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 .

Agenda

•Aim and importance of the project• Background – Hypothesis of the project• Experimental:

MicrobiologyChemical analysis of lignocellulose degradation in soilStructural analysis

• ModelingLignocellulose degradation in soilApplication

• Acknowledgements

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 !!

Natural biodegradation system in soil

CelluloseHemicellulose Other complex

compounds

Degraded into smaller sub units.

Chemically modified

Humus

Organic acids

Polyurinoids

Metal Ions

Microcosm

Lignin

Microcosm

Amino acids

Background

Background

Delignification, repolymerization

Humus formation

Proteins

in soil

Lignocellulosic system in soil

Possible lignin mechanism in soil

Background

Humus

Other complex

compounds

Polyurinoids

Organic acidsAmino acids

Chemically modified/partially degraded Lignin

Lignin

Microcosm

• To understand the biodegradation of lignocellulose in soil

• To model the biodegradation of lignocellulose in soil

Design the pretreatment system

Aim of the projectBackground

Methodology

•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

Scanning Electron Microscopy (SEM)

Aromatic carbons attached to methoxy groups in syringol unit

Amorphous and crystalline compounds attached to C4

C2,C3,C5 of cellulose

C4 of amorphous

cellulosePhenolmethoxyl of

coniferyl and sinapyl moities

4 weeks 8 weeks12 weeks16 weeks

Solid State NMR Analysis

Solid State NMR Analysis

•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.

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

Batch samples for every four weeks

% C

once

ntr

atio

n o

f th

e to

tal c

omp

oun

d

Py-GC/MS Analysis

Py-GC/MS Analysis

•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.

Cellulose and Hemicellulose are proportionately decreasing while the lignin concentration is stable

and increased after a period of 12 weeks

Py-GC/MS Analysis

δ 3.81

δ 3.81

Hα in β-structures

CONTROL

1H NMR analysis

16 week SAMPLE

•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.

1H NMR analysis

•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.

1H NMR analysis

TG Analysis

min0 5 10 15 200 250 300 350 40 45 50 55

Soil Sample S5 Soil sample S4,

After 20 weeks After 16 weeks After 12 weeks After 8 weeks After 4 weeks Sugars

Lignin

Modeling General Equation for the Soil Degradation system

[S]+[X]+O2 + H2O [P] + [S1] + CO2 +[X]Soil

pH

where in S = s1+ s2 + s3 .X = x1 +x2 +x3 .P = products ( glucose and other residual sugars ).S1 = modif ied lignin .

( s1 =cellulose , s2= hemicellulose, s3= lignin )

(Maximum microbial growth on the biomass respectively )

Water balance equation :

dm H2O /dt = dmbio H20/dt + dm H2O intake / dt - dmexhaust H2O/dt

mH20 = mass of H20 in soil mbio

H2O = mass of H2O evolution taking place as a result of the degradation dm H2O

intake = water intake via intake airdmexhaust H2O = Water outlet Via exhaust air here ,dmbio H20/dt = 0

Therefore , dm H2O /dt = dm H2O intake / dt - dmexhaust H2O/dt

(Input = output +accumulation - generation)

C02 Balance equation :

dm CO2 /dt = (dmCO2bio/dt- mCO2 dvexhaust /dt )/v

mCO2 = Mass of CO2 in soil dm CO2

bio = evolution of CO2 during Bioreaction V= free space in the soil dvexhaust /dt = flow of exhaust air

t = time dmCO2

bio/dt = negligible ;The change in the flow of the exhaust air is also negligible dmCO2/dt = Negligible Therefore not being considered .

Modeling

Microbial growth (X ) = x1+x2+x3

= max f temp * si / Ksi + si -

where in Si = s1/s2/s3 .dx/dt = x1

d(S1) / d(t) = -Vb1*S1*X1/(Ks1+S1) #Cellulose BalanceS1(0) = 0.71 # weight in gm/gm  

d(S2) / d(t) = -Vb2*S2*X2/(Ks2+S2) #Hemicellulose BalanceS2(0) = 0.48 #  

d(S3) / d(t) = -Vb3*S3*X3/(Ks3+S3) #Lignin BalanceS3(0) = 0.28 #  

Modeling

We derived an relation using polymath which defines the degradation pattern in the soil system.  

µ=µmax1*S1/(Ks1+S1)-∆1 

t(0) = 0t(f) = 3360 µ2=µmax2*S2/(Ks2+S2)- ∆ 2 µ3=µmax3*S3/(KS3+S3)- ∆ 3

Considering the values as follows ;µmax1=0.08µmax2=0.05μmax3=0.03 ∆ 1=0.001∆ 2=0.001∆ 3=0.001

Modeling

Modeling

Time (in hours )

Init

ial S

ubst

rate

co n

cent

r ati

on in

gm

/ gm

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.

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 .

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 !!

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 .

Acknowledgements

And

My Team …

Any Questions ?