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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 [1], 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 [6]. 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 [4]. 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 [7]. 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 [5] and xyloalleno-                    shown in Fig. 1. Further biological evaluation of them is in
lide A [6] 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: zxbwhy@163.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  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 [11] 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
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