Bioresource Technology 116 (2012) 2428

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Oil recovery from renery oily sludge using a rhamnolipid biosurfactant-producing

Pseudomonas
Ping Yan a,b, Mang Lu c, Qin Yang d, Hai-Ling Zhang d, Zhong-Zhi Zhang a,, Rong Chen e
a

State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 102249, China
Dalian Petrochemical Branch Company, PetroChina, Dalian 116000, China
c
School of Materials Science and Engineering, Jingdezhen Ceramic Institute, Jingdezhen 333403, Jiangxi, China
d
Oil & Gas Technology Research Institute, Changqing Oileld Company, PetroChina, Xian 710018, China
e
Beijing Green Oil Technology Services Co., Ltd., Beijing 100028, China
b

a r t i c l e

i n f o

Article history:
Received 18 January 2012
Received in revised form 7 April 2012
Accepted 9 April 2012
Available online 17 April 2012
Keywords:
Biosurfactants
Pseudomonas aeruginosa
Oil recovery
Oily sludge

a b s t r a c t
In this study, a rhamnolipid biosurfactant-producing strain, Pseudomonas aeruginosa F-2, was used to
recover oil from renery oily sludge in laboratory and pilot-scale experiments. The optimum values of
carbon to nitrogen ratio, temperature, sludgewater ratio and inoculum size for oil recovery were determined as 10, 35 C, 1:4 and 4%, respectively. An oil recovery of up to 91.5% was obtained with the equipping of draft tubes during the eld pilot-scale studies. The results showed that strain F-2 has the potential
for industrial applications and may be used in oil recovery from oily sludge.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Oily sludge is a complex mixture of petroleum hydrocarbons,
sediments, heavy metals and water, generated during the exploration and development of oilelds and also in the petroleum reneries. The annual output of oil sludge by Chinas renery industry
was estimated to be about 1,000,000 tons, mainly derived from
the cleaning process of oil storage tanks (Liu et al., 2011).
Oily sludge is listed as hazardous waste in Resource Conservation and Recovery Act (RCRA) (USEPA, 1989), and represents a major source of contamination for soil, air and ground water. Because
of its large yield, treatment difculties, and potential hazards to the
environment, oily sludge has become as a major problem plaguing
the petroleum and petrochemical industry (Chang et al., 2000).
Due to the numerous sources of oily sludge, there is no single
method for oily sludge treatment. Typically, oil sludge can be handled via various physical and chemical processes such as dewatering and incineration, stabilization, solvent extraction, washing by
hot water and surfactant, pyrolysis, and biodegradation (Jing
et al., 2011). However, each method has its own advantages and
disadvantages. In general, expensive reagents and complex equipments are needed during the oil recovery from oily sludge using
physical and chemical methods, complicating the processes and
Corresponding author. Tel.: +86 10 89734284; fax: +86 10 69744636.
E-mail address: zzzhang1955@hotmail.com (Z.-Z. Zhang).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.04.024

increasing operating costs. In addition, secondary pollution may

be caused when chemical approaches are applied. Microbial degradation of oily sludge typically saves energy, capital investment and
operating costs. However, it would be inadvisable to decompose oil
by microorganisms, since oily sludge is recognized as a valuable
energy resource that can be recycled as fuel (Shie et al., 2000).
Synthetic surfactants that are used to enhance contaminant solubility are often toxic, representing an additional source of contamination (Banat et al., 2004). Recently, extensive studies have
been conducted on the isolation and characterization of surfactant-producing microorganisms and their application in environment remediation (Lai et al., 2009; Saeki et al., 2009; Sarachat
et al., 2010; Ferhat et al., 2011).
Biosurfactants can be used not only for bioremediation processes, but also for cleaning oil storage tanks, increasing ow
though pipelines and enhancing oil recovery from oil reservoirs
(Desai and Banat, 1997). However, studies on the application of
biosurfactants for oil recovery from oily sludge are still very scarce.
Pornsunthorntawee et al. (2008) reported obtaining higher oil
recoveries using biosurfactants than that obtained using synthetic
surfactants on a laboratory scale.
In this study, studies on oil recovery from oily sludge using the
biosurfactant-producing microorganism, Pseudomonas aeruginosa
F-2, were carried out in laboratory and pilot-scale experiments.
The orthogonal experimental design was used to determine the
optimum conditions for oil recovery from the oily sludge. The

P. Yan et al. / Bioresource Technology 116 (2012) 2428

outcome of this work is expected to provide a basis for developing

environmentally friendly and economically competitive approaches for oily sludge treatment.
2. Methods
2.1. Microorganism
In this present study, a biosurfactant-producing microorganism,
identied as P. aeruginosa F-2, was used in the laboratory and eld
pilot tests. This strain was isolated from petroleum-contaminated
soil and stored in our laboratory (unpublished results). Preliminary
experiments revealed that the rhamnolipid biosurfactant produced
by strain F-2 could effectively emulsify crude oil and possessed stable surface activity at variable ranges of pH and salinity.

25

According to the levels listed in Table 1, oil sludge, MM, inoculum and water were added to a 250 mL Erlenmeyer ask to produce a total volume of 100 mL. The mixtures were then shaken
on a rotator at 50 rpm. The extent of oil removal contributed by
ushing with microorganism-free water was evaluated and considered as blank effects in comparison with that using inoculum to
recover oil. To demulsify the mixture, after 72 h of incubation, dilute sulfuric acid was added to the ask at a concentration of 0.33%
(w/v). Then the contents of the ask were allowed to settle for 2 h.
The supernatant and oil layer were decanted from the ask after
settling. The sludge was rinsed with distilled water and shaken
for 3 min at 50 rpm, and the rinse water was decanted after settling. All the decanted solutions were pooled in a separating funnel,
and the oil was extracted with dichloromethane. Oil recovery efciency was calculated gravimetrically after evaporating the solvent
under N2.

2.2. Oil sludge

2.4. Field pilot experiment
The oil sludge sample used in the study was derived from the
bottom sludge of oil separating tank in Dalian Petrochemical
Branch Company, PetroChina, Liaoning Province, north-east China.
The oil sludge sample appeared to be black, viscous and in the form
of semi-solid cake at ambient temperature (Supplementary
Fig. S1).
The organics containing oil and grease were represented by total extractable organics (TEO). The characteristics of the oil sludge
sample were: moisture, 14.46 wt.%; pH, 7.1; ash, 1.98 wt.%; volatile
matter, 95.62 wt.%; TEO, 46.80 wt.%; net caloric value, 28.27 MJ/
kg; bituminous, 5.18 wt.%; saturated hydrocarbon, 23.52 wt.%; aromatic hydrocarbon, 9.53 wt.%; non-hydrocarbon, 8.57 wt.%. The oil
sludge had lower moisture, lower ash content, higher volatile matter and oil content.
2.3. Laboratory batch experiments
To investigate factors impacting the recovery of oil using strain
F-2, batch tests were carried out under varying conditions. The effects of four factors, i.e. C/N ratio, temperature, inoculum size and
sludge/water ratio (v/v), on oil recovery were investigated using a
L9 (34) orthogonal experimental design (Table 1) (Taguchi, 1987).
The results for all nine experiments were analyzed using the statistical software SPSS 13.0 (IBM, Chicago, IL, USA) for the analysis of
variance (ANOVA).
The mineral medium (MM) consisted of (g/L): KCl, 1.1; NaCl,
1.1; KH2PO4, 3.4; K2HPO4, 4.4; plus 5 mL of a trace element solution containing (g/L): ZnSO4
7H2O, 0.29; CaCl2
4H2O, 0.24; CuSO4
5H2O, 0.25; MnSO4
7H2O, 0.17. Nitrogen was added as
(NH4)2SO4 in the proportions as shown in Table 1.
To obtain the inoculum, the bacterial colonies were transferred
into a nutrient broth. Then the culture was incubated at 30 C in a
shaking incubator at 150 rpm for 48 h. The microbial growth was
monitored as a function of cultivation time by measuring the
absorbance of the culture medium every 2 h. From the graph plotting of microbial concentration and time, the best cultivation time
was then determined. Then the culture medium was used as the
inoculum for the tests.
Table 1
Factors and levels for the orthogonal test.
Design variable

(A) C/N ratio

(B) Temperature (C)
(C) Sludgewater ratio (v/v)
(D) Inoculum size (%)

Levels
1

5
20
1:2
2

10
35
1:4
4

20
45
1:6
6

The pilot-scale experiment was carried out in the wastewater

treatment plant of Dalian Petrochemical Branch Company. The
schematic diagram and photos of the experimental system are
shown in Figs. 1 and S2 (Supporting information) respectively.
The system consisted of three identical stainless steel-made tanks
(1.1 m long, 0.8 m wide, 1.1 m high), supported by a steel frame.
They were laid side by side and the system has a total height of
about 2.0 m. Three pipelines were placed on the side of each tank
to introduce sludge, heating steam and tap water into the tanks,
respectively. In addition, a level gauge was mounted on the side
of each tank to control the addition of material and to monitor
the separation of oilwatersediment.
The tanks received oil sludge, inoculum solution, MM solution
and water, based on the optimum conditions determined in the
laboratory study. During the experiments, the local temperatures
were relatively low (1221 C), thus the contents of the tanks were
heated by the introduction of steam. The temperatures were controlled within the range of 3040 C by temperature controllers
connected to the valves of the steam pipelines.
The treatment was terminated after 72 h of incubation by adding sulfuric acid to the tanks at a concentration of 0.33% (w/v).
Then the treated sample was transferred into a centrifuge, and centrifuged at 5000g for 15 min. The oil and aqueous phase after centrifugation were separated, and the mass of oil layer separated was
then measured and considered as the oil recovery.
The concentration of organic material in the aqueous phase separated from the oil was measured as chemical oxygen demand
(COD). For this, samples were ltered through a 0.22 lm (pore
diameter) membrane, and then were reuxed with a known excess
of potassium dichromate for 2 h at about 150 C. After digestion,
the excess dichromate was titrated against ferrous ammonium
sulfate.
2.5. Analysis of sludge properties
Sludge was analyzed for pH, ash and volatile matter using standard methods (Lu, 1999). Water contents were measured using the
Dean and Stark method (ASTM D-95-05, 2005), which involves reux distillation with toluene and separation of the water phase.
The caloric value was determined using a PARR1281 calorimetric
bomb. The sludge settling ratio (SSR) was measured as follows:
Fresh sludge sample was mixed thoroughly, and then transferred
into a 1 L graduated measuring cylinder for SER measurement.
Sludge settling efciency in each cylinder was monitored after
2 h of settlement and expressed as the volume ratio of settled
sludge to mixed liquor.

26

P. Yan et al. / Bioresource Technology 116 (2012) 2428

Thermometer

Steam distributor
Air distributor

Steam distributor

Steam distributor

Air distributor

Air distributor

Oil sludge feeding pump

Steam
Air

Oil storage
Dewatered sludge
Wastewater
Centrifugation
Fig. 1. Schematic diagram of the pilot-scale experimental system.

2.6. Instrumental analysis

Five grams of sub samples of air-dried and pulverized sludge
were mixed with an equal amount of anhydrous Na2SO4. The mixture was extracted in a Soxhlet apparatus with 100 mL of dichloromethane for 8 h. The total amount of extract namely TEO was
quantied gravimetrically after evaporating the solvent under
nitrogen. The dry extracts were fractionated by silica-gel column
chromatography to separate saturate, aromatic, non-hydrocarbon
and bituminous fractions, and each fraction was quantied gravimetrically (Bastow et al., 2007). The hydrocarbon fractions were
analyzed by gas chromatographymass spectrometry (GCMS).
Fractions F1, F2 and F3 were dened as the group of hydrocarbons
from C10 to C16, C16 to C34, and C34 to C50, respectively. The details of the instrumental analyses have been given previously (Yan
et al., 2011).

sludgewater ratio, 1:4; and inoculum size, 4%. The formulation

was adopted in the following studies. To validate the proposed
optimum conditions, experiments on oil recovery were repeatedly
performed under the optimum conditions. The experimental oil
recovery obtained was close to the expected oil recovery estimated
by Taguchi software (Nutek Inc., USA) with a condence level of
95%.
Nitrogen plays an important role in the production of biosurfactants. In general, nitrogen limitation enhances biosurfactant production by Pseudomonas (Syldatk et al., 1985). It has been
demonstrated that the optimum C/N ratios for rhamnolipids production by P. aeruginosa might be 16 (Guerra-Santos et al., 1984)
or 12.24 (Silva et al., 2010). Nevertheless, some other studies have
reported no signicant change in the biosurfactant production by
P. aeruginosa for C/N ratios of 6.552 (Wu et al., 2008).

3.2. Oil recovery under eld conditions

3. Results and discussion
3.1. The orthogonal experiment results
Table 2 shows the orthogonal experimental design and the results obtained from the full 34 factorial experiment matrix. As
shown, the optimum formulation was found to be A1B2C2D2,
and it was obtained as follows: C/N ratio, 10; temperature, 35 C;
Table 2
Orthogonal experimental design and the results obtained from the full 34 factorial
experiment matrix. Control values have been subtracted from the data presented.
Set no.

1
2
3
4
5
6
7
8
9

Design variable

TEO recovery (%)

10
10
10
30
30
30
50
50
50

20
35
45
20
35
45
20
35
45

1:2
1:4
1:6
1:4
1:6
1:2
1:6
1:2
1:4

2
4
6
6
2
4
4
6
2

73.5
86.4
76.2
68.5
71.8
65.3
64.2
67.3
62.1

Based on the optimum conditions, eld experiments were performed from 22nd, August, 2011 to 26th, September, 2011. A total
of 12 batch trials were carried out. Each tank received 150 L sludge,
30 L inoculum solution, 570 L water and the corresponding MM.
Meanwhile, a control without inoculum was set under the same
conditions.
The TEO recovery efciencies of batchs 19 are shown in Fig. 2.
It can be found that the TEO recovery was in the range of 62.6
81.9% for batches 19, and was 11.6% for the control. The lower
TEO recoveries of batches 8 and 9 were attributed to the bad control of tank temperature caused by rain.
Considering the high cost and operational complexity of centrifugation separation, batches 1012 were conducted on a tank
equipped with four draft tubes. After adding the sulfuric acid, aeration was continued for about 2 h, and then the contents of the
tank were allowed to settle down. As shown in Fig. S3 (Supporting
information) most of the oil was enriched in the draft tubes, resulting in recovery efciencies of 86.9%, 91.5%, and 86.6% for batches
10, 11 and 12, respectively. Thus, it can be concluded that the combination of acid-induced demulsication, air oatation and enrichment with draft tubes has a good effect on oil recovery from oil

27

100

1000

80

800

COD concentration (mg/L)

TEO recovery (%)

P. Yan et al. / Bioresource Technology 116 (2012) 2428

60

40

20

600

400

200

0
0

3.3. COD concentration in separated aqueous phase

The aqueous phase separated consisting of oil and other organic
material could be considered as wastewater, and the COD concentration in such aqueous phase was analyzed. As shown in Fig. 4,
COD concentration in wastewater was in the range of 362
891 mg/L for the 12 batches of treatment. This wastewater could
be emitted into the wastewater treatment plant for further
remediation.

10

11

12

Fig. 4. COD concentration in wastewater.

Fig. 2. TEO recovery during the eld pilot-scale experiments. Batch 0 is the control,
prepared without the addition of the biosurfactant producing Pseudomonas.

sludge. In this study, demulsied oil droplets were continuously

centered to the draft tubes during air oatation (Fig. 3). The oil
droplets could not escape from the draft tubes once they were entered into the tubes.

Batch

Batch

8 and 9 (Fig. 5), due to the temperature control problem. Especially,

the SSR values for batches 10, 11 and 12 are lower than those of
other batches.
3.5. Hydrocarbon fraction analysis for recovered oil
Table 3 lists the hydrocarbon fraction distributions in the original oily sludge sample and recovered oil samples under eld treatment conditions. It can be found that the difference among the
proportions of hydrocarbon fractions in the oils was very small.
The F1 fraction in the recovered oil was slightly lower than that
in the original sludge, while the F2 and F3 fractions in the recovered oil were slightly greater than those in the original sludge. This
indicates that strain F-2 has a preference for the degradation of
some light oil compounds such as F1, leading to the relative enrichment of F3 and F4 fractions in the recovered oil.

3.4. Sludge settling efciency

3.6. Evaluation of sludge residue
SSR is an important parameter in the evaluation of the effectiveness of wastewater treatment. In this study, the SSR value was generally below 0.7 with the exception of higher values for batches 7,

Renery oil sludge is a hazardous waste, thus further treatment

is needed for the sludge residue after oil recovering. Incineration is
an effective approach for treating oil sludge residue. The content of
volatile matter and caloric value are the important parameters in

0.8

SSR

0.6

0.4

0.2

0.0
1

10

11

12

Batch
Fig. 3. Schematic diagram of draft tube used in this study.

Fig. 5. Sludge settling efciency of samples during eld pilot-scale experiments.

28

P. Yan et al. / Bioresource Technology 116 (2012) 2428

Table 3
Hydrocarbon fraction distribution for samples before and after treatment.
Samples

Fraction distribution (%)

Original sludge
Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Batch 6
Batch 7
Batch 8
Batch 9
Batch 10
Batch 11
Batch 12

F1

F2

F3

23.4
22.1
22.4
22.5
22.3
22.1
22.5
22.1
22.3
22.2
22.4
22.1
22.3

66.3
66.1
65.8
66.1
66.1
65.7
66.2
66.2
66.1
66.1
66.2
65.9
66.3

11.6
11.5
11.7
11.6
11.8
11.8
11.7
11.5
11.7
11.5
11.7
11.8
11.6

laboratory and eld conditions. By considering oil recovery efciency and hydrocarbon fraction distributions in the recovered
oil and in the separated wastewater, the biosurfactant assisted
oil recovery was identied as an effective method with satisfactory
performance. The sludge residue could be further treated by
incineration.
Acknowledgements
This work was funded by the National Key and Special Project
Foundation of China (2011ZX05009-004), and the National Natural
Science Foundation of China (Nos. 41102231, 41172333).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2012.04.
024.

Table 4
Sludge characteristics of sludge residue after treatment.

References

Samples

Moisture
(%)

Ash
(wt.%)

Wet-based volatile
matter (wt.%)

Dry-based volatile
matter (wt.%)

Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
10
Batch
11
Batch
12

92.23
93.24
94.58
87.47
92.55
93.32
92.56
92.56
88.73
91.88

2.25
1.89
1.15
2.26
2.55
1.69
1.71
2.37
2.55
1.78

5.52
4.87
4.28
10.27
4.90
4.99
5.73
5.07
8.72
6.35

71.04
72.04
78.82
81.96
65.77
74.70
77.02
68.15
77.37
78.11

92.55

2.47

4.98

66.85

91.38

2.16

4.83

69.42

1
2
3
4
5
6
7
8
9

Table 5
Net caloric values of sludge residue after treatment.
Samples

Wet-based caloric value

(103 kJ/kg)

Dry-based caloric value

(103 kJ/kg)

Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
10
Batch
11
Batch
12

1.88
2.05
2.28
4.25
1.68
1.19
4.61
2.14
3.13
2.51

25.07
25.60
24.98
25.48
22.49
24.3
25.94
24.78
24.78
26.33

3.43

29.16

3.32

28.75

1
2
3
4
5
6
7
8
9

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and 5 list volatile matter and caloric values of the sludge residue
after treatment. It can be found that the sludge residue has a relatively high dry-based volatile matter. The caloric values of the
sludge residue are high enough for incineration treatment.
4. Conclusions
In this study, a biosurfactant-producing microorganism, P. aeruginosa F-2, was used to recover oil from renery oily sludge under

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