Novel Methods for Producing Fermentable Sugars from Lignocellulosic

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					                             Novel Methods for Producing Fermentable
                               Sugars from Lignocellulosic Biomass
                                            Jill Burdette, Paresma Patel, Brian Vande Berg, Brian Carr,
                                                         Nicholas Duck and Michael Koziel
                                                                               Athenix Corp., Research Triangle Park, NC 27703

 Athenix has developed several novel, biologically-friendly pretreatments for hydrolysis of lignocellulose. These
 pretreatments utilize mild chemical reagents that are available in bulk quantities, and are carried out under
 moderate conditions. We have tested these pretreatments using corn stover to assess commercial feasibility:

                   Corn                                             Athenix                                                      Enzymatic
                                                                                                                                                                                                                                              Sugars
                  Stover                                         Pretreatments                                                   Hydrolysis

    •10-20% load                                     •Mild, Inexpensive Chemicals                                              •Commercial                                                                                                •Glucose
                                                     •Mild Conditions                                                            cellulases                                                                                               •Xylose
                                                         •Atmospheric pressure                                                 •pH 5.0, 40-50°C                                                                                           •Arabinose
                                                         •Temperatures < 100°C
                                                                                      Corn Stover Composition
                                                                   •Glucan               34.1%                  •Lignin 21.2%                                                                           Untreated                                          Pretreatment
Athenix Pretreatments                                              •Xylan                21.1%                  •Acetyl  1.0%                                                                            Stover                                             + Spezyme
                                                                   •Galactan              2.8%                  •Ash     6.5%
                                                                   •Arabinan              2.6%                  •Protein 3.9%
1. Are simple and flexible                                         •Mannan                0.7%                  •Water   6.3%
                                                                                                                                                                                                        •Simple, Flexible Method
                                                                                                                                                                                                        •Single Vessel for Pretreatment and
                                                                                                                                                                                                         Enzymatic Hydrolysis
                                                                                        Mass Reduction Following Pretreatment + Enzymatic Hydrolysis

                                                                                                                                                                           80%
                                                                                                                                                                           70%

 2. Are compatible with                                                                                                                   % Total Mass Lost
                                                                                                                                                                           60%
                                                                                                                                                                           50%
  enzymatic hydrolysis                                                                                                                                                     40%
                                                                                                                                                                           30%
                                                                                                                                                                           20%
                                                                                                                                                                           10%
                                                                                                                                                                                            0%
                                                                                                                                                                                                          Pretreatment     Spezyme only         Pretreatment +
                                                                                                                                                                                                              only                                Spezyme

                                                                               Untreated                         Pretreatment                                                      Samples were rinsed, dried, and weighed to determine change
                                                                                Stover                            + Spezyme                                                        in mass. Untreated samples were included to control for
                                                                                                                                                                                   mass lost from evaporation.


                                                                                              Sugar Release from Pretreatment + Enzymatic Hydrolysis
                                                                                                                                                                                             90%
                                                                                                                                                                                             80%
                                                                                                                                                              % Glucose Hydrolysis (HPLC)




                                                                                                 Glucose
                                                                                                                                                                                             70%

3. Produce viable sugars                                                                                                                                                                     60%
                                                                                                                                                                                             50%
                                                           HPLC Signal (uA)




       that support                                                                                          Xylose                                                                          40%
                                                                                                                                                                                             30%
   fermentative growth                                                                                                                                                                       20%
                                                                                  Arabinose                           Red = Hydrolyzate                                                      10%
                                                                                                                      Blue = Sugar Stds                                                           0%
                                                                                                                                                                                                               Untreated    Spezyme only          Pretreatment +
                                                                                               Retention Time (min)                                                                                             Stover                              Spezyme

                                                                                                                                           Stover hydrolyzates were diluted and filtered prior to injection. Sugars
                                                                                      Sugar Detection by HPLC                              were separated via anion exchange chromatography and quantified by
                                                                                                                                           pulsed amperometric detection. Standard curves were generated from
                                                                                                                                           sugar standards included in each run.

                                            Use of Unconditioned Hydrolyzate to Support Fermentative Growth

                 2.5                                                                                                                                                                                     0.2
                                                                                           Sugars released following enzymatic
                                          Stover Sugars                                    hydrolysis were adjusted with NaOH                                                                                                                   Stover
     nm)




                 2.0
                                                                                                                                                                                            nm)




                                                                                                                                                                                                        0.15
                                                                                           to neutral pH, filter-sterilized, and                                                                                                                Sugars
       600




                 1.5
                                                                                                                                                                                              600




                                                                                           added to sterile minimal growth                                                                               0.1
     Growth (A




                                                                                                                                                                                            Growth (A




                 1.0                                                                       media.      Control growth media
                                        Glucose/Xylose                                     contained glucose and xylose at                                                                              0.05
                 0.5
                                                                                           equivalent concentrations.                                                                                                                        Glucose/Xylose
                 0.0                                                                                                                                                                                      0
                   0.00%      0.50%             1.00%                         1.50%                                                                                                                       0.00%        0.25%            0.50%             0.75%    1.00%
                              Sugar Added to Media (%)                                                                                                                                                                         Sugar added to Media (%)


                           Escherichia coli                                                                                                                                                                       Saccharomyces cerevisiae
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