Appl Biochem Biotechnol

DOI 10.1007/s12010-013-0392-y

Pigment Production by Filamentous Fungi

on Agro-Industrial Byproducts:
an Eco-Friendly Alternative

Fernanda Cortez Lopes & Deise Michele Tichota &

Jamile Queiroz Pereira & Jéferson Segalin &
Alessandro de Oliveira Rios & Adriano Brandelli

Received: 2 April 2013 / Accepted: 8 July 2013

# Springer Science+Business Media New York 2013

Abstract The search for new sources of natural pigments has increased, mainly because of the
toxic effects caused by synthetic dyes used in food, pharmaceutical, textile, and cosmetic
industries. Fungi provide a readily available alternative source of natural pigments. In this
context, the fungi Penicillium chrysogenum IFL1 and IFL2, Fusarium graminearum IFL3,
Monascus purpureus NRRL 1992, and Penicillium vasconiae IFL4 were selected as pigments
producers. The fungal identification was performed using ITS and part of the β-tubulin gene
sequencing. Almost all fungi were able to grow and produce water-soluble pigments on agro-
industrial residues, with the exception of P. vasconiae that produced pigments only on potato
dextrose broth. The production of yellow pigments was predominant and the two strains of P.
chrysogenum were the largest producers. In addition, the production of pigments and myco-
toxins were evaluated in potato dextrose agar using TOF-MS and TOF-MS/MS. Metabolites as
roquefortine C, chrysogine were found in both extracts of P. chrysogenum, as well fusarenone
X, diacetoxyscirpenol, and neosolaniol in F. graminearum extract. In the M. purpureus extract,
the pigments monascorubrin, rubropunctatin, and the mycotoxin citrinin were found. The crude
filtrates have potential to be used in the textile industry; nevertheless, additional pigment
purification is required for food and pharmaceutical applications.

Keywords Agro-industrial wastes . Pigments . Eco-friendly . TOF-MS . Fungi

Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0392-y)

contains supplementary material, which is available to authorized users.
F. C. Lopes : D. M. Tichota : J. Q. Pereira : A. Brandelli (*)
Laboratório de Bioquímica e Microbiologia Aplicada, Departamento de Ciência de Alimentos (ICTA),
Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 91501-970 Porto Alegre, Brazil
e-mail: abrand@ufrgs.br

J. Segalin
Unidade Química de Proteínas e Espectrometria de Massas (UNIPROTE-MS), Centro de Biotecnologia,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

A. de Oliveira Rios
Laboratório de Análise de Alimentos, Departamento de Ciência de Alimentos (ICTA), Universidade
Federal do Rio Grande do Sul, Porto Alegre, Brazil
 Appl Biochem Biotechnol

Introduction

Microorganisms have been traditionally used to produce several substances with industrial
importance. Filamentous fungi have proved to be useful, because of their ability to produce
primary and secondary metabolites such as peptides, enzymes, organic acids, heterologous
proteins, antibiotics, and pigments [1, 2]. There is a growing preference for natural colorants
among consumers because of their advantages over synthetic colorants in terms of both
health and environmental aspects [3]. Microbial pigments are a promising alternative in
relation to other additives extracted from animals and plants. The advantages of producing
pigments from microorganisms include independence from weather conditions, colors of
different shades, and growth on inexpensive substrates, for example, agro-industrial residues
[4].
Large amounts of agro-industrial residues are generated from diverse economic activities,
increasing the biotechnological interest on the utilization of these residues as inexpensive
substrates to support the growth of microorganisms [5]. The utilization of agro-industrial
waste as growth substrates may represent an added value to the industry and meets the
increasing consciousness for energy conservation [6, 7]. Several processes have been
developed that utilize these as raw material for the production of bulk chemicals and
value-added fine products. Application of agro-industrial residues in bioprocesses on the
one hand provides alternative substrates, and also helps in solving pollution problems, which
their disposal may otherwise cause [8].
Therefore, the aim of this study was to select and to identify strains of filamentous fungi
as potential pigment producers using agro-industrial wastes as growth substrate and to
analyze the possible metabolites produced by these strains using mass spectrometry.

Materials and Methods

Microorganisms and Screening of Pigment-Producing Fungi

The fungi used for the screening belong to the mycology collection of the Laboratory of
Biochemistry and Applied Microbiology (UFRGS). The fungi were maintained on potato
dextrose agar (PDA) slants covered with mineral oil at 4 °C and subcultured periodically.
Twenty-four fungi were inoculated in the center of potato dextrose agar plates, using a
bacteriological needle: Alternaria solani (plant isolate—UNICRUZ), Apiosordaria sp. (plant
isolate—UNICRUZ), Aspergillus niger ATCC 16404 (UNISC), A. niger IFL5 (citrus
isolate—UFRGS), Aspergillus ochraceus (soil isolate—UFRGS), Aspergillus sp. (soil
isolate—UFRGS), Bipolaris sorokiniana (soil isolate—UFRGS), Cercosporina sojina (plant
isolate—UNICRUZ), Fusarium graminearum (plant isolate—UNICRUZ), Fusarium
oxysporum (plant isolate—UNICRUZ), Fus arium sporotrichioides (plant
isolate—UNICRUZ), Isolate B16 (soil isolate—UFRGS), Isolate P14 (soil isolate—UFRGS),
Monascus purpureus NRRL1992 (UFRGS), Penicillium L014 (soil isolate—UFRGS), Peni-
cillium expansum (food isolate—UFRGS), Penicillium sp.1 (soil isolate—UFRGS), Penicilli-
um sp. 2 (soil isolate—UFRGS), Penicillium nalgiovense (soil isolate—UFRGS), Penicillium
stoloniferum (soil isolate—UFRGS), Rhizoctonia sp (plant isolate—UNICRUZ), Rhizopus
oligosporus (soil isolate—UFRGS), Trichoderma ressei (plant isolate—UFRGS), and
Verticillium albanum (plant isolate—UNICRUZ). The plates were incubated for 7 days, at
30 °C. The visual pigment production and secretion was available and five fungi were selected
(supplementary Fig. S1).
Appl Biochem Biotechnol

DNA Isolation and PCR Assay

Genomic DNAwas extracted as described by Lopes et al. [9]. Fungal identification was based on the
partial sequencing of the intergenic region of the rDNA ITS and part of the β-tubulin gene. An ITS
region was selected as a target sequence for PCR using universal primers: ITS 1 (5′-TCCG
TAGGTGAACCTGCGG-3′) as a forward primer and ITS 4 (5′-TCCTCCGCTTTATTGATAT
GC-3′) as a reverse primer, according to Horisawa et al. [10]. Each 25 μL PCR reaction mix
contained 2.5 μL Taq buffer ×10, 1.0 μL MgCl2 25 mM, 0.5 μL dNTPs 25 mM, 0.2 μL Platinum
Taq DNA polymerase 5 U/mL (Invitrogen), 2.5 μL primer 20 mM, 50 ng genomic DNA, and
13.3 μL Milli-Q water. PCR assays were performed according to described previously [9].
The primers used for part of the β-tubulin gene were Bt2a (5′-GGTAACCAAATCGGT
GCTGCTTTC-3′) and Bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) as forward and
reverse, respectively. Conditions were 32 cycles of 94 °C for denaturation for 1 min, 58 °C
for annealing for 1 min, 72 °C for extension for 1 min, and a final extension for 10 s at 72 °C
[11]. The PCR products were analyzed on a 0.9 % agarose gel.

DNA Sequencing and Phylogenetic Analysis

Samples were sequenced in the ACTGene Laboratory (Centro de Biotecnologia, UFRGS, Porto
Alegre, Brazil) using the automatic sequencer ABI-PRISM 3100 Genetic Analyzer armed with
50 cm capillaries and POP6 polymer (Applied Biosystems). The sequences were edited using the
software BioEdit Sequence Alignment Editor (1997–2005 Tom Hall). After edition, the search for
homologous sequences was performed in the NCBI databank (http://www.ncbi.nlm.nih.gov/blast).
For phylogenetic reconstruction, a fragment of 585 bp of the ITS region and a fragment of
485 bp of the β-tubulin gene were used. The sequences obtained were edited in the software
BioEdit, compared with those from GenBank and data matrix aligned in the ClustalX software.
The ATCC strains were used for comparisons, when available in the NCBI database. The
phylogenetic tree was constructed by the Neighbor-joining method [12] according to the
Kimura’s two-parameter model of nucleotide evolution [13] using MEGA 5.01 software. To
access the confidence limits of the branching, 1,000 Bootstrap replications were performed.

Media and Culture Conditions

The selected fungi were cultivated on agro-industrial residues through submerged fermentation.
Feather meal, fish meal, cheese whey, grape waste, soybean protein, and soybean meal were
homogenized to a fine powder (100 mesh). Rice husk, chicken feathers, and pig hair were used
without milling. The source and the compositional features of each residue are found in Table 1.
All residues were used at 10 g/L concentration as only source of nutrients, salts were not added
and initial pH was set at 6.5, adjusted before the sterilization. The sterilization was performed
using autoclave for 15 min at 121 °C. The fungi were also cultured in potato dextrose broth.
Cultivations were performed for 7 days, at 30 °C and 125 rpm in 250-mL Erlenmeyer
containing 50 mL of the medium. The inoculum contained 106 conidia/mL and was prepared
as described previously [15]. Cultures were filtered through Whatman no. 1 filter to separate the
mycelium. All cultivations were performed in duplicate.

Pigments Analysis

The filtrates were analyzed in a spectrophotometer (UV-mini 1240, Shimadzu, Tokyo, Japan). A
scan was performed from 400 to 700 nm to determine the wavelength of maximum absorption.
 Appl Biochem Biotechnol

Table 1 Compositional features and source of the agro-industrial residues

Agro-industrial Source Compositional features

byproduct

Feather meal Bunge Alimentos S.A, Esteio, 7.8 % humidity, 4.4 % fat, 2 % ash, 83.7 %
Brazil protein
Fish meal Etna, Rio Grande, Brazil A waste of fish processing, contains 54 %
protein,
11 % lipids, 5 % carbohydrate, and 30 %
minerals
Cheese whey Parmalat, Porto Alegre, Brazil Waste from the production of mozzarella
cheese, contains 13 % protein, 1 % lipids,
77 % carbohydrate (lactose), and 9 %
minerals
Grape waste Obtained as described by Silveira 30 % fiber, 21 % carbohydrate, 10 % protein,
et al. [14] 10 % fat
Soybean protein Bunge Alimentos S.A, Esteio, 79 % protein, 3 % lipids, 12 % carbohydrates,
Brazil and 5 % minerals
Soybean meal Cerelus, Ijuí,Brazil 50 % protein, 3 % lipids, 35 % carbohydrate,
5 % fiber, and 7 % minerals
Chicken feather Avipal S.A, Porto Alegre, Brazil 89 % protein, 3 % fat, 8 % minerals
Rice husk Provided by Dr. L.A.D. Silva Not available

The results were expressed in units of absorbance (UA/mL) at a given wavelength (λ), multiplied
by the dilution factor. A blank with the autoclaved media was used to discount the own color of
the culture media, for the insoluble media a previous centrifugation (10,000×g for 10 min) was
performed, to avoid the interference of the particles. The color parameters L, a*, b*, and h from
the CIELab system were determined using a Minolta 40 equipment. The CIELAB colorimetric
system was interpreted as follows: L* indicates lightness read from 0 (black) to 100 (white) [16].
The positive a* value indicates the red color while the negative a* value represents the green
color. Similarly, positive and negative b* values indicate the yellow and the blue colors,
respectively. Chroma (C*) values denote the saturation or purity of color. Hue angle (h) values
represent the degree of redness, yellowness, greenness, and blueness; the maximum is at 0, 90,
180, and 270, respectively. All measurements were performed in duplicate.

Extraction of Metabolites and TOF-MS Conditions

The production of mycotoxins and other metabolites were investigated in potato dextrose agar, a
standard medium to fungi growth. Four samples of fungal hyphae, together with underlying culture
medium were taken by vertically cutting 6-mm diameter plugs from each quarter. The plugs were
transferred to 5-mL tubes and were extracted twice through ultrasonication in 3 mL acetonitrile and
2 mL 1 % formic acid in ethyl acetate for 10 min. The solvents were evaporated under N2 gas and
the residue was resuspended in 1 % formic acid aqueous solution and passed through a disposable
nitrocellulose filter with 0.2 μm pore size before TOF-MS analysis. A Waters Q-TOF micro was
used to determine the presence of secondary metabolites, the instrument is equipped with a nano-
electrospray ionization (nano-ESI) source. Data were analyzed with the Waters MassLynx soft-
ware. MS spectra were processed using background subtract followed by smoothing the spectrum
with Savitzky-Golay smoothing, and measuring the peak top with a centroid top of 80 %. Direct
injection with 1 μL/min flow was performed. The conditions of TOF-MS were capillary voltage
3,300 V, top flight tube 5,630 V, MS cone voltage 50 V, sample cone voltage 50 V, collision gas
Appl Biochem Biotechnol

argon, collision energy 6 eV, source temperature 100 °C, and reflectron 1,780 V. The TOF MS/MS
conditions were the same, changing only the collision energy to 25 V with modifications [17]. The
mycotoxins masses and the fragmentation profile of roquefortine C (in Penicillium chrysogenum
extracts) and citrinin (in M. purpureus extract) were evaluated.

Results and Discussion

Screening of Pigment-Producing Fungi

Fungi were selected as producers of water-soluble pigments according to their ability to produce
and secrete pigments to the PDA medium. Five strains of fungi that produced pigments on PDA,
Penicillium sp 1, F. graminearum, M. purpureus, P. expansum, and Penicillium sp 2 were selected
(Fig. S1). Among the selected fungi, only M. purpureus is a traditional producer of pigments.
Species of the genus Monascus are used in Asia for centuries as a source of pigments for the
coloring of traditional foods. This genus is subject of several studies, mainly due to the growing
interest in natural pigments to be used in the food industry [2, 7]. A screening performed by
Velmurugan et al. [18] for pigment producers resulted in four selected fungi among 153 isolates,
based on their ability to produce different shades (red, reddish brown, pink, and yellow) of
pigments. The four isolates were identified as Isaria sp., Emericella sp., Fusarium sp., and
Penicillium sp. The genera Penicillium and Fusarium were also found in the present work.

Fungi Identification and Phylogenetic Analysis

The identification of a natural pigment-producing fungus is important for industrial appli-

cation. The identification of the isolates selected in this work is summarized in Table 2.
The use of the ITS primers was more efficient to determine the species of the strains
comparing with the β-tubulin primers. The ITS region is more variable than the coding
protein genes, as β-tubulin. The ITS regions evolve rapidly and then are suitable for
discriminating closely related species or even varieties of the same species [19]. In addition,
genes encoding proteins accumulate fewer mutations and are less variable [20]. Sequences
were submitted to GenBank and the accession numbers are found in Table 2.
The ITS region is used as a molecular target for differentiation of fungal species and the
sequence variation within this area has also proved to be useful in phylogenetic studies of
many different fungi [21]. To obtain more precise phylogenetic trees, analyses of multiple
gene sequences, like protein genes, have been required [22]. The phylogenetic tree of the
ITS sequences is shown in Fig. 1. The phylogenetic tree of the β-tubulin sequences are not
shown due to the low resolution obtained in this study. It is possible to observe that the fungi
of the genera Penicillium and Monascus show higher similarity comparing with the fungus
of the genus Fusarium. Strains of Penicillium and Monascus have a common ancestor. This
can explain the production of the Monascus pigments by some strains of Penicillium
reported in literature. It has been hypothesized that Monascus or Monascus-like azaphilone
pigments are likely to be produced by species in the Penicillium subgenus biverticillium that
produce significant amounts of red to orange-red pigments [23].

Production of Pigments on Agro-Industrial Residues

The fungi were cultured on different agro-industrial byproducts to verify the production of
pigments using inexpensive substrates. This is an important feature for industrial application,
 Appl Biochem Biotechnol

Table 2 Molecular identification based in the ITS region and part of the β-tubulin gene

Fungi ITS-identification/ Fragment β-tubulin-identification/ Fragment

identity (bp) identity (bp)

Penicillium sp. 1 No similarity found 150 Penicillium vasconiae 480

JQ614058.1
94 %
Fusarium graminearum Gibberella zeae/Fusarium 489 Fusarium sp. 100 % 330
graminearum IFL3
JQ614060.1
100 %
Monascus purpureus Monascus purpureus 537 Monascus purpureus 505
NRRL1992 JQ614061.1 JQ614057.1
100 % 81 %
Penicillium expansum Penicillium chrysogenum 533 Penicillium chrysogenum 457
IFL2 IFL2
JQ614062.1 JQ614056.1
100 % 100 %
Penicillium sp. 2 Penicillium chrysogenum 537 Penicillium chrysogenum 450
IFL1 IFL1
JQ614063.1 JQ614055.1
100 % 100 %

showing also the capability of the fungi to grow in a wide variety of nutrients sources. The
media used and the colorimetric data are shown in Table 3. The fungi M. purpureus and F.
graminearum showed the production of pigments in a larger number of residues than the
other fungi. P. vasconiae produced pigments only on potato dextrose broth. The predomi-
nant colors of the pigments produced were yellow, as observed by the maxima absorption in
wavelengths around 400 nm. The strain of Monascus also produced red pigments on soy

Fig. 1 The neighbor-joining tree inferred from the partial sequence of the ITS region data set using Mega 5.0
according to Kimura two-parameter model. Bootstrap values from 1,000 replications are indicated at the nodes
Appl Biochem Biotechnol

Table 3 Colorimetric data of fungal pigments produced on different media

Colorimetric data

Filamentous fungi Culture medium Maximum absorption L* a* b* C* h

(λ: UA)

P. chrysogenum IFL1 GW 400 nm: 46.50 29.22 9.93 11.88 15.49 50.10
CW 403 nm: 74.7 38.20 0.96 17.49 17.52 86.85
PD 400 nm: 139.5 39.63 1.22 11.25 11.31 83.79
P. chrysogenum IFL2 CW 403 nm: 78.15 34.68 3.16 15.06 15.39 78.15
PD 400 nm: 137.05 38.02 −0.18 24.80 24.80 90.41
F. graminearum SM 400 nm: 23.45 35.30 3.52 12.00 12.50 73.67
FM 406 nm: 60.00 26.04 5.57 5.44 7.78 44.33
SP 400 nm: 17.8 29.04 3.35 8.43 9.07 68.34
PD 400 nm: 93.15 36.47 2.51 18.03 18.20 82.07
M. purpureus SM 400 nm: 21.45 31.50 6.85 6.62 9.52 44.02
CW 509 nm: 3.70 30.76 10.80 11.61 15.85 47.06
SP 430 nm:20.6/514 nm: 20.95 28.96 6.70 0.18 6.71 1.57
RH 400 nm: 7.20 39.38 4.69 3.00 5.57 32.63
PD 405 nm:26.30/500 nm:17.15 24.34 19.48 6.12 20.41 17.44
P. vasconiae PD 400 nm: 103.00 36.29 3.12 16.40 16.70 79.23

GW grape waste, CW cheese whey, PD potato dextrose, SM soybean meal, FM feather meal, SP soy protein,
RH rice husk

protein and cheese whey media. There is a great potential in the extraordinary color range of
pigments produced by ascomycetous fungi within the red and the yellow spectra [16].
Glucose showed to be an important carbon source for pigment production, because there
was a higher pigment production in PDA than in the other media tested.
The filtrate that had the more intense color is the potato dextrose broth from M.
purpureus, which is in agreement with the lower value of L* 24.34. Otherwise, the filtrate
with higher luminosity was the potato dextrose broth from P. chrysogenum IFL1 with the L*
value 39.63. The negative a* value of the potato dextrose broth from P. chrysogenum IFL2
(a* −0.18) represents the greener tones in the filtrate, and the higher a* value was obtained
in potato dextrose filtrate from M. purpureus. The positive b* values in all filtrates confirm
the predominance of yellow pigments. The hue angles of all of the fungal extracts ranged
from 1.57 to 90.41. This indicated the color of the filtrates varied from red to orange and
yellow to light green-yellow. The filtrate that showed higher purity (C* 24.80) was potato
dextrose broth from P. chrysogenum IFL2.
The pigment production by P. chrysogenum was earlier reported [24]. Until today there
are few reports on pigments produced by these species, because the pigments were usually
used as a chemotaxonomic tool. P. chrysogenum produces the yellow pigments sorbicillin
and xanthocillins [25] and chrysogenin was also reported as a yellow pigment produced by
this fungus [26]; these last authors also emphasized the few studies in this area. The
production of the reddish pigments rubrofusarin and aurofusarin by F. graminearum was
also described. M. purpureus produces a wide variety of pigments and, at least six molecular
structures are known: rubropunctatin, monascorubrin, rubropunctamine, monascorubramine,
monascine, and ankaflavine [14]. To our knowledge there are no reports about the produc-
tion of pigments by P. vasconiae.
 Appl Biochem Biotechnol

Table 4 List of ions found in the extracts commonly produced by these strains obtained by TOF-MS

Fungi Compound Precursor ion (m/z) References

P. chrysogenum IFL1 Chrysogine (M+H+) 191 [27]

Meleagrin (M+H+) 434 [27, 28]
Roquefortine C (M+H+) 390 [27, 28]
P. chrysogenum IFL2 Chrysogine (M+H+) 191 [27]
Roquefortine C (M+H+) 390 [29]
Xanthocillin X (M+H+) 288 [27]
F. graminearum Diacetoxyscirpenol (M+H+) 367 [27]
Fusarenone X (M+H+) 355 [27]
15-Acetoxyscirpenol (M+H+) 325 [27]
Neosolaniol (M+H+) 383 [27]
M. purpureus Citrinin (M+H+) 251 [30]
Monascorubrin (M+H+) 383 [30]
Rubropunctatin (M+H+) 355 [30]

Fig. 2 TOF-MS/MS spectra of selected precursor ions. Fragmentation profiles of the peak at m/z 390 in the
extract of P. chrysogenum IFL1 (a), peak at m/z 390 in the extract of P. chrysogenum IFL2 (b), and peak at m/z
251 in the extract of Monascus purpureus (c)
Appl Biochem Biotechnol

Secondary Metabolites

A footprinting was performed in order to find metabolites produced by these fungi in PDA.
The precursor ions of the metabolites commonly produced by these strains are found in
Table 4. The two strains of P. chrysogenum showed the ion precursor of roquefortine C, a
mycotoxin produced by this fungus. Roquefortine C was not abundant in the sample, but its
presence was confirmed by the fragmentation profiles (Fig. 2a and b) that are similar to a
previously reported pattern [27, 31]. Roquefortine has very low importance as a mycotoxin,
justifying the safe status of P. chrysogenum [32]. In addition, it is important to highlight the
presence of xanthocillin ion in the extract of P. chrysogenum IFL2.
The F. graminearum extract presented ions of three mycotoxins, namely diacetoxyscirpenol,
fusarenone X, and neosolaniol. The production of mycotoxins by this fungus has been widely
explored, and butenolide, fusarin C, trichotecenes, and zearalenone are the most common
mycotoxins produced by F. graminearum [25]. The extract of M. purpureus showed the ions
of orange pigments monascorubrin and rubropunctatin, besides the mycotoxin citrinin. We
performed the fragmentation of this peak (Fig. 2c) and confirmed the presence of this myco-
toxin in the extract, as we obtained the same fragmentation pattern reported by [33]. The extract
of P. vasconiae was not submitted to TOF-MS/MS because of the limited knowledge about the
possible metabolites produced by this strain.
The production of mycotoxins does not exclude these strains as pigments producers. It is
possible to avoid the mycotoxin production changing the culture conditions as culture media
and aeration. Roquefortine C was not detected in cheese whey filtrates of P. chrysogenum
IFL1 and the filtrate was also pigmented [34]. Furthermore, our future studies will focus the
capability of these pigmented filtrates to color leather as an alternative eco-friendly dying.

Conclusions

Four strains of filamentous fungi were capable to produce pigments during growth on different
agro-industrial byproducts. A selected P. vasconiae produced pigments only on potato dextrose
broth. Yellow pigments were predominant in the water-soluble extracts of all fungi, but M.
purpureus also produces red pigments on soy protein and cheese whey. The analysis by mass
spectrometry techniques allows the identification of both pigments and mycotoxins in the
extracts. The use of filamentous fungi that have capacity to growth on available agro-industrial
residues may represent an interesting alternative to obtain pigments or pigmented extracts.

Acknowledgments The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.
We also thank Dr. Patricia Valente da Silva, Dr. Charley Christian Staats, Dr. Fernanda Stanisçuaski (UFRGS), and
Dr. João Lúcio de Azevedo (ESALQ/USP) for the insightful suggestions on the manuscript and Dr. Nelson Netto
(UNICRUZ) for providing some fungi for our mycology collection.

References

1. Radzio, R., & Kück, U. (1997). Process Biochemistry, 32, 529–539.

2. Hajjaj, H., Blanc, P. J., Goma, G., & François, J. (1998). FEMS Microbiology Letters, 164, 195–200.
3. Mapari, S. A. S., Meyer, A. S., & Thrane, U. (2008). Biotechnology Letters, 30, 2183–2190.
4. Rajasekaran, R., Chandrasekaran, R., & Muthuselvam, M. (2008). Advances in Biotechnology, 7, 19–25.
5. Pandey, A., Soccol, C. R., Nigam, P., & Soccol, V. T. (2000). Bioresource Technology, 74, 69–80.
 Appl Biochem Biotechnol

6. Soccol, C. R., & Vandenberghe, L. P. S. (2003). Biochemical Engineering Journal, 13, 205–218.
7. Daroit, D. J., Silveira, S. T., Hertz, P. F., & Brandelli, A. (2007). Process Biochemistry, 42, 904–908.
8. Singhania, R.R.; Soccol, C.R.; Pandley, A. (2008) Application of tropical agro-industrial residues as
substrate for solid-state fermentation processes. In: Current developments in solid-state fermentation,
Springer Science+Business, New York.
9. Lopes, F. C., Dedavid e Silva, L. A., Tichota, D. M., Daroit, D. J., Velho, R. V., Pereira, J. Q., Côrrea, A.
P. F., & Brandelli, A. (2011). Enzyme Research. doi:10.4061/2011/487093.
10. Horisawa, S., Sakuma, Y., & Doi, S. (2009). Journal of Wood Science, 55, 133–138.
11. Glass, N. L., & Donaldson, G. C. (1995). Applied and Environmental Microbiology, 61, 1323–1330.
12. Saitou, N., & Nei, M. (1987). Molecular Biology and Evolution, 4, 406–425.
13. Kimura, M. (1980). Journal of Molecular Evolution, 16, 111–120.
14. Silveira, S. T., Daroit, D. J., & Brandelli, A. (2008). LWT Food Science Techonology, 41, 170–174.
15. Dedavid e Silva, L. A., Lopes, F. C., Silveira, S. T., & Brandelli, A. (2009). Applied Biochemistry
Biotechnology, 152, 295–305.
16. Mapari, S. A. S., Meyer, A. S., & Thrane, U. (2006). Journal of Agricultural and Food Chemistry, 54,
7027–7035.
17. Senyuva, H. Z., Gilbert, J., & Öztürkoglu, S. (2008). Analytica Chimica Acta, 617, 97–106.
18. Velmurugan, P., Kamala-Kannan, S., Balachandar, V., Lakshmanaperumalsamy, P., Chae, J. C., & Oh, B.
T. (2009). Carbohydrate Polymers, 79, 262–268.
19. Fungaro, M. H. P. (2000). Biotecnologica Ciencias Desenvol, 3, 12–16.
20. Einax, E., & Voigt, K. (2003). Organisms, Diversity and Evolution, 3, 185–194.
21. Bastola, D. R., Out, H. H., Doukas, S. E., Sayood, K., Hinrichs, S. H., & Iwen, P. C. (2004). Mycological
Research, 108, 117–125.
22. Inuma, T., Khodaparast, S. A., & Takamatsu, S. (2007). Molecular Phylogenetics and Evolution, 44, 741–
751.
23. Mapari, S. A. S., Hansen, M. E., Meyer, A. S., & Thrane, U. (2008). Journal of Agricultural and Food
Chemistry, 56, 9981–9989.
24. Wolf, F. T., Kim, Y. T., & Jones, E. A. (1960). Physiological Plant, 13, 621–627.
25. Mapari, S. A. S., Meyer, A. S., Thrane, U., & Frisvad, J. (2009). Microbial Cell Factories, 8, 1–15.
26. Asilonu, E., Bucke, C., & Keshavarz, T. (2000). Biotechnology Letters, 22, 931–936.
27. Nielsen, K. F., & Smedsgaard, J. (2003). Journal of Chromatography. A, 1002, 111–136.
28. Vishwanath, V., Sulyok, M., Labuda, R., Bicker, W., & Krska, R. (2009). Analytical and Bioanalytical
Chemistry, 395, 1355–1372.
29. Sulyok, M., Krska, R., & Schuhmacher, R. (2010). Food Chemistry, 119, 408–416.
30. Rasmussen, R. R., Rasmussen, P. H., Larsen, T. O., Bladt, T. T., & Binderup, M. L. (2011). Food and
Chemical Toxicololgy, 49, 31–44.
31. Tor, E. R., Puschner, B., Filigenzi, M. S., Tiwary, A. K., & Poppenga, R. H. (2006). Analytical Chemistry,
78, 4624–4629.
32. Paterson, R. R. M., Venâncio, A., & Lima, N. (2006). Revista Iberoamericana de Micologia, 23, 155–
159.
33. Prabha, D., D’Souza, L., Kamat, T., Rodrigues, C., & Naik, C. G. (2009). Indian Journal of Marine
Science, 38, 38–44.
34. Lopes, F. C., Tichota, D. M., Sauter, I. P., Meira, S. M. M., Segalin, J., Rott, M. B., et al. (2013). Annals of
Microbiology, 63, 771–778.