Sustainable Bioprocessing for Biopolymers and Fuel Precursors

AnthonyJ.SinskeyDept.ofBiology,HSTandESD

Massachuse>sIns@tuteofTechnology

asinskey@mit.edu

Overview

•  BriefoverviewoftheSinskeylabbiopolymerandbiofuelsresearch–  Overview–  Carbonstorage

•  FeedstockdevelopmentforBiofuels–  Glucose–  Xylose–  Glycerol–  Scalability

•  Gene@csandgenomics

Engineering Carbon Storage Biology

StorageMolecules Polymers(PHAs) Triglycerides(TAGs)

SubstrateRangeofBiocatalyst Glucose,xylose Glycerol Vola@leorganicacids Oils/fa>yacids

BioprocessDevelopment Mediumcomposi@on Fermenta@onstrategy Productrecovery Productprocessing

CarbonStorageBiology Biosynthesis Degrada@on Cellbiology Genomics/Gene@cs

CarbonStorageSystems

Polyhydroxyalkanoates (PHAs) •  Naturalpolyestersusedforcarbonandenergystorage•  Renewableandbiodegradablematerial

O

O

R

Ralstonia eutropha H16 electron micrograph

PHA chemical structure

n

Amorphous PHB

PHA Synthase

O

O

n

PHA Depolymerases

and Oligomerases

Phasins Regulatory Proteins

PhaA PhaB

PhaC

PhaZ

PhaP PhaR

2

PHB Homeostasis

The future is now for PHAs

1. PHAcommercializa@onisinprogress  Metabolix,others

2. Governmentregula@onswilldriveadop@onofrenewables  Bansoftradi@onalplas@cbagsinChina,partsofUSA

3. Consumersdemandenvironmentallyfriendlyproducts  MajorretailerssuchasWal‐Mart

Petrochemicals aren’t biodegradable

  Petroleum-based plastics persist in the environment

  Microscopic plastic particles can be detected inside fish and plankton

Figure: Thompson et al. (2004) Science 304: 838. Image: www.bluegg.com

Applications for PHA polymers

•  Drug Delivery •  Orthopaedic •  Hypoallergenic •  Nutrition •  Tissue

Engineering

•  PHA Latexes •  Film Forming •  Unique

Properties •  Solvent Free

•  Bulk polymer •  Sustainable •  Single use

disposable items •  Solvents/Chemicals •  Paper coatings

Compostable gift cards

  Target is selling gift cards made from a PHA polymer marketed by Telles, a joint venture between Archer Daniels Midland and Metabolix.

  The gift cards are biodegradable: right-hand image shows a card after 40 days in a compost pile

Images: www.treehugger.com, www.sustainableisgood.com, Metabolix

Commodity Plastic Applications

Gift Card

Metabolix & Target

Pipette Tip Refill Base

Metabolix & VWR

Other products: biodegradable compost bags, ground covers, etc.

Golf Tee

Metabolix

PHA production in plants

  Plants can be engineered to produce PHA   Host plants include switchgrass and sugar cane   Economically attractive (water + CO2 -> plastic)   Opportunity to integrate with biomass to fuels programs

Images: www.dailyherald.com, www.americanenergyindependence.com

Partnership with ADM, Corn Wet Mill Integration, Start-up December 2009

•  Tellessitedesignedfor110millionlbs/year

–  30AcrefacilityadjacenttoADMcornwetmillinClinton,Iowa

–  Integratedeconomics

•  Footprintdesignedfor4xexpansion($1+billionrevenuepoten@al)

Enabling the Integrated Biorefinery

Biomass for Electricity

Integrated Biorefinery

Polymer

Switchgrass 7.5% PHA

(Alternative) Biomass Model

$250 MM

100 MM lb

Biofuel

$90 MM

45 MM gal

Annual Revenue Potential

Elimination of Capital-Intensive Operations

BiomasstoBiofuel‐Mul@‐stepcollabora@on

Feedstocks Triacylglycerol

(TAG) production

TAG recovery

TAG to biofuel conversion

Prokaryo@ccarbonstorage

•  Majorityofprokaryotesstorecarbonaspolyhydroxyalkanoates(PHAs)

•  Storageistheresultofnutrientlimita@oninthepresenceofcarbon

•  Smallnumberofbacteriastorecarbonastriacylglycerol(TAG)

TAG

PHA monomer

R. opacusoverview

•  Gram+ac@nomycete

•  Capableofcatabolizingawidearrayoforganiccompounds

–  Alkanes–  Aroma@cs–  Cangrowonglucoseconcentra@onsashighas30%

•  Accumulates~50‐55%celldryweightastriacylglycerol(TAGs)

•  DoesnotappeartoaccumulatePHAs

•  Accumula@onoccursasaresultofhighCarbon:NH4ra@os

Alvarez, 1996

Lipid bodies in R. opacus PD630

WhyTAGs,R. opacus?

•  TAGsarereadilyconvertedtobiodieselthroughasimpletransesterifica@onreac@on

•  Biodieselisa“drop‐infuel”•  Biodieselismoreenergyrichthanethanol,hydrogenornaturalgas

•  TAGscanbeconvertedtobiojetfuel•  R. opacuscansynthesizeupto76%ofCDWasTAGs

FeedstocktoTAGs

•  Aimtoincreasethemetabolicplas@cityofR. opacus 

•  Wanttou@lizecellulosicfeedstockstoproduceTAGs

•  Wanttoop@mizeTAGproduc@ononvariousfeedstocks

•  WorkprimarilyperformedbyDr.KazuhikoKurosawaandJilUlrich

SixForsfermenta@onapparatus

•  Controlledparameters:–  Agita@onbetween300and1200rpm

–  Dissolvedoxygenlevelmaintainedat70%withpureO2

–  pHat6.9–  Temperatureat30°C

•  Analy@cs:–  Sugarconsump@on

–  Nitrogenconsump@on

–  Celldryweight(CDW)

–  Totalfa>yacids(tFA)

Glucosefermenta@on12.0% glucose and 0.67% (NH4)2SO4 (C:N ratio = 17.8 g/g).

Expansionofcarbonsourceu@liza@on

•  R. opacuscannaturallyu@lizeglucoseaswellasmanyothercarbonsources‐incapableofmetabolizingxylose

•  Usingagainoffunc@onscreenwepreviouslyconstructedstrainsofR. opacuscapableofu@lizingxylose

•  Allowsfortheproduc@onofTAGsfromhemicellulosicfeedstocks

Xylosefermenta@on12% xylose and 0.75% ammonium sulfate

Co‐u@liza@on

•  Mostorganismsaresubjecttocataboliterepression– Whenpresentedwithmul@plecarbonsourcespreferen@allyu@lizeonecarbonsourcethenthenext

– Canslowdownfermenta@onprocess,leadtobiosynthe@cbo>lenecks,etc.

Xylose/glucosecometabolism6.0% glucose, 6.0% xylose and 0.75% (NH4)2SO4

R. opacus fa>yacidspecies•  Comparisonofthefa>yacidprofilefromglucose,xylose,andmixedsugar

growncultures

Glycerol

•  ByproductofconversionofTAGstobiodiesel– Asbiodieselproduc@onincreases,glycerolpricesshoulddecrease

•  R. opacusdoesnotnaturallygrowusingglycerolasasolecarbonsource

•  U@lizedasimilarapproachtoengineerglycerolu@lizingstrains–  Isolatedseveralstrainsthatcanproduceupto34%CDWasTAGs

Cellulosicfeedstocks

•  Wehavetestedseveraldifferentcellulosicfeedstocks– Woodpulp–  Cornstover–  “Energycane”–  Sorghum

•  UsingsorghumasafeedstockwehavebeenabletoproducesignificantamountsofTAGs–  Upto8.6g/l–  Decreasedlagphase

Scaledupfermenta@ons

•  Previousworkfocusedon300mlscalefermenta@ons

•  Incollabora@onwiththeCenterforBiocatalysisandBioprocessingwehavebegunscalingupfermenta@ons – 100Lruns– 1000Leventually

100LiterBiostatDfermenta@onapparatus

•  Controlledparameters:–  Agita@onbetween300

and1200rpm

–  Dissolvedoxygenlevelmaintainedat70%withpureO2

–  pHat6.9–  Temperatureat30°C

•  Analy@cs:–  Sugarconsump@on

–  Nitrogenconsump@on

–  Celldryweight(CDW)–  Totalfa>yacids(tFA)

100Lfermenta@on

Genomicsandgene@cs

•  R. opacusPD630genomehasbeenfullysequencedandiscurrentlybeingannotated–  Largenumberofgenesdevotedtocarbonu@liza@onandfa>yacidbiosynthesis

–  Allowsustoengineertheorganismusingreversegene@cs– WorkperformedbyDr.JasonHolder

•  Severalgene@cscreenshavebeenimplementedtoiden@fyTAGbiosynthesisandstoragerelatedgenes

Gene@cs

•  Iden@fiedbothTAGbiosynthe@candstructural/storage‐relatedgenes– Havebeguntocharacterizeafamilyoflipidbodyforma@on/matura@ongenes

–  Iden@fiedgenesimportantforbalancingofredoxmolecules

•  HaveisolatedmutantsincapableofdegradingTAGs

Gene@cs(con@nued)

tadA tadB tadC tadR ro02107

WT WT + tadB WT + tadA WT + tadC

Glyceraldehyde 3-P

1,3-bisphosphoglycerate

3-phosphoglycerate

NAD+

NADH

ADP

ATP

GapA

Pgk

Glyceraldehyde 3-P

3-phosphoglycerate

NAD(P)+

NADPH

GapN/TadD vs.

Two branches of G3P dehydrogenase

GapN-His is sufficient for NP-G3P activity

Glyceraldehyde 3-phosphate

3-Phosphoglycerate

NAD(P)+ NADPH

Purified GapN

50kDa

20kDa

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Empty Vector

GapN-His

ΔO

D34

0

Glyceraldehyde 3-P

1,3-bisphosphoglycerate

3-phosphoglycerate

NAD+

NADH

ADP

ATP

NAD(P)+

NADPH

GapN

pgfp ptadD pgapA

TAGs

Polar lipids

TadD-GapA balance dictates TAG accumulation

Applica@onofGene@cs

•  U@lizingdatafromgene@cswehavebeguntoengineerstrainswithincreasedyields– TadDoverexpressingstrainproducesupto10%moreTAGsthanWT

– TadBandTadRoverexpressingstrainsdemonstrateincreasedTAGyieldsandaccumulateTAGsfasterthanWT

Goingforward

•  Fermenta@onwork•  Feedstockdevelopment

•  Fed‐Batchfermenta@ons

•  Con@nuewithgene@cscreens•  Furtherstrainengineering•  Extrac@ontechniques

TheSinskeyTeam