Investigation of Natural Biodegradation System in Soil for Designing Efficient Biological Pretreatment Technology for Bi

1. Investigation of the Natural Biodegradation System in Soil; Application for Designing an Efficient Biological Pretreatment Technology for Biofuel Production. A Research Proposal Presentation By Mythreyi Chandoor Biological And Agricultural Engineering Systems Washington State University,Pullman,WA. 29th June 2009.

2. Agenda • Purpose of the project , and its significance . • Methodology and its theoretical background • Preliminary results & Discussion • Program of study • Conclusion

3. Significance of the project Investigation of the Natural Biodegradation System in Soil; Application for Designing an Efficient Biological Pretreatment Technology for Biofuel Production. enzymes Hemicellulose Cellulose Lignin Other complex compounds Organic acids Degraded into smaller sub units. Chemically modified /partially degraded. Polyurinoids Unknown x Microcosm Amino acids Humus

4. The modification of Lignin ,in the extreme conditions ,with the various factors involved will help us in solving the best possible mechanism of lignin removal from Biomass for Bioethanol production. Possible Lignin Mechanism in soil Lignin Other factors ?? Microcosm Chemically modified/partially degraded Lignin Other complex compounds Organic acids Polyurinoids Amino acids Humus Humic acid

5. Methodology

6. Methodology

7. Preliminary results • Microbial Characterization • SEM (Scanning electron Microscopy) • Dye Degradation experiment • NMR(Soil State Nuclear Magnetic Resonance Spectroscopy) • FTIR (Fourier Transform Infrared Spectroscopy) • GC-MS (Gas Chromatography Mass Spectroscopy) • Compositional analysis

8. Microbial Characterization

9. SEM –Scanning electron microscopy

10. 24 hour dye experiment X-axis = Time period Y-axis = concentration of the dye

11. Solid State NMR ; Control Versus Four Weeks.

12. Control Versus Eight Weeks

13. Control versus Twelve Weeks

14. Overlay of all the Time intevals

15. FTIR 1039.63 0.025 C-H deformations (asymmetric in methyl, methylene, and methoxyl groups) Abs 0.02 Splitting of aliphatic side chain in lignin Also aromatic stretch Cleavage of acetyl side chain in Hemicellulose (carbonyl group) 0.015 0.01 phenol hydroxyl stretching 1259.52 3358.07 1159.22 0.005 2920.23 1510.26 1732.08 Methoxy l stretching 0 3500 3000 2500 2000 1750 1500 1250 1000 1/cm Biodegradation

16. Functional analysis Mean Value of OH groups=average (A3430,A1370, A1165, A1043/A1510(1600) Mean value phenolic OH groups=A1370/A1510(1600) Mean value of OCH3 groups=average (A2890,A1460,A1420)/A1510(1600) Mean value of C=O groups=A1720/A1510(1600) Mean value of aromatic ring= average (A1510, A1600, A844) Ratio of aliphatic to aromatic signals = A2936/A1510(1600) BioResources 3(1):13-20

17. GC-MS Analysis

18. Composition Analysis • Determination of Total solids • Determination of Ash content • Determination of Lignin

19. Method of Determination. • % Total Solids (T final) = ( w2 –w / w1)* 100 W = Dry dish weight ,g W1 = Initial sample weight ,g W2 = Sample weight plus dish weight after drying , g. • % of ash content = (w2 – w/ w1) * 100 W= Ignited dish weight ,g W1 = Initial moisture- free sample weight ,g W2 = sample weight plus dish weight after removal from furnace.

20. Determination of lignin • % Acid –Insoluble Lignin = [(w2 – w3) / w1 *( T final / 100% )] * 100 W1 = Initial weight of the sample W2 = Weight of the crucible, acid soluble lignin and acid insoluble lignin after drying in the oven W3 = weight of crucible and acid –insoluble ash after removal from furnace T final = % total solids content of shredded sample on a 105 0 C dry weight basis.

21. Determination of Lignin • %Acid –Soluble lignin = [[(A/ b*a) * df * v/1000ml ] /[ (w * T final) /100] ] * 100 A = Absorbance Df = Dilution factor b = cell path ,1 cm a = absorptivity ,equal to 110 L/g-cm V = filtrate volume W = Initial Biomass sample weight T final = % total solids content of Biomass sample.

22. Time line for the project

23. Conclusion • 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. • FTIR data analysis which showed the decreasing level of phenolic OH and –OCH3 groups in the successive incubation time.

24. Conclusion • The degradation of the biomass was due to the microbial activities in the soil and biomass. • To verify the presence of microcosm, 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.

25. Conclusion 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.