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. 2017 Mar 20;17(7):775–780. doi: 10.1002/elsc.201600228

Carotene production from waste cooking oil by Blakeslea trispora in a bubble column reactor: The role of oxidative stress

Konstantina Nanou 1, Triantafyllos Roukas 1,✉, Emmanuel Papadakis 2, Parthena Kotzekidou 3
PMCID: PMC6999405  PMID: 32624823

Abstract

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

Abbreviations

CAT

catalase

CSL

corn steep liquor

ROS

reactive oxygen species

SOD

superoxide dismutase

WCO

waste cooking oil

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, 2, 3, 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 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 (H2O2), hydroxyl radicals (HO•), and superoxide 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, inactivation of enzymes and ultimately cell death 12, 13. Aerobic organisms possess enzymatic and non‐enzymatic defense systems for protecting the cells from oxidative stress. In the enzymatic defense system, superoxide dismutase (SOD), catalase (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, trehalose, ubiquinol (UQH2), and metallothioneins. They act as 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 H2O2 during carotene production from a synthetic medium in shake flask culture was studied 15, 16, 17.

Recently, in our laboratory, the production of carotenes from WCO in shake flask culture was studied 18. However, the oxidative stress response of B. trispora induced by hydroperoxides of WCO and ROS in a bubble column reactor has not been studied. 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 fermentation 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).

2.4. Analytical techniques

At specific time intervals, 20 mL of the sample were removed from the reactor and analyzed. The dissolved oxygen concentration 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) official methods. For determination of carotene concentration, carotenes were extracted from the fungus biomass by ethanol as described by Roukas and Mantzouridou 20 and the composition 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 measuring 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 aeration rates of 4 and 5 vvm, while at aeration rate of 6 vvm the carotene concentration was increased up to 4th day of fermentation 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%, respectively. The above results show that β‐carotene was the major accumulated compound.

Figure 1.

Figure 1

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Change in carotene concentration (A) and dry biomass (B) during fermentation of WCO and CSL by Blakeslea trispora in a bubble column reactor. ‐□‐, ‐◯‐, and ‐Δ‐, 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.

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.

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.

Figure 2.

Figure 2

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Change in dissolved oxygen concentration during carotene production from WCO and CSL by Blakeslea trispora in a bubble column reactor. ‐□‐, ‐◯‐, and ‐Δ‐, 4, 5, and 6 vvm, respectively. Each point is the mean ± SD of three repetitions.

In all fermentation conditions, the pH of the medium decreased 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 degradation of proteins. In all cultures, the concentration of unconsumed 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, remained 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.

Figure 3.

Figure 3

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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 ‐Δ‐, 4, 5, and 6 vvm, respectively. Each point is the mean ± SD of three repetitions.

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).

Figure 4.

Figure 4

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Change in the projected area of pellets during carotene production from WCO and CSL by Blakeslea trispora in a bubble column reactor. □‐, ‐◯‐, and ‐Δ‐, 4, 5, and 6 vvm, respectively. Each point is the mean ± SD of three repetitions.

Figure 5.

Figure 5

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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.

4. Discussion

As shown in Fig. 1A and B the biosynthesis of carotenes was carried out during the growth phase of the microorganism (aeration rate 6 vvm) or in growth and static phase of the fungus (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 concentration 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. Therefore, the change in mycelium morphology might be a response to this stress. Pellets with small projected area supply a satisfactory 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, increasing 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 concentration 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 production (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 indicated 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, 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).

Figure 6.

Figure 6

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Factors causing oxidative stress in Blakeslea trispora during carotene production from WCO and CSL in a bubble column reactor.

The production of carotenes in fermentation medium containing 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 publication 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 easily and the mixing of the fermentation broth requires less energy. In addition, laboratory results obtained in bubble column reactors 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 deproteinized 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 concentration of carotenes (6.0, 46.0, and 89.0 mg/L, respectively) was obtained 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 culture. The maximum concentration of carotenes obtained when 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 compared with the amounts of carotenes (980 mg/L) produced from WCO and CSL. The toxic substances such as hydroperoxides, alcohols, 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 reduce 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 cooking 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.

The authors have declared no conflict of interest.

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