Modern College of Arts, Science and Commerce

Ganeshkhind, Pune
Department of Microbiology

Review Article:
Isolation of Lignin and Lignin Degraders

By- Shraddha Rakesh

Pardeshi
Guided by- Dr. Gauri Phadke
Class-M.SC
(Microbiology)
Roll
No.- 1940572
TABLE OF CONTENTS:

CONTENTS PAGE NO.

Abstract 2

Introduction 2
Isolation of lignin and its 3
significance

Isolation of lignin degraders 8

and its significance

References 12

1
 Isolation of Lignin and lignin Degraders
Abstract
Lignin is a major component of soil organic matter and also rich source of carbon dioxide in
soil. Lignin can be isolated from different sources like wheat straw, soil and marine
environment. However, the lignin has its complex structure and recalcitrant nature, lignin
degradation is a major challenge. The discovery of lignin degrading enzymes from bacteria
could provide advantages over fungal enzymes in terms of their production and relative ease of
protein engineering. In this review, it’s an overview of isolation of lignin and lignin degraders
from different sources and the applications of lignin

Keywords: Lignin, Kraft lignin, degradation, soil, Wheat straw

Introduction:
Lignin is a class of complex organic polymers that form key structural materials in the support
tissues of vascular plants and some algae. Lignin are particularly important in the formation of
cell walls, especially in wood and bark, because they lend rigidity and do rot easily. Chemically
lignin are cross linked phenolic polymers. It is the most abundant source of carbon in the soil
after cellulose. Lignin degradation can thus play a major role in improving earth’s biofuel
resources and also serve as an alternative to harsh technologies used in the paper and pulp
industry. Degradation studies are mainly biotic, aerobic, and co-metabolic. Studies have shown
that certain bacteria and fungi are able to break down various biopolymers in soil.
Lignocellulosic biomass degradation has been widely studied in wood-rotting Baciomycetes
fungi due to their potential to degrade lignin.[4]

Lignin structure and its biosynthesis

In the plant cell, lignin is biosynthesized by the combination of three basic hydroxycinnamoyl
alcohol monomers or monolignols:
1. p-Coumaryl alcohol;
2. Coniferyl alcohol;
3. Sinapyl alcohol.

These monolignols are often referred to as phenylpropanoids, which differ in the substitutions at
the 3-C and 5-C positions in the aromatic ring. Lignin synthesis starts with the random self-
replicating radical coupling of phenoxy radical to form an oligomeric product. After
polymerization, these polymers are referred as p-hydroxyphenyl (H), guaiacyl (G), and syringyl
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(S) (from p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, respectively).
Monolignols are linked either by C–C bond or c-O–C bond, and more than two third of
monolignols are joined by ether linkages. [4]

Different type of linkages in the lignin molecule [4]

[A] ISOLATION OF LIGNIN AND ITS SIGNIFICANCE:

Lignin can be isolated from different sources (i) Soil (ii) Wheat Straw

(i) Lignin in soil

Because of the high inflow of organic aromatic matter into the soil, lignin is considered a major
component of soil organic matter. The High stability and low degradability of lignin soil
contribute to increasing humus formation. The copper oxidation (CuO) method is commonly
used for the characterization and quantification of lignin in soil. Oxidation by CuO yields
phenolic compounds such as vanillyl, syringyl, and cinnamyl type compounds. These
compounds reflect the origin and extent of lignin decomposition in the soil. The sum of the
above three monomeric phenolic components gives an estimate of total lignin in the soil,
whereas the carboxylic acid to aldehyde ratio conveys the extent of lignin decomposition
3
Studies has shown that biotic, aerobic, and co-metabolic degradation are the main processes
involved in lignin degradation.

Lignin Distribution in Different Soil Horizons

Distribution of lignin in different soil limits has been discussed by many groups. The lignin
content in the soil decreases from the upper soil horizon to the lower soil horizon. However, in
a few cases, an increase in lignin content of soil organic matter (SOM) with depth has been
observed. Lignin distribution in soil could also vary with location
A relatively higher rate of lignin degradation is found in lower horizon of soil as compared to
the upper horizon because the acid-to-aldehyde ratios of the vanillyl and syringyl units are
greater in lower horizon. An increase in gradient is found from organic to mineral horizon. This
is in accordance with the decreasing vanillyl, syringyl, and cinnamyl phenolic-lignin contents
with the depth and limited supply of fresh organic materials in deep soil horizons. The particle
size of soil components also influences the lignin content and acid-to-aldehyde ratio. The lignin
content of SOM decreases from the coarse to the finest particle-size fractions while the acid-to-
aldehyde ratio increases with decreasing particle size and is the highest in the clay fraction.

Isolation of lignin from soil

Soil samples for isolation of microorganisms

1.Samples were taken at different altitudes from sites corresponding to the main soil types in
forest regions in the north of the island, where plant litter derives predominantly from trees and
shrubs of an endemic thermophilic laurel forest.
2. These soils are classified as ferrallitic and fersiallitic.
3. Analyzed soils have been formed on volcanic materials and are rich in clay and lime. The
organic matter content of the ferrallitic and fersiallitic soils varies from 2 to 6%, and the C-to-N
ratio between 8 and 17.
4. Soil samples in the south of the island were taken at 800-1200m, a zone comprised of brown
andic, modal and fersiallitic acidic soils.
5. Their organic matter content varies between 6 and 18%, with a C-to-N ratio between 6 and
14.
6. Plant litter on these tested sites is predominantly from Pinus canariensis.
7. Counts of microorganisms were carried out with 5 g aliquots of each soil sample,
resuspended into 95ml of 0.85% NaCl solution by 30min shaking; serial dilution of the
suspension and plating on specific media at 37 and 28°C.
8. Multiple C-N source agar (MCNS) supplemented with cycloheximide (50 µg ml-1), rose-
bengal medium (RB) supplemented with chloramphenicol (50 µg ml-1) and basidiomycete
selection medium (BS) supplemented with ampicillin (250 µg ml-1) according to Hunter-Cevera
et al. (1986), were used to isolate total aerobic bacteria and fungi, respectively.
9. To isolate actinomycetes, two 5 g samples from different soils were thermically treated at
l00-120°C for 1 h, or at 65°C for 2 h, treated as described above and then inoculated on
4
glucose-asparaginecasamino acids agar (GAC) and arginine-glycerol salts agar (AGS),
respectively.
10. These media were supplemented with cycloheximide (5Oµg ml-1) for AGA medium and
cycloheximide (5Oµg ml-1), polymyxin (2Oµg ml -1) and penicillin (40 µg ml-1) for GAC
medium.

Selection of microorganisms with ligninolytic potential

The basal mineral medium described by Janshekar et al. (1982) was used to isolate fungi and
that by Odier and Monties (1978) for bacteria and actinomycetes. These media were
supplemented with the purified Kraft lignin (PKL, 0.1 %, w/v) obtained from Indulin AT
(Westvaco, North Charleston, SC, U.S.A.), after thorough purification as described by Kadam
and Drew (1986), with minor modifications to remove low molecular weight impurities. These
consisted of multiple washings of the purified lignin with ethyl acetate, until no compounds
were detected by TLC with ethyl acetate-methanol (9: 1) at the solvent system and Folin-
Ciocalteu as developing reagent. Finally, PKL was washed with acidified water at pH 2.5 with
0.2 N HCI and vacuum dried to constant weight. The enrichment medium was made selective
for the growth of fungi, bacteria and actinomycetes, by
addition of antibiotics at similar concentrations to those applied in the isolation procedure on
nutritive media.

Biochemical tests

Microorganisms were subjected to the Bavendamm test to monitor phenoloxidase

(Bavendamm, 1928); to the Nobles test, to monitor extracellular oxidase (Nobles, 1958), along
with lactase, tyrosinase, peroxidase and hydrolysis of ferulic acid tests (Rayner and Boddy,
1988). The Bavendamm test was carried out using malt extract agar containing tannic acid
(0.5%, w/v). Extracellular oxidase, lactase, tyrosinase and peroxidase tests were performed over
growing microorganisms on malt extract agar, by adding to the tested colonies gum guaiac (0.5
g in 30ml 96% ethanol), guaiacol (0.1 M in 96% ethanol) and p-cresol (0.l%, w/v, in 96%
ethanol), to detect extracellular oxidase, lactase and tyrosinase, respectively (Rayner and
Boddy, 1988). Equal parts of 0.4% (v/v) H2O2 + 1% (w/v) pyrogallol in water was used to
detect peroxidase activity. Hydrolysis of the lignin related aromatic was determined in malt
extract media containing 0.06% (w/v) of ferulic acid (Rayner and Boddy, 1988). Cellulolytic
activity of microorganisms was detected on solid medium containing cellulose (0.5%, w/v)
stained with aniline blue-black (0.005%, w/v). Decolorization around growth zones indicates
cellulose degradation according to Kauri and Kushner (1988).

Identification of selected strains

Microorganisms were identified by the Laboratoire de Mycologic Systematique et Applique,

Universite Catholique de Louvain, (MUCL), Louvain-la-Neuve, Belgium. [1]

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(ii) Isolation of lignin from wheat straw

Wheat straw (Triticum aestiuium) was chosen as the forage source for development of the
lignin isolation procedure as it is high in lignin and is generally poorly digested. The straw was
ground through a 1-mm screen prior to analysis. Total cell wall fiber (neutral detergent fiber),
hemicellulose (neutral detergent fiber, minus acid detergent fiber), cellulose (acid detergent
fiber minus 72% H2S04 acid detergent lignin), and lignin (acid detergent lignin minus ash) were
determined by the sequential detergent procedure (Van Soest and Robertson, 1980). Crude
protein was measured as Kjeldahl N X 6.25 (AOAC, 1975). Neutral detergent fiber (Van Soest
and Robertson, 1980) was prepared from the wheat straw for subsequent analysis. Organic
matter was determined by ashing at 500 °C for 3 h.

Grinding Time: A 50-g portion of wheat straw neutral detergent fiber was ground in an
Attritor agitating ball-mill (Union Process, Akron, OH). The 750-mL pot was filled with the
sample and 1700 g of 3.06-mm stainless steel bails. Sufficient toluene was added to cover the
sample and grinding media. The sample chamber was cooled by a water jacket. Grinding speed
was set at half full range (~300 rpm). Samples were taken after 1,2,4,6,8,12, and 16 days of
grinding. The entire contents of the sample chamber were removed at each sampling time. The
sample was strained through a 1-mm screen to remove the stainless-steel balls, and then a 25-
mL aliquot of the sample slurry was removed for analysis. The rest of the sample was returned
to the sample chamber for continued grinding. The 25-mL samples were filtered through
Whatman 54 filter paper (Whatman Paper Ltd.). The ball-milled residue was then allowed to
air-dry prior to lignin extraction.

Cellulase Hydrolysis Time: The ball-milled residue of wheat straw ground for 8 days was used
to determine optimum hydrolysis time with cellulase. The cellulase was derived from
Aspergillus niger and is reported to contain substantial hemicellulose activity. The sample was
suspended in 20 mL of 33 mM acetate buffer, pH 4.5, per gram sample. The acetate buffer
contained 5 mg. rmL-1cellulase for a substrate to enzyme ratio of 10: l. This ratio of substrate to
enzyme was a compromise be- tween activity and amount of enzyme needed for large batches
of substrate. A few drops of toluene were added as a preservative. Samples were incubated at 39
°C for 1, 2, 4, 6, or 8 days. Samples were filtered, washed with water, and air-dried prior to
lignin extraction of the hydrolysis residue.

Lignin Extraction.: Wheat straw samples from the grinding time and hydrolysis time
experiments were extracted with 96% (v/v) dioxane-water. The samples were extracted with 10
mL-g-' sample weight at room temperature for 24 h. After filtration, the extracts were diluted
1:100 with 96% dioxane and absorbance at 280 nm was measured. The influence of solvent
composition on lignin extraction was determined by extracting samples ball-milled for 8
days only or ball-milled and hydrolyzed with cellulase for 4 days. Samples were extracted as
before with dioxane water mixtures containing 0, 20, 40, 60, 80, 96, or 100% dioxane.
Efficiency of lignin extraction was measured as absorbance at 280 nm.

6
Lignin Isolation: Wheat straw lignin’s were isolated for chemical characterization by ball-
milling the straw for 8 days, followed by hydrolysis with cellulase for 4 days, and successive
extraction of the residue with 96 and 50% dioxane. The extraction solvent was removed from
the 96 and 50% extracts by lyophilizing. The residues were then redissolved in a minimal
amount of 90% acetic acid. The 96 and 50 % dioxane-soluble, water-insoluble lignin’s were
precipitated by adding the acetic acid solutions to large volumes of water. The lignin’s were
recovered by centrifugation and lyophilized. The aqueous supernatants from the lignin
precipitation steps were also lyophilized to recover the dioxane-soluble, water-soluble lignin
fractions.

SIGNIFICANCE OF LIGNIN:
 Lignin is used in Adsorption of Heavy Metal Ions in Water
 Lignin was used to treat the surfaces of natural hemp fibers to utilize lignin’s natural
affinity for cellulosic fibers.
 Lignin as a potential carbon fiber precursor.
 Lignin’s natural antioxidant properties provides use in cosmetic and topical formulations.
 Lignin enhances performance of energy storage devices
 Alkaline fragmented/purified lignin mixed with diesel using surfactants/emulsifiers
 Phenols prepared by taking lignin reacting with a H‐supplying solvent at elevated
temperature/pressure.
 Polymerization of acrylamide and hydroxy methylated shown to enhance tensile strength
of paper by about 40%

The most important starting material for carbon fiber production is polyacrylonitrile (PAN), but
due to high costs, these carbon fibers remain a niche product for use in high-end industrial
applications, aerospace, sporting equipment and different luxury automotive products However,
in the recent times, the interest in lignin for its use as an inextensible raw material for the
production of carbon fibers of reasonable qualities is rapidly growing.

BIOMEDICAL APPLICATIONS OF LIGNIN

Anti-tumor activity
Cancer is a disease with the highest incidence and mortality worldwide. The biodegradability,
biocompatibility and chemical stability of lignin, as well as the possibility to obtain it as
nanoparticles, make lignin a potential candidate for applications in cancer therapy. In 1998, Lu
et al. reported that lignin could play a role in protecting against colon cancer. Podophyllotoxin
is a lignan known as the most active cytotoxic natural product, being used as starting compound
for the synthesis of anticancer drugs etoposide and teniposide. A recent study related to the in
vivo effect of deoxypodophyllotoxin on gastric cancer cells in a xenograft model evidenced its
inhibitory effect. Two carbohydrate–lignin metabolites with anti-tumor activities were separated
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from a hot-water extract of Inonotus obliquus. Cytotoxicity tests showed that these lignin
compounds induced cell apoptosis and inhibited the activation of the nuclear transcription factor
NF-κB in cancer cells. The cytotoxic effect of different lignin’s was studied using the cell
membrane of human keratinocyte HaCaT and murine fibroblast 3T3 cells. The report revealed
that lignin’s presented good antioxidant properties at low concentrations and cytotoxic effects at
high concentrations. Pancreatic lipase has an important role in the efficient digestion of
triglycerides, being responsible for the hydrolysis of 50-70% total dietary fats. Serum pancreatic
lipase is associated with many kinds of diseases. The inhibitory effect of lignin on pancreatic
lipase, through the disruption of substrate emulsification in a heterogeneous reaction system
was demonstrated, as well as its direct activation effect through the formation of a lignin–
pancreatic lipase complex in a homogeneous aqueous reaction system. The positive effect of
lignosulfonic acid for the treatment of inflammation-induced intestinal barrier dysfunction
observed in inflammatory bowel disease was reported. It enhanced the tight junction barrier
function and ameliorated the intestinal barrier disruption induced by the inflammatory
cytokine’s TNF-α and IFN-γ. [16]

[B] ISOLATION OF LIGNIN DEGRADERS AND ITS SIGNIFICANCE:

i)Lignin Degradation in soil. (ii) Lignin Degradation by Bacteria
Steps in Lignin Degradation

Lignin biodegradation involves both depolymerization and aromatic ring cleavage Extracellular
enzyme brought about oxidation of lignin in the following steps:

1. Oxidation of ß –O–4 linkages to aryl glycerol compounds;

2. Aromatic rings cleavage, mostly follows the ß–ketoadipate pathway;
3. Cleaved aromatic rings coupled with ß–O–4 oxidation leads to the formation of cyclic
carbonate structures.

Enzymes involved in lignin degradation:

1.Lignin Peroxidase (LiP, EC 1.11.1.14)

2.Manganese Peroxidase (MnP, EC 1.11.1.13)
3.Versatile Peroxidase (VP, EC 1.11.1.16)
4.Laccase (Lac, E.C. 1.10.3.2)

(i)Lignin Degradation in Soil:

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The Lignocellulosic complex in the plant cell wall contains approximately 40 to 60% cellulose,
20 to 40% hemicellulose, and 10 to 25% lignin, which provides rigidity to the cell wall
structure. Certain enzymes from specialized bacteria and fungi have been identified by
researchers that can catalyze a number of oxidative and hydroxylation reactions, depolymerize
the phenolic and non-phenolic lignin polymer, and also mineralize the insoluble lignin. The
orientation, adsorption, and diffusion of the ligninolytic enzymes in the soil solid phase affect
the lignin degradation in soil.

In laboratory studies, the impact of soil particle size on soil respiration was observed by Datta et
al., which can, in turn, affect lignin degradability in soil.

Illustration of ligninolytic enzymes and their selective action on lignin components [4]

(ii) Lignin Degradation by Bacteria

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Kraft lignin degradation by Pandoraea sp.
Bacterial Strain and culture conditions
The bacterial strain Pandoraea sp. B-6 was isolated from the steeping fluid of eroding bamboo
slips. The bacteria were grown in the Luria–Bertani broth medium at 30°C with shaking at 120
rpm until the optical density at 600 nm (OD600) of inoculum reached approximately 1.0. Two-
millimeter aliquots of this culture were aseptically inoculated into triplicate flasks containing
100 ml sterile KL mineral salt medium. The flasks were incubated at 30°C with shaking speed
of 120 rpm for 7 days. These cultures were used to investigate the effects of the temperature,
pH, and initial KL concentration on KL degradation by Pandoraea sp. B-6. The pH values were
monitored with a pH meter during adjustment of buffer to required pH

Bacterial growth and COD measurements

Degradation experiments were carried out on a rotary shaker (120 rpm) under aerobic
conditions at 30°C, pH 10 for 7 days. The rate of Pandoraea sp. B-6 growth was determined by
measuring the OD600 of cultured samples withdrawn at intervals on a Hitachi U-4100
spectrophotometer using centrifuged uninoculated medium as a control in 1.0-cm cuvette path
length cells. The control and cultured samples were centrifuged at 10,000 rpm for 10 min to
remove biomass and suspended solids, and the chemical oxygen demand (COD) of the
supernatant was measured by the fast digestion-spectrophotometric method (the Environmental
Protection Industry Standard of the People’s Republic of China, HJ/T 399-2007).

Measurement of the color removal

The intensity of color, before and after incubation, was

determined by the standard method of the Canadian Pulp and Paper Association. The amount of
color present was determined spectrophotometrically and was related to the absorbance of a
PtCo standard solution at the same wavelength. The samples were centrifuged at 10,000 rpm for
10 min to remove suspended solids. The pH of the supernatant was then adjusted to 7.6, and
thereafter the absorbance at 465 nm against distilled water was measured using the
spectrophotometer. The absorbance values (A) were then transformed into color units (PtCo) as
follows:
CU (PtCo) = 500×A2/A1
where A1 corresponds to the A465 of a 500-CU platinum cobalt
standard solution (0.132); and A2 is the absorbance of the effluent sample.
The ability of the Pandoraea sp. B-6 to efficiently degrade Kraft lignin. Pandoraea sp. B-6 grew
well in the sterile Kraft Lignin mineral salt medium without any co-substrate. For Kraft Lignin
degradation by Pandoraea sp. B-6, the optimum pH was 10 and the optimum temperature was
30°C. In the range from pH 7 to 12, the greatest COD and color removal and the highest levels
of activity of extracellular ligninolytic enzymes were observed at pH 10. Pandoraea sp. B-6 has
significant potential for use in applications requiring for lignin degradation and for treatment
10
of black liquor or other KL containing pollutants before their release into the environment.[12]

SIGNIFICANCE OF LIGNIN DEGRADATION:

Lignin is present in bound form it is necessary to degrade the lignin which can be degraded by
bacteria’s and enzymes as mentioned above for the following applications:

Degraded lignin fragments are building blocks of the humic compounds in soil. Hence, lignin
degradation has received vast attention. Lignin Degradation by various Fungi and bacteria
present in plant biomass and soils that are capable of producing ligninolytic enzymes such as
LiP, MnP, VP,DyP.Most of these enzymes are substrate specific, in contrast to Lac activity
which oxidizes a variety of substrates like polyphenols,diphenols,benzenethiol and aromatic
amines.lignn degraders also used to study genomics, biochemistry, and proteomics will uncover
the role of ligninolytic enzymes in the coming years. They are also used in paper pulp
bleaching, Detoxification ,Fiber modification, dye colorization, removal of phenols from wines
etc.

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