Crude Cacterial Consortium Biosurfactants Potent for Bioemulsification of Oils and Petroleum...

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International Journal of Research (IJR) Vol-1, Issue-7, August 2014 ISSN 2348-6848

CRUDE CACTERIAL CONSORTIUM BIOSURFACTANTS POTENT FOR BIOEMULSIFICATION OF OILS AND

PETROLEUM HYDROCARBONSJyotsna Kiran Peter, Abhishek Kumar Rao and Ritu Kumari

Crude Cacterial Consortium Biosurfactants Potent for

Bioemulsification of Oils and Petroleum Hydrocarbons

Jyotsna Kiran Peter1*, Abhishek Kumar Rao1and Ritu Kumari1

Department of Microbiology and Fermentation Technology (MBFT), Jacob School of Biotechnology and

Bioengineering (JSBB), Sam Higginbottom Institute of Agriculture Technology and Sciences (SHIATS),

Naini, Allahabad, Uttar Pradesh, India, 211007

*Corresponding author: Jyotsna Kiran Peter1*,[email protected]

AbstractCrude biosurfactants from Pseudomonas

aeruginosa, Serratia marscecens and Bacillus

subtilis along with the consortium of the three

mentioned bacteria were harvested after batch

mode submerged fermentation. The

emulsification was found highest by consortium

crude biosurfactants as compared to individual

bacterial crude biosurfactants. The stability was

also found persistant from 0h to 96 h in case of

crude consortium biosurfactants.

Key words: Pseudomonasaeruginosa,Serratia

marscecens, Bacillus subtilis, crude consortium

biosurfactants and emulsification

IntroductionHydrocarbons are commonly used as the

substrate for the production of biosurfactants. It

has been postulated that the biological function of

surface-active compounds is related tohydrocarbon uptake, and therefore a

spontaneous release occurs with these

substrates (Mu et al.,2009). Biosurfactants are

produced by several types of microorganisms,

such as bacteria, fungi and yeasts Namir et al.,

2009]. Bacillomycin F, D, L [Marion and Maget-

Dana, 1985], surfactin [Schneider et al., 1988],

lichenysin [Grangemard et al., 1999], iturin

[Peypoux et al.,1978], halobacillin [Grangemard

et al., 1999], and plipastatin [Nishikiori et al.,

1986] are lipopeptide biosurfactants produced by

Bacillus strain. Surfactin, one of the mosteffective biosurfactants, was isolated from the

cell-free culture medium after growth of Bacillus

subtilis on glucose [Cooper et al., 1981]. This

biomolecule is usually a cyclic compound

consisting of seven amino acids bonded to a lipid

moiety. Surfactin is effective in lowering the

surface tension of water to30 mN/m [Fox and

Bala, 2000], which is comparable with the values

obtained by conventional synthetic surfactants.

Additionally, surfactin preparations have other

interesting characteristics, including antibiotic

and antiviral properties [Davis et al., 1999].

Surfactin isoforms produced by Bacillus subtilis

HSO121 have been studied extensively in our

laboratory [Haddad et al.,2008; Liu et al.,2007].

Pseudomonas species are known to produce

different types of BS viz., rhamnolipids, cyclic

lipopeptides- putisolvins, lipopolysaccharide.

Two types of cyclic lipopeptides (putisolvins I and

II) are produced by P. putida PCL1445, which

possess surfactant activity and also plays

significant role in biofilm formation and

degradation. Serratia is one of the well-studiedbacterium in terms of molecular genetic studies of

BS production. Serratia, a Gram-negative or-

ganism is known to produce extracellular surface

active(Matsuyama et al., 1987) and surface

translocating agents (Horng et al., 2002) S.

marcescens produces a cyclic lipopeptide BS

Serrawettin which contains 3-hydroxy-C10 FA

side chain. BS production is correlated with

populational surface migration (Wei et al.,2004)


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Plate 1: Degradation of oil and

simultaneous production of biosurfactant

from Consortia



from Bacillus subtilis



from Pseudomonas aeruginosa



from Serratia marscecens

Drop collapse assay of crude biosurfactants

Crude biosurfactants from the individual strains of

Pseudomonasaeruginosa,Serratia marscecens

and Bacillus subtilis along with the consortium of

the three mentioned bacteria showed positive

drop collapse assay (Table1). The drop collapse

assay signifies the biosurfactant activity of crude

biosurfactants. An oil drop acquires a convex

shaped drop while resting on a calibrated glass

slide. The convex shape of the drop is distorted

or the drop collapse due to biosurfactants activity

as the nature of biosurfactant is to align and orient

them into various configurations at oil aqueous

interphase and reduces the surface tension of oil

drop so that the drop collapses. The assay has

been used by several authors to screen the

biosurfactant activity of either microorganism or

partially or crude or purified biosurfactants.

Table1: Drop collapse assay of crude biosurfactants

Biosurfactants source

P

etrol

D

iesel

Mo

biloil

Kerosene

Mu

stard

So

ybean

Jasmine

Almond

Pseudomonas aeruginosa+

+ + + + + + +

Serratia marscecens + + + + + + + +

Bacillus subtilis + + + + + + + +

Consortium+ + + + + + + +


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Emulsification index of crude biosurfactant

of Pseudomonas aeruginosa

Crude biosurfactants obtained fromPseudomonas aeruginosa was used for detection

of hydrocarbon (Kerosene, Petrol, Diesel and

Mobil oil) emulsification and vegetable

emulsification (Mustard oil, Coconut oil, Soy bean

oil and Jasmine oil). The emulsification stability

was observed from 0h to 96h as E0, E24, E48, E72

andE96. The highest emulsified hydrocarbon was

mobil oil followed by diesel and petrol while

kerosene was not emulsified by the crudebiosurfactant. The stability of the three emulsified

hydrocarbon gradually declined from 0-96h. All

the four vegetable oils were emulsified and found

stable till 96h of incubation. The highest

emulsisfid was mustard oil followed by soy bean

oil, jasmine oil and then coconut oil.

Table 2:Emulsification index of crude biosurfactant of Pseudomonas aeruginosa

Emulsification index of crude biosurfactant of Pseudomonas aeruginosa

Substrate E0 E24 E48 E72 E96

Hydrocarbons Kerosene 0 0 0 0 0

Petrol 25 22.22 17.64 12.5 5.88

Diesel 51.72 50 48.14 48 41.66

Mobil oil 65.71 44.11 40.62 34.37 33.33

Vegetable oils Mustard oil 56 36 30.43 27.27 22.22

Coconut oil 50 45.83 45.45 40.9 38.88

Soy bean oil 53.57 50 48.14 46.15 40.9

Jasmine oil 52 48 45.83 43.47 36.36

Fig:1 Emulsification index of crude biosurfactant of Bacillus subtilis for hydrocarbons

K

d0

20

40

60

80

E0 E24 E48 E2E!6

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emulsificationactivity(%

)

emulsification activity measured at different time intervals (h)

Ke&'(e)e Pe*&'+ de,(e+ M'-,+


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Plate:5 Emulsification activity of crude biosurfactant of Pseudomonas aeruginosa showing

emulsification of vegetable oils

Plate:6 Emulsification activity of crude biosurfactant of Pseudomonas aeruginosa showing

emulsification of hydrocarbons

Emulsification index of crude biosurfactant of

Serratia marscecens

Crude biosurfactants obtained from Serratia

marscecens was used for detection of

M.(*a&d

C'/').*

(' -ea)

1a(,)e

0

20

40

60

E0 E24 E48 E2E!6

"6

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emulsificationactivity(%)

emulsification activity measured at different time intervals (h

M.(*a&d C'/').* (' -ea) 1a(,)e


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hydrocarbon (Kerosene, Petrol, Diesel and Mobil

oil) emulsification and vegetable emulsification

(Mustard oil, Coconut oil, Soy bean oil andJasmine oil). The emulsification stability was

observed from 0h to 96h as E0, E24, E48, E72 and

E96. Among hydrocarbons highest emulsified was

mobil oil followed by diesel, petrol and kerosene.

Among vegetables most emulsified oil was

mustard while slightly less emulsified werecoconut and soybean oil. Jasmine was not

emulsified by crude Serratia marscecens

biosurfactant.

Table 3: Emulsification index of crude biosurfactant of Serratia marscecens

Emulsification index of crude biosurfactant of Serratia marscecens

Substrate E0 E24 E48 E72 E96

Hydrocarbons Kerosene 19.04 13.63 8.69 4.76 0

Petrol 10.52 9.09 0 0 0

Diesel 46.42 37.03 26.92 24 17.39

Mobil oil 70.83 56.52 52.17 45.45 44.44

Vegetable oilsMustard oil 60 46.15 40.74 40 29.62Coconut oil 51.72 48.27 44.82 40.74 36.36

Soy bean oil 50 45.45 42.85 40 33.33

Jasmine oil 0 0 0 0 0

Fig:3 Emulsification index of crude biosurfactant of Serratia marscecensfor hydrocarbons

Fig:4 Emulsification index of crude biosurfactant of Serratia marscecensfor hydrocarbons

Ke&'(e)e

D,e(e+

0

20

40

60

80

E0 E24 E48 E2 E!6

$!#04

$%#6% 8#6!4#6

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"6#"2 "2#$4"#4" 44#44

Emulsificationindex(%)

Emulsification activity at different time intervals (h)

Ke&'(e)e Pe*&'+ D,e(e+ M'-,+ O,+

M.(*a&d ',+C'/').* '',+

S' -ea) ',+

1a(,)e ',+0

$00

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40%%#%%

00000



M.(*a&d ',+ C'/').* '',+ S' -ea) ',+ 1a(,)e ',+


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Plate:7 Emulsification activity of crude biosurfactant of Serratia marscecensshowing emulsification

of vegetable oils

Plate:8 Emulsification activity of crude biosurfactant of Serratia marscecens showing emulsification

of hydrocarbons


Bacillus subtilis

Crude biosurfactants obtained from Bacillus

subtilis was used for detection of hydrocarbon(Kerosene, Petrol, Diesel and Mobil oil)

emulsification and vegetable emulsification

(Mustard oil, Coconut oil, Soy bean oil and

Jasmine oil). The emulsification stability was


E96. The most stable and highly emulsified oil was

mobil among the hydrocarbons the stability was

recorded till 96h. Lower emulsification activity

was recorded for kerosene petrol and dieselwhose stability declined to 0 at 96h of incubation.

Among vegetable oil jasmine was not emulsified

and soy bean was the highest emulsified oil with

stability showing till 96h as 42.85%.


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Table 4: Emulsification index of crude biosurfactant of Bacillus subtilis

Emulsification index of crude biosurfactant of Bacillus subtilis

Substrate E0 E24 E48 E72 E96Hydrocarbons Kerosene 14.81 11.11 7.69 3.84 0

Petrol 13.63 11.76 9.09 0 0

Diesel 9.09 6.25 3.57 3.33 0

Mobil oil 86.95 61.53 48 45.83 37.03

Vegetable oils Mustard oil 44 41.66 39.13 36.36 33.33

Coconut oil 16 12 8.33 4.54 4.54

Soy bean oil 51.85 48.14 46.15 44 42.85

Jasmine oil 0 0 0 0 0


Fig:6 Emulsification index of crude biosurfactant of Bacillus subtilis for vegetable oils

Ke&'(e)e

Pe*&'+

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Plate:9 Emulsification activity of crude biosurfactant of Bacillus subtilis showing emulsification of

hydrocarbons


consortium

Crude biosurfactants obtained from consortium

was used for detection of hydrocarbon

(Kerosene, Petrol, Diesel and Mobil oil)

emulsification and vegetable emulsification

(Mustard oil, Coconut oil, Soy bean oil and

Jasmine oil). The emulsification stability was


E96. The crude consortium biosurfactant was able

to emulsify all hydrocarbons and vegetable oils

that were found stable till 96h of incubation. The

stability of the emulsion formed from 0h remained

high till 96h in all cases. The consortium

biosurfactant showed high emulsification index as

well as stability in all hydrocarbons and vegetable

oils tested.

Table 5: Emulsification index of crude biosurfactant of consortium

Emulsification index of crude biosurfactant of consortiumSubstrate E0 E24 E48 E72 E96

Hydrocarbons Kerosene 71.42 69.23 68 65.21 59.09

Petrol 59.09 60 52.38 47.61 42.1

Diesel 75 73.07 72 70.83 65.21

Mobil oil 70 69.56 65.21 63.63 60

Vegetable oils Mustard oil 45.83 41.66 40.9 40 38.88

Coconut oil 57.14 43.33 39.28 34.61 25.92

Soy bean oil 66.66 66.33 60 58.62 55.55

Jasmine oil 68.96 67.85 64 60.86 57.14


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Fig:8 Emulsification activity of crude biosurfactant of consortia showing emulsification of hydrocarbons

Fig:9 Emulsification activity of crude biosurfactant of consortia showing emulsification of vegetable oils

Plate:10 Emulsification activity of crude biosurfactant of consortia showing emulsification of vegetable oils

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Plate:11 Emulsification activity of crude biosurfactant of consortia showing emulsification ofvegetable oils

DiscussionBiosurfactants can be synthesized using different

microorganisms and carbon sources and

production is influenced by the composition of the

medium and by culture conditions (Desai and

Banat, 1997; Franzetti et al., 2009). Techaoei et

al.(2007) did preliminary screening of preliminary

screening of biosurfactant producingmicroorganisms isolated from hot spring and

garages in northern Thailand and reported the

emulsification at 24 36 and 48 h of incubation.

The deposition of material at the edge of

evaporating droplets, known as the coffee ring

effect, is caused by a radially outward capillary

flow. This phenomenon is common to a wide

array of systems including colloidal and bacterial

systems. The role of surfactants in counteracting

these coffee ring depositions is related to the

occurrence of local vortices known as Marangoni

eddies. Here we show that these swirling flows

are universal, and not only lead to a uniform

deposition of colloids but also occur in living

bacterial systems. Experiments on Pseudomonas

aeruginosa suggest that the auto-production of

biosurfactants has an essential role in creating a

homogeneous deposition of the bacteria upon

drying (Sempels et al., 2013). Bodour et al.

reported that the drop collapse method may be

used to detect biosurfactant producing

microorganisms in natural environments. The

results of the present study also suggested that

the drop collapse method suits well as a primary

screening method for biosurfactant production

and the oil spreading method is good to quantify

the biosurfactant. The Biosurfactant production

may be also detected by using emulsification

index. Surface activity and emulsification activity

have direct correlation [Surachai et al., 2007].

Biosurfactants have the ability to reduce surface

tension and emulsify hydrocarbons, thereby,

boosting the bioavailability quotient of the

contaminating hydrocarbons. According to the

study of Chandran and Das, 2011, the stability of

formed emulsions was found to be more than one

month in room temperature without changing

emulsification activity. Interestingly, the crude

biosurfactants gave the highest emulsification

activity (E24) on diesel, probably because it was

produced by diesel oil as a carbon source. The

emulsification index values of biosurfactants were

also measured at different temperatures, pH andNaCl concentrations. The optimum temperature

for emulsification activity of biosurfactants was at

room temperature (28 C), even showing

emulsification activity at temperature of 10 -100

C. This mechanism is of importance when two

immiscible phases (oil and water) are present and

direct substrate uptake is plausible (Neu, 1996;

Franzetti et al. 2009). The presence of

biosurfactants may also lead to a potential

enhancement of biodegradation efficiency. In this


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concept, the biosurfactant molecules act as

mediators, which increase the mass transfer rate

by making hydrophobic pollutants morebioavailable for microorganisms (Inakollu et al.

2004; Whang et al. 2009). Alternatively,

biosurfactants may also induce changes in the

properties of cellular membranes, resulting in

increased microbial adherence. Hydrocarbons

are organic compounds made of carbon atoms

bound to each other forming a backbone with

hydrogen atoms attached to the remaining sites

on carbon. The carbon backbone can be straight

or normal, branched, or cyclic (Olah and Molnar,

1995). Most hydrocarbons used as an energy

source are degraded under aerobic conditions tocarbon dioxide and water. This degradation

process is a catabolic process and the

degradation to inorganic compounds is called

mineralization. However, some hydrocarbons are

not mineralized but transformed into simpler

compounds (Ferrari et al. 1996). The

hydrocarbons used as a carbon source are

degraded to smaller compounds and

incorporated into the cell materials. This

degradation process is a combination of catabolic

and anabolic processes (Brock et al., 1994).

Cometabolism is another mode of degradation,

which is observed in the degradation of

hydrocarbons. In cometabolism the hydrocarbon

is transformed, but the organisms does not gain

any energy or nutrients (Field et al.,1991, Juhasz

et al., 1996). The specific degradation

mechanisms are determined by the compound

structure. Linear alkanes degrade through b-

oxidation in which the backbone is broken up two

carbons at a time and the resulting acetyl-CoA is

mineralized in the TCA cycle. Some cyclic

alkanes degrade through cometabolism (Juhaszet al.1996). Aromatic compounds are generally

degraded via a dioxygenase enzyme, which

converts the compound to a catechol followed by

ring fission in the ortho or meta positions (Prince,

1993). Emulsification is a process that forms a

liquid, known as an emulsion, containing very

smalldroplets of fat or oil suspended in a fluid,

usually water. The high molecular weight

biosurfactants are efficient emulsifying agents.

They are often applied as an additive to stimulate

bioremediation and removal of oil substances

from environments. Micelles are capable of

dissolving hydrophobic contaminants in theirhydrophobic core, which results in an increased

apparent aqueous solubility of the pollutants

(Edwards et al., 1991; Prak and Pritchard, 2002).

Conclusion

Conclusively, the research revealed the

emulsifying potential of crude biosurfactants

from Pseudomonas aeruginosa, Serratia

marscecensand Bacillus subtilis along with theconsortium of the three mentioned bacteria.

The consortium was more potent of producing

stable emulsion in the aforementioned

hydrocarbons and vegetable oils.

Therefore it could be recommended from the

study that the crude consortium or individual

crode biosurfactants could be used as strong

emulsifying agents.

Acknowledgements

The authors offer gratuitous thanks to HonbleVice Chancellor, Most Rev. Prof. R.B. Lal,

SHIATS, Naini, Uttar Pradesh, India for

provision of research conductance. Heartfelt

thanks to Head, Department of Microbiology

and Fermentation Technology, JSBB,

SHIATS, Allahabad, Uttar Pradesh, India for

the kind cooperation towards the research.

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