A Project Report Submitted In Part Fulfilment Of The Requirement For The Degree Of Masters Of Science In Applied Microbiology & Biotechnology By Ashutosh Gupta Department of Applied Microbiology and BiotechnlogyDr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P) 470 003
Hydrocarbons are organic compounds that consist of only carbon andhydrogen atoms. HydrocarbonAliphatic Hydrocarbon Aromatic Hydrocarbon Term aromatic hydrocarbon or arene was assigned on the basis of factthat many of the compound have sweet essence. Polycyclic aromatic hydrocarbon (PAH), are neutral, non – polar organicmolcecules, consists of three or more benzene ring fused in linear,angular, or cluster arrangement, having alternate double and triple bond.•Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitousxenobiotic environmental pollutant.
Polycyclic aromatic hydrocarbon are more recalcitrant than lowmolecular weight PAHs to microbial attack because of their lipophilicnature, strong resonance energy, strong bonding.Many PAHs exhibit toxic, mutagenic, tumorigenic, and carcinogenicproperties on the basis their structure and mechanism of activation.Hydrophobicity increases and volatility decreases with the number offused benzene ring.The possible fates of PAH degradation in soil are volatilization,photooxidation, chemical oxidation, adsorption, adhesion, andbioaccumulation.Significant loss of PAHs with more than three rings in soil does nothappen abioticallyCompounds with more than five benzene rings, are more resistant tomicrobial breakdown.
NaphthaleneNaphthalene is a white, crystalline, volatile solid and simplest polycyclicaromatic hydrocarbon consist of a fused pair of benzene ring. Properties Description Solubility Benzene, ether, tetrahydronaphthalene, CCl4, Molecular formula & Weight C10H8 and 128.16 Melting Point 80 oC Boiling Point 217.9 oC Uses surfactant, fumigant, insecticides, cloth moths, toilet
Acenaphthene Acenaphthene is a white – light yellowish crystals like solid polycyclicaromatic hydrocarbon (PAH), consisting of ethylene bridge connectingposition 1 and 8. Also known as Ethylenenaphthalene. Properties Description Molecular formula & C10H6(CH2)2 and 154.21 Weight Solubility Chloroform, ether, Benzene Melting Point 95 oC Boiling Point 279 oC Uses Manufacturing plastics & an polyploidy inducing agent..
AnthraceneAnthracene is a tricyclic aromatic hydrocarbon derived from coal tar. Its molecular structure consists of three benzene like rings joined side byside . Properties Description Molecular formula CnH2n-18 Solubility Carbon sulphide, Benzene, Chloroform etc Melting Point 218 oC Boiling Point 354 oC Uses production of dyes, insecticides, wood preservatives etc.
Biological agents degrading Polycyclic aromatic HydrocarbonA diverse group of fungi such as Zygomycete Cunninghamella elegans,the Ascomycetes Aspergillus niger, & Penicillium sp. and the White rotbasidiomycetes Phanerochaete chrysosporium, Trametes versicolor,Pleurotus ostreatus has been demonstrated to oxidize PAHs rangingfrom 2 – 6 aromatic ring .Non – basidiomycete fungi oxidize PAHs rather than minearalizationduring the initial metabolism.Phanerochaete, Trametes, and Agaricales, Pleurotus ostreatus, & othermetabolize anthracene into anthraquinone which can further degradeby Trametes, & other white rot fungi.
Objectives of this study To isolate & identify PAHs degrading fungi from soil. To screen biodegradation of PAHs in isolated fungi. To determine the biodegradability of isolates in varyingconcentration of PAHs. To perform qualitative analysis of PAHs degradation on the basisof fungal biomass.
MATERIALS & METHODSCollection of soil samplesSix oil contaminated soil samples used for isolation of fungi ,were from sixdifferent sites.Each polybag was well labelled with the site of collection.• Petrol Pump Soil From Railway Station, Sagar (M.P.)• Garage Soil, Makroniya• Kabulapur Bus Stand Soil• Civil Lines Garage Soil• Makroniya Garage Soil – 1• Makroniya Petrol Pump Soil
Isolation of indigenous fungi : Primary screening Direct plate Method has been used for the isolation of fungi from soil Pinch of each fine soil sample was sprinkled on solid agar plates havinghydrocarbon as a sole carbon source ,with the help of sterilized spatula. Media & Chemicals For isolation of fungi, Bacto Bushnell – Haas agar media has been used.Special About Bacto Bushnell – Haas Media Chemically Defined, and Contain no any carbon source
Bacto Bushnell – Haas Agar MediaMedia Composition MgSO4 - 0.2 gm CaCl2 - 0.02 gm KH2PO4 - 1 gm FeCl2 - 0.05 gm NH4NO3 - 1 gm Agar – agar - 15 gm Distilled water - 1000 ml pH - 7.0
Preparation Of Stock Solution Naphthalene – 5 gm of naphthalene was dissolved in 10 mlof acetonitrile and heat for a while until crystals disappeared.Acenaphthene - 5 gm of acenaphthene was dissolved in10 ml of acetone and heat for a while until crystalsdisappeared.Anthracene – - 5 gm of acenaphthene was dissolved in 10ml of acetone and heat for a while until crystals disappeared.
Methodology• 0.5% each of ,Naphthalene, Acenaphthene & Anthracene from stocksolution were added in medium as a sole carbon source.• 0.1% of Tween 80 was added to the medium.• Medium were sterilized at 121˚C ,15 lbs pressure for 20 minutes.After sterilization, media was poured in sterilized petri plates aseptically,and all the plates were left for overnight for solidification.Pinch of each fine soil sample was sprinkled on media with the help ofsterilized spatula
All the plates were incubated at 28 oC temperature, for 20 – 25 daysPure isolates were maintained on Potato Dextrose Agar (PDA) slants.•Identification of isolated fungiThe spore bearing mycelia were then carefully sectioned , teased outand stained on a slide using lactophenol cotton blue stain and thenobserved with a light microscope.
Secondary screeningScreening in solid agar plate• Bacto Bushnell – Haas agar media of varying concentration of eachhydrocarbon was prepared.Four different concentration of hydrocarbon are as follows :-1.0% - 10 µg/ml - 1.00 gm dissolved in 100 ml distilled water0.75% - 7.5 µg/ml - 0.75 gm dissolved in 100 ml distilled water0.5% - 5 µg/ml - 0.50 gm dissolved in 100 ml distilled water
Methodology Bacto Bushnell – Haas agar media of four different concentration for each carbonsource was prepared separately , incorporated with 0.1% Tween 80. The medium was sterilized at 121˚C temperature,15 lbs pressure for 20 minutes. All fungal isolates were point inoculated at all plates of different concentration ofeach hydrocarbon All the plates were incubated at 28˚C for 12 - 15 days. After incubation all plateswere observed for fungal growth.
Screening in broth• On the basis of results obtained from Screening in solid agar plates, screening inbroth (Bacto Bushnell - Haas) was carried out.• Based on this, specific concentration of carbon source for screening of isolatesin broth for all three compounds were fixed to be 0.5% (5µg/ml)Preparation of Methylene blue solution (Redox indicator)• 2% (w/v) methylene blue solution as a redox indicator was prepared• 1 gm of methylene blue was dissolved in 50 ml of distilled water. The preparedsolution was sterilized separately at temperature 121˚C , 15 lbs pressure for 20minutes.
Media for Screening• Bacto Bushnell – Haas broth was used for the screening test. Medium weresterilized at 121˚C , 15 lbs pressure for 20 minutes•Methodology• Three agar discs of a pure culture of each isolate were inoculated into 50 ml ofsterilized Bacto Bushnell – Haas broth in well labelled 150 ml Erlenmeyer flask.• 0.5% of each hydrocarbon (Naphthalene, Acenaphthene & Anthraceneseparately),& Tween 80 (0.1%) along with 0.1% Tween – 80 and 0.05 ml ofmethylene blue solution was also transferred to media.••Control flask was also prepared having no organism.All the flask were incubatedat temperature 28˚C with constant shaking at 120 rev/min for 7 days. The aliquotsin the flask was monitored daily for colour change from deep blue to colourless.
Percentage of Methylene blue reductionAfter incubation, broth in flask were subjected to filteration by filter paper toseparate biomass & other, followed by centrifugation at 8000 rpm for 15 minutes.Supernatant thus obtained were analyzed spectrophotometrically at 609 nm (formethylene blue). Percentage of biodegradation was calculated by a formula –% of Degradation= [1- Absorbance of treated sample/Absorbance of control] 100
Biodegradation assay of the isolated culturesThree agar discs of fresh pure cultures of each isolates were inoculatedinto the Bacto Bushnell – Haas broth (50ml/150ml Erlenmeyer flask)containing 0.1% (v/v) Tween 80 & 0.5% (v/v).Each hydrocarbon (Naphthalene, Acenaphthene & Anthracene) was addedto different well labelled flask separately.Redox indicator (Methylene blue) was not added.Control tube without the organism was prepared accordingly.
All the flask were incubated in shaker incubator at temperature 28˚C,120 rev/minfor 7 days.After incubation , broth was filtered through filter paper. The retentate so obtainedcontained only fungal biomass,which was weighed for dry weight of fungalbiomass by constant weighing.
• A total of eight fungal isolates were obtained from Bacto Bushnell – Haasmedium identified as - Fusarium sp., Rhizopus sp., Absidia sp., Aspergillus niger,Aspergillus terreus, Aspergillus versicolor,Trichoderma harzianum & Aspergillus sp.• Among these biodegraders, few organism showed best potential forbiodegradation e.g Fusarium spp., Rhizopus sp., Trichoderma harzianum, &Aspergillus versicolor . It was observed during screening in agar plates, as well asin methylene blue reduction test for all three hydrocarbon compounds.• The ability of these isolates to produce a colour change in the medium ispresumably due to the reduction of the indicator by the oxidized products ofhydrocarbon degradation or due to the fungal growth which utilizes oxygen for theirmetabolism• Among the eight fungal isolates Fusarium sp., Rhizopus sp., Trichodermaharzianum & Aspergillus versicolor displayed the fastest onset of biodegradation.
•Table – I :- Reduction of methylene blue by fungal isolates after 7 days ofincubation in different hydrocarbon Organism Naphthalene Acenaphthene Anthracene Control 1.023 .722 .843 Fusarium sp. .671 .303 .504 Rhizopus sp. .817 .524 .621 Absidia sp. .732 .618 .611 Aspergillus niger .824 .603 .599 Aspergillus terreus .864 .706 .792 Aspergillus versicolor .702 .507 .667Trichoderma harzianum .658 .583 .498 Aspergiilus sp. .857 .318 .653
For NaphthaleneFusarium sp., Aspergillus versicolor, and Trichoderma harzianum gave best resultas compared to other isolates. 34.5 % , 31.37% and 35.68% of biodegradationwere notedRhizopus sp. also gave good results in terms of biodegradation,which wasestimated by their bulk biomass of .562 gm followed with Fusarium sp. with abiomass of .488 gm. Least amount of degradation was showed by Aspergillusterreus & Aspergillus niger with percentage of methylene blue reduction 15.54% and 19.43 %.For Acenaphthene Fusarium sp., and Aspergillus sp. showed best degradation potential . Reductionof methylene blue by 58.04% and 55.96% were recorded after incubation of 7days at 28˚C in shaking condition In terms of biomass, Fusarium sp. , Aspergillus terreus & Trichodermaharzianum having the bulk biomass of 0.766 gm, 0.589 gm, 0.665 gmrespectively.
For AnthraceneOn the basis of methylene blue reduction test and dry weight of fungal biomass,Fusarium sp. , Rhizopus sp. , Trichoderma harzianum and Aspergillus sp. gavebest result of degrading anthracene. 40.22% , 26.33% , 40.93% and 22.54% ofmethylene blue was reduced by these isolates respectively. Rhizopus sp. ,Trichoderma harzianum and Aspergillus sp. gave the biomass of 2.187 gm , 1.244gm and 1.015 gm respectively.
Field application of the isolatesOil spillage, accidental leakage of petroleum products , oil refineries , discharge ofspent oil from garages and others are the major sources of pollution. . Thesemicrobes can remove or reduce to atleast non –toxic level.One approach is to attach or stick these microorganism to some carriers orextenders in the form of powdered or liquid form using Tween 80 which alsoenhances the degradation process. In order to prevent run – off, ‘inert’ stickers oradhesives (such as molasses, corn syrup, latexes) may be incorporated into theformulation. A good sticker combined with charcoal can serve as a protectant.This will reduce the effects of ultra – violet light, dessication other environmentalfactors.The advantage associated with fungal remediation lay primarily in the versatility ofthe technology and its cost efficiency compared to other remediation technology(such as incineration, thermal desorption, extraction). The use of fungi is expectedto relatively economical as they can be grown on a no. of inexpensive forest oragricultural wastes such as corncobs, sawdust. More so, their utilization is agentle non – aggressive approach.
Current Trends In Mycoremediation Of PAHsFungal bioreactors for removal of PAHs is one of the contribution ofbiotechnological approach. During the last decades, fungal bioreactors fordegradation of PAHs have been developed. . These bioreactor are still indevelopmental phase, some of them are Immobilized bioreactor, closed batch feedbioreactor, compost bioreactors and others.Immobilized bioreactors are best known for removal of naphthalene.Goodremoval of naphthalene was attributed to maintenance of immobilized cells ofPhanerochaete chrysosporium in bioreactors.The system is closed batch feed design equipped with gas scrubbers thuspreventing toxic metabolites to escape in environment. Thus system allowssimultaneous determination of mineralization of PAHs and mass recovery.Composting is one of the most promising reactor system for hazardous soiltreatment. Composting can reduce the amount of extractable PAHs by stimulatingbiodegradation or binding of intermediate to organic matter in soil.
In general, fungi have demonstrated their ability to degrade low molecular weightPAHs faster in soil but higher molecular weight PAHs, degradation is limited. Thusit is important to screen isolate and apply fungi that can degrade high molecularweight PAHs efficiently.The use of white rot fungi in soil bioremediation of PAHs is an exciting andpromising technology, because of their ability to degrade highly condensed PAHsits high tolerance to substrate & a large capacity to compete with indigenous soilmicroflora.Fungal degradation of PAHs has been achieved successfully in bioreactors. . Anunderstanding of metabolic pathways and metabolite is crucial to the developmentof successfully strategies for mycoremediation in soil.Extensive research on enzymes involved in metabolic pathways is necessary tooptimize the process of biodegradation by fungi. Genes encoding enzymesinvolving degradation oh high molecular weight PAHs must be cloned, sequenced& characterized. The development of selective bioengineered fungi can open anew era in PAH environment