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High Performance Technologies for

Ethanol Production from Sweet Potato

Chengdu Institute of Biology, Chinese Academy

of Sciences, Chengdu 610041,China

EmailEmail

[email protected]@cib.ac.cn

Zhao Hai


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z 3E Principle

Energy

Environment

Economy

z Mode of Fuel Ethanol in ExistenceCorn Fuel Ethanol (American Mode)

Sugarcane Fuel Ethanol (Brazil Mode)

Cassava Fuel Ethanol (Thailand Mode)

Chinese Mode of Fuel Ethanol :3E+ Food Supplies Security

Mode of Chinese Fuel Ethanol Production


3/48

Feedstocks for bio-ethanol production

Cellulosic materialsCellulosic materials

Woody fiber grasses

Canna edulis Ker sweet potatosweet potato cassava

Sugar cropsSugar crops

Sugar beets sugar cane sweet sorghum

No grainNo grain

starch cropsstarch crops

Corn wheat

GrainsGrains
http://www.acclaimimages.com/usepolicy.html


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Why we use sweet potato to produce bio-ethanol?

99Hig h q u a n t i t y o f en er g y o u t p u tHig h q u an t i t y o f en e r g y o u t p u t

H al l an d Sm i t t l e. 1 9 9 3 . I n d u s t r i al - t y p e sw e et p o t a t o e s: Ar enew ab le ener gy r esou r ce fo r Geor g ia . UGA Res. Rpt . 42 9.

Feed s t o ck Ga l / A c r eWheat 340

Corn 400

Sweet Sorghum 600Sweetpotato 640

Sugarcane 650

Sugar Beets 700Switchgrass 1000Miscanthus 1250

Po t en t ia l Et h an o l Y ie ld s

Difficult tobe utilized


5/48

Growth

period(month)

Root yield

kg/mu

Starch

content(%)

Starch

yield(kg/mu)

Ethanol

yield(kg/mu

Yearly ethanol

yieldkg/Year/mu

Sweet

potato 5

Average 1500 20% 300 150 360

High 3000 25% 750 375 900

Cassava 10

Average 1500 22% 330 165 198

High 3000 28% 740 370 444

Corn(CK) 3

Average 328 64% 210 105 420

High 500 64% 320 160 640

99High speed o f ener gy ou t pu tH igh speed o f ene rg y ou t pu t


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99Be easy t o b e u t i l i zedBe easy t o b e u t i l i zed

Sweet potatoFuel

Ethanol


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The issue of fuel ethanol production fromsweet potato

99Sw eet p o t a t o i s a k i n d o f sm al l f ar m er co r p sSw eet p o t a t o i s a k i n d o f sm al l f ar m er co r p s - - -- - - I tI t

i s d i f f i cu l t t o be used as indu st r ia l f eeds tockis d i f f i cu l t t o be used as indu st r ia l f eeds tockMain usageMain usage

Feedstuff(50Feedstuff(50))

Decomposition(30Decomposition(30))

Seed (10%)Seed (10%)

Commercial usage(10%)Commercial usage(10%) 10% Seed

10%commercialusage

50feedstuff

30Decomposition


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99High v i scosi t y o f sw eet po t a toH igh v i scosi t y o f sw eet po t a to

Low efficiency of heat exchangers

Low efficiency of enzyme kinetics

Impact on the escape of CO2

Inhibit the activity of strain


10/48

99Low ethanol content, high energy consumptionLow ethanol content, high energy consumptionin existing production technologiesin existing production technologies

Ratio Between Material and Water 11

Ethanolconcentration

(% v/v)

Fermentation

(h)

Fermentationefficiency

(%)

Corn

(American) 15 50

90

Sugar Cane

(Brazil) 8-9 10 90

Sweet Potato 5-6 60 88

More energy and water consumption


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Our work

Sw eet p o t a t o M ic roo rgan i sm s

Energy-savingReactor

Fe rm e n t a t i o n

techno logy

Very highgravity

fermentation

RapidFermentation

Ethanoltolerance

Temperature

tolerance

Pressuretolerance

Mechanisms

of Tolerance

Demonstrationproject

+

Viscosity ReductionTechnology


12/48

Breeding of Stress Tolerance Yeast Strain

Objective: To carry out the very high gravity fermentation

Ethanol tolerance yeast

Very High GravityFermentationVHG

Reduce waterconsumption

Reduce energyconsumption

Reducewastewater

Improve productivity

of equipments

Avoid pollution

by other bacteria


13/48

8 ethanol tolerant strains of

yeast were obtained . With Y1

or Y5,more than 18% of

ethanol was produced within60h,and the fermentation

efficiency was 92%.

The charac te r i s t i c hy d r o l ys i s

enzym es m ap o f t h e st r a i ns

Ethano lt o le rance s t r a in

Ord ina rys t ra i n

0

2

4

6

810

12

14

16

18

20

0 20 40 60 80

Fermentation time (h)

RG

C

%(

w/v)

E

thanolconcentration%(v/v)

Changes of RGC

Changes of ethanol concentration


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Differential gene expressionDifferential gene expressionPartialPartial

Gene ex pr ess ion o ft he yeast i n t he

cour se o f ve ry h ighg r a v i t y f er m en t a t i on


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PathwayName Total PathwayName Total

Glycolysis / Gluconeogenesis 19 One carbon pool by folate 9

Purine metabolism 40 Terpenoid biosynthesis 4

Peptidoglycan biosynthesis 1 Ubiquinone biosynthesis 3

Pantothenate and CoA biosynthesis 5 Glycan structures - biosynthesis 2 7

Two-component system - Organism-specific 1 Valine, leucine and isoleucine degradation 8

Pyrimidine metabolism 26 Limonene and pinene degradation 6

Thiamine metabolism 1 Valine, leucine and isoleucine biosynthesis 10

Alanine and aspartate metabolism 11 Phosphatidylinositol signaling system 13

Glutamate metabolism 11 Bile acid biosynthesis 9

Aminoacyl-tRNA biosynthesis 23 Lysine biosynthesis 9

Ribosome 61 DNA polymerase 10

Main pathway involved in very high gravity ofethanol fermentation of Saccharomyces cerevisiae


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Gene folder change in glycolysis

GeneFolder change

ADH2 0.16

ADH4 0.19

ALD3 0.50

ALD4 0.29

ALD5 0.40

ALD6 0.14

FBP1 0.10

PYK2 0.17

PDA1 0.41

PDB1 0.43

PDC5 0.34

PDC6 0.23

LAT1 0.43

HXK1 0.49

HXK2 0.50

ACS2 0.09

GAL10 6.25GPM2 2.45

PGM1 3.59


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Gene Folder change

IDI1 0.32

ERG8 0.28

ERG1 0.22

ERG9 0.49

ERG7 0.09

MVD1 0.20

HMG1 0.44

HMG2 0.29

ERG20 0.20

ERG12 0.38

Gene folder change in steriod synthesis


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Genes folder change involved in heat shock protein

of Saccharomyces cerevisiae

-10

0

10

20

30

40

50

60

70

80

90

HSP26 FES1 SSA2 SSA4 HSP78 SSA3 HCH1 AHA1 HSC82 HSP82 SIS1 HSP10 SSE1 STI1 HSP42 YDJ1 ZIM17 SSZ1 SSB1 SSB2

Gene

Foldercha

nge


19/48

Objec t i ve To reduce water consumption in cooling

To maintain sustaining production in summer

To reduce pollution by other microorganisms which

could not be tolerant to temperature

To relieve the inconsistency between the fermentation

temperature of the yeast and process temperature of

cellulase or amyloglucosidase in the Simultaneous

saccharification and fermentation (SSF)

Temperature tolerance yeast


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There were some similar mechanisms for yeast to tolerance high

concentration of ethanol and high temperature

High

concentration of

ethanol

High

temperature

Increase of hsp protein + +

Increase of H+-ATPase protein in the membrane + +

Decrease of unsaturated fatty acid + +

Increase of trehalose + +

Increase of steriod + +

Partial references:Z.H.Liu. Appl Microbiol Biotechnol.(2007)77:901-908

Agustn Aranda. Arch Microbiol (2002) 177 :304312

Peter W.FEMS Microbiology Letters . ( 1995) 134 :121 127

Research st r a t eg ic


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Temperature tolerance strains were screened

from ethanol tolerance strains after heat

shock treatment. Taking into account of

ethanol concentration, fermentation time and

fermentation efficiency, a strain of yeast

could ferment at 40 normally. 13% of

ethanol could be produced within 33h, and

the fermentation efficiency was 92%

Sugar concentration(%,w/v)

Fermentationtime(h)

Fermentationtime(h)

Ethanol concentration(%,w/v)

Fermentationtime(h)

OD value


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Act i v i t y o f k eyendoenzym es o f t he

yeast i n t he cou r se o fet h a n o l f er m en t at i o n

at 4 0

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40 (h)

6-

(U/g

)

30

40

42

G-6-PD(U/g)

Fermentationtime(h)

0

0. 5

1

1. 5

2

2. 5

3

0 10 20 30 40 (h)

ATP

U/

30

40

42

ATPase(U/g)

Fermentation

time(h)

0

10

20

30

40

50

0 10 20 30 40 (h)

(U/g) 30

40

42

ADH(U/g)

Fermentationtime(h)


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PathwayName Total

Cell cycle 37

Purine metabolism 35

Pyrimidine metabolism 23

Ribosome 20

MAPK signaling pathway 19

Glycine, serine and threonine metabolism 18

Pyruvate metabolism 17

Glycerophospholipid metabolism 16

Phosphatidylinositol signaling system 15

Butanoate metabolism 14

Benzoate degradation via CoA ligation 14

Glycolysis / Gluconeogenesis 14

Citrate cycle (TCA cycle) 13

Starch and sucrose metabolism 13

Arginine and proline metabolism 13

Inositol phosphate metabolism 13

Glycerolipid metabolism 13

Tryptophan metabolism 12

Aminoacyl-tRNA biosynthesis 12

Alanine and aspartate metabolism 12


cour se o f et hano lf er m en t a t i o n at 4 0

Main pathway involved in ethanol fermentation at 40

Gene ex pr ess ion o ft he yeast i n e t hano lf er m en t a t i o n at 4 0


24/48

Gene Folder change

PGM1 18.27

ENO2 7.62

GAL10 6.28

GPM2 4.91

ALD5 3.73

PDC1 0.41

PDB1 0.40

ADH4 0.40

ALD4 0.34

PDC5 0.33

ALD6 0.25

ACS2 0.19

FBP1 0.15

PYK2 0.10

Gene Folder change

HSP26 21.90

SSA3 10.35

SSA4 8.38

SSA2 4.61

HSP104 3.00

HSP42 2.83

Genes folder change involved inheat shock proteinof Saccharomyces cerevisiae



25/48

Pressure tolerance strain

The more bigger fermentation scale, the lessmanufacture cost

High pressure coursed by high fermentation

mash could do damage to the strains

High co2 pressure may result in lowerfermentation parameters in scale-up

compared with lab scale


26/48

The strain we screened could

produce 9% of ethanol within

24h under the co2 pressure of

0.3Mpa,and the fermentation

efficiency was above 90%

React o r f o r e t hano l f e rm en t a t i onunder h i gh p ressu re

Ethanol(%,w/w)

Sugar(%,w

/w)

Fermentationtime(h)

Fermentationtime(h)


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Act i v i t y o f k ey endoenzym es o f t he yeast

0

1

2

3

4

5

6

10 15 20 25 30 h

0.1 Mp

0.2 Mp0.3 Mp

0.4 Mp

0

5

10

15

20

25

30

6 12 18 24 30 (h)

ADHU

/

0. 1 Mpa

0. 2 Mpa

0. 3 Mpa

0. 4 Mpa

0

0.2

0.4

0.6

0.8

11.2

1.4

1.6

1.8

6 12 18 24 30(h)

6-

U/

0. 1 Mp

0. 2 Mp

0. 3 Mp

0. 4 Mp

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 (h)

ATP

U

/

0. 1 Mp

0. 2 Mp

0. 3 Mp

0. 4 Mp

G-6PD(U/g)

A

DH(U/g)

A

TPase(U/g)

Hex

okinase(U/g)

Time(h) Time(h)

Time(h)Time(h)


28/48


cour se o f et hano lf er m en t a t i on a t

0 .2mPa

Main pathway involved in ethanol fermentation at 0.2mPa

Pathway Name Total

Ribosome 98

Cell cycle 32

Purine metabolism 26

Pyrimidine metabolism 18

Glycine, serine and threonine metabolism 17

Glutamate metabolism 15

MAPK signaling pathway 14

Starch and sucrose metabolism 14

Oxidative phosphorylation 13

Selenoamino acid metabolism 12

Glycolysis / Gluconeogenesis 11

Glycerophospholipid metabolism 11

DNA polymerase 11

Pyruvate metabolism 10

Lysine degradation 10

Sulfur metabolism 10


29/48

Gene Folder change

HSP26 33.76

SSA2 3.38HSP32 4.40

HSP30 3.07

HSP78 2.94

SSA4 2.87

HCH1 2.32

HSP82 2.20

HSP42 2.22

SIS1 2.03

Genes folder change involved inheat shock protein

of Saccharomyces cerevisiae


ALD5 3.27

PGM1 1.83

ALD3 0.40

CDC19 0.39

PDC5 0.33

PGK1 0.30

PDC6 0.29

FBP1 0.13

ADH4 0.13

ENO2 0.09

ACS2 0.08

Vi it d ti t h l


30/48

Viscosity reduction technology

Sweet potato and canna edulis ker are non-Newtonian fluid, the

viscosity of which are more than 10104 mPa.S, while the

viscosity of ordinary fermentation culture are below 100 mPa.SThe excessive addition of water can be useful to reduce mash

viscosity, however, the concentration of fermentable sugars in

fermentor is also decreased by the dilution, and more energy is

required for water evaporation.


31/48

Changes of polysaccharide and glucosidic

bond in sweet potato under the function of

viscosity reduction technology

To un ders t and

t h e h i g hv iscos i t ym ech a n i sm o fsw eet p o t a t o b yso l id -phasemo n co lo n i can t i b od y f o r

ca r b o n h yd r a t e


32/48


33/48

Fermentation technologies

To enhance ethanol concentration, reduce energy consumption,

decrease fermentation time and then reduce the production cost ofethanol

Objective


34/48

Rap id e t hano l f erm en t a t i on t echn o logy f r om f r esh sw eetp o t a t o

Objec t i ve To use the feedstock in

harvest season as soon as

possible

To avoid rot because ofoverstocking of sweet potato

To improve the productivity

of unit equipment

Overs t ocked sw eet po t a t o


35/48

0

20

40

60

80

100

120

140

160

180

200

0 3 6 9 12 15 18 21 24 27 30 Time (h)

Reducing

sugras

concentration(g/kg)

0

20

40

60

80

100

Ethanolcon

centration

(g/kg)

18% 20% 22% 24%

18% 20% 22% 24%

0

20

40

60

80

100

120

140

160

180

200

0 3 6 9 12 15 18 21 24 27 30 33

Time (h)

Reducingsugar(g/kg)

0

20

40

60

80

100

120

Ethanol(g/kg)

1:1 sugar 2:1 sugar 3:1 sugar 5:1 sugar

1:1 ethanol 2:1 ethanol 3:1 ethanol 5:1 ethanol

With the screened yeast anddeveloped fermentation

technique,12.35% of ethanol

was produced within 24h, the

fermentation efficiency was

92%and the ethanol

productivity was 4.06 g/kg/h

With the screened z.mobilis and

developed fermentation

technique,12.06% of ethanolwas produced within 21h,the

fermentation efficiency was

94%and the ethanol

productivity was 4.53 g/kg/h

Rapid ethanolfermentation of yeast

Rapid ethanol fermentationof z.mobilis


36/48

Very h i gh g r av i t y e t h ano l f e rm en t a t i on t echn o logy f o r f r eshsw eet p o t a t o

Initial sugar

concentration(w/g kg-1)270 300 330

Fermentation time

h28 39 48

Ethanol

concentration(w/g kg-1)132.86 146.30 151.19

Fermentation

efficiency(t)91.44 90.42 84.15

Ethanol productivity(g

kg-1 h-1)4.75 3.75 3.15

Reducing sugars(w/g kg-

1)5.64 6.82 20.68

Total reducing

sugars(w/g kg-1)15.19 18.38 31.53

Viscosity(mPa s) 1074.75 1798.25 3033.15

0

50

100

150

200

0 3 6 9 12 15 18 21 24 27 28

Time( /h)

Concent

ration(w

/g

kg-

1)

0

1

2

3

4

5

6

pH

pH

Value

pH

10L scale fermentation

A fermentation stimulant was developed to improve the activity of the

strain. Under optimal condition,16.84% of ethanol was produced within

30h,ethanol fermentation efficiency was 91.44%,and the ethanol productivity

was4.74 g kg-1 h-1


37/48

Demonstration project was carried out

in 10 thousand ton scale production line

of ethanol production factory at Sichuan

province.

Compared with existed fermentation

technique, fermentation time reduced

from more than 60 hours to less than 30

hours, ethanol concentration increased

from 5%-6%(v/v) to 12.41%(v/v), and

fermentation efficiency enhanced from

88% to more than 90% in average.

Technique integration and demonstration project


38/48

Batch 1 2 3 4 5 6 7 8

Ratio of sweet

potato to water11.1 31 3.681 41 4.51 31 31 41

Viscosity of

sweet potato

with water

15676 21231 29876 33164 41154 11206 21929 30758

Viscosity of

processedsweet potato

(mPa.S)

1108 1868 1654 1213 1384 1804 1453 1856

Ethanol

concentration

(%,v/v)

4.48 8.99 9.9 12.41 11.87 11.15 10.58 9.91

Time(h) 60 20 20 29 27 21 23 22

Final viscosity

(mPa.S) 40 914 406 340 473 1138 603 635

Reducing

sugars(%)0.23 0.52 0.23 0.67 0.45 0.53 0.5 0.65


39/48

0

24

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16 18 20

(h)

(%)

0

200

400

600

800

1000

1200

(mPa.S

)

0

3

6

9

12

15

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 29

h

%

0

200

400

600

800

1000

1200

1400

mPa.S

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10 12 14 16 18 20 22 24 26 27

h

%

0

200

400

600

800

1000

1200

1400

1600

mP

a.S

0

5

10

15

20

0 2 4 6 8 10 12 14 16 18 20 21

(h)

%

0

500

1000

1500

2000

mP

a.S

(%)

(%)

(mPa.S)

Sugars

Ethanol

viscosity

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16 18 20

(h)

(%)

0

200

400

600

800

1000

1200

(mPa.S

)

Sugars

Ethanol

viscosity

Sugars

Ethanol

viscosity

Sugars

Ethanol

viscosity

Sugars

Ethanol

viscosity

Time(h) Time(h)

Time(h)Time(h)

The third batch

The fourth batch

The fifth batchThe sixth batch

Concen

tration(%)

Concentratio

n(%)

Concentration(%)

Concentration(%)

Viscosity(mP

a.s)

Viscosity(mP

a.s)

Viscosity(mPa.s)

Viscosity(mPa.s)

Viscosity(mPa.s)


40/48

0

2

4

6

8

10

12

14

0 2 4 6 8 1012141618202223

h

%

0

200

400

600

800

1000

1200

1400

mPa.S

(%)

(%)

(mPa.S)

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16 18 20 22

h

%

0

500

1000

1500

2000

mPa.S

(%)

(%)

(mPa.S)

Sugars

Ethanol

viscosity

Sugars

Ethanol

viscosity

Concentration(%

)

Concentration(%

)

Viscosity(mPa.s)

Viscosity(mPa.s)

The seventh batch

The eighth batch


41/48

Ratio ofRatio of

sweetsweet

potatopotato

to waterto water

ViscosityViscosity

reductionreduction

FermentatFermentat

ion timeion time

EthanolEthanol

concentraconcentra

tiontion

FermeFerme

ntationntation

efficienefficien

cycy

PresentPresent

technologtechnolog

iesies11 11

By addingBy adding

excessiveexcessive

waterwater>60h>60h 55--6%(v/v)6%(v/v) 10000mPa.s

toto

90%

Comparison between present technologies and theseComparison between present technologies and these

technologiestechnologies

The advantage of our technologies

less water was needed to be

added to the sweet potato mash,

and 70% of water could be

saved.

ethanol concentration was

increased from 5-6% to

12%.Thus 40% of energy for

water evaporation was saved,

and wastewater could be

reduced from 16t to 9t.

the COD in the wastewater was

partially removed and reduced

from 64200mg/L to 41200 mg/L.


42/48

Technological breakthroughTechnological breakthrough

andandAchievementsAchievements

Fou r t echn o log ica l b r eak t h r ough o f


43/48

Fou r t echn o log ica l b r eak t h r ough o fn o n - g r a i n e t h an o l p r o d u c t i o n

HighHigh--efficient strains ofefficient strains of

ethanol fermentationethanol fermentation

Selective breeding out high-concentration fermentation strains ofwithstand pressure and temperatureresistance, which haveindustrialization value.

HighHigh--efficient fermentationefficient fermentation

of high viscosity materialsof high viscosity materials

Reducing viscosity of fermentedmash , raising material-water ratio,which can cut down 70% waterconsumption.

HighHigh--concentration ethanolconcentration ethanol

fermentation of fresh sweetfermentation of fresh sweet

potatopotato

Improving ethanol concentration,(from 5 to 12%)reducing energy cost,steam consume of ethanol distillation

can cut down and discharge of wastewater can cut down 40% at least.

Rapid ethanol fermentationRapid ethanol fermentation

ofof fresh sweet potatofresh sweet potato

Fermentation time will be shortenfrom 60h to 30h . It can enhanceethanol production capacity of unitequipment at least 1 times higher.

P


44/48

Paten ts

Patents


45/48

Joint Research Project of Chinese Academy of Sciences Ethanol production

from Canna edulis Ker

Knowledge Innovation Program of The Chinese Academy of SciencesHot-

tolerance strain for ethanol production

Key Technologies R & D of Sichuan Province Key technology of ethanol

production from sweet potato

Key Technologies R & D of Sichuan ProvinceKey biotechnology of ethanolproduction

Program of Development and Reform Commission of Sichuan Province

Establishment of the exclusive evaluation system of sweet potato for ethanol

production

Ou r w o r k w as m ai n ly su p p or t ed b y t h ef o l l ow i n g p r o j ect s

Acknowledgements


46/48

National Key Technologies R & DEnergy-saving preservation technology

for sweet potato and ethanol production

National Key Technologies R & DEthanol production from Canna edulis

Ker and sweet potato at the scale of 5000ton of ethanol per year

National High Technology Research and Development Program of China

(863 Program) High efficiency transformation techniques of Lignocellulose

International Cooperative Program of Sichuan ProvinceRelative

Characteristics of Sweet Potato for Bio-ethanol Production

National Key Technologies R & DKey technology of ethanol production

from sweet potato

National Key Technologies R & DBreeding of sweet potato for ethanol

production


47/48

National Key Technologies R & DSustaining supply of sweet potato for

ethanol prodcution

National High Technology Research and Development Program of China (863

Program) Rapid ethanol production from sweet potato with high viscosity

The earmarked fund for Modern

Agro-industry Technology Research

System Energy utilization of sweet

potato


48/48

Thanks for your attention !

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