INTRODUCTION
Orange is widely planted and is relatively less perishable than most
other tropical fruits. In some fruits, the discarded portion can be very
high, for example, orange 30-50% (Lerner, 2003). The recent thrust in
bioconversion of agricultural and industrial wastes to chemical feedstock
has led to extensive studies on cellulolytic enzymes produced by fungi
and bacteria (Baig et al., 2004). Cellulase production from different
agro-wastes have been reported using a variety of microorganisms. These
include banana peel, millet, guinea corn, rice husk and maize straw using
Saccharomyces cerevisae and Aspergillus niger (Baig et
al., 2004; Milala et al., 2005). Shahera and Sanaa (2002)
have also reported cellulase production by yeast strains from orange wastes.
Despite a worldwide and enormous utilization of natural cellulosic sources,
there are still abundant quantities of cellulose-containing raw materials
and waste products that are not exploited or which could be used more
efficiently. The problem in this respect is however to develop processes
that are economically profitable. Cellulose is a useful as a digestive
aid especially in animals. It is also useful in the production of glucose
syrups, paper milling, production of single-cell protein and improving
the palatability of poor quality vegetables. Cellulose-containing wastes
may be agricultural, urban, or industrial in origin, sewage sludge might
also be considered a source of cellulose since its cellulosic content
provides the carbon needed for methane production in the anaerobic digestion
of sludge. Cellulose which forms about 40-50% of plant`s composition is
the most abundant organic matter on earth. Proper biotechnological utilization
of these wastes in the environment will eliminate pollution and convert
them into useful by-products (Milala et al., 2005). Thus the agro
wastes left behind for natural degradation can be utilized effectively
to yield fermentable sugars which can be converted into other substance
like alcohol. The crystallinity and lignification limit the accessibility
and susceptibility of cellulose to cellulolytic enzymes and other hydrolytic
agents (Caritas and Humphrey, 2006).
Pretreatment of cellulose opens the structure and removes secondary interaction
between glucose chains (Tang et al., 1996). Various physical and
chemical treatments of lignocellulosic substrates are necessary for lignin
removal by effective disruption of the lignin carbohydrate linkage and
the highly ordered cellulose itself. Pretreatment of the substrate results
in the reduction of the particle size by increasing surface to volume
ratio and causes reduction in crystallinity. The chemical treatment of
lignocelluloses causes swelling leading to an increase in internal surface
area, decrease in the degree of polymerization, decrease in crystallinity,
separation of structural linkages between lignin and carbohydrates, thus,
increasing the cellulose hydrolysis (Ravindra and Bhaathi, 2006).
Nigeria possesses abundant cellulolytic wastes as leftovers from agricultural
practices. These wastes can be used to produce alcohol which can be exported
to gain much needed foreign exchange and boost our foreign reserves. Processing
of these wastes can also help provide jobs and assist in controlling environmental
pollution. This study therefore evaluates the possibility of the re-use
of agro-industrial wastes namely orange peel as a substrate for cellulose
production.
MATERIALS AND METHODS
Plant material and microorganisms: This study was carried out
in 2007. Orange fruit was washed thoroughly with water, peeled and sliced.
The juice was removed with the aid of a squeezer and the pulp separated
from the pericarp (albedo) and the three materials were sun-dried separately.
They were later oven-dried at 70°C, still being handled separately
and then pounded using a mortar and pestle. These materials were subjected
to alkali and steam pre-treatment, after which the materials were washed
and dried in the oven at 70°C. All waste substrates were then ground
separately using a blender and kept in containers on the shelf. The organisms
used for this study were Trichoderma longibrachiatum, Aspergillus
niger and Saccharomyces cerevisiae. These were isolated from
three sources: Aspergillus niger was isolated from rotten wood
(RW) picked up on the premises of Unilorin permanent site, Saccharomyces
cerevisiae was isolated from palm wine (PW) bought from a palm wine
tapper at Offa garage in Ilorin and Trichoderma longibrachiatum was
collected from the Faculty of Agriculture, University of Ilorin.
Screening and identification of microorganisms: Plate screening
of the isolated organisms for cellulase production was carried out according
to the method of Brown et al. (1987). A point of inoculation the
spores of each organism was grown on PDA supplemented with 2% (w/v) carboxymethylcellulose
(CMC). The plates were incubated at 29±1°C for 48 h after which
they were stained with Congo red stain for 15 min. Excess dye was removed
by washing with 1 M NaCl and the plates were fixed with 1 N HCl. The production
of extracellular cellulase by the organisms was indicated by a zone of
clearance around the colony.
The microorganisms were identified in Microbiology Laboratory in the
University of Ilorin according to Chaturvedi (2001). All organisms were
maintained on PDA slants. A spore suspension from 3 to 4 day old 10 mL
PDA slants of each culture in 10 mL sterile distilled water was made.
Mineral salts glucose medium was prepared and approximately 2.8x106
spores cells-1 of each culture were inoculated into 500 mL
flasks containing 100 mL of medium each. The spores cells-1
were counted using a Neubauer counting chamber. The flasks were incubated
for 24 h at 29±1°C on a Gallenkamp (England) rotary shaker
at 250 rpm to develop the inoculum. Mary Mandels` Mineral salts solution
was used for the fermentation and it was prepared as described by Jeffries
(1996). Culture conditions involved autoclaving the fermenting media containing
10 g L-1 of waste substrate at pH 5.0 and inoculating with
pure suspension of germinated spores of Trichoderma longibrachiatum,
Aspergillus niger and Saccharomyces cerevisiae.
Chemicals and reagents: Carboxymethylcellulose (CMC), Sodium potassium
tartarate, Glucose, Dinitrosalicylic acid, Sodium hydroxide and all other
salts used were of analytical grade and products of British Drug House
(BDH), England.
Enzyme assays: The waste substrates represent the carbon sources
in the fermentation media. These were combined with Mary Mandels` Mineral
Salts Medium to give Mineral salts glucose medium (MSGM) that was used
in the inoculum development, Mineral salts orange peel medium (MSOpeM),
Mineral salts orange pulp medium (MSOpuM) and Mineral salts orange albedo
medium (MSOalM).
All the media mentioned above were prepared separately and dispensed
in conical flasks. They were sterilized in the autoclave at 121°C
for 15 min.
The final pH was adjusted to 5.0 using a pH meter (Denver Instrument,
Model 20 pH/Conductivity meter).
The pH of the fermenting media containing the waste substrates at a level
of 10 g L-1 was adjusted to 5.0. The suspension of germinated
spores was inoculated at a level of 10% (v/v) into the production medium
contained in flasks. These were incubated at 29±1°C on a shaker
at 100 rpm. Glucose production in the medium was measured on Day 5 of
fermentation (Srivastava et al., 1987; Jeffries, 1996).
Cellulase activity was determined colorimetrically by measuring the increase
in reducing groups by the hydrolysis of a carboxymethylcellulose (CMC)
substrate. The procedure followed the 0.5 mL assay described by Jeffries
(1996).
Samples were withdrawn from the culture at 2 day intervals over a period
of 7-9 days and the supernatant that resulted following centrifugation
at 3,000 rpm for 15 min to remove solids, were assayed for total reducing
sugars using DNSA method of Miller (1959). Enzyme solutions were diluted
in 0.05 M citrate buffer, pH 4.8. The enzyme diluted in buffer and 1%
CMC (0.5 mL each) was mixed well and incubated for 30 min at 50°C.
Three milliliters of the DNSA was added and the tubes were placed in boiling
water bath for 5 min. The tubes were cooled and the reducing sugar, glucose
was determined (Jeffries, 1996). The sample, enzyme blank, glucose standard
and control were boiled together and absorbance was read at 540 nm using
a spectrophotometer (CamSpecM105). A control (substrate and buffer) otherwise
called spectro zero, was used to set the spectrophotometer at zero absorbance.
During the course of the experiments, the absorbance of the sample tube,
corrected by subtraction of the enzyme blank was translated into glucose
during the reaction using a glucose standard. The linear glucose standard
was used to translate the absorbance values of the sample tubes into glucose
i.e., mg glucose produced during the reaction. For a 30 min assay, 1 mg
of glucose equals 0.185 unit
Optimization experiments were carried out and each of the organisms were
grown on each of the substrates and hydrolyzed under conditions that produced
maximal activity of the enzyme from all the previous experiments. In accord
with the International Union of Biochemistry, one enzyme unit equals 1
μmol of substrate hydrolyzed per minute.
Determination of optimal conditions for enzyme production
Effect of varying time: Cellulase activity was measured at regular
intervals while fermentation was observed at 29±1°C for a period
of 9 days and the period of maximum enzyme production was determined.
Samples were withdrawn on Days 0, 1, 3, 5, 7 and 9.
Effect of varying pH: The pH of the fermentation media were adjusted
to various values ranging from 2.0-6.0 with 0.1 N NaOH or 0.1 N HCl. The
pH was determined using the pH meter (Denver Instrument, Model 20 pH/Conductivity
meter).
Effect of varying substrate concentration: Different concentration
of the waste substrates (orange pulp, albedo and peel), ranging from 1.0
to 5.0% were used in the fermentation media.Effect of varying temperature:
The fermentation was carried out at different temperatures ranging from
29±1 to 45°C.
Effect of varying inoculum size: Each cellulosic waste was fed
with varying sizes of inoculum of the organisms. The inoculum size was
varied from 2 to 10%.
RESULTS AND DISCUSSION
The three test organisms can produce cellulase from the orange wastes
(Table 1). In determining optimal conditions for glucose
production, T. longibrachiatum produced highest amounts of glucose
on day 7, A. niger on day 5 and from S. cerevisiae on day
3 for orange peel and albedo and day 5 for orange pulp (Table
2). Hydrolysis rates decline with time due to depletion of the more
amorphous substrates, product inhibition and enzyme inactivation (Ghose,
1987). Caritas and Humphrey (2006) and Narasimha et al. (2006)
also gave similar time course reports of maximum glucose yield on 5th
day of fermentation using A. niger. Effect of pH on glucose production
from the three waste substrates by the three microorganisms (Table
3) supports the findings of Lee et al. (2002) who reported
that CMCase, Avicelase and FPase activities exhibit a pH optimum
of approximately 4, while the pH optimum of β-glucosidase was between
pH 5 and 6.
Further increase in cellulose concentration beyond the level that gave
the optimum glucose did not result in proportionate increase in glucose
yield. Haapela et al. (1995) and Jeffries (1996) reported that
maximum endoglucanase activity was recovered on the medium with cellulose
at 10 g L-1. Mandels and Reese (1959) also reported that maximal
yields of cellulase were obtained on 1% substrate (cellulose, lactose,
cellobiose and glucose) using T. viride and Myrothecium verrucia.
These reports support the findings of this study as substrate concentration
of 10 g L-1 gave the highest amount of glucose from T.
longibrachiatum on orange albedo.
| Table 1: |
Fermentation of waste substrates by test fungi |
|
| Values are presented as Mean±SD (n = 3) |
| Table 2: |
Effect of varying time on glucose production by test
fungi |
|
| Values are presented as Mean±SD (n = 3) |
| Table 3: |
Effect of pH on the production of glucose by test fungi |
|
| (Substrate concentration: 1%, Temp: 29±1°C,
inoculum size: 10%), Waste substrate: 1%, Temperature: 29±1°C,
pH: 5.0, inoculum size: 10%, Values with different superscripts are
statistically different. Values are presented as Mean±SD (n
= 3) |
| Table 4: |
Effect of substrate concentration on the production
of glucose by test fungi |
|
| (pH: 5.0, Temperature: 29±1°C , Inoculum
size: 10%), Values are presented as Mean±SD (n = 3), All groups
are compared to each other at p<α = 0.05. Values with different
superscripts are statistically different |
Since the substrates contain different minerals apart from carbon which
may serve as nutrient supplements, increase in substrate concentration
leads to increase in these nutrients which may adversely affect the cell
concentration (Table 4). The increase in glucose production
until the optimum that was obtained was due to the availability of cellulose
in the medium; while a decrease in production beyond optimum concentration
is explained to be as a result of an inhibitory effect of accumulated
cellobiose and cellodextrins of low degree of polymerization to the growth
medium. It might also be due to the specific binding of the enzymes with
the substrates (Wang et al., 2006). Low glucose production after
optimum very probably highlights sugar depletion from the substrates into
the medium (Brien and Craig, 1996).
Decrease in amounts of glucose production resulted at inoculum sizes
above 6% for fermentations using A. niger (Table
5). This decrease in glucose production with further increase in inoculum
might be due to clumping of cells which could have reduced sugar and oxygen
uptake rate and also, enzyme release (Srivastava et al., 1987).
The optimum temperature for the synthesis of enzymes for saccharification
of agrowaste in all cases to enzymatic hydrolysis can be attributed to
lignin content of the material (Table 6). Pretreatment
of lignocellulosic material enhances enzyme activity and maximum saccharification
was achieved within the range 30-45°C coinciding with the characteristics
of mesophiles (Baig et al., 2004).
Optimum glucose from the waste substrates using T. longibrachiatum,
was produced at 10% inoculum size at 45°C on day 7 but at pH 5.0 and
3% substrate concentration for orange peel but 1% substrate concentration
for orange albedo and pH 4.0 and 3% substrate concentration for orange
pulp.
Optimum glucose from the waste substrates using A. niger was produced
at pH 4.5, 6% inoculum size on day 5 but at 2% substrate concentration
and 40°C for orange pulp; 4% substrate concentration and 45°C
for orange peel (Table 7).
For S. cerevisiae fermentation, optimum glucose was produced at
pH 4.5, 2% substrate concentration, 6% inoculum size, 45°C on day
3 for orange peel, pH 3.5, 4% substrate concentration, 2% inoculum size
and 45°C on day 5 for orange pulp and pH 3.5, 2% substrate concentration,
2% inoculum size and 45°C on day 3 for orange albedo.
These optimal conditions were combined in single fermentations for each
organism and cellulase activity was measured (Table 8).
Cellulase activity from orange peel was 1.64 U mL-1 when hydrolyzed
by T. longibrachiatum, 1.42 U mL-1 when hydrolyzed by
A. niger and 1.07 U mL-1 when hydrolyzed by S. cerevisiae.
Cellulase activity from orange pulp was 1.93 U mL-1 when hydrolyzed
by T. longibrachiatum, 1.58 U mL-1 when hydrolyzed by
A. niger and 1.15 U mL-1 when hydrolyzed by S. cerevisiae.
Cellulase activity from orange albedo was 1.75 U mL-1 when
hydrolyzed by T. longibrachiatum, 1.29 U mL-1 when hydrolyzed
by A. niger and 1.13 U mL-1 when hydrolyzed by S.
cerevisiae. Cellulase activity of S. cerevisiae was lowest.
The exo-β-1, 3-glucanases produced by S. cerevisiae yield
glucose as the end product, whereas endo-β-1, 3-glucanase releases
a mixture of oligosaccharides with glucose as the minor product. Because
β-1, 3-glucan is the main structural polysaccharide responsible for
the strength and rigidity of the yeast cell wall, β-1, 3-glucanases
have been suggested to play a role in important morphogenetic processes
involving the controlled autolysis of β-1, 3 glucan. During vegetative
growth, several endo-and exo-1, 3-β-glucanases are synthesized, some
of which are secreted only to remain entrapped in the cell wall whereas
others are released to the surrounding medium (Lee et al., 2002).
| Table 5: |
Effect of inoculum size on the production of glucose
by test fungi |
|
| (Temperature: 29±1°C, pH: 5.0, Substrate
concentration: 1%), Values are presented as Mean±SD (n = 3),
All groups are compared to each other at p<α = 0.05. Values
with different superscripts are statistically different |
| Table 6: |
Effect of temperature on the production of glucose
by test fungi |
|
| Values are presented as Mean±SD (n = 3), All
groups are compared to each other at p<α=0.05. Values with
different superscripts are statistically different |
| Table 7: |
Optimized glucose production by the test organisms
from the different waste substrates |
|
| Values are presented as Mean±SD (n = 3), All
groups are compared to each other at p<α = 0.05. Values with
different superscripts are statistically different |
| Table 8: |
Activity against CMC of T. longibrachiatum, A. niger
and S. cerevisae cellulase on orange wastes |
|
| Values are presented as Mean±SD (n = 3) |
In conclusion, this study revealed that orange peel, pulp and albedo,
which are examples of domestic and industrial agro-wastes, produce large
amounts of cellulase enzymes when hydrolyzed by cellulolytic microorganisms
and instead of being left behind for natural degradation can be utilized
effectively under these conditions, to produce cellulase.
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