Issuu on Google+

LOW MOLECULAR WEIGHT POLYCYCLIC AROMATIC HYDROCARBONS DEGRADATION POTENTIALS OF HIGH MOLECULAR WEIGHT POLYCYCLIC AROMATIC HYDROCARBON DEGRADERS

BY ONYENURU OKECHUKWU. P

i


DEDICATION This work is dedicated to God the Father, God the Son, and God the Holy Spirit for the understanding and ability to interpret the various literatures consulted. I also dedicate it to my parents Mr. and Mrs. I Onyenuru for their encouragement and moral support.

ii


ACKNOWLEDEGMENT My profound gratitude goes to my able supervisor Mr Femi Obayori whose contribution to this work cannot be overemphasized. My sincere thanks go to my unrelenting brother Mr Osborne Onyenuru, for his financial support. And also to my siblings Joy, Vivian, Edwin, Perpetual, and Patricia for their unflinching effort. Thanks a million. It is pertinent to say a big thanks to my friends Ayo, Oktabweblinks, Osho, Kenny, Seun, Vikky, Yemi, Busayo, Funke, T-money, Rafiu, Alex, and all the members of St Vincent the paul society of St brigid’s catholic church, to mention a but few. I love you all.

iii


ABSTRACT Bacteria capable of degrading polycyclic aromatic hydrocarbon were isolated from polluted soil by routine enrichment methods. Two Pseudomonas spp namely: Pseudomonas sp strain AA1 and Pseudomonas aeruginosa strain AA2 were isolated from mechanic workshop and Asphalt plant soil respectively. The isolates exhibited great degradative ability on the LWW PAHs dibenzothiophene, biphenyl, phenanthrene and anthracene. While none of the isolates was found to degrade phenol. The findings shows that, strain AA1 exhibited better degradative ability than strain AA2 indicated by higher turbidity in the AA1 cultures.

iv


INTRODUCTION AND LITERATURE REVIEW Polycyclic aromatic hydrocarbons (PAHs) are a group of over 100 different compounds formed during the incomplete combustion of coal, oil, garbage. The primary source of PAH in the environment is from the incomplete combustion of wood, and fuel for residential heating. PAHs are composed of fused aromatic rings, and are biochemically persistent in the environment due to the presence of dense cloud of π- electrons on both sides of the ring structure, making them resistant to nucleophilic attack. PAHs are heterogeneously distributed in the soil and may adsorb as crystalline on solid particles in the soil. PAHs generally have low solubility potentials in water because they lack free electrons to combine with water molecules, but slightly to completely soluble in organic solvent such as acetic acid, benzene and acetone. Several are soluble in toluene, xylene, 1,4-dioxane, and other organic solvents. This low solubility in water is responsible for the non-bioavailability of PAHs to PAH-degrading organisms and its consequent persistence in the environment. Solubility and vapour pressure of PAHs vary, however as molecular weight increases (i.e. number of aromatic rings), solubility and vapour pressure decreases, thus PAHs with more aromatic rings are more resistant to biodegradation. PAHs are of health significance since they have been implicated in several human and animal diseases. Benzo(a)pyrene induces trancheopapilloma and carcinogenecity in hamster and squamous cells of the lung in rat, while Benzo(a)anthracene causes hepatomas in mice. Their carcinogenicity, mutagenicity and teratogenicity have been linked to an increase in the number of aromatic rings (Cernilia 1992). Also worthy of consideration, is the production of toxic dead-end metabolites, metabolite repression, presence of other easily metabolisable substrates, and lack of metabolic and inducer substrates in the soil (Wand et al., 2003).

1


PAHs are classified as high or low molecular weight, based on the number of benzene rings. Low molecular weight PAHs have one to three aromatic rings, including naphthalene, anthracene, and phenanthrene. While high molecular weight PAHs have more than three aromatic rings, including pyrene, benzo(a)pyrene, coronene, and flouranthene.

BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS PAH biodegradation has been reported in a number of microorganisms, especially in bacteria, with a wide array of metabolites identified, and pathways proposed. Bacteria capable of degrading PAHs are limited to a number of taxonomic groups Pseudomonas such as Sphinghomonas, Burkholderia, Pseudomonas, Mycobacteria, and Rhodococcus (Kastner et al., 1994, Mueller et al., 1997). These bacteria species could either metabolize PAHs to les complex organic intermediates, which could then be used up by other organisms, or break it down to an assimilatable compound, thus using them as a source of carbon. The general pattern for the biodegradation of PAHs is by oxidation or aerobic oxidation of the benzene rings. The initial step is via the oxidation of the PAH into a highly unstable dihydroxylated intermediate, and in the presence of a dehydrogenase enzyme, dihydroxydiol is converted to cathecol, or protocathecol which are central intermediates. These may then by processed through either the ortho-cleavage type of pathway forming cis-cis-muconate or meta-cleavage type of pathway forming hydroxyl muconosemialdehyde, this is then transformed to a TCA cycle intermediate such as succinyl CoA ( Van der meer et al., 1992, Sutherland et al., 1992). Two oxygenase enzymes are involved in the degradation of PAHs which are the hydroxylating dioxygenase that catalyses the initial oxidation of the rings, and the other cleavage dioxygenase catalyses the breaking of the ring.

2


DEGRADATION OF HIGH MOLECULAR WEIGHT PAHs Degradation of LMW PAHs such as anthracene, phenanthrene, naphthalene, and biphenyl have been widely reported in the genera Pseudomonas, Sphingobium, Norcadia, Rhodococcus, and Mycobacterium (Cerniglia, 1984, Gibson, 1999) Anthracene is initially deoxygenated to 1,2-dihydroxyanthracene, which is then catabolised by dehydration to 3-hydroxy-2-naphthoic acid. Pseudomonas sp and Sphingomonas yanoikuyae B1 metabolize anthracene to 2-hydroxy-3-naphthoic acid, salicylate, and cathecol, while Mycobacterium degrades to give four metabolites cis-1,2 dihydroxy-1,2-dihydroxyanthracene, hydroxyanthracene,

and

6,7-benzocoumarine,1-methoxy-2-

9,10-anthraquinone,

with

ring

fission

product

3-(2-

carboxyvinyl)naphthalene-2-carboxylic acid. Fluoranthene has been reported to be degraded singly by Alcaligenes denitrificans strain WW1 (Weissefel et al., 1990), and by Mycobacterium specie strain PYR-1 (Kelly and Cerniglia, 1991) converting it to carbon dioxide, while a small amount of a ring cleavage metabolite was detected, and characterized as 9-fluorenone-1-carboxylic acid (Kelly et al., 1991). This isolate was also capable of degrading a six component synthetic mixture of three, four, and five ring PAHs (Kelly and Cerniglia, 1995). Mycobacterium species have also been reported to pyrene (Cerniglia and Heitkamp, 1990) with the detection of three products of ring oxidation cis-4,5-pyrenedihydrodiol, trans-4,5-pyrene dihydrodiol, and pyrenol, and four products of ring fission 4-hydropernaphthenone, 4-phenanthroic acid, phthalic acid and cummanic acid. The presence of cis and trans-4,5-dihydrodiol as one of the intermediates suggests a multiple pathway for the initial oxidative attack of pyrene (Kanaly and Harayama 2000). Studies confirmed the presence of a di and monooxygenase enzymes (Heitkamp and Cerniglia 1988, Heitkamp and Cerniglia 1999). Inducible enzymes seem to be responsible for pyrene catabolism, since lag phase in

3


pyrene mineralization was observed in cultures grown in the presence of pyrene and no pyrene mineralization was observed in non-induced cultures dosed with chloramphenicol as inhibitor of bacteria protein synthesis (Heitkamp et al., 1988). Chrysene is reported to be degraded by Rhodococcus strain WW1, while benzo(a)anthracene and chrysene is degraded by Pseudomonas putida, Pseudomonas aeruginosa, and flavobacterium (Trzesicka-mylnarz and Ward 1995). There is still limited information regarding bacteria degradation of PAHs more than five aromatic rings, both environmentally and pure or mixed cultures. Recently, a new enteric organism capable of degrading pyrene and phenanthrene leclercia adecarboxylata was isolated (Sarma et al., 2004).

BIODEGRADATION OF LOW MOLECULAR WEIGHT PAHs Pseudomonas putida degrades naphthalene to salicylic acid which is mediated by a single enzyme system(Holizoh et al., 1994) encoded by a naphthalene catabolic plasmid NAH7.the first operon (nahAa,Ab,Ac,Ad,B,C,D,E) encodes the pathway for naphthalene conversion to salicylate upper pathway), and the second (nah G,T,H,I,N,L,O,M,K,J) codes for conversion of salicylate via cathecol meta claevage to acetaldehyde and pyruvate (lower pathway) (ward et al., 2003). The regulator for both operons is encoded by a third operon containing nahR induced by salicylate. The naphthalene dioxygenase is a multi component non-heame iron oxygenase enzyme system consisting of a reductase, a putative Rieske(2 Fe-2S) iron-sulphur center and a ferrodoxin, and an iron-sulphur flavoprotein. The dioxygenase can catalase mono, dihydroxylation, and desaturation, O and N dealkylation, and sulfur oxidation against a variety of monocyclic and heterocyclic compounds (Ward et al., 2003). Naphthalene dioxyganase is a highly versatile enzyme that is able to mineralize other PAHs such as phenanthrene, and anthracene (Sanseverino et al., 1993).

4


Mycobacterium sp strain BB1 degraded phenanthrene, fluorine, fluoranthene, and pyrene, with the highest microbial growth recorded from the LMW phenanthrene. With the knowledge that solubility decreases with increase in number of aromatic rings, and a major limiting factor for an effective biodegradation is bioavailability of the substrate in the environment, bacteria species known to degrade HMW PAHs are likely to posses potentials of degrading LMW PAHs which have a better bioavailability and reduced ring number than the former provided the enzymes systems are present. Microbial degradation of P-Ahs is limited to the amount of the PAH dissolved in the water phase (Ogram et al., 1985), but with the crystalline and non-aqueous phase liquid nature of these compound, dissolve PAHs still remain unavailable to the degrading organisms (Ander et al., 2004). However a bacteria strategy which influences PAH transfer into the cell membrane of the degrading organism where detoxification occurs is the release of small, detergent-like molecules called biosurfactants with a hydrophilic head and a hydrophobic tail. These molecules form spherical micelles forcing the hydrophobic PAHs to become solubilized in the hydrophilic core of the micelles, leading to the transfer of the PAHs from solid, liquid, or adsorbed pool into the water phase. Microbial extracellular polymeric substances excreted by selected bacterial group Pseudomonas also helps in the bioavailability of PAHs and other hydrocarbons.

GENETICS OF PAH DEGRADERS The genetics of PAH degraders is highly dynamic. Pseudomonas sp degrades naphthalene to salicylate is encoded by a cluster of genes nah located on a plasmid. Anthracene, naphthalene, and phenanthrene degradation by Pseudomonas putida is also plasmid mediated pka1, Pka2, pka3 and the NAH7 plasmid (Sansevaring et al., 1993).There are indications that the NAH7 plasmid undergoes recombination with

5


chromosomes as well as plasmids since the phn naphthalene degrading gene of Burkholdeia shows similarities with the nah gene, and the chromosomally encoded nah upper and lower pathway genes in Pseudomonas stutzeri AN10 appear to have been recruited from other organism and combined. mycobacterium sp strain PYR-1 degrades Pyrene, benzo(a)pyrene, by a chromosomal gene nidA, while Rhodococcus also uses a chromosomally encoded gene nidB to degrade toluene, and nidC and nidD to degrade indene(Van hamme et al., 2003). Finally, the broad PAH-degrading capabilities found in some strains mentioned in this text may be attributed to relaxed enzyme specificity for PAHs (both low and high molecular weight and methyl substituted PAHs), the presence of multiple oxygenase, and presence of multiple pathways or multiple genes for iso-functional pathways (Van hamme et al., 2003).

AIMS AND OBJECTIVES The aims are as follows: Isolation of PAH degraders, from polluted soil 1) Identification of the PAH degraders 2) Determination of substrate susceptibility of the PAH degraders on a variety of PAHs 3) Determination of growth profile of the PAH degraders on anthracene and phenanthrene.

6


MATERIALS AND METHODS

SAMPLE COLLECTION Soil samples were collected from two sites: a mechanic workshop at Gowon Estate with a history of contamination of petroleum component such as gasoline, engine oil, brake oil, gear oil, diesel etc; while the second sample was taken for asphalts polluted plant at Berger yard. The surface of the site of collection was scraped to a depth of 5cm and the soil below this to a depth of 5 inches was mixed with a sterile spoon and then transferred to a sterile container, which was properly labeled and dated. Samples were collected at two to three different points at the contaminated site especially at points of high contamination i.e. soil areas where there is great amount of the petroleum product contamination. The samples were taken immediately to the laboratory for analysis.

ISOLATION AND ENRICHMENT OF PAH DEGRADERS PAH degraders were isolated by protein enrichment technique, by introducing 0.5g of the soil sample into sterile minimal salt medium (50ml), containing 50ppm each of phenanthrene, anthracene and pyrene. The cultures were incubated in the dark at room temperature to prevent photooxidation, with intermittent shaking and observed daily for signs of turbidity with a turbidometer. Upon appearance of appreciable turbidity, 0.5ml of the enrichment culture was transferred into a fresh minimal salt medium containing PAHS (this was also incubated under the same condition as above). This transfer was done three times upon appearance of turbidity. Some 0.1ml of the enrichment culture was used for serial dilution, Aliquots of the serial diluents inoculated on LB by spread plate technique. Colonies were picked and reintroduced into fresh medium as (MSM) as above.

7


The flask in which turbidity appeared were plated out again on Luria Berthani agar and several of the aliquot growing colonies were picked and preserved in 50:50 glycerol and Luria Berthani broth medium for further studies and stored in the freezer at 20oC

MEDIA PREPARATION The Media used in this study were Nutrient Agar (NA), Luria Berthani Agar and Minimal Salt medium. (Kastner et al., 1994)

NUTRIENT AGAR Nutrient Agar (14g) was weighed into 500 ml of distilled water in a conical flask enough to 20 plates containing 20ml each. It was shaken properly and kept in a boiling Water Bath to dissolve the Agar properly. The conical flask was plugged with clean cotton wool and wrapped in clean aluminum foil, and sterilized in an Autoclave at about 120oC for 15 minutes in an autoclave; plates were poured aseptically after cooling medium to 45oC. MINIMAL SALT MEDIUM (BROTH) The Minimal Medium described by Kastner et al., (1994) was used its composition are as follow per 1000mls of water: Na2HPO4 (2.13g), Yeast Extract (0.055g), KH2PO4 (1.30g), NH4CL (0.50g) and MgSO4.7H2o (0.2g) into 500 ml conical flask. Nyastatin (50mg) and Cycloheximide (20mg) and Nalidixic acid were added to suppress other bacteria fungi. Trace Element (1ml) was also added and the pH was adjusted within the range of 7.0-7.2.It was sterilized at 1210C for 15 minutes in an autoclave. The supplements were added as sterile filters.

LURIA BERTHANI MEDIUM (AGAR) This media is used for sub culturing organism with the tendency to lose their plasmid upon repeated sub culturing; Peptone (10g), Yeast extract (5g), Sodium Chloride (NaCl

8


10g), Agar (14g), all mixed in a conical flask containing 1000 ml of distilled water, allowed to dissolve for 10 minute after which the mixture is sterilized at 121oC for 15 minutes in an autoclave, the resulting mixture was poured under aseptic condition and left to solidify

TEST ORGANISMS IDENTIFICATION The test organisms were identified on the basis of cultural, morphological and biochemical characteristics according to the method of Cowan and Steel (1997) and Baron et al., 1995, Barrow and Feltham, (1995).

CULTURAL CHARACTERISTICS Isolates exhibited a number of colonial characteristics with respect to shape, size, elevation, translucence and pigmentation.

This provided clues to their probable

identification.

GRAM STAINING: Using aseptic techniques, smears of fresh cultures of 8-24 hours of the isolates were made by placing a drop of water on a clean slide with sterile flamed loop. The smears were air-dried, then the slides were flooded with crystal violet for 1 minute, iodine was added as mordant left for 1 minute and then rinsed off. The smear was then decolorized with 75% alcohol by adding the reagent drop wise until crystal violet failed to wash from the smears. The slides were flooded with safranin as counter stain for 30 seconds. The slides were air dried and examined under oil immersion light microscope to observe the organism for the gram reaction i.e. whether it is a gram positive or gram negative organism.

9


MOTILITY TEST This test was carried out to know if the organism possesses organelle of motility such as flagella or not.

The presence or absence of flagella hence, indicates whether the

organism is motile or non-motile. The hanging drop method was used. A depression cavity slide and a cover slip slide were used. Immersion oil was placed around the edge of a cavity slide. A loop full of each bacterium culture was transferred to the centre of a clean dry cover -slip placed on a staining rack. The cavity slide was the inverted over the drops hangs in between the cavity slide and the cover slide, with the culture now hanging motility was observed under the microscope.

SPORE STAINING: A smear was made from a 48 hours old culture of test organism. This was heat fixed on different glass slides. These slides were flooded with Malachite green stain and heated over a beaker of boiling water to avoid drying. The slides were then subsequently washed and counter stained with safranin for 20 seconds washed, air dried and examined under oil immersion lens. The vegetative portion of the organism stained pink to red, the spore portion was stained green.

BIOCHEMICAL CHARACTERISTICS The isolates were subjected to morphological and biochemical test for their identification according to the method of Baron et al., 1994

OXIDASE TEST This test was carried out to detect the presence of cytochrome oxidase in microorganism. Three drops of freshly prepared oxidase reagent was added to a filter paper and placed in a clean Petri dish, using a glass rod the test organism is smeared on the filter paper and colour changes were observed. The phenylenediamine in the reagent were oxidized to a deep purple colour.

10


CATALASE TEST: Most aerobic organisms are capable of oxidizing hydrogenperoxide to water and oxygen by the use of a catalase or superoxide dismutase enzyme, giving off fumes on contact with hydrogenperoxide. 2H2O2

2H2O + O2

catalase

A smear of fresh culture of (18-20 hours old) the test organism was prepared with the use of sterile distilled water on a clean glass slide. Few drops of hydrogen peroxide was added using a dropping pipette. Effervescence caused by the release of oxygen was observed indicating the presence of catalase enzyme.

INDOLE PRODUCTION Some microorganisms are capable of hydrolyzing Tryptophan, to Indole. Indole reacts with an indicator 4-Dimethylaminobenzaldehyde to form a dome red dye sheath. This test was carried out by growing the isolates in Trypton Broth for 48 hours at 37 oC, after which Chloroform (1-2 ml) was added to the Broth, mixed gently, and allowed to stand for 20 minutes. A cherry red colour at the reagent layer was an indication Indole production.

CITRATE UTILIZATION: The test is used to detect the ability of a microorganism to utilize citrate. In this study the specific medium used was Simon’s citrate medium, which comprises of ammonium salt as nitrogen source and citrate as sole source of carbon. The degradation of citrate leads to the alkalization of the medium, which is detected by the Bromothylmol Blue, a pH Indicator, which changes colour from green to blue.

11


Inoculums of the Isolates were introduced on Simon’s Citrate Agar Slants and incubation at 35oC for 5 days. Observation of colour change from green to blue indicated positive results.

METHYL-RED TEST This test is employed to determine the ability of an organism to ferment a desired sugar. Microorganism that ferment the desired sugars produce acids thus retains the Methyl red colouration (red) of the medium, while other microorganisms which cannot ferment the sugars don’t produce acids thus changes the methyl red colouration to yellow they produce other compounds like acetoin (Acetyl Methyl Carbinol), and diacetyl. The presence of these metabolic products is established by the means of Baritta reagent. Acetone and 2, 3 Butadiol are oxidized to diacetyl which in turn reacts with the reagent (Baritta reagent) to form Guanidine’s, this results in strong alkaline environments. The isolates were inoculated into 10 ml of the MR-VP medium and incubated at 35o C for 2 days. The test was performed after incubation.

VOGES-PROSKAEUR TEST: This test is based on the conversion of Acetyl methyl carbinol to diacetyl through the action of Potassium hydroxide (KOH) and atmospheric oxygen. Diacetyl is converted into a red complex under the catalytic action of two alpha- Naphtol and creatine. The organisms were activated in phosphate buffered glucose peptone medium at 37oC for 48 hours. After incubation, to 1ml of the culture, 0.6ml of alcoholic solution of aloha napthol and 0.2ml of KOH were added and shaken vigorously and placed in a sloping position for 20-30 minutes. The presence of a red coloration in the mixture indicated positive result.

12


HYDROGEN SULPHIDE PRODUCTION: This test involves the decomposition of organic sulphur compounds like Cysteine, Methionine or via reduction of inorganic sulphur compound such as Thiosulphate, Sulphate by microbial activity to release Hydrogen Sulphide. The release of Hydrogen Sulphide is detected by incorporating a heavy metal salt in the medium with which the gas reacts to form black metal sulphides, McCartney Bottles containing 9 ml of Nutrient Broth each were inoculated with test organism. Strips of lead acetate paper were inserted without contact with the medium and kept in the position by the screw up. After about 57 days incubation at 35oC positive result was shown with lead Acetate paper turning Black due to reaction with hydrogen Sulphide.

UREASE ACTIVITY This detects the ability of microorganisms to produce the urease enzyme while growing on an organic nitrogen source Urea. The metabolites formed (i.e. Ammonia and carbon (IV) oxide) produce an alkaline condition which can be detected by a colour change of the pH indicator. Plates of Christensen’s urea medium were inoculated with the isolates and incubated at 37 oC and observed daily for colour changes for 6-7 days. The observations of a colour change from yellow to pink showed a positive urease activity.

SUGAR FERMENTATION The sugar solutions tested for are 1% Glucose, Lactose, Sucrose, Maltose, Fructose, Manitol, Mannose, Ducitol, Sorbitol and Arabinose. The medium used was 1% Peptone water, Phenol red (0.1%) as indicator of acid production. The medium (10 ml) was dispensed into test tubes containing inverted Durham tubes to detect the presence of gas by the isolates (each of the sugars was put in a separate tube). The tubes were then inoculated with the isolates and incubated at room temperature for 5-7 days, the tubes

13


were observed daily for gas in the inverted durham tubes and also for colour changes. The production of acid was indicated by the appearance of a yellow colour in the tubes.

SUBSTRATE SPECIFICITY TEST This was done by the introduction of 0.1 ml of a 24 hours broth culture of the test isolates (AA1 and AA2) each into a separate 250 ml flask containing 99 ml of sterile Minimal Salt Medium and 50ppm of the respective low molecular weight polycyclic aromatic

hydrocarbon

which

include:

Biphenyl,

Naphthalene,

Phenanthrene,

Dibenzothiaphine, Anthracene and Phenol The cultures was incubated in the dark at room temperature and shaken intermittently daily, until turbidity appears. After several transfers, 0.1ml of culture was plated out in Luria Berthamine Agar and the organisms were purified by sub-culturing. Individual subcultures were introduced into a fresh flask to check for the ability to grade low molecular weight PAHs.

TIME COURSE EXPERIMENT The growth profile of the test isolate on low molecular weight PAH was carried out by aseptically introducing of 0.1 ml of the test isolates(AA1 and AA2) , into conical flasks in pairs (total 14 flask) containing 50 ml of sterile Minimal Salt Medium and 50ppm of the respective low molecular weight PAH (Phenanthrene and Anthracene). One ml of the culture (AA1 and AA2) was serial diluted. 0.1 ml of aliquots tubes (-2,-4,-5,snd -7) of culture organisms AA1 and AA2 was plated out on Nutrient Agar using the spread plate technique, observed after 24 hours, and the total Colony Forming Unit (CFU) was counted on plates with 30- 300 colonies. This was done daily for 2 weeks. The time course experiment was complemented by measuring pH changes and optical density which is a function of the turbidity (Monica, 1985).

14


RESULTS Table 1 Shows the cultural, cellular morphology and biochemical characteristics of the isolates AA1 and AA2. Isolate AA1 had a regular rough edges, spread on the line of inoculation, grew blood agar, chocolate agar and MacConkey producing beta haemolysis and non pigmented. Isolate AA2 had a round raised smooth edges, spread also on the line of inoculation and also produced beta haemolysis on blood chocolate and MacConkey agar, however, isolate AA2 had a bluish greenish pigmentation that diffused into the agar. Both isolate are gram negative rods motile, oxidase positive, catalase positive, failed to utilize

citrate,

indole

negative

and

15

failed

to

utilize

urea.


AA1

AA2 Urease Production

+ + +++ -

+ + + ++ + -

16

Fermentation Glucose Oxidation Pyoverdine Pigment Pyocyanine Pigment Pyorubine Pigment Pyomelanine

-

+ + + + + -

producing β-haemolysis.

Table 1: Cultural, Morphological and Biochemical Identification of Isolates AA1 and AA2

Pigment

Fermentation Lactose Oxidation Glucose

Lactose Fermentation

Sucrose Oxidation Sucrose

Fermentation Ducitol Oxidation

Fermentation Manitol Oxidation Ducitol

Manitol Fermentation

Arabinose Oxidation

and no gas as produced -

and no gas as produced

agar and MacConkey

KIA Arabinose

Haemolysis at 37OC

+ Strict Aerobe

CS

CHARACTERISTI

CULTURAL

ORGANISM

-

Strict Aerobe

blood agar, chocolate

Citrase Utilization

inoculation. It grew on

Growth at 42OC

spread on line of

Colour

with smooth edges. It -

Bluish-Green

colony is round, raised

Motility

3.4 mm per Colony. The

Indole Production

The organism measures

Oxidase

producing β-haemolysis.

Catalase

Agar and MacConkey

Spore Staining

Blood Agar, Chocolate

Gram Staining

Inoculation. Grew on

Shape

Spread on Line of

Rod

Irregular/rough edges.

Rod

2.3 mm per Colony.


+ = Positive

- = Negative

17

NR = Not Relevant


Isolate AA1 failed to ferment any of the sugar tested and also failed to produce pigment showing that it is a species of Pseudomonas but not aeruginosa.. On the other hand, isolate AA2 produced pigments pyoverdine and pyocyanin, it fermented arabinose, oxidize sucrose, lactose and glucose and grew at 42OC. These are attributes which conclusively identify it as Pseudomonas aeroginosa. Also isolate AA1 and AA2 show aerobiosis and produce no gas on KIA.

SUBSTRATE SPECIFICITY TEST OF ISOLATES AA1 AND AA2

Dibenzothiaphene

Biphenyl

Phenanthrene

Anthrancene

Naphthalene

Phenol

Substrate

TABLE 2:

AA1

+++

+++

+++

+++

_

_

AA2

++

++

++

++

++

_

Isolate

Table 2 shows the substrate specificity test result of the isolate AA1 and AA2 on the following

PAHs:

Diabenzothiaphene,

Biphenyl,

Phenanthrene,

Anthrancene,

Naphthalene and Phenol. Isolate AA grew very well on dibenzothiaphene, biphenyl, phenanthrene, anthracene but failed to grow on naphthalene and phenol while AA1 grew fairly on the dibenzothiaphene, biphenyl, phenanthrene, anthracene and naphthalene but failed to grow on phenol also. The microbial growth was measured by plate count and turbidity.

30


Fig 1-3 Shows pictures of the experimental cultures used in the project, all except the control which contains no sample organism showed turbidity, indicating a degradation of the PAHs since they are the only carbon sources in these medium. Fig 4-5 Shows the growth profile of the organisms in the substrate measured daily for 8 days. A sharp incline in the growth is noticed, proceeded by a slow decline indicating the usage of the nutrients of the lysed cells by the more adaptive cells. Most importantly the graph indicates a biodegradation of the PAHs in the medium.

31


FIG 1: 14 DAY OLD CULTIRE OF AA1 IN PHENANTHREN AND ANTHRACENE

32


FIG 2: 14 DAY OLD CULTURE OF AA1 IN DIBENZOTHIOPHENE AND BIPHENYL

33


FIG 3: 14 DAYS OLD TURBID CULTURE OF AA1 IN NAPHTHALENE ALONGSIDE A CONTROL MIXTURE

1.20E+08 1.10E+08 1.00E+08

1.00E+08

9.80E+07

9.50E+07 8.90E+07

9.20E+07

8.90E+07 8.30E+07

8.00E+07

8.80E+07

8.50E+07

8.10E+07

7.90E+07

plate count (cfu/ml)

6.80E+07

6.20E+07

6.00E+07

6.20E+07

AA1 4.00E+07

4.80E+07

AA2 2.78E+07

2.00E+07

0.00E+00 0

2.20E+07

1

2

3

4

5

6

7

DAYS (Time) AA1 (Plate Count) AA2 (Plate Count)

Fig. 4: Growth Profile of AA1 and AA2 on Phenanthrene

34

8


1.00E+08 9.40E+07 9.00E+07

8.90E+07

8.00E+07

8.50E+07 8.20E+07

8.40E+07

8.30E+07 7.90E+07

7.80E+07

9.20E+07 9.00E+07

8.70E+07

7.70E+07

7.00E+07

7.00E+07

plate count (cfu/ml

6.20E+07

6.10E+07

6.00E+07

5.70E+07 5.00E+07

AA1 4.00E+07 3.00E+07

AA2 2.52E+07

2.00E+07 2.00E+07 1.00E+07 0.00E+00 0

1

2

3

4

5

6

7

DAYS (Time) AA1 (Plate Count) AA2 (Plate Count)

Fig. 5: Growth Profile of AA1 and AA2 on Anthrancene

35

8


DISCUSSION The two organisms isolated on the enrichment procedure were identified as species of Pseudomonas: Pseudomonas sp. strain AA1 and Pseudomonas aeruginosa AA2. This tallies with earlier report which shows that Pseudomonas generally have good degradation ability on environmental pollutant (Kazunga and Alkins, 2000). Pseudomonades have been shown to have extended ability to degraded polycyclic aromatic hydrocarbon especially low molecular weight fractions (Kastner et al., 2000). The fact that the two Pseudomonas strains could degrade the low molecular weight PAHs tested can be attributed to evolution or the acquisition of this ability over a long period exposure. The high degradative ability of AA1 on PAHs may be attributed to the fact that low molecular weight PAHs are more likely to be encountered in oil polluted site than in asphalt polluted site which are known to contain more of higher fractions such as pyrene. The inability of the isolates to degrade phenol may be due to lack of previous exposure to the various environments from which the organism were isolated, or sensitivity to phenol which is toxic at certain concentration. The inability of AA1 to degrade naphthalene may not be unconnected with bioavailability of the substrate i.e. volatility or lack of genes for such. It is however suprising that the two organisms both degraded anthrancene and phenanthrene because often in literature, organism that been shown to have affinity for phenanthrene and pyrene, two PAHs which shared metabolic pathways (Krivobok et al.,2003) do not have the same affinity for anthrancene (Atlas, 1984; Cerniglia, 1992).

36


CONCLUSION From the results obtained, it is apparent that both Pseudomonas species, strain AA1 and Pseudomonas aeruginosa strain AA2 have good degradative abilities and promising bioremediating potentials. However there is need for further study on their ability to degrade higher fractions of petroleum hydrocarbons in large quantity, and also the physicochemical factors favouring their growth both on the field and in the lab, since culturing these organisms in their unnatural state has always been a great problem.

37


REFERENCES Atlas, R.M. (1984), Petroleum Microbiology. Macmillian publishing Co., New York. Baron, E. J, L. R. Peterson, and S.M. Fingold (1994), Bailey and Scott’s Diagnostic Microbiology 9th Edition. Mosby. St Louise. Barrow, G. I and Feltham, R. R. A. (1995), Cowan and Steels Manual for Identification of Medical Bacteria , 3rd Edition. Cambridge University Press, Cambridge. Boldrin, B; A Thiem and C, Fritzsche (1993), Degradation of Phenanthrene, Fluorine, Fluoranthene, and Pyrene by Mycobacterium sp. Appl. Environ. Microbiol. 23:457-459. Cerniglia, C. E. (1984), Microbial Metabolism of Poly Cyclic Aromatic Hydrocarbons. Adv. Appli. Microbiol. 30:31-71 Cerniglia, C. E. (1992), Biodegradation of Poly Cyclic Aromatic Hydrocarbons. Biodegradation 3:351-368 Cerniglia, C. E., and Heitkamp (1989), Microbial Degradation of Poly Cyclic Aromatic Hydrocarbons (PAH) in the Aquatic Environment, p.41-68. In U. Varanasi (cd), Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. CRC Press, Inc, Boca Raton, Fla. Chen. S. H, and M. D Aitken (1999), Salicylate Stimulates the Biodegradation of High Molecular Weight Polycyclic Hydrocarbons by Pseudomonas saccharophilia P15. Environ. Sci. Technol. 33:435- 439

38


Evans W.C, H. N. Ferneley, and E. Griffith (1965), Oxidative Metabolism of Phenanthrene and Anthracene by soil Pseudomonas. The Ring Fision Mechanism. Biochem. 95: 819-831 Gibson, D. T. (1999), Beijerinckia sp. strain B1: a strain by any other name. Jour. Ind. Microbiol. Biotechnol. 23:284–293. Churchill, S.A., J. P. Harper, and P. F. Churchill (1999), Isolation and Characterization of a Mycobacterium species Capable of Degrading three and four-ring Aromatic and Aliphatic Hydrocarbons. Appli. Environ. Microbiol. 65:549-552 Gibson, D. T., M. Venkatanayarama, D. M. Jerina, H. Yagi, and Yeh (1975), Oxidation of the Carcinogens Benzo(a)pyrene and Benzo(a)anthracene to a Dihydrodiol by a Bacterium. Science. 189:295-297 Heitkamp, M. A, and C. E. Cerniglia (1988), Mineralization of Polycyclic Aromatic Hydrocarbon by a Bacterium Isolated from Sediments Below an oil field. Appli. Environ. Microbiol. 54:1612-1614 Johnsen, A. R., L. Y. Wick, H. Harms (2005), Principle of Microbial PAH- Degradation in Soil. Environ. Pollu. 133:71-84 Kanaly, R. A, and Harayama. S. (2000), Biodegradation of High Molecular Weight Polycyclic Aromatic Hydrocarbons by Bacteria. Jour. Bacteriol. 182:2059-2067 Kastner ,M., M. Breuer-jammali, and B. Mahro (1998), Enumeration and Characterization of the Soil Microflora from Hydrocarbon-Contaminated soil Sites able

39


to Mineralize polycyclic aromatic hydrocarbons(PAH). Appli. Microbiol. Biotechnol. 41:267-273 Kazunga. C, Aitken. D. M. (2000), Products from The Incomplete Metabolism Of Pyrene By Poly Cyclic Aromatic Hydrocarbon-Degrading Bacteria. Appl. Environ. Microbiol. 66:1917-1922 Kelly, L., and C. E. Cerniglia (1991), The Metabolism of Fluoranthene by a species of Mycobacterium. Jour. ind. Microbial. 7:19-26 Kiyohara, H., S. Torigoe, N. Kaida, T. Asaki, T. Iindo, H. Hayashi, and N. Takizawa (1994), Cloning and Characterization of A Chromosomal Gene Cluster, Pah, that Encodes the Upper Pathway for Phenanthrene, and Naphthalene Utilization by Pseudomonas Putida OUS82. Jour. Bacteriol. 176:2439-2443 Krivobok, S.,Kuony, S.Meyer, C. Louwagie , M.Willison, J.C.,Joannaeau, Y. (2003), Identification of Pyrene-induced Proteins in Mycobacterium sp. Strain opy1: Evidence for Two Ring-hydroxylating dioxygenase. Jour. Bacteriol. 183: 3828-3841 Monica. C. (1985), Medical Laboratory Manual for Tropical Countries. Vol. 2: microbiology. First Ed. ELBS. Publishers. Ogram A. V., R. E. Jessup, L. T. Ou, P.S.C. Rao (1985), Effect of Absorption on Biological Degradation Rate of (2,4- Dichlorophenoxy) Acetic Acid in the Soil. Appli. Environ. Microbiol. 49:582-587 Parales R.E., N.C. Bruce, A. Schmid and L.P. Wackett (2002), Biodegradation, Biotransformation and Biocatalysis. Appl.Eviron.Microbiol. 68:4699-4709

40


Philip, W. E. (1983), Fifty years of Benz(a)pyrene. Nature 303:468-472 Sanseverino, J., B. M. Applegate, J. M. Henry King, and G. S. Sayler (1993), PlasmidMediated Mineralization of Naphthalene, Phenanthrene, and Anthracene. Appli. Environ. Microbial. 59:1931-1937 Sarma P. M., Bhattacharrya. D, Krishnans, and B Lal (2004), Degradation of Polycyclic Aromatic

Hydrocarbons

by

Newly

Discovered

Enteric

Bacteria

leclercia

adecarboxylata. Appl. Env. Microbiol. 70:3163-3166 Schneider, J., R. Grosser, K. Jayasimhulu, W. Xue, and D. Warshawsky (1996), Degradation of Pyrene, Benz(a)anthracene, Benz(a)pyrene by Mycobacterium sp. Strain RJGH-135 Isolated from a Former Coal Gasification Site. Appli. Environ. Microbiol. 62:13-19 Sutherland, J. B., F. Rafii, A. A. Khan, and C. E. Cerniglia (1995), Mechanisms of Polycyclic Aromatic Hydrocarbon Degradation, p. 269–306. In L. Y. Young and C. E. Cerniglia(ed.), Microbial Transformation and Degradation of Toxic Organic Chemicals. Wiley-Liss, New York, N.Y. Trezesicka-Mcynarrz, D., and O. P Ward (1995), Degradation of Polycyclic Aromatic Hydrocarbons by a Mixture Culture of and its Component Pure Culture, Obtained from PAH Contaminated Soil. Can. Jour. Microbial. 41:470-476. Van hamme, ,J. D., Singh, O. P. Ward (2003), Recent Advances in Petroleum Microbiology. Microbiol. Mol. Boil. Rev. 67:503- 549

41


Van der meer, J.R., W.. M. DE VOS, S. Harayama, and A. J. Zehnder (1992), Molecular Mechanism of Genetic Adaptation To Xenobiotic Compounds. Microbiol. Rev. 56:677694 Walter, U. M. Beyer, J. Klien and H.J. Relma (1991), Degradation of Pyrene by Rhodococcus Sp. UW1. Appli.Microbiol. Technol. 34: 671-676

42


APPENDIX I

CULTURE MEDIA 1.1

minimal salt medium (Kastner et al.,1994) Na2HPO4

2.13g

KH2PO4

1.3g

NH4CL

0.5g

MgSO4.7H2O

0.2g

Distilled water

500 ml

Trace element

1 ml

pH = 7.0

1.2

Trace Elements ZnSO4.7H2O

100mg/L

MnCL2.4H2O

25mg/L

H3BO3.

300mg/L

CoCl2.6H2O

346mg/L

CuCl2.6H2O

12.5mg/L

NiCl2.6H2O

21.0mg/L

NaMoO4.2H2O

30.0mg/L

pH = 7.0

1.3

Luria Berthani Agar Peptone Yeast extract

10g/L 5g/L

NaCl

10g/L

Agar

15g/L

43


1.4

Nutrient Agar Peptone

5g

Beef extract

3g

Distilled water

100 ml

pH = 8.2

44


APPENDIX II

2.1

Lugol’s Iodine Iodine

1g

Potassium iodide

2g

Distilled water

2.2

300 ml

Crystal violet Crystal violet

0.5g

Distilled Water

2.3

100ml

Methyl Red Methyl Red

2.4

0.4g

Absolute Ethanol

40ml

Distilled Water

100ml

Safranin Solution Safranin

0.25g

Ethanol(90%)

10ml

Distilled Water

2.5

100ml

Voges-Proskauer Reagent CuSO4.H2O

1g

Saturated NaOH

40g

40 ml of the above mixture should be added to 960 ml of

45

10 % KOH solution.


APPENDIX III

GROWTH PROFILE OF ISOLATES AA1 AND AA2 ON PHENANTHRENE AND ANTHRACENE substrate

Phenanthrene

Days

AA1 (cfu/ml)

Anthracene

AA2 (cfu/ml)

AA1 (cfu/ml)

AA2 (cfu/ml)

0

2.78x107

2.2 x107

2.52x107

2.0 x107

1

8.9 x107

6.2 x107

7.8x107

6.1 x107

2

9.5 x107

8.3 x107

8.3 x107

7.9 x107

3

9.8 x107

8.5 x107

8.9 x107

8.4 x107

4

1.0 x108

8.9 x107

9.4 x107

8.7 x107

5

1.1 x108

9.2 x107

9.2 x107

9.0 x107

6

8.8 x107

8.1 x107

8.5 x107

8.2 x107

7

7.9 x107

6.2 x107

7.7 x107

7.0 x107

8

6.8 x107

4.8 x107

6.2 x107

5.7 x107

46


LOW MOLECULAR WEIGHT POLYCYCLIC AROMATIC HYDROCARBONS DEGRADATION POTENTIALS PSEUDOMONAS