3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

The Effectiveness of Pseudomonas putida T1(8)

Biosurfactant in Bioremediation of Crude Oil
Contaminated Soil
Nur Hidayatul Alami *, Nimatuzahroh , Tini Surtiningsih
Department of Biology
Airlangga University, Surabaya, Indonesia
e-mail: Nh_daya@yahoo.com

Abstract
1 Introduction

Crude oil contaminated soil was a serious problem.

Bioremediation method was a viable alternative
method to remedy oil contaminated soil. A possible
way to increase bioremediation rate was the
application of biosurfactant. Pseudomonas putida
T1(8) was the native isolate which can produce
extracellular biosurfactant. The aim of this research
was to know the effectiveness of biosurfactant P.
putida T1(8) when it was added in bioremediation
test. The effects of biosurfactant addition in the
bioremediation can be known from the increasing
of the microbial heterotrophic total count (cfu/gsoil) and the decreasing of the oil residue (g/g-soil).
The type of the research was experimental with 3
controls, including non sterile soil without
biosurfactant (KI), sterile soil without biosurfactant
(KII), non sterile soil with biosurfactant at CMC
level (KIII) and 4 treatment, including non sterile
soil with adding of microbial consortium without
biosurfactant (B0), non sterile soil with adding of
microbial consortium and biosurfactant below the
CMC (B1), non sterile soil with adding of microbial
consortium and biosurfactant at CMC level (B2),
and non sterile soil with adding of microbial
consortium and biosurfactant above the CMC (B3).
The incubation times were 0, 2, and 4 weeks. The
resulted data of the logarithm microbial total count
and the crude oil residue (g/g-soil) were analyzed
descriptively and statistically by two-way analysis
of variances (two-way ANOVA) and Duncan test
(p=0.05). The results of this research indicated that
biosurfactant gave an effect on bioremediation
process. Biosurfactant at CMC value at final
incubation time (fourth week) was known to be the
most effective to increase microbial growth and to
decrease the oil residue with the amount of 12.68%.
Keywords:

As the increasing of explorative activities and oil

utilization, oil spills on the land has also been on
the increase (Ekpo and Udofia, 2008). The greatest
impact of oil spills is the disruption of land function
as a natural media to support vegetations, animals,
and human life. The disruption of land function will
influence all of the ecosystem stability (Lynch and
Poole, 1978; Reginawanti and Simarmata, 2004).
Bioremediation was developed to solve that
problem. Bioremediation has become a valuable
alternative technology as it is a cost effective,
cheap, and environmentally friendly treatment
(Alamri, 2009; Mariano et al., 2007). These
processes rely on the natural ability of
microorganisms to carry out the mineralization of
organic chemicals, leading ultimately to the
formation of CO2, H2O and biomass. There are
some methods in bioremediation, that are;
biostimulation,
bioaugmentation,
and
bioavailability.
Biostimulation is strategy to
accelerate
the
biological
breakdown
of
hydrocarbons in soil include stimulation of the
indigenous microorganisms by optimizing the
nutrients. Bioaugmentation is a strategy through
inoculation of an enriched mixed microbial
consortium into soil, whereas bioavailability is a
strategy to increase accessibility between substrate
and microbes (Mariano et al., 2007). The
combination of that all methods are necessary
needed to advance bioremediation process.
The effort to increase availability of hydrocarbon
substrate for microbes is extremely important, that
because of its hydrophobic characteristic. So, it is
difficult to absorb into the soil matrix. Providing a
way to reduce the sorption of the hydrophobic
organic contaminants to the soil matrix can increase
the rate and extent of biodegradation. For this
purpose, the addition of biosurfactant into the soil
aims to enhance the emulsification of hydrocarbons

soil bioremediation, biosurfactant,

Pseudomonas putida T1(8)

B018

Nur Hidayatul Alami, The Effectiveness of Pseudomonas putida T1(8) Biosurfactant in Bioremediation of Crude
Oil Contaminated Soil

Many
study
related
with
hydrocarbon
biodegradation test often used various biosurfactant
concentrations, which are: below the CMC, at
CMC, and above the CMC. Each concentration
give different response depends on the type of
biosurfactant. In some cases reported that
biosurfactant concentration above the CMC give
inhibition effect, thus another studies used
biosurfactant concentration below the CMC to
reduce inhibition effect (Rouse et al., 1994). While,
according to Kim et al. (2001), the most commonly
used of surfactant concentration are at CMC.
Hence, the aim of this study was to know the
effectiveness of biosurfactant P. putida T1(8) using
various concentration (below the CMC, at CMC,
above the CMC) when it was added in
bioremediation test.

and therefore they have the potential to solubilize

hydrocarbons and increase their bioavailability,
thus it possible to increase accessibility between
substrate and microbes. Finally, it can increase
microbial growth and biodegradation rate (Tuleva
et al., 2001; Jagadevan and Mukherji, 2004;
Mariano et al., 2007).
The advantage of biosurfactant if compared to their
chemically
synthesized
counterparts
is
environmentally friendly. Biosurfactant is low
toxicity, can be degraded naturally by microbes,
also can be produced from renewable sources
(microbes) (Kosaric, 2001).
In another country, application of biosurfactant has
already been developed to support bioremediation
process. This strategy is of paramount concern
(Banat et al., 2000). However in Indonesia, the
application of biosurfactant in bioremediation is
still rare. May be it is because of the cost of
biosurfactant production (Hamme et al., 2003).

Methodology

2.1 Microbial cultural

The bacteria producing biosurfactant which was
used in this study was Pseudomonas putida T1(8).
While, microbial consortium which was used as
hydrocarbonoklastics
microbes,
consist
of:
Aeromonas hydrophyla, Pseudomonas putida
T1(8), Pseudomonas aeruginosa, Acinetobacter
faecalis
type
II,
Pseudomonas
cepacea,
Actinobacillus
sp.,
Pseudomonas
stutzeri,
Pseudomonas
pseudomallei,
Pseudomonas
fluorescens-25, Azotobacter sp., Beijerinkia sp.,
Candida famata, Rhodotorulla mucilaginosa, and
Candida parapsilopsis. Those microbes were
storage in Microbiology Laboratory, Biology
Departemen Airlangga University Surabaya. The
bacterial isolate was maintained on nutrient agar
(NA), while yeast isolate on Sabouroud Dextrose
Agar (SDA).

In the previous study, Nimatuzahroh et al. (2009)

has been explored bacteria and yeast for
bioremediation of hydrocarbon contamination
purpose from contaminated area close to the storage
and distribution center of oil products in Surabaya
and Wonocolo refinery, East Java Indonesia. One
of those microbes is Pseudomonas putida T1(8).
Alami et al. (2010) has been reported that
Pseudomonas putida T1(8) which was grown on a
molasses substrate and was extracted by (NH4)2SO4
precipitation, gave the highest yield of biosurfactant
about 5.0467 g/l, had the lowest CMC value about
665 ppm, and decrease surface tension more than
10 mN/m (21.6 mN/m). It also showed the best
emulsifier activity in diesel oil and stable at the
temperature range 30-70C (100-93.18%), pH 4-7
(100-82.41%), and NaCl concentration 0-3 M (10098.60%).

2.2
Biosurfactant production
For biosurfactant production, a modification of a
mineral salts medium developed by Pruthi and
Comeotra (1997) was used. It contained per liter
distilled water, KH2PO4, 5.0g; K2HPO4, 2.0g;
FeSO4.7H2O, 0.0006g; (NH4)2SO4, 3.0g; NaCl,
10g; MgSO4. 7H2O, 0.2g; CaCl2, 0.01g;MnSO4.
H2O, 0.001g; H3BO3, 0.001g, ZnSO4. 7H2O,
0.001g; CuSO4. 5H2O, 0.001g; CoCl2.6H2O,
0.005g; and Na2M0O4. 2H2O, 0.001 g, pH was
adjusted to 7.0. The flasks containing molasses 2%
v/v as sole carbon source were inoculated with
Pseudomonas putida T1(8) at 4 % (v/v) OD=0.5 at
610. Cultivations were performed in 10 flasks (1000
mL), each flask contains 200 mL mineral salts
medium. The mixture was placed on a reciprocal
shaker at 150rpm for 3 days at 30C. Afterwards,
the culture was centrifuged and the supernatant was
extracted by (NH4)2SO4 precipitation. The crude
biosurfactant product will be added into

In addition, according to Bordoloi and Konwar

(2008), carbon source like molasses which was
used to produce biosurfactant Pseudomonas putida
T1(8) can be an alternative to compress
biosurfactant production cost. So, it can be of great
potential in the remediation of oil polluted soil.
The acceleration of bioremediation process adding
by biosurfactant is influenced with the effectiveness
of its biosurfactant. One of the factors that
influence the effectiveness of biosurfactant is CMC
value (Nimatuzahroh et al., 2004). Beside that,
another factors, like: biosurfactant toxicity, the
compatibility
of
biosurfactant
against
hydrocarbonoclastics
microbes,
hydrocarbon
component, and environmental factors also
influence the effectiveness of biosurfactant (Rouse
et al., 1994).

B018

3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

The microcosms were kept at room temperature,

approximately 30C for 0, 2, and 4 weeks.

bioremediation experiment with concentration

below the CMC (332.5 ppm), at CMC (665 ppm),
and above the CMC (997.5 ppm).

2.5
Biodegradation measurement
2.5.1 Heterotrophic microbial calculation
Total heterotrophic microbial were numbered by
using the pour plate technique on plate count agar
(NA for total bacteria and SDA for total yeast).
Plate count of the soil microbial population was
performed as follows: samples of 10 g of soil were
added with 90 mL sterile saline solution and
agitated mechanically, and then were precipitated
for 5 minutes. After appropriate serial dilutions, 1
mL of the suspension was added into the petri
dishes, and then NA and SDA were also added into
the Petri dishes. The cultures were then incubated
for 24 h (for bacteria) and 72 h (for yeast) at 35C.
At the end of the incubation time, the counting of
bacteria was performed for all treatments.

2.3
Preparation of soil
A soil sample in this study was obtained from
garden soil and sandy soil mixture. Before mixed
those, to both garden soil and sandy soil was
filtered by mess that has a measurement of 1
mm2.Then, each of those was weighted about 750 g.
After that, the soils were mixed and homogenized.
An initial determination of C:N concentrations was
performed to estimate the experimental supplement
of nutrients to ensure a C:N ratio of 100:10. Initial
pH was adjusted at 7. For moisture content,
additional water was added to bring the final
moisture content of soil mixture to 77%.
2.4 Bioremediation experiments
Bioremediation experiments were carried out in
flasks (100 mL). This study was assessed using the
full factorial experimental design, consists of 3
controls and 4 treatments, with the details like the
following sentences:
- Control I (KI) contains of non sterile soil +
crude oil
- Control II (KII) contains of sterile soil + crude
oil
- Control III (KIII) contains of non sterile soil +
crude oil + biosurfactant at CMC concentration
- Treatment I (B0) contains of non sterile soil +
crude oil + microbial consortium
- Treatment II (B1) contains of non sterile soil +
crude oil + microbial consortium +
biosurfactant with concentration above the
CMC
- Treatment III (B2) contains of non sterile soil +
crude oil + microbial consortium +
biosurfactant with concentration at CMC
- Treatment IV (B3) contains of non sterile soil
+ crude oil + microbial consortium +
biosurfactant with concentration below the
CMC

2.5.2 Total Petroleum Hydrocarbon (TPH)

analysis
The soil samples were dried with oven for 3 days
at 75 0C. After that, the dried soil was grinded.
About 1 gram of grinded soil was then added into
the tube. Afterwards, about 5 mL of toluene was
added into the tube. And then was agitated using
vortex for 10 seconds and leaving to stand for 10
minutes. The soluble crude oil within toluene was
then filtered using filter papers number 1. The
suspension was diluted by taking 0.5 mL, and then
was added with 4.5 mL toluene. After that, Optical
Density (OD) value of dilution suspension was
measured using spectrophotometer at 420. The
result of measurement were then calculate into
standard curve, so that will be obtained crude oil
content (mL/g tanah), then was converted into g/g
soil (Okolo et al., 2005).
3 Result
The type of heterotrophic microbial which was
analyzed in this study, consist of: bacteria and
yeast. The results of Total Plate Count of
heterotrophic microbial (log cfu/g) at each
treatment condition during incubation time 0, 2, and
4 weeks were showed in Figure 1. The figure
showed the increasing of total heterotrophic
microbial during incubation time in almost all of
the treatment condition (K1, K2, K3, BO, B2,
<CMC, >CMC).

Each of control and treatment consist of 3 replicates

for Total Plate Count (TPC) analysis and 3
replicates for Total Petroleum Hydrocarbon (TPH)
analysis. To ensure suitable moisture, each
microcosm was monitored periodically at the end of
weeks (0 week, 1st week, 2nd week, 3rd week, and 4th
week). Each of the flasks was filled by 10 g soil,
whereas for the control with a sterile soil, the soil
was dried by oven at 160C for 2 hours to sterilize
it completely. The flasks were then added by crude
oil 0.6 mL. Afterwards, 2 mL of consortium
suspension OD=1 at 600 also was added into the
flasks with consortium adding. Then for the groups
with biosurfactant adding, biosurfactant was added
into the flasks. The soil was then continuously hand
mixed for 5 minutes with a stainless steel spatula.
B018

Nur Hidayatul Alami, The Effectiveness of Pseudomonas putida T1(8) Biosurfactant in Bioremediation of Crude
Oil Contaminated Soil

CMC concentration (B2) against total heterotrophic

microbes.
The result of statistical analysis of incubation time
against total heterotrophic microbes had obtained F
value about 22.221 with the probability <0.05
(0.000), therefore H0 was refused, or there was an
influence of incubation time against total
heterotrophic microbes. So, that can be continued to
the Duncan test. From the Duncan test, it can be
seen that there is a significant difference on
treatment condition during two weeks and four
weeks of incubation time if compare to 0 week of
incubation time. But, there is no significant
difference between the both of two weeks and four
weeks.

T o ta l h e te ro tro p h ic
m ic ro b ia l (lo g c fu /g )

14
12
10
8
0 week

2 weeks

4 weeks

2
0
KI

KII

KIII

B0

B1

B2

B3

<CMC >CMC

Treatment condition

Figure 1: Total heterotrophic microbial (log

cfu/g) of each treatment condition during
incubation times of 0, 2, and 4 weeks.

Statistical analyses also showed the influence of

interaction of biosurfactant concentration and
incubation time against total heterotrophic
microbes, with F value about 3.851 and probability
<0.05 (0.008), therefore H0 was refused, or there is
an influence of interaction of biosurfactant
concentration and incubation time against total
heterotrophic microbes. So, that can be continued to
the Duncan test. From the Duncan test, it can be
seen that there is a significant difference on
treatment condition with the combination of
biosurfactant adding at CMC concentration (B2)
and incubation time of fourth week against total
heterotrophic microbes.

During 2 weeks of incubation time, Pseudomonas

aeruginosa was dominant at treatment condition of
KIII, B1, B2, and B3, although KIII was a
treatment group without consortium adding. This
showed that Pseudomonas aeruginosa was an
indigenous bacteria of the soil. From the yeast
group also showed the growth of Candida famata
and Candida parapsilopsis on treatment condition
of B0 and B2. In the other side, on treatment
condition of B1 and B3 only showed the growth of
Candida parapsilopsis. In almost all treatment,
Candida famata was less dominates (Data was not
shown). At fourth week of incubation time, it was
shown that Pseudomonas aeruginosa was dominant
at treatment condition of K1, KIII, B0, B3, <CMC,
and >CMC. Meanwhile, there is a change of
microbial dominance at treatment condition of B0,
B1 and B2, from Pseudomonas stutzeri at treatment
condition of B0 in the second week to
Pseudomonas aeruginosa in the fourth week, and
from Pseudomonas aeruginosa at treatment
condition of B1 and B2 in the second week to
Pseudomonas pseudomallei and Pseudomonas
putida T1(8) in the fourth week. At the fourth
week, it was also showed the existence of bacteria
that never exist before, like; Azotobacter sp. and
Beijerenkia sp. While, Pseudomonas aeruginosa
was begin exist at treatment condition of KI in the
fourth week. On the other hand, the highly growth
of Pseudomonas putida T1(8) was shown at
treatment condition of B2 in the fourth week (Data
was not shown).

The residual concentration of crude oil at different

treatment with various biosurfactant concentrations
and incubation times was shown descriptively on
Figure 2. From the figure was known the
decreasing of crude oil concentration since second
week on treatment condition of B1,B2, and B3. The
lowest residual concentration was on B2 with the
value of 87.32 %, meanwhile B1 also showed the
decreasing of crude oil but that was not better than
B2, with the crude oil residue was about 89.44 %,
whereas residual concentration on treatment
condition of B3 was higher than B1 and B2, but it
still lower than control (95.77 %). Afterwards
residual concentration of crude oil was then
analyzed statistically to know whether there is a
difference of crude oil residue in every treatment
condition with biosurfactant adding in various
concentrations. The result showed that F value was
about 9.318 with probability <0.05 (0.000),
therefore H0 was refused, or there was an influence
of biosurfactant concentration againts crude oil
residue. So, that can be continued to the Duncan
test. From the Duncan test, it can be seen that there
is a significant difference between treatment
condition without biosurfactant (B0) and treatment
condition with biosurfactant adding (B1, B2, B3).

The result of statistical analyses showed that there

is an influence of biosurfactant concentration
against total heterotrophic microbes, with F value
about 6.051 and probability <0.05 (0.003), therefore
H0 was refused, or there was an influence of
biosurfactant
concentration
against
total
heterotrophic microbes. So, that can be continued to
the Duncan test. From the Duncan test, it can be
seen that there is a significant difference on
treatment condition with biosurfactant adding at
B018

Percentage of crude oil

(%)

3rd International Conferences and Workshops on Basic and Applied Sciences 2010

to Rouse et al. (1994), biosurfactant at CMC

concentration have an ability to form micelle.
Hence, biosurfactant can emulsify the oil (disperse
a complex hydrocarbon molecule into simple
forms). Finally hydrocarbon component can soluble
and microbes can access to the substrate easily.
Therefore it can increase microbial growth. The
increasing of microbial growth in the treatment
condition with biosurfactant adding at CMC
concentration indicated the compatibility and low
toxicity of biosurfactant to the microbial
consortium (Figure 1). As crude oil can be
emulsified and mineralized by microbial activity,
crude oil content can be decreased.

100.00%
98.00%
96.00%
B0

94.00%

B1

92.00%

B2

90.00%

B3

88.00%
86.00%
84.00%
0 week

2 weeks

ISBN: 978-979-19096-1-7

4 weeks

Incubation time (week)

Figure 2: Percentage of crude oil at each

treatment condition during incubation times of
0, 2, and 4 weeks.

The results of descriptive and statistical analysis

showed the influence of biosurfactant adding in
various concentrations against total heterotrophic
microbial and crude oil residue. Biosurfactant at
CMC value was known to be the most effective to
increase microbial growth and to decrease the oil
residue.

Although from descriptive analysis was known that

there was a difference in crude oil decreasing
within B1, B2, and B3. But, from the statistical
analysis, there was no significant difference.
The statistical analysis of incubation time against
crude oil residue obtained F value about 0.048 with
the probability >0.05 (0.953), therefore H0 was
accepted, or there is no influence of incubation time
against crude oil residue. So, it did not need to be
continued to the Duncan test. Although there was
no difference between incubation time and crude oil
residue, but descriptively, there was a decreasing of
crude oil concentration until second week, after that
there was no decreasing of crude oil percentage
until fourth week.
The statistical analyses also showed the influence
of interaction of biosurfactant concentration and
incubation time against crude oil residue, with F
value about 0.329 and probability >0.05 (0.065),
therefore H0 was accepted, or there was no
influence of interaction
of biosurfactant
concentration and incubation time against crude oil
residue. Whatever the result of statistical analysis,
if that was analyzed descriptively, the best
combination was on combination of B2M2 and
B2M4, those was a treatment condition with
biosurfactant at CMC concentration and incubation
time two and four weeks.

The microbes were increasing significantly at

incubation time between 0 week and two weeks,
but the oil concentration was decreasing. Between
two weeks up to four weeks of incubation time, the
growth of microbes were decreasing, except to
treatment condition of B2 (biosurfactant
concentration at CMC value) (Figure 1). The
growth in B2 may because of the availability of C
substrate that was obtained by microbial activity
during 0 week up to second week. Beside that, the
microbes in B2 may be able to use biosurfactant as
its growth substrate. Overall, the decreasing of
microbes made static decreasing of oil
concentration up to fourth week (Figure 2). Beside
by microbial activity to degrade the substrate and
the availability of the substrate for microbes, the
rate and the completeness of bioremediation was
also influenced by environmental factors likes;
moisture, soil pH, oxygen content, nutrient content,
temperature, even by soil type (Vidali, 2001 and
Kosaric, 1996). That is way; the analysis of
environmental factors during bioremediation
process was extremely needed.

The monitoring of moisture during incubation time

was indicated that the moisture decreased from
77% to 59.41% at fourth week. It was mean that
moisture content still too high until fourth week.
While, from the monitoring of pH during
incubation time, showed that pH was still in
optimum range to support bioremediation process
(pH 6-7).
The results from to both descriptive analysis and
statistical
analysis
showed
that
various
concentrations of biosurfactant gave an influence to
the growth of heterotrophic microbes. The most
effective concentration was on CMC concentration
(B2) with fourth week incubation time. According

According to Vidali (2001), the range of optimal

moisture of bioremediation is in 30-90%. This is
possible in some cases, but in another cases, the
high moisture of the soil decreased bioremediation
rates. Baptista et al.(2005) and Cubitto et al. (2004)
reported that the high moisture (above 50%) gave a
negative effect to bioremediation rate, these
correlated with the less of aeration in the soil. The
lowest value of moisture in this study was 59.41 %
at fourth week. That moisture was still too high. At
the condition with too high moisture, the oil was
inclined at the top of soil surface where the water
content was also too high at the top of the soil. So,
it takes a more time for both oil and water to absorb
B018

Nur Hidayatul Alami, The Effectiveness of Pseudomonas putida T1(8) Biosurfactant in Bioremediation of Crude
Oil Contaminated Soil

Baldini, and F. Sineriz, Effects of Bacillus

subtilis
09
Biosurfactant
on
The
Bioremediation of Crude Oil-Polluted Soils.
Biodegradation. 15: 281-287, 2004.

into the soil. Thus, the effectiveness of

biosurfactant to increase the bioremediation process
also needs a more time. In addition, according to
Environmental Technologies Research Group and
Spanish Association of Fishering Cities (2002),
there is a complication of bioremediation in a
wetland or the soil with the high moisture; Oil can
penetrate into the anoxic layers of the sediment.
Below only a few centimeters of depth, the
environment becomes anaerobic, and crude oil
biodegradation is likely to be much slower even in
the presence of an adequate supply of nitrogen and
phosphorus. In this specific case, the addition of
oxygen may be required as part of the
bioremediation strategy.

[7] Ekpo, M.A. and U.S. Udofia, Rate of

Biodegradation
of
Crude
Oil
by
Microorganisms Isolated from Oil Sludge
Environment.
African
Journal
of
Biotechnology. 7(24): 4495-4499, 2008.
[8] Environmental Technologies Research Group
dan Spanish Association of Fishering Cities,
Marine
Bioremediation
Technologies
Screening Matrix and Reference Guide, 2002.
[9] Hamme, J.D.V., A. Singh, and O.P. Ward,
Recent Advances in Petroleum Microbiology.
Microbiology and Molecular Biology Reviews.
67(4): 503-549, 2003.

4 Conclusions
In conclusion, Pseudomonas putida T1(8)
biosurfactant gave an effect on bioremediation
process. Biosurfactant at CMC value was known to
be the most effective to increase microbial growth
and to decrease the oil residue about 12.68%, if
compared with the control at final incubation time
(fourth week).

[10] Jagadevan, S. and S. Mukherji, Successful In

Situ Oil Bioremediation Programmes-Key
Parameters. Indian Journal of Biotechnology.
495-501, 2004.
[11] Kim, I.S., J.S. Park, and K.W. Kim, Enhanced
Biodegradation of Polycyclic Aromatic
Hydrocarbons Using Nonionic Surfactants in
Soil Slurry. Applied Geochemistry. 16:14191428, 2001.

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ISBN: 978-979-19096-1-7