Ethanol is a clean burning, renewable resource that can be produced from fermentation of glucose rich substrates (Yu and Zhang, 2004). In many parts of the world, demand for ethanol as an alternative fuel source has steadily increased (Sheoran et al., 1998) due to efforts in decreasing the overall amount of greenhouse gases emitted into the atmosphere (Martin et al., 2002), dwindling fossil fuel resources, (Yu and Zhang, 2004) and increased gasoline prices. Since 1970s, it has become clear that availability of domestic natural gas and petroleum cannot meet the growing demand for these energy sources. Therefore, there has been serious concern for developing renewable energy sources in an effort to ease the severity of the expected shortage. One possibility is the conversion of waste or grown organic matter into liquid and gaseous fuels (Graham et al., 1976). Currently, the United States produces approximately three billion gallons of ethanol from corn annually (Potera, 2004).
Mango (Mangifera indica) is the most important fruit crop of the country,
India occupying about 60% of the total area under fruits. The total area under
mango is estimated at 2 million acres. It has been cultivated in India for at
least 4,000 years. The mango fruit is a large drupe exceedingly variable in
form and size, soft or juicy; sub acid or sweet with rich in aromatics and the
seed is solitary, ovoid-oblique encased in hard compressed fibrous endocarp
(stone). It has rich luscious, aromatic flavor and a delicious taste in which
sweetness and acidity delightfully blended. It contains good amount of sugar
(16-18% w/v) and many numbers of organic acids and also a good antioxidant,
carotene (as Vitamin A, 4,800 IU). Some of the varieties are very rich in sugar,
it s recorded as 20.5% in Nilambari. Sucrose, glucose and fructose are the principle
carbohydrates in ripe mango with small amount of cellulose, hemicellulose and
pectin. The green tender fruits are rich in starch, during ripening the starch
which is present is hydrolyzed into reducing sugars(Anonymous, 1962). The unripe
fruit contains citric acid, malic acid, oxalic acid and succinic and other organic
acids. In ripun fruits the main acid source is malic acid (Giri et al.,
In India, an effort to better appreciation of food processing industries, the nodal ministry, Ministry of food processing (MEPI) suggests alcoholic drink from non-molasses. Waste mango is defined as fruit that does not meet domestic and external organoleptic standards for acceptability on the international market. Waste mangoes include undersized, those with spotty or marked peel, or those that have suffered mechanical damage. Mangoes grown for export are typically harvested and transported to a packing shed for grading. The grading rejects then require disposal. Because they have already been collected and brought to a centralized location, the waste mangoes are low-cost, concentrated biomass feedstock. Such mangoes can be used as raw material in the generation of byproducts with increased added value, thus allowing farmers to diversify their harvests and receive higher income. In this context attempts for utilization of the surplus of mango fruits which are unattractive and of small size several applications have been studied, in which one of the possibility is ethanol production.
In the present investigation, the suitability of waste mango fruit juice for ethanol production was studied. We concentrated mainly on maximize the high quality juice extracted by improving the extracting techniques and optimize the fermentation process to improve the volumetric productivity of ethanol.
MATERIALS AND METHODS
Six varieties of mangoes were selected from the available ones grown in
the region of south India, Table 1. The mangoes are purchased
from local market (in the year of 2006) of Tirupati Andhra Pradesh, India.
Mangoes were washed with 1% HCl water and peeled off manually. The pulp was
divided into two portions. The first portion was left as control and the subsequent
samples of the second portion were treated with various concentrations of Trizyme
P50 pectinolytic enzyme (procured from Triton Chemicals Mysore), in order to
determine the most suitable and effective amount of enzyme and treatment time.
In all the above treatments 200 mL of pulp in 500 mL conical flask was placed
on a rotary shaker for intimate mixing.
|| Chemical characteristics of juice produced from different
|* Values presented is mean of triplicate experiments
|| Physical characteristics of wine produced from different
|*Values presented is mean of triplicate experiments
Juice extraction was made by pressing the treated pulp in cheese cloth and
the yields of juice were compared. The juice obtained in this manner was then
subjected for the analysis of total and reducing sugars, total acidity, pH and
soluble solids content. None of the varieties was ameliorated with sucrose.
The ethanol producing Saccharomyces cerevisiae 101 (wine strain)
obtained from CFTRI Mysore was used in the experiments. The culture was maintained
on MPYD (Malt extract 3 g L-1, Peptone 5 g L-1, Yeast
extract 3 g L-1 and Dextrose 20 g L-1 and agar 15 g L-1)
slants at 4°C. The inoculum was prepared by inoculating the slant culture
into 25 mL of the sterile MPYD liquid medium taken in 100 mL flask and growing
it on a rotary shaker (100 rpm) for 48 h. This inoculum (3x106 cells
mL-1) was transferred to 250 mL conical flask having 100 mL sterile
mango must (juice).
Batch fermentation of the inoculated must was carried out in a number of
flasks in order to determine the optimum pH and temperature by incubating at
different pH (3.5, 4, 5 and 6) and at different temperatures (20, 25 and 35°C),
respectively over a period of 3 days. The samples were collected by separation
of the cells by centrifugation at 5,000 g for 10 min. The samples were kept
at 20°C until analysis.
Sugar concentration was estimated by Shaffer and Somogyi (1995) method.
Sucrose was estimated by Dubios (1956) method Total dissolved solids were measured
by estimating specific gravity of water soluble portion of the mash obtained
by the centrifugation 10,500 x g for 15 min. The specific gravity was determined
at 20°C with Densitometer. With the aid of approximate tables the results
were converted to grams of dissolved solids per 100 mL and expressed as grams
Types of Sugars
The sugar types that are present in mango pulp were determined by paper
chromatography according to Trevelyan et al. (1950). Sugar spots were
detected with alkaline silver nitrate reagent. Mean of triplicate experiments
Ethanol and Other Volatile Compounds
Ethanol was determined with the help of Gas Chromatography (Antony, 1984).
Total acidity was determined by neutralization with 0.1 N NaOH expressed in
tartaric acid and volatile acidity within the distillate samples expressed in
acetic acid Mg 100 mL-1.
RESULTS AND DISCUSSION
Compared to the non- enzymatic method, the enzymatic extraction of pulp gave
higher quantity of juice. The effects of concentration of enzyme and incubation
period were shown in Fig. 1a and b. A maximum yield of juice
from the mango pulp was obtained in case of 0.6% v/v of pectinolytic enzyme
with10 hour treatment period. These results were in accordance with previous
investigations relate to wine and apple cider production. It is well known that
pectinase degrade the pectin and liberates more juice from pulpy fruits like
mango and banana (Joshi and Bhutani, 1991). In addition to this pectinase treatment
decrease the viscosity of the juice and increase the fermentation rate during
ethanol fermentation (Joshi and Bhutani, 1991). The total sugar present in the
different mango varieties varied according to the variety and ranges from 16-18%
(w/v) (Table 1a). Sugar content is different from different
variety of fruits and mainly depends up on the soil type and irrigation levels
(Kulkarni et al., 1980).
||Optimization of pectinase enzyme concentration for maximum
recovery of juice from mango pulp and (b) Optimization of pectinase enzyme
incubation time for maximum recovery of juice from mango pulp
With the aid of paper chromatography the sugar types were identified, mango
contained only 3 types of sugars, namely glucose fructose and sucrose, the major
part of sugar being sucrose (Fig. 2). The main sugar from
all the fruits is sucrose and followed by glucose and fructose. In mango fruits
fructose is in high content s compared to glucose, although the starch is the
main carbohydrate at unripe stage. Starch and sucrose are degrading to glucose
and fructose during ripening of the fruit. Due to the good amount of reducing
sugars available from the mango fruits it is possible to produce good amount
of ethanol by economically cheap through mango juice fermentation. In general
the sugar concentration from 15 to 20% (w/v) is preferable in industrial ethanol
productions. It has been proved that the high substrate content during ethanol
production slowdown the fermentation rate due to the increased osmotic stress
(Reddy and Reddy, 2005).
The pH of the six selected mango juice varieties is between 3.8-4.5. The low
pH 3.8 was found in Neelam (Table 1a). The physico-chemical
characteristics of mango juice after the fermentation were as shown in Table
1b. The percentage of alcohol was between 7-8.5% (w/v). The ethanol produced
from mango juice fermentation is near to the industrial level (Hammond et
al., 1996). The ethanol percentage mainly depending on sugar which is present
in the juice we used. All the sugar available was used by the organism and very
little residual sugar was remained in the media while completed the fermentation.
These results suggest that there are no inhibitory components in the mango juice
which is one of the advantages for ethanol production.
|| Chromatogram showing types of sugars present in mango fruit
|| Effect of pH on ethanol production from mango pulp
|| Effect of temperature on ethanol production from mango pulp
The pH of the produced wine had slightly increased when compared with the
mango must. This increment in pH after fermentation may be because of yeast
metabolites which are released into the medium during the fermentation. The
culture conditions like pH, temperature and inoculum size were optimized. The
effects of pH and temperature on ethanol production from mango juice were showed
in Fig. 3a and 3b. From the Fig.
3a and b it is clear that at pH 5.0 and temperature 30°C
ethanol production was high and also optimum conditions for mango juice fermentation.
The optimum pH of ethanol fermentation is very close the mango juice pH. In
addition to this, optimum pH is also near to the room temperature so need to
adjust the pH and special care for temperature maintenance. None of the varieties
were supplemented with nitrogenous sources like yeast extract, peptone and malt
extract. Mango juice contains around 0.6% proteins (data was not presented)
that only utilized by the used yeast, S. cerevisiae 101. This property
is very advantageous and intern it could reduce the ethanol production cost
from mango juice.
Around 2 kg of mangoes are requires for 1 L juice. The waste mangoes are
freely available we expend only transport charges to gather at one place and
the processing cost. Hence we calculated approximately 0.5 $ (US) is enough
per liter ethanol. However scale-up studies need to be carried out for knowing
concrete cost value of ethanol from mango juice.
Pectinolytic enzyme treatment is required to obtain high yield of juice from mango pulp. Fermentability also increased significantly with pectinase treatment. Banginapally variety is giving high ethanol compared to all other varieties. Fermentation conditions are also optimized and the selected yeast stain, Sachharomyces cerevisiae 101 is very much suitable for ethanol production from mango fruit juice. Using waste mangoes for ethanol production appears very promising. Ethanol yield (on a dry weight basis) from mangoes is higher than from most other agricultural wastes. The mangoes which, currently treated as a waste and as such, represent a very low cost feedstock. They ferment well and have a high carbohydrate concentration. Waste mangoes are already collected at packing areas and therefore transportation/harvesting costs will be minimal. No need of water for dilution and fermentation results in some what high concentration beer that would minimize distillation energy costs. Finally we conclude that mangoes are very important choicest fruits in India and an attempt to use this fruit in ethanol production would contribute considerably to the economy of the Indian as well as international mango producers.