Washington State University

Research Proposal

Submitted for Research and Academic plan

Acceptance for

Masters in Biological and Agricultural Engineering

29-06-2009

1 Mythreyi Chandoor, Masters Student, Biological Systems Engineering, Washington State University, Pullman, WA, 99164,E-mail: [email protected]

2Dr Shulin Chen, Professor/Scientist, Biological Systems Engineering, Washington State University, Pullman, WA, Phone number :( 509) 335-3743, E-mail: [email protected]

3Dr A.C,Kennedy ,Soil Scientist, USDA-ARS,Washington State University, Pullman, WA Phone number: (509)-335-1554 ,E-mail: [email protected]

4 Dr Greg Helms, Director, NMR center, Washington State University, Pullman, WA Phone number: (509)-335-7758, E-mail: [email protected]

5Dr Garcia Perez Manual, Assistant Professor, Washington State University, Pullman, WA. Phone number: (509) 335-3005, Email: [email protected] Investigation and modeling natural biodegradation system in soil; application for designing an efficient biological pretreatment technology for Biofuel production.

1 Mythreyi Chandoor, 2 Shulin Chen, 3, Kennedy, 4 Greg Helms, 5 Garcia Perez Manual. Washington State University, Pullman, WA.

Abstract:

In order to understand the natural biodegradation system in soil, apart from understanding the factors which are effecting the degradation system, the sequential chemical changes occurring in the lignocellulosic component of the biomass has to be analyzed which would provide the necessary information required to propose the lignocellulosic degradation pathway in soil. During this study, we used wood chips and wheat straw as substrates in the soil filled in pint jars. As the wood chips and wheat straw are the lignocellulosic sources, the analysis of these sources for every four week time interval about four months would provide us the systematic chemical changes occurring in the cellulose, hemicellulose and lignin components of the biomass samples. Soil used for this study has characteristics such as carbon content of 2.285 gm/Kg, sulfur content of 0.026 gm /kg and nitrogen content of 0.171 gm/kg. The 3 Gms of lignocellulosic biomass samples were incubated with 440 gms of soil at 20oC for four months and the moisture level was maintained to 15% which is same as in natural condition. The sampling of the biomass was done in every four weeks. The research work will include three different categories of study. The analysis of structural changes/modification of lignin and the sequential cleavage bond in the structure of lignin observed during the incubation of wood chips and wheat straw in soil for every set of four weeks until about twenty weeks by 13C CP/MAS NMR and FTIR. The second will be the biological analysis of the samples, microcosm isolation and characterization involved in the lignin degradation process in soil. 13C CP/MAS NMR analysis showed the structural modification in the area: 0-50 ppm indicating the changes in the phenolmethoxyl of coniferyl and sinapyl moieties and terminal methyl of alkyl group, 110-150 showing the changes in the O-substituted aromatic carbons of guaiacol, likewise 175-200 ppm region indicating the changes in aromatic carbons attached to methoxy groups in syringol units. These results were supported by the FTIR data analysis which showed the decreasing level of phenolic OH and –OCH3 groups in the successive incubation time. The degradation of the biomass was due to the microbial activities in the soil and biomass. To verify the presence of microcosm in that environment living in the wood particles, the electron microscopic analysis of the lignocellulosic biomass was done. It was clearly evidenced the presence of different types of bacterial and fungal organisms in the biomass. The microbial flora isolated from the biomass was additionally characterized on the basis of their ability to decolorize Azo dye. Dye discoloration assay was observed in A647 nm after the strains were grown in LB media with dye concentration of 0.002% incubating at 28oC for 24 hrs. Interestingly, some of the strains showed high discoloration activity within 16 hrs. The mechanism behind the discoloration and the strains identification is under investigation.

The aim of this research is to provide the information related to the possible lignin mechanism in soil ,which can be derived from different analytical techniques ,NMR would give us the changes in the specific bonds and aromatic rings of lignin, cellulose and hemicelluloses .FTIR would give us the change in the mean value composition of hydroxy,methoxy ,carboxy and other functional groups thus giving us the information regarding the percentage change in the functional group.GC-MS Pyrolysis would give us the concentration of different compounds. The relation between the change in the functional group, the kind of aromatic structure changed and the concentration of the compound would give us the basic idea of what chemical modification is happening in the lignocellulosic Biomass .As the basic structural units of all the three components is already known, the analysis of change in the chemical structure would probably give us an idea the lignocellulosic degradation pathway. As the process is taking place mainly due to the interaction between different sets of microcosm, thus with different chemical pathways and characterization and isolation of microcosm which shows related microbial activity resulting in the degradation of the lignocellulosic biomass, my research work would provide a new perspective of pretreatment technology.

1. Introduction:

Degradation of lignocellulosic biomass in soil is essential as it forms the major component of the plant cell wall and thus is abundant in nature. Among the different components of the lignocellulosic material, cellulose is the most abundant biological polymer where about every year approximately 28 billion tons of cellulose is formed as a result of photosynthesis where in it forms about 6% of atmospheric carbon dioxide fixed by land and sea plants (Smith, 1981). Another component, hemicellulose is a polysaccharide composed of pentoses, hexoses, and/or uronic acids. A variety of fungi and bacteria produce both endoenzymes (which cleave bonds within the polymer) and exoenzymes (which cleave monomers and dimers from the end of the polymer) (Perez et al, 2002). Decomposition products of hemicellulose include carbon dioxide, water, cell biomass, and a variety of small carbohydrates. Lignin is the most abundant aromatic polymer in nature. It is synthesized by higher plants, reaching levels of 20–30% of the dry weight of woody tissue (Sarkanen and Ludwig, 1971). It is composed of repeating benzene rings that are branched and complex. The aromatic structure of lignin makes it difficult to decompose (Sun and Cheng, 2002). Only a few fungi and bacteria have the capability to decompose lignin, requiring first depolymerization into smaller aromatic acids and alcohols, side chain removal and methoxyl group oxidation, and finally ring opening (Lee, 1997). Although white-rot fungi were long recognized as efficient lignin-degrading microbes, research on their enzymology and genetics is still going on. The importance for increased research interest can be traced to the discovery of “ligninases” and potential commercial applications in the pulp and paper industry and in the degradation of xenobiotics (Lee, 1997).Research on lignin biodegradation has accelerated greatly during the past 20 years, mainly because of the various potential applications of bioligninolytic systems in pulping, bleaching, converting lignin to useful products and treating of agricultural wastes using bacteria. Lignocellulosic-decomposing abilities of an actinomycete, streptomyces viridosporus T7A, was studied in relation to the potential utilization of this strain for the bioconversion of lignin to useful chemicals which included p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, p-coumaric acid, syringic acid, ferulic acid, and the ketol (1- hydroxy-3-(4-hydroxy-3-methoxyphenyl)-2-propanone)(Crawford,1981).In soil, the lignin forms a part of humus during the process of degradation (Sharma, 1998).

For the lignocellulose decomposition and production of ligninolytic enzymes research has been done in the fields of soil microbial characterization, where in they observed the effect of soil and its microcosm on the growth of white rot fungi. During the process of interaction of white rot fungi with soil microorganisms (Lang et al.,1996),the potential of soil which represents a diverse group of organisms that reside ,which range from macrofauna (earthworms, spiders,beetles,terminets, mice, moles etc) to micro and mesofauna (protozoa, nematodes ,etc) to microscopic forms of bacteria,fungi,and algae (Lartey and Robert, 2005).

The process of natural degradation of organic matter involves four reactions which occur in soil they are oxidation, reduction, hydrolysis and carbonation (Arora et al, 1991). Microorganisms play an important role during this process as in their absence accumulation of organic matter would take place till the total nitrogen, potassium and phosphorous, sulfur, and carbon would be locked up unavailable in the form of rock or gas. Due to the presence of microbes the elements from the organic matter are released, which adds them back into the circulations that they can be used again by the plant and animal life. The activity of the soil microbes is limited to availability of the energy, environmental conditions, and formation of certain detrimental substances which would create a resistance for their growth. They are 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 (Tescher and Adler, 1960).

Isolation, identification, characterization of environmental friendly microorganism for lignin degradation becomes essential, because the focus is on the efficiency of the lignin degradation. For the study of the microcosm in soil, apart from microbial characterization techniques such as atomic force microscope (AFM), metagenomics aid to give a clear idea about the microbial population. Anaerobic degradation of lignin in straw by ruminal microbes was directly observed using AFM (Hu et al, 2008). As the soil consist of various kinds of microorganisms some of which cannot be cultured in the laboratory, sequential extraction and DNA fingerprinting of the soil metagenome would give us the extractable soil DNA (Ascher,2009).

Primarily, colonization of the microbial community in soil is supported by the nutrients obtained as a result of lignocellulosic degradation. In order to understand the exact mechanism for lignocellulosic degradation in soil, the knowledge of the lignin, cellulose and hemicelluloses degradation pathway in soil has to be understood apart from the microbial analysis. Different analytical techniques are being used such as FTIR (Fourier transform infrared spectroscopy), NMR (Nuclear magnetic resonance spectroscopy), GC-MS (Gas chromatography-mass spectroscopy), (Nadji et al, 2009) give us a clear understanding about the chemical changes and complex formations in the biomass. Though the natural system is a slower process, the study of the soil degradation mechanism at 21o C provides an important data which would be useful in determination of the deconstruction mechanism of lignin in the plant cell wall. Thus degradation mechanism would be the most convenient way and feasible approach when conditions are optimized. The understanding the degradation mechanism will certainly help to construct the bioreactors for the biomass pretreatment. As the process is taking place mainly due to the interaction between different sets of microcosm, our research would provide a new perspective of pretreatment technology.

2. Methods:

The following diagramme gives us the basic understanding about the organization and application of how the methods adapted for the research would be useful in the understanding the mechanism of lignocellulosic degradation system in soil. 2.1 Finding out the microcosm involved in the biodegradation processes occurring in soil.

Microbial Characterization is carried on the soil and biomass samples. Biomass samples consist of wheat straw and wood chips (Pine wood). Soil if from Palouse area where in the deep fertile soil is a prime agricultural area known for wheat, lentil, and split pea production. The Microbial characterization was initially done on the soil and biomass samples before the incubation. Stock solution was prepared by weighing 0.1gm of soil and adding to 900µl of autoclaved water in autoclaved E-tube. Serial dilutions of the stock solution were done by adding it to 1st E-tube which had 900µl of autoclaved water .Then respective concentrations were made for 10-6 concentration. Media for bacterial and fungal growth were prepared using LBA (Lactose Broth Agar) and PDA (Potato Dextrose Agar) respectively. 6 plates for each media for all the 2 concentration of soil dilutions were used for inoculations. 100 µl of each concentration was used to inoculate on the plate. Wheat straw was weighed for 0.1 gm and was oven dried for one hour and desiccated for another hour .Later it was added to 100µl of autoclaved water.50 µl of each sample was plated in both LBA and PDA media plates.0.1gm of wood chips were weighed and mixed with 100 µl of autoclaved water. 50 µl of each sample was inoculated in LBA and PDA plates respectively. Fungal plates were kept in 30 oC and bacterial plates were kept in 37oC.

2.2 The 24-hour complex dye experiment:

In order to understand the microbial activity for discoloration of different complex dyes such as Poly R, Poly B. 0.002gm of the dye was dissolved in 1ml of autoclaved water, and 30µl of this solution was added to 3ml of LB and PDA different isolated strains, they were duplicates made for each strain. There were totally 6 strains from initial and first batch which should discoloration activity. Collection of samples for every four hours was done for a set of 24 hours. The collection of samples for every four hours helped us to understand the compositional changes occurring in the dye which is dissolved in the media ,which is due to the microbial activity .As the changes in the composition of the dye differs, the coloration of the media also changes which would reflect in the uv spectroscopy. The optical density recorded would give us an approximation of the amount of the dye degraded/modified due to the microbial activity.

2.3 SEM analysis of the Biomass samples:

Scanning electron microscopy was used to analyze the presence of wood and wheat straw degrading fungi .The samples analyzed were of initial (before incubation),1st batch (after 4 weeks).2nd batch (after 8 weeks),3rd batch (after 12 weeks).

2.4 Structural characterization of biomass reflecting the mechanism of biodegradation process.

The incubation of Biomass in soil samples was done; Weight of soil in each jar was weighed in plastic bags about 439gm and added to each Jar. The jars were numbered 1-49 and jars , 4,8,12,16,20,24,28,32,36,40 were filled with 1/4th of soil . These 1/4th filled jars were covered with 1 bag of biomass and then ½ of it was covered with soil .The other set of jars were half filled, and the biomass was covered with the rest of the soil filled in the remaining half jar. All the jars were incubated at 37oC.Soil was autoclaved in one jar and incubated along with these jars as a control. A measured volume of Biomass was kept in -20oC as a control. Weight of the empty jar was 262.00g.Weight of the bag filled with Biomass was 3.11gm.Weight of the soil in the jar was 439.35gm.

The three batches of biomass samples were collected for every four weeks .The structural characterization of biomass is analyzed using different analytical techniques such as GC-MS Pyrolysis, FTIR, and NMR. The biomass which consisted of wood chips and wheat straw were collected from the soil and filtered to remove the soil particles attached to it. Later these samples were grinded using 40x filters. These grinded samples were used to analyze the changes using the analytical techniques.

3. Results and Discussion:

3.1 microcosms involved in the biodegradation processes occurring in soil: Initial set of samples of Biomass and soil showed mixed colonies (Figure 1) .Tried to isolate and make pure cultures. After the isolation of strains, they were grown in plates with media and azure dye with a concentration of 0.002%. After a period of 24 hours discoloration was observed in few plates few showed discoloration after 48 hours. The samples after four weeks were collected and stored in -20oC.Based on the same protocol which was used for characterizing the initial set of samples, The number of mixed colonies observed were more than the ones which were observed from the initial set Later on the discoloration experiment was also carried out where in few plates showed discoloration and these were again stored and maintained.

3.2 The 24-hour complex dye experiment: Discoloration was observed and their respective values of OD (647nm) were measured by UV Spectroscopy (Figure 2). The following is the table of the measured values. Table 1gives us the concentration of the dye at various time periods for all the samples .Table 2gives us the OD values of different strains at different time periods

Time Period 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours Strain number 4.94 1 4.458 10.904 4.927 1.717 8.276 4.697 2 7.315 8.868 12.293 0.2 11.398 7.117 4 11.373 3.982 6.493 5.214 6.45 4.565 5 5.812 4.412 9.817 10.204 11.191 5.646 6 7.048 10.109 6.7 5.108 8.846 9.494 13 8.274 9.559 11.326 0.064 10.028 12.238 12 3.455 8.977 12.695 11.023 11.873 Time 4 8 hours 12 hours 16 hours 20 24 hours Period hours hours Strain number 1.461 1 1.498 1 1.462 1.679 1.203 1.48 2 1.277 1.157 0.893 1.809 0.962 1.293 4 0.964 1.535 1.341 1.104 1.344 1.49 5 1.393 1.285 1.084 1.054 0.978 1.406 6 1.298 1.061 1.273 1.404 1.159 1.109 13 1.203 1.104 0.967 1.837 1.068 0.897 12 1.576 1.149 0.862 0.746 0.925 Figure 3: Plates showing discoloration of azure dye (0.002 % concentration).

Control

Discoloration

3.3 SEM analysis of the Biomass samples:

There were fungal mycelia observed growing in the wood chips and wheat straw. And various other kinds of microorganism were observed which are yet to be understood(Figure 4). The presence of microorganisms which can feed on lignocellulosic biomass is being observed. The ability to degrade some of the complex dyes gives us a possibility that there might me chances of they being useful for commercial stain removers or other organic agents. The spectroscopy results showed that the values increased and decreased for every span of four hours and this was not being constant for all the strains. Based on this we might be able to predict that there might be a bond formation and breakage in the dye taking place as a result of microbial activity. In order to understand the exact mechanism, I am planning to do HPLC, where in I can separate the different compound in the media and then do LCMS to understand the change in the chemical structure of these separated compounds. SEM results for wheat straw control and wheat straw 3rd Batch samples (after12 weeks) :

Wheat straw control, magnification -3000x Wheat straw (after 12 weeks), magnification-3100x

SEM results for wood chips control and wheat straw 3rd Batch samples (after12 weeks) :

Wood chips control, Magnification -1400x Wood chips (after 12 weeks), Magnification -4000x

Figure 4: SEM analysis

3.4 Structural characterization of biomass reflecting the mechanism of biodegradation process.

During the NMR analysis, when the comparison is made between different batches including control (figure7), there are changes observed at 133,146,148,105, 173 ppm which shows the change in the syringyl, guaicyl and free phenolic structures of lignin .when the control and 8 week sample was compared, then there was a difference observed in the lignin aromatic structures, amorphous and crystalline compounds of C4 sugars and alkyl groups in aliphatic rings (figure 5).When twelve week samples and control was compared there were changes observed only in the lignin aromatic carbons and methoxy groups (figure 6). Figure 5: shows the comparison between the control and eight week sample.

Figure 6: Shows the comparison between the control and eight weeks. Figure 7: Comparison of all the three batches with the control.

FTIR results showed that there is a significant change in the hydroxy and phenolic OH groups of four week and twelve week samples(Table 3).The table shows that the change in the percentage composition of the different functional groups is probably because of the chemical modification of the compound.

CONTROL 4 WEEKS 8 WEEKS 12 WEEKS

Mean value of 16.608 9.0499 10.25 21.25 OH Groups

Mean value of 0.525 1.16 O.16 0.67 Phenolic OH groups

Mean value of 1.59 2.56 0.796 2.672 -OCH3 GROUPS Table 3: Functional Analysis

Based on the Microscopy results, the presence of fungal mycelia can be found .Based on the analytical techniques for the analysis of the sugars in the cell wall of the wheat straw and pine wood we can analyze the change in the structure of lignin in the twelve week compounds. When compared to the Control samples. The further analysis of the results of different analytical methods such as FTIR and Solid state NMR, GC –MS Pyrolysis and HPLC would basically give us information about the different kinds of compounds formed at different intervals of time and thus we can get a rough idea of how and where the bond breakage is occurring in the lignin structure.

My future work would include mainly characterizing the microcosm in different batches of soil samples and Biomass samples, and characterizing those set of microorganisms which are able to degrade any complex phenolic compounds. Apart from these I would further analyse the FTIR and NMR and GC-MS results to better understand the lignin pathway where in it chemically modification is taking place.

Acknowledgements:

This work so far has been supported by funds of Dr .Chen, Faculty of Biological systems Engineering department Dr Ann Kennedy who provided me with knowledge of the basic understanding of the soil system and its various factors which support it .Dr Greg Helms, who helped me in NMR analysis and also taught me the handling skills of NMR instrument and Dr Manuel, who provided me with the analytical instruments.

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