A Comparison of NaOH, Fenton, and Their Combined Pretreatments

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A comparison of NaOH, Fenton and their combined pretreatments for improving saccharification of corn stalks Menghui Yu, Sandra Chang, Dongsheng Li, Chengming Zhang, Li Jiang, Yaxin Han, Lisong Qi, Jihong Li, and Shizhong Li Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b02217 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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Energy & Fuels

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A comparison of NaOH, Fenton and their combined pretreatments for improving

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saccharification of corn stalks

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Menghui Yu a, Sandra Chang a, Dongsheng Li a, Chengming Zhang a, Li Jiang a, Yaxin Han a,

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Lisong Qi a, Jihong Li a,* and Shizhong Li a,*

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a

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Beijing 100084, PR China. Email: [email protected]

: Institute of Nuclear and New Energy Technology, Tsinghua University, Tsinghua Garden,

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*Corresponding author.

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Jihong Li and Shizhong Li

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Email: [email protected] [email protected]

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Tel.: +86 10 62772123; fax: +86 10 80194050

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Abstract

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In this study, the effectiveness of NaOH extraction (AE), Fenton oxidation (FO) and

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synergistic pretreatment using AE and FO for enzymatic saccharification of corn stalks (CS) was

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compared by statistical analyses. The results showed that AE-FO treatment resulted in statistically

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higher enzymolysis efficiency and glucose yield than single AE and single FO treatments.

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Compared with single AE and FO treatments, AE-FO resulted in statistically significant higher

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lignin removal and xylan removal, consequently improving the cellulose accessibility to cellulase.

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Among various synergistic pretreatment parameters, utilizing Plackett-Burman design determined

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the most effective factor improving cellulose accessibility to cellulase was NaOH loading. The

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results that we obtained could be extended to help further improve the synergistic pretreatment

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process by using CS for the production of biofuels and sugar-based chemicals.

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Key words

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Synergistic pretreatment; NaOH extract; Fenton oxidation; Enzymatic saccharification; Statistical

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method

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1.

Introduction

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Over the last few decades, lignocellulosic biomass has attracted much attention for the

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production of biofuels and sugars-based chemicals due to its abundant and renewable properties [1,

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2]. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. Generally,

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lignocellulosic biomass is hydrolyzed by enzyme saccharification, and the resulting hydrolysates,

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including fermentable sugars are then used to produce biofuels and sugar-based chemicals via

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microbial fermentation [3]. The carbohydrate with high content in lignocellulosic biomass could

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be converted into fermentable sugars by enzymatic saccharification. However, much of the

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elemental cellulose fibrils in the biomass are cross-linked with hemicelluloses and lignin, resulting

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in recalcitrant structures that are not easily accessible to enzymatic digestion [4, 5]. Therefore, a

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pretreatment process is necessary to break the lignin seal, in order to disrupt the recalcitrant

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structures and to increase the accessibility of enzymes to carbohydrate, such as cellulose

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hydrolysis [6].

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Alkaline pretreatments have been shown to be a promising method to overcome the

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recalcitrance of herbaceous crops for enzymatic hydrolysis. [3, 6, 7]. Compared with other

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pretreatments technologies, alkaline pretreatments are generally effective for lignin removal [8].

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Moreover, alkaline pretreatments do not cause excessive sugar degradation, but produce lower


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concentration of inhibitors with lower energy input compared with other thermochemical

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pretreatment methods [9-11]. For examples, Yu et al. reported that direct enzymatic

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saccharification of NaOH pretreated sweet sorghum bagasse without washing significantly

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improved the fermentable sugars conversion rate from 44.85% to 65.14% [3]. Kaar and

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Holtzapple during lime pretreatment of corn stover (75 mg g-1 dry biomass, 120 °C, 4 h) obtained

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88% cellulose conversion [12]. In recent years, other methods such as, Fenton oxidation (FO)

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which mimics the degradation of plant cell walls by fungi was used to pretreat lignocellulosic

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biomass [13]. The mechanism of action of FO is the generation of two kinds of hydroxyl radicals

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(HO• and HOO•) when Fe2+ catalyzes H2O2 decomposition under acidic conditions [14]. The

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generated hydroxyl radicals could both depolymerize carbohydrates and lignin to breakdown

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lignocellulosic biomass making it more accessible for enzymes [15]. Since FO and NaOH

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extraction (AE) operate under different mechanism of action, several works investigated the

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possibility that the combination of FO with NaOH extraction (AE) could be synergetic for

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herbaceous crops pretreatment [14, 16, 17]. Those previous studies optimized the process

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parameters of synergistic pretreatment by one-factor-at-a time approach, which were performed by

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changing one independent variable while fixing all others at a fixed level [14, 16, 17]. So far, no

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statistical analysis has been performed to compare the effectiveness of these pretreatments for

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enzymatic saccharification of CS. On the other hand, the one-factor-at-a time method was difficult

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to pick out significantly affected factors due to the interactions among factors [18]. Statistical

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experimental designs can effectively solve these issues and minimize the error in determining the

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effect of factors and interaction between factors [19]. Recently, statistical experimental designs

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have been applied to pick out significantly affected factors and optimize many processes [20-22].


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However, to the best of our knowledge, no statistical experimental design has been performed to

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screen key factors affecting synergistic pretreatments.

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In this study, a combination of AE with FO for CS pretreatment was investigated. The

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enzymolysis efficiency of synergistic pretreatment (AE-FO) was compared with single AE and

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single FO treatments using statistical analysis. Composition changes of CS involved in various

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pretreatments processes were compared by statistical methods. The key factor affecting synergistic

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pretreatment was evaluated by Plackett-Burman design. Moreover, influence of pretreatment

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sequence on enzymolysis efficiency and composition of CS was also compared via paired samples

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t-test.

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2. Materials and methods

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2.1. Materials

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Corn stalks (CS) were harvested in Hebei province. The CS was air dried, milled using a high

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rotated speed disintegrator (Taisite, Tianjin, China) to yield a particle with diameters of 0.8 and1.6

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mm. The composition of CS (on dry basis) was determined as 6.35 ± 0.26% extractives, 34.46 ±

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1.02% cellulose, 25.80 ± 1.06% hemicellulose, 20.61 ± 0.86% lignin and 7.29 ± 0.06% ash.

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Sodium hydroxide (NaOH), Ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2) were obtained

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from Yongda chemical reagent company (Tianjin, China). Cellulase enzyme Cellic CTec2 was

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generously provided by Novozymes Investment Co. Ltd (Beijing, China). The filter paper

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enzymatic activity was 48.60 ± 1.36 FPU/mL for Cellic CTec2. All the other chemical reagents

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were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Beijing, China).

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2.2. Pretreatment

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In this study, two single pretreatment processes were designated as Fenton oxidation (FO),


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NaOH extraction (AE). Additionally, two synergistic pretreatments process were designated as

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sequential NaOH extraction and Fenton oxidation (AE-FO), and sequential Fenton oxidation and

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NaOH extraction (FO-AE).

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FO was carried out in a 500 ml Erlenmeyer flask at a solid/liquid ratio of 1/20 (w/w). Ten

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grams of biomass (dry weight) was added to the Erlenmeyer flask, followed by supplementation

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of 50 ml of distilled water. Then Fenton reagent solution was added to the Erlenmeyer flask.

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Fenton reagent solution was prepared using FeSO4•7H2O and H2O2 (30%, w/w) at a mole ratio of

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1:25. The pH of the reaction mixture was adjusted to 3.0 by adding 5% oxalic acid. The

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Erlenmeyer flask was sealed with plastic thin-film to avoid H2O2 volatilization. FO was carried

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out at 35 °C, 150 rpm for 48 h. After FO, the whole slurry was collected and solid-liquid

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separation was performed by centrifuge. The separated solids were washed with deionized water

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until pH reached 7.0. The washed solids (called FO-CS) were used for enzymatic saccharification

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and AE. AE was performed in a 500 ml Erlenmeyer flask at a solid/liquid ratio of 1/15 (w/w), with

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CS soaked in NaOH solution and incubated for 1.5 h in a water bath at 90 °C. After AE, the

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suspension was centrifuged, and the obtained residue was washed with deionized water to neutral

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pH. The washed solids (called AE-CS) were used for enzymatic saccharification or FO. The

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composition of pretreated CS was determined by the NREL method [23]. Lignin removal (LR)

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and xylan removal (XR) after pretreatment were calculated by equation 1-2:

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Lignin removal LR, % = Xylan removal XR, % =

, ,

,

&', &',()*+)*,+*- &',

× 100

× 100

(1) (2)

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mCS and mPretreated CS was the mass of CS before and after pretreatment respectively. mlignin, CS

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represented the mass of lignin in CS. mlignin, pretreated CS represented the mass of residual lignin in


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pretreated CS. mxylan, CS represented the mass of xylan in CS. mxylan, pretreated CS represented the mass

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of residual xylan in pretreated CS. All the experiments were performed in triplicate.

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2.3. Enzymatic saccharification

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Enzymatic saccharification was conducted in 125 mL Erlenmeyer flasks, using 50 mM

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sodium citrate buffer (pH = 4.8) at 50°C and 150 rpm for 72 h. Two gram of pretreated CS was

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added to the flask, and then the buffer solution was added to the final solid concentration of 5%

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(w/w). Tetracycline (50 mg/L) was added to inhibit microbial growth. The cellulase was added at a

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loading of 28 FPU/g of pretreated CS. After 72 hours enzymatic hydrolysis, 1 mL of the sample

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was taken from reaction mixture and centrifuged at 14,000 rpm for 5 minutes. The supernatant

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was employed to assay the released monosaccharide. The enzymolysis efficiency (EE) and

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glucose yield (GY) were calculated using equation 3 and 4:

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Enzymolysis efficiency EE, % =

34567 ×8.:;3&'67 ×8.'

Glucose yield GY, % =

6'7 74'

45 ;&' 34567 × =>'

6'7 74' × 8.:

3CD' E>

× 100

× 100

(3) (4)

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Cglucose and Cxylose were the glucose and xylose concentration respectively in the hydrolysed slurry

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(g/L); Vhydrolysed slurry was the hydrolysed slurry volume (L); mglucan and mxylan were the mass of

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glucan and xylan in pretreated CS respectively (g); CSDry weight was the mass of CS; 0.9 and 0.88

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were the conversion factor of glucan and xylan to equivalent glucose and xylose, respectively. All

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the experiments were performed in triplicate. The mean of results was employed for analysis.

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2.4. Plackett-Burman experimental design

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From the literature, the basic parameters involved in combinative pretreatment were

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considered to include particle size, NaOH and Fenton loading [7, 13, 14, 16, 17]. A two-level

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Plackett-Burman experimental design (PBD) of 12 runs was introduced to screen the important

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variables significantly influencing the pretreatment efficiency. The Fenton loading was studied


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between 0.57 mmol/g-TS and 1.13 mmol/g-TS (on a Fe2+ concentration basis) to investigate effect

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of Fenton oxidation treatment. A NaOH loading between 1.06 wt% and 2.12 wt% was selected to

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identify the effect of alkali extraction treatment. The size of particles was test at 1.6 and 0.8 mm.

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Table 1 outlined the input variable levels in PBD. Each estimated variables was examined in two

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levels, low (-1) and high (+1) level. The glucan/(xylan+lignin) ratio in pretreated CS was set as

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response variables to represent the cellulose accessibility to cellulase. The influence of individual

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variable on glucan/(xylan+lignin) ratio was calculated by the following Equation 5: y FG = 2 ∑ JG; − JG /M

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(5)

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where, y (xi) was the influence of the tested variable (xi) and mi+ and mi− were responses of trials at

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which the variable is at its high or low levels respectively, n is the total number of trials. Each

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experimental run was performed in triplicate. The average response value in each experiment was

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employed for analysis. Table 1

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2.5. Analysis methods

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Concentration of monosaccharides (glucose and xylose) was measured by HPLC (Shimadzu

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LC-20AD, Tokyo, Japan) with a refractive index detector and a Bio-Rad HPX-87H column (250

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mm × 4.6 mm).

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The PBD experiments were planned using Design-Expert Software version 8.0.6 (Stat-Ease

152

Inc., Minneapolis, MN, USA). The statistical analysis was done by IBM SPSS statistics version

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21.0 for Windows (SPSS, Chicago, IL). A paired-samples t-test was used to compare the mean

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difference of data from two related groups with zero. The condition where there was no difference

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in means of data from two groups to be the null hypothesis. Results were considered statistically

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significant, when the obtained P-value was < 0.05.

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3.

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3.1 Comparison of different pretreated CSs’ enzymatic saccharification yields

Results and Discussions


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The capital cost for synergistic pretreatments is significantly higher than for single cases since

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it requires additional infrastructure investment and generates higher operating cost [24]. In order to

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offset the huge investment, the EE of CS by combinative pretreatments should be significantly

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higher than that by the single pretreatments. Therefore, there is a need to compare various

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pretreatment substrates’ EE by statistical analysis. Furthermore, in valuing the pretreatment

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methods, GY of various pretreatment substrates were also evaluated by statistical analysis. From

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Table 2, the result of Shapiro-Wilks (S-W) tests showed that each set of data followed normal

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distribution (P > 0.05). This can be seen from their p value that was greater than significance level of

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0.05. Moreover, when comparing the EE of AE-FO treated CS with that of CS treated by other

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pretreatments, the result of f-test showed the results of both groups conformed to the hypothesis of

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the homogeneity of variance (σ12 = σ22, P﹥0.05). Therefore, a paired samples t-test was deployed to

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check whether the difference between the two sets of date were significantly different from zero at

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99.5% confidence level. Table 2 showed that the EE of various pretreated CS under various

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experimental conditions. From Table 2, AE-FO treatment resulted in statistically significant higher

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EE of CS than AE (t= 9.32, P=0.00) and FO (t= 22.84, P=0.00) treatment. Under the various

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conditions of experiments, the average value of EE for AE-FO was 9.35%, 62.50% higher than that

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for single AE and FO treatment respectively. On the other hand, when comparing the GY of AE-FO

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treated CS (AE-FO-CS) with that of other pretreatments treated CS, the results of both groups

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conformed to the hypothesis of overall normal distribution (P > 0.05) and homogeneity of variance

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(P > 0.05). As shown in Table 2, AE-FO treatment resulted in statistically significant higher GY

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than AE (t = 12.85, P = 0.00) and FO (t = 20.62, P = 0.00) treatment respectively. These results

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implied the synergistic pretreatments using AE and FO was better for enhancing enzymatic

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saccharification of CS than single AE and FO treatment.

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Table 2 3.2 Comparison of different pretreated CSs’ composition

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Next the reason for synergistic pretreatment resulting in higher EE than corresponding single

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pretreatments was analyzed. According to Gabhane et al., an effective pretreatment protocol is to

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remove lignin and hemicellulose without doing much harm to cellulose [25]. Therefore, the


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structural changes of the AE-FO-CS compared with other pretreated CS were evaluated by

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statistical methods. From Table 3, when comparing the compositions of AE-FO-CS with that of

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other pretreatments treated CS, the results of both groups conformed to the hypothesis of overall

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normal distribution (P > 0.05) and homogeneity of variance (P > 0.05). Therefore, paired samples

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t-tests were used to determine how significance different pretreatments in changing the

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compositions of CS. From Table 3, AE-FO treatment resulted in statistically significant greater LR

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than single AE (t=22.46, P=0.00) and single FO (t=86.12, P=0.00) treatments. Under various

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conditions of experiments, the average value of LR for AE-FO was 20.15%, 66.68% higher than

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that for single AE and single FO treatment respectively. On the other hand, synergistic pretreatment

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also showed its advantage in xylan removal over single pretreatments. From Table 3, AE-FO

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resulted in statistically significant greater XR than AE alone (t=53.28, P=0.00) and FO alone

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(t=40.59, P=0.00). Under various conditions of experiments, the average value of XR for AE-FO

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was 26.61%, 35.94% higher than that for single AE and single FO treatment respectively. These

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statistical results suggested AE-FO mainly augmented the enzymolysis by further removing the

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lignin and hemicellulose compared with single AE and FO pretreatments, which consequently

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increased the cellulose accessibility to cellulase [26].

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Table 3

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3.3 Synergistic pretreatment using Fenton oxidation and NaOH extraction

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In order to determine if the sequence of whether pretreatment first with NaOH or Fenton has

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an effect on enzymatic saccharification yield of CS, EE and GY of two synergistic pretreatments

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(sequential Fenton oxidation and NaOH extraction, FO-AE and sequential NaOH extraction and

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Fenton oxidation, AE-FO) were evaluated by statistical analysis. As shown in Table 4, AE-FO

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treatment resulted in a significant increase of EE compared with FO-AE treatment (t=5.27, P =

210

0.00). Under the various conditions of experiments, the average value of EE for AE-FO was 7.67%

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higher than that for FO-AE treatment. On the other hand, from Table 4, AE-FO treatment resulted

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in a significant increase of GY compared with FO-AE treatment (t=8.74, P = 0.00). Under the


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various conditions of experiments, the average value of GY for AE-FO was 11.59% higher than

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that for FO-AE treatment. These results suggested the synergistic pretreatment sequence has a

215

significant influence on enzymatic saccharification yield of CS.

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Table 4

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In order to elucidate the mechanism of AE-FO pretreatment favored enzymatic

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saccharification of pretreated CS, composition changes of the AE-FO-CS in contrast to FO-AE

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treated CS (FO-AE-CS) were evaluated by statistical methods. From Table 5, the results of both

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groups conformed to the hypothesis of overall normal distribution (P > 0.05) and homogeneity of

221

variance (P > 0.05). The result of paired samples t-test showed there was no difference in LR

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between the two groups (t= -1.20, P=0.26). This result indicated the combinative pretreatment

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sequence had no significant effect on lignin removal. In contrast to LR, AE-FO treatment resulted

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in statistically significant greater XR than FO-AE (t=8.63, P=0.00). These results above suggested

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AE-FO treatment could result in higher cellulose accessibility than FO-AE. To verify the above

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hypothesis, statistical analysis were performed to evaluate the difference in glucan/(xylan+lignin)

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ratio between AE-FO-CS and FO-AE-CS. As shown in Table 5, each set of glucan/(xylan+lignin)

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ratio data fitted normality (P > 0.05) and homoscedasticity (P > 0.05). A paired samples t-test of

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the two sets of date showed that AE-FO treatment resulted in statistically significant higher

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glucan/(xylan+lignin) ratio in pretreated CS than FO-AE treatment (t= 3.86, P=0.00). Under

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various conditions of experiments, the average value of glucan/(xylan+lignin) for AE-FO-CS was

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15.54%, higher than that for FO-AE-CS. To our knowledge, cell wall structure is composed of

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elemental cellulose fibrils cross-linked with hemicelluloses and embedded in a non-cellulosic

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polysaccharide matrix of hemicelluloses and lignin [4, 5, 27]. Based on this understanding, it was


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expected that AE-FO resulting in higher glucan/(xylan+lignin) ratio in pretreated CS was

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attributed to further delignification and hemicellulose depolymerization, which could

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consequently improve the cellulose accessibility to cellulase [26]. Table 5

238 239

3.4 key factors affecting synergistic pretreatment

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The results above demonstrated the synergistic pretreatment might have a broad range of

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applications in pretreating lignocellulose since it resulted in higher EE than single pretreatments,

242

which could be attributed to its improvement in cellulose accessibility to cellulase. In order to

243

efficiently optimize the synergistic pretreatment process, there is a need to identify key process

244

parameters. In this study, the Plackett-Burman experimental design (PBD) was used to evaluate

245

the relative importance of various variables for synergistic pretreatment [28]. According to the

246

experimental analysis shown in Figure 1A and 1B, both AE-FO and FO-AE treatment, and the 95%

247

confidence interval of glucan/(xylan+lignin) implied that all three variables including NaOH and

248

Fenton loading and particle size showed positive effect on improving glucan/(xylan+lignin). From

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Table 6, for both AE-FO and FO-AE treatment, the most effective factor improving

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glucan/(xylan+lignin) ratio was NaOH loading (62.58% and 41.21% contribution for AE-FO and

251

FO-AE respectively), then by Fenton’s loading (24.56% and 36.76% contribution for AE-FO and

252

FO-AE respectively) and followed by particle size (12.86% and 22.04% contribution for AE-FO

253

and FO-AE respectively). The result from Table 6 suggested in studied extent of particle sizes, the

254

contribution of particle size to glucan/(xylan+lignin) increase was lower than NaOH and Fenton

255

loading.

256

glucan/(xylan+lignin) could be that NaOH extraction and Fenton oxidation act by two different

The

reasons

for

NaOH

loading

making

big

contributions


on

improving

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257

reaction mechanisms. Alkaline pretreatment mainly improved the glucan/(xylan+lignin) in

258

pretreated CS by disrupting the lignin-carbohydrate complex to resulting in the dissolution of

259

partial lignin and hemicellulose [29]. Cellulose was difficult to degrade under the alkaline

260

condition [30]. On the other hand, the generated hydroxyl radicals (HO• and HOO•) from Fenton

261

reaction both depolymerize polysaccharides including cellulose and hemicellulose as well as

262

lignin, which consequently reducing the pretreated CSs’ glucan/(xylan+lignin) [15]. Therefore, for

263

both AE-FO and FO-AE treatment, the most effective factor improving glucan/(xylan+lignin) ratio

264

was NaOH loading. Based on these understandings, among various process parameters of

265

synergistic pretreatment using AE and FO, the NaOH loading should be further optimized in

266

future studies to achieve high pretreatment efficiency. The results above provide guidance for

267

future process optimization in synergistic pretreatment of lignocellulosic biomass.

268

Figure 1

269

Table 6

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4. Discussions

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The results in study demonstrated the synergistic pretreatments using AE and FO resulted in

272

higher EE and GY than single AE treatment. On the other hand, during biofuel production,

273

pretreatment reactor contributes 44% of pretreatment cost [24]. The capital cost for synergistic

274

pretreatments was significant higher than single AE and FO treatments since synergistic

275

pretreatments needed extra capital investment for pretreatment reactor construction and energy

276

consumption [24]. In the future research, the synergistic pretreatments using AE and FO need to

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be further optimized to improve the efficiency and economics.

278

4. Conclusion


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In this study, the effectiveness of FO, AE and combined FO and AE pretreatments for

280

enzymatic saccharification of CS was investigated via statistical analyses. The following

281

conclusions could be drawn: AE-FO treatment resulted in statistically higher EE than single AE

282

and single FO treatments. In contrast to AE or FO alone, AE-FO resulted in statistically significant

283

higher LR and XR, consequently improving cellulose accessibility to cellulase. Among various

284

synergistic pretreatment parameters, the most effective factor improving cellulose accessibility to

285

cellulase was NaOH loading. Our findings will help further improve the synergistic pretreatment

286

process.

287

5. Acknowledgements

288

This research was funded by the Science and Technology Program for Public Wellbeing (Grant No.

289

2013GS460202-X).

290


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10. Behera, S.; Arora, R.; Nandhagopal, N.; Kumar, S. Importance of chemical pretreatment for bioconversion of lignocellulosic biomass. Renew Sust Energ Rev. 2014, 36(7), 91-106. 11. Whitfield, M. B.; Chinn, M. S.; Veal, M. W. Processing of materials derived from sweet sorghum for biobased products. Ind Crop Prod. 2012, 37(1), 362-375. 12. Kaar, W. E.; Holtzapple, M. T. Using lime pretreatment to facilitate the enzymic hydrolysis of corn stover. Biomass Bioenerg. 2000, 18(3), 189-199.

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13. Jung, Y. H.; Kim, H. K.; Park, H. M.; Park, Y. C.; Park, K.; Seo, J. H., et al. Mimicking the

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Technol. 2015, 179, 467-472.

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14. Zhang, C.; Pei, H.; Wang, S.; Cui, Z.; Liu, P. Enhanced Enzymatic Hydrolysis of Poplar after

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Combined Dilute NaOH and Fenton Pretreatment. Bioresources. 2016, 11(3), 7522-7536.

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15. Xie, S.; Qin, X.; Cheng, Y.; Laskar, D.; Qiao, W.; Sun, S., et al. Simultaneous conversion of

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all cell wall components by an oleaginous fungus without chemi-physical pretreatment.

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16. He, Y.; Yun, D.; Xue, Y.; Yang, B.; Liu, F.; Wang, C., et al. Enhancement of enzymatic

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extraction. Bioresource Technol. 2015, 193, 324-330.

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17. Zhang, T.; Zhu, M. Enhancing enzymolysis and fermentation efficiency of sugarcane bagasse

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by synergistic pretreatment of Fenton reaction and sodium hydroxide extraction. Bioresource

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Technol. 2016, 214, 769-777.

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18. Jo, J. H.; Lee, D. S.; Park, D.; Choe, W. S.; Park, J. M. Optimization of key process variables

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response surface methodology. Ann Microbiol. 2013, 63, 513-521.

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20. Guha, M.; Ali, S. Z.; Bhattacharya, S. Screening of variables for extrusion of rice flour employing a Plackett-Burman design. J Food Eng. 2003, 57(2), 135-144.

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21. Dayana, P. S.; Bakthavatsalam, A. K. Optimization of phenol degradation by the microalga

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Chlorella pyrenoidosa using Plackett-Burman Design and Response Surface Methodology.

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22. Elboughdiri, N.; Mahjoubi, A.; Shawabkeh, A.; Khasawneh, H. E.; Jamoussi, B. Optimization

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of degradation of hydroquinone, resorcinol and catechol using response surface methodology.

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Adv Chem Eng Sci. 2015, 5, 111-120.

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23. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D., et al. Determination

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of structural carbohydrates and lignin in biomass. Laboratory analytical procedure (LAP);

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2010. NREL/TP-510-42618, National Renewable Energy Laboratory, Golden, CO.

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24. Tao, L.; Aden, A.; Elander, R. T.; Pallapolu, V. R.; Lee, Y. Y.; Ong, R. G., et al. Process and

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technoeconomic analysis of leading pretreatment technologies for lignocellulosic ethanol

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production using switchgrass. Bioresource Technol. 2011, 102(24), 11105-11114.

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25. Gabhane, J.; William, S. P. M. P.; Vaidya, A. N.; Das, S.; Wate, S. R. Solar assisted alkali

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pretreatment of garden biomass: effects on lignocellulose degradation, enzymatic hydrolysis,

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crystallinity and ultra-structural changes in lignocellulose. Waste Manage. 2015, 40, 92-99.

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lignocelluloses: our recent understanding. Bioenerg Res. 2013, 6(2), 405-415. 27. Ding, S. Y.; Himmel, M. E. The maize primary cell wall microfibril: a new model derived from direct visualization. J Agric Food Chem. 2006, 54(3), 597-606.

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28. Borges, P. R. S.; Tavares, E. G.; Guimarães, I. C., Rocha, R. D. P.; Araujo, A. B. S.; Nunes, E.

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E., et al. Obtaining a protocol for extraction of phenolics from açaí fruit pulp through

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Plackett-Burman design and response surface methodology. Food Chem. 2016, 210, 189-199.

363

29. Hammel, K. E.; Kapich, A. N.; Jensen, K. A. Jr.; Ryan, Z. C. Reactive oxygen species as

364 365 366

agents of wood decay by fungi. Enzyme and Microb Tech. 2002, 30, 445-453. 30. Rabelo, S. C.; Filho, R. M.; Costa, A. C. Lime pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse. Appl Biochem Biotechnol. 2013, 169, 1696-1712.

367


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368

Figure Captions

369

Figure 1 The 95% confidence interval of glucan/(xylan+lignin)

370 371


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372

Table 1 Plackett-Burman experimental design using actual variables and coded variables Run

373

1

Particle size

Fenton loading 1 2+

NaOH loading

(mm)

(Fe , mmol/g-TS)

(wt %)

1

1.6 (+1)

1.13 (+1)

2.12 (+1)

2

0.8 (-1)

0.57 (-1)

1.06 (-1)

3

0.8 (-1)

0.57 (-1)

2.12 (+1)

4

1.6 (+1)

1.13 (+1)

1.06 (-1)

5

1.6 (+1)

0.57 (-1)

1.06 (-1)

6

0.8 (-1)

1.13 (+1)

2.12 (+1)

7

0.8 (-1)

1.13 (+1)

2.12 (+1)

8

1.6 (+1)

1.13 (+1)

1.06 (-1)

9

0.8 (-1)

1.13 (+1)

1.06 (-1)

10

1.6 (+1)

0.57 (-1)

2.12 (+1)

11

0.8 (-1)

0.57 (-1)

1.06 (-1)

12

1.6 (+1)

0.57 (-1)

2.12 (+1)

The Fenton reagent solution was prepared using FeSO4•7H2O and H2O2 at a mole ratio of 1:25.

374 375


Energy & Fuels

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Table 2 Enzymolysis efficiency (EE) and glucose yield (GY) of various pretreated CS1 Run

AE-FO (EE, %)

AE (EE, %)

FO (EE, %)

AE-FO (GY, %)

AE (GY, %)

FO (GY, %)

1

98.96

92.21 (+6.75)

32.61 (+66.34)

72.07

62.12 (+9.94)

17.13 (+54.94)

2

98.61

89.26 (+9.35)

29.26 (+69.34)

69.34

58.41 (+10.92)

15.06 (+54.27)

3

103.94

90.85 (+13.09)

42.32 (+61.62)

72.94

62.21 (+10.73)

21.69 (+51.25)

4

110.73

95.77 (+14.96)

52.95 (+57.78)

65.44

55.60 (+9.84)

31.32 (+34.11)

5

98.11

92.77 (+5.34)

28.82 (+69.29)

70.24

59.08 (+11.16)

15.41 (+54.83)

6

99.85

91.18 (+8.67)

30.11 (+69.74)

70.42

63.93 (+6.49)

15.27 (+55.16)

7

96.34

86.10 (+10.25)

29.46 (+66.88)

64.75

58.28 (+6.46)

9.75 (+54.99)

8

97.25

92.66 (+4.59)

21.62 (+75.63)

66.07

59.74 (+6.33)

10.61 (+55.47)

9

103.89

95.27 (+8.62)

57.27 (+46.61)

65.83

60.97 (+4.86)

29.70 (+36.13)

10

104.50

99.05 (+5.45)

60.64 (+43.85)

69.86

60.69 (+9.18)

31.08 (+38.78)

11

102.64

91.48 (+11.16)

43.57 (+59.06)

66.17

59.58 (+6.59)

22.04 (+44.13)

12

103.96

89.94 (+14.02)

40.89 (+63.87)

71.78

65.07 (+6.71)

20.17 (+51.61)

S-W tests

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

Homogeneity of variance

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

Significant difference




(t=9.32, P=0.00)

(t=22.84, P=0.00)

(t=12.85, P=0.00)

(t=20.62, P=0.00)

Paired samples t-test result

377

1

378

comparing EE and GY of AE-FO treated CS with other pretreatments treated CS. The value of EE exceeding 100% is due to detection error. Dates of groups

379

conformed to the hypothesis of overall normal distribution (P > 0.05) and homogeneity of variance (P > 0.05). Values were the means of triplicates.

S-W tests means Shapiro-Wilks test. EE means enzymolysis efficiency. GY means glucose yield. Dates in brackets denoted the incremental values while


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Energy & Fuels

Table 3 Influence of different pretreatments on pretreated CS characteristics 1 Run

AE-FO (LR, %)

AE (LR, %)

FO (LR, %)

AE-FO (XR, %)

AE (XR, %)

FO (XR, %)

1

87.07

63.43 (+23.63)

18.37 (+68.69)

76.70

53.65 (+23.04)

40.52 (+36.18)

2

83.80

62.24 (+21.55)

18.72 (+65.08)

74.33

46.55 (+27.77)

38.59 (+35.73)

3

87.13

62.60 (+24.53)

14.67 (+72.46)

78.71

51.50 (+27.21)

40.74 (+37.97)

4

81.58

61.47 (+20.11)

18.01 (+63.57)

74.57

47.77 (+26.80)

38.82 (+35.75)

5

83.55

62.09 (+21.46)

14.99 (+68.57)

75.08

47.94 (+27.13)

34.92 (+40.15)

6

85.51

66.79 (+18.72)

19.68 (+65.83)

78.69

52.13 (+26.56)

41.73 (+36.96)

7

80.39

62.39 (+18.00)

15.50 (+64.89)

75.03

48.26 (+26.77)

44.46 (+30.57)

8

80.16

62.11 (+18.05)

14.79 (+65.37)

74.37

47.45 (+26.91)

41.24 (+33.12)

9

88.20

66.79 (+21.41)

19.92 (+68.27)

79.71

52.13 (+27.58)

38.51 (+41.19)

10

85.86

63.43 (+22.43)

17.22 (+68.64)

76.90

53.65 (+23.25)

43.69 (+33.21)

11

85.18

62.24 (+12.94)

12.11 (+73.07)

75.46

46.55 (+28.91)

42.19 (+33.28)

12

85.46

66.49 (+18.96)

19.68 (+65.77)

78.84

51.48 (+27.36)

41.73 (+37.11)

S-W tests

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05


P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05





(t=22.46, P=0.00)

(t=86.12, P=0.00)

(t=53.82, P=0.00)

(t=40.59, P=0.00)


381

1

382

distribution (P > 0.05) and homogeneity of variance (P > 0.05). Values were the means of triplicates. Dates in brackets denoted the incremental values while

383

comparing LR and XR of AE-FO-CS with other pretreatments treated CS.

S-W tests means Shapiro-Wilks test. LR means lignin removal. XR means xylan removal. Dates of both groups conformed to the hypothesis of overall normal

384


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385

Page 22 of 25

Table 4 Influence of combined pretreatment sequence on enzymatic saccharification yields1 Run

AE-FO (EE, %)

FO-AE (EE, %)

AE-FO (GY, %)

1

98.96

98.81 (+0.14)

72.07

66.02 (+6.05)

2

98.61

93.88 (+4.72)

69.34

55.13 (+14.21)

3

103.94

95.18 (+8.76)

72.94

57.00 (+15.94)

4

110.73

95.79 (+14.94)

65.44

57.68 (+7.76)

5

98.11

93.21 (+4.91)

70.24

57.78 (+12.46)

6

99.85

87.57 (+12.28)

70.42

53.82 (+16.60)

7

96.34

90.47 (+5.87)

64.75

52.67 (+12.07)

8

97.25

90.89 (+6.86)

66.07

54.42 (+11.66)

9

103.89

104.09 (-0.21)

65.83

53.27 (+12.56)

10

104.50

95.85 (+8.56)

69.86

58.20 (+11.66)

11

102.64

92.14 (+10.50)

66.17

64.89 (+1.29)

12

103.96

88.87 (+15.09)

71.78

54.90 (+16.87)

S-W tests

P > 0.05

P > 0.05

P > 0.05

P > 0.05


P > 0.05

P > 0.05

P > 0.05

P > 0.05


(t = 5.27, P = 0.00)

FO-AE (GY, %)

(t = 8.74, P = 0.00)

386

1

387

Dates of both groups conformed to the hypothesis of overall normal distribution (P > 0.05) and

388

homogeneity of variance (P > 0.05). The value of EE exceeding 100% is due to detection error.

389

Dates in brackets denoted the incremental values while comparing EE and GY of AE-FO treated

390

CS with FO-AE treated CS. Values were the means of triplicates.

S-W tests means Shapiro-Wilks test. EE means enzymolysis efficiency. GY means glucose yield.


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Energy & Fuels

Table 5 Influence of combined pretreatment sequence on pretreated CS characteristics1 AE-FO

FO-AE

(glucan/(xylan+lignin))

(glucan/(xylan+lignin))

3.06

2.38 (+0.68)

Run

AE-FO (LR, %)

FO-AE (LR, %)

AE-FO (XR, %)

FO-AE (XR, %)

1

87.07

85.56 (+1.50)

76.70

72.94 (+3.75)

2

83.80

84.20 (+0.41)

74.33

72.25 (+2.08)

2.53

2.13 (+0.40)

3

87.13

86.01 (-1.12)

78.71

74.39 (+4.32)

3.19

2.44 (+0.76)

4

81.58

81.96 (-0.38)

74.57

71.09 (+3.48)

2.37

2.09 (+0.28)

5

83.55

83.97 (-0.42)

75.08

71.19 (+3.88)

2.63

2.03 (+0.60)

6

85.51

88.00 (-2.49)

78.69

78.05 (+0.65)

3.03

2.94 (+0.09)

7

80.39

82.97(-2.58)

75.03

70.74 (+4.28)

2.27

1.97 (+0.30)

8

80.16

82.89 (-2.73)

74.37

70.37 (+4.00)

2.24

1.97 (+0.28)

9

88.20

86.08 (+2.11)

79.71

74.28 (+5.42)

3.43

2.62 (+0.81)

10

85.86

85.03 (+0.84)

76.90

73.61 (+3.29)

2.75

2.49 (+0.27)

11

85.18

86.61 (-1.43)

75.46

72.77 (+2.69)

1.97

2.26 (-0.29)

12

85.46

87.75 (-2.29)

78.84

77.17 (+1.67)

2.99

2.88 (+0.11)

S-W tests

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05


P > 0.05

P > 0.05

P > 0.05

P > 0.05

P > 0.05


P > 0.05

No significant difference



(t=-1.20, P=0.26)

(t=8.63, P=0.00)

(t=3.86, P=0.00)

392

1

393

distribution (P > 0.05) and homogeneity of variance (P > 0.05). Dates in brackets denoted the incremental values while comparing compositions of AE-FO treated

394

CS with FO-AE treated CS. Values were the means of triplicates.

S-W tests means Shapiro-Wilks test. LR means lignin removal. XR means xylan removal. Dates of both groups conformed to the hypothesis of overall normal

395


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Table 6 Contribution of synergistic pretreatment variables to glucan/(xylan+lignin)

396

397

Page 24 of 25

1

Particle size

Fenton loading 1

NaOH loading

Contribution (%, AE-FO)

12.859

24.558

62.583

Contribution (%, FO-AE)

22.036

36.759

41.205

Fenton solution was prepared using FeSO4•7H2O and H2O2 at a mole ratio of 1:25.

398 399


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400

Figure 1

401 402 403 404


A Comparison of NaOH, Fenton, and Their Combined Pretreatments

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