Peiyi Li,Haozhe Lei,Boxing Jian,Ruiyan Liu,Miaomiao Zhou,Yumeng Wang,Hezhen Liu,Yun Wang,Bingyao Zhou

1.College of Bioresources Chemical and Materials Engineering,National Demonstration Center for Experimental Light Chemistry Engineering Education,Shaanxi University of Science and Technology,Xi'an,Shaanxi Province,710021,China

2.Key Laboratory of Paper Based Functional Materials of China National Light Industry,Xi'an,Shaanxi Province,710021,China

3.S h aanxi Pro vi nci al Ke y L ab orat or y o f P a permak i n g Tech n ol o g y and S peci al t y Pa per Development,Xi'an,Shaanxi Province,710021,China

4.Department of Chemical and Paper Engineering,College of Engineering and Applied Science,Western Michigan University,4651 Campus Dr,Kalamazoo,Michigan,49008,US

Abstract:In this study,a green,environmentally friendly method for rapid cellulose nanofribril(CNF)preparation with a significant cost advantage was developed.Pretreatment with a deep eutectic solvent(DES)synthesized from oxalic acid dihydrate and choline chloride(ChCl-O)was combined with various mechanical treatment methods to produce okara CNFs from agricultural waste,with different diameter distributions.The results showed that high-speed stirring produced CNFs with an average diameter of 27 nm.This method was advantageous because it consumed about 94%less energy than traditional high-pressure homogenization method.The DES recovery rate was more than 90%,and DES served as a highly effective treatment,indicating that DES pretreatment is an economical,convenient,and effective strategy for okara CNF preparation.

Keywords:okara;deep eutectic solvent(DES);cellulose nanofibril(CNF);high-speed stirring;high-pressure homogenization;recovery

1 Introduction

With the depletion of non-renewable petroleum resources,the development of high-value biologically sustainable resources is necessary.Natural cellulose,contained in various types of plants,is the most abundant,sustainable,and widely distributed biological resource in nature. Nanocellulose, or cellulose nanoparticles,comprises cellulose elements that are less than 100 nm[1]in at least one dimension.In recent years,cellulose nanofibrils (CNFs)and cellulose nanocrystals (CNCs)have been widely used in pharmaceuticals[2],biomedicine[3],electronic devices[4],and polymer nanocomposite reinforcement materials[5-7]due to their unique characteristics,such as large surface area[8-9],high modulus[10-12],highly porous structure[13],and biodegradability[14].CNFs can be separated from a variety of cellulose sources,including bleached kraft pulp,bleached sulfite pulp,bamboo pulp,balloon flower residue,straw,wheat straw,cocoa pod shell,bagasse,beet pulp,corncob,and Japanese cedar[15-16].In addition,raw banana,pineapple leaf fiber,and abaca leaf bast have also been used to produce CNFs[17].Although wood is the main raw material used for CNF production,industrial and agricultural waste have attracted attention in recent years as an alternative fiber source for CNF preparation because of their abundance,low cost,and renewability[18].Currently,China is a major soybean-processing country[19].As a major global food and oil crop,soybeans grown for protein,soy flour,and tofu production yield a large amount of by-product,called okara[20-21].Okara is a nutrient-rich biomass resource and its main components include dietary fiber(60%-70%),protein (13%-20%),fat(6%-19%),lignin(≈1%),and ash(3.5%-5.0%)[22].Okara also contains small amounts of soybean isoflavones,vitamins,soybean saponins,phytic acid,minerals,and other substances[23].In Asian countries,most okara is burned as waste or used as animal feed[24].Okara is derived from soybean cotyledons,which are the vegetative tissues of soybeans.Cotyledons are also called parenchyma,because they are composed of parenchyma cells.The fine fibers of parenchyma cells in the primary wall are loosely arranged and weakly bound together,facilitating separation for CNF preparation[25].The traditional method of CNF preparation is to remove lignin from the raw materials by acid or alkali pretreatment,followed by mechanical treatment[26-27].This method is not conducive to the recovery of lignin and chemicals and requires a lot of energy,failing to align with the current green manufacturing practices and environmental protection requirements.

Deep eutectic solvent(DES)synthesized by the reaction of two or more monomer coordination substances have a much lower melting point than either of the individual components and are considered green solvents[15,28-30].The use of a low-melting-point DES for okara CNF preparation can effectively reduce the impact on the structural properties of okara CNF[31-32].Furthermore,DESs can be effectively recycled and reused,significantly reducing the production cost and environmental burden[33-34].

The high-pressure homogenization process involves sending the fiber precipitation suspension into a container through a small nozzle,under high pressure.Due to the high speed,high pressure,and high fluid impact,high shear is generated in the suspension,reducing the fiber size to the nanometer scale.Although the microfibrillation degree proportionally increases with the number of homogenization steps and gradually decreases after reaching a certain threshold,the performance of the resulting CNFs remains constant[35].Ultrasonic treatment,another mechanical process,uses the cavitation caused by sound waves to generate strong mechanical vibrations that separate cellulose fibers[36].In this process,ultrasonic waves are transmitted through a liquid to generate tiny cavitation bubbles.When the cavitation bubbles form,expand,and finally burst,a strong mechanical shock is generated;this force is used to prepare CNFs.Highspeed stirring,a third mechanical process,involves the use of a high shear force generated by the rotation of the stirring blade to decompose the fiber cell walls and disrupt hydrogen bonding,which reduces the fiber size.These methods can all be used to produce CNFs but involve different levels of energy consumption[37-38].

Currently,high-pressure homogenization is the most commonly used method for CNF preparation.However,this method is not conducive to large-scale production because of its high energy requirement[39].In this study,we used DES for okara cellulose pretreatment and compared the performance of different mechanical dissociation methods for CNF production.Then,we evaluated the DES recovery rate and determined whether recovered DES could be recycled and used for okara pretreatment.This study proposes a green,environmentally friendly method(Fig.1)with low energy consumption,in which DES pretreatment is combined with high-pressure homogenization, ultrasonication, and high-speed stirring for the production of CNFs from okara.

Fig.1 Schematic process for preparing okara CNFs

2 Experimental

2.1 Materials

Okara was obtained from a soymilk factory in Heilongjiang Province, China. Choline chloride(C5H14ClNO)was purchased from Adamas Beta Chemical Reagent Co.,Ltd.(Shanghai,China).Oxalic acid (C2H2O4·2H2O) was purchased from Tianjin Zhiyuan Chemical Reagent Co.,Ltd.(China).All chemicals were of analytical grade and used without further purification.

2.2 DES pretreatment of okara cellulose

The DES was prepared by mixing choline chloride and oxalic acid in a 100-mL round-bottom flask at a molar ratio of 1:1.The reaction was conducted at a constant temperature of 80℃with continuous magnetic stirring until the solution became clear and transparent.The resulting DES was then mixed with the okara in a conical flask at a ratio of 1:20(w/V).The reaction was performed at 100℃for 30 min at a stirring speed of 40 r/min.At the end of the experiment,a reversephase solvent(distilled water)was used to precipitate the cellulose.The solid substrate was then washed with distilled water until the filtrate became neutral.The collected cellulose was made into a 2%suspension using distilled water,and the cellulose suspension was used as a precursor solution for the subsequent highpressure homogenization,ultrasonic treatment,or highspeed stirring.

2.3 Component analysis of okara cellulose

The water content and water activity of the fat component in okara raw material and DES pretreated okara cellulose samples were determined based on GB 5009.3—2010 and the water activity was measured using Soxhlet extraction;the procedure used to measure the activity of various adipose tissue proteins was mainly based on GB/T 14772—2008 and the Soxhlet extraction procedure.Measurement of activity via chitosan extraction,measurement of activity in adipose tissue proteins in fine samples,and measurement of cellular adipose content were mainly based on NYT 2007-2011 and the Dumas combustion method.Determination of ash content was mainly based on GB 5009.4—2010 and the combustion method;determination of holocellulose content was mainly based on GB/T 2677.10—1995 and the sodium chloride method.α-Cellulose content was determined based on GB/T 744.10—1989 and the sodium hydroxide method.

2.4 High-pressure homogenization

A 2%cellulose suspension was homogenized 15 times using a high-pressure homogenizer(AH-100D,ATS Engineering Limited,China,rated power:1500 W)at a pressure of 60 MPa. The duration of each homogenization period was 2 min.

2.5 Ultrasonication

A 2%cellulose suspension was dissociated for 30 min using a cell shredder(JY92-IIDN,Ningbo Xinzhi Biological Technology Co.,Ltd.,China,rated power:250 W)at an ultrasonic frequency of 25 kHz and an output power of 800 W.All samples were then freezedried at-47℃for 12-24 h.

2.6 High-speed stirring

A 2%cellulose suspension was poured into a highspeed mixer(JYL-CO12,Joyoung Company Limited,China,rated power:900 W)and processed for 3/6 min at 3000/6000 r/min. The mechanical energy consumption(EC,kWh)for the different mechanical treatment methods was determined using Eq.(1).

whereT(h)is the mechanical processing time,andP(W)is the output power of each machine at the specified setting.

2.7 Recovery of DES from the spent liquor

The DES was recovered by rotary evaporation.The evaporation temperature was 60℃,and the rotation speed was 80 r/min.

2.8 Characterizations

2.8.1 Scanning electron microscopy(SEM)

SEM(S-4800,Hitachi,Japan)was used to visualize the structures of the CNFs.SEM images were recorded at a low voltage(5 kV)and a working distance of 5 mm.The diameter of the CNFs was analyzed using Nano Measurer software(Department of Chemistry,Fudan University,China).

2.8.2 Fourier transform infrared(FT-IR)spectroscopy

FT-IR (Vertex70,Bruker,Germany)was used to identify the structural changes in the okara CNFs.Dried okara cellulose was mixed with KBr at a mass ratio of 1:100 and directly ground in an agate grinder.A certain amount of the mixture was compressed for 2 min at a pressure of 10 MPa.The tablets were placed in the infrared spectrometer and scanned 40 times from 500 cm-1to 4000 cm-1at a wave number resolution of 1 cm-1.Similarly,choline chloride and oxalic acid were dried and mixed with KBr at a mass ratio of 1:100,and the DES solution was analyzed using a liquid cell.

2.8.3 Recovery rate of DES and yield of cellulose

The recovery rate of DES was defined as the ratio of the amount of DES recovered from the spent liquor to the original amount used for okara treatment.The yield(%)of cellulose extract from recycled DES was determined using Eq.(2).

whereM1(g)is the mass of raw okara,andM2(g)is the mass of the DES-treated cellulose after freeze-drying.Both analyses were performed in triplicate for reproducibility.

3 Results and discussion

3.1 Effect of high-pressure homogenization on CNF morphology and diameter distribution

Fig.2(a)and Fig.2(d)show the morphology and diameter distribution of okara CNFs prepared by highpressure homogenization. As a conventional mechanical treatment method, high-pressure homogenization is usually used to mechanically dissociate the okara fibers.During the dissociation process,okara fibers gradually converted to CNFs.However,the mechanical energy consumption during high-pressure homogenization is very high,and the machine is prone to blockage,which negatively affects the CNF yield. During the high-pressure homogenization process,the pressure is continuously increased to 60 MPa to increase the mechanical shear force on the okara fiber,in order to fully achieve okara cellulose nanofibrillation.In previous research[25],the morphology of the prepared okara CNFs according to the number of high-pressure homogenization rounds was discussed in detail. Fifteen high-pressure homogenization rounds was determined to be the optimal number.The diameter distribution of CNFs prepared using fewer than 15 high-pressure homogenization rounds was not uniform,and more than 15 rounds produced no obvious difference compared to 15 rounds.Fig.2 illustrates that the diameter distribution of CNFs prepared by highpressure homogenization varied from 5 nm to 45 nm(relatively concentrated in the range of 25-30 nm),with an average diameter of 30 nm. The composition of okara pretreated with DES is shown in Table 1.

Table 1 Composition of okara raw material and okara pretreated with DES

3.2 Effect of ultrasonic treatment on CNF morphology and diameter distribution

Ultrasonic treatment was used to mechanically dissociate 2%cellulose for okara CNF preparation.The SEM image and diameter distribution presented in Fig.2(b)and Fig.2(e)indicate that okara cellulose formed a nanofibrous structure after 30 min of ultrasonic treatment.Individual CNFs with diameters of less than 50 nm can be clearly observed in the SEM image, indicating high nanofibrillation efficiency.The CNFs also had a more uniform diameter distribution than those obtained by highpressure homogenization. Compared with highpressure homogenization,the ultrasonic dissociation process required less mechanical energy,and the sample loss was small.Moreover,the ultrasonic treatment procedure was simple and evenly dispersed the suspension.The average diameter of the CNFs obtained after 30 min of ultrasonic treatment was 28 nm.

Fig.2 Morphology and diameter of CNFs prepared by high-pressure homogenization(a,d),ultrasonic treatment(b,e),and high-speed stirring(c,f)

3.3 Effect of high-speed stirring on CNF morphology and diameter distribution

A single-factor experiment was performed to determine the effect of stirring speed and mixing time on the average diameter of the high-speed stirring produced CNFs.Table 2 presents the results.The macroscopic morphology of the samples during the experiment exhibited different degrees of dispersion when different stirring durations were used.Less than 3 min of highspeed stirring was insufficient to fully disintegrate the cellulose,and some granular lumps were still observed.As shown in Table 2,when the stirring speed was 3000 r/min,the average diameter of the CNFs decreased from 44 nm to 27 nm as the stirring time increased from 3 min to 6 min.However,when the stirring speed was 6000 r/min,increasing the stirring time did not significantly change the average diameter of the CNFs.When the stirring time was 6 min,both stirring speeds resulted in similar CNF diameters,of～27 nm.Therefore,based on the energy consumption,CNF preparation via high-speed stirring was conducted at 6000 r/min for 3 min.

Table 2 Effect of stirring speed and mixing time on diameter of CNFs prepared by high-speed stirring

Fig.2(c)and Fig.2(f)show the morphology and diameter distribution of okara CNFs prepared by highspeed stirring.The CNFs prepared by high-speed stirring had an average diameter of 27 nm which accords with the sing-foutor experiment results shown in Table 2.

In summary,three different mechanical methods were used for okara cellulose treatment:high-pressure homogenization,ultrasonic treatment,and high-speed stirring.Nano Measurer software was used to analyze the SEM images to obtain the corresponding size distribution under different mechanical dissociation conditions.Thus,the properties of CNFs used for different applications can be controlled by adjusting the mechanical treatment parameters.This study also provides a variety of mechanical treatment options for the preparation of okara CNFs.

Table 3 lists the CNF diameter distributions for each mechanical treatment method.The energy consumed by the different mechanical treatment methods was calculated using Eq.(1).As shown in Table 3,highspeed stirring consumed much less energy than the other two mechanical treatment methods.Moreover,the processing time of the high-speed stirring method was much shorter,possibly because the okara raw material has a very low degree of crystallinity,owing to the large number of amorphous regions[19].Therefore,the energy required to separate cellulose fibers from okara is much lower than that required to separate fibers from wood raw materials[33].Thus,high-pressure homogenization,with high energy consumption,is not an appropriate method for CNF preparation.Highspeed stirring was also more energy-efficient than ultrasonic treatment,probably because the probe of the ultrasonication instrument releases a large amount of energy.Okara cellulose is defibrillated at a very high speed,forming CNFs that are physically entangled;thus,fiber agglomeration hinders defibrillation during ultrasonication.However,this problem does not occur with high-speed stirring. Therefore, the CNFs prepared by high-speed stirring(6000 r/min,3 min)were used for subsequent experiments.

Table 3 Comparisons of different mechanical treatment methods

3.4 FT-IR analysis of DES

The infrared spectrum of DES,shown in Fig.3,shows the following typical peaks.The absorption peakat 3272 cm-1is associated with a nonlinear stretching vibration[40].The two absorption peaks at 3020 cm-1are associated with the C—N and C—H bending vibrations,while the peak at 1637 cm-1is associated with the C—O and C—N stretching vibrations.The peak at 1480 cm-1is associated with the C—H bending vibration,the peak at 1410 cm-1is associated with the bending vibration of O—H,the peak at 1285 cm-1is associated with the C—N stretching vibration,and the peak at 870 cm-1is associated with the N—H bending vibration[41].

Fig.3 FT-IR spectra of oxalic acid,choline chloride,and DES

In the oxalic acid spectrum,the absorption peaks at 1128 and 1259 cm-1represent the C—C stretching vibration.The absorption peaks at 1446 and 1685 cm-1represent the C—O stretching vibration. The characteristic peak of the hydroxyl group stretching vibration is visible at 3429 cm-1.Compared with that in the oxalic acid spectrum,the peak representing the hydroxyl group stretching vibration in the DES spectrum clearly shifts and forms a broad peak,indicating the formation of intermolecular hydrogen bonds between choline chloride and oxalic acid.In summary,the results indicated the formation of a eutectic solvent(ChCl-O)from choline chloride and oxalic acid.

Fig.4 shows the FT-IR spectra of CNFs prepared from raw DES and from the DES recovered by rotary evaporation.The two categories of CNFs exhibit basically the same characteristic peaks.The absorption peak at 3411 cm-1corresponds to the stretching vibration of the hydroxyl group in okara CNFs,that at 1686 cm-1corresponds to the C—O stretching vibration in okara CNFs,and that at 1264 cm-1corresponds to the stretching vibration of C—N in okara CNFs.The stretching vibration associated with C—N in the okara CNFs produced an absorption peak at 783 cm-1due to the bending vibration of the O—H and C—H bonds in the CNFs.Thus it is concluded that the recovered DES is potent to dissolve okara cellulose there by to prepare CNFs.

Fig.4 FT-IR spectra of CNFs obtained from raw and recovered DESs

3.5 Effect of cycle number on DES recovery rate and cellulose yield

As shown in Fig.5,the recovery rate of the DES reached 90%,and the yield of okara cellulose was approximately 20%after the first three cycles.Unlike softwood thermomechanical pulp,okara raw material does not require a pulping process.Due to the 50%pulp yield after the pulping process,the yield after using DES pretreatment to prepare lignocellulose is approximately 28%[42],which is close to the yield of okara CNFs.In other words,the yield obtained after processing wood to wood pulp and then wood pulp to CNFs is similar to that obtained after processing raw okara directly to CNFs.As shown in Table 1,the cellulose content of okara significantly increased after DES pretreatment(theα-cellulose content increased from 62.9%to 92.6%).This shows that using a ChCl-O eutectic solvent system to pretreat okara is an effective method for purifying okara cellulose.With an increase in the number of cycles,the DES recovery rate decreased from 92%to 84%.This may be due to incomplete separation of DES from okara cellulose during the suction filtration process.Fresh DES was added to the reaction system to compensate for the DES loss after four cycles.The decrease in cellulose yield may be due to DES partially dissolving the protein and fat components in the okara raw material;the rotary evaporation cannot remove these components, resulting in a slightly inefficient pretreatment process.

Fig.5 DES recovery rate and cellulose yield

4 Conclusions

Through scanning electron microscopy(SEM)and diameter distribution analysis,it was concluded that high-pressure homogenization,ultrasonication,and high-speed stirring combined with deep eutectic solvent(DES)pretreatment can be used to prepare cellulose nanofibrils(CNFs)with an average diameter of less than 50 nm from okara.The effects of different mechanical treatments on CNF morphology and diameter distribution were evaluated.Compared with the high-pressure homogenization method,high-speed stirring demonstrated a prominent energy consumption advantage, providing a new avenue for CNF preparation.The average diameter of the prepared CNFs was 27 nm.The DES could be recycled up to three rounds of use,and the recovery rate exceeded 90%.The yield of okara cellulose was 20.1%-24.9%.

Acknowledgments

This research was funded by the Key Laboratory Research Open Fund of Shaanxi Provincial Department of Education(Grant No.17JS017),and the Research Initial Fund of Shaanxi University of Science and Technology(Grant No.BJ15-29).

Author contributions

Conceptualization, Li P Y and Wang Y M;experimental design,investigation,Lei H Z,Wang Y,Liu RY,Jian B X,and Zhou M M;mainly wrote the manuscript with contributions from Lei H Z and Liu H Z.All authors have read and agreed to the published version of the manuscript.

Data availability statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Conflicts of interest

The authors declare no conflict of interest.