10/3/2018DRAFT
Deciphering ligninocellulosic biodegradation in soil: Perspective and advances.
Mythreyi Chandoor, Deepak Singh, Dhrubojyoti D. Laskar, Ann Kennedy and Shulin Chen*
Department of Biological Systems Engineering, WashingtonStateUniversity, Pullman,
WA 99164
* Corresponding author. Tel.: +1509335 3743; fax: +15093352722
E-mail address: (S. Chen)
Abbreviations: Soil organic matter (SOM), Lignin peroxidase (LiP), Manganese peroxidase (MnP), Ammonia fiber explosion (AFEX).
Keywords:Lignocellulosics, Biofuels, Biological pretreatment, Bioconversion, Lignin degradation, Microorganisms and Humification.
Abstract:
"In all things of nature there is something of the marvelous.”—Aristotle.
The lignin degradation pathway in soil has the potential to improve biological pretreatment of the lignocellulosic biomass for efficient conversion to biofuel/bioproducts and chemicals. Soil is a natural reservoir for life that controls biogeochemical cycles through the regenerative and degradative processes, thus can be considered as an effective natural system for studying the lignin biodegradation. Critical structural changes and chemical modification/functionalization of lignin were associated with the enzymatic action of the soil microcosm, concomitantly; the resulted modified lignin forms a part of humus by reacting with the soil organic matter.The understanding of the formation of these humus-derived compounds will help in elucidating the lignin deconstruction/modification mechanism in soil. This could be important towards promotion of the biological degradation of lignocellulosic materials to employ and evaluate potential application as valuable chemicals. The purpose of this review is to confer thecurrent knowledge in soil biodegradation system particularly in the lignin degradation process, thereby, would provide us with new perspective of pretreatment technology during the process of biofuel production from lignocellulosic feedstock in a cost efficient and enhance the formulation of high value products from displaced lignin.
1. Introduction
The The U.S ethanol consumption is predicted to increase from 5.6 billion gallons (in 2008) to 13.5 billion gallons in 2012 (Thomson Reuters, 2009). If bioenergy is to become a viable means to reduce fossil fuel dependency, a balance between long-term sustainability and short-term productivity goals is needed. The history of bioenergy is often referred to first, second and later generation. A generation refers to the conversion technologies and substrate used for Biofuel production (Henry, 2010). Primarily the biofuels were derived from corn kernels, sugarcane or soybean oil, and later with the increased demand for the biofuel, there was a shift observed in the selectivity of the crop generating starch, sugar or bio-oil depending mainly on the environmental conditions specific to a particular area (Blottnitz, 2006). Soy oil is the largest feedstock for biodiesel, while the use of algae for the production of biodiesel, categorized under second generation biofuel, however, is not in competition with the land for food crops. In this regard, the biodiesel crops yield comparatively less energy per unit of crop area than that available for ethanol crops.
Compared to the total production of bioethanol in U.S, Biodiesel is about 0.45bg/y which is approximated to 7% of bioethanol (Hoekman, 2009). The accessibility biomass for subsequent utilization in the bioconversion process is adversely affected by the limited sources of agricultural land, water resources, and the food vs. fuel adjustment, which has amalgamated as one of the prime challenges towards increasing the efficiency in the production of bioethanol from biomass. Thus, this requires a need to focus more on development of cellulosic ethanol processes for long term sustainability (Hoekman, 2009). In the production of bioethanol, currently focus is directed for the utilization of lignocellulosic materials from abundant sources, such as wheat straw, corn husks, prairie grass, discarded rice hulls or trees (Schubert,2006).
The lignocellulosic biomass sources are considered a cheap and easily available material to make biofuels and, in general, comprised of 30 to 50% cellulose, 20 to 30% hemicellulose and 20 to 30% lignin. In this context, the continuous challenge in biofuels production is the inefficiency towards converting biomass into the desired bioproduct (Hoekman, 2009) and consequently recovering the biomass-derived components in a cost effective way, requires effective pretreatment process.The ethanol yield (g ethanol/ g biomass) from lignocellulose is less than that from sugar and starchcrops as the cell wall of lignocellulosic biomass contains lignin apart from cellulose and hemicelluloses along with protein and fats comparatively smaller proportion (Banerjee, 2009).Lignin is a complex aromatic macromolecular structure derived from hydroxycinnamyl alcohol or monolignols, such as p-coumaryl alcohol (H), coniferyl alcohol (G), and synapyl alcohol (S) and accordingly classified on the nature of monolignol present (H/G/S). These monomers are incorporated in the lignin primary structure in the form of various substructures and /or inter-unit linkages such as β-O-4, β-β, β-5, etc, as result of oxidative coupling process. The cellulose is protected in a matrix of lignin and hemicelluloses fibers (Ralph, 2004) which makes it difficult for the enzymes to access the surface area of cellulose. As the amount of sugars released is limited to the constraint that it is protected by lignin, thus, to utilize lignocellulosic biomass for biofuel production, the greatest challenge lies in the deconstruction of lignin, which protects the hemicellulose (source for C-5 sugars) and cellulose (source of C-6 sugars) fibrils. Though there are various pretreatment technologies which are used to treat the lignocellulosic biomass before the sugar utilization, they constitute about 18-20% of the total cost (Yang, 2008 and Aden, 2002 and Banerjee, 2009).
Till date, different kinds of pretreatment technologies were developed, depending on the composition and distribution of lignin, a factor which determines the nature of wood (Taherzadeh, 2008). For example, softwood is more recalcitrant than hardwood as it is composed of only guaiacyl units (G) which prevent the fiber swelling and thus reducing the exposure of cellulose fibers, where as the hardwood is composed of both guaiacyl and syringyl units(Ramos, 1992). The most extensively used pretreatment technologies and their details are briefly summarized in Table 1. At present, chemical and physicochemical pretreatments such as ammonia fiber explosion (AFEX), lime pretreatment, hot water pretreatment, acid pretreatment are industrially applied for the deconstruction of lignocellulose matrix.Lignin during the ammonia and lime pretreatment process is removed by modification in its chemical structure. However, with such pretreatment process, lignin is chemically altered thus making it unsuitable for to be used to produce high value products such as carbon fiber composites, resins, adhesive binders and coatings, polyurethane-based foams, rubbers and elastomers, plastics, films, paints, nutritional supplements, food and beverage additives. Moreover, the liquid hot water pretreatment, though the organic acids released due to the substitutions in hemicelluloses structure, account for removal of oligosaccharides, but the mechanism is hindered by the cleavage of O-acetyl and uronic acid substitutions from hemicellulose to generate acetic and other organic acids (Walsum et al, 1996).
Thus there is a need for anefficient lignin removal system which completely exposes the cellulose fibers, and retains lignin aromatic quality. Soil system offers to be a potential natural system as the process of deconstruction of lignin is a thermo-chemical and microbial process though the time takes for its complete degradation of the lignin is a point of concern. This review addresses the mechanistic and structural aspects of lignocellulosic biodegradation in the soil system, some of which are still only partly understood or resolved at the time of writing.
2. Soil as a potential environment for lignocellulose degradation
The degradation of the plant and other organic material occurs in the soil which forms as a medium for the growth of several microcosms playing a major role in the natural recycling mechanism of the complex organic component of soil into their respective elemental forms (Skipper et al, 2005). Several microorganisms (both aerobic and anaerobic) inhabiting soil can utilize lignocellulosic materials and animal bodies as carbon and nitrogen sources where they degrade the material sequentially to increase the soil organic matter. Microbial decomposition of plant biomass, the conversion of litter carbon to CO2 by microbial respiration, is one of the major processes controlling terrestrial CO2 fluxes and ecosystem carbon storage (Raich and Schlesinger, 1992, Couteaux et a, 1995, Aerts 1997).
Soil polysaccharides are contributed by the plant carbohydrates and microbial sugars. Time period of the presence of organic matter might significantly influence SOM composition (Nierop, 2000). 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). Apart from microbial action, temperature also plays a major role in decomposition of organic material. The relationship between the temperature sensitivity of decomposition in soil and carbon(C) quality can be realized as a complex interactions system, whilst acting between temperature and a range of other factors influencing the rate of decomposition(Fierer, 2005).
Sometime the photochemical affects also acts as a factor which helps lignin act as afacilitator of carbon turnover in terrestrial ecosystems (Swift, 1979). During this process, the factors which determine the net effect of lignin to be a driver-dependent might be other biotic factors present in soil apart from the photochemical effects (Austin, 2010). During degradation process of lignocellulosic materials in soil the products form as a result of carbon and energy cycle where in they breakdown into elemental forms through the process of humification (Miguel et al, 2002).Humification process and degree of oxidation is dictated by the increase in the O-alkyl C, resistant aliphatic components (partly from lignin polysaccharide and non-methoxyphenolics, such as tannins) and are determined by calculating the acid/aldehyde ratio.
Determination of factors which influence the soil organic matter and humification process generally is vegetation specific (Nierop, 2000). In any case, during the degradation of lignocellulosic biomass, the cellulose and hemicelluloses are easily degraded whereas lignin follows a specific pathway for its degradation (Stevenson, 1994). 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). This probably explains why the soil can be a potential system to depict the natural degradation of lignocellulosic biomass.
In addition to this, diversity in soil exceeds beyond that of eukaryotic organisms, apart from their role in formation of soil aggregates, they play a major role during nutrient cycle by being a hub for all kinds of interactions systems between different living organisms (from eukaryotes to unicellular microorganisms), and they form the major contributors’ in the complex interlinked food webs (Teuscher et al, 1960). Although the deconstruction of the lignin polymer in soil considered a slow process, the plausible chemical modification/functionalization within the lignin proper makes it feasible for the formation of a colloid. This would result in further cleavage of lignin primary structure, as the nature of formation of aggregate colloid and chemical modification are interrelated in the lignin degradation pathway.(Figure 1).
3. Soil microbes involved in lignocellulose degradation
Microorganisms are responsible for the decomposition of residue and the release of plant-available nutrients. The biomass in soil is first acted by the Dematicea, a family of dark pigmented soil fungi which belong to the class Hyphomycetes of division Ascomycota. These soft rot fungi produce enzymes such as xylanase, and very low amount of lignin degrading peroxidases and manganese peroxidases(Thurston, 1994, Orth, 1995 and Yanna, 2002) which act on the biomass to modify the lignin (biodegradation). At the site of action the soft rot fungi form cavities near the S2 woody cell wall layer near the vicinity of cellulose fibrils during the hyphae penetration, as a result of which the cellulose fibrils are exposed (Rayner ,1988 and Daniel, 1998). Apart from fungi, bacteria also participate in the biodegradation process of the plant litter. Bacteria and fungi competitively feed on biomass polysaccharides in reciprocated as well as other contemporary interactions (Wietse et al, 2004), although, most of the cellulose degradation occurs due to fungal action in soil.
Fungi responsible for cellulose degradation in soil are Hyphochytridiomycete and Oomycete classes of Eucomycota and Myxomycete (Arora et al, 1991). Apart from competitive relationship, there is also a communal interaction system (mutualism), where in bacteria derive their energy source from the degraded products released by fungi as a result of exoenzyme activity (Buyer, 2004). Bacteria such as nitrogen fixing bacteria, also acts on toxic solutes thereby, increase nitrogen content that positively effects and hinders fungal growth(Greaves, 1971) (Hendrickson, 1991). The biomass is the soil is consumed by different mesofauna such as Oribatida, Collembolan, Enchytraeidae which is infected by these soft rot fungi (Knight et al, 1967, Sadaka et al, 1998). Small chemical oxidizers such as activated oxygen species and enzyme mediatorsare probably involved in the initial steps of lignocellulosic degradation in soil (Angel et al, 2005).Basidiomycetes (White rot) and brown rot fungi produce extracellular lignin degrading enzymes (Singh et al, 2008) which are responsible for modifying lignin (Eriksson et al, 1990, Ten Have et al, 2001, Bennett et al, 2002 and Rabinovich et al, 2004, Carmen, 2009). In addition to lignin degradation, white rot fungi have the potential to degrade cellulose and hemicelluloses simultaneously or selectively. Apparently, different kinds of other fungi and insects which produce laccases are also documented, that have the potential to modify lignin structures (Mayer et al, 2002). Saccharomyces, Zymomonas mobilis, Pichia stipitis, Candida shehatae, Escherichia coli, Trichoderma reesei, Clostridium thermocellum, Clostridium papyrosolvens, Neospora crassa, Fusarium oxysporium degrade hexose sugars, while pentoses are degraded by Zymomonas mobilis, Trichoderma reesei, Clostridium Papyrosolvens, Fusarium oxysporium (Lee, 1996).
Some of the microbes considered important for the biomass dergradation process in soil and commercially, with their roles in context to soil system are depicted in Table1. Lignin peroxidase (LiP) and manganese peroxidase (MnP) produced by P. chrysosporium are described as true ligninases because of their high redox potential (Martinex, 2002, Gold et al, 2000). LiP acts on the non phenolic components where as MnP acts on both phenolic and non phenolic lignin components using Mn3+ as a catalytic oxidizer through a series of lipid peroxidation reactions (Jensen et al, 1996, Angel et al, 2005). It is noteworthy to mention here that, such processes also facilitates biodegradation of cellulose and other simpler organic compounds, in contrast to the extent and/or scope of biochemical modification of lignin structure in the biomass.
4. Biodegradation of lignocellulosic components in soil
It is well documented that, the microfungi and bacteria which initially feed on fresh litter in the soil are consumed by microfauna and macrofauna which consist of nematodes, protozoa and earthworms, snails, slugs and diplopods respectively (Mark et al, 2002). These macro and microfauna utilize the enzymes produced by the fungi and bacteria there by enhance the digestion in their gut (External rumen hypothesis) (Swift et al, 1979, Mark et al, 2002). In this context, carbohydrates, proteins, lipids, and modified lignin form part of soil organic matter after the initial microbial action by different bacteria and fungi present in soil and digestive system of animals present in soil (Tuomela et al, 1999). Whilst, cellulose being a simple chain of glucose monomer units, its cellulolysis forms the major contributor to the carbon and energy flux in soil (Lynch, 1981). In contrast, hemicellulose is a polysaccharide, composed of D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methyl-glucoronic, D-galacturonic and D-glucoronic acids linked by β-1,4- and sometimes by β-1,3-glycosidic bonds (Carmen, 2009 and Schwarz, 2001), whereas, lignin being considered 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 cleavage of the lignin side chains. 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. 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 polymers occurs in soil(Kovalev et al, 2008). A plausible degradation pathway indicative with the proposed cleavage sites of lignin macromolecular assembly (a representative lignin model with 8–O–4' and 8–1 inter-unit linkage) is depicted in Figure 2. The pathway of degradation of lignin proper is believed to proceed via an oxidative C–C bond cleavage, initiated at the C7 and C8 position of various substructures/inter-unit linkages (e.g. 8–O–4' and 8–1) within the lignin primary structure (Reference needed). As a consequence of such C–C bond cleavage, hydroxybenzaldehyde (1) derivatives were generated, namely, 4- hydroxybenzaldehyde (R1, R2 = –H, from H-unit), vanillin (R1 = –H, R2 = –OCH3, from G-unit) and syringylaldehyde (R1, R2 = –OCH3, from S-unit), respectively. The degradation pathway could also account for the formation of cleaved diol product (3) with subsequent oxidation to its corresponding ketal (4), however, these products (2, 3 and 4) would also be susceptible for undergoing further cleavage towards the conversion to hydroxybenzaldehyde derivative (1) (Figure 2).
In a representative manner, the parts of the soil components which are chemically modified along with modified or partially degraded lignin undergo a dehydrative condensation to form humus. Moreover, humic substances considered as a major reservoir of organic carbon in soils and aquatic environments (Aiken et al, 1985 and Sanchez-cortes et al, 2001) revealed that the polyphenolic compounds form as intermediates during the humus formation. Apart from the lignin molecule there are many kinds of other complex structures present in soil. As a result of chemical modification of lignin in soil, the partially degraded hydrophobic compounds in association with other available organic compounds and poly urinoids form macro aggregates. Thus, the degradative process in soil consists of incorporation of complex organic molecules and other related compounds with their transformation into humus (Figure 3).