ISOLATION, EXTRACTION AND PURIFICATION OF

BIOSURFACTANTS PRODUCING BACTERIA FROM

OIL CONTAMINATED SOIL
Harpreet Kaur, Kinjal Saha, Vaishnavi, Dr K.V. Bhaskara Rao* (corresponding author)
Department of Applied Microbiology and Biotechnology, School of Biosciences and
Technology, Vellore Institute Of Technology University, Vellore, India. 632014

ABSTRACT
Bioremediation using microorganisms that produce biosurfactants seems to be an effective
solution for oil pollution in soils, water or environment which is very prevalent these days.
Biosurfactants are metabolites that are produced by some microbes or are present in the cell
structure of some microbes. Biosurfactants contribute to a rise in the surface tension on
hydrophobic substrates that are water insoluble and make them available for degradation
through bioremediation. Oil contaminated soil was collected from Air Force Station,
Tambaram, Chennai (working area). The sample was first inoculated in engine oil and
petroleum enriched nutrient broth for 48 hours and later isolation was done by two methods;
hydrocarbon overlay agar method and oil enriched nutrient medium method. Seven distinct
isolates were isolated. Primary screening was done using blood hemolysis test to distinguish
the colonies which produce biosurfactants. Five out of the seven isolates showed hemolysis.
Biosurfactant production by these five isolates were screened using hydrocarbon overlay
method, drop collapse method, blue agar plate method and emulsification assay. Three
isolates (IS1, IS2 and IS4) out of five showed best results for biosurfactant production.
Biosurfactants were extracted from the isolates using chilled ethanol precipitation method
and isolates were characterized using standard biochemical tests to determine the genus.
Gram positive cocci which was identified as Staphylococcus and gram positive rod as
Bacillus.

KEYWORDS: Biosurfactant, hemolysis test, emulsification test, blue agar test and oil
collapse test

INTRODUCTION
Petrochemical industries serve as a major contributor towards environmental pollution
currently. The compounds that have an impact on the environment, predominantly being
hydrocarbons cannot be easily degraded and are also carcinogenic in some cases.
(*a)Mechanical and chemical methods of reducing the effects of these compounds do not
yield significant results and are often very expensive to be performed. A better alternative to
degrade the hydrocarbons would be through the use of biodegradation. Biodegradation
involves the degradation of pollutants with the help of microorganisms, converting toxic
pollutants into a non-toxic form.
Biosurfactants are amphipathic metabolites that are naturally synthesized by microorganisms
intracellularly or extracellularly (bacteria, fungi). They are composed of phospholipids,
glycolipids, lipoproteins and mycolic acid. They are surface active compounds which has
both polar (hydrophilic) and non-polar (hydrophobic) characteristics. Due to this property
they aggregate at the interfaces between fluids with different polarities such as hydrocarbons
and water, thereby helping in the recovery of oils. Due to their advantages over chemical
surfactants of biodegradability, less toxicity, environment friendly, high selectivity,
compatibility with human skin etc, the researches are going on for biosurfactant isolation and
its production at commercial levels.
The biosurfactants are classified into two broad classes :- 1. Low molecular weight surface
active agents (bio-surfactants) and 2.High molecular weight substrates (bio-emulsifiers).
Further they are divided into six classes based on the functional groups present in them:-
Glycolipids, Rhamnolipids, Lipopeptides, Liposaccharides, Phospholipids, Fatty acids and
Antibiotics.
(*b) Biosurfactants due to their variable characteristics find applications in various fields as
like food processing, recovery of oil, cosmetics, petroleum related industries and in the fields
of agriculture and medicine. Production of biosurfactants is dependent on economically
feasible and sustainable substrates as most of the vital carbon sources is derived from
different industries as like wastes from agricultural units (sugars, oils, molasses, compounds
containing starch and whey), fats from animal origin, distillery waste products etc. This aids
in amplification of the process of biosurfactant production and also suitable waste
management processes that not contribute much to the disposal and accumulation of
unwanted waste products from industries.

(*c) Some of the applications of biosurfactants are low carboxymethyl cellulose (CMC),
stability with respect to salt content and pH , biodegradable nature, foaming characteristics
with respect to food, Proper Hydrophobic lipophilic balance (HLB), efficiency of safety
mechanisms with regard to the environment, stability in terms of chemical aspects, surface
absorption, lower toxic content, biodegradability, affinity for contaminating agents,
absorption on particular ores: oil-bearing formations, formation of micro-emulsions and
solubilization, easier breakage of emulsions after recovery, biocompatible aspects.

MATERIALS AND METHODS

Collection of samples:
Soil samples contaminated with oil (waste or fresh engine oil and aviation oil thrown in the
soil) were collected from Air Force Station, Tambaram, Chennai in sterile autoclave bags.
Samples were transported to VIT Vellore and stored in the refrigerator till the time
experiment was started.
Preparation of media and isolation of colonies:
The sample was inoculated in 1% engine oil and petroleum enriched nutrient broth separately
for 48 hours. 0.85% sterile saline was taken and 1ml of broth was serially diluted with saline.
Using 3, 4 and 5 dilutions, isolation was carried by two method:
A) Hydrocarbon overlay method- The 1ml of the dilutions were spread plated on nutrient
agar plates and the plates were overlaid with sterile membrane filtered engine oil and
petroleum separately. The plates were examined for halo zone formation of oil displacement
around the colonies after 24, 48 and 72 hours of incubation at 37⁰C.
B) Oil enriched nutrient medium method – Sterile engine oil and petroleum (1%) were mixed
with the nutrient media separately before plating. This enriched media is then plated and 1ml
of the dilutions are inoculated by spread palate technique. These plates were kept for
incubation for 24 hours at 37⁰C.

Fig 1. OIL ENRICHED SEED CULTURE AND OIL OVERLAY METHOD

Fig 2. POURING PLATES

PRIMARY SCREENING :-
Blood hemolysis test: Isolates having different morphology were inoculated on freshly
prepared blood agar media (2-3%) to study the nature of hemolysis. The plates kept for
incubation for 24-48 hours at 37⁰C. Colonies which exhibited evident lysis were selected for
further analysis.

SECONDARY SCREENING :-
Production of bio surfactant:
All isolated colonies were inoculated in minimal salt medium in different conical flasks.
Composition of MSM (g/L) :- KH2PO4 20g, K2HPO4 5.0g, (NH4) 2PO4 30g, NaCl 0.1g,
FeSO4.7H2O 0.01g, MgSO4.7H2O 0.2g, CaCl2.2H2O 0.01g, MnSO4.7H2O 0.2g, Glucose
0.03g, Yeast extract 0.03g.
The culture was incubated for 5 days at 120rpm on shaker at room temperature and after
incubation it was centrifuged at 4⁰C at 8000rpm for 20 minutes. The cell free supernatant
was separated and used for further biosurfactant studies.

Drop collapse method :

Drop collapse method is used to test destabilization of oil by biosurfactant. In this method
petri-plate was coated with an oil drop, then cell free supernatant was added on it and
observed after one minute. Positive result are considered if drop spreads or collapses. All
the isolates were tested.

Blue agar plate method:

Blue agar was made by Minimal salt medium supplemented with methylene
blue(0.2mg/mL) and cetyl-trimethyl ammonium bromide(0.5mg/mL). This test is for
detecting anionic biosurfactants . 30µl of the supernatant (free from cell debris) was loaded
into the wells prepared in the blue agar plates. A negative control was maintained with
distilled water. Plates incubated at 37⁰C, for 48-72 hours. Positive result that is anionic
biosurfactant presence was shown by dark blue ring or light blue halo zone around the well
as cetyl-trimethyl ammonium bromide is cationic when reacts with anionic biosurfactant, it
forms blue colour complex.

Emulsification assay:
Emulsification activity of the biosurfactant was measured by vortexing 3ml of cell free
suspension with 2ml of engine oil and was kept overnight. After 24 hours the emulsion
index (EI) was calculated using the formula :-
EI = Height of emulsion layer/ Total height* 100

Biochemical characterization:
Potential isolates were partially characterized by performing standard biochemical tests to
determine the genus with reference to Bergey’s manual.
PURIFICATION OF BIOSURFACTANT
Biosurfactant was purified by taking one volume of extracted biosurfactant and adding
three volumes of acetone in it in a Eppendorf tube. Then the tubes were kept undisturbed for
10 hours at 4 ⁰C. After 10 hours, the tubes were centrifuged for 20 minutes at 11,000 rpm
and pellet was collected. Pellet after centrifuging was the purified biosurfactant which can
be then dried to remove residual acetone by evaporation.

RESULTS AND DISCUSSION

In primary screening, after incubation of plates with hydrocarbon overlay agar method
various colonies in engine oil overlay plates and few in petroleum plates overlay showed
halozones as biosurfactant producing colonies able to displace hydrocarbon(engine oil and
petroleum) and emulsify it. Out of these we got seven different isolates (IS1, IS2, IS3, IS4,
IS5, IS6, and IS7). These isolates were studied for biosurfactant presence.

Fig 3. HYDROCARBON (OIL) OVERLAY PLATES WITH COLONIES

 Fig 4. PERFORMING QUADRANT STREAKING

Fig 5. PURE CULTURE OF ISOLATES

Five isolates- IS1, IS2, IS4, IS6 and IS7 showed β-hemolysis in Blood agar hemolysis test.
The RBCs in the blood are lysed which can be linked to biosurfactant production. The assay
also predicts the surface activity of biosurfactant producing microbes.

TABLE 1:- BLOOD AGAR HEMOLYSIS RESULTS

ISOLATES IS1 I2 IS4 IS6 IS7

HEMOLYSIS Beta- Beta- Beta- Beta- γ–

OF BLOOD hemolysis hemolysis hemolysis hemolysis hemolysis
AGAR
Fig 6 and 7. HEMOLYSIS IN BLOOD AGAR

These five isolates were selected and tested for further secondary screening.
In Drop collapse method, out of five isolates, IS1, IS2, IS4 and IS6 showed drop collapsing
activity in 2minutes with engine oil. Presence of biosurfactant in culture supernatant can be
detected, if the oil drop collapses because of the reduction in interfacial tension. In case of no
surfactant in culture supernatant, the hydrophilic molecules are repelled from the
hydrophobic oil surface and the oil drop remains same or stable.

TABLE :- DROP COLLAPSE TEST RESULTS

Isolates IS1 IS2 IS4 IS6 IS7
Oil collapse + + + + -
time 80sec 40sec 30sec 95sec
Fig, 8 DROP COLLAPSE METHOD AT START Fig 9. AFTER 30SEC

Fig 10. AFTER 2 MIN

In Emulsification assay, three isolates (IS2 and IS4) exhibited more than 30% of
emulsification with engine oil and IS7 showed 37% emulsification with petroleum. Highest
emulsification index(EI) was shown by IS4. Higher the emulsification activity, higher is the
emulsification index.

TABLE 3:- EMULSIFICATION INDEX ASSAY

ISOLATE IS1 IS2 IS4 IS6 IS7
EI 33.1% 26.31% 68.42% 19.35%26. 37%
Fig 11. EMULSIFICATION ASSAY

In Blue agar, three isolates (IS1, IS2 and IS4) were positive for this test. Three halo zones or
ring were seen around the wells. Three potential isolates IS1, IS2 and IS3 which were found.
It method for detection of some glycolipids and anionic surfactants. Cetyl-trimethyl
ammonium bromide contains the cationic surfactant. In positive result case i.e anionic
biosurfactant production, the cationic surfactant reacts with biosurfactant and forms dark blue
coloured compound. Thus, wells get surrounded by dark blue halo-zones.

Table 4:- BLUE AGAR TEST RESULTS

BLUE AGAR IS1 IS2 IS4 IS4 IS7
TEST
+ + + - -

Fig 12. RING AROUND WELL IN BLUE AGAR METHOD

Biosurfactant produced by the isolate IS1, IS2 and IS4 were purified by acetone precipitation.
Pellet was obtained after centrifugation which served as purified biosurfactant.

Fig 13. BIOSURFACTANT PURIFICATION BY CHILLED ACETONE PRECIPITATION

MORPHOLGY AND BIOCHEMICHAL CHARACTERIZATION

Three potential isolates which exhibited positive results in most tests were selected for
observing colony morphology and biochemical characterization to identify the genus of the
isolates.
COLONY MORPHOLGY
ISOLATES IS1 IS2 IS4
SIZE Small (1mm) Small (1-2mm) Small (2-3mm)
SHAPE Circular Circular Round
MARGIN Entire Entire Entire
ELEVATION Convex Convex Slightly convex
COLOUR Golden-yellow Yellow Creamish-white
TEXTURE Smooth Smooth Smooth
OPACITY Opaque Opaque Translucent
BIOCHEMICAL TESTS

ISOLATES IS1 IS2 IS4

GRAM STAIN Gram positive Gram positive Gram negative rod

cocci cocci
MOTILITY Non-motile Non-motile Motile
CATALASE + + +
OXIDASE - - -
IMViC --++ -- ++ --++
ENDOSPORE - - +
CARBOHYDRATE
FERMENTATION
Glucose + + +
Sucrose + + +
Maltose + + -
Lactose + + +
GELATIN
LIQUEFACTION - - +

TRIPLE SUGAR IRON

TEST A/A A/A K/A

IS1 and IS2 were tentatively identified as Staphylococcus and IS4 is expected to be Bacillus

Fig 13. GRAM POSITIVE COCCI IN CLUSTERS

Fig 14. GRAM POSITIVE ROD

Fig15.INDOLE NEGATIVE Fig 16. MR NEGATIVE Fig 17. VP POSITIVE

CONCLUSION
This study aimed to screen and isolate different biosurfactant producing bacteria from oil
contaminated soil which can be produced at commercial level to rule out disadvantages faced
by chemical surfactant use. The result showed that Air Force Station Tambaram soil
contaminated with used aviation oil and engine oil (thrown in soil after use) has become
inhabitant of various biosurfactant producing microbes. Both gram positive cocci
Staphylococcus and gram positive rod Bacillus are isolated from the soil sample producing
biosurfactant.
Test with engine oil has given better results than petroleum like highest emulsification index,
positive blue agar results etc, the reason might be that the soil sample is regularly enriched
with engine oil and aviation oil as these oil thrown out in soil every day, therefore the
microbes are inhabited more to the engine oil than petroleum. Also petroleum composition
and and hydrocarbon amount is different than engine oil adding, therefore isolates may not
degrade that.
Bacillus(IS4) had given best results out of all the isolates, therefore can be optimized for
mass scale production of biosurfactant.
This research is important as although many microbes are identified for biosurfactant
production, but most of them which are identified belongs to pathogenic strains or species
like Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Rhodococcus etc ,
these pathogenic strains and species can be used for commercial mass production of
biosurfactants, but during production they have high chances of transfer of toxins, pathogenic
products and other pathogenic effects to biosurfactants and therefore causing harm to humans
and other living life. Therefore researches are going on to identify non-pathogenic
microorganism which can be used for mass production.

REFRENCES

*a - RESEARCH JOURNAL OF PHARMACY AND TECHNOLOGY

TITLE: Isolation, Screening and partial identification of Indigenous Biosurfactant Bacteria
from Oil Contaminated Soil
AUTHOR: Ammu Augustine, Pavana Prathap, Neethu Kamarudheen, K.V Bhasker Rao

*b - International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 7 Number 08 (2018)
TITLE: Review on Biosurfactant Production and its Application
AUTHOR: Priyam Vandana and Dinesh Singh
*c- RESEARCH PAPER
TITLE: Types, Production and Applications of Biosurfactants
AUTHORS: Catherine Mulligan and Bernard Gibbs(2002)

RESEARCH ARTICLE www.sciencedirect.com

TITLE: Isolation and characterization of hydrocarbon degrading
bacterial isolate from oil contaminated sites
AUTHORS: Geetha S.J., Sanket J. Joshia and Shailesh Kathrotiyab

SCIENCEDIRECT
TITLE: Isolation of biosurfactant producing bacteria from petroleum contaminated terrestrial
samples that collected in Bangkok, Thailand
AUTHORS: Tanakwan Budsabuna

Madame Curie Bioscience Databases

TITLE: Screening Concepts for the Isolation of Biosurfactant Producing Microorganisms
AUTHOR: Vanessa Walter, Christoph Syldatk, and Rudolf Hausmann.

Bioinformation. 2018 , PMCID: PMC6137570PMID: 30237676

TITLE: Screening, isolation and characterization of biosurfactant producing Bacillus
subtilis strain ANSKLAB03
AUTHORS: Anuraj Nayarisseri, and Sanjeev Kumar Singh.

ELSEVIER
TITLE : Isolation and characterization of hydrocarbon degrading
bacterial isolate from oil contaminated sites
AUTHORS : Geetha S.J, Sanket J Joshia and Shailesh Kathrotiyab

JOURNLAL OF APPLIED MICROBIOLOGY

TITLE : Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-
selected bacteria
S. Mnif, M. Chamkha, M. Labat and S. Sayadi
Laboratoire des Bioproce´de´ s Environnementaux, Poˆ le d’Excellence Re´ gional AUF

Brazilian Journal of Microbiology (2004) ISSN 1517-8382 81

TITLE : Selection of microorganisms for biosurfactant production using agroindustrial
wastes
AUTHORS : Marcia Nitschke, Cristina Ferraz, Gláucia Pastore
STUDENT NAMES :-
Harpreet Kaur – 19MSM0010
Kinjal Saha – 19MSM0005
S Vaishnavi – 19MSB0029

GUIDE NAME :- Dr K.V. Bhaskara Rao