ISOLATION, EXTRACTION AND PURIFICATION OF

BIOSURFACTANTS PRODUCING BACTERIA FROM

OIL CONTAMINATED SOIL

Department of Applied Microbiology and Biotechnology, SBST, VIT

University, Vellore, India
SET ID : 190655

GUIDE NAME : DR K.V. BHASKARA RAO

Harpreet Kaur – 19MSM0010
Kinjal Saha – 19MSM0005
S Vaishnavi – 19MSB0029 1
OBJECTIVES
• To isolate and extract biosurfactants producing bacteria from oil
contaminated soil
• To characterize the isolated bacterial strains
• To partially purify biosurfactants produced by bacteria

• INTRODUCTION
• Bio-surfactants are amphiphilic metabolites produced by
microorganisms on their cell surface
• Contain both hydrophobic and hydrophilic regions causing them to
aggregate at interfaces between fluids with different polarities such
as hydrocarbons and water. 2
• Predominantly produced by bacteria, yeast and fungi and are usually associated
with bacteria growing on water-immiscible substrates, using them as a source
of food.
• Due to their advantages over synthetic surfactants (biodegradability and
biocompatibility) they are being used as potential candidates for the
bioremediation of contaminated soils
• 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 and Fatty acids
• Various applications of biosurfactants are being used with several advantages in
laundry cleaning, food processing, cosmetic industry, petroleum industry,
microbe enhanced oil recovery, agriculture and medicine
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MATERIALS AND METHODS
COLLECTION OF SAMPLES

PREPARATION OF MEDIA AND ISOLATION OF

COLONIES

PRIMARY SCREENING
Blood hemolysis test

SECONDARY SCREENING
Production of bio surfactant

Drop collapse method

Blue agar plate method

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 Emulsification assay

Blue agar test

PURIFICATION OF BIOSURFACTANT

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 COLLECTION OF SAMPLES:
• Soil samples collected from Air Force Station, Tambaram, Chennai
• Samples were contaminated with oil (engine and aviation oil)

PREPARATION OF MEDIA AND ISOLATION OF COLONIES:

• Sample were inoculated in oil enriched nutrient broth (1% oil) for 48 hours
• 1ml of this broth was serially diluted with 0.85% sterile saline
• Using 3, 4 and 5 dilutions, isolation was carried by two method:
A) Hydrocarbon overlay method
B) Oil enriched nutrient medium method

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OIL ENRICHED SEED CULTURE AND POURING PLATES
OIL OVERLAY METHOD
PRIMARY SCREENING:-
Blood hemolysis test:
• Isolates were inoculated on freshly prepared blood agar media (2-3%)
• Incubated for 24-48 hours at 37⁰C.
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.
• Incubated for 5 days at 120rpm on shaker at 28⁰C
• Centrifuged at 4⁰C at 8000rpm for 20 minutes
• The cell free supernatant taken for further biosurfactant studies
 
Drop collapse method :
• petri-plate was coated with an oil drop 7

• then cell free supernatant was added on it and observed after one minute.
Blue agar plate method:
• for detecting anionic biosurfactants
• Blue agar was made by Minimal salt medium supplemented with methylene
blue(0.2mg/mL) and cetyl-trimethyl ammonium bromide(0.5mg/mL)
• 30µl of the supernatant was loaded into the wells
• A negative control was maintained with distilled water
• 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:
• 3ml of cell free suspension+ 2ml of engine oil- vortexed and kept overnight
• After 24 hours the emulsion index (EI) was calculated
EI = Height of emulsion layer/ Total height* 100

 Biochemical characterization: 8

• Potential isolates were partially characterized by performing standard

PURIFICATION OF BIOSURFACTANT

• one volume of extracted biosurfactant + three volumes of acetone

• The tubes were kept undisturbed for 10 hours at 4 ⁰C
• centrifuged for 20 minutes at 11,000 rpm and pellet was collected
• Pellet was the purified biosurfactant which can be then dried to remove residual
acetone by evaporation

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RESULTS AND DISCUSSION
PRIMARY SCREENING:-
 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

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 ENGINE OIL
OVERLAID
PLATES WITH
HALOZONES
AROUND
COLONIES

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QUADRANT STREAKING
Pure culture of isolates were made by sub culturing it three -
four times by quadrant streaking

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 Blood Agar Hemolysis Test
ISOLATES IS1 IS2 IS4 IS6 IS7
HEMOLYSI β- β- β- β- β-
S hemolysis hemolysis hemolysis hemolysis hemolysis

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  Drop Collapse Method

ISOLATE IS1 IS2 IS4 IS6 IS7

OIL COLLAPSE + + + + -
TIME 80 Sec 40Sec 30 Sec 95 Sec

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 Emulsification assay
ISOLATE IS1 IS2 IS4 IS6 IS7
EI 33.1% 26.31% 68.42% 93.35% -

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 Blue Agar Test
IS2
ISOLATE IS1 IS4 IS6 IS7
BLUE AGAR TEST + + + - -

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

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COLONY MORPHOLGY
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

ISOLATE IS1 IS2 IS4

SIZE Small (1mm) Small (1-2mm) Small (2-3mm)
SHAPE Circular Circular Circular
MARGIN Entire Entire Entire
ELEVATION Convex Convex Slightly Convex
COLOUR Golden-Yellow Yellow Creamish white
TEXTURE Smooth Smooth Smooth
OPACITY Opaque Opaque Translucent

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ISOLATE IS1 IS2 IS4
GRAM STAINING Gram positive Gram positive Gram positive
cocci cocci bacillus
MOTILITY Non-motile Non-motile Motile
CATALASE + + +
OXIDASE - - -
ENDOSPORE STAINING - - +
IMViC --++ --++ --++
CARBOHYDRATE
FERMENTATION
Glucose + + +
Sucrose + + +
Maltose + + -
Lactose + + +

GELATIN LIQUEFACTION - - +
TRIPLE SUGAR IRON A/A A/A A/K
TEST
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GRAM POSITIVE COCCI

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GRAM POSITIVE BACILLUS

 INDOLE NEGATIVE MR NEGATIVE VP POSITIVE

IS1 and IS2 were tentatively identified as Staphylococcus and IS4 is

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expected to be Bacillus
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
• Both gram positive cocci Staphylococcus and gram positive rod
Bacillus are isolated from the soil
• Test with engine oil has given better results than petroleum 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 and also petroleum composition is different
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• Bacillus(IS4) had given best results out of all the isolates, therefore
can be optimized for mass scale production of biosurfactant
• 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, during
production they have high chances of transfer of toxins, pathogenic
products and other pathogenic effects to biosurfactants
• Researches are going on to identify non-pathogenic microorganism
which can be used for mass production

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REFERENCES
• *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
TITLE: Review on
Biosurfactant Production and its Applicatio AUTHOR: Priyam Vandana
and Dinesh Singh
• *c- RESEARCH PAPER
TITLE: Types, Production and Applications of Biosurfactants
AUTHORS: Catherine Mulligan and Bernard Gibbs(2002)
 
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• 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
25
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
26
Nitschke, Cristina Ferraz, Gláucia Pastore 
Submission date: 23-Oct-2019
02:48PM (UTC+0530)

Submission ID: 1198653498

File name: SET_REPORT_1_-

converted_1.pdf (1.17M)

Word count: 2532

Character count: 14905

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THANK
YOU
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