ugi poster_for_open_house
TRANSCRIPT
Pretreatment Applica.on of Ligninoly.c Enzymes Faculty Sponsor: Dr. Chris.ne Kelly
School of CBEE Group Members: Uranbileg Daalkhaijav, Faraz Ebrahimi, Juissepp Rodriguez
Methodology Bioreactor Condi-ons: § 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 op.cal density using spectrophotometer at 600 nm wavelength. § Enzyma.c ac.vity is measured from the oxida.on 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 concentra.on is determined using mul.well plate colorimetric analysis.
2 liter BioFlo 110 bioreactor used for this study.
Colorimetric assay in mul.well plate.
Overall diagram of the MnP produc.on process in 2 L bioreactor.
The process diagram of the MnP produc.on experiment is seen in. The bioreactor is sparged air at constant rate while the pH is automa.cally regulated by addi.on of ammonium hydroxide. The reactor condi.ons are monitored using the integral bioreactor control system. Exit gas frac.ons 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 sensi.vity to the instrumental offset and methods of measurement introduces greatest possibili.es of errors. § The high cell density does not necessarily correlate with increased rMnP ac.vity. § Need to characterize the error in the instruments. Recommenda-ons: § Recreate dry weight analysis to get a more accurate rela.onship between absorbance and cell density. § Try to keep the cell density at a specific level in order to op.mize the rMnP output.
Breakdown of lignocelluloses to isolate fermentable sugars to dis.ll bioethanol.
Introduc.on
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 be_er 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) à glucose/ xylose ferment to ethanol
§ Lignin separa.on is an expensive process as currently prac.ced
Lignin Degrada-on via Enzymes
§ Enzyme manganese peroxidase (MnP) can degrade lignin
§ MnP produced from white rot fungi grows slow so li_le MnP is made
§ MnP gene cloned into yeast P. pastoris to produce large amounts of MnP
2catalystGlucose Ethanol +COuuuur
Overall Goal Improve characteriza.on of bioreactor experiment producing recombinent MnP.
Objec.ves 1. Install and operate off gas analyzer. 2. Perform carbon mass balance on the system. Examine the
yield change with cul.va.on .me. 3. Examine effects of pure oxygen on MnP .ter.
Pichia pastoris cells budding
§ Complete reac.on analysis require substrate and metabolite balances on the reactor system. § Current method lacks exhaust gas monitoring making reac.on analysis incomplete. § Off gas analyzer connected to reactor exhaust vent to measure the gas frac.ons in the exit gas. § Gas monitoring fills the major holes in our elemental balances and redistribu.on analysis.
EX-‐2000 Off-‐Gas CO2 /O2 Analyzer.
Objec-ve 1: Off gas analyzer
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Biom
as den
sity (g/L)
rMnP
ac-vity (u
nits/L)
Time (hrs)
biomass (O2 sparge)
Biomass (air sparge)
rMnP (air sparge)
rMnP (O2 sparge)
Pure oxygen was sparged in at 39 hours aier the start of the experiment. During this .me there is a 94% increase in biomass density compared to 42% seen in the reactor sparged with air during same .me period. There is rMnP ac.vity loss due to overhea.ng in bioreactor.
Objec-ve 3: Effects of pure oxygen
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Moles of O
xygen (m
ol O)
Time (hours)
Input Output Overall balance
Oxygen balance is most sensi.ve to instrumental errors. Unaccounted products and metabolites due to equipment limita.ons, 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 approxima.ng the cell density.
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Yield (m
o C / mol C su
bstrate)
Time (hours)
Y UA/s Yco2/s Yx/s Ave. Yx/s Ave. Yco2/s
As the condi.ons inside the reactor change, the growth and produc.on pa_erns of the cells change. § Biomass and carbon dioxide yield over the dura.on of the experiment is not constant.
§ Dissolved oxygen deple.on to zero corresponds with biomass density decrease.
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Enzyme Ac
-vity
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Cell De
nsity
(g/L,), [G
lucose] (g/L)
Time (hours)
Biomass
Glucose
MnP
The biomass goes through a lag phase, followed by an exponen.al growth phase and eventual leveling off. The substrate is consumed by biomass. During exponen.al growth phase of biomass, the substrate feed is quickly consumed by the cells so there is almost no glucose detected in the broth. Aier hemin was added at the start of fed-‐batch, rMnP produc.on 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)
Objec-ve 2: Elemental mass balance
§ 20% higher final cell density in reactor sparged with pure oxygen. § MnP ac.vity increase doesn’t always directly correspond with cell density increase.