Sustainable Bioprocessing for Biopolymers and Fuel Precursors
AnthonyJ.SinskeyDept.ofBiology,HSTandESD
Massachuse>sIns@tuteofTechnology
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)
[email protected]% 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
Top Related