Biodegradation of Lignocellulose in Soil: Basic Understanding of Degradation Mechanism

Review Draft

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. Thesestructures 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.Thelignin degradation pathway in soil provides us a new perspectivefor improvement of biological pretreatment of the lignocellulosic biomass for making biofuel and chemicals.

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, woodchips, 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.

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).

Lignocellulosic Biomass

Is this Possible?

Can this be cost effective???

Figure 1: Block description of the application of soil system, comparison with other technologies.

Soil can be a potentialsystem 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.

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.

2. 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 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. The soil forms a support system for the growth of all kinds of biotic organisms.

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 ismainly 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 microaggregates (< 250 #m diameter) bound into macro aggregates (> 250 #m diameter), 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.

Figure 2: The following figure describes the soil system and various factors involved in the lignocellulosic degradation process.

3. 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 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. The soil microenvironment provides the microcosm an atmosphere which makes it an interdependent system with an adaptability to adjust in any extreme conditions and degrade the organic matter present in soil.

4. Biodegradation system of lignocellulosic components in soil

Carbohydrates, proteins, lipids, andlignin 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 principalinput 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 intermediates are unstable and form hydrophobic partially degraded structures 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 amajor 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 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 available in the soil.

5. 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 mixed microbial activity where in bacteria also plays a major role (Arora et al, 1991).The action of cellulosytic enzymes is initiated by the presence of the Ca+2 ions and thiol donating molecules(Arora et al, 1991).

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.