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Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
217
A Study on Strategies Applied for Enhancing Anthraquinones Production
by Fusarium spp.
M.Z. El-Fouly 1, E.A. Hassan2, A.A.M Shahin1, H. A. El-Bialy1, E.M. Ramadan2
and A. A.Alsharqawey1. (1) Radiation Microbiology Department, National Center for Radiation Research and Technology
(NCRRT), Atomic Energy Authority, Cairo, Egypt (2)Faculty of Agriculture, Ain Shams University, Cairo, Egypt
Received: 3/2/2016 Accepted: 14/8/2016
ABSTRACT
Sixty Fusarium isolates were selected from different isolation sources and
screened for their ability to produce anthraquinones; seventeen of which showed
high or a moderate ability to produce anthraquinones. Selected Fusarium isolates
were screened for Fusaric acid production to exclude toxin-synthesis isolates. F.
arthosporoides and F. verticellioides showed the highest anthraquinones production
since their production yields were 649.1 and 275.7 µg/g; respectively. The
anthraquinones derivatives produced by selected Fusarium strains were
characterized by HPLC and GC-MS. The optimization of fermentation conditions
for F. arthosporoides revealed that the maximum anthraquinones titer was achieved
at 10 days of incubation period, pH 6.5 and 30°C and under shaking and light
conditions. For F. verticellioides, the highest anthraquinones yield was accomplished
after the same incubation period at pH 6.0, 25°C and under static and dark
conditions. Results evaluated the positive effect of ionizing (gamma) and non-
ionizing (UV) irradiations on the anthraquinones production by F. verticellioides
since 0.25 kGy and 50 J/m2 enhanced the anthraquinone yield by nearly 30%. The
antimicrobial and dyeing properties of the produced anthraquinone are also
studied. The present study succeeded to reduce the cost of anthraquinones
production by using kitchen garbage.
Keywords: Fusarium spp., Anthraquinones, Optimization, Ionizing and Non-ionizing
Irradiations, Kitchen Garbage, HPLC, Antimicrobial Activity andTextile
Dyeing.
INTRODUCTION
Anthraquinones are polycyclic organic compounds with chemical formula C14H8O2; consisting
of three fused rings of benzene with keto groups on the central ring. Natural anthraquinones vary due
to presence of different substituents; such as –OH, –CH3, –OCH3, –CH2OH, –CHO, –COOH, etc. A
group of anthraquinones derivatives have been identified from various species of fungi and lichens as
well as in plants.
Many aspects of environmental concerns are associated with anthraquinones including
manufacture of hydrogen peroxide, the base of many non-polluting oxidizing agents (1) and
manufacture of novel functional biofilm carrier and membrane materials with better hydrophilicity,
biocompatibility and stability than traditional ones (2).
Anthraquinones are used as catalysts with soda in Kappa Kraft during bleaching process of non-
wood pulps. They increase the rate of delignification, reduce the pulping time, temperature, or
chemical charge and decrease pulp crystanility so they improve the yield and viscosity of the pulp to
produce papers with fairly good brightness and tear resistance (3).
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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Anthraquinones are used as substrates in the chemical production of various dyes and pigments
as well as natural pigments with good colour-fastness for different textile materials especially wool
and silk since they have an affinity for these fibers without the aid of auxillary binding materials (4)
,they are also used in food industry (5). Because of the photophysical and electrochemical properties of
anthraquinones; they are used as sensitizers of thermodynamic dye-sensitizing solar cells (6) and
semiconductors conduction bands (7).
In addition, they are applied in aquaculture industry as algicides that prevent the growth of
cyanobacteria responsible for causing musty-off flavor in pond guest cultured catfish (8).
The present study aims to discuss some strategies applied for anthraquinones production from a
microbiological point of view. To accomplish this aim, the following steps are carried out: (1)
selecting new candidates capable of producing anthraquinones from local sources; (2) optimizing the
fermentation conditions for anthraquinones production by selected Fusarium spp; (3) Improving the
anthraquinones productivity by selected candidates using ionizing and non-ionizing radiations; and (4)
using a low cost media for reducing the cost of anthraquinones production process; and (5)
investigating the antimicrobial and dyeing properties of produced anthraquinones.
MATERIAL AND METHODS
1. Isolation of Fusarium spp.
Ten different soil samples representing different soil types available in Egypt; alkaline, clay,
saline and sandy were selected and kept inside sterilized plastic bags at 4°C until being used. Rotten
vegetables and fruits as well as infected plants belong to Allium sativum, Brahea armata, Capsicum
annuum, Cucumis sativus, Cynodon sp., Lactuca sativa, Matrecaria chamomina, Psidium guaja, Rosa
arabica, Sabal palmetto, Solanum lycopersicum and Triticum sp. were sampled under aseptic
condition. Fungal isolates, belonging to Fusarium species, were selected and purified using standard
isolation and purification methods. The highly anthraquinones producers were identified as Fusarium
arthosporoides and F. verticellioides at Mycological Center, Faculty of Science, Assuit University,
Egypt.
2. Cultivation Conditions and Optimizing the Anthraquinones Production
Sixty fungal isolates, belonging to Fusarium species, were preliminary screened for pigment
production by visual observation of red pigmentation on potato dextrose medium, as a preliminary
indication for anthraquinones production. For anthraquinones production, a defined mineral medium
containing K2HPO4, 1.4g; (NH4)2SO4, 1.0; KH2PO4, 0.6g; MgSO4·7H2O, 0.5g; ZnSO4·7H2O, 0.8mg;
Na2MoO4·2H2O, 0.8mg; CuSO4·5H2O, 0.8mg; FeCl3·6H2O, 0.8mg; MnSO4·H2O, 0.4mg was used and
supplemented with 30g of glucose (pH 5.6). Cultivation was performed in 250 ml conical flasks
containing 100 ml of the previously mentioned medium that inoculated with two disks of actively
growing margin of fungal cultures and then incubated in an orbital shaker (agitation speed 150 rpm) at
25±1 °C for 7 days.
The anthraquinones production by selected and identified fungal strains (Fusarium
verticellioides and Fusarium orthosporoides) was estimated at different incubation periods (ranged
from 4 d to 20 d), different temperatures (10, 15, 20, 25, 30, 35 and 40 °C) and various pH values
(ranging from 4.5 to 7.5). In an individual experiment, the selected Fusarium spp. were incubated at
the optimal cultivation conditions determined from previous experiments under shaking (150 rpm) or
static conditions at different illumination regimes (light or dark). Illumination condition was set up
using an incubator equipped with four fluorescent tube lamps. Flasks represented dark conditions were
covered with black carbon paper.
To reduce the cost of anthraquinones production process, seven kitchen wastes were collected
including banana peels, carrot peels, guava peels, lettuce leaves, tomato peels, watermelon peels
(white part) and zucchini peels and dried at 65°C for 36 h. Ten grams for each studied waste were
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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supplemented with 5ml of mineral solution (10x of minerals present in defined mineral media),
inoculated with spore suspension of selected fungal isolates and incubated at 28°C ± 2.0.
3. Analytical Analysis
Eleven Fusarium species showing high ability to produce anthraquinones in the preliminary
experiment were tested for toxin production to exclude the active Fusaric acid producer, the most
common toxin produced by Fusaium species. Selected fungal isolates were incubated in Czapek broth
medium and incubated in the dark at 25°C for 10 days. At the end of incubation period, fungal cultures
were filtered using whatman filter paper no. 1 and the filtrates were extracted three successive times
with an equal volume of chloroform. The combined chloroform extracts were concentrated and dried
using rotary evaporator; the residues were dissolved in 2 ml chloroform (9).
Anthraquinones was extracted by a method described by Itoh and his collaborates (10). To each
growing culture filtrate, an equal amount of chloroform was added with vigorous shaking for 15 min.
The chloroform layer containing anthraquinones was evaporated under reduced pressure at 30oC and
re-eluted with 5 ml of chloroform (HPLC-grade) and the absorbance of pigment was determined at
325 nm by spectrophotometer. A standard curve of anthraquinones was established using various
concentrations of purified anthraquinones (0.02-0.05 mg/l). The absorbance values are converted to
anthraquinones concentration using the standard curve and expressed in mg/l of fermentation medium.
The anthraquinones were characterized by HPLC located at Mycological center (Al-Azhar
Univ., Cairo) using GBC UV/Vis detector at 254 nm with the ODS C18 column (200 mm x 4.6mm x
5µm i.d.). The mobile phase was methanol : 0.1% phosphoric acid (75:25 V/V) and the flow rate was
1.0 ml min-1. Peak area was measured with a semicomputing integrator (Win Chrome Chromatography
Version 1.3).
Fungal growth was determined as dry weight; fungal mats were dried in an oven at 70ºC for 24
hrs till constant weights (11).
4. Irradiation Studies
The spore suspension of selected fungal isolates were collected under aseptic condition and
irradiated at room temperature using Cobalt-60 Russian model gamma cell (Issledovate) located at the
National Center for Radiation Research and Technology, Atomic Energy Authority of Egypt . The
dose rate at the time of experiment was 4.519 kGy/hour and the total activity of the source was 16134
cuire. Portions of defined mineral medium were inoculated by equal volumes of fungal suspension that
previously irradiated with low doses of gamma radiation (0, 0.25, 0.5, 0.75, 1.0 kGy) and incubated at
optimal cultivation conditions for each studied fungal isolate.
For UV irradiations, spore suspension for each fungal isolates was suspended in saline solution,
vortexed for 20 s, then aseptically irradiated with UV light from a germicidal lamp (254 nm) which
was placed 10 cm above the surface of the cells (UV source was warmed for 20 min before use). Five
milliliter of the irradiated solution was withdrawn after 10, 20, 25, 30, 35 and 40 min, and used as
inoculum for Erlenmeyer flasks containing the same mineral medium and incubated at optimal
conditions for each selected candidates. Fungal growth yield and anthraquinones production were
determined as previously mentioned. The intensity of the UV source was estimated by an intensimeter
code radiometer provided with a UV sensor at 254 nm wavelength. All the manipulations and the
incubation were carried out in the dark to avoid photoactivation (12).
5. Application Studies
The antimicrobial activity of crude anthraquinone was studied against clinical isolates of
Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa, Salmonells typhi and Staphylococcus
aureus as well as Candida albicans (identified and maintained in the Department of Microbiology,
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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National center for Radiation Research and Technology). The activity was estimated by pore diffusion
technique on Brain Heart Infusion medium for bacterial pathogens and on Sabroad’s dextrose agar
medium for pathogenic yeast (13). Plates were incubated for 18 h at 37°C; after which, the inhibition
zone was determined by milliliter and expressed as a percentage related to a broad spectrum antibiotic
(amoxicillin). The Dyeing properties of crude anthraqinones were studied for different types of textile
fabrics including cotton, acetate, aramid, polyester and wool in an individual experiment.
RESULTS
Ten soil samples represent all types of soil present in Egypt (alkaline, clay, salty and sandy) and
rotten fruits and vegetables were collected as well as infected plants having clear spots of fungal
infection with Fusarium spp. Sixty isolates belong to Fusarium spp were collected from previously
mentioned isolation resources (Table 1). As expected, poor capability of Fusarium spp to spread in
high alkalinity and salinity environments is observed since fungal growth favors acidic pH whereas the
infected Allium sativum (garlic) and Solanum lycopersicum (tomato) are excellent isolation resources
for Fusarium spp.
Table (1): Isolation of Fusarium spp from different sources
Seventeen Fusarium isolates were selected by visual observation of pigmentation in a
preliminary experiment (Data not shown); six from soil samples (symbolized as F6, F11, F13, F14, F21,
F25) and eleven from other isolation resources (symbolized as F40, F42, F45, F52, F53, F54, F55, F56, F57,
F58, F59). They were tested for toxin production to exclude the fungi able to produce Fusaric acid, the
most common toxin produced by Fusaium species. Five isolates which showed no capability to
Sample no. Origin No. of Fusarium isolates (symbols)
1 Alkaline soil (Al-Dakhlia governorate) 3 (F1, F2, F3)
2 Clay soil (Al-Sharkia governorate) 1 (F4)
3 Clay soil (Cairo governorate) 1 (F5)
4 Clay soil (Cairo governorate) 5 (F6, F7, F8, F9, F10)
5 Clay soil (Cairo governorate) 1 (F11)
6 Clay soil (Cairo governorate) 4 (F12, F13, F14, F15)
7 Salty soil (Al-Behera governorate) 3 (F16, F17, F18)
8 Sandy soil (Al-Kalibia governorate) 7 (F19, F20, F21, F22, F23, F24, F25)
9 Sandy soil (Al-Behera governorate) 1 (F26)
10 Sandy soil (Ismailia governorate) 2 (F27, F28)
11 Allium sativum (Garlic) 11 (F29, F30, F31, F32, F33, F34, F35, F36,
F37, F38, F39)
12 Brahea armata (Mexican blue palm) 1 (F40)
13 Capsicum annuum (Peeper) 1 (F41)
14 Cucumis sativus (Cucumber) 1 (F42)
15 Cynodon sp. (Bermuda grass) 2 (F43, (F44)
16 Hyphaene sp. (Doum palm) -
17 Juglans regia (Walnut) -
18 Lactuca sativa (Lettuce) 3 (F45, F46, F47)
19 Matrecaria chamomina (Chamomile) 1
20 Psidium guaja (Guava) 1 (F49)
21 Rosa arabica (Arabic rose) 1(F50)
22 Sabal palmetto (Cabbage) 1 (F51)
23 Solanum lycopersicum (Tomato) 8 (F52, F53, F54, F55, F56, F57, F58, F59)
24 Triticum sp. (Wheat) 1
25 Vitis vinifera (Grape vine) -
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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produce fusaric toxin were selected and individually screened for anthraquinones production. It is
clear from the data illustrated in Figure (1) that Fusarium isolate (F11) selected from clay soil is the
highest anthraquinone producer (650.0 µg/g) followed by isolate number (F42), the causal infection of
Cucumis sativus (275.8 µg/g). These isolates were identified at Mycological Center, Faculty of
Science, Assiut University as Fusarium arthosporoides and Fusarium verticellioides, respectively.
The production of anthraquinones by selected fungal strains was confirmed by HPLC using authentic
synthetic anthraquinones (Figure 2).
0
100
200
300
400
500
600
700
F11 F42 F45 F55 F58
An
thra
qu
ino
ne
s p
rosu
ctio
n
Selected Fusarium spp
μg/g
Fig. (1): Screening of selected Fusarium spp for anthraquinones production
Fig. (2): HPLC spectra of anthraquinones
A-Standard B-Produced by F . arthosporoides C- Produced by F verticellioides
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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From the industrial application point of view, establishing favorable fermentation conditions
including incubation period, incubation temperature, pH values, and aeration and illumination
conditions are considered critical criteria. Figure (3) illustrates the pattern of anthraquinones
production by selected fungal isolates; F. arthosporoides and F. verticellioides with time is similar to
the titer of anthraquinones production increased with time up to 10 days then slightly decreased. The
highest growth yield was achieved at the seventh day for F. arthosporoides and at the tenth day of
incubation period and F. verticellioides.
Fig. (3): Pattern of anthraquinones production by selected Fusarium strains with time under light
regime.
One of the most influential factors affecting the fungal community is pH. As expected, acidic
pH (5) favors the growth yield of F. verticellioides (Figure 4). On the other hand, the growth yield of
F. arthosporoides was enhanced at neutral pH values (6.5 and 7). Results also revealed that the
anthraquinones titer was attained at pH 6 and 6.5 values for F. arthosporoides and F. verticellioides,
respectively. The influence of different temperatures on the anthraquinones production by F.
arthosporoides and F. verticellioides were studied and illustrated in figure (5). It is clear that the
anthraquinones production by F. arthosporoides was almost similar in a broad range of temperature
degrees (20-35°C) whereas it obviously decreased by increasing temperature more than 25°C in case
of F. verticellioides. The highest growth yield of selected fungal strains was recorded at 25°C.
In an individual experiment, the dual effect of illumination and aeration conditions was studied.
Results revealed that illumination conditions has no effect on the anthraquinones production under
complete aerobic condition (shaking) in case of F. arthosporoides (Table 2) whereas under partially
aerobic condition(static), light enhanced anthraquinones production by verticellioides. Dark conditions
0
50
100
150
200
250
300
350
400
450
500
4 7 10 15 20
Incubation period (d)
Gro
wth
yie
ld (
mg
/100m
l)
0
50
100
150
200
250
300
350
400
450
500
An
thra
qu
ino
nes p
rod
ucti
on
(μg
/100m
l)
Anthraquinones production (µg/100ml) Growth yield (mg/100ml)
F. arthosporoides F. verticellioides
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
223
Fig. (4): The influence of different pH values on anthraquinones production by selected Fusarium
strains after ten days of incubation
Fig. (5): The influence of different temperatures on anthraquinones production by selected Fusarium
strains
nearly doubled the anthraquinones titer whatever the type of aeration condition (shaking or static) and
partially aerobic condition (static) nearly enhanced anthraquinones production by four folds in
comparison with fully aerobic condition (shaking). Growth yield of selected Fusarium strains follows
a similar pattern under a dual effect of illumination and aeration conditions.
0
50
100
150
200
250
300
350
400
450
500
4.5 5 5.5 6 6.5 7 7.5
pH values
Gro
wth
yie
ld (
mg
/100m
l)
0
50
100
150
200
250
300
350
400
450
500
An
thra
qu
ino
nes p
rod
ucti
on
(μg
/100m
l)
Anthraquinones production (µg/100ml) Growth yield (mg/100ml)
F. arthosporoides F. verticellioides
0
50
100
150
200
250
300
350
400
450
500
10 15 20 25 30 35 40
Incubation temperatures (°C)
Gro
wth
yie
ld (
mg
/100m
l)
0
50
100
150
200
250
300
350
400
450
500A
nth
raq
uin
on
es p
rod
ucti
on
(μg
/100m
l)
Anthraquinones production (µg/100ml) Growth yield (mg/100ml)
F. arthosporoides F. verticellioides
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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Table (2): The influence of dual effect of aeration and illumination conditions
Since a low cost cultivation media is a common strategy used by technologists to reduce the
cost of valuable products synthesized by different microorganisms, in the present study, seven kitchen
wastes available in Egypt were used as sole carbon and nitrogen sources for enhancing anthraquinones
production at the bench scale. Data in table (3) revealed that carrot peels followed by watermelon
peels were the best wastes for anthraquinones production by selected fungal strains; they supported
227.2 & 226.0 and 306.6 & 242.0 μg/100ml for F. arthosporoides and F. verticellioides, respectively.
Generally, zucchini peels, tomatoes peels and lettuce leaves also achieved good results. There is no
great difference among the growth yield of the selected fungal strains when they were grown on
different wastes under investigation but in lower yield than that achieved on synthetic minimal mineral
medium (Data not shown). In addition, guava peels achieved a high yield of anthraquinones, 210
μg/100ml for F. arthosporoides.
Table (3): The anthraquinones production of selected Fusarium strains grow on different kitchen
wastes*
Mutagenesis is a classical strategy to improve the activity of microorganisms to produce
valuable compounds. F. arthosporoides and F. verticellioides were exposed to increasing doses of
ionizing and non-ionizing radiations to enhance the anthraquinones production. Results revealed that
gamma irradiation doses below 1kGy and UV irradiation at 50 J/m2 increased the anthraquinones titer
of F. verticellioides where 0.25 kGy of gamma irradiation doubled the production process whereas
Growth yield
(mg/100ml)
Anthraquinones
production
(µg/100ml)
Dual effect of aeration and
illumination conditions Selected Fusarium strains
431.8 341.6 Light Shaking
F. arthosporoides 432.4 339.6 Dark
404.2 210.2 Light Static
325.2 117.8 Dark
327.8 160.0 Light Shaking
F. verticellioides 404.0 289.0 Dark
321.6 458.8 Light Static
324.4 921.8 Dark
Anthraquinones
production
(µg/100ml)
Different kitchen garbage Selected Fusarium strains
36.3 Banana peels
F. arthosporoides
227.2 Carrot peels
210.0 Guava peels
82.0 Lettuce refused leaves
104.0 Tomatoes peels
226.0 Watermelon peels
152.7 Zucchini peels
32.5 Banana peels
F. verticellioides
306.8 Carrot peels
126.3 Guava peels
239.1 Lettuce refused leaves
160.1 Tomatoes peels
242.0 Watermelon peels
222.0 Zucchini peels
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
225
all other irradiation doses either ionizing and non-ionizing radiations affected anthraquinones
production by selected F. arthosporoides negatively (Figures 6 &7).
Fig. (6): The influence of different doses of ionizing radiation (Gamma irradiation) doses on
theanthraquinones production by selected Fusarium strains
Fig. (7): The influence of different doses of non-ionizing radiation (UV irradiation) doses on the
anthraquinones production by selected Fusarium strains.
It’s worth mentioning that the concentration of anthraquinones used in the application studies
was determined by cytotoxicity assay against colon-9 normal cells (Data not shown). Figure (8) shows
that crude anthraquinones (50μg active ingredient) has a higher effect on the growth of G+ve bacteria
than on G-ve bacteria and pathogenic yeast. The highest antimicrobial activity was against Bacillus
cereus followed by Staphylococcus aureus. In case of G-ve bacteria, the highest antimicrobial activity
was against Salmonella typhi then Pseudomonas aeruginosa followed by Escherichia coli, moderate
antimicrobial activity against Candida albicans was observed. An experiment was conducted to test
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
226
the dyeing ability of anthraquinones to different types of textiles; cotton, acetate, aramid, polyester and
wool. Figure (9) reveals that the dyeing property of crude anthraquinones is more efficient for wool
and polyester followed by cotton and acetate fabrics.
0 50 100 150 200
Bacillus cereus
Candida albicans
Escherichia coli
Pseudomonas aeruginosa
Salmonella typhi
Staphylococcus aureus
Ffficiency of crude anthraquinones (%)*
Mic
rob
ial
pa
tho
gen
s
F. Arthsporioides F. Verticillioides
(*) Antimicrobial activity of crude anthraquinones compares to Amoxicillin® antibiotic
Fig. (8): The efficiency of crude Anthraquinones as an antimicrobial agent compared to a broad
spectrum Amoxicillin® antibiotic.
Fig. (9): The efficiency of crude Anthraquinones as a dyeing stuff.
DISCUSSION
Fungi produce a multitude of secondary metabolites, which have roles in a range of cellular
processes such as transcription, development and intercellular communications. In addition, many of
these compounds have industrial applications (14).
The genus Fusarium is widely distributed, occurring in most climatic regions of the world,
especially France, Northern Algeria, Spain, Morocco, Italy, Egypt, Germany, Syraia, Greece,
Lebanon, Turkey, Iran, Iraq and Pakistan. It generally persists in soil, plant residue and organic matter.
This genus includes a large and complex group of fungi ascomycete teleomorph involved numerous
species producing naxious secondary metabolites (15). In the present study, sixty Fusarium isolates
were isolated from different isolation sources including soil, rotten vegetables and fruits as well as
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
227
infected plants. Similarly, El-Kady and his collaborates isolated nearly three hundreds Fusarium
isolates from 25 cereals samples including wheat, barley, maize and sorghum (collected from different
places in Egypt). They found that Fusarium oxysporum was the most frequent species in all tested
samples and contributed about 78% of the total Fusarium isolates (16). One hundred and sixty-six
Fusarium isolates belonging to 24 species were isolated from different sources in Egypt (soil, air,
cereals etc) and differentiated according to their biochemical and physiological characteristics (17).
Industry is continuously looking for cheaper, more environmentally-friendly dyes instead of
traditionally used dyes. Anthraquinones have been isolated from a number of fungal strains belonging
to including Drechstera, Trichoderma, Aspergillus, Fusarium and Curvularia genus. Most of these
fungi produce a mixture of anthraquinones, this is a classic example of non-specific enzyme system
involved in the secondary metabolism (18). Two anthraquinone compounds are extracted from culture
filtrate of Fusarium oxysporum which were previously isolated from roots of citrus trees affected with
root infection. These anthraquinones were identified as 2-acetyl-3,8-dihydroxy or/and 3-acetyl-2,8-
dihydroxy-6-methoxy and are used as natural dyes for cotton fabrics (19).
Fungal synthesis of anthraquinones has several advantages over chemical methods since the
cultivation medium rarely contains expensive chemicals. Mostly, fungal anthraquinones production is
carried out at low temperature (30°C) and neutral pH, thus the expensive, fuel-consuming high
temperatures and environmentall- unfriendly strong alkalinity or acidity of the chemical synthesis are
not required 18. Anthraquinones production by F. oxysporum was studied in liquid mineral medium
containing 30% soluble starch, 3% (NH4)2(SO4)3, 0.3% MgSO4 and 4% KH2PO4 at pH 6, 28°C and
rotation speed (200 rpm) using orthogonal optimization experiment. In a previous study, optimization
of fermentation conditions succeeded to increase the pigment yield by about 1.8 folds compared with
the unoptimized conditions (20). Similarly, novel anthraquinone was biosynthesized by Fusarium sp.
using potato dextrose broth medium supplemented with 0.5% KH2PO4 at 28°C and pH 5.5 value under
shaking condition (250 rpm) (21).
Temperature is considered a critical factor affecting the fungal fermentation processes since it
has a significant influence on the fungal spore germination which determines the success of
fermentation process (22). The decrease in anthraquinones production at a high temperature (40.0 and
46.3 for F. arthosporoides and F. verticellioides, respectively) may be attributed to the negative effect
of high temperature on cell fluidity and ion exchange (23). Many investigators claimed the variation in
the pigment production at different pH values into changes in the morphology of the fungal
mycelium(24). In addition, pH influences the protein membrane charges with consequence nutrient
uptake, degree of mineral salts dissociation and the balance between CO2 and bicarbonate ions (23).
In the present study, a slight variation in the anthraquinones production was observed under
different aeration and light regimes. Many investigations have focused on the influence of aeration
rates on the morphology of fungal mycelia since vivid mixing modifies the hairy length of pellets, free
filamentous mycelia fraction and other rheology characteristics (25). Many of fungal metabolites
including anthraquinone gene clusters are silent under standard cultivation conditions. Some
regulatory elements may modulate transcription of genes involved in the secondary metabolism (14).
Fungi react to illumination in various ways including transcription of genes that is initiated within
minutes after exposure to light, activate and adjust metabolic enzymes. Thus the levels of secondary
metabolites are altered to proportionate with the harmful effects of light or to stimulate the
reproduction process; selecting the route depends mainly on light intensity in many cases (26). Light in
the visible part of the spectrum significantly reduced both the accumulation of biomass and pigment
production in certain filamentous fungi (27). The anthraquinones production by F. verticellioides
icreased by nearly six folds in dark and static conditions since anthraquinones and other phenolic
substances are precursors of dark colored humic pigments in soil (28).
Microorganisms used in industrial fermentation are rarely identical to the wild type, this is
because species are often genetically modified to maximize the yield of desired products. Mutation is
Arab Journal of Nuclear Science and Applications, 50 (1), (217-231) 2017
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often used, and is encouraged by mutagens such as X-ray and UV radiations or certain chemicals.
Selection and further reproduction of higher yielding strains over many generations can raise yields by
20-folds or more and using gene amplification, where copies of genes coding enzymes, involved in the
production process, are the main interesting routes (29). In the present study, the effect of ionizing and
non-ionizing radiations to enhance anthraquinones production was studied; results encourage this
route for increasing the anthraquinones yield.
Kitchen wastes are able to enter the mixed municipal waste system to be processed by standard
means such as incineration, due to their high moisture content. Furthermore, organic matter can be
trasformed into useful fertilizers (composting) and biofuel. These methods which are both
environmentally and economically efficient are now being developed which relying on various forms
of microbial decomposition or fermentation (30). The present study displays an alternative solution for
raw kitchen garbage by using it as sole carbon and nitrogen sources to produce industrially-valuable
compounds as anthraquinones. Similarly, F. equiseti (Corda) Sacc. 1886 produced various tones of
brown pigment when cultivated in media containing glucose, maltose, galactose, mannitol, sucrose,
fructose, lactose and starch (31). The activation of the plasma membrane H(+)-ATPase in F. oxsporum,
that synthesize dihydrofusarubins and javanicin was induced by glucose and maltose while lactose
exhibited a lower production. Enhancing the anthraquinones production by selected fungal strains on
kitchen wastes could be attributed to their mineral content (32). Different media were screened to select
a suitable complex media for the naphthoquinone production by locally isolated strain of F.
verticillioides and revealed revealed the metal ions such as K+, Na+, Mn2+, Cu2+ and Zn2+ less than 50
mg/l which were necessary for efficient naphthoquinone production by F. verticillioides (33). Two
anthraquinone-derivatives, chrysophanol and pachybasin were selected from Trichoderma harzianum
ETS 323 when cultivated on sugarcane bagasse as sole carbon source. It’s worth to mention that carrot
peels have the highest content of carbon source, in comparison with other tested kitchen wastes, that
gives the maximum anthraquinones yield (34).
The antimicrobial activity of anthraquinones against G+ve and G-ve bacteria as well as
pathogenic yeast is confirmed in the present study. Many hypotheses discuss the mechanism of
anthraquinones as antimicrobial agent. Antibacterial properties are exhibited by many anthraquinones,
such as emodin, physcion, questin, chrysophanol, catenarin, and altersolanols (35). Catenarin and
emodin inhibited the DNA dependent RNA polymerase of E. coli. They inhibit the incorporation of
certain nucleotides and amino acids in bacteria by inhibiting DNA-dependent RNA polymerase,
protein kinase C and protein tyrosine kinases (36). The antibacterial activity of 1,8-dihydroxy-
anthraquinone (Dan) on the Gram-positive bacterium Staphylococcus aureus is attributed to its
interaction with the cell wall and cell membrane by which it increases the permeability of the cell
envelope and leads to the leakage of cytoplasm and the deconstruction of cell (37).
Recently a revival of interest in the use of natural dyes in textile coloration has been growing as
a result of negative environmental impact of synthetic dyes on environment. Anthraquinone
derivatives have a great potential for dyeing textile with various shades of colors depending on the
nature and positions of auxochromic groups on their basic skeleton (38). The application of fungal
anthraqinones as a dyeing stuff for cotton, silk and wool has been reported in several studies (39, 40).
The anthraquinones that are used as substrates in the chemical production of various dyes and
pigments as well as natural pigments characterize with good color-fastness for different textile
materials especially wool and silk, since they have an affinity for these fibers without the aid of
auxiliary binding materials (4).
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