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International Journal of Microbiology & Parasitology
IJMP 19 | Volume1|Issue2|2014
1
Research Article
ISOLATION, SCREENING AND CHARACTERIZATION OF CRUDE
OIL AND n-HEXADECANE DEGRADING PSEUDOMONAS spp.
FROM INDUSTRIAL AREAS
Suja Augustine1 *, B.V. Pradeep
2
Department of Microbiology, Karpagam University, Coimbatore, Tamil Nadu, India
Correspondence should be addressed to Suja Augustine1
Received 3 November 2014; Accepted 20 November 2014; Published 20 December 2014
Copyright: © 2014 Suja Augustine et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACTS
Studies were conducted to know the occurrence and distribution of total degraders and alkane degraders in industrial areas
of Kerala, Tamil Nadu, Mangalore and Mumbai by collecting (water and sediment) hundred samples from 16 locations.
Total Heterotrophic Plate Count done in Soya bean Casein Digest agar showed more number of colonies in Kerala and
Mumbai samples. Using the most probable number (MPN) procedure separately enumerated total degraders and alkane
degraders and results showed more total and alkane degraders in Mumbai and Kerala samples. Biochemical potential was
assessed by conducting various morphological and biochemical analysis. Salt tolerance studies showed 6% to 9% of salt
tolerance. Out of the 16 isolates of Pseudomonas P1, P3, P7, P13 and P15 isolates were found to be more degrading than
MTCC 2975. Pseudomonas spp. isolated from all stations was found to be sensitive to Gatifloxacin, Ofloxacin, Gentamicin,
Amikacin, Co-Trimoxazole, Piperacillin, Ciproflaxacin and Chloramphenicol.
KEYWORDS: Crude oil, n-Hexadecane, Pseudomonas, Gas Chromatography
INTRODUCTION
Environmental pollution with petroleum and
petrochemical products (complex mixtures of
hydrocarbons) has been recognized as one of the most
serious current problems especially when associated
with accidental spills on large scale. If this occurs
hydrocarbons may reach the water table before
becoming immobilized in the soil. Bioremediation has
become an alternative way of remediation of oil
polluted sites (Karmen Plohl).
Pseudomonads are a large group of aerobic, nonsporing
gram negative motile bacillus belongs to the gamma
subclass of the proteobacteria and are
chemoorganotrophic. They are ubiquitous, mostly
saprophytic, found in water, soil or other moist
environments. Some of them are pathogenic to plants,
insects and reptiles. A few cause opportunistic human
infection (Pallerone,1984).
Pseudomonads are rapidly growing bacteria measuring
0.5 to 0.8 µm by 1.5 to 3.0 µm. Almost all strains are
motile by means of a single polar flagellum. It will
grow in the absence of O2 if NO3 is available as a
respiratory electron acceptor. The typical
Pseudomonas bacterium in nature might be found in a
biofilm or in a planktonic form. Pseudomonads possess
the metabolic versatility (Stanier et al., 1966). Organic
growth factors are not required for growth, and it can
use more than seventy five organic compounds for its
growth. Its optimum temperature for growth is 37°C
and it is able to grow at temperatures as high as 42°C.
It is resistant to high concentrations of salts and dyes,
weak antiseptics and commonly used antibiotics.
Crude oils are composed of mixtures of paraffin,
alicyclic and aromatic hydrocarbons. Hydrocarbons are
the simplest organic compounds and contain only
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carbon and hydrogen. They can be straight chain,
branched chain or cyclic molecules. Carbon tends to
form four bonds in a tetrahedral geometry.
Hydrocarbon derivatives are formed when there is a
substitution of a functional group at one or more of
these positions (Karmen Plohl)
Increasing petroleum exploration refining and other
allied industrial activities have led to the wide scale
contamination of most of the swamps, creeks, rivers,
streams and Oceans. The present study conducted on
different stations at Kerala, Tamil Nadu, Mangalore and
Mumbai. The aim of this study was to isolate the oil
degrading Pseudomonoas spp. from these stations.
MATERIALS AND METHODS
The studies were conducted to know the occurrence and
distribution of total oil degraders and alkane degraders in
water and sediment samples of industrial areas of Kerala,
Tamil Nadu, Mangalore and Mumbai.
Sample collection
Water and sediment samples were collected from selected
regions. Selected regions of Kerala for the present study
were Ambalamugal(P1), Fort Cochin(P2), Marine
Jetty(P3), Vypin(P4), Container Terminal(P5),
Vallarpadam(P6) and Cherai(P7). Selected regions of
Tamil Nadu were Velankanni(P8) and Chennai Port(P9)
and selected regions of Mangalore were Someshwaram
Beach(P10), Suratkal(P11), Panambur Beach(P12) and
Mangalore Airport(P13). Selected regions of Mumbai were
Panvel(P14),Gate way of India(P15) and Mumbai
Port(P16). In selected regions water was being influenced
by the tidal cycles and oil discharges from ships and boats.
It was also subjected to pollution by domestic discharges,
soil erosion, surface run-off and other human activities.
The reagent bottles were properly washed and sterilized.
100 Samples were collected for this study. The water
samples were collected from the surface without air
bubbles .The sediment samples were collected using
simple sampling technique. Care was taken in handling
and sampling to avoid contamination of the samples and
returned to the laboratory for bacterial extraction as soon as
possible.
Isolation of total heterotrophic flora
Total heterotrophic bacteria from water and sediment
samples were identified by serial dilution technique in
Soya bean casein digest agar. 10 gram of the sediment
sample collected was transferred to 100 ml of the distilled
water in a conical flask. The flask was shaken well. The
water and diluted sediment samples were membrane
filtered and the filter paper was put in 100 ml of sterile
water taken in different conical flasks. 1 ml of the above
sample transferred to the test tube to about 9 ml of blank
and pipettes were discarded. This was continued for the
required number of dilution (upto106
). 0.1 ml of the
samples of the required dilution was transferred into a petri
dish. The petri dishes were labeled correctly with glass
markers indicating the type of the sample, the medium and
dilution. About 15-20 ml of Soya bean Casein Digest Agar
medium was poured into petri dishes at an ear bearable
temperature (aseptically).The dishes were rotated
clockwise and anticlockwise direction for thorough mixing.
The dishes were left undisturbed for the agar to get
solidified and then incubated in an inverted position at
room temperature.
Microbial enumeration using most probable number
procedure
Improved most probable number method was done for
direct count of oil degrading microorganisms (Wrenn and
Venosa, 1996). Serial dilution of samples were inoculated
into mineral salt medium (MSM-Bushnell-Hass medium)
supplemented with 3% NaCl and adjusted to pH 7.8.
Crude oil degraders and n-Hexadecane degraders were
enumerated adding crude oil and n-hexadecane as sole
source of carbon and energy.
Identification of bacteria
Morphological, physiological and biochemical
characteristics of pure isolates were examined according to
the Bergey’s Manual of Determinative Bacteriology.
Primary identification was done on the basis of Gram
staining, colony and cell morphology. Biochemical
potential assessed by conducting various morphological
and biochemical analysis such as Nitrate reduction, OF
Test, Citrate utilization, Oxidase test, Catalase test, Starch
and Gelatin hydrolysis.
From the positive plates, the Pseudomonas species
(colonies were found to be yellowish brown, green
fluorescent, Bluish green which are transparent and
irregular) were picked and streaked onto Soya been casein
digest agar. Further sub culturing is done onto Nutrient
agar plates, Cetrimide agar, Nutrient agar slants and
peptone water and morphology were observed. From the
positive tubes 0.1 ml is transferred to Cetrimide agar plates
and spread plating done. From this colonies taken and
streaked onto nutrient agar plates and Soya bean casein
digest agar plates.
Isolation and cultivation of hydrocarbon degrading
bacteria
From the dilution, 0.1 ml of each one is spread onto B-H
agar supplied with hydrocarbons as sole carbon and energy
source by placing it in a vapour tube (Cut of micro pipette
tip, sealed at one end with heat in the lid of the
plate).Control plates without substrates were also
inoculated. The plates were sealed with paraffin and then
inoculated at 25°C for at least one month and colony
characteristics observed.
Antibiotic sensitivity test
Antibiotic sensitivity of oil degrading Pseudomonas spp.
were tested by Kirby and Bauer’s method (1966) with
young culture to find out the resistance pattern to various
antibiotics. The test cultures were plated onto Muller
Hilton Agar plates and the discs were placed on the surface
of the medium and the plates were incubated. The
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antibiotics were Ampicillin/Sulbactum, Co-Trimoxazole,
Cefotaxime, Piperacillin, Chloramphenicol, Ciprofloxacin,
Ceftizoxime, Tetracycline, Ofloxacin, gentamicin,
Amikacin and Gatifloxacin.
Salt tolerance studies
Studies were conducted to know the ranging tolerance of
organism, as it is an opportunistic pathogen, these levels of
studies are important to know whether it can survive in
high stress conditions. Inoculate 3ml Lactose Broth tubes
with a single along of bacterium. Add 1% concern of salt to
that broth. Allow to grow at 37°C for 24 hours. If turbidity
was shown transfer one loopful of organism to the LB
broth with 2% salt solution. Incubate at 37°C for 24 hours.
The process is repeated using different salt concentration
until there will not be any growth.
Biodegradation assay by spectrophotometric technique
The bacterial isolates of five selected regions P1, P3, P7,
P13 and P15 from overnight culture at the log phase of
growth were transferred to 250 mL conical flasks, each
containing 100 mL of sterile mineral salts medium with
(0.2% v/v) crude oil and n-Hexadecane separately. The
experiment was carried out in duplicate and uninoculated
flasks constituted the controls, accounting for abiotic
losses. All flasks were incubated at 22°C, 200rpm and pH
7 for 30days.Crude oil degradation and microbial growth
were determined spectrophotometrically at 510nm in
selected intervals of time (3, 6, 9,12, 15, 18, 21 and 24
days).
Gas chromatography analysis
Residual crude oil after degradation at the end of
incubation period was quantified chromatographically via
capillary gas chromatography using Chemito Gas
Chromatography 2000.
RESULTS AND DISCUSSION
Out of the hundred samples screened for crude oil and n-
Hexadecane degradation by Most Probable number
procedure (MPN) thirty two isolates obtained. Amongst
the thirty two isolates, five isolates were selected based on
degradation ability. Identification of the five isolates using
Gram staining, biochemical tests and Cetrimide agar
plating revealed the organism to be Pseudomonas spp.
Determination of microbial numbers -total plate count
Serial dilution was done to get the total plate count using
soybean casein digest agar and the result is given in Table
1. Bacteria that cannot grow on selective substrates do not
produce false positive responses even when the inoculum
density is very high. Thus this method, which is very
simple enough for use in this field, provided reliable
estimates for the density of the organisms.
Table 1: Total plate count
TOTAL PLATE COUNT
FOR HETEROTROPHIC FLORA
NAME OF STATIONS
SERIAL NUMBER
NATURE OF SAMPLES DILUTIONS NUMBER OF COLONIES
KERALA
AMBALAMUGAL 1 SEDIMENT 10-3
280
2 SEDIMENT 10-4
50
3 WATER 10-3
>300
4 WATER 10-4
>300
FORT COCHIN 5 SEDIMENT 10-3
50
6 SEDIMENT 10-4
20
7 WATER 10-3
60
8 WATER 10-4
28
MARINE JETTY 9 SEDIMENT 10-3
123
10 SEDIMENT 10-4
46
11 WATER 10-3
>300
12 WATER 10-4
>300
VYPIN 13 SEDIMENT 10-3
100
14 SEDIMENT 10-4
62
15 WATER 10-3
135
16 WATER 10-4
90
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CONTAINER TERMINAL
17 SEDIMENT 10-3
40
18 SEDIMENT 10-4
15
19 WATER 10-3
50
20 WATER 10-4
25
VALLARPADAM 21 SEDIMENT 10-3
45
22 SEDIMENT 10-4
19
23 WATER 10-3
65
24 WATER 10-4
30
CHERAI 25 SEDIMENT 10-3
250
26 SEDIMENT 10-4
45
27 WATER 10-3
>300
28 WATER 10-4
>300
TAMIL NADU
VELANKANNI 29 SEDIMENT 10-3
35
30 SEDIMENT 10-4
10
31 WATER 10-3
50
32 WATER 10-4
25
CHENNAI PORT 33 SEDIMENT 10-3
45
34 SEDIMENT 10-4
15
35 WATER 10-3
60
36 WATER 10-4
25
MANGALORE
SOMESHWARAM BEACH
37 SEDIMENT 10-3
90
38 SEDIMENT 10-4
53
39 WATER 10-3
125
40 WATER 10-4
80
SURATKAL 41 SEDIMENT 10-3
270
42 SEDIMENT 10-4
40
43 WATER 10-3
280
44 WATER 10-4
250
PANAMBUR BEACH
45 SEDIMENT 10-3
70
46 SEDIMENT 10-4
33
47 WATER 10-3
100
48 WATER 10-4
50
MANGALAORE PORT
49 SEDIMENT 10-3
290
50 SEDIMENT 10-4
60
51 WATER 10-3
>300
52 WATER 10-4
>300
MUMBAI
PANVEL 53 SEDIMENT 10-3
30
54 SEDIMENT 10-4
10
55 WATER 10-3
40
56 WATER 10-4
15
GATEWAY OF 57 SEDIMENT 10-3
>300
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INDIA 58 SEDIMENT 10-4
100
59 WATER 10-3
>300
60 WATER 10-4
>300
MUMBAI PORT 61 SEDIMENT 10-3
40
62 SEDIMENT 10-4
15
63 WATER 10-3
55
64 WATER 10-4
25
Microbial enumeration – mpn method
The most probable number (MPN) procedure was
conducted using BH media along with crude oil and n-
hexadecane separately to enumerate total and alkane
degraders and the results obtained were given on Table 2
for crude oil and on Table 3 for n-Hexadecane. Triphenyl
Tetrazolium chloride was used as an indicator of
degradation and all positive tubes showing red color
showed the oxidation of oil after 7 days of incubation.
These MPN procedures were accurate and selective. Out
of the 16 isolates, 5 isolates showed highest degradation in
MPN procedure. They were Ambalamughal(P1), Marine
Jetty(P3), Cherai(P7) of Kerala , Mangalore Port(P13) of
Mangalore and Gate way of India(P15) of Mumbai. They
showed an MPN index of 13x103
for crude oil and 13x103
for n-Hexadecane.
Most hydrocarbon degraders in MPN methods use
complex substrates such as crude oil or refined petroleum
products, as
the selective substrate (Mulkins Philip and Stewart, 1974 ;
Walker and Colwell, 1976 ; Brown and Breaddock, 1990 ;
Song and Bartha, 1990; Hrines et al., 1996).
The degradation of aromatic and alkane hydrocarbon was
tested with TTC with formazan production. The red colour
produced will increase with increase in cytochrome
oxidase production by bacteria (Premeela and Chandrika,
1997). After conducting MPN, the positive tubes were
taken and plated onto Bushnell-Hass agar (B-H agar)
medium to isolate hydrocarbon degrading bacteria. Only
bacteria which can utilize the hydrocarbon (Hexadecane or
crude oil) will grow on this media. Dr. Jaikie Aislabie
(Nexus Research group, 2002) worked same type of work
and also found that hydrocarbonoclastic bacteria alone can
grow in this media. The positive tubes were shown by
turbid and show disruption to the film of oil on the surface
of the medium were scored as positive and appearance of
colonies on the B.H. agar indicates hydrocarbon degrading
bacteria.
Table 2: Microbial Enumeration – MPN method for Crude oil
MICROBIAL ENUMERATION-MPN METHOD
FOR CRUDE OIL DEGRADATION
SL.NO STATIONS SAMPLE DILUTION MPN INDEX
100
10-1
10-2
10-3
10-4
10-5
KERALA
1 AMBALAMUGAL SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
2 FORT COCHIN SEDIMENT 5 5 5 5 3 0 7.9x103
WATER 5 5 5 5 3 0 7.9x103
3 MARINE JETTY SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
4 VYPIN SEDIMENT 5 5 5 5 3 0 7.9x103
WATER 5 5 5 5 3 0 7.9x103
5 CONTAINER TERMINAL SEDIMENT 5 5 5 4 2 0 2.2x103
WATER 5 5 5 4 2 0 2.2x103
6 VALLARPADAM SEDIMENT 5 5 5 4 3 0 2.7x103
WATER 5 5 5 4 3 0 2.7x103
7 CHERAI SEDIMENT 5 5 5 5 4 0 13x103
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WATER 5 5 5 5 4 0 13x103
TAMIL NADU
8 VELANKANNI SEDIMENT 5 5 5 3 1 0 1.1x103
WATER 5 5 5 3 1 0 1.1x103
9 CHENNAI PORT SEDIMENT 5 5 5 4 2 0 2.2x103
WATER 5 5 5 4 2 0 2.2x103
MANGALORE
10 SOMESHWARAM BEACH SEDIMENT 5 5 5 5 2 1 7x103
WATER 5 5 5 5 2 1 7x103
11 SURATKAL SEDIMENT 5 5 5 5 3 1 11x103
WATER 5 5 5 5 3 1 11x103
12 PANAMBUR BEACH SEDIMENT 5 5 5 5 2 0 4.9x103
WATER 5 5 5 5 2 0 4.9x103
13 MANGALAORE PORT SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
MUMBAI
14 PANVEL SEDIMENT 5 5 5 2 1 0 0.68x103
WATER 5 5 5 2 1 0 0.68x103
15 GATEWAY OF INDIA SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
16 MUMBAI PORT SEDIMENT 5 5 5 4 2 0 2.2x103
WATER 5 5 5 4 2 0 2.2x103
Table 3: Microbial Enumeration – MPN method for n-Hexadecane
MICROBIAL ENUMERATION-MPN METHOD
FOR n-HEXADECANE DEGRADATION
SL.NO STATIONS SAMPLE DILUTION MPN INDEX
100
10-1
10-2
10-3
10-4
10-5
KERALA
1 AMBALAMUGAL SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
2 FORT COCHIN SEDIMENT 5 5 5 5 3 0 7.9x103
WATER 5 5 5 5 3 0 7.9x103
3 MARINE JETTY SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
4 VYPIN SEDIMENT 5 5 5 5 3 0 7.9x103
WATER 5 5 5 5 3 0 7.9x103
5 CONTAINER TERMINAL SEDIMENT 5 5 5 4 2 0 2.2x103
WATER 5 5 5 4 2 0 2.2x103
6 VALLARPADAM SEDIMENT 5 5 5 4 3 0 2.7x103
WATER 5 5 5 4 3 0 2.7x103
7 CHERAI SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
TAMIL NADU
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8 VELANKANNI SEDIMENT 5 5 5 3 1 0 1.1x103
WATER 5 5 5 3 1 0 1.1x103
9 CHENNAI PORT SEDIMENT 5 5 5 4 3 0 2.7x103
WATER 5 5 5 4 3 0 2.7x103
MANGALORE
10 SOMESHWARAM BEACH SEDIMENT 5 5 5 5 2 1 7x103
WATER 5 5 5 5 2 1 7x103
11 SURATKAL SEDIMENT 5 5 5 5 3 1 11x103
WATER 5 5 5 5 3 1 11x103
12 PANAMBUR BEACH SEDIMENT 5 5 5 5 2 0 4.9x103
WATER 5 5 5 5 2 0 4.9x103
13 MANGALAORE PORT SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
MUMBAI
14 PANVEL SEDIMENT 5 5 5 3 1 0 1.1x103
WATER 5 5 5 3 1 0 1.1x103
15 GATEWAY OF INDIA SEDIMENT 5 5 5 5 4 0 13x103
WATER 5 5 5 5 4 0 13x103
16 MUMBAI PORT SEDIMENT 5 5 5 4 3 0 2.7x103
WATER 5 5 5 4 3 0 2.7x103
Identification of bacteria
The organism was concluded as Pseudomonas spp., as the
Gram character showed negative non spore forming rods
and answered positive for citrate utilization, catalase,
oxidase, nitrate reductase, and gelatin hydrolysis. The
organism was confirmed as Pseudomonas spp. by checking
the growth on cetrimide medium.
All the isolates were subjected to gelatin hydrolysis and
were found to liquefy gelatin, indicating the proteolytic
potential of the isolates. Isolates were subjected to sugar
fermentation by using various sugars such as glucose,
lactose, sucrose, maltose etc. and all were found to be
negative. The isolates were subjected to Triple sugar iron
test and were shown alkaline slants without any change in
the butt. . All the isolates were found to be indole
negative, methyl red negative and VP negative. All isolates
were found to be catalase and oxidase positive. All isolates
were found to reduce nitrate.
The families Pseudomonadales were found to be quite
predominant in the aquatic environment. They were found
to be commonly encountered group in the environment
(Alexandar, 1984) second to Bacillus. Pseudomonas
autogena and Pseudomonas perfectomarinus are the only
organisms among the 60 species described by Zobell and
Upahm (1994) which reduced nitrate to free nitrogen.
Pseudomonas can grow rapidly in ordinary medium and
very rarely it needs growth factors for development. 10%
of the isolates will need aminoacids, vitamins, 30% will
require complex mixture of growth factors (Alexander,
1984). Pseudomonas species have very simple nutritional
requirements. It is often observed “growing in distilled
water” which is evidence of its minimal nutritional needs.
In the laboratory, the simplest medium for growth of
Pseudomonas consists of acetate for carbon and
ammonium sulfate for nitrogen. In sediments
Pseudomonas acquired at
levels of 15-20%. The isolates were tested for their ability
to utilize citrate as sole source of carbon for growth.
Pseudomonas spp. produce two types of soluble pigments,
the fluorescent pigment pyoverdin and the blue pigment
pyocyancin. The latter is produced abundantly in media of
low iron content and functions in iron metabolism in the
bacterium. Pyocyanin(from “pyocyaneus”) refers to “blue
pus” which is a characteristic of suppurative infections
caused by Pseudomonas. It is well known that the
fluorescent pigment production depends on the nature of
medium for its manifestations (Seyleenas and Starck,
1943).
The fermentation of glucose in the presence of oxygen and
also in the absence of oxygen was tested by the O/F
activity. Pseudomonas are oxidative organisms, the
carbohydrate was utilized only in the presence of oxygen.
Organic growth factors were not required. They are
nutritionally versatile (Stanier et al., 1966). Pseudomonas
spp. not had enzyme potential to ferment all these sugars.
Catalase and oxidase was produced indicating the micro
aerophilic nature of Pseudomonas and production of
cytochrome oxidase.
After subculturing from B-H agar, the appearance of
Pseudomonas colonies on different media were noted and
results are given in Table 4 and Table 5.
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Table 4: Observation of Pseudomonas Spp. on different media
Sl. No. Media Observation
1 B-H agar Large, flat spreading and irregular, yellowish green colonies
2 Nutrient agar Large, flat, spreading and irregular, bluish green colonies.
Pigments see diffuses into the medium.
3 Soybean Casein digest agar Large, irregular, yellowish brown colonies. Pigment seen
diffuses into the medium.
4. Cetrimide agar Large, flat, spreading and irregular greenish yellowish
fluorescent colonies. Pigment seen diffuses into the
medium
Table 5: Morphological characterization in Cetrimide agar
Size
1.5 by 0.5
Elevation Flat
Colour Yellowish-brown, Green
Fluorescent, Bluish-green
Margin Irregular
Isolation of hydrocarbon degrading bacteria
After conducting MPN, the positive tubes were taken and
are plated onto B-H agar and hydrocarbon degrading
bacteria were detected. The colonies were appeared only
on the B-H plate and not on the substrate free control.
Antibiotic sensitivity test
Pseudomonas spp. isolated from all stations was found to
be sensitive to Gatifloxacin, Ofloxacin, Gentamicin,
Amikacin, Co-Trimoxazole, Piperacillin, Ciproflaxacin and
Chloramphenicol.
Pseudomonas spp. is naturally resistant to many
antibiotics due to the permeability barrier afforded by its
outer membrane LPS. Its tendency to colonize surface in a
biofilm form makes the cells impervious to therapeutic
concentrations of antibiotics. Since its natural habitat is
the soil, living in association with the bacilli,
actinomycetes and molds, it has developed resistance to a
variety of their naturally occurring antibiotics. Moreover,
Pseudomonas maintains antibiotic resistance plasmids,
both R-factors and RTFs, and it is able to transfer these
genes by means of the bacterial processes of transduction
and conjugation.
SALT TOLERANCE STUDIES
Pseudomonas spp. showed 6% to 9% of slat tolerance.
That means it can survive in stress conditions.
Pseudomonas spp. can be used widely for the degradation
of oils. In the case of oil spills these organisms can be
applied to the field. Because it can survive high stress
conditions and it possess oil degrading plasmids as well as
it has only minimal nutritional requirements. As this is the
naturally occurring one it can be introduced easily into the
oil spilled areas.
Biodegradation assay by spectrophotometric technique
Crude oil and n-Hexadecane degradation efficiency of the
isolates were compared with that of MTCC 2975 using
optic density measurements at 600nm and the results are
given in Figure 1 for crude oil degradation and Figure 2
for n- Hexadecane degradation. All the 5 samples show
high degradation efficiency than MTCC 2975. It was found
that all 5 isolates were able to degrade 40% of crude oil
within 24 days whereas MTCC 2975 took 30 days for
degradation. All the 5 isolates took 21- 24 days to degrade
50% of n-Hexadecane whereas MTCC 2975 took 30 days
to degrade 50% of n-Hexadecane.
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Figure 1: Crude oil degradation
Figure 2: n- Hexadecane degradation
Gas chromatography analysis
Based on the GC analysis, nitrogen was used as carrier gas
and the capillary column FID detector for the analysis, the
result was given as 88.91% and 54.88% of degradation for
crude oil and n-Hexadecane respectively after thirty days of
incubation. The results are given in Figure 3 for crude oil
and on Figure 4 for n-Hexadecane.
0
5
10
15
20
25
30
35
40
45
Day
s
Locations
Days % of Degradation
0
10
20
30
40
50
60
Day
s
Locations
Days % of Degradation
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Figure 3: GC Chromatogram for degraded crude oil
Figure 4: GC Chromatogram for degraded n-Hexadecane
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Biodegradation of oil spills is a major problem because it
usually occurs in marine water surface and seeding with
bacteria becomes difficult. Besides, there is no single
bacterium that can degrade all the components of oil which
are petroleum products.
The bacteria isolated here was able to degrade hydrocarbon
in both n-Hexadecane and crude oil. The isolation of such
degrading bacteria has been reported earlier.
Hasanuzzaman, M. etal., (2004) isolated a novel, oil
degrading bacterium from hot spring in Japan. It efficiently
degrades different types of oils, including edible oil wastes.
This strain is also gram negative rod, aerobic with a polar
flagellum.
Norman et al., (2004) inform that Pseudomonas aeruginosa
alkane degrader is frequently isolated from petroleum
contaminated sites and produces factors that enhances its
competitiveness and survival in many environments.
Szoboszlay et al., (2003) did comparative degradation
examination of Pseudomons aeruginosa and other
degraders on hydrocarbon polluted soil and proved that it
is a good oil degrader.
The earth has faced many disasters that have been caused
by human species throughout the history. Today one of
the most important hazards jeopardizing marine
environments would be marine oil spills. Studies on this
field may protect earth at least from this disaster.
Results indicate that this organism reserves the property to
prevent the contamination of oil polluted areas to a certain
extent.
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