Enhancement of Recyclable pH-Responsive Lignin-Grafted

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Article

Enhancement of Recyclable pH-Responsive Lignin Grafted Phosphobetaine on Enzymatic Hydrolysis of Lignocelluloses Feiyun Li, Cheng Cai, Hongming Lou, Yuxia Pang, Xinyi Liu, and Xueqing Qiu ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 30, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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

ACS Sustainable Chemistry & Engineering

Enhancement of Recyclable pH-Responsive Lignin Grafted Phosphobetaine on Enzymatic Hydrolysis of Lignocelluloses Feiyun Li†, Cheng Cai †, Hongming Lou*, †, Yuxia Pang†, Xinyi Liu † and Xueqing Qiu*,†, ‡ †

School of Chemistry and Chemical Engineering, Guangdong Provincial Engineering

Research Center for Green Fine Chemicals, South China University of Technology, Guangzhou, 510641, PR China ‡

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

Technology, Guangzhou, 510641, PR China *Corresponding author. Tel.: 86-20-87114722; E-mail: [email protected] (H.M. Lou); [email protected] (X.Q. Qiu)

1

ACS Paragon Plus Environment

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

ABSTRACT: Enzymatic conversion of lignocelluloses into fermentable sugar is a key step in the production of cellulosic ethanol. In this work, enzymatic hydrolysis lignin (EHL) grafted phosphobetaine (EHLPB) was prepared, and the phosphobetaine intermediate, 3-chloro-2-hydroxypropyl(2-(trimethylammonio)ethyl)phosphate, was synthesized from phosphocholine chloride calcium salt and epichlorohydrin. EHLPB showed a pH-sensitive response. When pH ≥ 5.0, it was completely dissolved in the buffer solution, whereas 95.5% of EHLPPB was precipitated when pH ≤ 3.0. Adding 1.2 wt% EHLPB-210 can increase the high-solid enzymatic digestibility of Eu-SPORL (sulfite pretreatment to overcome recalcitrance of eucalyptus) and CCR (corncob residue) from 33.6% and 52.6% to 71.5% and 73.6%, respectively. After the enzymatic hydrolysis of Eu-SPORL, 95% of EHLPB-210 was recovered by adjusting the slurry pH from 5 to 3. The recovered EHLPB-210 still kept the ability to enhance enzymatic hydrolysis of lignocelluloses. Adding 1.2 wt% recovered EHLPB-210 can increase the enzymatic digestibility of Eu-SPORL from 33.6% to 75.1%. Investigation of the adsorption of cellulase on lignin by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) showed that EHLPB can significantly reduce the non-productive absorption of cellulase on lignin, and therefore enhance the enzymatic hydrolysis of lignocelluloses. By adding recoverable pH-responsive EHLPB, the enzymatic hydrolysis efficiency of lignocelluloses improved and the value-added utilization of enzymatic hydrolysis lignin was realized.

KEYWORDS: Phosphobetaine, Enzymatic hydrolysis, Lignocellulose, pH-responsive, Enzymatic hydrolysis lignin. 2


Page 2 of 26

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


Introduction Energy security and environmental issues constrain sustainable development, and more countries are accelerating the transformation and utilization of renewable biomass resources.1,2 Developing biofuel ethanol and blending ethanol in gasoline have gradually become strategic choices in various countries.1,2 The successful conversion of biomass to cellulosic ethanol is mainly determined by an interplay of four important biorefinery aspects: (1) pre-treatment to overcome biomass recalcitrance; (2) deconstruction of polysaccharides into free monomeric sugars; (3) fermentation of the sugars for cellulosic ethanol production; (4) distillation and refining of ethanol.3 Several challenges in this filed include the low efficiency of cellulase4 and the high cellulase dosage required due to biomass recalcitrance and cellulase adsorption on lignin, and the low-value utilization of enzymatic hydrolysis lignin (EHL).3 These major obstacles may be responsible for the less competitive commercialization of cellulosic ethanol.

Although pH mediation can effectively reduce nonproductive binding of cellulase on lignin for some special substrates to improve the efficiency of enzymatic hydrolysis of lignocelluloses,5 the method is not very effective for most substrates.6 In contrast, adding additives can effectively reduce the non-productive adsorption of cellulase on lignin.3 Several studies have reported that bovine serum albumin (BSA) features high affinity to lignin and can reduce the adsorption of cellulase on lignin to improve the efficiency of enzymatic hydrolysis of lignocelluloses.7,8 Polyethylene

3



Page 4 of 26

glycol 8000 (PEG 8000) has resulted in significantly reducing cellulase loading by 50% while the efficiency of enzymatic hydrolysis of pretreated wheat straw retained 67% after 24 h.9 However, BSA and PEG are difficult to be recycled. Our group has recently reported a recyclable lignin-based amphoteric surfactant and it can effectively enhance

the

enzymatic

hydrolysis

of

pretreated

lignocelluloses.10,11

This

pH-responsive lignin carrier (pH-LC), which was obtained by the quaternization of sulfonated lignin, increased the enzymatic digestibility of CCR from 78% to 93% and a total of 97.2% of pH-LC could be recovered after the hydrolysis by adjusting the pH of the slurry from 4.8 to 3.2.11 Unfortunately, the preparation of pH-LC was complicated, which included firstly introducing a negatively charged sulfonate anion by sulfonation, and then, a positively charged quaternary ammonium cation. As the ratio of sulfonate anion to quaternary ammonium cation determines the pH responsive performance, the ratio must be precisely controlled to prepare products with better performance. As a result, the yield of pH-LC is low. Fortunately, zwitterionic phosphobetaine compounds with balanced charges have good hydrophilicity and can effectively resist proteins.12-14 We intended to directly prepare a pH responsive lignin-based amphoteric surfactant by grafting phosphobetaine intermediate onto EHL, which can be recycled through pH response and can enhance the enzymatic hydrolysis of lignocelluloses. The process is simple with no need for adjusting the ratio of anion and cation.

This study begun with the preparation of pH-responsive EHLPB from EHL by grafting with phosphobetaine intermediate, 3-chloro-2-hydroxypropyl- (2-(trimethyl4


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


ammonio)ethyl)phosphate, which was synthesized from phosphocholine chloride calcium salt and epichlorohydrin. The effect of EHLPB on the enzymatic hydrolysis of pretreated lignocelluloses, Eu-SPORL (sulfite pretreatment to overcome recalcitrance of eucalyptus) and CCR (corncob residue) was studied, along with pH-responsive performance and recovery performance. Finally, in order to understand the enhancement mechanism of pH-responsive EHLPB on enzymatic hydrolysis of pretreated lignocelluloses, the effect of EHLPB on the adsorption of cellulase on lignin was explored using SDS-PAGE analysis.

EXPERIMENTAL SECTION Materials. Phosphocholine chloride calcium salt tetrahydrate was purchased from Saan Chemical Technology (Shanghai) Co., Ltd. (China). Epichlorohydrin was bought from Shanghai Lingfeng Chemical Reagent Co., Ltd. (China). Sodium dodecyl sulfate (SDS, purity ≥ 99%) was obtained from Shanghai Aladdin Bio-Chem Technology Co.,Ltd. (China). Unstained protein molecular weight marker was purchased from Thermo Scientific (Lithuania). EHL was provided by Shandong Longlive Bio-Technology Co., Ltd. (China). Avicel (PH101) with a mean particle size of 50 μm was obtained from U.S. Sigma company (USA).

Eucalyptus wood (Eu) was bought from Qinbei district of Qinzhou city in Guangxi province. Pretreated lignocellulose Eu-SPORL was obtained by sulfite pretreatment to overcome recalcitrance of eucalyptus, the treatment process was referred to a previously reported method.15 The contents of glucan, acid-insoluble lignin, xylan, 5



and other components of Eu-SPORL accounted for 64.2%, 34.7%, 0.5% and 0.6% respectively of the total content.16 Eucalyptus lignin was obtained according to a previously reported method (Lou et al., 2018).17 CCR (corncob residue), enzymatic residue from the production of functional sugar, was purchased from Shandong Longlive Bio-Technology Co., Ltd. (China) and treated at 120 °C for 1 h by auto-clave prior to use. The contents of glucan, acid-insoluble lignin, xylan, and other components of pretreated CCR reached 77.9%, 18.9%, 0.1% and 3.1%, respectively.16

Commercial cellulase enzyme Cellic CTec2 (abbreviated CTec2), derived from the fungus Trichodermareesei (Hypocreajecorina) was supplied by Novozyme China (Shanghai, China). The protein concentration of cellulase is 73.6 mg/mL, and its cellulase activity is 147 filter paper cellulase units (FPU)/mL slurry of substrate. The determination of cellulase activity was referred to a previously reported method.18

All chemicals were of analytical grade and without further purification prior to use. Deionized water was used for the preparation of all solutions.

EHLPB-x from EHL. The preparation of EHLPB-x (x = 4, 10, 60, 120, 210, which denotes the percentage of mass ratio of phosphocholine chloride calcium salt tetrahydrate to EHL) was as follows. Figure 1 shows the reaction pathway.

6


Page 6 of 26

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


Figure 1. The scheme of the preparation of EHLPB-x.

2-(Trimethylammonio)ethyl hydrogen phosphate (compound 2) was obtained from phosphocholine chloride calcium salt tetrahydrate (compound 1) according to the method reported in literature.19 Next, compound 2 and epichlorohydrin were further used

to

yield

3-chloro-2-hydroxypropyl(2-(trimethylammonio)ethyl)phosphate

(compound 3) at 90 °C for 9 h in the presence of concentrated HCl aq. Finally, compound 3 was grafted onto EHL to obtain the desired production of EHLPB-x in the alkaline solution of pH = 12 at 85 °C for 5 h. The crude EHLPB-x product was further purified by acid precipitation, during which, the pH of the reaction mixtures was adjusted to 5.0 firstly, and then the solutions were stood and centrifugated, the pH of the supernatants was adjusted to 3.0 to precipitate EHLPB-x. Finally, the purified EHLPB-x was obtained by centrifugation.

Enzymatic Hydrolysis. EHLPB-x and cellulase were successively added to slurry

7



of substrate (0.6 g Avicel or lignocelluloses) of 30 mL acetate buffer (pH=5.0, 50 mM). The enzyme loading for Avicel and Eu-SPORL was 5 FPU/g glucan, and for CCR 3 FPU/g glucan. Enzymatic hydrolysis of the substrate under the conditions of 2% (w/v) solid concentration was carried out 50 °C, and 150 rpm for 72 h in a shaker (DDHZ-300, Jiangsu Taicang Equipment Factory, China). Enzymatic hydrolysis of lignocelluloses at 10% (w/w) of high-solid loading was also studied. The slurry of substrate measured 10 g, and the conditions were similar as 2% solid concentration except that the enzyme loading for substrate was 10 FPU/g glucan. Glucose in enzymatic hydrolysates at 72 h was detected by a commercial SBA-40E biosensor by a H2O2 electrode sensor (Institute of Biology of the Shandong Academy of Sciences, China). Glucose yield, which was defined as the molar percentage of substrate glucan enzymatically hydrolyzed to glucose in substrate, represents the substrate enzymatic digestibility at 72 h (SED@72h). Control experiments without EHLPB-x were also carried out for comparison. Data points were the average of three analyses. The deviations of datas were shown in the figures.

Measurement of Solubility of EHLPB-210. Ultraviolet (UV) spectrophotometer was used to obtain the solubility of EHLPB-210 at different pH according to literature method.10 A total of 1 g/L purified EHLPB-210 solutions were prepared with pH=5.0 50 mM acetate buffer. Then, the pH of the solutions was adjusted to 1.0, 2.0, 3.0, 3.5, 4.0, 5.0 and 6.0 by 6 M hydrochloric acid or 6 M NaOH solution. After the solutions were stood and centrifugated, and the supernatants were diluted for certain times with acetate buffer (pH=5.0, 50 mM), and the UV absorbance of solutions was further 8


Page 8 of 26

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


measured at 280 nm. The calculation method for the solubility of EHLPB-210 (SEHLPB-210) is as follows:

SEHLPB-210 =

 100%

A0 is the UV absorbance of the solution of pH = 5.0 at 280 nm, A1 is the UV absorbance at 280 nm of the supernatants of other experimental groups. The UV absorbance of the solution of pH = 5.0 was used as a control, the corresponding solubility is 100% as EHLPB-210 completely dissolves when p H ≥ 5.0.

Cellulase Adsorption on Lignin. A total of 1g (10 wt%) eucalyptus lignin was added to the solution of EHLPB-x (final concentration: 1 g/L) and CTec 2 (2 FPU/mL) in 9 mL of acetate buffer (pH 5.0, 50 mM). After incubation at 50 °C and 150 rpm for 24 h, 10 μL of supernatant was analyzed by 12% SDS-PAGE (Cai et al., 2018).11 Control experiments without EHLPB-x was also carried out for comparison, relative content of cellulase for CTec2 (2FPU/mL) was 100%.

Analytic Methods. Intermediate compound 3 was characterized by maxis impact ultra-high resolution time-of-flight mass spectrometry (MS) using a spectrometer in electrospray ionization mode (Bruker Daltonics, Germany) (Figure S1). The chemical structure of EHLPB was recorded with a Bruker AV 400 spectrometer (Bruker, Germany) in phosphorus-31 nuclear mangnetic resonance

31

PNMR (Figure S2) in

dimethyl sulfoxide-d6 and further characterized by a Vector 333 Fourier-transform infrared spectroscopy (FT-IR) spectrometer (Bruker, Germany) at an optical range of 400–4000 cm-1 and a resolution of 8 cm-1. The contents of elements of EHLPB-x were 9



measured by an elemental analyzer (VARIO EL, Elementar, Germany). The content of phosphobetaine in EHLPB-x was approximately calculated by the content of N (element). Zeta potentials of initial concentration of 1 g/L EHLPB-210 (50 mM HAc-NaAc) solution at different pH were measured by zeta potential analyzer (Brookhaven Zeta Plus). The contact angles of pure water on the lignin film and lignin film adsorbed EHLPB-210 were measured, respectively, according to the sessile drop method by using Powereach JC2000C1 (Shanghai, China).10

RESULTS AND DISCUSSION Characterization of EHLPB. EHLPB was synthesized from phosphobetaine intermediate and EHL.

phenolic hydroxyl group 1358cm-1 P-O-C

80

Transmittance (%)

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

Page 10 of 26

1090cm-1

70

60

EHL EHLPB-210

50 2000

1800

1600

1400

1200

1000

800

Wavenumber (cm-1)

Figure 2. Infrared absorption spectra of EHL and EHLPB-210.

The chemical structure of EHLPB was characterized by FT-IR (Figure 2) and 31

PNMR (Figure S2). The telescopic vibration absorption peak at 1358 cm-1 of

10


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


phenolic hydroxyl group in lignin was confirmed.20 In comparation with EHL, the intensity of the peak of EHLPB notably decreased to near zero owing to the grafting of phosphobetaine intermediate on the phenolic hydroxyl groups of lignin. The infrared absorption of EHLPB at 1090 cm-1 is attributed to telescopic vibration absorption peak of the P-O-C group of phosphobetaine.21 These findings indicate that phosphobetaine intermediate has been successfully introduced to EHL in combination with the peak at 0.36 ppm of P-O-C by 31PNMR spectrum analysis and MS spectrum analysis of intermediate compound 3 (Figure S1). The contents of elements of EHL and EHLPB-x were further measured by an elemental analyzer and

the content of

phosphobetaine intermediate grafted in EHL was calculated according to the content of N (Table 1). It can be found that the content of phosphobetaine intermediate of EHLPB-210 reached 1.12 mmol/g.

Table 1 Elemental analysis of EHL and EHLPB-x.

Sample

N (%)

C (%)

H (%)

S (%)

Content of phosphobetaine (mmol/g)

EHL EHLPB-4 EHLPB-10 EHLPB-60 EHLPB-120 EHLPB-210

0.52 0.55 0.70 1.08 1.38 2.09

51.68 58.93 58.28 58.02 58.08 58.97

5.33 5.13 5.54 5.35 5.40 5.47

0.43 0.49 0.42 0.35 0.33 0.31

0 0.02 0.13 0.40 0.61 1.12

Effect of EHLPB-x on The Enzymatic Hydrolysis of Lignocelluloses. The effect of EHLPB-x on the enzymatic hydrolysis of Eu-SPORL and CCR was investigated. As shown in Figure 3, when the dosage of EHLPB-x was less than 5 g/L, the 11



enzymatic hydrolysis of substrates was gradually strengthened with increasing concentration of EHLPB-x. However, the effect on the hydrolysis remained approximately unchanged when the concentration of EHLPB-x was more than 5 g/L. Adding 5 g/L EHLPB-210 raised SED@72h of Eu-SPORL from 41.2% to 82.5%, and for CCR from 39.1% to 93.7%. And besides, it can be found that all EHLPB-x could improve the enzymatic hydrolysis of lignocelluloses, but the ones with higher content of phosphobetaine intermediate performed better in enhancing the hydrolysis.

(a)

EHLPB-4 EHLPB-10 EHLPB-60 EHLPB-120 EHLPB-210

90 80

SED@72h (%)

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

Page 12 of 26

70 60 50 Eu-SPORL

40 0

2

4

6

Concentration of EHLPB-x (g/L)

12


8

Page 13 of 26

(b) 100

CCR

90 80

SED@72h (%)

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


70 60 EHLPB-4 EHLPB-10 EHLPB-60 EHLPB-120 EHLPB-210

50 40 30

0

2

4

6

8

Concentration of EHLPB-x (g/L)

Figure 3. (a) Effect of EHLPB-x on the enzymatic hydrolysis of Eu-SPORL; (b) Effect of EHLPB-x on the enzymatic hydrolysis of CCR (solid concentration: 2% (w/v); pH 5.0; ionic strength: 50 mM; cellulase loading: 5 FPU/g glucan for Eu-SPORL, 3 FPU/g glucan for CCR).

Effect of EHLPB-210 on the Enzymatic Hydrolysis of Lignocelluloses at High-Solid Loading. High-solid lignocellulose refinery shows many advantages, including high fermentable sugar and production concentration, low cost of separation, and less wastewater discharge. However, the efficiency of enzymatic hydrolysis of lignocelluloses at high-solid loading is notably low, and the reason is that constraint of the water has inhibited mass transfer rates of glucose and cellulase at high-sold enzymatic hydrolysis system22,23 and end product glucose inhibition. Because When EHLPB-210 was added, the enzymatic hydrolysis efficiency of lignocellulose was relatively higher, compared with adding other EHLPB-x. So the effect of EHLPB-210 on the high-solid enzymatic hydrolysis of lignocelluloses was also studied.

13



80

70 SED@72h (%)

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

60

50

40

30

Eu-SPORL CCR

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Concentration of EHLPB-210 (wt%)

Figure 4. Effect of EHLPB-210 on the high-solid enzymatic hydrolysis of lignocelluloses (solid concentration: 10% (w/w); pH 5.0; ionic strength: 50 mM; cellulase loading: 10 FPU/g glucan ).

Figure 4 shows the enzymatic digestibility of substrates was gradually increased with increasing concentration of EHLPB-210 under a lower dosage (< 1.2 wt%). Interestingly, when 1.2 wt% additive was used, the SED@72h for Eu-SPORL and CCR increased from 33.6% and 52.6 to 71.5% and 73.6%, respectively. However, the enhancement effect on hydrolysis remained approximately unchanged when the concentration of EHLPB-210 was more than 1.2 wt%.

Effect of EHLPB-210 on the Enzymatic Hydrolysis of Avicel and Lignocelluloses at Different pH. Figure 5 shows an overview of the effect of pH on the enzymatic hydrolysis of Avicel and lignocelluloses with or without 5 g/L EHLPB-210 as additive. As shown in the figure, the optimal pH of enzymatic hydrolysis of Avicel and lignocelluloses was 4.5 and 5.0, respectively, when an 14


Page 14 of 26

Page 15 of 26

additive was added. The SED@72h of Eu-SPORL and CCR reached 62% and 44.5% at pH = 4.5, respectively, when an additive was added. By contrast, when an additive was added, the SED@72h of Eu-SPORL and CCR increased to 82.5% and 93.7% at pH = 5.0, respectively. Compared with the optimum pH of enzymatic hydrolysis of Avicel, that of lignocelluloses was increased by 0.5. This is because the enhanced electrostatic repulsion between cellulase and lignin at elevated pH can result in reducing the non-productive adsorption of cellulase on lignin.6

100 80 SED@72h (%)

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


60 40 Avicel Avicel(EHLPB-210) CCR CCR(EHLPB-210) Eu-SPORL Eu-SPORL(EHLPB-210)

20 0 4.0

4.5

5.0

5.5

6.0

Initial pH

Figure 5. Effects of EHLPB-210 on the enzymatic hydrolysis of substrates at different pH (solid concentration: 2% (w/v); concentration of EHLPB-210: 5g/L; ionic strength: 50 mM; cellulase loading: 5 FPU/g glucan for Avicel and Eu-SPORL, 3 FPU/g glucan for CCR ).

pH-Responsive Performance and Recovery Performance of EHLPB. Figure 6 shows the zeta potentials and the solubility of 1 g/L EHLPB-210 in 50 mM acetate buffer at different pH, respectively. The zeta potential of EHLPB-210 solution

15



increased from -14.8 mV to -0.3 mV when the pH of solution decreased from 6.0 to 1.0, EHLPB-210 aggregated and precipitated due to weakened electrostatic repulsion between EHLPB-210 molecules. So when pH ≥ 5.0, EHLPB-210 was completely dissolved in the solution, and when pH ≤ 3.0, 95.5% of EHLPB-210 was precipitated out, that indicates the pH-sensitive response of EHLPB-210.

10

100

5 80

0

60

-5 -10

40 pH=1.0

pH=2.0

pH=3.0

pH=3.5

pH=4.0

pH=5.0

pH=6.0

20 0

Zeta (mV)

Solubility (%)

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

Page 16 of 26

-15 -20

1

2

3

4

5

6

-25

pH

Figure 6. The zeta potentials (1 g/L,50 mM) and solubility of EHLPB-210 at different pH.

As shown previously, when 1.2 wt% of EHLPB-210 was added, the SED@72h for Eu-SPORL at high-solid loading increased from 33.6% to 71.5%. After enzymatic hydrolysis, it was tested that 95% of EHLPB-210 could be recovered by adjusting the slurry pH from 5 to 3. The effect of recovered EHLPB-210 on enzymatic hydrolysis of Eu-SPORL was further investigated. The SED@72h for Eu-SPORL turned out to be 75.1% when 1.2 wt% recovered EHLPB-210 was added, which was better than that of adding fresh EHLPB-210. The reason might be the recovered EHLPB-210 contained some cellulase.

16


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


Enhancement Mechanism of EHLPB-x on the Enzymatic Hydrolysis of Lignocelluloses. As shown in Figure 6, the zeta potential of EHLPB-210 solution was -14.3 mv when pH=5.0, and this result meant that the absorption of cellulase on lignin absorbed EHLPB-210 might reduce, associated with the electrostatic repulsion interaction between EHLPB-210 and cellulase, because cellulase has a negative charge at pH = 5. Phosphobetaine is highly resistant to protein adsorption by forming a hydration layer due to balanced charge and minimized dipole.24,25 So EHLPB-210 might increase the hydrophilicity of lignin by absorbing on its surface. The effect of EHPB-210 on the hydrophilicity of lignin was further explored by measuring the contact angle of pure water on lignin film before and after absorbed EHLPB-210. The contact angle of pure water on the blank lignin film is 56°(Figure 7), and that on the lignin film adsorbed EHLPB-210 was reduced to 24°(Figure7). The result showed that adsorbed EHLPB-210 could increase the hydrophilicity of lignin, which might also lead to the absorption reduction of cellulase.

Figure 7. The contact angles of water on the lignin film which adsorbed EHL and EHLPB-210.

17



EHLPB-x

(a) CTec 2

Control

4

10

60

120

210

Marker Mw (KDa) 116

-GI CBHⅠ CBHⅡ

66.2

EGⅠ, EGⅡ 45.0 EG Ⅳ

35.0

EG Ⅲ EGⅤ

25.0

Xyn 18.4 14.4

(b)

50

Relative content of cellulase in solution(%)

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

Page 18 of 26

40

30

20

10

0 0 0 4 ol -60 -10 B21 12 ntr BBPB PB LP P P L L Co H L L H H E E E EH EH

Figure 8. (a) SDS-PAGE analysis of effect of EHLPB on the adsorption of cellulase on lignin; (b) Effect of EHLPB on the adsorption of cellulase on lignin.

Next, SDS-PAGE was used to examine the effect of EHLPB-x on the absorption of cellulase on lignin (Figure 8). As shown in Figure 8b, cellulase content in supernatant was only 11.1% in the absence of additives. When EHLPB-4, EHLPB-10,

18


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


EHLPB-60, EHLPB-120, and EHLPB-210 were introduced, cellulase content in supernatant rose to 19.2%, 29.5%, 35.2%, 40.8%, 46.4%, respectively. EHLPB-x with higher content of phosphobetaine intermediate became more negatively charged. The negatively charged surface of lignin absorbed EHLPB-x was less hydrophobic, which was not favorable for binding cellulase through hydrophobic interaction. Based on all these results, this study revealed that EHLPB-x improved the enzymatic hydrolysis of lignocelluloses by reducing the non-productive adsorption of cellulase on lignin. There were at least two reasons for the observed decrease in nonspecific cellulase binding to lignin: (1) EHLPB-x increased the hydrophilicity of lignin by absorbing on its surface. and (2) the electrostatic repulsion interaction between negatively charged EHLPB-x and cellulase (Figure 9).

Figure 9. Enhancing mechanism of EHLPB on the enzymatic hydrolysis of lignocelluloses.

CONCLUSIONS A lignin-based amphoteric surfactant EHLPB which can effectively enhance the

19



enzymatic hydrolysis of lignocellulose and achieve self-recovery by its pH-sensitive performance, was prepared by directly grafting phosphobetaine intermediate onto EHL with no need for adjusting the ration of anion and cation. The SED@72h of high-solid Eu-SPORL and CCR increased from 33.6% and 52.6% to 71.5% and 73.6%, respectively by adding 1.2 wt% EHLPB-210. 95% of EHLPB-210 could be recovered after enzymatic hydrolysis by adjusting the pH of the slurry from 5 to 3. Under the same experimental conditions, the efficiency of enzymatic hydrolysis increased from 33.6% to 75.1% for high-solid Eu-SPORL using 1.2 wt% recovered EHLPB-210 as additive. In addition, SDS-PAGE experiments showed that EHLPB could effectively reduce the non-productive adsorption of cellulase on lignin, which was attributed to the increased hydrophilicity of lignin after adsorbing EHLPB, and the electrostatic exclusion between negatively charged EHLPB and cellulase. In summary, the enzymatic hydrolysis efficiency of lignocellulose was improved by adding EHLPB and the recovery of lignin-based amphoteric surfactant was realized at the same time.

ASSOCIATED CONTENT Supporting Information

The supporting information are available free of charge. The HRMS spectra of the reaction suspension of including compound 3.(pdf)The 31

PNMR spectra of EHLPB-210.(pdf)

20


Page 20 of 26

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


AUTHOR INFORMATION Corresponding Author

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

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the financial support from the National Natural Science Foundation of China (21676109, 21878112), Science and Technology Program of Guangzhou (201707020025), Guangdong Special Support Plan (2016TX03Z298) and Science and Technology Program of Guangdong (2017B090903003).

21



REFERENCES (1) Hoffert, M.I.; Caldeira, K.; Benford, G.; Criswell, D.R.; Green, C.; Herzog, H.; Jain, A.K.; Kheshgi, H.S.; Lackner, K.S.; Lewis, J.S.; Lightfoot, H.D.; Manheimer, W.; Mankins, J.C.; Mauel, M.E.; Perkins, L.J.; Schlesinger, M.E.; Volk, T.; Wigley, T.M.L. Advanced technology paths to global climate stability: energy for a greenhouse planet. Science 2002, 298 (5595), 981-987, DOI 10.1126/science.1072357.

(2) Ragauskas, A.J.; Williams, C.K.; Davison, B.H.; Britovsek, G.; Cairney, J.; Eckert, C.A.; Frederick, W.J.; Hallett, J.P.; Leak, D.J.; Liotta, C.L.; Mielenz, J.R.; Murphy, R.; Templer, R.; Tschaplinski, T. The path forward for biofuels and biomaterials. Science 2006, 311 (5760), 484-489, DOI 10.1126/science.1114736.

(3) Jorgensen, H.; Pinelo, M. Enzyme recycling in lignocellulosic biorefineries. Biofuels. Bioprod. Bior. 2017, 11 (1), 150-167, DOI 10.1002/bbb.1724.

(4) Wang, B.; Shen, X.J.; Wen, J.L.; Xiao, L.; Sun, R.C. Evaluation of organosolv pretreatment on the structural characteristics of lignin polymers and follow-up enzymatic hydrolysis of the substrates from Eucalyptus wood. Int. J. Biol. Macromol 2017, 97, 447-459, DOI 10.1016/j.ijbiomac.2017.01.069.

(5) Lou, H.M.; Zhu, J.Y.; Lan, T.Q.; Lai, H.R.; Qiu, X.Q. pH-induced lignin surface modification to reduce nonspecific cellulase binding and enhance enzymatic saccharification of lignocelluloses. ChemSusChem 2013, 6 (5), 919-927, DOI 10.1002/cssc.201200859.

22


Page 22 of 26

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


(6) Lan, T.Q.; Lou, H.M.; Zhu, J.Y. Enzymatic saccharification of lignocelluloses should be conducted at elevated pH 5.2–6.2. BioEnerg. Res. 2013, 6 (2), 476-485, DOI 10.1007/s12155-012-9273-4.

(7) Wei, W.Q.; Wu, S.B. Enhanced enzymatic hydrolysis of eucalyptus by synergy of zinc chloride hydrate pretreatment and bovine serum albumin. Bioresour. Technol 2017, 245, 289-295, DOI 10.1016/j.biortech.2017.08.133.

(8) Yang, B.; Wyman, C.E. BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol. Bioeng. 2006, 94 (4),611-617, DOI 10.1002/bit.20750.

(9) Monschein, M.; Reisinger, C.; Nidetzky, B. Dissecting the effect of chemical additives on the enzymatic hydrolysis of pretreated wheat straw. Bioresour. Technol. 2014, 169, 713-722, DOI 10.1016/j.biortech.2014.07.054.

(10) Cai, C.; Zhan, X.J.; Zeng, M.J.; Lou, H.M.; Pang, Y.X.; Yang, J.; Yang, D.J.; Qiu, X.Q. Using recyclable pH-responsive lignin amphoteric surfactant to enhance the enzymatic hydrolysis of lignocelluloses. Green Chem. 2017, 19 (22), 5479-5487, DOI 10.1039/c7gc02571h.

(11) Cai, C.; Zhan, X.J.; Lou, H.M.; Li, Q; Qian, Y; Zhou, H.F.; Qiu, X.Q. Recycling cellulase by a pH-responsive lignin-based carrier through electrostatic interaction. ACS.

Sustainable

Chem.

Eng.

2018,

6

10.1021/acssuschemeng.8b02011.

23


(8),

10679-10686,

DOI


Page 24 of 26

(12) Dang, Y.; Quan, M.; Xing, C.M.; Wang, Y.B.; Gong, Y.K. Biocompatible and antifouling coating of cell membrane phosphorylcholine and mussel catechol modified multi-arm PEGs. J. Mater. Chem. B 2015, 3 (11), 2350-2361, DOI 10.1039/c4tb02140a.

(13) Goda, T.; Tabata, M.; Sanjoh, M.; Uchimura, M.; Iwasaki, Y.; Miyahara, Y. Thiolated 2-methacryloyloxyethyl phosphorylcholine for an antifouling biosensor platform.

Chem.

Commun

(Camb).

2013,

49

(77),

8683-8685,

DOI 10.1039/c3cc44357d.

(14) Zhang, R.N.; Liu, Y.N.; He, M.R.; Su, Y.L.; Zhao, X.T.; Elimelech, M.; Jiang, Z.Y. Antifouling membranes for sustainable water purification: strategies and mechanisms. Chem. Soc. Rev. 2016, 45 (21), 5888-5924, DOI 10.1039/c5cs00579e.

(15) Cai, C.; Qiu, X.Q.; Lin, X.L.; Lou, H.M.; Pang, Y.X.; Yang, D.J; Chen, S.W.; Cai, K.F.

Improving

prehydrolysates lignosulfonate.

enzymatic by

adding

Bioresour.

hydrolysis

of

lignocellulosic

cetyltrimethylammonium Technol.

2016,

substrates

bromide 216,

to

with

neutralize

968-975,

DOI

10.1016/j.biortech.2016.06.043.

(16) Luo, X.; Gleisner, R.; Tian, S.; Negron, J.; Zhu, W.; Horn, E.; Pan, X.J.; Zhu, J. Y. Evaluation of mountain beetle-infested lodgepole pine for cellulosic ethanol production by sulfite pretreatment to overcome recalcitrance of lignocelluloseind. Eng. Chem. Res. 2010, 49 (17), 8258–8266, DOI 10.1021/ie1003202.

24


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


(17) Lou, H.M.; Lin, M.L.; Zeng, M.J.; Cai, C.; Pang, Y.X.; Yang, D.J.; Qiu, X.Q. Effect of urea on the enzymatic hydrolysis of lignocellulosic substrate and its mechanism. BioEnergy. Res. 2018, 11 (2),456–65, DOI 10.1007/s12155-018-9910-7.

(18) Wood, T.M.; Bhat, K.M. Methods for measuring cellulase activities. Methods Enzymol. 1988, 160 (Biomass, Pt. A), 87-112.

(19)

Knopik,

P.;

Bruzik,

K.S.;

Stec,

W.J.

An

improved

synthesis

of

6-(O-phosphorylcholine)hydroxyhexanoic acid. Org. Prep. and Proced. In. 1991, 23 (2), 214-216, DOI 10.1080/00304949109458318.

(20) Boeriu, C.G.; Bravo, D.; Gosselink, R.J.A.; van Dam, J.E.G. Characterisation of structure-dependent functional properties of lignin with infrared spectroscopy. Ind. Crop. Prod. 2004, 20 (2), 205-218, DOI 10.1016/j.indcrop.2004.04.022.

(21) Ning, Y.CH. Foreword to interpretation of organic spectra. 2010, first ed. Science press, Beijing.

(22) Roberts, K.M.; Lavenson, D.M.; Tozzi, E.J.; McCarthy, M.J.; Jeoh, T. The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 2011, 18 (3), 759-773, DOI 10.1007/s10570-011-9509-z.

(23) Selig, M.J.; Thygesen, L.G.; Felby, C. Correlating the ability of lignocellulosic polymers to constrain water with the potential to inhibit cellulose saccharification. Biotechnol. Biofuels 2014, 7, 159-168, DOI 10.1186/s13068-014-0159-x. 25



(24) Chen, S.F.; Zheng, J.; Li, L.Y.; Jiang, S.Y. Strong resistance of phosphorylcholine self-assembled monolayers to protein adsorption: insights into nonfouling properties of zwitterionic materials. J. Am. Chem. Soc. 2005, 127 (41), 14473-14478, DOI 10.1021/ja054169u.

(25) Liu, P.SH.; Chen, Q.; Li, L.; Lin, S.C.; Shen, J. Anti-biofouling ability and cytocompatibility of the zwitterionic brushes-modified cellulose membrane. J. Mater. Chem. B 2014, 2 (41) ,7222-7231, DOI 10.1039/c4tb01151a.

Abstract

graphic

SYNOPSIS A novel recyclable pH-responsive EHLPB showed excellent enhancement effect on the enzymatic hydrolysis of lignocelluloses.

26


Page 26 of 26

Enhancement of Recyclable pH-Responsive Lignin-Grafted

Recommend Documents