applications of molecular applications of molecular ... · applications of molecular applications...

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Applications of Molecular Applications of Molecular Biotechnology:Biotechnology:Ethanol Production from Cellulosic BiomassEthanol Production from Cellulosic Biomass

David R. ShonnardDavid R. ShonnardDepartment of Chemical Engineering Department of Chemical Engineering

CM4710 Biochemical ProcessesCM4710 Biochemical ProcessesNovember 30, November 30, 20072007

Presentation OverviewPresentation Overview

nn Ethanol from Ethanol from LignocellulosicLignocellulosic Biomass and its Potential to Biomass and its Potential to Displace Petroleum in the Displace Petroleum in the USAUSA

nn Research Needs in Forest Resources, Bioconversion Research Needs in Forest Resources, Bioconversion Processing, Engines, and Decision AnalysisProcessing, Engines, and Decision Analysis

nn Dilute Dilute Acid Pretreatment of Tree Species from the Upper Acid Pretreatment of Tree Species from the Upper Midwest RegionMidwest Region

nn Enzymatic Hydrolysis of Pretreated Woody BiomassEnzymatic Hydrolysis of Pretreated Woody Biomass

nn Genetic Engineering of E. coli for ethanol production from Genetic Engineering of E. coli for ethanol production from woody biomasswoody biomass

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Managing the Carbon Cycle:Managing the Carbon Cycle:A Sustainable Energy ChallengeA Sustainable Energy Challenge

From http://www.bom.gov.au/info/climate/change/gallery/index.shtml

Combustion of Fossil Fuels acts as a Carbon PumpCombustion of Fossil Fuels acts as a Carbon Pump

COCO22 and Temperature in the and Temperature in the Northern Hemisphere are RisingNorthern Hemisphere are Rising

National Geographic, September 2004, pg 20, National Geographic Society, Washington, D.C.

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WoodWood--toto--Wheels (W2W) ConceptWheels (W2W) ConceptResearch Thematic AreasResearch Thematic Areas

Woody Biomass Resource Research

CO2

Bio-Processing ResearchPhoto: Glacial Lakes Energy

Vehicle Systems Research

SustainabilityAssessments /

Decision-Making

But, How Much Biomass is Available on But, How Much Biomass is Available on an Annual Basis in the USA?an Annual Basis in the USA?

Biomass as Feedstock for a Bioenergy and Bioproducts Industry: Technical Feasibility of a Billion ton Annual Supply: DOE/GO-102995-2135, April 2005.

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Forest Forest Biomass Biomass SourcesSources

Biomass as Feedstock for a Bioenergy and Bioproducts Industry: Technical Feasibility of a Billion ton Annual Supply: DOE/GO-102995-2135, April 2005.

AgricultureAgricultureBiomass Biomass SourcesSources Biomass as Feedstock for a Bioenergy and Bioproducts Industry: Technical

Feasibility of a Billion ton Annual Supply: DOE/GO-102995-2135, April 2005.

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How Much Petroleum is Used �.How Much Petroleum is Used �.Biomass as Feedstock for a Bioenergy and Bioproducts Industry: Technical Feasibility of a Billion ton Annual Supply: DOE/GO-102995-2135, April 2005.

.. and for What Purpose?.. and for What Purpose?Wang, Michael; Center for Transportation Research, Argonne National Laboratory

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How Much Gasoline Could be Replaced with How Much Gasoline Could be Replaced with Ethanol From 1B tons Ethanol From 1B tons LignocelluloseLignocellulose??

{ 1B tons biomass x 70{ 1B tons biomass x 70--100 gal Ethanol/ton biomass x 100 gal Ethanol/ton biomass x

.75 gal gasoline/gal Ethanol x 1.75 gal gasoline/gal Ethanol x 1--2 (efficiency of automobiles)} / 2 (efficiency of automobiles)} /

140B gal gasoline demand140B gal gasoline demand = 37.5= 37.5--75%75%

….displace 37.5-75% of current U.S. gasoline demand

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Are There Energy Benefits of Fuel Ethanol? Are There Energy Benefits of Fuel Ethanol? Fossil Energy and Petroleum UseFossil Energy and Petroleum Use

Energy in fuel

Energy for producing fuel

Uncertainty Range

Energy Use for Each Btu of Fuel Used

Wang, Michael; Center for Transportation Research, Argonne National Laboratory

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Changes in Greenhouse Gas Emissions Changes in Greenhouse Gas Emissions per Mile Driven (Relative to GVs)per Mile Driven (Relative to GVs)

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sWang, Michael; Center for Transportation Research, Argonne National Laboratory

Forest ResourcesForest ResourcesBiotechnology/Genetic Biotechnology/Genetic

engineeringengineering

Forest policy and Forest policy and managementmanagement

Carbon cyclingCarbon cycling

BioBio--processingprocessingEnzyme improvementEnzyme improvement

Pilot plant operationsPilot plant operations

Metabolic engineeringMetabolic engineering

Vehicle/EnginesVehicle/EnginesEngine researchEngine research

Engine tests w/emissionsEngine tests w/emissions

Hybrid vehicle designHybrid vehicle design

Vehicle dynamometerVehicle dynamometer

Michigan Tech�s Qualifications

Assessment/DecisionsAssessment/DecisionsTechnology evaluationTechnology evaluation

Logistics and facilitiesLogistics and facilities

LifeLife--cycle, environmental, ancycle, environmental, an

d policy assessmentsd policy assessments

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Microarray Gene Expression Analysis

Metabolite Profiling&

Chemical Fingerprinting

Our expertise:Micropropagation

Gene transformationMolecular biochemistry

Whole-genome microarrayand metabolite profiling

Research areas:Wood formation

Defense & fitnessNatural variations

Carbon sequestration

Forest Functional Genomics & Forest Functional Genomics & BiotechnologyBiotechnology

Cellulosic Cellulosic Biomass Biomass StructureStructure

Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda: DOE/SC 0095, June 2006.

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Composition of Woody BiomassComposition of Woody Biomass

Beguin, P., J.P. Aubert. 1994. �The biological Degradation of Cellulose�. FEMS Microbiology Reviews. 13:25-58

1. Cellulose2. Hemicellulose3. Lignin

Composition of Dry Cellulosic BiomassComposition of Dry Cellulosic Biomass

Cellulose(35-50%)

Dry Cellolosic Biomass

Hemicellulose(20-35%)

Glucose6-C sugars

Lignin(12-20%)

XyloseArabanoseMannoseGalactose(5-C sugars)

hydrolysishydrolysis

no hydrolysis

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Thermochemical ConversionsThermochemical Conversions�� Optimize Optimize biomassbiomass--toto--sugar sugar

reactionsreactions�� Reduce byproduct reactionsReduce byproduct reactions�� Evaluate timber species� mixtures Evaluate timber species� mixtures �� IncreaseIncrease biodiesel yieldsbiodiesel yields

Integrated Bioprocess FacilityIntegrated Bioprocess Facility�� IntegrateIntegrate fermentation and fermentation and

purification to increase fuel yields purification to increase fuel yields �� Test Test monitoring devices and monitoring devices and

process control schemesprocess control schemes�� MinimizeMinimize energy consumption and energy consumption and

waste generationwaste generation

Biochemical ConversionsBiochemical Conversions�� Develop/test highDevelop/test high--activity activity

cellulasescellulases for tree species mixturesfor tree species mixtures�� Optimize cellulose hydrolysis using Optimize cellulose hydrolysis using

peptidomimeticspeptidomimetics�� Improve fermentations for Improve fermentations for high high

yieldsyields of ethanol / other bioof ethanol / other bio--based based materialsmaterials

�� Use metabolic flux analysis to Use metabolic flux analysis to guide guide strain improvementstrain improvement

Product PurificationProduct Purification�� BoostBoost yields by coupling membrane yields by coupling membrane

separation with fermentation separation with fermentation �� ConserveConserve water by recovering and water by recovering and

recycling reactantsrecycling reactants

BioBio--processing Initiatives: processing Initiatives:

NREL Process to Convert NREL Process to Convert LignocellulosicLignocellulosic Biomass to EthanolBiomass to Ethanol

Saccharification is enzymatic hydrolysis of pretreated cellulose yielding Glucose using cellulase from Trichoderma reesei

Charles E. Wyman, “Biomass Ethanol: Technical Progress, Opportunities, and Commercial Challenges”, Annu. Rev. Energy Environ.,1999, 24: 189-226.

1 – 3 mm

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Pretreatment of Woody Biomass Pretreatment of Woody Biomass

nn Goals: Goals: Prepare cellulose fraction for enzymatic hydrolysisPrepare cellulose fraction for enzymatic hydrolysisnn Convert crystalline Convert crystalline cellulose cellulose to amorphousto amorphousnn Remove some lignin from the cell wallRemove some lignin from the cell wallnn Increase accessibility or enzymes to celluloseIncrease accessibility or enzymes to cellulosenn Convert Convert hemicellulosehemicellulose fraction of the wood to sugarsfraction of the wood to sugars

nn Dilute acid hydrolysis resultsDilute acid hydrolysis results

Dilute Acid PretreatmentDilute Acid Pretreatment

0.25-1.0%Diluted H2SO4

Minimal Degradation Product

Shu C. Yat, 2006

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Goals of Pretreatment ResearchGoals of Pretreatment Research

nn Investigate Mixture EffectsInvestigate Mixture EffectsMixtures of timber species plus Mixtures of timber species plus switchgrassswitchgrassHypothesisHypothesis: no synergistic or antagonistic effects due to : no synergistic or antagonistic effects due to use of mixturesuse of mixtures

nn Model pretreatment reactionsModel pretreatment reactionsDevelop kinetic parameters from single species Develop kinetic parameters from single species exptsexpts..Predict Predict monomericmonomeric sugar concentrations for single species sugar concentrations for single species and mixtures and compare with experimental yieldsand mixtures and compare with experimental yields

nn Small scale pretreatments using �mini� reactor Small scale pretreatments using �mini� reactor systemsystem

Analyze small scale (< 1/100x) samples of biomassAnalyze small scale (< 1/100x) samples of biomass

Experimental Strategy for Pretreatment Experimental Strategy for Pretreatment of 50:50 Biomass Mixturesof 50:50 Biomass Mixtures

SWITCHGRASS

ASPEN RED MAPLE

BALSAM BASSWOOD

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Duplicates for each experiment were performed.

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Methods: Reactor ExperimentsMethods: Reactor Experiments

nn Initial reactor temp. setpoint = 400Initial reactor temp. setpoint = 400ooCC

nn Initial temperature = Room TemperatureInitial temperature = Room Temperature

nn Initial pressure = 15 psiInitial pressure = 15 psi

nn Agitator speed = 50 rpmAgitator speed = 50 rpm

nn Record pressure and temperature readingsRecord pressure and temperature readings

nn Collect samples during experiment for HPLC analysisCollect samples during experiment for HPLC analysis

nn AspenAspennn BalsamBalsamnn BasswoodBasswoodnn Red MapleRed Maplenn SwitchgrassSwitchgrass

Shu C. Yat, 2006

Experimental Results Experimental Results -- ReproducibilityReproducibility

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Results Results �� Obtained by HPLC DetectionObtained by HPLC Detection

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Xylose MonomerXylan Oligomer

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Arabinose

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Biomass MixturesBiomass Mixtures

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Xylose Formation and Degradation

Red Maple

Balsam

Red Maple/Balsam Mix1

Red Maple/Balsam Mix2

Expermiental results for red maple and balsam single species experiments compared with red maple/balsam 50/50 mixture results.

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Modeling Modeling XyloseXylose Reaction KineticsReaction Kinetics

XyloseXylose Kinetic Model Formation Kinetic Model Formation

•• Some studies assume that the Some studies assume that the hemicellulosehemicellulose contains two types of contains two types of xylanxylan, a , a fast reacting fraction and a slow reacting fraction. One example of a kinetic fast reacting fraction and a slow reacting fraction. One example of a kinetic model assuming this relationship is shown in [1].model assuming this relationship is shown in [1].

•• Other studies assume there is an intermediate Other studies assume there is an intermediate xylanxylan oligomeroligomer formation step formation step that is necessary for kinetic that is necessary for kinetic modellingmodelling. An example of this approach is shown . An example of this approach is shown in [2].in [2].

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•A third kinetic modeling approach simplifies the above models and utilizes a pseudo first order rate constant. Experimental data in these studies suggest that the biphasic nature of the substrates is negligible. This approach is shown in [3].

•An improved application of the kinetic model in equation [3]was used in this work to determine the kinetic parameters for pure biomass pretreatment which takes into account the simultaneous mechanisms of xylose production and degradation and is shown in [4].

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XyloseXylose Kinetic Model Formation (cont.) Kinetic Model Formation (cont.)

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Single Species Single Species XyloseXylose Formation and Formation and Degradation ModelsDegradation Models

A B C

D ESingle species kinetic models and experimental data for xylose formation and degradation of: (A)Switchgrass (B) Balsam (C) Red Maple (D) Aspen and (E) Basswood. For all species: (♦) experimental data and (!) model data.

Single Species and Mixtures Data and Single Species and Mixtures Data and Mixtures Kinetic Model Predictions for Mixtures Kinetic Model Predictions for

XyloseXylose Formation and DegradationFormation and Degradation

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Figure 2: Single species and mixture experimental data plotted with mixture model prediction for xylose formation and degradation of (A)Basswood/Red Maple Mixture (B)Basswood/Switchgrass (C)Aspen/Balsam (D)Balsam/Switchgrass (E)Aspen/Basswood and (F)Red Maple/Switchgrass. For A-F: ■ experimental data mixture 1, ▲experimental data mixture 2, ▬ (thick line) model mixture 1, ! (thin line) model mixture 2. Single species experimental data: (A) * Aspen, ● Balsam (B) * Basswood, ● Switchgrass (C) * Balsam, ● Switchgrass (D) * Basswood, ● Red Maple (E) * Aspen, ● Basswood and (F) * Red Maple, ● Switchgrass.

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Single Species Theoretical YieldsSingle Species Theoretical Yields

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Mixtures Theoretical YieldsMixtures Theoretical Yields

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Mixtures ResultsMixtures Results

•The hypothesis that mixtures of the five biomass species should have no net effect on the individual species kinetics has held true.

•The kinetics of these individual species as generated by the new kinetic model are in excellent agreement with experimental data and can be used to accurately predict xylose concentrations obtained from mixtures of the biomass species.

•Mixtures of the five species studied can be pretreated simultaneously and maximum sugar yields, which are comparable to individual species yields, will still be obtained when the optimum temperature is reached.

Small Scale PretreatmentSmall Scale Pretreatment

nn System of mini reactors System of mini reactors SwagelockSwagelock SteelSteelAllows for greater than 100% reaction volume Allows for greater than 100% reaction volume decreasedecrease

nn Enzymatic hydrolysis can also be done on Enzymatic hydrolysis can also be done on small scalesmall scale

nn Forestry WorkForestry Work3 Wild Type Poplar Controls and 5 modified 3 Wild Type Poplar Controls and 5 modified Poplar samples (big leaf)Poplar samples (big leaf)Less than 4 grams total of each sampleLess than 4 grams total of each sample

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Similarities and Differences Similarities and Differences in Reaction Setupin Reaction Setup

nn Setup still allows for temperature controlSetup still allows for temperature controlnn Only one sugar sampleOnly one sugar samplenn Normal heat up with rapid coolingNormal heat up with rapid coolingnn Identified 170Identified 170°°C as optimum C as optimum

temperaturetemperaturenn Not enough liquid volume to study Not enough liquid volume to study

oligomersoligomers

Forestry Work ResultsForestry Work Results

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Future and Ongoing WorkFuture and Ongoing Work

nn Future work needs to be done to further study the Future work needs to be done to further study the oligomericoligomeric trends as well as the feasibility of converting trends as well as the feasibility of converting the the oligomersoligomers into monomers.into monomers.

nn Modeling Alternative Conditions of Reactor Operation Modeling Alternative Conditions of Reactor Operation --Different reactor configurations will be investigated in Different reactor configurations will be investigated in order to optimize process by maximizing products and order to optimize process by maximizing products and minimizing byproductsminimizing byproducts

CSTR: CSTR: Continuous Stirred Tank ReactorContinuous Stirred Tank Reactor

PFR: PFR: Plug Flow ReactorPlug Flow Reactor

nn Small scale enzymatic hydrolysis of hybrid poplarSmall scale enzymatic hydrolysis of hybrid poplar

Future and Ongoing Work (cont.)Future and Ongoing Work (cont.)

nn HeterologousHeterologous Expression and Mutagenesis of Cellulose Expression and Mutagenesis of Cellulose HydrolasesHydrolases for Improved Performancefor Improved Performance

nn Characterization of Improved Characterization of Improved CellulaseCellulase Enzymes for Cellulose Enzymes for Cellulose HydrolysisHydrolysis

nn Life cycle assessmentLife cycle assessment

nn Advanced imaging technology, including Scanning Electron Advanced imaging technology, including Scanning Electron Microscopy (SEM) and optical microscopy, of untreated, Microscopy (SEM) and optical microscopy, of untreated, pretreated, and pretreated, and enzymaticallyenzymatically hydrolyzed biomass samples to hydrolyzed biomass samples to view structural changes with fluorescent tagsview structural changes with fluorescent tags

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Scanning Electron MicroscopyScanning Electron Microscopy

SEM Image of Aspen prior to pretreatment

SEM Image of Aspen after pretreatment

Enzymatic Hydrolysis of Cellulose to Enzymatic Hydrolysis of Cellulose to Yield Glucose for FermentationYield Glucose for Fermentation

nn IntroductionIntroductionnn Technological issuesTechnological issues

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Building Blocks of CelluloseBuilding Blocks of Cellulose

ββββ-1,4 bonds

Difficult to hydrolyze

180 rotated

Repeat unit is cellobiose

100 � 14,000 glucose units

More easily hydrolyzed

Difficult to hydrolyze


Hydrogen bondingbetween glucose unitsin adjacent chains


Adsorption of cellulase componentsonto cellulose

Sequence of EventsSequence of Events

Endogluconases hydrolyze amorphous regions of celluloseyielding broken ended chains

Cellobiohydrolases attack the chainsfrom the non-reducing end yielding Cellobiose (2 glucose units)

ββββ-glucosidases break cellobiose into glucose units

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Components of Components of CellulasesCellulases

nn CBH CBH �� Cellobiohydrolases: Cellobiohydrolases: II attacks the nonattacks the non--reducing ends, reducing ends, IIIIattacks the the reducing ends of chainsattacks the the reducing ends of chains

nn EG EG �� endoglucanases: hydrolyze amorphous regionsendoglucanases: hydrolyze amorphous regionsnn ββ--glucosidases: split cellobiose to glucoseglucosidases: split cellobiose to glucosenn CBD CBD �� CelluloseCellulose--binding Domainbinding Domain

Valjamae, P. �The Kinetics of Cellulose Enzymatic Hydrolysis � Implications of the Synergism Between Enzymes�. ACTA Universitatis Upsaliensis. Uppsala. 2002

CellobiohydrolasesCellobiohydrolases

Valjamae, P. �The Kinetics of Cellulose Enzymatic Hydrolysis � Implications of the Synergism Between Enzymes�. ACTA Universitatis Upsaliensis. Uppsala. Sweden. 2002

Cellulose binding domain (CBD)

Tether portion

Active site

Crystalline cellulose

Cellobiose

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Observed rate of Cellulose HydrolysisObserved rate of Cellulose Hydrolysis


Hypotheses1. Consumption of

easily hydrolysable components

2. Inhibition by reaction product cellobiose

3. Inactivation of cellulase

Rate decreases over time

Surface ErosionSurface ErosionModelModel


1. Processivity of the CBH is hindered by the surface erosion pattern due to the strong binding of the CBD.

2. Mechanisms 2 and 3 from the previous slide are not important, as shown in the referenced work below.

Cellobiohydrolase (CBH)

Confronting other CBH

CBH confronts erosion features

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Enzyme Engineering of Enzyme Engineering of CellulaseCellulaseEnzymes for Enzymes for EEnhanced Activitynhanced Activity

nn Clone genes for Clone genes for cellulasescellulases into a suitable host cellinto a suitable host cellnn Perform random mutagenesis of on these genes Perform random mutagenesis of on these genes

using errorusing error--prone Polymerase Chain Reaction prone Polymerase Chain Reaction (PCR)(PCR)

nn Screen for enhanced activityScreen for enhanced activitynn Characterize �mutant� Characterize �mutant� cellulasescellulases for activity and for activity and

stabilitystabilitynn Sequence Sequence cellulasescellulases to determine the sites of to determine the sites of

mutationsmutationsnn MichaelMichael--BrodeurBrodeur Campbell and Jill Jensen (PhD Campbell and Jill Jensen (PhD

candidates)candidates)

Genetic Engineering of E. coli for Ethanol Genetic Engineering of E. coli for Ethanol Production from Woody BiomassProduction from Woody Biomass

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Process to Convert Cellulosic Biomass to Process to Convert Cellulosic Biomass to EthanolEthanol

1 –3 mm

0.8% H2SO4160ºC10 min

TrichodermareeseiFermentation

Genetically-engineered E. coli

Saccharification is enzymatic hydrolysis of pretreated cellulose yielding Glucose using cellulase from Trichoderma reesei

Charles E. Wymen, “Biomass Ethanol: Technical Progress, Opportunities, and Commercial Challenges”, Annu. Rev. Energy Environ.,1999, 24: 189-226.

History of Costs for Ethanol ProductionHistory of Costs for Ethanol Production

Sequential enzymatic hydrolysis then fermentation

Improved fungal strain for cellulaseproduction

Improved cellulase (150L) produced by Genencore

Charles E. Wymen, “Biomass Ethanol: Technical Progress, Opportunities, and Commercial Challenges”, Annu. Rev. Energy Environ.,1999, 24: 189-226.

Simultaneous Saccharification-Fermentation process

More efficient cellulase

Fermentation of 6C and 5C sugars using a single microorganism

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The cellulase responsible for enzymatic hydrolysis of pretreated cellulosic biomass is strongly inhibited by hydrolysis products: glucose and short cellulose chains. One way to overcome cellulase inhibition is to ferment the glucose to ethanol as soon as it appears in solution. SSF combines enzymatic hydrolysis with ethanol fermentation to keep the concentration of glucose low. The accumulation of ethanol in the fermenter does not inhibit cellulase as much as high concentrations of glucose, so SSF is a good strategy for increasing the overall rate of cellulose to ethanol conversion. It is important to keep the rate limiting step in mind. In SSF the ethanol production rate is controlled by the cellulase hydrolysis rate not the glucose fermentation, so steps to increase the rate of hydrolysis will lower the cost of ethanol production via SSF. The US Department of Energy, National Renewable Energy Laboratory (NREL) is funding Genercor International, Inc. to develop low cost cellulases that will reduce the cost of cellulose breakdown by a factor of 10.

Simultaneous Simultaneous SaccharificationSaccharification + + Fermentation (SSF)Fermentation (SSF)

The Challenge of Fermenting all Sugars in The Challenge of Fermenting all Sugars in BiomassBiomass

Saccharomyces cervisiae

Zymomonas mobilis

Ferment glucose to ethanolUtilize 6C sugars onlyTolerant to ethanol

Can these microorganisms be genetically engineered to utilize 5C sugars?

Escherichia coli

Can not ferment glucose to ethanolCan utilize 6C and 5C sugars

Is it easier to genetically engineer E. coli to ferment ethanol?

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GlycolysisGlycolysis: : EmbdenEmbden--MeyerhofMeyerhof--ParnasParnas(EMP) (EMP) PathwayPathway

�Principles of Biochemistry�Lehninger, Worth

This pathway is representative of a human muscle cell or E. coli

The end product is not ethanol

Is it Easier to Genetically Engineer This Is it Easier to Genetically Engineer This Pathway into Pathway into E. coliE. coli, or, or

Two genes are needed. One for pyruvatedecarboxylase and another for alcohol dehydrogenase. These enzymes working together in the cell will divert Pyruvate away from other fermentation products to ethanol. This would convert E. coli into an ethanol-producing microorganism, where before it was not! �Principles of Biochemistry�, Lehninger,

Worth

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Is it Easier to Genetically Engineer This Is it Easier to Genetically Engineer This Pathway into Pathway into S. cerviciaeS. cerviciae or or Z. mobilisZ. mobilis? ?

This is the Pentose Phosphate pathway in E. coli. This pathway is obviously more complicated, containing many more enzyme-catalyzed reactions than the two-step pathway on the previous slide. The pathway for other 5C sugars (arabanose, mannose, galactose) would be similar.

3 Xyloses would enter here

To Glycolysisand ethanol

ethanol

�Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002

Genetic Engineering of Ethanol Production Genetic Engineering of Ethanol Production in in E. coliE. coli

A plasmid for Pyruvatedecarboxylase (pdc)

Ingram, Conway, Clark, Sewell, and Preston, �Genetic engineering of ethanol production in E. coli�, App. Environ. Microbio., 1987, 53(10), 2420-2425.

A plasmid for Alcoholdehydrogenase (adh)

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Ethanol Ethanol Production Production in Sealed in Sealed Cultures Cultures of of E. coliE. coli TC4TC4

Ingram, Conway, Clark, Sewell, and Preston,�Genetic engineering of ethanol production in E. coli�,App. Environ. Microbio., 1987, 53(10), 2420-2425.

High Performance Liquid Chromatography Profiles

G = glucoseS = succinateL = lactic acidA = acetic acidU = unknownE = ethanol

Plasmid-free TC4 TC4withpLOI295

TC4withpLOI284

TC4withpLOI276

Questions?Questions?

Midwestern land cover (USFS North Central Research Station image)

applications of molecular applications of molecular ... · applications of molecular applications...

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