Effect of Nonionic Surfactants on Dispersion and Polar Interactions in

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Effect of Non-Ionic Surfactants on Dispersion and Polar Interactions in the Adsorption of Cellulases onto Lignin Feng Jiang, Chen Qian, Alan R Esker, and Maren Roman J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07716 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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The Journal of Physical Chemistry

Effect of Non-Ionic Surfactants on Dispersion and Polar Interactions in the Adsorption of Cellulases onto Lignin Feng Jiang,† Chen Qian,†,1 Alan R. Esker‡ and Maren Roman†,§* †

Macromolecules Innovation Institute, ‡Department of Chemistry, and §Department of Sustainable Biomaterials, Virginia Tech, Blacksburg, Virginia 24061, United States

CORRESPONDING AUTHOR: Email: [email protected], Phone: +1-540-231-1421, Fax: +1-540-231-8176

1

Current address: 5200 Bayway Dr, Baytown, TX 77520, current affiliation: Exxonmobil Chemicals 1 ACS Paragon Plus Environment


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ABSTRACT Residual lignin in pretreated biomass impedes enzymatic hydrolysis. Non-ionic surfactants are known to enhance the enzymatic hydrolysis of lignocellulosic biomass but their mechanisms of action are incompletely understood. This study investigates the effect of a non-ionic surfactant, Tween 80, on the adsorption of cellulases onto model lignin substrates. Lignin substrates were prepared by spin coating of flat substrates with three different types of lignin: organosolv lignin, kraft lignin, and milled wood lignin. The functional group distributions in the lignins were quantitatively analyzed by 31P NMR spectroscopy. The surface energies and surface roughnesses of the substrates were determined by contact angle measurements and atomic force microscopy, respectively. Tween 80 and cellulase adsorption onto the lignin substrates was analyzed with a quartz crystal microbalance with dissipation monitoring. Tween 80 adsorbed rapidly and primarily (≥85%) via dispersion interactions onto the lignin substrates and effected solubilization of lignin molecules, most notably with organosolv lignin, having the largest dispersive surface energy component and smallest molar mass. Cellulase adsorption onto the lignin substrates was mostly irreversible and had both a rapid and a gradual adsorption stage. Rapid cellulase adsorption was primarily (≥64%) mediated by dispersion interactions. The subsequent gradual mass increase is postulated to involve swelling of the lignin substrates. Adsorbed Tween 80 rendered lignin surfaces more hydrophilic by increasing their polar surface energy component and reduced both the extent of rapid cellulase adsorption as well as the rate of the subsequent gradual mass increase. The effect of Tween 80 on the rate and extent of the gradual mass increase depended strongly on the chemical properties of the lignin.

2 ACS Paragon Plus Environment

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INTRODUCTION As the most abundant natural organic material, lignocellulosic biomass has been subject to extensive research because of its potential value in the production of liquid biofuels. Lignocellulosic biomass is composed of a lignin and hemicellulose matrix encapsulating a cellulose scaffold. Functioning as support and protection for the cellulose scaffold against microbial degradation in living plants, lignin presents major challenges in the enzymatic hydrolysis of lignocellulosic biomass, including limiting the enzyme accessibility of the cellulose scaffold. Researchers have shown that the presence of lignin in the substrate significantly reduces enzymatic hydrolysis yields.1-5 Besides limiting access to the cellulose scaffold, lignin also provides non-specific binding sites, thus preventing effective cellulase adsorption onto the cellulose scaffold, a necessary step in the hydrolysis of the scaffold.6-12 To reduce the recalcitrance of lignocellulosic biomass to chemical and biochemical degradation, pretreatment methods have been developed that break down the lignin–hemicellulose–cellulose matrix and increase the enzyme accessibility of the cellulose scaffold. Chemical pretreatment methods encompass processes under acidic conditions,13,14 basic conditions,13,15,16 including kraft pulping, and organosolv pulping.17,18 Caustic conditions during biomass pretreatment inevitably cause changes in the lignin structure, such as the cleavage of β-O-4 ether bonds, similar to those observed during traditional pulping processes. Non-ionic surfactants have been studied extensively as potential additives in the biochemical conversion of lignocellulosic biomass because they increase enzymatic hydrolysis yields.19-21 Several hypotheses have been proposed for the mechanism of this enhancement, including alteration of the ultrastructure of the lignocellulosic substrate, increase of the thermal stability of the enzymes, and effects on enzyme–substrate interactions.20,22 Of the proposed mechanisms, the



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prevention of non-productive enzyme adsorption onto lignin is the most widely investigated and documented. Reduced non-productive adsorption of cellulases onto lignocellulosic substrates has been demonstrated for the non-ionic surfactants Agrimul,20 Triton X-100,20 Triton X-114,20 Tween 20,20,23 and Tween 80,20,24 as well as BSA8 and polyethylene glycol (PEG).25,26 However, most of these studies were bulk studies with real lignocellulosic substrates and did not involve real-time monitoring of the molecular interactions between the surfactant, cellulase, and lignin. Quartz crystal microbalances with dissipation monitoring (QCM-D) are relatively new analytical instruments that allow real-time detection of minute mass and viscoelasticity changes of adsorbed layers on oscillating quartz crystals by measuring changes in the resonant frequency, ∆f, and dissipation factor, ∆D, upon adsorption, which offer potential insight into these mechanisms. In the last decade, QCM-D has found increasing use in enzymatic degradation studies of model cellulose and lignocellulosic substrates.27-42 Pure and smooth lignin films of ~30 nm thickness, suitable for use as model substrates, have previously been prepared by Langmuir– Blodgett deposition.43-45 Only slightly higher surface roughnesses (0.6–1.5 nm) have been reported for lignin films prepared by spin coating.46-50 Such films have been used as model lignin surfaces to study lignin–cellulose interactions in aqueous electrolyte solutions,48 adsorption of polyelectrolyte or polyelectrolyte complexes onto lignin,47,49 and the surface energy and wettability of lignin.50 Lou et al.51 used model lignin substrates, prepared by spin coating of corn stover lignin, in combination with QCM-D to study the effect of commercial sodium lignosulfonate and three anionic polymers on cellulase adsorption. They observed a decrease in cellulase adsorption after treatment of the substrates with the lignosulfonate or anionic polymer and hypothesized that the decrease was due to blocking of adsorption sites, an increase in substrate hydrophilicity, and steric hindrance.


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This study was conducted to further enhance our understanding of the mechanisms by which surfactants prevent non-productive binding of cellulolytic enzymes onto lignin substrates. Because non-ionic surfactants have been shown to be most effective,20,52 Tween 80 (polyoxyethylene (20) sorbitan monooleate), consisting of PEGylated sorbitan as the hydrophilic headgroup linked to a monounsaturated, 18-carbon hydrophobic tail, was chosen as the surfactant. The adsorption of cellulases onto untreated and Tween 80-treated model lignin substrates was studied by QCM-D and atomic force microscopy (AFM). The lignin substrates for this study were prepared by spin coating of flat substrates with three different types of lignin having different chemical properties. Kraft lignin (KL) was chosen as a model for lignin from alkaline pretreatment methods, organosolv lignin (OL) was chosen as a model for lignin from acidic pretreatment methods, and milled wood lignin (MWL) was chosen as a model for native lignin. The results will be compared to those recently reported by Fritz et al.53 of a study resembling this one54 but using different lignins (softwood kraft (SWK), hardwood kraft (HWK), hardwood autohydrolysis cellulolytic (HWAH), maple cellulolytic (MPCEL) and wheat straw cellulolytic (WSCEL) lignin), cellulases, lower Tween 80 concentrations, and shorter adsorption periods. This study more closely investigates the effects of the surfactant on dispersion interactions and polar interactions, i.e. hydrogen bonding and dipole-involving interactions, between the enzymes and lignin substrates and elucidates their impacts on enzyme adsorption.

EXPERIMENTAL SECTION Materials KL (number average molar mass, Mn, = 5 kg·mol–1, weight average molar mass, Mw, = 28 kg·mol–1), Tween 80 (SigmaUltra), Celluclast 1.5L (Novozyme), and 2-chloro-4,4,5,5-



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tetramethyl-1,3,2-dioxaphospholane (CTMDP, 95%) were purchased from Sigma-Aldrich. Sulfuric acid (95.9 wt %, Certified ACS Plus), pyridine (Certified ACS), molecular sieves (4 Å), deuterated chloroform (Cambridge Isotope Laboratories, Inc. 99.8%), endo-N-hydroxy-5norbornene-2,3-dicarboximide

(e-HNDI,

Acros

Organics,

99+%),

chromium

(III)

acetylacetonate (Cr(acac)3, Acros Organics, 97%), diiodomethane (Acros Organics, 99+%, stabilized), formamide (Acros Organics, 99.5+%, extra pure), citric acid monohydrate (Certified ACS), sodium hydroxide (Acros Organics, 98.5%), hydrogen peroxide (34–37%, Technical Grade), aqueous ammonia (28.9%, Certified ACS Plus), ethanol (200 proof, Decon Labs) and PEG (“PEG 4000”, Bioworld, Biotechnology Grade) were purchased from Fisher Scientific. MWL from hemlock (Tsuga sp., Mn = 2.6 kg·mol–1, Mw=15.2 kg·mol–1)55 and OL from cottonwood (Populus trichocharpa, Mn < 1 kg·mol–1, Mw < 3 kg·mol–1)55 were kindly provided by Wolfgang Glasser. All water used was deionized (DI) water with a resistivity of 18.2 MΩ·cm, obtained from a Millipore Direct-Q 5 Ultrapure Water Systems.

Lignin Fractionation and Molar Mass Analysis Aqueous solutions of KL, MWL (both 1 wt % in 0.75 M ammonia), and OL (1 wt % in 1.5 M ammonia) were transferred to Spectra/Por 4 dialysis tubing (MWCO: 12–14 kg·mol–1, Spectrum Lab, Inc.) and dialyzed against water for the removal of low molar mass fractions and any watersoluble impurities. After two weeks of dialysis, the lignin suspensions were centrifuged at 25 °C and 4900 rpm for 15 min to remove any sedimenting larger particles. The aqueous lignin suspensions obtained upon centrifugation were stable, i.e. the dispersed lignin particles did not settle to the bottom of the glass vials upon storage. This observation has been previously reported


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for KL by Lindström.56 Finally, the lignin suspensions were concentrated to 1.5 wt % with a rotary evaporator under vacuum at 45 °C and stored in the refrigerator until use. The molar masses of the lignins were determined by gel permeation chromatography (GPC). To this end, the fractionated lignin suspensions were freeze-dried to yield lignin powders. For each sample, 40 mg of freeze-dried lignin was acetylated for 24 h at room temperature in 4 mL of 1:1 v/v pyridine/acetic anhydride, precipitated and washed with ethanol, and dried in a vacuum oven. Although acetylation of lignin samples can lead to errors due to incomplete dissolution in the acetylating reagent or organic eluent, it is still regarded one of the best methods for lignin molar mass determination.57 The GPC analysis was conducted at 30 °C with chloroform as the solvent using an Alliance Waters 2690 Separations Module equipped with a Waters HR 0.5 + HR 2 + HR 3 + HR 4 styragel column set and a Viscotek T60A dual viscosity detector and laser refractometer. The flow rate was 1.0 mg⋅mL–1. The absolute molar masses of the lignins were obtained with a polystyrene universal calibration.

Quantitative 31P NMR Lignin Analysis The functional group distribution in the lignins was analyzed by quantitative

31

P NMR

spectroscopy (Figure S1, Supporting Information) according to the method of Granata and Argyropoulos58 but with e-HNDI instead of cyclohexanol as the internal standard, which has been shown to circumvent the interference of the cyclohexanol phosphite peak during integration of the lignin phosphite peaks.59 In brief, a 1.6:1 v/v pyridine/deuterated chloroform mixture was prepared and dried over molecular sieves for several hours in the refrigerator. The solvent mixture was then used to prepare separate solutions of Cr(acac)3 (5 mg⋅mL–1), serving as a relaxation agent, and e-HNDI



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(10.85 mg⋅mL–1), which were subsequently stored in sealed containers in the refrigerator until use. For NMR sample preparation, 30 mg of freeze-dried lignin was suspended in 500 µL of the solvent mixture by vortexing for 1 min in an amber sample vial. Then, 150 µL of CTMDP was added, and the reaction mixture was vortexed for 1 min. Upon vortexing, a clear solution was obtained, indicating the phosphitylation of lignin hydroxyl and carboxyl groups by CTMDP (Scheme S1, Supporting Information). As the final step in the NMR sample preparation, 200 µL of the e-HNDI and 100 µL of the Cr(acac)3 solution were added to the phosphitylated-lignin solution, after which it was again vortexed for 1 min and transferred to a 5 mm o.d. NMR tube. Quantitative

31

P NMR spectra were acquired immediately after sample preparation with a

Varian Unity 400 NMR spectrometer, an observation sweep width of 6600 Hz, and a relaxation time of 25 s between successive pulses. For chemical shift calibration, the peak of the reaction product of water and CTMDP at 132.2 ppm was used as a reference signal.

Lignin Substrate Preparation Lignin substrates were prepared on silicon dioxide-coated AT-cut quartz crystals (Q-Sense). The quartz crystals were cleaned by immersion and shaking for 1 min in fresh piranha solution (7:3 v/v H2SO4/H2O2) (Caution: Piranha solution is highly corrosive and reacts violently with organic matter!), then rinsed with water, dried in a stream of nitrogen, and irradiated for 15 min with an ozone-producing mercury lamp (UV/Ozone cleaner, Bioforce Nanosciences). To minimize surface contamination, the quartz crystals were cleaned immediately prior to use. The 1.5 wt % aqueous lignin suspensions were diluted by addition of aqueous ammonia and water to reach a final lignin concentration of 1 wt % and ammonia concentration of 0.75 M for KL and MWL and 1.5 M for OL. A volume of 140 µL of 1 wt % lignin ammonia solution was


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spin-coated (WS-400B-6NPPLite, Laurell Technologies Corp.) for 1 min at 4000 rpm onto the cleaned quartz crystals. The lignin substrates were dried under vacuum at 65 °C for several hours for improved stability in aqueous media.

Surface Energy Measurements Lignin substrates for surface energy measurements were prepared by spin coating of 1 in. × 1 in. silicon wafer pieces as described above. Before spin coating, the wafer pieces, cut from a 15 cm wafer (Wafer World, Inc. West Palm Beach, FL), were cleaned by immersion for 1 h at 70 °C in 1:1:5 v/v NH3⋅H2O/H2O2/H2O and then for 1 h at ambient temperature in fresh piranha solution, rinsed with water, and dried in a stream of nitrogen. The surface energies of untreated and Tween 80-treated lignin substrates were determined from sessile drop contact angles, θ, measured with a video-based contact angle goniometer (FTA200, First Ten Angstroms, Inc., Table S1, Supporting Information). For Tween 80 treatment, lignin substrates were placed in polystyrene Petri dishes and covered with 1 mM Tween 80 solution in 50 mM sodium citrate buffer (pH 4.8). The Petri dishes were then shaken in a shaker for 140 min. After Tween 80 treatment, the substrates were rinsed with water and then dried in a stream of nitrogen and subsequently under vacuum at 65 °C for several hours. For surface energy measurements, sessile drops of 20 µL of water, diiodomethane, and formamide were placed onto untreated and Tween 80-treated lignin substrates, and the static contact angles of at least three different drops were measured for each liquid. As explained by Notley and Norgren,50 these three test liquids have Hansen solubility parameters that indicate that the liquids will not dissolve lignin.



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The surface energies of the lignin substrates, γs, were derived from the average of the measured contact angles, θ, with the Owens–Wendt–Rabel–Kaelble method,60-62 based upon the equation 1 + cos

⁄ 2

=

⁄

+

⁄

(1)

d where γl is the surface tension of the test liquid, γ l and

γ lp are the dispersive and polar

components of γl, respectively (Table S2, Supporting Information), and γ sd and dispersive and polar components of γs, respectively. γ sd and

γ sp were obtained from the intercept

and slope, respectively, of the line obtained by plotting 1 + cos ⁄2

⁄

⁄

. γs was obtained by summation of γ sd and

γ sp are the ⁄

versus

γ sp.

QCM-D Adsorption Experiments Adsorption of Tween 80 and cellulase onto lignin substrates was analyzed by QCM-D (QSense E4). Lignin substrates were placed in QCM-D flow modules, and a 0.25 mL⋅min–1 feed of 50 mM sodium citrate buffer was established with a peristaltic pump. The temperature inside the flow modules was controlled at 22.00 ± 0.02 °C. When the scaled rate of change of the 3rd overtone frequency, (∆f3/3), became less than 2 Hz⋅h–1 (after a period of at least 3 h), the substrates were considered saturated and equilibrated. In Tween 80 adsorption experiments, the buffer feed was then replaced with a feed of 1 mM Tween 80 solution in buffer with the same flow rate. After 140 min, the sodium citrate buffer feed was restored so that any loosely bound Tween 80 molecules would be rinsed from the substrate. Tween 80 adsorption experiments were terminated after 60 min of buffer rinse.


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In cellulase adsorption experiments on untreated lignin substrates, the initial buffer feed was replaced with a feed of cellulase solution in buffer, prepared by dilution of 0.5 g Celluclast 1.5L with 50 mM sodium citrate buffer to a volume of 100 mL. Celluclast 1.5L is a commercial cellulase solution derived from Trichoderma reesei, containing at least two exoglucanases, five endoglucanases, and two β-glucosidases, with a filter paper activity and protein concentration of 119 FPU·mL−1 and 143 mg·mL−1, respectively.28 After 4 h, the sodium citrate buffer feed was restored so that any reversibly bound cellulase molecules would be rinsed from the substrate. In cellulase adsorption experiments on Tween 80treated substrates, the initial buffer feed was first replaced with a feed of 1 mM Tween 80 solution in buffer for 140 min, restored for 60min, replaced with a feed of cellulase solution in buffer for 4 h, and again restored. Cellulase adsorption experiments were terminated after 30 min of the final buffer rinse. PEG adsorption experiments and cellulase adsorption experiments on PEG-treated lignin substrates were carried out as described above but with a 1 mM PEG solution in buffer instead of the 1 mM Tween 80 solution. The duration for the PEG solution feed was 20 min, as opposed to 140 min, used for the Tween 80 solution feed, because ∆f and ∆D remained constant after only a few minutes of adsorption. ∆f and ∆D were recorded in real time at the fundamental frequency (5 MHz) and 6 overtone frequencies (15, 25, 35, 45, 55, and 65 MHz) with QSoft 401 acquisition software (Q-Sense). The masses of the adsorbed layers on the lignin substrates were estimated with the QTools modeling center (v3.0, Q-Sense) by modeling ∆f and ∆D for the 3rd and 5th overtones with a single-layer Voigt element-based viscoelastic film model.63,64 For the density and viscosity of the



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bulk liquid, we used the density and viscosity of water at 22 °C, which are 998 kg⋅m–3

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65

and

0.955⋅10–3 kg⋅m–1s–1.66 The density of the viscoelastic film was fixed at 1100 kg⋅m–3.67

Substrate Imaging and Surface Roughness Determination Upon completion of the QCM-D adsorption experiments, the lignin substrates were taken out of the flow modules, thoroughly rinsed with water, dried in a stream of nitrogen and then under vacuum at 65 °C for several hours. The Tween 80- and cellulase-treated substrates as well as unused substrates on quartz crystals were imaged with an Asylum Research MFP-3D-BIO atomic force microscope. The substrates were scanned in air at ambient relative humidity and temperature in intermittent-contact mode with OMCL-AC160TS standard silicon probes (Olympus Corp.) with a scan frequency of 1 Hz, and 512 scans with 512 points/scan. RMS surface roughnesses were determined from the entire area of the AFM height image (2 µm × 2 µm or 5 µm × 5 µm, as specified).

RESULTS AND DISCUSSION Molar Mass and Chemical Properties of the Lignins The Mn values of OL, KL, and MWL were 2.3, 8.2, and 5.8 kg·mol–1, respectively, after dialysis. The increases in Mn from

Effect of Nonionic Surfactants on Dispersion and Polar Interactions in

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