Hydrolysate Fermentation Characterization for Xylose-Fermenting Saccharomycesy g y

Cerevisiae Strains Derived From Directed Evolution for Improved Xylose Utilization p y

and Tolerance to InhibitorsTongjun Liu Daniel L Williams Lucas S Parreiras Li QinTongjun Liu, Daniel L. Williams, Lucas S. Parreiras, Li Qin, Benjamin D. Bice, Donald Busalacchi, Ragothaman AvanasiNarasimhan, Rebecca J. Breuer, Trey K. Sato,  David B. Hodge

AIChE Annual Meeting

September 28 2012September 28, 2012

2

Tuesday, 12:30 pm. Room 334(260a) Characterization of Carbohydrate Accessibility and Enzyme Adsorption

Other Talks from Our Group

(260a) Characterization of Carbohydrate Accessibility and Enzyme Adsorption Capacity for Diverse Cell Wall Phenotypes Subjected to Alkaline Hydrogen Peroxide Pretreatment.

Tuesday, 12:55 pm. Room 334(260b) Lignin Structural Changes Associated with Oxidative Pretreatment Catalyzed by Cu-Diimine Complexes.

Wednesday, 12:30 pm. Room 335(498a) Hydrolysate Fermentation Characterization for Xylose-Fermenting Saccharomyces Cerevisiae Strains Derived From Directed Evolution for Improved X l U ili i d T l I hibiXylose Utilization and Tolerance to Inhibitors.

Wednesday, 2:10 pm. Room 303(471e) Characterization of Solubilized Biopolymers Fractionated From Alkali Pulping(471e) Characterization of Solubilized Biopolymers Fractionated From Alkali Pulping Liquors.

Thursday, 12:55 pm. Room 335(667b) Ph t i d M lti O i A h t Add M l l B ttl k i(667b) Phenotypic and Multi-Omic Approaches to Address Molecular Bottlenecks in the Fermentation of Lignocellulose Into Ethanol by Saccharomyces Cerevisiae.

Outline3

OutlineBackground: Pretreatment 

Xylose fermentation

Hydrolysate inhibitors 

Rationale/GoalsRationale/Goals

Results

Summary

Hemicellulose4

Ch i l P t t tEnzymatic Depolymerization of 

Cellulose

Chemical Pretreatment Polysaccharides

Biochemical Conversion of Plant Cell Wall Polysaccharides 

Lignocellulose Feedstock (Plant Cell Walls)

yto Biofuels

Biological Conversion of Monosaccharides

Microbial MetabolitesEthanol, Butanol, Carboxylic Acids, Alkanes, Isoprenoids, …

Plant Cell Wall Matrix Polymers• Lignin (10‐35%)

• Heteropolysaccharides(25 45%)(25‐45%)

• Cellulose (35‐55%)

Composition of Plant Cell WallsPentose utilization is important Xylose is the 2nd mostXylose is the 2nd most abundant sugar in biosphere

F li ll lFor lignocellulose conversion to biofuels, pentose fermentation is an attractive traitattractive trait

Other components in plant cell walls can be 

Monocots (Grasses)

Woody Plants

Hardwoods Softwoodsproblematic for fermentation

Source: Rydholm S, Pulping Processes. Wiley Interscience.

Microorganism Background: Xylose Utilization by S. cerevisiaeXylose Utilization by S. cerevisiae

Challenge: Convert all lignocellulose polysaccharide sugars to ethanol at g p y ghigh yields, titers, and productivities

Two approaches for xylose fermentationBacteria (and Piromyces): Xylose isomerase (XI)

Yeast: Xylose reductase (XR) + xylitol dehydrogenase (XDH)• Potential xylitol accumulation due to redox imbalance using XR, XDHpathway (NAD+/NADH vs. NADP+/NADPH)

NAD(P)H NAD(P)+ NAD+ NADH ATP ADP

Xylose XyluloseXylitolPentose Phosphate

Pathway

EthanolXR XDH

NAD(P)H NAD(P) NAD NADH

XK

ATP ADP

Xylulose -5-Phosphate

PathwayGlycolysisFermentationXI

Alkaline Hydrogen Peroxide PretreatmentBased on existing alkaline hydrogenBased on existing alkaline hydrogen peroxide pulp bleaching stages in the paper industry

Alkaline‐oxidative pretreatments as either standalone pretreatments ORdelignifying “finishing” post‐delignifying finishing postpretreatment  step

Unique advantagesWell‐suited for grasses

Current Challenges:Process integration

Economics

Water use/recycle

Alkaline hydrogen peroxide bleaching tower >1000 tpd capacity at Smurfit‐Kappa Kraftliner, Piteå, Sweden (photo / ycourtesy: Outokumpu Oy)

9Alkaline Hydrogen Peroxide PretreatmentPretreatment Liquefaction/Saccharificationq

90%

100%

Unquantified Solids

h

90%

100%

Unquantified Solids

A hSolids transferred to Solids transferred to the

50%

60%

70%

80%

onen

t Fraction

Ash

Water+EtOH Extractives

Acetate

Uronic Acids

Galactan50%

60%

70%

80%

onen

t Fraction

Ash

Water+EtOH Extractives

Acetate

Uronic Acids

GalactanInsoluble Insoluble

Solids transferred to the liquid phase

Solids transferred to the liquid phase (hydrolysate)

10%

20%

30%

40%

Compo

Galactan

Mannan

Arabinan

Xylan

Glucan10%

20%

30%

40%

Compo

Galactan

Mannan

Arabinan

Xylan

Glucan

FractionFraction

0%

0 3 6 12 18 30 36 42 48

Pretreatment Time (h)

Lignin (Klason)

ASL

0%

0 3 6 12 18 30 36 42 48

Pretreatment Time (h)

Lignin (Klason)

ASL0 4 8 12 16 20 24 28 32 36 40 44 48

Hydrolysis Time (h)Banerjee et al. (2012). BiotechnolBioeng. 109(4):922‐931. 

Fermentation Inhibitors 10

>1% of plant cell wall 10-15% of plant cell wall

FA

Other aromatics

Extractives: 8-15% of plant cell wall

pCA

2-5% of plant cell wall Xylan 

Other Polymers

(?)

Oxidative degradation products of sugars, lignin, extractives? >1%Acetatep-coumaric

acidFerulic

acid

Low MW FractionArabinanGlucan

Lignin (Klason)

90%

100%

Unquantified Solids

A h

Solids transferred to the liquid phase (pre-

Low MW FractionHemicellulose

Aggregates (minimal lignin?)

Low Mol. Wt. Hemicelluloses (?) (+ Pectin,starch,..?)

High MW Fraction

Polymers in hydrolysate

50%

60%

70%

80%

nent Fraction

Ash

Water+EtOH Extractives

Acetate

Uronic AcidsInsolubleInsoluble

the liquid phase (pre-hydrolysate)

ass

Abu

ndan

ce

10%

20%

30%

40%

Compo

n Galactan

Mannan

Arabinan

Xylan

Insoluble Insoluble FractionFraction

Pol

ymer

M

Lignin/aromatics (+ Hemicellulose?)

0%

10%

0 3 6 12 18 30 36 42 48

Pretreatment Time (h)

Glucan

Lignin (Klason)

ASL

11

Monocot Lignins pCA

Lignin

S

Monomer composition and structural organization significantly different than herbaceous and woody dicots or

Li i

Sherbaceous and woody dicots or gymnosperm lignins

Ferulates and p‐coumarate can comprise a significant fraction of grass lignins Lignin

FA

significant fraction of grass ligninsEster crosslinksHighly condensed (~85%)High phenolic hydroxyl content

High alkali solubility

12Quantification of Solubilizedp-Hydroxycinnamic Acids in Hydrolysatesp y y y yIdentification and quantification of ferulic acid and p‐coumaricacid in corn stover and switchgrass hydrolysates by LC‐MS 

Can represent more the 1.5% of the cell wall

Potential to reach concentrations of >1.0 g/L pCA and                                     >0.5 g/L FA in hydrolysates from 20% solids pretreatment

4.00E+07 NaOH only

1.0%1.60

g/g)

/L)

SG Ferulic Acid

SG pCoumaric Acid

Inhibitory to fermentation

2.50E+07

3.00E+07

3.50E+07

(cps)

pH 11.5 AHP (12.5% H2O2)

pH 11.5 AHP (25% H2O2)

pH 11.5 AHP (50% H2O2)

Ferulic AcidCoumaric Acid

0.6%

0.8%

0.80

1.20

d Yield on

 Biomass (g

cid Co

ncen

tration (g/

CS Ferulic Acid

CS pCoumaric Acid

5 00E 06

1.00E+07

1.50E+07

2.00E+07

Intensity

 

187m/z amupCA FA

0.2%

0.4%

0.40

Hydroxycinn

amicAcid

Hydroxycinn

amicAc

0.00E+00

5.00E+06

4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Time (min)

0.0%0.000 0.05 0.1 0.15 0.2 0.25

H

Hydrogen Peroxide Loading (g H2O2/g Biomass)

Outline13

Outline

Background:Background: 

Rationale/GoalsGenerate, screen, isolate Saccharomyces strains with:

• Xylose fermenting capacity

• Increased tolerance to AHP inhibitors

lResults

Summary

Strain Developmentf

14

46

10

20

d ce

ll de

nsity YB210

YIIc17_E5CEN.PK2S288c

p-Coumaric and Ferulic AcidspCAand FAAcetateNa2SO4

Screening of >100 WT Saccharomyces strains for tolerance to AHP

0 4 8 12 16 20 24Time (hrs)

0

2

4

Nor

mal

izedtolerance to AHP

hydrolysate inhibitorsNa+ Acetate p‐coumaric ai

ns

Acetate

46

10

20

lized

cel

l den

sity

Na , Acetate, p coumaricacid (pCA), ferulic acid (FA)

Parallel screening by OD  cyes

stra

0 4 8 12 16 20 24Time (hrs)

0

2

Nor

ma

measurement in microtiterplates

Q tifi d ifi acch

arom

c

46

10

20

lized

cel

l den

sity

Na2SO4Quantified as specific growth rate (μ)

Relative Specific Growth Rate

Sa

0 4 8 12 16 20 24Time (hrs)

0

2

Nor

maRelative Specific Growth Rate

LowHigh

Strain Developmentl f d Wild t pe

15

Evolution for improved xylose fermentation

b l

Wild‐type S. cerevisiae

Chromosomal integration of xylose reductase(XR), xylitol dehydrogenase (XDH), and 

YB210

Subsequent evolution in presence of p‐coumaricacid and ferulic acid for

Ferments xylose

( ), y y g ( ),xylulokinase (XK) from Pichia stipitis

Y2Aacid and ferulic acid for improved  hydrolysatetolerance

I d l

Aerobic evolution on YP‐xylosemedia for improved xyloseutilization

Improved xylosefermentationY35 Y56 Y60 Y73

Aerobic evolution on YP‐xylosedi ith h d i i

Improved tolerance to AHP

media with p‐hydroxycinnamicacids for improved hydrolysatetolerance

Y83 Y84 Y85 Y86Improved tolerance to AHPhydrolysate inhibitors

Y87

Outline16

Outline

Background:Background: 

Rationale/Goals

ResultsStrain performance on inhibitors, hydrolysatesp , y y

Characterization of hydrolysates

SummarySummary

0.12

Performance of Xylose‐Fermenting Yeasts14

0.06

0.08

0.1

Y2A8

10

12

Growth curve on YNB medium

Anaerobic growth in YNB media

0.02

0.04

Y56

Y73

Y87

Growth2

4

6

8

OD

600 Y2A

Y35 Y56 Y73 Y87 Evolution improves 

lose and ethanol0Specific xylose 

uptake rate, Qxyl (g/g/h)

Biomass yield (g/g)

50

40

Growth

0 20 40 60 80 100 1200

2

time (h)

xylose and ethanol rates

30

35

40

45

50

c. (g

/l)

20

25

30

35

c. (g

/l)

XyloseConsumption

Ethanol Production

Ferments xyloseY2A

Improved xylosefermentationY35Y56Y73

10

15

20

25

xylo

se c

onc

Y2A Y35 Y56 Y73 Y87

5

10

15

20

EtO

H c

on

Y2A Y35 Y56 Y73 Y87

fermentation

Improved tolerance to AHPhydrolysate inhibitors

Y87

0 20 40 60 80 100 1200

5

time (h)

0 20 40 60 80 100 1200

time (h)

18Impact of Individual Inhibitors on Y73All inhibitors impact biomass yield on glucose

Ferments xyloseY2A

Improvedxylose

Differing impacts on xyloseuptake

Improved xylosefermentation

Y73

Improved tolerance to AHPY87uptake

hydrolysate inhibitorsY87

Performance of Xylose-Fermenting YeastsValidation by improved growth and viability in rich media, y p g y ,microaerobic conditions

Significant improvement in tolerance for xylose media

75

1000 hrs

20 hrs

25 hrs

44 hrs Ferments xyloseY2A

50

iability (%

)

49 hrsImproved xylosefermentation

Y73

Improved tolerance to AHPhydrolysate inhibitors

Y87

25

Cell Vi hydrolysate inhibitors

0

Y73 YPD Y87 YPD Y73 YPX Y87 YPX

Glucose Xylose

Generation and Characterization of High‐Solids AHP Hydrolysates

20

High Solids AHP HydrolysatesThree corn stover and two switchgrass hydrolysates

Goal: Fermentable hydrolysates at sugar concentrations >100 g/L

140

160Ara

Xyl 70%

80%Glc

100

120

140

(g/L)

Xyl

Glc

50%

60%

%

nomers

Xyl

60

80

100

ncen

tration 

30%

40%

50%

rsion to M

o

20

40

60

Con

10%

20%

30%Co

nve

0

20

SG1 SG2 CS1 CS2 CS30%

10%

SG1 SG2 CS1 CS2 CS3

Generation and Characterization of High‐Solids AHP Hydrolysates

21

High Solids AHP HydrolysatesQuantification of fermentation inhibitorsImportant inhibitor pools: Inorganics, aliphatic acids, phenolic acids

9

101200 SG1

SG2

1

1.2

250

300

6

7

8

(g/L)

800

1000

)

CS1

CS2

CS30.8

1

 (g/L) 200

250

urry

4

5

6

oncentration

 

400

600

Na+

 (mM)

0 4

0.6

ncen

tration 

100

150

g solids / g sl

1

2

3Co

200

400

0.2

0.4Con

50

100g

0

1

Formate Acetate0

Na+0

Ferulate p‐coumarate0Solids to Hydrolysis

Effect of pH on Undetoxified High‐Sugar Corn Stover Hydrolysate Fermentation  

22

Engineered yeasts capable of fermenting high‐sugar, undetoxified AHP hydrolysates (CS2) to 4% ethanoly y ( )Minimal supplementation: 1.67 g/L YNB + 2.27 g/L  of urea

High pH is criticalg p

80

Glc Y87 (pH5.0) OD 1

80

90

Y87 (pH5.5) OD 18

pH 5.0 pH 5.5

60

(g/l)

Xly EtOH Xylitol glycerol OD 600

4

600 50

60

70

(g/l)

Glc Xly EtOH Xylitol glycerol 4

6

600

20

40

conc

.

2 OD

6

20

30

40

conc

. ( OD 600

2

4

OD

6

0 50 100 150 200 2500

time (h)

0

0 50 100 150 200 2500

10

time (h)

0

1645

Estimating Yields for Y73 and Y87: Diverse AHP Hydrolysates

23

10

12

14

16

ted (g/L)

Ethanol

Xylitol

Glycerol30

35

40

45

ted (g/L)

Ethanol

Glycerol

Metabolite Yields:Relatively unaffected

4

6

8

Metabolite Gen

erat

10

15

20

25

Metabolite Gen

erat Relatively unaffected 

by inhibitors

0

2

0 10 20 30 40 50

M

Xylose Consumed (g/L)

0

5

10

0 20 40 60 80 100

M

Glucose Consumed (g/L)Glucose Consumed (g/L)

1.2

1.4

1.6

1.8

g DCW

/L)

Detoxified CS1 or SG1, pH 5.0Undetoxified SG, pH 5.5Pure Xylose

5

6

7

DCW

/L)

Undetoxified SG2, CS2, CS3, pH 5.5

Detoxified CS1 or SG1, pH 5.0

Undetoxified CS1 or SG1, pH 5.0

Pure Glucose

Cell Mass Yields:Strongly affected by 

y = 0.0302x0.4

0.6

0.8

1

1.2

ll Mass Gen

erated

 (g

2

3

4

Mass Gen

erated

 (g D

inhibitors

R² = 0.0698

0

0.2

0.4

0 10 20 30

Cel

Xylose Consumed

0

1

0 20 40 60 80 100

Cell M

Glucose Consumed (g/L)

0 16 0 5

Impacts of AHP Hydrolysates on Fermentation: Rates and Yields

24

0.1

0.12

0.14

0.16

Rate (g/g/h)

Y2A

Y73

Y87

0 3

0.35

0.4

0.45

0.5

e Rate (g/L/h)

Y2A

Y73

Y87Inhibition of ratesStrong impact on xylose rate

0.04

0.06

0.08

cific Xylose Uptake 

0 1

0.15

0.2

0.25

0.3

olute Xylose U

ptake

Y87 shows improved xylosefermentation in hydrolysates

0

0.02

SG1 CS1 CS2 YNB G+X

Spec

0

0.05

0.1

SG1 CS1 CS2 YNB G+X

Abso

Ferments xyloseY2A

Improved xylosefermentation

Y73

Inhibition of biomass yields 0 06

0.07

0.08

0.09

0.1

ucose (g/g)

Y2A

Y73

Y87

Improved tolerance to AHPhydrolysate inhibitors

Y87

Inhibition of biomass yieldsAHP hydrolysates impact biomass yield

Mechanisms: ATP‐driven efflux of H+, Na+, or 0 02

0.03

0.04

0.05

0.06

omass Yield on

 Glu

Mechanisms: ATP driven efflux of H , Na , or phenolic acids decreases anaerobic ATP availability for growth?

0

0.01

0.02

SG1 CS1 CS2 YNB G+X

Bi

Summary / ConclusionsStrains initially selected for improved

25

Strains initially selected for improved hydrolysate inhibitor tolerance

Strains can completely fermentStrains can completely ferment glucose and xylose in AHPhydrolysates with no detoxification y yto > 4% ethanol

Hydrolysate inhibitors impact both y y pbiomass yields and xyloseconsumption rates

0.35

0.4

0.45

0.5

ate (g/L/h)

Y2A

Y73

Y87

0.07

0.08

0.09

0.1

se (g/g)

Y2A

Y73

Y87

Strains evolved on phenolic acids show improved xylose fermentation 

0.05

0.1

0.15

0.2

0.25

0.3

Absolute Xylose U

ptake Ra

0.01

0.02

0.03

0.04

0.05

0.06

Biom

ass Yield on

 Glucos

rates in hydrolysates 0SG1 CS1 CS2 YNB 

G+X

0SG1 CS1 CS2 YNB 

G+X

AcknowledgementsResearch Group: Collaborators:p

Trey SatoU. Wisc.U. Wisc.

Funding:gDepartment of Energy,BER DE‐FC02‐07ER64494David Hodge

Dan WilliamsMarc Hansen(†)

Muyang LiAlex Smith(†)Ryan Stoklosa

Dr. Tongjun LiuCharles Chen

Zhenglun Li

N t i t dNot pictured: Elizabeth Häggbjer, Natassa Christides, Genevieve Gagnier

Thank You!

Questions?

27