Kadarmoidheen et al.
ISSN-2277-6079
EFFECT
OF
CELLULOLYTIC
FUNGI
ON
THE
DEGRADATION OF CELLULOSIC AGRICULTURAL
WASTES.
M Kadarmoidheen1, P Saranraj*2, D Stella2
1 - Department of Microbiology, Thiruvalluvar Arts and Science College, Kurinjipadi – 607 302.
2 – Department of Microbiology, Annamalai University, Annamalai Nagar – 608 002.
Science Instinct Publications
Abstract
The fungal isolates bring about most of the cellulose degradation occurring in various
environments. The present study was conducted to study the biodegradation of cellulosic waste
materials viz., paddy straw, sugarcane bagasse and banana stalks using the cellulolytic fungi
isolates, Trichoderma viride, Aspergillus niger and Fusarium oxysporum. The cellulosic wastes
were subjected to various physical and chemical pretreatments for better susceptibility of
cellulosic wastes to fungal enzymes. Among the three fungal isolates examined for their relative
capacity to produce cellulase, Trichoderma viride showed the maximum C1 activity of 9.4 units
per 50 ml broth. Fusarium oxysporum showed the maximum Cx of 62.4 per cent loss in
viscosity. The cellulosic wastes were analyzed for the various biochemical constituents like
hemicelluloses and cellulose. The highest cellulose content of 36.23 per cent was observed in
banana stalks. The highest hemicellulose content of 24.82 per cent was recorded in sugarcane
bagasse. The effects of cellulolytic fungi on the biodegradation of cellulosic wastes were studied
at the periodical interval of 15, 30 and 45 days. Among the three fungal isolates studied
Trichoderma viride was found to be the most efficient in degrading the cellulosic wastes viz.,
paddy straw, sugarcane bagasse and banana stalks decreasing the cellulose content by 53.70,
51.59 and 55.28 per cent respectively. This was followed by Aspergillus niger and Fusarium
oxysporum in their efficiency to degrade the different cellulosic wastes.
Keywords: Cellulose, Cellulolytic fungi, Cellulosic waste and Hemicelluloses.
*
Corresponding Author:
P Saranraj,
Department of Microbiology,
Annamalik University,
Annamalik Nagar,
Chidambaram- 608 002,
Email-ID: microsaranraj@gmail.com
International Journal of Applied Microbiology Science 2012; 1(2): 13- 23
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Kadarmoidheen et al.
Introduction
gricultural wastes contain a high proportion of cellulosic matter which is easily
decomposed by a combination of physical, chemical and biological processes. The
bunch consists of 70% moisture and 30% solid; of which holocellulose accounts for 65.5%,
lignin 21.2%, ash 3.5%, hot water-soluble substances 5.6% and alcohol-benzene soluble 41% [1]. Lignin is an integral cell wall constituent, which provides plant strength and
resistance to microbial degradation [2]. The recognition that environmental pollution is a
worldwide threat to public health has given rise to a new massive industry for environmental
restoration. Biological degradation, for both economic and ecological reasons, has become an
increasingly popular alternative for the treatment of agricultural, industrial, organic as well as
toxic waste. These wastes have been insufficiently disposed off leading to environmental
pollution [3].
Fungi are the main cellulase producing microorganisms, though a few bacteria and
actinomycetes have also been reported to yield cellulase activity. Fungal genera like
Trichoderma and Aspergillus are known to be cellulase producers and crude enzymes
produced by these microorganisms are commercially available for agricultural use. The genus
Aspergillus species attack cellulose producing significant amount of cell free cellulase
capable of hydrolyzing cellulose into fermentable soluble sugars such as glucose; an
important raw material in chemical industries [4]. Aspergillus and Trichoderma specie are
well known efficient producers of cellulases [5]. Several studies by Mandles and Reese [6]
have been carried out to produce cellulolytic enzymes from biowaste degradation process by
many microorganisms including fungi such as Trichoderma, Penicillium and Aspergillus
species etc.
Many microorganisms are capable of degrading and utilizing cellulose and hemicellulose as
carbon and energy sources. During composting, the capacity of thermophilic microorganisms
to assimilate organic matter depends on their ability to produce the enzymes needed for
degradation of the substrate [7]. Both fungi and bacteria have been heavily exploited for their
abilities to produce a wide variety of cellulases and hemicellulases. Most emphasis has been
placed on the use of fungi because of their capability to produce copious amounts of
cellulases and hemicellulases which are secreted into the medium for easy extraction and
purification. In addition, the enzymes are often less complex than bacterial glycoside
hydrolases and can therefore be more readily cloned and produced via recombination in a
rapidly growing bacterial host such as Escherichia coli. However, the isolation and
characterization of novel glycoside hydrolases from Eubacteria are now being widely
exploited. There are several reasons for these shifts, for one, bacteria often have a higher
growth rate than fungi allowing for higher recombinant production of enzymes. Secondly,
bacterial glycoside hydrolases are often more complex and are often expressed in multienzyme complexes providing increased function and synergy. The present study was focused
on biodegradation of cellulosic waste materials viz., paddy straw, sugarcane bagasse and
banana stalks using the cellulolytic fungi isolates, Trichoderma viride, Aspergillus niger and
Fusarium oxysporum.
A
Materials and methods
Isolation of fungi from cellulosic and hemicellulosic materials
From manures
Representative samples of farm yard manure from pits of 6 to 9 inch depth were taken and
mixed well. Out of this sample, a known quantity of manure was added to 100 ml sterile
water blanks and shaken well for 15 minutes. Then serial dilutions were made and 1 ml of the
International Journal of Applied Microbiology Science 2012; 1(2): 13- 23
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Kadarmoidheen et al.
desired dilution was pipetted out into each sterile petridishes and poured with Dubos medium.
The dish was rotated in clockwise and anticlockwise directions so as to disperse the
suspension uniformly in the agar medium and the plates were incubated at room temperature
(28 ± 2°C) for 5 to 7 days. The fungal colonies developed were transferred to Rose Bengal
Agar slants.
From soil
Soil Sample were collected from the paddy field and from the pot culture yard of
Microbiology laboratory from a depth of 6 inches, mixed well and the representative sample
was taken. Required dilutions of the soil sample were plated in Dubos medium as detailed
earlier and the fungal colonies developed were transferred to Rose Bengal agar slants.
From decaying wood
The decaying wood was surface sterilized with 0.1 % mercuric chloride, small bits were cut
and placed in sterile petridishes containing Dubos medium. The dishes were incubated at
room temperature (28 - 30°C). The fungal colonies were transferred into Rose Bengal agar
slants.
Identification of the fungal isolates
Identification of the fungal isolates was carried out by the routine mycological methods i.e.,
Lactophenol cotton blue staining and plating on Sabouraud dextrose agar medium.
Pretreatment of paddy straw
Alkali pre-treatment
For Treatment-A, the rice straws were pre-treated with different percentage of sodium
hydroxide (NaOH), i.e., 5, 10, 15 and 20% each for different soaking time of 1, 2, 3 and 4
hours, respectively. Whereas, Treatment-B includes the treatment of rice straws with different
percentage of potassium hydroxide, i.e., 5, 10, 15 and 20% concentration each for soaking
time of 1, 2, 3 and 4 hours, respectively. After the alkali treatment, the treated rice straws
were then washed with distilled water as much as possible and the pH was adjusted to 7.8
using 1 M hydrochloric acid.
Heat pre-treatment
After the alkali treatment, the treated rice straws were subjected to the heat treatment for 1
hour at 121°C. Then, the fully treated rice straws were dried in the oven and kept for further
use.
Production Medium
The composition of production medium used was paddy straw-5g, KCl-2g, KH2PO4-1g,
MgSO4-0.05g, FeSO4-0.02g and Distilled water-1000ml. The pH was adjusted to 6.5 and the
media was sterilized in an autoclave for 15 min at 121°C. The media was inoculated with a
loop full of spore suspension of Aspergillus niger and then incubated at 30ºC in an orbital
shaker set at 100 rpm for 96 hours. The media were centrifuged at 5000 rpm for 15 minutes
to obtain crude enzyme solution.
Assay of Cellulase enzyme
Assay of Cellulase-C1
Cellulase C1 activity was estimated by the method described by Norkrans [8]. The reaction
mixture consisted of 1 ml of cellulose suspension (the concentration of which was adjusted
approximately to 0.85 absorbance at 610 nm), 4 ml of 0.2 M sodium acetate acetic acid buffer
International Journal of Applied Microbiology Science 2012; 1(2): 13- 23
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Kadarmoidheen et al.
at pH 5.6 and 5 ml of the culture filtrate. Absorbance was determined immediately at 610
nm in specrtronic-20 colorimeter and incubated at room temperature (28 ± 1°C) at the end of
24 hours, the absorbance was again measured and the difference was calculated. The enzyme
activity was expressed in terms of unit (1 unit = 0.01 absorbance at 610 nm).
Assay of Cellulase – Cx
The ability to reduce the viscosity of carboxyl methyl cellulose (CMC) in an Ostwald Fenske
Viscometer size 150 at 30°C was used as the assay for the activity of the enzyme Cx.
Carboxyl methyl cellulose of 0.5 per cent concentration was prepared in sodium acetate –
acetic acid buffer at pH 4.8. To 4 ml of carboxy methyl cellulose solution. 1 ml of acetate
buffer at pH 4.8 and 2 ml of the culture filtrate were added, contents transferred to Ostwald
Fenske Viscometer size 150 placed in water bath at 30 ± 1°C and viscosity measurement
were made at predetermined intervals upto 120 minutes. Per cent loss in viscosity was
calculated by employing the formula.
T0 – T1
Per cent loss of viscosity = ------------ × 100
T0 - Tw
Where,
T0 = flow time at ‘0’ time (seconds)
T1 = flow time at intervals (seconds)
Tw = flow time of double distilled water (seconds)
Screening of fungi for the degradation of cellulosic wastes
The relative efficiency of the fungal isolates to degrade sugarcane bagasse, paddy straw and
banana stalks ware studied in vivo. Modified Rose Bengal broth, standardized in the present
study was prepared without cellulose and dispersed in 50 ml quantities into 250 ml
Erlenmeyer flasks. The cellulosic wastes were separately incorporated into the medium at the
rate of 500 mg per flask. The flasks were sterilized at 10 lb pressure for 15 min inoculated
with 8 mm disc of the pure cultures of the test fungi and incubated at 28 ± 2°C for 45 days.
Following the incubation period of 15, 30 and 45 days, the fungi were killed by a mixture of
ethyl alcohol and formalin, the surface growth was scraped and washed of as far as possible
from the substrate and the proximate constituent viz., hemicelluloses and cellulose content
were determined following the method of Waksman and Stevens [9] as described below.
Estimation of hemicelluloses
The residue after hot water extraction was taken in 95 per cent ethyl alcohol and transferred
to 500 ml volumetric flask and made upto a volume of 150 ml. The extraction was continued
on a sand bath connected with a reflux condenser for 3 hours. The residue from alcohol
extraction was transferred to a flask, 150 ml of 2 per cent HCl added, connected with reflux
condenser and the contents boiled for 3 hours. The digested extract was filtered, thoroughly
washed with water until free from acid and made up the volume to 250 ml with water. The
extract was used to determine the amount of hemicelluloses as detailed below.
The HCl extract was neutralized with 10 per cent NaOH and reducing sugar content was
determined following the colorimetric method. To 1 ml of neutralized extract, one ml of
Nelson’s reagent was added to dissolve completely the cuprous oxide formed. The solution
was made upto 25 ml and the intensity of blue colour was read in a spectronic-20 at 495 nm.
The amount of hemicellulose present in the extract was calculated by multiplying the total
reducing sugars in 2 per cent HCl extract by 0.9 and the hemicellulose in original 250 mg
portion of the dried material was calculated.
International Journal of Applied Microbiology Science 2012; 1(2): 13- 23
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Kadarmoidheen et al.
Estimation of Cellulose
The residue left after HCl extraction was placed in 250 ml Erlenmeyer conical flask, 8 ml of
concentrated H2SO4 was added, mixed thoroughly and left in cold for 2 hours with
occasional stirring to convert cellulose to hydrocellulose. At the expiry of the time, 150 ml of
water was added and boiled under reflux condenser for 5 hours to transform hydrocellulose to
reducing sugars. The residue was thoroughly washed with water until free from acid and
made upto a volume of 250 ml. The reducing sugars were estimated and the amount of
reducing sugar was multiplied by 0.9 to obtain the amount of cellulose [9].
Results and discussion
Bioconversion of cellulose-containing raw materials is an important problem of current
biotechnology due to the increasing demand for energy, food and chemicals [10]. Cellulases
are enzymes which hydrolyse the β-1,4- glycosidic linkage of cellulose and is synthesized by
microorganisms during their growth on cellulosic materials [11]. The complete enzymatic
hydrolysis of cellulosic materials needs different types of cellulase; namely
endoglucanase,(1,4-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exocellobiohydrolase(1, 4-Dglucan glucohydrolase) and glucosidase (D-glucoside glucohydrolase) [12]. Agricultural
wastes contain a high proportion of cellulosic matter which is easily decomposed by a
combination of physical, chemical and biological processes. Lignin is an integral cell wall
constituent, which provides plant strength and resistance to microbial degradation.
In the present study, three fungal isolates were isolated and identified as Aspergillus niger,
Trichoderma viride and Fusarium oxysporum. The fungal isolates were examined to produce
cellulases, C1 and Cx, they were grown on Dubos medium and the results are presented in
Table-1. Among the three isolates, Trichoderma viride showed the maximum C1 activity of
9.4 units per 50 ml of the broth followed by Fusarium oxysporum and Aspergillus niger
which recorded 3.2 and 1.9 units respectively per 50 ml of the broth. The maximum Cx
activity of 62.4% loss in viscosity was observed in Fusarium oxysporum, followed by
Trichoderma viride and Aspergillus niger which showed 52.3% and 42.3% loss in viscosity
per 50 ml broth respectively. Both fungi and bacteria have been heavily exploited for their
abilities to produce a wide variety of cellulases and hemicellulases. Most emphasis has been
placed on the use of fungi because of their capability to produce copious amounts of
cellulases and hemicellulases which are secreted in the medium for easy extraction and
purification [13].
In this research, cellulosic wastes viz., paddy straw, sugarcane bagasse and banana stalks
were analyzed for the biochemical constituents viz., hemicelluloses and cellulose content
and the results are presented in Figure-1. The highest cellulose content of 36.23% was
recorded with banana stalks followed by sugarcane bagasse and paddy straw which contained
35.64%, 34.82% respectively. The highest hemicellulose content of 24.82% was recorded
with sugarcane baggase followed by banana stalks and paddy straw which contained 21.36%
and 21.24% hemicelluloses respectively.
Cellulose, being an abundant and renewable resource, is a potential raw material for the
microbial production of food, fuel and chemicals [14]. Various bacteria, actinomycetes and
filamentous fungi produce extracellular cellulases when grown on cellulosic substrates
though many actinomycetes have been reported to have less cellulase activity than moulds
[15]. Investigations on the extracellular cellulases of fungi have been concentrated mainly on
Trichoderma sp. [16] and studies on other mesophilic fungi suggested the possibility that
other cellulase systems could be utilized for the hydrolysis of cellulose [17]. Ajay Singh et al.
[18] studied cellulase production by Aspergillus niger AS-101, grown on alkali treated corn
cobs under various cultural conditions. The maximum yields of single cell protein and
International Journal of Applied Microbiology Science 2012; 1(2): 13- 23
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Kadarmoidheen et al.
cellulase, under solid-state fermentation were obtained when the culture was incubated at pH
5.5 for 18 d at 30°C with 12% substrate.
Plant lignocellulosics as organic substances are subjected to attacks by biological agents such
as fungi, bacteria and insects [19, 20]. Filamentous fungi which use cellulose as carbon
source possess the unique ability to degrade cellulose molecules in plant lignocellulose.
A laboratory experiment was conducted to find out the efficiency of cellulolytic fungi viz.,
Trichoderma viride, Aspergillus niger and Fusarium oxysporum to degrade the paddy straw
and the results was given in Table-2. Among the fungal isolate Trichoderma viride was
found to be the best in degrading cellulose of paddy straw which brought down the cellulose
content of paddy straw of 34.82% to 16.12% followed by Aspergillus niger and Fusarium
oxysporum which brought from 21.30% and 23.32% respectively, in a period of 45 days of
incubation. The highest percentage decrease of cellulose content was 53.70% by
Trichoderma viride followed by Aspergillus niger and Fusarium oxysporum which recorded
a decrease of 38.82% and 33.02% respectively after 45 days of inoculation. The results are
presented in Figure- 2. Hemicelluloses was also efficiently degraded by Trichoderma viride
which brought down 21.84% of hemicellulose to 14.12% in a period of 45 days of incubation
followed by Aspergillus niger and Fusarium oxysporum and which brought down 16.21%
and 17.35% respectively. The highest percentage decrease of hemicelluloses was 33.54% by
Trichoderma viride followed by Aspergillus niger and Fusarium oxysporum which recorded
a decrease of 23.68% and 18.31% respectively after 45 days of inoculation.
Among the three fungal isolates Trichoderma viride was found to be the best in degrading
cellulose of sugarcane bagasse which brought down the cellulose content of sugarcane
bagasse from 35.64% to 17.25% followed by Aspergillus niger and Fusarium oxysporum
which reduced to 21.36% and 25.39% respectively in a period of 45 days of incubation. The
results are presented in Table-3. The highest percentage decrease of cellulose content was
51.59% by Trichoderma viride followed by Aspergillus niger and Fusarium oxysporum
which recorded 40.06% and 28.75% respectively after 45 days of inoculation. The results are
presented in Figure- 3. Hemicellulose was most efficiently degraded by Trichoderma viride
which brought down 24.82% of hemicelluloses to 16.34% in a period of 45 days of
incubation followed by Aspergillus niger and Fusarium oxysporum which reduced the
hemicelluloses content to 19.32% and 20.49% respectively. The highest percentage decrease
of hemicellulose content was 34.16% by Trichoderma viride followed by Aspergillus niger
and Fusarium oxysporum which recorded a decrease of 22.15% and 17.56% respectively
after 45 days of inoculation.
The fungal isolate Trichoderma viride was found to be best the in degrading cellulose of
banana stalks, which brought down the cellulose content of banana stalks from 36.23% to
16.20% followed by Aspergillus niger and Fusarium oxysporum which reduced to 23.50%
and 26.79% respectively in a period of 45 days of incubation. The results are presented in
Table 4. The highest percentage decrease of cellulose content was 55.28% by Trichoderma
viride followed by Aspergillus niger and Fusarium oxysporum which recorded 35.13% and
26.05% respectively after 45 days of inoculation. The results are presented in Figure- 4.
Hemicellulose was most efficiently degraded by Trichoderma viride which brought down
21.36% of hemicelluloses to 15.72% in a period of 45 days of incubation followed by
Aspergillus niger and Fusarium oxysporum which reduced the hemicelluloses content to
15.98% and 16.75% respectively. The highest percentage decrease of hemicelluloses content
was 26.40% by Trichoderma viride followed by Aspergillus niger and Fusarium oxysporum
which recorded a decrease of 25.18% and 21.58% respectively after 45 days of inoculation.
A large number of microorganisms are capable of degrading cellulose, only a few of these
produce significant quantities of cell-free enzymes capable of completely hydrolyzing
International Journal of Applied Microbiology Science 2012; 1(2): 13- 23
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Kadarmoidheen et al.
crystalline cellulose in vitro [21]. Many microorganisms are capable of degrading and
utilizing cellulose and hemicellulose as carbon and energy sources. During composting, the
capacity of thermophilic microorganisms to assimilate organic matter depends on their ability
to produce the enzymes needed for degradation of the substrate [22]. Enzymatic hydrolysis
processing of cellulosic materials could be accomplished through a complex reaction of
various enzymes. Cellulases are inducible enzymes which are synthesized by microorganisms
during their growth on cellulosic materials.
Milila et al. [23] used rice husk, millet straw, guinea corn stalk and sawdust as fermentation
feed substrate for the evaluation of cellulase activity secreted by Aspergillus candidus. Rice
husk and millet straw had maximum enzyme activity at pH 5, while guinea corn stalk and
sawdust had maximum activity at pH 3 and 4, respectively. Enzymatic hydrolysis of
celluloses, the most abundant renewable resource on the earth, offers an attractive alternative
for the generation of sugars which can serve as the raw material for the production of various
products of commercial interest such as bioethanol [24, 25]. A number of approaches have
been adopted, aiming towards reducing the cost of enzyme production, these have included
the use of different lingo-cellulosic wastes including sawdust [26], corn cob [27], bagasse
[28], wheat straw [29, 30] and rice straw [31].
Table – 1:
Cellulolytic activity of fungal isolates
Fungal isolate
Trichoderma viride
Aspergillus niger
Fusarium oxysporum
Activity of enzymes in 50 ml broth
C1
CX
9.4
52.3
1.9
42.3
3.2
62.4
C1 – Expressed in units per 50 ml of the broth
CX – Expressed as per cent loss in viscosity
Fig.1.
Estimation of biochemical constituents of different cellulosic wastes
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Kadarmoidheen et al.
Fig.2.
Fig.3.
Effect of pretreatment of paddy straw by certain cellulolytic fungi on the
percentage content of different biochemical constituents
Effect of pretreatment of sugarcane bagasse by certain cellulolytic fungi on the
percentage content of different biochemical constituents.
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Kadarmoidheen et al.
Fig.4.
Effect of pretreatment of banana stalks by certain cellulolytic fungi on the
percentage content of different biochemical constituents
Table: 2.
Effect of certain cellulolytic fungi on the biodegradation of paddy straw.
Percentage contents after degradation
Percentage
of
Biochemical
constituent
in
constituents
original
Trichoderma viride
Aspergillus niger
Fusarium oxysporum
Sampling time in days
material
15
30
45
15
30
45
15
30
45
Hemicellulose
21.24
18.97
17.32
14.12
20.23
18.98
16.21
20.97
19.23
17.5
Cellulose
34.82
28.71
22.34
16.12
31.34
26.36
21.30
30.19
26.79
23.32
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Kadarmoidheen et al.
Table-3:
Effect of certain cellulolytic fungi on the biodegradation of sugarcane bagasse
Percentage contents after degradation
Percentage
of
Biochemical
constituent
in
constituents
original
Trichoderma viride
Cellulose
Table-4:
Fusarium oxysporum
Sampling time in days
material
Hemicellulose
Aspergillus niger
15
30
45
15
30
45
15
30
45
24.82
21.36
18.32
16.34
23.37
21.28
19.32
23.94
22.12
20.46
35.64
29.63
23.45
17.25
32.34
27.41
21.36
32.41
28.21
25.39
Effect of certain cellulolytic fungi on the biodegradation of banana stalks
Percentage contents after degradation
Percentage
of
Biochemical
constituent
in
constituents
original
Trichoderma viride
Cellulose
Fusarium oxysporum
Sampling time in days
material
Hemicellulose
Aspergillus niger
15
30
45
15
30
45
15
30
45
21.36
19.73
17.31
15.72
19.34
17.14
15.98
20.35
18.55
16.75
36.23
29.36
23.76
16.20
31.37
27.92
23.50
32.37
29.84
26.79
Conclusion
The research results confirmed that the rates of cellulose conversion are many times higher
than the rates of natural microbial decay, the reaction times needed for high conversion yields
are still relatively long, about 5-10 days. These results indicate that the cellulose fiber cannot
be treated as uniform substrates which are hydrolyzed to completion at the rate determined by
the most resistant member of the fiber population. Further research on cellulose and cell wall
chemistry will be needed to help illuminate differences in the susceptibility of cellulose fibers
to both chemical pretreatment and enzymatic hydrolysis. Pretreatment research has to be
performed in conjunction with the further development of better cellulase enzyme complexes,
since there is a strong interaction between the two systems. The most important criterion that
needs to be done is to work out the cost benefit ratio with pilot scale study.
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