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Investigation of Natural Biodegradation system for Lignocellulosic content in Biomass cell wall
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Mythreyi Chandoor, Deepak Singh, Dhrubojyoti D. Laskar, Ann Kennedy and Shulin Chen*
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Department of Biological Systems Engineering, Washington State University, Pullman,
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WA 99164
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* Corresponding author. Tel.: +1 509 335 3743; fax: +1 509 335 XXXX
E-mail address: chens@wsu.edu (S. Chen)
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Abbreviations: XXXX, XXXXX
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Keywords: xxxxx, XXXXXX.
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Abstract
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In order to understand the natural biodegradation system in soil, apart from understanding the
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factors that effect the degradation system, the sequential chemical changes occurring in the
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lignocellulosic component of the biomass has to be analyzed which would provide the necessary
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information required to propose the lignocellulosic degradation pathway in soil. We used wood
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chips and wheat straw as substrates in soil filled in pint jars. As the wood chips and wheat straw
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are the lignocellulosic sources, the analysis of these sources for every four week time interval
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about four months would provide us the systematic chemical changes occurring in the cellulose,
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hemicellulose and lignin components of the biomass samples. Soil used for this study has
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characteristics such as carbon content of 2.285 g/kg, sulfur content of 0.026 g/kg and nitrogen
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content of 0.171 g/kg. The 3 g of lignocellulosic biomass samples were incubated with 440 gms
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of soil at 20oC for four months and the moisture level was maintained to 15% which is same as
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in natural condition. The sampling of the biomass was done in every four weeks already stated.
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The research will include three different categories of study. The analysis of structural
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changes/modification of lignin and the sequential cleavage bond in the structure of lignin
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observed during the incubation of wood chips and wheat straw in soil for every set of four weeks
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until about twenty weeks by 13C CP/MAS NMR and FTIR. The second will be the biological
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analysis of the samples, microcosm isolation and characterization involved in the lignin
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degradation process in soil. 13C CP/MAS NMR analysis showed the structural modification in
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the area: 0-50 ppm indicating the changes in the phenolmethoxyl of coniferyl and sinapyl
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moieties and terminal methyl of alkyl group, 110-150 showing the changes in the O-substituted
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aromatic carbons of guaiacol, likewise 175-200 ppm region indicating the changes in aromatic
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carbons attached to methoxy groups in syringol units. These results were supported by the FTIR
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data analysis which showed the decreasing level of phenolic OH and –OCH3 groups in the
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successive incubation time. The degradation of the biomass was due to the microbial activities in
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the soil and biomass. To verify the presence of microcosm in that environment living in the
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wood particles, the electron microscopic analysis of the lignocellulosic biomass was done. The
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presence of different types of bacterial and fungal organisms were found in the residue. I do not
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see the data that really proves this.. The microbial flora isolated from the biomass was
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additionally characterized on the basis of their ability to decolorize Azo dye. Dye discoloration
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assay was observed in A647 nm after the strains were grown in LB media with dye concentration
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of 0.002% incubating at 28oC for 24 hrs. Interestingly, some of the strains showed high
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discoloration activity within 16 hrs. The mechanism behind the discoloration and the strains
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identification is under investigation.
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The aim of this research is to provide the information related to the possible mechanismsof lignin
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degradation in soil, which can be derived from different analytical techniques ,NMR would give
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us the changes in the specific bonds and aromatic rings of lignin, cellulose and hemicelluloses
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.FTIR would give us the change in the mean value composition of hydroxy,methoxy ,carboxy
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and other functional groups thus giving us the information regarding the percentage change in
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the functional group.GC-MS Pyrolysis would give us the concentration of different compounds.
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The relation between the change in the functional group, the kind of aromatic structure changed
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and the concentration of the compound would give us the basic idea of what chemical
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modification is happening in the lignocellulosic Biomass .As the basic structural units of all the
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three components is already known, the analysis of change in the chemical structure would
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probably give us an idea the lignocellulosic degradation pathway. As the process is taking place
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mainly due to the interaction between different sets of microcosm, thus with different chemical
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pathways and characterization and isolation of microcosm which shows related microbial
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activity resulting in the degradation of the lignocellulosic biomass, my research work would
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provide a new perspective of pretreatment technology.
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1. Introduction:
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Degradation of lignocellulosic biomass in soil is essential as it forms the major component of the
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plant cell wall and thus is abundant in nature. Sentence to longAmong the different components
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of the lignocellulosic material, cellulose is the most abundant biological polymer where about
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every year approximately 28 billion tons of cellulose is formed as a result of photosynthesis
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where in it forms about 6% of atmospheric carbon dioxide fixed by land and sea plants (Smith,
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1981). Another component, hemicellulose is a polysaccharide composed of pentoses, hexoses,
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and/or uronic acids. A variety of fungi and bacteria produce both endoenzymes (which cleave
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bonds within the polymer) and exoenzymes (which cleave monomers and dimers from the end of
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the polymer) (Perez et al, 2002). Decomposition products of hemicellulose include carbon
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dioxide, water, cell biomass, and a variety of small carbohydrates. Lignin is the most abundant
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aromatic polymer in nature. It is synthesized by higher plants, reaching levels of 20–30% of the
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dry weight of woody tissue (Sarkanen and Ludwig, 1971). It is composed of repeating benzene
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rings that are branched and complex. The aromatic structure of lignin makes it difficult to
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decompose (Sun and Cheng, 2002). Only a few fungi and bacteria have the capability to
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decompose lignin, requiring first depolymerization into smaller aromatic acids and alcohols, side
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chain removal and methoxyl group oxidation, and finally ring opening (Lee, 1997). Although
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white-rot fungi need latin names were long recognized as efficient lignin-degrading microbes,
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research on their enzymology and genetics is still going on. The importance for increased
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research interest can be traced to the discovery of “ligninases” and potential commercial
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applications in the pulp and paper industry and in the degradation of xenobiotics (Lee,
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1997).Research on lignin biodegradation has accelerated greatly during the past 20 years, mainly
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because of the various potential applications of bioligninolytic systems in pulping, bleaching,
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converting lignin to useful products and treating of agricultural wastes using bacteria.
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Lignocellulosic-decomposing abilities of an actinomycete, streptomyces viridosporus T7A, was
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studied in relation to the potential utilization of this strain for the bioconversion of lignin to
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useful chemicals, which included p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, p-
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coumaric acid, syringic acid, ferulic acid, and the ketol (1-hydroxy-3-(4-hydroxy-3-
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methoxyphenyl)-2-propanone)(Crawford,1981). In soil, the lignin forms a part of humus during
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the process of degradation (Sharma, 1998).
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For the lignocellulose decomposition and production of ligninolytic enzymes research has been
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done in the fields of soil microbial characterization (Reference), where in they observed the
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effect of soil and its microcosm on the growth of white rot fungi. Sentence runonDuring the
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process of interaction of white rot fungi with soil microorganisms (Lang et al.,1996),the potential
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of soil which represents a diverse group of organisms that reside ,which range from macrofauna
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(earthworms, spiders,beetles,terminets, mice, moles etc) to micro and mesofauna (protozoa,
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nematodes ,etc) to microscopic forms of bacteria,fungi,and algae (Lartey and Robert, 2005).
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The process of natural degradation of organic matter involves four reactions, which occur in soil.
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This is a long sentence and did not make sense together. They are oxidation, reduction,
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hydrolysis and carbonation (Arora et al, 1991). Microorganisms play an important (important is a
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hollow word, try to be m ore specific) role during this process as in their absence accumulation
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of organic matter would take place till the total nitrogen, potassium and phosphorous, sulfur, and
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carbon would be locked up unavailable in the form of rock or gas. Due to the presence of
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microbes, the elements from the organic matter are released, which adds them back into the
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circulations that they can be used again by the plant and animal life. The activity of the soil
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microbes is limited to availability of the energy, environmental conditions, and formation of
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certain detrimental substances which would create a resistance for their growth. They are mainly
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dependent on the supply pH oxygen, amount of organic matter present and the amount of
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inorganic compounds present with respect to the pH of the soil (Tescher and Adler, 1960).
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Isolation, identification, characterization of environmentally friendly microorganism for lignin
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degradation becomes essential (another hollow word), because the focus is on the efficiency of
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the lignin degradation. For the study of the microcosm in soil, apart from microbial
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characterization techniques such as atomic force microscope (AFM), metagenomics aid to give a
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clear idea about the microbial population. Anaerobic degradation of lignin in straw by ruminal
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microbes was directly observed using AFM (Hu et al, 2008). As the soil consist of various kinds
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of microorganisms some of which cannot be cultured in the laboratory, sequential extraction and
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DNA fingerprinting of the soil metagenome would give us the extractable soil DNA
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(Ascher, 2009).
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Primarily, colonization of the microbial community in soil is supported by the nutrients obtained
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as a result of lignocellulosic degradation. In order to understand the exact mechanism for
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lignocellulosic degradation in soil, the knowledge of the lignin, cellulose and hemicelluloses
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degradation pathway in soil has to be understood apart from the microbial analysis. Different
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analytical techniques are being used such as FTIR (Fourier transform infrared spectroscopy),
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NMR (Nuclear magnetic resonance spectroscopy), GC-MS (Gas chromatography-mass
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spectroscopy), (Nadji et al, 2009) give us a clear understanding about the chemical changes and
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complex formations in the biomass. Though the natural system is a slower process, the study of
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the soil degradation mechanism at 21o C provides an important data which would be useful in
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determination of the deconstruction mechanism of lignin in the plant cell wall. Thus degradation
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mechanism would be the most convenient way and feasible approach when conditions are
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optimized. The understanding the degradation mechanism will certainly help to construct the
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bioreactors for the biomass pretreatment. As the process is taking place mainly due to
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the interaction between different sets of microcosm, our research would provide a new
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perspective of pretreatment technology.
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Studying the ability of dry grass and wood degradation process during mulching and finding out
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the microbial mixture responsible for this soil enrichment process along with the soil
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environment: Although in the past years, great processes have been made to degrade the
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lignocellulosic content of the Biomass, the effective and efficient way in still in research .The
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soil contains a infinite number of Microcosm which has the potential to digest the mulch in few
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months. Studying the Lignocellulosic Biodegradation in soil and deriving the relation with other
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natural systems which can degrade the lignocellulosic content of Biomass and designing a
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Bioreactor
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Past works includes the work done on biodegradation of lignocellulosic material using thermo
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chemical conversion and enzymes produced by Microorganisms. The need for pretreatment and
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the latest technology for degradation of lignocellulosic content in feedstock. And emphasizing
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why soil has the potential to degrade the Lignocellulosic material
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Materials and experimental techniques
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Materials
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2.1. Materials
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Two biomass materials were used in this study: Pine wood, and wheat straw obtained from ???.
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These materials were incubated in soil as received, i.e. without drying, grinding, or sieving.
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Weight of soil in each jar was weighed in plastic mesh bags about 439gm and added to each jar.
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The jars were numbered, and the biomass was covered with the rest of the soil filled in the
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remaining half jar. All the jars were incubated at 37oC for 20 weeks.Soil was autoclaved in one jar
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and incubated along with these jars as a control. A measured volume of Biomass was kept in -
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20oC as a control. Weight of the bag filled with Biomass was 3.11gm.
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The biomass samples were collected every four weeks .The structural characterization of
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biomass was analyzed using different analytical techniques such as GC-MS Pyrolysis, FTIR, and
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NMR. The biomass which consisted of wood chips and wheat straw were collected from the soil
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and filtered to remove the soil particles attached to it. Later these samples were grinded using
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40x filters. These grinded samples were used to analyze the changes using the analytical
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techniques. Need references of protocols and more information on how you did these three
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things.
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Table1 (Biomass and Soil details with respect to Lignin,cellulose and hemicellulose and other
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nutrient and elemental condition)
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Results and discussion
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Conclusion
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Acknowledgement
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Reference
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