Accepted Manuscript

Facile pretreatment of lignocellulosic biomass using deep eutectic solvents

Cheng-Wu Zhang, Shu-Qian Xia, Pei-Sheng Ma

PII: S0960-8524(16)30990-7
DOI: http://dx.doi.org/10.1016/j.biortech.2016.07.026
Reference: BITE 16788

To appear in: Bioresource Technology

Received Date: 28 May 2016

Revised Date: 6 July 2016
Accepted Date: 7 July 2016

Please cite this article as: Zhang, C-W., Xia, S-Q., Ma, P-S., Facile pretreatment of lignocellulosic biomass using
deep eutectic solvents, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.07.026

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Facile pretreatment of lignocellulosic biomass using deep eutectic

solvents

Cheng-Wu Zhang, Shu-Qian Xia*, Pei-Sheng Ma

* Corresponding author. E-mail address: shuqianxia@tju.edu.cn (S.-Q. Xia).

Key Laboratory for Green Chemical Technology of Ministry of Education, School of

Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072,

China

Highlights

Three kinds of DESs used in the pretreatment of corncob.

Improved delignification and enzymatic hydrolysis efficiency.

Removal of lignin and hemicellulose.

Optimization of pretreatment temperature and time.

Abstract: In this work, three kinds of deep eutectic solvents (DESs) were facilely

prepared and used in the pretreatment of corncob, including monocarboxylic acid/

choline chloride, dicarboxylic acid/ choline chloride and polyalcohol/ choline chloride.

The enhanced delignification and subsequent enzymatic hydrolysis efficiency were

found to be related to the acid amount, acid strength and the nature of hydrogen bond

acceptors. The XRD, SEM and FT-IR results consistently indicated that the structures of

corncob were disrupted by the removal of lignin and hemicellulose in the pretreatment

process. In addition, the optimal pretreatment temperature and time were 90 °C and 24 h,

1
respectively. This study explored the roles of various DESs combinations, pretreatment

temperature and time to better utilize the DESs in the pretreatment of lignocellulosic

biomass.

Keywords: Pretreatment; Lignocellulosic biomass; Deep eutectic solvents;

Delignification; Enzymatic hydrolysis.

1. Introduction

The biorefinery prospect is integrating multiple biomass conversion processes to

produce energy, biofuels and high-valued chemicals(Ragauskas et al., 2006). Typically,

the integrative utilization and conversion of lignocellulosic feedstocks have been

extensively studied in the past decades. For its inherent complex constitutes and

structures, lignocellulose has the recalcitrance to chemical and biological

treatment(Himmel et al., 2007). Then the pretreatment process is necessary to

fractionate the lignocellulose components and enhance the accessibility of

polysaccharides (Brandt et al., 2013; Mora-Pale et al., 2011). Traditional pretreatment

methods have been widely practiced and their advantages and disadvantages have been

well reviewed(Mora-Pale et al., 2011).

Due to the exceptional solvent properties, ionic liquids (ILs) appears to be a kind of

efficient and promising solvent in the pretreatment of lignocellulosic biomass. In

particular, 1-ethyl-3-methyl imidazolium acetate ([Emim] [CH3COO]) is one

representative ionic liquid with high performance used in the pretreatment of various

lignocellulosic feedstocks. Besides, the biorenewable ionic liquids(Hou et al., 2012;

2
Hou et al., 2015; Liu et al., 2012), biocompatible ionic liquids(Wahlström & Suurnäkki,

2015) and protic ionic liquids(Achinivu et al., 2014) show their individual excellence in

the pretreatment process. In spite of this, the controversy over the ILs’ industrial

applications still exists for its disadvantages such as high price and toxicity. Then the

recycling and reuse of ILs appear critical for their applications in the practical

biorefinery(Brandt et al., 2013; Mora-Pale et al., 2011).

Deep eutectic solvents (DESs), also called ionic liquids alternatives, is a kind of nascent

green solvent with similar physicochemical properties to ILs(Dominguez de Maria &

Maugeri, 2011). Moreover, it has many other advantages such as easy to prepare,

biodegradable and biocompatible, attracting growing interests in academia and

industry(Zhang et al., 2012). Recently, DESs has exhibited its great capabilities in the

pretreatment of lignocellulose. Francisco firstly reported that DESs’ solubility of real

wheat straw biomass was promising(Francisco et al., 2012). Soon afterwards, many

groups put various DESs into the pretreatment trials and achieved amazing results

(Kumar et al., 2015; Nor et al., 2015; Procentese et al., 2015; Xu et al., 2016). However,

the pretreatment of lignocellulose using DESs is on its infant stage and the research isn’t

thorough and intensive yet. In this study, we systematically investigated the effects of

the DESs composed of choline chloride (ChCl) combined with renewable carboxylic

acids or renewable polyols on the pretreatment of corncob. Besides, the possible related

factors of DESs and pretreatment mechanisms were speculated and then testified.

Finally, the individual effects of pretreatment temperature and time on the pretreatment

3
were discussed in terms of lignin extractability, residue recovery, initial saccharification

rate and glucose yield.

2. Materials and Methods

2.1 Biomass and chemicals

The screening procedure and major components of raw corncob was specified in

previous literature(Zhang et al., 2010). ChCl (98%, Beijing J&K Scientific) was chosen

as the hydrogen bond acceptor and dried in vacuum oven for at least 48 h. The hydrogen

bond donors were lactic acid (98%; 85%), glycolic acid (99%), levulinic acid (99%),

malonic acid (99.5%), glutaric acid (99%), oxalic acid (99%), malic acid (>99.5%),

ethylene glycol (99.5%) and glycerol (99%). Cellulase from Trichoderma reesei was

purchased from Sigma Aldrich.

2.2 Pretreatment of corncob using the prepared DESs

6 g DESs was prepared by mixing the ChCl and carboxylic acid or polyalcohol at 90 °C.

Then 0.3 g corncob was added to the prepared DESs and the mixture was magnetically

stirred at a specific temperature for required time. Later, the mixture was diluted with 6

ml 0.1 M NaOH solution and then centrifuged. The supernatant was diluted to

determine the lignin content at 218 nm. The residues were washed with distilled water

for three times, lyophilized for 48 h and stored in sealed bags for the following use.

2.3 Enzymatic hydrolysis of corncob

8 ml citrate buffer (50 mM, pH 4.8) was added into the vial and preheated in the rotary

shaker at 50 °C. 20 mg corncob residue and 41 mg cellulase were subsequently added

4
into the vial. The enzymatic reaction system was stirred at 180 rpm. 0.2 ml sample was

removed at certain time intervals and boiled to quench the enzymatic reaction. After

centrifugation, the reducing sugar content was determined by the DNS method(Miller,

1959). The reaction was performed in triplicate.

Glucose yield (%) = released glucose amount/glucose amount in raw corncob× 100 (1)

2.4 Corncob characterization

2.4.1 X-ray diffraction (XRD) analysis

The corncob samples were scanned at 5°/min from 10° to 40° with a step size of 0.01°

using D8-Focus (Bruker, Germany). The scan voltage and current were 40 kV and 40

mA, respectively. The crystallinity index (CrI) was calculated by the peak height

method(Segal et al., 1959).

CrI = (I002-Iam)/I002×100% (2)

Where the I002 is the intensity of 002 lattice diffraction (2Ɵ=22.6°) and the Iam is the

intensity of amorphous section (2Ɵ=18°).

2.4.2 Fourier transform infrared spectroscopy (FTIR) analysis

The corncob samples were well mixed with KBr and then pressed into slices. They were

scanned from 4000 cm-1 to 400 cm-1 with the spectral resolution of 4 cm-1 using the

Vertex 70 (Bruker, Germany).

2.4.3 Scanning electron microscopy (SEM) analysis

The corncob samples were well scattered on the conducting resin and coated with Au.

And then they were scanned by the S-4800 (Hitachi, Japan) to observe the surface

5
morphology features.

3. Results and Discussion

3.1 Effect of DESs

DESs could be only formed by mixing two certain components in a specific range of

ratios (Florindo et al., 2014; Francisco et al., 2012). Taken the viscosity of DESs into

account as well (Florindo et al., 2014), we chose three kinds of DESs (monocarboxylic

acid/ ChCl, dicarboxylic acid/ ChCl and polyalcohol/ ChCl) and put them in the

pretreatment of corncob in this work. The effects of the chosen DESs on the

delignification extent and enzymatic hydrolysis of corncob were shown in Table 1 and

Fig. 1. It was notable that the delignification extent and enzymatic hydrolysis efficiency

were greatly enhanced after the pretreatment.

As for the lactic acid/ ChCl (shown in Table 1 and Fig.1 (a)), with the molar ratio of

lactic acid to ChCl increasing from 2:1 to 15:1, the lignin extractability increased

drastically (64.7%-93.1%) but the glucose yield scarcely changed (79.1%-83.5%). It

indicated that a larger amount of acid in the DESs can facilitate the extensive

delignification of corncob, resulting less residues recovery. Meanwhile, the initial

saccharification rates increased to 1.16-1.85 g/ (L*h) compared to 0.06 g/ (L*h) of raw

corncob. Once the lignin extractability reached over about 70%, the further increasing

delignification wouldn’t promote glucose yield. Besides, it has been reported that the

complete removal of lignin is not necessary to achieve better cellulose digestibility (Fu

et al., 2010; Hou et al., 2013; Hou et al., 2012; Lee et al., 2009). Compared to glycolic

6
acid and levulinic acid, lactic acid has a moderate acid strength (pKa=3.86). This may

lead to the relatively higher lignin extractability and glucose yield.

The group of dicarboxylic acid/ ChCl (shown in Table 1 and Fig.1 (b)) behaved less

effective in the process of pretreatment. Due to the relatively high temperature, the

carboxyl groups in the malonic acid and oxalic acid may get activated and release the

carbon dioxide. It could be confirmed by the massive bubbles generated in the

preparation process of corresponding DESs and the 1HNMR spectra of synthesized

DESs. The higher acid strength in malonic acid (pKa1=2.83, pKa2=5.69) and oxalic acid

(pKa1=1.25, pKa2=4.14) may bring about the limited lignin extractability and glucose

yield. In particular, the pulp became charred in the pretreatment by oxalic acid: ChCl

(1:1) due to the excessive acid strength.

The ethylene glycol: ChCl (2:1) and glycerol: ChCl (2:1) showed satisfactory

pretreatment results: good lignin extractability, high residues recovery and glucose yield

(shown in Table 1 and Fig.1 (c)). The free hydroxyl groups in the polyalcohol/ ChCl

may interact with the free and etherified hydroxyl groups in the lignin, leading to the

87.6% and 71.3% lignin extractability. Compared to carboxylic acid/ ChCl, the absence

of carboxylic group in polyalcohol/ ChCl could avoid the acid degradation of corncob

and result in higher residues recovery (73.4% and 71.2%). In addition, the lower

viscosity of polyalcohol/ ChCl can facilitate the interaction between DESs and corncob.

The combined factors made the highest glucose yield (85.3% and 96.4%).

3.2 Characterizations of corncob

7
The crystallinity index (CrI) values of corncob were shown in Table 1. It was notable

that the CrI values of corncob residues changed little. According to our unpublished

experimental data, the CrI values of cellulose declined significantly after the same

pretreatment process. Put these two results together, it could be deduced that the relative

contents of cellulose in corncob residues increased owing to the removal of amorphous

hemicellulose and lignin.

From the SEM pictures of raw corncob and corncob residues shown in Fig.S1, it was

clear that after the pretreatment the surface of corncob became roughened and

unordered. The changes could be ascribed to that DESs destructed the supramolecular

structures of corncobs by removing the hemicellulose and lignin in the pretreatment

process.

FT-IR spectra of raw corncob and corncob residues were shown in Fig.S2. The

absorbance near 3433 cm-1 in the raw corncob attributed to the hydrogen bonds in the

cellulose. After the pretreatment, this peak shifted to lower wavelength (from 3433 to

3433-3415 cm-1), indicating that DESs broke the hydrogen bonds in the cellulose to

some extent by the formation of hydrogen bonds with cellulose. The intensity of peak at

1737 cm-1 in corncob residues decreased after treatment by carboxylic acid/ ChCl and

further disappeared after treatment by polyalcohol/ ChCl. These changes may be

attributed to the breakage of ether linkages between hemicellulose and lignin(Hou et al.,

2012; Zhang et al., 2010). Besides, the band at 834 cm-1(C-H out of plane bending of

syringyl content in lignin) decreased after the pretreatment, indicating

8
delignification(Jahan & Mun, 2007).These changes in the FT-IR spectra were consistent

with the XRD data and SEM pictures. They strongly suggested that the DESs broke the

spatial structures of corncob by removal the hemicellulose and lignin. As a result, it

overcame the chemical and biological recalcitrance of corncob and achieved the

enhanced enzymatic hydrolysis efficiency.

3.3 Effect of pretreatment temperature and time

The effects of pretreatment temperature on the corncob pretreated by lactic acid: ChCl

(2:1) were shown in Table 2 and Fig.S3. With the pretreatment temperature rising from

70 °C to 110 °C, the lignin extractability increased significantly (18.1%-95.5%) and the

corncob residue recovery decreased correspondingly (69.7%-47%). At the same time,

the higher temperature facilitated the subsequent enzymatic hydrolysis digestion of

corncob(Nagoor Gunny et al., 2014). However, when the pretreatment temperature

reached 90 °C, the glucose yield remained constantly (77.8%-79.7%). It could be

attributed to the balanced effects of greater losses of polysaccharides and enhanced

enzymatic hydrolysis digestion (Procentese et al., 2015). Therefore, 90 °C appeared to

be the optimal temperature.

The effects of pretreatment time on the corncob pretreated by lactic acid: ChCl (2:1)

were shown in Table 3 and Fig.S4. With the elongation of pretreatment time from 1.5 h

to 36 h, both the lignin extractability and subsequent enzymatic hydrolysis efficiency

increased significantly. This prolonged contact between DESs and corncob ensured

more complete interactions. However, when the pretreatment time stretched longer than

9
24 h, its effects on the enzymatic digestion were slight. Hence the 24 h was considered

as the moderate pretreatment time. It ensured sufficient interactions between DESs and

corncob while reduced the energy needed to heat and stir the mixtures in the

pretreatment process.

4. Conclusions

Three kinds of DESs were prepared and used to pretreat the corncob. The acid amount,

acid strength and the nature of hydrogen bond acceptors exerted great influence on the

pretreatment results. The polyalcohol/ ChCl behaved relatively high lignin extractability,

residue recovery and glucose yield. The consistency of characterization results indicated

that the DESs deconstructed the structures of corncob by removing the hemicellulose

and lignin. In addition, the optimal pretreatment temperature and time showed to be 24

h and 90 °C. Further preliminary experiments of DESs’ recycling need to be carried out

for the practical industrial applications.

Acknowledgements

The authors sincerely acknowledged the National Basic Research (973) special

preliminary study program (2014CB260408) for the financial support and special

thanks to Rongxin Su’s team for providing the corncob for the research.

10
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Tables and Figures

Fig.1. Time courses of enzymatic hydrolysis of corncob pretreated by DESs

(monocarboxylic acid/ ChCl (a), dicarboxylic acid/ ChCl (b) and polyalcohol/ ChCl

(c)).

(a)

(b)

13
(c)

14
 Table 1 Effects of chosen DESs on the delignification extent and enzymatic

hydrolysis of corncob.

DESs Lignin Residues CrI Initial saccharification Glucose

extractedb recoveryb rate(g/(L*h))c yieldc

untreated / / 31.6% 0.06 22.1%

monocarboxylic acid: lactic acid: ChCl (2:1)a 64.7% 49.6% 38.6% 1.85 81.6%

ChCl lactic acid: ChCl (5:1)a 77.9% 48.2% 37.4% 1.45 83.5%

lactic acid: ChCl (10:1)a 86.1% 46.7% 32.3% 1.16 83.2%

lactic acid: ChCl (15:1)a 93.1% 45.5% 30.7% 1.49 79.1%

glycolic acid: ChCl (2:1) 56.4% 50.5% 30.8% 1.37 67.3%

levulinic acid: ChCl (2:1) 43.0% 71.2% 32.0% 0.51 62.0%

dicarboxylic acid: malonic acid: ChCl (1:1) 56.5% 53.6% 29.5% 0.75 61.5%

ChCl(1:1) glutaric acid: ChCl (1:1) 34.3% 74.2% 30.8% 0.42 40.7%

oxalic acid: ChCl (1:1) 98.5% 53.1% 31.6% 0.17 45.2%

malic acid: ChCl (1:1) 22.4% 63.5% 31.7% 0.24 37.4%

polyalcohol: ethylene glycol: ChCl (2:1) 87.6% 73.4% 27.9% 0.49 85.3%

ChCl(2:1) glycerol: ChCl (2:1) 71.3% 71.2% 33.1% 0.67 96.4%

a The concentration of lactic acid is 98wt%.

b 0.3g corncob was incubated in 6g DESs magnetically stirred at 90°C for 24h.

c The enzymatic hydrolysis condition: 20mg corncob, 8ml citrate buffer (50mM, PH 4.8), 41mg

cellulase from Trichoderma reesei, 50°C, 24h, 180 rpm. The initial saccharification rate is

calculated based on the initial first hour.

15
 Table 2 Effects of pretreatment temperature on the corncob pretreated by lactic acid: ChCl (2:1)a.

Pretreatment temperature (°C) Lignin extractedb Residue recoveryb Initial saccharification rate(g/(L*h))c Glucose yieldc

untreated / / 0.06 22.1%

70 18.1% 69.7% 0.17 44.9%

80 31.1% 69.2% 0.60 73.6%

90 42.7% 59.1% 1.03 79.7%

100 65.8% 50.9% 1.29 78.0%

110 95.5% 47.0% 1.44 77.8%

a The concentration of lactic acid is 85wt%.

b 0.3g corncob was incubated in 6g DESs magnetically stirred at certain temperature for 24h.

c The enzymatic hydrolysis condition: 20mg corncob, 8ml citrate buffer (50mM, PH 4.8), 41mg

cellulase from Trichoderma reesei, 50°C, 24h, 180 rpm. The initial saccharification rate is

calculated based on the initial first hour.

16
 Table 3 Effects of pretreatment time on the corncob pretreated by lactic acid: ChCl (2:1)a.

Pretreatment time (h) Lignin extractedb Residue recoveryb Initial saccharification rate(g/(L*h))c Glucose yieldc

untreated / / 0.06 22.1%

1.5 8.6% 77.1% 0.09 25.9%

3 15.7% 77.8% 0.20 39.2%

6 20.9% 70.9% 0.10 49.4%

12 25.7% 67.3% 0.36 65.4%

24 42.7% 59.1% 1.03 79.7%

36 48.5% 56.2% 1.32 80.7%

a The concentration of lactic acid is 85wt%.

b 0.3g corncob was incubated in 6g DESs magnetically stirred at 90°C for certain time.

c The enzymatic hydrolysis condition: 20mg corncob, 8ml citrate buffer (50mM, PH 4.8), 41mg

cellulase from Trichoderma reesei, 50°C, 24h, 180 rpm. The initial saccharification rate is

calculated based on the initial first hour.

17
18
Highlights

Three kinds of DESs used in the pretreatment of corncob.

Improved delignification and enzymatic hydrolysis efficiency.

Removal of lignin and hemicellulose.

Optimization of pretreatment temperature and time.

19