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High-Throughput Analysis of Lignin by Agarose Gel Electrophoresis

Christopher A. Holt, Betty Cottyn, Stephanie Baumberger, Krisztina Kovacs-Schreiner,
and A. John Blacker*
Cite This: J. Agric. Food Chem. 2020, 68, 14297−14306 Read Online

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sı Supporting Information

ABSTRACT: A high-throughput agarose gel electrophoresis (AGE) analytical method has been developed to separate lignin
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fractions according to their molecular weight (Mw), charge, and shape. Operating conditions to effect separation of species have been
evaluated along with imaging parameters. Kraft, soda (Protobind), and Organosolv lignins showed distinct differences in migration.
Bands were cut, extracted, and cross-analyzed by gel permeation chromatography (GPC), 1H NMR, and pyrolysis GC/MS to
confirm their identity as lignin. The band intensity was correlated with lignin concentration by running serially diluted samples and
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imaging each lane to produce a precise calibration curve. The AGE technique was used to monitor and compare enzymatic, bacterial,
chemical, and hydrothermal lignin digestions. Each method showed changes in lignin migration and band intensities over time. Low
Mw species were seen in samples collected from the anode buffer tank. Though requiring further development, the AGE method can
provide structural information about the lignin and is accessible to biological and chemistry laboratories.
KEYWORDS: lignin, agarose gel electrophoresis, depolymerization, electrophoretic migration, high-throughput analysis

1. INTRODUCTION requires no less than 48 h and can lead to artifacts due to only
Lignin is the second most abundant organic biopolymer on partial solubility. A further problem is that derivatization
Earth and is highly variable across different plant species.1 It is results in charge neutralization, and this can alter lignin’s
of intense current interest as a renewable source of aromatic tertiary structure and the apparent Mw and Mn.18 Moreover,
compounds with the idea that it might provide the chemical the GPC column is calibrated using polystyrene standards
industry with at least a partial replacement for related oil- which are considerably different from lignin and provide
derived products.2−5 Methods for its deconstruction are varied relative rather than absolute information. Nevertheless, this
and include thermal (pyrolysis), thermochemical (alkaline, method remains useful for lignin classification and detection of
acidic, oxidative, or reductive), hydrothermal (autocatalytic overall structural modifications. Direct analysis is possible by
acid hydrolysis), and biological (insecticidal, microbial, or aqueous and polar solvent-based GPC but only for soluble
enzymatic) methods.6,7 Because of its complex heterogeneous lignins.19,20 Detection modes combined with GPC are UV,
structure, developing analytical methods to compare the refractive index, or light-scattering detection.21,22 The Mw of
depolymerization performance of these methods is a challenge. lignins varies between species and with the method of isolation
To date, this has relied on discrete analytical techniques that and measurement. Reported ranges are from 4500 (pine/
are poorly suited to large numbers of samples that arise in spruce lignoboost lignins) to 78 400 g/mol (Norwegian spruce
studies relative to lignin depolymerization.8,9 A widely used lignosulfonates) with dispersities (Mw/Mn) around 4.23,24
method to determine the lignin content is the gravimetric Besides information on the Mw distribution, some structural
Klason procedure, which requires several hundred milligrams characteristics of lignin are useful to assess depolymerization,
of sample.10 Derivatization with acetyl bromide allows much such as interunit bonds and functional groups. A variety of
smaller quantities of lignin to be used but is unreliable without multidimensional 1H and 13C NMR techniques can be used for
a standard and suffers contaminant species also absorbing at this purpose.25−27 Once again, these techniques are time
280 nm.11 On the other hand, colorimetric determination consuming, require expertise to generate and interpret data,
methods like the Folin−Ciocalteu, ABTS, and DPPH and are not suited to high-throughput screening of
techniques measure indirectly the total phenolic content of depolymerization conditions.
lignin samples but are prone to overestimation when reducing Since agarose gel electrophoresis (AGE) was first
sugars are present, e.g., from (hemi)cellulose, and provide little described,28 it has been used commonly for characterization
structural information.12−14 Assessment of the lignin molecular
weight distribution is commonly performed through GPC,
which provides the apparent average molecular weight in Received: October 1, 2020
weight (Mw) and in number (Mn).15−17 However, this Revised: October 28, 2020
technique generally requires extraction of the phenolic Accepted: November 4, 2020
compounds from an aqueous reaction medium by organic Published: November 17, 2020
solvent, evaporation, derivatization, dissolution in the final
solvent (usually THF), and filtration before injection, which

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of polynucleic acid anions and proteins and even carbon by Aguié-Béghin et al.39 Alkali lignin low sulfonate content was
nanotubes.29−31 Lignin electrophoresis has been mentioned in purchased from Sigma-Aldrich. EasiCal polystyrene standards used for
only three reports,32,33 with neither exploring its potential for GPC molecular weight calibration were purchased from Agilent
Technologies. Rhodococcus jostii pTipQ2-ligAB was kindly provided
separation nor characterization of lignin samples. There is a
by Prof. Tim Bugg (Warwick University). Pseudomonas putida
report of isoelectric focusing of lignin34 and one describing KT2440 strain (DSM number 6125) was purchased from the
capillary zone electrophoresis (CZE) for determination of the DSMZ. The laccase enzymes used in this work were purchased
lignin content in black liquor.35 Agarose gel electrophoresis from MetGen.38 Sodium borate (SB) buffer was made of 36 mM
(AGE) involves applying an electric field across a cast gel boric acid and 10 mM sodium hydroxide. TAE buffer was made of 40
immersed in a conducting electrolytic buffer, Figure 1. mM Tris base, 20 mM acetic acid, and 1 mM EDTA.
For gel imaging, it is better to have low-intensity light produced on
a completely dark background than high emission on a light-emitting
background. Imaging was carried out with a ChemiDocTM MP
Imaging System by exposing to light from 650 to 675 nm and
measuring emission at 700−730 nm unless otherwise stated. Gel
analysis was carried out using ImageJ (a program developed by the
National Institutes of Health and accessible online, https://imagej.
nih.gov/ij/). Using the software, gel bands were selected (with fixed
selection dimensions and fixed vertical alignment) and processed
using the in-built gel functions (ESI 2).
Preparation of Lignin Samples for Electrophoresis. Samples
of Kraft lignin KL1 were diluted in water to 5g/L unless otherwise
stated. Samples of Kraft lignin KL2 were dissolved in water by
dropwise addition of 5 M NaOH before readjusting to pH 7 with
Figure 1. Agarose minigel electrophoresis tank with brown migrating HCl. Samples of Protobind were made by adding 2 mL of SB buffer to
lignin bands. 10 mg of lignin. Samples of Organosolv were made by adding 0.5 mL
of SB buffer to 30 mg of lignin. The lignin mixtures were sonicated for
10 min and then centrifuged at 2880g for 15 min to separate the
While CZE is rapid and requires only tiny sample sizes, AGE soluble from the insoluble fraction of the lignin. The soluble fraction
has several advantages that include the ability to simulta- was then used for electrophoresis.
neously separate large numbers of samples in a short time, Standard Conditions for Mini- and Maxiagarose Gels. A
making it a high-throughput method, direct comparison of Clarit-E Mini gel tank was used with a 10 × 2 mm comb for thick
migrating bands, the ability to cope with particulates and wells to enable high sample loading. A Clarit-E Maxi gel tank had 40
detect compounds that do not migrate, and extraction of × 1 mm comb for narrow wells. The agarose gels were made using 1%
separated samples and preparative methods. (w/v) agarose in SB buffer, heating the slurry to ∼100 °C until the
At higher pH’s, lignin becomes a polyanion as free phenolic solution became clear, and allowing it to cool to 60 °C before pouring
into a gel mold using a comb to define the loading wells. When using
groups ionize around pH 9−10 and like nucleic acids should
the Clarit-E Maxi gel tank, the gels were poured on a leveled
migrate toward the anode. Agarose gels consist of pores which Flexicaster to ensure a uniform gel thickness, which was found to
retard the movement of larger molecules with respect to markedly affect the fluorescence consistency (ESI S1.7). The gel was
smaller ones.36 Gels are typically produced by dissolving formed after around 30 min, and SB buffer was poured into the tank
purified agarose in a hot buffer and casting the solution in a to submerge the gel. Dust causes problems with the gel and image
rectangular tray that solidifies upon cooling. A comb placed in analysis and was minimized by ensuring all equipment and solutions
the liquid gel creates individual wells in which the analyte were clean using lens cleaning tissues to remove any residual dust.
solutions can be placed. As a result, compounds can be Lignin samples were prepared in Eppendorf tubes by mixing 40 μL of
separated based on Mw, charge, and shape, for example, linear, 50% (w/v) glycerol, 40 μL of 0.375 M NaOH (ESI S1.5), and 100 μL
circular, and supercoiled DNA which has been used to test of a 5 g/L lignin sample before mixing using a vortex mixer. The
minigel was charged with 20 μL of sample/lane using a Gilson pipet.
cleavage reagents.37,38 This paper assesses the possibility to The maxigel was charged using a multichannel pipet. The multi-
transfer the AGE technique to the investigation of various channel pipet channels do not directly line up with every gel well due
lignin depolymerization processes and to elucidate the lignin to the tight spacing of the wells, so every other lane was loaded at a
structural parameters governing their migration. With their low time, as shown in ESI S1.1. After loading, samples were equilibrated
environmental impact, enzymatic and microbial lignin with buffer in the pocket for at least 30 min to give more consistent
depolymerizations are being widely researched to access band migration (ESI S1.2 and S1.3). Power was supplied from a
aromatic monomers, while hydrothermal treatments are BioRad PowerPac 300 to apply a fixed voltage across the gel.
being evaluated for energy and material applications. Chemical Electrophoresis was typically carried out at a fixed voltage of 50 V for
treatments are used widely in paper and pulping operations. 60 min.
Preparative Methods and Extraction of Lignin from the
We report herein the use of electrophoresis in separating lignin Agarose. Electrophoresis was carried out at 50 V for 40 min in a
and its use in monitoring deconstruction processes.

■
minigel tank with SB buffer and a 0.75% agarose gel using a 2 mm
comb to load more lignin onto the gel. Alkali lignin was dissolved in
MATERIALS AND METHODS water (300 g/L) and Kraft KL1 (200 g/L). A 25 μL amount of the
General. Chemicals were purchased from Sigma-Aldrich, except lignin sample was then mixed with 10 μL of 50% glycerol before
agarose and boric acid which were from Thermo Fisher Scientific. loading onto the gel. Once complete, the main band, or fractions of it,
Kraft lignin samples KL2 and KL1, produced using high and low Mw was cut across the 8 lanes using a scalpel (ESI S1.10 and S3.3). The
cutoff membranes, were purchased from 8317267 Canada Inc. excised gel was then freeze dried and ground into a fine powder using
Protobind lignins PB1, PB2, and PB3 were purchased from a pestle and mortar. The powder was then added to a flask with 100
GreenValue Ltd. Organosolv lignins OS1, OS2, and OS3 were mL of distilled water, stirred overnight, and filtered through a glass
provided by the Energy Research Centre of The Netherlands (ECN). filter tube. Concentrated HCl was then added to precipitate the lignin,
They corresponded to three distinct pulping conditions, as described and the mixture was kept at 4 °C overnight. The solution was then

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Table 1. Variables Tested in Defining Conditions for Electrophoresis of Kraft Lignin KL1
entry variable range tested selected condition
1 sample 0.63−214 g·L−1 lignin 10 μL from a mixed Eppendorf containing 20 μL of 50% glycerol + 20 μL of 375 mM NaOH
+ 50 μL of 5 g·L−1 lignin
2 buffer tris-acetate-EDTA (TAE) pH 7.75, sodium borate 36 mM, pH 8.75
8.32, 8.75
sodium borate (SB) pH 7.75, 8.32,
8.75
3 agarose strength 0.5−2% (w/v) 1% (w/v)
4 voltage/current 50−120 V/∼1000−420 mA 50 V/∼1000 mA
5 time and retention 20−240 min 60 min; Rf ≈ 0.45
factor (Rf) 0.125−0.9 Rf
6 visualization 400−800 nm irradiation/emission irradiation 650−675 nm; emission 700−730 nm
7 gel and image ethidium bromide; SyBr safe; no dye no dye; ImageJ and ChemiDocMP
analysis

centrifuged at 4500 rpm for 15 min, the supernatant discarded, and time points and centrifuged for 15 min at 4500 rpm to pellet the
the pellet freeze dried ready for further analysis. The recovered yield bacteria; the supernatant was then used for electrophoresis.
of KL1 lignin was ∼83% based on the mass. To test removal of adsorbed proteins, 40 μL of 10 mg/mL
Evidence That the Migrating Bands Are Lignin. Preparation proteinase K solution was added to 1 mL of 1.5 g/L Ps. putida-treated
of samples for GPC was carried out by precipitating the soluble lignin KL1 lignin solution. The mixture was then incubated overnight in a
from water by adding 1 mL of 6 N HCl. After 24 h the Organosolv, shaking incubator at 200 rpm set at 37 °C. The sample was then
Protobind, and Kraft lignin solutions were centrifuged at 2880g for 15 centrifuged at 4500 rpm for 15 min, and the supernatant was used for
min at 4 °C, the supernatant was discarded, and the pellet was freeze electrophoresis. For the heat treatment test, 1 mL of the Ps. putida
dried. Acetylation was carried out on 10 mg of each lignin mixed with sample was heated to 95 °C for 5 min using a heat block. The sample
0.6 mL of a 2:1 solution of acetic anhydride and pyridine left at room was then centrifuged at 4500 rpm for 15 min, and the supernatant was
temperature for 24 h. A 0.2 mL amount of ice cold methanol was then used for electrophoresis.
added to quench the reaction, the solvent evaporated under vacuum, Microwave treatment of bacterially degraded lignin was done to
then 0.2 mL of toluene added, mixed, and evaporated. The solvent test removal of adsorbed protein by adding 2 mL of Ps. putida-treated
washing procedure was repeated twice more. The remaining lignin samples to a 10 mL pressure reaction vial using a lid to ensure
acetylated lignin sample was then freeze dried. A 1 mL amount of the reaction was sealed and to prevent water evaporation. Micro-
of THF was added to dissolve the sample which was then filtered waving was carried out with a CEM Discover SP automated
using a 0.45 μm PTFE filter. The sample was then ready for GPC. microwave using a stir bar to ensure proper mixing. Microwaving
GPC was carried out with THF as eluent at 1 mL/min using an was carried out at 95 °C for 30 min in order to denature any proteins
Agilent PLgel MIXED-C column using an UltiMate 3000 present without boiling the sample.
Autosampler Column Compartment for sample handling and an Hydrogen Peroxide Breakdown of Lignin. In 250 mL conical
UltiMate 3000 Photodiode Array Detector. Chromeleon software was flasks, control and test Kraft lignin KL1 samples (5 g/L) were mixed
used for sample analysis. GPC plots were normalized by expressing in 75 mM phosphate buffer with 2 mL of 30% (v/v) (0.35M)
hydrogen peroxide at 37 °C in a shaker incubator mixing at 200 rpm.
the arbitrary units (mAU) values as a percentage of the highest mAU
A 1 mL amount of sample was taken before and after 48 h incubation.
value for each respective sample.
Hydrothermal Lignin Degradation. A 210 mL amount of 100
Enzymatic Digestion of Lignin. Enzyme treatment was carried
g/L of alkali lignin in water was prepared before heating to 250 °C
out in a 250 mL conical flask with a total reaction volume of 50 mL
under self-generated pressure for 1 h. A 10 mL sample was taken
containing 5 g/L KL1 Kraft lignin, 70 mM pH 7 potassium phosphate
before heating, afterward the process water was filtered using paper to
buffer, 11.8 units of the MetZyme LIGNO laccase enzyme mixture,
remove solids, and the solution produced was then used for gel
and 0.55 mM syringaldehyde (100 μL of a 275 mM stock solution electrophoresis.
dissolved in methanol) used as laccase redox mediator.40 The control Analysis of Low Mw Species. The following samples were
sample was identical in composition and volume except for the investigated for low molecular weight products: 100 g/L Alkali lignin;
presence of enzyme. A 1 mL amount of sample was taken 100 g/L hydrothermally processed Alkali lignin; 100 g/L KL1 Kraft
preincubation and at the respective time points and centrifuged for lignin; H2O2 treated Alkali lignin; 10 g/L vanillin and 10 g/L guaiacol.
15 min at 4500 rpm; the supernatant was then used for The H2O2-treated Alkali lignin was produced by incubating at 37 °C
electrophoresis. Enzymatic digestion was carried out on a shaker in a 50 mL conical flask 10 mL of a solution containing 100 g/L Alkali
incubator at 180 rpm set at 37 °C. lignin, 70 mM phosphate buffer, and 0.35 M hydrogen peroxide,
Bacterial Digestion of Lignin. All bacterial growth was carried shaking at 180 rpm for 48 h. Electrophoresis was carried out in a
out with a working volume of 50 mL in 250 mL conical flasks in a minigel tank at 50 V. A 50 μL amount of each sample was mixed with
shaking incubator at 30 °C and 180 rpm. Degradation conditions 20 μL of 50% (w/v) glycerol and 20 μL of 0.375 M NaOH. A 20 μL
were pH 7 sterilized M9 media containing 6.78 g/L Na2HPO4, 3 g/L amount of this mixture was added to 8 of the gel wells (leaving the
KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, and 1.5 g/L KL1 Kraft first and last wells empty).
lignin.41 The following elements were added after sterilization with a Analysis of UV-absorbing species was carried out by taking 10 mL
syringe filter with a 0.2 μm pore size: 2 mM MgSO4, 100 μM CaCl2, samples from the stirred anode end buffer before electrophoresis and
100 μM CuSO4, 100 μM MnSO4, 100 μM FeSO4, 100 μM ZnSO4, every 15 min for 60 min (ensuring the current was switched off). The
and 0.1 g/L vanillic acid to supplement growth for Rh. jostii and 10 g/ absorbance was measured at 280 nm using SB buffer as a blank. The
L glucose for Ps. putida. For the Rh. jostii experiments, 50 μg/mL mass of vanillin and guaiacol in the buffer was calculated using
chloramphenicol dissolved in ethanol was added (without thio- absorbance readings taken at 274 nm. Extinction coefficients of ε =
streptone inducer). Prior to inoculation into the media, Rh. jostii and 2739/M/cm (274 nm, pH 12) and ε = 3488/M/cm (289 nm, pH 12)
Ps. putida were grown in LB medium for 24 h and used to inoculate were used to calculate the mass of vanillin and guaiacol present.42 As a
the above media to a starting OD600 of 0.1. No bacteria were added control, a gel with only glycerol samples was run and the anode end
to the control sample. A 1 mL amount of sample was taken at specific buffer sampled in the same way with no UV-active species detected.

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NMR analysis of the low Mw species was carried out by removing S1.4). To observe migrating species, gels are typically stained
all of the buffer from the gel tank (450 mL total, ensuring the current with dyes that interact to increase the UV fluorescence
was switched off). The solution was concentrated and extracted using spectrum, enabling imaging equipment to scan and quantify
ethyl acetate, and the solvent was evaporated in vacuo. The solids the band intensity. Lignin fluoresces at ∼715 nm upon
were then dissolved in CDCl3 and analyzed by 1HNMR (ESI S5.1 and
S5.2) with a Bruker Ascend 400 MHz instrument.
irradiation with light at ∼662 nm, a convenient setting in the
Effects of Surfactants on Electrophoresis. The gels were made imaging system (Epi-far red), avoiding the need for stains,
using either pH 8.3 TAE buffer (40 mM tris base, 20 mM acetic acid, entries 7 and 8 (ESI 2). Further work to improve the lignin
and 1 mM EDTA) or pH 8.75 SB buffer (36.4 mM boric acid and 10 separation might explore more basic running buffers and
mM sodium hydroxide). Gels were made with 1% agarose and run at hydrogels with different functionality and porosity.
100 V for 10 min. A 5 μL amount of of Kraft lignin KL2 or KL1 (1.5 Characterization of the Migration Band. A set of
g/L) was mixed with 2 μL of 50% (w/v) glycerol and 2 μL of either experiments was carried out to confirm the identity of the
10% (v/v) SDS, 10% (v/v) Triton X-100, 10% (v/v/) DTAB, or migrating band. A gel overloaded with lignin (5 mg/lane) was
water. Gels were imaged using UV trans excitation light and submitted to electrophoresis under the standard conditions
measuring emission at 602−650 nm. A gel containing SDS running
buffer and 1% agarose was mixed with 0.5 μg/mL ethidium bromide
(Figure 2; inset A). After running for 1 h, a brown streak was
(after the gel was molten due to safety reasons). The gel was run at 50
V, and images were taken after 15 and 30 min of electrophoresis.
Control experiments were run without lignin using TAE buffer and
1% agarose. SDS was added to both the gel and the gel buffer to a
final concentration of 0.1% (after microwaving, but before pouring to
reduce foaming). No samples were loaded onto this gel. Gel
electrophoresis was carried out at 100 V and stopped every 10 min,
and the gel was removed from the gel buffer in order to image.

■ RESULTS AND DISCUSSION

Electrophoresis Conditions. In developing a lignin
electrophoresis method, the first task was to define a set of
conditions that gave good separation and clearly visible bands.
Table 1 shows the ranges of variables tested and the preferred
migration conditions identified (see also ESI 1).
A stock solution of Kraft lignin KL143 was prepared by
dissolving between 0.63 and 10 g·L−1 in water and adding 50%
(w/v) glycerol to increase the density and viscosity to facilitate Figure 2. (A) Visible light image of minigel with Kraft KL1 lignin
charging of samples to the gel, entry 1. A lignin concentration samples run under preferred conditions and ruler with millimeter
of 5 g·L−1 was shown to give a clearly visible band, while that at graduations. (B) Second gel run with lignin extracted from the top
0.63 g·L−1 was faint and that at 10 g·L−1 was starting to smear, half and bottom half of the band shown. (C) ImageJ trace of gel B
Figure 4. While overloading with high lignin concentrations showing fluorescence intensity vs migrating distance (arbitrary units)
of lanes 1 (blue), 2 (empty), and 3 (red).
(up to 214 g·L−1) was possible, lateral diffusion occurred but
makes possible preparative separation. It was later found that
adding sodium hydroxide to the sample facilitated lignin seen, and two sections (top half and bottom half) were cut
dissolution and after charging to the gel and leaving 1 h to from the gel and extracted by buffer overnight. The soluble
equilibrate in the pocket gave sharper more visible bands (ESI extracts were then recharged to a second gel and submitted to
S1.3). Two different buffers were tested: Tris-acetate-EDTA electrophoresis for 1 h (Figure 2; inset B). The migration of
(TAE), pH 8.3 and sodium borate SB, pH 8.8. TAE gave a the material in lanes 1 and 2 and their intensities were different
diffuse smeared band with sample remaining in the pocket, and corresponded to the fractions cut from gel A. This was
while the borate buffer gave much sharper bands and better confirmed by the ImageJ trace showing different fluorescence
retention factor, entry 2 (ESI S1.8). Different pH adjustments intensity maxima and distances from the pocket, Figure 2, inset
to the buffer were made (ESI S1.9); however, the migration C. This confirmed that separation of the two lignin fractions
band was clearest at pH 8.8, presumably as a result of could be achieved and that there was consistency in the
ionization of the phenolic residues. The buffer strength migrating species. The cause of the bands overlapping is
selected was 36 mM, with one-half of this concentration unclear but may be related to a change in lignin structure or
giving less migration (ESI S1.6). Agarose concentrations of associative complex due to the extraction procedure.
0.5%, 1.0%, and 2.0% (w/v) were tested (ESI S1.4). The GPC was used to confirm the identity of the migrating band
lowest concentration gave the highest Rf but similar separation as lignin. Figure 3 shows lignin KL1 before and after
to the 1% gel (as measured by the length of the streaked band). electrophoresis with the entire band cut and extracted from
Migration in the 2% gel was slow and the separation poor. the gel.
Since the mechanical stability of 0.5% (w/v) gels was poor, the The profiles for both samples are similar; however the
1% (w/v) agarose gel was selected as the preferred electrophoresed sample lacks the earlier eluting population
concentration, entry 3. Voltages of 50−120 V were tested (9−16 min retention time). This population, corresponding to
with the powerpack automatically adjusting the current for the a higher apparent Mw, might result either from lignin
system’s resistance. Higher voltages resulted in overheating and conformational changes or from aggregation due to 2-fold
distortion of the bands. A run time of 60 min at 50 V gave freeze drying. The apparent Mw and Mn of the standard were
better resolution than 40 min at 120 V with a retention factor 12 243 and 1076 g/mol, respectively; while they were 95 929
(Rf) of around 0.3 and smearing between 0.125 and 0.45 (ESI and 1698 g/mol for the extracted sample. The dispersities for
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volume rather than the bottom of the pocket (ESI S1.2). This
result showed the electrophoresis and imaging techniques are
reliable for generating a standard curve to quantify the
concentration of lignin in an unknown sample, though they
should be repeated for each type of lignin.
Electrophoresis of Different Lignins and Comparison
with GPC. To assess the electrophoretic separation of
different types and Mw fractions of lignin, the migration was
compared to GPC elution. Figure 5, top row, shows a 38-lane
gel comparing the migrations of three Organosolv (OS1−3),
three Kraft (KL1, KL2, AL), and three Protobind (PB1−3)
Figure 3. GPC plots of lignin KL1 before electrophoresis and after lignins with four repeats of each. Figure 5, middle row, shows
with extraction from the gel. Plots normalized with respect to the peak GPC plots for each these lignins; the bottom row shows traces
maximum. of the same lignins scanned from the electrophoresis gel. In the
gel, differences in migration and fluorescence intensity were
KL1 before and after electrophoresis were 11.4 and 56.5, observed that presumably relate to differences in mass, charge,
respectively. In addition to GPC, 1H and HMBC NMR were and shape.
carried out on the extracted lignin and showed the character- The GPC and electrophoresis profiles showed high
istic sets of peaks for CHO (9.5 ppm), aromatic CH (∼7 similarity. In Figure 5A, the overlaid GPC plots for the
ppm), CHγ (∼5.5 ppm), CHα (∼4.5 ppm), and OMe (3.75 Protobind lignins PB1−3 show closely matched elution, which
ppm) (ESI 3.2). 31P NMR was attempted by derivatizing is mirrored in the electrophoresis in Figure 5D. A key
extracted and freeze-dried lignin with 2-chloro-4,4′,5,5′- difference, however, is the presence of additional peaks eluting
tetramethyl-1,3,2-dioxaphospholane (TMDP). The spectrum after 19 min of the GPC profiles. These peaks correspond to
showed an increase in side-reaction products (probably low Mw compounds assigned to phenolic monomers and
phosphite at around 15 ppm and hydrolyzed TMDP at 132 impurities arising from the derivatization procedure. In the
ppm) probably due to remaining traces of HCl that hydrolyzed electrophoresis traces, the closely matched overlay shows that
the reagent, making impossible the quantification of phenolic the three lignin samples had a similar mass to charge and
and aliphatic hydroxyl groups. MALDI-TOF MS was tried shape. Regarding Kraft lignins (Figure 5B and 5E), a similar
unsuccessfully; however, pyrolysis GC/MS of the extracted difference between KL2 and KL1 profiles was observed by
sample showed phenolic-related products guaiacol, 4-ethyl- GPC and electrophoresis. Thus, results obtained by both
and 4-propylguaiacol, and dimethoxy catechol and no agarose- methods were consistent, and because GPC is insensitive to
or carbohydrate-related products such as furans (ESI 3.3). This charge, it can be deduced that the mass-to-charge ratio and
combination of analytical results provided evidence that the shape of both KL2 and KL1 are similar. A distinct
migrating species was indeed lignin. electrophoretic behavior was observed with the Alkali lignin.
Quantification of Lignin Concentration. Figure 4 shows Indeed, it migrates significantly further than KL2 and KL1,
a concentration gradient of Kraft lignin KL1 prepared with a which suggests a higher anionic charge or a smaller size than
high Mw cutoff membrane on a 1% agarose minigel. for KL2 and KL1. Unfortunately, GPC analysis of this lignin
sample could not be performed as it was insoluble in THF and
failed to derivatize. Finally, good agreement was obtained
between the GPC and the electrophoresis profiles of the
Organosolv lignins OS1, OS2, and OS3 in terms of peak shape
and position (Figure 5C and 5F). The sample showing the
highest Mw, OS3, showed streaking from the pocket to the
main band on electrophoresis. In contrast, the lowest Mw
sample, OS1 (entry 7), showed a sharper, further migrating
band that correlated with its lower Mw. In conclusion, these
results indicate that electrophoresis could be used to segregate
lignin samples according to their migration profile and in
agreement with differences observed by GPC. Even slight
Figure 4. Serially diluted Kraft lignin KL1 run on a 1% agarose gel at
differences between the samples could be detected by this
pH 8.8, 50 V, for 60 min, and calibration curve with points taken from method. The GPC-determined values of Mw, Mn and
ImageJ fluorescence maxima in each lane. polydispersities for each lignin calibrated against polystyrene
standards are given in Table 2.
The set of data provides some degree of calibration for the
The serially diluted lignin produced a band which decreases electrophoresis gels and traces. Further work should develop a
in intensity with concentration. The gel was imaged, and lignin Mw ladder, as used in polynucleotide electrophoresis.
fluorescence emission scans of each lane gave an intensity area Denaturing Gel Electrophoresis. Surfactants, such as
that was plotted against concentration to obtain a calibration sodium dodecyl sulfate (SDS), are used commonly to denature
curve showing a best fit R2 of 0.988 (ESI S1.11). It was found proteins and reduce shape and charge effects, though usually in
that more consistent bands and accurate traces could be polyacrylamide (SDS-PAGE) rather than agarose gels. To test
obtained by casting onto a perfectly level gel bed avoiding dust, the effect of surfactants on lignin migration in gels, SDS
ignoring the outer lanes due to edge effects on the gel, and (anionic), Triton-X100 (neutral), or dodecyl trimethylammo-
allowing samples 0.5−1 h to distribute into the full pocket nium bromide (DTAB, cationic) was added to the samples and
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Figure 5. Comparison of different lignins by AGE and GPC. Top row: 38 lane lignin electrophoresis gel. Lanes: C = control; 1, 2, 19, 20 = PB1; 3,
4, 21, 22 = PB2; 5, 6, 23, 24 = PB3; 7, 8, 25, 26 = KL2; 9, 10, 27, 28 = KL1; 11, 12, 29, 30 = AL; 13, 14, 31, 32 = OS1; 15, 16, 33, 34 = OS2; 17, 18,
35, 36 = OS3. Middle row GPC plots: (A) Protobind lignins PB1, PB2 and PB3; (B) Kraft lignins KL2 and KL1; (C) Organosolv lignins OS1,
OS2, and OS3. Bottom row gel electrophoresis traces taken from top row gel; duplicate lanes averaged and normalized to 100% intensity: (D)
Protobind lignins PB1, PB2, and PB3; (E) Kraft lignins KL2, KL1, AL; (F) Organosolv lignins OS1, OS2, and OS3.

Table 2. Mn and Mw values (g/mol) for Kraft, Soda

(Protobind), and Organosolv Samples Determined from
GPC Chromatograms Using Polystyrene Standard
Calibration
entry lignin sample Mn Mw dispersity
1 Kraft KL2 1224 23 042 18.8
2 Kraft KL1 1076 12 243 11.4
3 Kraft AL N/A ∼10 000a N/A
4 Protobind PB1 560 5568 9.9
5 Protobind PB2 694 7657 11.0 Figure 6. (Left) Minigel without lignin showing the interaction
6 Protobind PB3 720 9888 13.7 between SDS and EtBr. Visible bands with SYBR safe and water are
due to residual EtBr in the tank interacting with the SDS. (Right)
7 Organosolv OS1 634 3198 5.0
Minigel, free from EtBr, showing the difference in migration and
8 Organosolv OS2 787 5919 7.5
fluorescence with SDS treatment of Kraft lignins KL2 and KL1.
9 Organosolv OS3 862 8802 10.2
a
Value provided by manufacturer.
samples, a sharper, more fluorescent band was observed
(Figure 6, right-hand gel). The effect of the surfactant was to
run as previously on a 1% agarose gel. SDS gave a single sharp reduce the difference between KL2 and KL1 migration,
band with an increased fluorescence, Triton-X gave a smear indicating interaction with lignin to reduce the mass to charge
centered around the pocket but extending to Rf ≈ 0.4, and and shape effects, but also separation of species. The nature of
DTAB gave fluorescence around the pocket indicating a the SDS−lignin complex is unclear, but it seems that
precipitate (ESI 6). SDS was evaluated further as neither the hydrophobic interactions are involved. More work is required
neutral nor the cationic surfactants improved separation. A to determine potential benefits of using surfactants in lignin
problem encountered early on was that even small residual electrophoresis.
quantities of ethidium bromide (EtBr) left in the electro- Investigation of Lignin Depolymerization. A series of
phoresis tanks complexed with the SDS to give fluorescent experiments was carried out on the Kraft KL1 lignin to
anionic bands that migrated down the gel, Figure 6 (left-hand compare the effects of different lignin deconstruction methods
gel). EtBr is a cationic dye used routinely in revealing DNA, through electrophoretic differences, Table 3. These methods
though now superseded by SYBR safe. The SDS EtBr band is were oxidation by a laccase enzyme preparation made
presumably SDS micelles ion paired with the EtBr. specifically for this purpose,36,44 conversion by two bacteria,
Despite repeated washing of the tank, the only solution was one of which was engineered to accumulate small molecule
to use a new one that had not been exposed to the dye. Now, intermediates,41,45 oxidation by hydrogen peroxide,46 and
when control samples were compared with SDS-treated hydrothermal treatment involving superheated water.
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Table 3. Comparison of Different Methods for Kraft KL1

Deconstructiona
starting
treatment concentration fluorescence
entry method time (h) (g/L) change (%)
1 laccase 72 5.0 −15
2 Ps.putida 264a 1.5 −6b,d
KT2440
3 Rh.jostii RHA1 240e 1.5 −26c,f
4 H2O2 48 5.0 −20
5g H2O2 48 5.0 −38
6g hydrothermal 1 95 −30h
a
For more details, see ESI 4.2. bAs determined from calibrated curve
areas from electrophoresis traces. cWorked up by microwave
treatment before electrophoresis. dIncrease in high Mw species.
e
With 0.1 g·L−1 vanillic acid. fIncrease in low Mw species. gAlkali
lignin. hTwenty-two percent of mass changed from soluble to
nonmigrating solid.

From these experiments, lignin depolymerization was

assessed by measuring the reduction in fluorescence area
under the curves generated by scanning each lane in the gel,
Figure 7.
Incubation of Kraft KL1 lignin with the laccase at 37 °C for
72 h (ESI S4.1) induced a reduction in the lignin band
fluorescence of 15% with respect to the controls, Table 3, entry
1. Interestingly, the trace showed a lower proportion of high
Mw species, a reduction in the main band intensity, but no
change in the further migrating, low Mw/charge, species. This
suggested that the enzyme might cleave interunit bonds but
that the released lower Mw compounds underwent reconden-
sation which is in agreement with laccase-related literature.47,48
The changes in electrophoretic profile matched those obtained
by GPC.
For the bacterial lignin digestion, the wild-type strain Ps.
putida KT2440 and blocked mutant Rh. jostii RHA1 were both
grown on lignin KL1 for 10 days; the former strain was
supplemented with glucose to initiate growth, while the latter Figure 7. Electrophoretic migration fluorescence traces of Kraft KL1
strain was supplemented with vanillic acid to induce enzymes after depolymerization treatment: C = control, T = treated, H = hour,
in the aromatic degradation pathways.45 Initial and final D = Day. Top to bottom: laccase enzyme; Rh. jostii bacteria; H2O2;
samples were centrifuged to separate the bacteria, and hydrothermal. See ESI 4 for gels. MW = microwaved.
supernatants were analyzed by electrophoresis. In contrast to
laccase, the bacterial treatments led to an increase in the species along with a general decrease in intensity across the
fluorescence intensity of the lignin band, ESI 4.2. This result whole lane. This indicates nonspecific structural modification
was unexpected but might be structural changes due to that affects the lignin fluorescence, a bleaching effect.
adsorption of extracellular enzymes. To test this, samples were Hydrothermal treatment of soluble Kraft AL lignin (95 g/L)
treated with proteinase K; however, no change in the lignin led to formation of a solid, 22% (w/w). Some carbonization
fluorescence was seen. It was found that a 30 min microwave was expected for the conditions used, 1 h at 250 °C. The solid
heat treatment was able to denature or desorb the attached was removed by filtration, and electrophoresis of the soluble
species, allowing the effect of lignin digestion alone to be seen. material revealed a decrease in the overall fluorescence
Comparison with the controls indicated that Ps. putida had intensity of 30% compared to the control (ESI 4.4). The
depolymerized 6% of the KL1 lignin and Rh. jostii 26% (Table trace showed two distinct bands: one of nonmigrating material
3, entries 2 and 3). The shape of the 10-day electrophoresis retained around the pocket (22% of the fluorescence) and a
trace for Rh. jostii showed a slight increase in the proportion of band less intense but migrating the same distance as the
low Mw species with a slightly shifted maximum and less untreated control (78% of the fluorescence), Table 3 (entry 6)
fluorescence than the control. and Figure 7. This implies that the mechanism of hydrothermal
Treatment of Kraft KL1 with H2O2 (0.35 M, 37 °C, 48 h) carbonization is through loss of aromaticity and deoxygena-
led to a 20% decrease in the fluorescence intensity of the lignin tion.49 Carbonization of lignin is well known,50 but it is novel
band (Table 3 (entry 4); ESI 4.3). This decrease was even to observe changes this way and may help in the development
more pronounced (38%) by treatment of the Alkali lignin of more selective methods.
(entry 5) tested for comparison, showing it was more Analysis of low Mw Species. Having observed changes in
susceptible to breakdown than Kraft lignin. In both cases, the main lignin band in each of the degradation methods,
the band profiles showed a lower proportion of higher Mw experiments were carried out to understand the mass balance
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Figure 8. UV absorptions of species eluting from electrophoresis gels with samples taken from the anode buffer tank at 0, 15, 30, 45, and 60 min
time intervals. Each sample loaded onto the gel contained 16.7 μL of 100 g/L lignin: KL1 untreated Kraft lignin KL1; AL alkali lignin; HTC H1
hydrothermally treated AL, 1 h; 0.35 M H2O2 treated KL1 after 48 h; 1% (w/v) vanillin; 1% (w/v) guaiacol. Error bars generated from four repeats
(ESI 5).

by seeing whether far-migrating species were formed that In summary, a useful AGE method has been developed to
accumulate in the anode end buffer. Electrophoresis was help characterize different lignins based on mass, charge, and
carried out with Kraft KL1, Alkali lignin, hydrothermal, and shape. As little as 10 μg of lignin is required in the sample, and
H2O2-treated samples along with controls of vanillin, guaiacol, this compares to 10 mg for GPC analysis Electrophoretic
and empty gel (ESI5.1). The current was switched off at 15 conditions have been identified to separate species according
min intervals to sample the buffer tank and measure the UV to their apparent Mw. These include gel density, buffer, pH,
absorbance. After 60 min the main lignin band remained in the sample loading, applied voltage, and migration time and for gel
gel, so only low Mw molecules were detected, Figure 8. imaging, excitation and fluorescence emission wavelengths and
In each experiment, the UV absorption increased with time, quantification of retention factors and band intensities. Once
indicating an accumulation of low Mw anionic species. Both edge effects, level gel casting, and equilibration of samples in
untreated lignins appeared to contain rapidly migrating species pockets were understood, both mini 8-lane, and maxi 40-lane
that absorb at 280 nm. Interestingly, KL1 hydrothermally gels were used with excellent reproducibility, as seen in the
treated for 1 h had a lower concentration, or UV inactive, fast small variations in repeat experiments. Overlaid curves were
migrating species which correlates with the observations in referenced to migration from the leading edge of the pocket. In
some cases, it was useful to normalize fluorescence values to
Figure 7. H2O2-treated KL1 gave an absorption that increased
100% so that small structural differences in lignin migration
over the hour to almost twice that of the KL1 control and is
could be observed. Fluorescence intensities were measured
commensurate with the decrease in fluorescence seen for the
from traces for serially diluted lignins, enabling calibration
main lignin band. It is useful to confirm that vanillin and curves with high linear correlation and subsequent determi-
guaiacol anions migrate rapidly through the gel but take >10 nation of unknown sample concentrations, proving that
min to emerge. On the basis of their reported extinction structural variations do not affect fluorescence. Use of SDS
coefficients,42 the concentrations in the tank are 19 and 24 μM surfactant identified a problem with the electrophoresis of
respectively, which provides an estimate of the concentration micelles and interaction with cationic dyes. Once these effects
of the species in the other samples. were eliminated, SDS was shown to complex with lignin by
One experiment was done in which the buffer solution from reducing mass−charge and shape effects. This was exemplified
the anode end of the KL1, H2O2 experiment was concentrated using Kraft lignins KL2 and KL1, whose structure was
and solvent extracted. 1H NMR of the sample showed modified to give the same migration velocities and less
unassigned signals at 1, 3.5, 4, and 7 ppm (ESI 5). smearing. The benefits of using SDS with lignin are not yet
Interestingly, the aromatic coupling patterns indicated a 3,4- clear, and more work is required to define these. Larger
disubstituted aromatic. Further work is ongoing to characterize quantities of purified lignin were prepared by overloading large
the breakdown products. This study shows promise in gels, cutting, and extracting the lignin band, and these were
separating small molecules from lignin digestates. used in GPC experiments to determine their mass and
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Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

polydispersity. Further work will look at analyzing the 1401C0066] and by the European Commission [ERA-IB-14-
extracted lignin by NMR, which has so far been unsuccessful 055] in the framework of the LIGBIO project. The IJPB
due to the size of sample required and residual acidity. research unit benefits from the support of the LabEx Saclay
Electrophoresis of different types of Kraft, Alkali, Organosolv, Plant Sciences-SPS (ANR-10-LABX-552 0040-SPS).
and soda (Protobind) lignins has shown differences in Notes
migration behaviors that correlate with GPC elution profiles
The authors declare no competing financial interest.

■
and can be used to assess differences in Mw and charge. For
example, Alkali lignin migrates more quickly than Kraft lignin
KL1 despite being similar in Mw, indicating a higher charge to ACKNOWLEDGMENTS
mass or different shape. It has not yet been possible to generate Wouter J. J. Huijgen (Energy Center of The Netherlands) is
a Mw ladder to correlate with migrating bands, as used acknowledged for providing the Organosolv lignin samples.
commonly in DNA and protein analysis. AGE was used to Amel Majira is sincerely acknowledged for technical support at
compare different methods of lignin breakdown. A 6−38% IJPB. This paper is dedicated with much gratitude to
decrease in mass/charge was seen in enzymatic, bacterial, Professors Jean-Marie Lehn and Jean-Paul Behr.

■
hydrothermal, and hydrogen peroxide treatments over different
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