Enhancement and Mechanism of a Lignin Amphoteric Surfactant on

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Enhancement and mechanism of lignin amphoteric surfactant on the production of cellulosic ethanol from high solid corncob residue Hongming Lou, Xiuxiu He, Cheng Cai, Tianqing Lan, Yuxia Pang, Haifeng Zhou, and Xueqing Qiu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01208 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 18, 2019

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Journal of Agricultural and Food Chemistry

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Enhancement and mechanism of lignin amphoteric surfactant on the

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production of cellulosic ethanol from high solid corncob residue

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Hongming Lou 1, 2, Xiuxiu He 1, Cheng Cai 1, Tianqing Lan *,3, Yuxia Pang 1, Haifeng

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Zhou 4, Xueqing Qiu *,1, 2

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1

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Research Center for Green Fine Chemicals, South China University of Technology,

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Guangzhou 510641, China

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2

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Technology, Guangzhou 510641, China

School of Chemistry and Chemical Engineering, Guangdong Provincial Engineering

State Key Laboratory of Pulp and Paper Engineering, South China University of

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3

11

Kunming 650500, China

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4

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Energy and Chemical Engineering, Shandong University of Science and Technology,

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Qingdao 277590, China

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*Corresponding Author:

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Xueqing Qiu:

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Tel.: 86-20-87114722, Fax: 86-20-87114721, E-mail: [email protected]

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Tianqing Lan:

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Tel：86-13095304980, E-mail: [email protected]

Yunnan Institute of Food Safety, Kunming University of Science and Technology,

College of Chemical and Environmental Engineering, Key Laboratory of Low Carbon

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1 ACS Paragon Plus Environment


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Abstract

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Lignin amphoteric surfactant and betaine could enhance the enzymatic hydrolysis of

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lignocellulose and recover cellulase. The effects of lignosulfonate quaternary ammonium

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salt (SLQA) and dodecyl dimethyl betaine (BS12) on enzymatic hydrolysis digestibility,

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ethanol yield, yeast cell viability and other properties of high solid enzymatic hydrolysis

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and fermentation of corncob residue were studied in this research. The results suggested

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that SLQA and 1 g/L BS12 effectively improved ethanol yield through enhancing

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enzymatic hydrolysis. SLQA had no significant effect on yeast cell membrane and the

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glucose fermentation. However, 5 g/L BS12 reduced ethanol yield due to that 5 g/L BS12

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damaged yeast cell membrane and inhibited the conversion of glucose to ethanol. Our

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research also suggested that 1 g/L BS12 enhanced ethanol yield of corncob residue

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fermentation, which was attributed to that lignin in the corncob adsorbed BS12 and

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decreased its concentration in the solution to a safe level for the yeast.

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Key word：corncob residue; high solid fermentation; lignin amphoteric surfactant; cell

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membrane damage; adsorption

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1. Introduction

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Lignocellulosic ethanol is renewable and eco-friendly, and has recently become a

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hot topic in the field of bioenergy research 1-4. Corncob residue was the by-product in the

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xylan-producing industry 5, 6 and its output in China reached 230,000 tons per year 7. In

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the xylan-producing industry, hemicellulose in corncob was removed and the structure of

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cell wall was degraded after diluted acid hydrolysis, the cellulose content in the corncob

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residue is as high as 60% or more 8. Therefore, corncob residue can be used as a cheap

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and abundant raw material for the production of bioethanol 9. Production of lignocellulosic ethanol mainly includes four steps: pretreatment,

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10, 11.

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saccharification, fermentation and ethanol separation

The serious problem in the

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enzymatic hydrolysis and fermentation of lignocellulose is poor efficiency of enzymatic

48

hydrolysis, which causes expensive cost of enzymatic hydrolysis and low ethanol yield

49

12, 13.

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processes to subtract lignin, which could enhance access of enzymes to cellulose

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developing more efficient cellulase 18, adding surfactants 19-21, and so on.

There are many ways to solve this problem, including: designing new pretreatment 14-17,

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The addition of surfactants was not only effortless to operate, but also could greatly

53

reduce the dosage of cellulase 22. There had been a lot of researches about that nonionic

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surfactants strengthened the enzymatic hydrolysis of lignocellulose

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surfactants could not only lessen the nonproductive adsorption of cellulase on lignin, but

56

also improve the stability of cellulase, and make it less susceptible to inactivation

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Additionally, nonionic surfactants had no inhibition against the yeast to convert glucose


23, 24.

Nonionic

25.


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to ethanol 26. Some anionic surfactants, such as sodium lignosulfonate, a by-product of

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sulfite pulping and sulfite pretreatment of lignocelluloses, could be adsorbed on cellulase

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aggregates, reduced nonproductive adsorption of cellulase on lignin and strengthened

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enzymatic hydrolysis 27. In addition, sodium lignosulfonate had no remarkable inhibition

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on the fermentation of lignocellulose

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quaternary ammonium surfactants which carries quaternary ammonium group, were

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widely used as sterilant 29, 30 and were not applicable in the microbial fermentation.

28.

However, the cationic surfactants, especially

65

Recent studies had found lignin amphoteric surfactant, synthesized by quaternization

66

of sodium lignosulfonate, could improve the enzymatic digestibility of corncob residue

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from 38% to 90% 31, which was more efficiently than sodium lignosulfonate. Besides, it

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could recover more than 50% of cellulase through adjusting pH after enzymatic

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hydrolysis 32, which was even superior to non-ionic surfactants such as Tween or Triton

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as they could not be used to recycle cellulase. In addition, the zwitterionic surfactant 3-

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sulfopropylhexadecyldimethylbetaine (SB3-16) could also increase the enzymatic

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digestibility of eucalyptus through dilute acid pretreatment from 28% to 73% and recover

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55% of protein content of the cellulase through cooling

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amphoteric surfactant and betaines are surfactant with quaternary ammonium groups that

75

which has widely been contained in the molecule of antiseptic 30, 34, 35.

33.

However, both the lignin

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Therefore, it is essential to study the effects of lignin amphoteric surfactant and

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betaines on the high solid enzymatic hydrolysis and fermentation of lignocellulose. In this

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study, the effects and mechanism of lignosulfonate quaternary ammonium salt (SLQA)


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and dodecyl dimethyl betaine (BS12, a classic and commonly used amphoteric surfactant)

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on the semi-simultaneous high solid enzymatic hydrolysis and fermentation of corncob

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residue were studied in order to provide basic data and guidance for the application of

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amphoteric surfactants in high solid enzymatic hydrolysis and fermentation of

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lignocellulose. By comparing the different effects of SLQA and BS12 and discussing the

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contributing factors, their mechanism on the production of cellulosic ethanol from high

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solid lignocellulose was proposed.

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2. Materials and Methods

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2.1 Materials

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Corncob residue used in this study was the by-product in the xylan-producing

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industry (Jinan Shengquan Group Share Holding Co., Ltd.). Its main components were

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79 wt.% cellulose, 9 wt.% acid-insoluble lignin, 1 wt.% acid-soluble lignin and 1 wt.%

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xylan. Commercial cellulase CTec2 was acquired from Novozyme China (Shanghai,

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China). Its protein content was 73.6 mg/mL, and cellulase activity was 147 FPU/mL 36.

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(3-Chloro-2-hydroxypropyl) trimethylammonium chloride (CHPTAC), 65 wt.% in

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H2O, was acquired from Shanghai Macklin Biochemical Co., Ltd (Lot#: C10035415).

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Sulfonated lignin (SL) was produced by Tongdao Shenhua Linhua Co., Ltd.

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Lignosulfonate quaternary ammonium salt (SLQA) was synthesized through the reaction

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of SL and CHPTAC 31, and CHPTAC was 30 wt.% of SL. The isoelectric point (pI) of

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SLQA which was tested by Zeta PALS instrument (Brookhaven Instruments Co.,

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America) was 2.0. Dodecyl dimethyl betaine (BS12) was purchased from Qingdao



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Yousuo Chemical Technology Co., Ltd and its pI was 6.6 determined by potentiometric

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titration.

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Lignin was produced by Shandong Longli Bio-Technology Co., Ltd. It was purified

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from the residue of enzymatic hydrolysis of corncob. The residue was disposed by

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alkaline dissolving and acid precipitating. The content of acid-insoluble lignin was 85

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wt.%. Methylene blue solution contained 0.6 g methylene blue (Shanghai Macklin

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Biochemical Co., Ltd), 30 mL 95% ethanol, 0.01 g KOH, 100 mL distilled water.

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Propidium iodide was acquired from Shanghai Yuanye Biotechnology Co., Ltd.

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2.2 High solid enzymatic hydrolysis of corncob residue

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In order to study the mechanisms of amphoteric surfactant on semi-simultaneous

110

enzymatic hydrolysis and fermentation of lignocellulose, the corncob residue was

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hydrolyzed at 50℃ for 12 h and at 35℃ for 60 h. This condition of enzymatic hydrolysis

112

was the same with fermentation experiment. 2% and 15% (w/v) substrate suspension of

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corncob residue were prepared in an Erlenmeyer flask (pH 4.8, 50 mM acetate buffer),

114

then sterilized at 115°C for 20 min. Amphoteric surfactants with different concentrations

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and CTec2 with different enzyme loading were added in the substrate suspensions, and

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then the substrate suspensions were hydrolyzed firstly at 50°C and 150 rpm for 12 h and

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subsequently 35°C and 130 rpm for 60 h. 1 mL of enzymatic hydrolysate was taken at

118

intervals, centrifuged for 10 min at 10,000 rpm (Sigma, model 1-14). And then the

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glucose content in the enzymatic hydrolysate was measured using Biosensor analyzer

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SBA-40E (Bioscience Research Institute of Shandong Academy of Sciences). In all


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enzymatic hydrolysis experiments, two replicates were done and the average values of

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data were given. The substrate enzymatic digestibility (SED) was calculated as follows:

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𝐺𝑙𝑢𝑐𝑜𝑠𝑒 (𝑔/𝐿）

2-1

SED = 𝐶𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 （𝑔/𝐿） × 1.11 × 100%

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2.3 Fermentation of different substrates

125

2.3.1 Inoculum preparation

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The dry yeasts (Hubei Angel Yeast Co., Ltd.) were incubated at 35°C for 12 h under

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150 rpm in the culture medium containing of: glucose, 20 g/L; yeast extract, 10 g/L;

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peptone, 20 g/L. After incubation, the yeasts were centrifuged and subsequently used for

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fermentation as inoculum.

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2.3.2 Semi-simultaneous enzymatic hydrolysis and fermentation of corncob residue

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2% and 15% (w/v) substrate suspensions of corncob residue were prepared in an

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Erlenmeyer flask (pH 4.8, 50 mM acetate buffer). After adding 1 g/L yeast extract and 1

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g/L ammonium chloride, the substrate suspension was sterilized at 115°C for 20 min.

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Amphoteric surfactants with different concentrations and CTec2 with different enzyme

135

loading were added in the substrate suspensions. The substrate suspensions were

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hydrolyzed in a shaker (50°C, 150 rpm, 12 h). After 12 h of enzymatic hydrolysis, 10%

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(v/v) of yeast inoculum was added in the substrate suspensions and then the substrate

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suspensions were cultured in a shaker (35°C, 130 rpm, 60 h). 1 mL of fermentation broth

139

was taken at intervals, then centrifuged for 10 min at 10,000 rpm. The biosensor analyzer

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SBA-40E was used to measure the ethanol content in the fermentation broth. In all

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enzymatic hydrolysis and fermentation experiments, two replicates were done and the



142 143 144

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average values of data were given. The ethanol yield was calculated as follows: 𝐸𝑡ℎ𝑎𝑛𝑜𝑙 (𝑔/𝐿)

Ethanol yield = 𝐶𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 (𝑔/𝐿) × 1.11 × 0.51 × 100%

2-2

2.3.3 Glucose fermentation

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10% (w/v) substrate solution of glucose was prepared in an Erlenmeyer flask (pH

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4.8, 50 mM acetate buffer). After adding 1 g/L yeast extract and 1 g/L ammonium

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chloride, the substrate solution was sterilized for 20 min at 115°C. Amphoteric surfactants

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with different concentrations and 10% (v/v) of yeast inoculum were put into the substrate

149

solution. The substrate solution was incubated in a shaker (35°C, 130 rpm, 72 h). At

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intervals 1 mL fermentation broth was taken, then centrifuged for 10 min at 10,000 rpm.

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The biosensor analyzer SBA-40E was used to measure the ethanol content in the

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fermentation broth. The ethanol yield was calculated as follows:

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𝐸𝑡ℎ𝑎𝑛𝑜𝑙 (𝑔/𝐿)

Ethanol yield = 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 (𝑔/𝐿) × 0.51 × 100%

154

2.3.4 Semi-simultaneous high solid enzymatic hydrolysis and fermentation of

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microcrystalline cellulose

2-3

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15% (w/v) substrate suspension of microcrystalline cellulose was prepared in an

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Erlenmeyer flask (pH 4.8, 50 mM acetate buffer). After adding 1 g/L yeast extract and 1

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g/L ammonium chloride, the substrate suspension was sterilized at 115°C for 20 min.

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Amphoteric surfactants with different concentrations and CTec2 (9 FPU/g glucan) were

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put into the substrate suspension. The substrate suspension was hydrolyzed in a shaker

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(50°C, 150 rpm, 12 h). After 12 h of enzymatic hydrolysis, 10% (v/v) yeast inoculum was

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inoculated in the substrate suspension and the substrate suspension was cultured in a


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shaker (35°C, 130 rpm, 60 h). At intervals 1 mL fermentation broth was taken, then

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centrifuged for 10 min at 10,000 rpm. The biosensor analyzer SBA-40E was used to

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measure the glucose and ethanol contents in the fermentation broth. The ethanol yield

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was calculated according to the formula 2-2.

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2.4. The cytotoxicity of amphoteric surfactants

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2.4.1 Yeast cell viability

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5 g/L amphoteric surfactants solutions were prepared in acetate buffer (pH 4.8, 50

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mM). 20% (v/v) of yeast inoculum was inoculated in the amphoteric surfactant solutions

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and the amphoteric surfactant solutions were incubated in a shaker (35°C, 130 rpm, 72

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h). After 72 h, 5 mL of the amphoteric surfactant solutions were taken. The yeasts in the

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amphoteric surfactant solutions were centrifuged and separated (4500 rpm, 5 min), then

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washed twice by acetate buffer (pH 4.8, 50 mM) and resuspended in this kind of buffer

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to get a yeast suspension. The yeast suspension was dropped on a glass slide and dyed

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with 6 μl methylene blue solution for 3 min. The dyed sample was observed with

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polarizing microscope (XPV-800E, Shanghai Bimu Instrument Co., Ltd, China) by

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magnifying 40 times.

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2.4.2 Yeast cell membrane damage

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The yeast resuspension was firstly prepared according to the experimental procedure

181

in Section 2.4.1, and added 100 μL of 1 g/L propidium iodide solution. After this mixture

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was incubated in the dark (35°C, 10 min), the yeasts in the solutions were separated (4500

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rpm, 5 min) and washed twice by acetate buffer (pH 4.8, 50 mM) to remove excess



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propidium iodide, then resuspended in acetate buffer (pH 4.8, 50 mM) to get the yeast

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suspension. The fluorescence intensity of the yeast suspension was measured using a

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fluorescence spectrophotometer (F7000, HITACHI, Japan) with 485 nm excitation light

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and 10 nm slit width.

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2.5 Determination of the adsorption of cellulase on lignin through SDS-PAGE

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Amphoteric surfactants solutions with different concentrations were prepared in 2

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mL acetate buffer (pH 4.8, 50 mM). The amphoteric surfactants solutions were incubated

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in a shaker (50°C, 150 rpm, 2 h) after adding 0.2 g lignin and 3 FPU CTec2, and then

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were separated (10,000 rpm, 5 min). The supernatant was used for the SDS-PAGE

193

experiment and the detailed experimental steps were described in the literature 37.

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2.6 Quartz Crystal Microbalance with Dissipation test

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Gold-coated QCM-D (Quartz Crystal Microbalance with Dissipation) crystals

196

sensor (thickness, 0.1 mm; fundamental resonance frequency, 5 MHz; sensitivity constant,

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0.177 mg∙m-2∙Hz-1) was acquired from Q-Sense AB (Sweden). Uniform lignin films were

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prepared by spin coating 38. Acetate buffer (pH 4.8, 50 mM) was added into the QCM-D

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flow module by a peristaltic pump (0.25 mL/min) to achieve an equilibrium state at 25°C.

200

After the base line was stable, 0.02 g/L of amphoteric surfactant solutions were pumped

201

into QCM-D flow module to measure the adsorption of amphoteric surfactants on the

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lignin films.

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3. Results and discussion

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3.1 Effects of amphoteric surfactants on enzymatic hydrolysis of corncob residue


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Fig. 1 shows the effects of two amphoteric surfactants on SED of corncob residue.

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It can be seen that SLQA and BS12 could enhance the enzymatic hydrolysis efficiency

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of corncob residue. Under the condition of low solid content and low enzyme loading

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(Fig. 1a), enzymatic hydrolysis of corncob residue was enhanced more by BS12 than by

209

SLQA. In addition, a higher concentration of SLQA (5 g/L) was more effective for

210

enzymatic hydrolysis than a lower concentration (1 g/L). A lower concentration (1 g/L)

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of BS12 was enough to obtain excellent hydrolysis-enhancing efficiency and increasing

212

its concentration to 5 g/L could hardly further enhance the SED of corncob residue.

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Under the condition of 15% solid content and 5 FPU/g glucan CTec2, adding 1 g/L

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or 5 g/L BS12 could not effectively enhance enzymatic hydrolysis (Fig. 1b). In contrast,

215

adding 5 g/L SLQA could effectively enhance enzymatic hydrolysis of corncob residue.

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However, the highest SED at 72 h was only about 40%. Considering that in the condition

217

of high solids, the enzymatic hydrolysis was difficult because of the high mass transfer

218

resistance and the ineffective adsorption of cellulase on lignin, a higher enzyme loading

219

of 9 FPU/g glucan was used to further investigate the effect of amphoteric surfactants on

220

SED of corncob residue.

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In Fig. 1c, we can see that under the condition of 15% solid content and 9 FPU/g

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glucan CTec2, both SLQA and BS12 could significantly enhance the enzymatic

223

hydrolysis of corncob residue and the high-dose of surfactants (5 g/L) were more

224

beneficial to enzymatic hydrolysis compared with the low-dose surfactants (1 g/L).

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In order to investigate the reason why SLQA and BS12 could enhance the SED of



226

corncob residue, the effects of SLQA and BS12 on the adsorption of cellulase on lignin

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were investigated by using SDS-PAGE method.

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CTec2 were composed of a variety of cellulase components. According to the

229

previous literature

230

molecular weight of each cellulase components (Fig. 2). Adding SLQA or BS12 could

231

decrease the adsorption of cellulase on lignin to some extent, and a higher concentration

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of amphoteric surfactant could significantly lessen the nonproductive adsorption of

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cellulase on lignin. Amphoteric surfactants decreased the adsorption of various cellulase

234

components on lignin such as CBH I, CBH II, EGI, EG II, EG III and EG IV, which

235

indicated that SLQA and BS12 could strengthen the enzymatic hydrolysis through

236

decreasing the nonproductive adsorption of cellulase on lignin.

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3.2 Effects of amphoteric surfactants on semi-simultaneous enzymatic hydrolysis

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and fermentation of corncob residue

37, 39

, the bands in the strip were allocated in accordance with the

239

Fig. 3 demonstrates the ethanol yield in the process of enzymatic hydrolysis and

240

fermentation of corncob residue adding SLQA or BS12. The effects of two amphoteric

241

surfactants were different under different conditions of enzymatic hydrolysis and

242

fermentation.

243

From Fig. 3a, we can find that when the solid content and the enzyme loading were

244

respectively 2% and 5 FPU/g glucan, adding SLQA or BS12 could increase the ethanol

245

yield compared with the control group and BS12 was more effective than SLQA. The

246

concentration of SLQA had little influence on its improving effect of ethanol yield, and


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1 g/L or 5 g/L SLQA had the similar enhancement. As for BS12, the concentration of 1

248

g/L had obvious promoting effect for the ethanol yield, but the concentration of 5 g/L

249

decreased the improvement effect and it even had an inhibition in the early enzymatic

250

hydrolysis and fermentation period.

251

In Fig. 3b, it can be shown that at high solid content and low enzyme loading (15%

252

solid content, 5 FPU/g glucan), SLQA had a more effective enhancement for enzymatic

253

hydrolysis and fermentation of corncob residue than BS12 and the enhancement increased

254

with the increasing of the SLQA dosage. This might be due to the effective enhancement

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of SLQA on the high solid content enzymatic hydrolysis of corncob residue (Fig. 1b). For

256

BS12, the low-dose of 1 g/L had a limited enhancement on enzymatic hydrolysis and

257

fermentation, but the high-dose of 5 g/L had an inhibition in the early enzymatic

258

hydrolysis and fermentation period and an enhancement at the end of enzymatic

259

hydrolysis and fermentation.

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Under high solid content and high enzyme loading (Fig. 3c), the ethanol yield was

261

improved compared with the control group when SLQA was added. However, when the

262

concentration of SLQA increased, the ethanol yield was not further improved. The

263

ethanol yield decreased at the end of enzymatic hydrolysis and fermentation, which was

264

consistent with other researches 40, 41. This was probably because yeasts converted ethanol

265

to other organic acids in the late stage of fermentation 42. 1 g/L BS12 had an effective

266

enhancement on enzymatic hydrolysis and fermentation of corncob residue, while 5 g/L

267

BS12 had an inhibition on enzymatic hydrolysis and fermentation.



268

3.3 Mechanism of the effects of amphoteric surfactants on semi-simultaneous high

269

solid enzymatic hydrolysis and fermentation of lignocellulose

270

Both SLQA and BS12 enhanced the enzymatic hydrolysis of corncob residue, while

271

their effects on the high solid enzymatic hydrolysis and fermentation of corncob residue

272

were different. The effects of these two surfactants on the conversion of glucose to ethanol

273

might be different. Therefore, it is necessary to study the effects of SLQA and BS12 on

274

the fermentation of glucose and microcrystalline cellulose to explore their mechanism on

275

the fermentation of high solid lignocellulose.

276

3.3.1 Effects and mechanism of amphoteric surfactants on glucose fermentation

277

Fig. 4 shows the ethanol yields in the process of glucose fermentation with different

278

concentrations of amphoteric surfactants. The results suggested that SLQA had no

279

significant effect on the ethanol yield, while BS12 strongly inhibited glucose fermentation.

280

The ethanol yield also decreased during the final fermentation period, which was similar

281

with the phenomenon shown in Fig. 3c and in the previous literatures 40, 42. As mentioned

282

above, this was probably because yeasts converted ethanol to organic acids in the final

283

stage of fermentation 42.

284

In addition, the effects of SLQA and BS12 on the yeast cell viability and cell

285

membrane damage were also studied to explore the mechanism of the effects of

286

amphoteric surfactants on glucose fermentation.

287

Fig. 5 shows the colors of the yeast cells observed under the microscope. In this

288

experiment, dead cells were usually stained blue by methylene blue, while live cells could


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43.

289

not be stained

The viability of yeast cells treated with SLQA was not significantly

290

different from the control group, while the viability of the cells treated with BS12

291

significantly decreased.

292

Propidium Iodide (PI) is a DNA staining reagent which was commonly used for

293

apoptosis detection and could emit red fluorescence after it embedded in double-stranded

294

DNA 44. Although PI could not get across live cell membranes, but it could get across

295

damaged cell membranes and then stained the nucleus 45. From Fig. 6, it could be seen

296

that cells treated with SLQA and BS12 had different affinity with PI. Cells treated with

297

SLQA showed no significant difference from the control group, while cells treated with

298

BS12 had a strong affinity with PI. This indicated that SLQA had little damage to the cell

299

membrane, while BS12 had obvious damage on the cell membrane.

300

Quaternary ammonium surfactants are widely used in the field of sterilization

301

because of their toxicity to microbial cells 46. In this study, SLQA containing quaternary

302

ammonium group had no significant effect on the glucose fermentation. This might be

303

due to that SLQA with an isoelectric point (pI) of 2.0 (Fig. S1) showed negatively charged

304

in acetate buffer (pH 4.8), and caused strong electrostatic repulsion between the yeasts

305

with negative charges and SLQA molecules, which could prevent the adsorption of SLQA

306

on yeasts. Besides, the three-dimensional structure of SLQA made it difficult to penetrate

307

and damage the cell membrane (Fig. 7). In contrast, BS12 had a higher isoelectric point

308

of 6.6 than SLQA (Fig. S1 and Fig. S2), which meant that BS12 had fewer negative

309

charges and more positive charges during fermentation than SLQA. The cationic



310

quaternary ammonium group of BS12 might promote the adsorption of BS12 on yeasts

311

by the electrostatic interaction. Meanwhile its small molecule and linear hydrophobic

312

alkyl chain made it easier to penetrate and damage the cell membrane, which thus

313

decreased the cell activity and inhibited the conversion of glucose to ethanol. This result

314

was accord with the earlier reports 47, 48.

315

3.3.2 Effects of lignin and amphoteric surfactants on semi-simultaneous high solid

316

enzymatic hydrolysis and fermentation of microcrystalline cellulose

317

For further revealing the mechanism of SLQA and BS12 effects on the high solid

318

enzymatic hydrolysis and fermentation of corncob residue, we also studied the effects of

319

amphoteric surfactants and lignin on semi-simultaneous high solid enzymatic hydrolysis

320

and fermentation of microcrystalline cellulose. It should be noticed that in the cases

321

without lignin, SLQA or BS12 could not enhance the enzymatic hydrolysis because they

322

could not increase the SED of microcrystalline cellulose through decreasing the

323

nonproductive adsorption of cellulase to lignin.

324

Fig. 8 illustrates the changes of ethanol yield with the concentration of SLQA and

325

BS12. In Fig. 8a, SLQA had no remarkable effect on ethanol yield of enzymatic

326

hydrolysis and fermentation of microcrystalline cellulose, and the concentration of

327

residual sugar in the solution was not higher than 1 g/L in the final stage of enzymatic

328

hydrolysis and fermentation, which meant that it was not affected by SLQA.

329

In Fig. 8b, BS12 decreased the ethanol yield to below 5%, and residual sugar in the

330

solution increased to more than 60 g/L. It meant that the yeasts could hardly ferment


Page 16 of 40

Page 17 of 40


331

glucose to ethanol after adding BS12. As mentioned above, BS12 in the solution would

332

damage the yeast cell membrane and inhibit the conversion of glucose to ethanol, which

333

decreased the ethanol yield and increased the concentration of residual sugar of enzymatic

334

hydrolysis and fermentation of microcrystalline cellulose.

335

The effect of lignin component of lignocellulose on the fermentation-inhibition

336

caused by BS12 was also studied through adding lignin to the system of enzymatic

337

hydrolysis and fermentation of microcrystalline cellulose. Fig. 8c demonstrates the

338

ethanol yields and the glucose contents in the fermentation broth. After adding 2.2% (w/v)

339

lignin, the inhibition of 1 g/L BS12 on enzymatic hydrolysis and fermentation of

340

microcrystalline cellulose was weakened. The ethanol yield was up to 30% at 60 h. The

341

yeasts could convert some of glucose to ethanol, but the content of residual glucose in the

342

broth was still much higher than that in the control group. When the concentration of

343

BS12 was 5 g/L or even higher, adding 2.2% (w/v) lignin could not effectively weaken

344

the inhibition of BS12 to the yeasts. Therefore, BS12 had a strong inhibition on the

345

conversion of glucose to ethanol and lignin could reduce this inhibitory effect to a certain

346

extent, which might be related to the adsorption of BS12 on lignin.

347

3.3.3 Mechanism of the effects of amphoteric surfactants on semi-simultaneous

348

high solid enzymatic hydrolysis and fermentation of lignocellulose

349

In order to discover the reason that lignin reduced the inhibitory effect of 1 g/L BS12

350

on the yeasts, the adsorption of amphoteric surfactants on lignin was determined through

351

QCM-D technology. In Fig. 9, - Δ F3 stands for the adsorption capacity of amphoteric



352

surfactants on lignin films. The -ΔF3 value of SLQA on lignin film was 15 Hz and that of

353

BS12 on lignin film reached 52 Hz, which suggested that more BS12 was adsorbed on

354

lignin than SLQA. This could be due to the different structures of these two kind

355

amphoteric surfactants. SLQA, which had three-dimensional structure and many

356

hydrophilic groups, was mainly adsorbed on lignin by hydrophobic interaction, so its

357

adsorption on lignin film would be less. Different from SLQA, BS12 with hydrophobic

358

tail chain and hydrophilic head was adsorbed more easily on lignin in the form of micelles.

359

According to all the results in this study, we put forward a hypothesis in the

360

enzymatic hydrolysis and fermentation of lignocellulose (Fig. 10). SLQA with more

361

negative charges (pI=2.0) and the three-dimensional structure in acetate buffer (pH 4.8)

362

had little undesirable effect on yeasts. Besides, SLQA could be adsorbed on lignin by

363

hydrophobic interaction and lessen the nonproductive adsorption of cellulase on lignin,

364

which strengthened the enzymatic hydrolysis, and subsequently improved the ethanol

365

yield.

366

On the contrary, BS12 with lower molecular weight had a different effect on

367

hydrolysis and fermentation from SLQA. Fig. 10 also demonstrates the mechanisms of

368

the low and high dosage BS12 of on hydrolysis and fermentation of lignocellulose

369

respectively. BS12 was absorbed on lignin component of lignocelluloses through the

370

electrostatic interaction between the cationic quaternary ammonium groups of the BS12

371

and the carboxyl group of lignin, and through the hydrophobic interaction between

372

hydrophobic chain of BS12 and hydrophobic groups of lignin, which made the lignin


Page 18 of 40

Page 19 of 40


373

surface more hydrophilic. Therefore, the nonproductive adsorption of cellulase on lignin

374

would be lessened and the enzymatic hydrolysis was strengthened. At the low-dose of 1

375

g/L, most BS12 was adsorbed on lignin and the solution concentration of free BS12 was

376

low, which weakened the inhibitory effect of BS12 on the yeast cell viability in the

377

process of fermentation and enhanced the ethanol yield of enzymatic hydrolysis and

378

fermentation of lignocellulose. However, at the high dosage such as 5 g/L, the lignin in

379

lignocellulose could only adsorb part of BS12 and most BS12 was still free in

380

fermentation broth. The free BS12 could be adsorbed on yeasts and caused the yeast cell

381

membrane damage, and further inhibited the enzymatic hydrolysis and fermentation of

382

lignocellulose.

383

In conclusion, SLQA which had no significant effect on yeast cell viability could

384

enhance ethanol yield through enhancing enzymatic hydrolysis. BS12 could improve

385

enzymatic hydrolysis efficiency, and at low-dose of 1 g/L it could improve ethanol yield

386

because most BS12 was adsorbed on lignin. However, at the high dosage such as 5 g/L,

387

BS12 would damage cell membrane and decrease the ethanol yield. This study illustrates

388

that though their effects are different, both lignin amphoteric surfactants such as SLQA

389

and betaines such as BS12 can promote the enzymatic hydrolysis and fermentation of

390

lignocelluloses by choosing appropriate dosage. Researches on developing lignin-based

391

enzymatic auxiliaries like SLQA could not only achieve the comprehensive utilization of

392

lignin, but also be conducive to the development of new biorefinery processes.

393

Corresponding authors



394

E-mail: [email protected] (X. Q. Qiu)

395

E-mail: [email protected] (T. Q. Lan)

396

Abbreviations

397

SLQA, lignosulfonate quaternary ammonium salt; BS12, dodecyl dimethyl betaine; SL,

398

sulfonated lignin; CHPTAC, (3-Chloro-2-hydroxypropyl) trimethylammonium chloride;

399

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; QCM-D,

400

quartz Crystal Microbalance with dissipation monitoring; β-GL, β-glucosidase; CBH

401

I, cellobiohydrolase I; CBH II, cellobiohydrolase II; EG II, endoglucanase II; EG IV,

402

endoglucanase IV; EG III, endoglucanase III; EG V, endoglucanase V; Xyn, xylanase.

403

Notes

404

The authors declare no competing financial interest.

405

Funding

406

The authors acknowledge the financial supports of the National Natural Science

407

Foundation of China (21676109, 21878112), Science and Technology Program of

408

Guangzhou (201707020025), Guangdong Special Support Plan (2016TX03Z298) and

409

Science and Technology Program of Guangdong (2017B090903003).

410

Supporting Information description

411

The test methods and data of isoelectric points of SLQA and BS12.

412


Page 20 of 40

Page 21 of 40

413


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414

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23. Kadhum, H. J.; Rajendran, K.; Murthy, G. S., Optimization of Surfactant Addition in

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derivatives to accelerate simultaneous saccharification and fermentation of unbleached

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27. Lin, X. L.; Cai, C.; Huang, J. H.; Lou, H. M.; Qiu, X. Q.; Lin, M. L.; Zheng, J. Y.;

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Appl. Mater. Interfaces 2016, 8, 4242-4249.

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30. Inacio, A. S.; Domingues, N. S.; Nunes, A.; Martins, P. T.; Moreno, M. J.; Estronca,

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towards bacteria: mechanisms of action and clinical implications in antibacterial

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X. Q., Using recyclable pH-responsive lignin amphoteric surfactant to enhance the

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enzymatic hydrolysis of lignocelluloses. Green Chem. 2017, 19, 5479-5487.

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Interaction. ACS Sustain. Chem. Eng. 2018, 6, 10679-10686.



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33. Cai, C.; Pang, Y. X.; Zhan, X. J.; Zeng, M. J.; Lou, H. M.; Qian, Y.; Yang, D. J.; Qiu,

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hydrolysis of lignocelluloses and recover cellulase by cooling. Bioresour. Technol. 2017,

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34. Peddie, B. A.; Wood, J. E.; Lever, M.; Happer, D. A. R.; de Zwart, F.; Chambers, S.

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Effect of Urea on the Enzymatic Hydrolysis of Lignocellulosic Substrate and Its

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Mechanism. BioEnergy Res. 2018, 11, 456-465.

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38. Cai, C.; Qiu, X. Q.; Lin, X. L.; Lou, H. M.; Pang, Y. X.; Yang, D. J.; Chen, S. W.;

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39. Ko, J. K.; Ximenes, E.; Kim, Y.; Ladisch, M. R., Adsorption of Enzyme Onto Lignins

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of Liquid Hot Water Pretreated Hardwoods. Biotechnol. Bioeng. 2015, 112, 447-456.


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40. Iram, M.; Asghar, U.; Irfan, M.; Huma, Z.; Jamil, S.; Nadeem, M.; Syed, Q.,

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Production of bioethanol from sugarcane bagasse using yeast strains: A kinetic study.

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Energy Sources Part A-Recovery Util. Environ. 2018, 40, 364-372.

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41. Arapoglou, D.; Varzakas, T.; Vlyssides, A.; Israilides, C., Ethanol production from

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potato peel waste (PPW). Waste Manage. 2010, 30, 1898-1902.

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42. Shen, J. C.; Agblevor, F. A., Ethanol production of semi-simultaneous

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43. Vairo, M. L., Methylene blue solutions for staining dead yeast cells. Stain Technol.

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Antibacterial Activity of Quaternary Ammonium Salt-Type Antibacterial Agents with a

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Phosphate Group. J. Oleo Sci. 2008, 57, 445-452.

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47. Vieira, D. B.; Carmona-Ribeiro, A. M., Cationic lipids and surfactants as antifungal

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48. Wessels, S.; Ingmer, H., Modes of action of three disinfectant active substances: A

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554



555

Figure captions

556

Fig. 1. Effect of amphoteric surfactants on SED of corncob residue under the conditions of

557

different solid content and enzyme loading. (a) solid content: 2%, CTec2: 5 FPU/g glucan;

558

(b) solid content: 15%, CTec2: 5 FPU/g glucan; (c) solid content: 15%, CTec2: 9 FPU/g

559

glucan.

560

Fig. 2. Effects of amphoteric surfactants on the adsorption of cellulase on lignin. CTec2:

561

1.5 FPU/mL cellulase solution before adsorption. Control: the adsorption without additives.

562

SLQA group and BS12 group: the adsorptions with different concentrations of amphoteric

563

surfactants.

564

Fig. 3. Effects of amphoteric surfactants on ethanol yield of semi-simultaneous enzymatic

565

hydrolysis and fermentation of corncob residue under the conditions of different solid

566

content and enzyme loading. (a) solid content: 2%, CTec2: 5 FPU/g glucan; (b) solid

567

content: 15%, CTec2: 5 FPU/g glucan; (c) solid content: 15%, CTec2: 9 FPU/g glucan.

568

Fig. 4. Effects of amphoteric surfactants on ethanol yield of glucose fermentation.

569

Fig. 5. Effects of amphoteric surfactants on yeast cell viability.

570

Fig. 6. Effects of amphoteric surfactants on the cell membrane damage.

571

Fig. 7. Mechanism of the effects of amphoteric surfactants on glucose fermentation.

572

Fig. 8. Effects of amphoteric surfactants on semi-simultaneous high solid enzymatic

573

hydrolysis and fermentation of microcrystalline cellulose. (a) SLQA; (b) BS12; (c) BS12

574

with 2.2% (w/v) lignin.

575

Fig. 9. Adsorption of amphoteric surfactants on lignin film.


Page 28 of 40

Page 29 of 40


576

Fig. 10. Mechanism of the effects of amphoteric surfactants on semi-simultaneous high

577

solid enzymatic hydrolysis and fermentation of lignocellulose.

578



(a)

80 control 1 g/L SLQA 5 g/L SLQA 1 g/L BS12 5 g/L BS12

SED (%)

70 60 50 40

(b)

Solid content: 2% CTec2: 5 FPU/g glucan

30 60

50

SED (%)

Page 30 of 40


40

30

20

SED (%)

(c)

80 70 60


50 40

579

10

20

30

40

50

60

70

80

Enzymatic hydrolysis time (h)

580

Fig.1. Effect of amphoteric surfactants on SED of corncob residue under the conditions

581

of different solid content and enzyme loading. (a) solid content: 2%, CTec2: 5 FPU/g

582

glucan; (b) solid content: 15%, CTec2: 5 FPU/g glucan; (c) solid content: 15%, CTec2: 9

583

FPU/g glucan.

584


Page 31 of 40


585 586

Fig. 2. Effects of amphoteric surfactants on the adsorption of cellulase on lignin. CTec2:

587

1.5 FPU/mL cellulase solution before adsorption. Control: the adsorption without

588

additives. SLQA group and BS12 group: the adsorptions with different concentrations of

589

amphoteric surfactants.

590



Ethanol yield (%)

(a)

80 70 60

control 1 g/L BS12 5 g/L BS12 1 g/L SLQA 5 g/L SLQA

50 40 Solid content: 2% CTec2: 5 FPU/g glucan

30

Ethanol yield (%)

(b)

Ethanol yield (%)

(c)

40 30 20 10 0 80


60

40

20

20

591


30

40

50

60

70

Enzymatic hydrolysis and fermentation time (h)

592

Fig. 3. Effects of amphoteric surfactants on ethanol yield of semi-simultaneous enzymatic

593

hydrolysis and fermentation of corncob residue under the conditions of different solid

594

content and enzyme loading. (a) solid content: 2%, CTec2: 5 FPU/g glucan; (b) solid

595

content: 15%, CTec2: 5 FPU/g glucan; (c) solid content: 15%, CTec2: 9 FPU/g glucan.

596


Page 32 of 40

Page 33 of 40


Ethanol yiled (%)

80

60 control 1g/L SLQA 5g/L SLQA 1g/L BS12 5g/L BS12

40

20

0 10

597 598

20

30

40

50

Fermentation time (h) Fig. 4. Effects of amphoteric surfactants on ethanol yield of glucose fermentation.

599



600 601

Fig. 5. Effects of amphoteric surfactants on yeast cell viability.

602


Page 34 of 40

Page 35 of 40


Fluorescence intensity

30

20 15 10 5 0

603 604

control SLQA BS12

25

560

580

600

620

640

Wavelength(nm)

Fig. 6. Effects of amphoteric surfactants on the cell membrane damage.

605



606 607

Fig. 7. Mechanism of the effects of amphoteric surfactants on glucose fermentation.

608


Page 36 of 40

Page 37 of 40


609 610

Fig. 8. Effects of amphoteric surfactants on semi-simultaneous high solid enzymatic

611

hydrolysis and fermentation of microcrystalline cellulose. (a) SLQA; (b) BS12; (c) BS12

612

with 2.2% (w/v) lignin.

613



0

ΔF3(Hz)

-10 -20 0.02 g/L SLQA 0.02 g/L BS12

-30 -40 -50 -60

614 615

0

500

1000

1500

2000

Time (s)

Fig. 9. Adsorption of amphoteric surfactants on lignin film.

616


Page 38 of 40

Page 39 of 40


617 618

Fig. 10. Mechanism of the effects of amphoteric surfactants on semi-simultaneous high

619

solid enzymatic hydrolysis and fermentation of lignocellulose.

620



621

Graphic for table of contents

622 623

Mechanism of the effects of amphoteric surfactants on enzymatic hydrolysis and fermentation of

624

lignocellulose.


Page 40 of 40

Enhancement and Mechanism of a Lignin Amphoteric Surfactant on

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