Professional Documents
Culture Documents
1987,40,213-218
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
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.
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).
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)
~~ -
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
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
REFERENCES