Supplemental Information
Supporting methods
Functional analysis of lignin structure using 2D nuclear magnetic resonance
2D-NMR analysis for the pretreated biomass was performed using the method previously
reported (Cheng et al., 2013). In brief, ball milled acid pretreated Miscanthus (15 mg) was
dissolved in 0.6 mL DMSO-d6 containing 10 μL of [EMIM][OAc]-d13. In the case of the
MWL, 15 mg was dissolved in 0.6 mL DMSO-d6. 2D NMR spectra were acquired on a
Bruker AVANCE 600 MHz spectrometer equipped with a gradient 5-mm TXI 1H/13C/15N
cryoprobe. The chemical shifts were referenced to the central DMSO solvent peak (δC 39.5
ppm, δH 2.49 ppm). 13C-1H correlation spectra (HSQC: heteronuclear single quantum
coherence) were measured with a Bruker standard pulse sequence. The experiments had the
following parameters: acquired from 10-0 ppm in F2 (1H) by using 2,048 data points for an
acquisition time (AQ) of 170 ms, 210-0 ppm in F1 (13C) by using 256 increments (F1
acquisition time 4.0 ms) of 200 scans with a 1s interscan delay (D1). HSQC cross-peaks were
assigned by combining the results and comparing them with the literature. Volume
integration of contours in HSQC plots were calculated using Bruker TopSpin 3.1
software. For quantification of S/G distributions, only the C2-H2 correlations from G units
and the C2-H2/C6-H6 correlations from S unitswere used, and the G integrals were logically
doubled. For rough estimation of the various inter-unit linkage types, the following wellresolved contours were integrated: Aα, Bα, Cα, and their percentages are expressed as a
percentage of the total common inter-units (A-C).
Spin-cast film thickness and surface morphology
Lignin film thickness was measured using QCM-D as described in the main text. Frequency
and dissipation measurements were taken before and after spin casting, and the change in
frequency was compared. Frequency shifts across the third through 11th overtone were
within 5% of each other before and after spin coating, and the dissipation shift upon the
addition of the lignin layer was also small compared to the frequency shift. Because these
criteria for use of the Sauerbrey equation were met, the frequency shift (7th overtone) was
converted to surface mass. Using an assumed lignin density of 1.3 g/L (Hu, 2002) the film
thickness was calculated.
Supporting results
Structural comparison of isolated milled wood lignin with acid-pretreated Miscanthus by
2D-NMR
To obtain the structural features of the isolated milled wood lignin we used 2D HSQC NMR.
This method allowed us to obtain a comprehensive structural characterization of the lignin,
used in our experiments, and also to compare the isolated lignin structural features to the
source lignin in the acid pretreated Miscanthus. The lignin structural features are apparent in
two regions: the aliphatic-oxygenated (δC/δH 50-90/2.5-6.0 ppm) and aromatic 13C-1H
correlations (δC/δH 100-150/6-8 ppm) regions. NMR spectra of the acid pretreated
Miscanthus and its isolated MWL are shown in Figure S8. The structural type of inter-unit
linkage patterns of lignin can be found in the aliphatic side chain region. In the case of the
acid pretreated Miscanthus, polysaccharide signals dominated the spectrum, and partially
overlapped with some lignin signals. However, at least one of the correlations for each of the
structures in lignin is well isolated. On the other hand, the spectrum of the MWL presented
mostly lignin signals that, in general terms, matched those observed in the HSQC spectrum of
the pretreated Miscanthus, but with improved detection of the more minor lignin structures.
The aliphatic-oxygenated region of the spectra gave information about the different
inter- unit linkages present in the lignin. In this region, correlation peaks were observed from
methoxyl groups, β-ether (β-O-4) units (A), phenylcoumaran (β-5) units (B) and resinol (β- β)
units (C). In general, substructures were more clearly visible in the HSQC spectrum of the
MWL since the sample is free from carbohydrate impurities. The relative abundances of the
main lignin inter-unit linkages, estimated from volume integration of contours in the HSQC
spectra, show similarity between the isolated lignin and the pretreated biomass. The data
indicated that the structures of both lignins consist of ∼ 60% β-O-4 linkages, ∼ 30−35%
phenylcoumaran and a lower amount of resinol. The main correlation peaks in the aromatic
region of the HSQC spectra corresponded to the aromatic rings and unsaturated side chains of
the different lignin units and hydroxycinnamates. This region emphasizes the differences in
the guaiacyl:syringyl (G:S) distribution in the lignin polymer. Signals from guaiacyl (G), and
syringyl (S) units were observed almost equivalently in the spectra of the pretreated
Miscanthus and in the isolated MWL, due to this region being purely derived from such
structures (and completely free of the polysaccharide correlations). The signals revealed
some heterogeneity among the G and S units that affected the correlation, possibly due to
different substituents (phenolic or etherified in different substructures), though when
comparing the aromatic region with the whole cell wall NMR of Miscanthus (Cheng et al.,
2013), it is apparent that those structures were formed during the pretreatment and
presumably indicate on condensed lignin. The S/G ratio estimated from the HSQC (S/G 0.5)
is similar for both MWL and pretreated Miscanthus, although signals of H-lignin units were
not detected in the HSQC spectrum of the isolated MWL. Accordingly, it could be concluded
that the common inter-unit linkages were still preserved in the isolated lignin and that the
isolation process simply removed the carbohydrates contamination and did not alter the lignin
structure.
Lignin film thickness and surface morphology
Lignin films were cast from a variety of solvents and the thickness of the
resulting films measured. Film thickness was reproducible and controllable by adjusting
the lignin concentration dissolved in solvent. At 1.0% w/v, the lignin film thickness
ranges from 70-80 nm. This is thick enough to ensure uniform and complete coverage of
the substrate, and thin enough to allow for sensitive mass measurements on the surface
of the film as proteins adsorb.
The lignin films produced when spin coated from a variety of solvents were
analyzed by atomic force microscopy. Previous reports suggested that dilute ammonium
hydroxide (0.5 − 3.2M NH4OH) is a suitable solvent for producing lignin films (Norgren
et al., 2006), however, dynamic light scattering data indicated that milled wood lignin
from acid pretreated biomass was not fully soluble in this solvent. No aggregation was
detected in either THF or 96% dioxane. While 96% dioxane is an excellent solvent for
milled wood lignin (it is, after all, the solvent by which the lignin was extracted from
biomass), films cast from this solvent exhibit micron-scale heterogeneity in both height
and phase data, with features protruding up from the surface. Films cast from THF onto a
bare gold substrate look much different: the heterogeneities present are in the form of
holes in the surface.
Addition of the cationic anchoring layer PDADMAC was to improve the
adhesion of the lignin and the gold substrate during spin coating. With the anchoring
layer, films cast from dioxane exhibited less extreme roughness, but continued to show
significant heterogeneity in the phase measurement, indicating some micron-scale
ordering. When THF was used to cast the films on PDADMAC-coated sensors, the
surface was much smoother and with neither holes nor roughness seen in the nonanchored films. While the surface does exhibit some small variation in height and phase,
it is the smoothest and most homogeneous surface cast.
Films cast from lignin dissolved in either THF or 96% dioxane, with and without
an anchoring layer of adsorbed polydiallyldimethylammonium chloride (PDADMAC)
are presented in Figures S10-S13.
Figure captions
Figure S8. Side chain (δC/δH 50-90/2.5-6.0) and aromatic (δC/δH 90-150/5.5-8.0)
regions in the 2D HSQC NMR spectra of pretreated Miscanthus (left) and of the
isolated MWL (right). Color-coded structures correspond to the major resonances in
the spectra.
Figure S9. Film thickness (nm) of lignin films, spin-cast from a THF solution of varying
lignin concentration. Error bars indicate one standard deviation of 3 samples.
Figure S10. AFM height (A) and phase (B) data for the lignin-coated surfaces used for
measuring cellulase binding kinetics. Gold QCM-D sensors were soaked in a 0.1% w/v
PDADMAC solution, followed by spin-coating with lignin dissolved in THF (1% w/v).
These conditions resulted in a smooth (roughness = 3.3 nm RMS) surface free of defects ,
holes, or surface aggregation.
Figure S11. AFM height (A) and phase (B) data for lignin coated sensors. Coating
conditions: bare gold surface coated with lignin dissolved in THF (1% w/v).
Figure S12. AFM height (A) and phase (B) data for lignin coated sensors. Coating
conditions: bare gold surface coated with lignin dissolved in 1,4 dioxane (1% w/v). The
appearance of aggregates on the surface is clear.
Figure S13. AFM height (A) and phase (B) data for lignin coated sensors. Coating
conditions: PDADMAC-soaked surface coated with lignin dissolved in 1,4 dioxane (1%
w/v). While the surface is smoother than the dioxane-coated samples, undesirable
heterogeneity in the phase data is apparent.