Biomass and Bioenergy 128 (2019) 105325

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Biomass and Bioenergy

journal homepage: www.elsevier.com/locate/biombioe

Review

Lignin degradation under anaerobic digestion: Influence of lignin T

modifications -A review
Muhammad Usman Khana, Birgitte Kiaer Ahringa,b,c,∗
a
Bioproducts, Sciences and Engineering Laboratory, Washington State University, Tri-Cities, Richland, WA, 99354, USA
b
Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99163, USA
c
Department of Sustainable Biotechnology, Aalborg University, Sydhavnen, 2450, Copenhagen, Denmark

A R T I C LE I N FO A B S T R A C T

Keywords: Large-scale implementation of Biorefineries for the substitution of oil-based fuels with biofuels and bio-products
Lignin will result in the production of large amounts of lignin.Furthermore,the paper and pulp industry produces lignin
Anaerobic lignin-degradation residues such as Kraft-lignin, which has limited use today. Lignin needs to be degraded before this high mole-
Wet oxidation cular weight molecule can be converted into valuable bio-products. Biodegradation of native lignin has been
Methoxylation
described for fungal strains such as white and brown rot fungi and for a variety of different bacterial genera.
Lignin-oxidizing enzymes
Anaerobic biodegradation of lignin is, however, far less understood today and most studies has been done using
Radical mediators
small molecular weight lignin degradation compounds, while little work has been done on the conversion of
native lignin. In this paper we will discuss the influence of lignin modifications pertaining to its degradation
during anaerobic digestion . Wet oxidation reactions using oxygen at a temperature of ca. 175 °C for 25 min was
found to increase the degree of methoxylation of lignin with simultaneous increment in conversion during
anaerobic conditions. Several studies have further found that de-methoxylation is the first step during anaerobic
degradation of lignin followed by ring cleavage and fermentation into methane and carbon dioxide. As lignin is
an important component of the materials that are fed to biogas plant, we have futher discussed the potential of
these findings in this paper.

1. Introduction sustainable and accessible for biofuels production [2].

Energy consumption using fossil fuels creates environmental pol-
lution including greenhouse gasses promoting climate changes
throughout the world. To levitate these problems, renewable energy
sources have been promoted as an alternative source. Moreover, for the
last decade, wind energy is being increasingly used for production of
electricity especially in Northern Europian countries as a substitute for
coal. Oil is, however, still the main energy source for the transport
sector both for the light and heavy transportation as well as aviation.
Electrification of the transport sector might solve some of the problems
in this sector along with biofuels produced from renewable biomass raw
materials. Biomass is produced in surplus amounts in most countries of
the world and a large part of this excessive amount can be utilized for
biofuels production without a negative impact on the ecosystem as a
whole [1]. The world total estimated production of lignocellulosic
biomass is 200 × 1011 tons/year of which 20 × 1011 tons/year could be
Biofuels are an attractive option to reduce the environmental im-
pacts of conventional fossil fuels but the first generation biofuels, which
are generally produced from the edible parts of the food crops has
created a competition between food and fuel. The competition between
food and fuel as well as the need for a higher reduction of greenhouse
gases has demanded alternative raw materials other than the food crops
to be used for biofuel production. Hence, residues from agricultural
production such as straw and husk as well as woody residues from
forestry have been in the center for biofuels production during the last
decade along with waste products such as municipal solid waste con-
taining high concentrations of lignocellulosic materials [3,4]. Lig-
nocellulose is the main component of biomass and it has been used for
the production of C5 and C6 sugars by pretreatment at high tempera-
ture and pressure, often with addition of either base or acids, followed
by enzymatic hydrolysis using cellulolytic enzymes [5]. Producing
biofuels and/or bio-products from cellulolytic sugars leaves the lignin

Abbreviations: L, Lignin; MSW, Municipal Solid Waste; LCC, Lignin Carbohydrate Complex; NMR, Nuclear Magnetic Resonance; HSQC, Heteronuclear Single
Quantum Coherence; AD, Anaerobic Digestion; APPL, Acid Precipitable Polymeric Lignin; SW, Soft Wood; HW, Hard Wood; LB, Lignocellulosic Biomass
∗
Corresponding author. Bioproducts Sciences and Engineering Laboratory, Washington State University, Tri-cities, 2710 Crimson Way, Richland, WA, 99354, USA.
E-mail address: bka@wsu.edu (B.K. Ahring).

https://doi.org/10.1016/j.biombioe.2019.105325
Received 15 January 2019; Received in revised form 24 July 2019; Accepted 31 July 2019
0961-9534/ © 2019 Elsevier Ltd. All rights reserved.
M.U. Khan and B.K. Ahring
fraction behind. The same is true for the paper making process, which
makes use of the cellulose, while the lignin residue is at best used for
steam production. As more bio-refineries will come into production,
more and more bio-refinery lignin residue will be produced. Utilization
of the entire lignocellulosic biomass can improve the economics of
using biomass materials in bio-refineries and will buy back the overall
cost of the products produced. While some paper producers such as
Borregaard Industry have found ways to produce a variety of products
from the lignin streams marketed through a special company Borre-
gaard LignoTech, most conventional paper and pulp industries have
still not implemented any valorization of this raw material besides
simple steam production [6]. The USA has a goal to produce 79 billion
liters of second generation biofuel annually by 2022. This will generate
62 million tons of lignin [7]. Therefore, there is a major interest in
finding ways to valorize lignin in bio-refineries and paper and pulp
industries. Focus has been on production of bio-oils from lignin using
pyrolysis or hydro-thermal liquefaction [8] -or on gasification to syngas
[9]. Work has further been done to investigate the possibility of using
lignin or lignin degradation products as a substrate for fermentation
[10]. The product(s) produced during fermentation could potentially be
the major product(s) in itself, or could be a platform molecule, which
can further be upgraded to the desired product(s).
A major limitation for biological conversion of lignin is the fact that
native lignin is more or less non-degradable without oxygen present,
while anaerobic conditions is a necessity for producing valuable pro-
ducts by fermentation [11]. Some but limited degradation of lignin is
generally seen in anaerobic environments such as the rumen of rumi-
nants [12] or after size reduction of straw fed to biogas reactors [13].
To make lignin a valuable raw material for fermentation it is, therefore,
important to understand the structural characteristics that makes this mo-
lecule recalcitrant under anaerobic conditions. In this review we will further
examine ways to make lignin degradable under anaerobic conditions as well
as the general pathways for anaerobic lignin degradation.
2. Structure of lignocellulosic materials
Cellulose, hemicellulose and lignin are the major building blocks of
lignocellulosic materials [14] as illustrated in Fig. 1. The origin, variety
and type of the plants are the main factors that determine the compo-
sition of these materials [15]. All the three components have been
discussed below.
2.1. Cellulose
Cellulose is the most abundant polymer and about 20–30% of the
primary cell wall's dry mass and nearly 50% of the mass in secondary
cell wall is composed of cellulose [16]. Cellulose is an unbranched
biopolymer of lignocellulose composed of glucose monomers bonded
together through β-1, 4-glycosidic linkage [16]. Cellulose has a general
formula (C6 H10 O5) n. The degree of polymerization (DP) of cellulose in
the primary cell wall is 250–500 and in the secondary cell wall it ranges
from 103-104 [17]. The glucose chains are assembled together by
Vander Waals interactions and hydrogen bonds. Through these bonds in
the molecule, three dimensional micro fibrils are formed. Every glucose
Fig. 1. Structure of lignocellulosic materials.
2
Biomass and Bioenergy 128 (2019) 105325
unit is linked with two intra chains and two to three inter chains of
hydrogen bonds. Due to these connections, the structure of cellulose
becomes stable and tightly packed. About 30–36 parallel chains of
cellulose are connected together to form each micro fibril, which are
embedded in other biopolymers making up lignocellulose i.e. lignin and
hemicellulose [18]. Cellulose contains both crystalline and amorphous
areas [19]. Cellulose is more resistant to thermal degradation than
hemicellulose because of the crystalline fraction [20]. The amorphous
fraction of cellulose is structurally less compact and can be degraded
easily by enzymes than the crystalline parts [20].
2.2. Hemicellulose
Hemicellulose is the other major polysaccharide after cellulose with
a lower degree of polymerization compared to the cellulose (100–200)
[21]. Lignocellulosic materials contain 20–40% hemicellulose on a
weight basis [21]. Hemicellulose is a mixture of pentoses, hexoses,
deoxy-hexoses and hexuronic acid but the most common type of sugar
present in hemicellulose is xylose [21]. Xylans and glucomannans are
the most abundant types of hemicellulose. Hemicellulose can be hy-
drolyzed easily as compared to cellulose by addition of dilute base or
acids as well as hemicellulase enzymes [22]. The contents and com-
position of hemicellulose vary between soft and hard wood. Even in the
same tree the contents and composition of hemicellulose vary con-
siderably in bark, stem, root and branches [22].
2.3. Lignin
Lignin is the third major component of lignocellulose and has a
complex, three dimensional structureconsisting of phenyl propane units
[23]. Lignin was described for the first time by Payen in 1838 and its
chemical structure was further described by Schulze in 1865 [24]. It is
the most abundant natural and renewable source of aromatic com-
pounds [23]. Lignin is an important component of lignocellulosic ma-
terials, which contributes up to 40% of the energy content and 30% of
the weight of these materials [24]. Lignin is an important constituent of
the cell wall because it provides the structural support and prevents
microbial attacks and decomposition of the cell wall [25]. By hindering
the permeation of water across the cell wall, lignin is also responsible
for the transport of nutrients and water in plants [25].
The recalcitrance of lignin has been an obstruction for the use of this
aromatic molecule for bio-products and solutions for degrading lignin
will allow for new industrial productions improving the economics of
lignocellulosic bio-refineries [26]. Lignin is both chemically and phy-
sically connected to cellulose and hemicellulose in lignocellulosic ma-
terials [27].
As a polymer, the bonding of lignin with cellulose and hemicellulose
are generally through a covalent connection with carbohydrates
(especially hemicellulose) leading to the so-called lignin carbohydrate
complex [28]. Lignin is structurally composed of cross-linking of three
hydroxycinnamyl alcohol monomers. These monomers include con-
iferyl alcohol (G-Type), sinapyl alcohol (S-type) and coumaryl alcohol
(H-type), and these units differ by the number of methoxy groups [28].
The arrangement of these methoxy groups affects the lignin structure
because these groups secure these positions from radical coupling re-
actions to form new bodings. The ratios of these monolignols vary
among different species of plants and affect the structure and properties
of the lignin [29]. Genetic alterations has often targeted the ratio of
hydroxyphenyl (H), guaiacyl(G) and syringyl (S) monomers, which in
turn will have an effect on for instance the pulping process. Generally
one type of monomer dominates depending upon the plant type; for
example the G-Type (G-Lignin) dominates in gymnosperms, whereas
dicotyledonous plants contains both G and S type units of lignin and all
three types of lignin monomers (GSH-Lignin) are present in the
monocotyledonous plants [30].
The three building blocks will be coupled together into a polymer
M.U. Khan and B.K. Ahring
Fig. 2. Different type of linkages in the lignin molecule.
through the action of radicals produced by redox reactions resulting
from the action of lignolytic enzymes [30]. The lignin content differs in
different species of lignocellulosic materials and hardwood contain
higher amount of lignin as compared to softwood [31].
The bindings of the different lignin sub-units uses various types of
carbon-carbon and carbon-oxygen bonds such as β-O-4 (phenylpropane
β aryl ether), β-β (dibenzodioxocin), α-O-4 (phenylpropane β aryl
ether), β-1 (1, 2 diaryl propane), 5-5 (biphenyl and dibenzodioxocin),
4-O-5 (diaryl ether) and β-5 (Phenylcoumaran) [32]. These linkages are
presented in Fig. 2 and their percentage in soft and hard wood is shown
in Table 1.
On the basis of reactivity the β-O-4 ether bond ranks highest com-
pared to the other bonds present in lignin [35]. Water soluble com-
pounds containing free phenolic groups are formed when the linkages
are broken [36]. The reactivity of lignin is affected by different func-
tional groups such as phenolic hydroxyl, carbonyl, noncyclic benzyl
ether, methoxyl and the aliphatic hydroxyl [28]. The chemical structure
of hardwood lignin vary among different plant species whereas soft
wood lignin does not vary much between different species but will be
significantly different from hardwood lignin [37].
3. Why lignin is important for biomass based bio-refinery
Currently the main challenge of biomass-based biorefineries is their
economics because of the fact that the biofuels are more expensive to
produce compared to fossil fuels owing to higher operational and per
capita cost of the production. The economics of biomass based bior-
efineries can be boosted if all parts of the biomass including the lignin
stream are used as a raw material for generation of high-value bio-
Table 1
Percentage of different types of linkages in Soft and Hardwood [28,33,34].
Linkage Type Soft Wood (%) Hard Wood (%)
β-0-4 45–50 60
α-O-4 2–8 6–8
β-5 9–12 7 cellulose fibrils, due to the tight connection between hemicellulose and
4-O-5 4–7 6.5
5–5 10–11 4.5
β-1 7–10 7
β-β 3 2
3
Biomass and Bioenergy 128 (2019) 105325
products and biofuels. Moreover, lignin is strongly bonded with the
other carbohydrates, which is a challenge for using the plant carbo-
hydrates for production of biofuel [38].
Lignin also interferes with the pretreatment process of lig-
nocellulose, and its degradation products can be inhibitors for the fer-
mentation process and further absorb hydrolytic enzymes used for hy-
drolysis of the pretreated material to sugars [31]. Therefore, lignin
removal is essential for efficient use of cellulose and hemicellulose.
Lignin has a high amount of aromatic constituents, which have many
applications for chemicals as well as polymer production, and many
studies have recently been done to find new optimal ways for making
use of this raw material [31].
4. Why pretreatment of lignocellulosic materials is necessary
The use of lignocellulosic materials as a feed stock for biorefineries
is gradually increasing. Degradation of lignocellulosic materials is,
however, hindered by the fact that the cellulose is strongly cross linked
and shielded by lignin, which prevent access of enzymes and microbes
to the cellulose [39]. Therefore, a pretreatment step is necessary for
opening the structure of the lignocellulosic materials by introducing
breakage in the lignin structure so that the cellulose fibrils become
exposed. An optimal pretreatment method is the one which can increase
the surface area, reduce the crystallinity of cellulose and depolymerize
lignin [40]. The largest operational cost for production of biofuels goes
to pretreatment of the lignocellulosic materials and it is important that
the pretreatment process can make all parts of the biomass accessible
for biofuels or bio-products formation during the process [20]. There
are many physical, chemical and, biological pretreatment methods,
which have been described for conversion of lignocellulosic materials.
These pretreatments include addition of acids such as dilute sulfuric
acid, alkaline pretreatment including ammonia fiber explosion or base
addition_, and use of different solvents [41]. During these pretreatment
methods a variety of chemicals are added having different effect on the
biomass materials such as dissolution of the hemicellulose with acid
pretreatment, which again will lead to removal of some lignin from the
lignin as shown in Fig. 1.
Besides fractionation of lignin, the combined dilute acid and steam
explosion pretreatment of biomass leads to acid catalyzed dehydration
of carbohydrates. During acidic conditions, the lignin will further re-
M.U. Khan and B.K. Ahring
condense and produce pseudo-lignin, when the pretreated material is
cooled after the pretreatment [42]. Often this re-condensation leads to a
more recalcitrant lignin fraction than the native lignin, which can be
prohibitive for cellulose hydrolysis as it often deposits on the cellulose
parts [43]. The base addition increases the solubilization of lignin more
effectively while the hemicellulose and cellulose solubilization is less as
compared to acidic or hydrothermal processes [44]. The addition of
NaOH decreases the crystallinity and polymerization of cellulose with
subsequent increase in lignin depolymerization [45]. Ethanol, Glycol,
ethylene, methanol and acetone are the main solvents which are used
during organosolve pretreatment, and these solvents will increase
lignin solubilization [46]. During the organosolv pretreatment the re-
condensation of lignin often occur as lignin droplets on the plant cell
wall's outer layer [47]. This lignin recondensation on the plant cell wall
further prohibits the enzyme action needed for production of biomass
sugars [43]. These side-effects indicate that during the pretreatment of
biomass with addition of chemicals, a number of unwanted reactions
often happens affecting the lignin structure. These changes in the lignin
structure as a result of the pretreatment can hinder the use of this lignin
for production of high value chemicals such as syringaldehyde, vanillin
and phenolics [43].
Anaerobic biogas plants for the production of biogas (methane and
carbon dioxide) from waste and lignocellulosic materials is a mature
technology. It is practiced world-wide and is in steep growth in Europe ,
especially in countries like Germany and Denmark, as an alternative to
phase out natural gas and substitute it with bio-natural gas, which has
been upgraded to the natural gas quality before being injected into the
natural gas grid. To boost the gas production, these biogas plants will be
supplied with agricultural residues such as straw, deep-litter and energy
crops in addition to manure and food waste. Due to the high con-
centration of lignocellulosics in these materials, pretreatment can be
benificial for the overall outcome of these plants. However, most of the
chemicals described above will have a negative impact on anaerobic
digestion [48]. Sulphur compounds , for instance will increase the
amount of H2S in the biogaswith further contamination of the residue,
which can limit its further use. Alkali based pretreatments can lead to
ammonia loss and pH problems for the process, while solvents will be
inhibitory for the methanogens and can lead to process failure [48]. It
is, therefore, desirable to use a pretreatment method which does not
rely on using polluting chemicals and which further produce a lignin
residue, which has not been negatively modified by chemicals inter-
actions.
4.1. Wet explosion pretreatment
During the Wet Explosion (WEx) pretreatment of lignocellulosic
materials (see Fig. 3), the material undergoes wet oxidation by addition
of oxygen followed by a steam explosion treatment. During this process
no chemical is added except oxygen as an oxidizing agent [43]. The
biomass will be instantaneously heated in the pretreatment reactor by
steam-injection to reach a temperature of between 140 and 210 °C and a
pressure of 0–3.5 MPa for a period of 10–30 min after addition of
oxygen [49]. The biomass is flashed into a pressure release tank after
the selected residence time in the pressure vessel. The flashing of the
pretreated biomass cause an abrupt decompression, which results in the
disruption of the biomass fibers.
The original WEx method was patented in 2004 [50] and was op-
erated in batch mode while the generation 2 of the process, the Ad-
vanced Wet Explosion pretreatment (AWEx) is a continuous process
with far more control [51]. The AWEx process has been operating in full
scale since 2016 to improve biogas yield of lignocellulosic materials at
Ribe biogas. In pilot scale the WEx process has been used for efficient
pretreatment of many types of lignocellulosic biomass materials such as
manure fibers [52], feedlot manure [53], lobally pine [42], bagasse
[49], corn stover and wheat straw [54]. The residence time, tempera-
ture and the oxygen-concentration used during the pretreatment are the
4
Biomass and Bioenergy 128 (2019) 105325
main factors controlling the severity of the pretreatment process. While
the Wet Explosion pretreatment method is superior for opening any
lignocellulosic biomass material tested so far it is by far the process of
choice for treating lignocellulosic material used as a substrate for
anaerobic digestion due to three mains reasons: (1) the Wet Explosion
pretreatment makes significant positive changes to the lignin fraction of
the lignocellulosic materials, which is the main barrier for accessing
cellulose during anaerobic degradation of lignocellosic materials, (2)
the pretreatment of lignocellulosic materials only makes use of oxygen
as an additive meaning that no harmful and costly chemical is added,
(3) there is no added cost for recovery of chemicals required after the
pretreatment process, which is necessary for pretreatments using sol-
vents or ionic liquids. The oxygen addition will act on the biomass to
relieve the lignin attachment both through depolymerization as well as
modifying the lignin along with hydrolysis of the hemicellulose fraction
into xylo-oligosaccharides and xylose [49].
4.2. Lignin structural changes by wet explosion pretreatment
Wet explosion of lignocellulosic materials will change the structure
both by solubilization of the hemicellulose and part of the lignin
whereas the cellulose fraction will be mainly retained with limited
structural changes. When wet exploded soft-wood was examined by one
dimensional 1H nuclear magnetic resonance spectroscopy it was seen
that the lignin fraction changed and somewhat depolymerized [43].
Furthermore,13C NMR spectra of the pretreated lignin was used to ex-
amine the composition of the different lignin units i.e. H, G and S within
the pretreated material and the spectra showed that the composition of
these lignin units significantly changes compared to the untreated
lignin. While the untreated softwood lignin primarily contains G lignin
with one methoxy group (-OMe) as well as acetyl groups, the wet ex-
ploded lignin has new linkages including carboxyl and C-H aromatic
linkages. The formation of these bonds indicate the formation of S-
lignin, as a result of the wet explosion process. Besides, it was found
that carboxyl groups are formed during the wet explosion treatment,
which might interact with the G-lignin and form S-structures. By 2-
dimensional HSQC NMR, it was further verified that the amount of S
lignin had increased significantly as a result of the pretreatment pro-
cess, while the amount of H units in the lignin was not detectable after
the pretreatment. It is, therefore, obvious to conclude that one of the
actions of the wet explosion pretreatment is to methoxylate the 3 and 5
positions of the aromatic ring structure of lignin and that these struc-
tural modifications can be the results of a combined de-polymerization
and oxidation reactions occurring within the lignin molecule. As a re-
sult of the pretreatment the fraction G lignin decreases while H lignin
gets undetectable-with simultaneous increase in the amount of S-lignin
through selective methoxylation [42]. During this process, some oxi-
dative cleavage of C-C bonds in the hemicellulose takes place resulting
in the production of acetic acid and acetate ion along with methyl
groups [42]. These acetate and methyl groups could be involved in the
methoxylation of H and G lignin when oxygen is present- but this ob-
servation needs further investigation [55].
As described earlier,the wet explosion pretreatment process results
in the formation of more S-like lignin structures. This was further ver-
ified after the testing of other biomasses such as hard wood and wheat
straw. The aromatic ring in these structures contain a methoxy group at
both the 3 and 5 positions, which makes this structure far more open
towards further degradation compared to the more condensed H lignin
structure. The β-O-4 linkages in S-like structures can be cleaved easily
as compared to G and H structures and making it a better raw material
for production of high value chemicals [56].
4.3. Effect of Wet Explosion pretreatment on the anaerobic degradation of
the lignin
Wet explosion pretreatment was further studied as a mean for
M.U. Khan and B.K. Ahring
Fig. 3. Effect of Wet Explosion pretreatment on lignocellulosic materials.
increasing lignin degradation during anaerobic digestion process.The
two types of lignin that are found in lignocellulosic materials are Acid
soluble and acid insoluble Acid insoluble lignin is the primary lignin
found in lignocellulosic materials. This lignin has a high molecular
weight-, normally greater than 850 kD. This type of lignin is recalcitrant
during anaerobic digestion because the anaerobic bacteria do not pos-
sess the oxidative enzymes and pathways, which are required for the
initial depolymerization of lignin [57]. To degrade lignin under anae-
robic conditions, the lignin has to be depolymerized into smaller frac-
tions, where the anaerobic bacteria can get access using their enzyme
pool to at least part of the molecule [58]. Alternatively, the lignin
structure has to be modified in such a way that the microorganisms can
get access even within the lignin molecule.
When lignin undergo pretreatment with the wet explosion process,
the lignin to carbohydrate ratio of the feed material increases, which is
an indication that hemicellulose as well as a small fraction of the cel-
lulose are solubilized during wet explosion process, while only small
fractions of the lignin is fully depolymerized. Therefore, most of the
lignin will remain behind in the solid fraction. However, the lignin to
carbohydrate ratio of the wet exploded material was found to decrease
far more after anaerobic digestion showing that both acid soluble and
insoluble lignin is consumed by anaerobic bacteria to produce methane
and carbon dioxide after wet explosion [53].
Not only the ratio between carbohydrates and lignin, but also the
extra methane production showed clearly that Wet Explosion makes
lignin available for anaerobic digestion as shown in Table .2. The di-
gested material was fed a lot of manure, which is mainly composed of
lignin after 6 month of decomposition including a composting step. It
was found that the methane yield increased considerably when the
biogas reactor was fed with wet exploded feed material compared to
untreated material.
In this study the authors further examined the influence of the
pretreatment on the composition of the materials in the feed and ef-
fluent of the AD process. The compositional analysis results of the feed
and effluent showed that the carbohydrates and lignin contents in the
5
Biomass and Bioenergy 128 (2019) 105325
Table 2
Effect of Wet Explosion pretreatment on lignin degradation during anaerobic
digestion [53].
Untreated Lignin Pretreated Lignin
Methane Yield∗ (L/kg-VS/Day) 70 ± 27 320 ± 36
Lignin Degradation (%) 12.6 44.4
Volatile Solid Destruction (%) 11.9 40.3
*Methane yield is reported as an average over 30 days during anaerobic di-
gestion of untreated feed lot manure in a CSTR at 55 °C and over 90 days during
anaerobic digestion of pretreated feed lot manure in a CSTR at 55 °C.
** Lignin degradation was measured by compositional analysis of the Feed and
Effluent.
*** Volatile Solids destruction was calculated by measuring the VS of the Feed
and Effluent.
untreated feed and effluent did not vary considerably because of a slight
conversion of carbohydrates and lignin into methane . This clearly
shows that both the carbohydrate and lignin fraction remaining in the
feedlot manure is highly resistant to further degradation, which fits
with the fact that the manure has been exposed to weather and further
degraded by composting after collection. In contrast, the carbohydrates
and lignin contents of the pretreated feed and effluent varied con-
siderably and the effluent had far lower lignin content and somewhat
lower carbohydrate content compared to the feed material, which is a
clear indication that a major change in the accessibility of the material
has occurred as a result of the wet explosion pretreatment. The de-
gradation of lignin was clearly improved during anaerobic digestion
after the wet explosion pretreatment process.
A total of 11.9% of the volatile solids fed to the AD reactor was
converted to methane in the untreated feed material, whereas the
conversion was 40.3% of the volatile solids, when the feed had been
wet exploded before the AD process [53]. Most of the volatile solids
degraded during the AD-process was lignin and overall 44.4% of the
lignin fraction was converted to biogas during the anaerobic digestion
process after wet explosion whereas only 12.6% was converted for the
M.U. Khan and B.K. Ahring
untreated feed as shown in Table 2. With our knowledge of the lignin
modification occurring during wet explosion like the methoxylation of
the lignin, it is obvious to assume that this specific modification is the
main reason that the wet exploded lignin is showing improved de-
gradability compared to the untreated lignin. As we will discuss later,
the methoxy group is the main point of attack for a lot of the anaerobic
microbes, which have been found today to be capable of converting
lignin degradation compounds.
When the lignin undergoes wet explosion pretreatment, lignin de-
composition products are formed along with a changed lignin structure.
As a result, products such as phenolics and aliphatic compounds are
formed. When the wet exploded material are anaerobically digested
and the effluent was analyzed most of these phenolic compounds
founds in the fed material were not detected in the effluent, which is an
indication that these lignin degradation compounds formed during the
wet explosion process were more or less totally converted into biogas
during the anaerobic digestion process besides a few exceptions: 2, 6
dimethyl phenyl isocyanate and 2-propyl phenol, which were not
consumed during AD [53]. For some unknown reasons these aromatic
substances were resistant in our reactor system. As mentioned pre-
viously, the soluble lignin degradation compounds will only account for
a minor fraction of the lignin converted after Wet Explosion while the
majority will be found a high-molecular insoluble lignin.
5. Biological degradation of lignin
5.1. General scheme for microbial lignin degradation
A large number of microbes have been described with lignin de-
grading capabilities. Some produces large lignin degradation fragments
whereas in other cases, smaller fragments with just a few aromatic ring
structures are produced [59]. Fungi is the fastest and most studied- but
a lot of new research has been done in bacterial degradation during the
last years. Anaerobic bacteria will not be capable of degrading native
lignin, however, lignin degradation compounds- or modified lignin can
be converted.
Importantly, there is a significant similarity between the overall
mechanisms used by all microbes studied so far. Overall, a number of
highly active oxidative enzymes are used in different combinations such
as Lignin Peroxidases, Managenes Peroxidases, Versatile Peroxidase,
Laccase and Dye-Decolorizing peroxidase [60]. These enzymes take
part in strong redox processes, wherein radicals or redox mediators are
produced [61]. These reactive molecules are small compared to en-
zymes and can get into the 3-dimensional lignin structure and perform
their functions on formation of bonds within the molecule. As a result
lignin degradation compounds will be formed, which can be attacked
by specific microbes either for further degradation or modification or
for final ring-opening of the aromatic ring structure. The final de-
gradation products of lignin depend on the availability of the electron
acceptors . In aerobic environments it will be CO2 while it could be H2S,
NH4 or methane, besides, CO2 in different anaerobic environments. An
illustration of the general model for microbial lignin degradation is
shown in Fig. 4.
5.2. Lignin degradation by fungi
Fungi is a major group of lignin degraders and does this far effi-
ciently than bacteria [62]. Furthermore, there has been more studies
done with fungal lignin degradation compared to bacterial degradation-
and this in particular for anaerobic conditions. As described, lignin is a
poly-phenolic, recalcitrant organic polymer with a cross linked com-
plicated structure, which is not degradable by direct enzymatic hy-
drolysis such as cellulose and hemicellulose. In spite of its recalcitrant
nature some microbes have the potential of degrading lignin through
the action of oxidative enzymatic reactions forming radicals and redox
mediators, which can act on the lignin molecule as described above
6
Biomass and Bioenergy 128 (2019) 105325
[63]. Some of the most studied microbes known for their lignin de-
grading capabilities are brown and white rot fungi [62]. These microbes
produces lignolytic enzymes such as Lignin peroxidase (LiP), containing
a heme cofactor, which has been isolated and described for Phaner-
ochate chrysporium [63]. Besides LiP, the white rot fungi produce a
variety of other enzymes, which can promote lignin degradations such
as laccases, lignin peroxidase, and manganese peroxidase. During lignin
degradation lignin fragments will be produced with different aromatic
structures. Different microbes can convert these lignin degradation
products and the final end-product is CO2 [62].
Even that both white and brown rot fungi are well-known fungi
capable of lignin degradation, the white rot fungi has been found to be
far superior compared to the brown rot fungi [64]. Studies have for
instance shown that white rot fungi can degrade lignin together with
cellulose ,carbon dioxide and water, whereas the brown rot fungi
mainly attack the cellulose [65]. The lignin degradation by white rot
fungi occur in two ways, a selective and a non-selective process.The
white rot uses a non-selective process where both polysaccharides and
lignin are degraded together [66]. In the selective process, the white rot
fungi attack mainly the lignin and hemicellulose and leave most of the
cellulose behind [67].
Brown rot fungi will only partially degrade the lignin and during
this degradation, the phenolic hydroxyl content of the reaction mixture
increases because of the partial oxidation and production of new car-
bonyl and carboxyl groups [68]. The brown rot fungi degrades the
lignin by the humification process wherein the lignin residues are
converted into fulvic and humic acids. Moreover the lignin degradation
by brown rot fungi also occurs by hydroxylation, demethylation and
oxidation of the side chains. During the lignin degradation by brown rot
fungi the aromatic rings remain conserved and the carbohydrates are
removed, leaving behind a dark brown lignin residue [69].
5.3. Lignin degradation by bacteria
Bacterial lignin degradation was not described before 1980's [70].
Since then a large number of studies have been done to examine the
potential of bacterial lignin degradation. These studies has revealed
that bacterial degradation can be slow- but even the wood surface can
be degraded at high moisture and low oxygen contents. It was further
shown that both hard as well as the soft wood can be degraded by
bacteria and that they can degrade all components of the cell walls,
fibers as well as vessel structures of plants [71].
Several bacteria have been found to be capable of degrading lignin
even under severe environmental conditions. Some of these bacteria are
thermo-tolerant having ligninolytic potential and can be interesting for
specific industrial processes using more extreme conditions [72]. For
instance, some soil bacteria belonging to the Actinomyces genus can
degrade lignin and produce metabolites of high molecular weight
during their degradation process. These products are known as acid
precipitable polymeric lignin [73].
Most thermal lignin degradation technologies are nonspecific and
will lead to a variety of products in low concentrations, which can be
difficult to separate from each other [74]. In contrast, the microbial
degradation of lignin is well controlled, and lead to specific products.
Several bacteria with lignin degradation potential have been identified
and in some cases their enzymes were also found useful for the de-
gradation of lignin in industrial scale [71].
Anaerobic digestion has not been extensively studied, when it comes
to degradation of lignin and its degradation products, but some studies
have shown conversion of lignin degradation products into methane
and carbon dioxide [75]. However, when it comes to native lignin, very
limited degradation will happen during anaerobic conditions while
anaerobic biodegradation has been demonstrated for several lignin
derived aromatics, phenols and benzoates showing up to 90% conver-
sion of these aromatic substrates and metabolic conversion into me-
thane and carbon dioxide [76]. During anaerobic degradation of
M.U. Khan and B.K. Ahring
Fig. 4. Microbial lignin degradation model.
methoxylated aromatic alcohol (coniferyl alcohol) it was found that
95% of the carbon was converted to methane and carbon dioxide [77].
Further it was shown, that the anaerobic degradation of methoxylated
aromatics (syringols) occurs by three distinct steps. During the first
step, de-methylation of the methoxylated aromatic molecule occurred
during which the methyl group from the methoxylated aromatic is re-
moved. During the second step, the de-methoxylated aromatic structure
is opened and converted to acetate, hydrogen and carbon dioxide,
which in the third step is converted into methane and carbon dioxide
[75].
Recently DeAngelis described lignin depolymerization under anae-
robic conditions by Tolumonas lignolytic, a facultative anaerobe bac-
terium. The strain was isolated from tropical forest soil and it was ob-
served that it utilized lignin as both a carbon and energy source during
the lignin degradation process. It was further determined that the lignin
degradation occur by several putative pathways [78]. The same re-
searchers have further isolated another facultative anaerobic bac-
terium, Klebsiella sp. which also degrade the lignin via a putative
pathway [79].
Lignin is an aromatic polymer, which cannot be degraded by low
redox potential oxidoreductases [80]. The three dimensional complex
bulky structure of lignin is the main barrier for bacterial degradation, as
was the case for the white and brown rot fungi . Like these microbes in
question, the ligninolytic enzymes produced by these bacteria cannot
directly attack the lignin molecule [80].
Instead, the ligninolytic bacteria generate extracellular oxidative
enzymes such as heme-peroxidases, which degrade the lignin through
oxidative reactions by forming highly active radicals. These radicals
generally have very high redox potential and will further be capable of
ring-opening of the aromatic ring [80]. The disintegration by radicals
will break the C4- ether and Cα-Cβ bonds in the lignin molecule. The
breakdown of these bonds results in the formation of aldehydes, which
is a major product formed during lignin depolymerization. Besides ring-
cleavage of the aromatic rings, radicals can further demethoxylate the
rings. The main product formed during demethoxylation process is
methanol and the cleavage of aromatic structures results in the
7
Biomass and Bioenergy 128 (2019) 105325
formation of muconate type structures [81].
Different studies on the anaerobic degradation of lignin and lignin
derived aromatics have proved that the native lignin under anaerobic
conditions will show only low lignin degradation. Modified lignin,
however, such as lignin with a high degree of methoxylation can be
degraded to a large extent under AD as describes earlier and give rise to
methane and carbon dioxide [53]. Besides, the soluble lignin can be
degraded to a high degree in anaerobic environments.
When the aromatic compounds are anaerobically digested by sulfate
reducing and fermentative bacteria, it is found that the anaerobic
bacteria utilize O-methyl group as carbon and energy source and the
methane yield increases when fermentative bacteria are present capable
of degrading the aromatic ring structure [82]. The fermentative, nitrate
and sulfate reducing anaerobic bacteria can also degrade the O-de-
methylated derivatives of syringate and vanillate i.e. gallate and pro-
tocatechuic acid (PCA), respectively. Studies of anaerobic digestion of
O-demethylated derivative of syringate i.e. gallate showed that the
fermentative bacteria Pelobacter acidigallici and Eubacterium oxidor-
educens both are capable of degrading these compounds through the
phloroglucinol pathway [83]. During the anaerobic degradation of
lignin, the β-aryl-ether bond and other bonds in the Lignin Carbohy-
drate Complexes will break into lower molecular weight compounds
which eventually end as methane and carbon dioxide [84]. When Kraft
lignin was used as substrate for AD it was found that only 15% of the
lignin was converted to methane and carbon dioxide [85]. Lignin can
also be degraded under anoxic conditions by many types of facultative
anaerobic and microaerophilic bacteria [86]. Some of the anaerobic
bacteria capable of degrading lignin and lignin derived aromatic have
been presented Table 3.
Overall, anaerobic lignin degradation studies have shown that the
anaerobic bacteria degrade the lignin in three steps as previously de-
scribed with demethoxylation as the initial step, followed by ring
cleavage and fermentation leading to precursors of the last step, me-
thanogenesis [75,88,89].
M.U. Khan and B.K. Ahring Biomass and Bioenergy 128 (2019) 105325

Table 3
Anaerobic Lignin degrading bacteria.
S.No. Bacteria Family Substrate Reference

1 Enterobacter lignolyticus SCF1 Enterobacteriaceae Alkali Lignin [87]

2 Methanomethylovorans Methanosarcinaceae Kraft Alkali Lignin [88]
3 Methanoculleus sp. Methanomicrobiaceae Kraft Alkali Lignin [88]
4 Tolumonas lignolytica Aeromonadaceae Alkali Lignin [78]
5 Sporomusa sp. Veillonellaceae Methoxylated Aromatics [79]
6 Klebsiella sp Enterobacteriaceae Alkali Lignin [83]
7 Magnetospirillum Rhodospirillaceae Poplar Wood Chips [86]
8 Clostridium methoxybenzovorans Clostridiaceae Vanillate [89]
9 Clostridium methoxybenzovorans Clostridiaceae Isovanillate [89]
10 Clostridium methoxybenzovorans Clostridiaceae Vanillin [89]
11 Clostridium methoxybenzovorans Clostridiaceae Vanillyl alcohol [89]
12 Clostridium methoxybenzovorans Clostridiaceae Syringate [89]
13 Acetoanaerobium sp Eubacteriaceae Kraft Lignin [90]
14 Rhodococcus jostii Nocardiaceae Kraft Lignin [91]

6. Understanding lignin degradation in the context of anaerobic
digestion
The overall understanding of anaerobic digestion and the general
scheme for the microbial conversion process is today limited to the
conversion of carbohydrates, proteins and lipids (see Fig. 5). In this
overall scheme, hydrolysis is the first step-a process involving a direct
action of enzymes produced by acidogenes on the different polymers
and degrading these materials into monomers, which can then be taken-
up by the microbes and fermented.
While this model has proven to give valuable information about the
processes active during AD in biogas plants for waste and wastewaters it
still lack some important informations about the processes active for
conversion of fibrous materials containing large amounts of lignin such

Fig. 5. Steps involved in anaerobic digestion of an organic material and modified lignin.

8
M.U. Khan and B.K. Ahring
as lignocellulosic biomass material, deep litter or even manure, where a
major part of the raw materials are composed of lignin. While most of
the native lignin will be left untouched during AD, lignin which has
been modified as described in this paper is available for degradation
(see Fig. 5). This will be true for lignin, which has selectively been
methoxylated containing S-structures with methoxy groups in both 3
and 5 position of the aromatic ring.
7. Future perspectives
Lignin is not inert under anaerobic conditions and the lignin waste
stream from a biorefinery can, after modification, be used as a substrate
for biogas plants. Lignin solubilization and depolymerization is the
main hindrance in lignin degradation during anaerobic digestion. An
oxidative pretreatments such as the wet explosion pretreatment can
increase the lignin depolymerization and solubilization. To understand
the necessary changes a detailed structural characterization is required
to study the lignin fate during anaerobic digestion for future exploita-
tion of lignin as a substrate for biogas production. Furthermore, this
study could be helpful in producing valuable aromatic products from
the lignin before final anaerobic digestion.
8. Conclusion
In this paper we demonstrate that lignin is not always inert under
anaerobic conditions. In this way anaerobic digestion can be used to
produce biogas from the lignin waste stream produced from the lig-
nocellulosic biorefineries at least if the material has been modified by
an oxidative pretreatments process such a Wet Explosion. During this
pretreatment process the lignin gets depolymerized and methoxylated
and is mainly composed of S- structures. As a result it can be attacked
by anaerobic bacteria through a chain of processes starting with de-
methoxylation followed by ring-clevage and fermentation leading to
production of methane and carbon dioxide. With this in mind, under-
standing the ways to improve the methoxylation process of lignin be-
fore its addition to ruminants or biogas systems might be an important
mean to improve the conversion of lignin in these systems.
Acknowledgements
This work was supported by a grant from Easterday Farm to support
research on Anaerobic Digestion and from The Government of Punjab
in collaboration with University of Agriculture, Faisalabad, Pakistan to
Muhammad Usman Khan for studies abroad.
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