Bioethanol Production from Prickly Pear (Opuntia ficus-indica (L) Mill.) Cladodes

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BIOETHANOL PRODUCTION FROM PRICKLY PEAR (OPUNTIA FICUS-INDICA (L) MILL.) CLADODES
F. Sánchez
(1)
, M.D. Curt
(1)
, J. Fernández
(1)
, J.M. Agüera
(2)
, M. Uceda
(2)
, G. Zaragoza
(2)
.
(1)
Dpt. Producción Vegetal: Botánica y Protección Vegetal. Universidad Politécnica de Madrid (UPM). 28040 Madrid (Spain)
Telephone nº: +34915492692. Fax nº: +34915498482. E-mail address: j.fernandez@upm.es.
(2)
Fundación CAJAMAR. Paseo de Almería 25, 2ª planta. (04001). Almería (Spain)
Telephone nº: +34950210189. Fax nº: +34950621660. E-mail address: fundacion@fundacioncajamar.com
ABSTRACT: The aim of the work is to find out the alcoholigenous potential of non-cellulosic carbohydrates from
prickly pear cladodes by fermentation with the yeast Saccharomyces cerevisiae (commercial strains). Different
hydrolysis pretreatments and process conditions are carried out in order to determine the best procedure for a
maximum ethanol concentration in the fermenting media.
Keywords: bioethanol, fermentation, hydrolysis, sugar crops.
1 INTRODUCTION
Prickly pear cactus is a crop cultivated mainly for its
fruits (pears) that are sold fresh or transformed in jellies,
juices and other products. Young stems (cladodes) are
also sold as vegetables in some world regions (mainly in
Mexico). Older cladodes, particularly those from
varieties without or with few or small thorns, can also be
used as fodder, and there are several other minor uses of
its fruits and cladodes, including medicinal and cosmetic
ones [1], [2].
Prickly pear is a crop that can be cultivated in arid
environments due to its CAM metabolism, high water
retention capability, and other cactaceae family
adaptative strategies. Cladode yields depend very much
on crop management as it is not frequently that crop
operations such as fertilizations or weedings are done
when cladodes (instead of pears) are the harvestable
product. As a few examples, values over 100 tn/(ha.year)
of fresh cladode can be considered for rainfed lands with
Opuntia ssp. plants with five years or more in central
Mexico [3]. In Brasil, Cordeiro et al [4] reported values
betw
een 22 and 50tn /(ha.year) for two different varieties
of O. ficus-indica in rainfed semiarid zones with an
adequate land management. Finally, yields of 3 – 9 and
15 – 22.5 tn/(ha.year) of dry matter have been reported
for O. ficus-indica cultivated in deep and sandy soils in
areas with 200 and 400 mm of rain, respectively, and an
adequate land management [5].
Due to its high content of carbohydrates (about 30%
d.m.b, not including the holocelullosic fraction) and low
content of lignin (<4%, d.m.b. [6]) cladodes can be
considered as an interesting bioethanol feedstock in arid
and semiarid regions.
Initially, ethanol production from prickly pear can
be consider as a high valuable by-product of crops mainly
destined to food production, or as the main product in
crops specifically destined to fuel production
(considering prickly pear as a so-called energy crop).
First option can be considered in regions where: A
significant demand of pears (or young cladodes) exists,
large areas of land are cultivated with this plant, and the
crop can be considered as a profitable one. Here, pruning
residues and those fruits that do not fit the market
standards (because of being: damage, too or not enough
mature, etc) can be used to produce ethanol. On the other
hand, there are several other regions where prickly pear is
cultivated but it is not, actually, a profitable crop. The
main reason for this is the small amount of demand of the
produ
cts and/or the high cost of harvesting them (as this,
in the case of fruits, must be done manually, nowadays).
In these regions (and in those where prickly pear could be
cultivated but it is not, for the same mentioned reasons)
crops could be grown specifically for fuel production. If
cladodes,–besides fruits-, can be processed to obtain
ethanol, as previously reported [7], mechanization of the
harvest will lead to reduce its costs, and, as demand for
feedstock would be steady, the crop could be profitable in
this areas.
2 MATERIALS AND METHODS
A selection of average prickly pear mature cladodes
from a spineless ecotype was picked up in September of
2008 from an experimental plot located in Almeria
(Spain). (Fig.1)
Figure 1: Picking up cladodes at the experimental plot
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Cladodes were then grinded, mixed, and frozen at
-18ºC in sampling bags until they were needed.
Total sugars (excluding those contained in
hemicellulose and cellulose) were determined by Nelson-
Somoghy method (as described in [7]) after hydrolyzing
fresh cladode with hydrochloric acid 1M (30 min).
Two kinds of different fermentative substrates were
prepared from the cladode mix. First of them (S1) was
obtained by hydrolyzing cladodes with boiling
hydrochloric acid 1M for 30 min, following the same
method used for sugar determination. Amounts of about
30 g of fresh cladode were boiled in aproximately 400 ml
of hydrochloric acid. Once filtration has been done, this
method provides us a liquid with almost all cladode sugar
content, but with a low concentration of sugar, because of
the low fresh cladode: acid solution ratio.
The other substrate (S2) was obtained by
hydrolyzing the cladode mix with concentrated sulphuric
acid (96%) in very small amounts (Table II), and in an
autoclave (121ºC, 20 min). Water was previously added
in o
rder to obtain and homogeneous and easily mixable
substrate (0.84 ml for each fresh cladode gram, as
determined in previous experiments). As sterilization of
the substrate has to be done, energy requirements for
hydrolyzation were covered by this process. The final
product is a pulp.
Both substrates were, after this, led to a pH of 3.8
(recommended for the fermentation of this substrate [7])
with KOH 3.61 M. Ammonium sulphate (0.15±0.02 g/l
in S1, and 0.1 g in each trial with S2, what means
0.34±0.04 mg/g fresh cladode ) was also added as a
nitrogen source.
Occasional extractions of substrate were made in
order to study the evolution of dry matter between the
different operations (results are not shown here, however,
these extractions have been considered in volumes,
weights, and other results).
Yeasts (Saccharomyces cerevisiae, commercial
strains) were bought fresh and frozen at -18ºC in their
original solid state. Two days before each fermentation a
portion (of about 2 grams) of yeast was introduced into a
sterile erlenmeyer flask containing: 100 ml of S1 at pH =
3.8 (obtained by leading 100 ml of S1 to a pH = 3.8, what
means a total volume of 137± 13 ml), 5 g of sucrose,
and 0.1 g/l of ammonium sulphate. Yeasts were then
cultivated during 48 hrs at a temperature of 25±1 ºC. An
air compressor supplied air through a gas filter with a 0.2
µm pore membrane in alternate periods of one hour (in
order to prevent foam from reaching the filters). A
magnetic stirring unit kept the substrate mixed at the
minimum speed needed for a complete mix of the
medium. The final product obtained by this process is
what we called “yeast inoculum”, also known by
enologists as “starter”.
Starter was inoculated using a sterile syringe at
26.7
±0.02 ml/l in S1, and 60 ml in each trial with S2,
what means 0.21±0.03 ml/g fresh cladode.
Concerning fermentations, two retentions times (4
and 5 days) and two temperatures (25ºC and 30ºC) were
tested. Blank fermentations, with 700 ml of distilled
water (led to pH = 3.8 with citric acid 1M), ammonium
sulphate (0.1 g) and yeast inoculum were also carried out
for each trial condition. Fermentations of S1 at 25ºC and
blank fermentations were carried out in erlenmeyer flasks
with water-repellent cotton-plugs. A magnetic stirring
unit in blank fermentations and an orbital shaker in S1
were use to kept the substrate mixed. Fermentations of S2
and S1 at 30ºC were carried out in a “Minifor Laboratory
Fermentor” (Lambda Instruments).
Samples were taken at the fourth and fifth day of the
process, cooled in a fridge, and frozen at -18ºC until
ethanol determinations were done. Fermented S1 was
then filtered through a 0.45 µm pore membrane.
Fermented S2 was centrifuged (7000 rpm, 10 min) to
separate solid particles and then filtered. Finally, ethanol
concentration was determined using a gas chromatograph
(GC 8000. CE Instruments)
Dry matter contents of S2 trials -once fermentation
had finished- were determined by drying samples at a
temperature of 103 – 105 ºC until constant weight. Total
carbon and nitrogen contents in these substrates were
determined using a NA 2000 analyzer (Finson
Instruments).
Trial conditions are shown in Table I. Amounts of
fresh cladode and sulphuric acid in fermentations with S2
are shown in Table II.
Table I: Trial conditions
Trial Substrate
Retention
time (days)
Temperature
(ºC)
S2-1a S2 4 30
S2-1b S2 5 30
S2-2a S2 4 25
S2-2b S2 5 25
S2-3a S2 4 25
S2-3b S2 5 25
S2-4a S2 4 30
S2-4b S2 5 30
S1-1a S1 4 30
S1-1b S1 5 30
S1-2a S1 4 25
S1-2b S1 5 25
S1-3a S1 4 25
S1-3b S1 5 25
S1-4a S1 4 30
S1-4b S1 5 30
B1* Water 4 25
B2* Water 5 25
B3** Water 4 25
B4** Water 5 25
B5* Water 4 30
B6* Water 5 30
B7** Water 4 30
B8** Water 5 30
*
20 ml inoculum added
**
60 ml inoculum added
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Table II: Amounts of fresh cladode and sulphuric acid
in fermentations with S2
3. RESULTS
Total amount of sugars in cladode mix from which
S2 is obtained was 32.4±1.8 % (d.m.b.). Dry matter
content in fresh cladode was 14.6±0.4 %.
Total volumes and sugar concentrations in S1 are
shown in Table III.
Table III: Volumes and sugar concentrations in S1
Fresh cladode and fermented products contents in total N
and total C are shown in Table IV.
Table IV: Contents in total C and total N.
Ethanol concentrations in fermented substrates are
shown in Table V.
Table V: Ethanol concentrations
Ethanol (ppm)
Substrate Mean value Standard desv.
S2-1a 8461.5 245.0
S2-1b 8428.7 1057.0
S2-2a 7400.7 654.1
S2-2b 7594.8 1097.7
S2-3a 5765.4 665.9
S2-3b 6283.8 23.9
S2-4a 5778.9 202.6
S2-4b 5628.4 334.7
S1-1a 1011.9 97.6
S1-1b 881.4 53.9
S1-2a 541.0 29.5
S1-2b 84.0 16.3
S1-3a 810.2 85.7
S1-3b 325.0 95.5
S1-4a 566.1 33.3
S1-4b 339.1 53.7
B1* 0.0 0.0
B2* 0.0 0.0
B3** 11.2 2.5
B4** 1.8 3.5
B5* 434.3 52.3
B6* 346.7 27.9
B7** 580.4 86.2
B8** 260.5 14.9
*20 ml inoculum added
** 60 ml inoculum added
Considering S2 dry matter content, the content in
reducing sugars previously referred, and volumes after
fermentation, ethanol yields are those shown in Table
VI.
Table VI: Ethanol yields in fermentations of S2
(weight/weight)
Considering S1 sugar concentrations and volumes
after fermentation, ethanol yields are shown in Table VII
Trial
Fresh cladode
initial weight
(g)
Sulphuric
acid (96%)
added for
hydrolisis
(ml)
Ammonium
sulphate
added (g)
S2-1 311.9 11.3 0.1
S2-2 277.3 4.7 0.1
S2-3 344.0 5.7 0.1
S2-4 277.4 4.7 0.1
Trial
Initial volume
(ml)
Initial sugar
concentration
(ppm)
S1-1 600 4820
S1-2 387 3681
S1-3 383 3681
S1-4 600 3681
Fresh
cladode
total
carbon
content
(% d.m.b.)
Fermented
product
total
carbon
content (%
d.m.b.)
Fresh
cladode
total
nitrogen
content
(%
d.m.b.)
Fermented
product
total
nitrogen
content
(% d.m.b.)
S2-1
33.25±0.46
16.93±0.62
0.39±0.01
0.31±0.01
S2-2 23.39±0.63
0.41±0.02
S2-3 23.31±0.58
0.30±0.03
S2-4 26.41±0.88
0.42±0.04
Substrate
Initial
sugar (mg)
Ethanol
(mg)
Fermentation
yield (%)
S2-1a 14752.7 6041.5 41.0
S2-1b 14752.7 6018.1 40.8
S2-2a 11835.9 3700.4 31.3
S2-2b 11835.9 3797.4 32.1
S2-3a 16273.0 4156.9 25.5
S2-3b 16273.0 4530.6 27.8
S2-4a 13123.5 3236.2 24.7
S2-4b 13123.5 3151.9 24.0
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Table VII: Ethanol yields in fermentation of S1
(weight/weight)
4. DISCUSSION
In principle, the used method for sugar determination
gives us the total content of reducing sugars that are not
contained in the structural polysaccharides cellulose and
hemicellulose. This means soluble monosaccharides and
those monosaccharides resulting from sucrose, starch and
mucilage hydrolysis.
According to values reported by Stintzing and Carle
[6], starch content in cladodes oscillates with seasons
between 8.5 and 17.1 % (d.m.b.). Ayadi, et al [8] , found
a total content of starch of 13.09±1.15 % (d.m.b.) in
spineless cladodes, with 6.01±0.88 % (d.m.b.) of soluble
sugars. Sepúlveda et a
l [9], reported that mucilage can
represent between 17.9 and 20.8 % (d.m.b.) of cladode
weight, and Goldstein et al [10] found a total amount
between 9 and 19 % (d.m.b.) of mucilage in cladodes.
An important issue to take into account is that a
significative fraction of reducing sugars from mucilage
hydrolysis is composed by pentoses, and thus, not
fermentable by the yeasts used in these experiments. It is
not easy to determine the carbohydrate composition of
mucilage, as this depends on various factors (including
the way of extraction and purification [11]). According to
Majdoub [12] about a 28 % (weight/weight) of mucilage
could be composed by carbohydrates, and Abraján [11]
found that a fraction up to 75.1 % (moles/moles) of total
sugar in the mucilage of one year old peeled and scalded
cladodes was composed by pentoses.
According to this information we can estimate (even
though in a highly imprecise way) that about 21 % of
mucilage is composed by pentoses, and this could mean
between 1.9 and 4 % of total cladode dry matter.
Considering the average result (32.4 % d.m.b.) for
total reducing sugars in S2, and the above exposed data,
total fermentable sugars could be between 5.9 and 12.3 %
less than total reducing sugars. Finally, this could lead
fermentation yield to 46.7 % (in case of S2-1a), what
would be closer to de 51.1 % theoretical yield.
Despite this fact, interesting ethanol yields have been
reached when sulphuric acid is added in an approximate
proportion of 1 ml for each 30 grams of fresh cladode
(trial S2-1), instead of 0.5 ml for the same amount of
fresh cladode.
A possible reason for low ethanol yields in some of
the fermentations, particularly in those with S1, is the
high concentration of potassium in the media. Volumes
around 150 ml of KOH 3.61M were needed to lead
original S1 to a pH=3.8. This means concentrations of K
+
over 21000 mg/l. Further investigation concerning this
issue is needed.
Statistical analysis of the data (ANOVA-
Bonferroni´s multiple comparison procedure) show no
significant differences between “a” and “b” fermentation
yields in all S2 trials, nor between S2-4 (“a” and “b”) and
S2-2,3 (“a” and “b”). This means no difference between
retention times and temperatures. Significant differences
between S2-1 and all the other fermentations have been
found, probably due to the different amount of acid used
in the hydrolysis.
Concerning S1, one remarkable issue is that
significant differences between “a” and “b” fermentations
can be seen in S1-2,3,4 fermentation results.
Chromatograms of these “b” fermentations shown peaks
of non-determined compounds with great areas (even
greater than ethanol peaks) appearing before
acetaldehyde (a small amount of acetaldehyde appears in
almost all chromatograms of all done experiments).
Substrate extractions done in fourth day (“a” trials) may
led to an oxygen excess in the medium, or even to a
microbiological contamination, despite the process was
carried out in adequate conditions (laminar flow
chamber, sterile material,…).
Regarding yeasts, commercial strains are easy to find
and inexpensive (so they could lead to a cost reduction of
the industrial process of fermentation), but –probably-
they are not the best option to reach the highest ethanol
yields of these substrates in the present conditions.
Further investigations with selected strains of yeasts must
be done.
Finally, it is interesting to mention that, considering
average cladode yields reported by Le Houerou, 1996 [5],
and the best of S2 fermentation yields, total ethanol
production could represent 984 l/ha (with 200 mm of
rain) and 3116 l/ha (with 400 mm).
5. CONCLUSIONS
Interesting concentrations of ethanol have been
reached in trials with S2, but far below the desired 5%
considered as economically feasible. In general terms,
higher fermentation yields are found in trials with S2, so
there is no reason to carry out S1 hydrolysis, as ethanol
concentration in the fermented media is much lower than
in S2. Finally, fermentation yields must be improved, and
selected strains of yeasts may play an important role in
this.
6. REFERENCES
[1] Sáenz, C. (2000) “Processing technologies: an
alternative for cactus pear (Opuntia spp.) fruits and
cladodes”. Journal of Arid Environments 46: 209–
225
[2] Sáenz, C et al. Arias, E (Coord). (2006) “Utilización
Agroindustrial del Nopal”. Estudio FAO Producción
y Protección Vegetal nº 162. Rome.
[3] López, J.J., Fuentes, J.M., and Rodriguez, A. (2001)
“Producción y uso de Opuntia como forraje en el
centro-norte de Mexico”. In “El nopal (Opuntia ssp.)
como forraje.”. Estudio FAO Producción y
Protección Vegetal nº169. Rome.
[4] Cordeiro, D., Gonzaga, S. (2001) “Opuntia como
forraje en el oeste semiárido de Brasil”. In “El nopal
Substrate
Initial sugar
(mg)
Ethanol
(mg)
Fermentation
yield (%)
S1-1a 2892.0 774.1 26.8
S1-1b 2892.0 674.3 23.3
S1-2a 1424.5 283.5 19.9
S1-2b 1424.5 44.0 3.1
S1-3a 1409.8 409.1 29.0
S1-3b 1409.8 164.1 11.6
S1-4a 2208.6 419.5 19.0
S1-4b 2208.6 251.3 11.4
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(Opuntia ssp.) como forraje.”. Estudio FAO
Producción y Protección Vegetal nº169. Rome.
[5] Le Houerou, H..(1996) “The role of cacti
(Opuntia.spp.) in erosion control, land reclamation,
rehabilitation and agricultural development in the
Mediterranean Basin” Journal of Arid
Environments. 33(2), 135-139.
[6] Stintzing, F.C., and Carle, R. (2005) “Cactus stems
(Opuntia spp.): A review on their chemistry,
technology, and uses” Molecular Nutrition Food
Research. 49, 175 – 194.
[7] Retamal, N.(1987) “Aprovechamiento
Agroenergético de la Chumbera (Opuntia ficus-
indica (L. Mill.))”. Doctoral Thesis. Universidad
Politécnica de Madrid (Spain).
[8] Ayadi. M.A., Abdelmaksoud, W., Ennouri, M.,
Attia, H.(2009) “Cladodes from Opuntia ficus indica
as a source of dietary fiber: Effect on dough
characteristics and cake making” Industrial Crops
and Products 30, 40–47.
[9] Sepúlveda, E. Sáenz, C., Aliaga, E. Aceituno, C.
(2007) “Extraction and characterization of mucilage
in Opuntia spp.” Journal of Arid Environments 68,
534–545.
[10] Goldstein, G., Andrade, J.L., Nobel, P. (1991)
“Differences in water relations parameters for the
chlorenchyma and parenchyma of Opuntia ficus
indica under wet Versus dry conditions”. Australian
Journal of Plant Physiology. 18, 95–107. Cit. in
Sepúlveda, E. Sáenz, C., Aliaga, E. Aceituno, C.
“Extraction and characterization of mucilage in
Opuntia spp.” Journal of Arid Environments 68
(2007) 534–545.
[11] Abraján, M.A. (2008) “Efecto del método de
extracción en las características químicas y físicas
del mucílago del nopal (Opuntia ficus-indica) y
estudio de su aplicación como recubrimiento
comestible”. Doctoral Thesis. Universidad
Politécnica de Valencia (Spain).
[12] Majdoub, H., Roudesli, S., Picton, L., Le Cerf, D.,
Muller, G. and Grisell, M. (2001). “Prickly pear
nopals pectin from Opuntia ficus-indica physico-
chemical study in dilute and semi-dilute solutions”.
Carbohydrate Polymers.46: 69-79. Cit. in :
Goycoolea, F. and Cárdenas, A. (2003) “Pectins
from Opuntia spp.: A Short Review” Journal of the
Professional Association for Cactus Development,
5, 17 – 30.
7. ACKNOWLEDGEMENTS
This work is framed within the agreement “Estudio
del cultivo de chumbera (Opuntia ficus-indica (L.)
Miller) y tabaco arbóreo (Nicotiana glauca
Graham) para la producción de bioetanol” between
Universidad Politécnica de Madrid and Fundación
CAJAMAR. Financial support from Fundación
CAJAMAR through the project CENIT I+DEA
“Investigación y Desarrollo del Etanol para
Automoción”, (
Ministerio de Ciencia e Innovación)
is gratefully acknowledged.
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