1. Investigation and modeling natural biodegradation system in soil; application for designing an efficient biological pretreatment technology for Biofuel production. Mythreyi Chandoor, Deepak Singh and Shulin Chen Bioprocessing and Bioproduct Engineering Laboratory, Department of Biological Systems Engineering Washington State University.

2. Agenda Aim and importance of the project Background – Hypothesis of the project Experimental:  Microbiology  Chemical analysis of lignocellulose degradation in soil  Structural analysis Modeling  Lignocellulose degradation in soil  Application Acknowledgements

3. Aim and importance of the project Demand for an Alternate fuel – The U.S. ethanol consumption is forecast to increase from 5.6 billion gallons last year to 13.5 billion gallons in 2012, (Thomson Reuters, 2009). What are the challenges ? The greatest challenge lies in the deconstruction of lignin part of the biomass to release sugars. Need for novel pretreatment technology !! Demand for an Alternate fuel – The U.S. ethanol consumption is forecast to increase from 5.6 billion gallons last year to 13.5 billion gallons in 2012, (Thomson Reuters, 2009). What are the challenges ? The greatest challenge lies in the deconstruction of lignin part of the biomass to release sugars. Need for novel pretreatment technology !!

4. Natural biodegradation system in soil Cellulose Hemicellulose Other complex compounds Degraded into smaller sub units. Chemically modified Humus Organic acids Polyurinoids Metal Ions Microcosm Lignin Microcosm Amino acids Background

5. Delignification, repolymerization Humus formation Proteins in soil Lignocellulosic system in soi l

6. Possible lignin mechanism in soil Background Humus Other complex compounds Polyurinoids Organic acids Amino acids Chemically modified/partially degraded Lignin Lignin Microcosm

7. To understand the biodegradation of lignocellulose in soil To model the biodegradation of lignocellulose in soil Design the pretreatment system Aim of the project Background

8. Methodology

9. SEM (Scanning Electron Microscopy) NMR(Solid State Nuclear Magnetic Resonance Spectroscopy) 1-D NMR (Nuclear Magnetic Resonance Spectroscopy ) TG (Thermogravimetric Analysis ) FTIR (Fourier Transform Infrared Spectroscopy) GC-MS (Gas Chromatography Mass Spectroscopy) Experimental results

10. Scanning Electron Microscopy (SEM)

11. Aromatic carbons attached to methoxy groups in syringol unit Amorphous and crystalline compounds attached to C4 C2,C3,C5 of cellulose C4 of amorphous cellulose Phenolmethoxyl of coniferyl and sinapyl moities 4 weeks 8 weeks 12 weeks 16 weeks Solid State NMR Analysis

12. The amount of syringol and guaicol units of lignin have increased after 16 weeks The Oxidation of syringyl and guaicyl units of lignin will give rise to syringol and guaicol units.

13. Quantitatively, syringyl and guaicyl units have decreased where as the syringol and guaicol amounts have increased which shows that there is change in the chemical nature of lignin structure Solid State NMR Analysis

14. Batch samples for every four weeks % Concentration of the total compound Py-GC/MS Analysis

15. The Change in the lignin polymer is observed after the completion of 12 weeks. The increase in the lignin content is attributed to the kind of subunits taken into consideration ; Syringol,guaicol, ethanone and others were considered which are formed as a result of oxidation or modification of lignin.

16. Cellulose and Hemicellulose are proportionately decreasing while the lignin concentration is stable and increased after a period of 12 weeks Py-GC/MS Analysis

17. δ 3.81 Hα in β-structures CONTROL 1 H NMR analysis 16 week SAMPLE

18. The signal at δ 3.81 ppm : methoxyl groups lower in sample.Indicates the enzymatic modification of the lignin molecules. Signals in δ 4.39 ppm: Hγ in β-O-4 structures and β-5 structures and Signals in δ 5.49 ppm : Hα in β-5 structures respectively. 1 H NMR analysis

19. The low intensity of the protons in ß-O-4 units with biodegradation confirms the ß-O-4 linkage degradation during the biological degradation process. δ 6.93 ppm, δ 7.41 ppm, δ 7.53 ppm corresponding to aromatic protons (certain vinyl protons), aromatic protons in benzaldehyde units and vinyl protons on the carbon atoms adjacent to aromatic rings in cinnamaldehyde units and aromatic protons in benzaldehyde units respectively were in low intensity in the 16 week samples. 1 H NMR analysis

20. TG Analysis Sugars Lignin

21. Modeling

22. C0 2 Balance equation : dm CO2 /dt = (dm CO2 bio /dt- m CO2 dv exhaust /dt )/v m CO2 = Mass of CO 2 in soil dm CO2 bio = evolution of CO 2 during Bioreaction V= free space in the soil dv exhaust /dt = flow of exhaust air t = time dm CO2 bio /dt = negligible ; The change in the flow of the exhaust air is also negligible dm CO2 /dt = Negligible Therefore not being considered. Modeling

23. d(S1) / d(t) = -Vb1*S1*X1/(Ks1+S1) #Cellulose Balance S1(0) = 0.71 # weight in gm/gm d(S2) / d(t) = -Vb2*S2*X2/(Ks2+S2) #Hemicellulose Balance S2(0) = 0.48 # d(S3) / d(t) = -Vb3*S3*X3/(Ks3+S3) #Lignin Balance S3(0) = 0.28 # Modeling

24. We derived an relation using polymath which defines the degradation pattern in the soil system. µ=µ max1 *S 1 /(Ks 1 +S 1 )-∆ 1 t(0) = 0 t(f) = 3360 µ 2 =µ max2 *S 2 /(Ks 2 +S 2 )- ∆ 2 µ 3 =µ max3 *S 3 /(KS 3 +S 3 )- ∆ 3 Considering the values as follows ; µmax1=0.08 µmax2=0.05 μmax3=0.03 ∆ 1=0.001 ∆ 2=0.001 ∆ 3=0.001 Modeling

25. Time (in hours ) Initial Substrate concentration in gm / gm

26. Application of the model The model developed is a relation drawn between the total initial concentrations of the cellulose, hemicellulose and lignin, defined in a specific proportion at any point of time. Further,the model would correlate the various factors involved parallel to the degradation rates of each component respectively. The model developed is a relation drawn between the total initial concentrations of the cellulose, hemicellulose and lignin, defined in a specific proportion at any point of time. Further,the model would correlate the various factors involved parallel to the degradation rates of each component respectively.

27. Conclusion Based on the different experiments conducted on the samples which were incubated for 4,8,12,16 and 20 weeks it has been observed that : The optimized conditions for lignin modification is obtained after a period of 16 weeks. These optimized conditions are in relation to various factors present in the soil system, with respect to the relative proportion of each component. Based on the different experiments conducted on the samples which were incubated for 4,8,12,16 and 20 weeks it has been observed that : The optimized conditions for lignin modification is obtained after a period of 16 weeks. These optimized conditions are in relation to various factors present in the soil system, with respect to the relative proportion of each component.

28. Conclusion The determination of the exact relation between these factors would be helpful in developing a model which would predict the specific ratio of cellulose, hemicellulose and lignin apart from other factors involved such as pH,temperature and other organic compounds. Thus providing a suitable mechanism for the pretreatment technology !! The determination of the exact relation between these factors would be helpful in developing a model which would predict the specific ratio of cellulose, hemicellulose and lignin apart from other factors involved such as pH,temperature and other organic compounds. Thus providing a suitable mechanism for the pretreatment technology !!

29. I would like to thank Dr. Ann Kennedy USDA-ARS Soil Scientist/ Adj. Prof. Crop and Soil Sciences,WSU. Dr. Greg Helms, NMR Center Director,WSU. Dr. Manuel Garcia-Perez. Assistant Professor / Scientist. Biological Systems Engineering,WSU. Dr. Bill, Assistant manager,NMR Center,WSU. And my Advisor … Dr. Shulin Chen, Professor/Scientist. Department of Biological Systems Engineering,WSU.

30. Acknowledgements And My Team … Acknowledgements

31. Any Questions ?