Set Report
Set Report
Set Report
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.
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.
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.
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
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.
ISOLATES IS1 I2 IS4 IS6 IS7
TABLE 1:- BLOOD AGAR HEMOLYSIS RESULTS
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.
Isolates IS1 IS2 IS4 IS6 IS7
TABLE :- DROP COLLAPSE
Oil collapse + TEST+RESULTS + + -
time 80sec 40sec 30sec 95sec
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.
ISOLATE IS1 IS2 IS4 IS6 IS7
EI 3:- EMULSIFICATION
TABLE 33.1% 26.31%
INDEX ASSAY68.42% 19.35%26. 37%
BIOCHEMICAL TESTS
ISOLATES IS1 IS2 IS4
GELATIN
LIQUEFACTION - - +
IS1 and IS2 were tentatively identified as Staphylococcus and IS4 is 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. 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
SCIENCEDIRECT
TITLE: Isolation of biosurfactant producing bacteria from petroleum contaminated terrestrial
samples that collected in Bangkok, Thailand
AUTHORS: Tanakwan Budsabuna
ELSEVIER
TITLE : Isolation and characterization of hydrocarbon degrading
bacterial isolate from oil contaminated sites
AUTHORS : Geetha S.J, Sanket J Joshia and Shailesh Kathrotiyab