Mineralization of Petroleum Contaminated Wastewater by Co-Culture of Petroleum-Degrading Bacterial Community and Biosurfactant-Producing Bacterium

doi:10.4236/jep.2011.27102

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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.

Email: *[email protected]

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 )

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)

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.

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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