Continuous Butanol Fermentation from Low-Value Sugar ... - Clostridia

Clostridium XII, 10 ‐ 12 Sept. 2012, Nottingham, UK
Butanol Fermentation from Low-Value
Sugar-Based Feedstocks by Clostridia
Ye Ni, Zhihao Sun
School of Biotechnology, Jiangnan University
Wuxi, China
2012.9.10

Clostridium XII, 10 ‐ 12 Sept. 2012, Nottingham, UK
Current Status of Bio-butanol in China
• Market
• Factories
• Research
Studies on ABE fermentation in Our Lab
• Continuous Fermentation by Clostridium saccharobutylicum
2

Market
Butanol consumption and import in China
70
Wt
60
50
40
30
20
10
0
1800
USD
1600
1400
1200
1000
800
600
2005 2006 2007 2008 2009 2010 2011 2012
Year
Butanol import
Average price
Year
Butanol import Average price
Ton
USD
2005 239926 870.5
2006 235834 1130.6
2007 290358 1473.6
2008 244661 1438.4
2009 450742 968.9
2010 623509 1448.9
2011 491246 1588.2
2012.1−6 235083 1318.5
During the period of 2005–2011, around 50% of total annual butanol consumption was
imported, and butanol consumption increased rapidly at an average rate of 6‐8% in China.
(Data sources :http://chem.chem99.com/)
3

Market
Butanol Price Trend in China
Butanol Price (Jilin Petrochemical)
2011
2012
11000
10000
2011/11/01 2012/01/01 2012/08/24
Market: butanol price downturn continuation …
( http://jiage.china.alibaba.com/price/trend/ )

Factories
ABE Factories in China (data by 2012.8)
Factory
Current
Capacity (t/y)
Starting
date
Current Status
Address
Laihe Rockley Bio-Chemicals Ltd 150,000 2007.12.5 − Songyuan, Jilin
Jilin Cathay Biotechnology Co. Ltd 100,000 2008.3.20 − Jilin, Jilin
Guangxi Guiping JinYuan Alcohol Co. Ltd 30,000 2007.8 − Guiping, Guangxi
Jimaoyuan Biochemical Co. Ltd 40,000 2008.3.2 producing ethanol Lianyungang, Jiangsu
Lianhai Biological Co. Ltd 50,000 2008.10 − Haimen, Jiangsu
Lianyungang Union of Chemicals Co. Ltd 40,000 2010.4 producing ethanol Lianyungang, Jiangsu
All ABE factories stopped production or switch to ethanol ...

Research
Choice of Feedstocks for ABE Fermentation
Starchy materials:
corn, cassava, wheat
Molasses:
cane and beet molasses
Cellulosic biomasses:
crop straw hydrolysate
Energy crops:
sweet sorghum, jerusalem artichoke
Food materials
Substitutes
Low-value
sugar-based
feedstocks
Cost issue
“Food versus fuel” debate

Corn material
Profit from corn utilization in ABE fermentation in China
Utilization of corn components (per ton corn)
Corn components
Output
(Kg)
Price
(Yuan/t)
Profits
(Yuan)
Starch (10% water) 600 2860 1700
Fiber 115~120 1350 160~200
Embryo 50~60 4500 270
Protein 60 4300 250
720
CSL (45% water) 350
Total 2420
*CSL: 10% solid content (3% protein), its profit was not considered.
‣ In 2012.2, corn price in Jilin is 2,030 yuan/t (14% water), including shipping;
‣ 4.5~4.7 t corn/t solvent, equal to a material cost of 10,080 yuan/t solvent;
‣ After utilization of corn component: 3,200 yuan/t solvent, the material cost: 7,000 yuan.
7

Corn Stover material
Corn Stover Butanol Biorefinery
8

Corn Stover Butanol Biorefinery
Corn stover cost when construction: 200 yuan/t; when production: 500−700 yuan/t
9

Studies on ABE in Our Lab
Continuous Butanol Fermentation from Low-Value Sugar-Based
Feedstocks by Clostridium saccharobutylicum DSM13864
10

Sugar Utilization of Clostridial Strains
Sugars utilization of various Clostridial strains in ABE fermentation
Total solvent (g/L)
18
16
14
12
10
8
6
4
2
0
Glucose Sucrose
Fructose Mixed sugars
DSM 6422 DSM 13864 DSM 1739 DSM 2152
Strain
C. saccharobutylicum DSM 13864
could be used for ABE fermentation
from lignocellulosic hydrolysates.
C. Beijerinckii DSM 6422、DSM 1739
C. Saccharobutylicum DSM 13864
C. Saccharoperbutylacetonicum DSM 2152
C. saccharobutylicum DSM 13864
could be used for ABE fermentation
from cane molasses.
Butanol, Total solvent (g/L)
16
14
12
10
8
6
4
2
Butanol Total solvent Residual sugar
0
Xylose
Arabinose
Glucose
Sugar mixture

Scheme of 4-Stage Continuous ABE fermentation
Feeding tank Blending vessel Bioreactor 1 Bioreactor 2 Bioreactor 3 Bioreactor 4 Collection tank

Substrate: glucose
Strain: DSM 13864
Batch fermentation in 3-L bioreactor
Solvent( g 稬
-1
)
20
16
12
8
4
0
Acetone
Ethanol
Butanol
Total solvent
Acetic acid
Butyric acid
0 10 20 30 40 50 60 70
Fermentation time( h)
20
16
12
8
4
0
Acid( g 稬
-1
)
-1
pH and Cell density( g 穕 )
9
8
7
6
5
4
3
2
1
pH
Cell density
Residual glucose
0
0
0 10 20 30 40 50 60 70
Fermentation time( h)
60
55
50
45
40
35
30
25
20
15
10
5
Residual glucose( g 稬
-1
)
Total solvent: 18.20 g/L (butanol 11.53 g/L)
Productivity: 0.379 g/L/h
Yield: 0.314 g ABE/g sugar
Fermentation time: 48 h
13

Substrate: glucose
Butanol, Total solvent (g/L)
Strain: DSM 13864
4-Stage continuous fermentation (D=0.03, 0.05, 0.1 h -1 )
16
14
12
10
8
6
4
2
D=0.05 h -1
?
D=0.1 h -1
D=0.03 h -1
0
0
0 100 200 300 400 500 600
Fermentation time (h)
Butanol Total solvent Acetic acid Butyric acid
16
14
12
10
8
6
4
2
Acetic acid, Butyric acid (g/L)
-1
pH and Cell density( g 稬 )
7
Ⅳ
D=0.1 h -1
6 D=0.05 h -1 D=0.03 h -1
5
4
3
2
1
5
0
0
0 100 200 300 400 500 600
Fermentation time( h)
pH Cell density Residual glucose
40
35
30
25
20
15
10
Residual glucose concentration( g 稬
-1
)
At D = 0.05 h -1 :
Total solvent: 11.57 g/L (butanol 7.29 g/L); Productivity: 0.145 g/L/h
At D = 0.1 h -1 :
Total solvent: 8.99 g/L (butanol 6.14 g/L); Productivity: 0.225 g/L/h
14

Substrate: cane molasses
Acetone, Butanol, Ethanol, Total solvent (g/L)
Strain: DSM 13864
Batch fermentation in 5-L bioreactor
20
18
16
14
12
10
8
6
4
2
0
Acid-producing
phase
Solvent-producing phase
0 10 20 30 40 50 60 70
Fermentation time (h)
Butanol Total solvent Acetone
Ethanol Acetate Butyrate
10
8
6
4
2
0
Acetate, Butyrate (g/L)
Sugar concentration (g/L)
Total solvent: 17.88 g/L (butanol 11.86 g/L)
Productivity: 0.50 g/L/h
Yield: 0.33 g ABE/g sugar
Fermentation time: 36 h
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70
Fermentation time (h)
Sucrose
Glucose
Fructose
Total residual sugar
15

Substrate: cane molasses
Strain: DSM 13864
4-Stage continuous fermentation
Solvent, Orignic acid(g/L)
16
12
8
4
D=0.1 h -1 60 20
Butanol
D=0.15 h -1
Total solvent
50
Acetic acid
16
Butyric acid
Total suger 40
12
30
20
10
Total sugar (g/L)
Solvent, Orignic acid (g/L)
8
4
Butanol
Total solvent
Acetic acid
Butyric acid
Total sugar
60
50
40
30
20
10
Total sugar (g/L)
0
0 40 80 120 160 200 240
Fermentation time (h)
0
0
0
0 30 60 90 120
Fermentation time (h)
150 180
At D = 0.1 h -1 (30−130 h)
Total solvent: 11.74 g/L (butanol 7.18 g/L); Productivity: 0.294 g/L/h
Fermentation time: 221 h
At D = 0.15 h -1 (30−100 h)
Total solvent: 13.75 g/L (butanol 8.37 g/L); Productivity: 0.439 g/L/h
Fermentation time: 170 h
16

Pretreatment conditions of various straw
Straw types
Corn straw
g straw/L
liquid
Glucose
g/L
Xylose
g/L
Arabinose
g/L
Total sugar
g/L
Enzymolysis ratio
%
60 33.34 11.16 1.04 45.54 72.41
70 42.88 14.32 1.33 58.52 80.51
80 48.52 16.18 1.59 66.29 80.54
90 57.19 18.84 2.03 78.06 85.08
60 37.09 11.70 0.65 49.43 78.59
Rice straw
70 44.00 13.63 0.68 58.30 80.21
80 49.53 14.71 0.59 64.84 78.78
90 53.83 17.15 1.59 72.56 79.10
60 38.34 12.50 0.44 51.27 81.52
Wheat straw
70 45.43 15.74 0.78 61.96 85.23
80 42.02 14.74 0.83 57.58 69.96
90 53.16 18.79 1.17 73.12 79.70
Alkaline Pretreatment: 1% NaOH, 120 o Cfor 2 h
Cellulase hydrolysis: 20−30 FPIU/g straw, pH 4.8 and 50 o Cfor 40 h
17

Substrate: corn stover hydrolyzate
Strain: DSM 13864
Batch fermentation in 3-L bioreactor
Acetone, Butanol, Ethanol, Total solvent (g/L
18
16
14
12
10
8
6
4
2
0
Acetone
Ethanol
Butanol
Total solvent
Acetate
Butyrate
Total acids
0 10 20 30 40 50
Fermentation time (h)
12
10
Acetate, Butyrate (g/L)
8
6
4
2
0
Sugar concentration (g/L)
60
55
50
45
40
35
30
25
20
15
10
5
0
0 5 10 15 20 25 30 35 40 45 50
Fermentation time (h)
Glucose
Xylose
Arabinose
Total residual sugar
Total solvent: 16.1 g/L (butanol 10.59 g/L)
Productivity: 0.40 g/L/h
Yield: 0.33 g ABE/g sugar
Fermentation time: 40 h
18

Substrate: corn stover hydrolyzate
5-stage temperature-shifting continuous fermentation in 500-mL tanks
(Stage 1, 2: 37 ℃, Stage 3−5: 30 ℃)
Butanol, Total sovent (g/L)
18
16
14
12
10
8
6
4
2
0
60
Total solvent Butanol Residual sugar
50
0
0 40 80 120 160 200 240 280
Fermentation time (h)
40
30
20
10
Residual sugar (g/L)
Total solvent: 12.28 g/L (butanol 8.50 g/L) after 80 h
Productivity: 0.25 g/L/h
Yield: 0.33 g ABE/g sugar
Fermentation time: 270 h (D=0.1 h -1 )
19

Substrate: corn stover hydrolyzate
Strain: DSM 13864
4-Stage Continuous fermentation
18
D=0.1 h -1 D=0.15 60
16
h-1
60
Butanol,Total solvent,
Acetic acid,Butyric acid (g/L)
15
12
9
6
3
0
0
0 30 60 90 120 150
Fermentation time (h)
Butanol Total solvent Acetic acid
Butyric acid OD660 Total sugar
50
40
30
20
10
OD 660
, Total sugar (g/L)
Butanol,Total solvent,OD 660
Acetic acid,Butyric acid (g/L)
12
8
4
0
0
0 40 80 120 160 200 240
Fermentation time (h)
Butanol Total solvent OD660
Acetic acid Butyric acid Total sugar
50
40
30
20
10
Total sugar (g/L)
At D = 0.1 h -1
Total solvent: 13.44 g/L (butanol 9.29 g/L); Productivity: 0.336 g/L/h
Fermentation time: 150 h
At D = 0.15 h -1 :
Total solvent: 11.43 g/L (butanol 7.81 g/L); Productivity: 0.429 g/L/h
Fermentation time: 220 h
20

Substrate: cassava
Strain: C. acetobutylicum A
Batch fermentation in 3-L bioreactor
Acetone, Butanol, Ethanol, Total solvent (g/L)
25
20
15
10
5
Acetone Butanol
Ethanol Total solvent
0
3.0
0 20 40 60 80 100
Fermentation time (h)
pH
5.5
5.0
4.5
4.0
3.5
pH
Total solvent: 20.9 g/L (butanol 12.0 g/L)
Productivity: 0.211 g/L/h
Yield: 0.33 g ABE/g sugar
Fermentation time: 96 h
21

Substrate: cassava
Strain: C. acetobutylicum A
4-Stage continuous fermentation in 3-L bioreactor
Butanol( g/L)
14
12
10
8
6
4
2
stage 1 stage 2 stage 3 stage 4
Residual sugar( g/L)
100
80
60
40
20
stage 1 stage 2 stage 3 stage 4
0
0 20 40 60 80 100
Fermentation time( h)
0
0 20 40 60 80 100
Fermentation time( h)
Total solvent: 22.8 g/L (butanol 12.38 g/L)
Productivity: 0.57 g/L/h
Yield: 0.33 g ABE/g sugar
Fermentation time: 96 h (D=0.1 h -1 )
22

Discuss
Batch and Continuous ABE fermentation
Operation mode Strain Substrate
Dilution
rate (h -1 )
Productivity
(g/L/h)
Total
solvent (g/L)
Reference
Batch Fermentation C. beijerinckii P260 Corn stover − 0.31 26.27 Qureshi et al. 2010
C. acetobutylicum 2N Beet molasses − 0.34 16.32 Fan et al. 2010
C. Saccharobutylicum
DSM 13864
Cane molasses − 0.50 17.88 Ni et al. 2012
C. acetobutylicum XY16 Glucose − 0.63 20.30 Guo et al. 2012
Continuous fermentation
Immobilized cell C. beijerinckii BA101 Glucose 2.0 15.80 7.9
Qureshi et al. 2000
C. beijerinckii ATCC 55025 Glucose 0.2 1.76 8.99 Zhang et al. 2009
Continuous fermentation
Membrane cell bioreactor
C. pasteurianum ATCC 6103 Glycerol 0.9 8.3 9.2 Malaviya et al. 2012
C. Saccharoperbutylacetonicum
N1-4
Glucose 0.11 7.55 8.58 Tashiro et al. 2005
Continuous fermentation
Free cell
C. saccharobutylicum
DSM 13864
Gelatinised
sago starch
0.05 0.46 9.10 Liew et al. 2006
C. acetobutylicum BCRC 10639 Glucose 0.054 0.37 6.85 Yen et al. 2011
C. saccharobutylicum
DSM 13864
Corn stover
hydrolysate
0.1
0.15
0.336
0.429
13.44
11.43
Ni et al.
Unpublished
23

Conclusions
Low-value sugar-based feedstock was utilized in batch and
continuous ABE fermentation, including: cane molasses, corn
stover hydrolyzate, etc.
Using cane molasses, the ABE fermentation could be steadily
operated for over 100 h at D=0.15 h -1 , the average total solvent of
13.75 g/L (butanol 8.37 g/L); and productivity of 0.439 g/L/h were
obtained in a 4-stage continuous fermentation.
Corn stover hydrolysate was prepared using alkaline pretreatment
and enzyme hydrolysis. In a 4-stage continuous fermentation, the
process was operated for 220 h at D=0.15 h -1 , the average solvent
was 11.43 g/L (butanol 7.81 g/L), and the average solvent
productivity was 0.429 g/L/h.
24

Acknowledgement
Graduate students:
Yun Wang
Ziyi Xia
Gang Song
Shan Sun