Bioresource Technology 203 (2016) 198–203

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Waste cooking oil: A new substrate for carotene production by

Blakeslea trispora in submerged fermentation
Konstantina Nanou, Triantafyllos Roukas ⇑
Laboratory of Food Engineering and Processing, Department of Food Science and Technology, Aristotle University, Box 250, 54124 Thessaloniki, Greece

h i g h l i g h t s

The production of carotenes from waste cooking oil by B. trispora was studied.

Waste cooking oil was found to be suitable for carotene production.

The specific activities of superoxide dismutase and catalase were studied.

The oxidative stress induced by hydroperoxides and BHT increased carotene production.

Blakeslea trispora formed only pellets during fermentation.

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to evaluate a waste, waste cooking oil (WCO) as substrate for carotene
Received 6 November 2015 production by Blakeslea trispora in shake flask culture. WCO was found to be a useful substrate for caro-
Received in revised form 15 December 2015 tene production. B. trispora formed only pellets during fermentation. The oxidative stress in B. trispora
Accepted 16 December 2015
induced by hydroperoxides and BHT as evidenced by increase of the specific activities of superoxide dis-
Available online 21 December 2015
mutase (SOD) and catalase (CAT) increased significantly the production of carotenes. The highest concen-
tration of carotenes (2021 ± 75 mg/l or 49.3 ± 0.2 mg/g dry biomass) was obtained in culture grown in
Keywords:
WCO (50.0 g/l) supplemented with CSL (80.0 g/l) and BHT (4.0 g/l). In this case the carotenes produced
Waste cooking oil
Blakeslea trispora
consisted of b-carotene (74.2%), c-carotene (23.2%), and lycopene (2.6%). The external addition in the
Carotenes above medium glucose, Span 80, yeast extract, casein acid hydrolysate, L-asparagine, thiamine. HCl,
Oxidative stress KH2PO4, and MgSO47H2O did not improve the production of carotenes.
Culture morphology Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction heterothallic zygomycota with two mating types (termed ‘‘plus’’

Carotenes are highly unsaturated isoprene derivatives. Natu-
rally occurring carotenes are tetraterpenoids consisting of eight
isoprene residues. Carotenes are used as antioxidants, coloring
agents for food products such as margarine, butter, cheese, soft
drinks, and baked goods, and for pharmaceutical, nutritional, and
feed applications (Tinoi et al., 2005; Valduga et al., 2014;
Varzakakou and Roukas, 2010; Yan et al., 2012). They have many
important physiologic functions, including the prevention of can-
cer, cardiovascular diseases, osteoporosis, and strengthened
immunity (Wang et al., 2012). Carotenes are produced primarily
by fungi, yeasts, and some species of bacteria, algae and lichens.
The greatest yield has been obtained from Blakeslea trispora, a
⇑ Corresponding author. Tel.: +30 2310 991635; fax: +30 2310 991650.
E-mail address: roukas@agro.auth.gr (T. Roukas).
and ‘‘minus’’) (Roukas, 2015).
The production of carotenes from different chemically defined
media by B. trispora has been described (Filotheou et al., 2012;
Liu et al., 2012; Wang et al., 2013; Nanou and Roukas, 2013). How-
ever, utilization of a synthetic medium is not economical and the
exploitation of a less expensive medium could prove beneficial.
Numerous agro-industrial by-products such as hydrolyzed mung
bean waste flour, fermented radish brine, whole stillage, grape
juice, wheat straw, parboiled rice water, crude glycerol, molasses,
and cheese whey have been considered as potential carbon sources
for biotechnological production of carotenes by different strains of
fungi, yeasts, and bacteria (Tinoi et al., 2005; Malisorn and
Suntornsuk, 2008, 2009; Ananda and Vadlani, 2010; Yan et al.,
2012; Petrik et al., 2013; Valduga et al., 2014; Zhai et al., 2014;
Cutzu et al., 2013; Goksungur et al., 2004; Varzakakou et al.,
2010a; Roukas et al., 2015).

http://dx.doi.org/10.1016/j.biortech.2015.12.053
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
 K. Nanou, T. Roukas / Bioresource Technology 203 (2016) 198–203
It has been well-reported that vegetable oils such as olive oil,
olive pomace oil, soybean oil, cottonseed oil, corn oil, sunflower
oil, and pure fatty acids such as oleic acid, linoleic acid, and linole-
nic acid act very well as substrates for carotene production
(Mantzouridou et al., 2004; Varzakakou and Roukas, 2010;
Varzakakou et al., 2010b; Nanou and Roukas, 2011; Nanou et al.,
2012). However, a major barrier in the commercialization of caro-
tene production from vegetable oil is its high manufacturing cost,
which is due to the high cost of virgin vegetable oil. Therefore,
the use of waste cooking oil (WCO) for the carotene production
is a promising alternative as it combines aspects, due to the oil
low cost of acquisition, with environmental preservation, as it pre-
vents water sources contamination at the same time producing a
less pollutant fuel. WCO is an oil-based substance consisting of edi-
ble vegetable matter that has been use in the preparation of foods
and is no longer suitable for human consumption (Sheinbaum-
Pardo et al., 2013). WCO is generated in several locations, mainly
when we consider urban areas with high consumption of refined
vegetable oils. More than 80% of the oil is consumed in households
making its final disposal a heavy burden due to its high volume
(Mandolesi de Araujo et al., 2013). There are basically two possible
destinations for WCO: sewage systems, causing water pollution
and encumbering its treatment, and processing equipment that
can transform waste into a new product. Considerable amounts
of this waste are still disposed of incorrectly in most places, as fast
food chains and restaurants have increased (Mandolesi de Araujo
et al., 2013). Compared with refined oils, WCO has different chem-
ical and physical properties due to the thermolytic, oxidative, and
hydrolytic reactions which occur during frying. In this process, oil
is heated in air and in the presence of light at temperatures of 160–
200 °C for relatively long periods of time. For economical reasons,
the same oil is used many times or continuously. Generally, in pub-
lic restaurants, frying is conducted in the same oil for several days;
however, in household frying, oil is exchanged after several weeks.
The quantity of WCO generated per year by any country is huge.
The disposal of WCO is problematic, because disposal methods
may contaminate environmental water (Kulkarni and Dalai,
2006). The production of carotenes from WCO is one of the better
ways to utilize it efficiently and economical. The huge abundance
and low cost of WCO are attractive for ensuring the economic via-
bility of the process as well as for preventing environmental pollu-
tion (Hama et al., 2013). The amount of WCO generated in each
country varies, depending on the use of vegetable oil. It is esti-
mated that the current world annual amount of WCO is about 29
million tons (Lisboa et al., 2014). It is used as an ingredient in ani-
mal feed, soap manufacture, oleochemical industries, and biodiesel
production (Sheinbaum-Pardo et al., 2013; Zou et al., 2013; Li et al.,
2014). The utilization of WCO as an inexpensive carbon source for
the production of carotenes has an industrial interest. The produc-
tion of carotenes from WCO has not investigated. To the best of our
knowledge, this is the first report where the production of carote-
nes from WCO by B. trispora was studied.
2. Methods
2.1. Microorganisms and culture conditions
The microorganisms used in this work were B. trispora ATCC
14271, mating type (+) and B. trispora ATCC 14272, mating type
(). Both strains were obtained from the American Type Culture
Collection (ATCC) (Rockville, MD, USA). The strains were grown
on potato dextrose agar (PDA) Petri dishes at 26 °C for 4 days for
sporulation. Eight ml of sterile distilled water was added to the
Petri dish and the spores were collected by scraping off the med-
ium surface. The spore suspension containing 1.5  106 and
199
4.0  105 spores/ml of the strains 14271 and 14272, respectively,
was used to inoculate the medium.
2.2. Pre-treatment of WCO
WCO collected by a local super market. It was a mixture from
household frying oils, mainly corn oil, sunflower oil, soybean oil,
and cotton seed oil. The free acidity of the used oil was 1.6% (w/
w, expressed as linoleic acid), while the peroxide value was
65.0 meq. peroxide/Kg of oil. The substrate consisting of WCO sup-
plemented with corn steep liquor (CSL) (Sigma, S-4648), butylated
hydroxytoluene (BHT) (Sigma, B-1378), glucose, Span 80, yeast
extract, casein acid hydrolysate, L-asparagine, thiamine. HCl, KH2-
PO4, and MgSO47H2O as shown in Fig. 1. The pH of the medium
was adjusted to 7.5 with 10 N NaOH.
2.3. Fermentation conditions
The fermentation was carried out in 250 ml conical flasks con-
taining 50 ml medium. The substrate was sterilized at 121 °C for
15 min. After cooling, the medium was inoculated with the appro-
priate ratio of (+) and () mating type of B. trispora in order to
obtain a ratio of 1:1 (1.5  105/1.5  105 spores/ml), 1:2
(1.5  105/3.0  105 spores/ml), 1:5 (1.5  105/7.5  105 spores/
ml), and 1:10 (1.5  105/1.5  106 spores/ml) of (+)/(), respec-
tively. The flasks were incubated at 26 °C in a rotary shaker incuba-
tor (Lab Line Orbit-Environ Shaker, Lab-Line Instr., Melrose Park, IL,
USA) at 250 rev/min.
2.4. Analytical techniques
At specific time intervals, fermentation broth was removed
from the flasks and analyzed. Carotene concentration was deter-
mined by extraction of carotenes from the cells with ethanol
according to Roukas and Mantzouridou (2001). Dry biomass and
residual sugars were determined as described by Varzakakou
et al. (2010a). The composition of carotenes was determined by
high-performance liquid chromatography (HPLC) as described by
Varzakakou and Roukas (2010). The specific activities of SOD and
CAT were measured as described by Nanou and Roukas (2011).
The free acidity and the peroxide value of the used oil were deter-
mined using the American Oil Chemists’ Society (AOCS) official
methods. The data are the average values ± SD of three indepen-
dent experiments.
2100
1800
1500
Carotenes (mg/l)
1200
900
600
300
0
0 2 4 6 8
Fermentaon me (days)
Fig. 1. Carotene production from WCO supplemented with different substances. 4
– medium 1: CSL 80 g/l; } – medium 2: medium 1 + WCO 50 g/l; s – medium 3:
medium 2 + BHT 4.0 g/l;  – medium 4: medium 3 + glucose 50 g/l + Span 80 10 g/l;
d – medium 5: medium 4 + yeast extract 1.0 g/l + casein acid hydrolysate 2.0 g/l
+ L-asparagine 2.0 g/l + thiamine. HCl 5.0 mg/l; h – medium 6: medium 5 + KH2PO4
1.5 g/l + MgSO47H2 O 0.5 g/l.
200 K. Nanou, T. Roukas / Bioresource Technology 203 (2016) 198–203
3. Results and discussion
3.1. Effect of composition of medium on carotene production and
biomass concentration
The production of carotenes from WCO supplemented with dif-
ferent substances is shown in Fig. 1. In culture grown in CSL and
WCO supplemented with CSL the biosynthesis of the carotenes
was carried out during the growth phase of the microorganism,
while in other cases the biosynthesis of pigments was carried out
in growth and static phase of the microorganism (Figs. 1 and 2).
In culture grown in CSL the carotene concentration was very low
(15.0 ± 0.8 mg/l) after 4 days of incubation. When the medium sup-
plemented with WCO the concentration of carotenes increased
drastically from 15.0 ± 0.8 to 900 ± 70 mg/l at the same time of
the fermentation. The above results show that a 60-fold increase
of the concentration of carotenes was obtained by the addition of
WCO into the medium. Zhang et al. (2015) reported that many
chemical reactions, such as hydrolysis, thermal oxidation, and
the Maillard reaction, occur in the frying oils under the conditions
of oxygen, high temperature, and complex food constituents. Con-
sequently, a series of homologous products, such as free fatty acids
(FAs), diacylglycerols, hydrocarbons, alcohols, aldehydes, ketones,
acids, oxidized triacylglycerol (TAG) monomers, cyclic fatty acid
monomers, polymerized TAGs, trans isomers, and nitrogen-
containing products are formed. Also, major changes are occurred
in the composition of original TAGs. In general, the amounts of sat-
urated FAs are increased while the concentrations of unsaturated
FAs are decreased during deep-fat frying. In addition, Aladedunye
(2015) reported that increase in frying temperature increases ther-
mal oxidation and oligomerization reactions not only of the fatty
acids or triacylglycerol molecules, but also of the unsaponifiable
minor components. Thus, antioxidant minor components in oil
are either thermally inactivated during frying or have their levels
severely reduced. However, the stability-enhancing capacity of
minor components differs one from another. These antioxidative
minor components (a, c-, and d-tocopherol, tocotrienols, c-
oryzanol, tocochromanols, phytosterols, sesamins, hydroxytyrosol,
carotenoids, and the phenolics) do exert significant influence on
the stability of frying oils and there are optimal concentrations
above which some of these antioxidant compounds become pro-
oxidative (Aladedunye, 2015). Based on the above knowledge, the
increasing amount of pigments in media containing WCO is
explained by the fact that the WCO contains free unsaturated fatty
acids, mainly oleic, linoleic and linolenic acid. The unsaturated
fatty acids are degraded into acetyl-CoA through b-oxidation cycle.
40
30
Dry biomass (g/l)
20
10
0 Rao of (+) and (-) mang type
0 2 4 6
Fermentaon me (days)
Fig. 2. Effect of medium composition on dry biomass. Symbols as in Fig. 1.
Then the acetyl-CoA is converted to carotenes. Also, acetyl-CoA is
an allosteric activator of pyruvate carboxylase, a key enzyme in
gluconeogenesis, ATP and NADH. The catabolism of the fatty acids
gives rise to intracellular energy excess and directs glucose toward
the biosynthesis of carotenes. The marked increase in carotenes
indicates stimulation of the cyclization reactions and, simultane-
ously, of carotenes by the produced fatty acids. In addition, the
increasing concentration of carotenes was an effect of change in
C:N ratio when WCO was added to the substrate (Varzakakou
et al., 2010b). Another reason resulted in a significant increase of
carotene concentration when the medium was supplemented with
WCO was the concentration of hydroperoxides in WCO. The perox-
ide value of the used oil was 65 meq peroxides per Kg of oil. This
concentration of hydroperoxides induced oxidative stress in B. tris-
pora as shown by the specific activities of the enzymes SOD and
CAT in Fig. 4. In a reaction catalyzed by temperature, light, or
metal, hydrogen atom is abstracted from the fatty acids resulting
in the generation of lipid alkyl radicals. The lipid radicals rapidly
react with triplet oxygen to produce peroxy radicals, which in turn
abstract hydrogen from another lipid molecule to form hydroper-
oxide and next lipid radical. Thus a chain reaction occurs at this
stage when free radicals are continually produced. Oxidation reac-
tion is terminated when free radicals react to form non-radical
products (Aladedunye, 2015). On the maximum carotene concen-
tration, the specific activities of SOD and CAT were 7.5 and
55.0 units/mg protein, respectively (Fig. 4). The activities of the
above enzymes showed that B .trispora is found under oxidative
stress during carotene production. Thus, the oxidative stress
response of B. trispora, induced by hydroperoxides, resulting in a
significant increase of carotene production.
The external addition of BHT into the medium increased signif-
icantly the carotene concentration from 900 ± 70 to 2021 ± 75 mg/l
after 6 days of the fermentation and then decreased. On the other
hand, the addition into the medium of glucose, Span 80, yeast
extract, casein acid hydrolysate, L-asparagine, thiamine. HCl, KH2-
PO4, and MgSO47H2O did not improve the production of carotenes
(Fig. 1). The significant increase of carotene concentration when
the medium was supplemented with BHT may be explained by
the fact that the morphology of microorganism changed from pel-
lets with large projected area to pellets with smaller projected area
(Fig. 5 and Supplementary data). This change of the pellets began
early during the growth phase and continued until the 6th day of
the fermentation compared with the medium consisting of WCO
2000
a
Carotenes (mg/l)
1500
1000
500
0
40 b
Dry biomass (g/l)
30
20
10
0
1:1 1:2 1:5 1:10
8
Fig. 3. Effect of the ratio of (+) and () mating type of B. trispora on carotene
concentration (a) and dry biomass (b) (values are reported at 6 days of
fermentation).
 K. Nanou, T. Roukas / Bioresource Technology 203 (2016) 198–203 201

increase of the carotene production. Also, the administration of

10 high dose of BHT may exert a prooxidant effect on the organism
a (Nanou and Roukas, 2010). In our case, BHT acting as prooxidant
interacts with molecular oxygen rather than with O
Specific acvity SOD
(units/mg protein)

8 2 radical. This
reaction yields phenoxyl radical and superoxide anion. Deeper oxi-
6 dation of BHT may result in formation of BHT quinone methide and
other products of BHT oxidation which can also generate superox-
4
ide anion caused oxidative stress to the fungus as indicated by the
2 specific activities of SOD and CAT (Fig. 4). On the maximum caro-
tene concentration, the specific activities of SOD and CAT were
0 6.5 and 25.0 units/mg protein, respectively indicated that during
0 2 4 6 8
the fermentation the fungus is found under oxidative stress result-
100
b ing in a significant increase in carotene concentration. Moreover,
80
BHT as antioxidant can operate as hydroperoxides scavenger of
Specific acvity CAT
(units/mg protein)

WCO. This means that in this case, the oxidative stress in B. trispora
60 induced mainly by the superoxide anion.
Goksungur et al. (2004) studied the production of b-carotene
40 from beet molasses supplemented with linoleic acid, kerosene,
and antioxidant in stirred-tank and bubble column reactors and
20
found that the maximum concentration of the pigment was 92.0
0 and 360.0 mg/l, respectively. Varzakakou et al. (2010b) studied
0 2 4 6 8 the production of carotenes from deproteinized hydrolyzed whey
Fermentaon me (days) supplemented with Tween 80, Span 80, and b-ionone in shake flask
culture and found that the highest concentration of the product
Fig. 4. Change in the specific activity of SOD (a) and CAT (b) during fermentation. }
was 1100 mg/l. Malisorn and Suntornsuk (2008), Tinoi et al.
– culture grown on WCO and CSL; s – culture grown on WCO supplemented with
CSL and BHT. (2005) and Cutzu et al. (2013) found that a maximum concentra-
tion of carotenes (0.2, 3.5, and 14.0 mg/l, respectively) was
obtained when Rhodotorula glutinis was grown in fermented radish
4 brine, hydrolyzed mung bean waste flour, and crude glycerol,
Mean projected area of pellets (mm2)

respectively. The cited results show that the amounts of carotenes

3
2 When the maximum concentration of carotenes was observed,
1 sisting of WCO and CSL and WCO supplemented with CSL and
0 percent of total carotenes) were 60.5%, 34.7%, 4.7% and 74.2%,
0 2 4 6
Fermentaon me (days)
Fig. 5. Change in the projected area of pellets during fermentation. } – culture
grown on WCO and CSL; s – culture grown on WCO supplemented with CSL and
BHT.
and CSL. Nanou and Roukas (2010) reported that BHT affect on
lipid membranes with particular emphasis on the mitochondrial
membrane. BHT is incorporated into the phospholipids of the
plasma membrane causing in modifications of the membrane
properties. Thus, the incorporation of exogenous BHT into the cell
membrane caused significant changes in the morphology of the
fungus forming pellets with small projected area (Fig. 5 and Sup-
plementary data), and this would result in a perceived stress to
the microorganism. Therefore, the change in cell wall morphology
might be a response to this stress. Pellets with small projected area
could be beneficial to improve the mass transfer characteristics
relating substrate, products/byproducts, and oxygen. This change
of cell morphology works in both directions; through better diffu-
sion, it helps to maintain a satisfactory supply of nutrients to the
cells, while it facilitates the removal of byproducts of catabolism
from the microenvironment of the cells. Also, pellets with small
projected area favor oxygen supply to the cells, and it is especially
important to maintain the concentration of ROS at high levels.
Thus, BHT may affect the intracellular generation of oxygen radi-
cals causing oxidative stress to the fungus resulting in a significant
produced from different agro-industrial byproducts were very low
compared with our results, i.e. 2021 mg/l using as fermentation
medium WCO supplemented with CSL and BHT.
the carotenes were analyzed by HPLC. Three carotene compounds
(b-carotene, c-carotene, lycopene) were identified. In media con-
BHT the proportions of b-carotene, c-carotene and lycopene (as
8 23.2%, and 2.6%, respectively. These results show that the major
accumulated compound was b-carotene.
As shown in Fig. 2 in all cultures (except the culture grown on
CSL) the biomass concentration increased during the first 4 days
of fermentation and then remained constant. In culture grown on
CSL the dry biomass increased up to 6th day of incubation and then
decreased. The maximum dry biomass (3.4 ± 0.2 g/l) was observed
after 6 days of fermentation. In other cases the dry biomass chan-
ged between 39.5 and 42.0 g/l after 4 days of fermentation. The
highest biomass concentration (42.0 ± 1.2 g/l) was obtained in cul-
ture grown in WCO supplemented with all the nutrients.
The pH of the substrates decreased from 7.5 to 5.5 during the
first 2 days, but then increased slowly up to 7.0 at the end of the
fermentation (data not shown) likely due to release of ammonia
from the degradation of proteins. The sugar concentration of the
medium decreased rapidly during the first 4 days followed by a
slower rate of decrease (data not shown). This was accompanied
with the rapid increase of biomass concentration observed at the
same time (Fig. 2). In all cultures, the lowest concentration of sug-
ars (1.0–4.0 g/l) was observed at the end of the fermentation.
3.2. Effect of the ratio of (+) and () mating type of B. trispora on
carotenes and biomass concentration
In medium consisting of WCO supplemented with CSL and BHT
the effect of inoculum ratio on carotenes and dry biomass concen-
tration was studied (Fig. 3a and b). The concentration of carotenes
202 K. Nanou, T. Roukas / Bioresource Technology 203 (2016) 198–203
increased with the increase of the inoculum ratio from 1:1 to 1:5 of
(+) and () mating type and remained constant beyond this value
(Fig. 3a). These results clearly showed that the ratio of (+) and ()
mating type had a significant effect on the production of carotenes.
The optimum ratio of (+) and () mating type for the maximum
carotenes concentration (2021 ± 75 mg/l) was found to be 1:5. This
means that one positive strain of B. trispora ATCC 14271 can be
characterized by its ability to mate with 5 negative strains of B. tris-
pora ATCC 14272 to form a mated culture capable of producing
2021 mg carotenes per liter substrate in 6 days. Wang et al.
(2012, 2015) who studied the effect of inoculation process on lyco-
pene production by B. trispora NRRL 2895 (+) and NRRL 2896 () or
B. trispora NRRL 2895 (+) and I5 () found that the maximum lyco-
pene concentration from synthetic medium was achieved with an
optimal ratio of 5:1 or 1:2 for the (+) to () strains, respectively. In
our previous work, it was found that the optimum ratio of B. tris-
pora ATCC 14271 (+) and ATCC 14272 () for the maximum caro-
tene production from cheese whey was found to be 1:10
(Varzakakou et al., 2010a). There are some possible reasons for
these differences, including the strain of the microorganism used,
the composition of the substrate, the fermentation system, and,
generally, the conditions under which the fermentation takes
place.
The dry biomass concentration remained almost constant with
the increase of the inoculum ratio from 1:1 to 1:10 of (+) and ()
mating type (Fig. 3b). The highest biomass concentration
(40.0 ± 0.8 g/l) was obtained in medium inoculated with 1:5 of
(+) and () mating type.
3.3. Change in the specific activity of antioxidant enzymes during
fermentation
In media consisting of WCO and CSL and WCO supplemented
with CSL and BHT the specific activities of antioxidant enzymes
were studied during fermentation (Fig. 4a and b). In examining
the processes of oxidative stress in B. trispora induced by hydroper-
oxides and BHT, we monitored the activities of SOD and CAT which
are the two key defensive enzymes to oxidative stress (Nanou and
Roukas, 2013). In medium consisting of WCO and CSL the specific
activity of SOD increased drastically during the first 2 days of incu-
bation, remained almost constant between 2nd and 6th day, and
increased at the end of fermentation (Fig. 4a). On the other hand,
the specific intracellular activity of CAT showed two maxima, one
on the 2nd day of the incubation and another at the end of the fer-
mentation. The minimum between the two maxima was observed
after 4 days of incubation (Fig. 4b). The highest specific activities of
SOD (8.5 units/mg protein) and CAT (55.0 units/mg protein) were
observed at the end of the fermentation. Thus, when the concen-
tration of hydroperoxides and reactive oxygen species (ROS)
formed during fermentation was high after the 4th day of incuba-
tion as indicated by the increased specific activities of antioxidant
enzymes (Fig. 4a and b), B. trispora possess primarily the nonenzy-
matic antioxidant system that protects the microorganism from
oxidative stress resulting in a decrease in carotene concentration
(Fig. 1).
In medium consisting of WCO supplemented with CSL and BHT
the specific activity of SOD increased during the first 4 days of
incubation, decreased between 4th and 6th day, and increased
until the end of the fermentation (Fig. 4a). On the other hand,
the specific activity of CAT increased up to the 2nd day of the fer-
mentation, decreased between 2nd and 4th day, remained almost
constant between 4th and 6th day, and increased until the end of
incubation (Fig. 4b). The highest specific activities of SOD (7.5
units/mg protein) and CAT (95.0 units/mg protein) were observed
at the end of the fermentation.
3.4. Culture morphology
In media consisting of WCO and CSL and WCO supplemented
with CSL and BHT the dispersed morphology of B. trispora consists
of compact aggregates (pellets) with different sizes on the basis of
their projected area (Fig. 5 and Supplementary data). In both
media, the projected area of pellets increased rapidly during the
first 2 days of the fermentation and then decreased until the end
of the incubation. In media consisting of WCO and CSL and WCO
supplemented with CSL and BHT the highest projected area of pel-
lets was 3.2 and 1.6 mm2, respectively after 2 days of the
fermentation.
4. Conclusions
A new substrate, WCO was evaluated for carotene production
by B. trispora in submerged fermentation. B. trispora formed only
pellets during fermentation. The oxidative stress in B. trispora
induced by hydroperoxides of WCO increased the carotene produc-
tion. The external addition of BHT into the medium changed the
morphology of microorganism from pellets with large projected
area to pellets with small projected area. This morphological differ-
entiation of the fungus was associated with high oxidative stress
resulting in a significant increase of carotene production. WCO
supplemented with CSL and BHT was an attractive medium for
the production of carotenes.
Appendix A. Supplementary data
Supplementary data related to the morphology of the pellets of
B. trispora grown on WCO and CSL (a) and WCO supplemented with
CSL and BHT (b) can be found at electronic annex. Supplementary
data associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.biortech.2015.12.053.
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