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Process Biochemistry 49 (2014) 569–575
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Bioprospecting of Amazon soil fungi with the potential for pigment
production
Jessyca dos Reis Celestino a , Loretta Ennes de Carvalho b , Maria da Paz Lima c ,
Alita Moura Lima d , Mauricio Morishi Ogusku e , João Vicente Braga de Souza e,∗
a
Post Graduate Program in Pharmaceutical Sciences, Universidade Federal do Amazonas – UFAM. R. Alexandre Amorin 330, 69010-300, Manaus,
Amazonas, Brazil
b
Post Graduate Program in Chemistry, Universidade Federal do Amazonas – UFAM. Av. General Rodrigo Octavio Jordão Ramos 3000, 69077-000, Manaus,
Amazonas, Brazil
c
Department of Natural Products, Instituto Nacional de Pesquisas da Amazônia – INPA. Av. André Araújo 2936, 69080-971, Manaus, Amazonas, Brazil
d
Post Graduate Program in Biotechnology, Universidade Federal do Amazonas - UFAM. Av. General Rodrigo Octavio Jordão Ramos 3000, 69077-000,
Manaus, Amazonas, Brazil
e
Mycology Laboratory, Instituto Nacional de Pesquisas da Amazônia – INPA. Av. André Araújo 2936, 69080-97, Manaus, Amazonas, Brazil
a r t i c l e
i n f o
Article history:
Received 17 July 2013
Received in revised form 19 January 2014
Accepted 19 January 2014
Available online 30 January 2014
Keywords:
Amazonian fungi
Pigments
Chemical characterisation
Optimisation
a b s t r a c t
The aim of this study was to isolate fungi able to produce pigments. Fifty strains were isolated from the
Amazon soil by the conventional technique of serial dilution. Submerged fermentation was performed
in Czapeck broth in order to select strains able to synthesise pigments. Five strains were able to produce
pigments and were identified by sequencing the rDNA (ITS regions). These fungi were identified as Penicillium sclerotiorum 2AV2, Penicillium sclerotiorum 2AV6, Aspergillus calidoustus 4BV13, Penicillium citrinum
2AV18 and Penicillium purpurogenum 2BV41. P. sclerotiorum 2AV2 produced intensely coloured pigments
and were therefore selected for chemical characterisation. NMR identified the pigment as sclerotiorin. In
this work, the influence of nutrients on sclerotiorin yield was also studied and it was verified that rhamnose and peptone increased production when used separately. These results indicate that Amazonian
fungi bioprospecting is a viable means to search for new sources of natural dyes.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Fungi are present in almost every environment on earth, with
the greatest diversity found in tropical regions that have a hot and
humid climate, which favours fungal multiplication [1]. Among the
tropical biomes, the Amazon rainforest contains the richest biodiversity, with a large number of plants, animals and microorganisms
that are not well known [2]. The soil of this forest, contrary to what
one might imagine, is poor in nutrients, and the maintenance of this
rich forest is ensured the innumerable microbial diversity present
in the soil, allowing the forest’s animals and plant components to
feed by recycling organic matter [3–5]. Soil fungi are metabolically
very active and are able to produce many substances of economic
value, including enzymes of industrial interest [6], metabolites with
pharmacological activity [7] and pigments [8].
Dyes derived from natural sources have been increasingly used
by pharmaceutical, textile and food industries due to the dyes’
∗ Corresponding author. Tel.: +55 92 9114 7815.
E-mail address: [email protected] (J.V.B.d. Souza).
1359-5113/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2014.01.018
lower toxicities to the environment and to man when compared to
synthetic dyes [9,10]. Several organisms, such as plants, animals,
bacteria, fungi and algae, are capable of synthesising pigments, but
fungi stand out for their potential to produce large amounts of dye
in small spaces [11,12]. Most pigment-producing fungi are of the
Aspergillus, Penicillium, Paecilomyces and Monascus species [8,13].
The majority of the pigments produced by fungi are quinones,
flavonoids, melanins and azaphilones, which belong to the aromatic
polyketide chemical group [14] and have been widely described for
medicinal uses and potential use as dyes [15,16]. Given that many
synthetic dyes used today are severely criticised for their longterm mutagenic and carcinogenic effects, legislation has imposed
increasingly stringent restrictions on synthetic dyes, especially
those that are food additives [11,17]. The chemical synthesis of natural products is not very viable because it generally leads to high
costs and low yields; consequently, the search for biological sources
that generate significant amounts of colourants has increased substantially [18].
In this context, with the aim of increasing the production
capacity of natural dyes by fungi, submerged fermentation was carried out, and the factors that influence the biosynthesis of these
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metabolites, such as the nutritional composition of the culture
medium, have been extensively studied [19]. The sources of carbon
and nitrogen influence the growth of fungus, the type of pigment
produced and the yield of the desired substance [20]. In this work,
to identify Amazonian soil fungi that have the ability to produce
pigments, it was (i) isolated and identified Amazon soil fungi that
produce pigment, (ii) chemically characterised the pigment produced by a fungal isolate, and (iii) evaluated the effect some sugars
and nitrogenous compounds had on the production of pigment.
2. Materials and methods
2.1. Isolation, identification and preservation of soil fungi
Four soil samples were collected from the surface (1–3 cm
depth) of the forest located at the Instituto Nacional de Pesquisas
da Amazônia (INPA), Manaus, Amazonas, Brazil (Latitude-South 03◦
09 39 and Longitude West 59◦ 98 77 ). For isolation, 1 g of soil
was transferred to a tube containing 9 mL of sterile distilled water,
which was serially diluted from 10−2 to 10−5 g/mL; 0.1 mL from
each dilution was inoculated in Petri dishes containing Potato Dextrose Agar (PDA) with chloramphenicol (250 mg/L). The experiment
was performed in triplicate. The plates were incubated at room
temperature (25 ± 2 ◦ C). Colonies that grew in 72 h were seeded
onto PDA plates until individual colonies appeared. The genera of
fungal species were identified based on the macro- and micromorphological characteristics, as suggested by Lacaz et al. [21] and
Barnett and Hunter [22].
A visual examination of the flasks was performed after 14 days
of fermentation and only fungi that released pigments in Czapeck
medium during this period were submitted to identify the species
by molecular biology. Specifically, fungal DNA was extracted from
mycelium using the QIAamp Tissue and Blood (Qiagen® , Hilden,
Germany) extraction kit according to the manufacturer’s recommendations. The Internal Transcribed Spacer (ITS) was amplified
using the primers ITS1/ITS4 [23]. PCR products were purified with
polyethylene glycol, based on the protocol described by Lis and
Schleif [24], with modifications of Lis [25] and Paithankar and
Prasad [26]. The sequencing reaction was performed with the
Big-Dye terminator cycle sequencing reagents (BigDye® , Applied
Biosystems, Foster City, CA, USA) kit, and sequencing was carried
out in the ABI Prism® Seq 3130 Genetic Analyzer (Applied Biosystems). The sequences obtained were compared to those in the
GenBank database (database incorporating DNA sequences of all
publicly available sources).
A method developed by the INPA laboratory of Medical Microbiology was used to preserve the isolated strains capable of producing
pigments. In this technique, 0.4 mL of distilled water, 0.025 mL of
DMSO dimethylsulphoxide (cryoprotector), 0.050 mL of glycerol
(cryoprotector) and 10 g of glass beads (with holes) were added to
autoclaved cryotubes. Approximately 250 mg of small fragments
collected from fungal cultures grown on PDA plates for 7 days,
at room temperature (25 ± 2 ◦ C), were transferred to microtubes
(2 mL). This procedure was performed in triplicate. The vials were
sent to the Mycology Collection Centre at INPA and were stored at
−70 ◦ C.
2.2. Screening for pigment production
Erlenmeyer flasks (250 mL) with 50 mL of Czapeck broth (3 g/L
NaNO3 , 1 g/L K2 HPO4 , 0.5 g/L MgSO4 .7H2 O, 0.5 g/L KCl, 0.01 g/L
FeSO4 ·7H2 O and 30 g/L sucrose), pH of 5.0 and concentration of
1 × 104 spores/mL medium. The flasks were incubated at room
temperature (25 ± 2 ◦ C) for 14 days and kept in a dark place, and
fermentation occurred in static conditions. Culture media containing pigments (50 mL) were subjected to successive extractions
with solvents of different polarities: hexane (30 mL), ethyl acetate
(30 mL) and butanol (30 mL). After extraction three fractions of
each solvent were obtained for each producing fungus. There was a
visual analysis of fractions derived and a fungal strain was selected
for further characterisation stage chemical, being chosen one that
had the highest number of coloured fractions and also fractions
containing pigments noticeably more intense.
2.3. Isolation and chemical characterisation of pigment produced
by selected fungus
The selected strain was fermented in a volume of 4 L, and
the pigment was extracted from the broth containing mycelium.
Successive extractions with 100 mL ethyl acetate were performed
until the total volume of 2 L. The extract was concentrated in
a rotary evaporator (IKA, RV10 digital, Santa Clara, CA, USA)
and then fractionated by column chromatography on a Sephadex
LH-20 (h × Ø = 52.0 cm × 3.0 cm) column (Sigma–Aldrich Co, St.
Louis, MO, USA), using 100% methanol as the eluent; 22 fractions
were collected. The thin layer chromatography profile allowed
to choose fraction 11 (197.3 mg) for further analysis by adding
it to a microcrystalline cellulose column (h × Ø = 25.0 cm × 2.0 cm)
(Merck, Darmstadt, Germany), eluted with a hexane:ethyl acetate
gradient. The first 9–11 fractions, combined, yielded a precipitate
that was purified with hexane:ethyl ether to give compound 1
(orange precipitate, 6 mg). The structural characterisation of the
pigment was performed by NMR on a Bruker Fourier 300 apparatus;
chemical shifts (ı) were expressed in ppm and coupling constants
(J) in Hertz. A UV/vis spectrophotometer (Model No. UV-1102 SP)
was used to identify the maximum absorbance (max ) of the pigment, ranging from 320 to 700 nm.
2.4. Effect of carbon and nitrogen sources on pigment production
To investigate the influence of the culture medium on pigment
production, the Czapeck broth was prepared with modified carbon
(30 g/L) and nitrogen (3 g/L) sources. The carbohydrates evaluated
were sucrose, glucose, fructose, lactose, galactose, rhamnose and
xylose. The nitrogen sources analysed were sodium nitrate, potassium nitrate, peptone (12% total nitrogen), yeast extract (8% total
nitrogen), malt extract (2% total nitrogen) and monosodium glutamate. All these substances had analytical grade and were obtained
by Sigma–Aldrich, USA. The experiments were performed in triplicate, where the bioprocess was conducted as described in section
2.2 [250 mL Erlenmeyer flasks, 50 mL of Czapeck broth, in static
conditions, kept in the dark at room temperature (±25 ◦ C), with
14 days of fermentation]. To extract the pigment, 30 mL of ethyl
acetate was added to the flask being collected after only 24 h. Pigment production was measured using a UV/vis spectrophotometer
(Model No. UV-1102 SP) through the maximum absorbance of the
pigment analysis (max ).
3. Results
To identify which fungi isolated from the soil had the potential
for pigment production, submerged fermentation was performed.
Initially, the fungal isolates were transferred to tubes containing PDA and incubated at room temperature (25 ± 2 ◦ C) for 72 h.
After 3 days, the fungus spores of each isolate were suspended in
sterile distilled water (approximately 2 mL) and counted using a
Neubauer chamber. This spore suspension was used to inoculate
3.1. Isolation and identification of Amazon soil fungi
To isolate microorganisms to be screened for pigment production, soil samples were serially diluted and plated on BDA to obtain
isolated colonies. The isolated cultures were determined to belong
to the Ascomycota phylum. It was obtained 50 filamentous fungi
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571
Table 1
Coding and genus identification of fungi isolated from samples of the Amazon soil.
Number
Code
Genus
Number
Code
Genus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2LIV1
2AV2
2AC3
2VV4
2LAC5
2AV6
2LAV7
2AC8
3BV9
3BV10
3NV11
5MV12
4BV13
4AV14
4MV15
3BV16
2CC17
2AV18
2NV19
2AC20
3AV21
2BV22
2LV23
2RV24
2AC25
Fusarium sp.
Penicillium sp.
Trichoderma sp.
Penicillium sp.
Aspergillus sp.
Penicillium sp.
Fusarium sp.
Paecilomyces sp.
Fusarium sp.
Verticillium sp.
Aspergillus sp.
Fusarium sp.
Aspergillus sp.
Penicillium sp.
Penicillium sp.
Penicillium sp.
Trichoderma sp.
Penicillium sp.
Aspergillus sp.
Trichoderma sp.
Trichoderma sp.
Penicillium sp.
Fusarium sp.
Aspergillus sp.
Fusarium sp.
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
2RV26
2VV27
2VV28
2LAC29
2RV30
2MV31
2MV32
2AC33
2RC34
3VV35
3BC36
3VV37
3BV38
4BV39
4BV40
2BV41
2VV42
2VV43
3AC44
3AC45
3BC46
3VV47
4BC48
4VV49
5VV50
Trichoderma sp.
Penicillium sp.
Trichoderma sp.
Fusarium sp.
Trichoderma sp.
Penicillium sp.
Aspergillus sp.
Fusarium sp.
Aspergillus sp.
Aspergillus sp.
Penicillium sp.
Fusarium sp.
Paecilomyces sp.
Scedosporium sp.
Trichoderma sp.
Penicillium sp.
Penicillium sp.
Penicillium sp.
Trichoderma sp.
Trichoderma sp.
Trichoderma sp.
Penicillium sp.
Fusarium sp.
Penicillium sp.
Penicillium sp.
Fig. 1. Pigments produced by Penicillium sclerotiorum 2AV2, Penicillium sclerotiorum 2AV6, Aspergillus calidoustus 4BV13, Penicillium citrinum 2AV18 and Penicillium purpurogenum 2BV41 extracted with solvents of different polarities (hexane, ethyl acetate and butanol) (for interpretation of the references to colour in this figure legend, the reader
is referred to the web version of the article).
of the genera Penicillium (17), Aspergillus (8), Trichoderma (11),
Fusarium (10), Paecilomyces (2), Verticillium (1) and Scedosporium
(one). There was a predominance of Penicillium sp. fungi composing the Amazon soil microbiota where the samples were collected,
as shown in Table 1.
3.2. Screening of pigment production by soil fungi and molecular
identification of dyes
To screen strains for pigment production, the strains in Table 1
were subjected to submerged fermentation in Czapeck broth. As
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Table 2
1H300 MHz and 13 C(75 MHz) NMR spectral data of the pigment produced from
Penicillium sclerotiorum 2AV2.
Position
ıH (300 MHz,
CDCl3 )
ıC a (75 MHz,
CDCl3 )
HMBC correlations
(300/75 MHz,
CDCl3 )
1
2
3
4
4a
5
6
7
8
8a
9
10
11
12
13
14
7.95 (s)
152.7
C-1, C-3, C-4a, C-9
15
16
17
18
COMe
COMe
6.66 (s)
6.09 (d; J = 15.6 Hz)
7.08 (d; J = 15.6 Hz)
5.72 (d; J = 9.8 Hz)
2.50 (m)
1.27 (sl)
1.40 (m)
0.88 (t; J = 7.4 Hz)
1.03 (d; J = 6.6 Hz)
1.86 (s)
1.59 (s)
2.19 (s)
157.9
106.7
138.8
110.6
191.3
84.4
185.8
114.0
115.7
142.9
131.8
148.8
34.9
29.6
12.2
20.2
12.2
22.3
170.1
19.9
C-3, C-5, C-8a, C-9
C-3, C-4, C-11
C-3, C-12
C-10
C-12
C-16
C-13, C-14
C-12, C-13, C-14
C-10, C-11, C-12
C-6, C-7, C-8
170.1
Chemical shifts are in ı (ppm) and coupling constants (J) in Hz.
a result, it was found that 5 of the 50 fungi were able to synthesise pigments. The pigments were extracted after fermentation by
successive partition with hexane, ethyl acetate and butanol. Fractions with colours ranging from yellow to red, as shown in Fig. 1,
were collected. The five pigment-producing fungi were identified
by ribosomal DNA sequencing (rDNA region) and showed similarity with the species Penicillium sclerotiorum (100%, strains 2AV2
and 2AV6), Aspergillus calidoustus (99%, strain 4BV13), Penicillium
citrinum (99%, strain 2AV18) and Penicillium purpurogenum (99%,
strain 2BV41).
3.2.1. Chemical characterisation
Penicillium sclerotiorum 2AV2 released intensely coloured substances in three of the used solvents; consequently, it was selected
for further chemical colourant characterisation. Purification procedures resulted in a yellow–orange pigment. NMR analysis allowed
the structural elucidation of the molecule. Carbons were assigned
based on HSQC and HMBC data. The long-range correlations
observed (HMBC experiment) confirmed the attribution of all the
carbon signals of the molecule. The spectral data of 1H and 13NMR
(Fig. 2; Table 2) were compared with (+)-sclerotiorin identified by
Paired et al. [27], and confirmed the identification of the pigment
sclerotiorin (Fig. 3). Scanning spectrophotometry showed that the
sclerotiorin pigment has a maximum absorbance (max ) in ethyl
acetate at 350 nm.
3.2.1.1. Effect of carbon and nitrogen sources on pigment synthesis. To study the effect of the culture medium on sclerotiorin
production, the selected fungus was grown in Czapeck medium
base modified with different sources of carbon or nitrogen in
each experiment. The carbon sources were composed of carbohydrates and nitrogen sources were represented by inorganic salts or
nitrogenated organics. The maximum absorbance at 350 nm was
achieved when rhamnose was used as sugar in the fermentation
base medium (data are shown in Fig. 4). Peptone and yeast extract
were the nitrogen sources that allowed the greatest absorbances
at the analysed wavelengths (max × 350 nm) (data are shown in
Fig. 4).
Fig. 2. 1 H(300 MHz) NMR spectral data (a), HSQC (Heteronuclear single-quantum
correlation spectroscopy) (b) and HMBC (Heteronuclear Multiple Bond Correlation)
(c) spectrum of the pigment produced by Penicillium sclerotiorum 2AV2.
Fig. 3. Chemical structure of the pigment produced by Penicillium sclerotiorum
2AV2 (Sclerotiorin). This chemical structure was achieved by 1 H(300 MHz) and
13
C(75 MHz) NMR spectral data and long-range correlation (HMBC, Table 1).
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573
Fig. 4. Effect of different carbon (a) and nitrogen (b) sources in production of Sclerotiorin (max in ethyl acetate at 350 nm) by Penicillium sclerotiorum 2AV2.
4. Discussion
Soil fungi have been widely isolated from nature for their ability
to produce pharmacologically active molecules [7]. These microorganisms have also been noted to produce natural pigments that
could be used in the dye industry, given the variety and intensity of
colours produced by different strains of environmental fungi and
the great value to natural colourants [8,28]. Despite the Amazon
rainforest being a home to a great diversity of microorganisms,
there are few published studies that explore the ability of these
fungi to produce pigments. Therefore, this work aimed to isolate
pigment-producing soil fungi from the Amazon.
In this work, the identification of microorganisms in soil samples revealed the existence of 50 fungi of the genera Penicillium,
Aspergillus, Fusarium, Trichoderma, Paecilomyces, Verticillium and
Scedosporium. These fungi have been isolated from environmental
samples of soil [6,29], water [30,31], interior plants (endophytic
fungi) [32,33], marine organisms [34,35] and even arctic glacial
ice [36,37]. Serial dilution was used for the isolation of soil fungi.
This is a very usual techniques for isolating microorganisms from
environmental samples, since these often harbour a large number
of viable microbial cells. Thus, it was possible to obtain isolated
colonies, Amazonian soil samples had to be diluted before plating on culture media. On the other hand, the dilution restricted
number of fungal strains and allow only a small fraction of fungi
present in the soil were identified where the samples were collected.
Of the all isolates, some fungi produce pigments in Czapeck
broth during submerged fermentation. Producing fungi were identified by rDNA ITS region as P. sclerotiorum 2AV2, P. sclerotiorum
2AV6, A. calidoustus 4BV13, P. citrinum 2AV18 and P. purpurogenum
2BV41.
P. sclerotiorum is a recognised producer of azaphilones, including rotiorin, isochromophilone and sclerotiorin, with the latter
being the most reported for these species [38,39]. The literature on A. calidoustus describes it as a rare pathogen that can
cause infection mainly in those immunocompromised [40] but
can also produce metabolites of industrial interest [41]. P. citrinum is a producer of anthraquinone chromophores [42] and is
often described by mycotoxin citrinin production, a yellow metabolite that induces nephrotoxicity and hepatotoxicity in humans
[43]. P. purpurogenum is a producer of azaphilones, such as
mitorubrin and mitorubrinol, and quinones as purpurogenone and
purpurquinones [44,45]. Amazon strains of P. purpurogenum have
also already been described for the production of coloured substances [16].
In this work, the pigments produced by P. sclerotiorum 2AV2
were chosen for chemical characterisation due to their intense
colouration in the three solvents, indicating a fungus with good production capacity. The substance was identified by one-dimensional
and two-dimensional NMR as sclerotiorin, a yellow–orange pigment that is soluble in organic solvents such as ethyl acetate and
ether but insoluble in water, being first reported its synthesis by
fungi of the Amazon.
Sclerotiorin was originally isolated from P. sclerotiorum [46];
however, it can also be produced by other fungi [47,48]. It has
important biological activities, including endothelin receptor binding activity [27], aldose reductase inhibition [48], antimicrobial
activity [39,48], antifungal activity [50], inducing apoptosis of
HCT-116 cancer cells [47] and inhibitory effect on human immunodeficiency virus HIV-1 integrase and protease [49].
Pigment synthesis by fungi is directly influenced by the nutrients available in the medium, such as carbon and nitrogen, as well
as the selection of the fungal strain due to interspecies variation
[19]. Some carbohydrates or compounds containing nitrogen can
be more easily assimilated by certain strains than for others, which
justifies the higher or lower yields of the desired product [20].
In this study, synthesis of sclerotiorin by Penicillium sclerotiourum
2AV2 was on average three times higher when the medium was
prepared with rhamnose sugar compared to sucrose (the usual
sugar in Czapeck medium). Galactose was the second choice to
obtain higher concentrations of the bioproduct. Lactose is a convenient carbon source for many microorganisms [50], but it caused
a large decrease in the production of sclerotiorin for this strain.
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These results show that unusual sources in culture media available
can significantly increase the synthesis of this pigment by the P.
sclerotiourum 2AV2 fungal strain isolated from an Amazonian soil
sample.
The results indicate that the effect of nitrogen sources on the
synthesis of sclerotiorin was far greater than that exerted by carbohydrate sources. The literature describes nitrogen sources as very
important in the synthesis of pigments for regulating the expression of genes of interest and activating important pathways in
the production of these compounds [51]. In the present study,
nitrogenated peptone and yeast extract most favoured sclerotiorin
production when compared to sodium nitrate (the usual nitrogen
source in Czapeck), with a yield more than six times higher than in
conventional Czapeck. Peptone is commonly used in various culture
media, and many fungi appear to have the ability to use it during
metabolism, leading to increased production of pigments [52]. In
addition to nitrogen, the organic nitrogen compounds provide carbon and sulphur to the culture medium and enable the fungus to
obtain energy. Yeast extract and malt also provide vitamins and
coenzymes, favouring the growth of the fastidious microorganisms.
However, it is known that pigment production is favoured when the
medium contains nitrogen sources, especially of organic origin [53].
In the present study, it was found that yeast extract showed results
consistent with peptone, increasing pigment synthesis. However,
the use of malt extract caused suppression of pigment production
for the fungal strain studied. In fermentation that evaluated the
use of peptone, malt and rice as a nitrogen source for P. sclerotiorum LAB18 isolated from Brazilian cerrado soil, peptone was also
the nutrient that caused the greatest increase in sclerotiorin pigment production, while the others showed low levels of production
[54].
In this work, when a new submerged fermentation was prepared
with peptone and rhamnose (the sources of carbon and nitrogen that increased production of sclerotiorin when the modified
base medium Czapeck), under the same conditions as previously described fermentation, the maximum absorbance (max ) at
350 nm was 69 ± 0.15, showing that the combination repressed
the synthesis of sclerotiorin when compared with the bioprocess in which the peptone was used without modifying the usual
way Czapeck sugar (sucrose). As mentioned above, peptone is
an organic nitrogen source; therefore, it also provides carbon
molecules, providing the culture medium with many nutrients. The
use of rhamnose in this case may have caused the phenomenon of
catabolite repression, in which the presence of two sources easily
assimilable by the fungus can reduce or even suppress the production of the metabolite of interest.
The aim of these experiments with different carbon and nitrogen
sources was to start a discussion about the nutritional conditions
related to production of the pigment. It is well known that for the
industrial purposes it is necessary inexpensive substrates and the
results presented in the present work can help selection of these
substrates.
Considering the results found in this study, it was concluded
that fungi from Amazon soil may be potential pigment producers.
Of the 50 fungal isolates, 5 species stood out by the synthesis of
coloured compounds in Czapeck broth, whether they belong to the
genera Penicillium sp. and Aspergillus sp. Sclerotiorin, which is an
important pigment that has been isolated previously, was isolated
from P. sclerotiorum 2AV2. Through the standardisation of carbon
sources and nitrogen, it was possible to increase the production
of this compound, though more studies are needed to optimise
the production by the selected strain. Given these findings, it was
concluded that the vast diversity of Amazon microorganisms with
biotechnological potential and the increasing need for new biocolourants demonstrate the importance of further bioprospecting
studies.
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