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Biofuel production from Water Hyacinth using various yeasts

Conference Paper · January 2011

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Aniket Prabhudesai Agnivesh Pandey

University of Mumbai Amity University
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Birla College
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 AniketPrabhudesai*,Agnivesh Pandey,
Annika Durve, Meeta Bhot and Jossy Varghese

Department of Biotechnology,
Birla College of Arts, Science and Commerce, Kalyan,
Dist Thane, MS.
 INTRODUCTION
 Need of an alternative source of energy

 Bio-ethanol

 Water Hyacinth (Eichhornia crassipes)

 Objective:
Use of Water hyacinth as biomass for production of alcohol
using three yeasts.
 MATERIALS & METHODS
Micro-organisms:
 Sacchromyces cerevisiae
 Candida shehatae (NCIM: 3500)
 Pichia stipitis (NCIM:3497)
Growth media:
 Sabouraud agar/broth
 MGYP agar/broth
Chemicals/Reagents:
 DNSA Reagent
 Phloroglucinol Reagent
 Absolute Alcohol
 Dichromate Reagent
Apparatus:
 Colorimeter
 Distillation unit
 Step I : ACID HYDROLYSIS
Water Hyacinth (WH): washed, cut, dried (oven), powdered

Acid Hydrolysis- 2%, 4%, 6%, 8%, 10% H2SO4 (WH:H2SO4; 1:20 w/v)

Autoclave and filter

WH hydrolysate Glucose, Xylose

determination

Step II: DETOXIFICATION OF HYDROLYSATE

WH hydrolysate
∆ 60˚C
Add NaOH(s) till pH 9.0-9.5

Overlimed with solid Ca(OH)2 to pH 10.0

Filter and collect supernatant Glucose, Xylose

determination
 Step III: MEDIA PREPARATION
Detoxified WH Hydrolysate (85-90ml)

Add 0.9g Peptone

Adjust pH to 5.6
Glucose, Xylose
Autoclave at 121˚C, 15lbs, 15mins determination

Step IV: FERMENTATION PROCESS

Inoculate yeast culture to medium Cell density:
Haemocytometer
Incubate at RT

Aliquoted samples at different time intervals (1,2,5,7days)

Distillation

Ethanol determination
Determination of Sugars:

 Glucose – DNSA method

 Xylose – Phloroglucinol method

 Total Carbohydrate- Phenol Sulfuric acid method

Effect of Detoxification on cell density: using Haemocytometer

Minimum Inhibitory Concentration (MIC) determination:

 Alcohol
 Glucose
 Xylose
 RESULTS & DISCUSSIONS
Phenol-sulfuric acid assay for total carbohydrates
Phenol Sulfuric acid assay for Total
Carbohydrate estimation

0.6
0.5
Absorbance at
510nm(units)

0.4
0.3
0.2
0.1
0
0 0.05 0.1 0.15
concentration of glucose (mg/ml)

Acid extract STEP I - Carbohydrate STEP II - Carbohydrate

(1g WH/20ml acid) content (mg/ml) content (mg/ml)
2% 1.33 mg/ml 1.28 mg/ml
4% 1.37 mg/ml 1.18 mg/ml
6% 1.39 mg/ml 1.16 mg/ml
8% 1.39 mg/ml 0.93 mg/ml
10 % 1.39 mg/ml 0.97 mg/ml
 Results of DNSA assay for Glucose estimation
DNSA assay for Glucose estimation
Absorbance at 540nm(units)

0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 0.2 0.4 0.6 0.8 1 1.2
concentration of glucose(mg/ml)

Samples Glucose content (mg/ml)

Step1 -
Step2 4.12 mg/ml
Step3 3.48 mg/ml
Results of Phloroglucinol assay for Xylose estimation
Phloroglucinol assay for Xylose estimation

0.8
Absorbance at
540nm(units)

0.6

0.4

0.2

0
0 0.5 1 1.5
concentration of xylose (mg/ml)

Samples Xylose content (mg/ml)

Step1 4.625 mg/ml
Step2 4.125 mg/ml
Step3 3.8 mg/ml
 Results of Effect of Detoxification on cell density

Organism Medium Initial cell Final cell Increase in

density density cell density
(cells/ml) (cells/ml) (cells/ml)
P.stipitis Non- 1.610 x 106 1.0610 x 107 9.0 x 106
detoxified
P.stipitis Detoxified 1.800 x 106 1.4780 x 107 12.98 x 106
S.cerevisiae Non- 1.990 x 106 9.400 x 106 7.41 x 106
detoxified
S.cerevisiae Detoxified 1.830 x 106 9.420 x 106 7.59 x 106
C.shehatae Non- 2.040 x 106 1.0240 x 107 8.2 x 106
detoxified
C.shehatae Detoxified 1.750 x 106 1.1140 x 107 9.39 x 106
 Minimum Inhibitory Concentration(MIC) results:
3% 6% 9% 12 % 15 % 18 % 21 % 24 % 27 % 30 % Positive Negative
control control
P.stipitis + + - - - - - - - - + -

S.cerevisiae + + + + + - - - - - + -

C.shehatae + + + - - - - - - - + -

Alcohol
10 % 20 % 30 % 40 % 50 % 60 % 70 % 80 % Positive Negative
control control
P.stipitis + + + + + - - - + -

S.cerevisiae + + + + + + + - + -

C.shehatae + + + + - - - - + -

Glucose
5% 10 % 15 % 20 % 25 % 30 % 35 % 40 % 45 % 50 % Positive Negative
control control
P.stipitis + + + + + + + - - - + -

C.shehatae + + + + + - - - - - + -

Xylose
 Alcohol estimation by Dichromate method
Dichromate assay standard graph Dichromate assay Dichromate assay

0.7 0.8
0.9
0.6 0.7
0.8

Concentration of
concentration of

Alcohol(mg/ml)
alcohol(mg/ml)
0.5 0.6
(Blank - B.R.)ml

0.7
0.5
0.6 0.4
0.4
0.5 0.3
0.3
0.4 0.2 0.2
0.3 0.1 0.1
0.2
0 0
0.1
0 2 4 6 8 0 2 4 6 8
0
Fermentation time(days) Fermentation time(days)
0 0.2 0.4 0.6 0.8 1 1.2
Concentration of Alcohol(mg/ml) P.stipitis(static) P.stipitis(shaker) S.cerevisiae(static) S.cerevisiae(shaker)

Dichromate assay P.stipitis P.stipitis S.cerevisiae S.cerevisiae C.shehatae C.shehatae

(static) (shaker) (static) (shaker) (static) (shaker)
0.5
Day 0.28 0.225 0.67 mg/ml 0.335 0.335 0.45 mg/ml
Concentration of

0.4
Alcohol(mg/ml)

1 mg/ml mg/ml mg/ml mg/ml

0.3

0.2
Day 0.39 0.28 0.56 mg/ml 0.56 mg/ml 0.335 0.45 mg/ml
0.1 2 mg/ml mg/ml mg/ml
0
0 2 4 6 8
Fermentation time(days)
Day 0.39 0.39 0.67 mg/ml 0.45 mg/ml 0.45 mg/ml 0.45 mg/ml
5 mg/ml mg/ml
C.shehatae(static) C.shehatae(shaker)

Day 0.61 0.56 0.67 mg/ml 0.56 mg/ml 0.39 mg/ml 0.45 mg/ml
7 mg/ml mg/ml
 CONCLUSION

 This method is a simple process for economical

bioconversion of water hyacinth to alcohol thus
helping clean up the environment and provide a
renewable source of energy.
 REFERENCES
 Ayudhya C. I., Tantimongcolwat T., Kongpanpee T., Prabkate P., Prachayasittikul V.,
(2007). ‘Appropriate Technology for the Bioconversion of Water Hyacinth (Eichhornia
crassipes) to Liquid Ethanol: Future Prospects for Community Strengthening and
Sustainable Development’ , EXCLI Journal, 6: 167-176

 Nigam J.N.,(2002). ‘Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose

acid hydrolysate to motor fuel ethanol by xylose–fermenting yeast’. Journal of
Biotechnology, 97: 107–116

 Ogawa Masami G. O., Usui I.Y., Urano N., (2008). ‘Ethanol production from the water
hyacinth Eichhornia crassipes by yeast isolated from various hydrospheres’, African
Journal of Microbiology Research, 2 :110-113.

 Sadashivam S. and Manickam A. (2004). ‘Biochemical Methods’, Chapter 1, Carbohydrates Pg.

10, 2nd edition. New Age publisher, New Delhi.
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