the Effect of Rhamnolipid Biosurfactant Produced

The Effect of Rhamnolipid Biosurfactant Producedby Pseudomonas fluorescens on Model Bacterial Strainsand Isolates from Industrial Wastewater

Evgenia Vasileva-Tonkova Anna Sotirova

Danka Galabova

Received: 11 June 2010 / Accepted: 20 July 2010 / Published online: 1 August 2010

Springer Science+Business Media, LLC 2010

Abstract In this study, the effect of rhamnolipid biosur-

factant produced by Pseudomonas fluorescens on bacterial

strains, laboratory strains, and isolates from industrial

wastewater was investigated. It was shown that biosurfac-

tant, depending on the concentration, has a neutral or det-

rimental effect on the growth and protein release of model

Gram (?) strain Bacillus subtilis 168. The growth and

protein release of model Gram (-) strain Pseudomonas

aeruginosa 1390 was not influenced by the presence of

biosurfactant in the medium. Rhamnolipid biosurfactant at

the used concentrations supported the growth of some slow

growing on hexadecane bacterial isolates, members of the

microbial community. Changes in cell surface hydropho-

bicity and permeability of some Gram (?) and Gram (-)

isolates in the presence of rhamnolipid biosurfactant were

followed in experiments in vitro. It was found that bacterial

cells treated with biosurfactant became more or less

hydrophobic than untreated cells depending on individual

characteristics and abilities of the strains. For all treated

strains, an increase in the amount of released protein was

observed with increasing the amount of biosurfactant,

probably due to increased cell permeability as a result of

changes in the organization of cell surface structures. The

results obtained could contribute to clarify the relationships

between members of the microbial community as well as

suggest the efficiency of surface properties of rhamnolipid

biosurfactant from Pseudomonas fluorescens making it

potentially applicable in bioremediation of hydrocarbon-

polluted environments.

Introduction

Petroleum hydrocarbons are serious environmental pollu-

tants due to their persistence and high toxicity to all bio-

logical systems. Biodegradation is one of the primary

mechanisms for elimination of hydrocarbon pollutants

[14]. One of the major factors limiting the degradation of

hydrocarbons such as n-alkanes is their low availability to

the microbial cells because of low water solubility. Many

microorganisms employ several strategies to enhance

availability of hydrophobic pollutants, such as biofilm

formation and biosurfactant production [7, 13, 20]. In this

sense, the growth of microorganisms on oil hydrocarbons

has often been related to their capacity of producing

polymers with surfactant activity. Biosurfactants are

structurally diverse group amphiphilic compounds which

are potentially useful, particularly due to their anionic

nature, low toxicity, biodegradability, and high surface and

emulsifying activity [10, 18, 21].

Microorganisms synthesize a wide variety of high- and

low-molecular mass biosurfactants with different natural

roles in the growth of the producing microorganisms [19].

As the chemical structures and surface properties of bio-

surfactants are quite different, they would have more or

less specific role in different ecological niches. Co-treat-

ment with adapted microbial isolates and produced surface

active complexes of their specific ecological niches or

other related environments would be more effective for

bioremediation. Moreover, treatment of polluted sites with

biosurfactants or their producers (seeding, bioaugmenta-

tion, etc.) may affect microbial strains as well as other

organisms presented in the treated site. The bioavailability

of the hydrophobic substrate depends on the biosurfactant

efficiency, therefore, there seemed to be good prospects to

isolate novel more active biosurfactants [3].

E. Vasileva-Tonkova (&) A. Sotirova D. GalabovaThe Stephan Angeloff Institute of Microbiology, Bulgarian

Academy of Sciences, Acad. G. Bonchev Str., Block 26,

1113 Sofia, Bulgaria

e-mail: [email protected]

123

Curr Microbiol (2011) 62:427433

DOI 10.1007/s00284-010-9725-z

Previously, 15 bacterial strains were isolated from

industrial wastewater samples and were screened for

growth on hydrocarbons and biosurfactant production [27,

29]. Isolate HW-6 identified as Pseudomonas fluorescens

produced rhamnolipid biosurfactant with effective surface

and emulsifying properties when grown on hexadecane as a

sole carbon source [28]. Various carbon sources were

assessed for their effectiveness in the growth and biosur-

factant production by Pseudomonas fluorescens HW-6

[26]. In this study, the effect of rhamnolipid biosurfactant

produced by Pseudomonas fluorescens HW-6 on some

microbial cells, model strains, and isolates members of the

tested ecosystem was investigated. Evaluation was made

on the efficacy of surface properties of rhamnolipid bio-

surfactant and its potential application in bioremediation of

hydrocarbon contaminated environment.

Materials and Methods

Biosurfactant

Rhamnolipid biosurfactant used in this study was isolated

from the culture supernatant of hexadecane grown Pseu-

domonas fluorescens HW-6 as described previously

[27, 28].

Microorganisms

Several bacterial strains isolated in this laboratory from

lubricant-polluted wastewater samples were used in the

present study [27]. Strains Bacillus subtilis 168 (Culture

Collection of the Institute of Microbiology, Bulgarian

Academy of Sciences) and Pseudomonas aeruginosa

1390 (National Bank of Industrial Microorganisms and

Cell Cultures) were used as model Gram (?) and Gram

(-) bacterial strains, respectively. Cultures were main-

tained at 4C on meat peptone agar slants and transferredmonthly.

Growth Conditions

Laboratory strains B. subtilis 168 and P. aeruginosa 1390

were grown in mineral salts medium (MSM) supplemented

with 2 mM CaCl2, 0.5% casein hydrolysate, and 0.5%

maltose, pH 7.2 [25]. For preparing inocula, strains were

cultivated for 18 h at 37C with agitation (200 rev min-1).The experimental cultures were inoculated with 1% (v/v)

inoculum and incubated in 250-ml flasks until late expo-

nential phase at the same growth conditions as those used

to prepare the inoculum.

Bacterial isolates Pseudomonas sp. HW-1, Bacillus sp.

HW-4, and Escherichia sp. HW-13 were cultivated in

Erlenmeyer flasks containing MSM supplemented with

1.5% n-hexadecane as a sole carbon source. Flasks were

inoculated with 2% (v/v) log phase culture grown in a

nutrient broth medium then incubated 7 days in the dark at

(28 2C) on a rotary shaker (130 rev min-1). An abioticcontrol was also prepared for comparison purpose. Growth

of cultures was monitored spectrophotometrically by

measuring the optical density at 570 nm (OD570). The

changes in the cell permeability of bacterial strains in the

presence of rhamnolipiod biosurfactant were followed by

the amount of protein released in the culture medium.

Protein content was determined by the method of Bradford

using human serum albumin as a standard [5].

Permeabilization of Bacterial Cells

Permeabilization procedure was performed with resting

cells of some Gram (?) and Gram (-) isolates. Cells were

cultivated until late exponential phase in MSM with 2%

glucose then separated by centrifugation. Cell pellet was

washed twice with distilled water and resuspended in

0.02 M phosphate buffer, pH 7.5 to a final OD of 0.250.30

(in a volume 1.5 ml). Cell suspensions were treated with

rhamnolipid biosurfactant at concentrations above the

critical micelle concentration (CMC) [28], 0.003, 0.006,

and 0.012%, and then incubated with shaking at room

temperature for 1 h. The extracellular protein content and

0

0.1

0.2

0.3

0.4

0

0.5

1

1.5

2

control 0.001 0.005 0.03

Pro

tein

(m

g/O

D57

0)

Gro

wth

(O

D57

0)

Biosurfactant concentration (%)

a

0

0.1

0.2

0.3

0.4

0

0.5

1

1.5

2

control 0.001 0.003 0.005

Pro

tein

(m

g/O

D57

0)

Gro

wth

(O

D57

0)

Biosurfactant concentration (%)

bFig. 1 Effect of differentconcentrations of rhamnolipid

biosurfactant from

P. fluorescens HW-6 on thegrowth (bars) and protein

release (drawings) of

Pseudomonas aeruginosa 1390(a) and Bacillus subtilis 168 (b)

428 E. Vasileva-Tonkova et al.: Rhamnolipid Biosurfactant Effect on Bacterial Strains

123

cell hydrophobicity were then measured and compared

with the control samples without biosurfactant.

Cell Surface Hydrophobicity Test

Cell hydrophobicity was measured by bacterial adherence

to hydrocarbons (BATH) according to a method of

Rosenberg et al. [22]. Hydrophobicity was expressed as the

percentage of adherence to hexadecane calculated as fol-

lows: 1009 (1-OD550 of the aqueous phase/OD550 of the

initial cell suspension).

Results and Discussion

Biosurfactant Effect on the Growth and Protein Release

of Model Bacterial Strains

The effect of different concentrations of rhamnolipid bio-

surfactant from P. fluorescens on growth and protein release

of two model bacterial strains, Gram (-) cells of P. aeru-

ginosa 1390 and Gram (?) cells of B. subtilis 168, was

studied. As can be seen in Fig. 1a, rhamnolipid biosurfactant

at all used concentrations not affect the growth of P. aeru-

ginosa 1390. About 30% decrease in the amount of secreted

protein was observed in the presence of 0.03% biosurfactant,

concentration that is about 15-fold higher than CMC.

It was found that rhamnolipid biosurfactant at concen-

tration below CMC (0.001%) did not alter the growth of B.

subtilis 168 (Fig. 1b). Biosurfactant at concentration above

CMC (0.003 and 0.005%) slightly inhibited the growth of

the strain, about 14 and 18%, respectively, compare to the

control. A complete inhibition of the growth of B. subtilis

168 was established at higher concentrations of biosur-

factant, 0.01 and 0.03%. A decrease in the amount of the

secreted protein was observed with increasing the amount

of added biosurfactant from 0.001 to 0.005% (Fig. 1b).

Similar results were obtained about the effect of

rhamnolipid biosurfactant produced by Pseudomonas sp.

PS-17 on bacterial cells [24]. Although biosurfactants are

known to have soft effect on microbial cells, there are some

reports which demonstrate that rhamnolipid biosurfactants

produced by P. aeruginosa have toxic effect against

Bacillus subtilis and other Gram-positive strains [1, 4]. The

observed differences in the response of Gram (-) and

Gram (?) bacterial strains to the effect of rhamnolipid

biosurfactant most probably were due to the different

composition of the bacterial cell surface. It is well known

that Gram (-) bacterial cells have a unique outer mem-

brane. Lipopolysaccharides which are the main component

of the outer membrane of Gram negative bacteria protect

the sensitive inner membrane and the cell wall from the

effect of hydrocarbons and other toxic compounds [8, 9].

Biosurfactant Effect on the Growth of Bacterial Isolates

Gram (?) isolate Bacillus sp. HW-4 and two Gram (-)

isolates, Pseudomonas sp. HW-1 and Escherichia sp. HW-

13, which showed slow growth in a previous study [27],

were chosen for studying the effect of rhamnolipid bio-

surfactant on their growth on hexadecane. It was found that

biosurfactant in concentrations below CMC (0.001%) and

0

0.5

1

1.5

2

2.5

3

1 2 3 4 7

Gro

wth

, OD

570

Time, days

Pseudomonas sp. HW-1

0

0.5

1

1.5

2

2.5

3

1 2 3 4 7

Gro

wth

, OD

570

Time, days

Escherichia sp. HW-13

hdhd+0.001% Pfhd+0.0024% Pfhd+0.005% Pf

0

1

2

3

4

5

1 2 5 7

Gro

wth

, OD

570

Time, days

Bacillus sp. HW-4

hdhd+0,0024% Pfhd+0,005% Pfhd+0.01% Pf

hdhd+0.001% Pfhd+0.0024% Pfhd+0.005% Pf

Fig. 2 Effect of different concentrations of rhamnolipid biosurfac-tant from P. fluorescens HW-6 on the growth on hexadecane of Gram(-) isolates (Pseudomonas sp. HW-1 and Escherichia sp. HW-13),and Gram (?) isolate Bacillus sp. HW-4

E. Vasileva-Tonkova et al.: Rhamnolipid Biosurfactant Effect on Bacterial Strains 429

123

over CMC (0.0024, 0.005, and 0.01%) shortened lag phase

and stimulated the growth of the tested isolates on hexa-

decane (Fig. 2). The stimulation effect was better expres-

sed with tested Gram (-) isolates at concentrations of

biosurfactant below and near CMC. Growth in the presence

of rhamnolipid biosurfactant was about sixfold higher in

comparison to the control, at concentration below CMC for

HW-1 isolate, and at concentration near CMC for HW-13

isolate. At the seventh day of growth, an inhibition of the

growth of HW-1 isolate was observed at all tested con-

centrations of biosurfactant. For the tested Gram (?)

Bacillus sp. HW-4, biosurfactant slightly stimulated the

growth on hexadecane (about 1.5-fold) (Fig. 2).

Stimulation of the growth of the tested bacterial isolates

in the presence of rhamnolipid biosurfactant could be due

to an increase of hexadecane availability to bacterial cells

by increasing the apparent aqueous solubility of the

hydrocarbon. Due to the amphiphilic nature, biosurfactants

could make contact between the cells and n-alkane [6].

Owsianiak et al. observed that rhamnolipids and nonionic

Triton X-100 surfactants increased diesel fuel biodegra-

dation by initially slow-degrading cultures and decreased it

by fast degraders [17]. This indicates that effectiveness of

surfactants depends on the specification of microorganisms

and not on the type of surfactant. The negative effect of

rhamnolipid biosurfactant of P. fluorescens HW-6 on the

growth of Pseudomonas sp. HW-1 isolate could be caused

by the hydrophobic part of the biosurfactant which binds to

the hydrophobic cell surface structures and the oilwater

interface, making both structures more hydrophilic. This

inhibitory interference may prevent the adhesion of cells to

hydrocarbon [30].

Biosurfactant Effect on Cell Surface Hydrophobicity

and Permeability of Bacterial Isolates

The effectiveness of cell surface properties of rhamnolipid

biosurfactant of P. fluorescens HW-6 was studied at con-

centrations over CMC. For this purpose, cell surface

hydrophobicity and permeability of some bacterial isolates,

members of the microbial consortium, were followed in

experiments in vitro. The following isolates were selected

possessing different cell surface hydrophobicity when

grown on glucose: Gram (-) strains Pseudomonas sp.

HW-1, Pseudomonas sp. HW-10, Pseudomonas sp. HW-12

and Escherichia sp. HW-13, and Gram (?) strains Bacillus

sp. HW-4, Arthrobacter HW-7, Streptococcus sp. HW-9

and Micrococcus sp. HW-11. The results revealed that

rhamnolipid biosurfactant modified cell surface hydropho-

bicity and increased membrane permeability of the tested

isolates. Bacterial cells treated with rhamnolipid biosur-

factant became more or less hydrophobic than untreated

cells depending on individual characteristics and abilities of

the strains (Figs. 3, 4). Due to its amphiphilic structure,

biosurfactant has different orientation in relation to the

hydrophobic and hydrophilic character of the bacterial

surface. For example, cell surface hydrophobicity of

Pseudomonas aeruginosa was greatly increased by the

presence of cell-bound rhamnolipid [30], whereas cell-

bound emulsifier decreased cell surface hydrophobicity of

Acinetobacter strains [23]. Therefore, biosurfactants altered

cell surface hydrophobicity of microorganisms thus enable

them to attach or detach from surfaces according to need. It

was reported that rhamnolipid and Triton X-100 surfactants

altered cell surface hydrophobicity of microbial consortia in

0

10

20

30

40

50

1 2 3 4

Hyd

rop

ho

bic

ity,

%


0

10

20

30

40

50

1 2 3 4

Hyd

rop

ho

bic

ity,

%


0

10

20

30

40

50

1 2 3 4

Hyd

rop

ho

bic

ity,

%


0

10

20

30

40

50

1 2 3 4

Hyd

rop

ho

bic

ity,

%


Fig. 3 Effect of differentconcentrations of rhamnolipid

biosurfactant from

P. fluorescens HW-6 on cellsurface hydrophobicity of Gram

(-) bacterial isolates. Axis:

1control; 20.003%

biosurfactant; 30.006%

biosurfactant; 40.012%

biosurfactant


123

similar manner: increased for the hydrophilic and decreased

for the hydrophobic cultures [17]. Nevertheless, no corre-

lations between cell surface hydrophobicity and biodegra-

dation of diesel fuel by the consortia were found.

Changes in permeability of bacterial cells were followed

by the levels of extracellular protein. For all tested strains,

an increase in the amount of the released protein was

observed with increasing the amount of the added biosur-

factant (Figs. 5, 6). The effect probably was due to

increased cell permeability as a result of changes in the

organization of cell surface structures. The biosurfactant

most likely forms molecular aggregates in surface bacterial

membranes, leading to the formation of transmembrane

pores that serve as channels to the periplasm [12]. This

effect was better expressed in the tested Gram (?) cells

than in Gram (-) cells (Fig. 6). A possible explanation

could be related to the different composition of the bac-

terial cell surface.

Bacterial strains used in this study were isolated from a

relatively closed ecosystem, the settler of mineral oil

fraction in the Electric Power Station, and represent well-

balanced microbial consortium where particular partici-

pants have different roles and support each other [27].

Some members in a consortium produce surfactants and

thus provide others non-producing bacteria helping them to

degrade hydrocarbons. Horizontal transfer of capsule

0

10

20

30

40

50

60

70

1 2 3 4

Hyd

rop

ho

bic

ity,

%

Bacillus sp. HW-4

0

10

20

30

40

50

60

70

1 2 3 4

Hyd

rop

ho

bic

ity,

%

Arthrobacter sp. HW-7

0

10

20

30

40

50

60

70

1 2 3 4

Hyd

rop

ho

bic

ity,

%Streptococcus sp. HW-9

0

10

20

30

40

50

60

70

1 2 3 4

Hyd

rop

ho

bic

ity,

%

Micrococcus sp. HW-11


biosurfactant from

P. fluorescens HW-6 on cellsurface hydrophobicity of Gram

(?) bacterial isolates. Axis as in

Fig. 3

0

0.5

1

1.5

Pro

tein

(m

g/O

D57

0)

Bacillus sp. HW-4

0

0.5

1

1.5

1 2 3 4 1 2 3 4

Pro

tein

(m

g/O

D57

0)

Arthrobacter sp. HW-7

0

0.5

1

1.5

Pro

tein

(m

g/O

D57

0)

Streptococcus sp. HW-9

0

0.5

1

1.5

1 2 3 4 1 2 3 4

Pro

tein

(m

g/)D

570)

Micrococcus sp. HW-11


biosurfactant from

P. fluorescens HW-6 on proteinrelease of Gram (?) bacterial

cells. Axis as in Fig. 3


123

polysaccharide has been demonstrated resulting in bacteria

coated with emulsifying capsule produced by bacteria of

another species [16]. The results suggest that rhamnolipid

biosurfactant produced by P. fluorescens HW-6, member of

the microbial consortium, possesses effective cell surface

properties and promotes assimilation of hydrocarbons by

slow growing isolates.

Pseudomonads are known for their broad nutritional

versatility which enables them to use many wastes and

pollutants as sources of carbon and energy. They are fre-

quently found as dominating in the microflora of oil-con-

taminated sites [11]. Pseudomonas fluorescens encompasses

a group of common, non-pathogenic saprophytes that col-

onize soil, water, and plant surface environment. Moreover,

fluorescent pseudomonads have often been used as biolog-

ical control agents and as biosensors in wastewater biore-

mediation [2, 15]. In view of this complex of possibilities,

Pseudomonas fluorescens HW-6 and produced biosurfactant

with effective surface properties represent a promising

potential for application in bioremediation of hydrocarbon-

polluted environments.

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c.284_2010_Article_9725.pdfThe Effect of Rhamnolipid Biosurfactant Produced by Pseudomonas fluorescens on Model Bacterial Strains and Isolates from Industrial WastewaterAbstractIntroductionMaterials and MethodsBiosurfactantMicroorganismsGrowth ConditionsPermeabilization of Bacterial CellsCell Surface Hydrophobicity TestResults and DiscussionBiosurfactant Effect on the Growth and Protein Release of Model Bacterial StrainsBiosurfactant Effect on the Growth of Bacterial IsolatesBiosurfactant Effect on Cell Surface Hydrophobicity and Permeability of Bacterial IsolatesReferences

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the Effect of Rhamnolipid Biosurfactant Produced