nanoscale interactions of polyethylene glycol with thermo-mechanically pre-treated ...

Nanoscale Interactions of Polyethylene GlycolWith Thermo-Mechanically Pre-TreatedPinus radiata Biofuel Substrate

Lloyd A. Donaldson, Roger H. Newman, Alankar Vaidya

Scion, Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua 3046, New Zealand;

telephone: þ64-7-343-5581; fax: þ64-7-348-0952; e-mail: [email protected]

ABSTRACT: Non-productive adsorption of cellulose degrad-ing enzymes on lignin is a likely reason for reduced rate andextent of enzymatic conversion of lignocellulosic substrate tosugars. Additives such as polyethyleneglycol (PEG) may act asblocking agents in this non-productive interaction. However,the exact molecular level interactions of PEG with lignin inpre-treated lignocellulosic substrates are not known. We haveused confocal fluorescence microscopy combined withFörster resonance energy transfer (FRET) to reveal molecularlevel interactions between lignin present in thermo-mechan-ically pre-treated Pinus radiata substrate, and fluorescentlylabeled PEG. It is demonstrated that PEG interaction withlignin is mainly associated with particles derived fromsecondary walls, with little or no penetration into fragmentsderived from the middle lamella. This nanoscale informationon the PEG–substrate interaction will assist in rationalizingpre-treatment methods to reduce the recalcitrance ofsoftwood biofuel substrates.

Biotechnol. Bioeng. 2014;111: 719–725.

� 2013 Wiley Periodicals, Inc.

KEYWORDS: fluorescence microscopy; FRET;polyethyleneglycol; cellulose; cellulase; radiata pine; biofuelsubstrate

Introduction

Research on biofuels is gaining momentum as a result ofdepletion of crude petroleum reserves, periodic increases inthe price of fossil fuels, and the need to mediate climatechange associated with man-made increases in atmosphericCO2 levels (Ryan et al., 2006; Xu et al., 2013). The mostpromising biofuel technology available today is biological or

thermo-chemical conversion of sugars to bioethanol orbiodiesel (Kumar et al., 2009). Lignocellulosic biomassderived from non-food resources provides an abundantfeedstock for the production of sugars. However, lignocellu-losic feedstocks derived from softwood trees are challengingsubstrates for effective conversion to sugars via enzymatichydrolysis (Ouyang et al., 2010; Ragauskas and Huang, 2013;Sipos et al., 2010). A key factor limiting the effectiveness ofenzymatic hydrolysis of softwood substrates is unproductivebinding of enzymes with lignin (Börjesson et al., 2007b;Eriksson et al., 2002; Rahikainen et al., 2011).One approach to overcome the recalcitrant nature of

softwood is to supplement the hydrolytic reaction mixturewith additives. Many additives such as surfactants, proteins,and synthetic polymers have been found to be effective insaccharification of lignocellulosic substrates such as cornstover and cobs (Zhang et al., 2011), wheat straw (Kristensenet al., 2007), and pre-treated softwood (Börjessonet al., 2007a,b). Polyethyleneglycol (PEG) is particularlyattractive among the additives currently available because ofits widespread availability and cheap price (Börjessonet al., 2007a; Zhang et al., 2011). Different mechanismshave been proposed for the PEG effect in improvingenzymatic saccharification. The most accepted mechanisminvolves PEG interaction with lignin which reduces non-productive binding of enzymes within lignocellulosic sub-strates (Börjesson et al., 2007a,b).Deactivation by lignin will remain a concern after scale-up

of enzymatic hydrolysis using real rather than modelsubstrates, so it seems important to elucidate the mechanismby which additives block this enzyme deactivation. Forexample, Sewalt et al. (1997) demonstrated a negativeinfluence when they added lignin during enzymatichydrolysis of filter paper. Eriksson et al. (2002) showedthat delignification of steam pre-treated spruce (SPS)improved the glucose yield from enzymatic hydrolysis, andthat adding non-ionic surfactant to the delignified SPS gaveno further improvement. Börjesson et al. (2007b) added 14C-labeled PEG to a suspension of SPS and reported a good fit toa two-site Langmuir adsorption isotherm. The amount oftightly bound PEG corresponded to a PEG:SPS mass ratio of

Correspondence to: L. Donaldson

Contract grant sponsor: New Zealand Ministry of Business, Innovation and

Employment (formerly the Ministry for Science and Innovation)

Contract grant number: CO4X0802

Received 11 September 2013; Revision received 10 October 2013; Accepted 21 October

2013

Accepted manuscript online 28 October 2013;

Article first published online 18 November 2013 in Wiley Online Library

(http://onlinelibrary.wiley.com/doi/10.1002/bit.25138/abstract).

DOI 10.1002/bit.25138

ARTICLE

� 2013 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 111, No. 4, April, 2014 719

approximately 0.02:1. This tightly bound PEG seemed moreeffective in blocking cellulase deactivation than the looselybound PEG, since pre-incubation of SPS with PEG for 5 hincreased the adsorption of loosely bound PEG withoutimproving the glucose yield from subsequent enzymatichydrolysis. Furthermore, Börjesson et al. (2007a,b) proposedhydrophobic interactions between PEG and lignin whichprevents binding of cellulose hydrolyzing enzymes to thelignin, thereby improving the hydrolysis.

Recently a number of studies have focused on thenanoscale interactions between enzyme and substrate ineither artificial cellulose substrates (Bubner et al., 2012;Igarashi et al., 2011; Jung et al., 2013; Luterbacher et al., 2013;Moran-Mirabal et al., 2008) or in cellulosic biomass(Gierlinger et al., 2008; Zhu et al., 2011). Studies have usedelectron microscopy (Donaldson, 1988; Donaldson et al.,1988), scanning probe microscopy (Bubner et al., 2012;Igarashi et al., 2011), FTIR (Gierlinger et al., 2008), andfluorescence techniques (Dagel et al., 2011; Luterbacher et al.,2013; Moran-Mirabal et al., 2008) to detect enzymeinteractions with substrate surfaces. Enzyme access tocellulose is limited by encrustation with matrix substancessuch as lignin and hemicellulose (Ding et al., 2008;Rahikainen et al., 2011) and pre-treatments such as steamexplosion or ball milling are designed to increase exposure ofthe substrate (Donaldson, 1988; Donaldson et al., 1988;Kumar et al., 2009; Wong et al., 1988). Enzymes will oftenpreferentially access areas of damage such as dislocations(Thygesen et al., 2011).

In contrast there are few reports on the interaction of PEGwith lignocellulosic substrates. Such interactions have beenstudied using XRD, ATR-FTIR, radioactive labeling, andother techniques (Börjesson et al., 2007a,b; Zhanget al., 2011), based on pure cellulosic substrates such asAvicel or modified substrates such as chemically de-lignifiedbiomass (Börjesson et al., 2007a,b; Sewalt et al., 1997; Siposet al., 2010; Ouyang et al., 2010). Hence the conclusionsdrawn from those studies may not be extended directly topre-treated lignocellulosic substrates. Furthermore, there areno direct molecular level investigations involving exactlocalization of PEG on the thermo-mechanically pre-treatedsubstrate. Nanoscale information on the PEG–substrateinteraction will help in rationalizing the pre-treatmentmethod to reduce recalcitrance of softwood substrates.

In this work for the first time we explore the molecularlevel interaction of PEG with thermo-mechanically pre-treated softwood (Pinus radiata) using confocal microscopy.Nanoscale interactions between labeled PEG and lignin areinvestigated using FRETmeasurements.

Materials and Methods

Enzymes (Celluclast 1.5L and Novozyme 188) were obtainedfrom Novozymes A/S (Bagsværd, Denmark). Filter paperactivity units (FPU) were determined according to the IUPACmethod (IUPAC, 1987), and the b-glucosidase activity usingp-nitrophenyl-b-glucopyranoside as a substrate following the

procedure described by Bailey and Nevalainen (1981).Celluclast 1.5L had an overall cellulase activity of 67 FPU/mL, and very low b-glucosidase activity. Novozyme 188 had492 IU/mL b-glucosidase activity. PEG (MW 3400) labeledwith rhodamine dye was purchased from NANOCS, Inc.(New York). A molecular weight of 3,400 was chosen becauseit was comparable to the values used in other studies of PEGas a saccharification additive (Börjesson et al., 2007a,b; Zhanget al., 2011). All other chemicals were purchased from Sigma-Aldrich (Milwaukee) and were used as received.

Pre-Treatment of the Wood Chips

Radiata pine wood chips were obtained from a local sawmilland were pre-treated with steam at 170�C for 72min, withoutaddition of chemicals, then converted to pulp in a single diskrefiner at Scion’s fiber and pulp processing pilot plant. Thepre-treatment wasmilder than, for example, steam-explosionprocessing, allowing increased opportunity for utilization oflignin as a co-product of saccharification. Extractives weredetermined using a FOSS Soxtec System 1043 extraction unitwith dichloromethane as the solvent. Lignin was determinedusing methods based on TAPPI Standard Method T222 om-88 and TAPPI Useful Method UM 250. Carbohydrates wereanalyzed by ion chromatograph using a Dionex ICS 3000instrument (Pettersen and Schwandt, 1991). The composi-tion of the pulp was: lignin 34.5%, glucan 51.5%, xylan 4.1%,mannan 6.4%, galactan 1.0%, arabinan 0.2%, and extractives1.8%. Holocellulose with lignin content of <3% wasprepared from the pulp by chlorite delignification(Uprichard, 1965).

A 2 kg portion of the pulp (58% solids content, w/w) wasdiluted to 6% consistency with water, and further processedfor 300min in a 40 L tumbling ball mill using 20mmdiameter porcelain balls. The substrate was stored in arefrigerator at 5�C until further use.

Confocal Fluorescence Microscopy

Preliminary experiments confirmed that rhodamine dye only(without PEG) did not bind and interact with the substrate,and that adding the PEG at the same time as the enzymeprovided an improved digestion of substrate. A similarimprovement in corn straw saccharification was reportedwhen simultaneous addition of PEG with enzyme wasperformed (Zhang et al., 2011).

In this work a labeled substrate was prepared as follows.PEG-rhodamine (5mg) was dissolved in 0.5mL of deionizedwater. The enzyme mixture was added (1.5 FPU) followed bythe substrate (75mg). The reaction volume was made up to5mL with 0.05M sodium citrate buffer pH 4.8 containing0.01% (w/v) sodium azide, and saccharified at 50�C for 24 h.The undigested substrate was centrifuged at 4,000 rpm for10min and the wet residual substrate was used, withoutwashing, for microscopy. The glucose in the filtrate wasmeasured using a YSI-2700 glucose analyzer, in order todetermine the degree of conversion of cellulose to glucose.

720 Biotechnology and Bioengineering, Vol. 111, No. 4, April, 2014

The following substrates were examined by confocalfluorescence microscopy:

� Substrate plus PEG-rhodamine, enzyme treated for 24 h at50�C (treated).

� Substrate without PEG-rhodamine, enzyme treated for24 h at 50�C (control-1).

� Substrate without PEG-rhodamine, treated with heatdeactivated enzyme for 24 h at 50�C (control-2).

� Holocellulose plus PEG-rhodamine without enzymetreatment (control-3).

Samples for confocal fluorescence microscopy weremounted in 50% glycerol in buffer at pH 9 and examinedusing a Leica TCS SP5 spectral confocal microscope withsequential excitation at 458 and 561 nm and emission at 470–550 and 570–700 nm in order to distinguish lignin andrhodamine fluorescence. Gain was optimized to fill the 255gray level dynamic range for PEG-rhodamine treatedsubstrate to allow assessment of intensity and color differ-ences between treated and control samples which wereimaged under identical conditions. Measurement of fluores-cence spectra was carried out using 458 nm excitation, withspectra collected from 470–700 nm at 10 nm intervals and abandwidth of 10 nm.

F€orster Resonance Energy Transfer (FRET)

FRET efficiency was measured using the acceptor photo-bleaching technique (Periasamy and Day, 2005) with thesame excitation and emission conditions as described above.In this experiment lignin was considered to be the donor andPEG-rhodamine was the acceptor. Acceptor photobleachingwas carried out on a region of interest within each of six fieldsof view, using a wavelength of 561 nm for 40 scans at 100%laser power. FRET efficiency, a measure of the proximity ofthe donor and acceptor molecules, was calculated for eachpixel in the field of view from images before and afterbleaching of the acceptor molecules within a region ofinterest using the following formula:

FRET Efficiency ¼ ðDpost � DpreÞ=Dpost

where Dpre is the intensity of the donor before photo-bleaching and Dpost is intensity after photo-bleaching. Anaverage FRET efficiency value for the acceptor bleachedregion of interest was also calculated.

Results and Discussion

Confocal Fluorescence Microscopy

Confocal microscopy in the presence of a fluorescent probe(PEG-rhodamine) was used to study molecular level inter-actions between additive (PEG) and thermo-mechanically pre-treated P. radiata substrate. When the substrate was treated with

enzymes (control 1—55% conversion of cellulose to glucose) orby heat deactivated enzymes (control 2—no conversion ofcellulose to glucose) in the absence of PEG-rhodamine,substrate showed natural green fluorescence from lignin withexcitation at 458 and 561nm (Fig. 1A). Our earlier work hasshown that cellulose and hemicellulose are non-fluorescent(Donaldson et al., 2010). In a separate experiment, holocellu-lose, which is devoid of lignin, when treated with rhodamine-labeled PEG (control 3), showed only red fluorescence due torhodamine. This fluorescence rapidly declined due to leachingof the label from the holocellulose indicating a lack of binding inthe absence of lignin. No such leaching was observed with thelignified substrate, indicating that most of the PEG-rhodamineis bound to the substrate. After extensive washing all of therhodamine label could be removed from the holocellulosesubstrate.Addition of rhodamine-labeled PEG together with the

enzymes resulted in 72.2% conversion of cellulose to glucoseindicating a 1.31-fold improvement over the control, andintroduced additional orange-red fluorescence in the undi-gested residue (Fig. 1B–D). Images of the PEG-rhodaminetreated substrate showed two distinct components. Green

Figure 1. Confocal fluorescence images of substrate with sequential excitation at

458 and 561 nm, and emission at 470–550 and 570–700 nm shown as projections. A:

Substrate treated with enzyme in the absence of PEG (control 1) showing green lignin

fluorescence. Scale bar¼ 24mm. B: Substrate treated with enzyme plus PEG-

rhodamine at the same gain as A showing green lignin fluorescence and red

rhodamine fluorescence. Scale bar¼ 24mm. C: Substrate treated with enzyme plus

PEG-rhodamine showing an enlarged view of orange secondary walls and green

middle lamella (arrow). The green stripe-like appearance originates from the highly

lignified cell corner middle lamella. Scale bar¼ 12mm. D: Substrate treated with

enzyme plus PEG-rhodamine showing an enlarged view of secondary wall revealing

different intensities of PEG-rhodamine labeling suggesting variable porosity (arrow).

Scale bar¼ 8mm.

Donaldson et al.: Nanoscale Interactions of PEG With Pinus radiata 721

Biotechnology and Bioengineering

fluorescing particles correspond to fragments of lignin-richmiddle lamella as determined by their size and shape(Fig. 1C). The width of the cell corner middle lamella inradiata pine is about 1–2mm (Donaldson, 2001) whichis comparable to the brightly fluorescent strips in thesubstrate. The brightness of the green fluorescence isconsistent with the highly lignified nature of middle-lamellamaterial, its low porosity making it resistant to infiltration bythe labeled PEG. The size and shape of the orange-redfluorescing particles indicated fragments of secondary cellwalls. Variable penetration of PEG was seen in secondary wallfragments suggesting variation in porosity of the substrate(Fig. 1D).

The proximity of PEG molecules to lignin in our substratewas measured by comparing fluorescence spectra of PEG-rhodamine treated substrate with that of a control specimentreated with enzyme in the absence of PEG (control 1), usingexcitation at 458 nm (Fig. 2). Rhodamine-labeled PEG-3400without substrate showed emission with a lmax of 590 nm(Fig. 2A). Control specimens treated with enzyme (control 1)or with heat deactivated enzyme (control 2) but without PEGshowed identical emission with a lmax of 540 nm corre-sponding to lignin fluorescence (Fig. 2B). The spectrumof secondary cell walls treated with enzyme plus PEG-rhodamine showed quenching of the lignin fluorescence at540 nm (Fig. 2B). Fluorescence quenching indicates proxim-ity of the rhodamine molecules to lignin, consistent withadsorption of the PEG-3400 on the lignin. The fluorescencespectrum of a middle lamella fragment treated with enzymeplus PEG-rhodamine showed only a small peak at 590 nm,consistent with trace amounts of adsorbed PEG-3400.Fluorescence quenching was absent in the spectra of themiddle lamella component (Fig. 2C), compared with spectraof secondary cell walls (Fig. 2B). These observations areconsistent with a low degree of penetration of PEG-rhodamine into middle lamella fragments.

F€orster Resonance Energy Transfer (FRET) Measurements

FRET is a technique for measuring molecular interactionbetween two fluorophores (Stryer and Haugland, 1967). Theconditions for FRET to take place include significant overlap(30% or more) between the fluorescence emission spectrumof the donor and the absorption spectrum of the acceptor,and the very close spatial proximity of donor and acceptormolecules—generally less than 10 nm (Periasamy andDay, 2005). FRET efficiency is a measure of the proximityof donor and acceptor molecules—the higher the FRETefficiency the closer the two molecules. When FRET is takingplace the fluorescence of the donor will be quenched in thepresence of the acceptor. Acceptor photobleaching measuresthe fluorescence of the donor before and after bleachingof the acceptor. If FRET is occurring then the donorfluorescence will increase after the acceptor is bleached.This energy exchange is non-radiative; the acceptor doesnot absorb fluorescence from the donor (Periasamy andDay, 2005). The obvious quenching of lignin fluorescence as

shown in Figure 2 provides tentative evidence that FRET isoccurring in the PEG-rhodamine labeled substrate.

Measurements indicate significant FRET efficiency in thePEG-rhodamine treated secondary wall fragments (Fig. 3).Average FRET efficiency varied from 42% to 57% in PEG-rhodamine treated substrates but was below 10% inuntreated controls (Table I). This 10% threshold representsa baseline error probably resulting from random variations inintensity between the pre and post bleaching images. FRETefficiency varied significantly within the area of measurementwith values at individual locations as high as 80% efficiency.This variation probably reflects local differences in porosity ofthe substrate where the PEG-rhodamine comes into closercontact with the lignin adjacent to the pores (Fig. 1D).Because the PEG-rhodamine molecule is a little smaller thanthe cellulase enzymes, the PEG molecules may access areas oflignin that would not be accessible to the enzyme. FRETefficiency measurements on the middle lamella particlesconfirmed conclusions from imaging (Fig. 1) and spectros-copy (Fig. 2), that these fragments contained little or no PEG-rhodamine. FRET efficiency in middle lamella particles wascomparable to the 10% baseline determined from untreatedsubstrate (control 1) suggesting that any PEG-rhodamineassociated with middle lamella particles may be located onthe surface where it has limited contact with few accessiblelignin molecules. Thus, microscopic investigation confirmed

Figure 2. A: Fluorescence emission spectrum of PEG-rhodamine excited at

458 nm with a lmax¼ 590 nm. B: Fluorescence emission spectra from a secondary wall

fragment of substrate with (solid circles) or without (open squares) PEG-rhodamine.

When PEG-rhodamine is present fluorescence emission from lignin at 540 nm is

reduced indicating quenching. C: Fluorescence emission spectra from amiddle lamella

fragment of substrate with (solid circles) or without (open squares) PEG-rhodamine

showing a very small peak due to rhodamine at 590 nm.


Figure 3. FRET analysis of PEG-rhodamine treated substrate. A: Untreated substrate (control 1) shows no significant FRET. Scale bar¼ 48mm. B: PEG-rhodamine treated

substrate shows very significant FRET. Scale bar¼ 48mm. C: PEG-rhodamine treated substrate comparing secondary wall (red) and middle lamella (green) with high and low FRET

efficiencies, respectively. Scale bar¼ 23mm. In each case the color scale bar indicates FRET efficiency, the higher the value the closer the proximity of lignin and PEG-rhodamine

molecules. The white rectangles highlight the region of interest within which FRET measurements were made. Pixels outside of the region of interest represent random variations in

brightness between the pre- and post-bleaching images which are not indicative of FRET and thus represent a control for comparison with the region of interest.

Table I. Average brightness of donor and acceptor images within a region of interest before (pre) and after (post) acceptor photobleaching, and

calculated FRET efficiency, for six replicate fields of view on control and PEG-rhodamine treated substrate.

Sample 1 2 3 4 5 6

ControlDonor pre 39.7 55.7 41.8 42.4 36.8 43.1Donor post 41.8 61.8 45.5 45.9 39.1 45.6Acceptor pre 33.0 34.0 26.4 26.4 21.5 26.9Acceptor post 19.6 17.3 14.4 13.9 11.9 15.3Efficiency (%) 5.0 9.9 8.0 7.8 6.1 5.6

PEG-rhodamineDonor pre 32.1 6.4 49.9 52.4 30.1 8.6Donor post 56.1 11.1 96.2 102.8 70.6 15.3Acceptor pre 52.8 40.6 63.8 78.3 43.4 26.9Acceptor post 49.3 38.8 22.2 76.4 16.8 19.3Efficiency (%) 42.7 42.1 48.2 49.0 57.4 43.8



the penetration of substrate by PEG and its association withmore exposed and accessible lignin. This observation suggeststhe more exposed and accessible lignin from poroussecondary cell wall could be responsible for non-productivebinding of enzymes compared to less accessible lignin fromthe middle lamella.

Middle lamella particles consist mainly of lignin withsmaller amounts of hemi-cellulose such as xyloglucan,glucuronoxylan, arabinoxylan, and pectins with little or nocellulose (Putoczki et al., 2008). Therefore, the inability ofPEG to penetrate these regions is almost irrelevant to glucoseyield. The very large surface area inside the porous secondarywall and binding of PEG to lignin lining the surface of poreswithin this region may account for improved yield bydecreasing the non-specific binding of enzyme to thesesurfaces.

Conclusions

Fluorescence microscopy demonstrates that adsorbed PEGmolecules are in contact with lignin molecules in particlesderived from secondary cell walls but with very little bindingto the lignin molecules from non-porous middle lamella. Asignificant FRET efficiency between lignin and PEG insecondary cell wall fragments demonstrates a close proximity(<10 nm) whereas in the middle lamella fragments, FRETresponse was localized to the surface of the particles, indi-cating that the low accessibility of the lignin limits PEGbinding in these fragments. These results confirm that PEGbinds to lignin within porous areas of the substrate therebyreducing non-specific binding of enzyme at these locations.

We wish to thank John Lloyd and Karl Murton for providing thethermo-mechanically pre-treated Pinus radiata substrate, and SylkeCampion for performing substrate treatments. Financial supportfrom the New Zealand Ministry of Business, Innovation andEmployment (formerly the Ministry for Science and Innovation)under contract CO4X0802 is gratefully acknowledged.

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