two step fermentation process for the production of ...

Journal of Environmental Research And Development

Vol. 4 No. 4, April-June 2010

TWO STEP FERMENTATION PROCESS FOR THE PRODUCTION OF ETHANOL FROM AGRICULTURAL WASTE Thippa Reddy K. S.* and Pushpa Agrawal Department of Biotechnology, R.V. College of Engineering, Vidyaneketan post, Bangalore, Karnataka, (INDIA)

Received September 12, 2009

Accepted June 01, 2010

ABSTRACT Renewable agricultural resources like crops residues- wheat straw, cotton stalks, corn cob, etc., are being used for the ethanol production to compensate the energy crisis. In the present study, corn cob - an agricultural waste is used to produce ethanol through two step fermentation process. First the material is subjected for pretreatment with Aspergillus niger at temperature 30°C and pH 4.5 to convert the lignocelluloses to reducing sugars. Then, the reducing sugars are subjected to Zymomonas mobilis fermentation at 30°C temperature. From the experiment, 3% ethanol production has been observed.

Key Words: Lignocelluloses, Crops residue, Renewable agricultural resources, Energy crisis, Fermentation, Reducing sugars

INTRODUCTION Growing population coupled with increased reliance on fossil fuels has led to severe energy crisis apart from environmental pollution. To substitute our depleting fossil fuels and to minimize the emission of toxic gases into atmosphere by burning the fossil fuel, it is imperative to find an alternative source of energy. Ethanol is one such alternative which can meet the growing demand for fuel. It also plays an important role in reducing green house gas emission and global warming. Ethanol can be * Author for correspondence

produced by biomass such as agricultural wastes of various crops, through 1 fermentation Corn cob - an agricultural waste is composed of cellulose-42% hemicellulose-39% and lignin-14%.2 In India, considerable geographical area is under maize cultivation and there is production of a good quantity of corn cob. Owing to its composition and difficulty in its degradation, in India, it is wasted. However, it can be hydrolyzed at basic pH, and subjected to two steps fermentation process to enhance the ethanol yield. In the first step above said corn components are converted to simple sugars and in the second step, those

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simple sugars are fermented to ethanol. The different pH level is having a marked influence on the quantity of the ethanol produced. The conversion of cellulosic material into sugars is, followed by biological transformation by microorganisms into variety of products. Among them ethanol, has provided a potential source of cheap raw material that may compete with petroleum derivatives.3 In addition to the potential economic incentive for biologically producing ethanol, ethanol fermentation has become a standard for the evaluation of improved process schemes for the fermentation industry. Ethanol is an ideal standard for the fermentation industry.4 The xylose fermenting yeasts has attracted widespread interest because of the economy of production of ethanol from lignocellulosic materials. It is much improved by the efficient fermentation of both hexose and pentose sugars which are converted to ethanol. Microorganisms like Peichia stiptiss, Candida shehataing, Z. mobilis and Saccharomyces cerevisiae can convert glucose to ethanol. 5 Degradation of cellulosic waste is done by enzymatic and subsequent fermentation of glucose by yeasts, for the production of ethanol. New technology for converting sugars to industrial alcohol was necessary to substantially reduce the overall base cost. Various techniques for increasing the productivity, such as continuous culture, cell recycle and vacuum distillation, have been evaluated. Also for the maximum


Productivity, Zymomonas mobilis is an important ethanol producing bacteria. Zymomonas mobilis produces 1.9 mol of ethanol from each mole of glucose fermented using a modification of the enter-Douddoroff pathway.6 Hemicellulose fraction can be easily hydrolyzed to manomeric sugars, like xylose and glucose under mild conditions. Dilute acid hydrolysis is one of the two major processes for hydrolyzing agricultural and wood wastes. During hydrolysis, several compounds such as furfural, hydroxymethylfurfural, acetic acid, syringic acid, vanillin, p-hydroxybenzeneoic acid are released as a result of sugar or lignin degradation processes. Lignin hydrolysis provide rich medium for fermentation of sugars into ethanol. The effectiveness of a detoxification method depends on the hemicellulosic hydrolysate and the microorganism employed for fermentation. due to large proportion of xylose in hemicellulosic hydrolysated. The inability of Saccharomyces cerevisiae to ferment xylose to ethanol, the use of xylose fermenting yeasts to co-ferment pentose and hexose sugars in hydrolysate offers an opportunity for the efficient utilization of hemicellullse component of agricultural residual.

MATERIAL AND METHODS Sun dried corn cob was used as substrate in this study. Physical treatment Corncob was grinded into small pieces of 5mm size and weighed ten gm dried in an oven at 60oC.

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Fig. 1 : The substrate- corncob- for ethanol production

Chemical treatment – hydrolysis The dried substrate was treated with 5% sodium hydroxide solution and then washed Using distilled water 6-7 after washing; substrate was again dried in oven at 60oC, then autoclaved at 15 psi for 20 minutes.10 I Step – Fermentation using Aspergillus niger Corncob was treated with Aspergillus niger (grown in PDA Media) in aseptic condition at 30oC for 7 days. The conversion to reducing sugar due to the activities of fungi been estimated by analyzing sugars by DNS method. 1-8 II Step – Fermentation using Zymomonas mobilis - 89 Thus, fermented substrate was sterilized and dissolved in 200 ml of distilled water. Then

aliquots of different pH grades in the range of 4.5 to 7.5 were developed. To each grade the microorganisms Zymomonas mobilis- 89 9 and cultured in media were inoculated o Table 1: of 100 ml and incubation at 30oC for 48 hours. Analytical method Ethanol concentration was measured using UV- visible-double beam spectrophotometer (Double Beam, Elico company, model no Elico -SL 164). Ethanol was analyzed by the dichromate oxidation method which is based on the complete oxidation of ethanol by dichromate in acidic 1-11

Medium. Sugars are determined photometrically using dintrosalicylic acid reagent (Systronics Colorimeter: model No 115).16

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Table 1 : Description of the media used for Zymomonas mobilis- 89 No.

Microorganism Type

Media

1.

Zymomonas mobilis- 89 Yeast Extract Glucose Magnesium Chloride Ammonium Sulphate Potassiumdihydrogen Phosphate

Concentration in 100 ml (mg) 1.0 2.0 1.0 1.0 1.0

RESULTS AND DISCUSSION Table 2: Ethanol production at different pH levels in one step fermentation i.e. with Zymomonas mobilis – 89 (II Step) Sl.No. pH *Ethanol % 1 pH 4.5 0.1683 e 2 pH 5.0 0.2577 d 3 pH 5.5 0.3327 c 4 pH 6.0 0.3923 b 5 pH 6.5 0.4477 a 6 pH 7.0 0.3650 b c 7 pH 7.5 0.2287 d Note: - In each column, mean value followed by same letters are not significantly different according to DMRT at P= 0.05 Table 3: Ethanol production at different pH levels in two step fermentation i.e. with Aspergillus niger and Zymomonas mobilis – 89 (I Step and II Step) Sl.No. 1 2 3 4 5 6 7

pH pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5

*Ethanol production in (Percentage ) % 1.17 b 1.60 a 1.55 a 1.37 a b 1.24 b 1.14 b 1.14 b

Note: - In each column, mean value followed by same letters are not significantly different according to DMRT at P= 0.05 Substrate Corn Cob was treated with Chemical 967



Table 4: Ethanol production at different pH levels in two step fermentation using hydrolysed substrate i.e. with hydrolysis followed by fermentation with Aspergillus niger and Zymomonas mobilis – 89. S.No. pH *Ethanol production in (Perctnage ) % 1 pH 4.5 2.02 b 2 pH 5.0 3.02 a 3 pH 5.5 2.92 a b 4 pH 6.0 2.96 a b 5 pH 6.5 3.13 a 6 pH 7.0 2.52 a b 7 pH 7.5 2.00 b Note:- In each column, mean value followed by same letters are not significantly different according to DMRT at P= 0.05 In the previous studies (Akin-osanaiye., et al 2007) on the degradation of wood and saw dust by lentinus squarrasulsu(Mont.) resulted in 3.84 mg/ml of ethanol 11. Humprey.C et al reported carica papaya (pawpaw) agro waste treated in different treatments and saccharification yielded 5.19 (%)12 was ethanol. Sanjeev K. Sharma et al., reported that maize husks, rice husks and bitter kola pulp after acid hydrolyisis with sulfuric acid and saccharifaction with Aspergillus produced 12.9g/l of ethanol13. Mujgan Telli Okur et al., reported which fermentation of enzymatically saccharified Sunflower stalks for ethanol production was 0.444 g/g.14--15. Thippareddy et al., reported that Deseeded sunflower head used for the production of ethanol resulted 2.00 (%) 17. From the results it is evident that the two step fermentation technique can yield relatively higher quantity of ethanol compared to any other combination of treatments used in the present experiment. Further, Zymomonas mobilis is having more affinity to convert the reducing sugars to ethanol by fermentation. It was also observed that the chemically treated substrate (Corn Cob), with sodium

hydroxide, is more amenable for Aspergillus niger activities and yields more reducing sugars compared to untreated substrate. Thus enhances the available reducing sugar for Zymomonas mobilis fermentation activity to produce ethanol.

CONCLUSION Corn cob after hydrolysis in basic medium of dilute sodium hydroxide, can be subjected to two step fermentation process with Aspergillus niger and Zymomonas mobilis sequentially yields good quantity of ethanol. (3.13%)

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