Pretreatment Application of Ligninolytic EnzymesFaculty Sponsor: Dr. Christine Kelly

School of CBEEGroup Members: Uranbileg Daalkhaijav, Faraz Ebrahimi, Juissepp Rodriguez

MethodologyBioreactor 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.

Sample Analysis:Cell density was determined by optical density using

spectrophotometer at 600 nm wavelength.Enzymatic activity is measured from the oxidation of 2,6-

dimethoxyphenol 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.

2 liter BioFlo 110 bioreactor used for this study.

Colorimetric assay in multiwell plate.

Overall diagram of the MnP production process in 2 L bioreactor.

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.

Results and Analysis

Special thanks to Kelsey Yee, Dr. Kelly, Dr. Harding, Shamon Walker and Andy Brickman.

Conclusion 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.

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.

Breakdown of lignocelluloses to isolate fermentable sugars to distill bioethanol.

IntroductionEmerging Demand for Ethanol and the Use of BiomassIncrease 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.

Producing Bioethanol

Biomass lignocellulose = lignin + cellulose + hemicellulose.

Separate cellulose from lignin in pretreatment stage of biomass processing

Cellulose and hemicellulose (polysaccharides) glucose/ xylose ferment to ethanol

Lignin separation is an expensive process as currently practiced

Lignin Degradation via Enzymes

Enzyme manganese peroxidase (MnP) can degrade ligninMnP produced from white rot fungi grows slow so little

MnP is madeMnP gene cloned into yeast P. pastoris to produce large

amounts of MnP

2catalystGlucose Ethanol +CO��������������

Overall GoalImprove characterization of bioreactor experiment producing recombinent MnP.

Objectives1. 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.

Pichia pastoris cells budding

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.

Gas monitoring fills the major holes in our elemental balances and redistribution analysis.

EX-2000 Off-Gas CO2 /O2 Analyzer.

Objective 1: Off gas analyzer

0 3 7 9 15 19 23 27 31 33 39 43 47 510

1000

2000

3000

4000

5000

6000

7000

8000

0

20

40

60

80

100

120

140

biomass (O2 sparge)

Biomass (air sparge)

rMnP (air sparge)

rMnP (O2 sparge)

Time (hrs)

rMnP

acti

vity

(uni

ts/L

)

Biom

as d

ensi

ty (g

/L)

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.

Objective 3: Effects of pure oxygen

0 3 7 9 15 19 23 27 31 33 39 43 47 51-20

0

20

40

60

80

100

120

140

160InputOutputOverall balance

Time (hours)

Mol

es o

f Oxy

gen

(mol

O)

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.

0 3 7 9 15 19 23 27 31 33 39 43 47 51

-0.8

-0.4

0.0

0.4

0.8

1.2

1.6Y UA/s Yco2/s Yx/s Ave. Yx/s Ave. Yco2/s

Time (hours)

Yiel

d (m

o C

/ m

ol C

sub

stra

te)

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.

Dissolved oxygen depletion to zero corresponds with biomass density decrease.

0 10 20 30 40 50 600

20

40

60

80

100

120

0

1000

2000

3000

4000

5000

6000

7000

8000

Biomass

Glucose

MnP

Time (hours)

Cell

Den

sity

(g/L

,), [G

luco

se] (

g/L)

Enzy

me

Activ

ity (u

nits

/L)

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 de-tected in the broth. After hemin was added at the start of fed-batch, rMnP production starts.

Batch Fed-batch

Carbon

Biomass (inoculate)

Glucose (media + feed)

Carbon Dioxide (off – gas)

Biomass (in broth)

Glucose (in broth)

Diagram of carbon sources and sinks in fed-batch bioreactor process. rMnP and Byproducts

(in broth)

Objective 2: Elemental mass balance

20% higher final cell density in reactor sparged with pure oxygen.

MnP activity increase doesn’t always directly correspond with cell density increase.