BIOETHANOL PRODUCTION FROM RICE & WHEAT HUSKS AFTER ACID …

www.wjpr.net Vol 7, Issue 13, 2018.

991

Shweta et al. World Journal of Pharmaceutical Research

BIOETHANOL PRODUCTION FROM RICE & WHEAT HUSKS

AFTER ACID HYDROLYSIS & YEAST FERMENTATION

Sabina Bano1, Dr. Shweta Sao*

2 and Dr. Harit Jha

3

1M.Phil. Scholar, Department of Microbiology, Dr. C.V.Raman University, Kargi Road, Kota

Bilaspur-495113(C.G.), India.

2Professor & Head, Department of Life Science, Dr. C.V.Raman University, Kargi Road,

Kota Bilaspur-495113(C.G.), India.

3*Assistant Professor, Department of Biotechnology, Guru Ghasidas University, Koni,

Bilaspur, (C.G.), India.

ABSTRACT

Bioethanol is a renewable resource that can be produced from

fermented cellulosic biomass. The use of lignocellulosic materials

from agricultural wastes provides a low-cost fermentative substrate.

The aim of this study is to produce bioethanol from rice & wheat husks

using fermentation process and to determine the effect of temperature

on bioethanol yield. The samples were pre-treated with conc. Sulphuric

acid at hydrolysis time (15-30 min.). After acid hydrolysis, the

fermentation is carried out by yeast Saccharomyces cerevisiae. Six

samples of rice & wheat were prepared at different temperatures to

determine the effect of temperature on ethanol yield, the pH was kept

constant at 6.0. Sugar concentrations were determined by

dinitrosalicylic acid (DNS) spectrophotometric method. Total

carbohydrates were determined by Anthrone test. The ethanol concentrations were

determined by hydrometer. Sugar concentrations after hydrolysis and bioethanol production

of rice and wheat husks were 1.39-2.10 mg/ml and 1.69-1.97 mg/ml, respectively. Total

carbohydrates after hydrolysis and bioethanol production of rice and wheat husks were 2.33-

2.69 mg/ml and 3.02-6.57 mg/ml, respectively. Highest ethanol concentrations were obtained

at temperature 350c. This indicates that pH 6.0 and 35

0c was the optimum parameter for the

yeast to produce ethanol. Data were analysed using 1 way Anova.

World Journal of Pharmaceutical Research SJIF Impact Factor 8.074

Volume 7, Issue 13, 991-1004. Research Article ISSN 2277– 7105

Article Received on

16 May 2018,

Revised on 07 June 2018,

Accepted on 28 June 2018,

DOI: 10.20959/wjpr201813-12772

*Corresponding Author

Dr. Shweta Sao

Professor & Head,

Department of Life

Science, Dr. C.V.Raman

University, Kargi Road,

Kota Bilaspur-495113

(C.G.), India.


992


KEYWORDS: Bioethanol, Biomass, Sugar concentration, Hydrometer.

INTRODUCTION

With rapid depletion of the world’s fuel, (petroleum, diesel etc.) in recent years, ethanol has

emerged as one of the alternative liquid fuel and has greater environmental impact. Bio

ethanol production is one of the best ways for crude oil reduction and environmental

compliance. It can be used as fuel having features like high octane number, low cetane

number and high heat of vaporization. It can be produced through fermentation of sugars.

Today worldwide interest is in the usage of bioethanol as an energy source & its concern on

the efficiency for bioethanol production. Development of fermentation processes using cheap

carbon sources is important for commercial scale production. Bioethanol is a renewable

resource produced from lignocellulosic biomass. This is today’s fuel which doesn’t contribute

to global warming. It reduces greenhouse gasses, as combustion of ethanol gives low

emission of carbon monoxide and nitrogen oxides (Park et al. 2010). Using less valuable

materials, like agricultural waste, could reduce the expense significantly, which means

requires less cost for its production.

The hydrolysis is carried out by conc. H2SO4 and the fermentation is carried out by yeasts or

bacteria. Pre-treatment of lignocellulosic biomass prior to hydrolysis can significantly

improve the hydrolysis efficiency by removal of lignin and hemicelluloses, reduction of

cellulose crystallinity and increase of porosity (McMillan, 1994; Palmqvist and Hahn-

Hagerdal,).

Lignocellulosic plant biomass is an important renewable carbon resource for the bio refinery

industry and is thus considered a sustainable and environment friendly alternative to the

current petroleum platform (Wongwilaiwalina et al., 2010). The use of lignocellulosic

materials from agricultural wastes provides a low-cost fermentative substrate. Using

agricultural wastes as lignocellulosic feed stocks for bioethanol production was greatly

promising. One of the advantages of the use of lignocellulosic biomass is not interfering food

production.

Ethanol represents closed carbon dioxide cycle because after burning of ethanol, the released

carbon dioxide is recycled back into plant material because plants use CO2 to synthesize

cellulose during photosynthesis cycle (Wyman, 1999). Ethanol production process only uses

energy from renewable energy sources; no net carbon dioxide is added to the atmosphere,


993


making ethanol an environmentally beneficial energy source. In addition, the toxicity of the

exhaust emissions from ethanol is lower than that of petroleum sources (Wyman and Hinman,

1990). Ethanol derived from biomass is the only liquid transportation fuel that does not

contribute to the green house gas effect.

Bioethanol produces only carbon dioxide and water as the waste products on burning, and the

carbon dioxide released during fermentation and combustion equals the amount removed

from the atmosphere while the crop is growing. It reduces greenhouse gas emissions by as

much as 59 percent relative to gasoline and keeps harmful chemicals like MTBE and benzene

out of air. As a renewable fuel, ethanol is doing more for the environment. Indeed, ethanol

seems to have many things going for it: it’s biodegradable, it produces slightly less

greenhouse emissions than fossil fuel, it can replace harmful fuel additives (e.g., methyl

tertiary butyl ether; MTBE).

The main objective of this study is to successfully produce bioethanol from rice and wheat

husks by acidic hydrolysis and fermentation process. It emerges as a demanding renewable

energy source. The demand for oil is expected to increase to 57% from 2002 to 2030.

MATERIALS AND METHODS

The rice husks were obtained from hanuman rice mill, Torwa, Bilaspur. Whereas wheat husks

were obtained from the agricultural farm land of Masturi & Ratanpur area. Baker's yeasts are

obtained from poonam bakery, in front of city kotwali, Bilaspur (c.g.).

Sample Preparation

The rice and wheat husks were washed properly. After washing, husks were allowed to sun-

dry for 7 days. The samples were packed in the seal bag or jute bag and stored at room

temperature (± 38°C). Each husks samples were treated by acid hydrolysis and followed by

yeasts fermentation.

Sample Hydrolysis

The samples were treated with conc. H2SO4 for 20-30 mins. The slurry was separated by

filtration; the filtrate was collected to determine sugars concentrations after being washed

with distilled water. Total carbohydrates were determined by Anthrone test.


994


Yeast Fermentation

The two hydrolysis samples (80 ml each) from hydrolysis were sterilized by autoclaves

(121°C, 15 min). In fermentation process, Saccharomyces cerevisiae (Baker’s yeasts) was

used to ferment the simple sugar to ethanol and carbon dioxide. To determine the effects of

temperature on ethanol yield, the pH was kept constant at 6.0 while the temperature was

varied at 350

C, 560C & at room temperature. The fermentation process continued for 6 days

(72 hrs).

Sugar determination

The hydrolysis samples of rice & wheat husks were used to determined the sugar

concentrations by dinitrosalicylic acid (DNS) spectrophotometric method. The samples of

two, four, and six fermentation days from each hydrolysis treatment previously were also

determined the reducing sugar by DNS method. Total carbohydrates were determined by

Anthrone test. The presence of sugar was tested by Benedict test.

Ethanol determination

The fermentation samples of rice and wheat husks at two, four, and six fermentation days,

each treatment were determined the ethanol by specific gravity methods with the help of

hydrometer. After every 48 hrs, the samples are measured its specific gravity of varied

temperatures with the help of Hydrometer.

Figure 1: Reading a Hydrometer A hydrometer.


995


Data analysis

Data were analysed using 1 way Anova method. Reducing sugar and bioethanol data from

fermentation samples were analyzed with 1 way Anova.

RESULTS AND DISCUSSION

The result of the investigation showed that the rice and wheat husks produced a significant

amount of ethanol. The volumetric production of ethanol varied according to the variations in

temperatures. It also varied according to fermentation time. The effects of sulphuric acid on

hydrolysis of rice and wheat husks were investigated. Hydrolysis time for the rice and wheat

husks were 20-30 min. Purpose of this optimization is to break the rigid lignin structure and

matrix conformation of cellulose. Each hydrolysis at specified acid concentration followed a

trend where concentration of total sugar increased when the hydrolysis time increasing.

Longer time is required by acid to carry out hydrolytic chemical reaction, which is the

cleavage of β-1-4- glycosidic bond, before converting cellulose into glucose.

The target is to produce maximum yield of sugar. The sugar concentration produced from

acid dose on pre treatment for 20-30 minutes. Pretreatment with an acid dose for 30 minutes

generated 2.10 mg/ml sugars from rice husks. The effects of acid dose and hydrolysis time

are illustrated.

In general, Yeast is an acidophilic organism and as such, grows better under acidic condition.

The optimum pH range for yeast growth can vary from pH 4.0 to 6.0, depending on the

temperature, the presence of oxygen and strain of yeast. Optimum pH values are required for

the activity of plasma membrane bound proteins. During growth, it is important for the yeast

to maintain a constant intercellular pH. There are many enzymes functioning during within

the yeast cell during growth and its metabolism.

Sugar Concentrations after Acid Hydrolysis

Sugar concentration of rice and wheat husks slurry after acid hydrolysis was low. Since the

substrate complexity, it is not easy to degrade both the substrates. Acid hydrolyze

lignocelluloses of rice and wheat husks into simple sugars. Therefore, there are increasing

sugar concentrations after hydrolysis treatment compared to before hydrolysis treatment.

Table 1 showed that sugar concentrations of rice after hydrolysis treatment was 2.10 mg/ml,

whereas sugar concentrations of wheat after hydrolysis treatment were 1.29 mg/ml.


996


Sugar concentration (Rice) by DNS method.

Sugar concentration (Wheat) by DNS method.

Table 1: Sugar concentration (mg/ml) of Rice and Wheat husks after Acid Hydrolysis.

Hydrolysis treatment Rice husk Wheat husk

Treatment with H2SO4 2.10 1.39

Total Carbohydrate In Rice After Acid Hydrolysis

The basic units of carbohydrates are the monosaccharides which cannot be split by hydrolysis

into more simple sugars. The carbohydrate content can be measured by hydrolyzing the

polysaccharides into simple sugars by acid hydrolysis and estimating the resultant

monosaccharides. Carbohydrates are dehydrated with concentrated H2SO4 to form

“Furfural”, which condenses with anthrone to form a green colour complex. Anthrone react

with dextrin, monosaccharides, disaccharides, polysaccharides, starch, gums and glycosides.


997


From Table 2, The total carbohydrate in rice after acid hydrolysis was 2.69 mg/ml, whereas

total carbohydrate present in wheat after hydrolysis was 2.33 mg/ml.

Total carbohydrates (Rice) after acid hydrolysis by Anthrone test.

Total carbohydrates (Wheat) after acid hydrolysis by Anthrone test.

Table 2: Total carbohydrates (mg/ml) of Rice and Wheat husks after Acid Hydrolysis.

Hydrolysis treatment Rice husk Wheat husk

Treatment with H2SO4 2.69 2.33

Sugar Concentrations after Acid Hydrolysis and Yeast Fermentation

After acid hydrolysis, all samples are fermented using yeast S. cerevisiae for six days. Sugars

are consumed by yeasts for growth and energy generation. Sterilization was stopped the

enzymatic and microbes activities, therefore, there is no hydrolysis of lignocelluloses to

sugar. The yeast S. cerevisiae lacked lignocelluloses hydrolytic enzymes. However the yeast

had disaccharides hydrolytic enzymes, such galactosidases or maltases, therefore there is no

additional sugar concentration during fermentation except hydrolysis of disaccharides into

monosaccharides. This condition was directed to the decreasing sugar concentrations


998


following increasing fermentation days (Table 2). Sugar concentrations of rice at varied

temperatures for six fermentation days were given in table 3. Decreasing sugar concentrations

following increasing fermentation days were due to the metabolism of the yeasts. The yeasts

were used sugars for growth and generating energy.

Table 3: Sugar concentration (mg/ml) of Rice and Wheat husks after acid Hydrolysis

and Yeast Fermentation.

S.No. Temperature Rice Wheat

1.

350c 1.97 1.84

560c 1.64 1.61

Room Temp. 1.80 1.74

Sugar concentration of Rice.

Sugar concentration of Wheat.

Table 4: Total carbohydrate (mg/ml) of Rice and Wheat husks after acid Hydrolysis

and Yeast Fermentation.

S.No. Temperature Rice Wheat

1. 35

0c 6.57 3.78

560c 3.02 3.28

Room Temp. 3.15 3.68


999


Total carbohydrate in Rice.

Total carbohydrate in Wheat.

Effects of temperature of fermentation process on ethanol concentration

Temperature is one of the major factors that determine the ethanol production. Table 5

showed the ethanol concentrations that are obtained at different temperatures. However, as

the temperature increases beyond 300c, it showed increase in production of ethanol. At 35

0c

ethanol concentration were maximum and followed by room temperature where ethanol was

obtained. Fermentation process required a suitable temperature for the yeast to react.

Temperature that is too high kills yeast, and low temperature slows down yeast activity.

Thus, to keep a specific range of temperature were required.

However, the ethanol concentration was decreased at 450c. This indicates that 35

0c were the

optimum temperature for ethanol production. This studies result denied the study of Yah et

al., (2010)[23]

, who found optimum temperature of ethanol production to be 250c.


1000


Table 5: Bioethanol concentration at different temperature measured by Hydrometer.

S.No. Agricultural Wastes Temperature Fermentation Days

2 Days 4 Days 6 Days

1. Rice Husk

350c 0.21 0.18 0.22

560c 0.25 0.27 0.24

Room Temp. 0.24 0.27 0.28

2. Wheat Husk

350c 0.26 0.24 0.27

560c 0.30 0.28 0.3

Room Temp. 0.28 0.26 0.28

From the result we can conclude that higher the temperature, lower the ethanol concentration.

The rate of enzyme catalyzed reaction increases with temperature up to a certain temperature

and then the enzyme begins to denature. Higher temperature inhibits the growth of the cells

and fermentation significantly decreases. In this study, ethanol concentration declined

considerably at above 500c, which showed the inhibition effects on the cell growth at higher

temperature.

Based on the high temperature might denature the ribosome and enzymes. Furthermore,

higher temperature would alter the structure of the membrane and decrease its functionality.

Above the optimum temperature, the enzyme reaction drops precipitously as the enzyme

denatures.

Enzymes are sensitive to temperature change. At temperature above 400C, the rate of

respiration slows down and drops. This was because all the enzyme are made up of the

protein chains of amino acid. It exists in the form of a helix structure with the hydrogen

bonds holding them together. When heat was applied to the enzyme, energy was given off.

The active enzyme cell deforms and the hydrogen bonds break, denature the yeast enzyme.

This process called as denaturizing. The optimum temperature in which yeast enzyme work

best is around 350C, below this temperature the rate of reaction was slow and above 40 or 45-

500C the yeast enzyme would denature.

At low temperature the cells showed no ethanol concentration. This may be due to the

enzymes low tolerance to produce ethanol at lower temperature. Furthermore, at low

temperature the enzyme deactivated and reaction slows down stop altogether. At low

temperature, the molecules moves slower than at higher temperature. These explain that the

enzyme may not have enough energy to cause chemical reaction. Overall we can conclude

that temperature 350c was the optimum temperature for ethanol production.


1001


Statistical analysis for effects of temperature on fermentation process of rice & wheat

husks

One way analysis of variance (ANOVA) was conducted to evaluate the relationship between

effects of temperature on ethanol concentration. The independent values were temperature

while the dependent values were ethanol concentration. ANOVA used to determine whether

there was a significance difference in ethanol concentration. Table 4 shows the ANOVA table

for the effect of temperature on ethanol concentration. The one way ANOVA results indicate

that there was a significant difference in temperature at level for three conditions.

Group Temperature Fermentation Days (Rice)


Group 1 350C 0.21 0.18 0.22

Group 2 560C 0.25 0.27 0.24

Group 3 Room Temp. 0.24 0.27 0.28

Analysis of Variance Results

F-statistic value = 8.45939

P-value = 0.01794

Table 5: Summary for one way ANOVA table for effect of Temperature and Ethanol

Concentration (Rice).

Data Summary

Groups N Mean Std. Dev. Std. Error

Group 1 3 0.2033 0.0208 0.012

Group 2 3 0.2533 0.0153 0.0088

Group 3 3 0.2633 0.0208 0.012

ANOVA Summary

Source

Degrees of

Freedom

DF

Sum of

Squares

SS

Mean

Square

MS

F-Stat P-Value

Between

Groups 2 0.0062 0.0031 8.4594 0.0179

Within Groups 6 0.0022 0.0004

Total: 8 0.0084

https://goodcalculators.com/standard-deviation-calculator/

https://goodcalculators.com/standard-error-calculator/


1002


Group Temperature Fermentation Days (Wheat)


Group 1 350C 0.26 0.24 0.27

Group 2 560C 0.30 0.28 0.3

Group 3 Room Temp. 0.28 0.26 0.28

Analysis of Variance Results

F-statistic value = 6.06246

P-value = 0.03628

Table 6: Summary for one way ANOVA table for effect of Temperature and Ethanol

Concentration (Wheat).

Data Summary

Groups N Mean Std. Dev. Std. Error

Group 1 3 0.2567 0.0153 0.0088

Group 2 3 0.2933 0.0115 0.0067

Group 3 3 0.2733 0.0115 0.0067

ANOVA Summary

Source

Degrees of

Freedom

DF

Sum of

Squares

SS

Mean

Square

MS

F-Stat P-Value

Between

Groups 2 0.002 0.001 6.0625 0.0363

Within Groups 6 0.001 0.0002

Total: 8 0.003

CONCLUSION

This study shows that temperature 35°C showed the highest ethanol content (F -value =

8.45939), (P-value = 0.01794). The lowest ethanol concentration was achieved at 56°C. The

study also shows that at 35°C ethanol concentration were maximum followed by room temp.

https://goodcalculators.com/standard-deviation-calculator/

https://goodcalculators.com/standard-error-calculator/


1003


(42°C). However there is very less ethanol production at 56°C. Statistical analysis one way

ANOVA results indicate that there was a significant difference in temperature at level for

three conditions (F -value = 8.45939). The one way ANOVA results for temperature indicate

that there was a significant difference in temperature at (p<o.o1) level for three conditions (F

-value = 8.45939). Further study should conduct on more parameter that effect the

fermentation process on ethanol production. There are other parameter such as amount of

substrate, time and glucose concentration which affects ethanol production during

fermentation. This will gives overall view how ethanol production affected. In conclusion, at

temperature 35°C is the optimum temperature or condition for ethanol production.

REFERENCE

1. Gong-Yuan Wel, Wa Gao, II-Hyuck Jin, Su-Young Yoo, Jai-Heon Lee, Chung-Han

Chung, and Jin-Woo Lee Pretreatment and Saccharification of Rice Hulls for the

Production of Fermentable Sugars, 14 edn., Busan, Korea: Biotechnology and Bioprocess

Engineering, 2009.

2. TJAHJADI PURWOKO, SITI LUSI ARUM SARI, EDWI MAHADJOENO,

SUNARTO Bioethanol production from rice and corn husks after enzymatic and

microbes hydrolysis and yeast fermentation, edn., Central Java, Indonesia:

BIOTEKNOLOGI, 2017; 14.

3. Y.C.WONG and V.SANGGARI Bioethanol Production from Sugarcane Bagasse using

Fermentation Process, edn., Kelantan, Malaysia: ORIENTAL JOURNAL OF

CHEMISTRY, 2014; 30: 507-513.

4. M N Salimi, S E Lim, A H M Yusoff and M F Jamlos Conversion of rice husk into

fermentable sugar by two stage hydrolysis, 908 edn., Perlis, Malaysia: IOP Publishing

2017; 012056.

5. Sanat Rath, Ajay Kumar Singh, Harison Masih,Yashab Kumar, Jyotsana Kiran Peter,

Pankaj Singh, Santosh Kumar Mishra Bioethanol production from wates potatoes as an

environmental waste management and sustainable energy by using cocultures Aspergillus

niger and Saccharomyces cerevisiae, edn., Uttar Pradesh: International Journal of

Advanced Research, 2014; 2: 553-563.

6. Tehmina Saleem Khan and Umarah Mubeen Wheat Straw: A Pragmatic Overview, edn.,

Pakistan: Current Research Journal of Biological Sciences, 2012; 4(6): 673-675.


1004


7. Diana L. Vullo and Mónica B. Wachsman A Simple Laboratory Exercise for Ethanol

Production by Immobilized Bakery Yeasts (Saccharomyces cerevisiae), edn., Buenos

Aires, Argentina: JOURNAL OF FOOD SCIENCE EDUCATION, 2005; 4: 53.

8. ARTHUR CAPUTI, JR., MASAO UEDA, and THOMAS BROWN

SPECTROPHOTOMETRIC DETERMINATION OF ETHANOL IN WINE, Modesfo,

California: E. & J. Gallo Winery, 1968; 95353.

9. JAMES A. HAWKISS AND DONALD D. VAN SLYKE. COMPARISON OF RATES

OF SUGAR DISAPPEARANCE AND CARBON DIOXIDE FORMATION DURING

FERMENTATION OF GLUCOSE, 84 edn., Rockejeller Institute for Medical Research,

New York.): J. Biol. Chem. 1929; 243-247.

10. Zhaoyang XuEmail authorFang Huang Pretreatment Methods for Bioethanol Production,

edn., china: National Natural Science Foundation of China, 2014; 174: 31300483.

11. Devendra Prasad Maurya Ankit Singla Email author Sangeeta Negi An overview of key

pretreatment processes for biological conversion of lignocellulosic biomass to

bioethanol, edn., Allahabad.: Springer Berlin Heidelberg, 2015; 5: 2190-572X.

12. Spyridon Achinas, Gerrit Jan willem Euverink Consolidated briefing of biochemical

ethanol production from lignocellulosic biomass, edn., Groningen, Netherlands:

ELECTRONIC JOURNAL OF BIOTECHNOLOGY, 2016; 23: 44-53.

13. Kexun Li, Shun Liu, Xianhua Liu An overview of algae bioethanol production, 38 edn.,

Tianjin, China: International Journal of Energy Research, 2014; 965-977.

14. Thippa Reddy K. S.* and Agrawal Pushpa STUDIES ON CHARACTERIZATIONS OF

AGRICULTURE WASTE (RICE HUSK) FOR THE PRODUCTION OF ETHANOL, edn.,

Bangalore, Karnataka: Journal of Environmental Research And Development., 2012; 7.

15. Mushimiyimana and P Tallapragada Bioethanol Production from Agrowastes by Acid

Hydrolysis and Fermentation Process, edn., Bangalore: Journal of Scientific & Industrial

Research, 2016; 75: 383-388.

16. www.newagepublishers.com/samplechapter/000091.pdf

Documents

BIOETHANOL PRODUCTION FROM RICE & WHEAT HUSKS AFTER ACID …