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Atmospheric and room temperature plasma mutagenesis and astaxanthin production from sugarcane bagasse hydrolysate by Phaffia rhodozyma mutant Y1
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Atmospheric and room temperature plasma mutagenesis and astaxanthin production from sugarcane bagasse hydrolysate by Phaffia rhodozyma mutant Y1 Yuan Zhuanga, Gui-Li Jianga, Ming-Jun Zhua,b,c,* a
Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, China b College of Life and Geographic Sciences, Kashi University, Kashi 844000, China c Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region, Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges & Universities under the Department of Education of Xinjiang Uygur Autonomous Region, Kashi University, Kashi 844000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Astaxanthin Phaffia rhodozyma Atmospheric and room temperature plasma Sugarcane bagasse hydrolysate Cell wall disruption
In this study, atmospheric and room temperature plasma and ultraviolet mutagenesis was studied for astaxanthin overproducing mutant. Phaffia rhodozyma mutant Y1 was obtained from the selection plate with 120 μmol/L diphenylamine as selection agent, and its carotenoid concentration and content were 54.38 mg/L and 5.38 mg/g, which were 19.02 % and 21.20 % higher than that of the original strain, respectively. Sugarcane bagasse hydrolysate was used for astaxanthin production by mutant Y1 at 22 °C and 220 rpm for 96 h, and the biomass and carotenoid concentration reached 12.65 g/L and 88.57 mg/L, respectively. Ultrasonication and cellulase were used to break cell wall and the parameters were optimized, achieving an astaxanthin extraction rate of 96.01 %. The present work provided a novel combined mutagenesis method for astaxanthin overproducing mutant and a green cell wall disruption process for astaxanthin extraction, which would play a solid foundation on the development of natural astaxanthin.
1. Introduction Astaxanthin (3,3′-dihydroxy-β,β'-carotene-4,4′-dione) [1], is a kind of lipophilic carotenoid with strong antioxidant activity, which is more than 550 times stronger than α-tocopherol [2]. Astaxanthin is widely used in food, health care products, cosmetics [3–7] and medicine, which can prevent and treat many diseases [8,9]. In addition, astaxanthin is widely added to the feed of fishes such as salmon or trout in aquaculture, allowing these fishes to accumulate astaxanthin to obtain red fish [10] and improve the juvenile survival rate of fishes as a nutrient [11]. The production of natural astaxanthin mainly comes from Haematococcus pluvialis and Phaffia rhodozyma [12,13]. Among them, H. pluvialis, considered to have the highest content of natural astaxanthin at present, has 15∼30 mg/g dry cell weight (DCW) of astaxanthin, which accounts for more than 99 % of the total carotenoids produced by H. pluvialis [14]. Even so, P. rhodozyma has also many advantages over H. pluvialis [15], such as fast growth rate, high-density fermentation, less heavy metal, simple culture conditions and no need for illumination. In addition, the yeast cells are rich in proteins,
minerals and vitamins, which might be processed to form yeast extract for use in food and pharmaceutical industries [16]. Previous research has indicated that astaxanthin synthesized by P. rhodozyma accounts for up to 84 % of its total synthesized carotenoids [1]. However, the average content of astaxanthin in wild P. rhodozyma was only 0.2∼0.4 mg/g DCW [17], which seriously hindered the commercial development of astaxanthin. At present, there are many methods for strain modification, such as physicochemical mutagenesis, protoplast fusion and genetic engineering [18,19]. It has been reported that the astaxanthin concentration of P. rhodozyma was greatly increased, and the astaxanthin content reached 9.7 mg/g using traditional mutagenesis combined with metabolic engineering [20]. However, genetic engineering is time-consuming, costly and technically difficult. On the contrary, mutation breeding is more economical, convenient and easy to operate. The carotenoid content of Rhodotorula glutinis PTCC 5256 strain irradiated with 254 nm ultraviolet (UV) rays was 890 μg/g, which was 1.508 times higher than that of wild strain [21]. In addition, there are ethyl methyl sulfonate mutagenesis [22], nitrosoguanidine mutagenesis [23], and atmospheric and room
⁎ Corresponding author at: Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, China. E-mail addresses: [email protected] (Y. Zhuang), [email protected] (M.-J. Zhu).
https://doi.org/10.1016/j.procbio.2020.01.003 Received 19 September 2019; Received in revised form 17 December 2019; Accepted 7 January 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Yuan Zhuang, Gui-Li Jiang and Ming-Jun Zhu, Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.01.003
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2.2. Microbial strain and cultivation
temperature plasma (ARTP) mutagenesis. ARTP mutagenesis is a popular method for microbial mutagenesis bio-breeding in recent years. The mutagenesis of cells mediated by ARTP has many advantages, such as fast, safe, high mutation rate and efficient. It is widely used in plants, fungi and bacteria to improve the yield of the target product or enhance substrate tolerance of the strain [24,25]. The present study has reported that astaxanthin accumulation in Saccharomyces cerevisiae was promoted by metabolic engineering and ARTP mutagenesis, and the astaxanthin concentration obtained by fermentation in a 5 L fermenter was 217.9 mg/L [26]. In order to screen the mutants with high astaxanthin concentration, the screening agent should be added to the culture medium after mutagenesis. Namthip et al. found that the most efficient screening agent among several screening agents tested was diphenylamine, by which a high titer astaxanthin mutant was screened out [27]. Diphenylamine is an inhibitor of phytoene desaturase, which can inhibit the synthesis of astaxanthin [28]. The mutants had stronger resistance to the screening agent, showing a redder color than the original strains on the screening plate. The mutants screened were found to produce more astaxanthin than the original strain [27]. In order to reduce the cost of astaxanthin production by P. rhodozyma and improve astaxanthin concentration, some researchers have used low-cost raw materials to culture P. rhodozyma, such as eucalyptus hydrolysate [29], Yucca fillifera date juice [30], pretreated barley straw [31], sugarcane juice [32], mussel processing wastewater [33] and sweet sorghum juice [1]. Among them, sugarcane is one of the most productive crops in the world. In 2010, the yield of sugarcane in the world was a total of 428.11 million tons, and the residue sugarcane bagasse (SCB) can be used to produce the second generation of ethanol [34]. In addition, some scholars have studied that after effective physical, chemical or biological pretreatment, SCB can be converted into different sugars by enzymolysis, such as glucose, cellobiose, and xylose, which can be further used as carbon source for microorganism [35]. The hard cell wall of P. rhodozyma prevented astaxanthin from being degraded by external factors, such as light, heat, acid and oxygen [36], but it also prevented the extraction and utilization of astaxanthin. Currently, some processes have been developed to break yeast cells [37], such as chemical method [38], enzymic method [39] and mechanical method [40]. Although the enzymatic method is mild and can reduce the damage to astaxanthin, the extraction rate of astaxanthin is not ideal and it is time-consuming [41]. However, the ultrasonication can help disruption of cell wall, thus strengthening the diffusion of the target components in the cell [42]. In addition, the toxicity of organic solvents used in the extraction of astaxanthin, such as dimethyl sulfoxide and acetone [42,43], seriously limits the application of astaxanthin in some products. In summary, it is required to find a simple and effective method to break the wall and extract astaxanthin. The present study aims to screen mutant with high yield carotenoid by a novel combined mutagenesis method (ARTP and UV). Then, based on the mutant and sugarcane bagasse hydrolysate (SCBH) as carbon source, the optimal medium and culture conditions are determined for the accumulation of carotenoids. A green and efficient cell wall disruption process using ultrasonication followed by enzymatic hydrolysis is developed to improve astaxanthin extraction.
Phaffia rhodozyma Y119 was stored in the Fermentation Engineering Laboratory of School of Biological Science and Engineering, South China University of Technology. It was maintained on the agar slant of Yeast Extract Peptone Dextrose Medium (YPD) at 4 °C and subcultured for further research work. Seed culture spore suspension was prepared from actively growing yeast in YPD Erlenmeyer flask (250 mL), containing 22.5 mL sterilized medium (20 g glucose, 20 g peptone and 10 g yeast extract in 1000 mL distilled water), was inoculated with 10 % v/v of the spore suspension of P. rhodozyma and incubated at 22 °C at 220 rpm. 2.3. Physical mutagenesis by ARTP and UV P. rhodozyma Y119 was incubated at 22 °C for 72 h–96 h on the agar slant of YPD. A ring of cells from the actively growing slant was inoculated to Erlenmeyer flask containing 25 mL YPD and incubated at 22 °C to the logarithmic phase. Then, 2 mL yeast suspensions were taken out from Erlenmeyer flask and centrifuged at 4000 rpm for 5 min. The yeast cells were harvested and washed twice with deionized water. Normal saline was added to make the yeast with a cell concentration of about 106∼107 CFU/mL. Ten μL yeast suspensions diluted with normal saline were taken out and evenly coated on the iron plate in ARTP mutagenesis system with 120 W operating voltage, 2 mm working distance, 12 L/min helium gas flow and different mutagenic time (30, 40, 50, 60, 70 and 80 s). After mutagenesis, 1 mL sterilized normal saline was used for washing the cells on the iron plate down. And then, the yeast suspensions were diluted to suitable concentration, coated on the YPD plate and cultured in the 22 °C incubator for four days to calculate lethality rate. The time under 85 % lethality rate was determined as ARTP mutagenic time. After determining the action time of ARTP mutagenesis, the yeast suspensions after ARTP mutagenesis were diluted and coated on the YPD plate. Then, the irradiation at 20 cm directly below the UV lamp (30 W electric power) was conducted for different time, and the lethality rate was calculated. 2.4. Screening of mutant strains by diphenylamine One hundred μL yeast suspensions, which had been diluted to suitable concentration, were taken out for spreading on the YPD plate with different concentrations of diphenylamine (10, 20, 40, 60, 80, 120, 140 and 160 μmol/L). After incubating at 22 °C for 5∼6 days, the colony growth and color changes were observed to determine the optimal concentration of diphenylamine. After mutagenesis with UV and ARTP, the yeast suspensions were diluted and coated on the YPD plate with optimal concentration of diphenylamine. Large and red single colonies on the YPD plate were selected out and numbered one by one. Total selected strains were inoculated on the agar slant of YPD, after which they were incubated in YPD liquid medium for the determination of biomass and carotenoid concentration. Method for measuring biomass concentration has been reported in previous study [44]. Total carotenoids were extracted with acetone from the cell pellets which had been treated by the method of hot acid, and analyzed at 474 nm using a UV spectrophotometer (Unic, UV-2150), as described by Jiang [15]. In addition, the selected mutants were subcultured for 9 times to test the hereditary stability.
2. Materials and methods 2.1. Chemicals and reagents
2.5. Preparation of sugarcane bagasse hydrolysate (SCBH)
Diphenylamine was purchased from Shanghai Miriel Chemical Technology Co., Ltd. Acetone, ethanol and n-hexane were obtained from Tianjin Damao Chemical Reagent Factory. Cellulase (Cellic® Ctec2) was obtained from Novozymes (China) Investment Co. Ltd. SCB was donated by Guangzhou Sugarcane Research Institute. All other reagents were analytically grade and purchased from Sigma-Aldrich, USA.
The pretreatment of sugarcane bagasse (SCB) was carried out in a 250 mL Erlenmeyer flask, of which the conditions (pretreatment with potassium peroxymonosulfate and NaOH) have been studied in previous study [45]. Then, enzymolysis was conducted with 30 % substrate, 50 mM sodium citrate buffer (pH 4.8) and cellulase (20 FPU/g 2
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pretreated SCB) in a 250 mL Erlenmeyer flask for 6 days (55 °C, 150 rpm). After vacuum filtration followed by filtration with 0.45 μm membrane, the obtained sugar solution of SCBH was determined by high performance liquid chromatography (Waters 2414, Milford, LA, USA).
2.10. Extraction of astaxanthin In order to improve the extraction rate of astaxanthin, the yeast cells cultivated with SCBH as carbon source were broken by ultrasonication and cellulase. The order of ultrasonication and cellulase was changed and the optimum condition for cell wall disruption was found through an orthogonal experiment design using the SPSS software. After cell wall disruption, ethanol was used for extracting carotenoid, and then carotenoid ethanol solution was mixed with n-hexane for further extracting astaxanthin, as described in previous studies [4,46]. Briefly, 2 mL n-hexane was added to 2 mL carotenoid ethanol solution with a sufficient oscillation in the vortex mixer for 30 s, then mixed with 6 mL deionized water for 30 s and stood for 5 min. The mixture was centrifuged at 800 x g for 5 min to obtain the supernatant, of which the absorbance was measured at 468 nm using a spectrophotometer. Astaxanthin concentration in n-hexane was calculated by Eq. (1):
2.6. Optimization of nitrogen source with SCBH as carbon source The high concentration sugar solution obtained from SCB was diluted to a total sugar concentration of 30 g/L, including 20.22 g/L glucose, 6.71 g/L xylose and 3.05 g/L cellobiose. The fermentation medium contained SCBH as carbon source, 1.0 g/L KH2PO4 and 0.5 g/L MgSO4 (the same below). And KNO3, urea, (NH4)2SO4, peptone and yeast extract at the concentration of 5 g/L were added as nitrogen source, respectively. The sterilized fermentation medium was inoculated with the secondary seed solution (10 % v/v) and incubated at 22 °C, 200 rpm (the same below). The biomass and carotenoid concentration of yeast were measured by sampling every 24 h.
Astaxanthin concentration(mg/mL) =
A 468 × D× P× V2 ε×V1× pathlength
(1)
Where A468 is absorbance, P is molecular molar mass of astaxanthin 597 g/mol, pathlength is the thickness of the light absorption pool which is 1 cm, ε is the molar absorptivity of astaxanthin 125100 L/mol/cm, D is dilution ratio, V2 is n-hexane volume (mL), and V1 is ethanol volume (mL). Finally, the extraction rate of astaxanthin was calculated on the basis of the carotenoid concentration extracted by acetone along with hot acid method.
2.7. Optimization of C/N ratio with SCBH as carbon source The optimal nitrogen source in Section 2.6 was selected as the nitrogen source. Then, SCBH with sugar concentration of 30 g/L was used as carbon source, and the concentration of nitrogen source was adjusted to obtain different C/N ratios, which were 2, 3, 4, 5, 6 and 7, respectively. The yeast cells were incubated under these conditions, of which the biomass and carotenoid concentration were measured by sampling every 24 h.
2.11. Statistical analysis
2.8. Optimization of mixed nitrogen source with SCBH as carbon source
The experiments were performed in triplicate, and results are presented as the mean ± standard deviation (SD). Statistical evaluations were performed on the basis of confidence intervals using the Tukey-b test. A value of p < 0.05 was considered significant. All statistical analysis were carried out using the SPSS software (Ver. 17.0, SPSS Inc., Chicago, IL, USA).
Yeast extract and urea were mixed at 6: 0, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5, 0: 6 (w/w) with a total concentration of 6 g/L. Other components of fermentation medium contained 1.0 g/L KH2PO4, 0.5 g/L MgSO4 and SCBH with sugar concentration of 30 g/L. The yeast cells were incubated under these conditions, of which the biomass and carotenoid concentration were measured by sampling every 24 h.
3. Results and discussion 3.1. Strain improvement by ARTP and UV
2.9. Effects of different carbon sources on cell growth and carotenoid accumulation
As shown in Fig. 1A, when ARTP mutagenic time was 50 s, the lethality rate of yeast was 84.2 %. It has been reported that the positive mutagenesis rate of yeast cells is higher when lethality rate by ARTP mutagenesis is close to 85 % [47]. Therefore, 50 s was selected as the mutagenic time of ARTP. In addition, when UV irradiation is carried out, high lethality rate probably causes negative mutagenesis of the strain. Thus, the time of UV irradiation for lethality rate of 70∼80 % is generally selected as mutagenic time [48]. When the yeast cells on the plate were irradiated by UV for 40 s, the lethality rate was 70.3 %
To compare the effects of SCBH and other carbon sources (30 g/L glucose, mixed carbon source of 20.22 g/L glucose, 6.71 g/L xylose and 3.05 g/L cellobiose) on carotenoid accumulation of yeast, the secondary seed solution (10 % v/v) was inoculated to three different sterilized fermentation media (22.5 mL), each of which contained one of the above three carbon sources with the same sugar concentration, respectively. The biomass and carotenoid concentration of yeast were measured by sampling every 24 h.
Fig. 1. Lethality rate of P. rhodozyma under different mutagenic time. (A) ARTP mutagenic time. (B) UV mutagenic time. 3
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Fig. 2. The growth and hereditary stability of mutant strain obtained by ARTP and UV mutagenesis. (A) The comparison of biomass and carotenoid accumulation between mutant strain and original strain. (B) Hereditary stability of mutant strain.
and urea (8.08 mg/g). Therefore, compared with other nitrogen sources tested, yeast extract is more conducive to the growth of yeast and the accumulation of carotenoids, which was mainly attributed to the abundant nutrients of yeast extract [16].
(Fig. 1B), which was in the range of the best lethality rate. Thus, 40 s was selected as the optimal UV mutagenic time. Briefly, the conditions for the combined mutagenesis were determined as follows: first, the cells were mutagenized by ARTP for 50 s, and then irradiated by UV for 40 s.
3.4. Effects of different C/N ratios on cell growth and carotenoid accumulation
3.2. Screening and hereditary stability of P. rhodozyma mutant When diphenylamine concentration gradually increased from 10 μmol/L to 100 μmol/L, the colonies on the screening plate gradually changed from red to light red, and even a few white colonies appeared. This indicated that the higher the concentration of diphenylamine, the stronger the inhibitory effect on the synthesis of carotenoids, which was consistent with previous research reports [27,28]. When diphenylamine concentration reached 120 μmol/L, only white colonies appeared on the plate. Therefore, the optimal concentration of diphenylamine selected in this experiment was 120 μmol/L. After repeated mutagenesis and screening, a light red colony was found, which was immediately preserved and its biomass and carotenoid concentration were determined. As shown in Fig. 2A, the biomass concentration, carotenoid concentration and carotenoid content of the original strain were 10.35 g/L, 45.69 mg/L and 4.41 mg/g, respectively, and the mutant strain was 10.1 g/L, 54.38 mg/L and 5.38 mg/g, respectively. In this paper, the mutant strain was numbered Y1. Although the biomass concentration of Y1 was slightly lower than that of the original strain, its carotenoid concentration and content were 19.02 % and 22.00 % higher than that of the original strain, respectively. After being subcultured for 9 times (Fig. 2B), the carotenoid concentration and content of the mutant strain were 54.97 mg/L and 5.33 mg/g, respectively, which were 20.31 % and 20.86 % higher than the original strain, indicating that the mutant strain had excellent hereditary stability. In a previous study, P. rhodozyma mutant by ARTP mutagenesis had good hereditary stability, whose carotenoid concentration was 1.67 mg/L and 40 % higher than the original strain [49]. Through ARTP mutagenesis, sodium nitrite mutagenesis, UV mutagenesis followed by UV-sodium nitrite mutagenesis, Liu [50] obtained a red yeast with carotenoid concentration of 14.47 mg/L, which was 1.67 times that of the original strain.
Proper C/N ratio can maintain the fast growth of yeast cells and the synthesis of carotenoid, only in this way can the maximum carotenoid concentration be guaranteed. The effects of different C/N ratios on the growth and carotenoid accumulation of P. rhodozyma mutant is shown in Fig. 4. When the C/N ratio was 2, the biomass concentration reached a maximum of 11.5 g/L. With the increase of C/N ratio, the biomass concentration showed a downward trend. As stated in earlier literature reports, high carbon loadings inhibited the growth of P. rhodozyma by reducing the amount of available nitrogen [51]. When the C/N ratio was 5, the carotenoid concentration reached the maximum value of 92.19 mg/L. With the increase of the C/N ratio from 2 to 7, the carotenoid concentration first increased and then decreased with a C/N ratio of 5 as the dividing line, which was consistent with previous research reports. A report proved that a C/N ratio below 5 has a negative effect on the carotenoid production of P. rhodozyma, suggesting that abundant nitrogen promotes cell growth but inhibits the activity of enzyme that converts β-carotene to astaxanthin, and in contrast, a high C/N ratio affects cell growth, thus affecting the carotenoid synthesis [51]. After comprehensive consideration of biomass and carotenoid concentration, the best C/N ratio was 5, that is, the best addition amount of yeast extract was 6 g/L. 3.5. Effects of mixed nitrogen source on cell growth and carotenoid accumulation Compared with the cost of yeast extract and peptone, urea is cheaper. In addition, P. rhodozyma showed high carotenoid content with urea as nitrogen source among all the tested nitrogen sources. Therefore, P rhodozyma was cultured by combining yeast extract with urea to obtain the ideal carotenoid concentration at a lower cost. The effects of mixed nitrogen source on the growth and carotenoid accumulation of P. rhodozyma mutant are shown in Fig. 5. As can be seen from the results in Fig. 5B, when the ratio of yeast extract to urea was 6: 0 (6 g/L of yeast extract), the yeast cells had the highest carotenoid concentration. However, when the ratio was 6: 0, 5: 1 and 3: 1, the difference in carotenoid concentration of P. rhodozyma was not significant (p > 0.05). Therefore, from the perspective of cost, mixed nitrogen source with a ratio of 3:1 between yeast extract and urea could be selected to culture the yeast, and at this time, the carotenoid concentration and content were 81.44 mg/L and 6.46 mg/g, respectively. There is a study reporting that compared with yeast extract and ammonium sulfate as mixed nitrogen source, yeast extract and
3.3. Effects of different nitrogen sources on cell growth and carotenoid accumulation In this study, five nitrogen sources were selected to explore the effects of different nitrogen sources on the growth and carotenoid accumulation of P. rhodozyma mutant. The experimental results are shown in Fig. 3. It can be seen from the figure that the yeast extract was most conducive to the growth of the yeast (9.45 g/L), followed by peptone and ammonium sulfate. When yeast extract was used as nitrogen source, the highest carotenoid concentration was 82.57 mg/L, and the highest carotenoid content was 8.73 mg/g, followed by peptone (8.67 mg/g) 4
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Fig. 3. Effects of different nitrogen sources on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content.
Fig. 4. Effects of different C/N ratios on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content. 5
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Fig. 5. Effects of mixed nitrogen source on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content.
promoting astaxanthin accumulation. However, whether this is also related to the cellobiose in SCBH needs to be further studied. Overall, the optimized SCBH medium shows obvious advantage over glucose medium. Compared with glucose medium, SCBH medium can promote yeast cells to accumulate more carotenoids without significantly inhibiting the growth of yeast cells. Similarly, it's been reported that the astaxanthin concentration and biomass concentration of yeast, cultivated with SCBH which was pretreated with ammonia, were 2.56 mg/L and 7.16 mg/L, respectively [55]. The carotenoid concentration of yeast was 60.75 mg/L in fermentation medium (pH 5.0) containing 30 g/L sugarcane molasses and 5 g/L yeast extract [56]. P. rhodozyma was cultivated in a 2 L bioreactor with sweet sorghum juice, of which the biomass and astaxanthin concentration were 29 g/L and 65.4 mg/L, respectively [1]. The biomass and astaxanthin concentration of P. rhodozyma cultivated in fruit and vegetable waste medium were comparable to those cultivated in the synthetic medium, reaching 5.3 g/L and 355 μg/g, respectively [57]. In addition, the study on the feasibility of producing astaxanthin by P. rhodozyma cultivated in the pretreated whey medium has been reported [58]. The above examples show that it has certain economic and ecological value to produce astaxanthin by cultivating P. rhodozyma using agro-industrial waste.
urea as mixed nitrogen source had more significant promotion effect on astaxanthin accumulation [1].
3.6. Effects of different carbon sources on cell growth and carotenoid accumulation Glucose, xylose and cellobiose as carbon sources for the culture of yeast have high cost, and only for use in the laboratory. In this study, the carotenoid concentration and content of P. rhodozyma cultured with three different carbon sources were compared. Three different carbon sources were glucose, SCBH, and mixed carbon source (containing glucose, xylose and cellobiose), respectively. Under the condition of guaranteeing the carotenoid concentration, it is expected to replace the pure sugar with the cheap SCBH to cultivate P. rhodozyma. As shown in Fig. 6, when SCBH was used as carbon source, the biomass concentration of yeast was comparable to that of glucose as carbon source, possibly because SCBH had no many toxic substances produced to inhibit the growth of yeast after potassium peroxymonosulfate pretreatment and enzymolysis. As is known to all, lignocellulose feedstocks after pretreatment will produce sugar solution, and depending on the pretreatment method, they will generate various by-products, such as furfural and phenolic compounds [52,53], which can affect yeast cultivation and product synthesis. It has been reported that P. rhodozyma was cultured to produce astaxanthin with detoxified sweet sorghum bagasse hydrolysate, because the hydrolysate without detoxification contained phenolic compounds that inhibited the yeast cultivation [54]. From the perspective of carotenoid concentration, the carotenoid concentration of yeast cultivated with SCBH as carbon source was 88.57 mg/L and mixed carbon source was 92.07 mg/L. There was no significant difference (p > 0.05) between the two carbon sources, but both were higher than 65.19 mg/L when 30 g/L glucose was used as carbon source. This result was mainly attributed to the xylose contained in both mixed carbon source and SCBH. Previous reports [29,54] have shown that xylose is more favorable than glucose in
3.7. Extraction of astaxanthin using enzymolysis followed by ultrasonication Both ultrasonication and enzymolysis are green methods of cell wall disruption, combined to obtain an environmental-friendly and efficient method of cell wall disruption. Constant parameters in this orthogonal experiment were enzymolysis time 6 h, enzymolysis temperature 50 °C, the ultrasonic power 361 W, 2 s on and 5 s off. The results are shown in Table 1. According to the range analysis, the order of influence of each factor on the extraction rate of astaxanthin after enzymolysis and enzymolysis followed by ultrasonication was as follows: cell concentration > 6
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Fig. 6. Effects of different carbon sources on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content.
enzyme amount > ultrasonic time. According to the results shown in Table 1, the highest Ⅰ (67.51 %) and Ⅱ (90.75 %) were observed in experiment No.6 under the optimal conditions: cell concentration, 0.5 %; enzyme amount, 64.6 FPU/g; enzymolysis time 6 h; ultrasonic time, 45 min. A previous study also showed that P. rhodozyma treated with βglucanase followed by ultrasonication can produce up to 435.71 ± 6.55 μg of astaxanthin/g cell, which was about twice the content of astaxanthin obtained by organic solvent extraction [59].
Table 1 The results of orthogonal design for the optimization of astaxanthin extraction using enzymolysis followed by ultrasonication. No.
Cell concentration (%)
Enzyme amount (FPU/g)
Ultrasonic time (min)
Ⅰ (%)
Ⅱ (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K¯1
1 1 2 1.5 1.5 0.5 0.5 0.5 1.5 0.5 2 2 1 1.5 2 1 60.24 47.28 40.88 39.02 21.22 84 72.43 65.27 56.04 27.96
72.20 79.80 79.80 72.20 79.80 64.60 79.80 57.00 57.00 72.20 72.20 57.00 57.00 64.60 64.60 64.60 44.66 47.18 45.26 42.14 5.04 68.58 72.07 68.78 68.32 3.75
45 25 45 15 35 45 15 25 45 35 25 35 15 25 15 35 46.32 42.86 42.95 47.12 4.26 71.28 69.44 67.55 69.46 3.73
45.58 49.40 29.87 47.43 31.72 67.51 57.57 55.52 45.52 60.36 27.66 31.6 46.01 38.86 34.25 48.11
66.69 77.78 54.92 72.55 57.59 90.75 83.01 80.47 65.47 81.77 54.10 56.97 71.39 65.47 58.16 73.88
K2 K¯3 K4 RⅠ K5 K6 K7 K8 RⅡ
3.8. Extraction of astaxanthin using ultrasonication followed by enzymolysis According to the parameters obtained from the above orthogonal experiment (cell concentration 0.5 %, enzymolysis temperature 50 °C, ultrasonic power 361 W, 2 s on, 5 s off), the order of ultrasonication and enzymolysis was changed. According to the values of K¯1, K¯2 , and K3 in Table 2, after 60 min of ultrasonication alone, the extraction rate of astaxanthin reached the maximum, which was 54.44 %. When the extraction rate of astaxanthin reached about 90 % (No.6), compared with the method of enzymolysis followed by ultrasonication (No.6 of Table 1), the amount of cellulase could be reduced from 64.6 FPU/g to 51.3 FPU/g by increasing the enzymolysis time from 6 h to 10 h and the ultrasonic time from 45 min to 60 min with the method of ultrasonication followed by enzymolysis. According to the range analysis, the order of influence of each factor on the extraction rate of astaxanthin using ultrasonication followed by enzymolysis was as follows: enzyme amount > enzymolysis time > ultrasonic time. According to the values of K¯ 4 , K5 , and K¯ 6 , the best combination of ultrasonication combined with enzymolysis was as follows: ultrasonic time, 60 min; enzymolysis time, 10 h; enzyme amount, 64.6 FPU/g. A confirmatory experiment performed under the optimal conditions, the results showed that the extraction rate of astaxanthin reached 96.01 %. There is a previous study [60] reporting that different cell
Ⅰ Extraction rate of astaxanthin using enzymolysis. Ⅱ Extraction rate of astaxanthin using enzymolysis followed by ultrasonication. K¯1, K2 , K¯3 and K 4 come from the results of Ⅰ. K5 , K 6 , K7 and K 8 come from the results of Ⅱ. RⅠ is the range of K¯1, K2 , K¯3 and K 4 ; RⅡ is the range of K5 , K 6 , K7 and K 8 .
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Author statement
Table 2 The results of orthogonal design for the optimization of astaxanthin extraction using ultrasonication followed by enzymolysis. No.
Ultrasonic time (min)
Enzymolysis time (h)
Enzyme amount (FPU/g)
Ⅲ (%)
Ⅳ (%)
1 2 3 4 5 6 7 8 9 K¯1
60 30 60 30 45 60 45 45 30 50.76 53.46 54.44 3.68 87.93 83.54 89.36 5.82
14 10 6 14 14 10 10 6 6 52.89 52.89 52.89 0 81.91 90.99 87.97 9.08
64.60 39.90 39.90 51.30 39.90 51.30 64.60 51.30 64.60 52.89 52.89 52.89 0 84.2 82.41 94.25 11.84
54.44 50.76 54.44 50.76 53.46 54.44 53.46 53.46 50.76
95.95 87.53 82.05 84.94 83.03 90.08 95.36 72.22 91.45
K2 K¯3 RⅢ K4 K5 K6 RⅣ
The study was designed by Gui-Li Jiang and Ming-Jun Zhu. The experiments were executed by Gui-Li Jiang. Data analysis and data interpretation were executed by Yuan Zhuang and Ming-Jun Zhu. All the authors contributed in writing the manuscript and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgments The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China, China [grant no. 51878291] and Guangzhou Science and Technology Program, China [grant no. 2014 Y2 -00515]. References
Ⅲ Extraction rate of astaxanthin using ultrasonication. Ⅳ Extraction rate of astaxanthin using ultrasonication followed by enzymolysis. K¯1, K2 , and K¯3 come from the results of Ⅲ. K 4 , K5 , and K 6 , come from the results of Ⅳ. RⅢ is the range of K¯1, K2 , and K¯3 . RⅣ is the range ofK 4 , K5 , and K 6 .
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disruption methods (including abrasion with celite, glass pearls in vortex agitator, ultrasonic waves, sodium carbonate and dimethyl sulfoxide) were tested to obtain astaxanthin, among which the dimethyl sulfoxide was found to be the most efficient method. Although the method of extracting astaxanthin by organic solvent has high efficiency and low cost, the application of astaxanthin is severely restricted due to safety problems [60]. Monks et al. [61] evaluated the efficiency of extracting carotenoids with the methods of chemical, enzymatic and ultrasound-assisted treatments, showing that the enzymolysis was the most effective method, and the methods of ultrasound-assisted cell disruption alone or combined were inefficient, especially ultrasound combined with enzymolysis simultaneously. However, the method with ultrasonication followed by enzymolysis was not tried [61]. In addition, recent biorefinery technologies, such as ionic liquids [62] and supercritical solvent extraction [63], have high extraction efficiency of astaxanthin, some of which can even over 99 % [64]. However, both have high costs, and what’s more, the ionic liquids are toxic [41]. In conclusion, the method of ultrasonication followed by enzymolysis was used to cell disruption, and ethanol was used for extraction. Compared with the other methods mentioned above, although the cost of enzyme was a bit high, the method in this study was green and efficient, which was conducive to the downstream application of astaxanthin.
4. Conclusion In this study, ARTP and UV was successfully used for the mutagenesis of P. rhodozyma, and a mutant Y1 was obtained from the diphenylamine selection plate, whose astaxanthin content was significantly improved. Compared with glucose, carotenoid concentration of mutant Y1 with SCBH as carbon source was significantly improved. A green and simple astaxanthin extraction method was well established, with which ultrasonication and cellulase were used to break cell wall and ethanol was used to extract astaxanthin, leading to a satisfactory astaxanthin extraction rate. The present study plays solid foundation for the industrial production and application of astaxanthin.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Atmospheric and room temperature plasma mutagenesis and astaxanthin production from sugarcane bagasse hydrolysate by Phaffia rhodozyma mutant Y1 Yuan Zhuanga, Gui-Li Jianga, Ming-Jun Zhua,b,c,* a
Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, China b College of Life and Geographic Sciences, Kashi University, Kashi 844000, China c Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region, Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges & Universities under the Department of Education of Xinjiang Uygur Autonomous Region, Kashi University, Kashi 844000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Astaxanthin Phaffia rhodozyma Atmospheric and room temperature plasma Sugarcane bagasse hydrolysate Cell wall disruption
In this study, atmospheric and room temperature plasma and ultraviolet mutagenesis was studied for astaxanthin overproducing mutant. Phaffia rhodozyma mutant Y1 was obtained from the selection plate with 120 μmol/L diphenylamine as selection agent, and its carotenoid concentration and content were 54.38 mg/L and 5.38 mg/g, which were 19.02 % and 21.20 % higher than that of the original strain, respectively. Sugarcane bagasse hydrolysate was used for astaxanthin production by mutant Y1 at 22 °C and 220 rpm for 96 h, and the biomass and carotenoid concentration reached 12.65 g/L and 88.57 mg/L, respectively. Ultrasonication and cellulase were used to break cell wall and the parameters were optimized, achieving an astaxanthin extraction rate of 96.01 %. The present work provided a novel combined mutagenesis method for astaxanthin overproducing mutant and a green cell wall disruption process for astaxanthin extraction, which would play a solid foundation on the development of natural astaxanthin.
1. Introduction Astaxanthin (3,3′-dihydroxy-β,β'-carotene-4,4′-dione) [1], is a kind of lipophilic carotenoid with strong antioxidant activity, which is more than 550 times stronger than α-tocopherol [2]. Astaxanthin is widely used in food, health care products, cosmetics [3–7] and medicine, which can prevent and treat many diseases [8,9]. In addition, astaxanthin is widely added to the feed of fishes such as salmon or trout in aquaculture, allowing these fishes to accumulate astaxanthin to obtain red fish [10] and improve the juvenile survival rate of fishes as a nutrient [11]. The production of natural astaxanthin mainly comes from Haematococcus pluvialis and Phaffia rhodozyma [12,13]. Among them, H. pluvialis, considered to have the highest content of natural astaxanthin at present, has 15∼30 mg/g dry cell weight (DCW) of astaxanthin, which accounts for more than 99 % of the total carotenoids produced by H. pluvialis [14]. Even so, P. rhodozyma has also many advantages over H. pluvialis [15], such as fast growth rate, high-density fermentation, less heavy metal, simple culture conditions and no need for illumination. In addition, the yeast cells are rich in proteins,
minerals and vitamins, which might be processed to form yeast extract for use in food and pharmaceutical industries [16]. Previous research has indicated that astaxanthin synthesized by P. rhodozyma accounts for up to 84 % of its total synthesized carotenoids [1]. However, the average content of astaxanthin in wild P. rhodozyma was only 0.2∼0.4 mg/g DCW [17], which seriously hindered the commercial development of astaxanthin. At present, there are many methods for strain modification, such as physicochemical mutagenesis, protoplast fusion and genetic engineering [18,19]. It has been reported that the astaxanthin concentration of P. rhodozyma was greatly increased, and the astaxanthin content reached 9.7 mg/g using traditional mutagenesis combined with metabolic engineering [20]. However, genetic engineering is time-consuming, costly and technically difficult. On the contrary, mutation breeding is more economical, convenient and easy to operate. The carotenoid content of Rhodotorula glutinis PTCC 5256 strain irradiated with 254 nm ultraviolet (UV) rays was 890 μg/g, which was 1.508 times higher than that of wild strain [21]. In addition, there are ethyl methyl sulfonate mutagenesis [22], nitrosoguanidine mutagenesis [23], and atmospheric and room
⁎ Corresponding author at: Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, China. E-mail addresses: [email protected] (Y. Zhuang), [email protected] (M.-J. Zhu).
https://doi.org/10.1016/j.procbio.2020.01.003 Received 19 September 2019; Received in revised form 17 December 2019; Accepted 7 January 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Yuan Zhuang, Gui-Li Jiang and Ming-Jun Zhu, Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.01.003
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2.2. Microbial strain and cultivation
temperature plasma (ARTP) mutagenesis. ARTP mutagenesis is a popular method for microbial mutagenesis bio-breeding in recent years. The mutagenesis of cells mediated by ARTP has many advantages, such as fast, safe, high mutation rate and efficient. It is widely used in plants, fungi and bacteria to improve the yield of the target product or enhance substrate tolerance of the strain [24,25]. The present study has reported that astaxanthin accumulation in Saccharomyces cerevisiae was promoted by metabolic engineering and ARTP mutagenesis, and the astaxanthin concentration obtained by fermentation in a 5 L fermenter was 217.9 mg/L [26]. In order to screen the mutants with high astaxanthin concentration, the screening agent should be added to the culture medium after mutagenesis. Namthip et al. found that the most efficient screening agent among several screening agents tested was diphenylamine, by which a high titer astaxanthin mutant was screened out [27]. Diphenylamine is an inhibitor of phytoene desaturase, which can inhibit the synthesis of astaxanthin [28]. The mutants had stronger resistance to the screening agent, showing a redder color than the original strains on the screening plate. The mutants screened were found to produce more astaxanthin than the original strain [27]. In order to reduce the cost of astaxanthin production by P. rhodozyma and improve astaxanthin concentration, some researchers have used low-cost raw materials to culture P. rhodozyma, such as eucalyptus hydrolysate [29], Yucca fillifera date juice [30], pretreated barley straw [31], sugarcane juice [32], mussel processing wastewater [33] and sweet sorghum juice [1]. Among them, sugarcane is one of the most productive crops in the world. In 2010, the yield of sugarcane in the world was a total of 428.11 million tons, and the residue sugarcane bagasse (SCB) can be used to produce the second generation of ethanol [34]. In addition, some scholars have studied that after effective physical, chemical or biological pretreatment, SCB can be converted into different sugars by enzymolysis, such as glucose, cellobiose, and xylose, which can be further used as carbon source for microorganism [35]. The hard cell wall of P. rhodozyma prevented astaxanthin from being degraded by external factors, such as light, heat, acid and oxygen [36], but it also prevented the extraction and utilization of astaxanthin. Currently, some processes have been developed to break yeast cells [37], such as chemical method [38], enzymic method [39] and mechanical method [40]. Although the enzymatic method is mild and can reduce the damage to astaxanthin, the extraction rate of astaxanthin is not ideal and it is time-consuming [41]. However, the ultrasonication can help disruption of cell wall, thus strengthening the diffusion of the target components in the cell [42]. In addition, the toxicity of organic solvents used in the extraction of astaxanthin, such as dimethyl sulfoxide and acetone [42,43], seriously limits the application of astaxanthin in some products. In summary, it is required to find a simple and effective method to break the wall and extract astaxanthin. The present study aims to screen mutant with high yield carotenoid by a novel combined mutagenesis method (ARTP and UV). Then, based on the mutant and sugarcane bagasse hydrolysate (SCBH) as carbon source, the optimal medium and culture conditions are determined for the accumulation of carotenoids. A green and efficient cell wall disruption process using ultrasonication followed by enzymatic hydrolysis is developed to improve astaxanthin extraction.
Phaffia rhodozyma Y119 was stored in the Fermentation Engineering Laboratory of School of Biological Science and Engineering, South China University of Technology. It was maintained on the agar slant of Yeast Extract Peptone Dextrose Medium (YPD) at 4 °C and subcultured for further research work. Seed culture spore suspension was prepared from actively growing yeast in YPD Erlenmeyer flask (250 mL), containing 22.5 mL sterilized medium (20 g glucose, 20 g peptone and 10 g yeast extract in 1000 mL distilled water), was inoculated with 10 % v/v of the spore suspension of P. rhodozyma and incubated at 22 °C at 220 rpm. 2.3. Physical mutagenesis by ARTP and UV P. rhodozyma Y119 was incubated at 22 °C for 72 h–96 h on the agar slant of YPD. A ring of cells from the actively growing slant was inoculated to Erlenmeyer flask containing 25 mL YPD and incubated at 22 °C to the logarithmic phase. Then, 2 mL yeast suspensions were taken out from Erlenmeyer flask and centrifuged at 4000 rpm for 5 min. The yeast cells were harvested and washed twice with deionized water. Normal saline was added to make the yeast with a cell concentration of about 106∼107 CFU/mL. Ten μL yeast suspensions diluted with normal saline were taken out and evenly coated on the iron plate in ARTP mutagenesis system with 120 W operating voltage, 2 mm working distance, 12 L/min helium gas flow and different mutagenic time (30, 40, 50, 60, 70 and 80 s). After mutagenesis, 1 mL sterilized normal saline was used for washing the cells on the iron plate down. And then, the yeast suspensions were diluted to suitable concentration, coated on the YPD plate and cultured in the 22 °C incubator for four days to calculate lethality rate. The time under 85 % lethality rate was determined as ARTP mutagenic time. After determining the action time of ARTP mutagenesis, the yeast suspensions after ARTP mutagenesis were diluted and coated on the YPD plate. Then, the irradiation at 20 cm directly below the UV lamp (30 W electric power) was conducted for different time, and the lethality rate was calculated. 2.4. Screening of mutant strains by diphenylamine One hundred μL yeast suspensions, which had been diluted to suitable concentration, were taken out for spreading on the YPD plate with different concentrations of diphenylamine (10, 20, 40, 60, 80, 120, 140 and 160 μmol/L). After incubating at 22 °C for 5∼6 days, the colony growth and color changes were observed to determine the optimal concentration of diphenylamine. After mutagenesis with UV and ARTP, the yeast suspensions were diluted and coated on the YPD plate with optimal concentration of diphenylamine. Large and red single colonies on the YPD plate were selected out and numbered one by one. Total selected strains were inoculated on the agar slant of YPD, after which they were incubated in YPD liquid medium for the determination of biomass and carotenoid concentration. Method for measuring biomass concentration has been reported in previous study [44]. Total carotenoids were extracted with acetone from the cell pellets which had been treated by the method of hot acid, and analyzed at 474 nm using a UV spectrophotometer (Unic, UV-2150), as described by Jiang [15]. In addition, the selected mutants were subcultured for 9 times to test the hereditary stability.
2. Materials and methods 2.1. Chemicals and reagents
2.5. Preparation of sugarcane bagasse hydrolysate (SCBH)
Diphenylamine was purchased from Shanghai Miriel Chemical Technology Co., Ltd. Acetone, ethanol and n-hexane were obtained from Tianjin Damao Chemical Reagent Factory. Cellulase (Cellic® Ctec2) was obtained from Novozymes (China) Investment Co. Ltd. SCB was donated by Guangzhou Sugarcane Research Institute. All other reagents were analytically grade and purchased from Sigma-Aldrich, USA.
The pretreatment of sugarcane bagasse (SCB) was carried out in a 250 mL Erlenmeyer flask, of which the conditions (pretreatment with potassium peroxymonosulfate and NaOH) have been studied in previous study [45]. Then, enzymolysis was conducted with 30 % substrate, 50 mM sodium citrate buffer (pH 4.8) and cellulase (20 FPU/g 2
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pretreated SCB) in a 250 mL Erlenmeyer flask for 6 days (55 °C, 150 rpm). After vacuum filtration followed by filtration with 0.45 μm membrane, the obtained sugar solution of SCBH was determined by high performance liquid chromatography (Waters 2414, Milford, LA, USA).
2.10. Extraction of astaxanthin In order to improve the extraction rate of astaxanthin, the yeast cells cultivated with SCBH as carbon source were broken by ultrasonication and cellulase. The order of ultrasonication and cellulase was changed and the optimum condition for cell wall disruption was found through an orthogonal experiment design using the SPSS software. After cell wall disruption, ethanol was used for extracting carotenoid, and then carotenoid ethanol solution was mixed with n-hexane for further extracting astaxanthin, as described in previous studies [4,46]. Briefly, 2 mL n-hexane was added to 2 mL carotenoid ethanol solution with a sufficient oscillation in the vortex mixer for 30 s, then mixed with 6 mL deionized water for 30 s and stood for 5 min. The mixture was centrifuged at 800 x g for 5 min to obtain the supernatant, of which the absorbance was measured at 468 nm using a spectrophotometer. Astaxanthin concentration in n-hexane was calculated by Eq. (1):
2.6. Optimization of nitrogen source with SCBH as carbon source The high concentration sugar solution obtained from SCB was diluted to a total sugar concentration of 30 g/L, including 20.22 g/L glucose, 6.71 g/L xylose and 3.05 g/L cellobiose. The fermentation medium contained SCBH as carbon source, 1.0 g/L KH2PO4 and 0.5 g/L MgSO4 (the same below). And KNO3, urea, (NH4)2SO4, peptone and yeast extract at the concentration of 5 g/L were added as nitrogen source, respectively. The sterilized fermentation medium was inoculated with the secondary seed solution (10 % v/v) and incubated at 22 °C, 200 rpm (the same below). The biomass and carotenoid concentration of yeast were measured by sampling every 24 h.
Astaxanthin concentration(mg/mL) =
A 468 × D× P× V2 ε×V1× pathlength
(1)
Where A468 is absorbance, P is molecular molar mass of astaxanthin 597 g/mol, pathlength is the thickness of the light absorption pool which is 1 cm, ε is the molar absorptivity of astaxanthin 125100 L/mol/cm, D is dilution ratio, V2 is n-hexane volume (mL), and V1 is ethanol volume (mL). Finally, the extraction rate of astaxanthin was calculated on the basis of the carotenoid concentration extracted by acetone along with hot acid method.
2.7. Optimization of C/N ratio with SCBH as carbon source The optimal nitrogen source in Section 2.6 was selected as the nitrogen source. Then, SCBH with sugar concentration of 30 g/L was used as carbon source, and the concentration of nitrogen source was adjusted to obtain different C/N ratios, which were 2, 3, 4, 5, 6 and 7, respectively. The yeast cells were incubated under these conditions, of which the biomass and carotenoid concentration were measured by sampling every 24 h.
2.11. Statistical analysis
2.8. Optimization of mixed nitrogen source with SCBH as carbon source
The experiments were performed in triplicate, and results are presented as the mean ± standard deviation (SD). Statistical evaluations were performed on the basis of confidence intervals using the Tukey-b test. A value of p < 0.05 was considered significant. All statistical analysis were carried out using the SPSS software (Ver. 17.0, SPSS Inc., Chicago, IL, USA).
Yeast extract and urea were mixed at 6: 0, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5, 0: 6 (w/w) with a total concentration of 6 g/L. Other components of fermentation medium contained 1.0 g/L KH2PO4, 0.5 g/L MgSO4 and SCBH with sugar concentration of 30 g/L. The yeast cells were incubated under these conditions, of which the biomass and carotenoid concentration were measured by sampling every 24 h.
3. Results and discussion 3.1. Strain improvement by ARTP and UV
2.9. Effects of different carbon sources on cell growth and carotenoid accumulation
As shown in Fig. 1A, when ARTP mutagenic time was 50 s, the lethality rate of yeast was 84.2 %. It has been reported that the positive mutagenesis rate of yeast cells is higher when lethality rate by ARTP mutagenesis is close to 85 % [47]. Therefore, 50 s was selected as the mutagenic time of ARTP. In addition, when UV irradiation is carried out, high lethality rate probably causes negative mutagenesis of the strain. Thus, the time of UV irradiation for lethality rate of 70∼80 % is generally selected as mutagenic time [48]. When the yeast cells on the plate were irradiated by UV for 40 s, the lethality rate was 70.3 %
To compare the effects of SCBH and other carbon sources (30 g/L glucose, mixed carbon source of 20.22 g/L glucose, 6.71 g/L xylose and 3.05 g/L cellobiose) on carotenoid accumulation of yeast, the secondary seed solution (10 % v/v) was inoculated to three different sterilized fermentation media (22.5 mL), each of which contained one of the above three carbon sources with the same sugar concentration, respectively. The biomass and carotenoid concentration of yeast were measured by sampling every 24 h.
Fig. 1. Lethality rate of P. rhodozyma under different mutagenic time. (A) ARTP mutagenic time. (B) UV mutagenic time. 3
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Fig. 2. The growth and hereditary stability of mutant strain obtained by ARTP and UV mutagenesis. (A) The comparison of biomass and carotenoid accumulation between mutant strain and original strain. (B) Hereditary stability of mutant strain.
and urea (8.08 mg/g). Therefore, compared with other nitrogen sources tested, yeast extract is more conducive to the growth of yeast and the accumulation of carotenoids, which was mainly attributed to the abundant nutrients of yeast extract [16].
(Fig. 1B), which was in the range of the best lethality rate. Thus, 40 s was selected as the optimal UV mutagenic time. Briefly, the conditions for the combined mutagenesis were determined as follows: first, the cells were mutagenized by ARTP for 50 s, and then irradiated by UV for 40 s.
3.4. Effects of different C/N ratios on cell growth and carotenoid accumulation
3.2. Screening and hereditary stability of P. rhodozyma mutant When diphenylamine concentration gradually increased from 10 μmol/L to 100 μmol/L, the colonies on the screening plate gradually changed from red to light red, and even a few white colonies appeared. This indicated that the higher the concentration of diphenylamine, the stronger the inhibitory effect on the synthesis of carotenoids, which was consistent with previous research reports [27,28]. When diphenylamine concentration reached 120 μmol/L, only white colonies appeared on the plate. Therefore, the optimal concentration of diphenylamine selected in this experiment was 120 μmol/L. After repeated mutagenesis and screening, a light red colony was found, which was immediately preserved and its biomass and carotenoid concentration were determined. As shown in Fig. 2A, the biomass concentration, carotenoid concentration and carotenoid content of the original strain were 10.35 g/L, 45.69 mg/L and 4.41 mg/g, respectively, and the mutant strain was 10.1 g/L, 54.38 mg/L and 5.38 mg/g, respectively. In this paper, the mutant strain was numbered Y1. Although the biomass concentration of Y1 was slightly lower than that of the original strain, its carotenoid concentration and content were 19.02 % and 22.00 % higher than that of the original strain, respectively. After being subcultured for 9 times (Fig. 2B), the carotenoid concentration and content of the mutant strain were 54.97 mg/L and 5.33 mg/g, respectively, which were 20.31 % and 20.86 % higher than the original strain, indicating that the mutant strain had excellent hereditary stability. In a previous study, P. rhodozyma mutant by ARTP mutagenesis had good hereditary stability, whose carotenoid concentration was 1.67 mg/L and 40 % higher than the original strain [49]. Through ARTP mutagenesis, sodium nitrite mutagenesis, UV mutagenesis followed by UV-sodium nitrite mutagenesis, Liu [50] obtained a red yeast with carotenoid concentration of 14.47 mg/L, which was 1.67 times that of the original strain.
Proper C/N ratio can maintain the fast growth of yeast cells and the synthesis of carotenoid, only in this way can the maximum carotenoid concentration be guaranteed. The effects of different C/N ratios on the growth and carotenoid accumulation of P. rhodozyma mutant is shown in Fig. 4. When the C/N ratio was 2, the biomass concentration reached a maximum of 11.5 g/L. With the increase of C/N ratio, the biomass concentration showed a downward trend. As stated in earlier literature reports, high carbon loadings inhibited the growth of P. rhodozyma by reducing the amount of available nitrogen [51]. When the C/N ratio was 5, the carotenoid concentration reached the maximum value of 92.19 mg/L. With the increase of the C/N ratio from 2 to 7, the carotenoid concentration first increased and then decreased with a C/N ratio of 5 as the dividing line, which was consistent with previous research reports. A report proved that a C/N ratio below 5 has a negative effect on the carotenoid production of P. rhodozyma, suggesting that abundant nitrogen promotes cell growth but inhibits the activity of enzyme that converts β-carotene to astaxanthin, and in contrast, a high C/N ratio affects cell growth, thus affecting the carotenoid synthesis [51]. After comprehensive consideration of biomass and carotenoid concentration, the best C/N ratio was 5, that is, the best addition amount of yeast extract was 6 g/L. 3.5. Effects of mixed nitrogen source on cell growth and carotenoid accumulation Compared with the cost of yeast extract and peptone, urea is cheaper. In addition, P. rhodozyma showed high carotenoid content with urea as nitrogen source among all the tested nitrogen sources. Therefore, P rhodozyma was cultured by combining yeast extract with urea to obtain the ideal carotenoid concentration at a lower cost. The effects of mixed nitrogen source on the growth and carotenoid accumulation of P. rhodozyma mutant are shown in Fig. 5. As can be seen from the results in Fig. 5B, when the ratio of yeast extract to urea was 6: 0 (6 g/L of yeast extract), the yeast cells had the highest carotenoid concentration. However, when the ratio was 6: 0, 5: 1 and 3: 1, the difference in carotenoid concentration of P. rhodozyma was not significant (p > 0.05). Therefore, from the perspective of cost, mixed nitrogen source with a ratio of 3:1 between yeast extract and urea could be selected to culture the yeast, and at this time, the carotenoid concentration and content were 81.44 mg/L and 6.46 mg/g, respectively. There is a study reporting that compared with yeast extract and ammonium sulfate as mixed nitrogen source, yeast extract and
3.3. Effects of different nitrogen sources on cell growth and carotenoid accumulation In this study, five nitrogen sources were selected to explore the effects of different nitrogen sources on the growth and carotenoid accumulation of P. rhodozyma mutant. The experimental results are shown in Fig. 3. It can be seen from the figure that the yeast extract was most conducive to the growth of the yeast (9.45 g/L), followed by peptone and ammonium sulfate. When yeast extract was used as nitrogen source, the highest carotenoid concentration was 82.57 mg/L, and the highest carotenoid content was 8.73 mg/g, followed by peptone (8.67 mg/g) 4
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Fig. 3. Effects of different nitrogen sources on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content.
Fig. 4. Effects of different C/N ratios on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content. 5
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Fig. 5. Effects of mixed nitrogen source on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content.
promoting astaxanthin accumulation. However, whether this is also related to the cellobiose in SCBH needs to be further studied. Overall, the optimized SCBH medium shows obvious advantage over glucose medium. Compared with glucose medium, SCBH medium can promote yeast cells to accumulate more carotenoids without significantly inhibiting the growth of yeast cells. Similarly, it's been reported that the astaxanthin concentration and biomass concentration of yeast, cultivated with SCBH which was pretreated with ammonia, were 2.56 mg/L and 7.16 mg/L, respectively [55]. The carotenoid concentration of yeast was 60.75 mg/L in fermentation medium (pH 5.0) containing 30 g/L sugarcane molasses and 5 g/L yeast extract [56]. P. rhodozyma was cultivated in a 2 L bioreactor with sweet sorghum juice, of which the biomass and astaxanthin concentration were 29 g/L and 65.4 mg/L, respectively [1]. The biomass and astaxanthin concentration of P. rhodozyma cultivated in fruit and vegetable waste medium were comparable to those cultivated in the synthetic medium, reaching 5.3 g/L and 355 μg/g, respectively [57]. In addition, the study on the feasibility of producing astaxanthin by P. rhodozyma cultivated in the pretreated whey medium has been reported [58]. The above examples show that it has certain economic and ecological value to produce astaxanthin by cultivating P. rhodozyma using agro-industrial waste.
urea as mixed nitrogen source had more significant promotion effect on astaxanthin accumulation [1].
3.6. Effects of different carbon sources on cell growth and carotenoid accumulation Glucose, xylose and cellobiose as carbon sources for the culture of yeast have high cost, and only for use in the laboratory. In this study, the carotenoid concentration and content of P. rhodozyma cultured with three different carbon sources were compared. Three different carbon sources were glucose, SCBH, and mixed carbon source (containing glucose, xylose and cellobiose), respectively. Under the condition of guaranteeing the carotenoid concentration, it is expected to replace the pure sugar with the cheap SCBH to cultivate P. rhodozyma. As shown in Fig. 6, when SCBH was used as carbon source, the biomass concentration of yeast was comparable to that of glucose as carbon source, possibly because SCBH had no many toxic substances produced to inhibit the growth of yeast after potassium peroxymonosulfate pretreatment and enzymolysis. As is known to all, lignocellulose feedstocks after pretreatment will produce sugar solution, and depending on the pretreatment method, they will generate various by-products, such as furfural and phenolic compounds [52,53], which can affect yeast cultivation and product synthesis. It has been reported that P. rhodozyma was cultured to produce astaxanthin with detoxified sweet sorghum bagasse hydrolysate, because the hydrolysate without detoxification contained phenolic compounds that inhibited the yeast cultivation [54]. From the perspective of carotenoid concentration, the carotenoid concentration of yeast cultivated with SCBH as carbon source was 88.57 mg/L and mixed carbon source was 92.07 mg/L. There was no significant difference (p > 0.05) between the two carbon sources, but both were higher than 65.19 mg/L when 30 g/L glucose was used as carbon source. This result was mainly attributed to the xylose contained in both mixed carbon source and SCBH. Previous reports [29,54] have shown that xylose is more favorable than glucose in
3.7. Extraction of astaxanthin using enzymolysis followed by ultrasonication Both ultrasonication and enzymolysis are green methods of cell wall disruption, combined to obtain an environmental-friendly and efficient method of cell wall disruption. Constant parameters in this orthogonal experiment were enzymolysis time 6 h, enzymolysis temperature 50 °C, the ultrasonic power 361 W, 2 s on and 5 s off. The results are shown in Table 1. According to the range analysis, the order of influence of each factor on the extraction rate of astaxanthin after enzymolysis and enzymolysis followed by ultrasonication was as follows: cell concentration > 6
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Fig. 6. Effects of different carbon sources on the growth and carotenoid accumulation of P. rhodozyma. (A) Biomass concentration. (B) Carotenoid concentration. (C) Carotenoid content.
enzyme amount > ultrasonic time. According to the results shown in Table 1, the highest Ⅰ (67.51 %) and Ⅱ (90.75 %) were observed in experiment No.6 under the optimal conditions: cell concentration, 0.5 %; enzyme amount, 64.6 FPU/g; enzymolysis time 6 h; ultrasonic time, 45 min. A previous study also showed that P. rhodozyma treated with βglucanase followed by ultrasonication can produce up to 435.71 ± 6.55 μg of astaxanthin/g cell, which was about twice the content of astaxanthin obtained by organic solvent extraction [59].
Table 1 The results of orthogonal design for the optimization of astaxanthin extraction using enzymolysis followed by ultrasonication. No.
Cell concentration (%)
Enzyme amount (FPU/g)
Ultrasonic time (min)
Ⅰ (%)
Ⅱ (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K¯1
1 1 2 1.5 1.5 0.5 0.5 0.5 1.5 0.5 2 2 1 1.5 2 1 60.24 47.28 40.88 39.02 21.22 84 72.43 65.27 56.04 27.96
72.20 79.80 79.80 72.20 79.80 64.60 79.80 57.00 57.00 72.20 72.20 57.00 57.00 64.60 64.60 64.60 44.66 47.18 45.26 42.14 5.04 68.58 72.07 68.78 68.32 3.75
45 25 45 15 35 45 15 25 45 35 25 35 15 25 15 35 46.32 42.86 42.95 47.12 4.26 71.28 69.44 67.55 69.46 3.73
45.58 49.40 29.87 47.43 31.72 67.51 57.57 55.52 45.52 60.36 27.66 31.6 46.01 38.86 34.25 48.11
66.69 77.78 54.92 72.55 57.59 90.75 83.01 80.47 65.47 81.77 54.10 56.97 71.39 65.47 58.16 73.88
K2 K¯3 K4 RⅠ K5 K6 K7 K8 RⅡ
3.8. Extraction of astaxanthin using ultrasonication followed by enzymolysis According to the parameters obtained from the above orthogonal experiment (cell concentration 0.5 %, enzymolysis temperature 50 °C, ultrasonic power 361 W, 2 s on, 5 s off), the order of ultrasonication and enzymolysis was changed. According to the values of K¯1, K¯2 , and K3 in Table 2, after 60 min of ultrasonication alone, the extraction rate of astaxanthin reached the maximum, which was 54.44 %. When the extraction rate of astaxanthin reached about 90 % (No.6), compared with the method of enzymolysis followed by ultrasonication (No.6 of Table 1), the amount of cellulase could be reduced from 64.6 FPU/g to 51.3 FPU/g by increasing the enzymolysis time from 6 h to 10 h and the ultrasonic time from 45 min to 60 min with the method of ultrasonication followed by enzymolysis. According to the range analysis, the order of influence of each factor on the extraction rate of astaxanthin using ultrasonication followed by enzymolysis was as follows: enzyme amount > enzymolysis time > ultrasonic time. According to the values of K¯ 4 , K5 , and K¯ 6 , the best combination of ultrasonication combined with enzymolysis was as follows: ultrasonic time, 60 min; enzymolysis time, 10 h; enzyme amount, 64.6 FPU/g. A confirmatory experiment performed under the optimal conditions, the results showed that the extraction rate of astaxanthin reached 96.01 %. There is a previous study [60] reporting that different cell
Ⅰ Extraction rate of astaxanthin using enzymolysis. Ⅱ Extraction rate of astaxanthin using enzymolysis followed by ultrasonication. K¯1, K2 , K¯3 and K 4 come from the results of Ⅰ. K5 , K 6 , K7 and K 8 come from the results of Ⅱ. RⅠ is the range of K¯1, K2 , K¯3 and K 4 ; RⅡ is the range of K5 , K 6 , K7 and K 8 .
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Author statement
Table 2 The results of orthogonal design for the optimization of astaxanthin extraction using ultrasonication followed by enzymolysis. No.
Ultrasonic time (min)
Enzymolysis time (h)
Enzyme amount (FPU/g)
Ⅲ (%)
Ⅳ (%)
1 2 3 4 5 6 7 8 9 K¯1
60 30 60 30 45 60 45 45 30 50.76 53.46 54.44 3.68 87.93 83.54 89.36 5.82
14 10 6 14 14 10 10 6 6 52.89 52.89 52.89 0 81.91 90.99 87.97 9.08
64.60 39.90 39.90 51.30 39.90 51.30 64.60 51.30 64.60 52.89 52.89 52.89 0 84.2 82.41 94.25 11.84
54.44 50.76 54.44 50.76 53.46 54.44 53.46 53.46 50.76
95.95 87.53 82.05 84.94 83.03 90.08 95.36 72.22 91.45
K2 K¯3 RⅢ K4 K5 K6 RⅣ
The study was designed by Gui-Li Jiang and Ming-Jun Zhu. The experiments were executed by Gui-Li Jiang. Data analysis and data interpretation were executed by Yuan Zhuang and Ming-Jun Zhu. All the authors contributed in writing the manuscript and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgments The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China, China [grant no. 51878291] and Guangzhou Science and Technology Program, China [grant no. 2014 Y2 -00515]. References
Ⅲ Extraction rate of astaxanthin using ultrasonication. Ⅳ Extraction rate of astaxanthin using ultrasonication followed by enzymolysis. K¯1, K2 , and K¯3 come from the results of Ⅲ. K 4 , K5 , and K 6 , come from the results of Ⅳ. RⅢ is the range of K¯1, K2 , and K¯3 . RⅣ is the range ofK 4 , K5 , and K 6 .
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disruption methods (including abrasion with celite, glass pearls in vortex agitator, ultrasonic waves, sodium carbonate and dimethyl sulfoxide) were tested to obtain astaxanthin, among which the dimethyl sulfoxide was found to be the most efficient method. Although the method of extracting astaxanthin by organic solvent has high efficiency and low cost, the application of astaxanthin is severely restricted due to safety problems [60]. Monks et al. [61] evaluated the efficiency of extracting carotenoids with the methods of chemical, enzymatic and ultrasound-assisted treatments, showing that the enzymolysis was the most effective method, and the methods of ultrasound-assisted cell disruption alone or combined were inefficient, especially ultrasound combined with enzymolysis simultaneously. However, the method with ultrasonication followed by enzymolysis was not tried [61]. In addition, recent biorefinery technologies, such as ionic liquids [62] and supercritical solvent extraction [63], have high extraction efficiency of astaxanthin, some of which can even over 99 % [64]. However, both have high costs, and what’s more, the ionic liquids are toxic [41]. In conclusion, the method of ultrasonication followed by enzymolysis was used to cell disruption, and ethanol was used for extraction. Compared with the other methods mentioned above, although the cost of enzyme was a bit high, the method in this study was green and efficient, which was conducive to the downstream application of astaxanthin.
4. Conclusion In this study, ARTP and UV was successfully used for the mutagenesis of P. rhodozyma, and a mutant Y1 was obtained from the diphenylamine selection plate, whose astaxanthin content was significantly improved. Compared with glucose, carotenoid concentration of mutant Y1 with SCBH as carbon source was significantly improved. A green and simple astaxanthin extraction method was well established, with which ultrasonication and cellulase were used to break cell wall and ethanol was used to extract astaxanthin, leading to a satisfactory astaxanthin extraction rate. The present study plays solid foundation for the industrial production and application of astaxanthin.
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