ugi poster_for_open_house

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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 fedbatch 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 offgas 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) 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 2 catalyst Glucose Ethanol +CO uuuur 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. EX2000 OffGas CO 2 /O 2 Analyzer. Objec-ve 1: Off gas analyzer 0 20 40 60 80 100 120 140 0 1000 2000 3000 4000 5000 6000 7000 8000 0 3 7 9 15 19 23 27 31 33 39 43 47 51 Biomas density (g/L) rMnP ac-vity (units/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 20 0 20 40 60 80 100 120 140 160 0 3 7 9 15 19 23 27 31 33 39 43 47 51 Moles of Oxygen (mol 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. 0.8 0.4 0.0 0.4 0.8 1.2 1.6 0 3 7 9 15 19 23 27 31 33 39 43 47 51 Yield (mo C / mol C substrate) 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. 0 1000 2000 3000 4000 5000 6000 7000 8000 0 20 40 60 80 100 120 0 10 20 30 40 50 60 Enzyme Ac-vity (units/L) Cell Density (g/L,), [Glucose] (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 fedbatch, rMnP produc.on starts. Batch Fedbatch Carbon Biomass (inoculate) Glucose (media + feed) Carbon Dioxide (off – gas) Biomass (in broth) Glucose (in broth) Diagram of carbon sources and sinks in fedbatch 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.

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Page 1: Ugi Poster_for_Open_House

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  

0  

20  

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140  

0  

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

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  

-­‐20  

0  

20  

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

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.  

-­‐0.8  

-­‐0.4  

0.0  

0.4  

0.8  

1.2  

1.6  

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

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.  

0  

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0  

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0   10   20   30   40   50   60  

Enzyme  Ac

-vity

 (units/L)  

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.