Journal of Environmental Protection, 2011, 2, 895-902
doi:10.4236/jep.2011.27102 Published Online September2011 (http://www.SciRP.org/journal/jep)
895
Mineralization of Petroleum Contaminated
Wastewater by Co-Culture of
Petroleum-Degrading Bacterial Community and
Biosurfactant-Producing Bacterium
Bo young Jeon1, Il Lae Jung2, Doo Hyun Park1*
1Department of Chemical & Biological Engineering, Seokyeong University, Seoul, Korea; 2Department of Radiation Biology, Envi-
ronmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon, Korea.
Received June 15th, 2011; revised July 21th, 2011; accepted August 26th, 2011.
ABSTRACT
Activity of a crude biosurfactant extracted from the culture fluid of Serratia sp. that was isolated from riverbed soil was
shown to increase in proportion to the cultivatio n time, and was higher at pH 8 than at pH 7. Serratia sp. grew in the
mineral-based medium with soybean oil but was not with kerosene-diesel. The petroleum-degrading bacte-
ria—Acinetobacter sp., Pseudomonas sp., Paracoccus sp., and Cupriavidus sp.—were isolated from a specially de-
signed enrichment culture. Th e efficien cy of min era lization o f wa stewater con taminated with keros ene a nd diesel (WKD)
by the petroleum-degrading bacterial community (PDBC) was enhanced significantly by addition of the crude biosur-
factant. The efficiency of mineralization of the WKD was also about 2 times boosted by co-culture of Serratia sp. and
PDBC. Bacterial community of Serratia sp. and PDBC co-cultivated in the WKD was maintained for at least 8 days
according to the TGGE pattern of 16S rDNA obtained from the bacterial cu lture. In con clusion, the co -cultu re of Serra -
tia sp. and PDBC is an applicable technique for the mineralization of wastewater contaminated with petroleum, which
may substitute for chemica l or biological surfactant.
Keywords: Biosurfactant, Serratia Sp. Petroleum-Degrading Bacteria, Mixed Culture, TGGE
1. Introduction
The petroleum hydrocarbons can be converted biologi-
cally or chemically to carbon dioxide, water, and inor-
ganic compounds by dissimilatory metabolism of petro-
leum-degrading bacteria or combustion technique. Both
biological and chemical conversion of petroleum hydro-
carbons to inorganic compounds is defined as minerali-
zation; however, the combustion technique can’t be applied
for mineralization of petroleum-contaminated wastewater
and soil.
A variety of petroleum-derived compounds have con-
taminated soil and water in specific areas surrounding
systems for the production, storage, distribution, and
processing of petroleum by accidental spills and leakages.
In particular, accidental petroleum leakages from under-
ground storage tanks causes significant pollution of wa-
ters and soils [1]. For the successful intrinsic and engi-
neered bioremediation of soil or water contaminated with
petroleum, the complex relationship existing among pol-
lutants and microorganisms involved in contaminant
degradation must be understood [2]. Light non-aqueous-
phase liquids composed of petroleum hydrocarbon float-
ing on water surface coagulate as the result of water
fluctuations [3,4]. This phenomenon may effect the inhi-
bition of hydrocarbon uptake by microorganisms or in-
duce the adsorption of microorganisms onto oil drops.
Theoretically, microorganisms within petroleum drops
larger than bacterial size can lose their biological activity
due to damage to the membrane. The emulsification
process of utilizable carbon sources may constitute the
rate-limiting step in the microbial degradation of petro-
leum hydrocarbon pollutants contaminating water.
Biosurfactants are unique amphipathic molecules that
are metabolically generated by a variety of oil-utilizing
microorganisms; they have been explored for possible
use in a broad range of industrial and bioremediation
applications [5-7]. Certain biosurfactants generated by

Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community 896
and Biosurfactant-Producing Bacterium
Bacillus species induce low interfacial tensions between
the hydrocarbon and the aqueous phases required for the
mobilization of petroleum hydrocarbons [8,9]. Several
groups of biosurfactants have been identified as prereq-
uisites for the formation of the fruiting body of Bacillus
subtilis and the biofilm produced by Pseudomonas
aeruginosa [10,11]. Nonaqueous-phase liquids, including
nonpolar hydrocarbons, chlorinated solvents or manmade
organic compounds, may be retained as relatively immo-
bile and discontinuous globules [12-14]. The solubility or
miscibility of the non-aqueous-phase liquids can be en-
hanced by chemically synthesized or biologically pro-
duced surfactants [15,16].
Four species of petroleum-degrading bacteria—Pseu-
domonas sp., Cupriavidus sp., Paracoc cus sp., and Acine-
tobacter sp.—were isolated from an enrichment culture
saturated with hydrocarbon vapors. Vapor of a hydrocar-
bon mixture was sparged into the enrichment culture
medium in order to increase the probability of contact
among hydrocarbon molecules and bacterial cells in
aqueous phase. However, the sparging of petroleum or
nonaqueous hydrocarbons into the bacterial culture is not
an appropriate technique for application to treatment
system for petroleum-contaminated wastewater.
In this study, a biosurfactant-producing bacterium iso-
lated from soil and PDBC (petroleum-degrading bacterial
community) isolated from an enrichment culture system
were employed to improve the efficiency of mineraliza-
tion of petroleum hydrocarbons-contaminated wastewa-
ter. The effect of a crude biosurfactant and a co-culture
of the biosurfactant-producing bacterium and the PDBC
on bacterial mineralization of WKD (wastewater con-
taminated with kerosene and diesel) was estimated and com-
pared based on the TOC (total organic carbons) variation. The
physiological stability of the biosurfactant-producing bacte-
rium and the PDBC during co-cultivated in the WKD was
analyzed using the TGGE technique.
2. Materials and Methods
2.1. Isolation of Microorganisms
Biosurfactant-producing bacterium was isolated from
riverbed soil using a mineral-based soybean oil medium
composed of 3 g/L of ammonium sulfate, 1 g/L of potas-
sium phosphate monobasic, 3 g/L of potassium phos-
phate dibasic, 5 g/L of soybean oil, and 2 ml/L of trace
mineral stock solution. The trace mineral stock solution
was composed of 0.01 g/L of MnSO4, 0.01 g/L of
MgSO4, 0.01 g/L of CaCl2, 0.002 g/L of NiCl2, 0.002 g/L
of CoCl2, 0.002 g/L of ZnSO4, 0.002 g/L of Al2[SO4]3,
0.001 g/L of CuSO4, 0.002 g/L of MoCl2, and 10 mM
EDTA [17]. Ten grams of river bed soil was put in 100
ml of mineral-based soy bean oil medium and incubated
at 30℃and 150 rpm in a shaking incubator for 7 days.
The bacterial culture grown in the mineral-based soybean
oil medium was serially diluted up to 108 times with the
fresh medium by 10-folded dilution method and then
cultivated in same condition for 5 days. Maximally di-
luted medium in which bacteria were grown was serially
diluted several times again. Finally, the purity of bacte-
rial culture grown in the maximally diluted medium was
estimated by TGGE technique.
Petroleum-degrading bacteria were isolated from a
bacterial culture enriched with petroleum, which was
grown using a variety of hydrocarbons according to the
method previously developed by Lee et al. [18]. A mix-
ture of volatile hydrocarbons was flowed into the soil
mixture suspension with air flow (3 L/min) by evapora-
tion, as shown in Figure 1. One hundred μl of the bacterial
culture enriched for more than 6 months was spread onto
agar plates containing a mineral-based medium without
organic compounds. The agar plates were placed into
glass desiccators containing kerosene and diesel in the
bottom. Air in the desiccator may be saturated naturally
with the petroleum vapor evaporated from kerosene and
diesel, by which the bacterial cells spread on the agar
plates may contact with petroleum molecules and ab-
sorbed those. Desiccator cap was opened a time a day for
5 min to supply fresh air. Colonies emerged on the min-
eral-based agar medium were transferred to a min-
eral-based broth medium containing 5 g/L of kerosene
and diesel, which was shaken vigorously at 250 rpm to
induce dispersion of petroleum molecules into aqueous
medium and incubated at 30℃.
2.2. Identification of Microorganisms
Chromosomal DNA was directly extracted from the bac-
terial isolates. 16S ribosomal DNA was amplified via
direct PCR using the chromosomal DNA template and
16S-rDNA specific universal primers as follows: forward
5’-GAGTTGGATCCTGGCTCA G-3’ and reverse
5’-AAGGAGGGGATCCAGCC-3’. The PCR reaction
mixture (50 μl) consisted of 2.5U of Taq polymerase,
250 μM of each dNTP, 10 mM Tris-HCl (pH 9.0), 40
mM KCl, 100 ng template, 50 pM primer, and 1.5 mM
MgCl2. Amplification was conducted for 30 cycles of the
following: 1 min at 95℃, 1 min of annealing at 55℃,
and 2 min of extension at 72℃ using a PCR machine (T
Gradient model, Biometera, German). Bacterial identity
was determined on the basis of 16S-rDNA sequence ho-
mology, via the GenBank database system.
2.3. Temperature Gradient Gel Electrophoresis
(TGGE)
TGGE technique is useful to effectively separate the

Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community 897
and Biosurfactant-Producing Bacterium
Figure 1. Schematic structure of bioreactor for the enrichment of a bacterial community capable of degrading petroleum
hydrocarbons. Various organic vapors can be transferred via air flow through the pipeline from the compressor to bacterial
cultures. A one-tenth concentration of fresh medium balanced with evaporated volume was automatically refilled [19].
variable region of 16S-rDNA by difference of tempera-
ture-dependent denaturation between AT and GC pair.
The 16S-rDNA amplified from chromosomal DNA was
employed as a template for the preparation of the TGGE
sample (16S-rDNA variable region). A variable region of
16S-rDNA was amplified using a forward primer
(eubacteria, V3 region) 341f 5-CCTACGGGAGGCA-
GC-AG-3’ and reverse primer (universal, V3 region)
518r 5’-ATTACCGCGGCTGCTGG-3’. A GC clamp
(5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGG
GCACGGGGGGCCTACGGGAGG-CAGCAG-3’) was
attached to the 5’-end of the GC341f primer [19]. The
procedures for PCR and DNA sequencing were identical
to the conditions used for 16S-rDNA amplification, with
the exception of an annealing temperature of 53℃. The
TGGE system (Bio-Rad, DcodeTM, Universal Mutation
Detection System, USA) was operated in accordance
with the manufacturer’s specifications. Aliquots (45 ml)
of the PCR products were electrophoresed in gels con-
taining 8% acrylamide, 8 M urea, and 20% formamide
with a 1.5 x TAE buffer system at a constant voltage of
100 V for 12.5 hr and then at 40 V for 0.5 hr, applying a
thermal gradient of 39℃ to 52℃. Prior to electrophoresis,
the gel was equilibrated to the temperature gradient for
30 min to 45 min.
2.4. Amplification and Identification of TGGE
Band
DNA was separately extracted from each TGGE band
and purified using a DNA gel purification kit (Accuprep,
Bioneer, Korea). The purified DNA was then amplified
with the same primers and procedures used for TGGE
sample preparation, in which the GC clamp was not at-
tached to the forward primer. The species-specific iden-
tity of the amplified variable 16S-rDNA was determined
based on sequence homology, according to the GenBank
database system.
2.5. Biosurfactant Activity Assay
Ten μl of n-decane containing Sudan III (0.001%, w/v)
was dropped into distilled water (surface diameter, 90
mm) in a petri dish, resulting in the formation of an oil
film. The surfactant can cause the oil film to spread and
form a ring on the surface of the water. Cell-free culture
fluid of Serratia sp., Cupriavidus sp., Pseudomonas sp.,
Paracoccus sp., and Acinetobacter sp. was dropped onto
the oil film to evaluate surfactant production by the pe-
troleum-degrading bacteria.
2.6. Separation of Biosurfactant from Culture
Fluid
Cell-free culture fluid was obtained by 30 min of cen-

Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community 898
and Biosurfactant-Producing Bacterium
trifugation at 5000 xg and 4℃. Five hundred ml of a
chloroform-methanol (1:1) mixture was mixed with 5000
ml of the cell-free culture fluid in a separation funnel and
then shaken for 100 strokes at 20℃ for 120 min. The
solvent phase was separated from the cell-free culture
fluid and then the solvent was evaporated via N2-flushing.
Finally, a viscous liquid remained, and was employed as
a crude biosurfactant for the petroleum mineralization
tests.
2.7. Effect of Biosurfactant on Mineralization of
WKD
Unpurified domestic sewage was obtained from a man-
hole of a pipeline flowing in an aerobic treatment reactor
in a terminal disposal plant of sewage (Jungrang plant,
Seoul, Korea). Two hundred ml of the sewage was pre-
pared in a 500ml-medium bottle (reactor), to which 20
g/L of kerosene-diesel mixture was added to prepare
WKD. The initial pH of the WKD was adjusted to 8 us-
ing ammonium hydroxide. PDBC that was previously
cultivated in the mineral-based kerosene-diesel medium
and harvested by centrifugation at 5000 xg and 4℃ for
30 min was used as an inoculum. Each 10 g/L of the
crude biosurfactant and PDBC based on wet weight was
inoculated into the prepared WKD. No biosurfactant was
added to but PDBC was inoculated into the WKD for the
control test. The bacterial reactor was cultivated at 30℃
in a 200 rpm of shaking incubator. All reactants em-
ployed in the bacterial petroleum mineralization test were
prepared in triplicate. The mineralization activity of the
petroleum-degrading bacteria was determined on the
basis of the TOC variation.
2.8. Effect of Co-Culture on Mineralization of
WKD
Serratia sp. that was previously cultivated in the min-
eral-based soybean oil medium and harvested by cen-
trifugation at 5000 xg and 4℃ for 30 min was used as an
inoculum. Each 10 g/L of the harvested Serratia sp. and
WKD based on wet weight was inoculated into the WKD
of which initial pH was adjusted to 8 using ammonium
hydroxide. No Serratia sp. but PDBC was inoculated
into the WKD for the control test. The bacterial reactor
was cultivated at 30℃ in a 200 rpm of shaking incubator.
All reactants employed in the bacterial petroleum miner-
alization test were prepared in triplicate. The mineraliza-
tion activity of the petroleum-degrading bacteria was
determined on the basis of the TOC variation.
2.9. TOC Measurement
The TOC was evaluated in accordance with the method
organized by the HACH Company (Loveland, Colo,
USA). All procedures for the measurement of TOC were
conducted in accordance with the instructions provided
in the HACH manual (US Patent 6,368,870). Samples
were diluted appropriately with distilled water within the
range of detection. All chemicals used for TOC meas-
urement were purchased from HACH. TOC was meas-
ured directly with a programmed spectrophotometer for
automatic calculation (HACH model, DR/2500).
3. Results
3.1. Bacterial Identity
The 16S-rDNA variable region amplified with genomic
DNA extracted from bacterial culture selected with min-
eral-based soybean oil medium was separated as a single
band in TGGE (data not shown), which is the sign that
the selected bacterial culture is pure. The purely isolated
bacterium was identified based on 16S-rDNA sequence
homology and registered in the GenBank database sys-
tem, from which the following accession number was
obtained: FJ971961, designating Serratia sp. SK090424.
The petroleum-degrading bacteria were previously regis-
tered [18].
3.2. Biosurfactant Activity
Biosurfactant production by Serratia sp. was propor-
tional to cultivation time and was significantly higher at
pH 8 than at pH 7, as shown in Figure 2. The relatively
higher biosurfactant activity at pH 8 may be caused by
the higher emulsification effect of alkaline condition for
soybean oil. The emulsified oil may be absorbed and
catabolized more actively by bacterial cells than the
droplet oil.
3.3. Effects of Biosurfactant on Mineralization of
Petroleum
Efficiency of mineralization of PDBC was about 2 times
higher with biosurfactant than without as shown in Fig-
ure 3. The emulsification activity of PDBC for kerosene
and diesel is presumed to be lower than Serratia sp.
based on the enrichment process. In the process for en-
richment or isolation, Serratia sp. was cultivated with
soybean oil droplet but PDBC was cultivated exclusively
with petroleum vapor that may be dispersed freely into
bacterial culture. The nonaqueous kerosene and diesel
droplet may be dispersed limitedly into bacterial cultures
under condition without biosurfactant, and this condition
permits the limited growth of PDBC, as well as the lim-
ited absorption of kerosene-diesel. The biosurfactant was
quite effective in facilitating the catabolism of petroleum
hydrocarbons by specific PDBC.

Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community 899
and Biosurfactant-Producing Bacterium
2 4 7 9 11 days
Figure 2. Oil spreading activity (diameter, mm) of biosurfactant produced by Serratia sp. SK090424 cultivated in the
mineral-based soybea n me dium for 2, 4, 7, 9, and 11 days at pH 7 (upper) and 8 (lower).
Incubation time (days)
0246810
TOC (mg/L )
0
2000
4000
6000
8000
Figure 3. Mineralization of WKD by PDBC in the condition
with () or without the biosurfactant ().
3.4. Effect of Co-Culture of Serratia sp. and
PDBC
Serratia sp. was not grown when cultivated with petro-
leum vapor evaporated from kerosene and diesel; how-
ever, the efficiency of mineralization of WKD was in-
creased by co-culture of Serratia sp. and PDBC, as
shown in Figure 4. At the initial reaction time from 0 to
4 days, the mineralization by the co-culture was rela-
tively lower than that by the addition of the biosurfactant;
however, the efficiency was recovered at the later reac-
tion time between 6 to 10 days. This result implies that
some metabolites generated from kerosene and diesel
hydrocarbons by PDBC metabolism may constitute use-
ful nutrient for the growth and biosurfactant production
of Serratia sp.
Incubation time (days)
0246810
TOC (mg/L)
0
2000
4000
6000
8000
Figure 4. Mineralization of WKD by co-culture of Serratia
sp. SK090424 and PDBC () or PDBC ().
3.5. Bacterial Community Variation during
WKD Treatment
Eight of distinguishable DNA bands were observed on
TGGE for the petroleum-degrading bacteria and the
biosurfactant-producing bacteria cultivated in the WKD,
as shown in Figure 5. In the TGGE pattern obtained on
the 2nd and 8th day of incubation time, the DNA band for
Serratia sp. was maintained. This result implies that
Serratia sp. incapable of catabolizing the petroleum hy-
drocarbons may grow on some of the metabolites pro-
duced by the PDBC. Five of the eight partial 16S-rDNAs
extracted from the TGGE bands were identified as the
five bacterial species that had been initially inoculated
into the AWKD, but the others were identified as uncul-
tured bacteria and Pseudomonas sp., which may have

Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community 900
and Biosurfactant-Producing Bacterium
A B
Figure 5. Diversity of the bacterial community cultivated in
the WKD at 2nd day (A) and 8th day (B) of incubation time.
Serratia sp. and PDBC were inoculated into the WKD at
initial time. The numbered DNA band was identified on the
basis of sequence homology as follow s: 1. uncultured bacte-
rium; 2. Pseudomonas sp.; 3. Paracoccus sp.; 4. Pseudomo-
nas sp.; 5. Acinetobacter sp.; 6. Serratia sp. ; 7. Cupriavidus
sp.; 8. Pseudomonas sp.
originated from the unpurified sewage.
4. Discussion
The biological mineralization of petroleum hydrocarbons
may be one of the solutions for the remediation of petro-
leum-contaminated environments [20]; however, the low
water miscibility or solubility of the petroleum hydro-
carbons limits their availability to specific microorgan-
isms capable of catabolizing petroleum hydrocarbons
[21]. The biodegradation efficiency of the petroleum
hydrocarbons may be increased in proportion to their
solubility. Chemically synthesized or biologically gener-
ated surfactants have been employed in order to increase
the solubility of the petroleum hydrocarbons and, by ex-
tension, to augment the efficiency of mineralization of
the hydrocarbons [22,23]. The synthesized surfactants
are relatively toxic for microorganisms and not particu-
larly biodegradable, but the biosurfactants are non-toxic
and biodegradable [24]. Both of the surfactants may
prove problematic for application to oil-contaminated
beaches, soils, or bioreactors based on the toxicity of
synthetic surfactants to microorganisms and the micro-
organism-induced degradation of biosurfactant [25-27].
The toxicity of the synthetic surfactant may inhibit the
ability of the bacterial community to mineralize hydro-
carbons, and the biodegradability of the biosurfactant
may also induce an abrogation of its activity. The con-
centration of the surfactant in the reactant or in contami-
nated environments must be maintained at above-minimum
levels for the successful emulsification of petroleum hy-
drocarbons, as both of the surfactants are effective to a
limited extent for low-concentration application to con-
taminated sites [24]. This is the limiting factor in the ap-
plication of both synthetic surfactants and biosurfactants to
the bioreactor for the treatment of petroleum-contaminated
wastewater. The ideal reaction conditions for the biosurfac-
tant-mediated petroleum mineralization may be accom-
plished by the co-culture of biosurfactant-producing bacte-
rium and petroleum-degrading bacteria, as the petro-
leum-degrading bacteria don’t produce actively biosur-
factant, but all of the biosurfactant-producing bacteria do
not consistently degrade petroleum hydrocarbons [28,29].
The data shown in Figure 3 demonstrate that the PDBC
depends on the biosurfactant for the effective mineraliza-
tion of petroleum hydrocarbons. The data shown in Fig-
ure 4 demonstrate that the biosurfactant-producing bac-
terium, Serratia sp., depend upon the growth of PDBC in
the WKD. Serratia sp. was shown to be unable to grow
in the presence of petroleum hydrocarbons, but did grow
and generate biosurfactant when co-cultured with the
PDBC during growth in WKD. We conducted no tests to
identify which bacterial strain of the PDBC produces
metabolites for the growth of Serratia sp., but we may
assume that the PDBC—Pseudomonas sp., Cupriavidus
sp., Paracoccus sp., and Acinetobacter sp.—can generate
a variety of metabolites, and some of these metabolites
may function as substrates for the growth of Serratia sp.;
additionally, certain of these metabolites may function as
a substrate for biosurfactant production.
1
2
3
4
5
6
7
8
5. Conclusions
A co-culture of biosurfactant-producing bacteria and
PDBC may function as an effective substitute for syn-
thetic surfactants or biosurfactants, as the limiting factors
of the synthetic surfactant and biosurfactant may be sup-
plemented by the biosurfactant continuously generated
by growing cells of the biosurfactant-producing bacteria.
The relationship between the biosurfactant-producing
bacteria and PDBC may fall short of a true symbiosis,
but is clearly sufficient for the synergetic mineralization
of petroleum hydrocarbons. The co-culture technique
tested in this study may be applicable to treatment sys-
tems for petroleum- or xenobiotics-contaminated waste-
water based on the synergetic growth of the biosurfac-
tant-producing bacteria and PDBC and the practical
monitoring techniques of specific bacterial community.

Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community 901
and Biosurfactant-Producing Bacterium
Practically, various techniques that are real time PCR,
DNA chip, and FISH (fluorescence in situ hybridization)
have been employed to monitor the bacterial communi-
ties growing in bioreactors and natural ecosystems.
6. Acknowledgements
This work was supported by the New & Renewable En-
ergy of the Korea Institute of Energy Technology
Evaluation and Planning (KETEP) grant funded by the
Korea government Ministry of Knowledge Economy
(2011T10011 00 334 ).
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