Pretreatment Application of Ligninolytic Enzymes
Introduction
Biomass lignocellulose = lignin + cellulose + hemicellulose.
Separate cellulose from lignin in pretreatment stage of
biomass processing
Objective 1: Off gas analyzer
Complete reaction analysis require
substrate and metabolite balances on
the reactor system.
Current method lacks exhaust gas
monitoring making reaction analysis
incomplete.
Off gas analyzer connected to reactor
exhaust vent to measure the gas
fractions in the exit gas.
Breakdown of lignocelluloses to isolate
fermentable sugars to distill bioethanol.
Gas monitoring fills the major holes
in our elemental balances and
redistribution analysis.
EX-2000 Off-Gas CO2 /O2
Analyzer.
Cellulose and hemicellulose (polysaccharides)  glucose/
xylose ferment to ethanol
Ethanol +CO2
Lignin separation is an expensive process as currently
practiced
Lignin Degradation via Enzymes
Enzyme manganese peroxidase (MnP) can degrade lignin
MnP produced from white rot fungi grows slow so little
MnP is made
Objective 2: Elemental mass balance
120
140
7000
biomass (O2 sparge)
6000
Biomass (air sparge)
5000
rMnP (air sparge)
4000
rMnP (O2 sparge)
120
100
80
60
3000
40
2000
20
1000
0
0
0
3
7
9
15
19
23
27
Fed-batch
60
Biomass
6000
Glucose
5000
MnP
4000
3000
40
2000
20
0
20
43
47
51
30
Time (hours)
40
50
The process diagram of the MnP production experiment is
seen in. The bioreactor is sparged air at constant rate while the
pH is automatically regulated by addition of ammonium
hydroxide. The reactor conditions are monitored using the
integral bioreactor control system. Exit gas fractions are
measured by the off-gas analyzer, and is read by the data
logger.
20% higher final cell density in reactor sparged with pure
oxygen.
MnP activity increase doesn’t always directly correspond with
cell density increase.
Carbon Dioxide (off –
gas)
Overall diagram of the MnP production
process in 2 L bioreactor.
Biomass (inoculate)
Sample Analysis:
Cell density was determined by optical density using
spectrophotometer at 600 nm wavelength.
Enzymatic activity is measured from the oxidation of 2,6dimethoxyphenol at 469 nm. The broth sample was spun down
at 10,000 rpm for 3 minutes to separate the MnP containing
supernatant from the cells.
Glucose concentration is determined using multiwell plate
colorimetric analysis.
Biomass (in
broth)
Carbon
Glucose (media +
feed)
0
10
39
2 liter BioFlo 110 bioreactor used
for this study.
Pure oxygen was sparged in at 39 hours after the start of the experiment.
During this time there is a 94% increase in biomass density compared to 42%
seen in the reactor sparged with air during same time period. There is rMnP
activity loss due to overheating in bioreactor.
1000
0
33
7000
100
80
31
Time (hrs)
8000
Batch
MnP gene cloned into yeast P. pastoris to produce large
amounts of MnP
8000
Enzyme Activity (units/L)
catalyst
Cell Density (g/L,), [Glucose] (g/L)
Glucose
Objective 3: Effects of pure oxygen
Biomas density (g/L)
Producing Bioethanol
Bioreactor Conditions:
2 liter BioFlo 110 bioreactor was
used
Inoculated reactor with Pichia
pastoris.
Cell density of 1 – 6 g/L marks the
start of fed-batch when hemin and
trace salts are added.
Samples from the broth taken every
4 – 6 hours.
Results and Analysis
rMnP activity (units/L)
Emerging Demand for Ethanol and the Use of Biomass
Increase in petroleum fuel prices are driving the demand
for renewable fuels. Cellulosic ethanol from waste biomass
such as switchgrass and woodchips can yield better net
energy than using corn or sugar canes.
Methodology
Faculty Sponsor: Dr. Christine Kelly
School of CBEE
Group Members: Uranbileg Daalkhaijav, Faraz Ebrahimi, Juissepp Rodriguez
Glucose (in
broth)
60
The biomass goes through a lag phase, followed by an exponential
growth phase and eventual leveling off. The substrate is consumed by
biomass. During exponential growth phase of biomass, the substrate
feed is quickly consumed by the cells so there is almost no glucose
detected in the broth. After hemin was added at the start of fed-batch,
rMnP production starts.
Diagram of carbon sources and
sinks in fed-batch bioreactor
process.
rMnP and Byproducts
(in broth)
Dissolved oxygen depletion to zero corresponds with biomass
density decrease.
Pichia pastoris cells budding
Improve characterization of bioreactor experiment producing
recombinent MnP.
Objectives
1. Install and operate off gas analyzer.
2. Perform carbon mass balance on the system. Examine the
yield change with cultivation time.
3. Examine effects of pure oxygen on MnP titer.
Yco2/s
Yx/s
Ave. Yx/s
160
Ave. Yco2/s
1.2
0.8
0.4
0.0
0
3
7
9
15
19
23
27
31
33
39
43
Input
Output
Overall balance
140
Moles of Oxygen (mol O)
Overall Goal
Yield (mo C / mol C substrate)
Y UA/s
1.6
Colorimetric assay in multiwell
plate.
47
51
120
100
Time (hours)
The data sensitivity to the instrumental offset and methods of
measurement introduces greatest possibilities of errors.
The high cell density does not necessarily correlate with
increased rMnP activity.
Need to characterize the error in the instruments.
80
60
40
20
0
-20
-0.4
Conclusion
0
3
7
9
15
19
23
27
31
33
39
43
47
51
Time (hours)
-0.8
As the conditions inside the reactor change, the growth and production
patterns of the cells change.
Biomass and carbon dioxide yield over the duration of the
experiment is not constant.
Oxygen balance is most sensitive to instrumental errors. Unaccounted
products and metabolites due to equipment limitations, may cause
unbalance.
Carbon and oxygen input and output is not balanced due
to missing byproducts, offset in the off gas analyzer, and
method of approximating the cell density.
Recommendations:
Recreate dry weight analysis to get a more accurate
relationship between absorbance and cell density.
Try to keep the cell density at a specific level in order to
optimize the rMnP output.
Special thanks to Kelsey Yee, Dr. Kelly, Dr. Harding, Shamon
Walker and Andy Brickman.