Renewable Chemicals and Fuels From Oleaginous Yeast

Abstract

The invention provides methods of manufacturing alkanes from triglyceride oils produced through fermentation of oil-bearing microbes. The processes provided herein can utilize a variety of carbohydrate feedstocks including cane bagasse, sugar beet pulp, corn stover, glycerol, corn starch, sorghum, molasses, waste glycerol, and other renewable materials. These processes further comprise hydrotreating, hydrocracking, isomerization, distillation, and other petrochemical processes for use with oil-bearing microbes and products derived therefrom to manufacture fuels. Particular embodiments include the manufacture of ASTM D975 and ASTM D1655 compliant fuels. Genetically engineered microbes provided herein can be used in the manufacture of renewable diesel and renewable jet fuel.

1 . A method of producing a fuel or a chemical, the method comprising the steps of:
 (a) heterotrophically culturing an oleaginous yeast in the presence of a fixed carbon source, wherein:
 (i) the fixed carbon source is selected from the group consisting of glycerol, depolymerized cellulosic material, sucrose, molasses, glucose, arabinose, galactose, xylose, fructose, mannose, and acetate; and 
 (ii) the oleaginous yeast accumulates at least 10% of its dry cell weight as triacylglyceride; 
   (b) isolating the triacylglyceride from the oleaginous yeast; and   (c) subjecting the isolated triacylglyceride to a chemical reaction selected from the group consisting of cracking, (hydro)deoxygenation, esterification, and isomerization, and optionally, distillation.   
     
     
         2 . The method of  claim 1 , wherein the depolymerized cellulosic material is selected from the group consisting of sugar cane bagasse, sugar beet pulp, corn stover, wood chips, sawdust, switchgrass, rice hull, corn fiber, wheat straw, rice straw, and citrus pulp. 
     
     
         3 . The method of  claim 1 , wherein the oleaginous yeast is selected from the group consisting of  Cryptococcus curvatus, Cryptococcus terricolus, Candida  sp.,  Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis , and  Yarrowia lipolytica.    
     
     
         4 . The method of  claim 1 , wherein the oleaginous yeast contains at least one exogenous gene encoding a lipid pathway enzyme. 
     
     
         5 . The method of  claim 4 , wherein the triacylglyceride pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, a fatty acyl-ACP thioesterase, and a glycerol-3 phosphate acyltransferase. 
     
     
         6 . The method of  claim 1 , wherein said fuel or chemical is selected from the group consisting of renewable diesel, fatty acid esters, and jet fuel. 
     
     
         7 . The method of  claim 6 , wherein said renewable diesel conforms to the specifications of the ASTM D975 standard. 
     
     
         8 . The method of  claim 6 , wherein said biodiesel conforms to the specifications of the ASTM D6751 standard. 
     
     
         9 . The method of  claim 6 , wherein said jet fuel conforms to the specifications of the ASTM D1655 standard. 
     
     
         10 . The method of  claim 1 , wherein the triacylglyceride is isolated by lysing the oleaginous yeast. 
     
     
         11 . The method of  claim 10 , wherein the oleaginous yeast is lysed by mechanical means. 
     
     
         12 . A method for producing a triacylglyceride, said method comprising the steps of culturing an oleaginous yeast in a culture media to provide an oleaginous yeast comprising at least about 10% triacylglyceride by dry weight; and extracting the triacylglyceride from the oleaginous yeast. 
     
     
         13 . The method of  claim 12 , wherein said oleaginous yeast is selected from the group consisting of  Cryptococcus curvatus, Cryptococcus terricolus, Candida  sp.,  Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, Saccharomyces cerevisiae  and  Yarrowia lipolytica.    
     
     
         14 . The method of  claim 12 , wherein the oleaginous yeast is cultured in media comprising a carbon source selected from the group consisting of glucose, fructose, sucrose, lactose, galactose, xylose, mannose, rhamnose, arabinose, glycerol, and acetate. 
     
     
         15 . The method of  claim 12 , wherein the oleaginous yeast is cultured in media comprising a carbon source selected from the group consisting of depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, sugar cane juice, switchgrass, milk whey, molasses, potato, rice, sorghum, and wheat. 
     
     
         16 . The method of  claim 12 , wherein said triacylglyceride is extracted by solvent extraction. 
     
     
         17 . A fuel or chemical produced by the method comprising the steps of:
 (a) heterotrophically culturing an oleaginous yeast in the presence of a fixed carbon source, wherein:
 (i) the fixed carbon source is selected from the group consisting of glycerol, depolymerized cellulosic material, sucrose, molasses, glucose, arabinose, galactose, xylose, fructose, mannose and acetate; and 
 (ii) the oleaginous yeast accumulates at least 10% of its dry cell weight as triacylglyceride; 
   (b) isolating the triacylglyceride from the oleaginous yeast; and   (c) subjecting the isolated triacylglyceride to a chemical reaction selected from the group consisting of cracking, (hydro)deoxygenation, esterification, and isomerization, and optionally, distillation.   
     
     
         18 . The method of  claim 17 , wherein the depolymerized cellulosic material is selected from the group consisting of sugar cane bagasse, sugar beet pulp, corn stover, wood chips, sawdust, switchgrass, rice hull, corn fiber, wheat straw, rice straw, and citrus pulp. 
     
     
         19 . The fuel or chemical of  claim 17 , wherein the oleaginous yeast is selected from the group consisting of  Cryptococcus curvatus, Cryptococcus terricolus, Candida  sp.,  Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis , and  Yarrowia lipolytica.    
     
     
         20 . The fuel or chemical of  claim 17 , wherein the oleaginous yeast contains at least one exogenous gene encoding a lipid pathway enzyme. 
     
     
         21 . The fuel or chemical of  claim 20 , wherein the triacylglyceride pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, a fatty acyl-ACP thioesterase, and a glycerol-3 phosphate acyltransferase. 
     
     
         22 . The fuel or chemical of  claim 17 , wherein said fuel or chemical is selected from the group consisting of renewable diesel, fatty acid esters, and jet fuel. 
     
     
         23 . The fuel or chemical of  claim 22 , wherein said renewable diesel conforms to the specifications of the ASTM D975 standard. 
     
     
         24 . The fuel or chemical of  claim 22 , wherein said biodiesel conforms to the specifications of the ASTM D6751 standard. 
     
     
         25 . The fuel or chemical of  claim 22 , wherein said jet fuel conforms to the specifications of the ASTM D1655 standard. 
     
     
         26 . The fuel or chemical of  claim 17 , wherein the triacylglyceride is isolated by lysing the oleaginous yeast. 
     
     
         27 . The fuel or chemical of  claim 26 , wherein the oleaginous yeast is lysed by mechanical means.