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J . Sci. Food Agric.

1987,40,213-218

Ethanol Production by Fermentation of Fruits and


Cladodes of Prickly Pear Cactus [Opuntiaficus-indica
(L.) Miller]

Norma Retamal," Jose M. Duran" and Jesus Fernandezb


"Department of Plant Physiology, and bDepartment of Botany, Escuela T.S. Ingenieros
Agr6nomos, Universidad Politecnica de Madrid, Ciudad Universitaria, 28040-Madrid,
Spain

(Received 14 November 1986; revised version received 6 February 1987;


accepted 13 February 1987)

ABSTRACT

Using prickly pear cladodes and fruits and different yeast strains of the
genus Saccharomyces, both the chemical composition of the plant material
and alcoholic fermentation were studied under controlled conditions.
Before fermentation, fresh or previously dried cladodes were hydrolysed
using cellulase or acid (HCl). The yield was determined by measurement of
ethanol production by gas-liquid chromatography. Two conversion
indices, representing either the percentage of reducing sugars or the energy
converted into ethanol, were considered. Using fresh or dried cladodes
without fruits, the best results were always obtained after performing both
types of hydrolysis. The enzymic method gave the highest sugar yield.
However, ethanol production was similar to that obtained by acid hydro-
lysis of fresh cladodes, and only slightly higher than that of previously
dried cladodes. The potential ethanol production that could be obtained
from prickly pear cultivation in various regions (arid, semi-arid and
irrigated) is discussed.
Key words: Biomass, energy, ethanol, fermentation, fuels, Opuntia,
prickly pear.

1 INTRODUCTION

The technology for large-scale production of ethanol is available. However,


conversion costs vary greatly depending on the feedstocks and processes used.
Five major feedstocks are available for ethanol production: (1) sugar from cane,
213
1. Sci. Food Agric. 0022-5142/87/$03.500 Society of Chemical Industry, 1987. Printed in Great
Britain
214 N . Retamal, J . M. Durun, J . Fernandez

beet or sorghum; (2) starch from corn (maize), wheat, barley and other cereals;
(3) cellulosic materials from wood and wastes; (4) carbohydrate by-products from
processing sulphite liquor wastes, whey or food industry wastes; and ( 5 ) inulin
from chicory, artichoke or other crops.
An alternative to these widely discussed sources is Opuntiaficus-indica, a CAM
(Crassulacean Acid Metabolism) plant ,* whose cultivation has been suggested for
arid lands because of its special adaptive mechanism and capacity to produce
bi~mass.~
The chemical composition of prickly pear fruit has been studied previously,
mainly for its possibilities in human nutrition." In order to better understand
CAM in cacti, the organic acids of a few Opuntia species have been investigated
by several investigators.'.* However, the chemical composition of the cladodes
(modified stems in cacti species) is not well-known, and its possible seasonal
variations have only recently been r e p ~ r t e d . ~
The release of monomers from complex polysaccharides is the key step in their
utilisation as a fermentation substrate for alcohol production. Consequently,
improved hydrolysis technologies are needed.

2 MATERIAL AND METHODS

Prickly pear (Opuntia ficus-indica) fruits and cladodes (pads, modified stems in
cacti) with fruits were collected from fieldgrown plants in Pelahustan (Toledo,
Spain). The fresh tissue was desiccated for 48 h in a drying oven (60C) with a cold
air current. When dried, it was ground with a hammer mill (Culati, model
DFH 48) and desiccated at 130C to a constant weight. With the exception of
moisture content, the chemical analyses were performed on dry material.
Fats (F, g 100 g-I) were extracted with diethyl ether (b.p. 35C) using a
Soxhlet-type extractor with a distillation speed of 4-5 drops s-l, for 4 h. Nitrogen
determination by semi-micro Kjeldahl method as outlined by the AOAC'O was
converted to crude protein (P, g 100 g-I) using the factor 6.25. Heat energy (HE,
kJ kg-I) was determined according to Maynard and Loosli,ll using a Parr oxygen
calorimeter. Carbohydrates (C, g 100 g-I) were determined according to
Maynard and Looslill using the following formula: HE=9.15 C+9.40 F+5.65 P.
Minerals (M, g 100 g-I) were determined as: M=lW-(C+F+P).
Before alcoholic fermentation, the dried or fresh cladodes (with or without
fruits) were hydrolysed by one or both of the following hydrolysis procedures: (a)
with cellulase E C 3.2.1.4 (Merck, 20 mU mg-I) at 47"C, p H 4.5, during 4 h; and
(b) with 1 N HCI at 100C for 30 min in a shaking bath. When both hydrolyses
were performed, the cellulase hydrolysis always preceded the acid hydrolysis.
The reducing sugars present in the fermentable juice before (S,) and after (S,)
alcoholic fermentation were determined using the methods described by NelsonL2
and Somogyi.I7
Alcoholic fermentation was carried out with 50 ml of fermentable liquid,
pH 3.8, at 25C in 100 ml Erlenmeyer flasks. Three strains of Saccharomyces
cerevisiae were compared: S . cerevisiae (No. 234, Reg. 3 ) , S. cerevisiae var.
Ethanol production by fermentation of prickly pear 215

ellipsoideus (No. 94, Reg. 8) and S. cerevisiae in commercial form. All strains
were obtained from the collection of the Department of Viticulture and Enology
of the Instituto Nacional de Investigaciones Agrarias (El Encin, Alcala de
Henares, Spain).
Ethanol (E, ml 100 ml-I) was determined by gas-liquid chromatography
(g.1.c.) according to the Stackler and Christensen method,14 and a minimum of
three replicates was used in each determination. Two indices were used to
evaluate the fermentation yield: sugar conversion [100 (Sl-S2)/Sl], and energy
conversion from the heat energy contained in the plant material (PM) to the heat
energy accumulated in the ethanol (ET) obtained by fermentation (100 ET/PM).
The energy from the ethanol was calculated according to the caloric power of the
absolute ethanol (7100 kcal kg-lx0.789 kg litre-l-5 600 kcal litre-I).

3 RESULTS AND DISCUSSION

Fresh cladodes exhibit a high moisture content (92%) (Table 1). This can be
explained by the fact that it is a CAM species2J5with stem tissues containing
numerous mucilaginous cells that store a large volume of water.16 From the dry
material examined, the most important fraction corresponded to the carbohy-
drates (63.9% in cladodes and 91.4% in fruits). The lipid fraction was the least
important (2.6% in cladodes and 1.8% in fruits). In dry cladodes, the mineral
fraction, determined as ash (23.5%), was more significant than the protein
fraction (10-0%),though the latter in dry fruit (443%) was higher than the mineral
content (2.0%). The heat energy was 1.22 times higher in the dry fruits than in the
dry cladodes.
As regards the final production of ethanol, the three Saccharomyces strains
used (Table 2) showed similar conversion capacities. Although the final concen-
tration of ethanol in the fermentation medium was slightly lower when using the
commercial strain (1.13 ml 100 ml-I) as opposed to the two wild strains (1.26 ml
100 rnl-l), the sugar conversion was very similar and even slightly higher in the
commercial strain (89%) than in the wild strains (86%). Considering the easier

TABLE 1
Chemical Composition of Prickly Pear (Opuntia ficus-indicu) Parts
Plant material Composition (g 100 g-1)
Water Carbo- Fats Proteins Minerals
(W) hydrates (F) (8 (M)
(C)
Fresh cladodes 5.1 0.2 0.8 1.9 1155
Dry matter from 63.9 2.6 10.0 23.5 14484
cladodes
Fresh cladodes+ 86.0 11.7 0.3 0.8 1.2 2339
fruit (1:l. w/w)
Fresh fruit 80.0 18.3 0.4 0.9 0.4 3 549
Dry fruit - 91.4 1.8 4.8 2.0 17719
216 N . Retamal, J . M . Duran, J . Ferncindez

TABLE 2
Comparison of Different Yeast Strains of Saccharomyces for Ethanol
Production (X-t SE) by Fermentation of Prickly Pear (Opuntia ficus-indica)
Cladodes
Yeast struins Ethanol Sugar
(ml100 ml-I) conversion
(%o)
~~ -

S . cerevisiae (No. 234, Reg. 3) 1.26"+0.05 86


S. cerevisiae var. ellipsoideus 1.26"k0.03 86
(No. 94. Reg. 8)
S . cerevisiae (commercial form) 1-13+0.05 89
The average values with the same superscript are not significantly different
(PSO.05).

access to the commercial strain and the results shown in Table 2, it seems logical to
recommend the use of commercial yeast found in the market, as opposed to other
strains that are more difficult to obtain.
The results from Table 3 show that ethanol production from fresh or previously
dried 0. ficus-indica cladodes is possible. Whereas the fruits (either fresh or
previously dried) can be directly submitted to alcoholic fermentation without
previous hydrolysis, the cladode carbohydrates must first be saccharified by some
type of hydrolysis. Of the two types of hydrolysis studied, the acid hydrolysis
(1 N HCI, 100C, 30 min) seems more convenient when fermenting fresh
cladodes, and the cellulase hydrolysis is suggested for the previously dried
cladodes. As expected, enzymic hydrolysis followed by acid hydrolysis consider-
ably improved the yield obtained over either of the hydrolyses alone. Neither of
them is capable of extracting the total amount of carbohydrates present initially.
According to different authors, to obtain a suitable yield in the conversion of

TABLE 3
Ethanol Production and Conversion Indices by Fermentation of Cladodes and Fruits of
Prickly Pear (Opuntia ficus-indica)
Plant material Hydrolysis Ethanol Conversion (%)
ml100 ml-' litres t-' Sugars Energy
Cellulase 0-65 5.20 62 11
Fresh cladodes Acid 0-71 5.68 57 12
Cellulase+acid 0.86 8.87 68 18
Cellulase 1-52 76.00 65 12
Dry matter Acid 1-55 71.00 59 11
from 'ladodes 1-77 88.50 61 14
Fresh cladodes+ Cellulase+acid 2.57 26.60 96 21
fruit
Fresh fruit Without 5.45 47.85 99 32
Dry fruit Without 4-93 24630 90 33
Ethanol production b y fermentation of prickly pear 217

biomass to alcohol, a final concentration of 5-10 ml ethanol per 100 ml solution is


necessary. We have shown that in Opuntia it is only possible if using fresh or dry
fruits.
Mechanical fruit harvesting is possible but in this case parts of cladodes will be
unavoidably introduced into the fermentative juice. When the cladodes are
combined with fruits (Table 3), the yield in ethanol is significantly reduced, and
previous hydrolysis is necessary. Actually, the acid hydrolysis is economically
more convenient and as efficient as the enzymic one. If cellulase hydrolysis is
performed, 0.1 g of protein extract (20 mU mg-l, EC 3.2.1.4) per 100 g of initial
dry matter is recommended.
Presently, it is not understood how biomass production from the prickly pear
can be obtained. According to Acevedo et al.,l7 normal prickly pear cultivation
found in Til-Til, a semi-arid region in northern Chile, produces a biomass of 4.2
and 1.26 kg (d.m.) plant-' of cladodes and fruits, respectively. Taking as
reference the biomass production calculated by Pinto and Acevedo,18estimating
the number of plants cultivated per hectare between 625 and 5000 in arid or
irrigated areas respectively, and considering the alcoholic fermentation yields
indicated in Table 2, we conclude that 300 (arid region) to 3000 (irrigated region)
litres of ethanol year-' could be obtained per hectare of Opunfiucrops (Table 4).
Although in irrigated regions other crops such as potatoes, beet, sweet corn, etc.
may be more profitable than prickly pear for ethanol production, prickly pear
cultivation could be advantageous in arid and semi-arid regions, thus making use
of an important area of land that hitherto has been dismissed as unusable. To
evaluate the potential for biomass production and its conversion into energy
(fuels or similar) in Mediterranean regions, several experimental plots have been
established in the last five years in Almeria and Jerez de la Frontera (Cadiz) in
Spain. The aim of this experiment is to determine the optimal area of prickly pear
crop necessary to supply an industrial fermentation plant processing 50 000 litres
day-' of ethanol for 5 months of the year. Assuming that the plant is located in the

TABLE 4
Theoretical Ethanol Production under Different Conditions of Land Use
Production Regions
Arid Semi-arid Irrigated
Plant material
Size (mxm) 4x4 2x2 2x 1
Plants ha-l 625 2 500 5 000
Biomass (t ha-')
Cladodes 2-63 10.50 21*00
Fruit 0-79 3-15 6.30
Ethanol (litres ha-' )
Cladodes 187 746 1491
Fruit 195 776 1553
Cladodes+fruit 382 1522 3 044
218 N . Reramal. J . M. Duran. J . Fernundez

centre of the area of cultivation a n d a production of 1500 litres of ethanol per


hectare, t h e theoretical radius of t h e cultivated land w o d d be approximately
4 km.

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