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 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 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 nHexadecane 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
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 INDIA 57 SEDIMENT 10-3 >300
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 nhexadecane 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 13×103 for crude oil and 13×103 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 13×103
WATER 5 5 5 5 4 0 13×103
2 FORT COCHIN SEDIMENT 5 5 5 5 3 0 7.9×103
WATER 5 5 5 5 3 0 7.9×103
3 MARINE JETTY SEDIMENT 5 5 5 5 4 0 13×103
WATER 5 5 5 5 4 0 13×103
4 VYPIN SEDIMENT 5 5 5 5 3 0 7.9×103
WATER 5 5 5 5 3 0 7.9×103
5 CONTAINER TERMINAL SEDIMENT 5 5 5 4 2 0 2.2×103
WATER 5 5 5 4 2 0 2.2×103
6 VALLARPADAM SEDIMENT 5 5 5 4 3 0 2.7×103
WATER 5 5 5 4 3 0 2.7×103
7 CHERAI SEDIMENT 5 5 5 5 4 0 13×103
WATER 5 5 5 5 4 0 13×103
TAMIL NADU
8 VELANKANNI SEDIMENT 5 5 5 3 1 0 1.1×103
WATER 5 5 5 3 1 0 1.1×10
9 CHENNAI PORT SEDIMENT 5 5 5 4 2 0 2.2×103
WATER 5 5 5 4 2 0 2.2×103
MANGALORE
10 SOMESHWARAM BEACH SEDIMENT 5 5 5 5 2 1 7×103
WATER 5 5 5 5 2 1 7×103
11 SURATKAL SEDIMENT 5 5 5 5 3 1 11×103
WATER 5 5 5 5 3 1 11×103
12 PANAMBUR BEACH SEDIMENT 5 5 5 5 2 0 4.9×103
WATER 5 5 5 5 2 0 4.9×103
13 MANGALAORE PORT SEDIMENT 5 5 5 5 4 0 13×103
WATER 5 5 5 5 4 0 13×103
MUMBAI
14 PANVEL SEDIMENT 5 5 5 2 1 0 0.68×103
WATER 5 5 5 2 1 0 0.68×103
15 GATEWAY OF INDIA SEDIMENT 5 5 5 5 4 0 13×103
WATER 5 5 5 5 4 0 13×103
16 MUMBAI PORT SEDIMENT 5 5 5 4 2 0 2.2×103
WATER 5 5 5 4 2 0 2.2×103

Table 3: Microbial Enumeration – MPN method for n-Hexadecane

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.

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.

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.

Figure 3: GC Chromatogram for degraded crude oil

Figure 4: GC Chromatogram for degraded n-Hexadecane

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.