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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.
•Purpose of the project , and its significance .
•Methodology and its theoretical background
•Preliminary results & Discussion
•Program of study
•Conclusion
Significance of the project
Investigation of the Natural Biodegradation System in Soil;
Application for Designing an Efficient Biological Pretreatment Technology
for Biofuel Production.
enzymes
Cellulose
Hemicellulose
Degraded into smaller sub units.
Amino
acids
Polyurinoids
Other
complex
compounds
Lignin
Organic
acids
Chemically modified /partially degraded.
Unknown x
Microcosm
Humus
Possible Lignin Mechanism
in soil
Lignin
Other factors
??
Microcosm
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.
Chemically modified/partially degraded Lignin
Other complex
compounds
Organic acids
Polyurinoids
Amino acids
Humus
Humic acid
Methodology
Microbiology
Degradation of the Biomass
NMR
FT-IR
GC-MS
Pyrolysis
Microbial
characterization
Leco analysis
SEM
NMR, FTIR.
Analyze different kinds of
organic acids
Metagenomics
Dye Degrading potential
Determination of :
Growth on culture media
Acid soluble lignin,
Plating using Poly
dyes.
16s RNA and 18s RNA
Acid insoluble lignin,
Ash content of Biomass
Soil Analyses
Literature search
Analyzing the data periodically,
and check for the different
protocols for analysis of the
various aspect of the soil
degrading system.
Analysis of humic acid,fulvic
and Humic with the help of FT- Abstract sent to AICHE, ASABE
IR,ESR (Schnitzer,1991)
based on the preliminary results
of the experiment.
Abstract accepted by AICHE.
Methodology
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
Microbial Characterization
SEM –Scanning electron microscopy
24 hour dye experiment
14
12
10
8
6
4
X-axis = Time period
Y-axis = concentration of the dye
2
0
4
8
12
16
20
24
Solid State NMR ; Control Versus Four Weeks.
Control Versus Eight Weeks
Control versus Twelve Weeks
Overlay of all the Time intevals
1039.63
FTIR
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
phenol
hydroxyl stretching
1159.22
1259.52
1510.26
1732.08
0.005
2920.23
3358.07
0.01
Methoxy l
stretching
0
3500
Biodegradation
3000
2500
2000
1750
1500
1250
1000
1/cm
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)
BioResources 3(1):13-20
Ratio of aliphatic to aromatic signals = A2936/A1510(1600)
GC-MS Analysis
Compound name
% of the
compound
Control
% of the
compound
After 4 weeks
% of the
compound
After 8 weeks
% of the
compound
After 12 weeks
Acetol
5.449
5.753
4.633
5.232
Coumarin
2.269
1.435
2.139
1.271
P-vinyl gluaicol
4.170
4.572
3.799
5.915
Syringyl
1.954
1.150
1.475
2.612
Ethanone
1.730
1.422
1.581
1.244
7
6
5
4
3
control
2
4 weeks
1
8 weeks
12 weeks
0
Acetol
Coumarin
P-vinyl
gluaicol
Syringyl
Ethanone
Composition Analysis
 Determination of Total solids
 Determination of Ash content
 Determination of Lignin
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.

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.
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.
Time line for the project
Tasks
Literatures searching and
reading
Experiment materials
preparation
Rate of degradation
measurement
Microbial characterization
Further analysis of lignin
degrading microcosm
Paper 1 preparation and
submission
Masters Thesis writing &
Defense
11/
08
×
12/0 1/0 2/0 3/0 4/0 5/0 6/0 7/0 8/0 9/0 10/0
8
9
9
9
9
9
9
9
9
9
9
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
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.
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.
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.