An environmentally friendly method for enzyme-assistedpreparation of microfibrillated cellulose (MFC) nanofibers

M. Henriksson a, G. Henriksson a, L.A. Berglund a, T. Lindstrom b,*

a Department of Fibre and Polymer Technology, Royal Institute of Technology, SE-10044 Stockholm, Swedenb STFI-Packforsk AB, Box 5604, SE-114 86 Stockholm, Sweden

Received 20 February 2007; received in revised form 4 May 2007; accepted 20 May 2007Available online 8 June 2007

Abstract

Microfibrillated cellulose nanofibers (MFC) provide strong reinforcement in polymer nanocomposites. In the presentstudy, cellulosic wood fiber pulps are treated by endoglucanases or acid hydrolysis in combination with mechanical shear-ing in order to disintegrate MFC from the wood fiber cell wall. After successful disintegration, the MFC nanofibers werestudied by atomic force microscopy (AFM). Enzyme-treatment was found to facilitate disintegration, and the MFC nano-fibers produced also showed higher average molar mass and larger aspect ratio than nanofibers resulting from acidicpretreatment.! 2007 Elsevier Ltd. All rights reserved.

Keywords: Microfibrillated cellulose; Nanofibers; Endoglucanase; Hydrolysis; Degree of polymerization

1. Introduction

Microfibrillated cellulose nanofibers (MFC) dis-integrated from wood pulp can be used in polymernanocomposites of high mechanical performance[1]. These nanocomposites show a Young’s modulusapproaching 20 GPa. The reported strength is alsohigh, although the data should be interpreted withcare since flexural strength of brittle materialsdepends strongly on specimen geometry. Still, nano-composites based on MFC nanofibers from woodpulp is a new class of materials with potential forexceptionally high mechanical performance.

MFC nanofibers are expected to show high sti!-ness since the Young’s modulus of the cellulosecrystal is as high as 134 GPa [2]. Its polymer mole-cules crystallize in extended-chain conformationduring biosynthesis and form microfibrils with a lat-eral dimension of around 4 nm [3]. In wood, cellu-lose microfibrils show preferred orientation andoften in a direction close to the axial fiber direction.Wood microfibrils form aggregates of 15–18 nmthickness in cellulosic wood pulp fibers [3].

Cellulosic pulp fibers can be disintegrated intofine fragments by mechanical refining methods.However, such methods tend either to damage themicrofibril structure by reducing molar mass anddegree of crystallinity or fail to su"ciently disinte-grate the pulp fiber. Herrick et al. [4] and Turbaket al. [5] reported on a method for producing

0014-3057/$ - see front matter ! 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.eurpolymj.2007.05.038

* Corresponding author. Fax: +46 8 214235.E-mail address: tom.lindstrom@stfi.se (T. Lindstrom).

European Polymer Journal 43 (2007) 3434–3441

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EUROPEANPOLYMERJOURNAL

microfibrillated cellulose. The MFC typically con-sist of disintegrated microfibril aggregates with alateral dimension in the scale of tens of nanometers.The method was to pass a dilute cellulosic woodpulp fiber/water suspension through a mechanicalhomogenizer, where a large pressure drop facili-tates microfibrillation. The function of a homoge-nizer is explained in detail by Rees [6]. When acellulosic pulp fiber suspension is homogenized,the procedure is often repeated several passes inorder to increase the degree of fibrillation [1,4]. Ahigher number of passes obviously increases theenergy required for disintegration. In order to facil-itate disintegration, one may reduce fiber length bymechanical cutting [4] or the fiber cell wall can betreated and embrittled by acid hydrolysis [7] priorto homogenization. However, acid hydrolysis cansignificantly lower the molecular weight of celluloseand result in short, fibrous crystallites in the form ofmicrocrystalline cellulose (MCC) [8]. As a conse-quence, acid hydrolysis may reduce the reinforce-ment e!ect [9].

Nakagaito and Yano demonstrated improveddisintegration as the number of passes through thehomogenizer was increased to 30 times [1]. How-ever, MFC nanofibers were recently disintegratedin a more e"cient process where 2,2,6,6-tetrameth-ylpiperidine-1-oxyl (TEMPO) oxidation was utilizedas a pretreatment step prior to mechanical treat-ment [10]. The use of enzymatic pre-treatmentmay also lower processing cost by lowering thenumber of passes through the homogenizer, andhas advantages from the environmental point ofview as compared with chemical methods.

In nature, cellulose does not degrade by singleenzymes. Instead aerobic fungi like Trichoderma-,Phanerochaete- and Aspergillus-species degrade cel-lulose by excreting a mixture of independently act-ing, but cooperating hydrolytic enzymes [11]. Sucha set of cellulases can contain seven or more di!er-ent enzymes belonging to di!erent protein families[12], but at least four functional types of cellulasescan be recognized: A- and B-type cellulases aretermed cellobiohydrolases, and are able to attackalso highly crystalline cellulose, whereas C- andespecially D-type cellulases, endoglucanases with acommon name, generally require some disorder inthe structure in order to attack the cellulose[13,14]. Cellobiohydrolases and endoglucanasesshow strong synergetic e!ects [11,15]. In the presentstudy, environmentally friendly pretreatment ofhigh cellulose content wood pulp fibers with a pure

C-type endoglucanase was investigated in order tofacilitate disintegration of MFC nanofibers.

2. Experimental

2.1. Materials

Two di!erent commercial bleached wood sulph-ite pulps based on Norway Spruce (Picea abies)from Domsjo Fabriker AB, Sweden, were used.One softwood sulphite pulp intended for specialpaper qualities, termed special paper pulp (SPP),delivered both as never dried and dried pulp, andone softwood dissolving pulp (DSP), delivered dry.The pulp fibers are about 40 lm wide and more than1 mm long. The pulps are close to lignin- andextractives-free and carbohydrate analyses showedthat SPP contained 85% cellulose while the cellulosecontent of DSP was 93%. The rest is mainlyhemicelluloses. The enzyme used is Novozym 476,an endoglucanase manufactured by NovozymesA/S, Denmark. The poly(diallyldimethylammoniumchloride) (poly-DADMAC) used in atomic forcemicroscopy, was Alcofix-109 purchased fromCDM, Sweden. Prior to use the sample was frac-tionated and the fraction above 500 k was used.This fraction had Mw 870,000.

2.2. Pretreatment procedures

The fibers were treated by four di!erent meth-ods. Three of the methods included beating in aPFI-mill, manufactured by HAM-JERN, Norway,according to EN 25 264-2:1994 [16] with one modi-fication: 40 g fibers diluted with water to a totalweight of 300 g (13.3% dry content) was used.

• The enzymatically treated fibers were firstmechanically beaten 1000 revolutions in a PFI-mill in order to increase the swelling in waterand make the cellulose more accessible for theenzyme. Enzymatic treatment was performedas follows: 3% pulp was dispersed in 50 mMtris(hydroxymethyl)aminomethane/HCl bu!erwith pH 7 and 3%, 1.5%, 0.5% and 0.02%enzyme, related to the weight of the pulp (SPP-3%, DSP-3%, DSP-1.5%, DSP-0.5%, and DSP-0.02%). The fibers were incubated at 50 "C for2 h, washed on a Buchner funnel, thereafterincubated again at 80 "C for 30 min, to stop the

M. Henriksson et al. / European Polymer Journal 43 (2007) 3434–3441 3435

activity of the enzymes, and then washed again.The fibers were finally beaten 4000 revolutionsin a PFI-mill.

• No-enzyme references termed SPP-ref, and DSP-ref were also prepared, and treated in the sameway as the enzymatically treated pulp, but with-out exposure to enzymes.

• DSP samples were also treated with the enzy-matic step replaced by mild hydrolysis and termedDSP-MH. A 3% fiber suspension was adjusted topH 1 with HCl and incubated at 50 "C for 1 h.After incubation, the fibers were washed on aBuchner funnel. DSP-MH was finally beaten4000 revolutions in a PFI mill.

• A much stronger hydrolysis was also performedto form DSP-SH. This method did not includeany beating. A 7% pulp suspension was firstswollen in 3% NaOH at 50 "C for 10 min andthen washed. Thereafter the hydrolysis was per-formed with 7% pulp concentration in 2.5 MHCl at 90 "C for 2 h. After incubation, the fiberswere again washed, but not beaten.

2.3. Homogenization

After treatments, 2% fiber suspensions inwater were subjected to the homogenizing actionof a high-pressure slit homogenizer, LaboratoryHomogenizer 15 M, Gaulin Corp., USA. The fiberswere passed 20 times through the slit. Processingwas performed in a similar way as described inRef. [4]. E!orts were made to keep the pressure con-stant for the di!erent test-runs. During the process,the suspension viscosity increased with increasingnumber of passes. Initially the suspension was atroom temperature but the temperature increased

with increasing number of passes to 70 "C. Herricket al. [4] found that increasing the temperature from20 "C to 70–80 "C facilitates homogenization.

2.4. Estimation of cellulose molecular weight

The molecular weight of cellulosic fibers can beestimated from an average intrinsic viscosity value.The measurements were performed on dissolvedfibers, prior to homogenization, according toSCAN-CM 15:99 [17] with cupriethylendiamine assolvent. Fibers were weighed in the wet state. Thewater content of the sample was taken into accountwhen adding water to the samples. Two samples weremade for each set of fibers. The intrinsic viscosity, g(ml/g), is related to the degree of polymerization(DP) and an empirical relationship for this poly-mer-solvent system is suggested as g = 2.28 DP0.76

[18]. The presence of any hemicelluloses is not consid-ered when calculating DP. The DP data after homog-enization for DSP-0.5% and DSP-0.02% reported inTable 1, were obtained from samples homogenized12 passes in a di!erent type of homogenizer, aMicro-fluidizer M 110-EH, Microfluidics Inc., USA.

Molecular weight distribution was estimatedfor three dissolving pulp samples (DSP, DSP-1.5%, DSP-SH) by size exclusion chromatography(SEC). The solvent system used was dimethylaceta-mide (DMAc)/LiCl. Pullulan was used for calibra-tion and a refractive index detector was used.Water was removed from the MFC suspensionsby solvent exchange with methanol and DMAcbefore being dissolved in DMAc/LiCl containingethylisocyanate. Sample preparation and measure-ments were carried out at MoRe Research AB,Sweden.

Table 1DP estimated at di!erent stages in the process and statement of whether the pulp was possible to homogenize into nanofibers or not(yes/no)

Pulp Method DP Possible to homogenize? DP after homogenization

SPP special paper pulp SPP 2930 – –SPP-ref, no-enzyme reference 2620 No –SPP-3%, 3% enzyme 910 Yes 640

DSP dissolving pulp DSP 1280 – –DSP-ref, no-enzyme reference 1200 No –DSP-3%, 3% enzyme 540 Yes 340DSP-1.5%, 1.5% enzyme 640 Yes 480DSP-0.5%, 0.5% enzyme 600 Yes 470DSP-0.02%, 0.02% enzyme 910 Yes 740DSP-MH, mild hydrolysis 1100 No –DSP-SH, strong hydrolysis 170 Yes Not measured

Please note that with the exception of DSP-SH, all treated pulps were beaten in a PFI-mill prior to homogenization (see Section 2).

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2.5. Microscopy

Optical micrographs were obtained by placing adrop of fiber suspension of low concentration on aglass slide.

Scanning electron microscopy (SEM) imageswere captured using a Hitachi s-4300 field emissionSEM operating at 2 kV. The sample was mountedonto a substrate with carbon tape and coated witha thin layer of gold. The image in Fig. 3 wasobtained from a sample of DSP-0.5% homogenized12 passes in a di!erent type of homogenizer, aMicrofluidizer M 110-EH, Microfluidics Inc., USA.

Atomic force microscope (AFM) images weremeasured using a Nanoscope IIIa, from VeccoInc., USA. The scan was performed in tappingmode in air with a standard silica cantilever. Ampli-tude images are presented. Silica plates were used assubstrate for the samples. The surfaces of the silicaplates were first oxidized at 1000 "C for 3 h and thenhydroxylized in a plasma oven to ensure that thesurfaces were hydrophilic. Thereafter the surfacecharge of the plates was modified by placing theplates into a 100 mg/l poly-DADMAC/1 mM NaClsolution for 10 min. The plates were rinsed with1 mM NaCl to remove excessive poly-DADMAC.The plates were placed in 0.01% fiber suspensionfor 5 s and rinsed with 0.5 ml, 1 mM NaCl solution.The samples were dried at room temperature priorto AFM analysis.

3. Results and discussion

3.1. Physical appearance of pretreated pulp

Two major grades of cellulosic fibers (SPP andDSP) were divided into three di!erent groups, thefirst pretreated with a commercial endoglucanaseat di!erent concentrations (SPP-3%, DSP-3%,DSP-1.5%, DSP-0.5%, and DSP-0.02%), the secondpretreated with mild or strong acid hydrolysis(DSP-MH, DSP-SH), and the third was not chemi-cally treated (SPP-ref, DSP-ref). The fibers werethen subjected to mechanical beating, except fromDSP-SH, and homogenization in order to disinte-grate MFC nanofibers. Beating is used in industryin order to increase the flexibility of the fiber as wellas its surface roughness. The physical appearance ofthe fibers after the di!erent treatments, was studiedby optical microscopy, see Fig. 1. Initially the fibersare about 40 lm wide and more than 1 mm long.Estimates of DP for the materials are presented in

Table 1. Fig. 1a presents the DSP-ref after mechan-ical beating in a PFI-mill. The length is unchangedalthough surface fibrillation and presence of finematerial (cell wall fragments) is observed. The e!ectof enzymatic treatment is apparent by comparisonof Fig. 1a and b. The fiber length is somewhatreduced for DSP-3% and the extent of fine materialis increased. However, corresponding observationsof DSP-0.02% show only limited fiber shorteningcompared with DSP-3%. For the case of DSP-3%,there is also a reduction in DP from 1280 to 540with the pretreatment, see Table 1. However, lowerenzyme concentration (1.5%, 0.5%, and 0.02%)results in an estimated DP of 640, 600, and 910,respectively. The high DP for DSP-0.02% is interest-ing. As high DP as possible is desirable for MFCnanofibers, since this is expected to increase theinherent tensile strength of the cellulose [19]. Proce-dures with low enzyme concentration are thereforepreferable from this point of view, since the reduc-tion in molecular weight is smaller. The stronglyhydrolyzed fibers in Fig. 1c show substantiallyreduced fiber length and DP (see Table 1). Thismaterial is closer to microcrystalline cellulose [8]and the resulting microfibrils are expected to havelow aspect ratio.

3.2. Ease of homogenization

Prior to homogenization, the two-phase natureof the water–fiber suspension is apparent since sed-imentation occurs rapidly for all of the suspensions.After successful homogenization, suspensions arestable. The viscosity also increases substantiallywith the number of passes through the homoge-nizer. This is related to the increasing degree ofMFC nanofiber disintegration from the cellulosicfiber cell wall.

The purpose of acid or enzymatic pretreatment isto facilitate microfibrillation during the homogeni-zation procedure. From Table 1, it is apparent thatnot all types of cellulosic fibers were possible tohomogenize. However, all fibers subjected toenzyme pretreatment were successfully homoge-nized. In several other cases, the fibers blocked theslit in the homogenizer. As a consequence, the flowof the suspension was stopped and microfibrillationwas prevented. When fibers agglomerate and blockthe slit, the equipment must be emptied, demountedand cleaned before the processing may continue.This occurred repeatedly with some cellulosic fibersand they were impossible to homogenize. There was

M. Henriksson et al. / European Polymer Journal 43 (2007) 3434–3441 3437

no di!erence in ease of homogenization between thespecial paper pulp SPP (85% cellulose) and dissolv-ing pulp DSP (93% cellulose). In addition, bothnever dried and dried SPP pulps were tried sincenever dried pulp has been reported to facilitatehomogenization [4], but no such e!ect wasobserved.

Previous work showed that MFC nanofiberswere easier to disintegrate from mechanically cutcellulosic fibers [4]. However, the specific procedureused is impractical, whereas the present PFI-millingand other mechanical beating methods can be usedindustrially. The observed fiber blockage problemsare caused by fiber agglomeration. The tendencyfor agglomeration is reduced with reduced fiberlength. Although the present cellulosic fibers of low-est molar mass are easy to disintegrate, molar massis not the major parameter influencing ease ofhomogenization. This is demonstrated by the fol-lowing case. The DSP-3% could not be homoge-nized directly after enzyme treatment. However,when this pulp was subjected to PFI-milling, withlittle change in DP but significant fiber shortening

and cell wall disintegration, homogenization waspossible. Ease of disintegration in the homogenizertherefore is influenced by fiber length, althoughother factors are also important. The combinationof enzyme treatment and mechanical beating cer-tainly provides an advantageous starting state forfiber disintegration. It is possible that the endoglu-canase treatment increases cell wall swelling in thewater suspension and, as a consequence, the cellwall is more easily disintegrated into MFC nanofi-bers as it is subjected to mechanical shearing duringthe homogenization process.

It was observed that cellulosic fiber suspensionspossible to homogenize, showed slower agglomera-tion and sedimentation in the inlet reservoir. Thepresence of smaller fiber fragments in the enzymat-ically treated and strongly hydrolyzed pulps seemedto act as a suspending agent. This is in agreementwith previous experience [4] where pulp dispersionin a 2% suspension of uncut pulp fibers wasimproved by the addition of MFC. The stabilityof the cellulosic fiber suspension therefore appearsto be another factor facilitating homogenization.

Fig. 1. Optical microscopy images of (a) DSP-ref, no-enzyme reference of dissolving pulp after beating, (b) DSP-3%, enzyme-treateddissolving pulp (3%) after beating, and (c) DSP-SH, strongly acid hydrolyzed dissolving pulp. Scale bar is 100 lm.

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3.3. After homogenization

There is a significant decrease in DP duringhomogenization, see Table 1. For instance, DP ofDSP-3% decreases from 540 to 340 after homogeni-zation (20 passes through the homogenizer). Thecellulosic fiber with the higher original DP (SPP)shows higher final DP. Herrick et al. [4] and Turbaket al. [5] observed a 27% reduction in cellulose DPdue to homogenization, similar to the present case.Boldizar et al. [7] found e!ects of DP on the rein-forcement e"ciency of MFC nanofibers in polyv-inylacetate. Higher DP gave better mechanicalproperties, although DP in the range 200–400 gavesignificant reinforcement e!ects. The MFC nanofi-bers produced using the present enzymatic methodswere therefore expected to function well as compos-ite material reinforcement. This was confirmed in arecent study [20].

Fig. 2 shows the molecular weight distributionfor DSP, DSP-1.5%, and DSP-SH. DSP-SH isseverely degraded. A shift in molecular weight dis-tribution towards lower values is also apparent forthe enzymatically treated molecules, although thehigher molecular weight fractions are well pre-served. Weight average molecular weights are pre-sented in the text of Fig. 2.

The MFC nanofiber samples were studied bySEM and AFM. In Fig. 3 there is a SEM imageof DSP-0.5%. The original pulp fiber structure is

destroyed and the resulting nanofibers are of largeaspect ratio. AFM images of the DSP-3% nanofi-bers show highly microfibrillated nanofibers of largeaspect ratio (length/diameter), see Fig. 4a. Thenanofibers are about 15–30 nm wide and severalmicrometer long. There is also a fraction of shorternanofibers with thickness of about 5–10 nm. Theacid hydrolyzed DSP-SH nanofibers show muchsmaller aspect ratio, see Fig. 4b. These nanofibershave thickness of about 5–15 nm. Battista demon-strated strong correlation between DP and lengthof cellulose nanofibers [8]. The DSP-SH also con-tains larger fragments of cellulosic fiber cell walls.At higher magnification, the DSP-SH nanofibersvaried significantly in thickness, whereas MFC fromDSP-3% showed more uniform nanofiber thickness.

3.4. Mechanisms facilitating disintegration of MFCnanofibers

Acid hydrolysis facilitates disintegration of MFCnanofibers from cellulosic pulp fibers. Boldizar et al.suggested that the reduced molar mass associatedwith hydrolysis embrittles the cell wall and facili-tates disintegration [7]. However, the reduction inmolecular weight appears di"cult to control. Inthe present study, the resulting MFC also shows awide distribution in aspect ratio and diameters.

Endoglucanase pretreatment in combinationwith mechanical beating greatly facilitates disinte-gration of MFC nanofibers from cellulosic woodfibers by mechanical shearing of a 2% fiber suspen-sion (homogenization). Although fiber cutting dur-ing the mechanical beating stage contributes to

Fig. 3. SEM image of freeze-dried DSP-0.5% after homogeniza-tion. Scale bar is 30 lm.

Fig. 2. Molecular weight distributions for MFC nanofibers basedon dissolving pulp. Samples are termed DSP, dissolving pulp,DSP-1.5%, enzyme treated dissolving pulp (1.5%) after homo-genization, and DSP-SH, strongly acid hydrolyzed dissolving pulpafter homogenization. The weight average molecular weights areDSP: 285,700, DSP-1.5%: 280,000, and DSP-SH: 63,600.

M. Henriksson et al. / European Polymer Journal 43 (2007) 3434–3441 3439

ease of disintegration for the higher enzyme concen-trations, the DSP-0.02% showed only limited fibershortening and yet was easily disintegrated. A recentstudy [21] suggests an additional mechanism facili-tating MFC disintegration. Endoglucanase treat-ment increased the reactivity of cellulosic pulpfibers, even after mild treatment where the molecu-lar weight distribution of cellulose was virtuallyunchanged. It was suggested that endoglucanasetreatment increases swelling of cellulosic pulp fibersin water suspension. A hypothesis is therefore thatthis e!ect also contributes to MFC nanofiber disin-tegration. Indirect support for this explanation isprovided by the limited fiber shortening and smallchange in DP for the sample subjected to 0.02%enzyme concentration. Despite little reduction inDP due to enzyme treatment (from 1280 to 910),the DSP-0.02% pulp was easily disintegrated.

4. Conclusions

Endoglucanase pretreatment facilitates disinte-gration of cellulosic wood fiber pulp into MFCnanofibers by mechanical shearing in an environ-mentally friendly process without solvents or chem-ical reactants. The MFC successfully produced fromenzymatically pretreated cellulosic wood fibersshowed a more favorable structure than nanofibersresulting from fibers subjected to hydrolysis bystrong acid. AFM images showed high aspect ratio

of the MFC nanofibers prepared after enzymaticpretreatment. These high aspect ratio nanofibersare of great interest as reinforcement in nanocom-posite materials. The acid hydrolyzed MFC showedinhomogeneous distribution of nanofiber geometryand a large extent of thick cell wall fragments oflow aspect ratio.

One e!ect of high concentration enzymatic pre-treatment and mechanical beating is to reduce fiberlength and increase the extent of fine material. How-ever, pretreated fibers subjected to the lowest enzymeconcentration, 0.02%, were also successfully disinte-grated although molecular weight and fiber lengthwere well preserved. The estimated DP was onlyreduced from 1280 to 910. This indicates that addi-tional mechanisms are in operation than loweredmolecular weight, associated ‘‘embrittlement’’, andfiber shortening. It is suggested that increased cellu-losic fiber swelling in water due to enzyme-treatmentfacilitates preparation of MFC nanofibers.

Acknowledgements

Biofibre Materials Centre (BiMaC) at KTH(GH) and the Swedish Research Council (MH) aregratefully acknowledged for financial support. DrS. Notley and Prof L. Wagberg at KTH are grate-fully acknowledged for help with the AFM work.Novozyme is acknowledged for the gift of theenzyme.

Fig. 4. AFM images (5 · 5 lm2) of MFC nanofibers, (a) DSP-3%, enzyme treated dissolving pulp (3%), (b) DSP-SH, strongly acidhydrolyzed dissolving pulp.

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