Fungi Isolated, Diagnosis and Pure from Two Highly Polluted Soils for Hydrocarbon Degradation

Fungi Isolated, Diagnosis and pure from Two Highly polluted soils for Hydrocarbon Degradation

Duaa Hessian HadeDr.Eman.M.Jarallah

Micro Biology /fungi

Abstract

Cultivale fungi were isolated from oily contaminated sites have revealed four fungal species which include :Aspergillus niger,Aspergillus terrus,Aspergillus flavus and penicillum sp .that utilize gasoline as asole carbon source and energy.the aim of the study was to determine the capacity of these fungi to degrade TPH.that changes in PH value,viable counts,optical density at 600nm and dry weights produced by fungal isolate during utilization of gasoline of the zero time to14 day.That fungal isolates showed positive correlation between viable counts and dry weights during incubation period .Hence dry weights increased with a concomitant increase in viable counts .there was also high negative correlation between dry weight and pH values ,the increasing in accompanied with decreasing of environmental pH.

Introduction

Petroleum spillage a source is of severe water and soil pollution that,inaddition to the known environmental problem, reduces microbial diversity through the phenomenon of selectivity. This response of microorganism to organic contaminants has been studied for many years .It has been found that individual microorganisms can mineralize only limited range of hydrocarbon substrates, so assemblages of mixed populations with an overall broad of enzymatic capacities are required to increase the rate and extent of petroleum biodegradation(Mancera-Lopez et al.,2007).The produced water along with the oil and gas during crude oil production .Is it called oil field brine effluent or formation water (Read,1978).Produce water is usually treated before disposal ,because it constitutes asource of chronic pollution since they are continuously discharged in to the open sea over along period of time . The all activities of using crude oil led to pollution risks that can be minimized ,but don’t totally eliminated ,causing sereval problem for the environment (Pala et al.,2006).the oil consists of four groups of hydrocarbons including aliphatics,aromatics,resins and asphaltines(Colwell and Walker,1997).Thepolycyclic aromatic hydrocarbon (PAHS)and derivaties are widespread products of incomplete combustion of organic materials arising from natural combustion such as volcanic eruption (Dasilva et al.,2003;Pang etal.,2003). The major (PAHS)pollutions are dustrial in activities,trallsporation ,refuse burning ,gasification and plastic waste incineration (Mrozik et al.,2003). TheBenzo(a) Pyrene(Bap)5-ring hydrocarbons its chemical structure is highly recalcitrant and resistant to microbial degradation.Crude oil including microorganisms and plants (Dariush etal.,2007). The various microorganisms are able to use some crude oil fraction as sole carbon source and change these component to non-toxic materials such as CO2 and H2O(Ewis etal.,1998). The major gasoline including (benzene, toluene and xylene-BTX)are soluble in water and are considered human carcinogens (Claudia and Selma,2000).Numerous microorganisms have their ability to degrade hydrocarbons .The biodegradation capabilities of fungi have been subject in many due to their capable to synthesize unspecific enzymesvolved in cellulose and lignin degradation (Colombo etal.,1996;Krivobok etal.,1998.,Salicis etal.,1999;Garcia etal.,2000;Santos and Linardi,2004;Potin etal.,2004). ,beside their ability of degrading high molecular weight, including aromatic structures. the degradation of hydrocarbons by filamentous fungi and yeasts has been investigated previously and it was concluded that most fungal species are excellent hydrocarbon degraders(Sutherland,2004.,Gadd,2001).

Materials and Methods

Isolation of strains from contaminated soils

Cultivable native microorganisms were isolated from the severely ,long –term contaminated soils. strains were isolated from polluted soil previously biostimulated with a mineral solution specific to bacterial or fungi cultivation .Biostimulation was performed in 125 ml Erlenmeyer flasks ,where 10g of polluted soil was humidified with 10ml of mineral medium . The Erlenmeyer flasks were incubated at environmental temperature for 7 days . The microorganisms were separated from the soil particles by gentle shaking of 1g soil (dry weight) with 10ml of sterile water for 30min .After sedimentation ,the supernatant suspension was used to prepare appropriate dilutions (from 1ҳ10-1to1ҳ10-3)with sterile water .Aliquots of 0.2 ml were spread –plated on the appropriate medium (Sabouraud maltose agar and Czapek agar). The plates were incubated at 28Cºfor 7 days . The microorganisms were allowed to spread unit purification and were then conserved in refrigerator ready for use in the production of inoculate for the hydrocarbon degradation experiments (Mancera-Lopez et al.,2007).

Determination of the ability of fungal isolates to utilize gasoline

Known volume of 150 ml of the basal medium (minimal salt medium, composition10 g NaCL,.29 g KCL,.42g MgSo4,.83g KH2PO4,.42g NaNO3 ,.1.25g NaHPO4,100ml distilled water ,pH 7.2)was dispensed in to 250 ml conical flasks and gasoline were introduced separately in to flasks at 1.0% v/v after sterilization (Okpokwasili and Okorie,1998).Overnight broth cultures (35g/l of malt extract broth for fungi) of each organism was seeded in to each flask and incubated in shaker incubator (New Brunswickm Scientific Incubator shaker) at 150rpm/min and 30Cº .Utilisation of gasoline was monitored at two days interval for 14 days by monitoring fungal growth measured by viable count on nutrient agar .the optical density was determined at 600 nm wavelength with PG T70 U.V/VIS spectrophotometer and changes in ionic concentration ,p H ,was determined with pH meter .Fungi was harvested on the filter paper by filtration and dried in the oven ,the weight was determined.

Results

The characterization of fungi isolated from oily derivties contaminated sites have revealed four fungal species which include :Aspergillus niger,A.flavus,A.trreus and Penicillium sp that could utilize gasoline as carbon source.No significant difference was observed in the changes in PH values obtained on gasoline by fungal isolates respectively (p=226).there was significant difference (p=002) in the pH values of the fungal isolates .thesevalues were dropped significantly during utilization of gasoline by all fungal isolates from the first to the 14 th day of incubation .A.terrus had the lowest pH of 5.29 after 14 days of incubation (fig1).A.flavus produced the lowest pH of 5.29 on gasoline while A.niger recorded the highest pH of 5.32 on gasoline (fig1) .Investigating the ability of four fungal species to utilize Gasoline as sole carbon source .

Fig1:changes in pH value Produced by fungalisolates during utili zation of gasolin

The viable counts of all fungal isolates increased significantly from 0h to the 14 th day of incubation during the utilization of gasoline .there was no significant difference observed for viable counts on gasoline for funal isolates respectively.A.niger had the highest viable count value of 30х10-3CFU\ml on gasoline after 14 days of incubation on crude oil while A.terrus and penicillum sp both recorded the lowest viable count value of 22х10-3 CFU\ml (fig.2) after the 14 th day of incubation .

changes in viable counts of N.A Produced by fungal isolates during utili zation of gasolin :2Fig

optical density values increased significantly from zero time to 14 th day of incubation during the utilization of gasoline by fungal isolates .A.terrus had the highest optical density value of 450nm on gasoline while penicillium sp had the lowest optical density valueof400 nm when grown ongasoline (Fig.3).

Fig 3:changes in optical density nm Produced by fungal isolates during utili zation of gasolin

Dry weight values increased significantly from 0h to the 14 th day of incubation during the utilization of gasoline by fungal isolates .Mean while dry weight values on gasoline showed no significant difference (p=212).there was significant difference in dry weights of fungal isolates .A.terrus and penicillium sp both had the highest dry weight value of 2.2 on gasoline while A.niger and A.flavus both had the lowest dry weight value of 1.9 on gasoline (Fig.4).

changes in dry weights Produced by fungalisolates during utili zation ofgasoline:Fig 4

correlation analysis results of the fungal isolates showed positive .the correlation between viable counts and dry weights .Hence dry weights increased with a concomitant increase in viable counts .There was also high negative correlation . between dry weight and pH ,hence as dry weight increased pH decreased concomitantly.

Discussion:

The fungi isolated from contaminated soils indicated the prevalence of Aspergillusflavus have shown the best abilities of utilize and degrade gasoline.Oboh et al.(2006) have reported the abilities of fungal species which included ;Aspergillus sp and Penicillum species to grow on gasoline as the sole carbon and energy source when screened for hydrocarbon utilization .the major genera of fungi active in polluted soils were Aspergillus sp and Penicillium but the said (Nkwelang et al.,2008).the major genera of fungi active in polluted soils were Aspergillus ,Penicillum and Mucor .Santos et al.(2008).reported the ability of Aspergillus sp to biodegrade gasoline.Uzoamaka et al .(2009). Reported that the eight isolates showed potentials for hydrocarbon biodegradation areAspergillusversicolor,A.niger,A.flavus,syncephalastrum spp.,Trichoderma spp.,Neurospora sitophila,Rhizopus arrhizus and Mucor spp. Atlas and Bartha(1972).observed thant both water in oil and oil in water emulsions are formed following oil spillage.the ability of the microorganisms to lower the interfacial tension will increase the interface and thus accessibility of the hydrocarbon substrate .the two phase liquid medium where the bulk of the carbon and energy source are found is water insoluble and all other minerals nutrients are dissolved in the water phase .the physical state of the petroleum hydrocarbon is known to have amarked effect on its biodegradation ;the more soluble it is in water the more liable it is to microbial attack.the hydrocarbon degrading organisms act mainly at the oil and water interface .the reduction in PH of the culture fluids in flasks within the 14 day incubation period confirmed chemical changes of the hydrocarbon substrates which must have been precipitated by microbial enzymes (Atlas and Bartha,1972).Hydrogen ion concentration is major variable governing the activity and composition of fungi many species can metabolise over awide pH range from the highly acidic to alkaline extremes .the growth profiles of the fungal isolates on gasoline revealed asharp drop in pH .The insensitivity of the fungi to high hydrogen ion concentration and narrow pH range of most bacteria account for the sharp drop in pH .microbial degradation of hydrocarbons often leads to production of organic acids and other metabolic products (Nwachukwu and ugoji,1995;Okpowasili and james,1995).the organic acid of produced account for the reduction in pH levels (Oboh et al.,2006).this may be followed by aconcomitant development of the ability of the organisms to emulsify petroleum hydrocarbons and which is a major factor in hydrocarbon up take and assimilation .Ptaek et al.,(1987). Reported that many of the petroleum degrading bacteria produce extracellular emulsifying agents .utilization of gasoline resulted in increase in cell densities with aconcomitant reduction in the oil layer complete disappearance of crude oil and gasolie with prolonged incubation.According to Dariush et al.(2007).increasing crude oil concentration decreased the reduction of crude oil in non vegetated and vegetated soil samples .In all the contaminated non vegetated soils ,In the higher concentration the difference of crude oil . the reduction was significant between the vegetated and non vegetated samples in concentrations up to 5%,while the reduction was non significant between the vegetated andnon vegetated samples in concentrations 7% to 10% (Dariush et al.,2007).the biodegradation of contaminants is the best means to completely remove oil pollutants .Biodegradation should be accelerated in order to develop a faster means of cleaning up pollutants .they key to increasing the rate of biodegradation of contaminant is to optimize the growth rate of indigenous soil degrading microflora.the finally increasing biodegradation rates of indigenous microorganisms is the best option to maximize contaminant clean up when all avenues have been exhausted .this observation has been reported previously (Oboh et al.,2006). And can be attributed to genetic make-up due to the constitutive expression of physiological owing to previous exposure of exogenous hydrocarbon or hydrocarbon catalyzing enzymes present in the contaminated soils.

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