Butanol Fermentation from Low-Value Sugar-Based Feedstocks by Clostridia

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1 Clostridium XII, 1 Sept. 2, Nottingham, UK Butanol Fermentation from Low-Value Sugar-Based Feedstocks by Clostridia Ye Ni, Zhihao Sun School of Biotechnology, Jiangnan University Wuxi, China 2.9.1

2 Clostridium XII, 1 Sept. 2, 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

3 Market Butanol consumption and import in China 7 Wt USD Year Butanol import Average price Year Butanol import Average price Ton USD During the period of , around 5% of total annual butanol consumption was imported, and butanol consumption increased rapidly at an average rate of 6 % in China. (Data sources : 3

4 Market Butanol Price Trend in China Butanol Price (Jilin Petrochemical) /11/1 2/1/1 2//2 Market: butanol price downturn continuation ( )

5 Factories ABE Factories in China (data by 2.) Factory Current Capacity (t/y) Starting date Current Status Address Laihe Rockley Bio-Chemicals Ltd 15, Songyuan, Jilin Jilin Cathay Biotechnology Co. Ltd 1, Jilin, Jilin Guangxi Guiping JinYuan Alcohol Co. Ltd 3, 27. Guiping, Guangxi Jimaoyuan Biochemical Co. Ltd, producing ethanol Lianyungang, Jiangsu Lianhai Biological Co. Ltd 5, 2.1 Haimen, Jiangsu Lianyungang Union of Chemicals Co. Ltd, 21. producing ethanol Lianyungang, Jiangsu All ABE factories stopped production or switch to ethanol...

6 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

7 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 (1% water) Fiber 115~ ~2 Embryo 5~ Protein CSL (5% water) 35 Total 22 *CSL: 1% solid content (3% protein), its profit was not considered. In 2.2, corn price in Jilin is 2,3 yuan/t (1% water), including shipping;.5~.7 t corn/t solvent, equal to a material cost of 1, yuan/t solvent; After utilization of corn component: 3,2 yuan/t solvent, the material cost: 7, yuan. 7

8 Corn Stover material Corn Stover Butanol Biorefinery

9 Corn Stover Butanol Biorefinery Corn stover cost when construction: 2 yuan/t; when production: 5 7 yuan/t 9

10 Studies on ABE in Our Lab Continuous Butanol Fermentation from Low-Value Sugar-Based Feedstocks by Clostridium saccharobutylicum DSM136 1

11 Sugar Utilization of Clostridial Strains Sugars utilization of various Clostridial strains in ABE fermentation Total solvent (g/l) Glucose Sucrose Fructose Mixed sugars DSM 622 DSM 136 DSM 1739 DSM 2152 Strain C. saccharobutylicum DSM 136 could be used for ABE fermentation from lignocellulosic hydrolysates. C. Beijerinckii DSM 622 DSM 1739 C. Saccharobutylicum DSM 136 C. Saccharoperbutylacetonicum DSM 2152 C. saccharobutylicum DSM 136 could be used for ABE fermentation from cane molasses. Butanol, Total solvent (g/l) Butanol Total solvent Residual sugar Xylose Arabinose Glucose Sugar mixture

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

13 Substrate: glucose Strain: DSM 136 Batch fermentation in 3-L bioreactor Solvent( g 稬 -1 ) 2 16 Acetone Ethanol Butanol Total solvent Acetic acid Butyric acid Fermentation time( h) 2 16 Acid( g 稬 -1 ) -1 ph and Cell density( g 穕 ) ph Cell density Residual glucose Fermentation time( h) Residual glucose( g 稬 -1 ) Total solvent: 1.2 g/l (butanol g/l) Productivity:.379 g/l/h Yield:.31 g ABE/g sugar Fermentation time: h 13

14 Substrate: glucose Butanol, Total solvent (g/l) Strain: DSM 136 -Stage continuous fermentation (D=.3,.5,.1 h -1 ) D=.5 h -1? D=.1 h -1 D=.3 h Acetic acid, Butyric acid (g/l) -1 ph and Cell density( g 稬 ) Fermentation time (h) Fermentation time( h) Butanol Total solvent Acetic acid Butyric acid ph Cell density Residual glucose D=.5 h -1 Ⅳ D=.1 h -1 D=.3 h Residual glucose concentration( g 稬 -1 ) At D =.5 h -1 : Total solvent: g/l (butanol 7.29 g/l); Productivity:.15 g/l/h At D =.1 h -1 : Total solvent:.99 g/l (butanol 6.1 g/l); Productivity:.225 g/l/h 1

15 Substrate: cane molasses Acetone, Butanol, Ethanol, Total solvent (g/l) Strain: DSM 136 Batch fermentation in 5-L bioreactor Acid-producing phase Solvent-producing phase Fermentation time (h) Butanol Total solvent Acetone Ethanol Acetate Butyrate Acetate, Butyrate (g/l) Sugar concentration (g/l) Total solvent: 17. g/l (butanol 11.6 g/l) Productivity:.5 g/l/h Yield:.33 g ABE/g sugar Fermentation time: 36 h Fermentation time (h) Sucrose Glucose Fructose Total residual sugar 15

16 Substrate: cane molasses Strain: DSM 136 -Stage continuous fermentation Solvent, Orignic acid(g/l) 16 D=.1 h Butanol D=.15 h -1 Total solvent 5 Acetic acid 16 Butyric acid Total suger Total sugar (g/l) Solvent, Orignic acid (g/l) Butanol Total solvent Acetic acid Butyric acid Total sugar Total sugar (g/l) Fermentation time (h) Fermentation time (h) 15 1 At D =.1 h -1 (3 13 h) Total solvent: 11.7 g/l (butanol 7.1 g/l); Productivity:.29 g/l/h Fermentation time: 221 h At D =.15 h -1 (3 1 h) Total solvent: g/l (butanol.37 g/l); Productivity:.39 g/l/h Fermentation time: 17 h 16

17 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 % Rice straw Wheat straw Alkaline Pretreatment: 1% NaOH, o Cfor 2 h Cellulase hydrolysis: 2 3 FPIU/g straw, ph. and 5 o Cfor h 17

18 Substrate: corn stover hydrolyzate Strain: DSM 136 Batch fermentation in 3-L bioreactor Acetone, Butanol, Ethanol, Total solvent (g/l Acetone Ethanol Butanol Total solvent Acetate Butyrate Total acids Fermentation time (h) 1 Acetate, Butyrate (g/l) 6 2 Sugar concentration (g/l) Fermentation time (h) Glucose Xylose Arabinose Total residual sugar Total solvent: 16.1 g/l (butanol 1.59 g/l) Productivity:. g/l/h Yield:.33 g ABE/g sugar Fermentation time: h 1

19 Substrate: corn stover hydrolyzate 5-stage temperature-shifting continuous fermentation in 5-mL tanks (Stage 1, 2: 37, Stage 3 5: 3 ) Butanol, Total sovent (g/l) Total solvent Butanol Residual sugar Fermentation time (h) Residual sugar (g/l) Total solvent:.2 g/l (butanol.5 g/l) after h Productivity:.25 g/l/h Yield:.33 g ABE/g sugar Fermentation time: 27 h (D=.1 h -1 ) 19

20 Substrate: corn stover hydrolyzate Strain: DSM 136 -Stage Continuous fermentation 1 D=.1 h -1 D= h-1 6 Butanol,Total solvent, Acetic acid,butyric acid (g/l) Fermentation time (h) Butanol Total solvent Acetic acid Butyric acid OD66 Total sugar OD 66, Total sugar (g/l) Butanol,Total solvent,od 66 Acetic acid,butyric acid (g/l) Fermentation time (h) Butanol Total solvent OD66 Acetic acid Butyric acid Total sugar Total sugar (g/l) At D =.1 h -1 Total solvent: 13. g/l (butanol 9.29 g/l); Productivity:.336 g/l/h Fermentation time: 15 h At D =.15 h -1 : Total solvent: 11.3 g/l (butanol 7.1 g/l); Productivity:.29 g/l/h Fermentation time: 22 h 2

21 Substrate: cassava Strain: C. acetobutylicum A Batch fermentation in 3-L bioreactor Acetone, Butanol, Ethanol, Total solvent (g/l) Acetone Butanol Ethanol Total solvent Fermentation time (h) ph ph Total solvent: 2.9 g/l (butanol. g/l) Productivity:.211 g/l/h Yield:.33 g ABE/g sugar Fermentation time: 96 h 21

22 Substrate: cassava Strain: C. acetobutylicum A -Stage continuous fermentation in 3-L bioreactor Butanol( g/l) stage 1 stage 2 stage 3 stage Residual sugar( g/l) stage 1 stage 2 stage 3 stage Fermentation time( h) Fermentation time( h) Total solvent: 22. g/l (butanol.3 g/l) Productivity:.57 g/l/h Yield:.33 g ABE/g sugar Fermentation time: 96 h (D=.1 h -1 ) 22

23 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 P26 Corn stover Qureshi et al. 21 C. acetobutylicum 2N Beet molasses Fan et al. 21 C. Saccharobutylicum DSM 136 Cane molasses Ni et al. 2 C. acetobutylicum XY16 Glucose Guo et al. 2 Continuous fermentation Immobilized cell C. beijerinckii BA11 Glucose Qureshi et al. 2 C. beijerinckii ATCC 5525 Glucose Zhang et al. 29 Continuous fermentation Membrane cell bioreactor C. pasteurianum ATCC 613 Glycerol Malaviya et al. 2 C. Saccharoperbutylacetonicum N1- Glucose Tashiro et al. 25 Continuous fermentation Free cell C. saccharobutylicum DSM 136 Gelatinised sago starch Liew et al. 26 C. acetobutylicum BCRC 1639 Glucose Yen et al. 211 C. saccharobutylicum DSM 136 Corn stover hydrolysate Ni et al. Unpublished 23

24 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 1 h at D=.15 h -1, the average total solvent of g/l (butanol.37 g/l); and productivity of.39 g/l/h were obtained in a -stage continuous fermentation. Corn stover hydrolysate was prepared using alkaline pretreatment and enzyme hydrolysis. In a -stage continuous fermentation, the process was operated for 22 h at D=.15 h -1, the average solvent was 11.3 g/l (butanol 7.1 g/l), and the average solvent productivity was.29 g/l/h. 2

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

26 26

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