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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



4

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



5

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



6

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



7

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.



9

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



10

Figure 3: GC Chromatogram for degraded crude oil

Figure 4: GC Chromatogram for degraded n-Hexadecane



11

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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1. Akhihiko Murakami, Kazumobu Zuzukit, Akiko

Yamne and Tadao Kusama, 1985. Degradation of crude

oil by Pseudomonas sp. in enriched sea water medium.

J. of Oceanographical Society of Japan. 41 : 337-359.

2. Alexandar, M., 1982. Most probable number method

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