Multistep Process to Produce Fermentable Sugars and

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A novel concept of utilizing residual solids from enzymatic hydrolysis in biorefinery for lignosulfonates and fermentable sugars production Yalan Liu, Jinwu Wang, and Michael P. Wolcott ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02328 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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ACS Sustainable Chemistry & Engineering

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A multi-step process to produce fermentable sugars and lignosulfonates from softwood

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enzymolysis residues

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Yalan Liua*, Jinwu Wangb, Michael P. Wolcotta

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a

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University, Pullman WA 99164-1806, United States

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b

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Flagstaff Road, Orono, ME 04469-5793, United States

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*Corresponding to: [email protected] (+1) 5093393158

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Abstract:

Composite Materials & Engineering Center, 2001 East Grimes Way, Washington State

United State Department of Agriculture, Forest Service, Forest Products Laboratory, 35

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The residual solids from enzymatic hydrolysis was usually burned to produce energy and have

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been explored as a feedstock for various products including activated carbon and lignin based

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polymers. These products require additional procedures unrelated to the existing biorefinery

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equipment. In the current study, we proposed successive sulfite treatments to utilize the

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enzymolysis residues for the producing fermentable sugars and lignosulfonates. Two sulfite

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methods were compared as an initial pretreatment. It was found that the acid bisulfite treatment

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achieved higher hemicellulose removal (90%), while neutral sulfite resulted in higher lignin

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removal (50%) and a greater lignosulfonate yield (26.6 g from 100 g raw material). Overall, the

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acid bisulfite treatment resulted in 66% glucan conversion by enzymatic hydrolysis, whereas

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only 37% for the neutral sulfite method. In addition, the neutral sulfite pretreated wood reached

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80% of the maximum sugar yield after 6 h of enzymatic hydrolysis, whereas 48 h for the acid

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bisulfite pretreated wood. The multi-step treatment process achieved 84% total sugar conversion

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and 58% lignin removal with 32.4 g lignosulfonates was produced from 100 g raw materials

ACS Paragon Plus Environment

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through this process. The results demonstrate that the multi-step treatment process has a potential

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to maximize the yields of fermentable sugars and lignosulfonates.

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Keywords:

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Acid bisulfite treatment, Neutral sulfite treatment, Lignosulfonates, Sugars, Enzymatic

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hydrolysis

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1. Introduction

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Pretreatment is a primary step to achieve adequate enzymatic hydrolysis yield of lignocellulosic

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materials. Currently, there are a variety of pretreatment methods for lignocellulosic conversion,

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such as mechanical, biological, and thermochemical methods1-2. Thermochemical methods are

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widely used for pretreatment processes such as dilute acid for agricultural biomass and sulfite

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pretreatment for woody biomass 3-5. Sulfite pulping has been used in pulp and paper industry

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since the 1840s 6. It can be carried out under acid, neutral and alkaline conditions aiming at

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hemicelluloses removal and/or lignin removal to obtain pulps with different characteristics.

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Hemicellulose and lignin removal can decrease enzyme absorption to cellulose and increase the

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accessibility of cellulose to the enzyme 7-8 resulting in increased enzymatic hydrolysis yield. In

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addition, hemicelluloses can be hydrolyzed into monomeric sugars, and the lignin sulfonated

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producing lignosulfonates, both of which are solubilized in the spent liquor. The acid bisulfite

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method was found works efficiently on softwood pretreatment compared to other pretreatment

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methods, such as dilution acid method4, 9-10. Neutral sulfite method was proved to achieve high

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lignosulfonates yield in the spent liquor6, which increases the utilization of lignin in the

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feedstock and reduced environmental issues.

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Zhu et al. 4, 10-13 carried out extensive studies employing the SPORL (sulfite pretreatment to

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overcome recalcitrance of lignocellulosic material) pretreatment process to achieve high sugar


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recovery form woody biomass. Initially, a high temperature in the range of 180-220 oC was

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adopted for SPORL treatments 14-16. However, the monomeric sugar degradation (such as

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furfural and hydroxymethylfurfural) existed to a large extent at these high temperatures.

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Therefore, a lower temperature (140 -165 oC) with long treatment time has also been explored.

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Studies 9-10 confirm the pretreatment at 145 oC for 3 hour achieved over 80% total carbohydrates

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conversion by enzymatic hydrolysis at a 4 - 6% (w/w) enzyme dosage based on the biomass. The

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enzymolysis residues contains more lignin than softwood (15 to 35% lignin) and hardwood

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(about 20% lignin), thus, it is a good resource for producing lignosulfonates 17. As lignin based

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polymer was widely investigated in current researches18-24, lignosulfonates can be another

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feasible product for utilizing lignin in the residues.

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The lignosulfonates can be separated and purified from spent liquors 25-26 and then used in many

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industries 27-28. Therefore, establishing lignosulfonates as a co-product can increase the

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profitability of a biorefinery and more efficiently utilize the feedstock. Neutral sulfite treatment

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has been found to produce high lignosulfonates yield with little degradation to the cellulose 6, 29.

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Moreover, the lignosulfonate production with current types of equipment in the biorefinery can

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be helpful for decreasing economic investment.

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The objective of this study is to explore a feasible way to utilize the enzymolysis residues with

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current equipment in biorefinery. Initially, we investigated the performance of two pretreatment

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methods: acid bisulfite and neutral sulfite treatment in producing both monomeric sugars and

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lignosulfonates. These two methods were first compared to delineate their performance and

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function mechanism as a sole pretreatment method for biomass. Then, the enzymolysis residues

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from the acid bisulfite pretreated wood was subjected to a neutral sulfite treatment. The spent

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liquors from the sequential treatments were analyzed for monomeric sugars and lignosulfonates


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to characterize overall sugar and lignosulfonates production. The second step enzymatic

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hydrolysis was carried out on neutral sulfite treated residues to investigate carbohydrates

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accessibility after two treatments. As well, the yields of enzymatic hydrolysis were investigated

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to optimize hydrolysis time. In all, the total mass balance of carbohydrates and lignin was

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investigated.

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2. Materials and Methods

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2.1 Materials preparation

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Douglas-fir (Pseudotsuga menziesii) micronized wood powders was prepared by an impact mill

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(Zhenzhou Tianyuan Environmental Protection Machinery Co. Ltd, China) with 30 min milling

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of nominal 2-mm wood chips. The median particle size (D50) of this sample is 32 µm with a

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standard deviation 1.2 µm measured by Malvern particle size analyzer (Mastersizer 3000 laser

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diffraction).

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The cooking liquor for acid bisulfite treatment has a 2.15% of calcium bisulfite and a 0.37% of

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free sulfur dioxide. The sodium carbonate (J.T. Baker, ACS Reagent, Fisher Scientific) and

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sodium sulfite (J.T. Baker, ACS Reagent, Fisher Scientific) were used for neutral sulfite

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treatment. 96% Sulfuric acid (ACROS Organics, Fisher Scientific) was diluted into 72% with

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distilled water for compositional analysis.

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2.2 Acid bisulfite treatment (AbS)

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The wood to chemical liquor ratio is 1:4 to obtain a 6.9% (w/w) equivalent sulfur dioxide

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loading on wood at 145 °C and 2h. A Parr reactor (No. 4845, 100 ml) was employed for all the

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treatments. It required approximately 30 min to reach 145 °C and 15 min to cool down to room

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temperature. After treatment, the slurry was separated by vacuum filtration. The spent liquor was


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collected for monomeric sugar and lignosulfonates (LS) analysis. The pretreated wood was

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washed with distilled water and used for enzymatic hydrolysis.

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2.3 Neutral sulfite treatment (NS)

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A neutral sulfite liquor consisted of 6% (w/w) sodium carbonate and 16% (w/w) sodium sulfite

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based on the oven dry wood weight. The solid to liquid ratio is 1:6 with a treatment temperature

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160 oC and duration time 180 min as was outlined elsewhere 29. It required approximately 35 min

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to heat up to 160 oC and 15 min to cool down. After treatment, the slurry was separated by

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vacuum filtration and the solids were washed thoroughly. The same analysis was carried out for

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the spent liquor and pretreated wood as described above.

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2.4 Multi-step treatment process

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The comparison results show acid bisulfite treatment is efficient in achieving high overall sugar

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recovery whereas neutral sulfite performs well on lignin removal and shortening enzymatic

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hydrolysis time to level-off. Therefore, we explored a sequential treatment by first applying the

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AbS treatment on the micronized wood powder at145 oC for 2 h and then enzymatically

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hydrolyzed the pretreated wood. The enzymolysis residues were subsequently treated with the

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NS liquor at160 oC for 3 h to produce additional lignosulfonates. Lastly, the secondary residual

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solids from the second-step treatment underwent additional enzymatic hydrolysis for subsequent

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carbohydrate conversion. The solids from each step were analyzed on chemical compositions.

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2.5 Enzymatic hydrolysis

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The pretreated wood from AbS and NS treatment were hydrolyzed with Cellic® CTec2 and

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HTec2, which were complimentarily provided by Novozymes North America (Franklinton, NC).

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The cellulase activity for CTec2 is 152.9 FPU /ml. The pretreated wood was hydrolyzed with a 4%

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(w/w) CTec2 and 0.4% (w/w) HTec2 (based on oven-dry wood basis) enzyme dosage. The solid


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loadings were 2% (w/v) of a total 50 ml volume with 0.5 ml 2% (w/w) sodium azide added for

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antibacterial function in hydrolysis slurry. A supernatant sample was collected after 0, 3, 6, 12,

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24, 48, and 72 h hydrolysis and then diluted for analysis of monomeric sugars.

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2.6 Fourier transform infrared (FTIR) analysis

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The untreated, AbS, and NS treated samples used for FTIR analysis were prepared by drying in

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the convection oven at 45 oC for 24 h. FTIR spectra were collected with a Thermo Nicolet

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Avatar 370 spectrometer operating in the attenuated total reflection (ATR) mode (Smart

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performance, ZnSe crystal performance, ZnSe crystal).

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2.7 Spent liquor analysis

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Monomeric sugars in spent liquor can be measured according to the NREL standard 30 by ion

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exchange chromatography (Dionex ICS-3000, CarboPac PA20 Guard column: 4 × 50 mm and

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IonPac AS11-HC analytical column: 4 × 250 mm, ED40 electrochemical detector, AS40

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autosampler, degassed E-pure water, 50mM and 200mM NaOH solution as eluent with a flow

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rate 0.5 ml/min ).

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The concentration of LS was measured by the UV-Visible spectrophotometer ( Perkin Elmer

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Lambda 35, PerkinElmer, Inc. Waltham, MA) and calculated from the absorbance at wavelength

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232.5 nm with an extinction coefficient of 24.5 31-32.

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2.8 Compositional analysis

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The samples for compositional analysis were prepared according to the NREL standard 33. Wet

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pretreated wood was dried in a convection oven at 45 oC for 24 h. A two-step acid hydrolysis

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was carried out based on the NREL standard 34. A 300 mg oven dried sample was hydrolyzed

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with 3 ml 72% sulfuric acid for 1h at 30 oC. Then, 84 ml water was added into the mixture and

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autoclaved for 1 h at 121 oC. Monomeric sugars were measured by Dionex ICS-3000, while acid


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soluble lignin was measured by the UV-Visible spectrophotometer. Due to the co-elution of

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xylose and mannose, the two sugars are reported as one value in this study as xylan/mannan or

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xylose/mannose.

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3. Results and discussion

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3.1 Comparison of AbS and NS treatment

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3.1.1 Treatment performance

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The performances of the AbS and NS pretreatment methods were compared in terms of lignin

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removal, hemicellulose removal, sugar recovery in spent liquor, as well as enzymatic hydrolysis

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yield of pretreated wood. As shown in Table 1, AbS treatment achieved 90% hemicellulose

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removal and 24% lignin removal, while NS treatment obtained 47% and 50%, respectively.

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However, monomeric sugar was not detected in NS spent liquor whereas 67% hemicellulose

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sugar was recovered in AbS spent liquor. The absence of free sugars in the NS spent liquor may

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be attributed to the high treatment temperature and long duration time, which are favorable for

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monomeric sugar degradation. Under this non-acidic conditions, the carbohydrate losses might

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be mainly attributed to the endwise peeling degradation into carboxylic acids, and the chain

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cleavage through hydrolysis with subsequent secondary peeling. Monomeric sugars are rapidly

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degraded due to a low protection from other moieties 35-37 resulting in a low overall sugar

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recovery by the NS treatment. But, the NS treatment resulted in 43% higher yield of LS and 26%

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more lignin removal than AbS treatment.

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When compared to the AbS and NS methods, the NS treatment performed better in lignin

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removal, while AbS treatment achieved better hemicellulose removal and monomeric sugar

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recovery in spent liquor. The mechanism for these two methods are hypothesized and diagramed

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in Figure 138. In the AbS treatment, the primary reaction is hemicellulose hydrolysis, while lignin


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sulfonation is the primary reaction in NS treatment accompanied by the secondary reactions of

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the edgewise peeling and hydrolysis with rapid degradation of monomeric sugars. AbS treatment

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is carried out under acid condition, which is favorable for hemicellulose hydrolysis. Whereas the

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SO32- in NS treatment under neutral condition is favorable for the breakage of lignin units6.

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3.1.2 Enzymatic hydrolysis

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Figure 2 compares the enzymatic digestibility of pretreated wood by the two treatment methods.

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Figure 2(a) shows the AbS resulted in 30% higher glucan yield than the NS pretreated wood at a

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4.4% enzyme dosage. Increasing of enzyme dosage to 8.8% did not increase glucan yield

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significantly for the NS pretreated wood. Since the AbS treatment hydrolyzed 13% glucan into

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the spent liquor, the total glucan conversion into monomeric sugars at 0 hour is set at this value

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in Figure 2(a). It is apparent that the NS treatment is more effective at lignin removal while

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hemicellulose conversion is more greatly facilitated by AbS treatment. As is shown in Figure 1,

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the combined lignin and hemicellulose complex provides protection to the cellulose fibrils,

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thereby imparting the recalcitrance to enzymatic conversion which is commonly found38.

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Therefore, from the results presented here, we can conclude that for softwoods, partial removal

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of both the lignin and hemicellulose are an important factor for affecting glucan yields. In

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addition, the acid hydrolysis of the hemicellulose, that occurs in the AbS method improves the

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overall sugar yield by liberating these C6 varieties during the pretreatment stage.

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Figure 2(b) shows the AbS method resulted in 40% higher xylan/mannan conversion compared

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to the NS method. The NS pretreated wood had 20% xylan/mannan conversion compared to 6%

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of AbS pretreated wood by enzymatic hydrolysis. Sixty-one percent of xylan/mannan have been

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hydrolyzed into the AbS spent liquor, while the NS method did not generate any free sugars in

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the spent liquor. However, the NS treated solid reached a plateau of sugar conversion (24 h for


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glucan and 12 h for xylan/mannan) sooner than the AbS pretreated wood. This may indicate that

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high lignin removal can reduce enzymatic hydrolysis time compared to hemicellulose removal.

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This behavior may result from the cellulase absorption on lignin that has been observed by

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others7-8.

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3.1.3 FTIR analysis

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Figure 3 shows band intensity changes for AbS and NS pretreated wood. The peak at 1731 cm-1

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for the untreated sample is attributed to ester linkages from hemicelluloses and lignin 39. The

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spectrum becomes flat at 1731 cm-1 for the NS pretreated wood but a small broad peak,

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indicating the higher percentage of ester linkage removal from the NS than the AbS treatment.

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The band 1508 cm-1 represent aromatic skeleton vibration from lignin 18, 40. The NS pretreated

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wood displayed a lower intensity of this band, indicating higher lignin removal. Bands 1158,

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1105, and 1055 cm-1 are assigned to C-O-C vibration and C-O vibration in cellulose and

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hemicellulose 41-42. After the hemicellulose removal by the treatments, the peaks of these two

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bands become more obvious for the two pretreated wood compared to those for the untreated

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sample. The AbS pretreated wood displayed higher intensity for these two bands compared to the

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NS pretreated wood indicating cellulose structure becomes more accessible due to high

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hemicellulose removal. This may account for the high glucan yield of AbS pretreated wood.

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3.2 Multi-step treatment process

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3.2.1 Sugar conversion and lignin removal

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Table 2 shows carbohydrate conversion and lignin removal in each step. This process achieved

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83.9% total sugar conversion and 58.3% lignin removal. The first two steps obtained

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approximately 69.5% sugar conversion, which is the major source of sugar conversion. The NS

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treatment step removed 35% lignin, which is 13% higher than AbS step. The final enzymatic


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hydrolysis step resulted in 15% additional sugar conversion. Considering the small amount of

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carbohydrates remained in the residual solids after two treatments, the second enzymatic

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hydrolysis may not prove cost-effective.

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3.2.2 Enzymatic hydrolysis

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The kinetics of enzymatic hydrolysis was also investigated. Figure 4 shows similar glucan and

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xylan/mannan yields were achieved at 72 h for the 1st and 2nd enzymatic hydrolysis. However, it

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took at least 48 h to achieve 80% of maximum yield in the first hydrolysis, whereas it only took

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6 h in the second hydrolysis as shown in Figure 4. Therefore, the second hydrolysis may be

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carried in a small amount of time, such as 6 h. This can save time occupancy of an equipment in

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a biorefinery resulting in saving energy and investments.

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3.3 Total mass balance analysis

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Figure 5 shows the total mass balance of carbohydrates and lignin in the multi-step treatment

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process. We can tell that the acid bisulfite pretreatment hydrolyzed a majority of hemicelluloses

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while producing 18.7 g LS. In the first enzymatic hydrolysis, 64% glucan and 69%

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xylan/mannan were converted from the AbS pretreated wood. Meanwhile, the neutral sulfite

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treatment removed 46% lignin from first enzymolysis residues and produced 13.7 g LS. Sixty-six

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percent glucan and 70% xylan/mannan from the NS treated residual solids were converted in the

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second hydrolysis. At last, the final residual solids comprised of 18% carbohydrates and 72%

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lignin. The majority of the final residual solids is lignin, it can be simply reused for lignin based

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polymers, activate carbon, or worked as binder in pellet production. In total, this process

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achieved 84% total sugar conversion and 32.4 g lignosulfonates production from 100 g raw

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materials. The AbS treatment and 1st enzymatic hydrolysis was aimed at maximizing

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carbohydrate conversion, while the NS treatment was in pursuit of maximizing lignosulfonates.


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By conducting the AbS treatment first, the rapid monomeric sugar degradation under neutral or

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alkaline sulfite conditions was avoided. Also, the second hydrolysis was completed in a short

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time period such as 6-12 h due to monomeric sugar yield plateau after 24 h.

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Acknowledgements

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The chemical liquor in this study was kindly provided and analyzed by Weyerhaeuser with help

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from Dr. Jonhway Gao. The authors gratefully acknowledge the Northwest Advanced

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Renewables Alliance (NARA), supported by the Agriculture and Food Research Initiative

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Competitive Grant no. 2011-68005-30416 from the USDA National Institute of Food and

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Agriculture, and the China Scholarship Council funding.

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Table Captions Table1. Comparison of acid bisulfite and neutral sulfite treatment method. Table 2. Total Sugar conversion and lignin removal in multi-step treatment process.

Figure Captions Figure 1. Proposed reaction mechanism of AbS and NS treatment (adapted from Hsu, 1980). Figure 2. The enzymatic hydrolysis of acid bisulfite and neutral sulfite pretreated wood: (a) glucan and (b)xylan/mannan. Figure 3. FTIR-ATR analysis of untreated, AbS, and NS pretreated wood. Figure 4. Enzymatic hydrolysis kinetics in the multi-step treatment process based on treated material. Figure 5. Total mass balance of carbohydrates and lignin of the multi-step treatment process.


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Table 1 Acid bisulfite treatment Component

Untreated (g)

Spent liquor (g)

Treated Solid (g)

Glucan

38.1

4.3

Xylan/Mannan

13.0

Arabinan

Neutral sulfite treatment

Removal (%)

Spent liquor (g)

Treated Solid (g)

Removal (%)

33.4

12.3

0

34.2

10.2

8.6

1.7

86.9

0

8.0

38.5

1.2

0.5

0

100

0

0.4

66.7

Galactan

3.0

2.5

0

100

0

0.7

76.7

Hemicellulosea

17.2

11.6

1.7

90.1

0

9.1

47.1

AILb

29.1

─

23.5

19.2

─

13.4

53.8

ASLc

4.9

─

2.3

53.1

─

3.6

26.5

Total Lignind

34.0

─

25.8

24.1

─

17.0

50.0

Lignosulfonates

─

18.7

─

─

26.6

a. b. c. d.

─

─

Hemicellulose includes xylan / mannan, arabinan, and galactan AIL short for acid soluble lignin ASL short for acid insoluble lignin Total lignin includes AIL and ASL


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Page 20 of 26

Table 2 Component

Acid bisulfite

1st Enzymatic hydrolysis

Neutral sulfite

2nd Enzymatic hydrolysis

Total (%)

Sugar conversion

28.6%

40.9%

0

14.3%

83.9

ASL removal

51.1%

0

17.3%

0

68.4

AIL removal

18.0%

0

37.4%

0

55.4

Total lignin removal

22.7%

0

35.6%

0

58.3


20

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Figure 1

Lignin

Cellulose

Amorphous Region Crystalline Region

Hemicellulose


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Page 22 of 26

Figure 2

\


22

Page 23 of 26

1055 1031

1158 1158

Untreated

897

1105

1262

1328

1371

1455 1421

1508

1639 1607

1031

Figure 3

1731

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NS treated

AbS treated

1800

1600

1400

1200

Wavenumbers (cm

800

1000

-

1


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Page 24 of 26

Figure 4


24

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Figure 5

Raw material:100g Glucan: 38.1g Xyl/mann: 13.0g Arabinan: 1.2g Galactan: 3.0g ASL: 4.9g AIL: 29.1g

Solids: 41.3g Glucan: 12.0g Xyl/mann: 0.6g ASL: 2.4g AIL: 23.9g

Solids: 63.9g Glucan: 33.4g Xyl/mann: 1.7g ASL: 2.4g AIL: 23.9g AbS treatment o 145 C and 2h

Spent liquor Glucan: 4.3g Xyl/mann: 8.6g Arabinan: 0.5g Galactan: 2.5g LS: 18.7g

st

Solids: 27.5g Glucan: 11.6g Xyl/mann: 0.4g ASL: 1.2g AIL: 13.0g nd

1 Enzymatic hydrolysis

NS treatment o 160 C and 3h

2 Enzymatic hydrolysis

1st hydrolysate: Glucan: 21.5g Xyl/mann: 1.2g

Spent liquor: Sugar: N.D. LS: 13.7g

2nd hydrolysate: Glucan: 7.6g Xyl/mann: 0.3g


Solids: 19.6g Glucan: 3.5g Xyl/mann: 0.1g ASL: 1.2g AIL: 13.0g

25


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Table of Content ; Title: A multi-step process to produce fermentable sugars and lignosulfonates from softwood enzymolysis residues; Authors: Yalan Liu, Jinwu Wang, Michael P. Wolcott ; Synopsis:Utilizing softwood enzymolysis residues with multi-step treatment process targeting at boost lignosulfonates and fermentable sugar production 338x190mm (300 x 300 DPI)


Page 26 of 26

Multistep Process to Produce Fermentable Sugars and

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