original scientific paper

ISSN 1330-9862
https://doi.org/10.17113/ftb.57.03.19.6068

Carotenoid Production by Rhodotorula mucilaginosa in Batch and

Fed-Batch Fermentation Using Agroindustrial Byproducts

Tábita Veiga Dias Rodrigues* , SUMMARY

Thalita D. Amore , Carotenoids are natural pigments that can be produced through biotechnological pro-
Erika Carvalho Teixeira and cesses. However, the costs are relatively high and can be minimized by using lower-cost
substrates as alternative nutrient sources. The fed-batch fermentation is one of the tech-
Janaina Fernandes de
niques used to obtain a high biomass concentration and/or maximum production. Thus,
Medeiros Burkert
the aim of this work is to produce carotenoids in batch and fed-batch fermentation with
School of Chemistry and Food, Federal the yeast Rhodotorula mucilaginosa CCT 7688 using agroindustrial byproducts in the cul-
University of Rio Grande, 96203-900 Rio ture medium. Carotenoid production was increased using experimental designs, which
Grande, Brazil modified the concentration of the agroindustrial medium. In batch production the highest
concentrations of total carotenoids (1248.5 μg/L) and biomass (7.9 g/L) were obtained in
Received: 3 October 2018
Accepted: 17 June 2019 the medium containing 70 g/L sugar cane molasses and 3.4 g/L corn steep liquor at 25 °C
and 180 rpm in 168 h, demonstrating an increase of 17 % when compared to the standard
yeast malt medium (1200 μg/L). In the fed-batch production, different feeding strategies
were tested with 30 g/L sugar cane molasses and 6.5 g/L corn steep liquor, reaching a total
carotenoid production of 3726 μg/L and biomass concentration of 16 g/L. Therefore, the
strategy of the fed-batch process resulted in an increase in the carotenoid production of
approx. 400 % compared to that in the batch process (740.3 μg/L). Thus, the R. mucilagi-
nosa strain has the potential to produce carotenoids in agroindustrial medium.

Key words: β-carotene, feeding strategies, corn steep liquor, sugar cane molasses, yeast

INTRODUCTION
Carotenoids are natural pigments found abundantly in nature whose isolation and char-
acterization have identified more than 600 molecules, allowing a great applicability of these
compounds, resulting in an increase in their use in the food, cosmetics and pharmaceutical
industries (1). These compounds are responsible for the intense colouring of fruits, vegeta-
bles, flowers, algae, bacteria and fungi (2), which varies from yellow to red, and are the most
studied natural pigments (3). The carotenoid world market in 2017 was US$ 1.5 billion with a
forecast of US$ 2.0 billion for 2022. β-Carotene is the most commonly consumed carotenoid,
with a market worth of US$ 261 million in 2010 and an estimation of US$ 334 million in 2018 (4).
The carotenoids, which are marketed as food additives and supplements, are mainly ob-
tained by synthetic methods (3). However, their production by natural processes has increased
due to the market demand for healthier foods with health benefits, in addition to the concern
of the consumers for the use of chemical additives in food (5). The antioxidant capacity (6),
the provitamin A activity, and the reduction of the risk of developing degenerative (7) and
cardiovascular diseases (8) are some of the beneficial health effects provided by carotenoids.
These compounds obtained in bioprocesses can be produced by a diversity of microor-
ganisms, such as microalgae (9-11) bacteria (12-14), fungi (15-16) and yeasts (17-19). However, the
production of carotenoids using biotechnological processes still has a high cost. Therefore, an
*Corresponding author: alternative to minimize the cost of this process is the utilization of agroindustrial byproducts
Phone: +555332336542 as sources of alternative nutrients (20). The genus Rhodotorula has been studied with the use
E-mail: tata.v.d.r@hotmail.com of low-cost substrates, such as sugar cane molasses (21), raw glycerol (22) and coffee grounds

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 Food Technol. Biotechnol. 57 (3) 388-398 (2019)

(23), to produce carotenoids. Although several microorganisms transferred to the same medium and incubated at 25 °C for 48
can biosynthesize carotenoids, not all of them are of interest for h. From the tubes containing the microorganisms in YM agar
industry. However, yeasts need a source of carbon and nitrogen slants, 1 mL of cell suspension in 0.1 % sterile peptone water
for biosynthesis, which is relatively simple when compared to were added to 9 mL of YM broth and incubated before inoc-
other microorganisms (24). ulation under the same conditions described previously (29).
The yeast Rhodotorula mucilaginosa utilized in this study was
isolated previously (25) and it demonstrated the ability to pro- Carotenoid production in shake flasks
duce carotenoids in media containing agroindustrial byprod-
The inoculum was prepared in 250-mL Erlenmeyer flasks
ucts, such as rice parboiling wastewater, raw glycerol, sugar cane
with 90 mL YM broth (29) and 10 mL previously grown culture
molasses and corn steep liquor (26), with antioxidant activity.
and incubated (incubator model TE-420; Tecnal, Piracicaba,
Another factor that may increase the production of carotenoids
Brazil) at 25 °C and 150 rpm for 48 h or for the time needed to
is the use of the fed-batch fermentation. This process controls
reach 108 cells/mL, counted using a Neubauer chamber (Lab-
the substrate concentration, timing its addition at moments in-
oroptik, Lancing, UK) (30). Carotenoid bioproduction in batch
dicated as favourable, minimizing the effects of inhibition of the
and fed-batch processes was conducted in 500-mL Erlenmey-
microorganism by the substrate and possibly improving the pro-
er flasks with 225 mL agroindustrial culture medium at an in-
duction of the biocompound (27,28).
itial pH=6.0 and 10 % inoculum (cultivation started with 107
Few studies are found in the literature that evaluate the ca-
cells/mL). The conditions of the process were 25 °C, 180 rpm
rotenoid production using agroindustrial byproducts as poten-
for up to 216 h (31). Culture samples were collected every 24 h
tial sources of nutrients in fed-batch process using the yeast
to determine the pH, biomass concentration, total reducing
Rhodotorula mucilaginosa. In this work we aim to maximize the
sugars and production of carotenoids.
carotenoid production by Rhodotorula mucilaginosa in shake
flasks with the use of agroindustrial byproducts and study dif-
ferent strategies of feeding in a fed-batch process. Selection of agroindustrial medium for the production
of carotenoids
MATERIALS AND METHODS Preliminary experiments were conducted to select the
agroindustrial medium. Two formulations of agroindustri-
Microorganism al byproducts were studied for the carotenoid production:
The yeast Rhodotorula mucilaginosa CCT 7688 used in this corn steep liquor (35.6 g/L) with raw glycerol (6.6 g/L) and
study was previously isolated (25) from environmental samples corn steep liquor (36.5 g/L) with sugar cane molasses (6 g/L)
obtained in the region of Escudo Sul-Rio Grandense, Rio Grande according to Cipolatti (26), and compared with YM medium
do Sul (Brazil), identified and deposited at the André Tosello (Table 1).
Tropical Culture Collection.
Experimental design for batch carotenoid production
Agroindustrial byproducts To maximize the batch carotenoid production, a study of
The agroindustrial byproducts used in this study were corn the composition of the agroindustrial medium formulated
steep liquor from Corn Products (Paraná, Brazil), sugar cane with corn steep liquor and sugar cane molasses was conduct-
molasses obtained from Melaços Brasileiros (São Paulo, Brazil) ed using two central composite designs (Table 2). The evalu-
and raw glycerol from the synthesis of biodiesel from BS Bios ated responses were the maximum volumetric concentration
Indústria e Comércio de Biodiesel Sul Brasil S/A (Rio Grande do of carotenoids (μg/L) throughout the process, the specific
Sul, Brazil). A partial characterization of raw glycerol and sub- mass fraction of carotenoids (μg/g) and biomass concentra-
strates for yeast malt (YM) medium was performed, and the tion (g/L), all determined at the same time. The experiments
mass fractions of carbon and nitrogen were determined using were performed in triplicate, under the incubation conditions
a CHNS/O analyzer (Perkin Elmer 2400, Rodgau, Germany). Car- described previously to validate the maximum carotenoid
bon in the sugar cane molasses was determined with the total production using agroindustrial substrates.
organic carbon and total nitrogen analyzer (TOC-VCSH model;
Shimadzu, Tokyo, Japan), and corn steep liquor was previously Fed-batch fermentation
characterized (26).
Based on the results of the batch fermentation, two pro-
duction media were defined to evaluate fed-batch process: the
Maintenance and reactivation of microorganisms first contained 3.5 g/L corn steep liquor and 70 g/L sugar cane
The microorganisms were maintained in YM agar slant molasses with one-pulse feeding in 168 h. The second culture
tubes with (in g/L): yeast extract 3, malt extract 3, peptone 5, medium contained 6.5 g/L corn steep liquor and 30 g/L sugar
agar 20 (all from KASVI, São José do Pinhais, PR, Brazil), glu- cane molasses with different feeding pulses (using the same
cose 10 (Synth, São Paulo, Brazil) and KNO3 0.2 (Synth) at 4 °C amount of each component in the pulse): run 1 (one pulse at
(29) for 3 months. For reactivation, the microorganisms were 96 h), run 2 (one pulse at 48 h), run 3 (one pulse at 72 h), run 4

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T.V. DIAS RODRIGUES et al.: Improvement of Carotenoid Bioproduction by Rhodotorula mucilaginosa

Table 1. Partial characterization of substrates, C:N ratio, carotenoid production by Rhodotorula mucilaginosa and productivity in different culture
media at 25 °C, initial pH=6.0 and 180 rpm
w/%
Substrate γ(medium)/(g/L) C:N ratio wSC/(μg/g) γVC/(μg/L) γ(biomass)/(g/L) rb/(g/(L·h)) rvc/(μg/(L·h))
C N H
CSL* 17.83 3.80 2.41 CSL 36.5 +SCM 6.0 6.04 (96.4±9.8)a (336±10)a (4.5±1.1)a (0.10±0.01)a (14.0±0.4)a
SCM 31.47 0.04 -
RG 31.50 <0.07 2.27 CSL 35.6 +RG 6.6 6.20 (78.7±7.2)a (297±15)b (4.2±0.4)b (0.07±0.00)b (6.2±0.7)b
Yeast extract 41.26 11.45 5.35
Malt extract 42.11 1.41 5.42 YM 8.28 176.3±4.5 1200±75 7.1±0.3 0.080±0.004 15.1±2.1
Peptone 12.16 2.70 1.46
CSL=corn steep liquor, SCM=sugar cane molasses, RG=raw glycerol, YM=yeast malt medium; *composition according to Cipolatti (26); wSC=specific
carotenoid mass fraction, γVC=volumetric carotenoid concentration, rb=biomass productivity and rvc=volumetric carotenoid productivity,
expressed as mean value±standard deviation (N=3); different letters in the same column indicate a significant difference (p<0.05) according to
Student’s t-test

Table 2. Coded levels and real values (in parentheses) of the first and second central composite design (CCD 2²) used for batch carotenoid pro-
duction on agroindustrial media
First central composite design
Assay X1 X2 R1 R2 tc/h R3 R4 R5
1 -1 (10) -1 (3.5) 2.6 479 168 177.9 0.0 27.5
2 +1 (50) -1 (3.5) 8.7 1040 144 119.2 5.1 106.9
3 -1 (10) +1 (9.5) 3.1 407 120 128.0 0.0 13.2
4 +1 (50) +1 (9.5) 7.2 1077 168 149.3 3.5 45.7
5 0 (30) 0 (6.5) 7.2 80 144 110.8 1.6 42.0
6 0 (30) 0 (6.5) 5.8 74 168 127.3 1.3 42.0
7 0 (30) 0 (6.5) 5.1 670 144 130.4 1.76 42.0
Second central composite design
Assay X1 X2 R1 DR1 R2 tc/h DR2 R3 DR3 R4 R5
1 -1 (50) -1 (0.58) 4.88 8.20 623 168 9.31 127.71 7.17 4.86 370.14
2 +1 (70) -1 (0.58) 6.74 0.89 909 144 8.14 134.98 1.57 15.66 435.75
3 -1 (50) +1 (3.41) 7.17 0.98 1077 168 6.87 163.62 6.50 3.14 109.26
4 +1 (70) +1 (3.41) 8.10 4.94 1404 168 3.49 173.39 0.35 12.69 143.65
5 0 (60) 0 (2) 7.28 5.36 1209 168 11.75 166.15 6.72 4.57 192.39
6 0 (60) 0 (2) 7.07 2.55 1104 168 3.35 156.22 0.74 3.67 192.39
7 0 (60) 0 (2) 6.98 1.29 1141 168 6.49 165.61 6.15 3.55 192.39
X1=γ(SCM)/g/L, X2=γ(CSL)/(g/L), R1=γ(biomass)/(g/L), R2=γVC/(µg/L), R3=wSC/(µg/g), R4=γ(total reducing sugar)/(g/L) and R5=C:N ratio (for
abbreviations see legend of Table 1); DR1=relative deviation of biomass concentration, DR2=relative deviation of volumetric carotenoid
concentration and DR3=relative deviation of specific carotenoids; tc=time of cultivation needed for maximum volumetric production of carotenoids

(one pulse at 48 and one at 96 h), run 5 (one pulse at 72 and After cell disruption, 6 mL of acetone (Neon, Suzano, SP, Brazil)
one at 120 h), run 6 (one pulse at 96 and one at 144 h) and run were added to stimulate the extraction and the suspension
7 (one pulse at 96 and one at 168 h). The feeding strategies for was centrifuged at 1745×g (CT-5000R; Cientec) for 10 min. The
the fed-batch process were based on the procedure described supernatant was separated, and the procedure was repeated
by Chang et al. (32). until the cells were totally bleached. A volume of 10 mL of 20
% NaCl solution (m/V) (Synth) and 10 mL of petroleum ether
(Neon) were added to the supernatants. After the formation of
Extraction and determination of total carotenoids
two phases, the apolar phase was filtered with Na2SO4 (Neon)
The biomass was recovered using centrifugation (Cientec to form the carotenogenic extracts (30).
CT-5000R; Belo Horizonte, Brazil) at 3439×g for 10 min and The total carotenoid mass fraction in the extracts was de-
dried for 48 h at 35 °C to extract the carotenoids (33). It was termined at 448 nm using spectrophotometer model SP-220
subsequently macerated with a mortar and pestle and stand- (Biospectro, Zhejiang, PR China) and expressed as its major
ardized with 115 mesh (26). The samples were frozen for 48 h component (β-carotene in petroleum ether with a specific
1%
at -18 °C (33). The cells were disrupted with dimethylsulfoxide absorbance of A1cm =2592 using the following equation (34):
(DMSO; Synth) as described by Michelon et al. (30). In tubes w TC = ( A × V ×10 6 ) / ( A11cm
%
×100 × msample ) /1/

containing 0.05 g of biomass, 2 mL of DMSO were added at
55 °C and homogenized for 1 min in a vortex (Biomixer QL- where wTC is the mass fraction total of carotenoids (μg/g), A is
901, Ningbo, PR China) at 15 min intervals for a total of 1 h. the absorbance, V is the volume of carotenoids (mL), msample

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 Food Technol. Biotechnol. 57 (3) 388-398 (2019)

1%
is the dried cell mass (g), and A1cm is the specific absorbance. RESULTS AND DISCUSSION
The volumetric concentration of carotenoids (μg/L) was cal- Characterization of substrates of the culture medium and
culated using the mass fraction of total carotenoids (μg/g) influence on the carotenoid production
multiplied by the biomass concentration (g/L).
The partial characterization, including carbon and nitro-
gen mass fractions, of the substrates used in the formulations
Determination of pH and biomass concentration of the culture medium to produce carotenoids (Table 1) is con-
Aliquots were taken from the fermentation and centri- sistent with the literature findings for these agroindustrial res-
fuged (1745×g for 10 min), the supernatants were separated idues (25,26,41). The production of various biomolecules, such
for pH determination with a potentiometer (Quimis Q400MT; as lipids, rhamnolipids (42), exopolysaccharides (43) and carote-
noids (44), can be influenced by the C:N ratio. The YM, corn steep
São Paulo, Brazil) as described by the AOAC official method
liquor with raw glycerol and corn steep liquor with sugar cane
972.44 (35). The biomass concentration throughout the pro-
molasses had a C:N ratio of 8.28, 6.20 and 6.04, respectively (Ta-
cess was estimated by measuring the absorbance at 620 nm
ble 1). A C:N ratio higher than 5.0 may positively influence carot-
(SP-220; Biospectro) using a previously constructed standard
enoid production as described for Phaffia rhodozyma (17,31,44).
biomass curve (g/L) (36).
Therefore, agroindustrial media with this C:N ratio are promis-
ing sources of alternative substrates for carotene bioproduction.
Determination of total reducing sugars The highest specific mass fraction and volumetric concen-
Total reducing sugars were determined spectrophoto- tration of carotenoids, as well as volumetric carotenoid produc-
metrically at 540 nm (SP-220; Biospectro) with 3,5-dinitrosali- tivity, were in the YM medium (Table 1), having a C:N ratio high-
er than that of the two agroindustrial media tested, which may
cylic acid (DNS; Vetec, São Paulo, Brazil) as described by Miller
have influenced the production of the carotenoids and biomass.
(37) using a standard glucose curve. The sugar determination
However, carotenoid synthesis was similar in both agroindustri-
was performed in the culture medium previously centrifuged
al media, with a C:N ratio close to 6.0 (Table 1), since there was
(CT 5000 R; Cientec) at 3439×g for 10 min. The agroindustrial
no significant difference (p>0.05) in the specific carotenoid pro-
medium containing sugar cane molasses was previously sub-
duction. The corn steep liquor with sugar cane molasses was
jected to hydrolysis with 2 mL of 2 mol/L HCl (Neon) in boiling
selected to maximize the carotenoid production because it re-
water (100 °C for 10 min), followed by the addition of 2 mL of sulted in a volumetric concentration, biomass concentration, bi-
2 mol/L NaOH (Neon) to neutralize the acid (38). omass productivity and volumetric productivity higher by 12.0,
6.7, 55.7 and 30.0 %, respectively, than of the corn steep liquor
Determination of the kinetic parameters with raw glycerol.
The volumetric carotenoid productivity, rvc (μg/(L·h)), and
biomass productivity, r b (g/(L·h)), were calculated using the Maximization of carotenoid production
following equations: The use of an experimental design enables the study of
rvc=(γmax–γ0)/tf /2/ the influence of the levels of one factor on the response vari-
able. Thus, the primary effects of such a design may be simply
and
calculated as the difference between the average value of the
rb=(Xmax–X0)/tf /3/ measurements made at the high level (+1) of the variable and
the average value of measurements at the low level (-1) (40).
where γmax and γ0 are the maximum and initial volumetric con- The first experimental design was conducted using a central
centrations of carotenoids (μg/L) respectively, tf is fermenta- composite design (CCD) to evaluate the primary effects of the
tion time (h) at which the maximum volumetric concentration concentrations of sugar cane molasses and corn steep liquor
of carotenoids was obtained, Xmax is biomass concentration in the medium on the production of carotenoids. The real and
(g/L) at tf, and X0 is initial biomass concentration (g/L). coded values of the investigated variables with the respective
responses are shown in Table 2.
In the first central composite design the volumetric con-
Statistical analysis
centration of carotenoids ranged from 407 μg/L (assay 3) to
Data were analysed using Statistica software v. 5.0 (18). 1077 μg/L (assay 4), the specific carotenoid mass fraction from
All analyses used a 95 % confidence level (p<0.05). An anal- 110.8 μg/g (assay 5) to 177.9 μg/g (assay 1), biomass concen-
ysis of variance (ANOVA) was used to estimate the statistical tration from 2.6 g/L (assay 1) to 8.7 g/L (assay 2), and the C:N
parameters. The mean value was compared by Tukey’s test ratio of the production medium from 13.2 (assay 3) to 106.9
at a 5 % significance level. For the comparison between two (assay 2). The final concentration of the total reducing sugars
treatments, the Student’s t-test (p<0.05) was used. Contour in assays 1 and 3 was not detected, indicating the total sugar
surfaces were drawn as described by Box et al. (39) and Rod- consumption by the yeast, while it was 5.1 g/L in assay 2 (ini-
rigues and Iemma (40). tial higher concentration of sugar cane molasses).

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T.V. DIAS RODRIGUES et al.: Improvement of Carotenoid Bioproduction by Rhodotorula mucilaginosa

The analysis of the primary effects (Fig. 1) indicated that biomass concentration from 4.88 g/L (assay 1) to 8.10 g/L (as-
the increase in sugar cane molasses concentration (from 10 say 4), C:N ratio from 109.26 (assay 3) to 435.75 (assay 2) and
to 50 g/L) positively influenced (p<0.05) the production of the final concentration of the total reducing sugars from 3.14
carotenoids (615 μg/L), biomass concentration (5.1 g/L), to- g/L (assay 3) to 15.66 g/L (assay 2).
tal reducing sugar concentration in the culture (4.3 g/L) and The C:N ratio plays an important role in the synthesis of sec-
the initial C:N ratio (60) in the production medium. A different ondary metabolites (45). However, a lower concentration of ni-
behaviour was observed with the increase of corn steep liq- trogen allows a higher C:N ratio in the culture medium, which
uor concentration (from 3.5 to 9.5 g/L), which did not signif- can affect the reduction in cell growth and the production of
icantly influence response variables (p>0.05), except the C:N carotenoids (31,44). The higher C:N ratio (370.14 and 435.75 in
ratio, which decreased to approx. 40. Variations in the con- assays 1 and 2 in the second CCD; Table 2) negatively influenced
centrations of sugar cane molasses and corn steep liquor in cell growth and carotenoid production. Similar results were ob-
the studied range did not influence the carotenoid synthesis tained by Saenge et al. (46), who studied different C:N ratios (140,
significantly (p>0.05), which was confirmed by specific carot- 160 and 180) and found that the C:N ratio of 180 allowed higher
enoid mass fractions. The maximum of biomass and volumet- carotenoid production, close to that obtained in this study (ra-
ric carotenoid concentrations was achieved in assays 2 (8.7 tios 110-190 in assays 3 to 7 in the second CCD, Table 2).
g/L and 1040 μg/L respectively) and 4 (7.2 g/L and 1077 μg/L A model fitting was accomplished in the second CCD (Ta-
respectively) (Table 2). In general, the initial C:N ratio in the ble S1) with the independent variables (corn steep liquor and
production medium above 40 increased the cell growth and sugar cane molasses concentrations) and responses (biomass
volumetric concentration of carotenoids. concentration, specific carotenoid mass fraction and volumet-
ric carotenoid concentration).
On the basis of the ANOVA (Table S1), Eqs. 4, 5 and 6 were
established resulting in first-order models to describe the vol-
umetric carotenoid concentration in µg/L, specific carotenoid
mass fraction in µg/g and biomass concentration in g/L, re-
spectively, as a function of corn steep liquor and sugar cane
molasses concentrations:
γvc=1067.7+151.50·X1+235.50·X2 /4/
wSC=154.85+18.75·X2 /5/
γbiomass=6.38+0.7·X1+0.91·X2 /6/
where X1 is sugar cane molasses concentration in g/L, and X2
is corn steep liquor concentration in g/L.
The pure error was low, indicating good reproducibility
of the experimental data. Based on the F-test, the models are
Fig. 1. Effects of the variables of sugar cane molasses (SCM) and
corn steep liquor (CSL) on the responses of volumetric carotenoid predictive, since the calculated F-value is higher than the criti-
(VC) concentration, specific carotenoid (SC) mass fraction, biomass cal F-value (3.4-, 2.48- and 2.91-fold for volumetric carotenoid,
concentration, total reducing sugars and C:N ratio in the first central specific carotenoid mass fraction and biomass concentration,
composite design (CCD). *significantly different (p<0.05)
respectively), and the regression coefficients (0.93, 0.87 and
0.96 for volumetric carotenoid concentration, specific carot-
The concentration of sugar cane molasses probably had
enoid mass fraction and biomass concentration, respectively)
a significant effect on the evaluated responses because its
are considered satisfactory (47). Coded models were used to
composition is considerably richer in carbohydrates than of
generate contour curves (Fig. 2).
corn steep liquor (Table 1). Carbon source is one of the most
frequently studied variables because it influences the produc- The maximization of the volumetric concentration of ca-
tion of carotenoids, by affecting acetyl coenzyme A (CoA) syn- rotenoids (Fig. 2a) and biomass concentration (Fig. 2b) oc-
thesis, which converts to mevalonic acid, the first precursor of curred with the increase in the concentration of corn steep
carotenoid production (20). liquor and sugar cane molasses. The maximum specific ca-
Thus, for the maximization of carotenoid production, a rotenoid mass fraction (Fig. 2c) was independent from the
second CCD was conducted, where the concentration range concentration range of the sugar cane molasses, but higher
of the sugar cane molasses was increased and that of the in concentrations of corn steep liquor.
corn steep liquor decreased (Table 2). This experimental de- These results are observable in assay 4 (second CCD, Ta-
sign demonstrated a variation in the responses, since the vol- ble 2), which used the maximum levels (level+1) of the stud-
umetric concentration of carotenoids varied from 623 µg/L ied variables, verifying the highest volumetric carotenoid con-
(assay 1) to 1404 µg/L (assay 4), mass fraction of specific ca- centration of 1404 µg/L, specific carotenoid mass fraction of
rotenoids from 127.71 µg/g (assay 1) to 173.39 µg/g (assay 4), 173.39 µg/g, and biomass concentration of 8.1 g/L.

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 Food Technol. Biotechnol. 57 (3) 388-398 (2019)

a) expected behaviour in carotenoid bioproduction. The initial

pH showed a small decrease in the first 24 h, reaching at the
end an average of 6.0. Culture medium had an initial C:N ra-
tio of 143.65, and the total reducing sugars decreased by an
average of 50 % in the first 96 h, not being fully consumed
at the end of the process (15 g/L). A maximum biomass con-
centration of 7.9 g/L was achieved, approx. 2.5 % less than the
model (Eq. 6) had predicted (8.2 g/L). The volumetric carote-
noid concentration reached a maximum of 1248.5 μg/L in 144
h, 14.2 % less than predicted by Eq. 4 (1454.7 μg/L). Under the
same conditions, the specific carotenoid mass fraction was
152.5 μg/g, 13.8 % less than predicted by Eq. 5 (173.6 μg/g).

a)

b)

b)

c)

Fig. 3. Kinetics of the carotenoid production by Rhodotorula mucilag-

inosa at 25 °C, 180 rpm and an initial pH=6.0: a) γ(sugar cane molas-
ses)=70 g/L and γ(corn steep liquor)=3.4 g/L used to validate the em-
Fig. 2. Contour curves of: a) volumetric carotenoid concentration, b) pirical models (N=3), b) γ(sugar cane molasses)=30 g/L and γ(corn steep
biomass concentration obtained by CCD and c) specific carotenoid liquor)=6.5 g/L in a fed-batch fermentation at feeding time t=96 h
concentration as a function of the corn steep liquor (CSL) and sugar
cane molasses (SCM) concentrations
The relative deviations obtained during the experimental
Validation of the model for the carotenoid bioproduction validation were less than 20 % from those predicted by the
Fig. 3 shows the kinetics of the carotenoid production models. Therefore, the experimental results fit well with the
by Rhodotorula mucilaginosa. To find the highest volumet- proposed models (40). The models generated for the other
ric concentration and specific mass fraction of carotenoids responses in this study were not predictive.
and biomass concentration, models generated by Eqs. 4, 5 The agroindustrial medium used by R. mucilaginosa in this
and 6 were validated with the following composition of the study was 70 g/L sugar cane molasses and 3.4 g/L corn steep
production medium: 70 g/L sugar cane molasses and 3.4 g/L liquor, with a C:N ratio of approx. 140, reaching a maximum
corn steep liquor (Fig. 3a). These assays demonstrated the of total carotenoid production 1248.5 μg/L (152.5 μg/g) and

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T.V. DIAS RODRIGUES et al.: Improvement of Carotenoid Bioproduction by Rhodotorula mucilaginosa

biomass concentration 7.9 g/L, at 25 °C and 180 rpm with in- significant increase (p<0.05) was observed during fed-batch
itial pH=6.0 in 144 h. Under these conditions similar results production by 78 % (trials 1 to 2 in Table 3) and 400 % (trials
to those on standard YM medium (1200 μg/L, Table 1) were 3 to 4 in Table 3). Therefore, the production medium contain-
achieved, using only two agroindustrial byproducts. In addi- ing 6.5 g/L corn steep liquor with 30 g/L sugar cane molasses
tion, a 270 % increase in the production of the volumetric ca- (run 4 in Table 3) was the best for carotenoid and biomass
rotenoid concentration and 75 % in the biomass production production in the fed-batch process with different feeding
was achieved compared to the initial agroindustrial medium strategies (Fig. 4).
with initial C:N ratio 6.0 (36.5 g/L corn steep liquor with 6.0 There was an increase (p>0.05) of about 150 % in biomass
g/L sugar cane molasses, Table 1). accumulation in all feeding strategies on medium contain-
The increase in the volumetric concentration of carote- ing 6.5 g/L corn steep liquor with 30 g/L sugar cane molas-
noids achieved in this study through experimental design ses, when compared to that of the batch fermentation (Fig.
was at least 3 and 5 times higher than that reached by Otero 4a). However, it was observed that there was no significant
(25) and Cipolatti (26), respectively, with the same yeast strain difference (p<0.05) in the carotenoid synthesis between the
and similar agroindustrial substrates in other concentrations. evaluated strategies (Fig. 4c). The highest volumetric carote-
noid concentration was in run 3 with the feeding pulse in 96
Carotenoid production in the fed-batch fermentation h of culture (3726.7 μg/L), when a significant increase (p<0.05)
Two culture media were selected for the study of the fed- compared to the other feeding strategies and from the batch
-batch fermentation. One was the previously optimized me- process was observed (Fig. 4b). In relation to the YM standard
dium (3.4 g/L corn steep liquor with 70 g/L sugar cane mo- medium (Table 1), there was a 3-fold increase in carotenoid
lasses), and the second culture medium (6.5 g/L corn steep production (1200 μg/L). The volumetric productivity reached
liquor with 30 g/L sugar cane molasses) in batch process 19.40 μg/(L·h) in run 3, which was significantly (p<0.05) higher
reached approx. 60 % of the optimum carotenoid produc- than under other conditions. The biomass productivity var-
tion (run 5 to run 7 in the first central composite design in ied from 0.05 to 0.1 g/(L·h), depending on feeding strategy.
Table 2). Additionally, in this process a depletion of total re- Colet et al. (48) obtained biomass productivity between 0.05
ducing sugars occurred in 96 h (data not shown), making it and 0.085 g/(L·h), which was similar to the results of this study.
interesting for fed-batch production with the pulse feeding Colet et al. (48) evaluated the production of carotenoids
with 25 mL of medium (32). The biomass concentration in- in fed-batch fermentation by Sporidiobolus salmonicolor in a
creased approx. by 70 % (run 1 to 2) and 168 % (run 3 to 4) in bioreactor with 1 L working volume. The composition of the
the fed-batch batch compared to the batch process (Table medium was: peptone 15 g/L, malt extract 5 g/L and glycer-
3) in both media. ol 80 g/L. The maximum total carotenoid concentration ob-
During the follow-up of the carotenoid production in tained in their studies was 4400 μg/L in 96 h with a 112.5 mL
fed-batch fermentation with feeding in 96 h (Fig. 3b), the feed every 12 h (Table 4 (18,48-50)). Dias et al. (51) studied the
pH decreased during the first 24 h of culture after a gradu- production of lipids and carotenoids by Rhodosporidium to-
al increase. With feeding, the pH dropped again in the first ruloides in a batch and fed-batch fermentation in a bench
hours and gradually increased throughout the process. This bioreactor with 5 L working volume. The maximum produc-
pH change was also observed by Cipolatti (26). The decline tion obtained was 33.4 mg/L under conditions of initial pH=5,
in the pH probably occurs as a consequence of cell devel- temperature 30 °C with strategy of one pulse feeding at the
opment and the release of compounds, such as acetic acid, end of the batch culture in the medium containing 9 g/L Mg-
alcohol or citric acid cycle intermediates during the adapta- SO4·7H2O, 20 g/L yeast extract and 60 g/L glucose.
tion phase (24). Table 4 summarizes the carotenoid production findings
The synthesis of the carotenoids was not significantly in- in the literature and compares them with the results obtained
fluenced (p>0.05) by the fed-batch process or the compo- in this work. The study demonstrated promising results con-
sition of the culture medium, since there was no variation sidering that the medium contains only two agroindustrial
in the mass fraction of specific carotenoids. Similar was ob- byproducts, sugar cane molasses and corn steep liquor, and
served for the volumetric carotenoid concentration, where a was conducted in shake flasks.

Table 3. Biomass concentration and carotenoid production on agroindustrial media in batch and fed-batch fermentation with different feeding
strategies
Trial γ(medium)/(g/L) Process Feeding pulse γ(biomass)/(g/L) wSC/(µg/g) γVC/(µg/L) tc/h
1 Batch - (7.9±0.9)b (152±13)a (1248± 94)c 144
CSL 3.4+SCM 70
2 Fed-batch t=168 h 50 (13.7±1.2)a (139±22)a (2229±1534)b 192
3 Batch - (6.0±0.8)b (123±9)a (740±55)d 144
CSL 6.5+SCM 30
4 Fed-batch t=96 h 25 (16.1±4.5)a (118.8±13.7)a (3726.7±506.0)a 192
CSL=corn steep liquor, SCM=sugar cane molasses; wSC=specific mass fraction of carotenoids, γVC=volumetric concentration of carotenoids, tc=time
of cultivation; different letters in the same column indicate a significant difference (p<0.05) according to Tukey’s test

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 Food Technol. Biotechnol. 57 (3) 388-398 (2019)

Table 4. Carotenoid production obtained by cultivation of red yeasts on different substrates and operating conditions
Cultivation
Strain Substrate Operating conditions γVC/(μg/L) Reference
method
Incubated in a shake flask at 25 °C,
Rhodotorula mucilaginosa Sugar cane molasses Batch 1248.5 This work
180 rpm and initial pH=6.0
Incubated in a shake flask at 25 °C,
Rhodotorula mucilaginosa Sugar cane molasses Fed-batch 3726.0 This work
180 rpm and initial pH=6.0
Crude glycerol, maceration Stirred tank bioreactor at 25 °C,
Sporidiobolus salmonicolor Batch 7388.0 (49)
water and rice parboiling water initial pH=4.0 and 180 rpm
Incubated in a shake flask at 25
Rhodotorula glutinis Wastewater food industry Batch 1200.0 (50)
°C,115 rpm and initial pH=5.5
Stirred tank bioreactor at 25 °C,
Sporidiobolus pararoseus Sugar cane molasses Batch 1969.3 (18)
initial pH=6.0, and 158 rpm
Incubated in a shake flask at 27.5 °C,
Sporidiobolus pararoseus Sugar cane molasses Batch 565.0 (18)
150 rpm and initial pH=4.0
Stirred tank bioreactor at 25 °C, 180
Sporidiobolus salmonicolor Glycerol Fed-batch 4400.0 (48)
rpm and initial pH=4.0

a)
CONCLUSIONS
In this study, it was possible to use agroindustrial byprod-
ucts (sugar cane molasses, corn steep liquor and raw glycerol) to
produce carotenoids by Rhodotorula mucilaginosa. The carote-
noid production in the batch fermentation in the medium con-
taining 70 g/L sugar cane molasses and 3.4 g/L corn steep liquor
reached a maximum of 1248.5 μg/L (152.5 μg/g), with 7.9 g/L
biomass concentration. The fed-batch fermentation using the
agroindustrial culture medium (6.5 g/L corn steep liquor with 30
g/L sugar cane molasses) with a 96-hour feed gave promising
b) results of 3726.7 μg/L carotenoids (118.8 μg/g). Therefore, this
demonstrates the potential of using agroindustrial byproducts
as an alternative source of nutrients in the batch and fed-batch
fermentation. In summary, our study demonstrated the feasi-
bility of minimizing costs of the production medium, adding
value to these byproducts, and possibly decreasing the gener-
ation of waste from industrial processes and reducing their en-
vironmental impact.

ACKNOWLEDGEMENTS
The authors are grateful for FAPERGS (Foundation Re-
c)
search Support in the state of Rio Grande do Sul), CNPq (Na-
tional Council of Science and Technological Development)
and the support of the Support Program for Academic Pro-
duction Publication (Programa de Apoio à Publicação da Pro-
dução Acadêmica)/PROPESP/FURG/2018. This study was fi-
nanced in part by the Coordination of Superior Level Staff
Improvement (CAPES) Brasil, Finance Code 001.

SUPPLEMENTARY MATERIAL
Fig. 4. Comparison of: a) biomass concentration in batch and fed-batch All supplementary material is availabe at www.ftb.com.hr.
fermentations with 6.5 g/L corn steep liquor and 30 g/L sugar cane
molasses medium, b) volumetric concentration of carotenoids and c)
specific carotenoid mass fraction. B=batch process, fed–batch run (t/h): ORCID IDs
1) 48, 2) 72, 3) 96, 4) 48 and 96, 5) 72 and 120, 6) 96 and 144, 7) 96 and
168. The results are expressed as mean values±standard deviations T.V.D. Rodrigues https://orcid.org/0000-0002-2482-5758
(N=3); different letters (lowercase for biomass and volumetric carote- T.D. Amore https://orcid.org/0000-0002-2260-4118
noid concentrations and uppercase for biomass and volumetric carot-
enoid productivity) indicate a significant difference (p<0.05) according E.C. Teixeira https://orcid.org/0000-0002-7381-1467
to Tukey’s test J.F.M. Burkert https://orcid.org/0000-0002-8504-5376

July-September 2019 | Vol. 57 | No. 3 395

T.V. DIAS RODRIGUES et al.: Improvement of Carotenoid Bioproduction by Rhodotorula mucilaginosa

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