is a yeast. ### Claim 3 (depends on 2) The method of claim 2, wherein the yeast is a Saccharomyces cerevisiae. ### Claim 4 (depends on 1) The method of claim 1, wherein the first concentration of nicotine ranges from about 0.5% w/v to about 1.5% w/v nicotine. ### Claim 5 (depends on 1) The method of claim 1, wherein the incrementally higher concentration of nicotine is an incremental increase of about 0.5% w/v. ### Claim 6 (depends on 1) The method of claim 1, wherein the microorganism is cultured in the medium conducive to growth for a length of time sufficient for the microorganism to reach a growth plateau. ### Claim 7 A method for producing ethanol from tobacco biomass, the method comprising: (i) fermenting a tobacco biomass extract with a nicotine resistant microorganism, wherein ethanol is produced from the fermentation and wherein the nicotine resistant microorganism is produced according to a process comprising: (a) culturing a microorganism in a medium conducive to growth, wherein the medium comprises a first concentration of nicotine that retards but does not inhibit growth of the microorganism; (b) selecting a single colony of the microorganism and inoculating the single colony into a fresh culture of the medium conducive to growth, wherein the fresh medium comprises a second concentration of nicotine that is incrementally higher than the first concentration and that retards but does not inhibit growth of the microorganism; and (c) repeating step (b) until the desired concentration of nicotine is achieved or until growth of the microorganism is completely inhibited, wherein the microorganism produced is resistant to the desired concentration of nicotine; and (ii) isolating the ethanol produced in step (i). ### Claim 8 (depends on 7) The method of claim 7, wherein the amount of ethanol produced from the fermentation is increased by 40% or greater due to use of the nicotine resistant microorganism. ### Claim 9 (depends on 7) The method of claim 7, wherein the amount of ethanol produced from the fermentation is increased by 80% or greater due to use of the nicotine resistant microorganism. ### Claim 10 (depends on 7) The method of claim 7, wherein the nicotine resistant microorganism is a yeast. ### Claim 11 (depends on 10) The method of claim 10, wherein the yeast is a Saccharomyces cerevisiae. ### Claim 12 (depends on 7) The method of claim 7, wherein the first concentration of nicotine ranges from about 0.5% w/v to about 1.5% w/v nicotine. ### Claim 13 (depends on 7) The method of claim 7, wherein the incrementally higher concentration of nicotine is an incremental increase of about 0.5% w/v. ### Claim 14 (depends on 7) The method of claim 7, wherein the microorganism is cultured in the medium conducive to growth for a length of time sufficient for the microorganism to reach a growth plateau.