Recovery of lipase derived from Burkholderia cenocepacia ST8 using sustainable

aqueous two-phase otation composed of recycling hydrophilic organic solvent

and inorganic salt
Pau Loke Show
a,
, Chien Wei Ooi
b
, Mohd Shamsul Anuar
c
, Arbakariya Ariff
d
, Yus Aniza Yusof
c
,
Soo Kien Chen
e
, Mohamad Sufan Mohamad Annuar
f
, Tau Chuan Ling
f
a
Manufacturing and Industrial Processes Division, Faculty of Engineering, Centre for Food and Bioproduct Processing, University of Nottingham Malaysia Campus, Jalan Broga,
Semenyih 43500, Selangor Darul Ehsan, Malaysia
b
Chemical and Sustainable Process Engineering Research Group, School of Engineering, Monash Universit, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia
c
Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
d
Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
e
Department of Physics, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
f
Institute of Biological Sciences, Faculty of Science, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia
a r t i c l e i n f o
Article history:
Received 8 November 2012
Received in revised form 27 February 2013
Accepted 8 March 2013
Available online 20 March 2013
Keywords:
Enzymes
Biocatalysis
Alcohols
Recycling aqueous two-phase otation
Salt effect
a b s t r a c t
Recycling hydrophilic organic solvent/inorganic salt aqueous two-phase otation (ATPF) is a novel, low
cost, green and high efcient techni que for recovery of biomolecules. Recycling ATPF composed of 2-pro-
panol and potassium phosphate was developed for sustainable separation, concentration and purication
of Burkholderia cenocepacia ST8 lipase from liquid fermentatio n broth. Thirteen parameters upon recy-
cling hydrophilic organic solvent/inorganic salt ATPF performance were investigated. The optimum con-
dition s for this recycling ATPF were determined to be 40 mL volume of 50% (w/w) 2-propanol, 1.0 L of
250 g/L of potassium phos phate, pH 8.5, 100% (v/v) of crude feedstock, 30 mL/min of N
2
ow rate for
30 min in a 8 cmradius of colorimeter tube with G4 porosity (515 lm) sintered glass disk. A purication
factor of 14.4 0.04 and a lipase yield of 99.2 0.03% were achieved in this optimized ATPF. The recycling
of phase-formi ng components employed at the end of recovery process was based on the principals of
green chem istry, with high efciency and economical viability. There was no gross variation of results
during the process of scaling-up. Therefore, this novel recycling ATPF is feasib le to be applied at indus-
trial-sc ale.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Aqueous two-phase system (ATPS) has been proven to be an
effective, environmental friendly, economically viable and biocom-
patible method for separation, concentratio n and purication of
biological material, particularly for macromolec ules like protein,
enzyme and DNA [1]. ATPS is predominantl y based on polymer/salt
system, such as polyethyl ene glycol (PEG)/potassiumphosphate, or
polymer/po lymer system, such as PEG/dextran, which have been
well-develop ed and documented for the recovery of biomolecule s.
In recent years, conventi onal ATPS has been enhanced by the utili-
zation of random copolymers of ethylene oxide propylen e oxide
(EOPO), which can be recovered through thermo-induct ion. How-
ever, the challenges to apply this thermose parating copolymer in
biotechnolo gical industry are still remained unresolved, owning
to the high cost of the polymer and the difculties in isolating
the target biomolecules from polymer phase. Furthermore, there
are several limitations in polymer/ salt ATPS and polymer/ polymer
ATPS. These include slow segregation of two-phase in polymer/
polymer ATPS, time consuming and complications associate d with
the recycling of phase-fo rming components. ATPS consists of a
hydrophi lic organic solvent and an inorganic salt solution has been
reported for the use in recovery of protein [2], amino acid [3], and
other natural products [4]. Hydrophilic organic solvent/inor ganic
salt ATPS has many advantag es, which include rapid phase-separ a-
tion, high extractio n efciency, low viscosity , high polarity, gently
environm ent, inexpensivene ss of phase-forming chemicals and
facile recycling of the hydrophilic organic solvent and inorganic
salt [5].
Solvent sublation (SS) is an adsorptiv e bubble mass transfer
techniqu e. In SS, surface-active compounds in aqueous phase will
be adsorbed and attached on the bubble surfaces of an ascending
gas stream owing to the top of bulk aqueous phase in the column.
These surface-a ctive compounds will be collected in an immiscible
liquid layer on the top of aqueous phase (Fig. 1). The advantages of
1383-5866/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2013.03.018

Corresponding author. Tel.: +60 3 8924 8605; fax: +60 3 8924 8017.
E-mail address: PauLoke.Show@nottingham.edu.my (P.L. Show).
Separation and Purication Technology 110 (2013) 112118
Contents lists available at SciVerse ScienceDi rect
Sepa ration and Purication Techn ology
j our nal homepage: www. el sevi er . com/ l ocat e/ seppur
this method include simple technique, high level of separation ef-
ciency, concentr ation coefcient and purication factor, low dos-
age of organic solvent and risk of downstream pollution. This
method has been widely utilized in the recovery of biomolecules,
for example metal ion [6], organic pollutant s [7] and active compo-
nents from natural products [8]. A sustainab le, green and low cost
hydrophilic organic solvent/i norganic salt aqueous two-phas e o-
tation (ATPF) is an integrati on of hydrophilic organic solvent/i nor-
ganic salt ATPS and SS, which are complementar y to each other and
comprise the benets from both methods . Furthermor e, this novel
ATPF composed of short-chain alcohol and inorganic salt is ex-
pected to overcome most of the limitations of ATPS, such as high
cost of phase-formi ng polymer, high energy input and complica-
tion associate d with recycling of chemicals. Our previous work
has shown that this novel recycling polymer/salt ATPF can be used
to recover lipase derived from Burkholderia cepacia [9]. However,
limitations such as difculties in recycling ammonium sulfate
[10], high cost of EOPO and slow segregation of two-phase, have
received less attention and thus restricted the application of ATPF
at large-scale. Therefore, the development of a sustainable and fac-
ile method to recover lipase efciently is strongly demande d in the
current trend market.
To the authors knowled ge, there is no literature regarding the
application of this novel and sustainab le ATPF that composed of
recycling hydrophilic organic solvent and inorganic salt in the
recovery of any protein. Lipases derived from Burkholderi a cenopa-
cia (B. cenopacia ) ST8 have attracted major interests for industrial
applications such as chemical, food, detergent, pharmaceutical ,
bioremediati on and cosmetic [11]. The enzyme has a molecular
weight around 43 kDa. The enzyme has high catalytic activity for
various substrates and partial solubility. A detailed experime nt
was conducted with an aim to recover of B. cenopacia ST8 lipase
from fermentation broth in sustainab le ATPF utilizing phase-form-
ing components with recycling capability (i.e., hydrophilic organic
solvent and inorganic salt). The inuences of 13 variables, namely
(1) type of hydrophilic organic solvent, (2) type of inorganic salt,
(3) concentratio n of hydrophilic organic solvent solution, (4) con-
centration of inorganic salt solution, (5) volume of hydrophilic or-
ganic solvent phase, (6) pH, (7) volume of inorganic salt phase, (8)
radius of colorimeter tube, (9) amount of crude feedstock, (10) type
of gas, (11) gas ow rate, (12) otation time and (13) porosity size
of sintered glass disk were investigated in detail. The sustainable
ATPF was evaluated based on the separation efciency (E), concen-
tration coefcient (a) and purication factor (PF) of lipase. Scaling-
up of the recycling ATPF was also attempted in this study. This is
the rst report about recovery of protein using recycling hydro-
philic organic solvent/inor ganic salt ATPF.
2. Materials and methods
2.1. Chemical s and apparatus
Methanol , ethanol, 1-propanol, 2-propanol, 1-butano l, ammo-
nium sulfate [(NH
4
)
2
SO
4
], sodium citrate (Na
3
C
6
H
5
O
7
), di-potas-
sium hydrogen phosphate (K
2
HPO
4
), potassium di-hydrogen
phosphat e (KH
2
PO
4
), sodium chloride (NaCl), di-sodium phosphate
(Na
2
HPO
4
), sodium di-hydrogen phosphate (NaH
2
PO
4
), magnesium
sulfate (MgSO
4
) were purchased from Merck (Darmstadt, Ger-
many), p-nitrophenylla urate (pNPL) and nutrient broth were
sourced from SigmaAldrich Co. (St. Louis, USA). All chemicals used
in the study were of analytica l grade. The ATPF apparatus was sim-
ilar to the ones in a previous publication [9]. The vacuum rotary
evaporator, Laborata model 4001, (Viertrieb, Germany ) was used
to recycle the alcohol of the systems.
2.2. Media and culture condition
B. cenocepac ia ST8 (a kind gift from Dr. S.L. Hii) isolated from
forest soil samples from Setapak, Malaysia, was used in this study.
This bacterial strain was cultivated using the media as described by
Lau et al. [12] and the cultivatio n condition was as reported in an
earlier publication [13]. After 72 h, the fermentation culture was
harvested and employed in the studies.
2.3. Optimiza tion of hydrophilic organic solvent/inorga nic salt ATPF
The ATPF parameters [type of hydrophilic organic solvent, type
of inorganic salt, concentration of hydrophilic organic solvent solu-
tion [hydrophilic organic solvent], concentratio n of inorganic salt
solution [salt], volume of hydrophilic organic solvent phase (V
solv
),
volume of aqueous phase (V
aq
), pH, radius of colorimeter tube (r),
amount of crude feedstock (C
F
), type of gas, gas ow rate (F
R
), o-
tation time (f
T
) and porosity size of sintered glass disk (P)] were
optimized to the maximum separation, concentr ation and purica-
tion of lipase. The initial system was set to 25 mL of V
solv
, 250 mL of
V
aq
at pH 7, 50% (w/w) of C
L
, 30 mL/min of F
R
and 30 min of f
T
in G4
porosity of sintered glass disk. The experiments were performed at
room temperature (RT) and expressed as the average of triplicate
readings with an estimated error of 5%.
2.4. Recycling of hydrophilic organic solvent and inorganic salt in ATPF
The top hydrophilic organic solvent-rich phase was isolated
from ATPF and subjected to evaporation in vacuum rotary evapora-
tor for 15 min (200 rpm) at 30 C. After the evaporation process,
target enzyme remained in the rotary ask, while the concentrated
hydrophi lic organic solvent obtained from the evaporation was re-
used in the preparati on of a new ATPF system. For the recycling of
inorganic salt from ATPF, suitable amount of concentr ated hydro-
philic organic solvent was added to the aqueous salt-rich bottom
phase in order to crystallize the inorganic salt [14]. The crystallized
inorganic salt was recovered through ltration. The hydrophilic or-
ganic solvent used to crystallize the aqueous salt-rich bottom
phase underwent evaporati on process in the vacuum rotary evap-
orator in order to separate the contaminan ts and the hydrophilic
organic solvent. The evaporated hydrophi lic organic solvent recov-
ered from crystallize of inorganic salt can be re-used to form a new
ATPF system. In this experiment, ve successive separation, con-
centration and purication of lipase from fermentation broth in
hydrophi lic organic solvent/inorgani c salt ATPF systems were
carried out. Two experiments were conducte d using the recycled
aqueous phase (i.e., inorganic salt) which was recovered from the
previous ATPF. After the recovery process of lipase, the enzyme
Products
Aqueous phase
= crude
feedstock +
inorganic
solvent
solution
Gas
Sintered glass
disk
Hydrophilic organic
solvent phase
Air
bubble
Hydrophobic side
Hydrophilic side
Fig. 1. Schematic view of recovery of surface-active compound in ATPF. The
surface-active (hydrophobic compound) in the aqueous phase will be absorbed on
the gas bubble surfaces of an rising gas stream and collected in the layer of
hydrophilic organic solvent phase on top of the aqueous phase [23,24].
P.L. Show et al. / Separation and Purication Technology 110 (2013) 112118 113
activity and total protein content of all the phases were deter-
mined and recorded.
2.5. Analytical methods
The enzyme activity was determined by employin g previously
described spectrophot ometric method [15], with minor modica-
tions [16] at room temperat ure. The protein concentratio ns were
measured by using Bradford method and bovine serum albumin
(BSA) was employed as a standard [17]. The results are expressed
as the average of triplicate readings with an estimated error of 5%.
2.6. Determinati on of separation efciency (E), concentrati on
coefcient (a), purication factor (PF), selectivity (S), yield of lipase
(Y), recovery of hydrophilic organic solvent (R
solv
) and inorganic salt
(R
aq
)
E and a were calculated using the following equation:
E 1 C
W
=C
Wi
10 1
a C
Solv
=C
Wi
2
where C
W
is the lipase concentrat ion of the aqueous phase (i.e. inor-
ganic salt) at time t, C
Wi
represe nts the original concentration of li-
pase in aqueous phase, and C
solv
is the lipase concentrat ion in
hydrophi lic organic solvent phase at time t.
Selectivity (S) was dened as the ratio of the enzyme partition
coefcient (K
e
) to the protein partition coefcient (K
p
):
S K
e
=K
P
A
T
=A
B
P
B
=P
T
3
where K
e
and K
p
are the ratios of lipase and protein concentrat ions
recorded in top and bottom phases, respective ly. A
T
and A
B
are the
lipase activity (units/mL) recorded in the hydrop hilic organic sol-
vent and the aqueous phases, respective ly. P
T
and P
B
are the total
protein concentrat ion (mg/mL) measured in hydrophilic organic
solvent and aqueou s phases, respective ly.
The purication factor (PF) was dened as the ratio of lipase-
specic activity (SA) found in hydrophilic organic solvent phase
to the original lipase-speci c activity recorded in the crude
feedstock:
PF SA hydrophilic organic solvent phase =SA crude feedstock
4
where the SA represe nts the ratio of lipase activity to the total pro-
tein concentrat ion of the sample taken.
Yield (Y) of lipase in the hydrophilic organic solvent phase was
measured using the following equation :
Y 1=1 1=V
R
K
e
100% 5
where V
R
is the volume ratio of the top phase to the bottom phase.
Hydrophilic organic solvent recovery (R
solv
) was dened as:
R
solv
V
solv
=V
isolv
100% 6
where V
solv
is the volume of hydrophili c organic solvent recovered
from the evaporation proces s, V
isolv
is the total volume of hydro-
philic organic solvent used in the hydrophilic organic solvent phase
of ATPF.
Inorganic salt recovery (R
aq
) was dened as:
R
aq
m
salt
=m
isalt
100% 7
where m
salt
is the mass of inorganic salt recove red from the ltra-
tion process, m
isalt
is the total mass of inorganic salt used in the
aqueous phase of ATPF.
2.7. Sodium dodecyl sulfatepolyacrylamide gel electrophore sis (SDS
PAGE)
SDSPAGE analysis was conducted in SE250 electrophor esis
unit (GE Healthcare) using polyacrylamid e gel made of a 4.5% (v/
v) stacking gel and a 12% resolving gel as described by Laemmli
[18]. The SDSPAGE procedure has been described in our previ-
ously publications [9,13,16].
3. Results and discussion
3.1. Selection of hydrophilic organic solvent/inorgan ic salt based on
ATPF
Similar to ATPS, the concentratio ns of both inorganic salt (i.e.,
magnesiu m sulfate, sodium phosphate, sodium chloride, potas-
sium phosphate, sodium citrate and ammoniu m sulfate) and
hydrophi lic organic solvent (i.e., methano l, ethanol, 1-propanol,
2-propanol and 1-butano l) play inuential roles in ATPF. In ATPF,
the volume of the aqueous phase is greater than the volume of
the hydrophilic organic solvent phase. Hence, the concentration
of inorganic salt is a key to the formation of an immiscibl e two-
phase system due to the salting-out effect. The experiments were
conducte d by mixing hydrophi lic organic solvent at different con-
centration with variant types of inorganic salt (salt concentration
ranged from 200 to 500 g/L). The partitioning of lipase in hydro-
philic organic solvent/i norganic salt ATPF was studied and the re-
sults are shown in Table 1. The results indicate that each system
has its own minimum concentration of hydrophilic organic sol-
vent/inorga nic salt for the formation of twophases system. The
immiscibl e two-phas e cannot exist if the hydrophi lic organic sol-
vent/inorga nic salt concentration is lower than this minimum con-
centration. For instance, the minimum concentratio ns of methano l
and ammonium sulfate for the formation of ATPF are 80% (w/w)
and 450 g/L, respectively . In general, the lipase is able to survive
in the presence of hydrophilic organic solvent and inorganic salt
at certain extend, which makes these phase forming-comp onents
ideal for the recovery of lipase by using hydrophilic organic sol-
vent/inorga nic salt ATPF.
All inorganic salts used in this study, except sodium chloride
(NaCl), can form stable and adjustable ATPF with methano l or eth-
anol. The partition coefcient, K and yield, Y (%) of lipase for ATPF
consisted of NaCl were signicantly low. This phenomenon was
attributed to the extremely low solubility of NaCl in methano l or
ethanol solution, which resulted in salt precipitatio n [19]. Findings
also demonst rated that incubation of lipase in solution containing
high inorganic salt concentratio n will reduce the K and Y values as
high salt concentr ation will inhibit lipase activity [20]. The results
(Table 1) show that the maximum K and Y values were achieved by
using ATPF composed of 50% (w/w) 2-propanol and 250 g/L potas-
sium phosphate. 2-propanol has a longer hydrocarbo n chain as
compare d to methanol, ethanol and 1-propanol, which facilitate s
the interaction with lipase, and thus improves the partitioning of
lipase to the hydrophilic organic solvent phase. However, 1-buta-
nol is not selected in this study even though it has a longer hydro-
carbon chain compare d to 2-propanol. This can be explained by the
partially solubility characterist ic of 1-butanol which make 1-buta-
nol unsuitable to be used in two-phase system. Based on the re-
sults presented in Table 1, 2-propanol/p otassium phosphate ATPF
was chosen for further optimization.
3.2. Optimiza tion of 2-propan ol/potassium phosphate concentratio n
Literatur es show that the system pHwill modify the partitioning
of desired protein and change the concentration of phase-forming
114 P.L. Show et al. / Separation and Purication Technology 110 (2013) 112118
components [2]. An experime nt was conducted by varying the pH
from 3 to 10 through the addition of potassium hydrogen phosphat e
(K
2
HPO
4
) and potassium di-hydrogen phosphate (KH
2
PO
4
) at differ-
ent ratio. Fig. 2 shows that high E and a were achieved in ATPF with
the pH ranging from 4.0 to 8.5. At pH < 4.0, the system exists as a
homogenous solution. This phenomeno n can be explained by the
fact that low ratio of K
2
HPO
4
to KH
2
PO
4
at lower pH has resulted
in poorer phase separation [21]. When the pH was increased from
4.0 to 8.5, the E and a showed less signicant variation , which are
in the range of 50.955.5% and 6.112.4, respectivel y. It is because
the ratio of inorganic salt composition in the system altered from
high KH
2
PO
4
:K
2
HPO
4
ratio when the pH value was increased. At
pH > 9.0, E and a could not be measured as the lipases were dena-
tured in strong alkaline condition. According to the results shown
in Fig. 2, pH 8.5 was chosen as the optimum pH for ATPF and this
pH8.5 is an approximat e pHvalue of the solution with K
2
HPO
4
only.
In other words, the solution with K
2
HPO
4
solely can be directly used
in ATPF without the need for adjusting pHand thus pre-treatment of
feedstock could be avoided. This would be benet from the aspect of
environment, economics and sustainable recovery process.
In comparison to ATPS, liquidliquid extraction process only
occurs at hydrophilic organic solvent layer in ATPF. Hence, the vol-
ume and concentratio n of hydrophilic organic solvent play a cru-
cial role in the whole system. In this part, the effects of volume
(3070 mL) and concentratio n [4080% (w/w)] of 2-propanol upon
the ATPF performance were investigated . Based on the results
shown in Fig. 3, stable E value was recorded in the ATPF composed
of 2-propanol ranging from 50 to 80% (w/w). At concentr ation of 2-
propanol <50% (w/w), the E value could not be measured as the
system existed as a homogen ous solution. Increasing the concen-
tration from 5080% (w/w) and the volume from 3070 mL would
cause the E value to be at invariant level of 5368%. The ndings
also show that E value increased when the concentratio n of 2-pro-
panol used in hydrophilic organic solvent phase was higher. This
might be due to the reason that more 2-propanol molecules partic-
ipated in the separation process causing the process to be more
efcient [22]. After reviewing the results in Fig. 3, a minimum con-
centration of 2-propanol [50% (w/w)] was selected because addi-
tion of 2-propanol beyond this point will not contribute any
signicant improvement to the overall E value (65%) of the
Table 1
Effect of type of alcohol and concentration of different salt upo n the performance of ATPF.
Type of salt [Salt] g/L 80% (w/w)
Methanol
50% (w/w)
Ethanol
50% (w/w) 1-
Propanol
50% (w/w) 2-
Propanol
10% (w/w) 1-
Butanol
K
a
Y
b
(%) K
a
Y
b
(%) K
a
Y
b
(%) K
a
Y
b
(%) K
a
Y
b
(%)
Ammonium sulfate [(NH
4
)SO
4
] 200 21.4 94.5
250 19.3 98.7 51.8 96.8 11.2 90.7
300 16.5 95.1 43.9 96.7 10.8 89.5
350 18.5 91.3 16.6 93.9 33.6 94.6 9.4 82.1
400 11.2 91.1 15.8 91.1 22.4 92.8 8.2 83.0
450 12.4 89.8 9.1 91.0 13.9 90.5 21.3 91.5 7.2 82.8
500 10.5 87.9 8.7 90.8 10.4 88.2 20.5 90.3 4.1 81.6
Sodium citrate (Na
3
C
6
H
5
O
7
) 200 11.1 91.1
250 10.3 80.8
300 18.5 95.9 44.9 99.5 9.5 80.3
350 17.6 94.3 31.4 92.3 8.3 81.1
400 17.4 93.2 25.4 91.5 7.9 80.0
450 19.6 90.5 12.3 92.1 21.8 90.5 7.1 79.8
500 11.3 81.4 18.3 90.1 12.4 90.6 11.1 89.2 5.6 79.6
Potassium phosphate (KH
2
PO
4
+ K
2
HPO
4
) 200 18.4 97.3
250 14.5 98.1 70.8 99.2 14.5 96.4
300 19.3 98.1 13.5 94.1 68.9 99.1 12.1 84.9
350 18.1 97.0 13.0 93.3 53.2 98.3 17.1 91.0
400 17.5 89.9 12.8 92.9 44.4 98.0 14.6 81.2
450 21.7 90.7 16.1 90.3 12.0 92.2 41.7 97.2 12.3 80.4
500 21.0 89.4 15.2 90.5 11.4 92.1 40.1 97.0 10.6 79.1
Sodium chloride (NaCl) 200 16.1 88.6
250 14.4 68.3 15.6 84.7
300 6.3 70.4 12.4 66.8 15.5 84.3
350 6.6 67.1 11.6 64.5 14.7 83.5
400 4.8 59.4 10.1 67.4 14.5 82.1
450 3.2 49.6 9.3 56.6 13.7 80.2
500 2.4 39.9 6.5 56.0 13.2 79.4
Sodium phosphate (Na
2
HPO
4
+ NaH
2
PO
4
) 200 16.4 94.9
250 14.9 90.7
300 18.7 94.0 36.9 95.6 14.8 89.7
350 19.5 85.7 18.6 93.1 36.6 96.4 13.9 90.5
400 17.4 83.9 16.8 92.4 31.4 95.6 13.2 89.1
450 19.4 89.6 16.6 84.0 12.2 92.6 21.3 94.6 11.9 88.8
500 17.8 87.4 15.7 81.5 12.4 90.9 21.3 92.6 11.1 87.9
Magnesium sulfate (MgSO
4
) 200 19.4 89.4
250 17.3 89.3 40.8 98.8 17.2 87.6
300 12.3 88.4 16.5 88.6 37.1 96.0 15.8 86.6
350 16.5 86.3 17.0 88.6 33.6 94.6 13.4 85.7
400 16.5 82.1 16.8 87.2 24.4 95.7 12.2 85.2
450 14.4 79.9 13.6 80.5 16.5 86.9 21.3 94.7 12.2 84.6
500 11.5 78.7 12.9 79.5 15.4 79.9 20.5 93.5 11.1 83.5
a
Partition coefcient, K.
b
Yield, Y (%) of lipase on the hydrophilic organic solvent phase were investigated. The results are expressed as a mean of triplicate readings with an estimated error of 5%.
P.L. Show et al. / Separation and Purication Technology 110 (2013) 112118 115
system. By taking economic aspect into the consideration, 40 mL of
50% (w/w) 2-propanol was chosen for further optimization.
3.3. The effect of otation time and gas ow rate
Flotation time and gas ow rate are the two important parame-
ters inATPF as they will directly affect the area of airwater interface
per unit volume of aqueous solution in unit time [14]. Fig. 4 shows
the effects of otation time (060 min) and gas ow rate (0
60 mL/min) on the E value. In general, increasing the otation time
would enhance the E value up to an optimum value when a plateau
was reached at certain time. The separation capacity began to reach
its limit beyond that time and E value did not increase further when
the operating time increased continuously. Moreover, increasing
the gas ow rate (P60 mL/min) continuo usly could inuence the
velocity of the mass transfer signicantly. Gas owat very high rate
would disturb the interface of the system. The bubbles could not
quickly rupture at very high gas owrate and thus the bubbles were
accumulate d onthe top of the contactor [23]. According to Fig. 4, it is
clearly seen that the E value increased sharply in the rst 15 min,
and all the curves reached separation and thermodyna mic equilib-
rium after 30 min. Therefore, 30 min of otation time at 30 mL/
min of gas owrate was selected as the optimum paramete rs for fur-
ther optimizati on. Long otation time and high gas owrate are not
economical for the practical uses at large-scal e.
3.4. The effect of the bubbles size
It cannot be denied that the size of bubbles is one of the key
parameters in ATPF, since it dictates the available interfacial area
for gasliquid mass transfer. When extremely small air bubbles
reach the interface of hydrophi lic organic solvent and inorganic
salt solution, small bubbles cannot enter the hydrophi lic organic
solvent phase immediately, and these small bubbles have to coa-
lesce into larger bubbles in order to overcome the interfacia l ten-
sion [24]. In ATPF, bubbles size distribution depends extensively
on column geometry (e.g., radius of colorimeter tube, r), type of
gas, and porosity size of the sintered glass disk. Fig. 5 shows the ef-
fects of radius of colorimeter tube, type of gas and porosity size of
the sintered glass disk upon performances of ATPF. Three different
porosity sizes of the sintered glass disk were tested, i.e., G3 (15
40 lm), G4 (515 lm) and G5 (102 lm). Porosity G4 showed
the optimum PF and E values. Among these three types of gases
(i.e., nitrogen, carbon dioxide and oxygen), nitrogen gas was pro-
vided better PF and E values. It might be due to the reason that
nitrogen gas acts like an inert gas and does not give any chemical
reaction during the otation. Furthermore, nitrogen gas has the
ability to improve the surface oxidation which eases the absorption
of lipase on the surface of the bubbles [25]. The results also show
that an increase in the radius of colorimeter tube would increase
the PF and E values. This is probably because the molecules diffu-
sive transport increased with an increase in colorimeter tube ra-
dius and the bubbles can spread more widely in aqueous phase
[26]. Therefore, maximum radius of colorimeter tube (r: 8 cm),
G4 porosity of the sintered glass disk and nitrogen gas were
selected for this studies.
3.5. Scale-up of 2-propan ol/potassium phosphate ATPF
Based on the results shown in Fig. 6, there is no variation in
applying different concentratio n of crude feedstock in ATPF system.
Hence, scale-up of recycling hydrophilic organic solvent/inor ganic
salt ATPF was conducte dusing unlteredfermentation broth at con-
centration of 100% (v/v). All the parameters which have been opti-
mized previously were kept constant ly throughout the various
scales. Fig. 6 shows that the amount of crude feedstock added in
hydrophi lic organic solvent phase has very minor effects onselectiv-
ity (S) and Y values. This investigatio n demonst rated the ease and
consisten cy in scaling-up of 2-propanol/potas sium phosphate ATPF.
After analyzing on the results shown in Fig. 6, the scale-up in recy-
cling ATPF has no effect on the efciency of the lipase recovery from
100% (v/v) unltered fermentation broth. Therefore, this method is
environm entally benign and economical (e.g. reduction of chemical
usage) for direct recovery of lipase from fermentation broth. Apart
from that, it would enable this sustainable ATPF to be used at indus-
trial scale for direct recovery of others bio-product as well as large
scale downstre amprocessing in biotechnologic al eld.
45
50
55
60
0
2
4
6
8
10
12
14
3 4 5 6 7 8 9 10
E
,

%
pH
E
H
o
m
o
g
e
n
e
o
u
s
s
y
s
t
e
m
L
I
p
a
s
e
d
e
n
a
t
u
r
a
t
i
o
n
Fig. 2. Effect of pH on the performance of ATPF. The pH of ATPF consisted of 50% (w/
w) 2-propanol and 250 g/L potassium phosphate were varied in the range of 310.
0
20
40
60
80
40 50 60 70 80 90
E
,

%
Concentration of hydrophilic organic solvent, % (w/w)
Vo=30mL
Vo=40mL
Vo=50mL
Vo=60mL
Vo=70mL
H
o
m
o
g
e
n
e
o
u
s
s
y
s
t
e
m
Fig. 3. Effect of concentration and volume of hydrophilic organic solvent upon the
performance of ATPF. The effects of volume (3070 mL) and concentration [4080%
(w/w)] of hydrophilic organic solvent phase were investigated in ATPF composed of
50% (w/w) 2-propanol and 250 g/L potassium phosphate at pH 8.5.
0
20
40
60
80
100
0 15 30 45 60
E
,

%
Flotation time, min
10mL/min 20mL/min 30mL/min
40mL/min 50mL/min 60mL/min
Fig. 4. Effect of gas ow rate and otation time upon the performance of ATPF. At
40 mL volume of 50% (w/w) 2-propanol and 250 g/L of potassium phosphate at pH
8.5, the effects gas ow rate (1060 mL/min) and otation time (060 min) on the
separation efciency of lipase in ATPF were investigated.
116 P.L. Show et al. / Separation and Purication Technology 110 (2013) 112118
3.6. Sustainable recovery of lipase and recycling of the phase-formi ng
component in ATPF
Table 2 outlines the performanc es of ATPF in ve successive cy-
cles of lipase recovery . Among all these ATPF systems, only the rst
ATPF system was prepared with fresh phase-formi ng chemicals.
The following four successive ATPF systems applied recycled 2-
propanol that was recovered from previous ATPF systems. How-
ever, only fourth and fth ATPF systems used aqueous phase that
was recovered from the previous ATPF. Table 2 shows that the rst
ATPF system indicates the highest a, E, PF and Y values among the
ve successive recoverie s of lipase in ATPF systems. This is because
fresh chemical enables the ATPF to perform better in the separa-
tion, concentration and purication of lipase [27]. From the second
to the third ATPF system, there is a sharp decrease in the efciency
of the recovery of lipase in ATPF. This phenomeno n is due to the
fact that there are contaminan ts saturated in aqueous phase and
it will cause a signicant decrease in a, E, PF and Y values [28].
When the fourth ATPF was carried out to recycle the aqueous
phase from the third APTF, a signicant improvem ent on the over-
all ATPF performanc e was shown. The purpose of conducting fth
ATPF (i.e., which is recycling both of hydrophi lic organic solvent
and aqueous phase) is to assess the practicabilit y of recycling of
the aqueous phase. Overall, the sustainable ATPF in recovery of li-
pase composed of recycling 2-propanol and potassium phosphate
was achieved in all the ve ATPF systems and the average recovery
of 2-propanol and potassium phosphate were 70% and 61%, respec-
tively. Therefore, this techniqu e is proven to be an effective, ideal,
sustainabili ty and both phases of ATPF are recyclability.
The purity of lipase recovered from ATPF was evaluated by 12%
SDSPAGE analysis [18] and the SDSPAGE prole is depicted in
Fig. 7. The multiple bands (Lane 1) represent contaminant proteins
in control crude feedstock . The Lanes 2, 3, 4, 5 and 6 showed the
samples from fth, fourth, third, second and rst ATPF systems,
respectively . A major dark band with molecular mass around
34 kDa was indicated as the B. cenopacia ST8 lipase [11]. The real-
ization of B. cenopacia ST8 lipase recovery at highly puried state
using sustainable recycling ATPF was conrmed by SDSPAGE
analyses, in line with highlight the crucial step of recycling aque-
ous phase in order to achieve an efciency lipase recovery in ATPF.
4. Conclusion
It is reported for the rst time for lipase derived from B. cenop-
acia ST8 was successfully separated, concentr ated and puried
from fermentation broth by using recycling hydrophilic organic
solvent/i norganic salt in sustainab le ATPF. At the optimized state
of sustainable recycling hydrophi lic organic solvent/inor ganic salt
ATPF: [(1) type of hydrophilic organic solvent: 2-propanol , (2) type
of inorganic salt: potassium phosphate, (3) concentration of hydro-
philic organic solvent solution: 50% (w/w), (4) concentration of
inorganic salt solution: 250 g/L, (5) volume of hydrophilic organic
solvent phase: 40 mL, (6) pH: 8.5, (7) volume of inorganic salt
phase: 1.0 L, (8) radius of colorimeter tube: 8 cm, (9) concentr ation
of crude feedstock : 100% (v/v), (10) type of gas: N
2
, (11) gas ow
rate: 30 mL/min, (12) otation time: 30 min and (13) porosity size
0
20
40
60
80
100
0
2
4
6
8
10
12
14
r:4cm r:6cm r:8cm r:4cm r:6cm r:8cm r:4cm r:6cm r:8cm
Porosity:G3 Porosity:G4 Porosity:G5
E
,

%
P
F
Fig. 5. Effect of gas type, radius of colorimeter tube (r) and porosity size of sintered
glass disk on the performance of ATPF. The optimum condition of ATPF at 40 mL
volume of 50% (w/w) 2-propanol, 250 g/L of potassium phosphate at pH 8.5 and
30 mL/min of gas ow rate for 30 min were performed by varying type of gas
[nitrogen (N
2
), oxygen (O
2
) and carbon dioxide (CO
2
)], radius of colorimeter tube
(48 cm) and porosity size of sintered glass disk (G3, G4 and G5).
0
50
100
150
200
250
300
0 20 40 60 80 100
S

a
n
d

Y
,


%
Concentration of crude feedstock, % (w/w)
Fig. 6. Effect of crude feedstock concentration on the performance of ATPF at
different scales under optimum condition. Different scales [40 mL volume of 50%
(w/w) 2-propanol, 250 g/L of potassium phosphate, pH 8.5, 100% (v/v) of crude
feedstock, 30 mL/min of N
2
ow rate at 30 min and G4 porosity with 8 cmradius of
colorimeter tube] were investigated under optimum condition.
Table 2
Separation efciency (E, %), concentration coefcient (a), purication factor (PF), yield
of lipase (Y, %), recovery of alcohol (R
alcohol
, %) and salt (R
salt
, %) were studied in ve
successive sustaina ble ATPF.
ATPF First
a
Second
b
Third
b
Fourth
c
Firth
c
a 16.1 13.4 5.4 14.9 15.2
E (%) 90.2 79.3 56.2 88.6 88.5
PF 14.4 12.2 8.9 12.9 13.6
Y (%) 99.2 89.9 73.2 96.7 97.3
R
alcohol
(%) 69.1 70.4 70.2 69.8
R
salt
(%) 59.9 61.7
a
Fresh chemicals were used in phase-forming components.
b
Hydrophilic organic phase (i.e., 2-propanol) was recycled by evaporation pro-
cess. However, aqueous phase was not recycled and direct used for next system
from the previous aqueous phase of ATPF.
c
Recycling of both phases from previous ATPF.
2 3 4 5 6 7
Lipase
kDa
175
83
62
48
33
25
Fig. 7. SDSPAGE analysis on recycling ATPF. The molecular weight of standard
protein marker ranged from 25 to 175 kDa. In the SDSPAGE, Lane 1: control crude
culture; Lane 2: fth ATPF; Lane 3: fourth ATPF; Lane 4: third ATPF; Lane 5: second
ATPF; Lane 6: rst ATPF and Lane 7: protein maker.
P.L. Show et al. / Separation and Purication Technology 110 (2013) 112118 117
of sintered glass disk: G4], an E value of 90.2%, a value of 16.1, PF of
14.4 and 99.2% yield of lipase were obtained in the system. It was
demonstrat ed that the average sustainable recovery of hydrophilic
organic solvent and inorganic salt were up to 70% and 61%, respec-
tively. Furthermor e, it is important to recycle the aqueous phase in
order to achieve an effective and sustainab le recovery of lipase in
recycling hydrophi lic organic solvent/inor ganic salt ATPF from fer-
mentation broth. The recycling of both phases in 2-propanol/p otas-
sium phosphat e ATPF is nearly ideal in terms of sustainability,
efciency, and operation time and cost-saving. This novel tech-
nique is suggested to be applied for the sustainable recovery of
other biomolecules.
Acknowled gments
This study was supported by University Malaya Research Grants
(RG055/11BIO) and UMRG Program Fasa 1/2012 (RP024-2012C)
from the University of Malaya, Malaysia.
References
[1] C.C. Ibarra-Herrera, O. Aguilar, M. Rito-Palomares, Application of an aqueous
two-phase systems strategy for the potential recovery of a recombinant
protein from alfalfa (Medicago sativa ), Sep. Purif. Technol. 77 (2011) 9498.
[2] C.W. Ooi, B.T. Tey, S.L. Hii, S.M.M. Kamal, J.C.W. Lan, A. Ariff, T.C. Ling,
Purication of lipase derived from Burkholderia pseudomallei with alcohol/salt-
based aqueous two-phase systems, Process Biochem. 44 (2009) 10831087.
[3] Z. Li, H. Teng, Z. Xiu, Aqueous two-phase extraction of 2,3-butanediol from
fermentation broths using an ethanol/ammonium sulfate system, Process
Biochem. 45 (2010) 731737.
[4] H.S. Ng, C.P. Tn, S.K. Chen, M.N. Mokhtar, A. Ariff, T.C. Ling, Primary capture of
cyclodextrin glycosyl transferase derived from Bacillus cereus by aqueous two
phase system, Sep. Purif. Technol. 81 (2011) 318324.
[5] V. Augugliaro, L. Palmisano, Green oxidation of alcohols to carbonyl
compounds by heterogeneous photocatalysis, ChemSusChem 3 (2010) 1135
1138.
[6] Y. Ma, Z. Chang, X. Hu, P. Yu, S. Wang, C. Lei, H. Liu, Separation of butyl acetate
from model emulsions by solvent sublation, Sep. Purif. Technol. 72 (2010) 77
84.
[7] K.T. Valsaraj, J.L. Porter, E.K. Liljenfeldt, C. Springer, Solvent sublation for the
removal of hydrophobic chlorinated compounds from aqueous solutions,
Water Res. 20 (1986) 11611175.
[8] Y. Wang, X.-h. Xu, J. Han, Y.-s. Yan, Separation/enrichment of trace tetracycline
antibiotics in water by [B
mim
]BF
4
(NH
4
)
2
SO
4
aqueous two-phase solvent
sublation, Desalination 266 (2011) 114118.
[9] P.L. Show, C.P. Tan, M.S. Anuar, A. Ariff, Y.A. Yusof, S.K. Chen, T.C. Ling, Direct
recovery of lipase derived from Burkholderia cepacia in recycling aqueous two-
phase otation, Sep. Purif. Technol. 80 (2011) 577584.
[10] X. Wang, M.M. Maroto-Valer, Integration of CO
2
capture and mineral
carbonation by using recyclable ammonium salts, ChemSusChem 4 (2011)
12911300.
[11] M. Yu, J.S.H. Tsang, Use of ribosomal promoters from Burkholderia cenocepacia
and Burkholderia cepacia for improved expression of transporter protein in
Escherichia coli , Protein Expression Purif. 49 (2006) 219227.
[12] H.L. Lau, A. Ariff, K.K. Woo, T.C. Ling, S.L. Hi, Production and optimization of
alkalostable lipase by alkalophilic Burkholderia cenocepacia ST8, Afr. J.
Biotechnol. 10 (2011) 70027009.
[13] P.L. Show, C.P. Tan, M. ShamsulAnuar, A. Ariff, Y.A. Yusof, S.K. Chen, T.C. Ling,
Extractive fermentation for improved production and recovery of lipase
derived from Burkholderia cepacia using a thermoseparating polymer in
aqueous two-phase systems, Bioresour. Technol. 116 (2012) 226233.
[14] M.R. Naimi-Jamal, H. Hamzeali, J. Mokhtari, J. Boy, G. Kaupp, Sustainable
synthesis of aldehydes, ketones or acids from neat alcohols using nitrogen
dioxide gas, and related reactions, ChemSusChem 2 (2009) 8388.
[15] C.W. Ooi, C.P. Tan, S.L. Hii, A. Ariff, S. Ibrahim, T.C. Ling, Primary recovery of
lipase derived from Burkholderia sp. ST8 with aqueous micellar two-phase
system, Process Biochem. 46 (2011) 18471852.
[16] P.L. Show, C.P. Tan, M.S. Anuar, A. Ariff, Y.A. Yusof, S.K. Chen, T.C. Ling, Primary
recovery of lipase derived from Burkholderia cenocepacia strain ST8 and
recycling of phase components in an aqueous two-phase system, Biochem.
Eng. J. 60 (2012) 7480.
[17] P.K. Smith, R.I. Krohn, G.T. Hermanson, Measurement of protein using
bicinchoninic acid, Anal. Biochem. 150 (1985) 7685.
[18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
of bacteriophage T4, Nature 227 (1970) 680685.
[19] W. Zhi, Q. Deng, Purication of salvianolic acid B from the crude extract of
Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two phase
system by counter-current chromatography, J. Chromatogr. A. 1116 (2006)
149152.
[20] A. Kamimura, Y. Oishi, K. Kaiso, T. Sugimoto, K. Kashiwagi, Supercritical
secondary alcohols as useful media to convert polyamide into monomeric
lactams, Chem. Sus. Chem. 1 (2008) 8284.
[21] J. Han, Y. Wang, C. Yu, C. Li, Y. Yan, Y. Liu, L. Wang, Separation, concentration
and determination of chloramphenicol in environment and food using an ionic
liquid/salt aqueous two-phase otation system coupled with high-
performance liquid chromatography, Anal. Chim. Acta 685 (2011) 138145.
[22] T. Sarachat, O. Pornsunthorntawee, S. Chavadej, R. Rujiravanit, Purication and
concentration of a rhamnolipid biosurfactant produced by Pseudomonas
aeruginosa SP4 using foam fractionation, Bioresour. Technol. 101 (2010)
324330.
[23] P.-y. Bi, D.-q.Li, H.-r. Dong, A novel technique for the separation and
concentration of penicillin G from fermentation broth: aqueous two-phase
otation, Sep. Purif. Technol. 69 (2009) 205209.
[24] P.-y. Bi, H.-r. Dong, J. Dong, The recent progress of solvent sublation, J.
Chromatogr. A 1217 (2010) 27162725.
[25] P. Yu, K. Huang, J. Zhao, C. Zhang, K. Xie, F. Deng, H. Liu, A novel separation
technique: Gas-assisted three-liquid-phase extraction for treatment of the
phenolic wastewater, Sep. Purif. Technol. 75 (2010) 316322.
[26] E.A. Quadrelli, G. Centi, J.-L. Duplan, S. Perathoner, Carbon dioxide recycling:
emerging large-scale technologies with industrial potential, ChemSusChem 4
(2011) 11941215.
[27] Y. Yanagishita, M. Kato, K. Toshima, S. Matsumura, Chemo enzymatic synthesis
and chemical recycling of sustainable polyurethanes, ChemSusChem 1 (2008)
133142.
[28] D. Dennewald, W.-R. Pitner, D. Weuster-Botz, Recycling of the ionic liquid
phase in process integrated biphasic whole-cell biocatalysis, Process Biochem.
46 (2011) 11321137.
118 P.L. Show et al. / Separation and Purication Technology 110 (2013) 112118