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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

3E PrincipleEnergyEnvironmentEconomy

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

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 grain No grain starch cropsstarch crops

Corn wheat

GrainsGrains

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

Why we use sweet potato to produce bio- ethanol?

High quantity of energy outputHigh quantity of energy output

Hall and Smittle. 1993. Industrial-type sweet potatoes: A renewable energy resource for Georgia. UGA Res. Rpt. 429.

Feedstock Gal/AcreWheat 340Corn 400

Sweet Sorghum 600Sweetpotato 640Sugarcane 650

Sugar Beets 700Switchgrass 1000Miscanthus 1250

Potential Ethanol Yields

Difficult to be utilized

Growth period

(month)

Root yield （kg/mu）

Starch content

(%)

Starch yield (kg/mu)

Ethanol yield (kg/mu）

Yearly ethanol yield（kg/Year/mu ）

Sweet potato 5

Average 1500 20% 300 150 360

High 3000 25% 750 375 900

Cassava 10Average 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

High speed of energy outputHigh speed of energy output

• China is the biggest sweet potato

producer in the world with the output

exceeded 100 M Ton (>80% world total)

in 2007(FAO, 2007).

• Sichuan province accounts for 16% of

total yield of sweet potato in China,

which is the biggest producer in China.

Abundant resourceAbundant resource

Plantation area of sweet potato

Be easy to be utilizedBe easy to be utilized

Sweet potatoFuel

Ethanol

The issue of fuel ethanol production from sweet potato

Sweet potato is a kind of small farmer corpsSweet potato is a kind of small farmer corps------ItIt

is difficult to be used as industrial feedstockis difficult to be used as industrial feedstock

Main usageMain usage

Feedstuff(50Feedstuff(50％％))

Decomposition(30Decomposition(30％％))

Seed (10%)Seed (10%)

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

10%commercial usage

50％feedstuff

30％Decomposition

High viscosity of sweet potatoHigh viscosity of sweet potato

• Low efficiency of heat exchangers

• Low efficiency of enzyme kinetics

• Impact on the escape of CO2

• Inhibit the activity of strain

Low ethanol content, high energy consumption Low ethanol content, high energy consumption in existing production technologiesin existing production technologies

Ratio Between Material and Water 1：1

Ethanol concentration

(% v/v)Fermentation

(h)

Fermentation efficiency

(%)

Corn (American) 15 50 ＞90

Sugar Cane (Brazil) 8-9 10 ＞90

Sweet Potato 5-6 ＞60 ≈88

More energy and water consumption

Our work

Sweet potato Microorganisms

Energy-saving Reactor

Fermentation technology

Very high gravity

fermentation

Rapid Fermentation

Ethanol tolerance

Temperature tolerance

Pressure tolerance

Mechanisms of Tolerance

Demonstration project

+

Viscosity ReductionTechnology

Breeding of Stress Tolerance Yeast Strain

Objective: To carry out the very high gravity fermentation

Ethanol tolerance yeast

Very High Gravity Fermentation（VHG）

Reduce water consumption

Reduce energy consumption

Reduce wastewater

Improve productivity of equipments

Avoid pollutionby other bacteria

8 ethanol tolerant strains of yeast were obtained . With Y1 or Y5,more than 18% of ethanol was produced within 60h,and the fermentation efficiency was 92%.

The characteristic hydrolysis enzymes map of the strains

Ethanol tolerance strain

Ordinary strain

02

46

810

1214

1618

20

0 20 40 60 80

发酵时间 Fermentation time (h)

糖浓

度 R

GC

% (w

/v)

乙醇

浓度

Eth

anol

con

cent

ratio

n %

(v/v

)

残糖浓度变化曲线 Changes of RGC

乙醇浓度变化曲线 Changes of ethanol concentration

Differential gene expression Differential gene expression （（PartialPartial））

Gene expression of the yeast in the

course of very high gravity fermentation

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 of ethanol fermentation of Saccharomyces cerevisiae

Gene folder change in glycolysis

Gene Folder 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 6.25

GPM2 2.45

PGM1 3.59

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

Genes folder change involved in heat shock proteinof 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

基因

与对

照相

比的

变化

倍数

基因表达量变化倍数

Gene

Folder change

Objective：• 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

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)77:901-908Agustín Aranda. Arch Microbiol (2002) 177 :304–312

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

Research strategic

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)

Fermentation time(h)


Ethanol concentration(%,w/v)


OD value

Activity of key endoenzymes of the

yeast in the course of ethanol fermentation

at 40℃

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40 发酵时间(h)

6-磷

酸葡

萄糖

脱氢

酶酶

活(U/g)

30

40

42

G-6-P

D (U

/g)


0

0. 5

1

1. 5

2

2. 5

3

0 10 20 30 40 发酵时间(h)

ATP酶

酶活

(U/g

)

30

40

42

ATPase(U

/g)


0

10

20

30

40

50

0 10 20 30 40发酵时间(h)

乙醇

脱氢

酶酶

活(U/g) 30

40

42

AD

H(U

/g)


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


course of ethanol fermentation at 40℃

Main pathway involved in ethanol fermentation at 40℃

Gene expression of the yeast in ethanol fermentation at 40℃

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 in heat shock protein

of Saccharomyces cerevisiae


Pressure tolerance strain

• The more bigger fermentation scale, the less manufacture cost

• High pressure coursed by high fermentation mash could do damage to the strains

• High co2 pressure may result in lower fermentation parameters in scale-up compared with lab scale

FermentorHeight:15m

Volume:5000 Cubic metere

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%

Reactor for ethanol fermentation under high pressure

Ethanol(%,w

/w)

Sugar(%

,w/w

)



Activity of key endoenzymes of the yeast

01

234

56

10 15 20 25 30 发酵时间(h)

己糖

激酶

活力

(U/g

) 对照

0.1 Mpa

0.2 Mpa

0.3 Mpa

0.4 Mpa

密闭0

5

10

15

20

25

30

6 12 18 24 30 发酵时间(h)

ADH酶

活(U

/g)

对照

0. 1 Mpa

0. 2 Mpa

0. 3 Mpa

0. 4 Mpa

封闭

00.20.40.60.8

11.21.41.61.8

6 12 18 24 30发酵时间(h)

6-磷

酸葡

萄糖

脱氢

酶酶

活(U

/g)

对照

0.1 Mpa

0.2 Mpa

0.3 Mpa

0.4 Mpa

封闭0

0.51

1.52

2.53

3.5

0 10 20 30 40 发酵时间(h)

ATP酶

活(U

/g)

对照

0. 1 Mpa

0. 2 Mpa

0. 3 Mpa

0. 4 Mpa

密闭

G-6P

D(U

/g)

AD

H(U

/g)A

TPase(U

/g)

Hexokinase (U

/g)

Time(h) Time(h)

Time(h)Time(h)


course of ethanol fermentation at

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

Gene Folder change

HSP26 33.76

SSA2 3.38

HSP32 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 in heat shock protein

of Saccharomyces cerevisiae


基因名

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

Viscosity reduction technology

Sweet potato and canna edulis ker are non-Newtonian fluid, the viscosity of which are more than 10×104 mPa.S, while the viscosity of ordinary fermentation culture are below 100 mPa.S

The 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.

Changes of polysaccharide and glucosidic bond in sweet potato under the function of

viscosity reduction technology

To understand the high viscosity mechanism of sweet potato by solid-phase moncolonic antibody for carbonhydrate

Viscosity reduction enzymes were developed according to polysaccharide and glucosidic bond which related to viscosity.

Under the function of optimal enzyme system, viscosity of fresh sweet potato mash reduced from 41154 mPa.S to 1384 mPa.S

After being processed with the enzyme system for viscosity reduction, the COD in the fermentation wastewater was partially removed and reduced from 64200mg/L to 41200 mg/L. Effect of enzymes on separation of liquid and solid in the

sweet potato mash

Effect of enzymes on viscosity reduction

Fermentation technologies

To enhance ethanol concentration, reduce energy consumption, decrease fermentation time and then reduce the production cost of ethanol

Objective：

Rapid ethanol fermentation technology from fresh sweet potato

Objective：•To use the feedstock in

harvest season as soon as

possible

•To avoid rot because of

overstocking of sweet potato

•To improve the productivity

of unit equipment

Overstocked sweet potato

0

20

40

60

80

100

120

140

160

180

200

0 3 6 9 12 15 18 21 24 27 30

时间 Time (h)

残糖浓度

Red

ucin

g su

gras

conc

entra

tion

(g/k

g)

0

20

40

60

80

100

酒精浓度

Eth

anol

con

cent

ratio

n(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)

Red

ucin

g su

gar (

g/kg

)

0

20

40

60

80

100

120

Eth

anol

(g/k

g)

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 and

developed 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 ethanol

was produced within 21h,the

fermentation efficiency was

94%，and the ethanol

productivity was 4.53 g/kg/h

Rapid ethanol fermentation of yeast

Rapid ethanol fermentation of z.mobilis

Very high gravity ethanol fermentation technology for fresh sweet potato

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

Fermentation time （h） 28 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时间Time( /h)

浓度

Con

cent

ratio

n(w

/g k

g-1)

0

1

2

3

4

5

6

pH值

pH

Val

ue

残还原糖乙醇 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

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

Batch 1 2 3 4 5 6 7 8

Ratio of sweet potato to water 1：1.1 3：1 3.68：1 4：1 4.5：1 3：1 3：1 4：1

Viscosity of sweet potato with water

15676 21231 29876 33164 41154 11206 21929 30758

Viscosity of processed

sweet 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

第三批试验发酵参数

02468

10121416

0 2 4 6 8 10 12 14 16 18 20

时间(h)

浓度

(%)

0

200

400

600

800

1000

1200

粘度

(mPa

.S)

还原糖

酒精

粘度

第四批试验发酵参数

0

3

6

9

12

15

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

时间（h）

浓度

（%）

0

200

400

600

800

1000

1200

1400

粘度

（m

Pa.S）

还原糖

酒精

粘度

第五批试验发酵参数

02468

1012141618

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

时间（h）

浓度

（%）

02004006008001000120014001600

粘度（

mPa

.S）

还原糖

酒精

粘度

第六批试验发酵参数

0

5

10

15

20

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

时间(h)

浓度（

%）

0

500

1000

1500

2000

粘度（

mPa

.S）

还原糖(%)酒精(%)粘度(mPa.S)

Sugars

Ethanol

viscosity

第三批试验发酵参数

02468

10121416

0 2 4 6 8 10 12 14 16 18 20

时间(h)

浓度

(%)

0

200

400

600

800

1000

1200

粘度

(mPa

.S)

还原糖

酒精

粘度

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 batch The sixth batch

Concentration(%

)C

oncentration(%)

Concentration(%

)C

oncentration(%)

Viscosity(m

Pa.s)

Viscosity(m

Pa.s)

Viscosity(m

Pa.s)

Viscosity(m

Pa.s)

Viscosity(m

Pa.s)

第七批试验发酵参数

02468

101214

0 2 4 6 8 1012141618202223

时间（h）

浓度（

%）

0200400600800100012001400

粘度（

mPa

.S）


第八批试验发酵参数

02468

10121416

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

时间（h）

浓度（

%）

0

500

1000

1500

2000

粘度（

mPa

.S）


Sugars

Ethanol

viscosity

Sugars

Ethanol

viscosity

Concentration(%

)

Concentration(%

)

Viscosity(m

Pa.s)

Viscosity(m

Pa.s)

The seventh batch The eighth batch

Ratio of Ratio of sweet sweet potato potato

to waterto water

Viscosity Viscosity reductionreduction

FermentatFermentat ion timeion time

Ethanol Ethanol concentraconcentra

tiontion

FermeFerme ntation ntation efficienefficien

cycy

Present Present technologtechnolog

iesies11：：11

By adding By adding excessive excessive

waterwater>60h>60h 55--6%(v/v)6%(v/v) <88%<88%

Our Our technologtechnolog

iesies4.54.5：：11

>10000mPa.s >10000mPa.s to to

<1000mPa.s<1000mPa.s<30h<30h >12%(v/v)>12%(v/v) >90%>90%

Comparison between present technologies and these Comparison 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.

Technological breakthrough Technological breakthrough and and

AchievementsAchievements

Four technological breakthrough of non-grain ethanol production

HighHigh--efficient strains of efficient strains of ethanol fermentationethanol fermentationSelective breeding out high- concentration fermentation strains of withstand pressure and temperature resistance, which have industrialization value.

HighHigh--efficient fermentation efficient fermentation of high viscosity materialsof high viscosity materialsReducing viscosity of fermented mash , raising material-water ratio, which can cut down 70% water consumption.

HighHigh--concentration ethanol concentration ethanol fermentation of fresh sweet fermentation of fresh sweet potatopotatoImproving ethanol concentration, (from 5 to 12%)reducing energy cost, steam consume of ethanol distillation can cut down and discharge of waste water can cut down 40% at least.

Rapid ethanol fermentation Rapid ethanol fermentation of of fresh sweet potatofresh sweet potatoFermentation time will be shorten from 60h to 30h . It can enhance ethanol production capacity of unit equipment at least 1 times higher.

Patents

Patents

Joint Research Project of Chinese Academy of Sciences “Ethanol production

from Canna edulis Ker ”

Knowledge Innovation Program of The Chinese Academy of Sciences”Hot-

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 Province“Key biotechnology of ethanol

production”

Program of Development and Reform Commission of Sichuan Province

“Establishment of the exclusive evaluation system of sweet potato for ethanol

production”

Our work was mainly supported by the following projects

Acknowledgements

National Key Technologies R & D“Energy-saving preservation technology

for sweet potato and ethanol production ”

National Key Technologies R & D“Ethanol 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 Province“Relative

Characteristics of Sweet Potato for Bio-ethanol Production”

National Key Technologies R & D“Key technology of ethanol production

from sweet potato”

National Key Technologies R & D“Breeding of sweet potato for ethanol

production“

National Key Technologies R & D“Sustaining 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”

Thanks for your attention !

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