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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
CAROTENE PRODUCTION FROM AGROINDUSTRIAL WASTES BY Arthrobacter globiformis IN SHAKE-FLASK CULTURE a
b
a
Yu-Gui Zhai , Mei Han , Wei-Guo Zhang & He Qian
a
a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University , Wuxi , JiangSu Province , P. R. China b
State Key Laboratory of Dairy Biotechnology , Technology Center Bright Dairy & Food Co., Ltd , Shanghai , P. R. China Accepted author version posted online: 06 Aug 2013.Published online: 09 Dec 2013.
To cite this article: Yu-Gui Zhai , Mei Han , Wei-Guo Zhang & He Qian (2014) CAROTENE PRODUCTION FROM AGRO-INDUSTRIAL WASTES BY Arthrobacter globiformis IN SHAKE-FLASK CULTURE, Preparative Biochemistry and Biotechnology, 44:4, 355-369, DOI: 10.1080/10826068.2013.829498 To link to this article: http://dx.doi.org/10.1080/10826068.2013.829498
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Preparative Biochemistry & Biotechnology, 44:355–369, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.829498
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CAROTENE PRODUCTION FROM AGRO-INDUSTRIAL WASTES BY Arthrobacter globiformis IN SHAKE-FLASK CULTURE
Yu-Gui Zhai,1 Mei Han,2 Wei-Guo Zhang,1 and He Qian1 1 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University, Wuxi, JiangSu Province, P. R. China 2 State Key Laboratory of Dairy Biotechnology, Technology Center Bright Dairy & Food Co., Ltd, Shanghai, P. R. China
& Industrial waste substrates, sugarcane molasses, and corn steep liquor were used for production of carotenes by Arthrobacter globiformis in this study. At the first stage, a one-factor-at-a-time approach was used for optimization of different media components such as carbon, nitrogen, MgSO4 7H2O, and KH2PO4, as well as pH, temperature, liquid medium volume, and inoculums level. The response surface method was further applied to determination of optimum values of process variables for maximum carotenes concentration. Results showed that the optimum combination for carotenes formation was as follows (g=L): sugarcane molasses, 40.00; corn steep liquor, 50.00; MgSO4 7H2O, 0.75; KH2PO4, 1.00. The maximum carotene concentration of 1.19 0.02 mg=g dry biomass, about 113% of 1.05 0.02 mg=g dry biomass growing in basal medium, was demonstrated by confirmatory experiments to be the optimum in liquid medium at 100 rpm, 30C, initial pH of 7.5, and cultivation for 60 hr. In a second stage, detailed studies showed about 1.64-fold and 1.43-fold increase in carotene concentration (mg=g dry biomass) in the presence of addition of ethanol (4%, v=v) and addition of hydrogen peroxide (4%, v=v) at 40 hr, and 32 hr in liquid medium, separately. Keywords Arthrobacter globiformis, carotenes, industrial waste substrates, oxidative stress, response surface method
INTRODUCTION Carotenes are naturally occurring lipid-soluble pigments, with the majority being C40 terpenoids. Carotenes have been proven to play important roles in human health as precursors of vitamin A, scavengers of active oxygen, antitumor substances, and enhancers in vitro antibody production; therefore, they are widely applied in food, medical, pharmaceutical, and Address correspondence to Wei-Guo Zhang, The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University, 1800# Lihu Road, Wuxi-214122, JiangSu Province, P. R. China. E-mail: [email protected]
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cosmetic industries as colorants and functional components.[1] Huge commercial demand for natural carotenes has focused attention on developing of suitable biotechnological techniques, including use of industrial wastes as carbon and nitrogen sources. Commercial production by plant extraction does not satisfy the market need because of its low content in the plants.[2] Chemical synthesis has many disadvantages, such as low yield, instability of product quality, and high cost.[3] Thus, interest in production of carotenes by microbial fermentation has recently increased. Production of carotenes using microbes is highly efficient because they are easily manipulated in the processing schemes, including the bacteria Flavobacterium, the algae Dunaliella and Haematococcus, the fungus Blakeslea trispora, and yeast of the genera Phaffia, Rhodotorula, and Sporidiobolus.[4–10] Increasing attention has been applied to finding suitable natural sources including biotechnological processes in the past several decades. In order to improve the yield of carotenes and subsequently decrease the cost, diverse studies have been performed by optimizing the culture conditions, including nutritional and physical factors. Factors such as concentration of carbon and nitrogen sources, metal ions, pH, aeration, temperature, and oxidative stress have a major influence on cell growth and yield of carotenes.[10,11] Numerous cheap industrial by-products have been considered as potential carbon and nitrogen sources for biotechnological production of carotenes.[7,12–16] Roukas and colleagues investigated the effect of aeration rate and agitation speed on b-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentator and optimization of the production of the pigment in a bubble reactor. Maximum b-carotene concentration (360.2 mg=L) was obtained in culture grown in a molasses solution containing 5% (w=v) sugar supplemented with linoleic acid (37.59 g=L), kerosene (39.11 g=L), and antioxidant (1.0 g=L).[7] Varzakakou and colleagues performed an experiment of identification for the carotenes in Blakeslea trispora during pigment production from deproteinized hydrolyzed whey supplement with plant oils. The composition of carotenes was 60.1% b-carotene, 32.5% c-carotene, and 7.4% lycopene.[12] Thus, it is essential to carry out the research on carotenes accumulation in A. globiformis in order to make full use of industrial waste substrates and thereby reduce environmental contamination. It is believed that the overall yield of carotenes is directly related to the total biomass yield, so keeping both high growth rates and high flow carbon efficiency for carotenes by optimal cultivation conditions is essential in order to achieve the maximal pigment productivity[17] The one-factor-ata-time (OFAT) method has been widely applied in many fields such as medium optimization, formulation of multicomponent designs, and others.[18,19] The response surface method (RSM) has shown to be an efficient and effective approach to systematic investigation of several target factors. RSM presents
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the attractive advantage that it needs much fewer trials when compared with the other statistical designs.[13,20,21] In the present work, a combination of RSM and OFAT was applied to optimize the concentrations of components in fermentative medium for carotenes production by A. globiformis. Several agents have been used to induce oxidative stress, including duroquinone, H2O2, and ethanol. Oxidative stress is known to enhance the biosyhthesis of carotenes in some red yeast, and carotenes are also associated with an antioxidant function.[22,23] The aim of the work presented here is to develop a moderate two-stage fermentation process for carotenes production by A. globiformis using industrial waste substrates. In the first stage, the influences of several factors such as carbon source, nitrogen source, sulfate, phosphate, temperature, pH, liquid medium volume, and inoculums level on the production of carotenes were studied by OFAT and subsequently by RSM. In the second stage, effects of ethanol and hydrogen peroxide on biomass and production of carotenes were investigated.
MATERIALS AND METHODS Microorganism and Culture Conditions In this study the strain A. globiformis was used, and it was conserved in glucose–peptone–agar in darkness at 4 C. Cells were grown under aerobic conditions in 500-m baffled conical flasks with 50 mL of the basal medium (initial pH of 7.5) containing the following (g=L): sugarcane (SCM), 30; corn steep liquor (CSL), 30; MgSO4 7H2O, 0.5; KH2PO4, 1. Cultivations were carried out at 30 C on a rotary shaker at 100 rpm for 60 hr. The cells were harvested by centrifugation (6000 rpm for 10 min) and stored at 20 C under a nitrogen atmosphere before use. SCM and CSL were obtained from Anhui BBCA International Co., Ltd., China. The composition of SCM was 57% (w=w) sucrose, 7% (w=w) ash, and 18% (w=w) total nitrogen. SCM also constitutes a valuable source of growth substrates, such as pantothenic acid, inositol, trace elements, and, to a lesser extent, biotin. The composition of CSL was 23% (w=w) total nitrogen, 9% (w=w) ash, and 34% (w=w) lactose or total sugars. This also includes amine acid, and some metal ions such as iron, calcium, zinc, and potassium.
Measurements of Dry Biomass and Carotene Concentration Dry biomass (g=L) and extraction of carotenes were carried out as reported earlier.[9] For all experiments, cell growth was measured as optical
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density (OD) at 600 nm, and then it was converted into dry biomass based on a standard curve of OD against dry biomass. The medium without SCM and CSL was used as the blank and as diluting solution.[22] The intensity of color of the supernatant was measured at 490 nm with a Mapada 6100 spectrophotometer. The concentration of carotenes presented in the sample was then determined by comparison with a standard calibration curve prepared from pure all-trans-b-carotene (Darmstadt, Germany).[14] The major carotenes were quantified by a Hitachi L-2000 high-performance liquid chromatopgraph (HPLC) equipped with a photodiode array detector with a C18 column (25 mm 4.6 mm; 4.6 mm particle size; Agilent, USA). Pigments were eluted with methanol at a flux rate of 1.00 mL=min and 30 C. Under these conditions, OH-spirilloxanthin, spirilloxanthin, anhydrorhodovibrin, and b-carotene were eluted within 5.4, 7.6, 10.1, and 13.4 min, respectively. Since OH-spirilloxanthin, spirilloxanthin, and anhydrorhodovibrin were commercially unavailable, these carotenes were identified by their absorption maxima (464, 494, 524 nm), (466, 495, 527 nm), and (454, 480, 513 nm), respectively, as indicated previously.[24] They were quantified against peak area calibrations calculated from standard curves of b-carotene with appropriate correction factors at 490 nm. For all the analyses, samples were measured in triplicate for each experiment. Experimental Design and Data Analysis In the first stage, effects of eight factors on dry biomass and carotene concentration were investigated using OFAT: SCM, CSL, MgSO4 7H2O, KH2PO4, initial pH, temperature, liquid medium volume, and inoculums level. Then RSM was used to maximize the carotenes production. A fractional factorial 27-point design involving a three-level, fourvariable array was employed. Coded values of variables, namely, concentrations (g=L) of SCM, CSL, MgSO4 7H2O, and KH2PO4, were applied. The basic equation for carotene concentration as a function of the already-mentioned four independent variables involved a secondorder polynomial of this form: Carotene concentration ¼ A0 þ A 1 X1 þ A2 X2 þ A 3 X3 þ A 4 X4 þ A 5 X1 X2 þ A 6 X1 X3 þ A 7 X1 X4 þ A 8 X2 X3 þ A9 X2 X4 þ A 10 X3 X4 þ A 11 X21 þ A12 X22 þ A13 X23 þ A 14 X24
ð1Þ
where X1 is the concentration of SCM, X2 the concentration of CSL, X3 the concentration of MgSO4 7H2O, X4 the concentration of KH2PO4, A0 is
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a constant, A1, A2, A3, and A4 are linear coefficients, A5, A6, A7, A8, A9, and A10 are cross-product coefficients, and A11, A12, A13, and A14 are quadratic coefficients. The commercial software Design Expert v6.06 (ATAT-EASE, Inc., Minneapolis, MN) was used for statistical analyses, regression analyses, and the significant difference (P 0.05) was assessed.[25] In flask experiments three parallel cultivations were carried out.
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Addition of Ethanol and Hydrogen Peroxide in Liquid Medium Several literature reports have revealed that adding stimulators can enhance the carotenes production by the yeast Dioszegia, the fungi Rhodotorula glutinis, and Blakeslea trispora grown under normal conditions.[22,23,27] Thus, we investigated the effects of two stimulators (ethanol and hydrogen peroxide) on biomass and carotenes production in the second stage. All experiments were done in triplicate.
RESULTS AND DISCUSSION Effects of SCM and CSL on Dry Biomass and Carotene Concentration The results are given as the units of dry biomass (g=L) and carotene concentration (mg=g dry biomass) at the end of growth. Figures 1a and 1b showed that carotenes formation and organism growth increased with increasing initial concentrations of SCM and CSL up to 40 and 50 g=L, respectively. The maximum dry biomass and carotene concentration were 26.11 0.80 g=L and 1.01 0.03 mg=g dry biomass, separately.
Effects of Metals Ions on Dry Biomass and Carotene Concentration Metals ions are involved in all aspects of microbial life. Metal ions can specially cause activation or inhibition mechanisms for specific carotenogenic enzymes, in particular, for specific desaturases involved in carotenes biosynthesis.[1] KH2PO4 is essential in compositions of many macromolecular substances (DNA, RNA, ATP, etc.). It also can maintain the pH value and keep suitable cytoplasmic osmotic pressure.[28] Effects of MgSO4 7H2O and KH2PO4 were assessed, and the best results, presented in Figures 1c and, were obtained at the concentrations of 0.5 or 1.0 g=L, and 1.0 g=L, separately.
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FIGURE 1 Effect of some parameters on carotene concentration and dry biomass: (a) sugarcane molasses; (b) corn steep liquor; (c) MgSO4 7H2O; (d) KH2PO4; (e) temperature; (f) pH; (g) liquid medium volume; (h) inoculums level.
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Effects of Temperature and pH on Dry Biomass and Carotene Concentration Temperature is another important factor affecting the performance of cells and product formation. It was reported to control the concentrations of enzymes involved in carotenes production, and changes in enzyme concentrations ultimately control carotenes levels in microorganism.[28] Biomass and carotenes formation increased with increasing temperature up to 30 C and decreased sharply above 30 C (shown in Figure 1e). The initial pH significantly affected the growth and total carotenes production as shown in Figure 1f. As the pH increased, biomass and carotenes production increased and reached a maximum level at pH 7.5 with a drop off at higher pH values. Effects of Liquid Medium Volume and Inoculums Level on Dry Biomass and Carotene Concentration Arthrobacter globiformils is an aerobic microorganism and therefore requires the provision of oxygen, so aeration is extremely important for the successful progress of fermentation. Aeration could beneficial to the growth and performance of microbial cells by improving the mass transfer characteristics with respect to substrate, product, and oxygen.[28] The best result was obtained with 30 mL of liquid medium (presented in Figure 1g). Carotenes accumulation was restricted largely in liquid medium volume lower than 30 mL (data not shown). Inoculums level is another factor affecting the growth. Low inoculums level will restrict the normal growth, and high inoculums level will make cells step into the stationary stage earlier and then reduce the accumulation of the secondary metabolites. The optimized condition was found to be 10% (v=v=) inoculums (presented in Figure 1h). Finally, dry biomass and carotene concentration reached up to 29.1 1.21 g=L and 1.05 0.02 mg=g dry biomass. Aliquots of fermentation broth removed from the conical flasks at 4-hr intervals (from 0 to 72 hr) were used for the determination of OD and carotene concentration.[11] As shown in Figure 2, carotenes were synthesized in two steps. In the first step (0 to 40 hr of liquid fermentation), called the ‘‘growth phase,’’ a rapid increase of biomass and a small amount of the pigment were obtained, and in the second step (40 to 60 hr of liquid fermentation), called the ‘‘production phase,’’ the biomass remained constant and the biosynthesis of carotenes was carried out.[28] Based on the results of OFAT, the maximum carotene concentration of 1.05 0.02 mg=g dry biomass was obtained with dry biomass of 29.1 1.21 g=L; however, maximum dry biomass of 32.24 1.32 g=L was
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FIGURE 2 Effect of fermentation time on carotene concentration and dry biomass.
obtained with a lower amount of carotene concentration of 0.89 0.02 mg=g dry biomass. OPTIMIZATION OF FERMENTATION CONDITIONS WITH RSM Table 1 presents the coded and real values. RSM was used to maximize the carotenes production, and the results are listed in Table 2. The data in Table 2 indicate that there was a wide variation from 1.21 0.03 mg=g dry biomass to 0.73 0.03 mg=g dry biomass of carotene concentration in the 27 trials. This variation reflects the importance of medium optimization to attain higher carotenes yield. According to anaylsis of variance (ANOVA) provided in Table 3, the results showed that the model F value 25.32 for carotenes production implied that the model was significant (P 0.05) with R2 0.967 indicating that model was in good agreement TABLE 1
Coded and Real Values in the Experimental Design Levels
Symbol
Independent Variables
1
0
þ1
Units
X1 X2 X3 X4
Sugarcane molasses Corn steep liquor MgSO4 7H2O KH2PO4
35 45 0.5 0.8
40 50 0.8 1.0
45 55 1.1 1.2
g=L g=L g=L g=L
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Carotene Production From Agro-Industrial Wastes TABLE 2
Design and Results of Response Surface Method
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Total Carotene Concentration (mg=g Dry Biomass) Standard Order
X1
X2
X3
X4
Actual Values
Predicated Values
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0
1 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0
0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0
0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0
0.73 0.03 0.85 0.04 0.81 0.04 0.92 0.03 0.75 0.04 0.96 0.02 0.86 0.02 0.88 0.03 0.79 0.02 0.95 0.04 0.89 0.02 0.95 0.03 0.73 0.04 0.70 0.03 0.81 0.01 0.99 0.04 0.71 0.04 0.81 0.02 0.74 0.01 0.95 0.03 0.86 0.04 0.83 0.02 0.95 0.03 0.95 0.04 1.21 0.03 1.20 0.04 1.19 0.05
0.72 0.05 0.86 0.05 0.80 0.05 0.92 0.05 0.75 0.05 0.96 0.05 0.85 0.05 0.87 0.05 0.80 0.05 0.93 0.05 0.90 0.05 0.95 0.05 0.72 0.05 0.70 0.05 0.81 0.05 0.98 0.05 0.74 0.05 0.79 0.05 0.70 0.05 0.92 0.05 0.84 0.05 0.82 0.05 0.94 0.05 0.95 0.05 1.20 0.05 1.20 0.05 1.20 0.05
with experimental data, and allowed the construction of the stereogram (presented in Figure 3). Nonsignificant lack of fit (P > 0.05) further validated the model. The regression equation for carotene concentration (mg=g dry biomass) as a function of four media components was
TABLE 3 Source Model Residual Lack of fit Pure error Total
Analysis of Variance for Carotene Concentration Sum of Squares
Degrees of Freedom
Mean Squares
F Value
P>F
0.49 0.016 0.016 2.00E-0.04 0.50
12 14 10 2 26
0.035 1.372E-0.03 1.626E-0.003 1.000E-0.004
25.32
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
CAROTENE PRODUCTION FROM AGROINDUSTRIAL WASTES BY Arthrobacter globiformis IN SHAKE-FLASK CULTURE a
b
a
Yu-Gui Zhai , Mei Han , Wei-Guo Zhang & He Qian
a
a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University , Wuxi , JiangSu Province , P. R. China b
State Key Laboratory of Dairy Biotechnology , Technology Center Bright Dairy & Food Co., Ltd , Shanghai , P. R. China Accepted author version posted online: 06 Aug 2013.Published online: 09 Dec 2013.
To cite this article: Yu-Gui Zhai , Mei Han , Wei-Guo Zhang & He Qian (2014) CAROTENE PRODUCTION FROM AGRO-INDUSTRIAL WASTES BY Arthrobacter globiformis IN SHAKE-FLASK CULTURE, Preparative Biochemistry and Biotechnology, 44:4, 355-369, DOI: 10.1080/10826068.2013.829498 To link to this article: http://dx.doi.org/10.1080/10826068.2013.829498
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Florida International University] at 20:53 07 September 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:355–369, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.829498
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CAROTENE PRODUCTION FROM AGRO-INDUSTRIAL WASTES BY Arthrobacter globiformis IN SHAKE-FLASK CULTURE
Yu-Gui Zhai,1 Mei Han,2 Wei-Guo Zhang,1 and He Qian1 1 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University, Wuxi, JiangSu Province, P. R. China 2 State Key Laboratory of Dairy Biotechnology, Technology Center Bright Dairy & Food Co., Ltd, Shanghai, P. R. China
& Industrial waste substrates, sugarcane molasses, and corn steep liquor were used for production of carotenes by Arthrobacter globiformis in this study. At the first stage, a one-factor-at-a-time approach was used for optimization of different media components such as carbon, nitrogen, MgSO4 7H2O, and KH2PO4, as well as pH, temperature, liquid medium volume, and inoculums level. The response surface method was further applied to determination of optimum values of process variables for maximum carotenes concentration. Results showed that the optimum combination for carotenes formation was as follows (g=L): sugarcane molasses, 40.00; corn steep liquor, 50.00; MgSO4 7H2O, 0.75; KH2PO4, 1.00. The maximum carotene concentration of 1.19 0.02 mg=g dry biomass, about 113% of 1.05 0.02 mg=g dry biomass growing in basal medium, was demonstrated by confirmatory experiments to be the optimum in liquid medium at 100 rpm, 30C, initial pH of 7.5, and cultivation for 60 hr. In a second stage, detailed studies showed about 1.64-fold and 1.43-fold increase in carotene concentration (mg=g dry biomass) in the presence of addition of ethanol (4%, v=v) and addition of hydrogen peroxide (4%, v=v) at 40 hr, and 32 hr in liquid medium, separately. Keywords Arthrobacter globiformis, carotenes, industrial waste substrates, oxidative stress, response surface method
INTRODUCTION Carotenes are naturally occurring lipid-soluble pigments, with the majority being C40 terpenoids. Carotenes have been proven to play important roles in human health as precursors of vitamin A, scavengers of active oxygen, antitumor substances, and enhancers in vitro antibody production; therefore, they are widely applied in food, medical, pharmaceutical, and Address correspondence to Wei-Guo Zhang, The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, JiangNan University, 1800# Lihu Road, Wuxi-214122, JiangSu Province, P. R. China. E-mail: [email protected]
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cosmetic industries as colorants and functional components.[1] Huge commercial demand for natural carotenes has focused attention on developing of suitable biotechnological techniques, including use of industrial wastes as carbon and nitrogen sources. Commercial production by plant extraction does not satisfy the market need because of its low content in the plants.[2] Chemical synthesis has many disadvantages, such as low yield, instability of product quality, and high cost.[3] Thus, interest in production of carotenes by microbial fermentation has recently increased. Production of carotenes using microbes is highly efficient because they are easily manipulated in the processing schemes, including the bacteria Flavobacterium, the algae Dunaliella and Haematococcus, the fungus Blakeslea trispora, and yeast of the genera Phaffia, Rhodotorula, and Sporidiobolus.[4–10] Increasing attention has been applied to finding suitable natural sources including biotechnological processes in the past several decades. In order to improve the yield of carotenes and subsequently decrease the cost, diverse studies have been performed by optimizing the culture conditions, including nutritional and physical factors. Factors such as concentration of carbon and nitrogen sources, metal ions, pH, aeration, temperature, and oxidative stress have a major influence on cell growth and yield of carotenes.[10,11] Numerous cheap industrial by-products have been considered as potential carbon and nitrogen sources for biotechnological production of carotenes.[7,12–16] Roukas and colleagues investigated the effect of aeration rate and agitation speed on b-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentator and optimization of the production of the pigment in a bubble reactor. Maximum b-carotene concentration (360.2 mg=L) was obtained in culture grown in a molasses solution containing 5% (w=v) sugar supplemented with linoleic acid (37.59 g=L), kerosene (39.11 g=L), and antioxidant (1.0 g=L).[7] Varzakakou and colleagues performed an experiment of identification for the carotenes in Blakeslea trispora during pigment production from deproteinized hydrolyzed whey supplement with plant oils. The composition of carotenes was 60.1% b-carotene, 32.5% c-carotene, and 7.4% lycopene.[12] Thus, it is essential to carry out the research on carotenes accumulation in A. globiformis in order to make full use of industrial waste substrates and thereby reduce environmental contamination. It is believed that the overall yield of carotenes is directly related to the total biomass yield, so keeping both high growth rates and high flow carbon efficiency for carotenes by optimal cultivation conditions is essential in order to achieve the maximal pigment productivity[17] The one-factor-ata-time (OFAT) method has been widely applied in many fields such as medium optimization, formulation of multicomponent designs, and others.[18,19] The response surface method (RSM) has shown to be an efficient and effective approach to systematic investigation of several target factors. RSM presents
Downloaded by [Florida International University] at 20:53 07 September 2014
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the attractive advantage that it needs much fewer trials when compared with the other statistical designs.[13,20,21] In the present work, a combination of RSM and OFAT was applied to optimize the concentrations of components in fermentative medium for carotenes production by A. globiformis. Several agents have been used to induce oxidative stress, including duroquinone, H2O2, and ethanol. Oxidative stress is known to enhance the biosyhthesis of carotenes in some red yeast, and carotenes are also associated with an antioxidant function.[22,23] The aim of the work presented here is to develop a moderate two-stage fermentation process for carotenes production by A. globiformis using industrial waste substrates. In the first stage, the influences of several factors such as carbon source, nitrogen source, sulfate, phosphate, temperature, pH, liquid medium volume, and inoculums level on the production of carotenes were studied by OFAT and subsequently by RSM. In the second stage, effects of ethanol and hydrogen peroxide on biomass and production of carotenes were investigated.
MATERIALS AND METHODS Microorganism and Culture Conditions In this study the strain A. globiformis was used, and it was conserved in glucose–peptone–agar in darkness at 4 C. Cells were grown under aerobic conditions in 500-m baffled conical flasks with 50 mL of the basal medium (initial pH of 7.5) containing the following (g=L): sugarcane (SCM), 30; corn steep liquor (CSL), 30; MgSO4 7H2O, 0.5; KH2PO4, 1. Cultivations were carried out at 30 C on a rotary shaker at 100 rpm for 60 hr. The cells were harvested by centrifugation (6000 rpm for 10 min) and stored at 20 C under a nitrogen atmosphere before use. SCM and CSL were obtained from Anhui BBCA International Co., Ltd., China. The composition of SCM was 57% (w=w) sucrose, 7% (w=w) ash, and 18% (w=w) total nitrogen. SCM also constitutes a valuable source of growth substrates, such as pantothenic acid, inositol, trace elements, and, to a lesser extent, biotin. The composition of CSL was 23% (w=w) total nitrogen, 9% (w=w) ash, and 34% (w=w) lactose or total sugars. This also includes amine acid, and some metal ions such as iron, calcium, zinc, and potassium.
Measurements of Dry Biomass and Carotene Concentration Dry biomass (g=L) and extraction of carotenes were carried out as reported earlier.[9] For all experiments, cell growth was measured as optical
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density (OD) at 600 nm, and then it was converted into dry biomass based on a standard curve of OD against dry biomass. The medium without SCM and CSL was used as the blank and as diluting solution.[22] The intensity of color of the supernatant was measured at 490 nm with a Mapada 6100 spectrophotometer. The concentration of carotenes presented in the sample was then determined by comparison with a standard calibration curve prepared from pure all-trans-b-carotene (Darmstadt, Germany).[14] The major carotenes were quantified by a Hitachi L-2000 high-performance liquid chromatopgraph (HPLC) equipped with a photodiode array detector with a C18 column (25 mm 4.6 mm; 4.6 mm particle size; Agilent, USA). Pigments were eluted with methanol at a flux rate of 1.00 mL=min and 30 C. Under these conditions, OH-spirilloxanthin, spirilloxanthin, anhydrorhodovibrin, and b-carotene were eluted within 5.4, 7.6, 10.1, and 13.4 min, respectively. Since OH-spirilloxanthin, spirilloxanthin, and anhydrorhodovibrin were commercially unavailable, these carotenes were identified by their absorption maxima (464, 494, 524 nm), (466, 495, 527 nm), and (454, 480, 513 nm), respectively, as indicated previously.[24] They were quantified against peak area calibrations calculated from standard curves of b-carotene with appropriate correction factors at 490 nm. For all the analyses, samples were measured in triplicate for each experiment. Experimental Design and Data Analysis In the first stage, effects of eight factors on dry biomass and carotene concentration were investigated using OFAT: SCM, CSL, MgSO4 7H2O, KH2PO4, initial pH, temperature, liquid medium volume, and inoculums level. Then RSM was used to maximize the carotenes production. A fractional factorial 27-point design involving a three-level, fourvariable array was employed. Coded values of variables, namely, concentrations (g=L) of SCM, CSL, MgSO4 7H2O, and KH2PO4, were applied. The basic equation for carotene concentration as a function of the already-mentioned four independent variables involved a secondorder polynomial of this form: Carotene concentration ¼ A0 þ A 1 X1 þ A2 X2 þ A 3 X3 þ A 4 X4 þ A 5 X1 X2 þ A 6 X1 X3 þ A 7 X1 X4 þ A 8 X2 X3 þ A9 X2 X4 þ A 10 X3 X4 þ A 11 X21 þ A12 X22 þ A13 X23 þ A 14 X24
ð1Þ
where X1 is the concentration of SCM, X2 the concentration of CSL, X3 the concentration of MgSO4 7H2O, X4 the concentration of KH2PO4, A0 is
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a constant, A1, A2, A3, and A4 are linear coefficients, A5, A6, A7, A8, A9, and A10 are cross-product coefficients, and A11, A12, A13, and A14 are quadratic coefficients. The commercial software Design Expert v6.06 (ATAT-EASE, Inc., Minneapolis, MN) was used for statistical analyses, regression analyses, and the significant difference (P 0.05) was assessed.[25] In flask experiments three parallel cultivations were carried out.
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Addition of Ethanol and Hydrogen Peroxide in Liquid Medium Several literature reports have revealed that adding stimulators can enhance the carotenes production by the yeast Dioszegia, the fungi Rhodotorula glutinis, and Blakeslea trispora grown under normal conditions.[22,23,27] Thus, we investigated the effects of two stimulators (ethanol and hydrogen peroxide) on biomass and carotenes production in the second stage. All experiments were done in triplicate.
RESULTS AND DISCUSSION Effects of SCM and CSL on Dry Biomass and Carotene Concentration The results are given as the units of dry biomass (g=L) and carotene concentration (mg=g dry biomass) at the end of growth. Figures 1a and 1b showed that carotenes formation and organism growth increased with increasing initial concentrations of SCM and CSL up to 40 and 50 g=L, respectively. The maximum dry biomass and carotene concentration were 26.11 0.80 g=L and 1.01 0.03 mg=g dry biomass, separately.
Effects of Metals Ions on Dry Biomass and Carotene Concentration Metals ions are involved in all aspects of microbial life. Metal ions can specially cause activation or inhibition mechanisms for specific carotenogenic enzymes, in particular, for specific desaturases involved in carotenes biosynthesis.[1] KH2PO4 is essential in compositions of many macromolecular substances (DNA, RNA, ATP, etc.). It also can maintain the pH value and keep suitable cytoplasmic osmotic pressure.[28] Effects of MgSO4 7H2O and KH2PO4 were assessed, and the best results, presented in Figures 1c and, were obtained at the concentrations of 0.5 or 1.0 g=L, and 1.0 g=L, separately.
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FIGURE 1 Effect of some parameters on carotene concentration and dry biomass: (a) sugarcane molasses; (b) corn steep liquor; (c) MgSO4 7H2O; (d) KH2PO4; (e) temperature; (f) pH; (g) liquid medium volume; (h) inoculums level.
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Effects of Temperature and pH on Dry Biomass and Carotene Concentration Temperature is another important factor affecting the performance of cells and product formation. It was reported to control the concentrations of enzymes involved in carotenes production, and changes in enzyme concentrations ultimately control carotenes levels in microorganism.[28] Biomass and carotenes formation increased with increasing temperature up to 30 C and decreased sharply above 30 C (shown in Figure 1e). The initial pH significantly affected the growth and total carotenes production as shown in Figure 1f. As the pH increased, biomass and carotenes production increased and reached a maximum level at pH 7.5 with a drop off at higher pH values. Effects of Liquid Medium Volume and Inoculums Level on Dry Biomass and Carotene Concentration Arthrobacter globiformils is an aerobic microorganism and therefore requires the provision of oxygen, so aeration is extremely important for the successful progress of fermentation. Aeration could beneficial to the growth and performance of microbial cells by improving the mass transfer characteristics with respect to substrate, product, and oxygen.[28] The best result was obtained with 30 mL of liquid medium (presented in Figure 1g). Carotenes accumulation was restricted largely in liquid medium volume lower than 30 mL (data not shown). Inoculums level is another factor affecting the growth. Low inoculums level will restrict the normal growth, and high inoculums level will make cells step into the stationary stage earlier and then reduce the accumulation of the secondary metabolites. The optimized condition was found to be 10% (v=v=) inoculums (presented in Figure 1h). Finally, dry biomass and carotene concentration reached up to 29.1 1.21 g=L and 1.05 0.02 mg=g dry biomass. Aliquots of fermentation broth removed from the conical flasks at 4-hr intervals (from 0 to 72 hr) were used for the determination of OD and carotene concentration.[11] As shown in Figure 2, carotenes were synthesized in two steps. In the first step (0 to 40 hr of liquid fermentation), called the ‘‘growth phase,’’ a rapid increase of biomass and a small amount of the pigment were obtained, and in the second step (40 to 60 hr of liquid fermentation), called the ‘‘production phase,’’ the biomass remained constant and the biosynthesis of carotenes was carried out.[28] Based on the results of OFAT, the maximum carotene concentration of 1.05 0.02 mg=g dry biomass was obtained with dry biomass of 29.1 1.21 g=L; however, maximum dry biomass of 32.24 1.32 g=L was
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FIGURE 2 Effect of fermentation time on carotene concentration and dry biomass.
obtained with a lower amount of carotene concentration of 0.89 0.02 mg=g dry biomass. OPTIMIZATION OF FERMENTATION CONDITIONS WITH RSM Table 1 presents the coded and real values. RSM was used to maximize the carotenes production, and the results are listed in Table 2. The data in Table 2 indicate that there was a wide variation from 1.21 0.03 mg=g dry biomass to 0.73 0.03 mg=g dry biomass of carotene concentration in the 27 trials. This variation reflects the importance of medium optimization to attain higher carotenes yield. According to anaylsis of variance (ANOVA) provided in Table 3, the results showed that the model F value 25.32 for carotenes production implied that the model was significant (P 0.05) with R2 0.967 indicating that model was in good agreement TABLE 1
Coded and Real Values in the Experimental Design Levels
Symbol
Independent Variables
1
0
þ1
Units
X1 X2 X3 X4
Sugarcane molasses Corn steep liquor MgSO4 7H2O KH2PO4
35 45 0.5 0.8
40 50 0.8 1.0
45 55 1.1 1.2
g=L g=L g=L g=L
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Design and Results of Response Surface Method
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Total Carotene Concentration (mg=g Dry Biomass) Standard Order
X1
X2
X3
X4
Actual Values
Predicated Values
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0
1 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0
0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0
0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0
0.73 0.03 0.85 0.04 0.81 0.04 0.92 0.03 0.75 0.04 0.96 0.02 0.86 0.02 0.88 0.03 0.79 0.02 0.95 0.04 0.89 0.02 0.95 0.03 0.73 0.04 0.70 0.03 0.81 0.01 0.99 0.04 0.71 0.04 0.81 0.02 0.74 0.01 0.95 0.03 0.86 0.04 0.83 0.02 0.95 0.03 0.95 0.04 1.21 0.03 1.20 0.04 1.19 0.05
0.72 0.05 0.86 0.05 0.80 0.05 0.92 0.05 0.75 0.05 0.96 0.05 0.85 0.05 0.87 0.05 0.80 0.05 0.93 0.05 0.90 0.05 0.95 0.05 0.72 0.05 0.70 0.05 0.81 0.05 0.98 0.05 0.74 0.05 0.79 0.05 0.70 0.05 0.92 0.05 0.84 0.05 0.82 0.05 0.94 0.05 0.95 0.05 1.20 0.05 1.20 0.05 1.20 0.05
with experimental data, and allowed the construction of the stereogram (presented in Figure 3). Nonsignificant lack of fit (P > 0.05) further validated the model. The regression equation for carotene concentration (mg=g dry biomass) as a function of four media components was
TABLE 3 Source Model Residual Lack of fit Pure error Total
Analysis of Variance for Carotene Concentration Sum of Squares
Degrees of Freedom
Mean Squares
F Value
P>F
0.49 0.016 0.016 2.00E-0.04 0.50
12 14 10 2 26
0.035 1.372E-0.03 1.626E-0.003 1.000E-0.004
25.32
