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.

What is claimed is:  
     
         1 . A method for producing an alcohol as an end-product comprising the steps of: 
 a) contacting a carbon substrate and at least one substrate-converting enzyme to produce an intermediate; and    b) contacting said intermediate with at least one intermediate-converting enzyme, wherein said intermediate is substantially all converted by said intermediate enzyme to said alcohol.    
     
     
         2 . The method of  claim 1 , wherein said intermediate-converting enzyme is a microbial enzyme.  
     
     
         3 . The method of  claim 2 , wherein said intermediate-converting microbial enzyme is secreted by a microorganism in contact with said intermediate.  
     
     
         4 . The method of  claim 1 , wherein said substrate-converting enzyme is a microbial enzyme.  
     
     
         5 . The method of  claim 4 , wherein said substrate-converting microbial enzyme is secreted by a microorganism in contact with said substrate.  
     
     
         6 . The method of  claim 1 , wherein said intermediate-converting enzyme and said substrate-converting enzyme are produced by microorganisms of the same species.  
     
     
         7 . The method of  claim 1 , wherein said intermediate-converting enzyme and said substrate-converting enzyme are produced by microorganisms of the different species.  
     
     
         8 . The method of  claim 1 , wherein concentration level of said intermediate is maintained at a level below that which triggers catabolite repression effects upon the conversion of said intermediate to said end-product.  
     
     
         9 . The method of  claim 1 , wherein concentration level of said intermediate is maintained at a level below that which triggers enzymatic inhibition effects upon the conversion of said intermediate to said end-product.  
     
     
         10 . The method of  claim 1 , wherein said intermediate is converted to said end-product at a rate sufficient to maintain the concentration of said at less than 0.25%.  
     
     
         11 . The method of  claim 1 , wherein said substrate is selected from the group consisting of biomass and starch.  
     
     
         12 . The method of  claim 11 , wherein said biomass comprises corn solids.  
     
     
         13 . The method of  claim 1 , wherein said intermediate is selected from the group consisting of hexoses and pentoses.  
     
     
         14 . The method of  claim 13 , wherein said hexose is glucose.  
     
     
         15 . The method of  claim 1 , wherein said contacting said substrate and substrate-converting enzyme further comprises bioconverting said substrate to produce said intermediate.  
     
     
         16 . The method of  claim 1 , wherein said alcohol end-product is ethanol.  
     
     
         17 . The method of  claim 1 , wherein the step of contacting said substrate and said at least one substrate-converting enzyme further comprises providing an amount of said substrate-converting enzyme at a concentration that produces said intermediate at a concentration that is less than or equal to the amount of said intermediate converted by said at least one intermediate-converting enzyme.  
     
     
         18 . The method of  claim 1 , wherein said at least one substrate-converting enzyme converts at least 50% of said substrate to said intermediate within 72 hours.  
     
     
         19 . The method of  claim 18 , wherein said at least one substrate-converting enzyme converts at least 90% of said substrate to said intermediate within 72 hours.  
     
     
         20 . The method of  claim 19 , wherein said at least one substrate-converting enzyme converts at least 95% of said substrate to said intermediate within 72 hours.  
     
     
         21 . The method of  claim 1 , wherein said at least one substrate-converting enzyme and said at least one intermediate-converting enzyme are obtained from a microorganism selected from the group consisting of Rhizopus and Aspergillus.  
     
     
         22 . A method for producing alcohol as an end-product comprising the steps of: 
 a) contacting a carbon substrate and at least one substrate-converting enzyme to produce an intermediate; and    b) contacting said intermediate with at least one intermediate-converting enzyme, wherein said intermediate is substantially all converted by said intermediate enzyme to said alcohol end-product, and wherein the presence of said end-product does not inhibit the further production of said alcohol end-product.    
     
     
         23 . The method of  claim 22 , wherein said intermediate-converting enzyme is a microbial enzyme.  
     
     
         24 . The method of  claim 22 , wherein said intermediate-converting microbial enzyme is secreted by a microorganism in contact with said intermediate.  
     
     
         25 . The method of  claim 22 , wherein said substrate-converting enzyme is a microbial enzyme.  
     
     
         26 . The method of  claim 22 , wherein said substrate-converting microbial enzyme is secreted by a microorganism in contact with said substrate.  
     
     
         27 . The method of  claim 22 , wherein said intermediate-converting enzyme and said substrate-converting enzyme are produced by microorganisms of the same species.  
     
     
         28 . The method of  claim 22 , wherein said intermediate-converting enzyme and said substrate-converting enzyme are produced by microorganisms of the different species.  
     
     
         29 . The method of  claim 22 , wherein said alcohol end-product is ethanol.  
     
     
         30 . A method for producing an alcohol end-product comprising the steps of: 
 a) contacting a carbon substrate and at least one substrate-converting enzyme to produce an intermediate; and    b) contacting said intermediate with at least one intermediate-converting enzyme, wherein said intermediate is substantially all converted by said intermediate enzyme to said alcohol end-product, and wherein the presence of said substrate does not inhibit the further production of said alcohol end-product.    
     
     
         31 . The method of  claim 30 , wherein said intermediate-converting enzyme is a microbial enzyme.  
     
     
         32 . The method of  claim 30 , wherein said intermediate-converting microbial enzyme is secreted by a microorganism in contact with said intermediate.  
     
     
         33 . The method of  claim 30 , wherein said substrate-converting enzyme is a microbial enzyme.  
     
     
         34 . The method of  claim 30 , wherein said substrate-converting microbial enzyme is produced is secreted by a microorganism in contact with said substrate.  
     
     
         35 . The method of  claim 30 , wherein said intermediate-converting enzyme and said substrate-converting enzyme are produced by microorganisms of the same species.  
     
     
         36 . The method of  claim 30 , wherein said intermediate-converting enzyme and said substrate-converting enzyme are produced by microorganisms of the different species.  
     
     
         37 . The method of  claim 36 , wherein said alcohol end-product is ethanol.