# US 10,308,908 B2 · Method of producing ethanol using continuous culture and continuous culture apparatus > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,908 B2 - Document ID: US10308908B2 - Status: Granted - Kind: B2 - Application number: 14/927,935 - Filed: 2015-10-30 - Published: 2016-05-05 - Granted: 2019-06-04 - Earliest claimed: 2014-10-31 - Assignees: Toyota Jidosha Kabushiki Kaisha - Applicants: Toyota Jidosha Kabushiki Kaisha - Inventors: Hibiki Matsushita; Emiko Tominaga - Web page: https://patentgrep.com/patent/US10308908B2 ## CPC - C12M 41/48: {Automatic or computerized control (automatic analysis G01N35/00)} - C12M 21/12: {for producing fuels or solvents (C12M21/04 takes precedence; liquid carbonaceous fuels C10L1/00, solid fuels C10L5/00)} - C12M 41/32: {of substances in solution} - C12P 7/10: substrate containing cellulosic material - C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source - Y02E 50/10: Biofuels, e.g. bio-diesel - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - 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)} - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract A method of producing ethanol includes: measuring a xylose concentration in a culture fluid that contains microorganisms having xylose utilizing ability, the culture fluid including a culture medium that contains saccharides derived from lignocellulose; and performing an addition control in which an additional culture medium is added to the culture fluid to conduct a continuous culture of the microorganisms, the additional culture medium containing saccharides derived from lignocellulose. ## Claims ### Claim 1 A continuous culture apparatus comprising: a culture tank in which microorganisms having xylose utilizing ability are continuously cultured in a culture medium containing saccharides derived from lignocellulose; a culture medium supply section that is programmed to perform a supply of an additional culture medium to the culture tank, the additional culture medium containing saccharides derived from lignocellulose, and wherein the additional culture medium does not contain the microorganisms; a xylose concentration measuring section that is programmed to measure a xylose concentration in a culture fluid in the culture tank; and a controller that is programmed to control the supply based on the measured xylose concentration, such that the xylose concentration is adjusted to be a preset threshold or lower, wherein said preset threshold is ⅓ or lower of the xylose concentration in the culture fluid before the start of culturing, wherein the controller is programmed to control a supply rate of the additional culture medium to the culture fluid such that the number of cells of microorganisms used for ethanol production produced per unit time and unit liquid volume in the continuous culture is more than the number of cells of microorganisms used for ethanol production produced per unit time and unit liquid volume in a batch culture in which the culture medium is used, and wherein the supply rate is higher than F which is obtained from the following equation F=V×(xmax−x0)/xs×T/(tbatch×(T−ti)) where F represents a supply rate of the additional culture medium, V represents a liquid volume of the culture fluid, xmax represents a maximum concentration of microorganisms used for ethanol production in the batch culture in which the culture medium is used, x0 represents a concentration of microorganisms used for ethanol predilection at a start of the batch culture in which the culture medium is used, xs represents a concentration of microorganisms used for ethanol production in a steady state of the continuous culture in which the culture medium is used, T represents a time for which the steady state is maintained in the culture fluid, tbatch represents a total time required for the batch culture in which the culture medium is used, and ti represents a total time required until the steady state is established in the continuous culture in which the culture medium is used. ### Claim 2 (depends on 1) The continuous culture apparatus according to claim 1, wherein the supply rate is controlled such that the xylose concentration in the culture fluid is 1 times or lower a xylose concentration in the culture fluid at an end of the batch culture in which the culture medium is used. ### Claim 3 (depends on 1) The continuous culture apparatus according to claim 1, wherein the culture medium contains a saccharified solution that is obtained by performing a saccharification treatment on lignocellulose after a steam treatment using a dilute acid. ### Claim 4 (depends on 1) The continuous culture apparatus according to claim 1, wherein the culture medium contains a saccharified solution that is obtained by performing a saccharification treatment on lignocellulose without performing a steam treatment using a dilute acid and in which a concentration of excessively decomposed saccharides in the saccharified solution is 400 ppm to 1500 ppm. ### Claim 5 (depends on 1) The continuous culture apparatus according to claim 1, wherein the xylose concentration is adjusted to be the preset threshold or lower by controlling the supply of an additional culture medium containing saccharides when the measured concentration is greater than the preset threshold. ### Claim 6 A continuous culture apparatus comprising: a culture tank in which microorganisms having xylose utilizing ability are continuously cultured in a culture medium containing saccharides derived from lignocellulose; a culture medium supply section that is programmed to perform a supply of an additional culture medium to the culture tank, the additional culture medium containing saccharides derived from lignocellulose, and wherein the additional culture medium does not contain the microorganisms; a xylose concentration measuring section that is programmed to measure a xylose concentration in a culture fluid in the culture tank; and a controller that is programmed to control the supply based on the measured xylose concentration, such that the xylose concentration is adjusted to be a preset threshold or lower, wherein said preset threshold is 10 g/L or lower. ### Claim 7 (depends on 6) The continuous culture apparatus according to claim 6, wherein the xylose concentration is adjusted to be the preset threshold or lower by controlling the supply of an additional culture medium containing saccharides when the measured concentration is greater than the preset threshold. ### Claim 8 A continuous culture apparatus comprising: a culture tank in which microorganisms having xylose utilizing ability are continuously cultured in a culture medium containing saccharides derived from lignocellulose; a culture medium supply section that is programmed to perform a supply of an additional culture medium to the culture tank, the additional culture medium containing saccharides derived from lignocellulose, and wherein the additional culture medium does not contain the microorganisms; a xylose concentration measuring section that is programmed to measure a xylose concentration in a culture fluid in the culture tank; and a controller that is programmed to control the supply based on the measured xylose concentration, wherein the controller is programmed to control a supply rate of the additional culture medium to the culture fluid such that the number of cells of microorganisms used for ethanol production produced per unit time and unit liquid volume in the continuous culture is more than the number of cells of microorganisms used for ethanol production produced per unit time and unit liquid volume in a batch culture in which the culture medium is used, and wherein the supply rate is higher than F which is obtained from the following equation F=V×(xmax−x0)/xs×T/(tbatch×(T−ti)) where F represents a supply rate of the additional culture medium, V represents a liquid volume of the culture fluid, xmax represents a maximum concentration of microorganisms used for ethanol production in the batch culture in which the culture medium is used, x0 represents a concentration of microorganisms used for ethanol production at a start of the batch culture in which the culture medium is used, xs represents a concentration of microorganisms used for ethanol production in a steady state of the continuous culture in which the culture medium is used, T represents a time for which the steady state is maintained in the culture fluid, tbatch represents a total time required for the batch culture in which the culture medium is used, and ti represents a total time required until the steady state is established in the continuous culture in which the culture medium is used. ### Claim 9 (depends on 8) The continuous culture apparatus according to claim 8, wherein the xylose concentration is adjusted to be a preset threshold or lower by controlling the supply of an additional culture medium containing saccharides when the measured concentration is greater than the preset threshold.