Methods for producing ethanol from carbon substrates

Abstract

The present invention provides means for the production of desired end-products of in vitro and/or in vivo bioconversion of biomass-based feed stock substrates, including but not limited to such materials as starch and cellulose. In particularly preferred embodiments, the methods of the present invention do not require gelatinization and/or liquefaction of the substrate. In particularly preferred embodiments, the present invention provides means for the production of ethanol.

1 . A method for producing an alcohol as an end-product comprising the steps of: 
 a) contacting a biomass carbon substrate which includes raw starch and at least two substrate-converting glucoamylase enzymes to produce an intermediate comprising pentoses. hexoses or combinations thereof; wherein one substrate-converting glucoamylase is a raw starch hydrolyzing (RSH) glucoamylase; and    b) contacting said intermediate with at least one intermediate-converting enzyme produced by an ethanologenic microorganism,    wherein said intermediate is substantially all converted by said intermediate-converting enzyme to said alcohol end-product and the presence of the alcohol end-product does not inhibit the further production of said alcohol end-product.    
     
     
         2 - 37 . (canceled)  
     
     
         38 . The method according to  claim 1 , wherein the biomass carbon substrate is obtained from corn or wheat.  
     
     
         39 . The method according to  claim 1 , wherein the biomass carbon substrate is a slurry having 10% to 35% starch by weight.  
     
     
         40 . The method according to  claim 1 , wherein the RSH glucoamylase is secreted by a microorganism in contact with the biomass carbon substrate.  
     
     
         41 . The method according to  claim 1 , wherein the RSH glucoamylase is obtained from a strain of  Humicola, Aspergillus  or  Rhizopus.    
     
     
         42 . The method according to  claim 41 , wherein the RSH glucoamylase is obtained from a  H. grisea  var.  thermoidea.    
     
     
         43 . The method according to  claim 41 , wherein the RSH glucoamylase is obtained from a strain of  R. niveus.    
     
     
         44 . The method according to  claim 1 , wherein one substrate-converting glucoamylase is derived from a fungal cell.  
     
     
         45 . The method according to  claim 44 , wherein the fungal cell is an  Aspergillus  cell.  
     
     
         46 . The method according to  claim 45 , wherein the  Aspergillus  is  A. niger.    
     
     
         47 . The method according to  claim 1 , wherein the at least two substrate-converting glucoamylases convert at least 90% of the raw starch of the biomass carbon substrate to the intermediate within 72 hours.  
     
     
         48 . The method according to  claim 1 , wherein the concentration level of the intermediate is maintained at less than or equal to 0.25% by weight volume.  
     
     
         49 . The method according to  claim 1 , wherein the intermediate is glucose.  
     
     
         50 . The method according to  claim 1 , wherein the alcohol end-product is ethanol.  
     
     
         51 . The method according to  claim 1 , further comprising adding an alpha amylase to the contacting step a).  
     
     
         52 . The method according to  claim 1 , further comprising adding a protease enzyme to the contacting step a).  
     
     
         53 . The method according to  claim 1 , further comprising adding a pullulanase to the contacting step a).  
     
     
         54 . The method of according to  claim 1 , wherein the intermediate-converting enzyme is secreted by a yeast.  
     
     
         55 . The method according to  claim 1 , wherein the intermediate-converting enzyme is an alcohol dehydrogenase or a pyruvate decarboxylase.  
     
     
         56 . The method according to  claim 1 , wherein 
 the RSH glucoamylase has a raw starch hydrolyzing unit (RSHU)/glucoamylase unit (GAU) ratio greater than 1:.5 and    the second glucoamylase has a RSHU/GAU ratio less than 0.6.    
     
     
         57 . The method according to  claim 1 , wherein the amount of glucoamylase is in the range of 0.2 to about 1.0 glucoamylase units (GAU) per gram of starch solids and the amount of RSH glucoamylase is in the range of 0.2 to about 1.0 raw starch hydrolyzing unit (RSHU) per gram of starch solids.  
     
     
         58 . A method for producing ethanol as an end-product comprising the steps of: 
 a) contacting a biomass carbon substrate, which includes raw starch, with at least two substrate-converting enzymes to produce an intermediate which includes hexoses, pentoses and combinations thereof, wherein the first substrate-converting enzyme is a starch hydrolyzing glucoamylase (RSH) enzyme and the second substrate-converting enzyme is an alpha amylase;    b) contacting said intermediate with at least one intermediate-converting enzyme obtained from an ethanologenic microorganism, wherein    the intermediate is substantially all converted by said intermediate-converting enzyme to an ethanol end-product; and    the presence of the ethanol end-product does not inhibit the further production of said ethanol.    
     
     
         59 . The method according to  claim 58 , wherein the biomass carbon substrate comprises corn solids.  
     
     
         60 . The method according to  claim 58 , wherein the biomass carbon substrate is obtained from corn, wheat or barley.  
     
     
         61 . The method according to  claim 58 , wherein the RSH enzyme is obtained from a  Rhizopus  or  Humicola.    
     
     
         62 . The method according to  claim 58 , wherein the alpha amylase is a bacterial alpha amylase.  
     
     
         63 . The method according to  claim 62 , wherein the bacterial alpha amylase is obtained from a  Bacillus.    
     
     
         64 . The method according to  claim 63 , wherein the  Bacillus  is a strain of  B. licheniformis.    
     
     
         65 . The method according to  claim 58 , wherein the alpha amylase is a fungal alpha amylase.  
     
     
         66 . The method according to  claim 65 , wherein the fungal alpha amylase is obtained from a  Rhizopus.    
     
     
         67 . The method according to  claim 58 , wherein the RSH enzyme and the alpha amylase convert at least 90% of the raw starch of the biomass carbon substrate to the intermediate within 72 hours.  
     
     
         68 . The method according to  claim 58 , wherein the ethanologenic microorganism is a yeast.  
     
     
         69 . The method according to  claim 58 , further comprising adding a pullulanase to the contacting step a).  
     
     
         70 . The method according to  claim 58 , further comprising adding a protease to the contacting step a).  
     
     
         71 . The method according to  claim 58 , further comprising adding a second glucoamylase to the contacting step a).  
     
     
         72 . The method according to  claim 71 , wherein the second glucoamylase is obtained from a strain of  Aspergillus.    
     
     
         73 . A one-step method for producing ethanol comprising, 
 a) adding to a conversion media a raw starch containing carbon substrate,    b) contacting the carbon substrate with a raw starch hydrolyzing (RSH) glucoamylase obtained from a strain of  Rhizopus, Humicola  or  Aspergillus  and a second glucoamylase obtained from a strain of  Aspergillus  or  Rhizopus,  to produce an intermediate comprising hexoses, pentoses and combinations thereof, and    c) converting the intermediate to ethanol in the presence of an ethanol producer in said conversion media,    wherein the intermediate is substantially all bioconverted to ethanol and the presence of the ethanol does not inhibit the further production of said ethanol in the culture.    
     
     
         74 . The method according to  claim 73 , wherein the ethanol producer is a yeast or bacteria.  
     
     
         75 . The method according to  claim 73 , wherein the raw starch containing substrate is obtained from corn or wheat.  
     
     
         76 . The method according to  claim 73  further comprising recovering the produced ethanol from the culture.  
     
     
         77 . The method according to  claim 73 , wherein the RSH glucoamylase is obtained from  Rhizopus.    
     
     
         78 . The method according to  claim 73 , wherein the second glucoamylase is obtained from  Aspergillus.    
     
     
         79 . The method according to  claim 73 , further comprising adding to the culture an enzyme selected from the group consisting of pullulanases, alpha amylases, proteases, and combinations thereof.  
     
     
         80 . The method according to  claim 72 , wherein the range of glucoamylase added to the culture is 0.2 to 1.0 GAU units of glucoamylase per gram of granular solids in the substrate and the range of RSH enzyme added to the culture is 0.2 to 1.0 RSHU per gram of granular solids.