Mini-review

Received: 7 August 2012

Revised: 25 October 2012

Accepted: 25 October 2012

Published online in Wiley Online Library: 12 December 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.3996

Role of lignin in a biorefinery: separation

characterization and valorization
Tong-Qi Yuan,a Feng Xua and Run-Cang Suna,b
Abstract
Lignin, a major component of the cell wall of vascular plants, has long been recognized for its negative impact and treated
as a by-product in a biorefinery. This highly abundant by-product of the biorefinery is undervalued and underdeveloped due
to its complex nature. The development of value-added products from lignin would greatly improve the economics of the
biorefinery. The inherent properties of lignin significantly affect the productivity of the biorefinery processes and its potential
applications. Although the structure and biosynthetic pathway of lignin have been studied for more than a century, they
have not yet been completely elucidated. In this mini-review, the primary obstacles to elucidating the structure of native
lignin, including separation and characterization, are highlighted. Several classical methods for separation and various NMR
techniques, especially 2D HSQC NMR, for characterization of lignin are reviewed. Some potential applications of lignin are
introduced. It is believed that a knowledge of the method to separate lignin from the cell wall and structural features of the
lignin polymer from lignocellulosic materials will help to maximize the exploitation of lignocelluloses for the biorefinery as well
as the utilization of lignin for novel materials and chemicals.
c 2012 Society of Chemical Industry


Keywords: lignin; biorefinery; separation; characterization; valorization; NMR

INTRODUCTION

346

Lignocellulosic materials are composed of three major

components: cellulose, hemicelluloses, and lignin.1 This abundant
renewable resource can serve as a source of carbonneutral or carbon-negative feedstock for the production of
biofuels, chemicals, and polymers, generally referred to as the
biorefinery.2 4 It is becoming a logical and promising alternative
to petroleum resource in light of the increasing economic and
environmental issues concerning the use of petrochemicals.5,6
However, most biorefineries currently concentrate on the
valorization of cellulose and hemicelluloses, a so-called sugar
platform, while lignin is usually considered a low-value residual.7
Numerous previous studies have pointed out that the inherent
properties of lignin significantly affect the productivity of the
biorefinery processes and its potential applications.7 11
Generally, lignin is considered as being formed by the
dehydrogenative polymerization of three hydroxycinnamyl
alcohols: p-coumaryl, coniferyl, and sinapyl alcohols (Fig. 1).12 Each
of these monolignols gives rise to different types of lignin units
called p-hydroxyphenyl, guaiacyl, and syringyl units, respectively.
However, it is now widely accepted that other monomers also
participate in coupling reactions that give rise to the lignin
macromolecule, such as coniferaldehyde, coniferyl alcohol, and
acylated monolignols.13 In addition, lignin is an amorphous
polymer with different linkages among the monolignol units, such
as -O-4, -, -5, -1, 5-5, 4-O-5, and dibenzodioxocin linkages,
of which the content of -O-4 linkage is over half of the whole
linkages of lignin.14 Besides, as lignin is biosynthesized after the
polysaccharide matrix has been laid down, lignincarbohydrate
complex (LCC) linkages are also formed in plant cell walls. The main
types of LCC linkages in lignocellulosic materials are believed to be
J Chem Technol Biotechnol 2013; 88: 346352

phenyl glycoside bonds, esters, and benzyl ethers.15,16 Although

the structural characterization of lignin and biosynthetic pathway
of monolignols have been investigated for over a century, they
have not yet been completely elucidated.17,18

INTERACTION OF LIGNIN WITH BIOREFINERY

A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power, and chemicals from
biomass. Lignocellulosic materials contain high amounts of carbohydrates (cellulose and hemicelluloses), which can be converted
into fermentable sugars.19 However, lignocellulosic materials are
resistant to microbial and enzymatic deconstruction, known as
biomass recalcitrance.20 Several natural factors are believed to
contribute to the recalcitrance of lignocellulosic materials to chemicals and enzymes. These include the degree of lignification, the
structural heterogeneity and complexity of cell-wall constituents,
and the inhibitors to subsequent fermentations that exist naturally
in cell walls or are generated during conversion processes.20 23
Among the various structural features affecting biomass
enzymatic digestibility, the presence of lignin is one of the most
significant contributors to biomass recalcitrance.24 The lignin
composition as well as lignin content have been considered to

Correspondence to: R-C. Sun, Institute of Biomass Chemistry and Technology,

Beijing Forestry University, Beijing 100083, China. E-mail: rcsun3@bjfu.edu.cn

a Institute of Biomass Chemistry and Technology, Beijing Forestry University,

Beijing, 100083, China
b State Key Laboratory of Pulp and Paper Engineering, South China University of
Technology, Guangzhou, 510640, China

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c 2012 Society of Chemical Industry

Separation, characterization, and valorization of lignin for biorefinery

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in a chemically unaltered form because of the tight physical
binding and chemical linkages between lignin and cell wall
polysaccharides.35 As lignin is synthesized after the polysaccharide
matrix has been laid down, isolation of pure lignin is problematic.36
The existence of the stable LCC linkages in cell walls is considered
one of the main reasons preventing selective separation of lignin
fraction. The other obstacle is the lack of ideal techniques, which
could provide adequate and quantitative information for the
entire lignin structure and various LCC linkages.37 Although the
exact structure of lignin is unknown, improvements in methods
for identifying lignin degradation products and advancements
in spectroscopic techniques have enabled scientists to elucidate
the major structural features of native lignin. Several classical and
modern techniques for separation and characterization of lignin
are discussed below.

Figure 1. The three primary lignin monomers, p-coumaryl alcohol, coniferyl

alcohol, and sinapyl alcohol and lignin structural p-hydroxyphenyl,
guaiacyl, and syringyl units.

affect the enzymatic hydrolysis of cellulose.25,26 The composition

of the functional groups of lignin27,28 and physical distribution,
such as syringyl (S) unit to guaiacyl (G) unit ratio,26,29 have
also been demonstrated to affect the enzymatic hydrolysis of
cellulose. In general, the yield of enzymatic hydrolysis is believed
to be enhanced by low lignin content and a high S:G ratio of
the substrate. Lignin appears to limit cellulose hydrolysis by
two distinct mechanisms: first by forming a physical barrier that
prevents enzyme access and second by non-productive binding
cellulolytic enzymes.30
The biomass recalcitrance must be overcome to explore biofuels
from lignocellulosic materials efficiently and economically. Indeed,
a pretreatment step is essential and is becoming the bottleneck
in the biorefinery process. The pretreatment technologies can
be classified into biological, physical, chemical, and physicochemical methods. The advantages and disadvantages of different
pretreatment methods have been previously reviewed,31,32 and
are outside the scope of the current mini-review. However, it
should be noted that almost all biological processing platforms
for the conversion of lignocellulosic materials to biofuels result in
the formation of a vast lignin process stream.33 The majority of
the lignin is generated mainly after the pretreatment stage, with
a small quantity of lignin being produced after fermentation. At
present, only a small proportion of lignin is utilized as an energy
source for power generation and limited value-added products,
therefore, a major part of this by-product could be used as a
feedstock. It is obvious that conversion of biorefinery lignin into
high-value co-products will help to offset the cost of biofuels
production. Unfortunately, as an embarrassing myth in industry
states You can make anything out of lignin, except money, the
high-value application of lignin is still a challenge.
The inherent complexity and heterogeneity of lignin and the
change of lignin structure during the biorefinery process make
it difficult to develop an efficient and economic conversion
technology of lignocellulosic materials and to provide high-value
application of lignin.34 Therefore, a comprehensive understanding
of lignin structure and composition is important for the whole
biorefinery process.

STRUCTURAL CHARACTERIZATION OF LIGNIN

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347

Elucidation of the structure of native lignin involves two major

obstacles. The primary one is that it cannot be separated

Separation
In the past several decades various methods have been developed to isolate lignin from plant cell walls. The first major method
38

was proposed by Bjorkman

who extracted milled wood lignin
(MWL) from finely ball-milled wood with aqueous dioxane. However, concerns exist over the similarity between MWL and native
lignin based on the low yields.39 The yields of MWL are limited,
range from 10% to 65%, and heavily dependent upon milling
time and the nature of the lignocellulosic materials.40 In order
to improve the yield of lignin, a method to isolate cellulolytic
enzyme lignin (CEL) was proposed by Pew.41 The CEL obtained
was found to be structurally similar to MWL,41,42 but at a higher
yield. Some improved methods to isolate CEL were subsequently
brought forward. A common goal is to swell cellulose or decrease
the cellulose crystallinity for a completely enzymatic hydrolysis.
For instance, ball-milled wood has been swelled or dissolved in
organic solvents, such as N,N-dimethylacetamide/lithium chloride
or dimethyl sulfoxide/N-methylimidazole, then regenerated in
water or ethyl acetate, and treated with cellulase.43,44 The yields of
CEL isolated from the regenerated ball-milled wood samples were
therefore higher than those obtained directly from the ball-milled
wood material. In addition, a novel procedure using the combination of enzymatic and mild acidolysis was proposed by Wu
and Argyropoulos.45 The enzymatic mild acidolysis lignin (EMAL)
obtained offered higher yields and purities than those of the corresponding MWL and CEL.37,45,46 Therefore, it is certain that EMAL
was more representative of the total lignin present in milled wood.
It should be noted that the structures of lignin are modified
to some extent during the ball-milling process. Previous research
confirmed that ball milling of wood degrades -O-4 structures in
lignin, and some condensation reactions may occur during this
process.35,39,47 In addition, all above mentioned methods separate
only a certain part of lignin in the plant cell walls, the lignin fraction
in the residual wood meal should be investigated. It is believed that
structural characterization of lignin in residual wood meal left after
extraction of MWL or CEL could be important for understanding
the structure of a greater proportion of lignin in the cell walls.48 An
ideal technique is to try to isolate as much of the lignin as possible
while minimizing the extent of chemical modification.
The largest amount of lignin is produced in the papermaking
industry. Lignin is separated by chemical processing techniques
known as pulping, such as sulfite, kraft, and soda pulping processes.
The proportion of lignin derived from the pretreatment process
of lignocellulosic materials for biorefineries is increasing. These
pretreatment processes include the organosolv process, steam
explosion, ammonia fibre explosion, acid and alkali pretreatments,

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T-Q Yuan, F Xu, R-C Sun

Figure 2. The 2D HSQC NMR spectra of MWL from Triploid of Populus tomentosa Carr.51 Symbols are taken from Fig. 3.

as well as ionic liquids pretreatment. The lignin obtained through

these processes is chemically different from that available from
the papermaking industry. More details about properties of lignin
from these processes, both pulping and pretreatments, have been
described in recent review articles.7 11 However, lignin separated
from these processes is not suitable for a structural study of native
lignin in the plant cell walls, as the structure of lignin will be
significantly changed during these processes.

348

Characterization
Since lignin is a very complicated polymer with no extended
sequences of regularly repeating units, its composition and
structure are generally characterized by the relative abundance of
H/G/S units, the distribution of inter-unit linkages, the functional
groups, as well as the degree of condensation in this polymer.
Various analytical methods have been developed for lignin
analysis, qualitatively and quantitatively, although there is still
a need to develop new methods capable of solving problems
associated with lignin.13
Over the last century, both destructive and nondestructive
methods have been developed for lignin characterization. The
destructive methods include acidolysis, hydrogenolysis, nitrobenzene oxidation, cupric (II) oxidation, permanganate oxidation,
ozonation, thioacetolysis, thioacidolysis, and derivatization followed by reductive cleavage (DFRC) proposed by Lu and Ralph
in 1997 as an alternative to thioacidolysis.49 All these destructive methods could provide information regarding the structure
of lignin through the generation of low-molecular weight compounds. Through careful analyses of these compounds, a detailed
picture of the original lignin can emerge. However, all these methods involve selective cleavage of the lignin backbone and liberate
only a fraction of the polymer for analysis.50 Thus, to date no
destructive method is available that allows the identification and
quantification of all structural features of lignin.
The nondestructive methods consist of various microscopic
and spectroscopic methods such as UV microscopy, interference
microscopy, Fourier transform infrared (FTIR) spectroscopy, Raman
spectroscopy, and NMR.13 As a mini-review, only the 2D NMR
technique will be discussed based on its unique usefulness for
lignin characterization. Details about other methods have been
described in several books and review articles.
NMR spectroscopy has been shown to be a reliable and
comprehensive technique to powerfully elucidate the lignin
structure. Although 1 H and 13 C NMR spectra have been applied

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for lignin characterization, these two conventional 1D NMR

spectra do not allow detailed assignment and quantification of
individual signals due to broad and overlapping NMR signals
resulting in low-resolution spectra.50 2D NMR, on the other hand,
such as Heteronuclear Single Quantum Coherence (HSQC) and
Heteronuclear Multiple Quantum Coherence (HMQC) experiments,
which provide correlations between directly bonded protons and
carbons in two dimensions, provide a great platform for lignin
characterization. 2D NMR spectroscopic technique combines the
sensitivity of 1 H NMR with the higher resolution of 13 C NMR and
can be used to reveal the ratio of the different lignin units and
the types as well as the percentage of the main interunit linkages.
For example, the 2D HSQC NMR spectra of MWL from Triploid
of Populus tomentosa Carr. are shown in Fig. 2.51 The detected
main substructures are depicted in Fig. 3.51 Unfortunately, the
quantification based on 2D HSQC NMR could provide just the
relative proportions of the detected structures rather than the
absolute values.
In 2003, a quantitative method to record 1 H-13 C correlation
NMR spectra (Q-HSQC) was developed by Heikkinen et al.52 by
modulating the polarization transfer delays of HSQC, where a
monomeric lignin model compound (apocynol) was used as an
internal standard. In 2007, a new quantification method with the
combination of quantitative 13 C and 2D HSQC NMR was proposed
by Zhang and Gellerstedt.53 The key point in this quantification
method is to select a suitable internal standard reference signal
originating from lignin with similar structural features. The selected
internal standard references can convert relative integration values
obtained from the corresponding 2D spectrum to the absolute
values. This method can be applied to identify and quantify
various LCC linkages.16,37 Recently, in 2011, a quick quantitative
HSQC (QQ-HSQC) was proposed by Sette et al.50 This new method
can be applied to quantitative evaluation of different inter-unit
linkages in an array of milled softwood and hardwood and
technical lignins by using the guaiacyl C2 and syringyl C2C6
signals as internal standards. It provides good resolution and
signal-to-noise ratio in a reasonable analysis time. It should be
mentioned that 2D HSQC NMR is a powerful technique for lignin
characterization but is not omnipotent. For example, the 4-O-5 and
55 subunits cannot be detected by 2D HSQC NMR. Therefore,
the traditional destructive methods are complementary to the 2D
HSQC NMR technique. A more comprehensive picture of lignin
can be obtained by combining destructive and nondestructive
methods.

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Figure 3. The detected main substructures of MWL from Triploid of Populus tomentosa Carr. (A) -O-4 aryl ether linkages with a free OH at the -carbon;
(A) -O-4 aryl ether linkages with acetylated OH at -carbon; (A) -O-4 aryl ether linkages with p-hydroxybenzoated OH at -carbon; (B) resinol
substructures formed by -, -O- , and -O- linkages; (C) phenylcoumarane substructures formed by -5 and -O-4 linkages; (D) spirodienone
substructures formed by -1 and -O- linkages; (I) p-hydroxycinnamyl alcohol end groups; (J) cinnamaldehyde end groups; (PB) p-hydroxybenzoate
substructures; (H) p-hydroxyphenyl units; (G) guaiacyl units; (S) syringyl units; (S) oxidized syringyl units with a C ketone; (G) oxidized guaiacyl units
with a -ketone.51

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further structural information. The DMSO/NMI solvent system has

been successfully applied for lignin characterization, especially
in tracing the structural changes of biomass during different
et al.55 combined this 2D NMR
treatments. Recently, Hedenstrom
of dissolved cell wall samples with multivariate analysis, which
was confirmed to be a new tool that will benefit cell wall research.
However, the in situ acetylation is time-consuming and leads
to the loss of some original information, such as the presence
of natural acetyl groups on wall components is masked when
the sample is per-acetylated. Therefore, a method to produce
non-derivatized cell walls in a form amenable to solution-state
NMR would be ideal.
The first method involves dissolution of ball-milled plant cell
walls in perdeuterated solvents (DMSO-d6 /NMI-d6 ), subsequently
facilitating solution-state 2D NMR application. Yelle et al.

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349

2D NMR of plan cell walls

In 2003, two solvent systems, dimethylsulfoxide/tetrabutylamm
onium fluoride (DMSO/TBAF) and dimethylsulfoxide/Nmethylimidazole (DMSO/NMI), were developed by Lu and
Ralph for cell wall dissolution to allow quite detailed structural
analysis of the whole cell walls.54 Ball-milled plant cell walls
can dissolve or swell in either of these two solvent systems
and in situ acetylation of the whole plant cell walls can readily
be conducted. The resultant acetylated cell walls can dissolve
in common NMR solvents (e.g. CDCl3 , DMSO-d6 ), allowing the
whole lignin fraction without separation to be analyzed by
high-resolution solution-state NMR methods for the first time.
In addition, the low viscosity of acetylated cell walls in CDCl3
allows the 2D HMBC (Heteronuclear Multiple Bond Correlation)
experiment to be acquired with satisfactory signals that provide

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synthesized the NMI-d6 and applied DMSO-d6 /NMI-d6 solvent
system to examine several lignocellulosic materials representative
of softwood (Loblolly pine), hardwood (Aspen), and herbaceous
plant (Kenaf).56 The 2D HSQC NMR spectra of these samples
were superior in some ways to those from the acetylated cell
walls, especially for revealing the naturally occurring acetates on
lignin or polysaccharides. However, two significant drawbacks of
this method are the high price of NMI-d6 and the overlap of
correlations from p-hydroxyphenyl units in lignin with residual
solvent peaks from incompletely deuterated NMI.57
Kim et al.36 found that the ball-milled cell walls can swell in
DMSO-d6 and form a gel, which is suitable for recording a 2D HSQC
NMR spectrum of decent quality. Gels from several lignocellulosic
materials, such as Loblolly pine, Aspen, Kenaf bast fiber, and
corn stalks, were used to acquire 2D HSQC NMR spectra. Most
interpretable structural fingerprints of polysaccharides and lignin
can be identified in the spectra. A significant advantage of this
method is the simple and rapid sample preparation. Besides, the
naturally occurring acetates on lignin and polysaccharides can be
easily observed. Moreover, in the case of ferulates, these important
cross-linking agents in grass cell walls are readily distinguished
for the first time in these spectra. Recently, this gel-sample 2D
NMR method has been improved by Kim and Ralph,40 while
utilizing pyridine-d5 in DMSO-d6 for ball-milled plant cell walls.
This new solvent system (DMSO-d6 /pyridine-d5 ) provides better
quality 2D NMR spectra than using DMSO-d6 alone and allows
considerably easier sample handling. Although with limitations,
such as ball-milling of sample, gel produced from ultrasonication,
unsuitability of long-range experiments (e.g. HMBC), and lack
proof of quantitative analysis by using these techniques, the
possibility of obtaining high-resolution 2D NMR spectra of
lignocellulosic materials in DMSO-d6 or DMSO-d6 /pyridine-d5
systems indeed opens the door to biorefinery.57
The other two potential methods are based on ionic liquids and
LiCl/DMSO-d6 . After dissolution and acetylation in ionic liquids,
the acetylated wood dissolved in DMSO-d6 can be analyzed by
2D HSQC NMR.58 Besides, perdeuterated pyridinium ionic liquids
were used to dissolve poplar cell walls in DMSO-d6 for 2D HSQC
NMR characterization.59 A doubt about the application of ionic
liquids for biomass characterization is whether the structure of the
components has been altered during the dissolution process.57
LiCl/DMSO, a novel solvent system, was developed by Wang
et al.60,61 for dissolving milled wood. This system completely
dissolved beech and spruce milled woods prepared from the
Wiley woods using only 2 h of planetary ball-milling.60 After being
treated with ethylenediamine (EDA), coarse (4080 mesh) wood
or kraft pulps can be dissolved in this new solvent system.61
Although no 2D HSQC NMR spectra of dissolved ball-milled wood
in LiCl/DMSO-d6 can be found in published literatures, this system

T-Q Yuan, F Xu, R-C Sun

still seems to have the potential to be a good solvent for 2D NMR
characterizing plant cell walls because of its excellent ability to
dissolve entire plant cell walls.57

VALORIZATION OF LIGNIN
Nowadays, technical lignins are mainly separated as by-streams
from conventional pulp mills (sulfite, kraft, and soda processes).
They differ from each other to a significant extent and the features
of these technical lignins have been investigated extensively.7 11
Furthermore, it is evident that massive amounts of lignin will be
generated from future lignocellulosic ethanol industries, both from
the pretreatment stage and after fermentation. The lignin obtained
from the lignocellulosic ethanol industries will be chemically
different from those currently available. However, most lignin
is burned as a fuel, despite the existence of a great number of
potential applications. The conversion of this low-value by-product
into high-value co-products will help to offset the costs of both
papermaking and bioethanol production.62
Current and potential application of lignin has been reviewed
extensively,7 11 and can be classified into three categories: (1)
power, fuel, and syngas products; (2) macromolecules; and (3)
low-molecular weight aromatic or phenolic compounds.63 Figure 4
summarizes the valuable products that could be obtained from
lignin with new and current technologies. A detailed overview
of technology and challenges for these valuable products was
provided in a report given by Holladay et al.63 As technical
lignins are different from each other, suitable applications for
them should be considered. For example, lignin obtained after
fermentation is not suitable for the production of lignin-based
biomaterials because of its low purity. However, this lignin-rich
solid residue can be converted to liquid fuels using various
pathways such as fragmentation, hydroprocessing or thermal
depolymerization.8 In addition, lignin is perhaps the largest
source of renewable material with an aromatic/phenolic skeleton.
Conversion of lignin to low-molecular-weight aromatic/phenolic
compounds offers a promising route for the high-value application
of lignin.11,64 Development of selective and robust catalytic
processes specifically designed for lignin conversion must be a
core effort in a biorefinery program.63
Similarly, four major classes of problems for lignin application
have been identified: (1) recovery of lignins from the product
stream; (2) purification of lignins; (3) the heterogeneous structure
of lignins; and (4) the unique reactivity of lignins.7 It must be noted
that all these four problems originate from the source and process
by which lignin was isolated. Therefore, more attention should
be paid back to the feedstocks and separation techniques before
converting lignin for high-value application.

350

Figure 4. Valuable products obtained from lignin with new and current technologies.63

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Separation, characterization, and valorization of lignin for biorefinery

Figure 5. Future process scenario for biorefinery.

CONCLUDING PERSPECTIVE
Lignin is one of the most significant contributors to biomass
recalcitrance and consequently increases the cost of biorefinery
processes. Several genetic engineering strategies, such as alter
the lignin content, composition, and structure, have been
demonstrated to develop tailored feedstocks designed for biofuels
production.34 These efforts are beneficial for biofuels production,
whereas less attention has been paid to the potential application
of the separated lignin from these tailored feedstocks. Therefore,
an ideal strategy is to change the role of lignin in a biorefinery
through genetic engineering strategy. Not only reduce cell wall
recalcitrance for biofuels production, but also open a window
of opportunities for valorization of lignin. Understanding the
biosynthesis and structure of lignin in plant cell walls and the
structural change of lignin during the biorefinery process are
useful for achieving this goal. In conclusion, a future process
scenario for a biorefinery is given in Fig. 5.

ACKNOWLEDGEMENTS
We are grateful for financial support of this research from National
Science Foundation of China (31110103902), Major State Basic
Research Projects of China (973-2010CB732204), and State Forestry
Administration (20100400706).

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