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Carotene production from waste cooking oil by Blakeslea trispora in a bubble

column reactor: The role of oxidative stress

Article  in  Engineering in Life Sciences · February 2017

DOI: 10.1002/elsc.201600228

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Konstantina Nanou1 Research Article

Triantafyllos Roukas1
Emmanuel Papadakis2
Parthena Kotzekidou3
1
Laboratory of Food Engineering
and Processing, Department of
Food Science and Technology,
Aristotle University,
Thessaloniki, Greece
2
Pesticide Science Laboratory,
Aristotle University,
Thessaloniki, Greece
3
Laboratory of Food Microbiology
and Hygiene, Department of
Food Science and Technology,
Aristotle University,
Thessaloniki, Greece
Carotene production from waste cooking oil
by Blakeslea trispora in a bubble column
reactor: The role of oxidative stress
The oxidative stress induced by hydroperoxides and reactive oxygen species (ROS)
during carotene production from waste cooking oil (WCO) and corn steep liquor
(CSL) by the fungus Blakeslea trispora in a bubble column reactor was investigated.
The specific activities of the intracellular enzymes superoxide dismutase (SOD) and
catalase (CAT) as well as the micromorphology of the fungus were measured in
order to study the response of the fungus to oxidative stress. The changes of the
morphology of microorganism leaded to pellets formation and documented using
a computerized image analysis system. As a consequence of the mild oxidative stress
induced by hydroperoxides of WCO and ROS a significant increase in carotene
production was obtained. The highest carotene concentration (980.0 mg/l or 51.5
mg/g dry biomass) was achieved in a medium consisted of CSL (80.0 g/L) and WCO
(50.0 g/L) at an aeration rate of 5 vvm after 6 days of fermentation. In this case the
carotenes produced consisted of β-carotene (71%), γ-carotene (26%), and lycopene
(3%). The strong oxidative stress in the fungus caused a significant increase of γ-
carotene concentration. Bubble column reactor is a useful fermentation system for
carotene production in industrial scale.

Keywords: Blakeslea trispora / Bubble column reactor / Carotenes / Oxidative stress / Waste
cooking oil
Received: November 6, 2016; revised: January 21, 2017; accepted: February 10, 2017

DOI: 10.1002/elsc.201600228

1 Introduction
Carotenes are highly unsaturated isoprene derivatives. They are
used as antioxidants, coloring agents for food products and for
pharmaceutical, nutritional, and feed applications [1]. A number
of industrial by-products such as cheese whey, molasses, corn
steep liquor (CSL), and crude glycerol have been used as carbon
sources for biotechnological production of carotenes by different
strains of fungi, bacteria, and yeasts [1–4].
Waste cooking oil (WCO) is a substance obtained after frying
of edible vegetable oil for long time that is not suitable for human
consumption [5]. It is used as an ingredient in animal feed,
soap manufacture, chemical industries, and biodiesel production
[5, 6]. The current world annual amount of WCO is estimated
about 29 million tons [7]. Chemical and physical properties of
Correspondence: Prof. Triantafyllos Roukas
(roukas@agro.auth.gr), Laboratory of Food Engineering and Pro-
cessing, Department of Food Science and Technology, Aristotle Uni-
versity, Box 250, 54124 Thessaloniki, Greece
Abbreviations: CAT, catalase; CSL, corn steep liquor; ROS, reactive
oxygen species; SOD, superoxide dismutase; WCO, waste cooking oil
WCO are different from refined oils due to the thermolytic,
oxidative, and hydrolytic reactions which occur during frying
[8,9]. Due to the high volume of the oil consumed in households
and restaurants its final disposal is a heavy burden [10]. The
utilization of WCO as an inexpensive carbon source for the
production of carotenes has an industrial interest. The great
availability and low cost of WCO ensure the economic viability
of the process and prevent environmental pollution [11].
In aerobic metabolism, reactive oxygen species (ROS) such
as hydrogen peroxide (H2 O2 ), hydroxyl radicals (HO• ), and su-
peroxide radicals (O2 •− ) are formed. Certain levels of ROS are
important for cell growth and cell wall biosynthesis. However,
excessive ROS, can cause lipid peroxidation, DNA damage, in-
activation of enzymes and ultimately cell death [12, 13]. Aero-
bic organisms possess enzymatic and non-enzymatic defense
systems for protecting the cells from oxidative stress. In the
enzymatic defense system, superoxide dismutase (SOD), cata-
lase (CAT), glutathione peroxidase (GPX), glutathione reductase
(GLR), alternative oxidase, and thiol peroxidases are included.
The most important non-enzymatic defense systems include
carotenes, glutathione (GSH), ascorbic acid, tocopherols, tre-
halose, ubiquinol (UQH2 ), and metallothioneins. They act as


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radical scavengers, being oxidized by ROS and thereby removing
oxidants from solution [13, 14]. The adaptive response of the
fungus Blakeslea trispora to the oxidative stress induced by iron
ions, liquid paraffin, and H2 O2 during carotene production from
a synthetic medium in shake flask culture was studied [15–17].
Recently, in our laboratory, the production of carotenes from
WCO in shake flask culture was studied [18]. However, the ox-
idative stress response of B. trispora induced by hydroperoxides
of WCO and ROS in a bubble column reactor has not been stud-
ied. The examination of oxidative stress in B. trispora was carried
out in two ways: (i) measuring the fermentation parameters such
as carotene concentration, dry biomass of the fungus, dissolved
oxygen concentration, and the specific activities of SOD and CAT,
and (ii) using image analysis system to study the morphology of
the fungus.
2 Materials and methods
2.1 Microorganisms and culture conditions
Two strains of B. trispora, i.e. B. trispora ATCC 14271, mating
type (+) and B. trispora ATCC 14272, mating type (-) were
obtained from the American Type Culture Collection (ATCC)
(Rockville, MD, USA) and used in this study. Potato dextrose
agar petri dishes were used for the growth of the strains at 26 °C
for 4 days. The spores formed were collected by scraping off the
medium surface after the addition of eight ml of sterile distilled
water per Petri dish. The substrate was inoculated with the spore
suspension containing 1.5 × 106 and 4.0 × 105 spores/mL of the
strains 14271 and 14272, respectively.
2.2 Fermentation medium
WCO (i.e. a mixture from household frying oils, consisted mainly
from corn oil, sunflower oil, soybean oil, and cotton seed oil)
obtained by a local WCO collection service. The free acidity of
the mixture was 1.6% (w/w, expressed as linoleic acid), while the
peroxide value was 65.0 meq. peroxide/Kg of oil. The fermenta-
tion medium consisted of WCO (50.0 g/L) as carbon source and
CSL (80.0 g/L, Sigma, S-4648) as sugar, nitrogen, amino acids,
and vitamins source. An appropriate amount of 10N NaOH was
added into the medium to adjust the pH to 7.5.
2.3 Fermentation conditions
A glass bioreactor (height 60.0 cm, diameter 5.5 cm, volume 1.4
L) [1] with a working volume of 0.7 L was used as fermentation
system for the production of carotenes from WCO and CSL.
The substrate and the reactor were sterilized at 121 °C for 20
min. A 1:5 ratio of (+) and (-) mating type of B. trispora (i.e.
1.5 × 105 /7.5 × 105 spores/mL of each strain, respectively) was
used for the inoculation of the substrate. The fermentation was
carried out in a controlled temperature chamber at 26°C. From
the bottom of the column was supplied sterile air at aeration
rates of 4, 5, and 6 vvm (2.8, 3.5, and 4.2 L/min).
2 
Eng. Life Sci. 2017, 0, 1–6
2.4 Analytical techniques
At specific time intervals, 20 mL of the sample were removed
from the reactor and analyzed. The dissolved oxygen concen-
tration during fermentation was measured according to Roukas
et al. [1]. The sugars concentration of the fermentation medium
was determined with the phenol-sulfuric acid method [19]. The
free acidity and the peroxide value of WCO were determined
according to the American Oil Chemists’ Society (AOCS) of-
ficial methods. For determination of carotene concentration,
carotenes were extracted from the fungus biomass by ethanol
as described by Roukas and Mantzouridou [20] and the com-
position of carotenes was determined as described in detail by
Roukas et al. [1]. The fermentation broth was filtrated through a
Whatman No 541 filter paper under vacuum and the mycelium
was washed with distilled water until the filtrate was colorless.
The dry biomass was determined by drying one gram of wet
biomass at 105 °C overnight to constant weight. The protein
content of the biomass and the specific activities of SOD and
CAT were measured as described by Schacterle and Pollack [21]
and Beauchamp and Fridovich [22] and Aebi [23], respectively.
The specific activity of the enzymes was expressed as units/mg
protein. The morphology of the fungus was studied by measur-
ing the projected area of pellets formed as described by Roukas
et al. [1]. Three independent experiments were carried out and
the data are the average values ± SD.
3 Results
3.1 Effect of the aeration rate on carotene production,
dry biomass, dissolved oxygen concentration,
pH, and sugar concentration
The production of carotenes from WCO supplemented with CSL
is shown in Fig. 1A. The concentration of carotenes increased
with the increase of fermentation time up to 6th day at aer-
ation rates of 4 and 5 vvm, while at aeration rate of 6 vvm
the carotene concentration was increased up to 4th day of fer-
mentation and then decreased. The maximum concentration of
carotenes (980±25 mg/L) was observed at aeration rate of 5 vvm
after 6 days of fermentation. In this case, the composition of
carotenes was studied by HPLC. The compounds of carotenes
which identified were β-carotene, γ-carotene, and lycopene. At
aeration rates of 4, 5, and 6 vvm, the proportions of β-carotene,
γ-carotene and lycopene (as percentage of total carotenes) were
72%, 25%, 3%, 71%, 26%, 3%, and 58%, 37%, and 5%, respec-
tively. The above results show that β-carotene was the major
accumulated compound.
As shown in Fig. 1B at aeration rates of 4 and 5 vvm, the
increase of the biomass concentration during the first 4 days
of fermentation, was followed by constant biomass between 4th
and 6th day and then decreased. On the other hand, at aeration
rate of 6 vvm the dry biomass increased up to 4th day and then
decreased. The highest biomass concentration (19.0±0.7 g/L)
was obtained at aeration rate of 4 vvm. In medium containing
only CSL the maximum dry biomass (2.0±0.2 g/L) was obtained
at aeration rate of 5 vvm after 4 days of incubation.
C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. Change in carotene concentration (A) and dry biomass
(B) during fermentation of WCO and CSL by Blakeslea trispora
in a bubble column reactor. --, --, and --, 4, 5, and 6 vvm,
respectively. -X-, carotene concentration and dry biomass when
the fungus was grown in CSL at aeration rate of 5 vvm. Each point
is the mean ± SD of three repetitions.
Figure 2. Change in dissolved oxygen concentration during
carotene production from WCO and CSL by Blakeslea trispora in
a bubble column reactor. --, --, and --, 4, 5, and 6 vvm,
respectively. Each point is the mean ± SD of three repetitions.
The concentration of dissolved oxygen fell rapidly during the
first 4 days of incubation (Fig. 2) as well as the sugar content of
the medium (data not shown); both due to the rapid increase of
biomass concentration observed at the same time (Fig. 1B). After
the 4th day of fermentation the dissolved oxygen concentration
increased until the end of fermentation. In cultures grown at
aeration rates of 4, 5, and 6 vvm the lowest dissolved oxygen
concentration was 10%, 14%, and 20%, whereas the maximum
75%, 86%, and 95%, respectively.

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Eng. Life Sci. 2017, 0, 1–6
Figure 3. Change in the specific activities of SOD (A) and CAT
(B) during carotene production from WCO and CSL by Blakeslea
trispora in a bubble column reactor. --, --, and --, 4, 5, and
6 vvm, respectively. Each point is the mean ± SD of three repeti-
tions.
In all fermentation conditions, the pH of the medium de-
creased from 7.5 to 5.0-5.5 during the first 2 days, but then
increased slowly up to 7.0 at the end of fermentation (data not
shown). This was due to release of ammonia from the degra-
dation of proteins. In all cultures, the concentration of uncon-
sumed sugars fluctuated between 1.0 and 1.5 g/L at the end of
fermentation.
3.2 Activities of antioxidant enzymes during
fermentation
At aeration rates of 4 and 5 vvm the specific activity of SOD
increased drastically during the first 2 days of incubation, then
decreased between 2nd and 4th day, and increased until the end
of fermentation. At aeration rate of 6 vvm, the specific activity of
SOD increased rapidly during the first 2 days of fermentation, re-
mained almost constant between 2nd and 6th day, and decreased
slightly at the end of fermentation (Fig. 3A). On the other hand,
in all cases the specific intracellular activity of CAT increased up
to the 2nd day of fermentation, and then decreased (Fig. 3B).
The highest specific activities of SOD (11.2 units/mg protein)
and CAT (52.5 units/mg protein) were observed after 2 days of
fermentation at aeration rates of 5 and 4 vvm, respectively.
3
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Figure 4. Change in the projected area of pellets during carotene
production from WCO and CSL by Blakeslea trispora in a bubble
column reactor. -, --, and --, 4, 5, and 6 vvm, respectively.
Each point is the mean ± SD of three repetitions.
Figure 5. Images showing the morphology of the pellets of B.
trispora grown on WCO and CSL in a bubble column reactor. (A,
B, and C) Aeration rate of 4, 5, and 6 vvm, respectively.
3.3 Culture morphology
The mycelium of B. trispora consisted of compact aggregates
(pellets) under all fermentation conditions. Thus, the dispersed
mycelium was evaluated on the basis of the projected area of
the pellets formed (Figs. 4 and 5). At aeration rate of 4 vvm,
the projected area of the pellets increased during the first 6 days
of fermentation and then decreased slightly until the end of the
incubation. On the other hand, at aeration rates of 5 and 6
vvm, the projected area of the pellets increased up to 4th day of
fermentation, and then decreased. At aeration rates of 4, 5, and
6 vvm, the highest projected area of pellets was 7.8, 3.7, and 1.7
mm2 , respectively (Fig. 4).
4 Discussion
As shown in Fig. 1A and B the biosynthesis of carotenes was
carried out during the growth phase of the microorganism
4 
Eng. Life Sci. 2017, 0, 1–6
(aeration rate 6 vvm) or in growth and static phase of the fun-
gus (aeration rates of 4 and 5 vvm). The carotene concentration
was very low (17.0 ± 1.0 mg/L) when B. trispora was grown
in CSL after 6 days of incubation at aeration rate of 5 vvm. A
drastic increase in carotene concentration was obtained from
17.0 ± 1.0 to 980 ± 25 mg/L at the same aeration rate when the
medium was supplemented with WCO (Fig. 1A). These results
show that a 57-fold increase of the concentration of carotenes
was obtained by the addition of WCO into the medium due to
the change in the composition of the substrate. The increasing
amount of carotenes was due to the high concentration of free
unsaturated fatty acids, mainly oleic, linoleic and linolenic acid
formed during frying of oils [9]. Through β-oxidation cycle, the
free fatty acids are degraded into acetyl-CoA that is converted to
carotenes [24]. In addition, during frying of oil, hydroperoxides
are continually produced (Fig. 6). They induced oxidative stress
in B. trispora as shown by the specific activities of the enzymes
SOD and CAT at aeration rate of 5 vvm (Fig. 3A and B) which
resulted in a significant increase in carotene concentration. In
medium containing WCO and CSL the carotene concentration
increased significantly from 687 ± 18 to 980 ± 25 mg/L after
6 days of fermentation increasing the aeration rate from 4 to 5
vvm and then decreased (Fig. 1A). The decline in the concen-
tration of carotenes after the 6th day of incubation was due to
the autolysis of the fungus. The high concentration of ROS is
indicated by the increasing activities of SOD and CAT (Fig. 3A
and B). The change of the morphology of the fungus from pellets
with large projected area to pellets with smaller projected area
as indicated in Figs. 4 and 5 leads to the increased amount of
carotenes at aeration rate of 5 vvm. Thus, at the aeration rate of 5
vvm the fungus formed pellets with small projected area, and this
would result in a perceived stress to the microorganism. There-
fore, the change in mycelium morphology might be a response
to this stress. Pellets with small projected area supply a satis-
factory amount of nutrients to the cells, facilitate the removal
of byproducts from the cells, and maintain the concentration of
ROS at high levels. Moreover, as shown in Figs. 1A and 2, increas-
ing the dissolved oxygen concentration up to aeration rate of 5
vvm the concentration of carotenes was increased, but at higher
aeration rates it was decreased. This means that the production
of carotenes is affected by oxygen supply of the fermentation
broth. At aeration rate of 5 vvm, ROS are mainly produced by
exposure of the microorganism to high dissolved oxygen con-
centration resulting an increase of oxidative stress of the fungus
as indicated by the increasing specific activities of SOD and CAT
(Fig. 3A and B) and a significant increase of the carotene pro-
duction (Figs. 1A and 6). On the other hand, further increase of
the aeration rate from 5 to 6 vvm, increased the production of
ROS due to the enhanced dissolved oxygen concentration (Fig.
2). In this case, the strong oxidative stress in the fungus as indi-
cated by the decreased activities of SOD and CAT (Fig. 3A and
B) resulted in a drastic decrease in carotene concentration (Fig.
1A). The decrease in the activities of the above enzymes was
due to the inactivation of the enzymes by the strong oxidative
stress [25]. Thus, at aeration rate of 6 vvm, the nonenzymatic
antioxidant system protects the fungus from the high oxidative
stress resulting in a decrease in carotene concentration (Fig. 1A).
Moreover, in this case the enhanced concentration of ROS react
with the components of the cell resulting in oxidation of proteins,
C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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nucleic acids, lipids, inactivation of enzymes, DNA damage, and
ultimately cell death [26]. Also, the composition of the carotenes
changed due to the strong oxidative stress in B. trispora. In this
case, a significant increase of γ-carotene ratio from 25% to 37%
at aeration rate of 4 and 6 vvm, respectively, was obtained (data
not shown).
The production of carotenes in fermentation medium con-
taining WCO and CSL in a bubble column reactor was higher
than the amount of carotenes produced in shake flask cultures
using the same medium (980 mg/L vs 900 mg/L) comparing the
results of the present study to those obtained in a previous pub-
lication [18]. In addition, bubble column reactor compared to
the shake flask fermentation system has several advantages. The
mixing of the fermentation broth is carried out inducing air from
the bottom of the column through the air compressor, whereas
in shake flasks mechanical agitation is used for the mixing of the
substrate. The fermentation conditions are controlled more eas-
ily and the mixing of the fermentation broth requires less energy.
In addition, laboratory results obtained in bubble column reac-
tors can be used for industrial scale-up but those of shake flask
cultures cannot. Goksungur et al. [27] studied the production
of carotenes from beet molasses supplemented with 1% (w/v)
of olive oil, 3% (w/v) of cottonseed oil, or 3% (w/v) of soybean
oil in shake flask culture and found that the maximum carotene
concentration was 34.0, 21.0, and 13.0 mg/L, respectively. In our
previous work, it was found that the maximum concentration
of carotenes was 640.0 mg/L when B. trispora was grown in de-
proteinized hydrolyzed whey supplemented with 0.1% (w/v) of
Tween 80, 1.0% (w/v) of Span 20, and 1% (w/v) of olive oil,
cotton seed oil, soybean oil, corn oil, and olive pomace oil in
shake flask culture [28]. Mantzouridou et al. [29] found that a
maximum carotene concentration of 85.0 mg/L was obtained
when B. trispora was grown in synthetic medium supplemented
with 1% (w/v) of olive oil, cotton seed oil, and soybean oil in fed-
batch culture. Buzzini and Martini [30], Marova et al. [31], and
Aksu and Tugba Eren [32] found that a maximum concentra-
tion of carotenes (6.0, 46.0, and 89.0 mg/L, respectively) was ob-
tained when Rhodotorula glutinis DBVPG 3853, R. glutinis CCY
20-2-26, and R. mucilaginosa NRRL-2502 were grown in grape
must, cheese whey, and molasses, respectively in shake flask cul-
ture. The maximum concentration of carotenes obtained when

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Eng. Life Sci. 2017, 0, 1–6
Figure 6. Factors causing oxidative stress in Blakeslea
trispora during carotene production from WCO and CSL
in a bubble column reactor.
Sporidiobolus pararoseus, Rhodotorula glutinis, and Arthrobacter
globiformis were grown in corn steep liquor supplemented with
glycerol and parboiled rice water, crude glycerol, and sugarcane
molasses in shake flask culture were 0.8, 14.0, and 27.5 mg/L,
respectively as reported by Valduga et al. [3], Cutzu et al. [4],
and Zhai et al. [2]. These results show that the concentrations of
pigments produced from the above byproducts were low com-
pared with the amounts of carotenes (980 mg/L) produced from
WCO and CSL. The toxic substances such as hydroperoxides, al-
cohols, aldehydes, and ketones contained in WCO are removed
from the fermentation broth during recovery of carotenes. At
the end of the fermentation, the mycelium is removed from the
fermentation broth by filtration. The filtrate contains a part of
the toxic compounds. The wet biomass is washed with water
and mixed with ethanol in order to extract the carotenes from
the mycelium. The extract is centrifuged and the supernatant is
evaporated under vacuum to separate carotenes from ethanol
and the rest of toxic compounds. Thus, the above measures re-
duce pollution of the carotenes in order to eliminate consumer
concerns about food safety.
In conclusion, the production of carotenes from WCO and
CSL by B trispora in a bubble column reactor is influenced by
the aeration rate. The hydroperoxides of WCO and ROS cause
oxidative stress in B. trispora and change the morphology of the
fungus resulting in a significant increase of carotene production.
Practical application
Waste cooking oil supplemented with corn steep liquor is
a low-cost substrate for the production of carotenes in a
bubble column reactor. The hydroperoxides of waste cook-
ing oil and reactive oxygen species cause oxidative stress
in B. trispora and change the morphology of the fungus
resulting in a significant increase of carotene production.
Bubble column reactor and waste cooking oil is a useful
combination to induce oxidative stress in B. trispora for
enhanced carotene production. The results of this work
can be applied for the production of carotenes in industrial
scale.
5
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