Fractionation of Oil-Bearing Microbial Biomass

Abstract

The invention generally relates to the production of hydrocarbon compositions, such as a lipid, in microorganisms. In particular, the invention provides methods for extracting, recovering, isolating and obtaining a lipid from a microorganism and compositions comprising the lipid. The invention also discloses methods for producing hydrocarbon compositions for use as biodiesel, renewable diesel, jet fuel, and other materials.

1 . A method of extracting a lipid from a recombinant microalga, the method comprising the steps of:
 (a) lysing recombinant microalgal cells to produce a lysate, wherein the recombinant microalgal cells:
 (i) have not been subjected to a drying step between culturing and lysing; and 
 (ii) contain a lipid; 
   (b) treating the lysate with an organic solvent for a period of time, wherein the organic solvent is an alcohol selected from methanol, ethanol, isopropanol or butanol; and   (c) separating the treated lysate into layers comprising a lipid layer and an aqueous layer and, optionally, a lipid:aqueous emulsion layer, and/or a cell pellet; and   (d) removing the lipid from the other layer(s).   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the lysing is accomplished by subjecting the recombinant microalgal cells to heating, sonication, mechanical lysis, osmotic shock, expression of an autolysis gene, exposure to pH above 8, exposure to an acidic pH, heating and exposure to an acidic pH, or digestion with an enzyme. 
     
     
         6 - 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the recombinant microalga produces a lipid with a lipid profile comprising at least 4% C8-C14. 
     
     
         12 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein step (d) comprises reducing the temperature of the mixture to below 25° C. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the recombinant microalga is of the genus  Chlorella  or  Prototheca.    
     
     
         19 - 36 . (canceled) 
     
     
         37 . The method of  claim 1 , wherein the recombinant microalgal cells comprise lipid that is at least 10% C14 triacylglycerols, at least 10% C12 triacylglycerols, or at least 10% C10 triacylglycerols. 
     
     
         38 . The method of  claim 1 , wherein the lysing is accomplished by subjecting the recombinant microalgal cells to sonication. 
     
     
         39 . The method of  claim 1 , wherein the recombinant microalgal cells comprise a polynucleotide encoding a sucrose invertase or a lipid pathway enzyme. 
     
     
         40 . The method of  claim 39 , wherein the polynucleotide encodes a sucrose invertase. 
     
     
         41 . The method of  claim 39 , wherein the polynucleotide encodes a lipid pathway enzyme. 
     
     
         42 . The method of  claim 41 , wherein the lipid pathway enzyme is a fatty acyl-ACP thioesterase. 
     
     
         43 . The method of  claim 39 , wherein the recombinant microalgal cells comprise one or more polynucleotides encoding a sucrose invertase and a lipid pathway enzyme. 
     
     
         44 . The method of  claim 43 , wherein the lipid pathway enzyme is a fatty acyl-ACP thioesterase. 
     
     
         45 . The method of  claim 18 , wherein the recombinant microalga is of the genus  Chlorella.    
     
     
         46 . The method of  claim 45 , wherein the recombinant microalga is  Chlorella protothecoides, Chlorella emersonii, Chlorella sorokiniana  or  Chlorella minutissima.    
     
     
         47 . The method of  claim 46 , wherein the recombinant microalga is  Chlorella protothecoides.    
     
     
         48 . The method of  claim 18 , wherein the recombinant microalga is of the genus  Prototheca.    
     
     
         49 . The method of  claim 48 , wherein the recombinant microalga is  Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis , or  Prototheca zopfii.    
     
     
         50 . The method of  claim 49 , wherein the recombinant microalga is  Prototheca moriformis.    
     
     
         51 . The method of  claim 39 , wherein the polynucleotide is codon-optimized for expression in the microalgal cells.