Journal of Applied Microbiology ISSN 1364-5072
O RIG I NA L ARTICLE
Optimization of fermentation condition for antibiotic
production by Xenorhabdus nematophila with response
surface methodology
Y.-H. Wang, J.-T. Feng, Q. Zhang and X. Zhang
Biorational Pesticides Research and Service Center, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, China
Keywords
antibiotic activity, fermentation condition,
optimization, response surface methodology,
Xenorhabdus nematophila.
Correspondence
X. Zhang, Biorational Pesticides Research and
Service Center, Northwest Sci-Tech University
of Agriculture and Forestry, Xinong Road
22, Yangling 712100, China.
E-mail: ylagri@126.com
2007 ⁄ 0686: received 27 April 2007, revised
24 August 2007 and accepted 25 August
2007
doi:10.1111/j.1365-2672.2007.03599.x
Introduction
Xenorhabdus nematophila is a Gram-negative bacterium,
belonging to the family Enterobacteriaceae. The bacterium
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 735–
744
Abstract
Aims: To evaluate the influence of environmental parameters on the produc-
tion of antibiotics (xenocoumacins and nematophin) by Xenorhabdus nemato-
phila and enhance the antibiotic activity.
Methods and Results: Response surface methodology (RSM) was employed to
study the effects of five parameters (the initial pH, medium volume in flask,
rotary speed, temperature and inoculation volume) on the production of anti-
biotics in flask cultures by X. nematophila YL001. A 25)1-factorial central com-
posite design was chosen to explain the combined effects of the five parameters
and to design a minimum number of experiments. The experimental results
and software-predicted values of production of antibiotics were comparable.
The statistical analysis of the results showed that, in the range studied, medium
volume in flask, rotary speed, temperature and inoculation volume had a sig-
nificant effect (P < 0Æ05) on the production of antibiotics at their individual
level, medium volume in flask and rotary speed showed a significant influence
at interactive level and were most significant at individual level. The maximum
antibiotic activity was achieved at the initial pH 7Æ64, medium volume
in 250 ml flask 25 ml, rotary speed of 220 rev min)1, temperature 27Æ8°C
and inoculation volume of 15Æ0%. Maximum antibiotic activity of 331Æ7 U
ml)1 was achieved under the optimized condition.
Conclusions: As far as known, there are no reports of production of antibiotic
from X. nematophila by engineering the condition of fermentation using RSM.
The results strongly support the use of RSM for fermentation condition opti-
mization. The optimization of the environmental parameters resulted not only
in a 43Æ4% higher antibiotic activity than unoptimized conditions but also
in a reduced amount of the experiments. The chosen method of optimization of
fermentation condition was efficient, relatively simple and time and material
saving.
Significance and Impact of the Study: This study should contribute towards
improving the antibiotics activity of X. nematophila. Integrated into a broader
study of the impact of environmental factors on the production of antibiotic,
this work should help to build more rational control strategy, possibly involv-
ing scale-up of production of antibiotics by X. nematophila.
is mutually associated with the infective juvenile (IJ)
nematodes of the genus Steinernema (Steinernematidae)
(Thomas and Poinar 1979). During IJ development, a
small number of ingested X. nematophila cells initiate
1
Antibiotic production by X. nematophila
colonization in the intravesicular structure of the IJs
intestinal vesicles and subsequently multiply within this
host niche until the vesicle niche is filled (Martens et al.
2003; Martens and Goodrich-Blair 2005). In the soil, IJs
seek out insect larvae and gain entrance into the larval
haemocoel and release intestinal X. nematophila cells by
defecation in response to an unknown signal(s) present in
insect haemolymph (Sicard et al. 2004). The bacteria mul-
tiply rapidly and produce a wide range of toxins and
hydrolytic exoenzymes, resulting in a bacterial septicaemia
and death of the host within 24–48 h (Forst et al. 1997).
The nematodes feed on the multiplying bacteria and
insect tissues degraded by the bacteria and reproduce
until the nutrient supply becomes limiting, at which time
they develop into IJs which are recolonized by the symbi-
otic bacterium. This nematode–bacteria complex has been
developed commercially as a biological control agent of
insect pests (Ehlers 1996).
The production of secondary metabolites with antibi-
otic properties is a characteristic common to entomo-
pathogenic bacteria, Xenorhabdus and Photorhabdus. More
than 30 bioactive secondary metabolites, belonging to
diverse chemical classes, have been reported from cultures
of Xenorhabdus and Photorhabdus. These include
hydroxystilbenes and indoles (Paul et al. 1981),
xenorhabdins (McInerney et al. 1991a), xenocoumacins
(McInerney et al. 1991b), xenorxides (Li et al. 1998),
nematophin (Li et al. 1997) and benzylineacetone (Ji et al.
2004). These metabolites not only have diverse chemical
structures, but also have a wide range of bioactivities of
medicinal and agricultural interest, such as antibiotic,
antimycotic, insecticidal, nematicidal, antiulcer, antineo-
plastic and antiviral (Webster et al. 2002).
Xenorhabdus nematophila has been known to produce
xenocoumacins (benzopyranone derivatives), nematophin
(indoles) and benzylineacetone (monoterpenoid). Xeno-
coumacins are highly active against Gram-positive bacte-
ria including Staphylococcus and Streptococcus species and
some Gram-negative bacteria, such as Escherichia coli.
However, most enterobacteria and Pseudomonas aerugin-
osa are resistant to xenocoumacins, as are the drug-resis-
tant strains of S. aureus. Xenocoumacins are also active
against the fungal species Aspergillus and Trichophyton
and the yeasts Candida and Cryptococcus. Xenocoumacins
dosed orally in rats have, in addition to their antimicro-
bial activity, potent activity against stress-induced ulcers
(McInerney et al. 1991b). The pharmacological activity of
xenocoumacins has not been tested, but it has been spec-
ulated to be similar to that of amicoumacins (McInerney
et al. 1991b), which have high acute toxicity to rats
(Shimojima et al., 1982). Nematophin is active against
Gram-positive bacteria, show strong activity against Bacil-
lus subtilis and Staphylococcus aureus. Nematophin also
Y.-H. Wang et al.
show significant antimycotic activity. In comparison, ben-
zylineacetone is active against Gram-negative bacteria. Xe-
nocoumacins are commonly produced by X. nematophila
(Webster et al. 2002).
Antibiotic production by the symbiotic bacteria differs
qualitatively and quantitatively depending on the strains
and species of bacteria and their culture conditions. Tem-
perature and aeration of the culture influence bacterial
growth and, consequently, affect antibiotic production
(Akhurst 1982; Chen et al. 1996). Also, a much lower anti-
biotic activity was observed for the X. nematophilus D1
strain cultured at 35°C than those at 15–30°C (Chen et al.
1996). Xenorhabdus spp. are facultative anaerobes, but aer-
ation is essential for antibiotic production (Akhurst 1982;
Chen et al. 1996). Yields of up to 300 and 100 mg l)1 of
xenocoumacin 1 and 2, respectively, were obtained from
X. nematophila strain All (ATCC 53200) cultured in TSB
(tryptic soy broth) (McInerney et al. 1991b). Li et al.
(1997) showed that the concentration of nematophin dif-
fered throughout the duration of culture and among
strains. Strain BC1 of X. nematophila produced two to five
times more nematophin over the whole period than did
strains D1 and ATCC 19061. The concentration of ne-
matophin in these bacterial cultures increased significantly
from the first to the second day, and remained relatively
high thereafter. The maximum nematophin concentrations
in BC1, D1 and ATCC 19061 were 605Æ34, 200Æ67 and
148Æ04 mg l)1, respectively. These results indicate that
the strains and culture conditions play an important
role in the onset and intensity of secondary metabolism.
To achieve high product yields, it is a prerequisite to
design proper culture conditions in an efficient
fermentation pro- cess. There is usually a relationship
between the culture conditions and the biosynthesis of
antibiotics (Chen et al. 1996; Li et al. 1997; Yang et al.
2001, 2006).
The application of statistical experimental design tech-
niques in fermentation process can result in improved
product yields, reduced process variability, closer confir-
mation of the output response to nominal and target
requirements and reduced development time and overall
costs. Conventional practice of single-factor optimization
by maintaining other factors involved at an unspecified
constant level does not depict the combined effect of all
the factors involved. This method is also a time-consuming
process and requires a number of experiments to deter-
mine optimum levels, which may be unreliable. These lim-
itations of a single-factor optimization process can be
eliminated by optimizing all the affecting parameters col-
lectively by statistical experimental design using response
surface methodology (RSM). RSM can be used to evaluate
the relative significance of several contributing factors even
in the presence of complex interactions (Rao et al. 2000;
Y.-H. Wang et al. Antibiotic production by X. nematophila
Dey et al. 2001; Hamseveni et al. 2001; Elibol 2004).
 Several factors (incubation temperature, pH, nutrients,
agitation, aeration and phase variation of the bacteria) are
known to affect the growth and production of antibiotics
of Xenorhabdus spp. (Webster et al. 2002). Some studies
have investigated the effects of these factors on the antibi-
otic activity of X. nematophila by one-factor-at-a-time
approach (Akhurst 1982; Chen et al. 1996; Yang et al.
2001, 2006), but little information exists on their com-
bined or interactive effects. Therefore, in this study, RSM
was used to optimize suitable fermentation conditions for
antibiotic production by X. nematophila YL001 and to
evaluate the relative significance of several affecting fac-
tors.
Materials and methods
Organism
Xenorhabdus nematophila YL001 was isolated from its
nematode symbiont, Steinernema sp. YL001 obtained
from the soil from Yangling, China (Wang and Zhang
2006). Xenorhabdus nematophila occurs in two phases
(Boemare and Akhurst 1988). Phase I exhibits a
significantly higher level of antibiotic activity, thus was
used in this study.
Culture conditions and media
Xenorhabdus nematophila YL001 was maintained on
nutrient agar (NA) medium and subcultured monthly.
Because of the instability of the phase I under normal
culture condition, glycerinated stocks of the bacteria fro-
zen at )70°C were frequently used as starting material for
culture. To ensure the presence of phase I, the glycerin-
ated stocks were incubated in the dark at 28°C on NA
supplemented with 0Æ004% triphenyltetrazolium chloride
(w ⁄ v) and 0Æ0025% bromothymol blue (w ⁄ v)
(NBTA). Phase I is distinguished from phase II by its
adsorption of NBTA to produce a red core colony
overlaid by dark blue and surrounded by a clear zone
after 3–5 days of incubation.
Seed culture for antibiotic production was prepared by
inoculating a loopful of phase I X. nematophila YL001
from a 72 h culture growing on an NBTA plate into a
250 ml flask containing 50 ml fresh YSG medium (glyc-
erol and yeast extract at 5 and 15 g l )1; 1 mol l)1 MgSO4,
5 ml; (NH4)2SO4, 2 g l)1; 1 mol l)1 KH2OP4, 5 ml;
1 mol l)1 K2HOP4, 5 ml; and 1 mol l)1 Na2SO4, 10 ml)
(Sundar and Chang 1993), which was used in all seeding
experiments. Media were adjusted to a final pH of 7Æ0
with 1 mol l)1 NaOH solution to provide optimal condi-
tion of growth for X. nematophila YL001. The flasks were
incubated in the dark at 28°C on an Eberbach rotary sha-
ker at 150 rev min)1 for 18–24 h until the optical density
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 735–
744
(600 nm) and pH readings were approx. 2Æ0 and
7Æ0, respectively. Then seeds culture was transfered into
differ- ent volume sterile medium in 250 ml flask and
allowed to grow for 72 h, each flask containing 8Æ0%
inoculums. The cultures were centrifuged (22 400 g, 20
min, 4°C) to sep- arate the bacterial cells and the
supernatants were stored at 4°C until required.
Assay of antibiotic activity
Antibiotic activity was measured by an agar diffusion plate
assay with B. subtilis (Maxwell et al. 1994). Briefly, 1 ml of
medium containing 107–108 colonies of B. subtilis was
applied to an NA plate. After 2 h incubation at room tem-
perature, the supernatants were filter sterilized (0Æ22 lm
syringe microfilter). The filtrates (100 ll) were placed on 6-
mm disc filters (Whatman 3-mm paper; Whatman, Clif-
ton, NJ) and air dried. The dried discs were put on the
NA plate and incubated at 28°C for 24 h to determine the
relationship between the size of the zones of inhibition of
bacterial growth and the concentration of the antibiotic.
Antibiotic activity was expressed as units of activity per
millilitre of the supernatants, where 1 U was defined as a
1Æ0-mm annular clearing around the antibiotic disc.
Maxwell et al. (1994) confirmed the assumption that
the changes in the size of the zones of inhibition
(expressed as units of activity per gram of insect tissue)
represented changes in antibiotic concentration, the
antibiotics were extracted from insect larvae killed by
X. nematophila by homogenizing the insects in distilled
water. So, the size of the zones of inhibition served as a
measure of antibiotic titer of X. nematophila YL001.
Experimental design and optimization by RSM
RSM consists of a group of statistical techniques devoted
to the evaluation of relations existing between a cluster of
controlled experimental factors and the measured
responses, according to one or more selected criteria
(Silva and Roberto 2001). A prior knowledge and under-
standing of the process and the process variables under
investigation are necessary for achieving a more realistic
model (Akhnazarova and Kefarov 1982). Therefore, RSM
was applied in two stages, first to identify the significant
variables for antibiotic production using one-factor-at-a-
time approach, orthogonal or Plackett–Burman designs,
and later the significant variables were optimized by using
a central composite design (CCD).
Earlier one-factor-at-a-time approach had been fol-
lowed to identify the parameters (variables) having signif-
icant effect on antibiotic production by X. nematophila. It
was found that temperatures, aeration, initial pH and
inoculation volume had profound effects on production
73
7
 Table 1 Experimental range and levels of the independent variables
the axis of each design variable at a distance of 2Æ0
Range and levels (2n)1 ⁄ 4 = 2Æ0 for n = 5) from the design centre (10).
Hence, the total number of design points (36) is
Variable
N = 2n)1 0
+ n0, which indicated)2that)136 experiments
+ 2n Parameter 1 2
were Xrequired
1 pH for this procedure. The
4Æ0 ‘s
5Æ5 T a TI s7Æ0 6.0’
TI ca 8Æ5
10Æ0
X2 Medium volume (ml) 25 50 75 100 125 software (StatSoft, Inc., Tulsa, OK, USA) was used for
X3 Rotary speed (rev min)1) 80 115 150 185 220 regression and graphical analyses of the data obtained.
X4 Temperature (°C) 20Æ0 24Æ25 28Æ5 32Æ75 The optimum combinations of independent variables
37Æ0 were determined by optimizing the second-order polyno-
X5 Inoculation volume (%) 1Æ0 4Æ5 8Æ0 11Æ5 15Æ0
mial equation using the ‘sTaTIsTIca 6.0’ software and
xi = coded value of the variable Xi; x1 = (pH )7) ⁄ 1Æ5; x2 = also by analysing the response surface contour plots
(medium volume )75) ⁄ 25; x3 = (rotary speed )150) ⁄ 35; x4 = (Khuri and Cornell 1987).
(temperature
The statistical analysis of the model was performed in
)28Æ5) ⁄ 4Æ25; x5 = (inoculation volume )8) ⁄ 3Æ5.
the form of analysis of variance (anova). This analysis
included the Fisher’s F-test (overall model significance),
of antibiotics (Chen et al. 1996; Yang et al. 2001, 2006). its associated probability P(F), correlation coefficient R
Hence, in flask fermentation experiments using X. ne- and determination coefficient R2 that measures the good-
matophila YL001, the initial pH (X1), medium volume in ness of fit of regression model. The analysis also include
flask (X2, ml), rotary speed (X3, rev min)1), temperature the Student’s t-value for the estimated coefficients and
(X4, °C) and inoculation volume (X5, %) were selected to associated probabilities, P(t). For each variable, the qua-
find the optimized conditions for antibiotic production dratic models were represented as contour plots.
using CCD and RSM.
The range and the levels of the variables investigated in
Results
this study are given in Table 1. The central values (zero
level) chosen for experimental design were: pH, 7Æ0; In order to search for the optimum combination of the
med- ium volume, 75 ml; rotary speed, 150 rev min )1; variables, experiments were performed according to the
temper- ature, 28Æ5°C; inoculation volume, 8Æ0%. In CCD experimental plan (Table 2) and the corresponding
developing the regression equation, the test variables experimental data were shown in Table 2. By applying
were coded according to the equation.
ðXi — multiple regression analysis on the experimental data, the
x ¯¼
X iÞ i ¼ 1; 2; 3; :::; k
ð1Þ
i
DXi experimental results of the CCD design were fitted with a
second-order polynomial equation. The results of the
where xi is the independent variable coded value, Xi the regression analysis are shown in Table 3. The Student’s t-
independent variable real value, X¯ i the independent test and P-values were used as a tool to check the signif-
vari- able real value on the centre point and DXi the step icance of each coefficient, which also indicated the inter-
change value. The response variable (antibiotic activity action strength between each independent variable. The
units) was fitted by a second-order model in order to larger the magnitude of the t-value and smaller the P-
correlate the response variable to the independent vari- value, the more significant is the corresponding coeffi-
ables. The general form of the second degree polynomial cient (Elibol 2004). It can be seen from the degree of sig-
equation is nificance (Table 3) that the linear term regression
X X X X
Y¼b
þ bx þ þ b ð2Þ coefficients of b2, b3, b4 and b5 were significant at 5%
b x x2
0 i i ij ij ii i
i i j
level. Among the linear terms, the main effects of med-
ium volume in flask and rotary speed on the antibiotic
where Y is the measured response, b0 the intercept term, employed to fit the second-order polynomial model. The
bi, bij and bii the measures of the effects of variables xi, CCD primarily consists of (i) a 2n)1-factorial experimen-
xixj and x2i, respectively. The variable xixj represents the tal design, where n is the number of test variables, (ii) n0
first-order interaction between xi and xj (i < j). centre points (10) (n0 ‡ 1) and (iii) two axial points on
For the conditions of fermentation optimization, CCD
for five independent variables each at five levels with 10
axial points and 10 replicates at the centre points were
activity were highly significant as was evident from their
respective P-values (Pb2 = 0Æ0000 and Pb3 = 0Æ0000). Tem-
perature also had significant effect on the antibiotics
activity at linear terms (Pb4 < 0Æ0008). Rotary speed had
highest impact on the production of antibiotics as given
by highest linear coefficient (19Æ91), followed by
medium volume in flask ()18Æ21), temperature ()9Æ54)
and inocu- lation volume (5Æ98). Quadratic coefficients
of b11 and b44 (pH and temperature) were significant at
1% level and showed negative effects on the production of
antibiotics, indicating that the production of antibiotics
increased as
 Table 2 Central composite design plan in coded value, the observed Table 3 Regression results from the data of central composite
and predicted response designed experiments

Observed Predicted Model Parameter Computed

value value parameter estimate SE† t-value P-value
Runs X1 X2 X3 X4 X5 (U ml)1) (U ml)1) Residual
b0 231Æ3306 3Æ545080 65Æ2540 0Æ00000**
1 1 1 1 1 1 153Æ3 152Æ0 1Æ25278 b1 3Æ47 2Æ320799 1Æ49374 0Æ15470
2 1 1 1 )1 )1 146Æ7 145Æ8 0Æ86111 b2 )18Æ21 2Æ320799 7Æ80981 0Æ00000**
3 1 1 )1 1 )1 121Æ7 108Æ4 13Æ28611 b3 19Æ91 2Æ320799 8Æ57822 0Æ00000**
4 1 1 )1 )1 1 150Æ0 152Æ8 )2Æ78889 b4 )9Æ54 2Æ320799 4Æ11137 0Æ00082**
5 1 )1 1 1 )1 179Æ2 199Æ5 )20Æ25556 b5 5Æ98 2Æ320799 2Æ57813 0Æ02022*
6 1 )1 1 )1 1 245Æ0 243Æ8 1Æ16944 b11 )32Æ83 2Æ009871 16Æ33604 0Æ00000**
7 1 )1 )1 1 1 166Æ7 163Æ5 3Æ24444 b22 )3Æ56 2Æ009871 1Æ77043 0Æ09570
8 1 )1 )1 )1 )1 179Æ0 157Æ2 21Æ75278 b33 )2Æ67 2Æ009871 1Æ32886 0Æ20254
9 )1 1 1 1 )1 140Æ0 155Æ1 )15Æ05556 b44 )27Æ35 2Æ009871 13Æ60576 0Æ00000**
10 )1 1 1 )1 1 170Æ0 172Æ8 )2Æ78056 b55 )2Æ42 2Æ009871 1Æ20447 0Æ24592
11 )1 1 )1 1 1 136Æ7 135Æ4 1Æ34444 b12 )7Æ49 2Æ842387 2Æ63423 0Æ01804*
12 )1 1 )1 )1 )1 143Æ3 155Æ8 )12Æ49722 b13 )4Æ67 2Æ842387 1Æ64474 0Æ11952
13 )1 )1 1 1 1 176Æ7 196Æ4 )19Æ74722 b14 0Æ53 2Æ842387 0Æ18470 0Æ85578
14 )1 )1 1 )1 )1 233Æ3 216Æ9 16Æ41111 b15 6Æ66 2Æ842387 2Æ34398 0Æ03232*
15 )1 )1 )1 1 )1 121Æ7 136Æ5 )14Æ81389 b23 )10Æ74 2Æ842387 3Æ77763 0Æ00165**
16 )1 )1 )1 )1 1 136Æ7 154Æ2 )17Æ53889 b24 5Æ71 2Æ842387 2Æ00975 0Æ06163
17 2 0 0 0 0 100Æ0 94Æ2 5Æ76944 b25 2Æ9 2Æ842387 1Æ02027 0Æ32278
18 )2 0 0 0 0 100Æ0 94Æ2 5Æ76944 b34 )5Æ23 2Æ842387 1Æ83824 0Æ08466
19 0 2 0 0 0 177Æ5 189Æ3 )11Æ81389 b35 1Æ34 2Æ842387 0Æ47056 0Æ64431
20 0 )2 0 0 0 256Æ7 261Æ8 )5Æ11389 b45 4Æ46 2Æ842387 1Æ56998 0Æ13598
21 0 0 2 0 0 268Æ0 265Æ4 2Æ61944
22 0 0 )2 0 0 173Æ3 185Æ7 )12Æ44722 *Significant at 5% level.
23 0 0 0 2 0 116Æ7 97Æ1 19Æ60278 **Significant at 1% level.
24 0 0 0 )2 0 127Æ2 135Æ3 )8Æ06389
†
Standard error.
25 0 0 0 0 2 240Æ0 237Æ5 2Æ46944
26 0 0 0 0 )2 203Æ3 213Æ6 )10Æ29722 bacterial growth and antibiotic production (Akhurst 1982;
27 0 0 0 0 0 223Æ3 225Æ6 )2Æ26389 Chen et al. 1996).
28 0 0 0 0 0 220Æ0 225Æ6 )5Æ56389 Using the designed experimental data (Table 2), the
29 0 0 0 0 0 226Æ7 225Æ6 1Æ13611 polynomial model for the antibiotics activity (Y) was
30
regressed0by 0
only 0considering
0 0 230Æ0 225Æ6
the significant terms 4Æ43611
and was shown as below:
31 0 0 0 0 0 233Æ3 225Æ6 7Æ73611
32 0 0 0 0 0 256Æ7 225Æ6 31Æ13611
33 0 0 0 0 0 230Æ0 225Æ6 4Æ43611 Y ¼ 231·3305 — 18·1250x2 þ 19·9083x3 — 9·5416x4
þ 5·9833x
35
34 05 — 0 0 1x20 þ
7·4875x 6·6625x
0 233Æ31 x5 — 10·7375x
225Æ6 2x3 7Æ73611
36 0 0 0 0 0 226Æ7 225Æ6 1Æ13611
— 32·8333x12 — 27·3458x4 ð3Þ
where Y is the response (the antibiotic activity units) and
x1, x2, x3, x4 and x5 are the coded values of the indepen-
the level of these factors increased and decreased as the dent variables, viz., initial pH, medium volume in flask,
level of these parameters increased above certain values. rotary speed, temperature and inoculation volume,
The result also indicated that pH and temperature could respectively.
act as limiting factors and small variations in their values The results of the second-order response surface model
will considerably alter either growth rate or product for- fitting in the form of anova are given in Table 4. The
mation rate or both. Interactive terms coefficients of b12, goodness of fit of the model based on RSM can be
b15 and b23 were also significant at 5% level. The interac- checked by the coefficient of determination (R2), which
tion between medium volume in flask and rotary speed provides a measure of how much variability in the
had significant effects on the production of antibiotics observed response values can be explained by the experi-
(Pb23 < 0Æ001). These results suggested that medium vol- mental factors and their interactions. The R2 value is
ume in flask and rotary speed had a direct relationship always between 0 and 1. The closer the R2 value is to
with the antibiotic activity in this condition. The result 1Æ00, the stronger the model is and the better it
fitted in the previous work, in which aeration influence predicts the response (Kaushik et al. 2006). In this case,
the coeffi-
Table 4 Analysis of variance for the quadratic model 300
280
Source of

Predicted values / U ml–1

260
variations d.f. SS MS F-value P>F 240
Model 9 82319Æ07 9146Æ563 49Æ10156 0Æ0000* 220
200
Residual 26 4843Æ240 186Æ2785
180
Lack of fit 17 3948Æ699 232Æ2764 2Æ336939 0Æ098155
160
Pure error 9 894Æ5410 99Æ39344 92Æ02381 0Æ00000 140
Total 35 87162Æ31 120
100
Coefficient of correlation (R) = 0Æ9718; coefficient of determination
80
(R2) = 0Æ9444; adjusted R2 = 0Æ9252. 60
*Significant at 1% level. 80 100 120 140 160 180 200 220 240 260 280 300
SS, sum of squares; MS, mean square. Observed values / U ml–1

cient of determination R2 = 0Æ9444, which implied that Figure 1 Residual diagnostics of contour surface of the quadratic
antibiotic activity2 was attributed to the given independent model of the predicted vs observed by replication of antibiotic
activity (U ml)1).
variables. The R also indicated that only 5% of the total
variations was not explained by the model. The value between the
of the adjusted determination coefficient (adjusted
R2 = 0Æ9252) was also high to indicate a high
significance of the model. These measures indicated that
the accuracy and general ability of the polynomial model
was good and that analysis of the response trends using
the model was reasonable. The anova of the quadratic
regression model demonstrated that the model was highly
signifi- cant, as was evident from the low P-value of the
Fisher’s F-test (Fmodel, mean square regression ⁄ mean
square residual = 49Æ1016) [(Pmodel>F) = 0Æ0000].
Moreover, the computed F-value was much greater than
the tabulated F-value (F0Æ01 (9, 9) = 5Æ35), indicating that
the treatment differences were highly significant. This
proved that the model equation as expressed in eqn (3)
provides a suit- able model to describe the response of the
experiment pertaining to antibiotic activity. The model
also showed a statistically insignificant lack of fit, as is
evident from the lower calculated F-value (2Æ34) than the
tabulated F-value (F0Æ01 (9, 17) = 3Æ68) even at the 0Æ01
confidence level. The model was found to be adequate
for prediction within the range of variables employed. A
higher value of the correlation coefficient (R = 0Æ9718)
indicates a good agreement between the experimental
and predicted values of antibiotic activity (Table 2).
Analyses of the observed vs predicted yields are shown
in Fig. 1. Point above or below the diagonal line
represented areas of over or under prediction. This figure
showed that no significant violations of the model were
found in the analysis, with a good correlation of the model
with the experimental data obtained.
The 3D response surface and the 2D contour plots
described by the regression model were drawn to
illustrate the effects of the independent variables, and
interactive effects of each independent variable on the
response variables. The shape of the corresponding
contour plots indicates whether the mutual interactions
independent variables are significant or not. An elliptical
nature of the contour plots indicates that the interactions
between the independent variables were significant. From
the 3D response surface plots and the corresponding con-
tour plots, the optimal values of the independent vari-
ables and the corresponding response could be predicted,
and the interaction between each independent variable
pair could be understood. The maximum predicted value
is indicated by the surface confined in the smallest ellipse
in the contour diagram. Figure 2a–h shows the 2D con-
tour plots of antibiotic activity for each pair of variables
by keeping the other three variables constant at its middle
level. It could be seen that the antibiotic activity increased
upon increasing the initial pH from 4Æ0 to 7Æ0, but any
further increase in its values resulted in decreased antibi-
otic activity. Therefore, the optimal initial pH was around
7Æ0. Similarly, the antibiotic activity increased with
increasing the temperature from 20Æ0 to 28Æ5°C, and any
further increase in its values resulted in decreased antibi-
otic activity. Therefore, the optimal temperature was
around 28Æ5°C (Fig. 2c,f–h). There was a significant
mutual interaction between medium volume in flask and
rotary speed (Fig. 2e). Under moderate initial pH and
temperature, the antibiotic activity increased with increas-
ing the rotary speed, and decreased with increasing the
medium volume in flask (Fig. 2a,b,f,g). The optimal med-
ium volume in flask and rotary speed were around 25 ml
and 200 rev min)1. Figure 2d,h shows that the optimal
inoculation volume was around 15Æ0%.
The optimal levels of fermentation conditions were cal-
culated by solving the regression equation (3).
The optimal values of the test variables in coded unit
area are as follows:
x1 ¼ 0·42979; x2 ¼ —2·0000; x3 ¼ 2·0000;
x4 ¼ —0·17619; x5 ¼ 2·0000
 (a) 2·0
(b) 2·0
1·5
1·5
1·0
Medium volume
1·0
0·5

Rotary speed
0·5
0·0
0·0
–0·5
–0·5
–1·0 220
180 –1·0 220
–1·5 140 180
100 –1·5 140
–2·0 60 100
20 –2·0 60
–2·0 –1·5 –1·0 –0·5 0·0
pH 0·5 1·0 1·5 2·0 –2·0 –1·5 –1·0 –0·5 0·0 0·5 1·0 1·5 2·0
pH
(c) 2·0
(d) 2·0
1·5
1·5
1·0
1·0

Inoculation volume
Temperature

0·5
0·5
0·0
0·0
–0·5
–0·5
–1·0
200 –1·0
–1·5 150 180
100 –1·5 140
–2·0 50 100
–2·0 –1·5 –1·0 –0·5 0·0 0 –2·0 60

pH 0·5 1·0 1·5 2·0 –2·0 –1·5 –1·0 –0·5 0·0 0·5 1·0 1·5 2·0
pH

(e) 2·0 (f 2·0

1·5
) 1·5
1·0 1·0
Rotary speed

Temperature

0·5 0·5
0·0 0·0
–0·5 260 –0·5
240
–1·0 220 –1·0 220
200 180
–1·5 180 –1·5 140
140 100
–2·0 120 –2·0 60
–2·0 –1·5 –1·0 –0·5 0·0 0·5 1·0 1·5 2·0 –2·0 –1·5 –1·0 –0·5 0·0 0·5 1·0 1·5 2·0
Medium volume Medium volume

(g) 2·0 (h) 2·0

1·5 1·5
Inoculation volume

1·0 1·0
Temperature

0·5 0·5
0·0 0·0
200
–0·5 –0·5 180
160
–1·0 220 –1·0 140
180 120
–1·5 140 –1·5 100
100 80
–2·0 60 –2·0 60
–2·0 –1·5 –1·0 –0·5 0·0 0·5 1·0 1·5 2·0 –2·0 –1·5 –1·0 –0·5 0·0 0·5 1·0 1·5 2·0
Rotary speed Temperature

Figure 2 Contour plot of antibiotic activity of Xenorhabdus nematophila YL001 (U ml)1): (a) the effect of pH and medium volume on antibiotic
activity, (b) the effect of pH and rotary speed on antibiotic activity, (c) the effect of pH and temperature on antibiotic activity, (d) the effect of
pH and inoculation volume on antibiotic activity, (e) the effect of medium volume and rotary speed on antibiotic activity, (f) the effect of
medium vol-
ume and temperature on antibiotic activity, (g) the effect of rotary speed and temperature on antibiotic activity and (h) the effect of
temperature and inoculation volume on antibiotic activity.
withthe corresponding Y = 363Æ5 U ml)1. The natural val-
ues obtained by uncoding them using eqn (1) are initial
pH 7Æ64, medium volume in flask 25 ml ⁄ 250 ml,
rotary speed 220 rev min)1, temperature 27Æ8°C and
inoculation volume 15Æ0%. The model predicted that the
maximum antibiotic activity unit that could be obtained
using the above optimum concentrations of the
variables was 363Æ5 U ml)1. The verification of the
results using the optimized conditions was
accomplished by carrying out shake-flask experiments.
The maximum antibiotic activity unit obtained
experimentally was found to be 331Æ7 U ml)1. This was
obviously in close agreement with the model prediction.
After optimization, the antibiotic activity was improved
by 43Æ4%, compared with that (231Æ3 U ml)1) at the
condition before optimization (Table 5).
Discussion
In this study, we focused on the optimization of culture
conditions for production of antibiotics by X. nematophila
YL001 through factorial design and response surface
analy- sis, although nutritional constituents also play an
impor- tant role and could as well be improved. The
approach allowed the determination of the culture
conditions that give the highest antibiotics activity for X.
nematophila YL001. In the cases, suitable model was
found to describe the response of the experiments
pertaining to the produc- tion of antibiotics, as the values
obtained experimentally are in accordance with the
expected values determined by the model. The model was
validated by comparing the observed and predicted values
in the optimum point. The optimization procedure allowed
an increase in the antibi- otics activity of X. nematophila
YL001.
Production of antibiotics by Xenorhabdus spp. is gener-
ally believed to be an aerobic process (Akhurst 1982;
Chen et al. 1996), so dissolved oxygen (DO) is an impor-
tant factor in the fermentation of X. nematophila. The cell
growth and production of antibiotics by X. nematophila
YL001 in 7-l fermentor are strongly affected by DO con-
Table 5 Experimental verification of combined effect of optimized condition on the response of antibiotic activity
Levels after optimization
Levels before
Variables optimization Coded
pH 7Æ0 0Æ42979
Medium volume (ml) 50Æ0 )2Æ0000
Rotary speed (rev min)1) 150 2Æ0000
Temperature (°C) 28Æ0 )0Æ17619
centration. Controlling the DO concentration above 30%,
the dry cell weight and antibiotic activity were improved
by 24Æ0% and 7Æ0%, respectively, compared with
fermen- tation without DO control (Wang and Zhang
2007). In shaken flasks, oxygen supply is related to
medium volume in flask and rotary speed. Decreasing
medium volume in flask or increasing rotary speed of
flasks could improve the DO level in medium. In this
study, medium volume in flask and rotary speed were
found to be the most important variable influencing
antibiotic activity of
X. nematophila YL001 at individual level and showed a
significant influence at interactive level (Table 3). Antibi-
otic activity increased as culture volume decreased or
rotary speed of flasks increased (Fig. 2a,b,f,g), indicating
that a higher DO level is beneficial to antibiotic produc-
tion. The results are in agreement with those obtained in
a 7-l fermentor.
The analysis of significance of each coefficient of the
quadratic regression model indicated that initial pH and
temperature have significant effect on antibiotic activity
of X. nematophila YL001 (Table 3). Higher pH and lower
pH are not beneficial to antibiotic production (Fig. 2a–
d). This result suggested that the broth pH could play a
crucial role in the behaviours of secondary metabolite
production, which showed a good agreement with previ-
ous works (Buckland et al. 1989; Yang et al. 2001, 2006).
One possible reason was that the key enzymes that domi-
nate antibiotics synthesis become inactivated or degener-
ated. Moreover, it is widely accepted that secondary
metabolism of micro-organisms represents an important
pathway for survival. A lower temperature could be
viewed as one type of environmental pressures, and sec-
ondary metabolism production might therefore be
enhanced (Lai et al. 2005). But in this study, when the
initial pH, medium volume, rotary speed and inoculation
volume were kept at its middle level, the maximum anti-
biotic activity (256Æ7 U ml)1) was produced at
middle level of temperature (28Æ5°C). Under 20Æ0°C and
37Æ0°C, the antibiotic activities were 127Æ2 and 116Æ7
U ml)1, respectively (Fig. 2d,f–h). The results indicate
that higher
Antibiotic activity units (U ml)1)
After optimization
Before
Uncoded optimization Predicted Experimental
7Æ64
25Æ0
220 231Æ3 363Æ5 331Æ7
27Æ8
Inoculation volume (%) 8Æ0 2Æ0000 15Æ0
and lower temperatures are not beneficial to antibiotic Nematode’ is gratefully acknowledged.
production. This is in good accordance with the previous
work (Chen et al. 1996) and the result of the significance
analysis of regression coefficients. The reason may be that
the influence of temperature on antibiotic production
varies from strain to strain. So, we could reasonably spec-
ulate that the optimum temperature might induce the
associated genes expression and ⁄ or activate the key
enzymes that dominate antibiotics synthesis by X. ne-
matophila YL001.
It is important to provide an optimum inoculum level
in fermentation process. A lower inoculum density may
give insufficient biomass causing reduced product forma-
tion, whereas a higher inoculum may produce too much
biomass leading to the poor product formation (Mudgetti
1986). In this study, the inoculation volume has signifi-
cant effect on antibiotic activity of X. nematophila YL001
(Table 4). In the range studied, the antibiotics activity
increased with increasing the inoculation volume, higher
inoculation volume is beneficial to antibiotic production
(Fig. 2h). However, Yang et al. (2006) reported for
another symbiotic bacteria, Xenorhabdus sp. D43, that
inoculation volume (2–10%) has insignificant effect on
antibiotic activity. Since the two results were obtained for
two different strains and different conditions, no
definitive conclusion on the effect of inoculation vol-
ume on antibiotic activity of Xenorhabdus spp. could be
made.
In conclusion, using the CCD and RSM, the fermenta-
tion conditions for production of antibiotics by X. ne-
matophila YL001 in shake flask system were optimized.
The optimal conditions were initial pH 7Æ64,
medium volume in 250 ml flask 25 ml, rotary
speed of 220 rev min)1, temperature 27Æ8°C and
inoculation volume of 15Æ0%. Higher
antibiotic activity (331Æ7 U ml)1) was obtained under
the optimization of the environmental parameters, which
was obviously higher than that under unoptimized
conditions. The chosen methods were proved to be a
powerful tool for the optimization of production of
antibiotics by X. ne- matophila YL001. Furthermore, the
information obtained is considered fundamental and
useful for the development of X. nematophila YL001
cultivation process for efficient production of antibiotic
on a large scale.

Acknowledgements
The financial support by the Ministry of Science and
Technology of the People’s Republic of China to the
National High Technology Research and Development
Program (863 program) ‘Research on the Pesticides of the
Bacterial Symbionts Associated with Insect-Pathogenic
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