ethanol from banana peels

Agricultural Wastes 16 (1986) 135 146

Ethanol from Banana Peels

H. K. Tewari, S. S. M a r w a h a & K. R u p a l

Department of Microbiology, College of Basic Sciences & Humanities, Punjab Agricultural University. Ludhiana (Pb.) 141004, India

A BSTRA CT

Comparative studies on the sacchar!Ifcation of banana peels" by acid, enzyme and steam were carried out to examine the potential of banana waste .for ethanol fermentation by Saccharomyces cerevisiae var. ellipsoideus. Hydrolysis of the substrate using 2"5% sulphuric acid at 15 psi /br 15 min enhanced the reducing sugar content more than ten-fold. The maximum saccharOqcation was 26.7% and 28.3% (wt basis) and 56.4% and 59.9% (CH20 basis) with 2.5% acid at 10 and 15psi for 15 rain. Further increases in sulphurie acid concentration and treatment duration had adverse effects on the hydrolysis. Steaming without pressure enhanced the sacchar(lqcation si.r-l~old, but steaming under pressure (at lO psi) fi)r 30 min gave better saccharification.

Maximum saccharification was achieved b), hydrolysing banana-waste cellulose with a cellulase enzyme from Trichoderma reesei QM 9414. A vield o/ l.38 and O.78% ( v/v) and44"5 and61.1% ethanol (mg g ~ reducing sugars) was achieved from cellulose and acid hydrolysed (2.5% at 15 psi.for 15 rain) banana peels, respectively.

I N T R O D U C T I O N

In India, with the alarming increase in growth rate of the population and depleted stocks of accessible reserves of coal and gas at present, it may be impossible to meet the energy requirement of the country by the end of this century. Moreover, the escalating prices of oils during the last decade are indicative of the priority need to develop an alternative

135

A~ricultural Wastes 0141-4607/86/$03.50 (!', Elsevier Applied Science Publishers Ltd. England. 1986. Printed in Great Britain

136 H. K. Tewari, S. S. Marwaha, K. Rupal

energy source to replace petroleum liquid fuels. Ethanol, in particular, has been considered as one of the most suitable substitute energy sources.

Recently, India has appreciated the importance of ethanol fermentation as a strategic matter in its economy. The ethanol can be produced from a variety of fermentable substrates by yeast culture under suitable conditions. It is estimated that the net world-wide amount of organic matter photosynthesised annually is about 1014t (Spano, 1978) and there is greater need to better utilise cellulosic wastes, after suitable treatment, for energy production. Karaki (1974) and McCann & Prince (1978) have reviewed ethanol production from various cellulosic waste materials. Recently, Tewari et al. (1982a,b, 1983, 1984) reported ethanol production from waste potatoes and groundnut shell.

The wastes from fruits also have a great potential for ethanol fermentation (Cooper, 1976). Ethanol production from apple pomace and pine- apple waste has been documented by Hang et al. (1981) and Manzola & Bartholomew (1979). Sendlewski (1980) used pumpkin as a possible substitute for potatoes and cereals as distillary raw material. The production of ethanol from banana has been reported by Pontiveros et al. (1978).

The present investigations were aimed at determining the suitability of banana peels, a waste, for ethanol production. Saccharification of the banana peels was carried out by acid, enzyme and steam to optimise the conditions of hydrolysis of the waste to reducing sugars. The saccharification material was fermented for alcohol production by Saccharomyces cerevisiae var. ellipsoideus.

METHODS

Preparation of substrate

Banana peels collected from a local market were chopped into small pieces and dried in an oven at 65°C for 48 h. The dried substrate was powdered with an electric grinder to a mesh size of 40, packed in poly- ethylene bags and stored at room temperature.

Organisms

Trichoderma reesei T. reesei QM 9414 obtained from the Army Department, US Army Natick Research Development Laboratories, Natick, USA, was used for

Ethanol J?om banana peels 137

cellulase production. The fungus was subcultured regularly on a medium containing glucose (1%), potato extract (250 g of peeled potatoes were chopped into small pieces, boiled and filtered through muslin; the filtrate was used as potato extract) and agar (2%) at fornightly intervals. The pH was adjusted to 5.0.

Preparation o f inoculum The medium containing glucose (1%), yeast extract (0-2%) and peptone (0'5%), adjusted to pH 5"0, inoculated with macerated mycelium (1% w/ v) was incubated at 28 + I 'C on a rotary shaker (220 rpm) for 3 days.

Production o f cellulase enzyme The medium of Shewale & Sadana (1978) was used for the production of cellulase by T. reesei. The medium (50ml) containing cellulose (1%) sterilised at 15 psi for 20 rain and inoculated with 2 ml of a culture of T. reesei was incubated at 28+ 1 C for 8 days on a rotary shaker at 220 rpm. The contents of the flasks were filtered through glass wool. The supernatant, containing the cellulase, was stored at 4~C.

Saccharomyces cerevisiae S. cerevisiae var. ellipsoideus obtained from the Department of Micro- biology, Punjab Agricultural University, Ludhiana, was maintained on glucose yeast-extract (GYE) medium containing glucose (1%), yeast extract (0"5%), peptone (0.5%) and agar (2%). The pH of the medium was adjusted to 5.0. The culture was stored in a refrigerator and subcultured at 15-day intervals.

The inoculum was prepared in 100ml of GYE medium, in 250ml capacity conical flasks, by transferring a loopful of yeast cells from a 24 h, actively growing, culture. The culture was incubated at 27 + 1 C for 20, h on a rotary shaker at 200 rpm.

E~'hyl alcohol fermentation The 250 ml capacity conical flasks containing 150 ml of saccharified and sterilised substrate were inoculated with 3% (v/v) yeast inoculum. The flasks were incubated at 30 + 1 C .

Saccharification of banana peel

Acid Peels were hydrolysed at 10 and 15psi for 15, 30, 45 and 60rain using


sulphuric acid at concentration ranging from 2-5 to 15.0%. To 1-0g of powdered peels, 8 ml of sulphuric acid was added. The acid hydrolysates were cooled to room temperature and diluted to 200ml. The solution was neutralised with sodium hydroxide (5% w/v).

Autoclaving To 1-0 g of powdered peels, 8 ml of distilled water was added and the resultant solution saccharified by autoclaving at 10 and 15 psi for 15, 30, 45 and 60 min in 250 ml capacity conical flasks.

Enzyme Fifteen millilitres of culture filtrate from T. reesei (as above) and 50 ml of citrate buffer (0.1 M, pH 4.8) were added to 5 g of powdered peels and incubated at 50 4- I°C for 72 h with shaking (220 rpm) in a water bath. The reducing sugars liberated were estimated using 3, 5 dinitrosalicylic acid (DNS) (Miller, 1959)) and the saccharification was calculated following Dhawan & Gupta (1977) as a percentage by weight.

Analysis

Carboxymethyl cellulase (CMC) activity The CMC activity was determined according to the procedure of Mandels & Sternberg (1976). The sugar liberated by the action of enzyme on substrate was estimated as Miller (1959).

Filter paper cellulase activity (FPA) The FP cellulase activity was measured according to Mandels & Stern- berg (1976) using Whatman No. 1 filter paper and citrate buffer (0.I M, pH 4.8).

Cellobiase activity The cellobiase activity was assayed by the method of Toyama & Ogawa (1977).

The unit of each of the above enzymes was defined as the amount of enzyme needed to liberate reducing sugars equivalent to l#mole min - 1 m g - 1 of substrate.

Proteins The method of Lowery et al. (1951) was followed.

Ethanol Jrom banana peels 139

Reducing sugars These were estimated according to the procedure of Miller (1959) using 3, 5 dinitrosalicylic acid (DNS). The absorbance was measured on a Bausch and Lomb spectronic-20 spectrophotometer at 575 nm.

Starch Starch in the substrate was determined according to Clegg (1956) using anthrone reagent. Absorbance was recorded at 630 nm.

Cellulose The procedure of Updegraff (1969) using anthrone reagent was employed. The absorbance was recorded at 620 rim.

Ethyl alcohol The method of Caputi and Wright (1969) using steam distillation of alcohol in a Microkjeldahl apparatus was followed.

RESULTS AND DISCUSSION

The peels contained 31, 205 and 255mgg -1 peels of reducing sugars, starch and cellulose, respectively. To carry out alcoholic fermentation, saccharification (can be achieved either by acid, steam or enzymic treatment) of the cellulose substrates is an essential step. However, there is scanty information available on the saccharification of fruit wastes.

The results of saccharification of the substrate with sulphuric acid at 10 psi are shown in Table 1. The reducing sugars produced ranged from 86.0 to 180.5mgg -~ and the saccharification of substrate from 7-9 to 16.7% (weight basis) and 17.0 to 35-2% (CH20 basis) when the acid treatment was carried out without subjecting the substrate to pressure and with immediate neutralisation (0 rain, Table 1). However, for treatment carried out with autoclaving the maximum saccharification was attained with 2.5% HzSO 4 and 15rain autoclaving and thereafter saccharification decreased both with increased autoclaving time and acid concentration.

The results of saccharification of banana peels using sulphuric acid at 1.5 psi are shown in Table 2. The maximum reducing sugar and sacchari-


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fication was achieved with 2"5% H 2 S O 4 autoclaved for 15min. Ponti- veros et al. (1978) achieved 55.8% saccharification of banana fruit pulp and peels with 2% H2SO 4 at 24psi for 30min. Other workers (Evack- lund et al. (1976) and Tewari et al. (1983)) have reported variable levels of saccharification of other cellulosic materials (sunflower seed husk and waste potatoes) using 3.4 and 4% H2SO 4 at 120 and 63°C for variable time intervals. The comparative studies on acid hydrolysis of cellulosic wastes by Taso (1979) have shown that sulphuric acid resulted in better saccharification than did HC1.

The reducing sugars liberated on saccharification of peels without acid treatment (0%) (Tables 1 and 2) may be mainly due to hydrolysis of starch and small amounts of cellulose or hemicellulose.

Saccharification of banana peels with eellulase

Trichoderma reesei QM 9414 grown as in 'Methods' yielded 2-40,.0.96 and 0-11 units of CMC, FPA and cellobiase activities ml - ~, respectively.

TABLE 3 Enzymatic Saccharification of Banana Peels

Incubation period Reducing sugars ~ Saccharification b (h) (mgg 1) (%)

24 131 51-3 48 203 79.6 72 224 87"8

aInitial reducing sugars: 31 mg g- 1. bSaccharification %: on cellulose basis.

The maximum CMC activity (2.40 units ml - 1) was achieved after 6 days incubation, whereas the highest FPA and cellobiase activity values were achieved after 8 days incubation. Thereafter, a decrease in activities was observed. The culture filtrate of T. reesei was incubated with substrate at 50°C for 72h and a maximum saccharification of 87.8% (on cellulose basis) was observed (Table 3). Chahal et al. (1981a,b) have also achieved 80-95% hydrolysis of exploded wood with a culture filtrate of T. reesei.

Ethanol from banana peels 143

Saecharification by steam

The maximum saccharification (36.5%) was attained after 45min of steaming of the substrate (Table 4). However, saccharification was less efficient than when carried out with autoclaving at 10 and 15 psi (Tables 1 and 2) without acid treatment of the substrate. Similar observations were made by Wayman (1980) on the saccharification of cellulosic wastes.

TABLE 4 Effect of Hea t ing on Saccharif icat ion of Banana Peels a

Time Steaming (rain)

Reducing Saecharification c sugars b (%)

(mg g- 1)

Wt basis CH,O basis

0 30 --- - - 15 179 16.6 34-9 30 181 16.8 35.4 45 187 17.3 36.5 60 187 17.3 36.5

React ion mixture = 1 "0 g substrate , 8 ml distilled water. bReducing sugars (mg g ~ )= to t a l reducing s u g a r s - initial reducing sugars. c% Sacchar i f ica t ion--see Table 1.

Ethanol fermentation

The results in Table 5 show the ethanol production efficiency of both enzymatically and acid hydrolysed banana peels. The low conversion efficiency of acid hydrolysed banana peels recorded may be assigned to the formation of furfural and methylfurfural or presence of excess of SO 4 ions which inhibit ethanol production by yeast, as documented by Banerjee & Vishwanathan (1974).

From the present results on the saccharification of banana peels by cellulase and the efficiency of S.cerevisiae var. ellipsoideus in converting hydrolysed banana peels to ethanol, a yield of 150 ml of alcohol kg- ~ of


TABLE 5 Production of Ethyl Alcohol from Banana Peels ~

Observations Saccharifying agent

H2S04 (2.5%) Cellulase ~

Reducing sugars 3.10 1.28 (g( 100 ml)- ') Alcohol % (v/v):

24h 1.38 0.78 48h 1.35 0.77

Alcohol yield (ml(100g reducing sugars) l)

24h 44.5 61-1 48 h 43"5 60"0

"Fermentation period: 24 and 48 h. Temperature: 30°C. pH: 4.5. bCellulase: crude filtrate of 7". reesei.

banana peels can be obtained. This is three times the yield of 43 g of ethanol kg -1 of apple powder recorded by Hang et al. (1981). Goewert & Nicholas (1980) have also reported the suitability of banana peels for alcohol fermentation since after enzyme treatment they contain sugars suitable for growth and alcohol production by yeast.

R E F E R E N C E S

Banerjee, N. & Vishwanathan, L. (1974). Effect of furfural and 5-hydroxy methyl furfural on growth and alcohol production by yeast. Proc. 40th Ann. Cony. Sugartechn., 1974, pp. 1-4.

Caputi, A., Jr. & Wright, D. (1969). Collaborative study of the determination of ethanol in wine by chemical oxidation. J. Assoc. Of f Anal, Chem., 52(1), 85 8.

Chahal, D. S., McGuire, S., Pikor, H. & Noble, G. (1981a). Production ofcellu- lase complex by T. reesei Rut C-30 on lignocellulose and its hydrolytic potential. Paper presented at the symposium Fuel and Chemicals f rom Bio- mass, American Society Annual Meeting, New York, August 1981.

Chahal, D. S., McGuire, S., Pikor, H. & Noble, G. (1981b). Iotech process for cellulase production by T. reesei on lignocellulose and its hydrolytic potential. Abstr. of communications, Second European Congress of Biotech- nology, Eastbourne, England, p. 217.

Ethanol Jrom banana peels 145

Clegg, K. M. (1956). The application of starch in cereals. J. Sci. Food Agri., 7, 40.

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Tewari, H. K., Singh, L. & Sethi, R. P. (1982b). A role on utilization of waste potatoes for the production of ethanol. The Punjab Horticultural Journal, 22 (July-December), 214-16.

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ethanol from banana peels

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