Abstract:
A process for hydrolyzing sucrose to glucose and fructose using succinic acid is described. The hydrolysate can be used to produce purified glucose and/or fructose or can be used as a carbon source for fermentations to produce various chemicals including succinic acid.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     Not Applicable  
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002]     Not Applicable  
       STATEMENT REGARDING GOVERNMENT RIGHTS  
       [0003]     Not Applicable  
       BACKGROUND OF THE INVENTION  
       [0004]     (1) Field of the Invention  
         [0005]     The present invention relates to a process for producing glucose and fructose from sucrose by hydrolysis using succinic acid. The hydrolysate can be used to produce the glucose/fructose or can be used as a carbon source for fermentations.  
         [0006]     (2) Description of the Related Art  
         [0007]     The use of inorganic acids, such as hydrochloric acid, to hydrolyze sucrose to glucose and fructose is known to those skilled in the art. An illustrative process is described in published U.S. application 2004 0231662 to De Mendonca Ferreira et al. The prior art has generally focussed on enzymatic hydrolysis of sucrose to produce glucose and/or fructose as evidenced by U.S. Pat. Nos. 5,998,177 and 6,660,502 to Catani et al.  
         [0008]     Succinic acid is an organic acid with two carboxylic groups. It is produced today mainly petrochemically from butane through maleic anhydride. Succinic acid is currently a low volume chemical. By production of succinic acid using biomass instead of petrochemicals as raw material, many new applications are possible. The production does not contribute to the accumulation of greenhouse gases since the feedstocks are renewable. A report from the U.S. Department of Energy designates succinic acid as a top twelve building block chemical produced from biomass (Werpy T., et al., Top Value Added Chemicals from Biomass. U.S. Department of Energy: Oak Ridge (2004)). Succinic acid can be used as a commodity or specialty chemical according to the report. As a commodity chemical it can substitute chemicals based on benzene and other intermediate petrochemicals to for instance produce polyester, solvents and other acids. Food ingredients, fuel additives and environmentally benign deicers are examples of specialty chemicals from succinic acid. The feedstocks of interest for producing succinic acid contain starch, hemicellulose or cellulose and can come from agricultural residues like corn fibres, forest products or beat and cane sugar.  
         [0009]     Fermentation can be accomplished with a number of different organisms.  E. coli  mutant AFP184 (described elsewhere) is used. The sugar available in sugar beet or cane is sucrose. It is a disaccharide of glucose and fructose.  E. coli  are not able to utilize sucrose, but can utilize glucose and fructose. Therefore, it is necessary to hydrolyze the sucrose before fermentation. Hydrolysis is normally performed with concentrated or dilute mineral acids. These acids are added to the process and require separate and costly recovery processes.  
         [0010]     Bacteria which enable the production of succinic acid are well known. Examples are U.S. Pat. No. 6,265,190 to Yedur, U.S. Pat. No. 6,743,610 to Donnelly et al. This acid has not been used for the production of glucose and fructose.  
       OBJECTS  
       [0011]     There is a need for an improved process for the preparation of glucose and fructose from sucrose. It is therefore an object of the present invention to provide a process for hydrolysis of sucrose which uses unique acid hydrolysis step for producing glucose and sucrose. It is further an object of the present invention to provide a unique hydrolysate which can be used for fermentation processes. Further still, it is an object of the present invention to provide a process for the isolation of pure glucose and fructose. Further still, it is an object of the present invention to provide a process which provides high yields and is economical. These and other objects will become increasingly apparent by reference to the following description and the drawings.  
       SUMMARY OF INVENTION  
       [0012]     The invention provides a process which uses succinic acid to hydrolyze (invert) sucrose into fructose and glucose. The resulting sugar mixture is then used as the substrate for an  E. coli  or other bacterial fermentation to produce succinic acid or other organic compounds. The process is novel in that: a) it uses succinic acid for sucrose hydrolysis, which has not been previously demonstrated, and b) the resulting sugar mixture can be fermented by succinate producing organisms that are otherwise unable to produce succinic acid from sucrose. If it is desired to recover the succinic acid, it can be accomplished in the same process used for the purification of the fermentation broth to produce pure succinic acid and there would be no need for building a new recovery plant.  
         [0013]     Thus the present invention relates to a process for producing glucose and fructose which comprises:  
         [0014]     hydrolyzing a composition comprising sucrose in an aqueous solution with succinic acid to produce glucose and sucrose in a hydrolysate. Preferably the hydrolysis is at a temperature between about 25° C. and below a boiling point of the solution. Preferably the temperature is between about 60° and 100° C. Preferably the sucrose is at a weight ratio of between about 10 to 1 to 1 to 10 in the aqueous solution. Preferably the sucrose is at a weight ratio of between 1 to 10 and 4 to 10. Preferably the succinic acid is at a concentration between about 0.1 and 10 percent by weight in the aqueous solution. Preferably the succinic acid is at a concentration of 0.5 to 5% by weight of the aqueous solution. Preferably the glucose and fructose are produced at a yield above 95% by weight based upon a weight of glucose and fructose available in the sucrose. Preferably the hydrolysis is over a time period of up to about 24 hours. Preferably in addition the hydrolysate is fermented by a microorganism which metabolizes the glucose and fructose to produce a fermentation product. Preferably the microorganism produces succinic acid from the hydrolysate as the fermentation product. Preferably the microorganism is an  Escherichia coli  strain.  
         [0015]     The present invention also relates to a fermentation process wherein a microorganism ferments a carbohydrate to produce a fermentation product, the improvement which comprises fermenting a succinic acid hydrolysate of a composition comprising sucrose to produce the fermentation product. Preferably the fermentation product is succinic acid and the microorganism is an  Escherichia coli  strain which with the carbohydrate produces succinic acid. 
     
    
     BRIEF DESCRIPTION OF DRAWINGS  
       [0016]      FIG. 1  is a graph showing sucrose hydrolysis at 60° C. and 1, 3, and 5% succinic acid by weight. SA is succinic acid.  
         [0017]      FIG. 2  is a graph showing sucrose hydrolysis at 80° C. for 0.5, 1, 3, and 5% catalyst.  
         [0018]      FIG. 3  is a graph showing sucrose hydrolysis at 100° C. for 0.5, 1, 3, and 5% catalyst.  
         [0019]      FIG. 4  is a graph showing sucrose hydrolysis at 80° C. and 100° C. for acid concentrations of 1 and 5%.  
         [0020]      FIG. 5  is a graph showing glucose and fructose concentrations as a function of time. 
     
    
     DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0021]     The following Examples 1 to 4 show the use of succinic acid to hydrolyze sucrose to glucose and fructose in high yields.  
       EXAMPLE I  
       [0000]     Effect of Catalyst Concentration at Low Temperatures.  
         [0022]     Sucrose and water are mixed in E-flasks in ratio liquid:solid 10:1. Temperature is kept constant at 60° C. in an oven equipped with a shake table. Succinic acid is added to the flasks to give concentrations of 1, 3, and 5% acid by weight. The data is shown in Tables 5 to 7. An increase in the catalyst loading increases the rate of hydrolysis significantly at low temperatures. Yields after ten (10) hours hydrolysis for glucose and fructose are summarized in Table 1. The yields are based on the amount of glucose or fructose formed divided by the amount of sucrose hydrolyzed. For glucose at higher acid concentrations some product degradation takes place. For fructose some degradation is evident at all acid concentrations.  
                                                             TABLE 1                           Yield of fermentable sugars in weight-%.                Yield (weight-%)                    Fructose   Glucose                            1% SA   88.4   99.3           3% SA   86.5   95           5% SA   87.2   94.9                      
 
       EXAMPLE 2  
       [0000]     Effect of Catalyst Concentration at Elevated Temperatures  
         [0023]     Sucrose and water were mixed in E-flasks in ratio liquid:solid 10:1. Temperature was kept constant by heating on a heating plate. Agitation was obtained from a magnetic stirrer. Temperatures investigated were 80° C. and 100° C. The catalyst loading (succinic acid added) corresponds to 0.5, 1, 3, and 5% by weight as in Example 1. Sucrose hydrolysis as a function of time for the different catalyst loadings at 80° C. and at 100° C. The data is shown in Tables 5 to 7. At higher temperatures the hydrolysis rate was greatly increased. At 80° C. acid concentrations above 1% resulted in almost complete hydrolysis after two hours. A concentration of 1% needed three hours to complete the hydrolysis and 0.5% was not finished until after six hours. At 100° C. the hydrolysis was completed in one hour for all acid concentrations. Hydrolysis products were formed in yields 95-100% based on the sucrose hydrolyzed with less degradation of fructose.  
       EXAMPLE 3  
       [0000]     Hydrolysis of Liquid:Solid Ratios 10:4  
         [0024]     To produce a media with high enough sugar concentration for fermentation purposes higher liquid:solid ratios (higher percentages of sucrose) must be used. Using the same experimental setup as in Example 2, but with liquid:solid ratio 10:4, acid concentrations of 1%, and 5% were investigated at 80° C. and 100° C. Sucrose hydrolysis results are presented in Table 14. Yields after finished hydrolysis are shown in Table 2. The yields are expressed on a weight basis as the mass of produced monosaccharides per mass of hydrolyzed sucrose. From the yields it can be seen that the best results are achieved by low acid loadings and high temperatures. After one hour the yields were close to 100%, and heating for another hour did not generate any significant sugar degradation.  
                                                             TABLE 2                           Yield of fermentable sugars in weight-%.                Yield (weight-%)                    Glucose   Fructose                             80° C., 1% SA   81.8   86.2            80° C., 5% SA   82.3   86.2           100° C., 1% SA   95.9   99.4           100° C., 5% SA   88.8   92.3                      
 
       EXAMPLE 4  
       [0000]     Fermentation of Glucose-Fructose Mixture in Ratio 50-50  
         [0025]     A 12 L fermenter was used. A media constituted by the substances given in Table 3 was mixed and sterilized in the fermenter. 0.5 L inoculum of a pure AFP184 (an  E. coli  mutant described in U.S. published application 200300/7559 to Donnelly et al), grown for 16 hours in sterile Tryptic Soy Broth, was added together with a 2 L solution with a concentration of 400 g/L of glucose and fructose in ratio 1:1. The total starting volume of the fermentation was 8 L. The fermentation was aerated for eight hours with filter sterilized air. After eight hours a high cell density was achieved and the fermentation conditions were changed to anaerobic by cutting of air and adding carbon dioxide. This then proceeds for 16 hours producing succinic acid. The final succinic acid concentration, yield per gram and mole consumed sugar during the anaerobic phase and the productivity per gram, litre and hour is shown in Table 4. The sugar concentration change during the fermentation is presented in Table 15.  E. coli  mutant AFP184 can ferment mixtures of glucose and fructose as produced in Examples 1 to 3.  
                                               TABLE 3                           Mineral and growth factor substrates for fermentation.                Substrate   Amount (g or L)                            Corn Steep   266   g           Liquor           K 2 HPO 4     11.2   g           KH 2 PO 4     4.8   g           (NH 4 ) 2 SO 4     26.7   g           MgSO 4     1.6   g           Antifoam agent   3   ml                      
 
         [0026]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                   
               
               
                 Final succinic acid concentration, yields, and 
               
               
                 productivity after 16 hours of production. 
               
             
          
           
               
                   
                 Succinic Acid 
                 Yield 
                 Yield 
                 Productivity 
               
               
                   
                 (g/L) 
                 (g/g) 
                 (mole/mole) 
                 (g/L/h) 
               
               
                   
                   
               
               
                   
                 51.95 
                 0.73 
                 1.12 
                 2.89 
               
               
                   
                   
               
             
          
         
       
     
       Data—Example 1  
       [0027]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                   
               
               
                 Glucose formation (g/L) at 60° C. 
               
               
                 for acid concentrations 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 Glucose, 1% SA 
                 Glucose, 3% SA 
                 Glucose, 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 0 
                 0.665254592 
                 1.433727361 
               
               
                 2 
                 2.647401928 
                 8.319265668 
                 12.49919376 
               
               
                 3 
                 8.943332524 
                 15.28128695 
                 20.67329705 
               
               
                 4 
                 11.81570429 
                 22.30937982 
                 27.23962543 
               
               
                 5 
                 19.77152651 
                 26.05398002 
                 35.49323757 
               
               
                 6 
                 17.31541166 
                 33.45268288 
                 38.71977609 
               
               
                 7 
                 27.57577628 
                 35.48615065 
                 40.78236791 
               
               
                 8 
                 28.83985555 
                 34.19917824 
                 42.36255161 
               
               
                 9 
                 30.72163142 
                 39.90952235 
                 43.57539008 
               
               
                 10 
                 33.34785223 
                 41.47563175 
                 44.24743298 
               
               
                   
               
             
          
         
       
     
         [0028]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                   
               
               
                 Fructose formation (g/L) at 60° C. 
               
               
                 for acid concentrations 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 Fructose, 1% SA 
                 Fructose, 3% SA 
                 Fructose, 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 0.67972 
                 2.33298 
                 3.65876 
               
               
                 2 
                 3.837 
                 9.80845 
                 12.9257 
               
               
                 3 
                 8.35472 
                 16.32199 
                 20.89805 
               
               
                 4 
                 12.95957 
                 22.18015 
                 26.83508 
               
               
                 5 
                 16.86656 
                 26.67031 
                 32.28647 
               
               
                 6 
                 20.14169 
                 30.14726 
                 34.66879 
               
               
                 7 
                 24.08336 
                 32.67965 
                 37.59368 
               
               
                 8 
                 25.38129 
                 34.36781 
                 39.20524 
               
               
                 9 
                 27.23786 
                 36.60941 
                 39.96632 
               
               
                 10 
                 29.69138 
                 37.77904 
                 40.66125 
               
               
                   
               
             
          
         
       
     
         [0029]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                   
               
               
                 Sucrose degradation (g/L) at 60° C. 
               
               
                 for acid concentrations 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 Sucrose, 1% SA 
                 Sucrose, 3% SA 
                 Sucrose, 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 100 
                 100 
                 100 
               
               
                 1 
                 91.47598829 
                 87.58720289 
                 88.40802667 
               
               
                 2 
                 82.73131168 
                 73.10737983 
                 63.0472783 
               
               
                 3 
                 77.27112921 
                 58.94906893 
                 48.72138111 
               
               
                 4 
                 64.26281948 
                 50.48299621 
                 35.88196376 
               
               
                 5 
                 57.96055181 
                 36.76323165 
                 27.96802436 
               
               
                 6 
                 48.86978287 
                 31.59578454 
                 20.82431382 
               
               
                 7 
                 47.39983256 
                 24.79683883 
                 15.29118304 
               
               
                 8 
                 41.42066148 
                 18.43986528 
                 11.84848076 
               
               
                 9 
                 35.90348212 
                 15.58531013 
                 8.835096554 
               
               
                 10 
                 32.80338213 
                 12.68737568 
                 6.722507349 
               
               
                   
               
             
          
         
       
     
       Data—Example 2  
       [0030]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                   
               
               
                 Sucrose degradation at 80° C. for 
               
               
                 acid concentrations 0.5, 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 0.5% SA 
                 1% SA 
                 3% SA 
                 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 100 
                 100 
                 100 
                 100 
               
               
                 1 
                 48.53388 
                 19.12333554 
                 16.81258471 
                 14.88987095 
               
               
                 2 
                 27.26248 
                 8.703267151 
                 3.326631595 
                 2.8984055 
               
               
                 3 
                 14.79558 
                 0.881246481 
                 0.881246481 
                 0.881246481 
               
               
                 4 
                 8.854452 
               
               
                 6 
                 2.418129 
               
               
                   
               
             
          
         
       
     
         [0031]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                   
               
               
                 Glucose formation at 80° C. for acid 
               
               
                 concentrations 0.5, 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 0.5% SA 
                 1% SA 
                 3% SA 
                 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 18.7314 
                 39.52014 
                 41.16771 
                 35.40328 
               
               
                 2 
                 33.2953 
                 43.74562 
                 50.29046 
                 44.26493 
               
               
                 3 
                 40.46413 
                 45.00098 
                 48.96617 
                 45.14757 
               
               
                 4 
                 48.98223 
               
               
                 6 
                 48.27418 
               
               
                   
               
             
          
         
       
     
         [0032]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                   
               
               
                 Fructose formation at 80° C. for 
               
               
                 acid concentrations 0.5, 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 0.5% SA 
                 1% SA 
                 3% SA 
                 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 17.85198 
                 37.612 
                 42.35413 
                 38.71786 
               
               
                 2 
                 34.52418 
                 47.15255 
                 48.16839 
                 47.37839 
               
               
                 3 
                 42.22377 
                 47.65608 
                 49.9901 
                 48.4048 
               
               
                 4 
                 51.25258 
               
               
                 6 
                 51.51636 
               
               
                   
               
             
          
         
       
     
         [0033]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                   
               
               
                 Sucrose degradation at 100° C. for 
               
               
                 acid concentrations 0.5, 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 0.5% SA 
                 1% SA 
                 3% SA 
                 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 100 
                 100 
                 100 
                 100 
               
               
                 1 
                 2.44425 
                 0.88125 
                 0.88125 
                 0.88125 
               
               
                 2 
                 0.881246 
                 0.88125 
                   
                 0.88125 
               
               
                   
               
             
          
         
       
     
         [0034]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                   
               
               
                 Glucose formation at 100° C. for 
               
               
                 acid concentrations 0.5, 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 0.5% SA 
                 1% SA 
                 3% SA 
                 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 52.84148 
                 52.02847 
                 53.60736 
                 46.82761 
               
               
                 2 
                 56.9705 
                 49.00942 
                   
                 47.37418 
               
               
                   
               
             
          
         
       
     
         [0035]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                   
               
               
                 Fructose formation at 100° C. for 
               
               
                 acid concentrations 0.5, 1, 3, and 5%. 
               
             
          
           
               
                 time 
                 0.5% SA 
                 1% SA 
                 3% SA 
                 5% SA 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 55.07813 
                 54.49409 
                 55.61529 
                 49.07266 
               
               
                 2 
                 59.56918 
                 51.47787 
                   
                 50.42975 
               
               
                   
               
             
          
         
       
     
       Data—Example 3  
       [0036]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                   
               
               
                 Sucrose degradation, glucose and fructose formation at 80° C. and 100° C., 
               
               
                 1% and 5% succinic acid and liquid solid ratio 10:4 in g/L. 
               
             
          
           
               
                   
                 Time 
               
             
          
           
               
                   
                 0 
                 0.5 
                 1 
                 1.5 
                 2 
                 2.5 
                 3 
                 4 
               
               
                   
                   
               
             
          
           
               
                 Sucrose 80 C., 1% SA 
                 400 
                 205.58 
                 127.32 
                 77.34 
                 54.09 
                 38.98 
                 30.49 
                 18.14 
               
               
                 Sucrose 80 C., 5% SA 
                 400 
                 67.60 
                 32.44 
                 16.95 
                 13.81 
                 12.59 
                 12.59 
               
               
                 Sucrose 100 C., 1% SA 
                 400 
                 28.41 
                 13.09 
                 12.59 
                 12.59 
               
               
                 Sucrose 100 C., 5% SA 
                 400 
                 14.52 
                 12.80 
                 12.59 
               
               
                 Glucose 80 C., 1% SA 
                 0 
                 57.60 
                 104.19 
                 121.73 
                 137.28 
                 147.30 
                 169.42 
                 156.22 
               
               
                 Glucose 80 C., 5% SA 
                 0 
                 145.39 
                 152.11 
                 171.20 
                 177.72 
                 175.63 
                 159.33 
               
               
                 Glucose 100 C., 1% SA 
                 0 
                 145.47 
                 179.85 
                 187.19 
                 185.72 
               
               
                 Glucose 100 C., 5% SA 
                 0 
                 174.43 
                 177.97 
                 172.02 
               
               
                 Fructose 80 C., 1% SA 
                 0 
                 61.36 
                 101.61 
                 124.35 
                 141.85 
                 154.89 
                 177.06 
                 164.61 
               
               
                 Fructose 80 C., 5% SA 
                 0 
                 151.39 
                 159.09 
                 178.22 
                 185.40 
                 184.48 
                 166.88 
               
               
                 Fructose 100 C., 1% SA 
                 0 
                 152.44 
                 187.22 
                 195.90 
                 192.62 
               
               
                 Fructose 100 C., 5% SA 
                 0 
                 181.21 
                 186.07 
                 178.86 
               
               
                   
               
             
          
         
       
     
         [0037]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
               
               
                 Time 
                 Glucose (g/L) 
                 Fructose (g/L) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0 
                 48.96 
                 53.18 
               
               
                 2 
                 48.9 
                 53.19 
               
               
                 4 
                 48.57 
                 53.65 
               
               
                 6 
                 41.92 
                 37.65 
               
               
                 8 
                 26.83 
                 19.23 
               
               
                 24 
                 0 
                 1.68 
               
               
                   
               
             
          
         
       
     
       Data—Example 4  
       [0000]     Various sources of sucrose can be used. In some instances the source can contain other materials as in the case of molasses.  
         [0038]     It is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the hereinafter appended claims.