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Process Biochemistry 41 (2006) 293–298 www.elsevier.com/locate/procbio Short communication Medium optimization of carbon and nitrogen sources for the production of eucalyptene A and xyloketal A from Xylaria sp. 2508 using response surface methodology Zeng Xiaobo a,*, Wang haiying b, He Linyu c, Lin Yongcheng c, Li Zhongtao a a College of Pharmacy, Wuhan University, 430072 Wuhan, PR China b College of Life Science, South-center University for Nationalities, 430074 Wuhan, PR China c School of Chemistry & Chemical Engineering, Sun Yat-sen University, 510275 Guangzhou, PR China Received 30 March 2005; received in revised form 6 June 2005; accepted 7 June 2005 Abstract To optimize the production of novel ketal compounds and allenic ethers from a mangrove fungus Xylaria sp. 2508, optimization of the culture medium was carried out using a two-step approach. A quick identiﬁcation of a suitable source of carbon and nitrogen was obtained by a simple screening experiment, which was followed by an application of response surface methodology (RSM) for further optimization design. A second order model was found to ﬁt the eucalyptene A production. Response surface analysis revealed that the optimum values of the tested variables for the production of eucalyptene A were: glucose 2.86% (w/v), peptone 0.78% (w/v) and yeast extract 0.35% (w/v). A production of 4.89 Â 10À7 g mlÀ1 for eucalyptene A, which was close to the predicted response, was observed in veriﬁcation experiment. The production of xyloketal A was 2.20 Â 10À7 g mlÀ1. In comparison to their production of basal medium, 1.53- and 1.32-fold increase had been obtained, respectively. The 3-D response surface curves implied that high yeast extract concentration repressed the growth of Xylaria sp. # 2005 Elsevier Ltd. All rights reserved. Keywords: Medium optimization; Ketal compounds; Allenic ethers; Xylaria sp.; Response surface methodology 1. Introduction fungus. The xyloketals represent a series of novel ketal compounds having a close biogenetic relationship. Xyloalle- After almost 40 years of investigation, the number of nolide A has a novel cyclic peptide containing an allenic ether compounds isolated from various marine organisms has of a N-( p-hydroxycinnamoyl) amide. Together with xyloal- virtually soared and exceeds 10,000 , with hundreds of lenolide A there were two other allenic ethers found from the new compounds still being discovered every year [2,3]. In spent culture medium . One of them, a known compound 2001 the number of new compounds and new stereo- eucalyptene A, exhibited antifungal activity. This was chemical structures reported is 683 . Some of these revealed by means of bioautography on Cladosporium marine nature products hold great potential for the cladosporioides for amounts as low as 50 mg . In development of new and much needed drugs. the primary bioassay, xyloketal A inhibited acetylcholine The mangrove fungus Xylaria sp. 2508, which was esterase at 1.5 Â 10À6 mol lÀ1 and xyloallenolide A depress- collected from seeds of an angiosperm tree and identiﬁed as ed the growth of fungus [5,6]. The chemical conﬁgurations Xylaria species (Ascomycota), was found to produce rich of xyloketal A, xyloallenolide A and eucalyptene A are secondary metabolites. Xyloketals A–E  and xyloalleno- shown in Fig. 1. Further biological evaluation of them is in lide A  were isolated from the spent culture medium of this progress. Considering the potential bioactivities of the secondary * Corresponding author. Tel.: +86 27 6875 2339/139 7168 2309; products of the fungus Xylaria sp. 2508 we have attempted to fax: +86 27 6875 4629. study and maximize the production of them. Preliminary E-mail address: email@example.com (Z. Xiaobo). experiments indicated that eucalyptene A had a yield about 10 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.06.002 294 Z. Xiaobo et al. / Process Biochemistry 41 (2006) 293–298 Fig. 1. Chemical conﬁguration of xyloketal A, xyloallenolide A and eucalyptene A. times of xyloallenolide A. It was also found that among and repeated twice at least. The mean values and the xyloketals A–E, xyloketal A has the maximum outcome. standard deviation were calculated from the data obtained Thus, in this experiment, we chose eucalyptene A and with these trials. The standard deviation in results was within xyloketal A as targets to determine the production of ketal 10%. compounds and allenic ethers from Xylaria sp. 2508, respectively. 2.2. Micro-organism The ‘one-at-a-time-approach’ is an operation frequently used in biotechnology to obtain high yields of the desired The strain of the fungus Xylaria sp. (no. 2508) was metabolic products in a microbial system. This method is not isolated from seeds of a mangrove angiosperm tree in Hong only time consuming, but disregards the complex interac- Kong. It was stored at the College of Life Science, Sun Yat- tions among various physicochemical parameters [8,9]. To sen University, Guangzhou, China. overcome this difﬁculty, experimental factorial design and response surface methodology (RSM) can be employed to 2.3. Inoculum preparation optimize the medium components. The response surface methodology, which includes factorial design and regression Starter cultures were maintained on cornmeal seawater analysis, can build models to evaluate the effective factors agar (glucose 0.010 g mlÀ1, cornmeal 0.010 g mlÀ1, pep- and study their interaction and select optimum conditions in tone 0.001 g mlÀ1 and agar 0.015 g mlÀ1, dissolved in fresh a limited number of experiments [10–12]. Here, we report seawater). The agar with mycelium was transferred statistical optimization of culture medium for enhanced aseptically to a 250 ml Erlenmeyer ﬂask containing production of ketal compounds and allenic ethers from 100 ml of basal liquid medium (glucose 0.010 g mlÀ1, Xylaria sp. 2508. peptone 0.002 g mlÀ1, yeast extract 0.001 g mlÀ1, NaCl 0.030 g mlÀ1). The ﬂask was incubated at 30 8C on a rotary shaker (150 rpm) for 5–7 days. The mycelium was 2. Materials and methods aseptically transferred to a 1 l ﬂask containing 300 ml of liquid medium for production experiments. 2.1. Chemicals and experimental statistics 2.4. Analytical methods Isopropyl alcohol for HPLC was purchased from Merck (Darmstadt, Germany). All other chemicals used were of Growth of Xylaria sp. 2508 was determined by weighting analytical grade commercially available in China. All the the mycelia which was centrifuged (3000 Â g 10 min, at experiments were carried out independently in triplicates ambient temperature) and washed twice with distilled water. Z. Xiaobo et al. / Process Biochemistry 41 (2006) 293–298 295 Xyloketal A and eucalyptene A were analyzed by HPLC. levels (À1, 0, +1). For glucose, the corresponding actual Culture medium from one ﬂask was centrifuged and the values of three coded levels were 1, 3 and 5%. For peptone volume of supernatant was metered to 300 ml. Then 3 ml of they were 0.2, 1, 1.5% and for yeast extract they were this liquor, 3 ml methanol and 50 ml isopropyl alcohol 0.2, 0.5, 1%. A series of 15 experiments including three solution (4.187 Â 10À4 g mlÀ1, dissolved in methanol) were center points were performed. The minimum and maximum mixed and centrifuged (5000 Â g, at ambient temperature, range of independent variables investigated and the full 5 min) for detection. HPLC (Agilent 1100 system) was experimental plan with respect to their coded forms are listed carried out with a C-18 column (3.9 mm Â 300 mm, in Table 1. Nova-Pak, Waters) and 80% MeOH (v/v, methanol/water) Cultures were stably incubated at 30 8C for 25 days. A as the mobile phase at the ﬂow rate of 0.5 ml minÀ1. multiple regression analysis of the data was carried out to To determinate the absorbance, the UV–vis detector was set obtain empirical models that deﬁne response (Y1 and Y2) in to 220 nm. Isopropyl alcohol was used as an internal terms of the independent variables. For a three-factor standard substance. Its relative correction factors to system, the following second-order polynomial equation xyloketal A and eucalyptene A were 0.5288À1 and was then applied to the data by the multiple regression 1.3609À1, respectively. procedure: 2 2 2 2.5. Optimization of xyloketal A and eucalyptene A Y ¼ a0 þ a1 X1 þ a2 X2 þ a3 X3 þ a11 X1 þ a22 X2 þ a33 X3 production from Xylaria sp. 2508 þ a12 X1 X2 þ a13 X1 X3 þ a23 X2 X3 (1) 2.5.1. Selection of most suitable carbon and nitrogen with Y, being the predicted response; a0, intercept; a1, a2, a3, sources by one-variable-at-a-time approach linear coefﬁcients; a11, a22, a33, squared coefﬁcients; a12, For selection of the best source of carbon and nitrogen for a13, a23, interaction coefﬁcients. xyloketal A and eucalyptene A production, various simple Using the above model, a SAS software package was and complex carbon sources (glucose, sugar, lactose, used to analyze the variance and to obtain the optimum maltose, glycerol, dextrin, starch, glyceryl monostearate) concentration of the medium components. The response and inorganic and complex nitrogen sources (sodium nitrate, surface graphs were plotted to understand the effect of the ammonium sulphate, urea, peptone, yeast extract, casein) selected variables individually and in combination and to were used as substitute individually for carbon or nitrogen determine their optimum levels for maximal xyloketal A and source in the basal liquid medium. In one test, one of the eucalyptene A production. All the experiments were carried source of carbon (glucose) and nitrogen (peptone and yeast out independently in triplicates and the average values were extract) in the basal liquid medium was replaced by matters presented. mentioned above with a ﬁnal concentration of 2% (w/v) for carbon source and 0.2% (w/v) for nitrogen source, while other ingredients kept original concentrations. Before 3. Results autoclaving, the pH value of the medium was adjusted to 8.0 by adding 1 mol lÀ1 NaOH or 1 mol lÀ1 HCl. 3.1. Selection of most suitable carbon and nitrogen Erlenmeyer ﬂasks (1000 ml) containing 300 ml of the sources by one-variable-at-a-time approach culture medium were inoculated with 1% (w/v) fresh mycelium. Yields of xyloketal A and eucalyptene A were Among the various carbon sources studied, Xylaria sp. determined after 25-day incubation at 30 8C under stationary 2508 produced more eucalyptene A and xyloketal A in the culture. presence of glucose and sucrose. This is shown on Table 2. It is also shown that complex organic nitrogen sources, viz., 2.5.2. Experimental design and optimization by RSM peptone, yeast extract and casein are observed to induce the Based on the results obtained in the previous experiments, production eucalyptene A and xyloketal A while simple glucose, peptone and yeast extract were found to be inorganic sources support no production of them. In medium determining variables in xyloketal A and eucalyptene A containing simple inorganic nitrogen sources and some production. Hence, these variables were selected for carbon sources, Xylaria sp. 2508 could scarcely grow. In all optimizing a higher lever of production of xyloketal A and carbon sources being tested, Xylaria sp. 2508 could only eucalyptene A. For this purpose, the response surface yield eucalyptene A and xyloketal A in the medium methodology by using a 3 Â 3 factorial central composite containing glucose and sucrose. Considering that the sucrose design (CCD) proposed by Box and Behnken  was molecule is composed of glucose and fructose and sucrose adopted. Three independent variables were glucose (w/v %, supported more mycelium growth but didn’t induce more X1), peptone (w/v %, X2) and yeast extract (w/v %, X3), and the yield of product than glucose (Table 2), glucose was selected dependent variables were production of eucalyptene A as the source of carbon. And peptone and yeast extract were (10À7 g mlÀ1, Y1) and xyloketal A (10À7 g mlÀ1, Y2). Each selected for the source of nitrogen in the following independent variable was studied at three different coded experiment. 296 Z. Xiaobo et al. / Process Biochemistry 41 (2006) 293–298 Table 1 Experimental designs used in RSM studies and observed responses Test number X1 (glucose) X2 (peptone) X3 (yeast extract) Mean observed responses Eucalyptene A (10À7 g mlÀ1) Xyloketal A (10À7 g mlÀ1) Mycelium weight (g) 1 À1 À1 0 2.610 0.97 16.40 2 À1 0 À1 2.047 1.02 16.98 3 À1 0 +1 0.121 n.d. 15.96 4 À1 +1 0 1.121 n.d. 16.22 5 0 À1 À1 3.058 1.39 20.92 6 0 À1 +1 0.254 0.13 15.02 7 0 +1 À1 2.610 1.16 23.74 8 0 +1 +1 0.356 n.d. 14.08 9 +1 À1 0 0.506 0.27 18.12 10 +1 0 À1 2.847 0.92 23.96 11 +1 0 +1 3.210 n.d. 11.24 12 +1 +1 0 1.015 n.d. 14.54 13 0 0 0 4.738 1.33 19.81 14 0 0 0 4.310 1.27 16.76 15 0 0 0 4.201 1.10 18.69 ‘n.d.’ indicates that no xyloketal A was detected. To ﬁnd the optimum rang of their concentrations for the cannot be got by response surface methodology. For production of eucalyptene A and xyloketal A, Xylaria sp. eucalyptene A, the regression equation was obtained by 2508 was cultured in the basal liquid medium with varying applying the response surface methodology. one parameter at a time and keeping the others constant. The coefﬁcients of the regression equation were Data obtained (not reported here) was used for design of calculated using the software SAS. The eucalyptene A response surface methodology. production (Y1) can be expressed in terms of the following regression equation: 3.2. Optimization by response surface methodology Y1 ¼ 0:772357 þ 1:250454X1 þ 5:448226X2 þ 0:144725X3 The observed responses of RSM experiments for 2 2 2 À 0:360308X1 À 4:967917X2 À 4:798811X3 studying the effects of three independent variables, viz., þ 0:656866X1 X2 þ 0:856881X1 X3 glucose, peptone and yeast extract, on production of eucalyptene A and xyloketal A are presented together with þ 1:052876X2 X3 (2) the mycelium weight in Table 1. Because xyloketal A cannot be detected in several experiments where concentrations of where Y1 is the eucalyptene A production; X1, glucose nitrogen sources are higher relatively, its regression equation concentration; X2, peptone concentration; X3, yeast extract concentration. The results of the second order response Table 2 surface model ﬁtting in the form of analysis of variable Effect of various carbon and nitrogen sources on eucalyptene A and (ANOVA) are given in Table 3. A good ﬁt of the regression xyloketal A production by Xylaria sp. 2508 model was checked by the R2 and R value, signiﬁcance of Nutrient source Eucalyptene A Xyloketal A Mycelium ‘‘total regress F-value’’ (5.36) and non-signiﬁcance of ‘‘lack (10À7 g mlÀ1) (10À7 g mlÀ1) weight (g) of ﬁt F-value’’ (13.87). Carbon sources The 3-D response surface curves were then plotted to Glucose 3.81 1.64 15.79 explain the interaction of medium components and the Sucrose 3.30 1.81 20.61 optimum concentration of each component required for the Lactose n.d. n.d. 9.37 Maltose n.d. n.d. 7.92 production of eucalyptene A (Fig. 2). In response surface Glycerol n.d. n.d. 3.36 curves two factors varied at a time when the other factor Dextrin n.d. n.d. 16.58 maintained at a ﬁxed level (zero level). As showing in graph Starch n.d. n.d. 4.04 A, B and C in Fig. 2, the eucalyptene A production is Glyceryl monostearate n.d. n.d. 10.89 inﬂuenced by all three independent variables. The maximum Nitrogen sources point was achieved when glucose, peptone, and yeast extract Sodium nitrate n.d. n.d. 3.67 concentrations were near their zero level. Ammonium sulphate n.d. n.d. 4.05 Urea n.d. n.d. 4.13 Casein 1.61 1.05 15.62 3.3. Validation of the experimental model Peptone 3.28 1.79 24.76 Yeast extract 3.42 1.54 25.62 The regression equation of eucalyptene A production was ‘n.d.’ indicates that no eucalyptene A or xyloketal A was detected. solved by using SAS software. The optimal values of each Z. Xiaobo et al. / Process Biochemistry 41 (2006) 293–298 297 Table 3 10À7 g mlÀ1) increase in production of eucalyptene A was Analysis of variance for the regression model of medium optimization of achieved compared with that in the basal medium eucalyptene A production obtained from the experimental results (3.19 Â 10À7 g mlÀ1). In veriﬁcation experiment, a 1.32- Sources Degree of Sum of Mean F-value fold increase of xyloketal A production (2.20 Â 10À7 freedom squares square g mlÀ1) was obtained in comparison to the basal medium Regression (1.67 Â 10À7 g mlÀ1). The result indicated that the opti- Linear 3 7.3840 2.4613 3.50 Quadratic 3 21.1858 7.0619 10.03a mized medium favors the production of xyloketal A. Crossproduct 3 5.3850 1.7950 2.55 Total regress 9 33.9549 3.7728 5.36a Residual 4. Discussion Lack of ﬁt 3 3.3583 1.1194 13.87 Pure error 2 0.1614 0.0807 This work has demonstrated the use of a multifactorial Total error 5 3.5198 0.7040 R2 0.9061 statistical approach for determining the conditions that lead R 0.9519 to the optimum yield of secondary metabolites production. a Signiﬁcant at 95% level. Central composite design maximizes the amount of information that can be obtained, while considering the interaction of independent variables and limiting the numbers test component in coded units are as follows: X1 = of individual experiments required. The response surface À0.067275, X2 = À0.114614 and X3 = À0.610045. Their methodology, a smaller and less time consuming experi- actual values were: 2.86% (w/v) glucose, 0.78% (w/v) mental design, could generally satisfy the optimization of peptone and 0.35% (w/v) yeast extract. The model predicted many microbial processes [8,10]. that the production of eucalyptene A could reach Though the RSM experiments was not designed for 4.70 Â 10À7 g mlÀ1 by using the above optimized concen- Xylaria sp. 2508 growth, the 3-D response surface curves trations of the variables. Veriﬁcation experiments were were plotted to understand the interaction of the medium accomplished by using the optimized conditions and gave a components and their effect on mycelium growth. Graph D production of 4.89 Â 10À7 g mlÀ1 of eucalyptene A, which in Fig. 2 shows that high concentration of yeast extract was closer to the predicted response of 4.70 Â 10À7 g mlÀ1. repressed the growth of Xylaria sp. At all levels (+1, 0, À1) of This result therefore corroborated the predicted values, yeast extract concentration, a change of peptone concentra- and the effectiveness of the model. A 1.53-fold (4.89 Â tion cannot signiﬁcantly affect growth. Since Xylaria sp. Fig. 2. Response surface curve of eucalyptene A production and mycelium weight of Xylaria sp. 2508. Graph A, B, C and D showed the effect of yeast extract, peptone and glucose concentration and their interaction. The 3-D response surface curves were plotted with two independent variables at a time, the other factor was maintained at the zero level. 298 Z. Xiaobo et al. / Process Biochemistry 41 (2006) 293–298 2508 was isolated from seeds of a mangrove angiosperm (863 Program), no. 2001AA624010. The authors thank Judy tree, it might be a symbiote of this tree and might absorb Van Bruggen from Canada for the English reviewing. some nutrients, especially those that couldn’t be synthesized by itself, from the tree tissue. Yeast extract contains complex nutrients such as vitamin, nucleic acid, lipid and other References substances, which might be necessary for Xylaria sp. 2508. Proper yeast extract concentration is necessary for growth  Proksch P, Edrada RA, Ebel R. Drugs from the seas—current status and microbiological implications. Appl Microbiol Biotechnol 2002; and secondary metabolites of Xylaria sp. 2508. 59:125–34. As with many other experiments have mentioned,  Faulkner DJ. Marine pharmacology. Antonie Van Leeuwenhoek insufﬁcient oxygen supply is one of the most frequently 2000;77:135–45. occurring problem associated with the application of  Faulkner DJ. Marine natural products. Nat Prod Rep 2002;19:1–48. Erlenmeyer ﬂask cultivation. In microbial cultures, the  Blunt JW, Copp BR, Munro MG, Northcote PT, Prinsep MR. Marine typical reaction behaviors during oxygen limitation may be natural products. Nat Prod Rep 2003;20:1–48.  Lin YC, Wu XY, Feng S, Jiang GC, Luo JH, Zhou SN, Vrijmoed LLP, the slowing down of the whole metabolism of the ¨ Jones EBG, Krohn K, Steingrover K, Zsila F. Five unique compounds: microorganisms and the microorganisms sensitively react- xyloketals from mangrove fungus Xylaria sp. from the South China ing to the oxygen supply with their product metabolism Sea coast. J Org Chem 2001;66:6252–6. [13,14]. In our experiment we encountered this problem.  Lin YC, Wu XY, Feng S, Jiang GC, Zhou SN, Vrijmoed LLP. 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