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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
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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
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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
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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
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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
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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
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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
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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
Gene ex pr ess ion o ft he yeast i n t he
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
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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
Gene folder change in glycolysis
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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
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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)
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Gene ex pr ess ion o ft he yeast i n t he
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
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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
Gene folder change in glycolysis
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
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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.
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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
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Fermentation technologies
To enhance ethanol concentration, reduce energy consumption,
decrease fermentation time and then reduce the production cost ofethanol
Objective
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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
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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
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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
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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
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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
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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)
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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
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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.
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Technological breakthroughTechnological breakthrough
andandAchievementsAchievements
Fou r t echn o log ica l b r eak t h r ough o f
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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
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Paten ts
Patents
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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
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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
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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
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Thanks for your attention !