Southeastern Sun Grant Center Quarterly Progress Report Project Title: Elucidation of Feedstock-Microbe Interactions in Butanol Production from Lignocellulosic Biomass Recipient Organization: Auburn University Principal Investigator: Dr. Maobing Tu Project Location: Forest Product Laboratory, Auburn University Department of Chemical Engineering, Auburn University Center for Renewable Carbon, University of Tennessee Reporting Period: April 1, 2015 – June 30, 2015 Date of Report: July 24, 2015 Written by: Maobing Tu, Y. Y. Lee, Nicole Labbe

1. Planned Activities: This Sun Grant project consists of three specific objectives: 1) Establish the relationship between enzymatic hydrolysis rate and butanol production rate and yield; 2) Identify feedstock characteristics that govern initial enzymatic hydrolysis rate and 72 h hydrolysis yield; 3) construct a predictive model between feedstock characteristics and butanol production rate and yield. In the last quarter, we focused on research objectives 2, the specific activities planned are as following:

1) Establish the relationship between enzymatic hydrolysis rate and butanol production

rate and yield a. Perform butanol production by SSF with organosolv pretreated woody

biomass b. Perform butanol production by SSF with pretreated switchgrass c. Build an initial model between hydrolysis rate and solvent yield in SSF

2) Identify feedstock characteristics that govern initial enzymatic hydrolysis rate and 72

h hydrolysis yield a. Generate more lignin for analysis and carbon fiber evaluation b. Identify effect of lignin and Avicel on butanol production in SSF process c. Characterize pretreated biomass/lignin with SEM

2. Actual Accomplishments:

1) Pretreated pure cellulose (Avicel) with NaOH-Fenton

2) Determined cellulase adsorption isotherms on NaOH-Fenton pretreated Avicel

3) Determined the enzymatic hydrolysis of NaOH-Fenton pretreated Avicel

4) Analyzed the structural difference of NaOH-Fenton pretreated Avicel with NMR

5) Characterized EOL lignin from Aspen, Black Willow and Cottonwood with SEM

The specific activities accomplished are presented below:

2.1 Pretreated pure cellulose (Avicel) with NaOH-Fenton

Avicel PH101 was mixed with 2% NaOH at 65 oC for 2 h, followed by an oxidative treatment with H2O2 and ferric sulfate, in which FeSO4·7H2O of 8-4 mM/g Avicel and H2O2 at 30% concentration from 1-8 mL/g Avicel were evaluated. In a solid to liquid ratio of 1:10, Fenton oxidative treatment was conducted at pH 3 adjusted with 50% acidic acid. And the reaction mixture was continuously agitated at 80 rmp and incubated at room temperature for 24 h, followed by filtration, water washing and stored at 4 ℃. Effects of the oxidative treatment conditions were evaluated based on the subsequent glucose yield, defined as percent yield of glucose from total cellulose in the Avicel. Fenton pretreated NaOH-Avicel under different conditions was defined based on H2O2 and FeSO4·7H2O. For example, Fenton pretreated NaOH-Avicel obtained under H2O2 at 30% concentration from 4 mL/g Avicel and FeSO4·7H2O of 16 mM/g Avicel was assigned as NaOH-Fenton Avicel 4/16.

2.2 Determined cellulase adsorption isotherms on NaOH-Fenton pretreated Avicel

To establish the adsorption isotherm onto pretreated Avicel, various concentrations of cellulase enzymes were incubated with a 2% suspension of pretreated Avicel in an acetate buffer at 4℃ for 3 h to reach equilibrium. The protein content in the supernatant was then determined and enzymatic distribution coefficient (R) and its correlation with cellulase effects were further estimated. As shown in Table 1 and Fig. 1, the maximum adsorption capacity of Celluclast onto NaOH-Fenton pretreated Avicel was Γmax =48.48 mg/g of cellulose, which was 2.38 times higher than those of control, Avicel (Γmax =20.37 mg/g of cellulose). It suggested that the NaOH-Fenton pretreated Avicel had a greater amount of adsorption sites when compared to Avicel. Compared with pure cellulose of Avicel (R = 0.22 L/g) and NaOH pretreated Avicel (R = 0.20 L/g), Fenton treated Avicel and NaOH-Fenton treated Avicel showed much lower enzyme distribution coefficient (R = 0.09and 0.10 L/g, respectively). It was observed the R value from Avicel was 5-folds higher than NaOH-Fenton pretreated Avicel. Similarly, the Langmuir constant of cellulase on Avicel (K = 10.74 mL/mg) and NaOH pretreated Avicel (K = 8.13 mL/mg) was 4-5 folds higher than those on Fenton pretreated Avicel (K = 2.39 mL/mg) and NaOH-Fenton pretreated Avicel (K = 2.08 mL/mg). This indicated there was a higher affinity between cellulase and Avicel than that between cellulase and NaOH-Fenton pretreated Avicel. Langmuir constants (K) and maximal adsorbed protein (Γm) from adsorption isotherm have often been used to evaluate the affinity and accessibility of cellulase enzymes on different cellulose substrates. On the basis of Langmuir constants of Celluclast onto Avicel and NaOH-Fenton pretreated Avicel (Table 1), it was observed that the cellulase enzyme had a higher affinity (higher K value) on Avicel than on NaOH-Fenton pretreated Avicel. However, NaOH-Fenton pretreated Avicel showed a higher Γm than that on Avicel. It is estimated that some oxidized groups in the NaOH-Fenton pretreated could interfere the binding between cellulose substrates and enzyme.

                                   Fig.  1.  Cellulase  adsorption  isotherms  on  Avicel,  NaOH  pretreated  Avicel,  and  NaOH-­‐Fenton  pretreated  Avicel  at  4℃. For  the  adsorption  isotherm,  different  concentrations  of  cellulases  were  incubated  with  2%  of  cellulose  substrates  in  50  mM  acetate  buffer  for  3  h  to  reach  equilibrium.        Table1  Langmuir  adsorption  isotherm  parameters  of  enzyme  adsorption  on  different  cellulose  substrates.    

                           

Cellulases Γm (mg/g) K (mL/mg) R2 (L/g)

Cellulases on Avicel 20.37 10.74 0.219

Cellulases on NaOH Avicel 24.56 8.13 0.200

Cellulases on Fenton Avicel 38.13 2.39 0.091

Cellulases on NaOH –Fenton Avicel 48.48 2.08 0.101

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2.3 Determined the enzymatic hydrolysis of NaOH-Fenton pretreated Avicel

If only alkaline was used in the pretreatment, the higher pretreatment temperature, the higher the enzymatic digestibility was observed on NaOH pretreated Avicel (Fig. 2). The final glucose yield was 69%, 70% and 78% when Avicel was pretreated at 55, 65 and 75 °C respectively. Previous works indicated that NaOH pretreatment could increases the surface area by swelling cellulose substrate and increasing carbohydrate accessibility to enzymes, while reducing the degree of polymerization (DP) and crystallinity of the cellulose fraction. When Avicel were treated by both NaOH and Fenton reaction, the enzymatic digestibility of pretreated Avicel was further enhanced. However, the initial hydrolysis rate of pretreated Avicel was much lower than the control, Avicel. It suggested that there was an inhibition of enzymatic hydrolysis for NaOH-Fenton pretreated Avicel at the beginning, and the inhibited effect could be from the structure change of the pretreated cellulose substrate.

                                                 

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Fig.2 Enzymatic hydrolysis of Avicel PH101 pretreated with sodium hydroxide (A), followed by an oxidative treatment with 4 mL 30% H2O2 per gram cellulose H2O2, 16 mM Fe2+concentration ferric sulfate and 24 h reaction time (B).

2.4 Analyzed the structural difference of NaOH-Fenton pretreated Avicel with NMR

The pretreated Avicel was compared and analyzed by 13C NMR. Avicel, NaoH pretreated Avicel, Fenton pretreated Avicel and NaOH-Fenton pretreated Avicel was dissolved in NaOD/D2O solution (Fig. 4). The peak at 104 ppm is assigned to C-1, the strong signal at 74 ppm to C-2, C-3, and C-5, and the two doublets appearing in the regions 81-90 ppm and 60-65 ppm to C-4 and C-6 resonances. 13C NMR spectrum of NaOH pretreated Avicel shows five sharp peaks, same as control, Avicel. It suggested that NaOH pretreatment, 2% NaOH at 65 oC for 2 h, had not changed the structure of cellulose, but swelled it. Spectra of oxidized samples show a decrease of the peak corresponding to –CH2OH group(C-6) and present the subsequent appearance of the peak corresponding to the carboxylic acid function, around 180 ppm.

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A                    B C D Fig.4. 13C NMR spectra of Avicel(D), NaOH pretreated Avicel (C), Fenton pretreated Avicel (B) and NaOH-Fenton pretreated Avicel (A) in 8% NaOD/D2O solution.

2.5 Characterized EOL lignin from Aspen, Black willow and Cottonwood with SEM

The effect of EOL lignin on enzymatic hydrolysis of biomass has been identified in our group. Here we characterized the EOL lignin particles with SEM Fig. 5). The results indicated EOL-AS, EOL-BW and EOL-CW from hardwood showed similar spherical particles (1 µm), which are much bigger than the EOL-LP from softwood. The physical size difference of lignin samples probably was associated with properties on enzyme adsorption in enzymatic hydrolysis.

Fig. 5 SEM images of EOL lignin samples from Organosolv pretreated Aspen (left), Black Willow (middle) and Cottonwood (right).

3. Explanation of Variance: N/A

4. Plans for Next Quarter: 1) Identify feedstock characteristics that govern butanol production rate and yield of

pretreated biomass 2) Characterize the organosolv lignin with NMR

3) Writing the report and manuscript for publication.

5. Budget: Please report the following budget information

a. Funds Expended to Date (End of Reporting Period): $116,502 b. Remaining Balance of Funds: 0

6. Patents: N/A

7. Publications / Presentations:

1) M. Tu (2015) Redefine the Role of Lignin in Enzymatic Hydrolysis of Lignocellulosic Biomass. SunGrant Conference, oral presentation, February 2-4, Auburn, AL.

2) W. Guan, S. Shi, M. Tu and Y. Lee (2015) Acetone-Butanol-Ethanol (ABE) production from pulp mill sludge by simultaneous saccharification and fermentation. Manuscript in review.

3) J. Yang, M. Tu (2015) Enhancing enzymatic digestibility of Avicel by NaOH and Fenton pretreatment. Manuscript in preparation.

8. Name of Students Funded on Project, including Department, Institution, Thesis/Dissertation Title (if applicable), Degree Obtained (if applicable), and Program Area:

Student Name

Department Institution Thesis/Dissertation Title

Degree Obtained/Date

Program Area

Pixiang Wang

SFWS Auburn University

Biomass

Wenjian Guan

Chemical Engineering

Auburn University

Bioenergy