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Assessment of different screening methods for selecting biosurfactant producing marine bacteria

Authors:
  • Dr. Babasaheb Ambedkar Marathwada University, Aurangabad - 431004, Maharashtra, India

Abstract and Figures

Different screening methods namely, hemolytic assay (HA), modified drop collapse (MDC), tilted glass slide (TGST), oil spread method (OSM), blue agar plate (BAP), hydrocarbon overlaid agar (HOA) plate, emulsification index (EI), emulsification assay (EA) were assessed for their efficiency to detect biosurfactant producing marine bacteria. Forty-five strains of bacteria, comprising 18 Acinetobacter and 27 other bacteria along with positive MTCC reference strains were examined. HA, MDC, TGST efficiently detected 15, 17 and 14 biosurfactant producers respectively. Five hemolytic cultures did not show any biosurfactant production in MDC, TGST, and/or OSM. The emulsification of kerosene was also poorer. These results suggest that HA is not totally reliable. Six bacterial isolates produced biosurfactant in OSM, and MDC as well as TGST. MDC and TGS tests demonstrated good activity for nine isolates and proved to be the essential methods. None of the bacteria produced glycolipid on BAP. Cultures showing >30% of emulsification with kerosene were found to be positive in at least one of the above mentioned screening methods. The reference strains, Gram negative bacterium MM73b produced 68% the highest emulsification and demonstrated biosurfactant production in modified drop collapse, tilted glass slide test with highest emulsification units of 213.8 (EU/ml) for petrol. In case of xylene, Acinetobacter spp. MM74 demonstrated 187.5, Acinetobacter spp. WB42 demonstrated 170.4 emulsification units. HOA plate identified 31 and 22 bacteria for diesel and crude oil degradation respectively. Thus, this method proved to be significant one. We suggest that single method is not suitable to identify all type of biosurfactants, and recommend that drop collapse, tilted glass slide test, oil spread method followed by emulsification assay are more suitable for primary screening.
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Indian Journal of Marine Sciences
Vol. 37(3), September 2008, pp. 243-250
Assessment of different screening methods for selecting biosurfactant
producing marine bacteria
S K Satpute1, B D Bhawsar1, P K Dhakephalkar2 & B A Chopade1*
1Department of Microbiology, *Institute of Bioinformatics and Biotechnology,
University of Pune 411 007, Maharashtra, India
2 Microbial Science Division, Agharkar Research Institute, Pune 411 004, Maharashtra, India
*[E-mail: chopade@unipune.ernet.in]
Received 22 August 2006, revised 11 February 2008
Different screening methods namely, hemolytic assay (HA), modified drop collapse (MDC), tilted glass slide (TGST),
oil spread method (OSM), blue agar plate (BAP), hydrocarbon overlaid agar (HOA) plate, emulsification index (EI),
emulsification assay (EA) were assessed for their efficiency to detect biosurfactant producing marine bacteria. Forty-five
strains of bacteria, comprising 18 Acinetobacter and 27 other bacteria along with positive MTCC reference strains were
examined. HA, MDC, TGST efficiently detected 15, 17 and 14 biosurfactant producers respectively. Five hemolytic cultures
did not show any biosurfactant production in MDC, TGST, and/or OSM. The emulsification of kerosene was also poorer.
These results suggest that HA is not totally reliable. Six bacterial isolates produced biosurfactant in OSM, and MDC as well
as TGST. MDC and TGS tests demonstrated good activity for nine isolates and proved to be the essential methods. None of
the bacteria produced glycolipid on BAP. Cultures showing >30% of emulsification with kerosene were found to be positive
in at least one of the above mentioned screening methods. The reference strains, Gram negative bacterium MM73b produced
68% the highest emulsification and demonstrated biosurfactant production in modified drop collapse, tilted glass slide test
with highest emulsification units of 213.8 (EU/ml) for petrol. In case of xylene, Acinetobacter spp. MM74 demonstrated
187.5, Acinetobacter spp. WB42 demonstrated 170.4 emulsification units. HOA plate identified 31 and 22 bacteria for diesel
and crude oil degradation respectively. Thus, this method proved to be significant one. We suggest that single method is not
suitable to identify all type of biosurfactants, and recommend that drop collapse, tilted glass slide test, oil spread method
followed by emulsification assay are more suitable for primary screening.
[Keywords: Acinetobacter, biosurfactants, bioemulsifiers, hydrocarbon, screening methods, bacteria, microbial cultures]
Introduction
Oil spills, effluents of petrochemical industries,
refineries and their hazardous substances lead to
pollution of marine ecosystem1. The hydrocarbon
moieties from such spills persist for long time in the
marine ecosystem. Under such conditions, many
marine microorganisms produce surface active
substances like biosurfactants/bioemulsifiers to
solubilize and assimilate hydrophobic compounds2.
These amphiphilic moieties bind to water insoluble
hydrocarbons3. Thus, due to the constant exposure,
marine microorganisms develop biodegrading
machinery and degrade various toxic compounds4.
Realizing such activity has led to number of screening
methods such as hemolysis of erythrocytes5, oil
spread6, modified drop collapse7, tilted glass slide8,
blue agar plate method9, emulsification index10,
emulsification assay11-12 and hydrocarbon degradation
on agar plates13 for detection of biosurfactant/
bioemulsifier producers.
Although different screening methods are
available, it is however, difficult to detect the types of
biosurfactant/bioemulsifier produced by the microbes
using a single method owing to the chemical and
functional properties. In view of this, it appears that
several screening methods are needed to understand
the ability of a single hydrocarbonclastic microbe in
producing biosurfactant. Hence, for efficient detection
of potential biosurfactant producers, combination of
various screening methods are required which was
evaluated during this study.
Materials and Methods
Seawater, sediment, shells samples were collected
at high and low tides from eastern (West Bengal off
Fraserganj), western (off Mumbai, Shivree) and
__________
*For correspondence:
Tel: (+91) 020 25690442
Fax: (+91) 020 25690087
INDIAN J. MAR. SCI., VOL. 37, NO. 3, SEPTEMBER 2008
244
southern (off Chennai and Calicut) Indian coasts
during October 2003 to September 2004. In addition
to this, samples were also procured from Iran,
Caspian Sea (Anzali Port). Samples were enriched in
500 ml Erlenmeyer flasks containing 100 ml of
Baumann’s14 medium with 3.5% NaCl (w/v),
incubated under 200 rpm at 28OC up to one week.
After observation of turbidity in medium, samples
were plated on various media viz., Zobell marine
medium (ZMM, Himedia), Acinetobacter minimal
medium (AMM)15, cysteine lactose electrolyte
deficient (CLED Himedia) and Holton’s media. These
media were prepared in artificial seawater (ASW)
containing NaCl, 35.0; NH4NO3, 4.0; KH2PO4, 6.0;
MgSO4.7H2O, 0.2; CaCl2, 0.01; FeSO4.7H2O, 0.01
and also natural sea water (NSW collected from
Mumbai coastal area). Plating of enriched samples
was done at regular interval of 24 hrs up to four to
five days in order not to miss slow growers. About
112 bacterial isolates were obtained and maintained
on ZMM at 4oC until taken up for further studies.
Among the 112 isolates 45 isolates were non-motile,
encapsulated coccobacilli that were oxidase negative,
catalase positive were assigned to genus
Acinetobacter. Identification of genus Acinetobacter
was confirmed by chromosomal DNA trans-
formation assay15. About 18 such confirmed strains
along with 20 Gram negative and seven Gram
positive marine isolates were selected for further
work.
Biosurfactant producing reference cultures viz.,
Bacillus subtilis MTCC1427, Bacillus pumilus
MTCC1456, Bacillus subtilis MTCC2422, Bacillus
subtilis MTCC2423; Bacillus sphaericus MTCC2473
and Pseudomonas aeruginosa MTCC2297 were also
included in this study. These cultures were procured
from Microbial Type Culture Collection (MTCC),
Chandigarh, India. Cultures were maintained on
nutrient agar (NA) containing (g/L) beef extract: 1.0,
yeast extract: 2.0, peptone: 5.0, NaCl: 5.0, agar: 15,
pH 7.2. Cultures were grown aerobically at respective
temperatures suggested by the supplier.
The following screening assays were carried out
for detecting biosurfactant production by
Acinetobacter spp. and other marine bacteria. ZMM
and Zobell marine broth (ZMB), nutrient agar and
minimal medium with 3.5% NaCl (w/v) were used as
the basal media. Biosurfactant production by both
marine isolates and reference cultures from MTCC
was examined.
Hemolytic activity (HA)
Bacterial cultures were streaked on ZMM
supplemented with 5% fresh human blood5 and
incubated at 28oC for 48-72 hrs. Observation was
made for , β and ϒ hemolysis. Hemolytic
activity was correlated with the production of
biosurfactant5.
Oil spread method (OSM)
Twenty-four hour old inoculum grown in ZMB
was used. Petriplate base was filled with 50 ml of
distilled water. On this water, twenty microlitre of
crude oil was layered uniformly. Further, ten
microlitre of culture was added at different spots on
the crude oil which is coated on water surface.
Occurrence of clear zone was an indication of a
biosurfactant producer6.
Modified drop collapse (MDC) method
Wells of microtitre plate were thinly coated
with Pennzoil ZDX (SAE 20W-40, India). The
microtitre plate was left undisturbed for
15 minutes for forming the uniform thin coating
of Pennzoil in the well. Five microlitre bacterial
culture grown in ZMB at 28oC under 200 rpm for 24
hrs were added individually to the centre of the well.
The biosurfactant producers were detected from the
drop collapsing within a minute from the oil
coated well7.
Tilted glass slide (TGS) test
A colony each of marine bacterial cultures was
grown for 24 hrs on ZMA mixed with a drop of 0.9%
NaCl at one end of the glass slide which was not
coated with any oil. The slide was tilted and the
biosurfactant production confirmed when the drop
began dipping down8.
Blue agar plate (BAP) method
Anionic biosurfactant, specifically rhamnolipids
were detected by this technique. Mineral salts
agar medium (MSA)9 supplemented with carbon
sources (2%) and cetyltrimethylammonium
bromide (CTAB: 0.5 mg/ml)-methylene blue
(MB: 0.2 mg/ml) were prepared. Carbon sources
tested were mannitol, glycerol, sodium citrate,
sodium acetate, peptone and glucose. For marine
bacteria 3.5% (w/v) NaCl was added in the above
MSA preparation. A dark blue halo around the culture
was considered as positive for biosurfactant
production.
SATPUTE et al.: SCREENING METHODS FOR BACTERIA
245
Emulsification index (EI)
Emulsification activity was measured by vortexing
1 ml of culture supernatant grown in ZMB at 28oC for
24 hrs. Further, 4 ml of water and 6 ml of kerosene
for 2 minutes to obtain maximum emulsification.
After 48 hrs emulsification index10 was calculated by
measurement of the height of the emulsion layer (a),
divided by the total height (b), multiplied by 100
(EI = a/b × 100). This assay was performed in same
size glass test tubes.
Hydrocarbon overlay agar (HOA) method
ZMA plates were coated individually with 40
microlitre of kerosene, hexadecane, benzene, toluene,
diesel or crude oil. Pure bacterial isolates were spotted
on these coated plates. Plates were incubated for 7-10
days at 28oC. Colony surrounded by an emulsified
halo was considered positive for biosurfactant
production13. As petrol and xylene were evaporating
very fast from the plates, their emulsification was
checked out by an assay described by Patil &
Chopade11.
Emulsification assay (EA)
Culture inocula were prepared by growing them
overnight in 10 ml ZMB in 100 ml Erlenmeyer flasks
at 28oC and 200 rpm. Cultures were centrifuged at
10,000 rpm for 15 min at room temperature. Three
mililitre of supernatant was mixed with 0.5 ml of
petrol. This mixture was mixed vigorously for 2 min.
This mixture was left undisturbed for one hour at
28oC to separate aqueous and oil phase. Aqueous
phase was removed carefully with the help of 1 ml
micropipette and absorption was measured. Same
procedure was followed for xylene also. Uninoculated
broth was taken as a blank. Absorbance of aqueous
phase was measured by using spectrophotometer
(UV1601 Shimadzu Corporation, Japan) at
wavelength of 400 nm. Emulsification activity per ml
(EU/ml) was calculated11 by using the formula, 1
Emulsification unit = 0.01 × Dilution factor.
Results
All 45 strains comprising Acinetobacter and other
marine bacterial isolates were examined for a variety
of biosurfactant producing and hydrocarbonclastic
activities. Results from various screening protocols
are listed in Tables 1 and Table 2. Fifteen isolates,
comprising six of Acinetobacter spp. (Table 1) and
nine others (Table 2) were positive for biosurfactant
production in the hemolytic assay. Among 15
hemolytic isolates, 12 showed α hemolytic activity.
Five hemolytic cultures did not show biosurfactant
production in the remaining seven screening methods.
Low emulsification activity was also observed in
these cultures suggesting that the assay is not totally
reliable. Results from other tests including OSM,
DCM, TGST, BAP are also shown in Table 1 and
Table 2. The MDC demonstrated eight Acinetobacter
spp. and nine other bacteria as biosurfactant
producers. Only six isolates showed biosurfactant
activity in the OSM. Same cultures were positive in
MDC and TGST. The MDC and TGST were good
equally indicating biosurfactant production by nine
isolates. The TGST detected four Acinetobacter spp.
and ten other bacterial isolates as biosurfactant
producers. From the reference cultures, Bacillus spp.
did not show hemolytic activity. However,
biosurfactant producing activity of these strains could
be confirmed by MDC, OSM and TGST.
Pseudomonas aeruginosa MTCC 2297, on contrary,
demonstrated α hemolytic activity, positive for MDC
and BAP method. However, it showed negative test in
OSM and TGST. Thus the hemolytic activity must be
considered as an unreliable criterion for the detection
of biosurfactant activity of a bacterial culture.
Comparatively, MDC, OSM and TGST are consistent.
The BAP method is a highly special technique for
detection of glycolipid producing microorganisms.
Glycolipid production was not detected in any of the
tested strains in BAP method. High EI were observed
for Acinetobacter spp. MM74 (Table 1) and other
bacterial isolates was WB64 and MM3b7 (Table 2).
The culture showing good emulsification activity
(>30%) was seen to be positive in at least one of the
above mentioned screening methods. The lowest
emulsification activity (5%) was observed in MSS96.
Most of the isolates demonstrated 20-35% of
emulsification.
The HOA plate method identified hydrocarbon-
clastic bacteria efficiently. Detailed qualitative
assessment for biosurfactant/bioemulsifier for all
bacteria studied are given in Tables 3 and 4. Diesel
and crude oil were respectively utilized by 31 and 22
marine isolates. This number is significant as
compared with other hydrocarbons. Kerosene was
utilized by four Acinetobacter spp. and four Gram
negative bacteria. Three species of Acinetobacter and
two others degraded hexadecane. More number of
reference cultures than marine isolates utilized
INDIAN J. MAR. SCI., VOL. 37, NO. 3, SEPTEMBER 2008
246
hexadecane. Only a few isolates could utilize benzene
and toluene. Among the reference strains only
Pseudomonas aeruginosa MTCC 2297 degraded
benzene. Whereas Bacillus spp. MTCC 2422 and
MTCC 2423 utilized toluene and diesel. The EA
identified maximum numbers of isolates that
emulsifies xylene and petrol efficiently. The
emulsification units of 213.8 (EU/ml), the highest, by
MM73b. For xylene, the EU of 187.5 by was
followed by WB42 (170.4 EU/ml). Bacillus Subtilis
MTCC 2422 produced high emulsification of 400.0
and 291.4 EU/ml for xylene and petrol respectively.
Pseudomonas aeruginosa MTCC 2297 utilized all six
tested hydrocarbons however, showed least
emulsification units for petrol and xylene.
Discussion
Biosurfactants/bioemulsifiers play a key role in
emulsifying hydrocarbons. Biosurfactants and
bioemulsifiers are thought to be very suitable
alternatives to chemical surfactants due to their
properties like eco-friendly, less/no toxicity,
biodegradability, high specificity, selectivity at
temperature, pH, salinity and synthesis from cheaper
renewable substrates16. The functional properties such
as emulsification, wetting, foaming, cleansing, phase
separation, surface activity, and reduction in viscosity
of crude oil for transportation17 are interesting.
Therefore, search of biosurfactant producing
microorganisms is an important area of research is
particular for bioremediation.
In this study, eight different screening methods
were assessed for selecting biosurfactant producing
marine bacteria. Through confirmation of hemolytic
activity is a commonly preferred method to screen
biosurfactant producing culture, it was seen in this
study that it is not a very useful test for marine
bacterial cultures. Further same reference cultures
negative for hemolytic activity did show biosurfactant
production in MDC, OSM and TGST. Similar
observations were true with Acinetobacter spp.
MM74 and Gram negative bacteria viz., MM73b and
Table 1 — Biosurfactant production by marine Acinetobacter spp.
Organism Hemolytic
activity
Modified
drop collapse
Oil
spread
Tilted glass
slide
Blue agar
plate*
Emulsification#
(%)
Acinetobacter spp.
CS 1 Nil Nil + Nil Nil 20
CS 3 Nil Nil Nil Nil 29
CS 5 + Nil Nil Nil Nil
CS 6 Nil Nil + + Nil 32
CS 9 Nil + Nil Nil Nil 20
CS 11 + Nil Nil Nil Nil
CS 20 β + ++ Nil Nil 25
CS 23 Nil Nil Nil Nil Nil 39
WB 40 β Nil Nil Nil Nil 44
WB 42 Nil ++ Nil + Nil Nil
WB 45 Nil + Nil + Nil Nil
WB 55 Nil + Nil + Nil Nil
MM 74 Nil + Nil Nil Nil 68
MSS 96 Nil Nil Nil Nil 5
Reference strains positive for biosurfactant production
Bacillus subtilis MTCC1427 Nil Nil + + Nil 40
Bacillus pumilus MTCC 1456 Nil Nil Nil + Nil 24
Bacillus subtilis MTCC 2422 Nil ++ ++ + Nil 75
Bacillus subtilis MTCC 2423 Nil ++ ++ + Nil 78
Bacillus sphaericus MTCC 2473 Nil ++ ++ Nil Nil 36
Pseudomonas aeruginosa MTCC 2297 + Nil Nil + 42
CS = Caspian Sea; WB = West Bengal; MM = Mumbai mussel; MSS = Mumbai, Shivree sediment; = Reduction of hemoglobin to met
= hemoglobin (medium becomes greenish); β = Lysis of RBC, medium around the colony becomes colourless; Nil = Negative test;
+ = Positive test; ++ = Good activity; * = CTAB, blue agar plates = Marine bacteria were checked with and without NaCl (3.5% w/v);
# = Values are the mean of three readings. Emulsification >30% is indicated in bold to denote high emulsification activity.
SATPUTE et al.: SCREENING METHODS FOR BACTERIA
247
WB64. As also noticed by other investigators18-20.
Confirmation of biosurfactant production through
other screening methods becomes essential to select
potent biosurfactant producers as proven in this study.
However, none of the tested marine bacteria produced
glycolipids.
Emulsification activity is one of the criteria to
support the selection of potential biosurfactant
producers. Emulsifying activities (E24) determine
productivity of bioemulsifier21. Ellaiah et al.10
screened 68 bacterial isolates from soil and found
only 6% of isolates with good emulsification activity
of up to 61%. During this study, emulsification of
kerosene by MM73b and MM74 was up to 68%. This
observation is important to suggest that potent
biosurfactant producing cultures can be detected
through such assays. The cultures showing >30%
emulsification activity were also positive for
biosurfactant production in two or three other
methods. It is also possible to detect biosurfactant
producing and hydrocarbon degrading activity
simultaneously on agar plate by overlaying with
hydrocarbon12. Maximum number of isolates positive
for kerosene, hexadecane, benzene, toluene and crude
oil degradation were also positive for diesel
utilization. Measurement of emulsification units help
to choose the carbon and energy source for
biosurfactant/bioemulsifier production. Patil and
Table 2 — Biosurfactant production by marine Gram negative and Gram positive bacterial isolates
Organism Hemolytic
activity
Modified
drop collapse
Oil
spread
Tilted
glass slide
Blue agar
plate*
Emulsification#
(%)
Gram negative bacteria
CS 2 Nil Nil Nil Nil Nil 27
CS 7 Nil Nil Nil Nil Nil 20
CS 13 Nil + + Nil Nil 20
WB 64 Nil + Nil + Nil 57
WB 68 Nil Nil Nil + Nil 34
MM 76 Nil Nil + Nil Nil
WB 69 β Nil Nil Nil Nil 40
MSS 86 Nil Nil Nil Nil 37
MSS 89 Nil Nil Nil Nil 19
IW 106 Nil Nil + Nil Nil
IW 108 Nil + Nil + Nil Nil
IW 112 Nil Nil Nil Nil Nil Nil
CS 2a Nil Nil Nil Nil Nil Nil
WB 28a + + + Nil Nil
WB 28b Nil Nil + Nil Nil
CS 3b + Nil Nil Nil 18
WB 68a Nil Nil + Nil Nil 42
WB 68b Nil Nil Nil Nil Nil Nil
MM 73b Nil + Nil + Nil 68
Gram positive bacteria
WB 41 Nil + Nil + Nil Nil
WB 63 Nil Nil Nil Nil Nil Nil
MG 77 ++ Nil + Nil Nil
MS 85 Nil + Nil Nil Nil 50
MSS 90 Nil Nil Nil Nil Nil 22
IW = Iran water; IOW = Iran oily water; Emulsification >30% is indicated in bold to denote high emulsification activity.
Other abbreviations as defined in Table 1 legend.
INDIAN J. MAR. SCI., VOL. 37, NO. 3, SEPTEMBER 2008
248
Chopade11 introduced emulsification assay based on
emulsification units of the tested oils. They selected
Acinetobacter junii SC14 for bioemulsifier
production. Thus, by examining emulsification units,
it is possible to select a potent biosurfactant/
bioemulsifier producer. We recommend these assays
as one of the important and effective assay for
screening the biosurfactant/bioemulsifier producers.
It is important to note that most of the researchers
have used maximum two or three screening methods
for selection of biosurfactant producers19. We suggest
a single method is not suitable to identify all type of
biosurfactants. Therefore, a combination of various
methods is required for effective screening. To the
best of our knowledge, this is the first report assessing
eight different screening methods for selecting
biosurfactant producing marine bacteria. In
conclusion, we recommend that DCM, TGST, OSM
followed by EA are more suitable for primary
screening.
Table 3 — Qualitative assessment of biosurfactant/bioemulsifier production by marine Acinetobacter spp.
Hydrocarbon overlaid agar plate method# Emulsification activity* (EU/ml) Marine isolates
Kerosene
Hexadecane
Benzene
Toluene Diesel Crude oil Petrol Xylene
Acinetobacter spp.
CS 1 Nil Nil Nil Nil ++ Nil 102.4 109.4
CS 3 + Nil Nil Nil ++ Nil 58.1 79.2
CS 5 Nil Nil Nil Nil + Nil 52.5 138.4
CS 6 Nil Nil Nil Nil +++ Nil 88.0 64.2
CS 9 Nil +++ Nil Nil Nil +++ 77.1 15.0
CS 11 Nil Nil Nil Nil Nil ++ 62.4 90.6
CS 12 Nil Nil Nil + Nil ++ 62.6 79.3
CS 15 Nil Nil Nil Nil ++ +++ 146.7 90.6
CS 20 Nil Nil Nil + + Nil 75.9 134.6
CS 23 Nil Nil Nil + ++ ++ 102.7 108.6
WB 40 ++ ++++ Nil Nil ++ +++ 67.3 102.6
WB 42 Nil +++ Nil Nil + +++ 37.0 170.4
WB 45 Nil Nil Nil Nil Nil +++ 21.9 148.7
WB 55 Nil Nil Nil Nil Nil Nil 73.0 90.3
MM 74 Nil Nil Nil Nil ++ Nil 168.1 187.5
MSS 96 Nil Nil Nil Nil +++ Nil 2.5 28.0
CS 2b + Nil Nil Nil ++ +++ 38.8 20.3
MM 73a ++ Nil Nil Nil + Nil 17.2 31.5
Reference strains positive for biosurfactant production
Bacillus subtilis MTCC1427 +++ ++ Nil Nil Nil + 161.2 158.5
Bacillus pumilus MTCC 1456 Nil +++ Nil Nil Nil +++ 125.2 156.5
Bacillus subtilis MTCC 2422 Nil +++ Nil +++ +++ ++ 291.4 400.0
Bacillus subtilis MTCC 2423 Nil +++ Nil +++ +++ ++ 245.1 361.2
Bacillus sphaericus MTCC 2473 Nil Nil Nil Nil ++ Nil 31.2 133.2
Pseudomonas aeruginosa MTCC 2297 +++ ++ +++ +++ + +++ 59.0 68.8
CS = Caspian Sea; WB = West Bengal; MM = Mumbai Mussel; MSS = Mumbai Shivree Sediment; Nil = Negative; + = Positive;
++ = Weak positive; +++; ++++ = Good activity; # Hydrocarbons used were AR grade and smeared on to Zobell marine agar plates;
positive signs indicate the cultures with emulsified halos for various hydrocarbons; *Emulsification units (EU/ml) mean of three
experiments; Figures in bold indicate high emulsification units. Emulsification activity (EU/ml) = 1 Emulsification unit = 0.01 O. D.
multiplied by dilution factor of absorbance at 400 nm.
SATPUTE et al.: SCREENING METHODS FOR BACTERIA
249
Acknowledgement
First author thanks the UGC, Govt. of India, for
financial support {(F.17-37/98(SA-I)}. This work is
also supported by the research project sanctioned by
DBT (BT/PR304/AAQ/03/155/2002), Govt. of India.
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Table 4 — Qualitative assessment of biosurfactant/bioemulsifier by marine bacterial isolates
Hydrocarbon overlaid agar plate method # Emulsification activity* (EU/ml)
Marine isolates
Kerosene Hexadecane
Benzene Toluene Diesel Crude oil Petrol Xylene
Gram negative bacteria
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... The solution was mixed with 1ml of a substrate (soybean or diesel oils). After a vigorous vortex for 2 min, the tubes were allowed to stand for one hour to separate aqueous and oil phases, before measuring the absorbance at 540 nm (Satpute et al., 2008). Aqueous phase was removed carefully and was measured at 450 nm and compared with un-inoculated broth which was used as negative control. ...
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Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane). Biosurfactants can reduce the stress between solids and liquids on the surface and at the end. Biosurfactants have several properties, i.e. they are stable, less harmful, as well as readily degradable, and extremely eco-friendly. Biosurfactants also have a wide range of industrial uses because they are a versatile category of chemical substances. The principal justification for conducting such research was the isolation of possible biosurfactants containing bacteria. Sampling was performed for the isolation of bacteria producing biosurfactants from different oil-polluted sites That is to say, experiment for emulsification, test for oil spreading, test for drop collapse, and measure for hemolysis. The capability to produce biosurfactants was seen in 22 different isolates from polluted sites B1, B2, and B3. Through different biochemical tests and Gram staining, it was identified that isolated bacterial strains are Pseudomonas spp and that is Pseudomonas aeruginosa. The procedure used as characterizing biosurfactants was the TLC plate’s procedure, by using TLC plates process yellow dots emerged after spraying on silica gel plates with an throne and ninhydrin reagents. These yellow spots confirmed the presence and production of rhamnolipid in the biosurfactant. Hence, it was concluded that identified strains in the study can be helpful in the heavy metals, pesticides, and hydrocarbons bio-degradation and bioremediation. These may also be used as biological control agents to protect plants from various pathogens, resulting in improved crop yields. Introduction Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane) (Chen et al., 2007; Ghayyomiet al., 2012). Main purpose of the bio-surfactantsgeneration or production is a consequence of financial availability (Van Dyke et al., 1993 It is reported that almost 50 percent of the world's surfactants are used because of the need for cleaning agents as well as the rate of growth grows every day (Deleu and Paquot, 2004). Appropriate use of bio-surfactants will control environmental emissions what these are the most dangerous, constantly rising gradually and disrupting the routine maintenance of life every day. Awareness campaign initiatives have been introduced and also increase for environmental laws, various innovative approaches need to be implemented and even the issue of pollution focused entirely. Developing appropriate advanced technologies to help clear up chemicals and toxins from the ecosystem, like hydrocarbons (both inorganic and organic). Studies on biosurfactants are being launched by scholars and researchers with significant health issues like adverse environmental effects, air contamination, environmental change, and waste management (Makkar and Cameotra, 2002 Biosurfactants contribute to expanded demand for such microbial products as alternatives to chemical surfactants (Benatet al., 2000). Microbes seem to have the capability to degrade contaminants, but their biodegradation is limited leading to hydrophobicity, low solubility in water, and inadequate bioavailability, of such pollutants (Patil, et al., 2012). GhayyomiJazeh, Mishraet. al (2001) those bacteria that produce biosurfactants were isolated from the site of petroleum spills and afterward, 160 strains and as well as 59 strains were able to produce biosurfactants have shown better performance in a test for hemolysis of blood, and 45 strains with positive findings within oil spread experiment were applied in the laboratory to isolate and segregate the media cultured Banat process (Rahman et al., 2002) These were observed and researched that biosurfactants of Pseudomonas aeruginosa spp are most likely to disrupt the bonding of hydrocarbons like nonadecane, octa, Hexa, and hepta, in marine Water contaminated with oil spills up To approximately 47%, 53%, 73% and 60%(Abrar et al., 2020). Current study concluded that the isolated strain having the ability to degrade hydrocarbon as well as the ability to degrade the heavy metal. The strain also can protect the plant from various diseases. The present research found that the isolated strain is capable of degrading hydrocarbon while also being capable of degrading the heavy metal. As well as the strain does have the capability to defend plants from different diseases. Material And Methods Area of Study The investigation was conducted at HazaraUniversity(HU) Microbiology Laboratory, MansehraPakistan. Assemblage of Samples Thehomestay area of the city Mansehra Pakistan which is named as a township, where oil spills arose, oil spills soil samples were obtained as well as sampling from various Mansehra automobile workshops were also done. Sterilized bags of polythene were being used to collect samples of the soil, after thatthe sample was taken towards the Hazara University (HU) Mansehra Microbiology Laboratory to examine and extract bacterial strains that could develop biosurfactants. The soil temperature at the time of sample selection was around 30 ° C. The pH was also verified by Galvano science companies at the time of selection by pH meter, and the pH being reported was 7. Preparation of Media 15 x 100 mm Petri dishes were being used to prepare the media. Agar plates were thoroughly cleaned with water from the tap and then carefully covered in aluminum foil following cleaning then placed within autoclave at 121°C for about 15 min at 15 psi for sterilization. The nutrient agar which contains 0.5% NaCl, 0.3% beef extract, 0.5% peptone, and 1.5% agar, in 500 ml of distilled water, 14 g of the nutrient agar media (Merck) were dissolved. The nutrient level used mainly for the production of non-fastidious species. Nutrient agar is widely known as it's capable of growing a variety of bacteria types and provides nutrients required for the growth of bacteria. Upon sufficient dissolution of such nutrient agar in distilled water, these were then sterilized by autoclaving for 15 min at 15 psi in the autoclave and held at 121 °C Upon autoclaving, pouring of the media was done in laminar flow hood, and then packed and placed for yet more use in a fridge at 4°C. 2.4 Preparation of serial dilution The bacteria are isolated using the serial dilution process. During this process, 10 test tubes were taken and distilled water (9ml) was added in each tube. After that tubes were put for 15 minutes in the autoclave machine at 121°C. After that 1gm of a crude oil sample from the soil was added in a test tube containing distilled water. Further, 1 ml of the solution was taken from the first test tube and poured to the adjoining tubes for the preparation dilution as under . Afterward, 10μl of the solution was pipetted from both the dilution of and shifted for spread culture techniques, then incubated the plates at 37°C for 48hrs. Biosurfactants extraction Firstly, in nutrient broth solution theculture of bacteria was added and inoculated with oil, the bacterial colony was then incubated at the temperature of 25°C in a shaking incubator just for 7 days. Incubation after seven days of trembling. Thebacterial Crop was then taken and centrifuged at 5000rpm at temperature 4°C for 20minutes. Following centrifugation, the supernatant was collected and then mixed in the equivalent amount in Methanol: Chloroform. White sediment was then retained and collected for further use . Bacterial Colonies Isolation 1 g of the soil polluted with oil was diluted serially up to 106 dilutions.10 μl of 104 and 106 dilutions for spread culture were transferred to the MSM agar plates and nutrient agar. The plates were then incubated at 37°C for 48hrs. Twenty-two morphologically separate colonies were separated for further specific examination just after the incubation and processed by using the technique of streak plate. Screening of Isolates’ Biosurfactants Behavior To check the activity of biosurfactants produced by the bacterial species the following methods of screening were done. Hemolytic Activity of Biosurfactants for Erythrocytes Blood agar containing 5% of blood was prepared as after the fresh isolates were added and inoculated on blood agar plates, then the plates were taken and placed in the incubator at temperature 37°C for 48hrs (Rashediet al., 2005). Thereafter the observation of clear zone in the colonies indicated the existence of bacterial species that produce biosurfactants. This experiment was undertaken to control the ability of isolated bacteria to induce blood agar hemolysis. Three forms of hemolysis usually involve; alpha, beta, and hemolysis of the gamma. The agar underneath the species is dark greenish, then it is Alpha, the yellowish color produced in beta hemolysis and gamma hemolysis does not affect the bacterial sppwhichadded on the plates (Anandaraj and Thivakaran, 2010). Bio-surfactant identification with process of CTAB MSM (Mineral salt agar medium) with (2%) of glucose serving both as carbon source, (0.5 mg / ml) acetyl-tri-methyl-ammonium-bromide (CTAB), and methylene blue (MB: 0.2 mg/ml) are used to detect anionic bio-surfactants (Satpute et al., 2008). For this method, thirty microliters (30μl) of cell-free supernatant were added to each of the wells of the methylene blue agar plate that comprises of borer (4 mm in diameter). after that, the incubation of the plates was done for 48-72 hrs at 37°C. Just after incubation in each of the wells, a dark blue halo zone was being used to show the successful anionic bio-surfactant production. Table 1: Composition of MSM Media S. No Ingredients Amount (gm/L) I Potassium dihydrogen phosphate (KH2PO4) II Magnesium Sulfate (MgSO4) III Iron Sulfate (FeSO4) IV Sodium Nitrate (NaNO3) V Calcium Chloride (CaCl2) VI Ammonium Sulfate (NH4)2SO4 Technique for Spreading of Oil A sufficient number of isolated bacteria were inoculated into a solution of 100ml nutrient broth. Over 3 days, the culture was incubated at 37 ° C in a rotating shaker incubator (150 rpm). After that biosurfactants synthesis was checked in culture suspensions (Priya and Usharani, 2009; Anandaraj and Thivakaran, 2010). For this process, thirty milliliters (30ml) of distilled water was added in a Petri dish. In the middle of the distilled water, 1 milliliter (1ml) of diesel oil was added, and then a centrifuged twenty microliter (20μl) culture was introduced to the middle of a plate, which was isolated from oil spilled soil or local oily groundwater. The species producing the bio-surfactant displace the hydrocarbons and disperse it even in the water. Then it was calculated and analyzed within 1 mint (Ali et al., 2013). Technique for Drop collapse In this process, 96-wellsformed in each of the plates of nutrient agar. Afterwards, all the 96-wells of microliter plates was then filled withmineral oil of about 2ml. Then stabilized the plate at 37oC for 1 hour, after which the oil surface was filled with 5μl of supernatant culture. Therefore, the drop shape was taken to be observed on the oil surface after 1min. The drop which was collapsed, generated by the supernatant culture which is used to signify positive(+ive) outcome and the drops which stayed the same and displayed no changeindicates negative(-ive) outcome. And was taking distilled water as a control(Plaza et al., 2006). Emulsification index The emulsification index was calculated, as stated by the process followed by Cooper and Goldenberg (1981) In this process, 2 ml of kerosene oil was taken and inserted in each of the test tubes to the same amount of cell-free supernatant, and then homogenized for 2 min in a vortex at high speed and allowed for 24 hours to stand. The emulsification steadiness was then determined after the 24 hours, and the emulsification value was estimated by measuring the emulsified layer height by the total liquid layer height, then multiplied by 100. Quantification for the Dry weight of Biosurfactants The bacterial colony was inserted and inoculated in the nutrient broth medium, followed by oil and centrifuged at 5000rpm and after that, the supernatant was clutched and treated with chloroform and methanol and mixed. The white colored deposits were taken and used for the furtherprocess of dry weight. Afterwards, took the clean Petri plate and determined the empty plate weight. Next, the sediment was poured onto Petri plates. Now, for the drying process the hot air oven was used and set the 100ºC of temperature for 30minutes and the plates were put in the oven. After the drying process, the plates were weighted again. The dry weight was calculated for the biosurfactants using the formula which described below: Selected strains Identification and their characterization Instead, various basic biochemical methods were used to identify the isolated bacterial strains. Various biochemical tests, such as Gram staining, Oxidase test, Urease test.Catalase test, Methyl red test, Motility test, Indole test, Starch hydrolysis, Citrate test, Spore staining, Gelatin hydrolysis. Then afterwards, for the preliminary characterization of the biosurfactant, the thin layer chromatography process was used. Physical characterization of the strains selected Gram staining First, on the slide, using the wire loop the bacterial pure culture was taken, and smear was prepared on the slide, and then a drop of purified water was applied. Then, the sterile loop or needle was correctly mixed the bacterial colony and purified water, then mixed up until it is somewhat turbid. Then, spirit lamp was used to fixed the bacterial smear on slide and cooled to room temperature. With this glass slide was loaded with solution of crystal violet and stood for 1minute anddistilled water was applied on slide. Meanwhile the slide was submerged for 1 minute with the iodine solution, and then flushed and rinsed with water. Therefore, decolorizer of about 1 to 2 drops(5 percent acetone and 95 percent alcohol) were added to the slide’s smear and stand for 30seconds, and then treated with water. After then slide was rinsed with safranin for 60seconds, and then treated with water anddry in air. Microscopic analysis was done with 100x objective lenses using emersion oil on smear. Cell morphology The isolates of the bacterial cell were gram stained on slides and then the slides were observed under the light microscope, showing the shape and color of the cells. Biochemical characterization of the selected strains Catalase test Aim of this study is to identify, evaluate and examine that, whether or not the microbes are capable of producing catalase enzymes, while catalase is a protective enzyme, i.e. catalase has the potential to protect against the lethal chemicals known as (H2O2). In this study a bacterial culture that was clarified overnight was used. This culture has been smeared on a glass slide, and 3 percent hydrogen peroxide (H2O2) has been applied and observed on smear. Effects have been observed for bubble formation. Citrate test This study was performed to check the amount or ingest the citrate as the carbon and energy supply for growth and metabolism. Medium containing bromothymol blue and sodium citrate as pH indicator, bacterial was introduced. Ammonium chloride is also present in this medium used as a nitrogen source. Results were noted with variations of color from green to blue. Urease test The capability of urease enzyme for degrading urea was calculated in this bacterial capacity test. Bacterial culture was taken and inoculated for 48 hours at 37 ° C in urease broth, and then color was observed. Methyl red test Through using the process known as mixed acid fermentation which is used to evaluate the bacteria's acid production. The bacterial culture was taken and introduced in the broth of MR-VP and then incubated for 3days at a temperature of 37°C. Two (2) to three (3) drops of Methyl red were added in the broth medium after the incubation period. The change in broth color was observed for final results after a few seconds. Indole test Through using the process to assess the bacteria 's capability to crash indole from tryptophane molecules. After the 24 hours of incubated, taken the fresh inoculum of bacteria and then inserted into the tryptone medium, 24 hours of incubation of about 30oC, 2ml of the tryptone broth medium was added into a sterile test tube. Kovac's reagent was taken to be added (few drops) in sterile test tube and stimulated for a few minutes, and variations of color were detected. Gelatin test It is the approach assess to figure out the use of enzymes known as gelatins from bacterial organisms that precipitate the gelatin. Fresh inoculum of bacteria was taken after 24 hours, and inserted into the media of gelatin agar. This was incubated for around 24 hours, so the temperature did not exceed 30 ° C. Media was observed after incubation time. Starch hydrolysis Several of the micro-organisms that use the starch as a carbon energysource. Therefore, this method has been used to assess whether or not bacteria may use starch as a source of carbon. The bacterial fresh inoculum was spread on the petri starch agar plates, and after that the plate was incubated for 24 hours andmaintained the temperature at 30 to 35 ° C, then gradually applying the supplements of iodine to the plates to flow the change, and then examining the plates. Preliminary characterization of the strains selected Experimental characterization of the bio-surfactant was performed by using the process of TLC (Anandaraj et al., 2010). On a silica gel plate, crude portion of the rudimentary bio-surfactant was separated using Methanol: Chloroform: water (CH3OH: CHCl3: H2O) in the ratio of as an eluent with a different color producing reagents. Ninhydrin reagent (0.5 g ninhydrin in 100ml anhydrous acetone) was used to find bio-surfactant lipopeptide as red spots and anthrone reagent (1 g anthrone in 5ml sulfuric acid combined with 95ml ethanol) as yellow spots to identify rhamnolipid bio-surfactant (Yin et al., 2008). Results and Discussion Isolation of bacteria At first, twenty-two (22) strains from a polluted soil sample were isolated from nutrient agar media.Mixed culture provided by these colonies, so they were taken and smeared on the plates of nutrient agar and then fresh inoculum was collected and stored at temperature of 4oC for the further analysis. Bio-surfactants (surface-active compounds)are formed by a variety of amphiphilic bacterial and fungal organisms that are extracellular (a part of the cellular membrane) (Chen et al., 2007). Screening of Isolated strains for biosurfactant producing colonies Different experiments were carried out to identify, isolate and screen bacteria that are capable of generating bio-surfactants and that is Oil spreading technique(OST), blood hemolysis test(BHT), CTAB test, Emulsification operation. There were twenty-two distinct isolates observed in the current research. And the B1, B2 and B3culture were taken and selected from the twenty-two (22) strains isolated from the polluted spot, which were found to produce biosurfactant. And the oil spreading technique showed promising results for these strains. And strain B2 showed a greater displacement of oil and this is 4 mm. Oil spreading method is quick and often easy to handle, and this technique requires no particular equipment, only a very small amount of sample is used. This approach can be applied when the production and quantity of biosurfactant is small (Plaza et al., 2006) and (Youssef et al., 2004) Only bacterial cultures have been allocated and screened for bacterial species that can generate or use biosurfactants. Just three (3) strainsamong them presented the best results.Those 3 strain,s (B1, B2 and B3) were selected as an additional analysis. Blood hemolysis test On the petri plates of blood agar, the . Isolated bacteriaof B1, B2 and B3 were taken andstreak at the temperature about 37°C for 48 hours. Strain B1 demonstrated β (Beta) hemolysis after the incubation cycle and B2 and B3strains demonstrated γ (Gamma) hemolysis. The B1 strain had an emulsification index of about 74 percent and that was very high as compared to 70 percent for B2 and about 53 percent for B3 respectively. Around the same time, B1 strain showed β (Beta) hemolysis and γ (Gamma) hemolysis was shown bystrains B2 and B3 on the platesof bloodagar. The β hemolysisshowed by the strain B1 in the blood agar test, and the strain B2 and B3 showed γ (Gamma) hemolysis. It is determined that 20 percent strains that are the bestproducer of rhamnolipid have not fully lysed the blood, because the ability of the producer strains capacity not be responsible for the hemolytic activity. According to many researchers, who have shown that this is not such an effective tool for biosurfactant detection due to many bioproducts that may also induce red blood cell lysis, that is not so sufficient to be the surface-active molecule (Youssef et al., 2004). (Rashedi and others, 2005). Table2 Blood Hemolysis Test CTAB agar plate test This test confirms the anionic biosurfactants development. After plate incubation at a temperature of 37 ° C for 72 hours, dark blue hollow zone was existedaround each of the B1 strains wells, which clearly indicated the positive (+ive) development of anionic Biofactant. In addition, the B1 and B2 strains showed positive (+ I ve) results and, in the CTAB analysis, the B3 strain was found to be negative (-ive). The growing microorganisms when secreted the anionic biosurfactants on the plates of CTAB (cetyl-tri-methyl-ammonium-bromide) and methylene blue, then as a result the dark blue-purple insoluble ion pairs formed on the plates. The halo zone around each of the colonies was developed that can recognize rhamnolipid production and that was dark blue in colour, and could correlate with production of rhamnolipid (Siegmund et al., 1991). As indicated in (Fig1) Fig1: B1 positive on CTAB agar plate Oil Spreading Technique The oil was displaced by B1, B2and B3 strains in this test strain and showed a zone that was so clear. The bacterial strains capable of developing biosurfactant were tested and separated from the sample of soil which was oil spilled and brought from the District of Mansehra, Pakistan and from automobile workshops of Mansehra. As shown in (Fig.2). Fig.2: Results of Oil Spreading by B1, B2 and B3Table 3;.Test for oil spreads Bacterial culture Formation of zone (mm) Readings B,1 B,2 B,3 Drop-collapse technique During this process the drop shape was observed at the oil surface. As seen in Fig 3, the collapsed drop was provided by the supernatant culture B1 , B2 and B3.. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Fig 3: Result of Drop-collapse test Table 4: The activity of Biosurfactant emulsification Dry weight of bio-surfactants In this examination, white-colored sediment was collected. Then measured the weight of the sterile Petri plate which was empty in the first step. Then, the sediment was poured into plates. The plates were taken and weighted after 30 minutes of drying on a hot air oven, following the process of drying. The weight of biosurfactants (dry weight) was measured using the following formulations: Fig 4: Dry weight of biosurfactants Table: 5: Dry weight of the biosurfactants Bacterial Culture Weight of the plate (g) biosurfactant in The plate after drying (g) Dry weight of Biosurfactant (g) B,1 B,2 B,3 Identification of selected strains and their characterization Gram staining For structural applications, and stroke analysis gram staining method was used.(Fig.5) shows findings from the process of gram staining. Fig 5: Microscopic view of Gram staining Biochemical identification of bacterial strains and their characterization Specific biochemical studies were performed to identify the species for further recognition and characterization. The bio-surfactant producing microorganism was found to be Pseudomonas aeruginosa after conducting various characterizations and the biochemical tests(Eric Deziel et al., 1996), Which can be used to further analyze and study the industrial development of the biosurfactant. Rhamnolipid is also isolated and produced from the Pseudomonas aeruginosa species on the silica gel plate (Rashedi et al., 2005), a form of biosurfactants highly recommended for processes of bioremediation. All the findings collected from biochemical testing were labeled as Berge 's Manual and it revealed that the protected microorganism was (Pseudomonas aeruginosa). Results of biochemical test were tabulated in (Table.5) Table 6: Bacterial strain identification Tests B1 B2 B3 Gram staining Negative Negative Negative Oxidases Positive e Positive Positive Catalase Positive Positive Positive Indole Positive Negative Negative Citrate Positive Negative Negative Urease Negative Positive Negative Nitrate Positive Positive Positive Motility Positive Positive Positive Gelatin hydrolysis Positive Negative Negative Lactose Negative Positive Positive Methyl red Negative Positive Positive Voges Proskauer Negative Negative Negative Fig 6: Results of biochemical tests(A) Methyl red and Voges Proskauer tests (b) catalase tests (c) oxidase tests (d) indole tests (e) citrate tests (g) lactose tests (h) urease tests Preliminary bacterial strain’s characterization The plates showed yellow dots, when sprayed with anthrone reagent. It indicated the existence of biosurfactants of rhamnolipid in the organism on the plate of TLC as seen in theFig.7 Fig 7: Biosurfactant characterization by TLC Conclusion Biosurfactant development is exciting and perceptible across industries to clean up oil waste and pollutants, particularly in the ecosystem.Compared with chemical surfactants, the biosurfactants are less harmful. It plays an important role in defining the advantages and the importance of industrial applications. Therefore, it is not possible to disregard the growing role and importance of biosurfactants in environmental sustainability.Biosurfactant formulations which can be used for bacterial, fungal, and viral organisms as growth inhibitors. Such biosurfactant inhibition properties can make them components that are applicable to Numerous illnesses that are used as medicinal agents. Therefore it was decided that the described strain could be used as a potential source for heavy metal bioremediation pesticide and hydrocarbon polluted sites. And also used as shielding the plant from different pathogens, contributing to improved crop yields. There is no doubt that the biosurfactants are a multifunctional, advanced, versatile, long-lasting and updated type not only for the twenty-first century but beyond. Conflict of interest The authors declared that they have no conflict of interest and the paper presents their own work which does not been infringe any third-party rights, especially authorship of any part of the article is an original contribution, not published before and not being under consideration for publication elsewhere. References Ali, S.R.; Chowdhury, B.R.; Mondal, P. and Rajak, S. “Screening and characterization of biosurfactants producing microorganism from natural environment (Whey spilled soil)”. Nat. Sci. Res. 2013, 3(13), 34–64. 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An update on the use of unconventional substrates for biosurfactant production and their new applications. Applied microbiology and biotechnology. 2002, 58(4), 428-434. Mishra, S.; Jyot, J.; Kuhad, R. C.; & Lal, B. Evaluation of inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil. Environ. Microbiol. 2001, 67(4), 1675-1681. Patil, T. D.; Pawar, S.; Kamble P. N. & Thakare, S. V. “Bioremediation of complex hydrocarbons using microbial consortium isolated from diesel oil polluted soil”. Der ChemicaSinica Journal of Biotechnology. 2012, 3(4), 953-958. Plaza, G.; Zjawiony, I.; and Banat, I. “Use of different methods for detection of thermophilic biosurfactants producing bacteria from hydrocarbon contaminated bioremediation soils”. Petro. Sci. Eng. 2006, 50(1), 71–77. Priya, T.; Usharani, G. “Comparative study for bio-surfactant production by using Bacilus subtilis and Pseudomonas aeruginosa”. Res. Int. 2009, 2(4), 284–287. Rahman, K.S.M.; T.J. 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