Biochemical Engineering Journal 161 (2020) 107698

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Biochemical Engineering Journal

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Regular article

Medium design from corncob hydrolyzate for pigment production by T

Talaromyces atroroseus GH2: Kinetics modeling and pigments
characterization
Lourdes Morales-Oyervidesa,b,1, Juan Pablo Ruiz-Sánchezb,1, Jorge C. Oliveiraa,
Maria J. Sousa-Gallaghera, Thelma K. Morales-Martínezc, Ambrogina Albergamod, Andrea Salvoe,
Daniele Giuffridad, Laurent Dufosséf, Julio Montañezb,*
a
School of Engineering, University College Cork, Cork, Ireland
b
Department of Chemical Engineering, Autonomous University of Coahuila. Saltillo, Coahuila, Mexico
c
Biotechnology Department, Autonomous University of Coahuila. Saltillo, Coahuila, Mexico
d
Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali, University of Messina, Messina, Italy
e
Dipartimento di Chimica e Tecnologie del Farmaco, Facoltà di Farmacia e Medicina, Sapienza Università di Roma, Roma, Italy
f
Chimie et Biotechnologie des Produits Naturels & ESIROI Agroalimentaire, Université de la Réunion, Ile de la Réunion, France

H I GH L IG H T S

• Corncob-based media is a potential option for pigment production by T. atroroseus GH2.

• The acid hydrolysis treatment released high xylose yields for culture media design.
• Co-utilization of xylose-glucose paves the way to study various agricultural wastes.
• Characterized pigments produced by T. atroroseus GH2 differed among evaluated media.
• Pigments produced by T. atroroseus GH2 are homologous of Monascus’ pigments.

A R T I C LE I N FO A B S T R A C T

Keywords: The genus Talaromyces has gained attention due to its ability to produce pigments with potential industrial
Hydrolysis applications in different areas. Prosperous application of fungal pigments has challenges to overcome, like de-
Biorefinery veloping a cost-effective bioprocess. Using agroindustrial wastes could provide inexpensive substrates and it
Fungal pigments contributes to maximize sustainability. Therefore, this study evaluated the feasibility of using corncob as a low-
Kinetics
cost substrate for pigment production by Talaromyces atroroseus GH2. An acid hydrolysis treatment was used to
Characterization
release sugars from corncob. Corncob liquors with enough xylose concentration (> 20 g/L) were investigated as
fermentation media with and without the addition of nutrients. Process kinetic modeling was applied and
pigments produced in corncob and control media were characterized. The diluted hydrolyzate without nutrient
supplementation showed a pigment production (16.17 ± 0.37 OD500nm) comparable to the control medium
(17.26 ± 0.41 OD500nm). Talaromyces atroroseus GH2 was able to co-utilize xylose and glucose in the corncob-
based medium. However, growth kinetics patterns differed in both media. In the hydrolyzate medium, biomass
growth presented an extended lag phase, which requires reduction for future process optimization. Finally,
characterized pigments differed among evaluated media, but the pigments produced by Talaromyces atroroseus
GH2 were mostly Monascus’ pigments homologous like monascorubrin and rubropunctamine. Talaromyces
atroroseus GH2 ability to produce pigments using corncob hydrolyzate makes it a pigment-producing strain for
an economically competitive large fermentation scale.

⁎
Corresponding author.
E-mail address: julio.montanez@uadec.edu.mx (J. Montañez).
1
The first two authors contributed equally to work.

https://doi.org/10.1016/j.bej.2020.107698
Received 2 April 2020; Received in revised form 7 June 2020; Accepted 22 June 2020
Available online 27 June 2020
1369-703X/ © 2020 Elsevier B.V. All rights reserved.
L. Morales-Oyervides, et al.
Nomenclature
PDA Potato dextrose agar
CDM Czapek-dox modified medium
HM Hydrolyzate based medium
HPLC High-performance liquid chromatography
DAD Diode array detector
ESI Electrospray ionization
MS Mass spectrometry
OD Optical density
HE Extracellular pigments fromhydrolyzate medium
HI Intracellular pigments fromhydrolyzate medium
CE Extracellular pigments from controlmedium
CI Intracellular pigments from controlmedium
dSi/dt Consumption rate of substratei (g/L.h)
1. Introduction
The toxicity of synthetic colorants used in foods, pharmaceuticals,
and cosmetic preparations led to increased research of natural sources
of pigments [1]. Fungi represent an alternative source of natural pig-
ments with potential industrial applications due to the wide range of
colors that they can produce under scalable, controlled conditions [2].
Up to now, the most well-documented pigment-producing fungus is
Monascus, which is known to produce at least 50–60 secondary meta-
bolites, from which six pigmented molecules are the most studied [3].
Nevertheless, certain Monascus strains co-produce citrinin with the
pigments under various cultivation conditions [4], which makes Mon-
ascus food products forbidden by international organisms such as the
“Food and Drug Administration” (FDA). Consequently, different my-
cotoxin-free fungal pigment producers are required to satisfy the need
for natural pigments without the presence of harmful side effects.
Talaromyces fungal species are known to produce pigments and
other bioactive compounds similar to those produced by Monascus [5].
Such pigments come in a broad range of colors with diverse structures
that form a class of natural molecules of high importance to several
industries. In most recent studies, the strategy has focused on opti-
mizing process conditions to achieve high yields in terms of color in-
tensity [6–9]. However, the regulatory approval by international or-
ganisms via pigment characterization represents a critical step to ensure
that the utilization of these pigments does not pose any risk for human
intake or the environment. Consequently, there is an increased interest
in the isolation and characterization of the pigments produced by Ta-
laromyces [10–12].
Additionally, the industrial application of natural pigments remains
uncompetitive compared to synthetic colorants due to high production
costs [13]. Efforts are necessary to develop a cost-effective bioprocess
for the production of such pigments. This need to increase production
yields at lower costs requires the use of inexpensive raw materials and
the utilization of cheap residues as a substrate is a reasonable strategy
[14]. Suitable waste selection will depend on the availability of raw
materials for any geographic zone and the nutrient requirements of the
bioprocess.
It has been demonstrated that Talaromyces species can produce high
pigment yields using xylose as a carbon source [6]. Thus, lignocellulosic
hydrolyzates, which contain high levels of xylose [15], represent a
potential source that can be microbiologically converted into pigments.
One option is the waste generated by corn crops. Worldwide, maize,
one of the most widely cultivated crops, produces a significant amount
of corncob residues that are either burned, discarded or used as animal
feed at least [16]. Notably, in Mexico, corn is the most important crop
since it is a principal constituent of the Mexican diet; for instance, in
2019, the cultivated area of maize was 7,143,102 ha [17]. Thus, it is
mandatory to find a better use for such waste in order to ensure a
2
Biochemical Engineering Journal 161 (2020) 107698
dX/dt Biomass growth rate (g/L.h)
dP/dt Production rate (OD/h)
Si Carbon sourcei concentration (g/L)
Sires Residual substrate i (g/L)
SL Limiting substrate (g/L)
Si o′ ratio between the initial substrate consumption rate and
the initial substrate concentration (h−1)
ki Lag time before a substrate starts being consumed (h)
YX/Si Yield of biomass formed by each substrate (g/g)
X Biomass concentration (g/L)
P Total pigment production (OD500nm)
YP/X Yield of total pigment production per biomass (OD.L/g)
α Production related to growth
β Production related to maintenance
R2 Correlation coefficient
biobased economy [18].
On the other hand, it is crucial to note that Talaromyces growth and
pigment production is profoundly affected by media components like
carbon and nitrogen sources [11,19]. Hydrolyzate liquors obtained
from lignocellulosic materials usually contain a mixture of carbohy-
drates (pentoses and hexoses) [15]. Thus, it is important to understand
the microorganism's capability to co-utilize pentoses and hexoses and
its effect on the produced pigmented molecules in order to maximize
process yields.
In this sense, the present work aimed to provide knowledge that will
contribute to the development of a cost-effective bioprocess for the
production of pigments by Talaromyces atroroseus GH2 using a lig-
nocellulosic waste such as corncob as raw material. The study was,
thus, designed in four tasks. Initially, acid hydrolysis conditions were
evaluated to obtain a liquor with high levels of xylose to be used as a
fermentation medium. Then, selected liquors were studied for the
production of pigments with and without nutrients supplementation.
Further, process kinetics were assessed to describe pigment production
when using a corncob-based medium composed of different carbon
sources. Finally, it was performed a partial characterization of the
pigments produced with a chemically defined medium and the designed
corncob-based medium.
2. Materials and methods
2.1. Corncob hydrolyzate preparation
Corncob was donated by the Agricultural Autonomous Antonio
Narro University (UAAAN, Mexico). Corncob was milled using a cutting
mill (Retsch SM 100, Germany), and particles in a range of 1−3 mm
were used for the hydrolysis process. The acid hydrolysis was carried
out at a constant temperature and pressure (121 °C, 15 psi), and a solid
to liquid ratio of 1:10 was used. Two factors were varied, H2SO4 con-
centration (0.5, 1.0, 1.5 % v/v) and residence time (30, 60, 90 min).
The liquors were recovered by filtration using a muslin-cloth and de-
toxified according to conditions reported in the literature [20].
2.2. Microorganism and inoculum preparation
Talaromyces atroroseus GH2 was used for the production of pigments
(Department of Food Science and Technology strain repository,
Autonomous University of Coahuila. Saltillo, Mexico). The strain was
isolated from a Mexican semi-desert plant (Larrea tridentata tissue,
Coahuila, Mexico) and it was genetically identified. The strain was
maintained at 4 °C on Potato Dextrose Agar (PDA) slants. According to
previously reported conditions, the inoculum was propagated in liquid
culture using Potato Dextrose Broth (ATCC medium: 336) [6].
L. Morales-Oyervides, et al.
2.3. Culture media formulation
A synthetic Czapek-dox modified medium (CDM) was used as a
control [21]. The medium contained (g/L): D-xylose (15.0), NaNO3
(3.0), K2HPO4 (1.0), KCl (1.0), MgSO4·7H2O (0.5), FeSO4· 7H2O (0.1),
and ethanol (20.0). The corncob liquors obtained by acid hydrolysis
were used for fermentation as a) liquor without nutrient supple-
mentation, b) liquor supplemented with CDM nutrients, c) liquor
without nutrient supplementation adjusted to a xylose concentration of
15.0 g/L, and d) liquor adjusted to a xylose concentration of 15.0 g/L
and supplemented with CDM nutrients. CDM nutrients include all
Czapek-dox medium components except xylose and ethanol.
2.4. Cultivation conditions
Pigment production was carried out according to previously re-
ported conditions [7]. The initial pH of all the studied media was ad-
justed to 5.0 before sterilizing by using 0.22 μm sterile membranes
(Millipore, USA). Erlenmeyer flasks (125 mL with 25 mL of working
volume) were inoculated with a mycelial suspension (10 % v/v) of
Talaromyces atroroseus GH2. The inoculated flasks were incubated at
30 ± 2 °C on an orbital shaker (Inova 94, New Brunswick Scientific,
USA) at 200 rpm for 8 days.
2.5. Process kinetics and model fitting
Fermentation kinetics in selected corncob hydrolyzate medium
(HM) and control medium were studied and described by unstructured
models. Substrate-dependent models were applied to describe growth
and production of pigments as a function of the consumption of the
carbon sources presented in both media [22]. The consumption rate of
substrate (dSi/dt, g/L.h) was estimated with the model:
'
dSi (Sio − Sires ) Sio Sires Si o' e Sio (t − ki )
=− ' tracellular pigments using a rotary evaporator. Then, intracellular and
dt [Sio − Sires + Sires e Sio (t − ki ) ]2 (a)
where the subscript i stands for analyzed substrate, Sires represents the
residual concentration of substrate i (g/L), Si o′ is the ratio between the
initial substrate consumption rate and the substrate concentration at
the beginning of the process (h−1) and ki is the lag time (h) before the
carbon source i starts being consumed.
Biomass growth rate (dX/dt, g/L.h) was expected to be a function of
the carbon sources concentration (Si, g/L) presented in both media and
the yield of biomass formed by each substrate i (YX / Si , g/g):
n
dX dS
= −∑ YX / Si i
dt i=1
dt (b)
Please note that in Eq. b, each substrate will have its individual con-
tribution of biomass per substrate and substrate consumption rate. The
Luedeking-Piret model described the rate of pigments production (dP/
dt, OD/h):
dP dX SL, res ⎞
=α + βX ⎛1 − ⎜ ⎟
dt dt ⎝ SL ⎠ (c)
where X is the instantaneous biomass concentration (g/L), SL and SL, res
are the instantaneous and residual limiting substrate (g/L), respec-
tively. The model includes two parameters with significant biological
meaning, α for production related to growth, and β for production re-
lated to maintenance. Carbon sources for model fitting were selected
after obtaining the kinetic studies results and, thus, they are described
in the results section (3.3).
2.6. Analytical methods
The liquors obtained from hydrolysis were analyzed for xylose,
3
Biochemical Engineering Journal 161 (2020) 107698
cellobiose, glucose, arabinose, and acetic acid concentration by high-
performance liquid chromatography (HPLC, Agilent, USA). The column
used was a Hi-Plex (300 mm x 8 mm x10 μm, Agilent, USA). The flow
rate and solvent phase were 0.5 mL /min and 5.0 mM H2SO4. Detection
was carried out by refractive index (RI) detector and an Agilent Hi-Plex
column at 45 °C, using 5.0 mM H2SO4 as eluent. The retention time and
peak areas were compared with external standards.
Extracellular pigment recovery and intracellular pigment extraction
were performed as previously reported [7]. Briefly, samples were cen-
trifuged at 6236 g for 20 min at 4 °C (Sorvall, Primo R Biofuge Cen-
trifugation Thermo, USA) and the extracellular pigmented extracts were
filtered (Cellulose filter, 0.45 μm. Millipore, USA). The collected bio-
mass was rinsed with distilled water and it was used for intracellular
pigments extraction with an aqueous ethanol solution (70 % v/v) using
the same fermentation working volume (25 mL). Extraction was carried
out in an orbital shaker (Inova 94, New Brunswick Scientific, USA) at
200 rpm at 30 °C for 1 h and biomass was again removed by cen-
trifugation (6236 g for 20 min at 4 °C). Pigment concentration (extra-
cellular and intracellular) was quantified indirectly by measuring the
optical density at the maximum absorbance wavelength (500 nm)
[8,9,23]. Total pigment concentration consisted of the sum of extra-
cellular and intracellular pigments. The gravimetric method was used
to measure biomass concentration. The substrate consumption analysis
was carried out by HPLC, as previously mentioned.
2.7. Partial characterization of colored extracts
The extracts obtained with different production media (HM and
CDM) were used for determining CIELAB color coordinates (Minolta CT
310, Konica Minolta, Mahwah, NJ). The absorbance for all the extracts
was adjusted to 5.0 at 500 nm for the colorimetric studies [24].
The pigment characterization was carried out following the meth-
odology previously described by Venkatachalam et al. with some
modifications [10]. Before analysis, ethanol was removed from in-
extracellular extracts were frozen (−20 °C) and lyophilized, yielding a
red-colored powder. For each sample, 100 mg of red powder was dis-
solved in 1 mL of methanol, then filtered through a 0.45 μm syringe
PTFE filter, followed by a second filtration through a 0.20 μm syringe
PTFE filter. The crude filtrates were stored at 4 °C in a vial until HPLC
analysis. Then, Briefly, HPLC-DAD-ESI-MS analyses were performed on
a Shimadzu Prominence UFLC XR system, and data obtained were
analyzed using the Shimadzu Lab Solution Software (Shimadzu, Kyoto,
Japan). The chromatographic procedure was carried out using a Kinetix
C18 column (150 × 2.1 mm; 1.7 μm, Phenomenex, Torrence, CA, USA).
Mobile phases were water for eluent A and acetonitrile for eluent B.
Both phases were acidified with 0.1 % formic acid. A flow ware of
0.2 mL/min, 1 μL for sample injection, and 30 °C oven temperature
settings were used. ESI-MS was carried out following the same settings
as described by the authors above mentioned [10]. Identification of
azaphilones was made by comparing the retention time, DAD, and MS
spectra obtained from analyzed samples with those already described in
literature.
2.8. Data and statistical analysis
Total pigment production (P, OD500nm), biomass (X, g/L), and yield
of total pigment production per biomass (YP/X, OD.L/g) were the re-
sponses considered for the media formulation from corncob hydro-
lyzate. YP/X was defined as the ratio between the total produced pig-
ment and biomass at the end of the fermentation [21].
Composition of the corncob liquors for hydrolysis conditions and P,
X and YP/X for media formulation were analyzed with an ANOVA to test
statistical differences (p < 0.05), followed by post-hoc analysis with
Tukey’s test at 5 % probability to define homogeneous groups.
Eqs. a, b, and c where integrated between X = Xo, P = Po, Si = Sio at
L. Morales-Oyervides, et al.
time t = 0 and X = X, P = P, Si = Si at time t = t. Model parameters
from resulted equations were jointly estimated by non-linear regression
analysis using Excel (Microsoft, USA). Statistical analyses were made
with Statistica 7.0 software (StatSoft, USA).
3. Results and discussion
3.1. Selection of hydrolysis conditions
Preliminary studies in this work included the evaluation of corncob
hydrolysis by two proven methods for hemicellulose breakdown, acid
hydrolysis and autohydrolysis [15]. However, with the autohydrolysis
treatment, the xylose concentration of the control medium (15 g/L) was
not even reached (data not shown). Therefore, the evaluation of acid
hydrolysis was only continued.
Nine different liquors from corncob hydrolysis at different condi-
tions were obtained. The evaluated factors at different levels, sugar
composition and acetic acid concentrations obtained are shown in
Table 1. The analysis revealed that both factors (time and acid con-
centration) and their interaction significantly affected the sugars and
acetic acid contents (p < 0.05). Xylose was the primary mono-
saccharide released in all the obtained liquors, representing up to 70 %
of recovered sugars, which is explained by the high ratio of hemi-
cellulose in corncob composition (≈ 44 %). Hemicellulose is a het-
erogeneous polymer constituted by different sugars such as L-arabinose,
D-galactose, D-glucose, D-mannose, and D-xylose; depending on its
structural composition, hemicellulose is classified in different groups
like xylans, glucans, mannans, etc [15]. Corncob hemicellulose is con-
sidered xylan-type; thus, it represents a potential source of xylose [25].
The monomers arabinose and glucose were recovered at an average of
12 % and 15 % of total sugars in all liquors. The xylose and arabinose
levels obtained in this study were similar to those reported by Cai et al.
[25]; however, the glucose level was slightly higher with the acid hy-
drolysis conditions studied here. It has been reported that glucose re-
covered from hydrolyzates can be obtained from both hemicellulose
and cellulose breakage [26]. It is possible that the cellulose was hy-
drolyzed at a lower level, resulting in higher glucose concentration and
solubilized cellobiose (3 %), which is a disaccharide obtained from the
hydrolysis of cellulose. Regarding the acetic acid content in all the li-
quors, the reached concentration was in the range of 2.53–4.18 g/L,
which is similar to that obtained by Cai et al. [25]. Previous studies
reported that acetic acid could negatively impact the microorganism
growth in a fermentation process [27,28], yet the concentration toler-
ated by Talaromyces atroroseus is still unknown and worth studying.
In any case, all the liquors presented a higher concentration of xy-
lose than the one of the chemically defined control medium (15 g/L).
Therefore, in theory, any set of conditions presented in Table 1 could be
selected to design a waste-based medium by adjusting the xylose con-
centration according to the control medium. From an economic point of
view, a liquor with a high concentration of xylose obtained in less time
Table 1
Composition of the corncob liquors obtained after acid hydrolysis.
Corncob Conditions Means of responses, g/L
Liquor
H2S04, v/v% Time, min Cellobiose
e e
1 0.5 30 0.52
2 0.5 60 1.13c
3 0.5 90 1.43a
4 1.0 30 1.07cd
5 1.0 60 1.33ab
6 1.0 90 1.20bc
7 1.5 30 1.28b
8 1.5 60 1.13c
9 1.5 90 0.97d
Different letters in each column indicate significant differences (Tukey’s post-hoc comparison, P < 0.05).
4
Biochemical Engineering Journal 161 (2020) 107698
and with low acid concentration would be preferred. However, the
highest concentration of xylose (33.66 ± 0.60 g/L) was obtained using
the most severe hydrolysis conditions tested (90 min, 1.5 % H2SO4).
Moreover, under those conditions, acetic acid concentration was also
high (4.18 ± 0.06 g/L), which could negatively affect fungal growth.
On the other hand, the concentration of glucose, arabinose and
cellobiose varied significantly among all evaluated treatments
(p < 0.05). Reported carbon sources to increase pigment production
by Talaromyces strains are xylose [29], sucrose [30] and starch [31],
while simple carbohydrates such as glucose might induce fungal growth
with reduced production of pigments [29,32]. It is difficult to select
only one treatment (i.e., high xylose – low glucose) because, mostly, the
concentration of these two substrates similarly increased under the
applied hydrolysis conditions. Moreover, it was unknown how such
obtained mixture of carbohydrates and their concentration could affect
pigment production by Talaromyces atroroseus GH2.
Therefore, liquors obtained using the low (30 min, 1.0 % H2SO4),
intermediate (60 min, 1.0 % H2SO4), and high levels (90 min, 1.5 %
H2SO4) of the hydrolysis conditions presented in Table 1 were selected
for the subsequent experiments of the pigment production process. At
these conditions, the liquours differed significantly in the composition
of all the carbohydrates and acetic acid (Tuckey test different homo-
genous groups, p < 0.05).
3.2. Media formulation from corncob hydrolyzate
As shown in section 2.3, the liquors were used as obtained or diluted
to achieve a xylose concentration similar to the one utilized in the
control medium. Also, they were evaluated with and without nutrient
supplementation. Results of total pigment production (extracellular and
intracellular), biomass growth, and pigment per biomass yield obtained
for all designed media with the different liquors are presented in
Table 2. ANOVA for pigment production, biomass growth and pigment
per biomass yield can be viewed as supplementary material.
The total production of pigments was significantly affected by all
the factors understudy (p < 0.05). The addition of nutrients was the
factor with the highest effect, which negatively affected the production
of pigments; the production was negligible in all the hydrolyzates
supplemented with nutrients (0.17–1.01 OD500nm). It has been reported
that Talaromyces pigments are secondary metabolites produced under
different stress conditions [21]. Therefore, the low production of pig-
ments when nutrients were added may be due to a low chemical sti-
mulation by the medium components when nutrients are widely
available. Also, it is possible that due to the hydrolyzate glucose com-
position, it is required a different nitrogen source (organic) than the
utilized in this study (NaNO3), as recently proposed for the pigment
production by Talaromyces amestolkiae [33]. Xylose adjusted con-
centration affected the production of pigments significantly
(p < 0.05). When the hydrolyzate was used without the addition of
nutrients and xylose adjustment, the microorganism produced a low
Glucose Xylose Arabinose Acetic Acid
e f
3.30 20.77 3.90 2.53a
4.88d 26.63d 4.27e 3.21b
7.11b 29.21cd 4.94de 3.72d
4.71d 26.53d 4.34e 3.16b
6.82c 29.98c 5.12d 3.74d
7.30b 31.83abc 5.49c 3.81d
6.58c 30.34bc 4.72d 3.45c
8.20a 32.74ab 6.02b 3.84d
8.31a 33.66a 6.39a 4.18e
L. Morales-Oyervides, et al. Biochemical Engineering Journal 161 (2020) 107698

Table 2
Designed media for fermentation studies from corncob liquor using selected hydrolysis conditions. Biomass, total pigment production (extracellular and in-
tracellular), and yield of pigment per biomass obtained.
Hydrolysis conditions Nutrient Additioni Xylose Adjustedii X, g/L P, OD500nm YP/X, OD.L/g
Time, min -H2SO4, % v/v

30−0.5 No No 3.64 ± 0.28 c,d 7.45 ± 0.55 c 2.06 ± 0.32 c,d

Yes No 3.11 ± 0.34 d,e,f 0.81 ± 0.28 e 0.26 ± 0.07 e
No Yes 3.41 ± 0.15 c,d 7.51 ± 0.53 c 2.21 ± 0.25 c
Yes Yes 3.92 ± 0.30 b,c 0.69 ± 0.02 e 0.18 ± 0.02 e
60−1.0 No No 6.25 ± 0.46 a 5.29 ± 0.19 d 0.85 ± 0.09 e
Yes No 2.47 ± 0.27 f,g 0.36 ± 0.03 e 0.15 ± 0.01 e
No Yes 3.28 ± 0.14 c,d,e 12.82 ± 0.34 b 3.92 ± 0.24 b
Yes Yes 3.65 ± 0.37 b,c,d 1.01 ± 0.20 e 0.28 ± 0.05 e
90−1.5 No No 4.43 ± 0.13 b 4.86 ± 0.40 d 1.10 ± 0.12 d,e
Yes No 0.88 ± 0.14 h 0.17 ± 0.03 e 0.20 ± 0.05 e
No Yes 1.99 ± 0.15 g 16.17 ± 0.37 a 8.18 ± 0.70 a
Yes Yes 2.67 ± 0.45 e,f 0.82 ± 0.08 e 0.31 ± 0.05 e
Control CDM 2.56 ± 0.23 17.26 ± 0.41 6.77 ± 0.67

i Corncob liquor supplemented with all CDM medium nutrients except xylose and ethanol.
ii corncob diluted with distilled sterile water to obtain a xylose concentration of 15 g/L.
Different letters in each column indicate significant differences between responses obtained using different hydrolysis conditions, nutrient addition and xylose
adjustment (Tukey’s post-hoc comparison, p < 0.05).

pigment yield (4.86–7.45 OD500nm), which confirms that there is some
level of inhibition by an elevated substrate.
The corncob liquor obtained at different hydrolysis conditions pre-
sented the lowest effect on pigment production even though it had
statistical significance (p < 0.05). The highest production of total
pigments was achieved by the diluted liquor obtained at the most se-
vere hydrolysis conditions and without the addition of any nutrient
(16.17 ± 0.37 OD500nm).
Regarding the microorganism growth, it was significantly affected
by all the analyzed variables (p < 0.05). It can be seen that
Talaromyces atroroseus GH2 was able to grow in all studied media ex-
cept for the liquor obtained at the maximum hydrolysis conditions
without adjusting the xylose concentration and with the addition of
nutrients. The ANOVA showed that the combined effect of the addition
of nutrients and the xylose adjustment was the highest effect on the
microorganism's growth. When the liquors were diluted, the addition of
nutrients promoted an increment in biomass growth; however, the
addition of nutrients negatively affected the growth of the micro-
organism when the concentrated liquors were used.
The maximum biomass was obtained with the liquor obtained at
intermediate hydrolysis settings without adjusting the xylose con-
centration and without adding any nutrient (6.25 ± 0.46 g/L), and it
was significantly different from the liquor obtained at the maximum
hydrolysis conditions. Thus, higher inhibitory compounds may be af-
fecting the microorganism despite the detoxification step used for the
hydrolyzates. As for the yield of pigment production per biomass, the
addition of nutrients was the factor with the highest effect; YP/X was
significantly reduced with the addition of nutrients. Also, adjusting the
xylose concentration promoted a significant increment in this response.
The maximum yield of pigment production per biomass was also ob-
tained with the diluted liquor obtained at maximum hydrolysis condi-
tions and without any nutrient addition.
The results obtained here differed from other authors, where fungal
pigments were produced using a hemicellulose hydrolyzate based
medium [29,34]. Zhou et al. studied the production of pigments by
Monascus spp. on a corncob hydrolyzate medium and reported the
highest production when the medium was supplemented with nutrients
(salts, xylose, and glucose) to achieve 70.0 g/L of the substrate, which is
3.5 higher than the concentration used in this study [34]. Sopandit
et al. studied the pigments' production by Penicillium resticulosum using
a waste stream cellulose medium and reported the highest production
with the hydrolyzate supplemented with xylose or carbox-
ymethylcellulose (30 g/L), salts, and a nitrogen source [29]. Pandit
et al. evaluated the production of pigments by Talaromyces
purpureogenus CFRM02 utilizing Bengal gram husk and reported that an
acid pretreatment of the material did not promote the production of
pigments; which implied the need of adding other nutrients such as
peptone, glucose and trace elements to the medium to achieve a high
production yield [12].
In this work, Talaromyces atroroseus GH2 was capable of achieving
high pigment yields by consuming only the diluted hydrolyzate without
any nutrient supplementation. The diluted liquor obtained at the
maximum levels for hydrolysis conditions without the addition of any
nutrient was selected for further fermentation studies (HM). Total
pigment production obtained with this medium was only 6 % lower
than the pigments produced CDM medium (17.26 ± 0.41 OD500nm ). In
terms of YP/X, it was 21 % higher when HM was used in comparison
with CDM (6.77 ± 0.67 OD/g.L).
Production of pigments using the diluted corncob hydrolyzate
without any nutrient supplementation makes corncob a potential low-
cost raw material towards the development of a cost-effective biopro-
cess. Nonetheless, it will be worth studying other hydrolysis methods
that comply with the green biorefinery concept [35] and thus be able to
develop a sustainable and environmentally friendly process.
3.3. Process kinetics and model fitting
It was of particular interest to understand how the microorganism is
consuming the different carbohydrates presented in the corncob liquor.
Most recently, it has been demonstrated that double substrate kinetics
models provide a better understanding of how nutrients conditions af-
fect the performance of biological systems [36]. Fig. 1 shows the per-
centage of each carbohydrate consumed by the microorganism during
the pigment production process. The main carbon sources consumed
were xylose and glucose, while arabinose and cellobiose were con-
sumed only less than 20 %. Thus, only xylose and glucose were con-
sidered for the modeling assessment. Each medium contained two
carbon sources for the microorganism growth and metabolites bio-
synthesis (xylose and ethanol, CDM; xylose and glucose, HM). For fu-
ture process development using a waste-based medium, it is crucial to
get an insight into how the microorganism consumes different sub-
strates in a medium and the effect on biomass growth and pigment
production. Therefore, the process kinetics using CDM and HM media
were studied. The models presented in section 2.5 were used to describe
the carbon source utilization in each medium. Biomass growth was
expected to be a function of the consumption of both carbon sources in
each medium.
The model fitting of biomass growth using the CDM medium was

5
L. Morales-Oyervides, et al.
Fig. 1. Carbohydrates consumed during the production of pigments by
Talaromyces atroroseus GH2 using HM medium. Cellobiose (square), glucose
(circle), xylose (triangle) and arabinose (diamond).
made without taking death cell points into account. For the HM
medium, it was assumed that pigments were produced during the
second phase of the growth kinetics. Xylose was considered as limiting
substrate for both mediums. Results are presented in Table 3, and the
model fittings for both CDM and HM media are shown in Fig. 2. The
models described the experimental data accurately with correlation
coefficients (R2) of 0.97 and 0.99 for CDM and HM media, respectively.
When CDM was used, it can be seen that the microorganism showed a
short lag phase (less than 24 h), which indicated that it rapidly adapted
to the medium. The stationary phase was reached at 120 h, and finally,
the death phase was observed. Si o′ significantly differed from xylose
(SXyl o′ = 0.14 h−1) and ethanol (SEtOH o′ = 0.05 h−1) which corre-
sponded to the initial consumption rates of 2.11 g/L.h and 1.10 g/L.h,
respectively. Thus, even though the lag time obtained for the utilization
of ethanol was low (kEtOH = 25.47 h), its assimilation started at a lower
rate than xylose. It is interesting to see that the microorganism con-
sumed both xylose and ethanol for its growth and it showed a diauxic
growth. The biomass yield on ethanol was higher than the obtained
with xylose (YX / SEtOH = 0.11, YX / SXyl = 0.08, respectively), which could
be explained by the high assimilation of the xylose and its rapid ex-
haustion. Previous studies from the research group demonstrated that
the addition of ethanol to the culture medium promoted the production
of pigments by Talaromyces atroroseus [37]; however, until now, it was
unclear if the ethanol served as a carbon source or as a stress factor.
Obtained results here demonstrated that Talaromyces atroroseus was
capable of simultaneously consuming ethanol and xylose, yet, xylose
was consumed at a higher rate. Similar behavior has been explained for
Monascus purpureus in regards to pigment production where the mi-
croorganism could uptake ethanol, inducing a shift in the metabolism
from growing to maintenance, thus favoring the production of pigments
[38,39].
Pigments were produced since the first 24 h, and the microorganism
continued the production until reaching the stationary phase. The
production of the pigments using CDM was associated with micro-
organism growth (α = 4.92, β = 0). It has been extensively reported
that fungal pigments are secondary metabolites produced under stress
conditions and mostly produced during the stationary phase or at the
end of the exponential growth [21,29,32]. Differences obtained with
CDM might be attributed to the utilization of mycelium as inoculum,
which required less adaptation time, so the microorganism showed a
reduced lag phase [7].
On the other hand, when the HM medium was used, the micro-
organism growth, production, and substrate consumption presented
different patterns. The microorganism required more time for adapting
to the medium by showing an extended lag phase. The stationary phase
was reached after 96 h (24 h less than in CDM); however, the death
6
Biochemical Engineering Journal 161 (2020) 107698
phase was observed until 168 h. The microorganism first used the most
assimilable and simple carbohydrate, glucose. Similar findings were
reported by Zhou et al. during the production of pigments by Monascus
in a corncob liquor; glucose was first assimilated than xylose [34]. Even
though initial glucose concentration was low (3.65 ± 0.34 g/L), the
total consumption of glucose was observed at 96 h. This was better
explained by the Si o′ values which were similar in xylose (SXyl o′ = 0.05
h−1) and glucose (SGluc o′ = 0.09 h−1); however, the corresponded initial
consumption rates varied from 0.32 g/L.h in glucose to 0.74 g/L.h in
xylose. These consumption rates are much lower than those obtained in
the CDM medium, which confirmed the low adaption of the micro-
organism to the hydrolyzate. Lower substrate consumption rates could
be attributed to the presence of hydrolysis by-products like furfural or
hydroxymethylfurfural despite the hydrolyzate detoxification step. So-
pandi et al. demonstrated that furfural concentration (1–5 g/L) could
significantly reduce glucose consumption by Penicillium resticulosum
during the pigment production process [29]. To overcome this issue, it
will be required to evaluate the effect of those hydrolysis by-products
on Talaromyces atroroseus growth, pigment production and substrate
uptake. Furthermore, the lag time (kxyl) for xylose assimilation was
64.29 h, and because the microorganism was at the end of the ex-
ponential phase, it started with rapid assimilation.
Furthermore, the highest biomass-substrate yield was achieved with
glucose (YX / SGluc = 0.54), while with xylose was nearly negligible
(YX / SXyl = 0.02). Therefore, glucose was mainly consumed for micro-
organism growth and xylose for cell maintenance. Pigments production
started after 72 h, which mostly corresponds to the exhaustion of glu-
cose. Moreover, when HM was used, pigment production was asso-
ciated with cell maintenance with values of α = 0 and β = 2.01. Thus,
in HM, the pigments showed characteristics of secondary metabolites;
that is, they were produced at the end of the logarithmic growth or by
nutrients exhaustion [32]. Differences obtained with CDM could be
attributed to the conditions utilized for inoculum preparation. The in-
oculum was prepared using PDA (with glucose added); thus, by trans-
ferring the microorganism to a medium with a different carbon source
than glucose (xylose), the microorganism immediately changed its
metabolism from growth to pigment production.
Regarding the reported kinetics for the production of pigments by
Talaromyces strains (Formerly Penicillium) using wastes, authors have
reported that pigments are produced either at the end [23] or at the
beginning of the logarithmic growth stage [29]. Kantifedaki et al.
evaluated the production of pigments by Penicillium purpurogenum using
wastes in solid and submerged fermentation and reported a negligible
production of pigments in submerged fermentation when using a
medium designed from hydrodistillation of orange peels [23]. Those
authors reported that their medium contained 25 g/L of total sugars
(mainly glucose, fructose, and sucrose); thus, carbon composition could
be the reason for the low production levels in that study. Moreover, in
their study, pigment production was observed at the end of the loga-
rithmic growth and after sugars exhaustion. Similarly, in the study for
the production of pigments by Penicillium resticulosum using a waste
stream cellulose medium, authors reported the need to add xylose or
carboxymethylcellulose to achieve high pigment production yields
Table 3
Model parameters obtained by least-squares regression of all data for biomass,
substrate consumption and pigment production kinetics in CDM and HM media.
Parameter CDM HM
Ethanol Xylose Xylose Glucose
Si o′ , h−1 0.06 0.14 0.05 0.09
ki, h 25.47 0.00 64.29 1.07
YX / Si 0.11 0.08 0.02 0.54
α 4.92 – 0.00 –
β 0.00 – 2.01 –
L. Morales-Oyervides, et al.
Fig. 2. Model fits of growth (a), total pigment production (b), and substrate consumption (c1, CDM; c2, HM) by Talaromyces atroroseus GH2 using CDM (squares) and
HM (circles) as media. Different symbols in c1: xylose (dark grey squares) and ethanol (light grey squares) and in c2: xylose (dark grey circles) and glucose (light grey
circles). Dashed lines represent model curves.
[29]. Such authors observed an extended lag phase (3 days) and the
microorganism started producing the pigments at the beginning of the
logarithmic phase. In another study for the production of pigments by
Penicillium sp. using a potato extract, authors reported a higher pro-
duction with the extract added with xylose and mannose rather than
with glucose [40]. In that study, kinetics were not evaluated but agreed
with our results and those obtained by Sopandi et al. [29], where
glucose is a good carbon source for growth but might interfere with the
production of pigments by Talaromyces strains. On the other hand, our
findings differ from the results obtained by Pandit et al., where they
obtained higher productivities with the addition of glucose and peptone
to the waste-based medium for the production of pigments by Talar-
omyces purpureogenus CFRM02 [41]. It has recently been pointed out
that glucose could be utilized for pigment production by Talaromyces
amestolkiae by using a neutral pH and complex nitrogen sources such as
meat peptone and meat extract [33].
The ability of Talaromyces purpureogenus GH2 to co-utilize a mixture
of carbon sources such as xylose and glucose opens the door to evaluate
various agricultural wastes. The key aspect is understanding how the
utilization of these two substrates affects pigment production and the
process kinetics. Supplementary experiments included the addition of
ethanol to the HM medium; however, ethanol was not consumed and its
addition did not increase the production of pigments. Those results
were attributed to the excess of carbon sources; for instance, for HM the
microorganism did not exhaust xylose concentration (Fig. 2c2). Thus,
future process optimization should focus on optimizing the glucose to
xylose ratio to avoid catabolite repression and to reduce the lag time
observed when the corncob-based medium was used. Knowledge about
7
Biochemical Engineering Journal 161 (2020) 107698
the mixed xylose/glucose utilization by pigment-producing strains is
limited and literature has demonstrated that microorganism behavior
on such nutrient conditions is quite contrasting [42,43]. Kinetic studies
using a synthetic defined medium of xylose and glucose could be useful
for a full understanding of the microorganism uptake of these two
substrates and then be able to optimize the xylose/glucose ratio in the
hydrolyzate. In any case, our findings highlight that the carbon source
composition of raw materials is essential for selecting a suitable waste
for developing a sustainable and cost-effective bioprocess for the pro-
duction of pigments by Talaromyces strains.
3.4. Color determination of the pigmented fungal extracts and partial
characterization of the pigments using LC–MS
The pigment extracts obtained in CDM and HM media were quali-
tatively perceived to be different. Pigments produced in HM medium
look like bright red, while pigments produced in CDM were dark red. A
quantitative description of the color is fundamental for industrial ap-
plications. CIELAB plot for pigmented extracts obtained with CDM
(Extracellular, CE; intracellular, CI) and with HM (Extracellular, HE;
intracellular, HI) is presented in Fig. 3, and it is interpreted as a positive
a* value indicating a red color, while a positive b* value indicates a
yellow color. The a* and b* values were positive for all the extracts
evaluated.
CE and CI exhibited values of a* (54.33–67.91) and b*
(49.27–50.32), indicating that these extracts were red. Extracts HE and
HI had lower values for a* (46.00–47.45) and higher for b*
(56.12–56.94), signifying that these values are “less red” than that
L. Morales-Oyervides, et al. Biochemical Engineering Journal 161 (2020) 107698

Fig. 3. CIELAB color space for pigmented extracts obtained with CDM medium
(CE, square; CI, triangles) and HM medium (HE, diamond; HI, circle).
obtained in CDM. Chroma values (color saturation) for all the extracts
evaluated were near to the circumference, indicating that they are vivid
and bright colors (CI, 74.05; CE, 83.90; HI, 73.20; HE 73.20). Hue
values for CE and CI were 35.96–42.80, respectively, while for HE and
HI were 49.79–51.07, respectively. Hue values near 0 indicated the
degree of redness; meanwhile, values near to 90 represent the level of
yellowness. Results for both medium were higher in terms of chroma
values (more vivid and bright colors) than those reported for the pro-
duction of pigments by Talaromyces purpureogenus using Bengal gram
husks as substrate [12]. Those authors reported improvements in terms
of color saturation (chroma) by adding peptone and other salts to the
waste-designed medium. On the other hand, our results were mostly
similar in terms of all CIELAB color coordinates (a*, b*, hue, and
chroma) reported for Talaromyces amestolkiae [8] and Talaromyces al-
bobiverticillius [44]. Regarding the production of pigments by Talar-
omyces amestolkiae, authors followed a step-wise statistical approach
varying nutrients and process conditions from which it was possible to
increase chroma values and to reduce the hue angles achieving a deep
red color [8]. As for the production of pigments by Talaromyces albo-
biverticillius using a potato dextrose broth medium supplemented with
sea salts, authors reported that a variation in added salt concentration
altered the color of the pigmented extracts [44]. Also, in a recent study,
it was reported that the pigments profile obtained by Talaromyces
atroroseus could vary with the addition of different amino acids as a
nitrogen source [11]. Thus, it is hypothesized that besides the sugar
composition in the corncob liquor, the nitrogen source could also be
affecting the metabolism of the strain and inducing the production of
orange-red pigments [4].
Table 4 presents all the tentatively identified compounds, their re-
tention time, DAD and MS data, tentative name and formula based on
the spectroscopic information obtained and the comparison with lit-
erature. Fig. 4 shows the three chromatograms of each sample, showing
the overall compounds detected by HPLC-DAD-MS. According to the
results, 10 different compounds were found in either extra or in-
tracellular crude extracts from the control medium or extracellular
crude extract from corncob hydrolyzate fermentation. Seven of them
were tentatively identified as Monascus-like pigments. Among the seven
identified compounds, four of them, viz. N-threoninerubropunctamine,
PP-R, PP-V, and Monascorubrin, were previously found either on Ta-
laromyces spp. or Talaromyces atroroseus extracts. While the other three
have been only described for Monascus spp. One more molecule iden-
tified as Cribrarione B has been only described from Cribraria cancellata,
and the last two compounds remained unidentified.
Compound 1. This compound was tentatively identified as
Manophilone B in agreement with Yuliana et al. [45]. Compound 1
exhibited the 337 [M−H]− m/z pseudomolecular ion and the UV–vis
spectra 206, 270, and 356.
Compound 2. In accordance with the reported by Iwata et al.,
compound 2 was tentatively identified as Cribrarione B [46]. This
compound showed the 249 [M+H]+ m/z and 248 [M−H]− m/z
pseudomolecular ions, and the UV–vis spectra 206 and 361.
Compound 5. Compound 5 was tentatively identified as N-threoni-
nerubropunctamine, accordingly with various results previously re-
ported [47–49]. It showed the 456 [M+H]+ m/z and 454 [M−H]− m/
z pseudomolecular ions, and the abduct 478 [M+H+Na]+ m/z. This
compound showed the UV–vis spectra 224, 273, 425 and 512.
Compound 6. Compound 6 results showed the 426 [M+H]+ m/z
pseudo molecular ion, along with the abducts 448, [M+H+Na]+ m/z
and 489 [M+H+Na + CH3CN]+ m/z, and the UV–vis spectra 221,
358, and 516. These results are in accordance with the values reported
for the tentative identification of this molecule as PP-R [5,24].
Compound 7. This compound showed the 412 [M+H]+ m/z and
410 [M−H]− m/z pseudomolecular ions, and the UV–vis spectra 221,
309, 409 and 522. This result, in agreement with the reported by

Table 4
Overall compounds detected by HPLC-DAD-ESI/MS in pigmented extracts produced with control medium, and corncob hydrolyzate by Talaromyces atroroseus GH2.
Compound RT DAD λ nm MS/ESI Tentative Formula Sample Reference
identification

1 5.376 206, 270, 356 337 [M-H]− Monaphilone Cn C20H32O4 CE, CI [45]
2 5.548 206, 361 249 [M+H]+; Cribrarione Bn C12H10O6 CE, CI, HE [46]
248 [M-H]−
3 9.664 219, 273, 425, 488 [M+H]+; n.i. |
CI
514 486 [M-H]−
4 9.689 224, 273, 425, 297 [M+H]+; n.i. |
CE, HE
513 296 [M-H]−
5 10.763 224, 273, 425, 456 [M+H]+; N-threoninerubropunctamineb C25H29NO7 CE, CI, HE [47,48,49]
512 454 [M-H]−; 478[M+H+Na]+
6 12.459 221, 358, 516 426 [M+H]+; 448[M+H+Na]+; PP-R a
C25H31NO5 CI [5,24]
489[M+H+Na + CH3CN]+
7 12.594 221, 309, 409, 412 [M+H]+ ; 410 [M-H]− PP-V a
C23H25NO6 CI [50,51]
552
8 13.161 224, 470 383 [M+H]+; Monascorubrinn C23H26O5 CE [47]
446 [M+H+Na + CH3N]+
9 13.483 223, 471 427 [M-H]− N-alaninerubropunctaminen C24H27NO6 HE [48]
10 14.165 223, 355 332 [M+H]+ Monaphilone Bn C20H28O4 CI [52]

a
Reported in Talaromyces atroroseus. b Reported in Talaromyces sp. n Non reported in Talaromyces spp. n.i., non identified. CE and CI, extracellular and intracellular
pigments respectively from control medium. HE, extracellular pigments from corncob hydrolyzate.

8
L. Morales-Oyervides, et al.
Fig. 4. Chromatograms of each sample showing the overall compounds detected by HPLC-DAD-MS. a) control medium, extracellular pigments; b) control medium,
intracellular pigments; c) corncob hydrolyzate medium, extracellular pigments.
research groups, permitted to identify the molecule as PP-V [50,51].
Compound 8. This compound was tentatively identified in ac-
cordance with the reports of Mapari et al. as Monascorubrin [47]. It
showed the 383 [M+H]+ m/z pseudo molecular ion, and the abduct
446 [M+H+Na + CH3N]+ m/z, and the UV–vis 224 and 470.
Compound 9. The results of compound 9 are in agreement with the
values reported by Jung et al. and identified tentatively as N-alani-
nerubropunctamine [48]. This molecule exhibited the 427 [M−H]− m/
z pseudo molecular ion, and UV–vis spectra of 223, and 471.
Compound 10. This molecule was tentatively identified as
Monaphilone B. The molecule showed the 332 [M+H] + m/z pseudo
molecular ion and UV–vis spectra 223, and 355. Such results are in
agreement with the values reported by Hsu et al. [52].
Finding similar pigments produced among different fungal species
can be well explained in accordance that the polyketide pathway,
which is the metabolic pathway that yields the azaphilone pigments, is
found among several species including Monascus, Penicillium, and
Talaromyces [47,53]. This phenomenon is of major importance because
it permits the production and obtention of pigments without the co-
production of harmful components, which is the reason that Monascus
9
Biochemical Engineering Journal 161 (2020) 107698
pigments are still forbidden in Europe and the USA for commercial use
[10].
The differentiation of azaphilone pigments may depend on the
structural components [24]. For red extract, while some molecules are
conformed by one component, some others can be constituted by two or
more components. This mixture allows the azaphilone pigments to ex-
hibit a variety of hues depending on the other moiety, more likely to be
an amine due to its remarkable affinity to ammonia that leads to the red
derivatives pigments [54].
Modifying the media components provokes a change in the pattern
of pigment production, as demonstrated by Mapari et al. They de-
monstrated how the type of pigments produced might vary in asco-
mycetous filamentous fungi not only among different species closely
related but also within the same species by growing the fungi in dif-
ferent media [24].
Although the metabolic pathways of pigments produced by
Talaromyces have not been fully elucidated [33], it is believed that
pigments biosynthesis is similar to Monascus. It was reported that or-
ange pigments are first synthesized; then, the orange pigments can be
reduced to yellow pigments; meanwhile, amination of the orange
L. Morales-Oyervides, et al.
pigments leads to red pigments [55]. Thus, varying the nitrogen source
has been extensively applied to modify the pigments produced by both
Monascus and Talaromyces [48,50,56]. Similarly, this technique has
been successfully applied for the production of azaphilone pigments
derivatives by Talaromyces atroroseus by utilizing amino acids as the
sole nitrogen source, thus modifying the produced pigments [11,19].
The difference between the amino derivatives pigments produced
with control and corncob hydrolyzate media may be explained by the
quantity and type of amino acids available in each medium.
Unfortunately, we were unable to characterize the hydrolysate in terms
of nitrogen composition. Nevertheless, studies have shown that despite
the relatively low corncob protein (≈ 5 %) [57], such components
could still be present in the hydrolyzate as a nitrogen source. Thus, we
assume that in the case of HM medium, the pigments could have re-
acted with possible free amino acids in the medium. On the other hand,
for the control medium, the amino derivatives may have formed from
the amino acids presented in the biomass, as previously reported by
Mapari et al. [47]. However, a nitrogen content characterization of the
hydrolyzate is required for understanding and controlling the produced
molecules. Also, the fact that amino acid derivatives were obtained in
the extracellular extracts could be explained due to such pigments ex-
hibit major hydrophilicity, so they are easier to be solubilized and ex-
creted into the medium [48].
Regarding the pigments PP-V and PP-R found in CDM but not in
HM, it could also be attributed to the nitrogen source, particularly the
one utilized in the control medium (NaNO3). PP-V and PP-R are mon-
ascorubramine derivatives and it has been demonstrated that they can
be produced upon sodium nitrate addition in the culture medium [56].
4. Conclusions
This study showed the promising use of corncob hydrolyzate for the
production of pigments by Talaromyces atroroseus GH2. The dilute hy-
drolyzate without any nutrient supplementation was the best medium
for pigment production, comparable to the obtained with a synthetic
medium. However, the pigments profile with the corncob-based
medium differed from the medium used as a control. In general, the
evidence from this study validates that carbon and nitrogen composi-
tion significantly affects the microorganism growth and the pigments
produced by Talaromyces atroroseus GH2. Agroindustrial wastes char-
acterization is crucial in order to be able to induce the microorganism's
secondary metabolism and to control the pigmented molecules pro-
duced. Future process optimization using a corncob hydrolyzate should
consider the reduction of the extended initial lag phase. Lastly, the
ability of Talaromyces atroroseus GH2 to grow and to produce a variety
of pigments with a corncob hydrolyzate medium without any nutrient
supplementation makes it a promising pigment-producing micro-
organism for economically competitive large fermentation scale.
CRediT authorship contribution statement
Lourdes Morales-Oyervides: Writing - original draft. Juan Pablo
Ruiz-Sánchez: Writing - original draft. Jorge C. Oliveira: Supervision,
Data curation. Maria J. Sousa-Gallagher: Writing - review & editing.
Thelma K. Morales-Martínez: Investigation. Ambrogina Albergamo:
Validation. Andrea Salvo: Validation. Daniele Giuffrida: Writing -
review & editing. Laurent Dufossé: Writing - review & editing. Julio
Montañez: Conceptualization, Funding acquisition.
Declaration of Competing Interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
10
Biochemical Engineering Journal 161 (2020) 107698
Acknowledgments
Authors L. Morales-Oyervides and J.P. Ruiz-Sanchez acknowledge
CONACyT-México for the financial support provided for conducting
their PhD and Master studies, respectively. Authors acknowledge
Autonomous University of Coahuila (Postgraduate and Research Office)
for the financial support (Seed project UADEC 2019 CGPI-UADEC C01-
2019-54).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.bej.2020.107698.
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