BIOREMEDIATION PROSPECTS OF FUNGI ISOLATED FROM WATER SOLUBLE FRACTION OF CRUDE OIL SAMPLES. Edema, N.E. and Okungbowa, F.I.

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1 Ife Jurnal f Science vl. 14, n. 2 (2012) 385 BIOREMEDIATION PROSPECTS OF FUNGI ISOLATED FROM WATER SOLUBLE FRACTION OF CRUDE OIL SAMPLES 1 2* Edema, N.E. and Okungbwa, F.I. 1 Department f Btany, Faculty f Science, Delta State University, Abraka. 2 Department f Plant Bilgy and Bitechnlgy, Faculty f Life Sciences,University f Benin, Benin City. *Authr fr crrespndence. fikun2002@yah.cm. Phne: (Received: September, 2012 ; Accepted: Nvember, 2012 ) ABSTRACT The fungi assciated with water sluble fractin (WSF) f crude il frm tw different lcatins were investigated. The samples were cllected frm Ezibin il well (Sample A), Okwagbe village in Ughelli Suth Lcal Gvernment Area f Delta State and frm NPDC labratry (Sample B) in Benin City, Ored Lcal Gvernment Area f Ed State. The WSF f Samples A and B were used at full strength (100%). Bth samples were cultured n saburaud dextrse agar (SDA). The salinity level, inic cntents, heavy metals and physical characteristics were determined. The fungi islated and identified were Trichderma harizianum (fund in bth samples), Aspergillus accileatus frm Sample A nly and Trichderma reesei frm Sample B nly. The values f sum f ins, physical characteristics (Sum f EC + TDS) and the analyzed values were higher in Sample A than Sample B. Sample A was mre acidic (ph5.6) than Sample B (ph6.5). Sample A shwed significant difference at (P<0.005) in the level f ins, while Sample B shwed n significant difference (P>0.05), but had significant difference at P<0.05 in the level f ph, EC and TDS. The ability f the fungi t adapt t these cnditins indicates their ptential as a tl fr biremediatin f crude il plluted water. Keywrds: Biremediatin, Crude Oil, Fungi, Plluted Water, Ptential. INTRODUCTION Crude il is a naturally ccurring cmplex mixture f hydrcarbn and nnhydrcarbn cmpunds. Several studies n spills n the envirnment in the NigerDelta area and ther trpical areas thrughut the wrld cnsistently shwed that the areas which are directly expsed t large r repeated il spills exhibit lng term envirnmental prblems. Oil pllutin is ne f the envirnmental cnsequences f crude il explratin and explitatin activities prducing aquatxiclgical effects which are deleterius t aquatic life (Agbgidi et al., 2005; Edema et al. 2007). Water sluble fractin (WSF) is a single phase slutin cmprised f individual disslved mlecules. Organisms expsed t water sluble fractin f crude il take up the disslved hydrcarbn and react t their effects (Overtn et al., 1994), and these effects n the grwth and develpment f different rganisms have been studied (Edema et al., 2008). Fungi have evlved a high degree f metablic versatility that allws them t use a diverse range f rganic substrates fr grwth, including simple cmpunds such as nitrate, ammnia, acetate r ethanl. The grwth f fungi as hyphae and single cells in aquatic envirnment is adapted fr the efficient extractin f nutrients, because these grwth frms have high surface area t vlume rati. Fungi are diverse in their eclgical adaptatin; sme f these have been said t help in biremediatin (Kacprzak et al., 2005). They have ptential fr cleaning up the envirnment in prcesses such as detxifying nxius chemicals and remving il spills. Fungi are knwn t degrade armatic hydrcarbns. They adapt mre readily t diverse cnditins fr lw misture and lw ph as cmpared t mst bacteria. They are thus useful in predicting the impact f a particular stress n the envirnment by their ability t respnd t these adverse cnditins thrugh a change in their number (Alexpuls et al., 1996). Uzamaka et al. (2009) reprted that sme fungal islates that were recvered frm il cntaminated sils shwed ptentials fr bidegradatin. Andrea et al. (2001) reprted that fungal species such as Trichderma and Phanerchaete have been implicated in hydrcarbn degradatin f crude il cntaminated sils. Amng these islates, Aspergillus versiclr and Aspergillus niger were shwn t display the fastest and highest extent f bidegradatin and the implicatin f these tw rganisms in hydrcarbn degradatin has been reprted by April et al. (2006). Salt level r salinity is the saltiness r disslved salt

2 386 Edema and Okungbwa.: Biremediatin Prspects f Fungi Islated frm Water Sluble cntent f a bdy f water. It is a general term used t describe the levels f different salts such as sdium chlride, magnesium, calcium, sulphates and bicarbnates. Salinity is nt expressed as percent, but as part per thusand ( / ) Mantyla (1987), which is apprximately amunt f salt (in grams) per kilgram f slutin. Salt is a natural element f sils and water. The ins respnsible fr salinizatin are Na, K, Ca, Mg and Cl. Salinity is the sum weight f many different elements within a given vlume f water (Mantyla, 1987). The salt fund in crude il is disslved in water f the crude il and nt in crude il itself. The salts that are mst frequently present in crude il are calcium, sdium and magnesium chlride (Pinet, 1996). The envirnment cntains a wide range f different filamentus fungi that degrade hydrcarbn (Bijfp, 2003). Micrbita ppulatins shw a rapid increase in respnse t mderate level f il, much f the ppulatin increase is due t increase in il degrading micrflra. Mst f the restratin f plycyclic armatic hydrcarbn (PAH) cntaminated sites depends n the use f plants fr phytremediatin r the use f fungi fr mycremediatin. The aim f this study was t determine the salinity, inic cntent and physical prperties f water sluble fractin f crude il as well as t islate and identify the fungi assciated with it, in rder t ascertain the pssible use f the fungi in biremediatin. MATERIALS AND METHODS Cllectin f crude il samples The crude il samples were cllected frm Ezibin il well, Okwagbe Village, Ughelli Suth Lcal Gvernment Area, Delta State (Sample A) and Ored flwstatin, Natinal Petrleum Develpment Crpratin (NPDC) labratry in Benin City, Ed State. The experiments were cnducted in Btany, Chemistry and Micrbilgy labratries f the Delta State University, Abraka. Preparatin f WSF The WSF was prepared accrding t the methd by Andersn et al. (1974). A sample f crude il (500 ml) was slwly mixed in 500 ml deinized water in a 2 litre cnical flask. Using an rbital magnetic stirrer with a magnetic rd, stirring was dne fr 24 hurs at rm temperature (28±2 C) fr bth samples (A and B) f crude il. After 24 hurs f stirring, the ilwater mixture was allwed t stand fr anther 24 hurs in a separating funnel; the lwer phase f the ilwater mixture was cllected and used as 100% strength fr bth samples. Preparatin f WSF culture The WSF samples were cultured n ptat dextrse agar (PDA) (Oxid, England) using the pur plate methd. A vlume f 1ml f each WSF cncentratin was transferred int a Petri dish. Then 20 ml f PDA, prepared accrding t manufacturer's instructins and cntaining 50µg/ml chlramphenicl t inhibit bacteria grwth, was pured int the plate. The plate was swirled rund fr even mixing and allwed t slidify. Cntrl plates cntained 1ml deinized water instead f WSF. All culture plates were incubated at rm temperature (28±2 C) fr 7 days. Culture samples were examined under lw (x40) and high (x100) pwers f an ptical micrscpe fr fungal grwth. Fungal identificatin was dne by CABI, U.K. Determinatin f prperties f WSF The physical characteristics, inic cntents and heavy metals determinatins were dne accrding t the methds described by Ademrti (1996) and Trivedi and Raji (1997). Statistical analysis The data were subjected t statistical analysis f variance (ANOVA) using mean ± standard errr, cmparisn test (Turkey Multiple Cmparisn Test). The significance was tested at 5% prbability level. RESULTS Three fungal species were islated (Table 1) and identified as Trichderma harziarium, Trichderma reesei and Aspergillus acculeatus. Trichderma harziarium and Aspergillus aculeatus were assciated with Sample A, while Trichderma harzianum and T. reesei were assciated with Sample B. The study shwed that the WSF cntained sme amunt f salt (Table 2). The ttal salinity in Sample B was higher than Sample A. Table 3 shws that the ph values fr Samples A and B were 5.60 and 6.50, respectively. Sample A was mre acidic than

3 Edema and Okungbwa.: Biremediatin Prspects f Fungi Islated frm Water Sluble Sample B. Samples A and B shwed a significant difference (P<0.05) in the level f EC and TDS (Table 4). The sum f physical characteristics had the least values. Heavy metals namely, Fe, Cu, Cr, Pb, Va, Zn and Table 1: Fungi Islated frm WSF Samples Fungi Islated Sample A Sample B Trichderma + + harzianum Trichderma + reesei Aspergillus + aculeatus Fungi identificatin was dne by CABI, UK. Mn were fund t be present (Table 5). Zinc had the highest value (2.46) in Sample B fllwed by Mn (2.45) in Sample A. Vanadium had the least value (0.01) in Sample A while Cr was nt detected in Sample B. Table 2: Ttal Salinity f WSF f Samples A and B using three Measurements Ttal Salinity Sample A Sample B / 00 / 00 ( parts per thusand) Sum f Ins Sum f physical characteristics EC + TDS Analysed values Table 3: Ins in WSF f Samples A and B Ins Sample A Sample B 2+ Mg 0.82± ± Ca 0.86± ± K 0. 78± ± Na 1.25± ±0.01 Cl 2.04± ±0.01 NO ± ± SO ± ±0.01 HCO ± ±0.02 PO ± ±0.00 Sum f Ins *Values in mean ± S.E. (P<0.05) Table 4: Physical Characteristics f the WSF f Crude Oil f Samples A and B Physical characteristics Sample A Sample B ph 5.60± ±0.00 EC 0.31± ±0.00 TDS 0.07± ±0.00 Sum (EC + TDS) 0.38± ±0.00 *Values f mean ± S.E. (P<0.05) 387

4 388 Edema and Okungbwa.: Biremediatin Prspects f Fungi Islated frm Water Sluble Table 5: Heavy Metals in WSF f Crude Oil f Samples A and B Heavy Metals Sample A Sample B Fe 0.16± ±0.00 Cu 0.16± ±0.00 Cr 0.02±0.00 ND Pb 0.03± ±0.00 Va 0.01± ±0.00 Zn 2.10± ±0.00 Mn 2.45± ±0.00 *Values in mean ± S.E. (P<0.05) ND: Nt Detected DISCUSSION Trichderma species have been implicated in hydrcarbn degradatin f crude il cntaminated sil (Andrea et al., 2001). Als, sme fungal islates such as Aspergillus species and Trichderma species have been reprted by April (2000) t shw ptentials fr hydrcarbn bidegradatin. Als, April (2000) shwed that Aspergillus species display the fastest and highest extent f bidegradatin and implicatin f the rganism in hydrcarbn degradatin. Trichderma and Aspergillus spp amng ther genera f fungi are knwn t metablize hydrcarbn r thrive in il cntaminated sites (Llans and Kjller, 1976; Bartha and Atlas, 1977). It has als been reprted that Aspergillus and Penicillium species were mre efficient metablizers f hydrcarbn than ther fungal species such as Candida, Mucr, Fusarium, Getrichum, Rhdtrula and Trichderma (Obire et al., 2008). The islatin f Trichderma and Aspergillus species frm the WSF samples therefre, supprts these previus findings. The salinity result was in agreement with Edema (2006). The salts that are mst frequently present in crude il are calcium, sdium and magnesium chlride (Pinet, 1996) and these were amng the detected salts in the current reprt. The ins respnsible fr salinizatin are Na, K, Ca, Mg and Cl. These ins invariably cntributed t the high salinity f the samples recrded in this wrk and partly explains the higher salinity f sample B. Salinity is the sum weight f many different elements within a given vlume f water (Mantyla, 1987). Salinity is an eclgical factr f cnsiderable imprtance influencing the type f rganisms that live in a water bdy (Lewis, 1982). Several elements were detected. Hwever, the salinity result may be attributed t the absence f inized cmpnents f ther elements such as N, P, Fe and numerus minr elements which are f immense bilgical imprtance but are usually minr cntributrs t ttal salinity (Wetzel, 2007). Ttal disslved slids and inic cnductivity f water are generally used measurements (Cvich, 1993). These prperties regulate the ph. The lw ph cnditins under which the fungi thrived is in agreement with (Alexpuls et al., 1996) that fungi adapt mre readily t diverse cnditins f lw ph. Sites cntaminated by rganic cmpunds have been shwn t be characterized by heavy metals (Buchez et al., 2000). In such a difficult case, sme wrkers pine that the use f filamentus fungi may give sme advantage ver bacterial biaugmentatin. Fungi display a high ability t immbilize txic metals (Baldrian, 2003). Fungal biremediatin has been successful fr cleanup f pentachlrphenl (PCP), a wd preservative and plycyclic armatic hydrcarbn (Andrea et al., 2001).. REFERENCES Ademrti, C.M.A Standard Methds fr Water and Effluent Analysis, March Publishers, Benin City, pp Agbgidi, O. Oknta, B. and Dlr, D Sciecnmic and envirnmental impact f crude il explratin and prductin n agricultural prductin: A case study f Edjeba and Kkri cmmunities in Delta State, Nigeria. Glb. J. Envirn. Sci. 4(2): Alexpuls, C., Mims, C. and Blackwell, M. th (1996). Intrductry Myclgy (4 editin). Jhn Wiley and Sns, 30pp. Andersn, J.W., Neff, J.M., Cx, B.A, Tatan, H.E. and Hightwer, G.M

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