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Biodegradation of agro-industrial orange waste under solid state fermentation and natural environmental conditions

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valuation of the possibility of the re-use of agro-industrial orange peel and pulp wastes under solid state fermentation and natural environmental condition as a source of enzymes production [? & ß amylase, cellulase, pectinase(s), lipase(s), esterase(s) and peroxidase(s)] the physiological enzymes of lysis and total protein. Different microorganisms such as fungi, bacteria and yeast which were charged of waste analyse and have ability to produce previous enzymes and protein. These microorganisms were isolated from the fermented waste and preliminarily identified to test each one of them for their enzymes production and also to test them for inducing the fermentation process under the natural conditions, or by using one of the present enzyme producer strain. The protein and physiological enzymes production were electro-phoretically patterned in different bands. And finally, the possibility to use the fermented waste as a bio-fertilizer was done and it stimulated the growth of wheat plant using water culture, especially after the toxicity of the fermented waste was investigated.
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Content Preview
Egyptian Journal of Biology, Volume 4,, 2002, pp. 23-30
? Printed in Egypt. Modern Press, Cairo. Egyptian British Biological Society (EBB Soc)


Biodegradation of agro-industrial orange waste under solid state fermentation and natural
environmental conditions

Shahera H. Attyia and Sanaa M. Ashour*
Botany Department, Women's College for Arts, Science and Education, Ain Shams University, Cairo, Egypt

ABSTRACT
Evaluation of the possibility of the re-use of agro-industrial orange peel and pulp wastes under solid
state fermentation and natural environmental condition as a source of enzymes production [? & ?
amylase, cellulase, pectinase(s), lipase(s), esterase(s) and peroxidase(s)] the physiological enzymes of
lysis and total protein. Different microorganisms such as fungi, bacteria and yeast which were
charged of waste analyse and have ability to produce previous enzymes and protein. These
microorganisms were isolated from the fermented waste and preliminarily identified to test each one
of them for their enzymes production and also to test them for inducing the fermentation process
under the natural conditions, or by using one of the present enzyme producer strain. The protein and
physiological enzymes production were electro-phoretically patterned in different bands. And finally,
the possibility to use the fermented waste as a bio-fertilizer was done and it stimulated the growth of
wheat plant using water culture, especially after the toxicity of the fermented waste was investigated.

KEYWORDS:
Solid state, peel & pulp waste, enzymes, protein, bio-fertilizer, bacteria, fungi, yeast.

INTRODUCTION
Solid State Fermentation methods are widely used for the industrial production of microbial
enzymes and metabolites. A variety of solid substrates have been identified for use in the
production of several enzymes such as cellulase, ? amylase and pectinase (Toyama 1963) and
lipase (Yamada 1977). The citrus processing residues are rich in both soluble and insoluble
carbohydrates (Kesterson & Braddok 1976) which makes them an attractive potential feedstock
for biological conversion to value added products. The traditional re-use of orange pulp and peel
waste ential a high cost, impractical for Egyptian citrus industries, and therefore these industries
have been accumulating peel and pulp waste in the soil, causing serious invironmental problems.

Peel, pulp and membranes from oranges and other related citrus fruits are highly
susceptible to hydrolysis by mixtures of cellulolytic and pectinolytic enzymes (Grohmann &
Baldwin 1992). Mixtures of cellulase and pectinase enzymes are necessary for the complete
conversion of all carbohydrates to monomeric sugars (Grohmann et al. 1994) special
attention was paid to carbohydrate enzymes produced by fungi which are industrially
important in the clarification and extraction of fruit juices and in degamming of natural fibers
in the textile industry. The enzyme pectinase was of particular interest because it is involved
in the phytopathogenic process caused by fungi (Fogarty & Kelly 1983). The re-use of agro-
industrial orange waste as organic fertilizers seems to be a low cost technology for the
recycling of the nutrients contained in this waste. Therefore the objective of this work is not
only to overcome the problems of the orange peel and pulp waste accumulation in the
environment by converting them under solid state fermentation into useful products such as
the production of carbohydrate lyses enzymes, (? & ?) amylase, cellulase, pectinase(s) and
lipase(s), physiological enzymes, esterase(s), peroxidase(s) in band profile and the total
protein, but also to minimize the time of fermentation. In addition to use the fermented waste
as a bio-fertilizer.

MATERIALS AND METHODS
Raw materials and preparation of precursor culture for solid state fermentation:
Fresh
orange peel and pulp waste was collected separately from Kaha factory. The so called "starter
________________________
* Address for Correspondence

Attyia and Ashour: Biodegradation of agro-industrial wastes

culture" was prepared as follows: 25 g of fresh orange waste was incubated in two types of
bottles (glass and polyethene) with capacities of 300, 500, and 1500 ml. Incubation continued at
room temperature for 15-20 days under natural environmental conditions. At the end of the
incubation period, the orange waste was a completely solubilized mixture, which was used as an
inoculum "precursor" for fresh new agro-industrial orange waste to minimize the time of
fermentation. Triticum vulgare C.V. "Giza 164" was obtained from the Crop Research Institute,
Ministry of Agriculture, Egypt, Cairo and was used as a bioassay seedling growth.
Growth media: Nutrient agar, Sabouraud and yeast-extract agar media were used for the
isolation and purification of bacteria, mould and yeast respectively.
Identification of micro-floral isolates: The bacterial isolates were preliminarily identified using
Bergey's Manual (Williams et al. 1989), while Samson (1979) were used for fungal
identification, various yeast strains were isolated but could not be identified. All the pre-
mentioned micro-floral isolates were isolated from the agro-industrial fermented waste of orange
peel and pulp.
Extraction of lysis enzymes: The enzymes examined in the present study were extracted from
fermented orange waste and their identified micro-floral organisms according to the procedure of
Kar and Mishra, (1976).
Lysis enzyme assays: Quantitative ?, ? amylase and cellulase activities were determined as
according to Malik & Singh (1980) while qualitative and quantitative activities of pectinase(s)
and lipase(s) were carried out using the clearing-zone technique described by Ammar et al.
(1995, 1994) and Elwan et al. 1977) respectively.
Isozymes and protein electrophoresis: One gram from each of fermented orange peel and pulp
waste samples were pelleted by centrifugation at 13000 rpm. for 10 min. The protein extracted
using protein extraction buffer contains 5 ml tris/borate pH 8.9 containing 5% SDS "sodium
deodocyle sulfate" and 5 ml cold distilled. H2O for isozymes and 2 h. shaking according to
Hussein & Stegemann (1978). Polyacralamide standard gel (5%) was performed on vertical slabs
using the biored gel electrophoresis system. The run was performed at 300 v for 2 h.
Isozymes and protein visualization: Coomassi brilliant blue R 250 was used in protein
staining, where 5 ml of 1% stain were added to 200 ml of a mixture of 60 g trichloroacetic acid,
800 ml water, 200 ml ethanol and 70 ml acetic acid. The gels were stained over night. The
destaining was composed of 300 ml methanol, 700 ml water, and 50 ml acetic acid. For
esterase(s) and peroxidase(s) isozyme visualization was achieved following the methods
Scandalios (1964) and Larsen & Beuson (1970), respectively.
Orange peel and pulp fermented applications on Triticum vulgare seedling:
Triticum vulgare seeds were treated with mixture of soluble fermented waste (peel or pulp)
and water (1:10 ml v/v) for 24 h. untreated and treated seeds were germinated in 15 cm petri
dishes (20 seeds per dish) then transferred into growth chamber under 12 h. light/day at
30±2°C and left to grow for 3 weeks with three replicates for each treatment. After that,
observations were made of shoot and root length, number of leaves, fresh and dry weight.
Statistical analysis:
All results were submitted to variance analysis (ANOVA), the significant
differences were measured at p< 0.05.

RESULTS AND DISCUSSION
Table 1 shows the different micro-floral strains which were isolated and preliminarily identified
from the fermented orange peel and pulp waste. Two different fungal strains, Aspergillus flavus
and Nigrospora sp. were found in orange pulp but were absent from the fermented orange peel
waste. For the isolated yeast, there were three different isolates from the peel and pulp waste,
Ashbell et al. (1987) showed that the orange waste often contain large numbers of yeasts. Two
24

Attyia and Ashour: Biodegradation of agro-industrial wastes

bacterial strains were found in the fermented orange peel waste (rod and cocci, gram -ve and non
spore former), and streptobacilli strain (gram +ve and non spore former) were isolated from the
fermented orange pulp waste but were absent from the fermented peel waste (Table 1).

Table 1: Preliminary identification of the isolated microflora from the fermented orange peel and pulp wastes.
The three yeast isolates were not identified but they were different according to their difference in their
enzymatic production.
The isolated strains from orange peel
The isolated strains from orange pulp
Fungi: Aspergillus nidulans & fumigatus
Fungi: Aspergillus nidulans, fumigatus & flavus,
Nigrospora sp
Yeasts: Y1 isolate
Yeasts: Y2 isolate & Y3 isolate
Bacteria: Strain No. 1: Streptobacilli, gram +ve,
Bacteria: Strain No. 1: Streptobacilli, gram +ve, terminal
terminal spore former.
spore former.
Strain No. 2: Streptobacilli, gram +ve, central
Strain No. 2: Streptobacilli, gram +ve, central spore
spore former.
former.
Strain No. 3: Cocci, gram -ve, non spore former.
Strain No. 3: Streptobacilli, gram +ve, non spore former.
Strain No. 4: Rod, gram -ve, non spore former.


The results in tables 2 & 3 include a comparative study between carbohydrate and lipase
enzymes that had been extracted from the fermented peeled and pulp and micro-floral organisms.
In the peeled fermented waste, A. nidulans was the best microorganism for ?-amylase production
(103.83 µg/ml) while yeast strain 2 found in the pulp was the best for ?-amylase production
(631.00 µg/ml), representing six times the value of that produced by A. nidulans in the peel. For
the production of ? amylase in the micro-floral enzymes from the peel, yeast strain 1 produced
the highest value (4566.00 µg/ml), while in the pulp yeast strain 3 showed the highest value
(9695.70 µg/ml) which is twice the value produced in the peeled waste. In addition yeast strain 1
showed the highest value of cellulase production (221.76 µg/ml), while yeast strain 2 in the pulp
showed the best yield (201.60 µg/ml). It can be deduced that we obtained two unusual strains of
cellulytic activity, because yeasts in general do not show an affinity towards cellulase production,
while microflora in the peel showed slight activity of cellulase production and its corresponding
in the pulp. For the last carbohydrate enzyme pectinase(s), A. fumigatus produced the highest
quantity of micro-floral pectinase(s) enzyme (1258.90 unit/ml) in the peel, which is in agreement
Kester & Visser (1990) which showed that Aspergillus strains were synthesizing a mixture of
several enzymes. A. fumigatus strain used in our experiment probably excreted several additional
enzymes, while Nigrospora sp. in the pulp showed the highest activity (28183.80 unit/ml) in
pectinase(s). Further more, Nigrospora sp. recorded high activity (1000 unit/ml) in lipase
production, while the highest activity in the peel was recorded by bacterial strain 1
(Tables 2 & 3). In addition, there is a high productivity of cellulase in contrast with the
pectinase(s) activity and do not produce appreciable amounts of pectinase which shows
agreement with Grohmann & Baldwin (1992). Using enzymatic hydrolysis much better than the
acidic hydrolysis of polysaccharride tissue, this gives agreement with Grohmann et al. (1995)
which showed that enzymatic hydrolysis is more sensitive than acidic hydrolysis of
polysaccharide in plant tissues because the hydronium is a less selective catalytic agent for this
reaction. Numerous glycosidic bonds can be broken at similar rates (Timell 1964) with the
exception of cellulose, which is relatively resistant to acid-catalyzed hydrolysis. This is due to its
insolubility and crystallinity (Philipp et al. 1979). In addition, pectin, in which glycosidic bonds
between galacturonic acid units appear to be more resistant due to a combination of inductive and
conformation effects (de Vries 1988).

When using cellulase and pectinase(s) enzymes approximately 90% of the waste is
solubilised (Grohmann et al. 1995). Higher solubilization of the orange waste by enzymatic
rather than by acid treatment is mainly caused by enzymatic depolymerization and

25

Attyia and Ashour: Biodegradation of agro-industrial wastes

solubilization of cellulose. Hydrolysis of untreated substrate is a very efficient treatment for
the solubilization of total wastes. Peel is highly susceptible to enzymatic hydrolysis.
Penetration by enzymes is aided by the disintegration of tissues and soluble forms of
polysaccharides are generally easier to hydrolyse than in soluble, particulate forms.
Table 2: A comparative study for the determination of peeled micro-floral enzymes
Micro-floral
?-amylase
?-amylase
Cellulase
Pectinase(s)
Lipase
strain tested
µg/ml
µg/ml
µg/ml
unit/ml
unit/ml
A. nidulans
103.83
2462.40
185.47
588.80
800.10
A. fumigatus
72.58
2846.50
211.68
1258.90
690.100
Y1 sp.
10.48
4566.00
221.76
not detected
585.00
Strain (1)
7.66
30.80
14.11
39.80
1000.00
Strain (2)
18.14
100.20
16.12
25.11
191.30
Strain (3)
11.29
100.30
10.08
19.05
182.60
Strain (4)
10.08
46.20
18.14
not detected
68.50
F (for enzymes) =1277.87, p< 0.05. F (for strains) = 600.89, p< 0.05. Value are the means of three replicate enzymes.
Table 3: A comparative study for the determination of pulp micro-floral enzymes
Micro-floral strain
?-amylase
?-amylase
Cellulase
Pectinase(s)
Lipase
tested
µg/ml
µg/ml
µg/ml
unit/ml
unit/ml
1- A. nidulans
332.64
1744.20
139.10
3.98
80.00
2- A. fumigatus
106.44
3898.80
171.36
25.11
90.00
3- A. flavus
436.75
4719.60
153.22
2.23
200.00
4- Nigrospora sp.
4.032
205.20
72.58
28183.80
1000.00
5- Y2 sp.
631.00
179.50
201.60
63.09
191.30
6- Y3 sp.
82.66
9695.70
38.28
not detected
100.00
7- Strain (1)
8.26
35.90
9.88
not detected
100.00
8- Strain (2)
9.88
102.60
14.11
not detected
182.60
9- Strain (3)
11.89
153.90
23.99
not detected
200.00
F (for enzymes) =148402.02, , p< 0.05. F (for strains) =141709.90, p< 0.05. Value are the means of three replicate enzymes


The results recorded in Table 4 show that the enzymes produced from the fermented
orange peel waste corresponds to the type and capacity of the bottles were used. The maximal
production obtained from each of the following enzymes were: ? amylase 219.744 µg/ml,
? amylase 1641.60 µg/ml, cellulase 84.67 µg/ml, pectinase(s) 1584.89 unit/ml (carbohydrate
enzymes) and lipase(s) 1233.00 unit/ml, while the corresponding bottles were used green plastic
(1500 ml), transparent plastic (300 ml), transparent glass (500 ml), green glass (300 ml) and
transparent glass (500 ml). In Table 5 showed the highest value from each of: ? amylase
94.652 µg/ml, ? amylase 1898.10 µg/ml, and cellulase 78.62 µg/ml and the transparent plastic
(300 ml) bottle was the best bottle for their production while for production of pectinase(s)
1548.90 unit/ml, green glass (300 ml) had been used and for lipase(s) 690.10 unit/ml, transparent
plastic (500 ml) was used. From tables 4 and 5, it can be deduced that for the best production of
? amylase, cellulase, pectinase(s) and lipase(s), the orange peel was used, in contrast to the
production of the highest value for ? amylase, where orange pulp was used.

Results obtained for the production of enzymes as a parameter for solid state fermentation
from a substrate of orange peel and pulp demonstrated clearly the impact of the process
parameter on the gross yield of enzymes as well as the independent nature in influencing the
organisms ability to synthesize these enzymes. In general a reduction in the amount of micro-
floral enzymes production in the fermented waste than their formation in their specific media, it
might be due to competition between the micro-foloral and the same substrate and impaired
oxygen transfer (Sandhya & Lonsane 1994). A lower moisture content also resulted in a decline
in enzyme yield. This may result from sub-optimal growth, reduced substrate swelling and high
water tension during low moisture (Lonsane et al. 1985).
26

Attyia and Ashour: Biodegradation of agro-industrial wastes


The particle size (specific surface area) is a critical factor in solid state fermented. A
similar trend was reported for most of the isolated enzymes gluco-amylase from wheat bran
(Pandey 1991) and for cellulase production with coir pith of small particle size (Muniswaran &
Charyulu 1994). With smaller particles the surface area for growth is greater but the inter-particle
porosity is less, whereas with large size the porosity is greater but the saturated surface area is
less. These two opposing factors (a decrease in surface area and increase in porosity) probably
interact to determine the value corresponding to optimum growth and enzyme production
(Muniswaran & Charyulu 1994).

The present investigation was carried out to increase the micro-flora of orange peel and
pulp waste and enhance fastening its natural biodegradation for incubation under natural
environmental conditions. On using the mentioned precursor it is noted that the apparent decay
was always higher when using a plastic bottles as compared to glass bottles, taking only 5 days.
This is probably due to the nature and capacity of the bottles themselves, which were made of
polyethene which can retain the heat temperature and allow the passage of O2 which stimulates
the growth of the micro-floral strains and so minimizes the time of solubilization and
liquefaction. As far as advantages of having a higher solubilization this must be related to the
microorganisms found in the subsequent fermented. By employing fungi capable of utilizing
quite complex compound, also yeasts pushed depolymerization taking place (Vacarino et al.
1989).
Table 4: A comparative study for the determination of the peeled fermented enzymes
Bottle capacity
?-amylase
?-amylase
Cellulase
Pectinase(s)
Lipase
Type & volume (ml)
µg/ml
µg/ml
µg/ml
unit/ml
unit/ml
1- Green glass 300ml
102.816
1179.90
54.43
1584.89
400.00
2- Transperant 300ml (plastic)
145.152
1641.60
80.64
891.20
1166.00
3- Transperant 500ml (glass)
107.654
1385.10
84.67
not detected
1233.00
4- Transperant 500ml (plastic)
42.336
1026.00
52.42
6.30
1100.00
5- Green 1500ml (plastic)
219.744
923.40
76.61
not detected
1000.00
F (for enzymes) = 2694.87, p< 0.05. F (for bottles) = 216.72, p< 0.05. Value are the means of three replicates
Table 5: A comparative study for the determination of the pulp fermented enzymes
Bottle capacity
?-amylase
?-amylase
Cellulase
Pectinase(s)
Lipase
Type & volume (ml)
µg/ml
µg/ml
µg/ml
unit/ml
unit/ml
1- Green glass 300ml
15.52
513.00
46.37
1548.90
182.60
2- Transparent plastic 300ml
94.752
1898.10
78.62
6.00
300.00
3- Transparent glass 500ml
36.88
1026.00
44.35
588.80
312.50
4- Transparent plastic 500ml
60.48
1077.30
48.38
63.09
690.10
5- Green plastic 1500ml
80.64
820.80
34.27
6309.50
312.00
F (for enzymes) = 1645.66, p< 0.05. F (for bottles) = 731.23, p< 0.05. Value are the means of three replicates

Peroxidase isozyme: The banding patterns of peroxidase isozymes as illustrated in Fig. 1A & B,
represents three bands only. Band No. 1 was found in all types of samples for both pulp and
peeled micro-floral peroxidase isozymes with different density and intensity, while band No. 2
was observed in the pulp micro-floral isozyme only (except sample No. 3) with the same density
and intensity. The last band (No. 3) was found for the pulp samples No. 1 and 3 only, while in
peeled samples it was found for sample No. 8 only and with a higher density (concentration).
Comparison indicated partial effectiveness of peroxidase isozymes in the identification the total
micro-flora which is responsible for the biodegradation of both orange pulp and peel waste in
different types and capacities of bottles and as a biochemical genetic marker.
Esterase isozyme: The banding patterns of esterase isozymes were studied in 10 samples of pulp
and peel agro-industrial orange wastes. In Fig. 2 A & B, zymogram analysis by polyacrylamide
gel electrophoresis for esterase isozymes showed three distinct zymogram patterns which

27

Attyia and Ashour: Biodegradation of agro-industrial wastes

provided good markers for identification and characterization and also elucidated reliable
biochemical markers. Band No. 1 was found in sample No. 9 only for the peeled waste, while
band No. 2 is the most variable band and sample No. 3 shows the highest density and intensity.
Samples 1, 2 and 3 for the pulp have the highest density and intensity, especially sample No. 3
for the pulp, while at the same time the third band was present in sample No.1 for the pulp waste
only with very high density.

The result as shown in Fig. 3A & B represents six different bands over all the samples.
The total micro floral protein of the pulp (1---5) and peeled (6---10) fermented wastes are shown
in Fig 3 (A & B) in SDS protein electrophoresis with a molecular weight of 200, 170, 100, 70,
50 and 37 K.D. a band of 200 KD. was present in all samples except No. 4 with different
intensities according to the key below. The band of 170 KD was also present in all 10 samples
(except No. 9) with different intensities, while the band of 100 KD was present in samples
No. 5 & 10 only with the same intensities. The band of 70 KD was present in samples No. 4 & 5
only "pulp" micro-flora, the band of 50 KD was present in all samples except sample No.
4, 5 and 9 with different intensities, but the last band of 37 KD was present in samples No.
1, 6, 8 and 10 with different intensities.

From these results it is evident that the protein electrophoretic bands could be considered
as a useful tool for the identification and characterization of the total micro-floral protein that are
responsible for the biodegradation for the pulp and peel agro-industrial waste and according to
the different bottles capacities.



1 2 3 4 5 6 7 8 9 10
1 2 3 4 5
6 7
8 9 10

















Fig. 2: Banding pattern of esterase of pulp 1-5
Fig. 1: Banding pattern of peroxidase of pulp

and peeled 6-10 orange fermented waste
1-5 and peeled 6-10 orange fermented waste

1 2 3 4 5 6 7 8 9 10









200 KD









170 KD





100 KD





70 KD







50 KD




37 KD

Fig. 3: Banding pattern of total protein of pulp 1-5 and peeled 6-10 orange fermented waste in 200, 170,
100, 70, 50 and 37 KD.

very high density
high density
moderate density
slight density



Starch consists of amylose and amylo pectin, ?-amylase hydrolyses the linear amylose
chain producing a mixture of maltose and glucose, while ?-amylase attacks amylose to produce
successive units of maltose. As the seeds germinate, the process of mobilization of food reserves
starts. There are some soluble reserves in the seeds which are utilized during the early phase of
germination. However, for the later stages of growth, conversion of insoluble reserves to soluble
forms is needed to supply the required nutrition for the embryo growth. Starch is one of the main
food reserves in whose mobilization ?-amylase plays the main role. Specific activity of
?-amylase activity increased with seedling growth (Jain & Khanna 1986). ?-amylase activity is
28

Attyia and Ashour: Biodegradation of agro-industrial wastes

quite high in our treatment (Table 6 for peeld and pulp). The results in Table 6 showed a great
stimulating effect on the seedling growth of Triticum vulgares seeds.

The maximum growth parameters on using transparent glass bottles for peeled
fermentation (500 ml) and green plastic bottles (1500 ml) respectively when compare with the
control (water treatment), while the maximum growth parameters on using the fermented pulp in
transparent plastic bottles (300 & 500 ml) respectively (Table 6). These can be referred to the
activity of ? and ?-amylase enzymes (Jain & Khanna 1986).

In conclusion, from the previous data in the present investigation, it can be concluded
that the production of the best amount of the corresponding enzymes, ? and ? amylases and
pectinase(s) we must inoculated the orange pulp waste with the corresponding strains, Yeast
strain 2, Yeast strain 3 and Nigrospora sp. respectively. However, the maximal production of
the cellulase and lipase enzymes we use the orange peel waste and inoculated with yeast
strain 1 to produce cellulase enzyme, in contrast for lipase(s) production we use the fermented
peel waste. This conclusion can be applied on a large scale especially all types of the
fermented wastes are free from any type of toxins, we deduced this notice experimentally.
Further more, the solubilized fermented orange peel and pulp waste can be used as a bio-
fertilizer which enhance the seedling growth of wheat.

Table (6): Effect of fermented peeled and pulp on seedling growth parameters of Triticum vulgare.

Fresh wt.
Dry wt.
No. of
Shoot
Root

(gm)
(gm)
leaves/
length
length
Treatment
plant
(cm)
(cm)
Untreated (control)
3.0
0.34
5
12.1
4.3
Peeled fermented in green glass bottle (300 ml).
3.0
0.83
6
16.8
10.0
Peeled fermented in transparent plastic bottle (300 ml)
2.1
0.50
6
14.6
6.80
Peeled fermented in transparent glass bottle (500 ml)
3.5
0.95
6
18.5
17.0
Peeled fermented in transparent plastic bottle (500 ml)
2.9
0.80
6
16.1
8.8
Peeled fermented in green plastic bottle (1500 ml)
3.2
0.84
6
17.6
11.8
Pulp fermented in green glass bottle (300 ml).
1.91
0.30
6
6.6
1.3
Pulp fermented in transparent plastic bottle (300 ml).
5.10
1.14
6
19.5
8.0
Pulp fermented in transparent glass bottle (500 ml).
2.68
0.93
5
16.8
8.0
Pulp fermented in transparent plastic bottle (500 ml).
3.86
1.10
5
19.0
16.0
Pulp fermented in green plastic bottle (1500 ml).
1.95
0.61
5
13.3
9.6
F (for parameters) = 227.61, p< 0.05. F (for treatment) = 9.71, p< 0.05.
Value are the means of three replicate for the treatments (control, treated) and the parameters

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30

Document Outline
  • ABSTRACT
  • MATERIALS AND METHODS
  • RESULTS AND DISCUSSION
  • ????? ???? ???? - ???? ???? ?????

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