International Journal of

Molecular Sciences

Review
Lignin, the Lignification Process, and Advanced,
Lignin-Based Materials
Maria Balk 1 , Pietro Sofia 2,3 , Axel T. Neffe 1, * and Nicola Tirelli 2, *

1 Institute of Functional Materials for Sustainability, Helmholtz-Zentrum Hereon, Kantstrasse 55,

14513 Teltow, Germany; maria.balk@hereon.de
2 Laboratory of Polymers and Biomaterials, Fondazione Istituto Italiano di Tecnologia, Via Morego 30,
16163 Genova, Italy; pietro.sofia@iit.it
3 The Open University Affiliated Research Centre at the Istituto Italiano di Tecnologia (ARC@IIT),
Via Morego 30, 16163 Genova, Italy
* Correspondence: axel.neffe@hereon.de (A.T.N.); nicola.tirelli@iit.it (N.T.)

Abstract: At a time when environmental considerations are increasingly pushing for the application
of circular economy concepts in materials science, lignin stands out as an under-used but promising
and environmentally benign building block. This review focuses (A) on understanding what we
mean with lignin, i.e., where it can be found and how it is produced in plants, devoting particular
attention to the identity of lignols (including ferulates that are instrumental for integrating lignin
with cell wall polysaccharides) and to the details of their coupling reactions and (B) on providing
an overview how lignin can actually be employed as a component of materials in healthcare and
energy applications, finally paying specific attention to the use of lignin in the development of organic
shape-memory materials.

Keywords: lignin; lignols; lignification biochemistry; wood ultrastructure; healthcare application;

supercapacitors; shape-memory materials

1. Introduction
Citation: Balk, M.; Sofia, P.; Neffe, Lignin is a naturally abundant polymeric material. As a major component of the walls
A.T.; Tirelli, N. Lignin, the of plant cells (see Section 2), it is virtually ubiquitous and is estimated to be produced
Lignification Process, and Advanced, by plants at the tune of around 20 billion tons/year [1]; this makes lignin one of the
Lignin-Based Materials. Int. J. Mol. most common macromolecular systems on our planet. Although naturally it is always
Sci. 2023, 24, 11668. https://doi.org/ intermixed with other biomass components, a number of processes—such as the extraction
10.3390/ijms241411668 of polysaccharides (chiefly cellulose) to yield paper or textile fibers or the production
Academic Editor: Andreas Taubert
of bioethanol—provide lignin as a byproduct, making it in principle usable for other
downstream applications. For example, in the mid-2010s paper/pulp production alone
Received: 7 June 2023 produced 50–70 million tons of lignin p.a. [2]. The growing use of biofuels suggests that
Revised: 10 July 2023 bioethanol production can be an even larger lignin source in a very close future.
Accepted: 12 July 2023 Chemically, lignin is a (variably) cross-linked polyphenol; it is produced during the
Published: 19 July 2023
radical/oxidative oligo- or polymerization of a variety of compounds, which predominantly
share a common p-hydroxycinnamic skeleton. Section 3 specifically focuses on the identity
of lignin precursors and on the mechanisms presiding their polymerization, which needs
to be understood in detail to rationalize structure and properties of lignin-based materials
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
(including wood) and to potentially engineer them by design.
This article is an open access article
Despite our reasonably good grasp over the synthesis, structure, and natural role of
distributed under the terms and lignin and notwithstanding its very wide availability, its exploitation has always been an
conditions of the Creative Commons issue. Firstly, its chemical identity (molar mass distribution, monomer composition, and
Attribution (CC BY) license (https:// degree of branching) is quite variable and sometimes difficult to control. Secondly, lignin
creativecommons.org/licenses/by/ has always been considered a low-value waste material: its thermo-mechanical properties
4.0/). are unattractive, making its processing difficult, and despite being a natural material, it

Int. J. Mol. Sci. 2023, 24, 11668. https://doi.org/10.3390/ijms241411668 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 11668 2 of 47

is poorly degradable and can also reduce the degradability of materials it is connected
to, such as polysaccharides. Indeed, lignin typically reduces their digestibility in animals
and more in general hampers the direct transformation of plant biomasses into biofuels or
sugars through (bio)chemical methods [3] or pyrolysis [4]. Currently, only about 2% of the
produced lignin finds commercial use, predominantly in specialty chemicals [2], while the
rest is being either burned or added to animal fodder essentially as a bulking agent.
Notwithstanding these issues, its inherently sustainable origin and low production
costs (from tens to hundreds USD/ton, depending on the purity [1]) are both undeniable
advantages and powerful drives for the development of new materials based on lignin. An
additional advantageous factor is that the costs associated with the disposal of a byproduct
disappear when lignin is used as a building block for added-value materials, which lends
even more economic sense to this perspective.
The following lines list the three main approaches to lignin processing, on the one
hand highlighting those discussed in the present review and on the other hand pointing
the reader to literature reviews for all others:
(A) Engineering the lignification process in order to modify lignin composition, in
this way to improve its fermentability to produce biofuels [5] or its digestibility in animal
fodder [6] or to allow its easier incorporation into biomaterials [7]. Although this is not the
focus of this review, these points are touched upon at the end of Section 3.4.
(B) Production of high(er)-value chemicals (e.g., vanillin [8], flavonoids [9], and many
other (methoxy)phenols and catechols and non-aromatic carboxylates and ketones) via
thermal, reductive, oxidative, or basic/acid-catalyzed depolymerization. Here, this ap-
proach is not discussed in detail, and we refer the reader to excellent and specialized recent
reviews [10,11].
(C) Use of lignin (directly in its native form or after chemical derivatization at phe-
nol/alcohol OH groups) in a variety of materials, where lignin (1) undergoes dramatic
chemical changes, e.g., being converted into carbonaceous materials via graphitization [12];
(2) becomes a building block for macromolecular products such as polyurethanes [11],
which then undergo their own processing; and (3) is integrated in nanostructured materials
as a functional component. Section 4 reviews the third case, specifically focusing on appli-
cation areas that confer lignin the highest added value, i.e., healthcare (Section 4.1), energy
storage (Section 4.2), and shape-memory materials (Section 4.3).

2. Plants, Wood, and Where to Find Lignin

Wood is the prototypical lignified tissue. The following discussion focuses therefore
on wood, but the reader should bear in mind that not all plants contain wood; for example,
grasses are non-woody plants. However, virtually all plants (bar bryophytes) produce
lignin and have lignified tissues that bear significant structural and biochemical similarities
to wood, although with important compositional and mechanical differences.
The defining feature of wood and lignified tissues is to be found at a (supra)molecular
level: their hardness is mostly related to the presence and the structural details of in-
terconnected three-dimensional networks, which are made of two classes of partially
phase-separated macromolecular components: polysaccharides such as cellulose, hemicel-
luloses, and pectins and the rather ill-defined, part-aromatic material referred to as lignin.
Polysaccharides usually make up 65–75% of wood’s weight; lignin makes up 18–35% [13].
The relative ratio of these components is used to classify trees into the limit definitions
of softwood (typically from gymnosperms, i.e., non-flowering trees) [14] and hardwood
(typically from angiosperms, i.e., flowering trees [15]). Coniferous species are typical
representatives of gymnosperms, and their softwood is made up of 40–45 wt% cellulose,
26–34 wt% lignin, and 7–14 wt% of other (cell wall) polysaccharides; the hardwood of
deciduous species has a similar cellulose content (38–49 wt%) but is typically lower in
lignin (23–30 wt%) and higher in cell wall polysaccharides (19–26 wt%) [16]. However,
many plants are commonly categorized as intermediate types (mixedwoods).
Int. J. Mol. Sci. 2023, 24, 11668 3 of 47

Wood also includes a fraction of low molecular weight (MW) organic compounds
(polyphenols such as stilbenoids, flavonoids or tannins, or terpenes [17]) as minor compo-
nents; their amount typically does not exceed 5–10% of the dry wood mass, but occasionally,
most commonly in tropical and sub-tropical plants, they may reach up to 20 wt% [18]. Apart
from being the main determinants of wood smell and color, the polyphenolic nature of these
molecules makes them capable of scavenging free radicals and also polymerize oxidatively;
on the one hand, this confers them a protective role but on the other hand may allow a
certain degree of integration in lignification processes, therefore blurring the distinction
between these low MW extractives and strictu sensu lignin precursors (see Section 3), above
all those with flavonoid or stilbenoid structure. All the above, however, should be con-
sidered indications rather than precise data: the actual composition of wood depends on
a variety of factors, such as the specific location in the plant’s body, its age, and on the
environmental conditions the plant has experienced during the production of wood (affect-
ing its biosynthesis) and at later stages (modifying it after its production). For example,
polysaccharides may undergo hydrolysis in the long term [19]. It is also noteworthy that
herbaceous (=non-woody) plants have lignin too, but in lower amounts than woody plants;
their lignin content ranges from 0.4 (maize flour) through 1.2 (white lupins), 3.5 (sugar beet
fibers), to up to 12–15 wt% (whole grasses or cereal husks) [20]. The following sections
refer mostly to woody plants (because of their higher lignin content), but most informa-
tion on cell walls and lignification (bio)chemistry applies to both them and non-woody
(herbaceous) organisms, and references will be made accordingly.

Structural Features of Lignified Tissues (Wood)

At a macroscopic level, woody plants are structured into roots, stems, and crowns.
Here, we will focus on stems (their trunks, Figure 1A), which are the most wood-rich
regions in woody plants, but they are also the largest parts of the bodies of herbaceous
plants.
Transversally, the stem’s innermost part is referred to as the pith or medulla, a spongy
and rather soft tissue. Although accurate measurements of its modulus are hard to find, it
increases stiffness for very soft stems such as maize stalks (where the pith has the largest
volume fraction) [21], while in hard stems such as those of pine trees it has a (minor)
softening effect [22]. It is a moderately lignified tissue (8–15 wt% in herbaceous plants
such as maize [23] or thistle [24], although occasionally, e.g., in coconut husk, may reach
25 wt% [25]).
Surrounding the pith, stems feature a system of concentric and tapering layers (the an-
nual rings) named xylem, which are enclosed in the external protective multilayer of the
wood bark (that contains the phloem, see later). The xylem takes care of the long-range
(upward) water-transport from roots to appendages (leaves). Within any annual ring of the
xylem, a lighter-colored part named earlywood is distinguished from a darker and denser
part delimiting the ring, called latewood; the vascular system of the latter presents smaller
cavities, and its cells have thicker, more lignified walls, making it denser and harder than
earlywood [26,27]. The xylem rings can be grouped into two distinct concentric regions,
sapwood and heartwood. Sapwood (or young xylem) is the younger, softer, physiologically
active outer portion of the wood trunk. Besides being the main contributor to the stem
mechanical support, sapwood also acts as a storage reservoir for water, as well as for
polysaccharides such as starch that reduce water loss [28].
Int. J. Mol. Sci. 2023, 24, 11668 4 of 47
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 48

Figure 1. (A). Cross-section of a wood trunk. The innermost layer is the spongy pith, surrounded by
Figure 1. (A).
the inner Cross-section
vascular of a wood
tissue (xylem); trunk.is The
the latter innermost
divided into thelayer is the
inactive spongy pith,
heartwood (for surrounded
mechanical by
thesupport) and the active
inner vascular tissue sapwood
(xylem); the(for latter
long-range waterinto
is divided transport). More heartwood
the inactive externally, cambium pro-
(for mechanical
duces both
support) and new sapwood
the active and the
sapwood (formore externalwater
long-range vascular tissue ofMore
transport). phloem, and finally
externally, the highly
cambium produces
lignified bark provides a protective barrier. (B). A multi-layer structure wraps around the plant cell
both new sapwood and the more external vascular tissue of phloem, and finally the highly lignified
cytoplasm (the lumen), starting with the plasma membrane, developing with three layers (S1-S2-S3)
bark provides a protective barrier. (B). A multi-layer structure wraps around the plant cell cytoplasm
of lignin-rich secondary cell walls and ending with the more external primary walls that are sur-
(the lumen),
rounded bystarting with themiddle
the pectin-rich plasma membrane,
lamella, which developing
also provideswith three layers
connection (S1 -S2 -S
to adjacent 3 ) ofThe
cells. lignin-
rich secondary
microfibril anglecell wallsofand
(MFA) ending
cellulose varieswith the more
radially external primary
and correlates walls that properties
with the mechanical are surrounded of
bythethelayers.
pectin-rich cell walls,
(C). Inmiddle cellulose
lamella, which microfibrils (MF, gray
also provides rods) are
connection toembedded in a matrix
adjacent cells. com-
The microfibril
posed
angle of hemicelluloses
(MFA) (in orange)
of cellulose varies radiallyandandpectin (in green,
correlates withonlythepresent in the properties
mechanical primary walls).
of theThelayers.
inset shows how the crystalline domains of hemicellulose bind to cellulose via interfacial hydrogen
(C). In cell walls, cellulose microfibrils (MF, gray rods) are embedded in a matrix composed of
bonding, while its amorphous chain portions bridge among them and at the same time covalently
hemicelluloses
connect to lignin (in microparticles
orange) and pectin (in green, only present
(lignin–carbohydrate complexes, in the primary
LCC). In thewalls). The inset
better known shows
“teth-
howered the crystalline
network” domains
model, of hemicellulose
hemicellulose bind the
chains fulfill to cellulose via interfacial
role of bridges betweenhydrogen bonding,
distant elements while
(cel-
itslulose bundleschain
amorphous and/or lignin), bridge
portions but actually
among crystalline
them and hemicellulose
at the samecan timealso bridge cellulose
covalently connectmicro-
to lignin
fibrils from within
microparticles their bundles, which
(lignin–carbohydrate makes LCC).
complexes, them both
In themechanically
better known active and inaccessible
“tethered network” model,to
hemicellulases [29]. Pectin is the main component of the middle lamella and is also abundantly pre-
hemicellulose chains fulfill the role of bridges between distant elements (cellulose bundles and/or
sent in the primary cell walls (30–50%) [30] but is basically absent in the secondary ones; i.e., its
lignin), but actually crystalline hemicellulose can also bridge cellulose microfibrils from within their
concentration profile is almost opposite to that of lignin. Please note that the relative thickness of
bundles, which
the various makes
layers them
is not both(secondary
in scale mechanically wallsactive
beingandmuch inaccessible
thicker than to all
hemicellulases
other elements). [29]. Pectin
is the main component of the middle lamella and is also abundantly present in the primary cell walls
(30–50%) With[30]age,
butsapwood
is basically cells gradually
absent in the die, and theones;
secondary tissues i.e.,become darker, producing
its concentration profile is the
almost
central cylinder known as heartwood, distinguished from a rather thin
opposite to that of lignin. Please note that the relative thickness of the various layers is not in scale (1 cm at most)
(secondary walls being much thicker than all other elements).
Int. J. Mol. Sci. 2023, 24, 11668 5 of 47

With age, sapwood cells gradually die, and the tissues become darker, producing
the central cylinder known as heartwood, distinguished from a rather thin (1 cm at most)
transition zone. This cell death causes the local release of many low MW compounds
(extractables, also known as extractives, typically polyphenolics), whose oxidative polymer-
ization and integration with lignin darken and provide durability to this xylem area [31,32].
The stem’s growth is regulated by a thin layer located between the xylem and phloem, the
vascular cambium, which forms a circular front of precursor cells differentiating into both
the internal xylem and the external phloem, the latter being responsible for the downward
transport of materials from sites of photosynthesis. Taxonomically, the phloem is part of
the external layer of the stem, i.e., the bark, which further comprises the outermost cork
and an intermediate layer of cork cambium (containing cork immature cells). The cork
acts as a protective layer against drying and other environmental conditions, as well as
pathogens, and is highly lignified and rich in extractives. Detailed comparisons between
xylem, phloem, and cork are rather rare, but it can be said that lignification increases in
that order, with significant variations also in lignin composition [33,34] (see Section 3.4).
At a cellular level, a structural feature is common to both woody and herbaceous
plants: their cells have walls with a helically reinforced, multi-layer composite structure
(Figure 1B). Their inner part is the cavity hosting the actual cell body (lumen), which in
mature, specialized cells is surrounded firstly by a three-layered (S1 , S2 , and S3 ) secondary
cell wall, then by a primary cell wall, and finally by a middle lamella, which separates
neighboring cells.
This middle lamella has a variable thickness (from as thin as 0.2 µm to in excess
of 1 µm, depending also on hydration), and its main (but not exclusive [35]) role as
an intercellular ‘glue’ is due to it being predominantly made of (calcium-)gelled pectin.
Proceeding inwardly, the thin (most commonly < 0.1 µm) primary walls feature rather
disorganized cellulose microfibrils, with lignin as a minor component, and still significant
amounts of pectin. Secondary walls are typical of cells having concluded their expansion
phase; they are thick, up to 13 µm [36], and typically divided in layers, which differ
in the orientation of cellulose microfibrils and in thickness, with S2 being the thickest
(1–10 µm, 75–85% of the secondary wall thickness [36,37]). All three layers have cellulose
as the major component (≈50%), followed by hemicelluloses and lignin in variable but
comparable amounts, while pectin is typically absent (although phloem secondary walls
may occasionally present it [38]). Of note, gymnosperm cells may further have a thin
(<0.1 µm) “warty layer” on the innermost lamella of S3 [39]. Its name derives from the
presence of wart-like protuberances, which are not responsible for the strengthening of the
plant structure but affect permeability. Secondary cell walls are the largest part and most
lignified cell wall compartment, owing to lignin rigidity and also hydrophobicity (hence
barrier properties). Lignin can also be found in the middle lamella but in relatively small
amounts and as non-interconnected aggregates [40], thereby not appreciably contributing
to the mechanical properties of this layer. This 3D organization is summarized in Figure 1C.

3. The Lignification Process: Building Blocks and (Bio)Chemistry

3.1. Monolignols (Lignin Building Blocks) from the Phenylpropanoid Pathway
In this section, we discuss the biosynthesis of compounds that will eventually act as the
lignin building blocks, i.e., the monolignols; please note that a detailed description of the
corresponding units in lignin is to be found in Section 3.4 “Lignin units and relative lignin
composition”. The most common set of lignin precursors is referred to as the canonical
monolignols; these three C9 units are sinapyl alcohol (producing the so-called S units
in lignin), coniferyl alcohol (G units), and p-coumaryl alcohol (H units), which is also
known under the name of p-hydroxycinnamyl alcohol (Figure 2A, left). These monolignols
have a common molecular motif, i.e., a phenol para-conjugated to a trans (E) double bond
terminating in an hydroxymethylene groups, and they differ for the presence and number
of methoxy groups flanking the phenolic OH. Although strictly speaking non-canonical,
two structurally related classes of lignin precursors exist (Figure 2A, right):
Int. J. Mol. Sci. 2023, 24, 11668 6 of 47

(1) γ-O-acylated compounds (esters), including acetates [41,42], p-coumarates (more

common in grasses than woody plants) [42], and p-hydroxybenzoates [43];
(2) catechols such as caffeyl alcohol (yielding the so-called C units in lignin) or 5-
hydroxyconiferyl alcohol (5HC units). Although structurally very similar to (methoxy)phenols,
the presence of two neighboring OH groups produce a peculiar reactivity during lignification
(see benzodioxane groups in Section 3.4) [44].
The structural similarities among the canonical and the above-described non-canonical
lignols are due to a common biosynthetic route, which is referred to as phenylpropanoid
pathway (Figure 3). The key intermediate of this process is p-coumaric acid, which is
derived from phenylalanine (Phe) via the reductive deamination into cinnamic acid op-
erated by phenylalanine ammonia lyase (PAL), followed by hydroxylation by cinnamate
4-hydroxylase (C4H). Unsurprisingly, a reduced expression of these enzymes leads to lower
lignin production [45].
Another common point of all phenylpropanoid processes is that cinnamate carboxy-
lates are always converted into primary alcohols by transforming them into coenzyme A
(CoA) derivatives (through 4-coumarate: CoA ligase, 4CL) and by first reducing them to
aldehydes (through cinnamoyl-CoA reductase, CCR) and then to alcohols (through cin-
namyl alcohol dehydrogenase, CAD). Of note, the aldehydes are present in traces in most
lignins, but they become particularly abundant in CAD-deficient plants [46], which tend to
produce also some saturated non-canonical lignols (e.g., guaiacylpropane-1,3-diol) [47].
Other defining features of the phenylpropanoid pathway are as follows:
(A) The cytochrome P450 (CYP450)-dependent nature of hydroxylating enzymes [48], such
as p-coumarate 3-hydroxylase (C3H, also known as coumaroyl shikimate 3-hydroxylase) [49] and
ferulate 5-hydroxylase (F5H, also known as coniferyl hydrolase). Curiously, the downregulation
of C3H and C4H genes in Populus trichocarpa increases the presence of lignol benzoate esters [50],
which may be due to cross-talks between the benzoate and the phenylpropanoid pathways.
(B) The pervasive presence of 3- or 5-O-methylating enzymes, i.e., caffeic acid O-
methyltransferase (COMT) or caffeoyl-CoA O-methyltransferase (CCoAOMT), which also
show a certain degree of redundancy [51], since coniferyl (G) units can be produced
by methylating caffeyl alcohol via COMT or caffeoyl-CoA via CCoAOMT. It is worth
mentioning that 5HC units (in the second group of non-canonical lignols, i.e., catechols)
are abundant in COMT-deficient plants [52–54]. A loss or reduction in COMT and/or
CCoAOMT expression has also been invoked to explain C-lignin structure peculiar to
cactus seed-coat, which is rich or uniquely composed of caffeic acids (C units) [55,56].
(C) A redundant biosynthesis of sinapyl alcohol, whose two paths see enzymes used
in the sequence CAD-F5H-COMT-CAD or alternatively in the sequence F5H-COMT-CAD,
i.e., producing first coniferyl alcohol or branching out at the level of its precursors aldehyde.
In both cases, however, the production of sinapyl alcohol (leading to “S units” in lignin)
requires that of coniferyl aldehyde (the precursor of the so-called “G unit”); this means that
synapyl alcohol levels will not alter the quantity but the quality (the S/G ratio) of lignin.
Indeed, the absence of F5H and COMT via selective mutations does not affect the overall
amount of lignin but reduces the S/G ratio [57], which is conversely increased by F5H
overexpression [58].
(D) A central role of caffeic acid. When its production is reduced by mutating caffeoyl
shikimate esterase (CSE), the biosynthesis of all lignols except p-coumaryl is hampered,
which means both a reduction in the total amount of lignin and its higher presence in lignin
(“H units”) [59]. Of note, a certain degree of redundancy is present here too: caffeoyl-
CoA can be produced directly from caffeolyl shikimate (through p-hydroxycinnamoyl-
CoA:quinate/shikimate, HCT) [59], thereby bypassing caffeic acid.
(E) The common occurrence of γ-O-acylated ester conjugates (Figure 3, bottom). For
example, acetates and p-coumarates make up to 80% of monolignols in (herbaceous) an-
giosperms such as Hibiscus cannabinus and Agave sisalana [42], while up to 45% acetylation
has been found in (woody) gymnosperms such as Carpinus betulus [41]. Reportedly, the
presence of esters is more common in the external layers of wood, e.g., the cork in Quer-
 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 48

Int. J. Mol. Sci. 2023, 24, 11668 7 of 47

angiosperms such as Hibiscus cannabinus and Agave sisalana [42], while up to 45% acetyla-
tion has been found in (woody) gymnosperms such as Carpinus betulus [41]. Reportedly,
thesuber
cus presence
[34]. of esters is more
Importantly, common
to our in the external
knowledge layers responsible
the enzymes of wood, e.g.,tothe
thecork in
synthesis
Quercus suber [34]. Importantly, to our knowledge the enzymes responsible to the
of these two esters have not been uncovered, whereas they have been identified for fer- synthe-
sis of these two esters have not been uncovered, whereas they have been identified for
uloylation [60] and p-hydroxycoumaration [61]. Last, there are diagnostic signatures
feruloylation [60] and p-hydroxycoumaration [61]. Last, there are diagnostic signatures
for the presence of acetates, at least in the case of sinapyl derivatives: in their absence,
for the presence of acetates, at least in the case of sinapyl derivatives: in their absence, β-
β-β’ coupling of sinapyl alcohols produces bicyclic structures, the resinols (see later in
β’ coupling of sinapyl alcohols produces bicyclic structures, the resinols (see later in Sec-
Section 3.4 and Figure 6B), whereas in their presence the same reaction yields substituted
tion 3.4 and Figure 6B), whereas in their presence the same reaction yields substituted
tetrahydrofurans [62].
tetrahydrofurans [62].

Figure 2. (A). The three canonical monolignols (left: p-coumaryl, coniferyl and synapyl alcohol) are
Figure 2. (A).very
structurally Therelated
three canonical monolignols
to other lignols (left: p-coumaryl,
(right) produced through the coniferyl and synapyl alcohol)
same phenylpropanoid path- are
structurally very related
way (see Figure to other
3). Of note, lignols (right)
the catechol groups produced through
in the caffeyl the same
alcohol phenylpropanoid
(producing pathway
C units in lignin)
andFigure
(see 5-hydroxyconiferyl
3). Of note, the alcohol allow
catechol for a different
groups radical/oxidative
in the caffeyl reactivity.C(B).
alcohol (producing A few
units flavo- and
in lignin)
noids (based on a polyphenolic
5-hydroxyconiferyl alcohol allowα,β-unsaturated cyclic ketone structure
for a different radical/oxidative (chromone);
reactivity. (B). Aleft)
fewand hy-
flavonoids
droxystilbenes and their glycolides (1,3-diphenolic ring linked to a phenolic, catecholic or 2-meth-
(based on a polyphenolic α,β-unsaturated cyclic ketone structure (chromone); left) and hydroxystilbenes
oxyphenolic ring through an ethylene residue; right) have been found to be monolignols too and
and
aretheir glycolides
produced through(1,3-diphenolic ring linked
the acetate/malonate to a phenolic,
polyketide pathway. catecholic or 2-methoxyphenolic
Taxonomically, tricin is a flavon;ring
through
dihydrotricin and naringenin are flavanones, and naringenin chalcone is—as suggested through
an ethylene residue; right) have been found to be monolignols too and are produced by the the
acetate/malonate
name—a chalcone. polyketide
(C). Twopathway. Taxonomically, tricin
hydroxycinammamides behave is aasflavon; dihydrotricin
monolignols. They areandferulic
naringenin
acid are
derivatives, which derive from the amino acid metabolic pathway. (D). Flavonolignans
flavanones, and naringenin chalcone is—as suggested by the name—a chalcone. (C). Two hydroxycinam- (left of the
vertical dashed line) and stilbenolignans (right of the dashed line) are low MW
mamides behave as monolignols. They are ferulic acid derivatives, which derive from the amino acid products of reaction
between a flavonoid (e.g., tricin) or a hydroxystilbene (e.g., piceatannol) and a phenylpropanoid
metabolic pathway. (D). Flavonolignans (left of the vertical dashed line) and stilbenolignans (right of the
lignol, whose sub-structures are separated by a red dashed line in the panel. While typical mecha-
dashed
nisms line) are reactions
for such low MWare products
listed inofFigure
reaction between
4. The readeraisflavonoid
addressed(e.g., tricin) for
elsewhere or amore
hydroxystilbene
compre-
(e.g., piceatannol) and a phenylpropanoid lignol, whose
hensive lists of flavono- [63] and stilbenolignan [64] structures. sub-structures are separated by a red dashed
line in the panel. While typical mechanisms for such reactions are listed in Figure 4. The reader is
addressed elsewhere for more comprehensive lists of flavono- [63] and stilbenolignan [64] structures.
Int. J. Mol. Sci. 2023, 24, 11668 8 of 47
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 48

Figure 3. The phenylpropanoid pathway leads to the biosynthesis of both canonical (no back-
Figure
ground)3. The
andphenylpropanoid pathway(light
non-canonical monolignols leadsyellow
to the background),
biosynthesis of both
while canonical
other (no background)
non-canonical mon-
and non-canonical
olignols are produced monolignols
through different(light but
yellow background),
connected pathwayswhile other non-canonical
(pink background). monolig-
Phenylalanine
(Phe)
nols areisproduced
sequentially converted
through to p-coumaryl
different alcoholpathways
but connected (H unit) by phenylalanine
(pink background). ammonia-lyase
Phenylalanine
(PAL),
(Phe) cinnamate 4-hydroxylase
is sequentially converted to(C4H), 4-coumarate:CoA
p-coumaryl alcohol (Hligase
unit)(4CL), cinnamoyl-CoA
by phenylalanine reductase
ammonia-lyase
(CCR), and cinnamyl alcohol dehydrogenase (CAD). p-coumaric acid (red circle) is the ‘hinge’ of all
(PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), cinnamoyl-CoA reductase
these biosynthetic paths, since not only it is the precursor of p-coumaryl alcohol, but directly (via p-
(CCR), and cinnamyl alcohol dehydrogenase (CAD). p-coumaric acid (red circle) is the ‘hinge’ of all
coumarate 3-hydroxylase, C3H) or through p-coumaryl-CoA (the so-called shikimate shunt (blurred
these biosynthetic
red arrow). Enzymes paths, since not
involved: only it is the precursor of p-coumaryl alcohol,
p-hydroxycinnamoyl-CoA:quinate/shikimate (HCT), but directly (via
p-coumaroyl
p-coumarate 3-hydroxylase,
shikimate 3-hydrolase C3H)
(C3′H), and orcaffeoyl p-coumaryl-CoA
throughshikimate (the so-called
esterase (CSE)) also leadshikimate shunt (blurred
to the production of
caffeic acid and then to that of all non-canonical phenylpropanoid monolignols.
red arrow). Enzymes involved: p-hydroxycinnamoyl-CoA:quinate/shikimate (HCT), p-coumaroyl There, multiple and
redundant
shikimate pathways lead
3-hydrolase (C3to coniferyl
0 H), (G unit)shikimate
and caffeoyl and sinapyl alcohols
esterase (S unit)
(CSE)) andlead
also involve
to thecaffeic acid
production
O-methyltransferase (COMT), caffeoyl-CoA O-methyltransferase (CCoAOMT), and ferulate 5-hy-
of caffeic acid and then to that of all non-canonical phenylpropanoid monolignols. There, multiple
droxylase (F5H). Please note that among the non-canonical, phenylpropanoid-derived monolignols
and redundant
here we considerpathways lead to coniferyl
also compounds absent in(G unit) 2,
Figure and sinapyl
such alcohols
as ferulic (S unit)
acid (used andformation
in the involve caffeic
of
acid O-methyltransferase caffeoyl-CoA O-methyltransferase
polysaccharide-lignin complexes, see later) or dihydroconiferyl alcohol, which is presentand
(COMT), (CCoAOMT), ferulate
in the lig- 5-
nin of CAD-deficient
hydroxylase treesnote
(F5H). Please [65].that
PMT: p-coumaroyl-CoA
among monolignol
the non-canonical, transferase (PMT). FMT:
phenylpropanoid-derived feru-
monolignols
loyl-CoA with feruloyl-CoA monolignol transferase. For the non-phenylpropanoid
here we consider also compounds absent in Figure 2, such as ferulic acid (used in the formation pathway, hy- of
droxystilbenes are produced through stilbene synthase (STS), and flavonoids are produced through
polysaccharide-lignin complexes, see later) or dihydroconiferyl alcohol, which is present in the lignin
chalcone synthase (CHS), whereas hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltrans-
offerase
CAD-deficient trees [65]. PMT: p-coumaroyl-CoA monolignol transferase (PMT). FMT: feruloyl-
(THT) and hydroxycinnamoyl-CoA:putrescine hydroxycinnamoyltransferase (PHT), respec-
CoA with feruloyl-CoA monolignol
tively, mediate the biosynthesis transferase. For theand
of diferuloylputrescine non-phenylpropanoid
of feruloyltyramine. pathway, hydroxystil-
benes are produced through stilbene synthase (STS), and flavonoids are produced through chalcone
synthase (CHS), whereas hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT)
and hydroxycinnamoyl-CoA:putrescine hydroxycinnamoyltransferase (PHT), respectively, mediate
the biosynthesis of diferuloylputrescine and of feruloyltyramine.
Int. J. Mol. Sci. 2023, 24, 11668 9 of 47

3.2. Monolignols from Other Pathways

Besides the three canonical and the several non-canonical monolignols derived from
phenylpropanoid pathway (see previous section), in recent decades several other phenolic
compounds have been recognized as true monolignols [66]. They are grouped in the cat-
egories of flavonoids and hydroxystilbenes (Figure 2B) and of hydroxycinnamic amides
(Figure 2C); the former two are derived from coumaroyl-CoA [67,68], and the latter is
derived from feruloyl-CoA [69] (pink boxes in Figure 3), thus by combining the phenyl-
propanoid pathway, respectively, with the acetate/malonate-derived polyketide (flavonoids
and hydroxystilbenes) or the amino acid biosynthetic pathway (hydroxycinnamamides).
Tricin [70] and resveratrol [71], either in their free form or in their many glycosides [72,73],
are, respectively, the most commonly encountered flavonoid and hydroxystilbene in lignins.
Tricin was the first not directly phenylpropanoid-related compound to be recognized as
a component of low MW flavonolignan structures (Figure 2D) [74] and then as an authentic
monolignol. Tricin is almost ubiquitous in grasses or related plants, e.g., reeds such as
Arundo donax [75], sugar cane [76], or Cyperus papyrus [77], bamboos such as Phyllostachys
pubescens [78], and cereals such as Sorghum bicolor [79], where it typically concentrates in
the aerial parts.
Piceatannol can be produced through processes unrelated to lignification; for example,
in mammals it is the first metabolite of resveratrol [80]. Relatively recently it was also
recognized as a lignol, and more specifically the first hydroxystilbene, during a careful
analysis of the monomeric structures recovered from palm fruit endocarp lignin [71] after
derivatization (conversion of alcohols to bromides with acetyl bromide) and reduction
(elimination of bromide and phenol with acidic zinc dust to yield cinnamyl alcohols) [81].
Of note, and as it happens for flavonoids, several soluble low MW adducts hydroxystilbenes
with lignols, i.e., stilbenolignans [82] such as aiphanol [83] (Figure 2D), have also been
isolated.
Last, it is worth pointing out that while feruloyltyramine was recognized as a lignol
almost 40 years ago, after being found in the Nicotiana tabacum lignin [84], the discovery of
diferulyolputrescine as a lignol in maize kernels is considerably more recent [85].

3.3. Cell Wall Polysaccharides Involved in Lignification (Polysaccharidic Lignols)

Lignin would easily phase separate in cells walls, should it not be integrated with
non-cellulosic polysaccharides, which include only some selected (feruloylated) members
of the hemicellulose and pectin families.
Hemicelluloses. Hemicelluloses are typically seen as the internal ‘glue’ that binds
together the structural elements of plant cell walls, i.e., cellulose microfibrils and lignin
aggregates.
Hemicelluloses are a rather heterogeneous class of polysaccharides, common to wood
and non-woody plants (up to 20–30% of the dry weight of the former and up to 40% in
the latter [86]), which contribute to cell wall strength and rigidity through interactions
with cellulose and lignin. In comparison to other plant-derived polysaccharides, e.g.,
cellulose, starch, pectins, and various gums, hemicelluloses have a significantly lower
commercial value. However, they lack significant toxicity and are readily biodegradable,
which has spurred some interest in applications such as food packaging [87] or biorefinery
processes [88].
Structurally, all hemicelluloses share the same glycosidic bond configuration β-(1,4) of
cellulose but are shorter (from about 100 in softwood to up to 200 in hardwood) [89] and
above all largely vary in composition (Figure 4A) and architecture, whereas cellulose is
strictly a linear homopolymer of glucose (Glc). Hemicelluloses, on the contrary, not only
also include mannose (Man) and xylose (Xyl) repeating units in their main chain but also
feature side chains (=they are branched), which are based on uronic units (mostly α-D-
glucuronic acid, possibly methylated) and irregular patterns of α-L-arabinofuranose (Araf),
α-D-xylopyranose (Xylp), α-galactopyranose (Galp), or α-L-fucopyranose (Fucp) units as
side chains. Depending, therefore, on their composition, polysaccharides categorized as
Int. J. Mol. Sci. 2023, 24, 11668 10 of 47

“hemicelluloses” are more properly termed xylans, xyloglucans, and galactoglucomannans

(all depicted in Figure 4A), as well as glucomannans, mannans, and β-(1,3:1,4)-glucans. We
here do not consider other similar cell wall polysaccharides, e.g., galactans, arabinans, or
arabinogalactans, as hemicelluloses, because they may participate in pectin biosynthesis
and/or because of the different glycosidic configurations.
Xyloglucans are mainly present in primary cell walls and make up about 20 wt%
of these structures in hardwoods, around 10 wt% in softwoods [90]; due to the limited
thickness of the primary walls, however, xyloglucans are generally not the major com-
ponents of the hemicellulose pool. The most common hemicelluloses in secondary walls
are galactoglucomannans in softwoods and xylans in hardwoods; for example, 20 wt% of
the hemicellulose is composed of O-acetyl-4-O-methylglucuronoxylan (a xylan) in birch
(hardwood) but of O-acetyl-galactoglucomannan in spruce (softwood) [91]. Last, it is
also worth pointing out that while glucuronoxylans are major constituents of hardwood
hemicelluloses, those in grasses (and commelinids such as bananas, gingers, palms, etc.)
are predominantly made of the structurally similar arabinoglucuronoxylans [90].
It is noteworthy that, in addition to the differences in the sugar residues of their
backbones and side chains, another source of hemicellulose heterogeneity comes from their
continuous modification by a variety of enzymes (main enzymes and their modification
sites depicted in Figure 4A). For example, the acetylation degree of xylans (high: they
account for the vast majority of cell wall acetyl esters [92]) is inversely proportional to their
hydrogen bonding capabilities and therefore to their interactions with cellulose. Among
the other forms of side chain derivatization, it is worth mentioning the introduction of
methoxyphenol groups, typically in the form of ferulic esters O-2 [93] and O-5 [94] linked to
arabinose residues in the side chains of xylans [95]; such residues can oxidatively dimerize
and/or react through a variety of coupling geometries, which will be discussed in more
detail in the next section.
In summary, the heterogeneity of hemicelluloses is not only due to the synthetic modal-
ities of their backbone but also to their enzymatic post-processing, which also provides the
capacity for them to interact with cellulose and lignin.
Pectins. Pectins (pectic polysaccharides) are the major component of the middle
lamella but are also abundant in primary walls; both this localization and the timing of their
deposition (early growth phases) would appear to make them almost mutually exclusive
with lignin, which is predominantly localized in secondary walls and is produced after the
cells have reached their final dimensions.
The main building block of pectins is α-(1,4)-linked D-galacturonic acid (GalA), which
is often acetylated or methylated and combined with a variety of comonomers, such as α-
and β- anomers of L-Fucp, L-Araf, L-Rhap, D-Manp, and D-Xylp (refer to Figure 4B for full
names and structures). The following pectin structures typically exist as independently,
but in principle are also potentially present as separate domains of larger macromolecular
structures:
(A) Unbranched homogalacturonans (HG), often referred to as “smooth” pectins. HGs
have a linear backbone with about 100 α-(1,4)-linked GalA units, which can be methylated
and/or acetylated. Of note, HG may be produced in a highly methylated form and then
demethylated in a random or blocky fashion, with the former organization being useful
to reduce ‘stickiness’, e.g., during cell division [96], and the latter being on the contrary
capable of calcium-mediated gelation. Two rather recently discovered derivatized forms
of poly(galacturonic acid) are xylogalacturonan (XGA), which is predominantly found in
leaves [97], and apiogalacturonan (AP), which has been found in aquatic plants such as
duckweeds [98] and seagrasses [99].
Int. J. Mol. Sci. 2023, 24, 11668 11 of 47
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 11 of 48

Figure 4. Hemicellulose and pectins; structures and symbols of the sugar monomers are reported in
Figure 4. Hemicellulose and pectins; structures and symbols of the sugar monomers are reported in
the left part of the panels; acetylated and methylated positions are highlighted with ‘Ac’ and ‘Me’
thelabels,feruloylation
left part of the panels; acetylated
sites with and methylated
orange arrows. positions classes
(A). Most important are highlighted with ‘Ac’
of hemicelluloses andandsites‘Me’
labels, feruloylation
for the actions of some sitesofwith orange
the most arrows.
common (A). Most
enzymes important
that modify classes
them. of hemicelluloses
In detail: in xylans, back-and
bone
sites forbiosynthesis
the actions is ofmediated
some of theby Irregular
most common Xylem enzymes
9, 10, andthat
14 (IRX9-10-14),
modify them. arabinosylation by
In detail: in xylans,
Xylan Arabinosyl
backbone biosynthesisTransferases1
is mediated andby2 Irregular
(XAT1-2),Xylemand further
9, 10,chain
and 14elongation with xylosyl
(IRX9-10-14), residues by
arabinosylation
onto arabinosyl side chains by Xylosyl Arabinosyl Substitution of Xylan1 (XAX1); finally, the addi-
Xylan Arabinosyl Transferases1 and 2 (XAT1-2), and further chain elongation with xylosyl residues
tion of glucuronic acids is mediated by Glucuronic acid substitution of Xylan1 (GUX1). Other mod-
onto arabinosyl
ifying enzymesside chains
include by Xylosyl Arabinosyl
Acetyltransferases (XOATs Substitution
and OsTBL1)ofand Xylan1 (XAX1); finally,
Acetylesterases (BS1 andthe ad-
dition
DARX1)of glucuronic
that introduceacidsandisremove
mediated by groups.
acetyl Glucuronic acid substitution
In xyloglucans, of Xylan1
xylosyl residues are(GUX1).
added onto Other
modifying
the glucan enzymes includeXyloglucan
chain through Acetyltransferases (XOATs and
Xylosyl Transferase 1, OsTBL1) and Acetylesterases
2, and 5 (XXT1-2-5, respectively,(BS1forand
isolatedthat
DARX1) xylosyl residues,
introduce andorremove
those condensed with a In
acetyl groups. galactosyl or withxylosyl
xyloglucans, galactosyl-fucosyl
residues areunits),
added areonto
added onto Xyl-Gal dimeric side chains through Xyloglucan L-side chain galactosyl
the glucan chain through Xyloglucan Xylosyl Transferase 1, 2, and 5 (XXT1-2-5, respectively, for Transferase po-
sition 2 and through Murus3 (XLT2 or MUR3), and are added onto Gal-Fuc through FUcosyl Trans-
isolated xylosyl residues, or those condensed with a galactosyl or with galactosyl-fucosyl units), are
ferase 1 (FUT1). The formation of Xyl-Araf side chains is mediated by arabinoSylTransferases 1 and
added onto Xyl-Gal
2 (XST1-2), dimeric side
and the acetylation chains
pattern through Xyloglucan
is, respectively, regulated L-side chain galactosyl
by Xyloglucan Transferase
O-acetyltransferase
position 2 and through
1 (AXY4-4L) Murus3 (XLT2
on fucosylated or MUR3),
galactosyl and byand are addedBackbone
Xyloglucan onto Gal-Fuc through
O-Acetyl FUcosyl Trans-
Transferase 1
(XyBAT1)
ferase 1 (FUT1).on backbone
The formation of residues.
glucosyl Xyl-Araf In galactoglucomannans,
side chains is mediated by thearabinoSylTransferases
introduction of galactosyl 1 and
side chains
2 (XST1-2), is mediated
and by GalactoMannan
the acetylation GalactosylTransferase
pattern is, respectively, regulated (GMGT), and theO-acetyltransferase
by Xyloglucan acetylation pat-
tern mannan is mediated by O-acetyltransferases (MOAT1-2-3-4). (B). “Smooth” pectins typically
1 (AXY4-4L) on fucosylated galactosyl and by Xyloglucan Backbone O-Acetyl Transferase 1 (Xy-
have a partially methylated, linear homogalacturonan (HG) backbone. If HG chains feature short
BAT1) on backbone
branches in the form glucosyl residues.
of β-(1,3) In galactoglucomannans,
and β-(1,2)-linked the introduction
D-xylose or β-(1,5)-linked D-apioseof galactosyl
residues, theyside
chains is mediated by GalactoMannan GalactosylTransferase (GMGT), and
are respectively referred to as xylogalacturonan (XGA) and apiogalacturonan (AP). “Hairy” pectins the acetylation pattern
mannan is mediated
are heavily branched O-acetyltransferases
bymacromolecules. (MOAT1-2-3-4). (B).
Rhamnogalacturonan “Smooth”
I (RG-I) pectins typically
has alternating have a
(acetylated)
GalA and α-(1,2)-rhamnosyl residues, the latter bearing oligo(galactose)
partially methylated, linear homogalacturonan (HG) backbone. If HG chains feature short branches and oligo(arabinose)
in branches,
the formwhere feruloyl
of β-(1,3) andresidues may be present.
β-(1,2)-linked D-xyloseRhamnogalacturonan
or β-(1,5)-linked IID-apiose
(RG-II) typically
residues,features
they are
a short (7–9 units) HG main chain with a large variety of branches (four types of side chains, named
respectively referred to as xylogalacturonan (XGA) and apiogalacturonan (AP). “Hairy” pectins are
heavily branched macromolecules. Rhamnogalacturonan I (RG-I) has alternating (acetylated) GalA
Int. J. Mol. Sci. 2023, 24, 11668 12 of 47

and α-(1,2)-rhamnosyl residues, the latter bearing oligo(galactose) and oligo(arabinose) branches,
where feruloyl residues may be present. Rhamnogalacturonan II (RG-II) typically features a short
(7–9 units) HG main chain with a large variety of branches (four types of side chains, named
A to D) made up of up to 12 different saccharides including uncommon monomers, e.g., β-D-
apiofuranose (Api), aceric acid (Ace), 2-keto-3-deoxy-D-mannooctanoic acid (Kdo), and 3-deoxy-D-
lyxo-heptopyran-2-ularic acid (Dha). Chains C and D are dimeric (Dha-Rha and Kdo-Ara), whereas
chains A and B show a large architectural diversity.

(B) Rhamnogalacturonans I (RG-I). [α-D-GalA-(1,2)-α-L-Rha-(1,4)-] dimeric units form

GalA/Rha alternating polymers, which are typically acetylated at GalA, and C-4 branched
with linear or branched oligo(α-(1,5)-L-arabinose) and oligo(β-(1,4)-D-galactose).
(C) Rhamnogalacturonans II (RG-II) [100]. RG-IIs are low MW (5–10 KDa) and highly
branched polymers of more than twelve different sugars. The main chain is made of
GalA residues with a low degree of methylation or acetylation, while four different types
of side chains exist, all with a complex composition (A to D in Figure 4B). A and B are,
respectively, 7- and 6- to 9-residue-long, branched chains comprising at least five different
sugar types. C and D are disaccharides composed of peculiar sugar residues (e.g., 3-deoxy-
lyxo-2-heptulosaric acid (DHA) and 3-deoxy-manno-2-octulosonic acid (KDO)).
In terms of the relative ratio between the various components, this is very variable.
Most sources report 60–65% of pectin being generally made up of HG [101], and this
may also comprise the 6–7% of XGA recorded in leaves [97] or large amounts of AP
in some aquatic plants (where it may even replace HG, whereas in others is possibly
replaced by XGA [102]. Of note, these data may also be affected by the sample treatment
for sugar analysis: for example, AP may be under-represented due to its recalcitrance
to pectinases [103]. However, general pectin compositional data should be taken with
a pinch of salt, and these “smooth” pectin components may not always account for the
majority of pectin: for example, the content of RG I has been measured from as low as
11% (in Arabidopsis [104]) to up to 85% (in Okra pods [105]) of the total pectin content
(itself very variable). Further, since RG-I is also associated with water-holding capacity and
firmness of plant tissues and its degradation to fruit maturation processes [106], its content
would further vary throughout the plant life cycle.
Pectins associate intermolecularly to the point of producing gels; mechanistically, the
association proceeds through (1) Ca2+ bridges, in HGs (provided that sufficiently long
demethylated sequences are available [107]); (2) formation of borate-diol esters, which are
most typical for RG-II (e.g., causing its dimerization [108,109] and contributing significantly
to cell wall modulus [108,109]) but have been shown to be operational also in the formation
of mixed HG/RG-I/RG-II aggregates [110]; and (3) oxidative coupling reactions involving
the side chains. Feruloyl esters are indeed present in pectins, where they are linked to
arabinan and galactan sequences, i.e., the side chains of RG-I [111,112], with an overall
content that can be as high as the 0.8 wt% of pectin [113].
Feruloylation as a route to hemicellulose and pectin integration with lignin. Both
hemicelluloses and pectins can therefore bear ferulate side chains [114]: the presence of
ferulic acid polysaccharide esters in cell walls is known since the ‘70s [115], and the first
reports of arabinose and galactose-linked ferulates (hinting therefore to xylans–and specifi-
cally arabinose-containing ones-and RG-I as the carrier components) go back to the early
1980s [116]. Ferulates are well known to undergo oxidative dimerization [115], which can al-
ready cross-link the polysaccharides they are part of [117], and both monomeric and dimeric
ferulates can undergo further oligomerization [43,118,119]. These oxidative processes are
well-known to strengthen hemicellulose networks and allow them to covalently bridge to
lignin through the formation of polysaccharide-lignin complexes [120–122] and thereby
also to act as a template for lignin deposition [123] and to decrease the degradability of cell
walls [119,124]. The occurrence of other covalent interactions in these complexes, such as
reactions on p-coumarates and transesterification reactions [125], cannot be discounted. In-
deed, p-coumarates possibly play a role similar to ferulates (e.g., dimerizing) [116,126] but
are less studied due to their lower amounts. It is also worth pointing out that ferulate-based
Int. J. Mol. Sci. 2023, 24, 11668 13 of 47

cross-linked structures are often seen as a marker of the switch between the deposition of
primary cell wall (relatively poorer in ferulate) and that of the secondary one [127]. Since
the latter is essentially devoid of pectin, this may be taken as an (erroneous) indication of
a generally poor pectin–lignin integration, which would tally with their opposite concen-
tration profile (see Figure 1C). However, lignification actually starts from the pectin-rich
areas of middle lamella and cell corners [40], and feruloylated cell walls have often been
suggested as potential nucleation sites [114], along with tricin (tricin deficiency leads to
poor lignification [128]), which may actually mean that pectin integrates to an initial and
less aggregated form of lignin.

3.4. Lignification (Bio)Chemistry

This discussion initially focuses on the biochemical/biophysical environment of lig-
nification (Figure 5); for the sake of simplicity, here we only discusss phenylpropanoid
monolignols but identical concepts can be applied to other lignols too.
A short summary of the processes. Intracellularly, most phenylpropanoid monolig-
nols have a very similar biosynthesis: they have a common precursor (p-coumaric acid,
see Figure 3) and a common intermediate (caffeic acid, the path leading to p-coumaryl
alcohol being the exception). At the end of their biosynthesis, monolignols are moved
across the cell membrane into the extracellular space (the apoplast) through a variety of
mechanisms [129]: (1) passive diffusion, if they are sufficiently hydrophobic to solubilize
in the membrane [130]; (2) vesicular transport, which has been shown to be operational
for the very hydrophilic glycosylated lignols [131]; (3) active transport through membrane
transporters [132], although - to our knowledge - to date only one transporter has been
identified, which selectively acts on p-coumaryl alcohol [133]; and (4) transport through
channels, which have been postulated [129] but to our knowledge not experimentally
verified yet (thus not shown in Figure 5). Extracellularly, under the assistance of laccases
and peroxidases, monolignols are activated, typically producing radicals at phenol OH
groups, and then polymerize oxidatively. Of note, the very same enzyme classes are also
those capable of lignin degradation [134,135], always through oxidative mechanisms.
Hydrogen peroxide plays a key role throughout the lignification process. H2 O2
fulfills a variety of roles in plants [136], but it is specifically pivotal during lignification.
Intracellularly, the biosynthetic routes to monolignols are mostly based on the cytosolic
C3H enzyme, which is not only a 3-hydrolase but also an ascorbate peroxidase and is
hydrogen peroxide-dependent [137]. Also CSE, i.e., the key enzyme of the so-called
shikimate shunt to caffeic acid, is hydrogen peroxide-dependent [138]. Extracellularly, all
peroxidases require H2 O2 as a co-factor [139]; NADPH Oxidases (NOX) are a major source
of this extracellular hydrogen peroxide (through superoxide dismutation by SuperOxide
Dismutase, SOD). Of note, NOX have been reported to be activated in a RAC1-mediated
fashion by CCR [140], which on its turn reportedly can associate to CAD in a single,
membrane-localized complex [138] that converts monolignols’ CoA thioesters into primary
alcohols, thereby providing a putative link between intra- and extracellular processes.
Another important source of apoplastic (=extracellular) H2 O2 are indeed laccases, which
produce it during lignol activation (generation of phenol radicals). Also in this case, the
liberation of hydrogen peroxide usable in lignol polymerization is therefore tied to events
with an upstream position in the lignification chain.
The enzymes: laccases and peroxidases. The polymerization of lignols is the final
phase of the lignification process. It is assisted by two classes of enzymes, laccases (multi-
copper oxidases, which depend on molecular oxygen) and peroxidases (iron-heme oxidases,
which employ hydrogen peroxide), both capable of catalyzing/assisting a wide variety
of oxidation reactions. Their significance for plants is witnessed by their number and
level of expression: in Arabidopsis thaliana, 17 laccase [141] and 73 class III peroxidase
genes [142] (several linked to lignification [143]) have been found; among them, LAC4
laccase and PRX64 peroxidase (the latter involved in the build-up of the Arabidopsis Cas-
parian strip [144]) are the most highly expressed oxidative genes in that plant. Interestingly,
Int. J. Mol. Sci. 2023, 24, 11668 14 of 47

Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 14 of 48

the expression of these two enzymes is topologically different, with the former preferen-
tially located in secondary cell walls, the latter confined to the middle lamella and cell
corners
located[145], but thiscell
in secondary is walls,
not a general
the latterfeature:
confinedatoperoxidase
the middlesuch as PRX72
lamella and celliscorners
localized
in[145], but thisand
cell walls, is not a general
LAC4 itselffeature: a peroxidase
is transiently such asinPRX72
expressed is localized
cell corners, andinthere
cell walls,
are also
and LAC4
laccases anditself is transiently
peroxidases expressed
that localize in cell corners,tissues
in non-lignified and there
[146].are also laccases and
peroxidases that localize in non-lignified tissues [146].

Figure 5. The biosynthesis of monolignols, the formation of lignin via enzymatic oxidative coupling,
Figure 5. The biosynthesis of monolignols, the formation of lignin via enzymatic oxidative coupling,
and the hydrogen peroxide (H2O2) detoxification system are summarized by using canonical struc-
and thealthough
tures, hydrogen theperoxide
scheme is(Hvalid
2 O2 )in
detoxification system
principle for most areall
if not summarized
monolignols. byPlease
usingnote
canonical
that thestruc-
tures, although
extracellular the scheme
space, is valid in
here generically principle
defined forapoplast,
as the most if not allsite
is the monolignols. Please note
for the production that the
not only
of lignin, butspace,
extracellular also suberin, cutin, anddefined
here generically other biopolymers. Acronyms:
as the apoplast, C3H—p-coumarate
is the site for the production3-hydrox-
not only of
ylase but
lignin, (acting
alsoalso as a cytosolic
suberin, cutin, andascorbate peroxidase and
other biopolymers. thereforeC3H—p-coumarate
Acronyms: also referred to as C3H-APX);
3-hydroxylase
CCR—cinnamoyl-CoA reductase; CAD—cinnamyl alcohol dehydrogenase; ABC—ATP-binding
(acting also as a cytosolic ascorbate peroxidase and therefore also referred to as C3H-APX); CCR—
cassette transporter; RAC1—the Rho GTPase Ras-related C3 botulinum toxin substrate 1; NOX—
cinnamoyl-CoA reductase; CAD—cinnamyl alcohol dehydrogenase; ABC—ATP-binding cassette
NADPH oxidases, also known as respiratory burst oxidative homologs (RBOHs); SOD—superoxide
transporter; RAC1—the
dismutase; LAC—laccases; Rho GTPase Ras-related
PRX—class III peroxidasesC3 botulinum toxin substrate
(Class I: microbial 1; NOX—NADPH
or intracellular plant pe-
oxidases,
roxidases.also known
Class as respiratory
II: extracellular burst
fungal oxidative homologs
peroxidases. (RBOHs); SOD—superoxide
Class III: extracellular plant peroxidases).dismutase;
LAC—laccases; PRX—class III peroxidases (Class I: microbial or intracellular plant peroxidases. Class
The first fungal
II: extracellular step inperoxidases.
the chain ofClass
reactions leading to polymerized
III: extracellular lignin is the introduc-
plant peroxidases).
tion of free radicals in monolignols, i.e., their activation, typically at the phenol OH. Evi-
denceThe firstArabidopsis
from step in theshows
chain that
of reactions
lignols are leading to polymerized
activated by laccases andlignin is the
not by introduction
peroxidases
of[147]. The latterinare
free radicals heavily involved
monolignols, in lignol
i.e., their oligo/polymerization,
activation, typically at the although
phenollaccases are
OH. Evidence
involved
from in this shows
Arabidopsis phase too:
thatinlignols
Arabidopsis, double mutants
are activated by laccasesdeficient
and notin LAC4 and LAC17
by peroxidases [147].
have
The shown
latter arehypolignified
heavily involved fibers and collapsed
in lignol xylem vessels [148], and
oligo/polymerization, the additional
although laccaseslossare in-
of function
volved in this ofphase
LAC11too: hasinled to growth defects
Arabidopsis, double and failure
mutants [147]. Furthermore,
deficient in LAC4 andalso singlehave
LAC17
or double mutants in several peroxidase genes have shown
shown hypolignified fibers and collapsed xylem vessels [148], and the additional reduced lignin, even if typi-
loss of
cally not as much as in laccase mutants; for instance, the prx72 mutant
function of LAC11 has led to growth defects and failure [147]. Furthermore, also single or results in a lignin
contentmutants
double reduction in up to 35%
several [149].
peroxidase genes have shown reduced lignin, even if typically
In short, it is widely accepted
not as much as in laccase mutants; for that both laccases
instance, and mutant
the prx72 peroxidases
resultsparticipate
in a lignin in content
the
polymerization phase;
reduction up to 35% [149]. however, the level of involvement of these (or other) enzymes has
been long debated, with the two options of them directly participating in the process or
In short, it is widely accepted that both laccases and peroxidases participate in the
having a more distant, assistive role. As nicely summarized in a review by Ralph et al.
polymerization phase; however, the level of involvement of these (or other) enzymes has
Int. J. Mol. Sci. 2023, 24, 11668 15 of 47

been long debated, with the two options of them directly participating in the process or hav-
ing a more distant, assistive role. As nicely summarized in a review by Ralph et al. [150],
the current consensus opinion is that lignol activation/polymerization processes are largely
combinatorial, chemically controlled processes; i.e., the large number of different monomers
(up to 35 [151]) react in a fashion that is predominantly dependent on their molecular ac-
cessibility reactivity, absolute concentrations and relative stoichiometric ratios, their supply
rate [152], and the local pH [153], as much as the enzyme concentration and activity [154],
rather than specific interactions with the active sites of the involved enzymes. This tallies
with the fact that peroxidases may operate on lignols not only by direct catalysis reactions
on monolignols but also through hydroxy radicals that they are known to generate [155].
There is, however, evidence of protein-driven direct assistance, although not mediated by
laccases or peroxidases; for example, these enzymes in vitro provide racemic resinols via
β-β’ coupling (see Figure 6B), whereas in vivo the same reaction products are optically
active [156]. This has led to the discovery of so-called dirigent proteins [157–160], which at
least for this specific reaction orientate the stereochemistry of the product, although not
being directly capable of chemical catalysis.
Finally, it is noteworthy that lignin degradation (e.g., by fungi) is based on laccases and
peroxidases too. From the first finding of a peroxidase capable of lignin degradation [161]
and that other oxidases (laccases) shared this capacity [162,163], it has gradually become
clear that laccase may have possibly a greater role [164], but peroxidase has a wider variety
(lignin peroxidases, manganese peroxidases, and versatile peroxidases) [134].
Chemical reactions of lignol oligo/polymerization. In the early stage of lignification,
monolignol units dimerize to form dilignols; in this phase, β-O-40 coupling leads to alkyl
aryl ethers, e.g., the canonical lignin units, that may later undergo β-50 reactions to phenyl-
coumarans or β-10 reactions to spirodienones (all in Figure 6A). Another common coupling
mechanism is the β-β’, leading to resinols (Figure 6B). It is noteworthy that 40 -O-β is also
the typical coupling mechanism detected for non-canonical tricin, and the apparent lack of
homoligomers [70] and its preferential presence in low MW lignins [165] suggest tricin to
possibly act as a nucleation site [166].
The general mechanism for the β-O-40 coupling (Figure 6A, top) sees first the attack
of a phenoxy radical to the β atom of a lignol; this leads to a quinone methide, which is a
strong Michael-type acceptor and rapidly undergoes a second reaction with a nucleophile.
When the latter is a water molecule (in red), the canonical H, G and S lignin structures are
produced.
The canonical H, G, and S units produced via 40 -O-β coupling can then undergo
further reactions: if the phenol OH is free in the ortho position, the radical attacks onto other
lignols’ double bonds, and the subsequent β-50 ring closure produce phenylcoumarans.
Of note, the same kind of reaction is also used to yield some piceatannol dimers [83]
(in brackets in Figure 6A, middle right). Should such positions not be available, e.g., in S
units, the ring closure employs an aliphatic alcohol, thereby generating spirodienones due
to hindered rearomatization. Of note, these units were discovered only rather recently [167],
because their lability during lignin extraction processes led to the isolation only of their
1,2-diarylpropan-1,3-diol degradation products [168] (see Figure 6A top right).
A different reaction output would occur if in the original β-O-40 coupling the attacking
phenol was actually a catechol (Figure 6A, bottom), e.g., a non-canonical monolignol,
such as 5-hydroconiferyl alcohol, piceatannol, or astringin, or catechol-containing lignin
units (aka C units)). The quinone methide would not react with water but with the
neighboring OH group, producing benzodioxanes, which indeed have been first found in
COMT-deficient and therefore 5HC (catechol)-rich plants [52,169]. Of note, a number of
piceatannol-based benzodioxane found in alcoholic extracts of the grass Cyperus longus are
produced following this mechanism [170].
Int. J. Mol. Sci. 2023, 24, 11668 16 of 47
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 16 of

Figure 6. All new bonds formed during lignol couplings, and the numbering of all relevant atom
Figure 6. All new bonds formed
are shown during
in red. lignol couplings,
(A). Reaction paths and and the numbering
structures of all
of lignin units relevant
deriving atoms
from β-O-4′ couplin
are shown in red. (A). Reaction paths and structures 0 coupling.
Please note that G (guaiacyl) units areofstructurally
lignin units deriving
related from β-O-4
to guaiacol but actually are derived fro
coniferyl alcohol,
Please note that G (guaiacyl) units areandstructurally
“S” stands for “syringyl”
related although
to guaiacol butthe corresponding
actually are derived monolignol
from is sinap
alcohol.
coniferyl alcohol, and (B). β-β’for
“S” stands couplings
“syringyl”produce bis(quinode
although methide) intermediates,
the corresponding monolignol which may react with i
is sinapyl
tramolecular alcohols and/or water, producing bicylic resinols or various tetrahydrofurans. The s
alcohol. (B). β-β’ couplings produce bis(quinode methide) intermediates, which may react with
reochemistry of these products is typically ascribed to the action of dirigent proteins [157]. (C). Tw
intramolecular alcohols and/or
relatively less water,
common producing bicylic resinols
coupling mechanisms (5-5′orandvarious
4-O-5′)tetrahydrofurans. The and diar
lead to dibenzodioxocin-
stereochemistry of these products is junctions.
ether-containing typically ascribed
Please noteto that
the action
5-5′ canofjoin
dirigent
two G proteins
units but [157]. (C). Two
is operational also in linki
together
relatively less common a G and
coupling mechanisms (5-50(D).
an H unit [171]. 4-O-50 ) lead
andExamples of coupling reactions involving
to dibenzodioxocin- ferulic esters [11
and diaryl
which also
ether-containing junctions. account
Please noteforthatferulate dimerization
5-50 can join two G and unitsconjugation to lignin also
but is operational of hemicellulose/pectin.
in linking
note, the β carbon of canonical monolignols is referred as the 8 carbon in ferulates.
together a G and an H unit [171]. (D). Examples of coupling reactions involving ferulic esters [117],
which also account for ferulate dimerization and conjugation to lignin of hemicellulose/pectin. Of
note, the β carbon of canonical monolignols is referred as the 8 carbon in ferulates.
Int. J. Mol. Sci. 2023, 24, 11668 17 of 47

Another important class of reactions is the β-β’ dimerization, leading to bicylic

pinoresinol or monocylic tetrahydrofuran structures previously mentioned in relation
to the stereochemical assistance by provided by the so-called dirigent proteins [156,158].
This pathway always starts through the β-β’combination of two coniferyl or sinapyl rad-
icals, leading to a bis(quinone methide); it is worth noting that, besides Michael-type
addition of either water or intramolecular alcohols (as in Figure 6B), this intermediate
has also been found to potentially undergo a reduction and produce its saturated analog
secoisolaricinol [172].
Other noticeable lignol reactions are the 5,50 and 4-O-50 couplings (Figure 6C), which
produce groups (respectively, biphenyls and diaryl ethers) particularly resistant to enzy-
matic degradation, e.g., via β-etherase [173] or pyrolysis [174]. Although diaryl ethers
are likely easier to dissociate than biphenyls, they still have much higher dissociation
energies than all other alkyl aryl ethers [175]. Since 5-50 coupling requires at least one ortho
position to phenolic OH to be free, this reaction is common in woods with a low content
of S units (softwoods), where it occurs on G units [176] or also on ferulates [177]. If the
5-50 biphenyls bear non-etherified phenols, they may further react with monolignols and
produce 8-membered cyclic structures referred to as dibenzodioxicins [178–180].
Finally, it is worth mentioning that ferulates undergo essentially the same kind of
reactions (Figure 6C), with the only significant difference being the initially formed struc-
tures are predominantly dhydrodimers; i.e., they still contain cinnamoyl double bonds
(i.e., conjugated to both phenyl and carboxylate groups) [117,181], whereas in most other
lignol dimers this unsaturation is lost. This is likely due to the different reactivity of the
quinone methides, which appear to favor a rearrangement that recovers the conjugation to
ester carboxylates rather than Michael-type addition.
Lignin units and relative lignin composition. Firstly, a warning: lignin composi-
tional data should be used with considerable caution, due to possible biases of both isola-
tion and analytical procedures. For example, isolation of lignin through thioacidolysis from
milled wood has shown to very considerably reduce the level of phenol etherification [182],
whereas the presence of 4-O-50 products may have been historically underestimated, due
to their difficult analysis via NMR [183,184], and the same applies to β-10 spierodienones
due to their above-mentioned acid lability [168].
Having said this and further adding that the specific make-up of lignin units may
depend not only on the plant species but also on the environmental conditions experi-
enced by the individual plant, a consolidated point is that β-O-40 alkyl aryl ethers are by
far the most common products encountered in a vast range of lignins. For example, in
grasses and grains they range from being present in 45–49% of the units in bamboo (β-β’
resinols 3.6–7.4%, β-β’ tetrahydrofurans 2.0–2.3%, β-50 phenylcoumarans 2.8–4.5%, β-10
spirodienones 1.3–2.3%, and 4-O-50 diaryl ethers 2.8–2.9%) [78], through 58% in flax fibers
(β-50 phenylcoumarans 11%, β-β’ resinols 9%, and lower amounts of spirodienones and
dibenzodioxocins) [185], 72% in jute (β-β’resinol 16%, β-50 phenylcoumarans 4%, and β-10
spirodienones 4%) [186], 75% in straw (β-50 phenylcoumarans 15% and 5-50 /β-O-40 diben-
zodioxocins 3%) [187], and 77–79% in spent grain from brewing (β-50 phenylcoumarans
11–13%, β-β’ resinols 5–6%, and 5-50 /β-O-40 dibenzodioxocins 3–5%) [188], to up to 82%
in elephant grass [189] and 83% in sugarcane (β-50 phenylcoumarans 6%) [187]. Also, in
woody plants β-O-40 alkyl aryl ethers have a similarly dominant position: for example,
they account for 46–50% of lignin units in Eucalyptus globulus (β-β’ resinols 10–14%) [190]
and 68–77% in the cork oak Quercus suber [34] in hardwoods and from 40 up to 50% in
various softwoods [191,192]. Of note, while 5,50 biphenyls are the second most common
linkages in softwood (up to 26% of the linkages, with an average of 10% for the third most
common β-50 phenylcoumarans), they are comparatively much less common in hardwood
(up to 9%) [192].
It has been suggested that β-O-40 couplings are more common in the early stages
of lignification, and reportedly this may not be due to them being favored in the initial
production of dilignols (β-50 and, to a lesser extent, β-β’ may be favored) but to β-O-40 being
Int. J. Mol. Sci. 2023, 24, 11668 18 of 47

preferred for the chain extending reaction of monolignols with di- and monolignols [192].
Equally interestingly, under conditions of limited supply of monolignols, e.g., because
of their difficult diffusion in an already lignified region, the dominant reactions may be
5-50 and, less, 4-O-50 cross-linking between lignin oligomers [192], which explains why
the corresponding units are quantitatively etherified [182] (i.e., the reaction should not
preferentially involve monolignols, which have free phenol groups). This would mean a
lower content of β-O-40 in older, more lignified plants or in the tissues more distant from
their centers (cork being most lignified, xylem least). Indeed, in the cork oak Quercus suber,
the amount of β-O-40 units has been found to decrease radially from 77% in xylem, through
71% in phloem, down to 68% in cork, and in the latter condensed structures are most
abundant (β-50 phenylcoumarans 20%, 5-50 /β-O-40 dibenzodioxocins 5%) [34]. Further, in
Eucalyptus globulus β-O-40 units have also been found to slightly decrease with age (and β-β’
resinols marginally increase) [190]. These features, however, may be more common in
woody plants than in the less lignified grasses; indeed, in, e.g., elephant grass (as the name
suggests, a grass), pith and cortex appear to be similar in composition [189].
In terms of the structural details of the units produced via β-O-40 coupling, it is
noteworthy that:
(A) H units are by far the least common, typically between 0.5 and 10% of total
lignin [151], although in some grasses they may reach up to >30% [193].
(B) G units are particularly frequent in softwood; for example, spruce lignin can have
up to 99% of G units [194], and similar values are seen also in non-woody plants such as
the Musa textilis banana [195].
(C) Hardwoods have a more variable S/G ratio, typically between 1:4 and 4:1; for
example, the ratio is 1.2:1 throughout heartwood, sapwood, and bark of teak (with H units
increasing, respectively, from 2 to 5%) [196], ≈3.5:1 in the heartwood and sapwood of
Eucalyptus globulus [197], 1.4:1 in magnolia, 2.5:1 in birch, and 3.3:1 in beech [198]. There are
also reports of significant variations within the body of a wood plant; for example, the S/G
ratio varies from 1.2:1 in the heartwood and sapwood, through 1:1.4 in the phloem to 1:6.5
in the bark of the cork oak [34]. Importantly, however, this does not imply that wherever
lignin has significant amount of S units, the younger the lignin, the higher is its S content:
for example, in grasses such as tall fescue, the S/G ratio tends to decrease (higher S content)
during stem development [199].
A final note refers to the couplings involving ferulates, since they may significantly
depend on the structural details of their lignols: 80% of feruloyltyramine undergoes β-O-40
and β-50 (couplings), with the remaining 20% being attached to cell walls through their
phenolic moiety [200], while diferuloylputrescine is almost exclusively incorporated via
β-50 linkages [85].
Targeted alterations to lignification. Lignin has traditionally been seen as an obstacle
for the valorization of plant biomasses, e.g., reducing their degradability and therefore
providing a less efficient energy source in animal foraging. However, this general view does
not take into account that lignin composition is a major determinant of this: for example,
with appropriate extraction/treatment processes, higher S/G ratios are linked to, e.g.,
much increased enzymatic degradability of bamboo residues [201], easier digestibility of
roughage from various sources in ruminants [202], or better methane production following
anaerobic digestion of birch biomass [203,204]. Therefore, in several studies it has been
tried to modify the lignification process in order to reduce the overall lignin content or to
alter its composition, in order to improve its later properties.
Hereafter, we provide examples of three biotechnological strategies employed to
achieve these targets (lignin reduction or lignin modification), but firstly it is important
to stress that the overall amount of lignin appears not to be really critical for plant devel-
opment; for example, when in Arabidopsis, Thaliana genes involved in lignol production
(e.g., C4H, 4CL, CCoAOMT, CCR, and CAD; see Figure 3 for an overview of their roles)
were individually mutated, lignin production decreased, with large increases in that of
Int. J. Mol. Sci. 2023, 24, 11668 19 of 47

hemicelluloses and no significant effect on that of cellulose but no major effect on plant
growth [205].
(1) Alteration of lignol biosynthesis. This approach utilizes the lignin-modifying ef-
fects of the reduced expression/activity of enzymes involved in lignol production. Possibly
the best example is offered by CAD, whose decreased activity impairs the reduction of
all aldehydes to canonical monolignols and thereby greatly increases the aldehyde lignin
content [206,207]. A lower CAD activity appears to have a disproportionate effect on S
units, as shown in CAD-mutated Arabidopsis (S/G ratio decreased from 1.5:1 to 1:2 [205],
linked to much increased degradability [206]) or in alfalfa treated with anti-CAD antisense
(S/G ratio decreased from 1:1 to 1:2–3) [208], which is likely ascribed to higher specificity
of these enzymes for sinapylaldehyde [209]. These findings fit with the observations that
naturally occurring CAD mutant plants (whose lignin is particularly rich in aldehydes
but appear otherwise rather normal [65,210,211]) appear to have increased digestibility, as
shown for the CAD-mutant varieties of Sorghum bicolor [212], and may be the case for
the specifically silkworm-friendly Sekizaisou mulberry trees [213]. Of note, some other
Sorghum bicolor varieties are natural COMT mutants [214], as much as some varieties of
poplar [215]. The better degradability of their biomass [216] is also in this case associated
with a lower S/G ratio, in addition to a much lower overall lignin content.
Another important example is offered by the hydroxylating enzyme F5H, which is
a key player in the production of synapyl alcohol. In rice, its downregulation enriched
lignin in G units (thereby making it in principle more degradable), while its upregulation
does the contrary [217]. In poplar genetically engineered to express an Arabidopsis F5H,
the resulting higher-S lignin showed an increased resistance to wood-decaying fungal
attacks [218] (although in other cases, such as the resistance of Brassica napus to Sclerotinia
sclerotiorum, it may be associated with a higher G content produced by F5H knockout [219]).
Of note, while reduced F5H and COMT both have the potential to reduce the S/G ratio
(comparatively seen, e.g., in sugarcane [220]), when an F5H overexpression was induced in
the presence of a nonsense mutation of COMT, the former did not compensate for the latter,
and the final lignin was still low in S units [221].
Besides changing the S/G ratio, lignol synthetic enzymes can also affect other descrip-
tors of lignin composition. For example, the downregulation of the caffeic acid-producing
C3H has led to a lignin abnormally high (up to 65%) in H units; this was accompanied by
the apparent absence of β-10 coupling products, since their ortho reactivity, e.g., via β-50
coupling, is favored for coumaryl alcohol-derived units [222].
Last, it is worth mentioning that, by silencing the flavonoid-producing CHS, a lignin
with highly reduced tricin was obtained, whose higher density of β-β’ and β-50 units
indicated a higher likelihood of monolignol dimerization and therefore supported the
hypothesis that tricin predominantly acts as an initiator group [223].
(2) Alteration of lignol transport. Knockout mutants lacking the only confirmed mono-
lignol transporter (AtABCG29, reportedly a selective exporter p-coumaryl alcohol and
therefore in principle affecting only H units) showed a 25–30% lower content in all lignin
units, which the authors of the study interpret mainly as a consequence of cross-talks
between the phenylpropanoid pathways [133] but may also be the result of the transporter
acting also on p-coumaroyl-CoA, the general precursors of all canonical lignols (Figure 3).
(3) Alteration of lignol polymerization. Peroxidases are a positive determinant of the
deposition of lignin in cell walls; for example, in tobacco plants, the introduction of addi-
tional peroxidase genes has been shown to both significantly enhance hydrogen peroxide
production and increase the lignin content [224]. However, the large number of these
enzymes, and the resulting redundancy, means that it is difficult to obtain significant effects
simply by altering only one of them. For example, in Arabidopsis plants the simultaneous
deficiency of three couples of lignin-involved peroxidases (prx2/prx25, prx2/prx71, and
prx25/prx71) lowered lignin content much more than in single mutants (without affecting
the growth) [225]. Similar multiple mutants (prx2/prx25 and prx2/prx25/prx71) were
Int. J. Mol. Sci. 2023, 24, 11668 20 of 47

needed to produce Arabidopsis seeds with thinner protective layers and lower polyphenol
content and to make them more sensitive to accelerated ageing and more permeable [226].
Similar effects have been recorded with laccases. For example, two laccase double
mutants, lac4-1 lac17 and lac4-2 lac17, caused, respectively, a 20% and 40% lignin reduction,
while deposition was completely prevented with the triple mutant lac4 lac11 lac17 [147].

4. Advanced Applications of Lignin-Based Materials

While Section 3 of this article is focused on biosynthesis, here advanced applications
of lignin-based materials will be discussed, where mainly technical lignin as source was
used. The advantage of utilizing technical lignin is that it is directly available on a larger
scale as a by-product of the papermaking and biofuel industries. To utilize lignin for
material design, it has to be isolated from biomass using different processes. The different
types of technical lignin are briefly discussed here, and we refer the reader to excellent
and specialized recent reviews for more in depth covering of this subject [227–229]. The
method for lignin extraction affects the native structure of lignin through the cleavage of
bonds between different lignin monomers and can result in chemical modifications. Here,
the content of functional groups in the lignin (phenolic hydroxyl, carboxyl, and sulfonate
groups) varies with the pulping process applied. Kraft lignin (KL) is obtained by Kraft
pulping. It is the major chemical pulping process, which is performed at high pH value,
and the lignin is precipitated from black liquor by the addition of acidifying agents (mineral
acid or carbon dioxide) [230]. Depending on the pH value which was utilized, different
compositions and yields of the KL can be obtained. Lignosulfonate can be isolated by a
sulfite pulping process, which is performed between pH = 2 and pH = 12 (depending on the
cationic composition of the pulping liquor). The resulting lignosulfonate includes a high
amount of sulfonate groups. Another class of technical lignin is the organosolv lignin. Here,
biomass components can be fractionated by means of an organic solvent (ethanol, ethylene
glycol, acetone, and tetrahydrofuran). The resulting organosolv lignin is free of sulfur
components and provides a higher homogeneity in contrast to KL or lignosulfonate [231].
Soda lignin (or alkali lignin) is obtained by soda pulping or delignification using strong
alkali. For this purpose, the biomass is heated in the presence of 13–16% alkali [227]. The
main difference in comparison to the Kraft pulping process is the utilization of a sulfur-free
medium of the cooking liquor, and for this reason, the soda lignin is sulfur-free [232].
The substantial development in lignin pretreatment and processing technologies has
enabled the design of lignin-based materials for advanced applications (Figure 7). In
this section, we present how lignin is utilized to highlight its potential for health care
applications, for energy storage, and as smart materials, as well as demonstrating how
lignin-based materials can be equipped with additional functionalities such as self-healing,
recyclability, and adhesiveness. It has to be noticed that lignin-based materials were also
successfully integrated into material systems indented for other types of applications, e.g.,
for the agroindustrial field or as bioplastics, and we refer the reader to specialized recent
reviews [233–235].
 Int.
Int. J. Mol. Sci. 2023, J. 11668
24, Mol. Sci. 2023, 24, x FOR PEER REVIEW 21 of 47 21 of 48

Figure 7. Schematic
Figureillustration
7. Schematicofillustration
(A) advanced applications
of (A) of lignin-based
advanced applications materials and
of lignin-based (B) addi-
materials and (B) ad-
tional functionalities,
ditionalwhich can be integrated.
functionalities, which can1.beDrug delivery,
integrated. 2. wound
1. Drug healing,
delivery, 3. energy
2. wound storage,
healing, 3. energy stor-
age, 4.
4. smart materials, 5. smart materials,
self-healing, 5. self-healing, 6.
6. adhesiveness, andadhesiveness, and 7. recyclability.
7. recyclability.

4.1. Lignin-Based Materials forMaterials

4.1. Lignin‐Based HealthcareforApplications
Healthcare Applications
Processes of increased sophistication (above all those
Processes of increased sophistication (abovethat allowthat
all those to obtain
allow to sulfur-free
obtain sulfur-free
lignin) [236] have progressively allowed to obtain lignin with sufficient reproducibility
lignin) [236] have progressively allowed to obtain lignin with sufficient reproducibility in in
chemical composition and overall and
chemical composition architecture to allow the
overall architecture to development of lignin-based
allow the development of lignin-based
materials formaterials
applicationsfor applications
in the health in care
the health
sector,care sector, including
including for tissuefor tissue engineering
engineering and and
drug/gene
drug/gene delivery delivery
[237]. [237].
It is of It that
note is of note that
this is in this
partisdue
in part due to physical
to physical properties
properties and and in
part to chemical
in part to chemical reactivity: reactivity: the abundance
the abundance of phenols
of phenols and and catechols
catechols enables
enables lignin to act
lignin
as a reasonably
to act as a reasonably strong strong antioxidant
antioxidant andand thereforescavenge
therefore scavengebiologically
biologically relevant
relevantoxidants
oxidants (ROS,(ROS, reactive
reactive oxygen
oxygen species),
species), which—althoughthe
which—although the correct
correct level
levelofofscavenging
scavenging matters—
matters—cancan be be instrumentalfor
instrumental foran
an accelerated
accelerated wound
woundhealinghealing[238]. In addition,
[238]. In addition,the inherent
the am-
phiphilicity of lignin enables the formation of nanomaterials
inherent amphiphilicity of lignin enables the formation of nanomaterials with phenolic with phenolic (hence a neg-
ative charge
(hence a negative charge at atphysiological
physiologicalpH) pH) and aliphatic
and aliphaticalcohols covering
alcohols the surface
covering and aromatic
the surface
and aromatic and methyl groups composing the bulk and thereby allowing for ligninbecome
and methyl groups composing the bulk and thereby allowing for lignin to to the
basis for carriers of hydrophobic molecules [239]. Hence, lignin-based
become the basis for carriers of hydrophobic molecules [239]. Hence, lignin-based particles particles can be
equipped with diverse therapeutics for many pharmaceutical applications including an-
can be equipped with diverse therapeutics for many pharmaceutical applications including
ticancer treatment [240].
anticancer treatment [240].
4.1.1. Lignin-Based Nanoparticles
4.1.1. Lignin-Based Nanoparticles
On the one hand, lignin and its derivatives can be loaded in nanoparticles (NPs) as
On the one hand, lignin and its derivatives can be loaded in nanoparticles (NPs)
presented with particular systems based on dextran and glycol, which were used for in
as presented with particular systems based on dextran and glycol, which were used for
vivo wound healing [241]. On the other hand, lignin-based NPs can be prepared using
in vivo wound healing [241]. On the other hand, lignin-based NPs can be prepared using
different synthesis methods, which affect the size distribution [239,242]. Nanoprecipita-
different synthesis methods, which affect the size distribution [239,242]. Nanoprecipitation
tion is a widely used process to create lignin-based NPs [243]. It is a solvent shifting
is a widely used process
method, to create
in which lignin-based
supersaturation of NPs [243].
a lignin It is a(organic
solution solventsolvent
shifting method,
is used) is obtained
in which supersaturation of a lignin solution (organic solvent is used) is obtained by a
controlled shifting into a non-solvent rich medium (e.g., water) resulting in particles with
spherical shape, diameter ≥ 100 nm, and negative surface charge. The characteristics of
Int. J. Mol. Sci. 2023, 24, 11668 22 of 47

NPs (morphology, size, and surface charge) can be controlled by the parameters of the
formation process (solvents, concentration, and type of lignin).
As example, when alkali lignin (AL) is precipitated under acidic conditions, monodis-
perse size distribution can be realized [244]. In contrast to that, the precipitation of AL
using water results in the formation of heterogeneous particle sizes [245]. A size distri-
bution between 50 and 350 nm could be obtained for NPs, which were generated by the
precipitation of lignin (dissolved in organic solvents) in water [246]. Here, NP sizes were
affected by the molar mass, content of hydroxy groups, volume of water, and the stirring
rate during preparation. The pure lignin-based NPs showed an advanced tissue formation
in the treatment of skin wounds as investigated in a mouse model. The particles did not
interfere with cell proliferation during wound healing.
Lignin-based NPs for tissue engineering can be equipped with calcium peroxide
(enabling a controlled release of oxygen) for an enhanced vascularization and maturation
of collagens [247]. Here, around 500 ppm oxygen per day could be released by calcium
peroxide, which was incorporated in sodium lignosulfonate (LS)-based NPs. These lignin
composite precursors could be injected together with an alkene-functionalized gelatin
matrix, which is photo-crosslinkable. These in situ forming lignin-based composites in-
cluding NPs (which can release oxygen) improved blood vessel formation and supported
the infiltration of smooth muscle actin and fibroblasts into the wound defects. Recently,
an accelerated wound healing could be realized with organosolv lignin-based NPs that
act as drug nanocarriers [248]. The NPs were loaded with curcumin (wound-healing
active, antioxidant, and anti-inflammatory property) as a promising small molecule for
the treatment of wounds. In vitro investigations indicated a strong antibacterial activ-
ity against Gram-positive bacterial pathogens (S. aureus). Fibroblast cell migration was
detected using a scratch assay. Here, wounded keratinocytes exhibited increased cell mi-
gration upon treatment with curcumin loaded nanocarriers. An enhanced dermal wound
closure (nearly full wound contraction after 12 days) in comparison to an untreated control
(wound size reduction of 43% after 12 days) could be observed in in vivo experiments
using wounded rats.
Drug delivery using pH-sensitive and amphiphilic lignin-based copolymers as carrier
materials showed adjustable release profiles as function of pH value [249], using AL
functionalized with methacrylate groups. In a second step, graft copolymerization with
methyl methacrylate (MMA) was performed resulting in AL-g-PMMA copolymers. NPs
with a hydrodynamic diameter of about 180 nm could be obtained by self-assembly. When
the particles were loaded with ibuprofen as drug, a release of about 82 wt% of the load
could be detected at a pH value of 7.4 (simulating intestinal fluid), whereas at a pH of
1.5 (simulating gastric fluid) less than 16 wt% of the drug was released within 72 h. The
designed NPs were able to inhibit the survival of human colon cancer cells HT-29 with a
final survival rate of only 5.3%.
Lignin-based NPs successfully demonstrated the ability to encapsulate ascorbic acid
(AA; can act as antioxidant additive to contrast skin aging processes and to protect the
cell against photo-damage) in order to improve stability and antioxidant activity against
temperature and pH-dependent degradation [250]. An AA ester was synthesized and was
efficiently encapsulated inside NPs from KL by a solvent shifting procedure (KL and AA
derivatives were dissolved in dimethyl isosorbide; after addition of water NPs precipitated).
The loaded NPs showed improved stability towards temperature and pH changes, and
the included AA derivatives were preserved towards degradation. After 24 h, the AA
derivatives could be released up to 84% at pH 3.0 and up to 78% at pH 5.4.
The treatment of fungal diseases (exemplarily shown in plants) was investigated with
lignin-based nanocarriers with a diameter of 200–300 nm, which were synthesized by
miniemulsion polymerization (based on aza-Michael addition reaction) utilizing methacry-
lated kraft lignin (KL) and bio-based amines as a crosslinker [251]. When fungicides were
encapsulated in situ during the miniemulsion polymerization (encapsulation efficiencies
between 70 and 99%), the growth of Phaeomoniella chlamydospora and Phaeoacremonium mini-
Int. J. Mol. Sci. 2023, 24, 11668 23 of 47

mum (lignase-producing fungi) could successfully inhibited (proved for 4 years in planta
studies).
It has to be noticed that the essential characteristics of lignin-based NPs utilized as
drug carriers are the capacity of drug loading and the efficiency of drug encapsulation.
As an example, when doxorubicin hydrochloride (DOX) was loaded into lignin-based
NPs during NP preparation, better encapsulation efficiency than for introducing the drugs
into NP colloidal solution was obtained as result of stronger interactions between lignin
and the utilized drug [252]. According to the chemical structure of lignin, strong inter-
actions (hydrogen bonding and π-π stacking) especially with hydrophobic drugs can be
realized [253–255]. As result of these strong interactions, an extremely slow release profile
can be obtained as presented for curcumin loaded lignin-based NPs in simulated gastric
conditions (<30% release within 6 days) [254]. Positively charged drugs can interact with
lignin-based NPs as they provide a negative surface charge enabling ionic bonding [256],
but the drug loading using hydrophilic molecules (such as capecitabine) is still challeng-
ing [257]. In addition, the translation of described materials for health care applications
requires reproducibility of material characteristics in accordance with structure–property
relationships. As the lignin structure (e.g., molar mass, dispersity, and degree of branching)
and functionality (e.g., content of aromatic hydroxy groups and aliphatic hydroxy groups)
are highly dependent on the lignin source and technical process for lignin production,
systematic investigations about chemical composition and macromolecular structure of
lignin as function of the different types in reference to drug delivery and tissue engineering
characteristics are essential.

4.1.2. Lignin-Based Hydrogels

The many functional groups of lignin can be employed to integrate lignin fragments in
sustainable and environmentally friendly hydrogels, which are recognized for their interest-
ing applicability in biomedicine [258]. As example, an antioxidant hydrogel was created by
crosslinking KL (acting as antioxidant agent) and gelatin in water [259]. The hydroxy group
from KL and the primary amine groups from gelatin were reacted with epichlorohydrin
under alkaline condition, whereby covalent bonds were formed in a ring-opening reaction.
These hydrogels showed the reduction of oxidative stress as investigated by in vitro natural
antioxidant expression tests and high antibacterial activities against E. coli. When AL
was crosslinked with agarose to generate hydrogels and extracted silk fibroin (enhanc-
ing mechanical tensile properties) as well as when zinc chromite NPs (as anti-infective
agent) were incorporated into the hydrogel matrix, lignin-nanobiocomposite scaffolds for
wound healing were generated [260]. For these gels, considerable hemocompatibility and
antibacterial activity was indicated. An almost completely healed wound within five days
was observed in a mouse model. As the desired shape of a biomaterial is always depen-
dent on, e.g., the size and shape of a wound, shape adaptivity can be highly demanded.
A lignin-incorporated nanogel was presented, which can be injected as a solution [238].
In that work, a strong antioxidant activity was determined from extracted organosolv
lignin (from coconut husks). The lignin was integrated into thermoresponsive nanogels
based on polyurethane (PU) copolymers of poly(ethylene glycol) (PEG), poly(propylene
glycol) (PPG), and poly(dimethylsiloxane) (PDMS) (Figure 8). These nanogels provided
a sol-gel transition between 22 ◦ C and 24 ◦ C (dependent on the lignin content) enabling
the formation of a polymeric network at body temperature, which can accommodate the
shape of a wound. Here, the burned wound of the mouse treated with lignin-incorporated
nanogels showed a complete healing within 25 days (comparable with performance of
other antioxidants like vitamin C and trolox) while a slower healing rate was observed
when lignin-free nanogels were used.
Int.Int.
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Figure 8. Schematic
Figure Schematicillustration
illustrationof the synthesis
of the of lignin-incorporated
synthesis nanogels
of lignin-incorporated for wound
nanogels healing heal-
for wound
application. (A). Extraction of lignin from coconut husk. (B). Synthesis of the thermoresponsive
ing application. (A). Extraction of lignin from coconut husk. (B). Synthesis of the thermorespon-
nanogel based on poly(ethylene glycol) (PEG, brown), poly(propylene glycol) (PPG, grey), and
sive nanogel based on poly(ethylene glycol) (PEG, brown), poly(propylene glycol) (PPG, grey),
poly(dimethysiloxane) (PDMS-diol, blue). (C). Preparation of temperature-sensitive lignin-incorpo-
and
ratedpoly(dimethysiloxane) (PDMS-diol,
nanogel. (D). Wound healing blue).
application of the(C). Preparation
nanogel. of with
Reprinted temperature-sensitive
permission from ref.lignin-
incorporated nanogel.
[238] Copyright (D). Wound
2021, American healing
Chemical application of the nanogel. Reprinted with permission
Society.
from ref. [238] Copyright 2021, American Chemical Society.
Lignin-based hydrogels can be utilized as a drug delivery platform. In one example,
Lignin-based
organosolv hardwood hydrogels
lignin was can be to
used utilized
design as a drug by
hydrogels delivery platform.
crosslinking In one
with poly exam-
(eth-
ple,
yleneorganosolv hardwood
glycol) diglycidyl etherlignin was used
(PEGDGE) [261].toAsdesign hydrogels
a hydrophilic by crosslinking
model with poly
drug, paracetamol
(ethylene
was selectedglycol) diglycidyl
for release ether
studies. The(PEGDGE)
paracetamol[261].
release Aswas
a hydrophilic
affected by themodel drug, parac-
composition
etamol was selected
of the hydrogels. Withfor release crosslinker
increasing studies. The paracetamol
content, drug releaserelease was affected
was reduced by the
as result
of increasing of
composition molecular interactions
the hydrogels. With(hydrogen
increasing bonds) between
crosslinker the drug
content, andrelease
drug the cross-
was re-
linker. as
duced However,
result ofthe composition
increasing of the reported
molecular interactionshydrogels has tobonds)
(hydrogen be optimized
between as they
the drug
showed a fast burst release profile with paracetamol as hydrophilic drug.
and the crosslinker. However, the composition of the reported hydrogels has to be opti- The efficiency
of lignin-based
mized hydrogels
as they showed as drug
a fast burstdelivery
releasematrix
profile forwith
hydrophobic
paracetamol drugsassuch as curcu-drug.
hydrophilic
min was investigated with hydrogels synthesized by crosslinking
The efficiency of lignin-based hydrogels as drug delivery matrix for hydrophobic LS lignin with PEG and drugs
such as curcumin was investigated with hydrogels synthesized by crosslinkingfacili-
poly [(methyl vinyl ether)-co-(maleic acid)] [262]. The hydrophobic nature of lignin LS lignin
tatedPEG
with curcumin loading,
and poly and the
[(methyl prepared
vinyl hydrogels were
ether)-co-(maleic acid)]able to sustain
[262]. the release for
The hydrophobic nature
up to 4 days. Another way to incorporate drugs in lignin-based
of lignin facilitated curcumin loading, and the prepared hydrogels were able hydrogels was presented
to sustain
by means
the releaseoffor
generating inclusion
up to 4 days. complexes
Another waywhen lignin is equipped
to incorporate drugs inwith cyclic oligo-
lignin-based hydro-
saccharides. Here, crosslinked LignoBoost lignin was functionalized with β-cyclodextrin
gels was presented by means of generating inclusion complexes when lignin is equipped
(β-CD) to create a drug carrier matrix [263]. Active components such as ketoconazole and
with cyclic oligosaccharides. Here, crosslinked LignoBoost lignin was functionalized with
piroxicam form inclusion complexes in an aqueous environment with β-CD, as β-CD pro-
β-cyclodextrin (β-CD) to create a drug carrier matrix [263]. Active components such as
vides a hydrophobic core cavity. The obtained drug carriers showed fast drug release ki-
ketoconazole and piroxicam form inclusion complexes in an aqueous environment with
netics (>80% release within 10 h), which fitted well in the Korsmeyer–Peppas model. Alt-
β-CD, as β-CD provides a hydrophobic core cavity. The obtained drug carriers showed
hough great progress was made for lignin-based hydrogels intended for drug delivery,
fast drug release kinetics (>80% release within 10 h), which fitted well in the Korsmeyer–
the ability of changing the swelling behavior when external stimuli are applied to obtain
Peppas model.release
an on-demand Although wouldgreat progress was
be important made for
to facilitate thelignin-based
transition fromhydrogels intended
fundamental
for drug delivery, the ability of changing
research into real health care applications. the swelling behavior when external stimuli are
applied to obtain an on-demand release would be important to facilitate the transition from
fundamental research
4.1.3. Lignin-Based into real health care applications.
Foams
An additional application area for lignin is the design of foam materials, as they pro-
4.1.3. Lignin-Based Foams
vide a low density, which leads to lower material costs. Lignin-based polyurethane foams
An additional application area for lignin is the design of foam materials, as they
provide a low density, which leads to lower material costs. Lignin-based polyurethane
foams (PUFs) were reported, which effectively promote wound healing of full-thickness
Int. J. Mol. Sci. 2023, 24, 11668 25 of 47

skin defects [264]. In a first step, enzymolysis lignin was functionalized with PEG and
glycerol. Subsequently, a one-step foaming process was applied with a silver nitrate
solution and hexamethylene diisocyanate. Silver nanoparticles were created in situ during
the foaming process as the phenolic hydroxy groups included in the liquefied lignin acted
as reducing and capping agent to silver ions. The created PUFs exhibited more than 99%
antibacterial rate against E. coli within 1 h and S. aureus within 4 h. An increase of the
silver nitrate concentration for the forming procedure increased the antibacterial ability
of PUFs, indicating that the silver nanoparticles are significantly contributing to the effect
against bacteria. Evaluations in a mouse wound healing model indicated that the designed
lignin-based foams could effectively promote wound healing of a full-thickness skin defect.
Lignin-based materials with a 3D structure were obtained by means of coaxial electro-
spinning resulting in scaffolds for wound dressing application [265]. Scaffolds formed from
core–shell fibers based on a chitin–lignin gel (as core using BioligninTM ) with PCL (as shell).
The presence of a PCL shell layer decreased the dissolution time of the chitin–lignin gel
fiber and provided in this way sustainable release of methylene blue, which was utilized
as model drug. When penicillin and streptomycin (showing bacterial inhibitory effect)
were introduced in the lignin-based gel, a clear inhibition zone against S. aureus and E. coli
bacterial strains could be obtained.
As the properties of lignin-based foams/scaffolds are highly related to the 3D structure
(pore size, density, and wall thicknesses), the processing can determine the characteristics
of the materials [266]. Hence, investigations focusing on the impact of micro-/nano-porous
structure of lignin-based foams on wound healing ability and drug delivery capacity would
be of interest.

4.1.4. Multifunctional Lignin-Based Materials

An additional feature of lignin-based materials for health care applications was de-
scribed for lignin/poly(ionic liquids) composite hydrogels utilized as dressing, which
provided self-healing properties [267]. The mechanical strength of the hydrogel dressing
was effectively improved by the introduction of lignin while the poly(ionic liquid) based on
(3-butyl-1-isopropyl-1H-imidazol-3-ium bromide) enabled good antibacterial activity and
self-healing. As result of supramolecular interactions within the lignin/poly(ionic liquids)
compounds, the dressing could be reused many times also after simple high-temperature
disinfection.
Lignin-based materials showing accelerated wound healing and providing repeatable
adhesiveness to a variety of substrates were described using Ag-lignin (AL was used) NPs
loaded in a poly(vinyl alcohol) (PVA) hydrogel matrix together with cellulose nanocrys-
tals [268]. Here, phenol or methoxy groups in lignin can reduce silver ions to metallic
silver NPs, while functional lignin groups will be oxidized to the corresponding quinone
or hydroquinone. The conversion of these groups into catechol groups is enabled in the
presence of photogenerated electrons (provided by Ag NPs) [269]. The hydrophilic hydroxy
groups of PVA could interact with various surfaces, whereas the generated catechol groups
provide strong adhesion and quinone groups can create physical cross-links with PVA and
enhance cohesion.
A strong adhesion capability was determined for multifunctional hydrogels created
by crosslinking of phenylboronic acid-modified hydroxypropyl cellulose (PAHC) using
Ag-AL NPs (lignin reduced in situ a [Ag(NH3 )2 ]+ complex resulting in Ag-lignin NPs)
(Figure 9) [270]. The introduced particles acted as antibacterial nanostructure and crosslink-
ing agent (catechol of lignin can form dynamic borate ester bonds with PAHC). The phenolic
hydroxy groups endowed lignin with a strong adhesion capability, and the complexation
of lignin and Ag can generate a quinone/catechol structure (dynamic redox environment)
enabling a repeatable adhesion. Therefore, the designed materials could adhere to a variety
of organic (including wound tissue) and inorganic substrates, could close a wound surface,
and could achieve a good hemostasis effect. As the hydrogel dressings included dynamic
borate ester bonds and hydrogen bonding, an excellent self-healing ability could be demon-
 Int.J.J.Mol.
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2023, 24,
24, 11668
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included dynamic
strated. The borate
materials ester bonds
provided andthinning,
a shear hydrogen bonding,
which enabledan excellent
hydrogelself-healing
injection as well
ability could be demonstrated. The materials
as the adaption of the hydrogel system to a mold shape. provided a shear thinning, which enabled
hydrogel injection
A high as wellfor
potential as the
the adaption
applicationof the
in hydrogel system
personalized to a mold was
healthcare shape.addressed by
A high potential for the application in personalized healthcare was addressed by
multifunctional on-chip electrochemical sensors, which were obtained by a laser-scribing
multifunctional on-chip electrochemical sensors, which were obtained by a laser-scribing
process that converts lignin-based (lignosulfonate) precursors into conductive nitrogen-
process that converts lignin-based (lignosulfonate) precursors into conductive nitrogen-
doped graphene patterns [271]. These electrodes were modified with an MXene/Prussian
doped graphene patterns [271]. These electrodes were modified with an MXene/Prussian
blue composite and with catalytic enzymes for selective detection of glucose, lactate, and
blue composite and with catalytic enzymes for selective detection of glucose, lactate, and
alcohol (markers of diabetes and indicator of athletic performance and of alcoholism)
alcohol (markers of diabetes and indicator of athletic performance and of alcoholism) re-
resulting
sulting in enhanced
in an an enhanced electrochemical
electrochemical activity
activity towardtoward the detection
the detection of biomarkers.
of these these biomarkers.
As lignin and lignin-based NPs are of interest for the health
As lignin and lignin-based NPs are of interest for the health care sector and care sector and in addition
in addi-
could act as a platform for the immobilization of different enzymes
tion could act as a platform for the immobilization of different enzymes as presented withas presented with the
example of peroxidases [272], we believe that new types of multi-functional
the example of peroxidases [272], we believe that new types of multi-functional lignin- lignin-based
materials
based will be
materials willdesigned
be designedconsidering
consideringalso enzyme
also enzymesupported
supportedpathways
pathways to to treat
treat skin
diseases
skin or infections
diseases in near
or infections in nearfuture.
future.
Thestrategies
The strategiesofofhow
howdifferent
differentfunctions
functions(self-healing,
(self-healing,adhesiveness,
adhesiveness, shape
shape adaptation,
adapta-
and and
tion, biochemical
biochemical sensing)
sensing)cancanbebe introduced
introduced ininlignin-based
lignin-based materials
materials openopen
up a up
newa new
directionofofhigh
direction highvalue-added
value-added products
products for for health
health carecare applications
applications and hopefully
and will will hopefully
inspireother
inspire otherscientist
scientist
toto utilize
utilize lignin
lignin as source
as source for for multifunctional
multifunctional biomaterials.
biomaterials.

Figure
Figure9.9.(A).
(A).Synthesis
Synthesisof of
multifunctional hydrogels
multifunctional as wound
hydrogels dressing
as wound materials
dressing basedbased
materials on lignin
on lignin
(light green), Ag nanoparticles (blue), and phenylboronic acid-modified hydroxypropyl cellulose
(light green), Ag nanoparticles (blue), and phenylboronic acid-modified hydroxypropyl cellulose
(PAHC, gren). (B). Adhesion on organic and inorganic substrates. (C). Self-healing capability, (D).
(PAHC, gren). (B). Adhesion on organic and inorganic substrates. (C). Self-healing capability,
(D). shape adaptivity, and (E). Demonstration of injectability of the created lignin-based hydrogels.
Adapted with permission from ref. [270]. Copyright 2021, American Chemical Society.
Int. J. Mol. Sci. 2023, 24, 11668 27 of 47

4.2. Supercapacitors Based on Lignin

Supercapacitors are one of the most effective and practical technologies for energy stor-
age. They enable the spanning of the power/energy gap between batteries and conventional
dielectric capacitors by providing a short charge time, high power density, and long cycle
life [273]. The underlying energy storage mechanisms are connected to reversible faradic
reactions or to electrostatic charge accumulation (at the interface of electrolyte/electrode).
Porous carbon materials, carbon based nanotubes, and ordered mesoporous carbon mate-
rials were considered to be the most suitable electrode materials for supercapacitors due
to their high specific surface area, developed pore structure, high electronic conductivity,
and excellent stability [274]. In comparison to other common precursors such as polyani-
line, pitch, and rayon, the most suitable precursor for the design of high-performance
carbon fibers is polyacrylonitrile (PAN), as it provides a high melting temperature (Tm ),
provides high carbon content, and can be pyrolyzed very quickly resulting in fine and
regulated fiber diameters. As disadvantage, PAN and other petroleum-derived polymers
are expensive and release toxic components during a carbonization process [275]. Hence,
lignin as green carbon source for the development of supercapacitors presents a promising
alternative [276].

4.2.1. Lignin as Electrode Material

Lignin fragments are not easily spinnable into fibers as they have a low molar mass. For
this reason, polymers with a high molar mass (PAN, PEG, and PVA) can be added to a lignin
solution, whereby viscosity and spinnability can be improved [274]. Organosolv lignin can
be used for supercapacitor applications as it includes a low ash content resulting in carbons
with very few metal impurities (which have to be removed for long-term cyclability).
A lignin-derived carbon nanofiber (CNF) electrode was created by electrospinning of a
lignin/PEG solution (90 wt% organosoly hardwood lignin) [277]. The obtained CNF mats
were densified by uniaxial compression (between 40 and 120 bar) and were carbonized at
800 ◦ C. The densification occurred by reducing the inter-fiber pore size. The improvement
of packing density was directly proportional to the performances of the created carbon
nanofiber electrode (reaching a volumetric capacitance of 130 F·cm−3 and energy density
of 6 Wh·L−1 at 0.1 A·g−1 ).
Conductivity of lignin-based electrode materials can be improved upon by integra-
tion of polypyrrole particles into the electrode material. Lignin/polypyrrole composite
electrode films were created with microporous and mesoporous structures by means of
electrospinning, carbonization, and in situ polymerization methods [274]. The specific
surface area could be increased (up to 872.60 m2 g−1 ) by carbonization, which induced
the removal of carbonyl and phenolic functional groups of lignin. Afterwards, polypyr-
role particles were added to the lignin nanofibers. The synthesized lignin/polypyrrole
composite anode provided good electrochemical performance with a large specific capac-
itance of 213.7 F·g−1 (at a current density of 1 A·g−1 ) in 1 M H2 SO4 as the electrolyte.
The incorporation of multi-channels in lignin-based CNFs enabled the design of high-
performance energy storage devices with an excellent cycling stability (5% capacitance
decay over 10,000 cycles at 10 A·g−1 ) [278]. Here, CNFs nanocomposites were synthesized
by means of co-electrospinning using poly(pyrrolidone)-SnCl2 ·2 H2 O as a shell material
and lignin/PMMA as core materials. A following heat and acid treatment resulted in a
pore-forming effect of SnCl2 ·2 H2 O and the generation of SnO2 . When the lignin/PMMA
composition was optimized, nanocomposites with hierarchical internal channels, porous
surface, and high specific surface area could be produced. Besides the great cycling stability,
supercapacitor electrodes based on these type of CNFs nanocomposites exhibited high
specific capacitance (406 F·g−1 at current density of 0.5 A·g−1 ).
Instead of electrospinning, a dual template technique can be used for the design of
porous lignin-based materials with supercapacitor application [279]. A hierarchical porous
carbon monolith was fabricated via dual templates (Pluronic P123 and silica NPs) with
a following carbonization procedure. When silica NPs of different sizes and amounts
Int. J. Mol. Sci. 2023, 24, 11668 28 of 47

were utilized, the resulting 3D structure of the carbon monolith could be tuned. For an
optimized porous morphology (using NPs of 7 nm) good electrochemical performance was
realized. The symmetric cell assembling with this type of carbon electrode resulted in a
large amount of energy (131 µWh·cm−2 ) at high power densities (1368 µW·cm−2 ) and good
cycle stability (~93% after 10,000 cycles). Porous lignin-derived carbon quasi-nanosheets
intended for the application as a supercapacitor were designed by a green and facile in
situ carbonization technique [280]. For this purpose, industrial waste LS/zinc oxalate
composites were fabricated from ethanol/water solution, and carbon quasi-nanosheets
could be obtained by a following co-pyrolysis with gas-exfoliation and templating of zinc
oxalate. For such materials, a long cycling stability (93.5% of the original capacitance after
10,000 cycles at 5.0 A·g−1 ) could be realized. An excellent electrochemical performance
(high specific capacitance, excellent rate capability, and high specific energy density) was
shown when the prepared materials were assembled into symmetric supercapacitors in
PVA/KOH gel electrolytes.
As promising method for the design of lignin-derived electrodes, direct laser writing
was introduced [273]. Laser induced graphene was synthesized from KL via direct laser
writing resulting in the formation of a hierarchical structure with a 3D interconnected net-
work. Laser writing in this process photothermally converts KL into few-layered graphene.
Soft electrodes could be fabricated by transferring the created few-layered graphene onto
PDMS. The produced flexible supercapacitors exhibited good electrochemical performance
and good cyclic stability (>90% capacitance was retained after 10,000 cycles). As an advan-
tage of their flexibility, the supercapacitors were able to withstand bending deformations
without significantly losing capacitance.
An important upgrading step of lignin-based materials for energy storage applica-
tion is the physical or chemical activation to enlarge surface area and facilitate efficient
permeation of the electrolyte [281]. Physical activation can be performed with carbon
dioxide, steam, or a combination of them. The creation of pore structures includes a
pore drilling mechanism, which increases the diameter of pores, and a pore deepening
mechanism, which influences the pore depth [282]. While carbon dioxide activation will
result in the formation of micropores, meso- and macropores can be obtained by means
of steam activation [283]. An improved quality consistency and shorter residence time
required for activation can be realized by chemical activation through incorporation of,
e.g., KOH, H3 PO4 , or ZnCl2 in lignin-based materials [281]. As an example, a regular
and well-developed porous network was formed by the incorporation of ZnCl2 , as it can
diffuse throughout the carbon matrix (molten above 290 ◦ C) and can migrate through
the microporous network at high temperatures (boiling point ~730 ◦ C) [284]. The effect
of different chemical activation components, which were impregnated on AL, on porous
structure and resulting electrochemical properties was analyzed [285]. Here, ZnCl2 - and
KOH-activated carbons contained mesoporous structures, while K2 CO3 as activating agent
resulted in the formation of micropores. For the series of activated-carbon materials, a
specific capacitance of 142.09, 251.04, and 263.46 F g−1 for an activation using ZnCl2 , KOH,
and K2 CO3 , respectively, was obtained.
A carbonization step or thermal treatment is not necessary for the design of lignin-
based electrode materials as reported for CNFs with an inter-fiber bonding structure.
Here, CNFs were developed by esterification of organosolv lignin with butyric anhydride
followed by electrospinning with PAN [286]. The resulting nitrogen–oxygen co-doped
CNFs provided a high heteroatom content and a good wettability. Lignin-based CNFs
were utilized as electrode materials, which showed a high specific capacitance (320 F·g−1
at 1 A·g−1 with 6 M aq. KOH as electrolyte). A high coulombic efficiency of 112.5%, good
energy density of 17.92 Wh·kg−1 , and good cycling stability (<6% loss after 5000 cycles)
were obtained when assembled CNFs//CNF symmetric supercapacitors were analyzed.
It has to be noticed that the specific surface area and pore size distribution is determin-
ing the performance of porous carbon materials for energy storage. As lignin provides a
complex molecular structure, the pyrolysis behavior and the design of electrode materials
Int. J. Mol. Sci. 2023, 24, 11668 29 of 47

are only limitedly controllable. In addition, the molar masses and content of impurities are
dependent on the lignin source and type of production, which will influence the physic-
ochemical properties of the final product [287]. Accordingly, a uniform strategy for the
development of lignin-based supercapacitors could not be achieved the inconsistency
of industrial lignin needs to be further considered; and the relationship between lignin
structure/resource and product properties have to be systematically investigated.

4.2.2. Lignin as an Electrolyte Material

As specific functional groups within the lignin structure (benzyl and phenolic groups)
could act as active reaction sites for ions, lignin-based materials are of interest for the design
of electrolytes. In addition, the numerous present oxygen atoms are important for pro-
moting electrolyte ion adsorption and redox reactions [230]. Hence, an approach to utilize
lignin for potential application in energy storage devices is the creation of lignin-based
hydrogels as electrolytes, which are flexible and compression-resistant [288]. A double-
crosslinked lignin hydrogel synthesized by crosslinking lignin and forming hydrophobic
lignin aggregates (obtained when the hydrogel is treated with H2 SO4 solution) was used
to create lignin-based hydrogels serving as electrolyte. In combination with polyaniline-
deposited carbon cloth as the electrode, a high specific capacitance of 190 F·g−1 , excellent
energy density, and good cycling stability were realized. This flexible supercapacitor was
able to retain high specific capacitance after 500 cycles of 180◦ bending or 80% compres-
sion strain. As lignin can be used as electrolyte material and as electrode material, the
supercapacitors based on a lignin-derived hydrogel electrolyte and a lignin-based elec-
trode were investigated [289]. The chemically cross-linked AL hydrogel electrolytes were
synthesized by a base-catalyzed ring-opening polymerization and crosslinking reaction
with poly(ethylene glycol) diglycidyl ether. The obtained hydrogels were flexible, provided
dimensional stability even in a highly swollen state (523% swelling capacity), and had
high ionic conductivity (10.35 mS·cm−1 ). Electrodes were fabricated by electrospinning
using a mixture of lignin with PAN in DMF followed by a stabilization process in air
at 250 ◦ C (formation of an N-containing ladder-type structure). The resulting stabilized
nanofiber mats were carbonized at 900 ◦ C and provided interconnected porous channels.
The created supercapacitor devices (including lignin hydrogel electrolyte and electrospun
lignin/PAN nanofiber electrodes) demonstrated a high capacitance (129.23 F·g−1 ) and
capacitance retention (95% over 10,000 cycles), demonstrated flexibility and durability
under diverse bending angles, and delivered a maximum energy and power density of
4.49 W·h·kg−1 and 2.63 kW·kg−1 , respectively. The mentioned examples of how lignin can
be utilized for energy storage application highlight promising strategies to reduce product
costs and to contribute to the development of more sustainable and greener energy devices.
Unfortunately, the practical application of such lignin-based materials is still greatly limited,
but the research progress is an important step toward green energy technology.

4.3. Lignin-Based Shape-Memory Materials

The next generation of value-added applications is the development of lignin-based
smart materials. Such materials can convert specific external stimuli to defined outputs
and change or activate a functional property of the material [290]. Properties can be
designed to change in a controlled fashion when, for example, temperature, moisture, pH,
or electricity are applied [291]. Shape-memory polymers (SMPs) especially have received
an increasing attention because of their potential applications in biomedical treatment,
sensors, and actuators [292]. These kinds of materials are capable of translating changes
in the environment into a directed geometric movement. The resulting function of the
material is denominated as a shape-memory effect (SME). To activate an SME, a stimulus
as an input signal causes the removal of an internal structural barrier (created during a
programming procedure) and initiates the recovery of the original shape [293,294]. As
an example, the programming process of a material providing shape memory properties
involves a shape deformation (e.g., in an amorphous state of the material), a shape fixation
 in the environment into a directed geometric movement. The resulting function of the
material is denominated as a shape-memory effect (SME). To activate an SME, a stimulus
Int. J. Mol. Sci. 2023, 24, 11668 as an input signal causes the removal of an internal structural barrier (created during 30 a of 47
programming procedure) and initiates the recovery of the original shape [293,294]. As an
example, the programming process of a material providing shape memory properties in-
volves a shape deformation (e.g., in an amorphous state of the material), a shape fixation
(obtained when temporary crosslinks are generated, e.g., when crystallization is induced
(obtained when temporary crosslinks are generated, e.g., when crystallization is induced
by decreasing the temperature), and the release of the external stress (Figure 10). As result,
by decreasing the temperature), and the release of the external stress (Figure 10). As result,
aa higher entropic state is created as polymer chains which will be oriented according to the
higher entropic state is created as polymer chains which will be oriented according to
deformation
the deformation direction (stretched
direction polymer
(stretched polymerchains), andand
chains), thethe
movement
movement of polymer
of polymerchains
back to their random coil formation is blocked by the generated temporary
chains back to their random coil formation is blocked by the generated temporary cross- crosslinks.
Finally, a directed
links. Finally, movement
a directed back back
movement to the tooriginal shape
the original (driven
shape by by
(driven entropy elasticity)
entropy elastic- can
be
ity)realized when a when
can be realized stimulus is applied
a stimulus (e.g., heat).
is applied (e.g., heat).

Figure 10. Shape-memory effect; here shown for a material using crystallization for fixing the
Figure 10. Shape-memory effect; here shown for a material using crystallization for fixing the tem-
temporary shape, though other options exist. The original shape (A) of a material is heated above
porary shape, though other options exist. The original shape (A) of a material is heated above the
the melting
melting temperature
temperature of crystalline
of crystalline domains domains (red,
(red, acting as acting as temporary
temporary netpoints).
netpoints). Then, Then, the
the material
material can be deformed
can be deformed into shapeinto
(B)shape (B) resulting
resulting in the orientation
in the orientation of thechains.
of the polymer polymer chains.
When When the
the tem-
perature is decreased,
temperature crystallization
is decreased, is induced,
crystallization and theand
is induced, temporary shape (C)shape
the temporary can be(C)
obtained
can beafter
obtained
removal
after of external
removal stress.stress.
of external The recovery processprocess
The recovery is initiated by temperature
is initiated increase increase
by temperature enabling enabling
the
polymer chains to regain their random coil formation. As result, a directed movement to the original
the polymer chains to regain their random coil formation. As result, a directed movement to the
shape (D) can be realized.
original shape (D) can be realized.
4.3.1. Temperature-Induced
4.3.1. Temperature-Induced SME
SME
Lignin can
Lignin can be
be utilized
utilized as
as crosslinker
crosslinkerin
inSMPs.
SMPs.Thermosets
Thermosetsbased
basedon onKL
KL(as
(asaacrosslinker),
cross-
linker), PEG (Mn = 400 g·mol -1, creating temporary netpoints), and citric acid (acting as a
PEG (Mn = 400 g·mol−1 , creating temporary netpoints), and citric acid (acting as a crosslinker)
crosslinker) were synthesized by a green one-pot heat curing method, in which only water
were synthesized by a green one-pot heat curing method, in which only water was produced
was produced as byproduct [295]. An increase of the lignin content (from 20 to 40 wt%)
as byproduct [295]. An increase of the lignin content (from 20 to 40 wt%) resulted in increasing
resulted in increasing crosslinking density, which drastically increased the storage mod-
crosslinking density, which drastically increased the storage modulus from 5.7 MPa to 2 GPa
ulus from 5.7 MPa to 2 GPa and the glass transition temperature (Tg) from −0.3 °C to 102
and the glass transition temperature (Tg ) from −0.3 ◦ C to 102 ◦ C. Excellent shape-memory
°C. Excellent shape-memory properties with a shape fixation of 95% and a thermally in- ◦
properties
duced shapewithrecovery
a shape of
fixation
99% at of 80
95%°Cand a thermally
(when 35 wt%induced shape
lignin was recovery
utilized) of 99%
could at 80 C
be ob-
(when 35 wt% lignin was utilized) could be obtained. Instead of PEG, acrylonitrile
tained. Instead of PEG, acrylonitrile butadiene rubber can be crosslinked with organosolv butadiene
rubber can be crosslinked with organosolv hardwood lignin [296]. The thermal annealing at
180 ◦ C results in the creation of reactive sites within lignin promoting crosslinking reactions,
and the Tg of the rubber matrix could be enhanced by 18 ◦ C. Shape-memory experiments
were carried out with a deformation of the lignin-based composites at 100 ◦ C, a fixation
at 0 ◦ C, and the thermally induced recovery at 50 ◦ C and/or 100 ◦ C. In this case, tunable
chemical and physical crosslinks within lignin and the utilized rubber resulted in good shape
programmability and a quick shape recovery. Directed shape shifts utilizing a physiological
relevant temperature range could be realized by means of PUs based on castor oil, LS, and
sophorone diisocyanate [297]. Here, the implementation of LS into the PUs increased the
Int. J. Mol. Sci. 2023, 24, 11668 31 of 47

Tg from 9.7 ◦ C (pure castor oil) to 18.6 ◦ C (3 wt% LS). The designed PU-LS films could be
programmed at 37 ◦ C and showed an excellent SME with a recovery to the original state
within 17 s at 37 ◦ C.
In contrast to utilizing lignin only as crosslinker, it can be incorporated in polymeric
networks to provide temporary netpoints as demonstrated for lignin-based PUs with 100%
biobased carbon content [298]. Here, non-isocyanate PUs were created using a non-toxic
cyclocarbonation scheme with KL. First, unmodified KL was oxyalkylated using glycerol
carbonate. In a subsequent step, the resulting 1,2-diol functionalized macromolecule
underwent a transesterification reaction with dimethyl carbonate resulting in the formation
of cyclocarbonate groups. Finally, the PUs were obtained after the addition of a fatty
acid-based dimer diamine as curing agent. The resulting materials possessed a broad
temperature transition region (related to lignin and to the dimer diamine) between the
glassy and rubbery state (0–100 ◦ C) indicating numerous microstates of the polymeric
material, which could be related to the large range of molar masses within the utilized
lignin (PDI = 4.1). When the PUs were bent at 105 ◦ C and cooled to RT, the deformation
was maintained. The reheating under stress-free conditions initiated the shape-memory
recovery process to the original shape.
As different lignin sources could provide a high difference in functional groups,
material properties will be affected. In this context, it was detected that the miscibility
of lignin with a polyol for PU synthesis correlated with the number of functional groups
(COOH and aliphatic OH) within different types of lignin (softwood KL, organosolv pine
bark lignin, and organosolv oak bark lignin) [299]. It has to be noted that the efficiency
of the crosslinking reaction for PU synthesis depends on the ratio and reactivity between
isocyanate groups and alcohol groups. As result of a lower reactivity of aromatic OH
groups with isocyanates, a less uniform cellular structure in PUs was reported resulting in
lower compressive moduli when lignin with a high content of aromatic OH groups was
used. While softwood KL with a content of aromatic OH groups of 3.75 mmol·g−1 showed
a compressive modulus <0.4 MPa, organosolv oak bark lignin had a lower aromatic OH
content of 3.12 mmol·g−1 and provided a compressive modulus >0.6 MPa. The resulting
PUs exhibited an increase of Tg s by about 20 ◦ C (dependent on the lignin source, Tg s
between 120 and 160 ◦ C were obtained), excellent shape fixation (100%), and good shape
recovery behavior (≥80%). As material properties are also highly dependent on molar mass
and branching degree, the investigation of the structure–property relationship for lignin is
complicated. In this context, a promising strategy using a solvent fractionation of technical
organosolv hardwood lignin was followed to separate lignin molar masses and functional
groups based on different solubilities [300]. Acetone-soluble fractionated lignin provided a
high amount of aliphatic moieties and an aromatic structure with a low branching degree. In
contrast, a higher molar mass was determined for the insoluble lignin fraction. In this way,
lignin functionalities and physical characteristics can be enriched enabling the control of
thermomechanical properties and shape-memory performance in lignin-based multiphase
polymers. The fractionated lignins were reacted with acrylonitrile butadiene rubber in the
melt-phase to generate partially crosslinked elastomers. Elastomers including the soluble
lignin fraction showed enhanced thermal stability (and an increase of Tg by about 19 K
when lignin is added), which was related to strong molecular interactions between lignin
and the rubber. Hence, a less pronounced influence on thermal properties (increase of Tg
by about 4 K) was detected when the insoluble lignin fraction was incorporated in the
rubber matrix. By means of high-resolution SEM measurements, large phase separation and
poor interfacial molecular interactions between the insoluble lignin fraction and the rubber
were detected. In contrast, elastomers including the soluble lignin fraction showed a good
dispersion of small phase-separated lignin domains within the matrix. The shape-memory
performance was affected by the type of integrated lignin. Elastomers with the soluble
lignin fraction demonstrated an excellent shape fixation at ambient temperature (after
programming at 70 ◦ C, 5% strain loss was measured), whereas a strain loss above 20% was
detected for the material including the insoluble lignin component. An almost complete
Int. J. Mol. Sci. 2023, 24, 11668 32 of 47

recovery process of both systems was obtained when the temperature was increased to
150 ◦ C.

4.3.2. Light-Induced SME

The molecular structure of lignin contains a large number of aromatic rings and con-
jugated functional groups. Hence, strong conjugation and π–π molecular interactions
among lignin molecules can be created enabling lignin-based materials with unique optical
properties including aggregation-induced emission, UV absorbance, and great potential
for sustainable photothermal conversion [301]. Copolymerization of enzymatically hy-
drolyzed lignin (EL) with epoxy soybean oil (ESO) and PEG gave materials with increased
tensile strength from 11.3 to 30.8 MPa and Tg (93 ◦ C to 115.7 ◦ C) when increasing the
EL content [302]. In dependence of the EL content, simulated solar irradiation resulted
in an increased surface temperature of the copolymer network as lignin has an excellent
photothermal property (can convert light energy into heat). As result of this indirectly
induced heating process, directed movements of the materials could be obtained within
20 s (for an EL content of 50 wt%) with excellent shape-memory properties (fixation and
recovery >97%). Furthermore, lignin-based castor oil-derived polyamide elastomers were
reported, which provided a stretched-induced crystallization of the polyamide elastomer
(enables the fixation of a macroscopic shape) and a shape shift triggered by indirectly
induced heating processes when near-infrared (NIR)-light was applied [303]. A surface
temperature of 200 ◦ C could be obtained after 5 s of NIR laser irradiation. In addition,
when standard sun irradiation (100 mW·cm−2 ) was utilized, a thermoelectric generator
could be powered. As an application for a light-induced SME for lignin-based materials, an
information encryption device was reported [304], Here, lignin was embedded in a cellu-
lose acetate (providing stimuli-sensitivity) matrix. Lignin was utilized as a photo-thermal
converter and enabled a rapid photo-thermal response. An information carrier could be
realized by a two-step programming procedure at 170 mW/cm2 and 90 mW/cm2 , in which
two different temporary shapes could be stabilized by cooling and alternating the light
source. The encryption was realized stepwise (two recovery processes-triple-shape effect)
by a photo-assisted shape-memory behavior.

4.3.3. Water-Induced SME

Water uptake may trigger shape recovery in SME by cleaving the temporary net-
points, e.g., by acting as softener reducing the Tg . Lignin can be linked to water-responsive
shape memory materials in order to avoid the loss of wet strength during excessive hydra-
tion [305]. For this purpose, a cellulose nanofiber nanocomposite membrane was prepared
including covalently crosslinked PVA and dealkali lignin. The lignin and PVA addition
enabled the regulation of water responsiveness and wet mechanical properties (dependent
on the lignin content, tensile modulus between 60 MPa and 0.7 GPa could be reached in
the swollen state). A recovery of 100% could be obtained within 4 s for a water-induced
SME. In addition, the nanofiber-based nanocomposites including lignin provided excel-
lent UV blocking performance with a transmittance value of the membrane <8%. Water
responsiveness can further be realized using sodium LS as swelling segment. Here, a 3D
porous network with an aligned wall structure was formed in a hydrothermal treatment
of single-wall carbon nanotubes and sodium LS (creating a lignin-based hydrogel) [306].
During a vacuum-assisted filtration process, a dispersion of LS and holey graphene oxide
gradually penetrated the hydrogel. After a second hydrothermal treatment (resulting
in the generation of reduced graphene oxide) and freeze drying, lignin-based ultralight
aerogels with a density of 6.9 mg·cm−3 were obtained. The aerogels exhibited an excellent
shape-memory performance. When the lignin-based aerogel was compressed to 86.2%,
a temporary shape could be obtained as result of a high densification degree. Once, the
aerogel was placed in water at 25 ◦ C, 94% of its initial height could be recovered within
8 s, and a hydrogel was generated. Here, the hydrophilic LS enabled the swelling, and
when enough water filled the pores of the aerogel, the shrunken walls stretched back again
Int. J. Mol. Sci. 2023, 24, 11668 33 of 47

into the aligned walls. The aerogel was utilized as water-driven artificial muscle. The
demonstrator showed a powerful driving force and could lift 1030.6 times its own weight,
which was attributed to the strong aligned wall structure. When the prepared aerogel was
utilized as a pressure sensor, a high sensitivity of about 2.28 kPa−1 and a wide detection
region of 0.27–14.1 kPa could be detected. In addition, the aerogel was assembled into
a symmetric flexible supercapacitor with a cellulose/H2 SO4 hydrogel electrolyte. Here,
excellent stored energy performance that can tolerate 5000 cycles of bending was shown.

4.3.4. Multifunctionality in Lignin-Based Shape-Memory Materials

An additional feature of lignin-based shape-memory materials was reported for a new
kind of PUs based on a polyhydroxyurethane including EL, which were prepared using a
green and environmentally friendly method (isocyanate-, solvent-, and catalyst-free) [307].
The designed material system possessed a dual network structure consisting of a dynamic
covalent network and a hydrogen bonding network. In this case, the lignin-based PUs
provided multifunctional characteristics including reprocessability/recyclability, self-healing
capability (crack width of a scratched film can heal 90% in 10 s), and thermally induced
shape-memory function. The ability of directed movements was attributed to the dynamic
covalent network in PUs and based on thermally triggered trans-carbamoylation exchange
reactions (carried out at 160 ◦ C). Potential applications as smart packaging label fabrication
(for heat-sensitive commodities) and as paper-based electromagnetic shielding materials
(when combined with natural cellulose paper) were demonstrated. Metal–ligand coordination
bonds present an alternative to induce multifunctional characteristics in lignin-based materials
(Figure 11) [308]. An ionomeric elastomer composite including commercial carboxyl elastomer
and a high content of EL provided recyclability, self-repair, and an SME. The addition of
zinc oxide enabled the creation of metal–ligand coordination bonds between the elastomer
(carboxyl group) and the EL (oxygen-bearing groups). As result of the reinforcing effect
of lignin and the generated coordination bonds, excellent mechanical properties could be
obtained (tensile strength up to 19.6 MPa and elongation at break up to 397%). As the
addition of zinc oxide enabled the creation of ionic bonds and ionic aggregates acting as
physical crosslinks, recyclability was realized (mechanical properties could be retained or
were improved after reprocessing cycles). The ionomeric elastomer composites provided good
self-repairable ability. After repairing at 100 ◦ C for 2 h, samples could recover ≥90% of the
initial tensile strength and ≥85% of the initial elongation at break. A thermally induced SME
could be obtained as a third functionality. While the ionic crosslinks (interaction between Zn
ions with the elastomer) acted as permanent netpoints, coordination interaction (elastomer–
Zn–lignin bonds) acted as temperature-sensitive temporary crosslinks. Here, the directed
movement to the original shape could be enabled within 80 s using a temperature of 80 ◦ C
with a fixation and recovery ratio above 85%.
Multifunctional lignin-based materials providing light sensitivity were realized based
on maleic anhydride grafted polyethylene elastomer and AL as an efficient photo-thermal
agent [301]. Electron transitions from low-energy orbitals to high-energy states were
possible by the conjugated structures in lignin resulting in a high photothermal conversion
efficiency of 53.7% (a temperature of about 280 ◦ C was reached under NIR irradiation). The
smart elastomer composites were able to provide excellent light-controlled self-repairing
properties (self-repairing efficiency regarding the tensile strength reached 98.2% within
20 min), a light-triggered shape-memory performance with good fixity and recovery ratios
above 80%, and strong photothermal bactericidal activity. Hence, possible applications as
smart materials for precise remote driving of robots, machines, sensors, sterilization, and
self-repairing equipment were suggested.
Int. Int. J. Mol.
J. Mol. Sci.Sci. 2023,
2023, 24,24, x FOR PEER REVIEW
11668 34 of 34
48 of 47

Figure11.
Figure 11.Multifunctionality
Multifunctionality inin lignin-based
lignin-basedthermoplastic
thermoplasticelastomer
elastomercomposites. Reprinted
composites. withwith
Reprinted
permissionfrom
permission fromref.
ref. [308]
[308] Copyright
Copyright 2022,
2022,American
AmericanChemical
Chemical Society.
Society.

InMultifunctional
summary, lignin lignin-based
has emerged materials providing
as a promising optionlightforsensitivity
the design were realized
of shape-memory
based on maleic anhydride grafted polyethylene elastomer and AL
materials with advanced applications as result of its diversity in functional properties. as an efficient photo-
thermal agent [301]. Electron transitions from low-energy orbitals
Lignin-based materials are not limited to a temperature trigger to initiate directed to high-energy states
move-
were possible by the conjugated structures in lignin resulting in a high
ments; also, water- or light-induced shape shifts could be realized. Hence, SMPs including photothermal con-
version
lignin asefficiency of 53.7% (a
natural resource aretemperature
comparable of in
about 280of
terms °Cversatility
was reached tounder NIR irradi-
conventional smart
ation). The smart elastomer composites were able to provide excellent light-controlled
materials. Although often the stimuli-sensitivity is not directly provided by lignin, lignin
self-repairing properties (self-repairing efficiency regarding the tensile strength reached
significantly contributes to the shape shifting output (e.g., by adjusting Tg to a physio-
98.2% within 20 min), a light-triggered shape-memory performance with good fixity and
logically relevant range or by converting light into heat). Thus, research on lignin-based
recovery ratios above 80%, and strong photothermal bactericidal activity. Hence, possible
materials with a SME and with SME-related applications will continue rapidly. Not all pub-
applications as smart materials for precise remote driving of robots, machines, sensors,
lished work contains sufficient information about the chemical structure of the lignin used.
sterilization, and self-repairing equipment were suggested.
This isIna major
summary, drawback
lignin when trying toasestablish
has emerged structure–property
a promising option for the or structure–function
design of shape-
relationships.
memory materials It is highly recommended
with advanced to authors
applications as resultand reviewers
of its in functional
diversity in this field to ensure
prop-
that such information is always included in published work.
erties. Lignin-based materials are not limited to a temperature trigger to initiate directed
movements; also, water- or light-induced shape shifts could be realized. Hence, SMPs in-
5. Conclusions
cluding lignin as natural resource are comparable in terms of versatility to conventional
Lignin
smart structure
materials. is, due
Although to the
often the variable biosynthesis,
stimuli-sensitivity is notalternative pathways,
directly provided and par-
by lignin,
tially stochastic reaction mechanism, complex. This complexity is at least
lignin significantly contributes to the shape shifting output (e.g., by adjusting Tg to a phys- retained when
extracting
iologically relevant range or by converting light into heat). Thus, research on lignin-based and
it, and extraction processes may add further facets as different parts of wood
different
materialstypes
with of woods,
a SME andwhich may significantly
with SME-related differ in
applications lignin
will composition,
continue are mixed.
rapidly. Not all
The detailed
published firstcontains
work part of this articleinformation
sufficient summarizes lignin
about theoccurrence and biosynthesis
chemical structure of the ligninpath-
used.including
ways, This is a major drawbackand
the synthesis when trying toof
structures establish
canonicalstructure–property
and non-canonical or structure–
monolignols
function
and relationships. It is highly
lignin–polysaccharide hybridrecommended to authors and
structures. Lignin-based reviewershave
materials in this field
been to in
used
ensure that
advanced such information
applications is always
exploiting included
lignin’s in published
complex molecular work.
structure with a variety of
functional groups that show antioxidant and antimicrobial properties. We summarized the
5. Conclusions
latest approaches for its use for health care applications, in energy storage devices, and
Lignin
for the design structure
of smartis,materials
due to thesuch
variable biosynthesis,
as SMPs. alternative
Especially for woundpathways, andand
healing par-drug
tially stochastic reaction mechanism, complex. This complexity is at least retained
delivery applications, much of the performed research is still at the proof-of-concept stage, when
extracting
and it, and
the medical extraction
products processes
have may addby
to be approved further facetsbodies.
regulatory as different parts
As the of wood
lignin structure
and different types of woods, which may significantly differ in lignin composition,
(e.g., molar mass, dispersity, and degree of branching) and its functionality (e.g., content are
of aromatic hydroxy groups and aliphatic hydroxy groups) are highly dependent on the
lignin source and technical process for lignin production, systematic investigations about
chemical composition/macromolecular structure of lignin as function of the different types
Int. J. Mol. Sci. 2023, 24, 11668 35 of 47

in reference to drug delivery and tissue engineering characteristics are essential before
pursuing clinical trials. We believe that the implementation of lignin as a natural source for
more technical related applications such as supercapacitors presents a promising strategy,
which can be translated into real products in the near future. As the electrochemical perfor-
mance of these kind of bio-based energy storage devices depends on pore characteristics,
surface chemistry, and the structural features of the utilized lignin, a uniform strategy for
the development of lignin-based supercapacitors should be considered, and the relationship
between lignin structure/resource and product properties has to be elucidated. It is also of
importance to attract industrial activity for these new materials, to demonstrate the scale-up,
and to better predict batch to batch properties to realize standardized lignin-based products.
Last but not least, there is lots of potential for future work to incorporate multiple stimuli in
smart materials to address switching “on” and “off” a corresponding lignin-based device.
Here, one focus could be how stimuli-sensitivity can be directly provided by lignin to
reduce integration of non-bio-based components in order to increase the green fingerprint.

Author Contributions: Conceptualization, A.T.N. and N.T.; writing—original draft preparation,

M.B., P.S., A.T.N. and N.T.; writing—review and editing, A.T.N. and N.T.; visualization, M.B., P.S.
and N.T.; supervision, A.T.N. and N.T. All authors have read and agreed to the published version of
the manuscript.
Funding: This work is supported by the Helmholtz Association through program-oriented funding
and received funding from the European Union’s Horizon 2020 research and innovation program
under Grant Agreement No. 824074 (GrowBot). The Open University Affiliated Research Centre at
Istituto Italiano di Tecnologia (ARC@IIT) is part of the Open University, Milton Keynes MK7 6AA,
United Kingdom.
Data Availability Statement: Data sharing not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Hodasova, L.; Jablonsky, M.; Skulcova, A.B.; Haz, A. Lignin, potential products and their market value. Wood Res. 2015, 60,
973–986.
2. Bajwa, D.S.; Pourhashem, G.; Ullah, A.H.; Bajwa, S.G. A concise review of current lignin production, applications, products and
their environmental impact. Ind. Crops Prod. 2019, 139, 111526. [CrossRef]
3. Dien, B.S.; Jung, H.J.G.; Vogel, K.P.; Casler, M.D.; Lamb, J.F.S.; Iten, L.; Mitchell, R.B.; Sarath, G. Chemical composition and
response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass Bioenerg.
2006, 30, 880–891. [CrossRef]
4. Burhenne, L.; Messmer, J.; Aicher, T.; Laborie, M.-P. The effect of the biomass components lignin, cellulose and hemicellulose on
TGA and fixed bed pyrolysis. J. Anal. Appl. Pyrolysis 2013, 101, 177–184. [CrossRef]
5. Chen, F.; Dixon, R.A. Lignin modification improves fermentable sugar yields for biofuel production. Nat. Biotechnol. 2007,
25, 759–761. [CrossRef]
6. Halpin, C. Lignin engineering to improve saccharification and digestibility in grasses. Curr. Opin. Biotechnol. 2019, 56, 223–229.
[CrossRef]
7. Mahon, E.L.; Mansfield, S.D. Tailor-made trees: Engineering lignin for ease of processing and tomorrow’s bioeconomy. Curr.
Opin. Biotechnol. 2019, 56, 147–155. [CrossRef]
8. Fache, M.; Boutevin, B.; Caillol, S. Vanillin Production from Lignin and Its Use as a Renewable Chemical. ACS Sustain. Chem. Eng.
2016, 4, 35–46. [CrossRef]
9. Lan, H.-N.; Liu, R.-Y.; Liu, Z.-H.; Li, X.; Li, B.-Z.; Yuan, Y.-J. Biological valorization of lignin to flavonoids. Biotechnol. Adv. 2023,
64, 108107. [CrossRef]
10. Schutyser, W.; Renders, T.; Van den Bosch, S.; Koelewijn, S.F.; Beckham, G.T.; Sels, B.F. Chemicals from lignin: An interplay of
lignocellulose fractionation, depolymerisation, and upgrading. Chem. Soc. Rev. 2018, 47, 852–908. [CrossRef]
11. Deng, W.; Feng, Y.; Fu, J.; Guo, H.; Guo, Y.; Han, B.; Jiang, Z.; Kong, L.; Li, C.; Liu, H.; et al. Catalytic conversion of lignocellulosic
biomass into chemicals and fuels. Green Energy Environ. 2023, 8, 10–114. [CrossRef]
12. Sagues, W.J.; Jain, A.; Brown, D.; Aggarwal, S.; Suarez, A.; Kollman, M.; Park, S.; Argyropoulos, D.S. Are lignin-derived carbon
fibers graphitic enough? Green Chem. 2019, 21, 4253–4265. [CrossRef]
13. Pettersen, R.C. The Chemical Composition of Wood. In The Chemistry of Solid Wood; American Chemical Society: Washington, DC,
USA, 1984; Volume 207, pp. 57–126.
Int. J. Mol. Sci. 2023, 24, 11668 36 of 47

14. Asif, M. 2—Sustainability of timber, wood and bamboo in construction. In Sustainability of Construction Materials; Khatib, J.M.,
Ed.; Woodhead Publishing: Soston, UK, 2009; pp. 31–54. [CrossRef]
15. Rennel, J.; Dillén, S. Pulp and Paper: Wood Sources. In Encyclopedia of Materials: Science and Technology; Buschow, K.H.J., Cahn,
R.W., Flemings, M.C., Ilschner, B., Kramer, E.J., Mahajan, S., Veyssière, P., Eds.; Elsevier: Oxford, UK, 2001; pp. 7913–7917.
[CrossRef]
16. Roger, M.R.; Roger, P.; Mandla, A.T. Cell Wall Chemistry. In Handbook of Wood Chemistry and Wood Composites; CRC Press: Boca
Raton, FL, USA, 2012. [CrossRef]
17. Sjöström, E. (Ed.) Chapter 5—EXTRACTIVES. In Wood Chemistry, 2nd ed.; Academic Press: San Diego, CA, USA, 1993; pp. 90–108.
[CrossRef]
18. Lehr, M.; Miltner, M.; Friedl, A. Removal of wood extractives as pulp (pre-)treatment: A technological review. SN Appl. Sci. 2021,
3, 886. [CrossRef]
19. Kránitz, K.; Sonderegger, W.; Bues, C.-T.; Niemz, P. Effects of aging on wood: A literature review. Wood Sci. Technol. 2016, 50, 7–22.
[CrossRef]
20. Knudsen, K.E.B. Carbohydrate and lignin contents of plant materials used in animal feeding. Anim. Feed Sci. Technol. 1997,
67, 319–338. [CrossRef]
21. Al-Zube, L.A.; Robertson, D.J.; Edwards, J.N.; Sun, W.; Cook, D.D. Measuring the compressive modulus of elasticity of pith-filled
plant stems. Plant Methods 2017, 13, 99. [CrossRef]
22. Dahlen, J.; Jones, P.D.; Seale, R.D.; Shmulsky, R. Sorting lumber by pith and its effect on stiffness and strength in southern pine no.
22 × 4 lumber. Wood Fiber Sci. 2014, 46, 186–194.
23. Morrison, T.A.; Jung, H.G.; Buxton, D.R.; Hatfield, R.D. Cell-wall composition of maize internodes of varying maturity. Crop Sci.
1998, 38, 455–460. [CrossRef]
24. Gominho, J.; Fernandez, J.; Pereira, H. Cynara cardunculus L.—A new fibre crop for pulp and paper production. Ind. Crops Prod.
2001, 13, 1–10. [CrossRef]
25. van Dam, J.E.G.; van den Oever, M.J.A.; Teunissen, W.; Keijsers, E.R.P.; Peralta, A.G. Process for production of high density/high
performance binderless boards from whole coconut husk—Part 1: Lignin as intrinsic thermosetting binder resin. Ind. Crops Prod.
2004, 19, 207–216. [CrossRef]
26. Schneider, R.; Zhang, S.Y.; Swift, D.E.; Begin, J.; Lussier, J.M. Predicting selected wood properties of jack pine following commercial
thinning. Can. J. For. Res. 2008, 38, 2030–2043. [CrossRef]
27. Buyuksari, U.; As, N.; Dundar, T. Mechanical Properties of Earlywood and Latewood Sections of Scots Pine Wood. Bioresources
2017, 12, 4004–4012. [CrossRef]
28. Pratt, R.B.; Tobin, M.; Jacobsen, A.; Traugh, C.; Guzman, M.; Hayes, C.; Toschi, H.; MacKinnon, E.; Percolla, M.; Clem, M.; et al.
Starch storage capacity of sapwood is related to dehydration avoidance during drought. Am. J. Bot. 2020, 108, 91–101. [CrossRef]
29. Park, Y.B.; Cosgrove, D.J. A Revised Architecture of Primary Cell Walls Based on Biomechanical Changes Induced by Substrate-
Specific Endoglucanases. Plant Physiol. 2012, 158, 1933–1943. [CrossRef] [PubMed]
30. Cosgrove, D.; Jarvis, m. Comparative structure and biomechanics of plant primary and secondary cell walls. Front. Plant Sci.
2012, 3, 204. [CrossRef]
31. Grabner, M.; Muller, U.; Gierlinger, N.; Wimmer, R. Effects of heartwood extractives on mechanical properties of larch. Iawa J.
2005, 26, 211–220. [CrossRef]
32. Taylor, A.M.; Gartner, B.L.; Morrell, J.J. Heartwood Formation and Natural Durability—A Review. Wood Fiber Sci. 2002,
34, 587–611.
33. Faleva, A.V.; Belesov, A.V.; Kozhevnikov, A.Y.; Falev, D.I.; Chukhchin, D.G.; Novozhilov, E.V. Analysis of the functional group
composition of the spruce and birch phloem lignin. Int. J. Biol. Macromol. 2021, 166, 913–922. [CrossRef]
34. Lourenco, A.; Rencoret, J.; Chemetova, C.; Gominho, J.; Gutierrez, A.; del Rio, J.C.; Pereira, H. Lignin Composition and Structure
Differs between Xylem, Phloem and Phellem in Quercus suber L. Front. Plant Sci. 2016, 7, 14. [CrossRef]
35. Zamil, M.S.; Geitmann, A. The middle lamella-more than a glue. Phys. Biol. 2017, 14, 015004. [CrossRef]
36. Plomion, C.; Leprovost, G.; Stokes, A. Wood formation in trees. Plant Physiol. 2001, 127, 1513–1523. [CrossRef] [PubMed]
37. Gorshkova, T.; Morvan, C. Secondary cell-wall assembly in flax phloem fibres: Role of galactans. Planta 2006, 223, 149–158.
[CrossRef] [PubMed]
38. Haughn, G.W.; Western, T.L. Arabidopsis seed coat mucilage is a specialized cell wall that can be used as a model for genetic
analysis of plant cell wall structure and function. Front. Plant Sci. 2012, 3, 5. [CrossRef]
39. Heady, R.D.; Cunningham, R.B.; Donnelly, C.F.; Evans, P.D. Morphology of warts in the tracheids of cypress pine (Callitrus Vent.).
IAWA J. 1994, 15, 265–281. [CrossRef]
40. Donaldson, L.A. Lignification and lignin topochemistry—An ultrastructural view. Phytochemistry 2001, 57, 859–873. [CrossRef]
[PubMed]
41. Del Rio, J.C.; Marques, G.; Rencoret, J.; Martinez, A.T.; Gutierrez, A. Occurrence of naturally acetylated lignin units. J. Agric. Food
Chem. 2007, 55, 5461–5468. [CrossRef]
42. Del Rio, J.C.; Rencoret, J.; Marques, G.; Gutierrez, A.; Ibarra, D.; Santos, J.I.; Jimenez-Barbero, J.; Zhang, L.M.; Martinez, A.T.
Highly Acylated (Acetylated and/or p-Coumaroylated) Native Lignins from Diverse Herbaceous Plants. J. Agric. Food Chem.
2008, 56, 9525–9534. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 37 of 47

43. Ralph, J. Hydroxycinnamates in lignification. Phytochem. Rev. 2010, 9, 65–83. [CrossRef]

44. Ando, D.; Lu, F.C.; Kim, H.; Eugene, A.; Tobimatsu, Y.; Vanholme, R.; Elder, T.J.; Boerjan, W.; Ralph, J. Incorporation of catechyl
monomers into lignins: Lignification from the non-phenolic end via Diels-Alder cycloaddition? Green Chem. 2021, 23, 8995–9013.
[CrossRef]
45. Sewalt, V.J.H.; Ni, W.; Blount, J.W.; Jung, H.G.; Masoud, S.A.; Howles, P.A.; Lamb, C.; Dixon, R.A. Reduced lignin content and
altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalanine ammonia-lyase or cinnamate
4-hydroxylase. Plant Physiol. 1997, 115, 41–50. [CrossRef]
46. Kim, H.; Ralph, J.; Lu, F.C.; Ralph, S.A.; Boudet, A.M.; MacKay, J.J.; Sederoff, R.R.; Ito, T.; Kawai, S.; Ohashi, H.; et al. NMR
analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into
lignins. Org. Biomol. Chem. 2003, 1, 268–281. [CrossRef] [PubMed]
47. Lapierre, C.; Pollet, B.; MacKay, J.J.; Sederoff, R.R. Lignin structure in a mutant pine deficient in cinnamyl alcohol dehydrogenase.
J. Agric. Food Chem. 2000, 48, 2326–2331. [CrossRef] [PubMed]
48. Reddy, M.S.S.; Chen, F.; Shadle, G.; Jackson, L.; Aljoe, H.; Dixon, R.A. Targeted down-regulation of cytochrome P450 enzymes for
forage quality improvement in alfalfa (Medicago sativa L.). Proc. Natl. Acad. Sci. USA 2005, 102, 16573–16578. [CrossRef] [PubMed]
49. Franke, R.; Humphreys, J.M.; Hemm, M.R.; Denault, J.W.; Ruegger, M.O.; Cusumano, J.C.; Chapple, C. The Arabidopsis REF8
gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J. 2002, 30, 33–45. [CrossRef]
50. Kim, H.; Li, Q.; Karlen, S.D.; Smith, R.A.; Shi, R.; Liu, J.; Yang, C.; Tunlaya-Anukit, S.; Wang, J.P.; Chang, H.-M.; et al. Monolignol
Benzoates Incorporate into the Lignin of Transgenic Populus trichocarpa Depleted in C3H and C4H. ACS Sustain. Chem. Eng.
2020, 8, 3644–3654. [CrossRef]
51. Do, C.-T.; Pollet, B.; Thevenin, J.; Sibout, R.; Denoue, D.; Barriere, Y.; Lapierre, C.; Jouanin, L. Both caffeoyl Coenzyme A
3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and
sinapoyl malate biosynthesis in Arabidopsis. Planta 2007, 226, 1117–1129. [CrossRef]
52. Ralph, J.; Lapierre, C.; Marita, J.M.; Kim, H.; Lu, F.C.; Hatfield, R.D.; Ralph, S.; Chapple, C.; Franke, R.; Hemm, M.R.; et al.
Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry 2001, 57, 993–1003.
[CrossRef]
53. Marita, J.M.; Ralph, J.; Hatfield, R.D.; Guo, D.J.; Chen, F.; Dixon, R.A. Structural and compositional modifications in lignin
of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase.
Phytochemistry 2003, 62, 53–65. [CrossRef]
54. Weng, J.K.; Mo, H.P.; Chapple, C. Over-expression of F5H in COMT-deficient Arabidopsis leads to enrichment of an unusual
lignin and disruption of pollen wall formation. Plant J. 2010, 64, 898–911. [CrossRef]
55. Chen, F.; Tobimatsu, Y.; Havkin-Frenkel, D.; Dixon, R.A.; Ralph, J. A polymer of caffeyl alcohol in plant seeds. Proc. Natl. Acad.
Sci. USA 2012, 109, 1772–1777. [CrossRef]
56. Chen, F.; Tobimatsu, Y.; Jackson, L.; Nakashima, J.; Ralph, J.; Dixon, R.A. Novel seed coat lignins in the Cactaceae: Structure,
distribution and implications for the evolution of lignin diversity. Plant J. 2013, 73, 201–211. [CrossRef] [PubMed]
57. Vanholme, R.; Storme, V.; Vanholme, B.; Sundin, L.; Christensen, J.H.; Goeminne, G.; Halpin, C.; Rohde, A.; Morreel, K.; Boerjan,
W. A Systems Biology View of Responses to Lignin Biosynthesis Perturbations in Arabidopsis. Plant Cell 2012, 24, 3506–3529.
[CrossRef]
58. Stewart, J.J.; Akiyama, T.; Chapple, C.; Ralph, J.; Mansfield, S.D. The Effects on Lignin Structure of Overexpression of Ferulate
5-Hydroxylase in Hybrid Poplar. Plant Physiol. 2009, 150, 621–635. [CrossRef] [PubMed]
59. Vanholme, R.; Cesarino, I.; Rataj, K.; Xiao, Y.; Sundin, L.; Goeminne, G.; Kim, H.; Cross, J.; Morreel, K.; Araujo, P.; et al.
Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis. Science 2013, 341, 1103–1106.
[CrossRef] [PubMed]
60. Smith, R.A.; Cass, C.L.; Mazaheri, M.; Sekhon, R.S.; Heckwolf, M.; Kaeppler, H.; de Leon, N.; Mansfield, S.D.; Kaeppler, S.M.;
Sedbrook, J.C.; et al. Suppression of cinnamoyl-CoA reductase increases the level of monolignol ferulates incorporated into maize
lignins. Biotechnol. Biofuels 2017, 10, 109. [CrossRef]
61. Petrik, D.L.; Karlen, S.D.; Cass, C.L.; Padmakshan, D.; Lu, F.; Liu, S.; Le Bris, P.; Antelme, S.; Santoro, N.; Wilkerson, C.G.;
et al. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium
distachyon. Plant J. 2014, 77, 713–726. [CrossRef]
62. Lu, F.; Ralph, J. Novel tetrahydrofuran structures derived from beta-beta-coupling reactions involving sinapyl acetate in Kenaf
lignins. Org. Biomol. Chem. 2008, 6, 3681–3694. [CrossRef]
63. Song, K.N.; Li, M.C.; Yang, Y.Q.; Zhang, Z.; Zhu, Q.; Liu, J.Y.; Wang, A.D. Natural flavonolignans as potential therapeutic agents
against common diseases. J. Pharm. Pharmacol. 2022, 74, 337–350. [CrossRef]
64. Pilkington, L.I.; Barker, D. Synthesis and biology of 1,4-benzodioxane lignan natural products. Nat. Prod. Rep. 2015, 32, 1369–1388.
[CrossRef]
65. Ralph, J.; MacKay, J.J.; Hatfield, R.D.; Omalley, D.M.; Whetten, R.W.; Sederoff, R.R. Abnormal lignin in a loblolly pine mutant.
Science 1997, 277, 235–239. [CrossRef]
66. del Río, J.C.; Rencoret, J.; Gutiérrez, A.; Elder, T.; Kim, H.; Ralph, J. Lignin Monomers from beyond the Canonical Monolignol
Biosynthetic Pathway: Another Brick in the Wall. ACS Sustain. Chem. Eng. 2020, 8, 4997–5012. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 38 of 47

67. Tropf, S.; Lanz, T.; Rensing, S.A.; Schroeder, J.; Schroeder, G. Evidence that stilbene synthases have developed from chalcone
synthases several times in the course of evolution. J. Mol. Evol. 1994, 38, 610–618. [CrossRef]
68. Eudes, A.; Dutta, T.; Deng, K.; Jacquet, N.; Sinha, A.; Benites, V.T.; Baidoo, E.E.K.; Richel, A.; Sattler, S.E.; Northen, T.R.; et al.
SbCOMT (Bmr12) is involved in the biosynthesis of tricin-lignin in sorghum. PLoS ONE 2017, 12, e0178160. [CrossRef] [PubMed]
69. Onkokesung, N.; Gaquerel, E.; Kotkar, H.; Kaur, H.; Baldwin, I.T.; Galis, I. MYB8 Controls Inducible Phenolamide Levels by
Activating Three Novel Hydroxycinnamoyl-Coenzyme A:Polyamine Transferases in Nicotiana attenuata. Plant Physiol. 2012,
158, 389–407. [CrossRef]
70. Lan, W.; Lu, F.C.; Regner, M.; Zhu, Y.M.; Rencoret, J.; Ralph, S.A.; Zakai, U.I.; Morreel, K.; Boerjan, W.; Ralph, J. Tricin, a Flavonoid
Monomer in Monocot Lignification. Plant Physiol. 2015, 167, 1284–1295. [CrossRef] [PubMed]
71. del Rio, J.C.; Rencoret, J.; Gutierrez, A.; Kim, H.; Ralph, J. Hydroxystilbenes Are Monomers in Palm Fruit Endocarp Lignins. Plant
Physiol. 2017, 174, 2072–2082. [CrossRef] [PubMed]
72. Rencoret, J.; Neiva, D.; Marques, G.; Gutierrez, A.; Kim, H.; Gominho, J.; Pereira, H.; Ralph, J.; del Rio, J.C. Hydroxystilbene
Glucosides Are Incorporated into Norway Spruce Bark Lignin. Plant Physiol. 2019, 180, 1310–1321. [CrossRef]
73. Zhou, J.M.; Ibrahim, R.K. Tricin-a potential multifunctional nutraceutical. Phytochem. Rev. 2010, 9, 413–424. [CrossRef]
74. Bouaziz, M.; Veitch, N.C.; Grayer, R.J.; Simmonds, M.S.J.; Damak, M. Flavonolignans from Hyparrhenia hirta. Phytochemistry
2002, 60, 515–520. [CrossRef]
75. You, T.T.; Mao, J.Z.; Yuan, T.Q.; Wen, J.L.; Xu, F. Structural Elucidation of the Lignins from Stems and Foliage of Arundo donax
Linn. J. Agric. Food Chem. 2013, 61, 5361–5370. [CrossRef]
76. Duarte-Almeida, J.M.; Negri, G.; Salatino, A.; de Carvalho, J.E.; Lajolo, F.M. Antiproliferative and antioxidant activities of a tricin
acylated glycoside from sugarcane (Saccharum officinarum) juice. Phytochemistry 2007, 68, 1165–1171. [CrossRef]
77. Rencoret, J.; Rosado, M.J.; Kim, H.; Timokhin, V.I.; Gutierrez, A.; Bausch, F.; Rosenau, T.; Potthast, A.; Ralph, J.; del Rio, J.C.
Flavonoids naringenin chalcone, naringenin, dihydrotricin, and tricin are lignin monomers in papyrus. Plant Physiol. 2022,
188, 208–219. [CrossRef] [PubMed]
78. Wen, J.L.; Sun, S.L.; Xue, B.L.; Sun, R.C. Quantitative structural characterization of the lignins from the stem and pith of bamboo
(Phyllostachys pubescens). Holzforschung 2013, 67, 613–627. [CrossRef]
79. Kwon, Y.S.; Kim, C.M. Antioxidant constituents from the stem of Sorghum bicolor. Arch. Pharmacal Res. 2003, 26, 535–539.
[CrossRef] [PubMed]
80. Potter, G.A.; Patterson, L.H.; Wanogho, E.; Perry, P.J.; Butler, P.C.; Ijaz, T.; Ruparelia, K.C.; Lamb, J.H.; Farmer, P.B.; Stanley, L.A.;
et al. The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme
CYPIBI. Br. J. Cancer 2002, 86, 774–778. [CrossRef]
81. Lu, F.; Ralph, J. Derivatization followed by reductive cleavage (DFRC method), a new method for lignin analysis: Protocol for
analysis of DFRC monomers. J. Agric. Food Chem. 1997, 45, 2590–2592. [CrossRef]
82. Kobayashi, M.; Mahmud, T.; Yoshioka, N.; Hori, K.; Shibuya, H.; Kitagawa, I. Indonesian medicinal plants Part.18. Kompasinol A,
a new stilbeno-phenylpropanoid from the bark of Koompassia malaccensis (Fabaceae). Chem. Pharm. Bull. 1996, 44, 2249–2253.
[CrossRef]
83. Lee, D.; Cuendet, M.; Vigo, J.S.; Graham, J.G.; Cabieses, F.; Fong, H.H.S.; Pezzuto, J.M.; Kinghorn, A.D. A novel cyclooxygenase-
inhibitory stilbenolignan from the seeds of Aiphanes aculeata. Org. Lett. 2001, 3, 2169–2171. [CrossRef]
84. Negrel, J.; Jeandet, P. Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum. Phytochemistry
1987, 26, 2185–2190. [CrossRef]
85. Del Rio, J.C.; Rencoret, J.; Gutierrez, A.; Kim, H.; Ralph, J. Structural Characterization of Lignin from Maize (Zea mays L.) Fibers:
Evidence for Diferuloylputrescine Incorporated into the Lignin Polymer in Maize Kernels. J. Agric. Food Chem. 2018, 66, 4402–4413.
[CrossRef]
86. Benaimeche, O.; Seghir, N.T.; Sadowski, Ł.; Mellas, M. The Utilization of Vegetable Fibers in Cementitious Materials. In
Encyclopedia of Renewable and Sustainable Materials; Hashmi, S., Choudhury, I.A., Eds.; Elsevier: Oxford, UK, 2020; pp. 649–662.
[CrossRef]
87. Chen, G.-G.; Qi, X.-M.; Guan, Y.; Peng, F.; Yao, C.-L.; Sun, R.-C. High Strength Hemicellulose-Based Nanocomposite Film for
Food Packaging Applications. ACS Sustain. Chem. Eng. 2016, 4, 1985–1993. [CrossRef]
88. Dietrich, K.; Hernandez-Mejia, C.; Verschuren, P.; Rothenberg, G.; Shiju, N.R. One-Pot Selective Conversion of Hemicellulose to
Xylitol. Org. Process Res. Dev. 2017, 21, 165–170. [CrossRef]
89. Abu Ghalia, M.; Dahman, Y. 15—Synthesis and utilization of natural fiber-reinforced poly (lactic acid) bionanocomposites. In
Lignocellulosic Fibre and Biomass-Based Composite Materials; Jawaid, M., Md Tahir, P., Saba, N., Eds.; Woodhead Publishing: Soston,
UK, 2017; pp. 313–345. [CrossRef]
90. Scheller, H.V.; Ulvskov, P. Hemicelluloses. Annu. Rev. Plant Biol. 2010, 61, 263–289. [CrossRef] [PubMed]
91. Berglund, J.; Mikkelsen, D.; Flanagan, B.M.; Dhital, S.; Gaunitz, S.; Henriksson, G.; Lindstrom, M.E.; Yakubov, G.E.; Gidley, M.J.;
Vilaplana, F. Wood hemicelluloses exert distinct biomechanical contributions to cellulose fibrillar networks. Nat. Commun. 2020,
11, 16. [CrossRef]
92. Xiong, G.; Cheng, K.; Pauly, M. Xylan O-acetylation impacts xylem development and enzymatic recalcitrance as indicated by the
Arabidopsis mutant tbl29. Mol. Plant 2013, 6, 1373–1375. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 39 of 47

93. Ishii, T.; Tobita, T. Structural characterization of feruloyl oligosaccharides from spinach-leaf cell walls. Carbohydr. Res. 1993,
248, 179–190. [CrossRef]
94. Levigne, S.V.; Ralet, M.C.J.; Quemener, B.C.; Pollet, B.N.L.; Lapierre, C.; Thibault, J.F.J. Isolation from sugar beet cell walls of
Arabinan oligosaccharides esterified by two ferulic acid monomers. Plant Physiol. 2004, 134, 1173–1180. [CrossRef]
95. Chiniquy, D.; Sharma, V.; Schultink, A.; Baidoo, E.E.; Rautengarten, C.; Cheng, K.; Carroll, A.; Ulvskov, P.; Harholt, J.; Keasling,
J.D.; et al. XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan. Proc. Natl. Acad. Sci. USA 2012,
109, 17117–17122. [CrossRef]
96. Willats, W.G.T.; Orfila, C.; Limberg, G.; Buchholt, H.C.; van Alebeek, G.; Voragen, A.G.J.; Marcus, S.E.; Christensen, T.; Mikkelsen,
J.D.; Murray, B.S.; et al. Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell
walls—Implications for pectin methyl esterase action, matrix properties, and cell adhesion. J. Biol. Chem. 2001, 276, 19404–19413.
[CrossRef]
97. Zandleven, J.; Sorensen, S.O.; Harholt, J.; Beldman, G.; Schols, H.A.; Scheller, H.V.; Voragen, A.J. Xylogalacturonan exists in cell
walls from various tissues of Arabidopsis thaliana. Phytochemistry 2007, 68, 1219–1226. [CrossRef]
98. Sowinski, E.E.; Gilbert, S.; Lam, E.; Carpita, N.C. Linkage structure of cell-wall polysaccharides from three duckweed species.
Carbohydr. Polym. 2019, 223, 115119. [CrossRef] [PubMed]
99. Pfeifer, L.; van Erven, G.; Sinclair, E.A.; Duarte, C.M.; Kabel, M.A.; Classen, B. Profiling the cell walls of seagrasses from A
(Amphibolis) to Z (Zostera). BMC Plant Biol. 2022, 22, 63. [CrossRef] [PubMed]
100. Atmodjo, M.A.; Hao, Z.; Mohnen, D. Evolving Views of Pectin Biosynthesis. Annu. Rev. Plant Biol. 2013, 64, 747–779. [CrossRef]
[PubMed]
101. Mohnen, D. Pectin structure and biosynthesis. Curr. Opin. Plant Biol. 2008, 11, 266–277. [CrossRef]
102. Avci, U.; Pena, M.J.; O’Neill, M.A. Changes in the abundance of cell wall apiogalacturonan and xylogalacturonan and conservation
of rhamnogalacturonan II structure during the diversification of the Lemnoideae. Planta 2018, 247, 953–971. [CrossRef]
103. Hart, D.A.; Kindel, P.K. A novel reaction involved in the degradation of apiogalacturonans from Lemna minor and the isolation
of apibiose as a product. Biochemistry 1970, 9, 2190–2196. [CrossRef]
104. Zablackis, E.; Huang, J.; Muller, B.; Darvill, A.G.; Albersheim, P. Characterization of the cell-wall polysaccharides of Arabidopsis
thaliana leaves. Plant Physiol. 1995, 107, 1129–1138. [CrossRef]
105. Sengkhamparn, N.; Verhoef, R.; Schols, H.A.; Sajjaanantakul, T.; Voragen, A.G.J. Characterisation of cell wall polysaccharides
from okra (Abelmoschus esculentus (L.) Moench). Carbohydr. Res. 2009, 344, 1824–1832. [CrossRef]
106. Pena, M.J.; Carpita, N.C. Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm
texture and cell separation during prolonged storage of apple. Plant Physiol. 2004, 135, 1305–1313. [CrossRef]
107. Powell, D.A.; Morris, E.R.; Gidley, M.J.; Rees, D.A. Conformations and interactions of pectins. II. Influences of residue sequence
on chain association in calcium pectate gels. J. Mol. Biol. 1982, 155, 517–531. [CrossRef]
108. O’Neill, M.A.; Eberhard, S.; Albersheim, P.; Darvill, A.G. Requirement of borate cross-linking of cell wall rhamnogalacturonan II
for Arabidopsis growth. Science 2001, 294, 846–849. [CrossRef] [PubMed]
109. O’Neill, M.A.; Warrenfeltz, D.; Kates, K.; Pellerin, P.; Doco, T.; Darvill, A.G.; Albersheim, P. Rhamnogalacturonan-II, a pectic
polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. In vitro conditions
for the formation and hydrolysis of the dimer. J. Biol. Chem. 1996, 271, 22923–22930. [CrossRef] [PubMed]
110. Ishii, T.; Matsunaga, T. Pectic polysaccharide rhamnogalacturonan II is covalently linked to homogalacturonan. Phytochemistry
2001, 57, 969–974. [CrossRef] [PubMed]
111. Ralet, M.-C.; Thibault, J.-F.; Faulds, C.B.; Williamson, G. Isolation and purification of feruloylated oligosaccharides from cell walls
of sugar-beet pulp. Carbohydr. Res. 1994, 263, 227–241. [CrossRef]
112. Colquhoun, I.J.; Ralet, M.C.; Thibault, J.F.; Faulds, C.B.; Williamson, G. Structure identification of feruloylated oligosaccharides
from sugar-beet pulp by nmr-spectroscopy. Carbohydr. Res. 1994, 263, 243–256. [CrossRef]
113. Buchholt, H.C.; Christensen, T.; Fallesen, B.; Ralet, M.C.; Thibault, J.F. Preparation and properties of enzymatically and chemically
modified sugar beet pectins. Carbohydr. Polym. 2004, 58, 149–161. [CrossRef]
114. Ralph, J.; Grabber, J.H.; Hatfield, R.D. Lignin-ferulate cross-links in grasses—Active incorporation of ferulate polysaccharide
esters into ryegrass lignins. Carbohydr. Res. 1995, 275, 167–178. [CrossRef]
115. Harris, P.J.; Hartley, R.D. Detection of bound ferulic acid in cell-walls of gramineae by ultraviolet fluorescence microscopy. Nature
1976, 259, 508–510. [CrossRef]
116. Fry, S.C. Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach
polysaccharide. Biochem. J. 1982, 203, 493–504. [CrossRef]
117. Bunzel, M.; Ralph, J.; Marita, J.M.; Hatfield, R.D.; Steinhart, H. Diferulates as structural components in soluble and insoluble
cereal dietary fibre. J. Sci. Food Agric. 2001, 81, 653–660. [CrossRef]
118. Hatfield, R.D.; Ralph, J.; Grabber, J.H. Cell wall cross-linking by ferulates and diferulates in grasses. J. Sci. Food Agric. 1999,
79, 403–407. [CrossRef]
119. Ishii, T. Structure and functions of feruloylated polysaccharides. Plant Sci. 1997, 127, 111–127. [CrossRef]
120. MacAdam, J.W.; Grabber, J.H. Relationship of growth cessation with the formation of diferulate cross-links and p-coumaroylated
lignins in tall fescue leaf blades. Planta 2002, 215, 785–793. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 11668 40 of 47

121. Grabber, J.H. How do lignin composition, structure, and cross-linking affect degradability? A review of cell wall model studies.
Crop Sci. 2005, 45, 820–831. [CrossRef]
122. Ralph, J.; Hatfield, R.D.; Grabber, J.H.; Jung, H.-J.G.; Quideau, S.; Helm, R.F. Cell Wall Cross-Linking in Grasses by Ferulates and
Diferulates. In Lignin and Lignan Biosynthesis; American Chemical Society: Washington, DC, USA, 1998; Volume 697, pp. 209–236.
123. Aminzadeh, S.; Zhang, L.; Henriksson, G. A possible explanation for the structural inhomogeneity of lignin in LCC networks.
Wood Sci. Technol. 2017, 51, 1365–1376. [CrossRef]
124. Buanafina, M.M.d.O.; Langdon, T.; Hauck, B.; Dalton, S.; Morris, P. Expression of a fungal ferulic acid esterase increases cell wall
digestibility of tall fescue (Festuca arundinacea). Plant Biotechnol. J. 2008, 6, 264–280. [CrossRef]
125. Grabber, J.H.; Ralph, J.; Lapierre, C.; Barriere, Y. Genetic and molecular basis of grass cell-wall degradability. I. Lignin-cell wall
matrix interactions. Comptes Rendus Biol. 2004, 327, 455–465. [CrossRef]
126. Hartley, R.D.; Morrison, W.H.; Himmelsbach, D.S.; Borneman, W.S. Cross-linking of cell wall phenolic arabinoxylans in gramina-
ceous plants. Phytochemistry 1990, 29, 3705–3709. [CrossRef]
127. Jung, H.-J.G. Maize stem tissues: Ferulate deposition in developing internode cell walls. Phytochemistry 2003, 63, 543–549.
[CrossRef]
128. Lam, P.Y.; Tobimatsu, Y.; Takeda, Y.; Suzuki, S.; Yamamura, M.; Umezawa, T.; Lo, C. Disrupting Flavone Synthase II Alters Lignin
and Improves Biomass Digestibility. Plant Physiol. 2017, 174, 972–985. [CrossRef]
129. Xin, A.; Herburger, K. Mini Review: Transport of Hydrophobic Polymers Into the Plant Apoplast. Front. Plant Sci. 2021, 11, 590990.
[CrossRef] [PubMed]
130. Vermaas, J.V.; Dixon, R.A.; Chen, F.; Mansfield, S.D.; Boerjan, W.; Ralph, J.; Crowley, M.F.; Beckham, G.T. Passive membrane
transport of lignin-related compounds. Proc. Natl. Acad. Sci. USA 2019, 116, 23117–23123. [CrossRef] [PubMed]
131. Tsuyama, T.; Matsushita, Y.; Fukushima, K.; Takabe, K.; Yazaki, K.; Kamei, I. Proton Gradient-Dependent Transport of p-
Glucocoumaryl Alcohol in Differentiating Xylem of Woody Plants. Sci. Rep. 2019, 9, 8900. [CrossRef]
132. Miao, Y.-C.; Liu, C.-J. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and
vacuolar membranes. Proc. Natl. Acad. Sci. USA 2010, 107, 22728–22733. [CrossRef] [PubMed]
133. Alejandro, S.; Lee, Y.; Tohge, T.; Sudre, D.; Osorio, S.; Park, J.; Bovet, L.; Lee, Y.; Geldner, N.; Fernie, A.R.; et al. AtABCG29 Is a
Monolignol Transporter Involved in Lignin Biosynthesis. Curr. Biol. 2012, 22, 1207–1212. [CrossRef]
134. Wong, D.W.S. Structure and Action Mechanism of Ligninolytic Enzymes. Appl. Biochem. Biotechnol. 2009, 157, 174–209. [CrossRef]
135. Martinez, A.T. Molecular biology and structure-function of lignin- degrading heme peroxidases. Enzym. Microb. Technol. 2002,
30, 425–444. [CrossRef]
136. Smirnoff, N.; Arnaud, D. Hydrogen peroxide metabolism and functions in plants. New Phytol. 2019, 221, 1197–1214. [CrossRef]
137. Barros, J.; Escamilla-Trevino, L.; Song, L.H.; Rao, X.L.; Serrani-Yarce, J.C.; Palacios, M.D.; Engle, N.; Choudhury, F.K.; Tschaplinski,
T.J.; Venables, B.J.; et al. 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. Nat.
Commun. 2019, 10, 11. [CrossRef]
138. Gao, W.; Li, H.-Y.; Xiao, S.; Chye, M.-L. Acyl-CoA-binding protein 2 binds lysophospholipase 2 and lysoPC to promote tolerance
to cadmium-induced oxidative stress in transgenic Arabidopsis. Plant J. 2010, 62, 989–1003. [CrossRef]
139. Polle, A.; Otter, T.; Seifert, F. Apoplastic peroxidases and lignification in needles of Norway spruce (Picea abies L.). Plant Physiol.
1994, 106, 53–60. [CrossRef] [PubMed]
140. Kawasaki, T.; Koita, H.; Nakatsubo, T.; Hasegawa, K.; Wakabayashi, K.; Takahashi, H.; Urnemura, K.; Urnezawa, T.; Shimamoto,
K. Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice.
Proc. Natl. Acad. Sci. USA 2006, 103, 230–235. [CrossRef]
141. Turlapati, P.V.; Kim, K.-W.; Davin, L.B.; Lewis, N.G. The laccase multigene family in Arabidopsis thaliana: Towards addressing the
mystery of their gene function(s). Planta 2011, 233, 439–470. [CrossRef] [PubMed]
142. Duroux, L.; Welinder, K.G. The peroxidase gene family in plants: A phylogenetic overview. J. Mol. Evol. 2003, 57, 397–407.
[CrossRef] [PubMed]
143. Herrero, J.; Esteban-Carrasco, A.; Miguel Zapata, J. Looking for Arabidopsis thaliana peroxidases involved in lignin biosynthesis.
Plant Physiol. Biochem. 2013, 67, 77–86. [CrossRef] [PubMed]
144. Lee, Y.; Rubio, M.C.; Alassimone, J.; Geldner, N. A Mechanism for Localized Lignin Deposition in the Endodermis. Cell 2013,
153, 402–412. [CrossRef] [PubMed]
145. Chou, E.Y.; Schuetz, M.; Hoffmann, N.; Watanabe, Y.; Sibout, R.; Samuels, A.L. Distribution, mobility, and anchoring of
lignin-related oxidative enzymes in Arabidopsis secondary cell walls. J. Exp. Bot. 2018, 69, 1849–1859. [CrossRef] [PubMed]
146. Hoffmann, N.; Benske, A.; Betz, H.; Schuetz, M.; Samuels, A.L. Laccases and Peroxidases Co-Localize in Lignified Secondary Cell
Walls throughout Stem Development. Plant Physiol. 2020, 184, 806–822. [CrossRef]
147. Zhao, Q.; Nakashima, J.; Chen, F.; Yin, Y.; Fu, C.; Yun, J.; Shao, H.; Wang, X.; Wang, Z.Y.; Dixon, R.A. Laccase is necessary
and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis. Plant Cell 2013,
25, 3976–3987. [CrossRef]
148. Berthet, S.; Demont-Caulet, N.; Pollet, B.; Bidzinski, P.; Cezard, L.; Le Bris, P.; Borrega, N.; Herve, J.; Blondet, E.; Balzergue, S.;
et al. Disruption of LACCASE4 and 17 Results in Tissue-Specific Alterations to Lignification of Arabidopsis thaliana Stems. Plant
Cell 2011, 23, 1124–1137. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 41 of 47

149. Fernandez-Perez, F.; Pomar, F.; Pedreno, M.A.; Novo-Uzal, E. Suppression of Arabidopsis peroxidase 72 alters cell wall and
phenylpropanoid metabolism. Plant Sci. 2015, 239, 192–199. [CrossRef] [PubMed]
150. Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P.F.; Marita, J.M.; Hatfield, R.D.; Ralph, S.A.; Christensen, J.H.; et al.
Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids. Phytochem. Rev. 2004, 3, 29–60. [CrossRef]
151. Vanholme, R.; De Meester, B.; Ralph, J.; Boerjan, W. Lignin biosynthesis and its integration into metabolism. Curr. Opin. Biotechnol.
2019, 56, 230–239. [CrossRef] [PubMed]
152. Demont-Caulet, N.; Lapierre, C.; Jouanin, L.; Baumberger, S.; Mechin, V. Arabidopsis peroxidase-catalyzed copolymerization of
coniferyl and sinapyl alcohols: Kinetics of an endwise process. Phytochemistry 2010, 71, 1673–1683. [CrossRef] [PubMed]
153. Tobimatsu, Y.; Takano, T.; Kamitakahara, H.; Nakatsubo, F. Reactivity of syringyl quinone methide intermediates in dehydrogena-
tive polymerization. Part 2: pH effect in horseradish peroxidase-catalyzed polymerization of sinapyl alcohol. Holzforschung 2010,
64, 183–192. [CrossRef]
154. Hwang, H.; Moon, S.-J.; Won, K.; Kim, Y.H.; Choi, J.W. Parameters affecting in vitro monolignol couplings during dehydrogenative
polymerization in the presence of peroxidase and H2 O2 . J. Ind. Eng. Chem. 2015, 26, 390–395. [CrossRef]
155. Chen, S.X.; Schopfer, P. Hydroxyl-radical production in physiological reactions—A novel function of peroxidase. Eur. J. Biochem.
1999, 260, 726–735. [CrossRef]
156. Davin, L.B.; Wang, H.-B.; Crowell, A.L.; Bedgar, D.L.; Martin, D.M.; Sarkanen, S.; Lewis, N.G. Stereoselective bimolecular phenoxy
radical coupling by an auxiliary (Dirigent) protein without an active center. Science 1997, 275, 362–366. [CrossRef]
157. Davin, L.B.; Lewis, N.G. Dirigent proteins and dirigent sites explain the mystery of specificity of radical precursor coupling in
lignan and lignin biosynthesis. Plant Physiol. 2000, 123, 453–461. [CrossRef]
158. Davin, L.B.; Lewis, N.G. Lignin primary structures and dirigent sites. Curr. Opin. Biotechnol. 2005, 16, 407–415. [CrossRef]
159. Paniagua, C.; Bilkova, A.; Jackson, P.; Dabravolski, S.; Riber, W.; Didi, V.; Houser, J.; Gigli-Bisceglia, N.; Wimmerova, M.; Budinska,
E.; et al. Dirigent proteins in plants: Modulating cell wall metabolism during abiotic and biotic stress exposure. J. Exp. Bot. 2017,
68, 3287–3301. [CrossRef] [PubMed]
160. Ralph, S.; Park, J.Y.; Bohlmann, J.; Mansfield, S.D. Dirigent proteins in conifer defense: Gene discovery, phylogeny, and differential
wound- and insect-induced expression of a family of DIR and DIR-like genes in spruce (Picea spp.). Plant Mol. Biol. 2006, 60, 21–40.
[CrossRef] [PubMed]
161. Tien, M.; Kirk, T.K. Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds. Science 1983,
221, 661–663. [CrossRef] [PubMed]
162. Niku-Paavola, M.L.; Karhunen, E.; Salola, P.; Raunio, V. Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem. J.
1988, 254, 877–883. [CrossRef]
163. Bourbonnais, R.; Paice, M.G. Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS
Lett. 1990, 267, 99–102. [CrossRef]
164. Baldrian, P. Fungal laccases—Occurrence and properties. Fems. Microbiol. Rev. 2006, 30, 215–242. [CrossRef]
165. Lan, W.; Rencoret, J.; Lu, F.C.; Karlen, S.D.; Smith, B.G.; Harris, P.J.; del Rio, J.C.; Ralph, J. Tricin-lignins: Occurrence and
quantitation of tricin in relation to phylogeny. Plant J. 2016, 88, 1046–1057. [CrossRef]
166. Lan, W.; Morreel, K.; Lu, F.; Rencoret, J.; Carlos del Rio, J.; Voorend, W.; Vermerris, W.; Boerjan, W.; Ralph, J. Maize Tricin-
Oligolignol Metabolites and Their Implications for Monocot Lignification. Plant Physiol. 2016, 171, 810–820. [CrossRef]
167. Zhang, L.M.; Gellerstedt, G.; Ralph, J.; Lu, F.C. NMR studies on the occurrence of spirodienone structures in lignins. J. Wood
Chem. Technol. 2006, 26, 65–79. [CrossRef]
168. Gellerstedt, G.; Zhang, L. Reactive lignin structures in high yield pulping: Part 1. Structures of the 1,2-diarylpropane-1,3-diol
type. Nord. Pulp Pap. Res. J. 1991, 6, 136–139. [CrossRef]
169. Ralph, J.; Lapierre, C.; Lu, F.C.; Marita, J.M.; Pilate, G.; Van Doorsselaere, J.; Boerjan, W.; Jouanin, L. NMR evidence for
benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into lignins of O-methyltransferase-deficient
poplars. J. Agric. Food Chem. 2001, 49, 86–91. [CrossRef] [PubMed]
170. Morikawa, T.; Xu, F.; Matsuda, H.; Yoshikawa, M. Structures of Novel Norstilbene Dimer, Longusone A, and Three New Stilbene
Dimers, Longusols A, B, and C, with Antiallergic and Radical Scavenging Activities from Egyptian Natural Medicine Cyperus
longus. Chem. Pharm. Bull. 2010, 58, 1379–1385. [CrossRef] [PubMed]
171. Hirayama, H.; Akiyama, T.; Kimura, S.; Nawawi, D.S.; Syafii, W.; Yokoyama, T.; Matsumoto, Y. Influence of the p-
hydroxyphenyl/guaiacyl ratio on the biphenyl and beta-5 contents in compression wood lignins. Holzforschung 2019, 73, 923–935.
[CrossRef]
172. Zhang, L.M.; Henriksson, G.; Gellerstedt, G. The formation of beta-beta structures in lignin biosynthesis—Are there two different
pathways? Org. Biomol. Chem. 2003, 1, 3621–3624. [CrossRef] [PubMed]
173. Masai, E.; Katayama, Y.; Kubota, S.; Kawai, S.; Yamasaki, M.; Morohishi, N. A bacterial enzyme degrading the model lignin
compound beta-etherase is a member of the glutathione-S-transferase superfamily. FEBS Lett. 1993, 323, 135–140. [CrossRef]
174. Lei, M.; Wu, S.; Liu, C.; Liang, J.; Xiao, R. Revealing the pyrolysis behavior of 5-5? biphenyl-type lignin fragment. Part I:
A mechanistic study on fragmentation via experiments and theoretical calculation. Fuel Process. Technol. 2021, 217, 106812.
[CrossRef]
175. Houston, R.W.W.; Abdoulmoumine, N.H.H. Investigation of the thermal deconstruction of beta-beta’ and 4-O-5 linkages in lignin
model oligomers by density functional theory (DFT). RSC Adv. 2023, 13, 6181–6190. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 42 of 47

176. Day, A.; Dehorter, B.; Neutelings, G.; Czeszak, X.; Chabbert, B.; Belingheri, L.; David, H. Caffeoyl-coenzyme A 3-O-
methyltransferase enzyme activity, protein and transcript accumulation in flax (Linum usitatissimum) stem during development.
Physiol. Plant. 2001, 113, 275–284. [CrossRef]
177. Quideau, S.; Ralph, J. Lignin-ferulate cross-links in grasses. Part 4.1-3 Incorporation of 5-5-coupled dehydrodiferulate into
synthetic lignin. J. Chem. Soc.-Perkin Trans. 1 1997, 16, 2351–2358. [CrossRef]
178. Argyropoulos, D.S.; Jurasek, L.; Kristofova, L.; Xia, Z.C.; Sun, Y.J.; Palus, E. Abundance and reactivity of dibenzodioxocins in
softwood lignin. J. Agric. Food Chem. 2002, 50, 658–666. [CrossRef]
179. Karhunen, P.; Rummakko, P.; Sipila, J.; Brunow, G.; Kilpelainen, I. Dibenzodioxocins; A novel type of linkage in softwood lignins.
Tetrahedron Lett. 1995, 36, 169–170. [CrossRef]
180. Kukkola, E.M.; Koutaniemi, S.; Pollanen, E.; Gustafsson, M.; Karhunen, P.; Lundell, T.K.; Saranpaa, P.; Kilpelainen, I.; Teeri, T.H.;
Fagerstedt, K.V. The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce
and silver birch xylem. Planta 2004, 218, 497–500. [CrossRef] [PubMed]
181. Ralph, J.; Quideau, S.; Grabber, J.H.; Hatfield, R.D. Identification and synthesis of new ferulic acid dehydrodimers present in
grass cell-walls. J. Chem. Soc.-Perkin Trans. 1 1994, 23, 3485–3498. [CrossRef]
182. Nanayakkara, B.; Manley-Harris, M.; Suckling, I.D. Understanding the Degree of Condensation of Phenolic and Etherified C-9
Units of in Situ Lignins. J. Agric. Food Chem. 2011, 59, 12514–12519. [CrossRef]
183. Li, Y.; Akiyama, T.; Yokoyama, T.; Matsumoto, Y. NMR Assignment for Diaryl Ether Structures (4-O5 Structures) in Pine Wood
Lignin. Biomacromolecules 2016, 17, 1921–1929. [CrossRef]
184. Yue, F.; Lu, F.; Ralph, S.; Ralph, J. Identification of 4-O-5-Units in Softwood Lignins via Definitive Lignin Models and NMR.
Biomacromolecules 2016, 17, 1909–1920. [CrossRef]
185. Del Rio, J.C.; Rencoret, J.; Gutierrez, A.; Nieto, L.; Jimenez-Barbero, J.; Martinez, A.T. Structural Characterization of Guaiacyl-rich
Lignins in Flax (Linum usitatissimum) Fibers and Shives. J. Agric. Food Chem. 2011, 59, 11088–11099. [CrossRef]
186. Del Rio, J.C.; Rencoret, J.; Marques, G.; Li, J.B.; Gellerstedt, G.; Jimenez-Barbero, J.; Martinez, A.T.; Gutierrez, A. Structural
Characterization of the Lignin from Jute (Corchorus capsularis) Fibers. J. Agric. Food Chem. 2009, 57, 10271–10281. [CrossRef]
187. Del Rio, J.C.; Lino, A.G.; Colodette, J.L.; Lima, C.F.; Gutierrez, A.; Martinez, A.T.; Lu, F.; Ralph, J.; Rencoret, J. Differences in the
chemical structure of the lignins from sugarcane bagasse and straw. Biomass Bioenerg. 2015, 81, 322–338. [CrossRef]
188. Rencoret, J.; Prinsen, P.; Gutierrez, A.; Martinez, A.T.; del Rio, J.C. Isolation and Structural Characterization of the Milled Wood
Lignin, Dioxane Lignin, and Cellulolytic Lignin Preparations from Brewer’s Spent Grain. J. Agric. Food Chem. 2015, 63, 603–613.
[CrossRef]
189. Del Rio, J.C.; Prinsen, P.; Rencoret, J.; Nieto, L.; Jimenez-Barbero, J.; Ralph, J.; Martinez, A.T.; Gutierrez, A. Structural Characteriza-
tion of the Lignin in the Cortex and Pith of Elephant Grass (Pennisetum purpureum) Stems. J. Agric. Food Chem. 2012, 60, 3619–3634.
[CrossRef] [PubMed]
190. Rencoret, J.; Gutierrez, A.; Nieto, L.; Jimenez-Barbero, J.; Faulds, C.B.; Kim, H.; Ralph, J.; Martinez, A.T.; del Rio, J.C. Lignin
Composition and Structure in Young versus Adult Eucalyptus globulus Plants. Plant Physiol. 2011, 155, 667–682. [CrossRef]
[PubMed]
191. Capanema, E.A.; Balakshin, M.Y.; Kadla, J.F. A Comprehensive Approach for Quantitative Lignin Characterization by NMR
Spectroscopy. J. Agric. Food Chem. 2004, 52, 1850–1860. [CrossRef] [PubMed]
192. Chang, H.-M.; Jiang, X. Biphenyl structure and its impact on the macromolecular structure of lignin: A critical review. J. Wood
Chem. Technol. 2020, 40, 81–90. [CrossRef]
193. Monteil-Rivera, F.; Phuong, M.; Ye, M.; Halasz, A.; Hawari, J. Isolation and characterization of herbaceous lignins for applications
in biomaterials. Ind. Crops Prod. 2013, 41, 356–364. [CrossRef]
194. Pereira, A.; Hoeger, I.C.; Ferrer, A.; Rencoret, J.; del Rio, J.C.; Kruus, K.; Rahikainen, J.; Kellock, M.; Gutierrez, A.; Rojas, O.J.
Lignin Films from Spruce, Eucalyptus, and Wheat Straw Studied with Electroacoustic and Optical Sensors: Effect of Composition
and Electrostatic Screening on Enzyme Binding. Biomacromolecules 2017, 18, 1322–1332. [CrossRef]
195. Martinez, A.T.; Rencoret, J.; Marques, G.; Gutierrez, A.; Ibarra, D.; Jimenez-Barbero, J.; del Rio, J.C. Monolignol acylation and
lignin structure in some nonwoody plants: A 2D NMR study. Phytochemistry 2008, 69, 2831–2843. [CrossRef]
196. Lourenco, A.; Neiva, D.M.; Gominho, J.; Marques, A.V.; Pereira, H. Characterization of lignin in heartwood, sapwood and bark
from Tectona grandis using Py-GC-MS/FID. Wood Sci. Technol. 2015, 49, 159–175. [CrossRef]
197. Lourenco, A.; Gominho, J.; Marques, A.V.; Pereira, H. Variation of Lignin Monomeric Composition during Kraft Pulping of
Eucalyptus globulus Heartwood and Sapwood. J. Wood Chem. Technol. 2013, 33, 1–18. [CrossRef]
198. Kishimoto, T.; Chiba, W.; Saito, K.; Fukushima, K.; Uraki, Y.; Ubukata, M. Influence of Syringyl to Guaiacyl Ratio on the Structure
of Natural and Synthetic Lignins. J. Agric. Food Chem. 2010, 58, 895–901. [CrossRef]
199. Chen, L.; Auh, C.; Chen, F.; Cheng, X.F.; Aljoe, H.; Dixon, R.A.; Wang, Z.Y. Lignin deposition and associated changes in anatomy,
enzyme activity, gene expression, and ruminal degradability in stems of tall fescue at different developmental stages. J. Agric.
Food Chem. 2002, 50, 5558–5565. [CrossRef] [PubMed]
200. Negrel, J.; Pollet, B.; Lapierre, C. Ether-linked ferulic acid amides in natural and wound periderms of potato tuber. Phytochemistry
1996, 43, 1195–1199. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 43 of 47

201. Huang, C.; Lin, W.; Lai, C.; Li, X.; Jin, Y.; Yong, Q. Coupling the post-extraction process to remove residual lignin and alter the
recalcitrant structures for improving the enzymatic digestibility of acid-pretreated bamboo residues. Bioresour. Technol. 2019,
285, 121355. [CrossRef]
202. Zhong, H.; Zhou, J.; Abdelrahman, M.; Xu, H.; Wu, Z.; Cui, L.; Ma, Z.; Yang, L.; Li, X. The Effect of Lignin Composition on
Ruminal Fiber Fractions Degradation from Different Roughage Sources in Water Buffalo (Bubalus bubalis). Agriculture 2021,
11, 1015. [CrossRef]
203. Mulat, D.G.; Dibdiakova, J.; Horn, S.J. Microbial biogas production from hydrolysis lignin: Insight into lignin structural changes.
Biotechnol. Biofuels. 2018, 11, 61. [CrossRef]
204. Barakat, A.; Monlau, F.; Steyer, J.-P.; Carrere, H. Effect of lignin-derived and furan compounds found in lignocellulosic hy-
drolysates on biomethane production. Bioresou. Technol. 2012, 104, 90–99. [CrossRef]
205. Van Acker, R.; Vanholme, R.; Storme, V.; Mortimer, J.C.; Dupree, P.; Boerjan, W. Lignin biosynthesis perturbations affect secondary
cell wall composition and saccharification yield in Arabidopsis thaliana. Biotechnol. Biofuels 2013, 6, 46. [CrossRef]
206. Kim, K.H.; Eudes, A.; Jeong, K.; Yoo, C.G.; Kim, C.S.; Ragauskas, A. Integration of renewable deep eutectic solvents with
engineered biomass to achieve a closed-loop biorefinery. Proc. Natl. Acad. Sci. USA 2019, 116, 13816–13824. [CrossRef] [PubMed]
207. Yamamoto, M.; Blaschek, L.; Subbotina, E.; Kajita, S.; Pesquet, E. Importance of Lignin Coniferaldehyde Residues for Plant
Properties and Sustainable Uses. ChemSusChem 2020, 13, 4400–4408. [CrossRef]
208. Baucher, M.; Bernard-Vailhe, M.A.; Chabbert, B.; Besle, J.M.; Opsomer, C.; Van Montagu, M.; Botterman, J. Down-regulation of
cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility.
Plant Mol. Biol. 1999, 39, 437–447. [CrossRef]
209. Sibout, R.; Eudes, A.; Pollet, B.; Goujon, T.; Mila, I.; Granier, F.; Seguin, A.; Lapierre, C.; Jouanin, L. Expression pattern of two
paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants.
Plant Physiol. 2003, 132, 848–860. [CrossRef] [PubMed]
210. Zhao, Q.; Tobimatsu, Y.; Zhou, R.; Pattathil, S.; Gallego-Giraldo, L.; Fu, C.; Jackson, L.A.; Hahn, M.G.; Kim, H.; Chen, F.; et al.
Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect
in Medicago truncatula. Proc. Natl. Acad. Sci. USA 2013, 110, 13660–13665. [CrossRef] [PubMed]
211. MacKay, J.J.; Omalley, D.M.; Presnell, T.; Booker, F.L.; Campbell, M.M.; Whetten, R.W.; Sederoff, R.R. Inheritance, gene expression,
and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 1997,
94, 8255–8260. [CrossRef] [PubMed]
212. Sattler, S.E.; Saathoff, A.J.; Haas, E.J.; Palmer, N.A.; Funnell-Harris, D.L.; Sarath, G.; Pedersen, J.F. A Nonsense Mutation in
a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype. Plant Physiol. 2009,
150, 584–595. [CrossRef]
213. Yamamoto, M.; Tomiyama, H.; Koyama, A.; Okuizumi, H.; Liu, S.; Vanholme, R.; Goeminne, G.; Hirai, Y.; Shi, H.; Nuoendagula;
et al. A Century-Old Mystery Unveiled: Sekizaisou is a Natural Lignin Mutant(1 OPEN ). Plant Physiol. 2020, 182, 1821–1828.
[CrossRef]
214. Sattler, S.E.; Palmer, N.A.; Saballos, A.; Greene, A.M.; Xin, Z.; Sarath, G.; Vermerris, W.; Pedersen, J.F. Identification and
Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum.
Bioenergy Res. 2012, 5, 855–865. [CrossRef]
215. Van Doorsselaere, J.; Baucher, M.; Chognot, E.; Chabbert, B.; Tollier, M.-T.; Petit-Conil, M.; Leple, J.-C.; Pilate, G.; Cornu, D.;
Monties, B.; et al. A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity.
Plant J. 1995, 8, 855–864. [CrossRef]
216. Dien, B.S.; Sarath, G.; Pedersen, J.F.; Sattler, S.E.; Chen, H.; Funnell-Harris, D.L.; Nichols, N.N.; Cotta, M.A. Improved Sugar
Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents. Bioenergy
Res. 2009, 2, 153–164. [CrossRef]
217. Takeda, Y.; Koshiba, T.; Tobimatsu, Y.; Suzuki, S.; Murakami, S.; Yamamura, M.; Rahman, M.M.; Takano, T.; Hattori, T.; Sakamoto,
M.; et al. Regulation of CONIFERALDEHYDE 5-HYDROXYLASE expression to modulate cell wall lignin structure in rice. Planta
2017, 246, 337–349. [CrossRef]
218. Skyba, O.; Douglas, C.J.; Mansfield, S.D. Syringyl-Rich Lignin Renders Poplars More Resistant to Degradation by Wood Decay
Fungi. Appl. Environ. Microbiol. 2013, 79, 2560–2571. [CrossRef]
219. Cao, Y.R.; Yan, X.Y.; Ran, S.Y.; Ralph, J.; Smith, R.A.; Chen, X.P.; Qu, C.M.; Li, J.N.; Liu, L.Z. Knockout of the lignin pathway gene
BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus. Plant Cell
Environ. 2022, 45, 248–261. [CrossRef] [PubMed]
220. Bewg, W.P.; Poovaiah, C.; Lan, W.; Ralph, J.; Coleman, H.D. RNAi downregulation of three key lignin genes in sugarcane
improves glucose release without reduction in sugar production. Biotechnol. Biofuels 2016, 9, 270. [CrossRef] [PubMed]
221. Tetreault, H.M.; Gries, T.; Palmer, N.A.; Funnell-Harris, D.L.; Satoz, S.; Ge, Z.X.; Sarath, G.; Sattler, S.E. Overexpression of ferulate
5-hydroxylase increases syringyl units in Sorghum bicolor. Plant Mol. Biol. 2020, 103, 269–285. [CrossRef] [PubMed]
222. Ralph, J.; Akiyama, T.; Kim, H.; Lu, F.C.; Schatz, P.F.; Marita, J.M.; Ralph, S.A.; Reddy, M.S.S.; Chen, F.; Dixon, R.A. Effects of
coumarate 3-hydroxylase down-regulation on lignin structure. J. Biol. Chem. 2006, 281, 8843–8853. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 11668 44 of 47

223. Eloy, N.B.; Voorend, W.; Lan, W.; Saleme, M.D.S.; Cesarino, I.; Vanholme, R.; Smith, R.A.; Goeminne, G.; Pallidis, A.; Morreel, K.;
et al. Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content.
Plant Physiol. 2017, 173, 998–1016. [CrossRef]
224. Kim, Y.H.; Kim, C.Y.; Song, W.K.; Park, D.S.; Kwon, S.Y.; Lee, H.S.; Bang, J.W.; Kwak, S.S. Overexpression of sweetpotato swpa4
peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco. Planta 2008, 227, 867–881.
[CrossRef]
225. Shigeto, J.; Itoh, Y.; Hirao, S.; Ohira, K.; Fujita, K.; Tsutsumi, Y. Simultaneously disrupting AtPrx2, AtPrx25 and AtPrx71 alters
lignin content and structure in Arabidopsis stem. J. Integr. Plant Biol. 2015, 57, 349–356. [CrossRef]
226. Renard, J.; Martinez-Almonacid, I.; Sonntag, A.; Molina, I.; Moya-Cuevas, J.; Bissoli, G.; Munoz-Bertomeu, J.; Faus, I.; Ninoles, R.;
Shigeto, J.; et al. PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis. Plant Cell
Environ. 2020, 43, 315–326. [CrossRef]
227. Ekielski, A.; Mishra, P.K. Lignin for Bioeconomy: The Present and Future Role of Technical Lignin. Int. J. Mol. Sci. 2021, 22, 63.
[CrossRef]
228. Berlin, A.; Balakshin, M. Chapter 18—Industrial Lignins: Analysis, Properties, and Applications. In Bioenergy Research: Advances
and Applications; Gupta, V.K., Tuohy, M.G., Kubicek, C.P., Saddler, J., Xu, F., Eds.; Elsevier: Amsterdam, The Netherlands, 2014;
pp. 315–336. [CrossRef]
229. Matsushita, Y. Conversion of technical lignins to functional materials with retained polymeric properties. J. Wood Sci. 2015,
61, 230–250. [CrossRef]
230. Yu, O.; Kim, K.H. Lignin to Materials: A Focused Review on Recent Novel Lignin Applications. Appl. Sci. 2020, 10, 4626.
[CrossRef]
231. Norgren, M.; Edlund, H. Lignin: Recent advances and emerging applications. Curr. Opin. Colloid Interface Sci. 2014, 19, 409–416.
[CrossRef]
232. Vishtal, A.G.; Kraslawski, A. Challenges in industrial applications of technical lignins. Bioresources 2011, 6, 3547–3568. [CrossRef]
233. Ariyanta, H.A.; Sari, F.P.; Sohail, A.; Restu, W.K.; Septiyanti, M.; Aryana, N.; Fatriasari, W.; Kumar, A. Current roles of lignin for
the agroindustry: Applications, challenges, and opportunities. Int. J. Biol. Macromol. 2023, 240, 124523. [CrossRef]
234. Tran, M.H.; Lee, E.Y. Green Preparation of Bioplastics Based on Degradation and Chemical Modification of Lignin Residue. J.
Wood Chem. Technol. 2018, 38, 460–478. [CrossRef]
235. Yang, J.; Ching, Y.C.; Chuah, C.H. Applications of Lignocellulosic Fibers and Lignin in Bioplastics: A Review. Polymers 2019,
11, 751. [CrossRef] [PubMed]
236. Zevallos Torres, L.A.; Lorenci Woiciechowski, A.; de Andrade Tanobe, V.O.; Karp, S.G.; Guimarães Lorenci, L.C.; Faulds, C.;
Soccol, C.R. Lignin as a potential source of high-added value compounds: A review. J. Clean. Prod. 2020, 263, 121499. [CrossRef]
237. Kumar, R.; Butreddy, A.; Kommineni, N.; Reddy, P.G.; Bunekar, N.; Sarkar, C.; Dutt, S.; Mishra, V.K.; Aadil, K.R.; Mishra, Y.K.;
et al. Lignin: Drug/Gene Delivery and Tissue Engineering Applications. Int. J. Nanomed. 2021, 16, 2419–2441. [CrossRef]
238. Xu, J.; Xu, J.J.; Lin, Q.; Jiang, L.; Zhang, D.; Li, Z.; Ma, B.; Zhang, C.; Li, L.; Kai, D.; et al. Lignin-Incorporated Nanogel Serving as
an Antioxidant Biomaterial for Wound Healing. ACS Appl. Bio Mater. 2021, 4, 3–13. [CrossRef]
239. Wijaya, C.J.; Ismadji, S.; Gunawan, S. A Review of Lignocellulosic-Derived Nanoparticles for Drug Delivery Applications: Lignin
Nanoparticles, Xylan Nanoparticles, and Cellulose Nanocrystals. Molecules 2021, 26, 676. [CrossRef]
240. Garg, J.; Nee Chiu, M.; Krishnan, S.; Kumar Tripathi, L.; Pandit, S.; Farasati Far, B.; Kumar Jha, N.; Kumar Kesari, K.; Tripathi,
V.; Pandey, S.; et al. Applications of lignin nanoparticles for cancer drug delivery: An update. Mater. Lett. 2022, 311, 131573.
[CrossRef]
241. Dai, M.; Zhang, J.; Liu, N.; Zhang, X.-H. Precise Engineering of Lignin Incorporated Dextran/Glycol Nanomaterials for Wound
Dressings for the Care of Anorectal Surgery. J. Polym. Environ. 2022, 30, 206–216. [CrossRef]
242. Beisl, S.; Adamcyk, J.; Friedl, A. Direct Precipitation of Lignin Nanoparticles from Wheat Straw Organosolv Liquors Using a
Static Mixer. Molecules 2020, 25, 1388. [CrossRef] [PubMed]
243. Leskinen, T.; Smyth, M.; Xiao, Y.; Lintinen, K.; Mattinen, M.-L.; Kostiainen, M.A.; Oinas, P.; Österberg, M. Scaling up Production
of Colloidal Lignin Particles. Nord. Pulp Pap. Res. J. 2017, 32, 586–596. [CrossRef]
244. Agustin, M.B.; Penttilä, P.A.; Lahtinen, M.; Mikkonen, K.S. Rapid and Direct Preparation of Lignin Nanoparticles from Alkaline
Pulping Liquor by Mild Ultrasonication. ACS Sustain. Chem. Eng. 2019, 7, 19925–19934. [CrossRef]
245. Dai, L.; Liu, R.; Hu, L.-Q.; Zou, Z.-F.; Si, C.-L. Lignin Nanoparticle as a Novel Green Carrier for the Efficient Delivery of Resveratrol.
ACS Sustain. Chem. Eng. 2017, 5, 8241–8249. [CrossRef]
246. Du, B.; Li, W.; Bai, Y.; Pan, Z.; Wang, Q.; Wang, X.; Ding, H.; Lv, G.; Zhou, J. Fabrication of uniform lignin nanoparticles with
tunable size for potential wound healing application. Int. J. Biol. Macromol. 2022, 214, 170–180. [CrossRef]
247. Balaji, S.; Short, W.D.; Padon, B.W.; Prajapati, T.J.; Farouk, F.; Kaul, A.; Belgodere, J.A.; Jimenez, S.E.; Deoli, N.T.; Guidry, A.C.;
et al. Engineering Antioxidant and Oxygen-Releasing Lignin Composites to Accelerate Wound Healing. bioRxiv 2022. [CrossRef]
248. Alqahtani, M.S.; Alqahtani, A.; Kazi, M.; Ahmad, M.Z.; Alahmari, A.; Alsenaidy, M.A.; Syed, R. Wound-healing potential of
curcumin loaded lignin nanoparticles. J. Drug Deliv. Sci. Technol. 2020, 60, 102020. [CrossRef]
249. Cheng, L.; Deng, B.; Luo, W.; Nie, S.; Liu, X.; Yin, Y.; Liu, S.; Wu, Z.; Zhan, P.; Zhang, L.; et al. pH-Responsive Lignin-Based
Nanomicelles for Oral Drug Delivery. J. Agric. Food Chem. 2020, 68, 5249–5258. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 45 of 47

250. Capecchi, E.; Piccinino, D.; Nascimben, C.; Tomaino, E.; Ceccotti Vlas, N.; Gabellone, S.; Saladino, R. Biosynthesis of Novel
Ascorbic Acid Esters and Their Encapsulation in Lignin Nanoparticles as Carriers and Stabilizing Systems. Int. J. Mol. Sci. 2023,
24, 9044. [CrossRef] [PubMed]
251. Machado, T.O.; Beckers, S.J.; Fischer, J.; Müller, B.; Sayer, C.; de Araújo, P.H.H.; Landfester, K.; Wurm, F.R. Bio-Based Lignin
Nanocarriers Loaded with Fungicides as a Versatile Platform for Drug Delivery in Plants. Biomacromolecules 2020, 21, 2755–2763.
[CrossRef] [PubMed]
252. Zhou, Y.; Han, Y.; Li, G.; Yang, S.; Xiong, F.; Chu, F. Preparation of Targeted Lignin–Based Hollow Nanoparticles for the Delivery
of Doxorubicin. Nanomaterials 2019, 9, 188. [CrossRef] [PubMed]
253. Lee, J.H.; Im, J.S.; Jin, X.; Kim, T.M.; Choi, J.W. In Vitro and In Vivo Evaluation of Drug-Encapsulated Lignin Nanoparticles for
Release Control. ACS Sustain. Chem. Eng. 2022, 10, 5792–5802. [CrossRef]
254. Alqahtani, M.S.; Alqahtani, A.; Al-Thabit, A.; Roni, M.; Syed, R. Novel lignin nanoparticles for oral drug delivery. J. Mater. Chem.
B 2019, 7, 4461–4473. [CrossRef]
255. Chai, Y.; Wang, Y.; Li, B.; Qi, W.; Su, R.; He, Z. Microfluidic Synthesis of Lignin/Chitosan Nanoparticles for the pH-Responsive
Delivery of Anticancer Drugs. Langmuir 2021, 37, 7219–7226. [CrossRef]
256. Alqahtani, M.S.; Kazi, M.; Ahmad, M.Z.; Syed, R.; Alsenaidy, M.A.; Albraiki, S.A. Lignin nanoparticles as a promising vaccine
adjuvant and delivery system for ovalbumin. Int. J. Biol. Macromol. 2020, 163, 1314–1322. [CrossRef]
257. Figueiredo, P.; Lintinen, K.; Kiriazis, A.; Hynninen, V.; Liu, Z.; Bauleth-Ramos, T.; Rahikkala, A.; Correia, A.; Kohout, T.; Sarmento,
B.; et al. In vitro evaluation of biodegradable lignin-based nanoparticles for drug delivery and enhanced antiproliferation effect
in cancer cells. Biomaterials 2017, 121, 97–108. [CrossRef]
258. Rico-García, D.; Ruiz-Rubio, L.; Pérez-Alvarez, L.; Hernández-Olmos, S.L.; Guerrero-Ramírez, G.L.; Vilas-Vilela, J.L. Lignin-Based
Hydrogels: Synthesis and Applications. Polymers 2020, 12, 81. [CrossRef]
259. Kim, B.; Kim, Y.; Lee, Y.; Oh, J.; Jung, Y.; Koh, W.-G.; Chung, J.J. Reactive Oxygen Species Suppressive Kraft Lignin-Gelatin based
Antioxidant hydrogels for Chronic Wound Repair. Macromol. Biosci. 2022, 22, 2200234. [CrossRef]
260. Eivazzadeh-Keihan, R.; Moghim Aliabadi, H.A.; Radinekiyan, F.; Sobhani, M.; Farzane, k.; Maleki, A.; Madanchi, H.; Mahdavi,
M.; Shalan, A.E. Investigation of the biological activity, mechanical properties and wound healing application of a novel scaffold
based on lignin–agarose hydrogel and silk fibroin embedded zinc chromite nanoparticles. RSC Adv. 2021, 11, 17914–17923.
[CrossRef] [PubMed]
261. Culebras, M.; Pishnamazi, M.; Walker, G.M.; Collins, M.N. Facile Tailoring of Structures for Controlled Release of Paracetamol
from Sustainable Lignin Derived Platforms. Molecules 2021, 26, 1593. [CrossRef] [PubMed]
262. Larrañeta, E.; Imízcoz, M.; Toh, J.X.; Irwin, N.J.; Ripolin, A.; Perminova, A.; Domínguez-Robles, J.; Rodríguez, A.; Donnelly,
R.F. Synthesis and Characterization of Lignin Hydrogels for Potential Applications as Drug Eluting Antimicrobial Coatings for
Medical Materials. ACS Sustain. Chem. Eng. 2018, 6, 9037–9046. [CrossRef] [PubMed]
263. Anghel, N.; Melinte, V.; Spiridon, I.; Pertea, M. Antioxidant, Antimicrobial, and Kinetic Studies of β-Cyclodextrin Crosslinked
with Lignin for Drug Delivery. Pharmaceutics 2022, 14, 2260. [CrossRef]
264. Li, S.; Zhang, Y.; Ma, X.; Qiu, S.; Chen, J.; Lu, G.; Jia, Z.; Zhu, J.; Yang, Q.; Chen, J.; et al. Antimicrobial Lignin-Based
Polyurethane/Ag Composite Foams for Improving Wound Healing. Biomacromolecules 2022, 23, 1622–1632. [CrossRef]
265. Abdullah, T.; Gauthaman, K.; Mostafavi, A.; Alshahrie, A.; Salah, N.; Morganti, P.; Chianese, A.; Tamayol, A.; Memic, A.
Sustainable drug release from polycaprolactone coated chitin-lignin gel fibrous scaffolds. Sci. Rep. 2020, 10, 20428. [CrossRef]
266. Ma, X.; Chen, J.; Zhu, J.; Yan, N. Lignin-Based Polyurethane: Recent Advances and Future Perspectives. Macromol. Rapid Commun.
2021, 42, 2000492. [CrossRef]
267. Zhang, Y.; Yuan, B.; Zhang, Y.; Cao, Q.; Yang, C.; Li, Y.; Zhou, J. Biomimetic lignin/poly(ionic liquids) composite hydrogel
dressing with excellent mechanical strength, self-healing properties, and reusability. Chem. Eng. J. 2020, 400, 125984. [CrossRef]
268. Yang, W.; Xu, F.; Ma, X.; Guo, J.; Li, C.; Shen, S.; Puglia, D.; Chen, J.; Xu, P.; Kenny, J.; et al. Highly-toughened PVA/nanocellulose
hydrogels with anti-oxidative and antibacterial properties triggered by lignin-Ag nanoparticles. Mater. Sci. Eng. C 2021,
129, 112385. [CrossRef]
269. Gan, D.; Xing, W.; Jiang, L.; Fang, J.; Zhao, C.; Ren, F.; Fang, L.; Wang, K.; Lu, X. Plant-inspired adhesive and tough hydrogel
based on Ag-Lignin nanoparticles-triggered dynamic redox catechol chemistry. Nat. Commun. 2019, 10, 1487. [CrossRef]
270. Deng, P.; Chen, F.; Zhang, H.; Chen, Y.; Zhou, J. Conductive, Self-Healing, Adhesive, and Antibacterial Hydrogels Based on
Lignin/Cellulose for Rapid MRSA-Infected Wound Repairing. ACS Appl. Mater. Interfaces 2021, 13, 52333–52345. [CrossRef]
[PubMed]
271. Lei, Y.; Alshareef, A.H.; Zhao, W.; Inal, S. Laser-Scribed Graphene Electrodes Derived from Lignin for Biochemical Sensing. ACS
Appl. Nano Mater. 2020, 3, 1166–1174. [CrossRef]
272. Capecchi, E.; Piccinino, D.; Bizzarri, B.M.; Botta, L.; Crucianelli, M.; Saladino, R. Oxidative Bio-Desulfurization by Nanostructured
Peroxidase Mediator System. Catalysts 2020, 10, 313.
273. Mahmood, F.; Zhang, H.; Lin, J.; Wan, C. Laser-Induced Graphene Derived from Kraft Lignin for Flexible Supercapacitors. ACS
Omega 2020, 5, 14611–14618. [CrossRef] [PubMed]
274. Hu, Z.-R.; Li, D.-D.; Kim, T.-H.; Kim, M.-S.; Xu, T.; Ma, M.-G.; Choi, S.-E.; Si, C. Lignin-Based/Polypyrrole Carbon Nanofiber
Electrode With Enhanced Electrochemical Properties by Electrospun Method. Front. Chem. 2022, 10, 841956. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 11668 46 of 47

275. Adam, A.A.; Ojur Dennis, J.; Al-Hadeethi, Y.; Mkawi, E.M.; Abubakar Abdulkadir, B.; Usman, F.; Mudassir Hassan, Y.; Wadi, I.A.;
Sani, M. State of the Art and New Directions on Electrospun Lignin/Cellulose Nanofibers for Supercapacitor Application: A
Systematic Literature Review. Polymers 2020, 12, 2884. [CrossRef] [PubMed]
276. Zhu, J.; Yan, C.; Zhang, X.; Yang, C.; Jiang, M.; Zhang, X. A sustainable platform of lignin: From bioresources to materials and
their applications in rechargeable batteries and supercapacitors. Prog. Energy Combust. Sci. 2020, 76, 100788. [CrossRef]
277. Hérou, S.; Bailey, J.J.; Kok, M.; Schlee, P.; Jervis, R.; Brett, D.J.L.; Shearing, P.R.; Ribadeneyra, M.C.; Titirici, M. High-Density
Lignin-Derived Carbon Nanofiber Supercapacitors with Enhanced Volumetric Energy Density. Adv. Sci. 2021, 8, 2100016.
[CrossRef]
278. Cao, M.; Cheng, W.; Ni, X.; Hu, Y.; Han, G. Lignin-based multi-channels carbon nanofibers @ SnO2 nanocomposites for
high-performance supercapacitors. Electrochim. Acta 2020, 345, 136172. [CrossRef]
279. Li, H.; Zhao, Y.; Liu, S.; Li, P.; Yuan, D.; He, C. Hierarchical porous carbon monolith derived from lignin for high areal capacitance
supercapacitors. Microporous Mesoporous Mater. 2020, 297, 109960. [CrossRef]
280. Fu, F.; Yang, D.; Zhang, W.; Wang, H.; Qiu, X. Green self-assembly synthesis of porous lignin-derived carbon quasi-nanosheets for
high-performance supercapacitors. Chem. Eng. J. 2020, 392, 123721. [CrossRef]
281. Li, W.; Shi, J. Lignin-derived carbon material for electrochemical energy storage applications: Insight into the process-structure-
properties-performance correlations. Front. Bioeng. Biotechnol. 2023, 11, 1121027. [CrossRef]
282. Wigmans, T. Industrial aspects of production and use of activated carbons. Carbon 1989, 27, 13–22. [CrossRef]
283. Rodríguez-Reinoso, F.; Molina-Sabio, M.; González, M.T. The use of steam and CO2 as activating agents in the preparation of
activated carbons. Carbon 1995, 33, 15–23. [CrossRef]
284. Gonzalez-Serrano, E.; Cordero, T.; Rodríguez-Mirasol, J.; Rodríguez, J.J. Development of Porosity upon Chemical Activation of
Kraft Lignin with ZnCl2 . Ind. Eng. Chem. Res. 1997, 36, 4832–4838. [CrossRef]
285. Wu, Y.; Cao, J.-P.; Hao, Z.-Q.; Zhao, X.-Y.; Zhuang, Q.-Q.; Zhu, J.-S.; Wang, X.-Y.; Wei, X.-Y. One-step Preparation of Alkaline
Lignin-based Activated Carbons with Different Activating Agents for Electric Double Layer Capacitor. Int. J. Electrochem. Sci.
2017, 12, 7227–7239. [CrossRef]
286. Dai, Z.; Ren, P.-G.; He, W.; Hou, X.; Ren, F.; Zhang, Q.; Jin, Y.-L. Boosting the electrochemical performance of nitrogen-oxygen
co-doped carbon nanofibers based supercapacitors through esterification of lignin precursor. Renew. Energy 2020, 162, 613–623.
[CrossRef]
287. Zhang, W.; Yin, J.; Wang, C.; Zhao, L.; Jian, W.; Lu, K.; Lin, H.; Qiu, X.; Alshareef, H.N. Lignin Derived Porous Carbons: Synthesis
Methods and Supercapacitor Applications. Small Methods 2021, 5, 2100896. [CrossRef] [PubMed]
288. Liu, T.; Ren, X.; Zhang, J.; Liu, J.; Ou, R.; Guo, C.; Yu, X.; Wang, Q.; Liu, Z. Highly compressible lignin hydrogel electrolytes via
double-crosslinked strategy for superior foldable supercapacitors. J. Power Sources 2020, 449, 227532. [CrossRef]
289. Park, J.H.; Rana, H.H.; Lee, J.Y.; Park, H.S. Renewable flexible supercapacitors based on all-lignin-based hydrogel electrolytes and
nanofiber electrodes. J. Mater. Chem. A 2019, 7, 16962–16968. [CrossRef]
290. Moreno, A.; Sipponen, M.H. Lignin-based smart materials: A roadmap to processing and synthesis for current and future
applications. Mater. Horiz. 2020, 7, 2237–2257. [CrossRef]
291. Kai, D.; Tan, M.J.; Chee, P.L.; Chua, Y.K.; Yap, Y.L.; Loh, X.J. Towards lignin-based functional materials in a sustainable world.
Green Chem. 2016, 18, 1175–1200. [CrossRef]
292. Zhao, S.; Abu-Omar, M.M. Materials Based on Technical Bulk Lignin. ACS Sustain. Chem. Eng. 2021, 9, 1477–1493. [CrossRef]
293. Lendlein, A.; Balk, M.; Tarazona, N.A.; Gould, O.E.C. Bioperspectives for Shape-Memory Polymers as Shape Programmable,
Active Materials. Biomacromolecules 2019, 20, 3627–3640. [CrossRef] [PubMed]
294. Xia, Y.; He, Y.; Zhang, F.; Liu, Y.; Leng, J. A Review of Shape Memory Polymers and Composites: Mechanisms, Materials, and
Applications. Adv. Mater. 2021, 33, 2000713. [CrossRef]
295. Xu, Y.; Odelius, K.; Hakkarainen, M. One-Pot Synthesis of Lignin Thermosets Exhibiting Widely Tunable Mechanical Properties
and Shape Memory Behavior. ACS Sustain. Chem. Eng. 2019, 7, 13456–13463. [CrossRef]
296. Nguyen, N.A.; Meek, K.M.; Bowland, C.C.; Naskar, A.K. Responsive lignin for shape memory applications. Polymer 2019,
160, 210–222. [CrossRef]
297. Madbouly, S.A. Bio-based castor oil and lignin sulphonate: Aqueous dispersions and shape-memory films. Biomater. Polym. Horiz.
2022, 1, 1–12. [CrossRef]
298. Sternberg, J.; Pilla, S. Materials for the biorefinery: High bio-content, shape memory Kraft lignin-derived non-isocyanate
polyurethane foams using a non-toxic protocol. Green Chem. 2020, 22, 6922–6935. [CrossRef]
299. Liu, L.-Y.; Karaaslan, M.A.; Hua, Q.; Cho, M.; Chen, S.; Renneckar, S. Thermo-Responsive Shape-Memory Polyurethane Foams
from Renewable Lignin Resources with Tunable Structures–Properties and Enhanced Temperature Resistance. Ind. Eng. Chem.
Res. 2021, 60, 11882–11892. [CrossRef]
300. Nguyen, N.A.; Bowland, C.C.; Bonnesen, P.V.; Littrell, K.C.; Keum, J.K.; Naskar, A.K. Fractionation of Lignin for Selective Shape
Memory Effects at Elevated Temperatures. Materials 2020, 13, 1940. [CrossRef] [PubMed]
301. Li, J.; Liu, W.; Qiu, X.; Zhao, X.; Chen, Z.; Yan, M.; Fang, Z.; Li, Z.; Tu, Z.; Huang, J. Lignin: A sustainable photothermal block for
smart elastomers. Green Chem. 2022, 24, 823–836. [CrossRef]
302. Jin, X.; Liu, X.; Li, X.; Du, L.; Su, L.; Ma, Y.; Ren, S. High lignin, light-driven shape memory polymers with excellent mechanical
performance. Int. J. Biol. Macromol. 2022, 219, 44–52. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11668 47 of 47

303. Chen, J.; Qi, J.; He, J.; Yan, Y.; Jiang, F.; Wang, Z.; Zhang, Y. Biobased Composites with High Lignin Content and Excellent
Mechanical Properties toward the Ingenious Photoresponsive Actuator. ACS Appl. Mater. Interfaces 2022, 14, 12748–12757.
[CrossRef] [PubMed]
304. Yang, H.; Wang, D.; Bi, H.; Ren, Z.; Xu, M.; Huang, Z.; Cai, L. Photo-assisted shape memory lignin/cellulose acetate composites
for information encryption. J. Appl. Polym. Sci. 2022, 139, 51997. [CrossRef]
305. Meng, D.; Zhao, Q.; Cheng, X.; Ma, J.; Kong, L.; He, X.; Li, J. Water-induced shape memory cellulose nanofiber-based nanocompos-
ite membrane containing lignin with quick water response and excellent wet mechanical property. Eur. Polym. J. 2022, 171, 111204.
[CrossRef]
306. Peng, Z.; Yu, C.; Zhong, W. Facile Preparation of a 3D Porous Aligned Graphene-Based Wall Network Architecture by Confined
Self-Assembly with Shape Memory for Artificial Muscle, Pressure Sensor, and Flexible Supercapacitor. ACS Appl. Mater. Interfaces
2022, 14, 17739–17753. [CrossRef]
307. Zhao, W.; Liang, Z.; Feng, Z.; Xue, B.; Xiong, C.; Duan, C.; Ni, Y. New Kind of Lignin/Polyhydroxyurethane Composite: Green
Synthesis, Smart Properties, Promising Applications, and Good Reprocessability and Recyclability. ACS Appl. Mater. Interfaces
2021, 13, 28938–28948. [CrossRef]
308. Zhang, G.; Tian, C.; Shi, J.; Zhang, X.; Liu, J.; Tan, T.; Zhang, L. Mechanically Robust, Self-Repairable, Shape Memory and
Recyclable Ionomeric Elastomer Composites with Renewable Lignin via Interfacial Metal–Ligand Interactions. ACS Appl. Mater.
Interfaces 2022, 14, 38216–38227. [CrossRef]

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