Journal of Environmental Chemical Engineering 6 (2018) 4144–4150

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Journal of Environmental Chemical Engineering

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Investigation of the activity of Acinetobacter calcoaceticus biodemulsifier to T

break stable water in oil emulsions
⁎
N. Akbari, D. Biria
Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Biodemulsifier production by Acinetobacter calcoaceticus and its capability to break the stable water in oil
Biodemulsifier emulsions were investigated. The chemical structure of the isolated biodemulsifiers from cultivation on various
Molecular structure nitrogen sources were preliminarily analyzed by Fourier-transform infrared spectroscopy (FT-IR). The cultiva-
O/W emulsion tion conditions such as temperature, pH and carbon to nitrogen source concentration (C/N) ratio were optimized
Stability
for maximum demulsification activity through response surface methodology. It was disclosed that the com-
Optimization
position of the isolated biodemulsifiers was depended on the utilized nitrogen source in the medium and altered
from a cyclic lipopeptide (on soybean) to a lipopolysaccharide (on ammonium salts). The obtained optimum
conditions for the biodemulsifier produced on ammonium nitrate (as the nitrogen source) were 35 °C for tem-
perature, 10 for C/N ratio and 5 for pH. Results indicated that the produced extracellular biodemulsifier can
reduce the surface tension to 38.6 mN/m and break 95% of a surfactant stabilized emulsion at the optimum
conditions.

1. Introduction
One of the important problems associated with the petroleum in-
dustry is the formation of water in oil emulsions in various stages of oil
production and recovery [1,2]. In fact, several undesirable con-
sequences such as higher transport and storage costs, corrosion of the
equipment and poisoning of the refining catalysts can be intensified by
the crude emulsions [3]. Therefore, destabilization of emulsions to
obtain a single oil phase is quite necessary. To this end, employing the
synthetic and biological demulsifiers has been suggested [4–8]. De-
mulsifiers are surface active molecules which can displace the emulsi-
fier molecules at the oil/water interface and modify the emulsion
properties leading to its destabilization. Recently, the significant ad-
vantages of biodemulsifiers (e.g. a more diverse chemical structure,
higher biodegradability, and low cost and toxicity) make them an in-
teresting alternative to the synthetic counterparts [5,9,10]. Biode-
mulsifiers can be divided to two main groups of cell bound and extra-
cellular metabolite demulsifiers. In the cell bound group, the cell wall
surface molecules such as proteins are considered as the demulsifying
agent [11]. The extracellular metabolites mainly consist of lipopeptide
and glycolipid compounds secreted by the cells [11]. There are several
demulsifier producing microorganisms such as the Acinetobacter cal-
coaceticus [2], Micrococcus sp. [6], Ochrobactrum anthropi [12], Bacillus
mojavensis [10], Nocardia [13], Corynebacterium [14], Alcaligenes sp.
[15], Dietzia sp., [16], Streptomyces sp., [17] and Pseudomonas aerugi-
nosa [18] that have been reported in the literature. It should be noted
that microorganisms which can grow on low cost carbon sources (e.g.
hydrocarbons in oily waste waters), tolerate the extreme conditions
such as the high salinity and produce an extracellular demulsifier are
more appropriate for industrial production of biodemulsifiers. There-
fore, the above mentioned list will be shortened to just a few options
like Acinetobacter calcoaceticus. Moreover, the produced biodemulsifier
should be highly active to destabilize the emulsions effectively. Un-
fortunately, the demulsification activity of many reported biodemulsi-
fiers is not high enough for practical and environmental applications.
Therefore, any effort to increase the activity of a biodemulsifier with
proper characteristics can be extremely advantageous.
The activity of biodemulsifiers can be related to the culturing con-
ditions of the producing microorganisms in two ways. Firstly, favorable
conditions stimulate and increase their production by the microorgan-
isms. For instance, it is believed that the hydrocarbon assimilating
bacteria produce biodemulsifier compounds to detach the biphasic oil/
water interface as a result of environmental stresses (e.g. lack of ni-
trogen source or accumulation of toxic compounds) [19]. Consequently,
composition of the culturing media (e.g. carbon and nitrogen sources)
can affect the demulsification production and activity [6,10,16]. For
example, Mutalik et al. reported that organic nitrogen sources were
effectively enhanced the production of biosurfactant rather than

⁎
Corresponding author at: Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, P.O. Box: 8174673441, Iran.
E-mail address: d.biria@ast.ui.ac.ir (D. Biria).

https://doi.org/10.1016/j.jece.2018.05.057
Received 17 January 2018; Received in revised form 5 May 2018; Accepted 31 May 2018
2213-3437/ © 2018 Elsevier Ltd. All rights reserved.
N. Akbari, D. Biria
inorganic nitrogen sources [20]. In another study, Li et al. improved the
demulsification ratio of a biodemulsifier produced by Bacillus moja-
vensis XH1 more than 35% through optimization of the production
medium [10]. Secondly, the chemical structure of the produced bio-
demulsifier can be changed by alteration of the culturing conditions. It
should be noted that even minor modifications in the chemical struc-
ture of biosurfactants can significantly influence their activity. Mor-
ikawa et al. showed that chemical modification of cyclic lipopeptide
biosurfactants can alter their activity drastically [21]. The dependency
of structure to chemical composition of culturing media has been re-
ported for biodemulsifiers. For example, Huang et al. showed that the
composition and activity of the produced biodemulsifier by Alcaligenes
sp. S-XJ-1 was depended on the utilized carbon source in the medium
[22]. Therefore, the optimization of the cultivation conditions can be
considered as an effective approach to increase the biodemulsification
activity.
On this basis, the purpose of the present study is the isolation and
preliminary characterization of the biodemulsifier produced by
Acinetobacter calcoaceticus to investigate the influence of various ni-
trogen sources on its structure and activity. Then, the demulsification
activity of the produced biodemulsifier will be maximized through
optimization of the culturing conditions using Box-Benken response
surface methodology (RSM).
2. Materials and methods
2.1. Materials
In this work, Span 80 was purchased from Daejung Chemicals &
Metals Co. Ltd (S. Korea). Kerosene and normal paraffin were obtained
from Esfahan Oil Refining Co. (Iran). Tween 80 was prepared from
Sigma–Aldrich. Analytical grades of the mineral salts such as
MgSO4·7H2O, KH2PO, K2HPO4, NH4NO3, CaCl2·2H2O, FeSO4·7H2O,
KNO3, (NH4)2SO4, NaCl and Ethylenediaminetetraacetic acid (disodium
salt dihydrate ≥97.0%) (EDTA) were obtained from Merck.
2.2. Microorganism and the growth conditions
2.2.1. Bacterial cultivation and biodemulsifier production
Acinetobacter calcoaceticus (ATCC 23055) which was previously
proved to have the capability of biodemulsifier production was used in
this study [2]. It was initially cultured in 100 mL of a nutrient broth
medium which enriched by 3.0 g/L beef extract and 5.0 g/L peptone
(pH = 7). After 17 h of growth, 5 mL of the obtained broth was added to
100 mL erlenmeyers containing 50 mL of a modified mineral salts
(MMS) medium and 4% (v/v) kerosene as the carbon source. The
composition of MMS medium was MgSO4·7H2O 0.2 g/L, KH2PO 6 g/L,
K2HPO4 4 g/L, NH4NO3 4 g/L and 1 mL of a trace mineral solution at pH
7. Trace mineral solution contained CaCl2·2H2O 1 g/L, FeSO4·7H2O 1 g/
L and EDTA 1.4 g/L [10]. The flasks were incubated at 30 °C and
180 rpm in a rotary shaker incubator for 24 h. Next, the remaining oily
phase was removed from the system and bacterial cells and debris were
separated by centrifugation at 12,000 rpm and 4 °C for 15 min. The
obtained supernatant was used as the cell free biodemulsifier solution
at the consequent experiments.
The dependence of the produce biodemulsifier to the bacterial cells
was also investigated. To this end, the supernatant was separated
carefully and the harvested cells were re-suspended in a phosphate
buffer solution. Next, the demulsifying activities of the cell free bio-
demulsifier solution and the suspended cells were determined.
The time course study of the biodemulsifier production by the
bacteria was performed within a 96 h period. At time intervals of 24 h,
dry weight of the bacterial cells, demulsifying activity and surface
tension of the biodemulsifier samples were measured and reported.
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Journal of Environmental Chemical Engineering 6 (2018) 4144–4150
2.2.2. Effects of the nitrogen source type on the produced biodemulsifier
The influence of various nitrogen sources on the structure and ac-
tivity of the produced biodemulsifier was studied. In this way, the
ammonium nitrate in the MMS medium was displaced by each of the
examined nitrogen sources (i.e. soybean meal, potassium nitrate and
ammonium sulfate) and the demulsification ratio of the produced bio-
demulsifiers were measured. The nitrogen sources were selected based
on the complexity and solubility in water (nitrate and ammonium salts
vs. soybean meal). It has been reported in the literature that the low
soluble nitrogen sources can stimulate the production of some bio-
surfactants [23] while there are some reports that the nitrate has been
found as the most effective nitrogen source [5]. Chemical structure of
the isolated biodemulsifier from each case was studied as well. Finally,
the nitrogen source with the most active biodemulsifier was selected for
the optimization experiments.
2.2.3. Effects of the carbon source type on the produced biodemulsifier
To evaluate the influences of various carbon sources on the activity
of the produced biodemulsifier, several flasks of the MMS medium were
supplemented with 4% v/v of carbon sources such as kerosene, bitter
almond oil, glucose and liquid paraffin mixture. It is reported that the
carbon source characteristics such as hydrophobicity and chemical
composition can be effective on the produced biodemulsifier by the
bacteria [5]. The studied carbon sources were selected from a wide
range of options such as glucose as a simple water soluble carbohydrate
to linear and cyclic compounds in the paraffinic mixture and bitter
almond oil. The demulsification activities of the obtained biodemulsi-
fier solutions were measured.
2.2.4. Effects of the salinity on the produced biodemulsifier
Breaking the stable brine in oil emulsions in upstream of the pet-
roleum industry is of the great importance. Therefore, a suitable bio-
demulsifier should have a high activity in higher salinities. To in-
vestigate the effect of salinity on the activity of the biodemulsifier, NaCl
was added to the MMS culturing media to obtain salt concentrations
equal to 5, 10, 15 and 20% w/v. Kerosene was added to media at 4%
(v/v) as the carbon source and all of the cultivation criteria were the
same as described above. The corresponding blank tests with the same
salt concentrations were carried out to ensure the effectiveness of the
produced biodemulsifier. The reported data are the mean value of tri-
plicates.
2.2.5. Emulsion preparation and demulsification tests
Water in oil (W/O) model emulsion was prepared according to the
suggested method of Huang et al. with some modifications [24]. The
oily phase was consisted of Span 80 (as the surfactant) in 80 mL of
kerosene at a final concentration of 1.9% w/v. 120 mL of water with
0.1% w/v Tween 80 was used as the aqueous phase. The aqueous phase
was slowly added to the oily phase at a rate of 2 mL/s as they were
vigorously mixed by a homogenizer (T25 IKA, Germany) at 10,000 rpm
for 5 min.
The emulsion type was identified using a water soluble dye (Navy
Blue) through microscopic observation. A small amount of Navy Blue
was added to the emulsion and the resultant mixture was observed
under the microscope. The scattered globules (aqueous phase) was
appeared blue while the continuous phase (oil) was colorless [12].
Therefore, the prepared emulsion can be considered as water in oil
emulsion.
In the demulsification tests, 2 mL of the produced biodemulsifier
solution was added to 18 mL of the prepared model emulsion in a 25 mL
graduated test tube [25] and mixed by a vortex mixer for 1 min. Then, it
was left undisturbed in a water bath at 30 °C for 24 h. After this time,
three liquid phases (i.e. separated oily phase, separated aqueous phase
and the remaining emulsion phase) can be observed in the tube. The
demulsification activity of the biodemulsifier can be calculated through
the following equation:
N. Akbari, D. Biria Journal of Environmental Chemical Engineering 6 (2018) 4144–4150

Demulsification ratio% Table 1

Box-Benken experimental design and the obtained results.
Oil Volume + Water Volume
= ⎜⎛ ⎞⎟
Original Emulsion Volume + Added Biodemulsifier Volume ⎠ Run FactorA: Factor B: C/ Factor C: Demulsification ratio
⎝ (1)
temperature N pH (%)

The blank test was conducted with 2 mL of MMS medium instead of 1 45 10 5 93.150
the biodemulsifier solution, and the emulsion breaking after 24 h for 2 25 15 7 79.720
this test was less than 5% which indicated that the synthetic emulsion 3 35 10 7 85.300
4 35 10 7 85.300
model was very stable.
5 45 5 7 82.600
6 25 10 9 86.229
7 35 15 5 93.300
2.3. Characterization of the demulsifying agent 8 35 15 5 94.660
9 45 10 5 86.930
2.3.1. Surface tension measurement 10 45 10 9 89.740
11 35 15 9 88.300
The surface tension (ST) of the biodemulsifier solution was mea-
12 35 5 5 93.300
sured by a platinum ring tensiometer (K20 Easy Dyne, Kruss, Germany) 13 35 15 9 90.600
at ambient temperature according to the procedure described by 14 45 15 7 72.000
Bodour and Miller-Maier [26]. Each reported value is the average of 15 35 10 7 86.600
16 35 10 7 86.600
five replicated measurements.
17 25 5 7 52.074
18 25 15 7 76.000
19 35 10 7 89.041
2.3.2. Biodemulsifier isolation and identification of the chemical functional 20 35 10 7 80.820
groups 21 25 10 5 83.780
The isolation of produced biodemulsifier from the bacterial solution 22 35 5 5 90.540
23 45 15 7 74.600
was carried out according to the method described by Kim et al. [27]. 24 25 10 5 86.600
After cultivation for 24 h, cells were removed from the fermented broth 25 35 5 9 70.600
medium by centrifuge (12,000 rpm at 4 °C for 15 min). Then, the col- 26 35 5 9 70.000
lected supernatant acidified with 4N HCL solution to pH 2. The pre- 27 45 5 7 73.000
28 25 5 7 52.074
cipitated demulsifying agent was settled at 4 °C overnight and re-
29 25 10 9 86.229
covered by centrifuge (12,000 rpm at 4 °C for 20 min). The obtained 30 45 10 9 90.600
solids were dissolved in chloroform/methanol (2:1, v/v) to remove the
mineral impurities. Finally, the organic solvents were separated in a
rotary evaporator to obtain a dry biodemulsifier powder. 3. Result and discussion
To investigate the effect of various nitrogen sources on the produced
biodemulsifier, the chemical structure of the isolated dried biode- 3.1. Biodemulsifier production
mulsifiers was preliminarily identified by Fourier transform infrared
spectrometer (FT-IR, JASCO 6300, Japan). The contributing functional Fig. 1 shows the time course of the produced biodemulsifier activity
groups in their structures were determined by the resultant spectra. by Acinetobacter calcoaceticus in 96 h. As can be seen, the maximum
demulsification ratio (68.67 ± 0.325%) and dry weight of the pro-
duced cells (1.384 ± 0.8 g) were achieved after 24 h of cultivation.
2.3.3. Optimization of biodemulsifier activity Moreover, surface tension of the demulsifier solution reduced to
Response surface methodology (RSM) is a statistical technique of 38.6 ± 0.6 mN/m after the first day and stayed constant. This in-
design of experiments to analyze and optimize the effects of the im- dicated that the biodemulsifier is a primary metabolite that produced in
portant independent variables on the system response [28,29]. In this the exponential growth phase of the bacteria.
work, the optimum conditions for the maximum activity of the pro- Generally, the surface activity of the biological demulsifiers can be
duced biodemulsifier by Acinetobacter calcoaceticus were determined related to the cell surface molecules or derived from the production of
through Box-Behnken response surface methodology. In Box-Behnken extra cellular metabolites. In this work, the origin of demulsifying ac-
method, each factor contains three equally spaced level values and a tivity of the system was determined by measuring the demulsification
quadratic model will be estimated to describe the obtained results. Box ratio for both the bacterial cells suspension and the cell free solution.
Behnken design is more efficient than the other response surface The results have been illustrated in Fig. 2. It was revealed that the cell
methods (e.g. Central Composite Design) at the same accuracy level suspension demulsifying activity (35%) was much lower than the ac-
because it requires less experimental runs [30]. tivity of the cell-free solution which showed a demulsifying ratio equal
Among the various parameters affecting the activity of the produced to 67.5 ± 1.1%. These results confirmed that the demulsification ac-
biodemulsifier, it was found that the temperature, pH and C/N ratio are tivity of the bacteria is mostly related to their extracellular metabolites.
the most important parameters. The levels of the selected variables This is an advantageous feature that facilitates the isolation of the
were designed in such a way that a wide range of variation could be produced demulsifier and reduces the cost of production.
covered. In fact, Acinetobacter calcoaceticus is a mesophilic bacterium
which can grow in moderate temperatures of the selected range. C/N
ratio from 5 to 15 covers a wide domain from high to low nitrogen 3.2. Chemical structure of the produced biodemulsifiers
source concentrations and pH from 5 to 9 includes the tolerable con-
ditions for the bacterial growth. The parameters and their levels in form The influence of various nitrogen sources (ammonium nitrate as a
of the designed experiments have been shown in Table 1. soluble source and soybean meal as an insoluble source) on the struc-
The demulsification ratios of the obtained biodemulsifier solutions ture of the produced biodemulsifiers was investigated and the obtained
were calculated and considered as the response. A mathematical model FT-IR spectra of the isolated biodemulsifiers have been shown in Fig. 3.
was fitted to the results and analysis of variance (ANOVA) was utilized The presence of peaks such as 3429 cm−1 for OH and NH, 1538 cm−1
to study the significance of the experimental factors as well as the ob- for NH, 1656 cm−1 for amide stretched CO, 1735 cm−1 for CO lactone
tained model on a basis of a 0.95 confidence level. ring, 1105 cm−1 for CN, 1015 cm−1 for esteric CO, 1450 cm−1,

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N. Akbari, D. Biria
Fig. 1. Time-course profiles of bacterial growth (left), surface activity and demulsification ratio of the produced biodemulsifier (right).
Fig. 2. Demulsification activities of the blank samples (Distilled water and
MMS medium) vs. the cultured medium and biodemulsifier solution.
2932 cm−1 and 2878 cm−1 for CH groups in the obtained biodemulsi-
fier from the soybean containing medium revealed that it should be
classified as a cyclic lipopeptide. Although the production of lipopep-
tides with demulsifying activity by a few bacteria such as Paenibacillus
alvei, Dietzia and Alcaligenes sp. has been previously reported [3,15,16],
the production of a lipopeptide demulsifier by Acinetobacter calcoace-
ticus has been hardly mentioned.
On the other hand, the biodemulsifier produced on the soluble
Fig. 3. FTIR spectra of the demulsifier produced by Acinetobacter calcoaceticuson various nitrogen sources.
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Journal of Environmental Chemical Engineering 6 (2018) 4144–4150
nitrogen source was identified as a lipopolysaccharide because of the
observation of the related peaks such as 3254 cm−1 for OH, 1294 cm−1
and 1764 cm−1 for stretched carbonyl CO, 1102 cm−1 for stretched CO,
1385 cm−1, 2867 cm−1 and 3124 cm−1 for CH groups. In fact,
Acinetobacter calcoaceticus has been previously well known for produ-
cing lipopolysaccharides such as the emulsan. Obviously, more analysis
needed to exactly determine the chemical structure of these molecules.
The demulsification activities of the two compounds were measured
and the results have been shown in Fig. 4. Apparently, the activity of
the demulsifier from ammonium nitrate containing medium is slightly
higher than that of produced in the soybean mill medium. However, the
demulsification activities of the obtained biodemulsifiers from the other
water soluble nitrogen sources such as ammonium sulfate and po-
tassium nitrate were lower and ammonium salt had a better outcome.
In fact, the solubility of biodemulsifiers in oil/aqueous phases is af-
fected by their chemical structure which is of the great importance to
the demulsification activity. The emulsion destabilizing is suggested to
be a result of demulsifier molecules diffusion into the two phase in-
terface and increasing the interfacial surface energy which in turn
makes the tiny emulsion droplets to coalesce into larger ones and fa-
cilitates the phase segregation [31,32].
3.3. Effects of carbon source on the production of biodemulsifier
The biodemulsifier solutions produced by the examined carbon
N. Akbari, D. Biria
Fig. 4. Demulsification activity of the produced biodemulsifiers on various
nitrogen sources.
Fig. 5. The influence of various carbon sources on demulsification ratio of the
produced biodemulsifier.
sources were tested for demulsifying activity and the results have been
shown in Fig. 5. Evidently, the glucose showed the highest demulsifi-
cation ratio (74.75%) and paraffin (67.4%) and kerosene (68%) came to
the next. The activity of biodemulsifier on almond oil was the least
(48%).
Although the production of biodemulsifier on both the hydrophilic
and hydrophobic substrates has been previously represented [5,10],
few reports are available on the production of highly active biode-
mulsifiers on glucose as the sole carbon source. Huang et al. [22]
suggested that the presence of glucose in the culturing medium will
cause a high contribution of the oxygen containing functional groups in
the biodemulsifier molecules (i.e. more polysaccharides on the cell
surface) and they concluded that it can reduce the demulsification ac-
tivity. However, the production of the lipopolysaccharide biodemulsi-
fier by the utilized Acinetobacter calcoaceticus in this work led to a dif-
ferent result as the most active biodemulsifier attained in a glucose
containing medium. On the other hand, the produced biodemulsifier in
this work was found out to be a growth dependent metabolite and
glucose can produce a higher cell density because of its high solubility
and simple structure. The similar activities of the biodemulsifier solu-
tions produced on kerosene or liquid paraffin mixture were not unusual
because a large fraction of kerosene consists of paraffinic materials. In
fact, it is suggested that paraffin can promote the activity of biode-
mulsifiers by modifying their lipid moieties [10]. Almond oil which is
rich of cyclic compounds (benzaldehyde) exhibited the lowest activity
which means that the cyclic hydrocarbons are not a good option to
produce biodemulsifiers by Acinetobacter calcoaceticus.
It should be noted that the aim of this work was to produce a highly
active biodemulsifier for petroleum industry which can be potentially
produced in oily waste waters. Therefore, kerosene was selected as the
carbon source for the optimization experiments.
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Journal of Environmental Chemical Engineering 6 (2018) 4144–4150
Fig. 6. Demulsification activity of the saline MMS medium (control tests) and
the effect of salinity on the produced biodemulsifier activity.
3.4. Effects of salinity on the biodemulsifier activity
The effects of salinity on biodemulsifier production as well as the
control tests (the saline medium without carbon source and bacteria)
have been shown in Fig. 6. It was revealed that the salt has a weak
demulsification activity as the maximum demulsification ratio of the
saline medium was obtained about 7 ± 1% for the 20% salt con-
centration. Evidently, the addition of NaCl had a positive effect on the
demulsifying activity of the produced biodemulsifier because the ob-
served increase in the demulsification ratios of the biodemulsifier so-
lution samples was higher than that of the blank tests without biode-
mulsifier. The maximum biodemulsification activity was obtained equal
to 82.25 ± 3.18 with 10% NaCl supplementation to the culturing
medium. Clearly, this means that the utilized microorganism in this
work is halotolerant which can be extremely beneficial to break the
brine in oil emulsions in a desalting process.
3.5. Optimization of biodemulsifier activity
The optimization of the biodemulsifier activity was carried out
through finding the maximum points of the response surface equation.
The experimental results of Box-Behnken design were employed to fit a
quadratic model to the obtained responses (percent of the demulsifi-
cation ratio) by linear regression. The resultant equation has been re-
presented below:
%DR = 37.7386 − 7.84173
(A) + 9.37073(B) − 41.239(C) − 0.177083
(A × B) + 0.268792(A × C) + 0.42725(B × C) + 0.10338
(A2) + 1.75696 (B2) (2)
Where DR% is percentage of the demulsification ratio and A, B and C
are coded variables for the temperature, C/N and pH, respectively. The
significance of the obtained model and its terms was investigated by
analysis of variance (ANOVA), as shown in Table 2. Accordingly, all of
the studied main and interactive effects were significant as their p-va-
lues were less than 0.05. Moreover, the small P-value of the model
(0.0001) with the high adjusted determination coefficient
(R2 = 0.9018) and its non significant lack of fit (P-value = 0.156) ap-
proved the adequacy of the proposed model. The comparison between
the model coefficients revealed that although all of the main and in-
teractive effects were proved to be significant, the main effects were
stronger than interactions as their absolute coefficient values were
higher. Moreover, the negative sign of the interaction coefficient be-
tween temperature and C/N ratio (A x B) indicated that the increase in
temperature reduced the effect of C/N ratio. The other interactions
coefficients had positive values and therefore had a unidirectional in-
fluence on demulsification ratio. The optimum criteria were determined
N. Akbari, D. Biria Journal of Environmental Chemical Engineering 6 (2018) 4144–4150

Table 2 Fig. 7a. As can be seen, the lowest activity of the biodemulsifier was
Analysis of variance (ANOVA) for the obtained response surface quadratic observed in neutral pH values. This can be explained by the fact that the
model. ionization of the polar functional groups in the biodemulsifier molecule
Source DF Adj SS Adj MS F P will take place at either low or high pH values and the ionized molecule
has a much stronger interaction with the emulsion structure. On the
Regression 9 1282.62 142.514 16.32 0.000 other hand, the increase of the medium temperature enhanced the
Linear 3 573.60 191.198 21.90 0.000
demulsifying activity and the maximum activity was occurred at higher
A 1 275.52 275.521 31.55 0.000
B 1 335.97 335.972 38.47 0.000 temperatures. In fact, higher temperatures seem to be beneficial to
C 1 336.92 336.919 38.58 0.000 production of higher amounts of biodemulsifier. However, when tem-
Square 3 840.78 280.260 32.09 0.000 perature passed the limit of 37 °C, the demulsifying activity decreased
A*A 1 410.07 410.066 46.96 0.000
because Acinetobacter calcoaceticus is a mesophilic species and the
B*B 1 218.86 218.862 25.06 0.000
C*C 1 278.91 278.911 31.94 0.000
bacterial cell growth at higher temperatures would be suppressed.
Interaction 3 445.06 148.354 16.99 0.000 The interactions between C/N ratio and temperature have been
A*C 1 99.08 99.084 11.35 0.004 shown in Fig. 7b. Clearly, higher demulsification activities have been
A*B 1 268.79 268.787 30.78 0.000 observed at high C/N ratios. This is in agreement with previous re-
C*B 1 146.03 146.034 16.72 0.001
search that reported biosurfactants with higher activities would be
Residual Error 16 139.72 8.732
Lack-of-Fit 1 18.06 18.057 2.23 0.156 produced at a low nitrogen source concentration or using a nitrogen
source with limited water solubility [23]. Apparently, rising the tem-
perature can fix the low activity of the biodemulsifier at low C/N ratios
as 35 °C for temperature, 10 for C/N ratio and 5 for pH to produce the to some extent. Therefore, the high demulsification activity at the low
biodemulsifier with the highest efficiency (97%). These conditions were C/N ratios was only attainable at high temperatures.
examined experimentally and a demulsification ratio equal to 95% was The joint effects of C/N ratio and pH have been shown in Fig. 7c. At
obtained which is in a good agreement with the optimum prediction of higher C/N ratios, both the acidic and alkaline pH values were favor-
the model. able to the biodemulsifier activity while the activity at the neutral pH
The interactive effects of the important factors were illustrated values reached to the minimum. At lower C/N ratios, the biodemulsifier
through the response surface plots (Fig. 7). The interactive effects of activity was higher at more acidic pH values. In fact, the lipopoly-
temperature and pH on demulsifying activity have been shown in saccharide demulsifier produced by Acinetobacter calcoaceticus contains

Fig. 7. The surface plots for interactive effects: (A) Temperature and pH, (B) Temperature and C/N ration and (C) pH and C/N ratio.

4149
N. Akbari, D. Biria
varying quantities of non-covalently bound proteins which their
quantity, electrical charge and structure would be changed by the al-
teration of pH and C/N ratio of the culturing medium [33]. The ob-
served variation in the demulsification activity can be related to these
proteins in the system. Clearly, the higher C/N ratios intensified the
biodemulsifier activity all over the studied pH range which can be re-
lated to the higher yield of biodemulsifier production at the lower ni-
trogen source concentrations as mentioned before.
4. Conclusions
Demulsification activity of the biodemulsifier produced by
Acinetobacter calcoaceticus was investigated. It was revealed that the
bacteria can produce an effective extracellular demulsifier to break the
water in oil emulsions even at the saline medium. The chemical
structure of the biodemulsifier was altered by the change in the utilized
nitrogen source, from a cyclic lipopeptide to a lipopolysaccharide in
soybean and ammonium nitrate containing media respectively.
Moreover, the demulsification activity was maximized by tuning the
operating conditions (i.e. temperature, pH and C/N ratio) and a high
activity equal to 95% was achieved at the optimum conditions. The
high activity in combination with the ability of Acinetobacter calcoace-
ticus to grow and produce the biodemulsifier at extreme conditions (e.g.
high salinity, acidic or alkaline pH, low water soluble carbon and ni-
trogen sources) makes it an appropriate option to be applied in the
environmental applications.
Declaration of conflict of interests
The authors confirm that there are no conflicts of interest regarding
the publication of this article.
Acknowledgements
This work has been partially supported by the National Iranian Oil
Refining & Distribution Company (NIORDC). The authors are also
grateful to University of Isfahan Research Council for financial support
of this project.
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