Biotechnology and Bioprocess Engineering 2009, 14: 302-306

DOI/10.1007/s12257-008-0207-0

Development of Fermentation Process for

PLA-degrading Enzyme Production by a New
Thermophilic Actinomadura sp. T16-1
Sukhumaporn Sukkhum1, Shinji Tokuyama2, and Vichien Kitpreechavanich1*
1
Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
2
Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan =
^~= The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-
degrading ^~~ sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at
50C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH4)2SO4,
0.4% K2HPO4, 0.2 % KH2PO4, and 0.02% MgSO4 7H2O. The optimal concentration of PLA film and gelatin obtained by re-
sponse surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238%
(w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of
the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-
degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter. KSBB

hW=Actinomadura=KI=~I=mi^J~=I==~=

INTRODUCTION new thermophilic isolate of Actinomadura sp. T16-1 was

Biodegradable plastics have several excellent properties
and may provide solutions to global environmental problems
[1]. Poly (L-lactide) (PLA) is synthetic polyester produced
from agricultural products such as cassava, rice, and corn [2].
Biological processes by both microbial and enzymatic activi-
ties are currently considered as sustainable recycling meth-
ods for PLA [1]. Actinomycetes belong to the family Pseu-
donocardiaceae and have been reported to produce PLA-
degrading enzyme, based on clear zone formation method
but the temperature for the degrading activity on plates is
about 30oC [3,4]. Various inducers as silk fibroin, elastin,
and gelatin were effective for producing the PLA-degrading
enzyme from the actinomycetes [5-7]. Lately, the plastic
compost by thermophilic microorganisms became a topic of
interest in plastic waste treatment [8]. Several thermophilic
bacteria such as Bacillus brevis, B. stearothermophilus, and
Geobacillus thermocatenulatus have been re- ported to pos-
sess PLA-degrading ability at 60oC [8-10]. Recently, a
G`=~= (Whatman No.1), washed twice, and re-suspended in dis-
Tel: +66-2-942-8389 Fax: +66-2-579-2081
e-mail: fsciwck@ku.ac.th
selected based on its ability of clear zone formation on emul-
sified-PLA agar plates at 50C [11]. However, the optimiza-
tion of the PLA-degrading enzyme production by thermo-
philic strains using statistical method has not been studied so
far. In this study, factors affecting the enzyme production
and medium optimization using response surface methodol-
ogy by this strain were investigated.
MATERIALS AND METHODS
_~~=p~=~=f=m~~
Actinomudura sp. T16-1, among 6 strains of actinomy-
cetes isolated from the soil of Thailand, showed the widest
clear zone on emulsified PLA agar plate at 50C, and was
used in this study. A loop of the strain grown on yeast-malt
extract agar slant was inoculated in 100 mL of yeast extract-
malt extract broth at 50C and 150 rpm for 96 days. The
cells were harvested by filtration through filter paper
tilled water. The cell suspension was used as the inoculum.
 Biotechnol. Bioprocess Eng. PMP=

c~=^=mi^J~=b=m q~=NK Experimental range and levels of the independent vari-

ables used in central composite design
A 10% (v/v) of the cell suspension was inoculated in 250-mL Independent Level
flasks containing 100 mL of the basal medium which consisted variables -1.41 -1 0 1 1.41
of (w/v) 0.1% PLA film, 0.1% yeast extract, 0.4% (NH4)2SO4,
PLA film (u1) 0 0.03 0.05 0.07 0.10
0.2% K2HPO4, 0.1% KH2PO4, and 0.05% MgSO4 7H2O, with
Gelatin (u2) 0 0.12 0.20 0.28 0.40
an initial pH of 7.0. PLA film for the enzyme production was
prepared by dissolving a 50 mg PLA pellet (LACEA, Japan)
in 50 mL of dichloromethane and then dried overnight at 30oC.
^= =
Factors affecting PLA-degrading enzyme production were
investigated using one factor at the time method with one
independent variable, while maintaining all the other factors at
a fixed level. The culture was incubated at 50oC in a rotary
shaker at 150 rpm for 96 h. The culture was then filtered
through the weighed filter paper (Whatman No.1) and the
obtained filtrate was used for assay of PLA-degrading enzyme
activity. Dry cell weight was determined after dried at 105oC,
overnight. The results were analyzed variance (ANOVA) by
using SPSS for windows (Version 10.0, USA).

`~=`=a=E``aF
_=
Response surface methodology was used to optimize the
medium composition for enhancing PLA-degrading enzyme
production. In this study, the CCD with two factors (PLA film
and gelatin) and five levels, including three replicates at the
center point, was used for fitting a second order response sur-
face [12]. Table 1 showed the factors and their values. The
data from the experimental design shown in Table 2 were used
for determining the regression coefficients of the second-order
multiple regression models. SPSS for windows and Statistica
5.0 software (Statsoft, USA) were used for regression analysis
and graphical analysis of the data, respectively.

_~=c~==c cK=NK Effect of medium compositions on the PLA-degrading en-

The batch fermentation was carried out in a 3-L airlift
fermenter with 2-L of working volume of the optimized me-
dium. A 10% (v/v) of the cell suspension was inoculated and
the fermentation was performed at 50oC, initial pH of 6.9,
and an aeration rate of 0.5 vvm. The culture was 24 h inter-
val sampled for assay PLA-degrading enzyme activity.
zyme production by ^~~ sp. T16-1 after 96 h culti-
vation. (A) Carbon sources and (B) organic nitrogen source.
Different letters indicate significant differences at = 0.05
level between treatments.
under the assay condition described.

mi^J~=b=^~
PLA-degrading enzyme activity was assayed based on the
decrease of turbidity at wavelength of 630 nm by a modified
method of Nakamura et al. [13] at 60C for 30 min, except
using at pH 9.0 because a preliminary study found that at this
pH gave the highest activity [14]. A final concentration of
0.1% (w/v) emulsified PLA (LACEA, Japan) in 100 mM
Tris-HCl buffer (pH 9.0), using an ultrasonic processor
model VCX 500 (Sonic and Materials, Newtown, USA) was
used as a substrate. One unit of the PLA-degrading activity
was defined as a 1 unit decrease in optical density per min
RESULTS AND DISCUSSION
c~=^=mi^J~=b=m
The effect of carbon sources on the production of the
PLA-degrading enzyme by Actinomadura sp. T16-1 are
shown in Fig. 1A. A significantly increase of the enzyme
production at a 95% confidence level was observed with an
increase in PLA concentration up to 0.05% (w/v) (data not
shown). It has been well established that higher substrate
concentration can lead to catabolic repression [15].
PMQ=

q~=OK Experimental design used in response surface methodology of 2 independent variables, PLA film (u1) and gelatin (u2), with three cen-
ter points with the observed and predicted PLA-degrading activity
Treatment Level Actual level PLA-degrading enzyme activity (U/mL)
number u1 u2 u1 u2 Observed Predicted
1 1 1 0.07 0.28 22.78 25.71
2 1 -1 0.07 0.12 19.78 24.16
3 -1 1 0.03 0.28 37.52 39.08
4 -1 -1 0.03 0.12 26.90 29.92
5 1.41 0 0.10 0.20 6.74 5.11
6 0 1.41 0.05 0.40 18.93 18.51
7 -1.41 0 0 0.20 27.57 27.10
8 0 -1.41 0.05 0 7.22 5.59
9 0 0 0.05 0.20 40.73 38.05
10 0 0 0.05 0.20 40.70 38.05
11 0 0 0.05 0.20 40.47 38.05

The effect of various organic nitrogen sources on the en-
zyme production was also investigated. Dry weight obtained
from the medium using peptone gave higher biomass, but
lesser PLA degrading activity compared to gelatin (data not
shown). Maximum amount of PLA-degrading enzyme was
obtained by using gelatin (Fig. 1B). The enzyme production
was not correlated with the dry weight formation (data not
shown). This indicated that gelatin acted as inducer as well
as an organic nitrogen source. Jarerat et al. [6] also demon-
strated that protein, peptide, and amino acids induced the
enzyme production but not cell growth. Gelatin and elastin
have been reported to be the best inducers for the production
of PLA-degrading enzyme from T. album [5]. In addition,
silk fibroin powder was an effective culture substrate for
inducing the enzyme production by Amycolatopsis strain 41
[16] and A. orientalis [7]. A significantly increase in enzyme
production at 95% confidence level was observed with in-
creasing gelatin concentration up to 0.2% (w/v) (data not
shown).
The enzyme production in the medium at different con-
centration of (NH4)2SO4, MgSO47H2O, and phosphate
buffer was investigated. The results indicated that the pro-
duction was not significantly influenced by 0.4~1% (w/v) of
(NH4)2SO4, 0.02~0.08% (w/v) of MgSO47H2O, and 1~4
fold concentration of phosphate buffer in the basal medium
(data not shown). However, the maximum activity (33.9
U/mL) was obtained under the medium consisting of (w/v)
0.05% PLA film, 0.2% yeast extract, 0.4% (NH4)2SO4, 0.4%
K2HPO4, 0.2% KH2PO4, and 0.02% MgSO47H2O. It was
found that PLA degrading enzyme production was not re-
pressed by ammonium sulfate.
o=p~=j showed an increase in PLA-degrading enzyme production as
Based on one factor at a time method, the results indi-
cated that PLA film and gelatin were identified as the major
factors affecting the PLA-degrading enzyme production of
Actinomadura sp. T16-1. The CCD experiments were de-
signed to obtain the best condition for the maximum PLA-
degrading enzyme production by the strain. Table 1 showed
the maximum and minimum levels of variables chosen for
trials in the CCD. The experimental design matrix and re-
sults obtained for enzymes activities were shown in Table 2.
Treatment runs 9~11 were the center points in the design,
which were repeated three times for estimation of error. By
applying multiple regression analysis on the experimental
data, the following second order polynomial equation (Eq. 1)
was used to explain the enzyme production.
Y = -16.444 + (890.993 X1) + (347.718 X2) -
(9004.569 X12) - (650.037 X22) - (1108.419 X1X2) (1)
The statistical significance of the regression model
checked by F-test and the analysis of variance (ANOVA)
demonstrated that the model was highly significant. The
determination coefficient (R2) implied that the sample varia-
tion of 95.7% for PLA-degrading enzyme production was
attributed to the independent variables, suggesting a satisfac-
tory representation of the process. There also was a good
correlation between the experimental and predicted values.
The parameter estimates and the corresponding P-values
demonstrated that among the independent variables, X1 (PLA
film) and X2 (gelatin) had a significant effect on enhance-
ment the PLA-degrading enzyme production. Positive coef-
ficients for X1 and X2 showed a linear effect to increase the
enzyme production. While, the quadratic term of these two
variables had a negative significant effect. However, no in-
teractions between the two variables were found to contrib-
ute to the response at a significant level. The contour and
three-dimensional plots of interactions among the variables
the concentration of PLA film and gelatin increased up to
0.035% (w/v) and 0.238% (w/v), respectively, in which the
predicted value was 40.4 U/mL (Fig. 2). The model was
validated by repeating the experiment under the optimized
conditions. The maximum experimental response for PLA-

^=
_=
cK=OK Response surface described by the model, representing
PLA-degrading enzyme activity (U/mL) as a function of PLA
film and gelatin concentrations.
degrading enzyme production was 44.6 U/mL after 96 h
cultivation with productivity of 0.46 U/mL/h. The enzyme
activity obtained was 1.32 folds higher than the activity pre-
dicted by the optimized medium by one factor at a time
method.
_~=c~==^=c
The feasibility of the regression models was also carried
out in a 3-L airlift fermenter. Figure 3 showed the batch pro-
file of PLA-degrading enzyme production in the optimized
medium. The maximum PLA-degrading enzyme production
at 72 h cultivation was 150 U/mL with the productivity of
2.08 U/mL/h. A significant increase of 3.36 and 4.50 folds in
PLA-degrading enzyme production and productivity, respec-
tivly, was observed in an airlift fermenter. This indicated that
the higher air supply in an airlift fermenter may stimulate the
growth and enzyme production by this strain.
CONCLUSION
The production of PLA-degrading enzyme by the thermo-
Biotechnol. Bioprocess Eng. PMR=
cK=PK= Time course of batch fermentation for PLA-degrading en-
zyme production by ^~~ sp. T16-1 in the optimized
medium. , 3-L airlift fermenter; , 250 mL shaking flasks.
philic strain, newly isolated Actinomadura sp. T16-1, was
optimized in this study. PLA film and gelatin were deter-
mined to be the best carbon and organic nitrogen sources,
respectively, as well as an inducer. The strain produced 33.9
U/mL of enzymatic activity after cultivation at 50C under
shaking of 150 rpm for 96 h using one factor at a time
method.
Central composite design was a useful tool for medium
optimization on PLA-degrading enzyme production. The
enzyme production increased to 44.6 U/mL in the basal me-
dium consisted of 0.035% (w/v) PLA film and 0.238% (w/v)
gelatin. The maximum PLA-degrading activity of 150 U/mL
was obtained within 72 h in the optimized medium in 3-L
airlift fermenter. This study is the first report for the optimi-
zation of PLA-degrading enzyme production by the thermo-
philic strain, Actinomadura sp. T16-1 using the response
surface method.
^= This work was financial supported by
the Thailand Research Fund through the Royal Golden Jubi-
lee Ph.D. Program (Grant No. PHD/0193/2547).
Received September 16, 2008; accepted December 21, 2008
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