icrisat 2 march 2016ksiconnect.icrisat.org/wp-content/uploads/2016/03/01032016.pdf · u ni te d s...

• Wilfred Vermerris • Department of Microbiology and Cell Science-IFAS

• University of Florida Genetics Institute

Sustainable production of

fuels and chemicals

from sorghum

ICRISAT 2 March 2016

Agriculture’s Challenge

ENERGY

POPULATION CLIMATE

www.nasa.gov

Florida as a test case for sustainable bioenergy production

• Sunny and warm climate plus long

growing season conducive to crop

production

• High pest and disease pressure

• Much of the Florida peninsula can be

classified as marginal land

• Low organic matter content

• Low water retention capacity

• Limitations on use of irrigation,

pesticides and fertilizers

Climate change and the need to avoid competition with food production will require the development of reliable crop production systems under increasingly challenging conditions

Plant

Shut-Down

Maintenance

Sweet sorghum can extend the processing window of

sugarcane

Jan Feb Mar April May June July Aug Sept Oct Nov Dec

Sweet Sorghum

Planting

S.S. Harvest

1st Crop

S.S. Harvest

2nd Crop Biofuel Cane Biofuel Cane

Sweet Sorghum

Planting

Planting and harvest logistics for a biorefinery that

processes sugarcane and sweet sorghum

United States Department of Agriculture

Topics

Animal HealthBiotechnologyEmployment ResourcesEnergyEnvironment and Natural ResourcesEmergency Preparedness andResponseFarm BillFood and NutritionFood SafetyForestryHomeland SecurityLaws and RegulationsMarketing and TradeOutreachPlant HealthResearch and ScienceRural and Community DevelopmentTravel and Recreation

Programs and Services

ASSISTING RURAL COMMUNITIES

BroadbandGrants and LoansDisaster AssistanceInsurance Programs

FOOD AND NUTRITION

SNAPWICFood SecurityChild Nutrition ProgramsNational Organic Program

CONSERVATION

Restoration and ConservationEnvironmental Markets

Next-generation sweet sorghums: Sustainable production of

fuels and chemicals from juice and bagasse

pentoses

fuels

fuels

chemicals

chemicals (e.g. D-and L-lactate)

CO2

ferm

en

tation

ferm

en

tatio

n

pre

treatm

ent

hexoses

chemicals

CBP

Sweet sorghum


Topics





FOOD AND NUTRITION


CONSERVATION




pentoses fuels

chemicals

fuels


CO2

ferm

en

tation

ferm

en

tatio

n

pre

treatm

ent

hexoses

lignin

nanotubes

chemicals

heat

CBP

Sweet sorghum

enhanced

plastics

jet fuel

Sweet sorghum in Florida…..

• Commercial sorghum breeding programs have

traditionally been based in Texas and the Great Plains

– Primary focus has been on grain and forage

sorghums

• Sweet sorghum breeding programs have traditionally

been based in MS, TN, KY, later also TX

• Use of existing sweet sorghum germplasm in Florida

does not generate the yields reported in the regions

where the germplasm originated

– Pests and diseases prevalent in the region

– Low-fertility soils, low water retention capacity, storms

• The UF sorghum breeding program is focusing on developing regionally adapted sweet sorghums that give high yields with limited inputs

Our number 1 target: Anthracnose resistance

• Colletotrichum sublineolum

– Fungal pathogen

– Thrives in humid

conditions

• Major problem in

southeastern USA

– Infects all aerial parts of

sorghum

– 70% yield reduction

Source: http://www.plantwise.org

/KnowledgeBank/Datasheet.aspx

Source: Crouch and Beirn, Fungal Diversity, 2009

Adapting sorghum to Florida

Commercial Improved UF cultivars

Dry weight yields: 15-20 t/ha

Mapping anthracnose resistance loci

• Bk7

– Florida inbred line

– Anthracnose resistant

– Unknown resistance mechanism

• Early Hegari-Sart

– Anthracnose

susceptible

• Biparental mapping population

– 135 lines

– F4 and F5

Bk7 Early Hegari-Sart

Terry Felderhoff

Mapping population

• Selection of inbred parents with

contrasting phenotypes

– Cross parents

– Heterozygous F1 selfed

– Multiple F2 progeny grown

– Continued selfing within lines

– 135 F4 and F5 lines

• Increased recombination

– Genetic mosaics of parental alleles,

and mostly homozygous

• Observe disease phenotypes among

lines within the population

– Replicate across years and locations

• Identify resistance loci by association

Genotyping by Sequencing

• Truncated genome sequencing

• Fast, inexpensive, data rich

• Single Nucleotide Polymorphisms (SNPs) determined

base on sequence comparisons among mapped

fragments derived from the parents

• GBS performed at Cornell University

Source: http://cbsu.tc.cornell.edu/lab/doc/GBS_overview_20111028.pdf

The raw GBS data are dirty!

A custom filtering procedure:

Terry Felderhoff

Determining the association between

SNP markers and disease resistance

• Determine distribution of marker alleles across infected and

uninfected lines

– Fisher’s exact test, -log10 of p-value

– H0: no difference in frequency of allele between contrasting

disease phenotypes

– Ha: high frequency of Bk7 alleles in resistant plants

– False discovery rate (FDR) with α=0.05 used as threshold

Marker Allele

Early Hegari Bk7

Disease

Phenotype

Resistant a b a+b

Susceptible c d c+d

a+c b+d

a+b+c+d

=n

Terry Felderhoff

Association analysis

1 3 2 4 5 6 7 8 9 10

(Too many) Candidate genes….

• Chromosome 7 locus

– No reported anthracnose resistance loci in literature

– 44.9 Mbp containing 740 transcripts

• Chromosome 9 locus

– Known Cs1B resistance gene on chromosome 9 • 1.8 Mb from closest edge of QTL

• Multiple recombination events between QTL and Cs1B

– 3.2 Mb containing 342 transcripts

• Similarity search of transcripts with BLASTX followed by BLAST2GO ontology analysis

– Chrom 7 locus has 46 candidate genes

– Chrom 9 locus has 39 candidate genes

Fine-mapping resistance loci

× Susceptible parent

Resistant parent

Resistant cultivar 1

• The five newly released sweet sorghum cultivars share the

anthracnose resistant parent ‘Bk7’ in their pedigree

• Selection criteria for these sweet sorghums included anthracnose

resistance

• Seven generations of inbreeding will have increased the probability of

recombination events in and around the QTL

×

F7

P

Chr 9 QTL




Inbreeding with selection for resistance

Validating candidate genes

• As a result of the fine mapping, the resistance QTL

was reduced from 3.2 Mb to 0.6 Mb, with just 6

candidate resistance genes

• Virus-induced gene silencing will be used to validate

the role of these genes in anthracnose resistance

Brome Mosaic Virus

RNA1

RNA2

RNA3

Tim Nelson, Samuel Roberts Noble Foundation

Silencing of resistance gene

Colletotrichum conidia

Resistant line

Improving biomass conversion with

brown midrib mutants • Generated in the 70’s at Purdue University via chemical

mutagenesis – 19 mutants

• Nine spontaneous bmr mutants discovered later on

• Initially used as forage because of improved intake and

palatability

Brown midrib sorghums

• What is the impact of the various bmr

mutations on cell wall composition and

biomass conversion?

• If we clone the most ‘valuable’ bmr

genes, we will not only generate

convenient molecular markers, but also

learn something about biochemical

targets

• Ultimately: enzyme engineering based

on detailed structural models

Saballos et al. (2008) Bioenerg. Res. 1: 198

OGO

OCH3S

O

HO

OH

OH

OH

OH3CO

G

OG OH

OH

OCH3

G

O

OCH3

G HO

OH

H3CO

S

O

GOH

HO

O

O

O

G

O

OCH3

S

HO OH

OH

HO

O

OCH3

O

H3CO G

OHO

HO

OCH3

H3CO

OCH3H3CO

OCH3

OCH3

O

H3CO

G

OH

H S G p-hydroxyphenyl guaiacyl syringyl

COOH COOH

NH2

OH

OH

OH OH OH

OH

OCH3 H3CO

OH

H2 COH

OH

OCH3 H3CO

H2 COH

OH

H2 COH

O O O

OCS-CoA OCS-CoA

OCH3

OCH3

OCH3

bmr12

bmr6

bmr2

0

5

10

15

20

25

N-bmr 2 N-bmr 3 N6-bmr N-bmr 12 N-bmr 19

Kla

so

n l

ign

in (

mg

/g )

Wt-bmr isolines

Wt bmr

Klason lignin (Purdue collection)

The bmr mutations reduce lignin content by ~15-20%


Unpretreated

Pretreated

(dilute sulfuric acid)

Enzymatic saccharification of stover (Purdue)


Glucose yields of sorghum stover from bmr and wild-type lines without and

with pretreatment after 48 h of enzymatic saccharification (60 FPU/g cellulose)

Lignin content vs. subunit composition

Lignin content vs. glucose yield (USDA)

• Lignin content

alone does not

explain variation in

glucose yield

• The S/G ratio

appears to

influence glucose

yield following a

threshold model

• The threshold lies

somewhere

between 0.08 and

0.4

Kla

son lig

nin

(m

g/g

)

S/G 0.63 0.03

Glu

cose y

ield

(m

g/g

)

0

50

100

150

200

250

300

BTx623 bmr12-ref bmr12-30 bmr12-34 bmr12-35

0

5

10

15

20

25

BTx623 bmr12-ref bmr12-30 bmr12-34 bmr12-35

0.07 0.08 0.42

c c b b

b

ab a

c b

bc

Sattler et al. (2012) BioEnerg. Res.

Plant Physiology 165: 1440 (2014)

s-trans s-cis

productive non-productive

s-trans s-cis


H3CO

Can we engineer COMT for broader or

more specific substrate specificity?

• Tailor lignin subunit composition

• Minimize negative impacts on plant

growth

Substrate inhibition of COMT

Substrate orientation

matters

s-trans s-cis


The high rotational energy associated with the conformation of the propenal side chain prevents the spontaneous switch of the non-productive isomer to the productive isomer

Can we use these structural features to engineer COMT for broader or more specific substrate specificity without inhibition? • Tailor lignin subunit composition • Minimize negative impacts on plant

growth

Industrial-scale processing

University of Florida Stan Mayfield Biorefinery

• L+SScF

– Liquefaction through

phosphoric acid/ steam

pretreatment (190C, 5 min)

and saccharification

• 3 x 10,000 Gal fermentors

Techno-economic analysis

Assumptions:

2.5% enzyme, $1.00 kg enzyme, $40/ ton dry bagasse, 0.24 g EtOH/g DW I. Nieves/ R. van Rijn

‘Smart delivery’ of drugs or DNA

• Use of carriers that target specific organs or tissues

– Viral vectors (DNA) – Adeno associated virus

– Carbon nanotubes (drugs, DNA) - $500/gram

• Benefits:

– Lower dose, reduced side effects

– Reduced degradation of therapeutics

• Challenges

– Viruses: immunogenic

– CNTs: cytotoxic

lbl.gov sciencephoto.com

gaia3d.co.uk

Lignin nanotubes (LNTs)

Lignin base-layer

Deposition of DHP liner

Dissolution of membrane

membrane

109 pores cm-2

109 nanotubes cm-2

Caicedo et al. (2012) Nanotechnology 23: 105605

Nanotubes Nanowires

200 nm 200 nm

1 µm 1 µm

COOH

OH

COOH

OH

OCH3

TEM

SEM

Caicedo et al. (2012)

ferulic acid : p-coumaric acid

5 : 1

ferulic acid : p-coumaric acid

1 : 5

Lignin nanotubes have low cytotoxicity

Carbon nanotubes: 50% cell loss at 5-10 mg/mL (Pantarotto et al., 2004)

Thioglycolic acid lignin NaOH lignin

Ten et al. (2014) Biomacromolecules

Pine TGA LNTs

15 μm

Confocal microscope images showing the nuclei of human

HeLa cells in cell culture.

Pine NaOH LNTs

LNT-mediated transfection of HeLa cells with GFP gene

• Gene expression levels vary

as a function of lignin source

and isolation method

Blank (-) PEI (+)

Sorghum-KL

Poplar-KL

Poplar-TGA 100 x magnification 1.4 mg LNTs per well

Mechanism of DNA uptake in the cell?

endocytosis endocytosis

MAGNET Streptavidin-paramagnetic bead

Biotinylated DNA

Is there a physical interaction between LNT

and DNA?

Native DNA

LNT

1 μm bead

Biotinylated DNA

Ten et al. (2014) Biomacromolecules

Delivery of therapeutic agents

Therapeutic agent

Ligand for cell-specific receptor Labile bond

Labile bond

So what are the prospects for sweet sorghum?

• There is plenty of land available to produce bioenergy

without competing with food production

– Requires adapted germplasm

• Sweet sorghum cultivars and hybrids are/will be

available, even for regions where sweet sorghum is a

new crop

– brown midrib hybrid sweet sorghums with

anthracnose resistance

– Plant breeding can benefit from and contribute to

genomics-assisted crop improvement methods

– Structural biology and genome editing as tools to

redesign metabolic pathways

• The bioprocessing technology is available to use both

the juice and the bagasse as a source of fermentable

sugars for fuels and chemicals

Challenges for biorefineries in the US

• The original three drivers for renewable fuels were

– Energy independence (control of supplies)

– Finite supplies of petroleum

– Mitigation of climate change

• Hydraulic fracking in the US effectively took care of

the first two drivers

• The prices of oil and natural gas are at record lows

– Difficult for biofuels to compete

This has had a major impact on the construction of new biorefineries We need a level playing field (tax policies) and a mechanism to reward sustainably produced biofuels from home-grown crops

Acknowledgements


Topics





FOOD AND NUTRITION


CONSERVATION


ChulHee Kang (WSU)

Scott Sattler (USDA-ARS)

John Ralph (UWisc)

Hugo Cuevas (USDA-ARS)

Terry Felderhoff

Alejandra Abril

Ji Wang

Ana Saballos

Rick van Rijn

Sanyukta Shukla

John Erickson

Lonnie Ingram

Amelia Dempere

Julene Tong

KT Shanmugam

Jim Preston

Randy Powell

Main features of LNTs

• Ability to customize

– Optical properties

– Dimensions (tubes vs. rods)

– Nano-mechanical properties

– Site of delivery: cytosol vs. nucleus

• Physical and chemical properties vary as a

function of lignin source

– Can be tailored via genetic approaches!

• Potential as a high-value co-product from a

biorefinery

Sorghum: A versatile C4 grass

• Annual, seed-propagated, naturally self pollinating

• Originated in Africa, cultivated across the globe

• Genetically diverse

• Robust: adaptable to many different conditions

– Tolerates hot and dry environments

– Limited input requirements

• Used for the production of grain, sugars and biomass

• Biomass:

– 40% cellulose

– 30% hemicellulose (glucuronoarabinoxylans >

xyloglucans)

– 20% lignin: H (3%), G (59%), S (38%)

– 10% pectin, waxes, ‘extractives’, minerals

Production of fuels and chemicals from

sweet sorghum in Florida

• Tall plants that accumulate soluble sugars

in the stem juice (15-22%)

• Stress tolerant (drought, heat) and limited

input requirements compared to sugarcane

• Minimal competition with food production

• Compatible with sugarcane production in

South Florida

• Ideal crop for transition from sugar-based to

lignocellulosic fuels and chemicals

Cultivar breeding strategy: Pedigree method

F5: estimate sugar yield; compare

performance against checks; look

for homogeneity; bulk seed

F4: as F3 plus select for sugar

based on destructive sampling of

sibs

F3: select 5-10 individuals among

and within families for biomass,

maturity, disease resistance

F2: select 5-20 biggest and cleanest

plants

Parents: high-sugar x other trait

400 F2 plants

5-20 F3 families

1-5 F5 families

5-10 F4 families

Each year we evaluate 10-20 sweet

sorghums from around the world

Release top performers

F6: replicated yield trials

(two years, three locations) 1-3 F6 families

Sweet sorghum hybrids • Hybrids are the product of a cross between two different inbred

parents; the progeny is planted and harvested

• Benefits of hybrids:

– You need the parents to make more seed (happy breeder)

– Potential for superior yield

– Seed production is combine-compatible (maybe….)

• Challenge 1: Since sorghum is a self-pollinating species, you

need a male-sterile female ‘A-line’ to ensure cross-pollination

• Challenge 2: The only way to propagate the A-line is to cross it

with a fertile B-line that is otherwise genetically identical

• Challenge 3: In order to get fertile hybrid seed (necessary for

sugar accumulation!), a fertility-restoring ‘R-line’ needs to be

available as a pollen donor

Seed

Parent Maintaine

r

Pollinator Seed

Parent Maintaine

r

Pollinator Seed

Parent Maintaine

r

Pollinator

Seed

Parent Maintaine

r

Pollinator

A B R

Sanyukta Shukla

Hybrid seed

produced from

fertilizing A-line

with R-pollen

Combine-compatible hybrid seed production

More challenges:

• Sugar accumulation is an additive trait

– both hybrid parents need to be sweet in order to

produce sweet offspring

• Sugar yield is the product of sugar concentration and

juice volume

– big stalks contain more juice

• If we select the parents carefully based on their dwarf

genotype, we can get tall offspring from short, combine

compatible parent lines

• But given that all known sweet sorghums are tall, is

height a prerequisite for being sweet?

• Four independent loci

– dw1, dw2, dw3, dw4

• Recessive mutations that

reduce height in an additive

fashion by ~ 40 cm per

locus

• Height reduction enables

combine harvest of

sorghum grain and

improves harvest index

From: Quinby 1967

Dwarf genes control height in sorghum

Tall sorghums tend to be sweeter…..

y = 0.017x + 8.4817 R² = 0.222

0

2

4

6

8

10

12

14

16

18

20

0 50 100 150 200 250 300 350 400

Sugar concentration as a function of plant height °

Brix

Plant height (cm)

Data based on 250 F3 families derived from grain x sweet sorghum

Ana Saballos, Terry Felderhoff, Sanyukta Shukla

But….

Production of combine harvestable hybrid sweet sorghum seed

• Tall sorghums tend to be sweeter than short sorghums, but

short sorghums can be sweet

– Height is not a prerequisite in sweet sorghums

• Good news: It is possible to produce short sweet inbred

parents for hybrid sweet sorghums

• Short, sweet inbred parents for hybrid production under

development:

– 22 R-lines

– 73 B-lines (to be converted to A-lines)

Sanyukta Shukla dw1 dw2 Dw3 Dw4 Dw1 Dw2 dw3 dw4

Dw1 Dw2 Dw3 Dw4

Catalytic mechanism of sorghum COMT

• What is the catalytic mechanism of COMT?

• What determines substrate specificity?

• Can we engineer COMT for broader or more

specific substrate specificity?

– Tailor lignin subunit composition

– Minimize negative impacts on plant growth

• Purify recombinant SbCOMT protein in E. coli

• X-ray crystallography of apo-enzyme and

ternary complex (= with substrate)

• Determine 3D structure of enzyme, including

substrate binding pocket

Green et al. (2014) Plant Physiol.

X-ray crystallography

• GBS: ‘brute force’

– Many markers over entire genome

– Number of samples (in increments of 96 samples)

– Service fee for libraries and sequencing ($35/sample)

– Labor cost for data analysis

• PCR

– Fewer markers, only at loci of interest

– Cost is a function of • Number of samples

• Number of markers

• Primer success rate and specificity

– Labor cost primarily for experiments

GBS vs PCR-based genotyping

A

C

T

A

A customizable model to determine the

most cost-effective genotyping method

Fine-mapping resistance loci

× Backcross Susceptible

parent

Resistant inbred

S

R

R

R

R R

S

S

×

BC1

BC1S1

Bio-functionalization

Figure 7

Figure 7

Can we make a lignin nanotube recognize a target?

Figure 7

Figure 7

Glass slide with Concanavalin A

Figure 7

Figure 7

Glass slide under UV-fluorescence

Functionalized nanotubes Native nanotubes

Caicedo et al. (2012)

Dose-dependent gene expression

100 ng 300 ng 1100 ng

Fixed amount of Pine-TGA LNTs with increasing amounts of plasmid DNA

100 x magnification 1.4 mg LNTs per well

Mechanical properties vary as a function of lignin source

• Nano-indentation: 5 nm

• Measure hardness (H) and

Young’s modulus (E)

0

1

2

3

4

5

6

7

8

0

10

20

30

40

50

60

70

80H (GPa) E (GPa)


Topics





FOOD AND NUTRITION


CONSERVATION




pentoses

fuels


fuels

chemicals

CO2

ferm

en

tation

ferm

en

tatio

n

pre

treatm

ent

hexoses

chemicals

CBP

Sweet sorghum

Nanotubes versus nanowires

Pine NaOH LNT

Pine TGA LNT

Pine NaOH LNW

Pine TGA LNW

Entry into the nucleus:

wires versus tubes made of TGA lignin

NUCLEUS

CYTOSOL

TUBE WIRE

Vetenskap SR P1

http://www.bio4energy.se/component/content/article/84-latest-news/1371-latest-in-lignin-rad-swedish-national-public-radio-at-lignin-2014.html

Closing remarks

• A fully integrated processing scheme for

biomass can generate multiple main products

(fuels, chemicals) as well as waste products

with potential value

• Volume versus value

• Market size

• Local versus global demand

• Cost of production: Lab > Pilot > Industrial

volu

me (

m3)

value ($/m3)

high value, low volume low

va

lue h

igh v

olu

me High value, high volume

Indian consortium

IICT, Hyderabad

ICRISAT, Hyderabad

DSR, Hyderabad

JNTU, Hyderabad

CESS, Hyderabad

IIT-Delhi

IIT-Madras

TNAU, Coimbatore

RVSKVV, Gwalior

Abellon Clean Energy Ltd.,

Ahmedabad*

Hindustan Petroleum

Corp. Ltd., Bangalore*

US consortium University of Florida,

Gainesville, FL

University of Missouri,

Columbia, MO

Virginia Tech, Blacksburg,

VA

Montclair State University,

Montclair, NJ

Texas A&M University,

College Station, TX

Green Technologies, LLC

Gainesville, FL*

Tiger Energy Solutions,

LLC, Columbia, MO*

*Industry partners

US funding: $12 million over 5 years

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCIOf8tfC5McCFQxwjgodyNkNYg&url=http://www.govtjobsearch.in/indian-institute-of-chemical-technology-iict-recruitment-2015-www-iictindia-org/&psig=AFQjCNGw6wfg9ieAjx3y7DYChTjrvixWoA&ust=1441701472711212

Technical Objectives

• Improve feedstocks using genomics, breeding tools and identify locally

adapted cultivars, and their optimization for large-scale production

• Develop production logistics and identify soil and environmental criteria to

ensure a commercially successful advanced feedstock production system

Feedstock development and supply (WP1)

Conversion technologies (WP2)

• Develop biocatalysts for production of advanced biofuels and co-products; optimize pretreatment and fermentation processes

• Develop products and applications from biorefinery waste streams that minimize environmental impact of biorefinery operations and maximize the revenues

Sustainability, marketing and policy (WP3)

• Analyze and develop certification protocols and sustainability standards

• Assess energy requirements and emissions, and perform economic analyses

• Undertake supply chain management analysis

Field Studies

• 2013 Live Oak

– 2 reps, natural inoculum

• 2013 Citra

– 3 reps, natural inoculum

• 2015 Live Oak

– 2 reps, cultured inoculum

• Observed during soft

dough stage

Primer Optimization and Marker

Evaluation

66 ° 65° 64° 63° 62°

EH Bk7

EH Bk7 EH Bk7 EH Bk7

• Optimization with temperature-gradient PCR

• PCR performed with parent DNA

– Performed at optimum temperature

– Four out of seven markers able to detect SNP differences

Courtesy of Lauren Stutts

UF Stan Mayfield Biorefinery Pilot Plant

18,000 sq. ft.

3,500 sq. ft.

service labs 8,500 sq. ft. process

6,000 sq. ft.

client space

chiller

Distillation

Phosphoric acid steam

pretreatment

Gravity-assisted removal of

pretreated biomass slurry

Fermentation tanks

Comparison of conversion processes

Corn Steam Cooker

Fermentation + amylase & glucoamylase

Purification

C. Mature Corn to Ethanol Industry

Lignocellulose

cc-Washing

Dilute Acid Hydrolysis

Liquid/solid Separation

Hemicellulose Fermentation

Cellulose +Lignin

Purification

Hemicellulose

Syrup & inhibitors

Hemicellulose Syrup Detox

A. Sulfuric Lignocellulose Process

Lignocellulose Dilute Acid Hydrolysis

Purification Fermentation + cellulase & hemicellulase

B. Phosphoric Lignocellulose Process (goal)

Fermentation Cellulose+ Cellulase

Liquefaction + amylase & glucoamylase

(Zirconium Hydrolyzer)

Side products of hydrolysis (hydrolysate inhibitors) drive process complexity!

(Stainless Steel Hydrolyzer)

(Stainless Steel Cooker)

Gypsum

Co-products

Co-products

CapX

L+SScF Process (eliminates steps) CapX

CapX

Liquefaction + cellulase & hemicellulase

300 to 500 U/mg protein

Cellulase 0.7 U/mg protein

L.O. Ingram

icrisat 2 march 2016ksiconnect.icrisat.org/wp-content/uploads/2016/03/01032016.pdf · u ni te d s...

Documents