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Optimization of medium composition and culture conditions for agarase

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DOI: 10.1016/j.procbio.2009.06.012

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Optimization of medium composition and culture conditions for agarase

production by Agarivorans albus YKW-34
Xiao Ting Fu a,b, Hong Lin a, Sang Moo Kim b,*
a
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
b
Faculty of Marine Bioscience and Technology, Kangnung-Wonju National University, 120 Gangneungdaehangno, Gangneung 210-702, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-
Received 7 February 2009 34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture
Received in revised form 16 June 2009 temperature were agar, yeast extract, and 25 8C, respectively, for agarase production by one-factor-at-a-
Accepted 18 June 2009
time design. The nutritional components of the medium and culture conditions were analyzed by
Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had
Keywords: significant effects on agarase production (p < 0.05). The optimum levels of these key variables were
Agarase
further determined using a central composite design. The highest agarase production was obtained in
Agarivorans albus
the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization
Enzyme production
Optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium
Plackett–Burman design composition and culture condition as well as the dominant occupation of agarase with high activity in
Central composite design culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.
ß 2009 Elsevier Ltd. All rights reserved.

1. Introduction optimization. For example, it can be applied to select the best

Agar is a mixture of polysaccharides extracted from the cell wall
of red algae (Rhodophyta), especially the genus of Gracilaria and
Gelidium [1]. Agarases, including a-agarases (EC 3.2.1.158) and b-
agarases (E.C. 3.2.1.81), catalyze the hydrolysis of agar. Agarases
were applied to produce agar-derived oligosaccharides, which
inhibited the growth of bacteria, slowed down the degradation of
starch, exhibited anticancer and antioxidative activities [2], and
had a moisturizing effect on skin and a whitening effect on
melanoma cells [3]. Due to these characteristics, agarases have
potential applications in food, pharmaceutical, and cosmetics
industries. Agarases were also used to prepare protoplasts of
marine algae [4] and to recover DNA from agarose gel [5]. So far,
several agarases have been isolated from different microorgan-
isms, including Alteromonas [6,7], Pseudoalteromonas [8], Vibrio [9],
Cytophaga [10], and Acinetobacter [11].
One-factor-at-a-time design was a traditional method for
optimization. The disadvantages of this method are that it is time-
consuming and potential interaction effects among factors are
ignored. However, it is useful to select the best experimental
treatment with a certain factor in the preliminary stage of
* Corresponding author. Tel.: +82 33 640 2343; fax: +82 33 647 9535.
E-mail address: smkim@kangnung.ac.kr (S.M. Kim).
carbon source among various carbohydrates for the growth of
microorganism. Statistical designs, such as Plackett–Burman design
and central composite design, overcome the disadvantages of one-
factor-at-a-time design by reducing the number of the tests while
giving meaningful results. Thus, the statistical designs have been
efficiently used for enhancing the yield of various bioprocesses [12].
Some researchers combined advantages of both methods to optimize
bioprocess by applying one-factor-at-a-time design followed by
statistical designs of the Plackett–Burman design and central
composite design [13,14]. For the optimization of agarase produc-
tion, one-factor-at-a-time method was reported in some literature
[11,15,16]. To the best of our knowledge, the application of statistical
method to optimize the production of agarase has not been reported.
Strain YKW-34 isolated from the gut of a turban shell [17] could
degrade the cell walls of both brown algae Laminaria japonica and
red algae Gelidium amansii [18,19]. In our previous study, the strain
was identified as Agarivorans albus. An alginate lyase [18] and two
agarases (AgaA34 [19] and AgaB34 [20]) with excellent properties
were purified and characterized, and the production of alginate
lyase was optimized [21]. In this paper, one-factor-at-a-time
design and two stages of Plackett–Burman design and central
composite design were applied to establish the effect of different
medium composition and culture conditions on agarase produc-
tion and to establish the optimum cultivation for agarase
production by A. albus YKW-34.

1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.06.012

Please cite this article in press as: Fu XT, et al. Optimization of medium composition and culture conditions for agarase production by
Agarivorans albus YKW-34. Process Biochem (2009), doi:10.1016/j.procbio.2009.06.012
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2. Materials and methods Table 2

Experimental design and results of Plackett–Burman design.
2.1. Microorganism and growth medium
Run X1 X2 X3 X4 X5 X6 X7 X8 X9 Agarase activity
The strain YKW-34 used in this study was originally isolated from the gut of the (U/ml)
turban shell, Turbinidae batillus cornutus [17]. It was identified as A. albus based on its
16S rRNA gene sequence (GenBank accession number EU084496) [19]. The seed was 1 +  +    + + + 0.64  0.10
maintained at 80 8C in a marine broth medium (Difco Laboratories, Detroit, MI, USA) 2 + +  +    + + 0.75  0.03
containing 20% (v/v) glycerol. The inoculum was prepared by incubating the seed 3  + +  +    + 0.66  0.04
culture at 20 8C with a shaking speed of 120 rpm for 12 h to reach a cell density of 4 +  + +  +    0.50  0.02
1.0  107 CFU/ml. For agarase production, the prepared inoculum was transferred to a 5 + +  + +  +   0.62  0.04
fermentation medium at a ratio of 1:10, and incubated at 25 8C with a shaking speed of 6 + + +  + +  +  0.61  0.06
120 rpm for 12 h. The fermentation medium was prepared by synthetic seawater 7  + + +  + +  + 0.63  0.11
(2.5% NaCl, 0.5% MgSO47H2O, 0.1% KCl, 0.02% CaCl2, 0.01% K2HPO4, 0.002% 8   + + +  + +  0.30  0.03
FeSO47H2O) supplemented with 0.1% agar and 0.1% yeast extract. The initial pH of 9    + + +  + + 0.46  0.05
the medium was adjusted to pH 7.0 by 1 M NaOH. Flasks (250 ml) containing 50 ml of 10 +    + + +  + 0.68  0.07
medium were used for both seed culture and agarase production. 11  +    + + +  0.48  0.11
12          0.32  0.04
2.2. Analytical methods

The cell growth was monitored by measuring the absorbance of culture fluid at Table 3
600 nm. The culture fluid was centrifuged at 12,000  g for 10 min, and then the Results of regression analysis of Plackett–Burman design.
supernatant was used for the determination of agarase activity and protein
concentration. Agarase activity was quantified by spectrometric determination of Factor code Coefficient Standard error t-Value p-Value
reducing sugar using Nelson method [22]. After incubating 50 ml of supernatant of
Intercept 0.55 0.0758
the culture fluid with 200 ml of 0.1% agarose in 20 mM Tris–HCl (pH 8.0) at 40 8C for
X1 0.079 0.0758 10.43 0.0091a
30 min, the amount of reducing sugar was determined by Nelson reagent. Enzyme
X2 0.071 0.0758 9.42 0.0111a
activity (U/ml) was defined as the amount of enzyme required to liberate 1 mmol
X3 0.00275 0.0758 0.36 0.7515
reducing sugar per min. Protein concentration in the culture fluid was determined
X4 0.00942 0.0758 1.24 0.3400
by Bradford method [23] using bovine serum albumin as the calibration standard.
X5 0.00058 0.0758 0.077 0.9457
X6 0.00692 0.0758 0.91 0.4578
2.3. One-factor-at-a-time design X7 0.00458 0.0758 0.60 0.6069
X8 0.014 0.0758 1.79 0.2150
Effect of carbon and nitrogen sources and culture temperature on agarase
X9 0.083 0.0758 10.92 0.0083a
production by A. albus YKW-34 was estimated using one-factor-at-a-time design by
a
the software package of Design Expert 7.0 (Stat-Ease Co., Minneapolis, MN, USA). Indicates model terms are significant.
Effect of various kinds of carbon source, i.e. agar, alginate, CM-cellulose (CMC),
fucoidan, glucose and galactose, each (0.1%) or in combination with agar (0.1% and points, 6 axial points and 6 trials at the center point (Table 5). All experiments were
0.1%), were investigated. Various kinds of nitrogen source, i.e. yeast extract, beef conducted in triplicate. The mean value of agarase activity (U/ml) was taken as the
extract, peptone, KNO3, and NH4Cl were applied individually (0.1%) to the response. A multiple regression analysis was applied to the data obtained. The
fermentation medium. The above cultures were carried out at 25 8C. Agarase behavior of the system was explained by the following quadratic equation:
activity was determined after cultivation for 12 h. To evaluate the effect of culture
temperature, the cultures were conducted at 20, 22.5, 25, 27.5 and 30 8C, X
k X
k X
k
respectively. Agarase activity was determined every 3 h, and the highest activity at Y ¼ b0 þ bi X i þ bi j X i X j (1)
i¼1 i¼1 j¼1
each culture temperature was noted. Triplicate experiments were carried out for
each treatment. Data were pooled and analyzed by Design Expert 7.0.
where Y is the predicted response (agarase activity, U/ml); b0, bi and bij are constant
coefficients; Xi and Xj are the coded independent factors. Statistical analysis of the
2.4. Statistical designs model was performed to evaluate the analysis of variance (ANOVA) by Design
Expert 7.0.
2.4.1. Plackett–Burman design
Effect of nine factors, including the carbon (agar) and nitrogen (yeast extract)
sources selected in the above experiment, components of synthetic seawater, and 2.5. In situ detection of agarase
the initial pH, on agarase production by A. albus YKW-34 was evaluated by Plackett–
The culture fluid was centrifuged at 12,000  g for 10 min, and then the
Burman design. The levels of each factor are listed in Table 1. A 12-run experiment
supernatant was concentrated by ultrafiltration (10-kDa cutoff membrane, Millipore,
was generated by Design Expert 7.0 (Table 2). All experiments were conducted in
Bedford, MA, USA). An aliquot of 10-ml enzyme preparation (0.32 mg/ml crude
triplicate. Data were pooled and analyzed by Design Expert 7.0. The factors which
protein) was applied to SDS-PAGE as described by Laemmli [24] on a 10%
were significant at 5% level (p < 0.05) were considered to have a significant effect on
polyacrylamide gel. After electrophoresis, in situ detection of agarase was performed
agarase production (Table 3).
as described in previous work [19]. To determine the molecular mass, the gel was
stained by Coomassie Brilliant Blue R-250 after electrophoresis. The molecular marker
2.4.2. Central composite design (Sigma Chemical, St. Louis, MO, USA) consisted of albumin (66 kDa), ovalbumin
Central composite design (CCD) was applied to determine the optimum level of the (45 kDa), carbonic anhydrase (29 kDa), and trypsin inhibitor (20 kDa).
significant factors (agar, yeast extract, and initial pH) identified by Plackett–Burman
design. The factors and respective coded and actual levels are given in Table 4. A 20-
run experiment generated by Design Expert 7.0 were carried out with 8 factorial 3. Results and discussion

Table 1 3.1. Evaluation of three factors by one-factor-at-a-time design

Range of variables of Plackett–Burman design.

Factor code Factor Levels The agarase production before optimization was 0.32 U/ml,
which was obtained by using the medium and culture conditions
1 +1

X1 (%) Agar 0.1 0.2 Table 4

X2 (%) Yeast extract 0.1 0.2 Range of variables of the central composite design.
X3 (%) NaCl 2.5 4.0
Factor code Factor Levels
X4 (%) MgSO47H2O 0.5 1.0
X5 (%) KCl 0.1 0.3 1.682 1 0 1 1.682
X6 (%) CaCl2 0.02 0.06
X7 (%) K2HPO4 0.01 0.03 X1 Agar (%) 0 0.10 0.25 0.40 0.50
X8 (%) FeSO47H2O 0.002 0.006 X2 Yeast extract (%) 0 0.10 0.25 0.40 0.50
X9 Initial pH 6.0 9.0 X3 Initial pH 4.98 6.00 7.50 9.00 10.02

Please cite this article in press as: Fu XT, et al. Optimization of medium composition and culture conditions for agarase production by
Agarivorans albus YKW-34. Process Biochem (2009), doi:10.1016/j.procbio.2009.06.012
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Table 5
Experimental design and results of the central composite design.

Run Coded level Agarase activity (U/ml)

X1 X2 X3 Actual value Predicted

value

1 1 1 1 0.30  0.02 0.19

2 1 1 1 0.23  0.04 0.14
3 1 1 1 0.40  0.02 0.32
4 1 1 1 0.15  0.03 0.20
5 1 1 1 0.46  0.02 0.33
6 1 1 1 0.25  0.04 0.25
7 1 1 1 0.44  0.03 0.47
8 1 1 1 0.28  0.02 0.31
9 1.682 0 0 0.18  0.02 0.31
10 1.682 0 0 0.16  0.01 0.13
11 0 1.682 0 0.08  0.03 0.24
12 0 1.682 0 0.46  0.02 0.40
13 0 0 1.682 0.17  0.08 0.27
14 0 0 1.682 0.49  0.06 0.49
15 0 0 0 0.80  0.07 0.82
16 0 0 0 0.79  0.07 0.82
17 0 0 0 0.84  0.06 0.82
18 0 0 0 0.81  0.11 0.82
19 0 0 0 0.85  0.05 0.82
20 0 0 0 0.85  0.03 0.82

described in Section 2.1. One-factor-at-a-time design was used in

the preliminary stage of optimization. A few reports described
selection of optimal carbon and nitrogen sources by this method
prior to using statistical optimization [25,26]. Time course of cell
growth and enzyme production associated with culture tempera-
ture [27], which indicated that the optimal harvest time varies
with culture temperature. Therefore, the effect of culture
temperature on agarase production was also evaluated by this
method. After the culture temperature is fixed, the optimal harvest
time can be consequently decided.

3.1.1. Evaluation of carbon source for agarase production

A. albus YKW-34 degraded the cell walls of Laminaria japonica
and Gelidium amansii. Therefore, the cell wall carbohydrates of
Laminaria japonica including alginate, cellulose, and fucoidan, and
those of Gelidium amansii including agar and cellulose, together
with their enzymatic products of glucose and galactose, were used
to induce agarase production. Furthermore, the combined effects
of agar with other kinds of carbon source were also investigated.
The effect of various kinds of sole carbon source on agarase
production was shown in Fig. 1a (carbon sources 1–7). No agarase
activity was detected without carbon source, while various kinds
of sole carbon source could induce agarase production by A. albus
YKW-34. It was different from the report of Lakshmikanth et al.
[15], in which Pseudomonas aeruginosa AG LSL-11 produced
agarase only when agar was used as the carbon source. The
maximum agarase production was obtained when agar was used
as the sole carbon source, while the agarase production decreased
to 50% and 25% of the maximum, respectively, when polysacchar-
ides (alginate, CMC, and fucoidan) and monosaccharides (glucose
and galactose) were used as a sole carbon source. The result
indicated that agar was the most effective carbon source for
agarase production by A. albus YKW-34. The combination effect of
different kinds of carbon source on agarase production is shown in
Fig. 1a (carbon sources 8–12). The combinations of agar with other
polysaccharides (alginate, CMC, and fucoidan) decreased agarase
production to 90% of the maximum, while the combinations of agar
with monosaccharides (glucose and galactose) significantly
decreased agarase production to 50% of the maximum. These
results indicated that enzymatic products exhibited repressive
effect on agarase production. The combinations of agar with other
polysaccharides were reported to completely repress agarase
Fig. 1. Effect of carbon and nitrogen sources, and culture temperature on agarase
production by Agarivorans albus YKW-34. (a) Effect of carbon source. 1: no carbon
source; 2: agar; 3: alginate; 4: CMC; 5: fucoidan; 6: glucose; 7: galactose; 8:
agar + alginate; 9: agar + CMC; 10: agar + fucoidan; 11: agar + glucose; 12:
agar + galactose. (b) Effect of nitrogen source. 1: no nitrogen source; 2: yeast
extract; 3: beef extract; 4: peptone; 5: KNO3; 6: NH4Cl. (c) Effect of culture
temperature. Bars represent the standard deviation from triplicate experiments.
production by P. aeruginosa AG LSL-11 [15], but to enhance agarase
production by Acinetobacter sp. AG LSL-1 [11]. A similar observa-
tion in catabolite repression was reported when agar was
combined with monosaccharide, in which the supplement of
glucose decreased agarase production to 20% of that obtained
when agar was used as the sole carbon source [16].
3.1.2. Evaluation of nitrogen source for agarase production
The effect of commonly used nitrogen source on agarase
production by A. albus YKW-34 is shown (Fig. 1b). No agarase
activity was detected without nitrogen source. The maximum
agarase activity was obtained when yeast extract was used as the
nitrogen source. Other two kinds of organic nitrogen source (beef
extract and peptone) decreased agarase production to 80% of the
maximum. The inorganic nitrogen source of KNO3 and NH4Cl
decreased agarase activity to 50% of the maximum. These results
indicated that nitrogen source was essential for the production of
agarase, and organic nitrogen source was better than inorganic one
for agarase production by A. albus YKW-34. The preference for
nitrogen source varies with microorganisms. The highest agarase

Please cite this article in press as: Fu XT, et al. Optimization of medium composition and culture conditions for agarase production by
Agarivorans albus YKW-34. Process Biochem (2009), doi:10.1016/j.procbio.2009.06.012
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production was obtained when yeast extract was used as the
nitrogen source by P. aeruginosa AG LSL-11 [15], while NaNO3 and
NH4NO3 were the optimal nitrogen source for agarase production
by Acinetobacter sp. AG LSL-1 [11] and Cytophaga flevensis [16],
respectively.
3.1.3. Evaluation of culture temperature for agarase production
A. albus YKW-34 could produce agarase at temperatures
ranging from 20 8C to 30 8C (Fig. 1c). The maximum agarase
activity was obtained when A. albus YKW-34 was cultured at 25 8C.
When cultured at temperatures above and below 25 8C, agarase
production was lower than that obtained at 25 8C. Culture
temperature was also found to significantly affect agarase
production in other reports, in which the optimal culture
temperature for agarase production was 20 8C by Cytophaga
flevensis [16], 30 8C by P. aeruginosa AG LSL-11 [15], and 37 8C
by Acinetobacter sp. AG LSL-1 [11]. The time course of agarase
production by A. albus YKW-34 at 25 8C was determined (data not
shown) and 12 h was found to be the optimal harvest time.
3.2. Screening of significant factors by Plackett–Burman design
After the optimal carbon and nitrogen sources, culture
temperature, and agarase harvest time were selected, Plackett–
Burman design was used to screen the significant factors
responsible for agarase production. A 9-factor-12-run Plackett–
Burman design was conducted, and the response for each run is
present in Table 2. Regression analysis was performed to evaluate
the significance of each factor (Table 3). Among the nine factors
studied, salts in synthetic seawater had no significant effect on
agarase production by A. albus YKW-34 (p > 0.2). Hence no further
optimization of these factors was carried out. Therefore the values
described in Section 2.1 were used as the optimized values for
these factors. Other three factors, i.e. agar, yeast extract, and initial
pH were found to have a significant effect on agarase production by
A. albus YKW-34 (p < 0.05). Plackett–Burman design can only
screen significant factors, but it is not adequate to determine the
optimum levels of these three factors, thus further optimization
was carried out by central composite design in this study. These
three factors had positive effects (t-value > 0) on agarase produc-
tion in the range of evaluated levels in Plackett–Burman design
(Table 1). Therefore their optimal levels were evaluated in a wider
range of levels by central composite design (Table 4).
3.3. Optimization of significant factors by central composite design
The actual and predicted values of agarase activity based on
CCD experimental design are shown in Table 5. Different
combinations of three factors yielded agarase activity ranging
from 0.08 U/ml to 0.85 U/ml. The p-values of liner, quadratic, and
cubic terms of the model were 0.682, <0.0001, and 0.0612,
respectively, which indicated the significance of the quadratic
terms. Thus, the following quadratic regression equation was
obtained to describe the agarase production:
Table 6
ANOVA table of the central composite design.
Source SSa DFb MSc F-Value p-Value
Model 1.34 9 0.15 13.78 0.0002
Residual 0.11 10 0.011
Total 1.44 19
Coefficient of variation 23.10
R2 0.93
Adjusted R2 0.86
Adequate precision 9.35
a
Sum of squares.
b
Degree of freedom.
c
Mean square.
model p-value of 0.0002 (desired <0.05) indicated that the model
terms were significant. The coefficient of variation of 23.10%
(desired <30%) indicated a good precision and reliability of the
experiments carried out. The adequate precision value measures
the signal-to-noise ratio. In this case, the value was 9.35 (desired
>4), which suggested an adequate signal. Those values indicated a
satisfactory fitness of the quadratic model.
The significance of each coefficient was determined by its p-
value. The smaller p-value indicated the higher significance of the
corresponding coefficient. As shown in Table 7, the second-order
terms of both agar (X12 ) and yeast extract (X22 ) (p < 0.0001), the
second-order term of initial pH (X32 ) (p = 0.0002), followed by the
first-order term of initial pH (X3) (p = 0.0444) had significant effects
on agarase production. Among these significant terms, each second-
order term showed a negative effect on agarase production (t-
value < 0). Thus a lower value of second-order term attributed to a
higher agarase production. The value of second-order term was low
when the level of each factor was close to the zero level. Therefore,
the optimum level of each factor was close to its respective zero level
which corresponded to the level of central point.
The three-dimensional response surface curve was plotted to
explain the interaction of three independent variables and to
determine the optimum condition (Fig. 2a–c). Each curve
represented a combined effect of two tested variables on the
response with the other variable maintained at its zero level. The
response surfaces were inverted paraboloids, which indicated that
agarase production gradually increased with increasing the value
of each factor up to each optimal point, thereafter gradually
decreased with further increasing the value of each factor. The
optimal levels of the three variables determined by Design Expert
7.0 were 0.23% of agar, 0.27% of yeast extract, and 7.81 of initial pH,
corresponding to the maximum agarase production of 0.83 U/ml.
The result indicated that lower or higher concentration of agar and
yeast extract showed negative effect on agarase production. It was
suggested that lower concentration of agar or yeast extract could
not fully support the cell growth, thus the agarase production was
Table 7
Results of regression analysis of the central composite design.

Factor code Coefficient Standard error t-Value p-Value

Agarase acti ¼ 0:820:052X 1 þ 0:048X 2 þ 0:065X 3 0:215X12 Intercept 0.82 0.042
0:18X220:16X32 0:0176X 1 X 2 0:006X 1 X 3 0:00006X 2 X 3 (2) X1 0.052 0.028 1.85 0.0945
X2 0.048 0.028 1.73 0.1152
where X1, X2 and X3 are the coded values of agar, yeast extract, and X3 0.065 0.028 2.30 0.0444a
X21 0.215 0.027 7.75 <0.0001a
initial pH, respectively. X22 0.18 0.027 6.42 <0.0001a
The statistical significance of the second-order model Eq. (2) X32 0.16 0.027 5.69 0.0002a
was evaluated by ANOVA (Table 6). The determination coefficient X1X2 0.0176 0.037 0.47 0.6508
(R2) of the model was 0.93, which indicated 93% of the variability in X1X3 0.006 0.037 0.17 0.8701
X2X3 0.00006 0.037 0.0015 0.9988
the response could be explained by this model. The R2 value of 0.93
was reasonably agreed with the adjusted R2 value of 0.86. The a
Indicates model terms are significant at 95% of confidence level.

Please cite this article in press as: Fu XT, et al. Optimization of medium composition and culture conditions for agarase production by
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Fig. 2. Response surface plots of agarase production by Agarivorans albus YWK-34.
(a) Interaction effect of agar and yeast extract on agarase production at fixed initial
pH of 7.81; (b) interaction effect of agar and initial pH on agarase production at fixed
yeast extract concentration of 0.27%; (c) interaction effect of yeast extract and
initial pH on agarase production at fixed agar concentration of 0.23%.
consequently lower than the optimum production. Higher con-
centration of agar caused higher viscosity of the culture medium,
thus the cell growth was hindered and agarase production was
consequently depressed. Higher concentration of yeast extract also
depressed agarase production. Mehta et al. reported that yeast
extract rich in amino acids and peptides displayed a repressive
effect on enzyme production when it was used at a higher
concentration [28]. The pH value of the medium affected the cell
growth of A. albus YKW-34 and stability of its agarase. The resulted
indicated that lower or higher pH than the optimal pH might cause
growth repression of this microorganism or degradation of its
agarase. The optimal initial pH values for agarase production
varied with the type of microorganisms. The optimal initial pH
Please cite this article in press as: Fu XT, et al. Optimization of medium composition and culture conditions for agarase production by
Agarivorans albus YKW-34. Process Biochem (2009), doi:10.1016/j.procbio.2009.06.012
5
Fig. 3. Time course of the cell growth and agarase production in optimized medium.
Samples were removed from the medium every 3 h up to 48 h. The optimized
medium was composed of 0.23% agar and 0.27% yeast extract at initial pH of 7.81.
Bars represent the standard deviation from triplicate experiments.
values for the production of agarase by P. aeruginosa AG LSL-11 [15]
and Acinetobacter sp. AG LSL-1 [11] were 8.0 and 7.0, respectively.
Further experiment was performed to confirm the predicted
agarase production using a medium representing the optimal
levels of the three variables. Agarase production with the activity
of 0.87 U/ml (n = 3) (Fig. 3) was obtained under the optimal
conditions. The good correlation between predicted and experi-
mental results confirmed the validation of the response model.
3.4. Determination of agarase production in optimized medium
Time course of the cell growth and agarase production by A.
albus YKW-34 in the optimized medium was determined (Fig. 3).
The amount of agarase production increased rapidly from 9 h to
12 h, maintained constantly from 12 h to 21 h, and then decreased
gradually. The maximum agarase production was 0.87 U/ml at
12 h, which was about three times higher than 0.32 U/ml obtained
with no optimization.
Purification and characterization of agarase were reported in
many literatures. However, there are only a few works related to
the optimization of agarase production by wild type microorgan-
isms [11,15]. The maximum agarase activities were 0.32 U/ml at
38 h by P. aeruginosa AG LSL-11 [15] and 0.45 U/ml at 18 h by
Acinetobacter sp. AG LSL-1 [11], respectively. It was noteworthy
that A. albus YKW-34 produced agarase efficiently with a higher
activity in a shorter fermentation period. Therefore, A. albus YKW-
34 can be a good potential for the production of agarase.
3.5. In situ detection of agarase
A clear zone on the agar sheet was observed after activity
staining (Fig. 4, lane 3), which indicated the degradation of agar by
agarase. Coomassie Brilliant Blue staining indicated that A. albus
YKW-34 produced an agarase with a molecular mass of 50 kDa
(Fig. 4, lanes 1 and 2). It was noteworthy that the agarase was the
dominant protein in the crude enzyme preparation, while other
proteins were only in a trace amount (Fig. 4, lane 2). The optimal
harvest time was determined to be 12 h, which was the middle
logarithmic phase of the microorganism (Fig. 3). At this stage, other
metabolites besides agarase were rarely produced. Microorganism
was supposed to produce agarase to hydrolyze agar for supporting
its growth. The dominant occupation of agarase in the crude
enzyme preparation is believed to facilitate further purification of
the agarase. Moreover, direct application of the crude agarase
preparation in oligosaccharide production is possible.
G Model
PRBI-8682; No of Pages 6
6 X.T. Fu et al. / Process Biochemistry xxx (2009) xxx–xxx
Fig. 4. SDS-PAGE electrophoresis of crude agarase preparation by Agarivorans albus
YKW-34. Lane 1: protein makers; lane 2: crude enzyme preparation, Coomassie
Brilliant Blue staining; lane 3: agar sheet, Lugol’s iodine staining.
4. Conclusions
Agarase production by A. albus YKW-34 was significantly
enhanced by optimization of medium composition and culture
conditions. The highest agarase production of 0.87 U/ml was
obtained in the medium consisting of synthetic seawater, 0.23%
agar, and 0.27% yeast extract, when the strain was cultured at 25 8C
for 12 h. The economic medium constitution and culture condition,
the high agarase activity, and the dominant occupation of agarase
in culture fluid enlighten the potential application of A. albus YKW-
34 for the production of agarase.
Acknowledgements
This work was supported by the grant of No. RTI05-01-02 from
the Regional Technology Innovation Program of the Ministry of
Knowledge Economics (MKE), Republic of Korea. Xiao Ting Fu was
a recipient of a graduate fellowship provided by Brain Korea (BK21)
program sponsored by the Ministry of Education, Science and
Technology, Republic of Korea. We would like to thank Professor Il
Shik Shin (Faculty of Marine Bioscience & Technology, Kangnung-
Wonju National University, Korea) for giving us the strain of A.
albus YKW-34.
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Please cite this article in press as: Fu XT, et al. Optimization of medium composition and culture conditions for agarase production by
Agarivorans albus YKW-34. Process Biochem (2009), doi:10.1016/j.procbio.2009.06.012
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