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Biodegradation of lignocellulose in soil: basic understanding of degradation mechanism.
Abstract:
"In all things of nature there is something of the marvelous.”—Aristotle.
Soil is a natural reservoir for all kinds of living being which controls the biogeochemical cycles
through the regenerative and degradative process. Understanding the lignocellulosic breakdown in
the soil system could be a valuable source which can be of great benefit for the utilization of
lignocellulosic materials to extract the valuable chemicals for the welfare of human beings. This paper
discusses about the current knowledge on the soil biodegradation system particularly in the lignin
degradation process. Lignin degradation in soil system occurs in two stages, modification in its
chemical structure and aggregate formation. The degradation of the complex aromatic rings in the
lignin polymer occurs as a result of aggregate formation called humus, further degradation of these
colloid results in humic acid, fulvic acid and humin. These structures resemble with the lignin
aromatic structures. Therefore, the understanding of the formation of humus could help in elucidating
the lignin deconstruction mechanism in soil. The present challenge lies in understanding the detailed
structural change of the humic acid, fulvic acid, and humin with respect to lignin. The lignin
degradation pathway in soil provides us a new perspective for improvement of biological pretreatment
of the lignocellulosic biomass for making biofuel and chemicals.
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1. Introduction:
According to the latest survey made by Thomson Reuters (2009), the U.S. ethanol consumption is
forecast to increase from 5.6 billion gallons last year to 13.5 billion gallons in 2012, far more than the
7.5 billion gallons in 2012 originally estimated. In order to decrease the green house gas emissions
,an alternative energy source that is capable of producing reduced amount of CO2 and also has being
cost effective is needed(Groom et al, 2008). The CO2 emissions estimated increase from 5,890
million tons in 2006 to 7,373 million tons in 2030.12 % of the green house gases emissions are
decreased by the production and combustion of bioethanol (Jason et al, 2006). Biofuel can be
produced from sugarcane, corn, lignocellulosic feedstock such as wheat straw, wood chips, and other
oil yielding plants such as pongamia and jatropa. Among most of the sources for used for the
production of biofuel, the lignocellulosic based biofuels has shown a great capability of replacing the
fossil fuel as they help in green house gas emissions (gas that contributes to the greenhouse effect by
absorbing infrared radiation) and also reduced release of CO2. Apart from these they would not
intense effect the ecological diversity when compared to others (Powlson et al, 2005; Farrell et al,
2006; Groom et al, 2008). When there is such rapid increase in the demand for biofuel, the need for a
breakthrough is necessary to meet the demands and reduce the green house gas emissions.
In order to produce biofuel from lignocellulosic biomass, the greatest challenge lies in the
deconstruction of lignin polymer for the release of sugars, which are used for the biofuel production.
Basically, lignocellulosic materials are the plant cell wall composites which are composed of 40-55%
of cellulose, 24-40% of hemicelluloses and lignin of about 18-25% in hardwood (Howard et al,
2003). In wheat straw the amount of lignin is about 18-20% (Tuomela et al, 1999) and about 25-35%
in soft wood (Nausbaumer et al, 1996). In the current context, bioethanol production from
lignocellulosic biomass would be mainly from the cellulosic (source of C-6 sugars) and hemicellosic
(source for C-5 sugars) components, and other useful organic components can be derived from the
lignin component which can be used for making bioplastics, dispersants in cement industry, additives
in agricultural chemicals, textile dyes, and carbon fibers (Jerfrey at al, 1983; Howard et al, 2003;
Roberto et al, 2003; Parajo et al, 1998). Recovery of cellulose portion from the biomass needs the
disruption of lignin and hemicellulose matrix. However, this process is considered as a challenge
making it more efficient, cheapest and safest technology.
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Lignin is a structure polymer of the vascular plants which helps in protecting the plant cell wall.
Degradation of this lignin compound is a major task because covalent lignin carbohydrate linkage
prevents the enzymatic degradation of lignocellulose and this is due to the fact that lignin can
depolymerize to oxidative units or co –polymerize and form complex aromatic structure.
Pretreatment is the process of releasing the cellulosic sugars by using various technologies such as
physical, physico-chemical, chemical, and biological processes. These different technologies that
have been used for pretreatment of lignocellulosic (Ye Sun et al, 2001) materials are acid treatment,
ammonia fiber/freeze explosion treatment, liquid hot water treatment, lime pretreatment etc. The acid,
lime and (ammonia fiber explosion) AFEX pretreatment technologies remove lignin significantly,
decrystallizing the cellulose which can be utilized for the bioethaol production. (Mosier et al,
2005).During the acid treatment dilute sulfuric acid is mostly used (Grohmann et al, 1985). For the
AFEX pretreatment, it is suitable for only hardwood rather than soft wood (Millan, 1994; Mosier et
al, 2005). As a part of pretreatment, the hemicelluloses are hydrolyzed during acid pretreatment and
alkali pretreatment .Thus decreasing the enzyme usage, making it cost efficient (Hahn-Ha et al,
2006).Though the present technologies are efficient enough to release cellulose sugars significantly,
for the technology to be cost effective the removed lignin components have to be made useful for
producing high value products which yet remains a challenge.
Biological pretreatment of the biomass is considered as one of the safest method to lignin
degradation, despite of the slow process (Akin et al, 1995; Gold et al, 1993). Biological pretreatment
is mainly focused on the use of enzymes (cellulases) produced by bacteria or fungi (e.g., Trichoderma
reesei) to hydrolyze cellulose (Hamelinck et al, 2005).As lignin is a complex organic polymer, in all
cases of pretreatment technologies, there is lack of enough information about the degradation
mechanism of the lignin which had become a potential barrier for an efficient deconstruction
mechanism. Due to the expensive pretreatment technologies, the bioethanol production is becoming
expensive process. For the production of bioethanol being cost effective, the scope lies in improving
the pretreatment technology in a way that the deconstructed lignin yields high value by-products
(Figure 1).
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1. Pretreatment Technologies
removal of lignin to release fermentable
sugars
Lignocellulosic Biomass
Thermo chemical Treatment
2. Hydrolysis of
fermentable sugars
(Release of C-6 and C-5
sugars)
Biological Treatment
3. Fermentation
and Biethanol
Production
Alkali Treatment
I.
Soil System
Lignin chemical modification
due to Microcosm, pH,
Temperature and other factors
((yet to be found).
Is this Possible?
Can this be cost effective???
II.
Aggregate formation and
addition of chelating agents
4. Lignin
based high
value
products
Figure 1: Block description of the application of soil system, comparison with other technologies .
Soil can be a potential system to depict the natural degradation of lignocellulosic biomass, as the
degradation of the plant and other organic material occurs in the soil which forms as a medium for the
growth of several microcosms. It plays a major role in the natural recycling mechanism of the complex
organic component of soil into their respective elemental forms (Skipper et al, 2005). During the
process of natural degradation all the complex materials in nature degrade into the elemental forms
through the process of composting (Miguel et al, 2002).
During the deconstruction mechanism of lignocellulosic biomass, the cellulose and hemicelluloses are
easily degraded where as lignin follows a specific pathway for its degradation (Stevenson, 1994) .The
chemical modification of lignin polymer allows the enzymes released by the microcosm to enter the
lignocellulosic matrix and digest the cellulose and hemicelluloses.
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The net desired compost is a result of micro environmental factors such as variations in temperature,
pH, pressure, and also due to the microbial interactions which have a direct or indirect effect on the
enzyme production system of the microorganism (Philippe et al, 2005).
Diversity in soil exceeds beyond that of eukaryotic organisms, they play a very important role during
nutrient cycle apart from their role in formation of soil aggregates, and they form the major
contributors’ in the complex interlinked food webs (Teuscher et al, 1960).These have the potential to
degrade any complex organic substance into simpler and more natural elemental forms in nature.
There potential for degradation can be observed in the cases where soil microcosm has shown to
degrade soil applied pesticides. The microbe could degrade the chemical before the chemical showed
its effect- enhanced degradation. Microorganisms living in the soil environment are responsible for
moderating the microenvironment by their enzymatic activity (Hatfield et al, 1994).
The enzymes released by the microorganism not only affect the biomass directly but also indirectly act
as inhibitors and activators for other microorganisms (Tuomela et al, 1999).These groups of organisms
have the ability to modify the microenvironment as the enzymes and other byproducts’ released due to
the metabolic activity effects the temperature, pH and other elements which act as key enhancers for
degradative reactions, thus playing an important role in the soil degradation system. Though the
deconstruction of the lignin polymer in soil is a slow process, the chemical modification of the lignin
makes it feasible for the formation of a colloid resulting in its further degradation. As the nature of
formation of aggregate colloid and chemical modification are interrelated in the lignin pathway, thus
the detailed understanding of the lignin degradation mechanism in soil would provide a novel
pretreatment system for the bioethanol production from lignocellulosic biomass.
I. Soil as a potential source for a novel pretreatment technology.
Soil consists of different kinds of elements which support all kinds of life forms. For plant growth, it has
14 elements which are considered to be essential because they are absorbed by plants in relatively large
amounts (Arora et al, 1991).Organic component of the soil is mainly distinguished into
undecomposed, decomposed and decomposing organic matter, where in the newly formed
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organic matter is the end product. The dead organic matter in the soil system is subjected to
different kinds of thermo chemical and pressure induced mechanisms where in microbial activity
also takes place (Teusher et al, 1960).The soil system provides a mixed system which involves
various sets of activities and reactions provided by the different sectors of soil. This includes
biotic and abiotic factors such as temperature, pH, enzymes released by the plants, water content,
and external environment. Biotic factors include soil flora and fauna, insects, termites,
earthworms, microorganisms.
There are mainly four kinds of chemical reactions which occur during the process of degradation
in soil, ie oxidation, reduction, hydrolysis, and carbonation. Apart from the chemical reactions,
the microorganisms activity is also involved which is limited by the presence of different
availability of the energy, environmental conditions, and formation of certain detrimental substances
which would create a resistance for their growth. Microorganisms in soil are mainly found near the
areas of humified plant debris, cell wall remenants, fibrous materials, granular and amorphous
materials (foster, 1988).
The Microbial activity in soil is mainly dependent on the supply pH oxygen, amount of organic matter
present and the amount of inorganic compounds present with respect to the pH of the soil. The
decomposition of cellulose in soil occurs at pH 6.8 to 7.5; therefore the formation of spring turf in
acidic soils takes place (Teusher et al, 1960). The properties of organic soil components manly depend on
pH, as cations such as H+,Ca++,Mg
++
,K+,Na+ are attached to colloids which changes the charge of that
colloids and thus form aggregates.
The organic composition in soil is mainly fixed in the form of micro aggregates (< 250 #m
diameter) bound into macroaggregates (> 250 #m), the bond strength in microaggregates are
stronger when compared to micro-aggregates (Tisdall, 1994). Macro-aggregates are stabilized by
saprophytic fungi where as micro-aggregates are stabilized by live or dead roots, fungi,
invertebrates and microorganisms (Lynch et al, 1985). These are degradation products as a result
of series of reactions. Though soil provides a complex system, its interaction mechanism
involving different sets of soil system provides a method of discovering novel degradation
pathways for lignocellulosic pretreatment of biomass.
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Wind
Sunlight
radiation
Cellulose
Hemicellulos
es
Other complex
compounds
Lignin
Enzymes
Organic acids
Chemically modified/partially degraded
Degraded into smaller sub units.
Polyurinoids
Unknown x ??
Amino acids
Microcosm
Humus
Soil biology and the biological micro-environment
The microenvironment in the soil is not consistent with the overall soil system. It varies with the
layer of the soil, penetration of sunlight, amount of moisture and presence of nutrient sources,
CO2, O2, and oxidation –reduction potential .these limit the boundaries of microbial activity on
the available substrate at specific micro-sites in the soil. Degradation of organic compounds
which are insoluble in water takes place with the help of the presence of H+ concentration,
where in various elements due to the H+ concentration where in it helps in solubility of these
elements (Arora et al, 1991). The main contributors of H+ ions in soil are the compounds which
have the hydrogen on reaction with water release hydrogen into the soil resulting in an acidic
environment. The microflora in soil includes the algae where in it utilizes the sunlight, present in
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the upper zone of soil.Heterotrphs which includes bacteria and fungi are responsible for the
initial degradation of the organic compounds which are easily degradable. In the soil ecosystem
their exists two kinds of communities, primary microcosm which are responsible for the initial
degradation of simple carbonaceous nutrients and secondary community which degrades the
substrates produced by the ecosystem. Functionally all the organisms present in the soil are
interdependent through the process of degradation of different forms of organic material
.Animals such as nematodes and few fungi directly derive their food from the living plants where
as bacteria and other fungi have litter as their substrate. Foster (1988) reviewed the location of
the various types of soil-dwelling organisms and found that fungi, which constitute about 80% of
the biomass in many soils, tend to be restricted to the rhizosphere of roots, to larger pores
between aggregates and to the surface of aggregates.
Biodegradation system of lignocellulosic components in soil
Carbohydrates, proteins, lipids, and lignin majorly form the organic matter of the soil system
(Tuomela et al, 1999).Among the lignocellulosic components of the plant cell wall, Cellulose is
an unbranched polymer made of glucose subunits, it forms the major constituent of the plant cell
wall, thus its degradation forming the major component of the carbon and energy flux in soil
(lynch, 1981). During the process of hydrolysis the cellulose with the help of cellulase enzyme is
broken down into glucose units. In soil system, during the process of cellulolysis a group of
enzymes synergistically act on the different binding sites as a result of which the polymer
degrades. (Jeewon et al, 1997). Lignocellulosic crop residues, such as cereal straw, provide the
principal input of cellulose to arable soils (Lynch, 1979).
The spontaneous crystallization of cellulose is attributed to its uniform chemical structure where
in the glucose residue is tilted by 1800C (Schwarz, 2001) where as hemicellulose has an extremely
heterogeneous chemical composition. Lignin is a complex, variable, hydrophobic, cross-linked,
three-dimensional aromatic polymer of p-hydroxyphenylpropanoid units connected by C–C and
C–O–C links (Jeewon et al, 1997). Chemical modification of lignin structures takes place in the
presence of oxygen where the microorganisms produce enzymes of the peroxidase type .These
enzymes in the presence of hydrogen peroxidase chemically modify the structure by breaking the
lignin side chains .Thus the intermdiates are unstable and form hydrophobic partially degraded
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structures due in the presence of water or oxygen. In the absence of oxygen and water lignin is
not degraded and accumulation of these complex polymer occur in soil (Kovalev et al, 2008)
After the chemical modification of lignin, the hydrophobic partially degraded compounds form
aggregates with other available organic compounds and polyurinoids form macro aggregates
which are called humus. They form a major part of humus composition which is required for the
production of humic acid. Colloid humus compounds consist of humic acids and water insoluble
salts which have very high affinity towards calcium and magnesium humate which act as
polyelectrolyte which makes the compound attach to more cations .During the process of organic
decomposition cellulose produce polyurinoides which are mucilaginous substances, aid in humus
congregation. Organic acids, such as humic acid and fulvic acid which form an intermediary
compound of organic matter decomposition also react in the similar way. The process of
degradation of humus substances is very slow despite of the availability of organic nutrients in
the soil environment.The slow degradation of the phenolic ,amino and sugar constituents of the
humus takes plays where in the depolymerisation form the rate limiting step as the concentration
of the monomeric structures is relatively low when compared to organic monomers which are
readily vailable in the soil.
Different sets of microorganisms which are present in soil
Microorganisms play an important role during the process of degradation in soil, as in their absence total
nitrogen, potassium and phosphorous, sulfur, and carbon would be locked up unavailable in the form of
rock or gas and thus degradation of the organic matter would not take place. Due to the presence of
microbes the elements from the organic matte are released, which adds them back into the circulations
that they can be used again by the plant and animal life. The class of fungi which is mainly
responsible for cellulose degradation in soil are Hyphochytridiomycete and Oomycete classes of
Eucomycota and Myxomycetes.Though fungi initiates the cellulolysis in soil, it’s a consoritium
activity where in bacteria also palys a major role (Arora et al,1991).the action of cellulosytic
enzymes is intiated by the presence of the ca+2 ions and thiol donating molecules .
Fungi such as Trichoderma, penicillium are leaf –litters decomposers which decrease the organic
macromolecules in the soil .Some of the plant pathogens feed on lignocellulosic biomass present
in the soil , in order to get sufficient energy to attack the plant host (Arora et al,1991).
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The microenvironment in soil is mainly controlled by the metabolites metabolites such as acids,
bases and ligands produce by the microcosm. These components interact directly and indirectly
with soil system thus changing the properties of the soil with respect to the chemical
environment. Thus the microcosm in soil system is an interdependent, organized system where
the enzymes released by the microcosm help in altering the microenvironment which suits the
degradation process. Though understanding the intricate microbial system is a challenge, it
provides us a basic understanding about how the soil system works with the lignocellulosic
degradation mechanism.
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(Jeewon et al,1997)
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Lignin mechanism -soil as its potential source.
Soil consists of different kinds of materials which undergo degradation continuously.
Lignocellulosic materials form one of the major component of the soil degradation system as it
forms the main component for the cell wall composition of most of the plant cells. During the
biodegradation process, among the lignocellulosic biomass which mainly consist of cellulose,
hemicelluloses, pectin, and lignin the cellulose and hemicelluloses are comparatively easily
degraded into monomer sugar units. Cellulose during the process of degradation forms
polyurinoid components which act as mucilaginous units for humus formation.Lignin is a
complex aromatic polymer which is composed of hydroxyl, methoxy and carboxyl groups. The
random synthesis of lignin formation makes it more strenuous task for its degradation as the
bond formed between the two lignin monomers is not similar to others. During the
biodegradation process of lignin, the lignin structure is modified with the help of enzymes such
as laccase, peroxidases and esterases which are released by lignin degrading fungi initially as the
breakage of bonds is not a feasible. Apart from the lignin molecule there are many kinds of other
complex structures present in soil .The part of the soil components which are chemically
modified along with modified or partially degraded lignin undergo a dehydrative condensation to
form humus aggregate. These aggregates are colloids which are composed of mainly polyuriods,
partially degraded lignin, and other complex aromatic structures. After the humus formation, it
undergoes dehydration and demethylation reactions which lead to decrease hydrogen to carbon
ration .During the dehydration and demethylation process, the humic acids and the protein get
separated. The complex of humus aggregate consists of humic acids, fulvic acids and humin. The
lignin compound mostly is a part of humic acids. During the process of dehydrative condensation
of the humus formation the chemically modified lignin further undergoes a chemical
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modification which results in a compound made of the other aromatic structures .These basically
has the capability to provide the necessary factors for further lignin degradation process .
Monolignol polymerization is another important step during lignification which remains poorly
understood and needs to be explored further in the context of lignin engineering. The
dehydrogenative polymerization of monolignols is thought to be catalyzed by peroxidases and
laccases
The further understanding of different compounds which are involved in the humic acids ,their
activity towards the further degradation of lignin ,whether it is forming a more complex or
simpler structure would give us a chance of understanding the chemical structure, its nature of
reactivity with other aromatic compounds and in turn its degradation .
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5. Conclusion: to be edited
Bioethanol production comparatively decrease the green house gas emissions because the
amount of carbon released during the fuel combustion is proportional to the amount of carbon
dioxide the plant consumes during the process of pHotosynthesis. Brazilian Bioethanol which is
produced from bagasse shows a reduction of 90% for green house gas emissions. In Europe
Bioethanol production is from different source like sugar beet, wheat comparatively has lower
GHG emissions when compared to gasoline. The most promising area for interest for the
production of bioethanol is lignocellulosic biomass in terms of GHS, availability of raw material
(Jeremy Woods, http://www.best-europe.org/Pages/ContentPage.aspx?id=482_)????
6. References:
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