Bioethanol production from lignocellulosic biomass by co-fermentation using Zymomonas mobilis and Pichia stipitis on bioreactor #
Kartik Gehlot and Sanjoy Ghosh*
Biochemical Engineering Lab, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India # Presenting Author: gehlot.kartik67@gmail.com *Corresponding email: ghoshfbs@iitr.ac.in
RESULTS & DISCUSSION
INTRODUCTION
Bioethanol production from lignocellulosic biomass is most popular and promising technology nowadays to reduce pollution (Fu et al., 2009).
Kans Grass Biomass contain up to 60% fermentable sugars in the form of
glucose
and xylose that can be converted to bioethanol (Zabed et al., 2016). Kans
Grass Biomass can be hydrolyzed by acid hydrolysis process into glucose rich
fraction (GRF) and xylose rich fraction (XRF) (Chaudhary et al., 2014). Glucose
fermentation is mainly performed by using bacteria (Zymomonas mobilis)
organisms with addition of nutrients just before P.stipitis inoculation at 14th hour of fermentation (Liu et al., 2018). The
Batch
Step feed 1
Step feed 2
Step feed 3
Overall
60
60
60
60
240
Initial Xylose (g/l)
26.03
45.26
73.38
101.87
101.87
Max. Ethanol Conc. (g/l)
Max. Biomass Conc. (g/l)
2.52
2.81
3.07
3.74
3.74
Fermentation Time (h)
6
4
5
24
39
Glucose Consumption (%) Ethanol Yield Coefficient Yp/s (g/g) Biomass Yield Coefficient Yx/s (g cells/g substrate) Max. Ethanol Productivity rp (g/l/h) Max. Biomass productivity rx, (g cells/g/h)
96%
92%
95%
61.6%
86.15%
0.433
0.32
0.468
0.47
0.424
0.042
0.004
0.004
0.011
0.015
4.33
4.80
5.622
1.18
2.61
0.42
0.07
0.052
0.028
0.095
Max. Ethanol Conc. (g/l)
present study, we studied the co--fermentation of glucose and xylose using above
above study resulted in complete utilization of both the sugars with
ethanol yield of 0.46g/g and ethanol productivity of 1.56 g/l/h on synthetic media
Table 5: Kinetic parameters of Fed-Batch process of P.stipitis
Table 4: Kinetic parameters of Fed-Batch process of Z.mobilis
Initial Glucose (g/l)
and xylose fermentation is mainly done by yeast (Pichia stipitis). In
Fig.7: Fed-batch process of P.stipitis on synthetic media with xylose on 7L bioreactor
Fig.6: Fed-Batch process of Z.mobilis on synthetic media with glucose on 7L bioreactor
METHODS
Batch
Step feed 1
Step feed 2
Overall
60
60
60
180
13.32
26.23
40.32
39.65
Max. Biomass Conc. (g/l)
31.25
45.26
46.81
46.13
Fermentation Time (h)
13
17
24
58
Xylose Consumption (%) Ethanol Yield Coefficient Yp/s (g/ g) Biomass Yield Coefficient Yx/s (g cells/g substrate) Max. Ethanol Productivity rp (g/ l/h) Max. Biomass productivity rx (g cells/g/h)
53.33%
43.33%
60%
81.11%
0.22
0.21
0.23
0.22
0.52
0.23
0.02
0.25
1.02
0.75
0.58
0.68
2.4
0.82
0.06
0.79
Table 6: Kinetic parameters of co-fermentation of sugars with Z.mobilis and P.stiptis on synthetic media at different RPM for P.stipitis
Parameters
400 rpm for P.stipitis
200 rpm for P.stipitis
Initial Glucose + Xylose (g/l)
60 + 40
60 + 40
Max. Ethanol conc. (g/l)
46.84
Max. Biomass Conc. (g/l)
1.31 (ZM) + 29.31 (PS)
1.22 (ZM) + 27.22 (PS)
Fermentation Time (h)
30
60
Sugar Consumption (Glucose + Xylose) (%)
100% + 100%
Ethanol Yield Coefficient Yp/s (g/g) Biomass Yield Coefficient Yx/s (g cells/g substrate) Max. Ethanol Productivity rp (g/l/h) Max. Biomass productivity rx (g cells/g/h)
0.468 (91.66%) 0.021 (ZM) + 0.732 (PS) 1.56 0.043 (ZM) + 0.0.977 (PS)
300C Fig.8: Co-fermentation of Glucose & Xylose by Z.mobilis & P.stipitis in 7L Bioreactor without distillation
23.666
100% + 100% 0.394 0.0385 0.394 0.0385
Table 7: Kinetic parameters of co-fermentation of Glucose & Xylose by Z.mobilis & P.stipitis on 7L Bioreactor with distillation and multi-step addition of xylose Fig.9: Co-fermentation of Glucose & Xylose by Z.mobilis & P.stipitis on 7L Bioreactor with distillation
Glucose
Initial Sugar (g/l)
Table 1: Chemical composition of various non-edible lignocellulosic biomass Major Components
Kans Grass (%)
Cellulose
44.0
Hemicellulose
Baggasse (%)
39.2
21.5
Acid Soluble Lignin
Wheat Straw (%)
37.8
25.4
26.37
40
40
40
180
28.78
18.87
17.97
3.02
68.64
Max. Biomass Conc. (g/l)
1.31
34.1
8.206
1.13
1.31 (ZM) + 41.14 (PS)
Fermentation Time (h)
14
20
18
20
72
Sugar Consumption (%)
100%
88.34%
91.7%
21.97%
82.66%
Ethanol Yield Coefficient Yp/s (g/g)
0.479 (93.8%)
0.471 (92.17%)
0.449 (87.86%)
0.075 (14.77%)
0.38 (74.62%)
Biomass Yield Coefficient Yx/s (g cells/g substrate)
0.021
0.852
0.205
0.0755
0.021 (ZM) + 0.344 (PS)
Max. Ethanol Productivity rp (g/l/h)
2.05
0.943
0.998
0.151
0.953
Max. Biomass productivity rx (g cells/g/h)
0.093
1.705
0.455
0.056
0.093 (ZM) + 0.709 (PS)
20.08
Acid Insoluble Lignin
2.12
1.4
1.1
Ash Content
3.35
6.5
4.3
Parameters
200 RPM
400 RPM
600 RPM
Initial Xylose (g/l) Max. Ethanol conc. (g/l) Max. Biomass Conc. (g/l) Fermentation Time (h) Xylose Consumption (%) Ethanol Yield Coefficient Yp/s (g/g) (% theoretical yield) Biomass Yield Coefficient Yx/s (g cells/g substrate) Max. Ethanol Productivity rp (g/l/h)
60 27.8 10.07 48 100 0.46 (90 %)
60 28.96 32.9 18 100 0.48 (94.1 %)
60 29.07 36.67 18 100 0.48 (94.1%)
0.16
0.54
0.611
0.58
1.6
1.61
Max. Biomass productivity rx
0.21
1.82
2.03
Table 8: Literature review of fermentation of sugars with Z.mobilis and P.stiptis on synthetic media and hydrolysate
26.3
21.2
Table 2: Mass Transfer studies on P.stipitis on 7L bioreactor with synthetic media
Fig.10: Co-fermentation of Glucose & Xylose by Z.mobilis & P.stipitis on 7L Bioreactor with distillation and multi-step addition of xylose
Table 3: Mass Transfer studies on P.stiptis on 7L bioreactor with hydrolysate Parameters
200 RPM
400 RPM
600 RPM
Initial Xylose (g/l) Max. Ethanol conc. (g/l) Max. Biomass Conc. (g/l) Fermentation Time (h) Xylose Consumption (%)
60 27.10 10.79 48 100
60 27.65 30.65 18 100
60 28.16 35.65 18 100
Ethanol Yield Coefficient Yp (g/g) (% theoretical yield)
0.45 (88.23%)
0.46 (90.2%)
0.47 (92.1%)
0.18
0.51
0.594
0.564
1.53
1.56
0.22
1.7
1.98
Biomass Yield Coefficient Yx/s (g cells/g substrate) Max. Ethanol Productivity rp (g/l/h) Max. Biomass productivity rx (g cells/g/h)
Overall
60
Max. Ethanol Conc. (g/l)
Fig.1: Overview of Bioethanol production from lignocellulosic biomass (Ref.: http://www.deinove.com/en/programmes-applications/deinol)
Xylose Step feed Step feed 1 2
Combination of micro-organisms
Media
Initial Glucose (g/l)
Initial Xylose ( g/l)
Max. Ethanol Conc. (g/l)
Max. Ethanol Yield (g/g)
Max. Ethanol productivity (g/l/h)
Reference
Zymomonas mobilis + Pichia Stipitis
Synthetic
30
20
-
0.49
1.277
Fu et al., 2009
Zymomonas mobilis + Pichia Stipitis
Synthetic
300
100
56.9
0.469
1.016
Singh et al., 2014
Zymomonas mobilis + Pichia Stipitis
Hydrolysate
200
60
62.3
0.474
1.416
Singh et al., 2014
Zymomonas mobilis + Pichia Stipitis
Synthetic
60
40
46.84
0.468
1.56
Present Study
CONCLUSIONS Z.mobilis and P.stipitis have considerable potential for large-scale ethanol production from lignocellulosic biomass hydrolysate because of high ethanol productivity.
The combination of above organisms are efficient in utilizing both glucose and xylose from synthetic media as well as natural hydrolysate.
In the present study, co-fermentation of glucose and xylose was performed on 7L NBS Bioreactor with 2L working volume that resulted in ethanol yield of 0.46 g/g and ethanol productivity of 1.56 g/l/h.
Addition of nutrients together with distillation after glucose fermentation with Z.mobilis resulted in enhanced ethanol productivity by P.stipitis in co-fermentation of glucose and xylose in synthetic media. REFERENCES
Fig.1: Batch process of P.stipitis on 7L bioreactor at 200 RPM with synthetic media
Fig.2: Batch process of P.stipitis on 7L bioreactor at 400 RPM with synthetic media
Fig.4: Batch process of P.stipitis on 7L bioreactor at 200 RPM with hydrolysate
Fig.5: Batch process of P.stipitis on 7L bioreactor at 400 RPM with hydrolysate
- Nan Fu, Paul Peiris, Julie Markham, John Bavor (2009). “A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures”. Enzyme and Microbial Technology, 45, 210–217. -Yanli Chen (2010). “Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review”. J Ind Microbiol Biotechnol, 38, 581–597 -Lalit K. Singh, Chandrajit B. Majumderb, Sanjoy Ghosh (2014). “Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass”. Biochemical Engineering Journal, 82, 150– 157 - Zabed et al. (2016). “Fuel ethanol production from lignocellulosic biomass: An overview on feedstocks and technological approaches.” Renewable and Sustainable Energy Review, 66, 751-774.
ACKNOWLEDGEMENT The authors are thankful to the financial assistance provided by Indian Institute of Technology Roorkee and MHRD for carrying out this research work. Fig.3: Batch process of P.stipitis on 7L bioreactor at 600 RPM with synthetic media
Fig.6: Batch process of P.stipitis on 7L bioreactor at 600 RPM with hydrolysate
Biochemical Engineering Lab Raikamal Bhattacharya, Sharath, Pritam Singh, Vasundhra Jain, Rohith, Sharika, Abhinav Jain, Pratibha, Rajshree, Preeti, Rashmi, Somesh Bhambi