Ethanol Production from Starch by Hydrolysis and Fermentation

Bhaskar G. Gaikwad¹*, Varda V. Kolapkar² and Manasi Shrouti³

¹C.E. Division, National Chemical Laboratory, Pune-411008, India

²Department of Biotechnology, Dr. D.Y. Patil B.B.I., Tathawade, Pune

³ Department of Biotechnology, Modern College of Arts, Science & Commerce, Ganeshkhind, Pune

*Corresponding Author E-mail: bg.gaikwad@ncl.res.in

ABSTRACT:

At present amylase is used for hydrolysis of starch before alcohol fermentation. Ethanol production from starch after hydrolysis of starch by acid was studied in this paper. Hydrochloric acid was used for hydrolysis. Different acid concentration and time were used for hydrolysis. We have used four substrates: soluble starch, insoluble starch, jwar (sorghum) and sweet potato. Jwar (sorghum) and sweet potato are starchy substrates available in plenty.These substrates we have used. We observed 92.79% hydrolysis with 13.33% soluble starch, 16.66% HCl concentration for 30 minutes. We observed 96.51% hydrolysis of insoluble starch (14.28%) by 20.63% HCl when 20 minutes heating time was used. In case of jwar we found 69.23% hydrolysis where 17.85% jwar (sorghum) was treated with 20.63% HCl for 30 minutes heating. We got 62.08% hydrolysis for 14.28% sweet potato starch treated with 20.63% HCl for 30 minutes heating. pH adjustment of hydrolyzate was studied. NaOH, NaOH with potassium phosphate for buffering action, calcium hydroxide, calcium carbonate were studied for pH adjustment. These hydrolyzate were subjected to fermentation after adjusting pH. Baker’s yeast was used for fermentation. Fermentation was carried out at temperature 28⁰C without shaking for 24h. We have obtained 4.37, 4.57 and 3.61% ethanol from fermentation soluble starch, insoluble starch and jwar (sorghum) hydrolyzate respectively.

KEYWORDS: Starch, Jwar, Bakers’ yeast, fermentation, hydrolysis, hydrochloric acid.

INTRODUCTION:

Starch is produced from plant source. It is abundant in nature. It is mainly used for edible purpose. Some portion is also used for liquor production. It is observed that lot of quantity of starch is not utilized, goes in waste. Sometimes it is not of edible quality due to various reasons. Such starch can be utilized for alcohol production. Presently amylase is used routinely in for hydrolysis of starch^1,2 . Amylase is expensive and not available everywhere. Acid hydrolysis can be done in simpler way. Study of hydrolysis of starch by acid is reported in literature by many researchers^3-5. Some researchers have hydrolyzed starch by acid, amylase and used for fermentation. In this research work, we have attempted to optimize acid hydrolysis followed by fermentation by bakers’ yeast. We have used hydrochloric acid for hydrolysis and hydolyzate arising is used for alcohol production using baker’s yeast.

MATERIALS AND METHODS:

Chemicals:

Ethanol was purchased from Thomas Baker Mumbai. Other chemicals were purchased from S.D. Fine Chemicals, Mumbai. Remaining material was purchased from local market.

Cultures:

Bakers’ yeast was purchased from local market.

Analysis of sugars :

Sugar is analyzed by DNSA reagent⁶. Samples of acid hydrolysis were neutralized by NaOH and analyzed.

Analysis of Ethanol

Ethanol was estimated by dichromate method⁷

Hydrolysis of starch:

Starch was weighed. Distilled water was added and mixed well. Hydrochloric acid is added. Mixture was heated in boiling water bath for different time. Sugar was estimated by DNSA method.

Fermentation:

Hydrolyzate was brought to pH 5.5. 5% . Bakers’ yeast was added and fermentation was carried out at 28⁰C for 24h. Sugar and ethanol were analyzed.

RESULTS AND DISCUSSION:

Starch concentration

Soluble starch was subjected to acid hydrolysis. We have tried different concentrations. But we observed higher concentrations are not useful. 13.33% soluble starch is suitable for study. In case of jwar starch increasing substrate concentration is not useful. 14.28 % concentration is useful (table 1). We observed 62.08% hydrolysis where 14.28% sweet potato starch treated with 20.63% HCl for 30 minutes heating. We got lower results when substrate concentration was increased to 17.46%.

Table 1: Effect of concentration of starch : soluble starch, jwar starch.

Starch	Starch %	HCl%	Time (Minutes)	Hydrolysis %
Soluble starch	13.33	16.66	20	65.88
	15	16.66	20	69.90
	16.66	16.66	20	61.61
Jwar starch	14.28	20.63	30	62.88
Jwar starch	17.85	20.63	30	69.23

Acid concentration

We have used 16.66 and 19.35% HCl for hydrolysis of soluble starch (13.33%) (figure1). 14.51% insoluble starch used for study. 19.35 and 20.63% HCl concentrations were used ( figure 1).

We observed increase in hydrolysis by increasing concentration. Insoluble starch was studied for higher concentration with higher concentration of HCl where 96.51% hydrolysis was observed with 20.63% HCl for 20 minutes. Acid hydrolysis of jwar was studied and we found there is not much differnce in hydrolysis when 20.63 and 21.87% HCl (figure 1). In case of sweet potato, although we have increased HCl concentartion i.e. 23.72 and 25%, we had lower hydrolysis i.e. 53.04 and 40.25% respectively.

Hydrolysis time:

We observed 92.79% hydrolysis with 13.33% soluble starch, 16.66% HCl concentration for 30 minutes (figure 2). Whereas 90.95% hydrolysis was observed for insoluble starch. In case of jwar we found 69.23% hydrolysis where 17.85% jwar was treated with 20.63% HCl for 30 minutes heating (figure 3). We observed 62.08% hydrolysis where 14.28% sweet potato starch treated with 20.63% HCl for 30 minutes heating.

Fermentation:

Soluble starch (13.33% starch, 16.66% HCl, 30 minutes heating); insoluble starch (1428% starch, 20.63% HCl , 20 min. heating) were hydrolyzed. In case of jwar we have used 14.28% starch, 20.63% HCl, 30 minutes heating because using higher HCl concentration, substrate and time not much improvement was observed in hydrolysis. pH of hydrolyzate was adjusted to 5.5 by NaOH but we found buffering is necessary. Therefore we have used 0.003M K₂HPO₄ before adjusting pH. Afterward we have tried CaCO₃ for adjusting pH. Fermentation of hydrolyzate arising from soluble starch, insoluble starch and jwar starch was done using 5% baker’s yeast at 28⁰C for 24h (table 2). Results of alcohol are presented in figure 4.

Osemwengie and Omorodion (2013)⁸ have reported glucose preparation from cassava starch by acid hydrolysis. Cao et al. (2014)¹, Preez et al. (1985)² have studied ethanol fermentation of sorghum where they have used amylase for hydrolysis. Muller and Miller (1983)⁹ have produced sugar from starch using acid hydrolysis at high temperature and pressure where hydrolyzate used for ethanol preparation. Monsalve et al. (2006)¹⁰ have produced ethanol from cassava where they have used sulphuric acid for hydrolysis. Scholz et al. (2012)¹¹ studied alcohol production from algal mass where sulphuric acid was used for hydrolysis. Some literature survey is cited in table 3. Although we have got less alcohol, we are reporting simple process for production of alcohol. The present work will be useful in developing feasible technology at higher scale where we can have patent.

Table 2: Hydrolysis and ferrmentation of starch

Starch	Starch (%)	HCl (%)	Time (minutes)	Hydrolysis (%)	Sugar utilization in fermentation (%)	Ethanol formation efficiency
soluble	13.33	16.66	30	92.01	89.29	84.33
insoluble	14.28	20.63	20	96.51	87.59	71.08
jwar	14.28	20.63	30	62.86	85.79	76.58

Table 3: Literature survey

Substrate	Acid (%)	Conditions	Hydrolysis (%)	References
Pinho starch, corn starch	10.8% HCl	25% starch, 44⁰C 6h	solubility increased	Thys et al. (2013)³
Algal biomass	H₂SO₄	30% biomass, 121⁰C	Hydrolysis till no further breakdown	Scholz et al. (2013)¹¹
Dioscorea alata starch	8% HCl	3.3% starch, 35⁰C, 32 days	63.66	Jiang et al. (2012)⁴
Quick sweet starch Normal sweet potato	8.04% HCl	1.5% starch, 37⁰C, 16-18 days	85 50	Genkina et al. (2009)⁵
Cassava	0.8 pH by H₂SO₄	17% starch, 5h	100	Monsalve et al. (2006)¹⁰
Bakery waste	2% H₂SO₄	132⁰C, 40 min.	92	Choi and Mathews (1996)¹²

CONCLUSION:

We observed 92.79% hydrolysis with 13.33% soluble starch, 16.66% HCl concentration for 30 minutes heating. By increasing starch (soluble) concentration hydrolysis was not increased. By increasing acid concentartion, hydrolysis of soluble and insoluble starch increased. For 13.33 % insoluble starch we have not tried more acid concentration because already such study was done for soluble starch. We observed 96.51% hydrolysis of insoluble starch (14.28%) by 20.63% HCl when 20 minutes heating time was used. In case of jwar we found 69.23% hydrolysis where 17.85% jwar was treated with 20.63% HCl for 30 minutes heating. We got 62.08% hydrolysis for 14.28% sweet potato starch treated with 20.63% HCl with 30 minutes heating. We have obtained 4.37, 4.57 and 3.61% ethanol from soluble, insoluble and jwar starch respectively.

REFERENCES:

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Received on 05.12.2014 Modified on 21.01.2015

Research J. Science and Tech. 7(1): Jan.-Mar. 2015; Page 14-18

DOI: 10.5958/2349-2988.2015.00003.0