ISOLATION AND CHARACTERISATION OF AQUATIC FUNGI …

Nigerian Journal of Mycology Vol. 9 (2017) 40

ISOLATION AND CHARACTERISATION OF AQUATIC FUNGIFROM OPEN DRAINS IN PORT HARCOURT MUNICIPALITY,

SOUTHERN NIGERIA.

*Ogbonna D. N. and Okorie S.Department of Microbiology

Rivers state University of Science and TechnologyNkpolu-Oroworukwo, P.M.B 5080, Port Harcourt, Nigeria.

E-mail: ogbonna. david@ust. edu. ng**Correspondencing author

AbstractGari slide culture media was used to isolate fungi from open drains in Port HarcourtMunicipality. Fungi grown onto Sabouraud Dextrose Agar (SDA) were purifiedby repeated sub-culturing unto gari slide culture media. Microcultures of the fungiisolated on SDA were made by transferring spore suspensions of each isolate ontoa 1 cm disc of the same medium placed on a microscope slide in a Petri dish. Allinoculated slides were incubated at 30±20C for 5 days, and then examined underthe microscope. Fungi isolated on SDA and gari medium were Aspergillus niger,Aspergillus tamarii, Aspergillus versicolor, Cryptococcus neoformans, Mucor spp,Penicillium chrysogenum, Penicillium marneffei, Phoma spp, Rhizopus spp,Scopulariopsis spp and Torulopsis glabrata. Also a micrograph of Torulopsis sppand Cryptococcus spp as yeasts on Gari slide culture medium are presented.However, the patterns and rate of growth of the fungal species were more insediments than in water samples which could be responsible for the degradationof receiving water bodies.

Keywords: Aquatic fungi, open drains, Gari slide culture media

INTRODUCTIONMicroorganisms are present in all

natural habitats, as well as in human-made environments such as opendrainage channels. Such reservoirs havefeatures such as darkness, long retentiontimes and stagnation zones that favourthe proliferation of microorganisms

(especially fungi) and biofilm formation,as well as, toxin production (Hageskalet al., 2009). Apart from their significantecological role in helping nutrientturnover, water can becomecontaminated or highly impacted bytheir activities (Hageskal et al., 2006;Besner et al., 2011; Douterelo et al.,

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2014, EPA, 2016 ) causing bad taste andodour. Through the inhalation of sporesafter aerosolisation of water, especiallywhen water passes through taps andshowers water loses its potability(Warris et al., 2001; Hageskal et al.,2009; Anaissie et al., 2011; Richardsonand Richardson, 2015).

Studies of the structuralcharacteristics of moulds could besimplified by placing on microscopeslides (about 1 cm square) blocks of agarcut from appropriate culture media; theupper edges of the agar blocks areinoculated with the desired moulds; thenafter adequate incubation of inoculatedblocks, the slides along with the agarblock are examined under themicroscope (Sokari et al., 1996). Thisis because moulds can be identified tothe generic level often without specialstaining or biochemical techniques sincetheir structural details could be readilyvisible microscopically. However, thestructural differences can be readilyobserved only if the mould colony is notdisturbed during preparation ormounting for microscopic examination(Sokari et al., 1996), and this is achievedby means of monocultures onmicroscopes slides. The aim of thepresent study, therefore, was to identifyfungi isolated from open drainagechannels in Port Harcourt using garimedia as an inexpensive, but effectivealternative culture medium to

conventional dehydrated mycologicalmedia for isolating and identifyingmoulds.

MATERIALS AND METHODSCollection of sample

Wastewater samples were collectedfrom open drains along the Ntanwogbacreek with sterilized plastic bottles. Eachsample bottle was rinsed with theappropriate sample before the finalcollection according to the standardmethods (APHA, 2012). To collect thewater sample, base of the sterilizedsample bottle was held with one hand,plunged about 30cm below the watersurface with the mouth of the samplecontainer positioned in an oppositedirection to water flow (APHA, 2012).The container was filled with wastewatersamples from different locations startingfrom the upstream (Afam /Kaduna streetbehind the Winners chapel) to thedownstream (at Abacha road, all sites inPort Harcourt Rivers State Nigeria )leaving a gap of about 2cm and thencovered.

Sediment samples for analysis werealso collected along the same watercourse. To collect the sediment samples,the bottles were opened and held withthe left hand while using the right handwith a plastic scooper to scoop thesediment sample. The sample bottleswere filled with sediment sample andcovered immediately. Thereafter, each

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sample was immediately labelled andtransported in a cooler packed with iceblocks for analysis. Sample collection

was carried out from February to August2016.

Fig 1. Map of Port Harcourt showing sampling stations

Mycological StudySerial dilution

Ten-fold serial dilutions of thesamples were made according toOliveira et al. (2016).

Inoculation and incubationOne milliliter of appropriate ten -

fold serial dilution of the sample wasinoculated onto appropriate Sabaurauddextrose agar in triplicates using pour

plate method of Hageskal et al. (2009)and spread plate method of Oliveira etal. (2016). Inoculated plates wereincubated at 28±20C for 48-72 hours.Visible discrete colonies on incubatedplates were counted and expressed ascolony forming unit per gram (cfu/g) ofsediment samples and colony formingunits per milliliter (sfu/ml) of wastewater samples.

Isolation and Characterisation of Aquatic Fungi Isolated from Open Drains in Port Harcourt Municipality, Southern Nigeria


Maintenance of pure cultureFungi grown onto SDA were

purified by repeated sub-culturing untogari slide culturing media. Pure cultureswere preserved on gari slide culturemedia at ambient temperature (28±20C)for further tests.

Garri slide culture methodAccording to the method of Sokari

et al. (1996), individual, fairly largegrains of gari were placed in a glass Petridish and autoclaved at 1210C. Using aflamed forceps, one granule of the gariwas transferred from the glass Petri dishunto a glass slide. Thereafter, fungalgrowth was touched with a wireinoculating needle and then placed ongari granule on glass slide. Inoculatedgranule was transferred into a sterilePetri dish layered with moistened cottonwool, and incubated at 300C for 3-5days.After incubation, structures of fungalgrowth were enhanced by touching edgeof coverslip with cotton blue inlactophenol. Identification of fungalspecies was done phenotypically basedon macroscopic and microscopicmorphological features of cultivation ingari slide culture medium.

Characterization and identification offungal isolates.

Pure cultures of fungal isolates wereidentified based on cultural parameters,microscopic technique and biochemical

tests including carbohydrate utilizationas described by Cruickshank et al.(1975). Characterization and identifi-cation of fungal isolates was doneaccording to Kelly et al. (2003) andSamson et al. (2004)

RESULTS AND DISCUSSIONPredominant fungal isolates from the

open drains were characterized andidentified as Aspergillus niger,Penicillium chrysogenum, Aspergillustamarii, Cryptococcus neoformans,Aspergillus versicolor, Torulopsisglabrata, Rhizopus spp, Mucor spp,Scopulariopsis spp Penicilliummarneffei and Phoma spp (Table 1; Figs1-9 a and b). Species of Torulopsis andCryptococcus (Table 2; Figs 10-11 a andb) were the only yeast isolates from theopen drainage system. Fungi areubiquitous in soils and wastewaterpreferring cool and moderate climate,especially with the abundance of organicmaterials (Nicoletti et al., 2009).However, Cryptococcus neoformans andRhizopus species were isolated morefrequently from both water and sedimentsamples in the open drains. The patternsand rate of growth of the fungal specieswere more in sediments than in watersamples. Doggett (2000) had earlierreported densities of filamentous fungiand yeasts in similar systems. Theirpresence in wastewater indicates thatthere is possible contamination by fungal

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pathogen (Prescott et al., 2005; Oliveiraet al., 2016) which may be responsiblefor the degradation of receiving waterbodies. Their presence was most likelythrough the runoff of fertilizers orsewage which contain excess nutrientthat plants, algae and fungi can utilizefor growth. Their abundance andactivities in drainage systems have

profound effect on the physicochemicalcharacteristics of the wastewaterespecially at the banks of the creek. Thisis as a result of drastic narrowing of thecreek channels induced by the decliningriparian vegetation along the systemparticularly at the degrading wastewateropen drains.

Table 1. Morphological Characteristics and identity of fungal Isolates



Plate 1(a) Morphological features ofPenicilium chrysogenum as seen on garimedium

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CONCLUSIONFungi isolated from all the open drainsexamined are considered to producemycotoxins and/or are opportunistichuman pathogens. Water storage indrainage systems generates stagnation,stratification, particle accumulation,dead zones, depletion of residualdisinfectant, and biofilm formation.These parameters combined withchemical, physical characteristics of thewater system (like high turbidity andtemperature, pH, total organic carbonand dissolved oxygen), are favorable tomicrobial growth making reservoirs apotential high risk of water qualitydegradation by fungi. In this currentwork, all sampling sites presentedAspergillus and other species, and thesespecies are recognized producers ofaflatoxins. Paterson et al [1997) andOliveira et al (2016) demonstratedproduction of aflatoxins in a cold waterstorage tank. Water subjected to storage

(e. g., reservoirs, cisterns and bottles) forlong periods, may contain increasedmycotoxin concentrations. In the longterm, daily consumption of largeamounts of water containing low levelsof mycotoxin, may pose a health hazard(Paterson et al., 1997; Skaar andHageskal, 2015; DEFRA, 2016, Oliveiraet al., 2016). Therefore, frequentsurveillance for fungi and the setting oflimits are required to improve themycological quality of drinking water.Presence of microorganisms in suchenvironments affects human health,causing various diseases from allergicreaction to more serious systemicinfections. The choice of Gari toSabouraud media was not meant to beselective in separating clearly fungaltaxa. This could be explained by thefungi not being under stress conditionsdue to the low or zero residual chlorineand the presence of considerableamounts of dissolved oxygen and

Plate 11

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organic matter in water samples.Furthermore, Sabouraud, as a richermedium, gave better conditions for thewater fungi (conidia and propagules) togrow, which is reflected by the numberof isolates obtained in this study.

REFERENCESAnaissie, E. J., Kuchar, R. T., Rex, J. H.,

Francesconi, A., Kasai, M., Muller,F. M. and Walsh, T. J ( 2 0 0 1 ) .Fusariosis associated with patho-genic Fusarium species colonizationof a hospital water system: Anew paradigm for the epidemiologyof opportunistic mold infections.Clinical Infectious Diseases 33:1871–1878.

American Public Health Association(APHA); American Water WorksAssociation (AWWA); W a t e rEnvironmental Federation (WEF)(2012). Standard Methods for theExamination of Water and Waste-water, 22nd ed. ; American PublicHealth Association; AmericanWater Works Association; WaterEnvironmental Federation: Washi-ngton, DC, USA,

Besner, M. -C., Prévost, M. and Regli,S. (2011). Assessing the publichealth risk of microbial intrusionevents in distribution systems:Conceptual model, available data,and challenges. Water Resources 45:961–979.

Cruickshank, R., Duguid, J. P., Mami-on, R. P. and Swain, R. H. A.,(1975). Medical Microbiology II,Churchill Press, London, p. 32

Demain, A. L. and Davies, J. E. (1999).Manual of Industrial Microbiologyand Biotechnology, 2nd Ed, Ameri-can Society for Microbiology Press,Washington D. C.

DEFRA (2016). Review of Fungi inDrinking Water and the Implica-tions for Human Health. Availableonline: http://dwi. defra. gov.uk/research/completed-research/reports/dwi70-2-255.pdf (accessed on9 February 2016).

Doggett, M. S. (2000). Characterisationof fungal biofilms within amunicipal water distribution

system. Applied and EnvironmentalMicrobiology, 66 (3): 1249-1251.

Douterelo, I., Boxall, J. B., Deines, P.,Sekar, R., Fish, K. E. and Biggs, C.A. (2014). M e t h o d o l o g i c a lapproaches for studying themicrobial ecology of drinkingwater distribution systems. WaterResources. 65: 134–156.

Environmental Protection Agency(EPA) (2016). Health Risk fromMicrobial Growth and Biofilmsin Drinking Water DistributionSystems. Available online:http://www.epa.gov/sites/product-ion/f i les/2015-09/documents/



2007_05_18_disinfection_tcr_whitepaper_tcr_biofilms. pdf

Hageskal, G., Knutsen, A. K., Gaustad,P., de Hoog, G. S and Skaar, I. (2006).Diversity and significance of moldspecies in Norwegian drinking water.Applied Environmental Microbiology 72: 7586–7593.

Hageskal, G., Lima, N. and Skaar, I.(2009). The study of fungi in drinkingwater. Mycological Research. 113:165–172.

Kelley, J.. Kinsey, G., Paterson, R. andBrayford, D. (2003). Identificationand Control of Fungi in Distribu-tion Systems; AWWA ResearchFoundation and American WaterWorks Association: Denver, CO,USA

Nicoletti, R. Manzo, E. and Ciovatta,M. L. (2009). Occurrence andbioactivities of funicone relatedcompound.

Oliveira, H. M. B; Santos, C; Russell, R;Paterson, M; Gusmão, N. B and Lima,N (2016). Fungi from a Groundwater-Fed Drinking Water Supply Systemin Brazil. International Journal ofEnvironmental Research and PublicHealth 13: 304-315

Paterson, R. R. M., Kelley, J. and Gal-lagher, M. (1997). Natural occur-rence of aflatoxin and Aspergillusflavus (Link) in water. Letters of Ap-plied Microbiology 25: 435–436.

Prescott, L. M., Harley J. P. and Klein D.A (2005). Microbiology. 6th ed.McGraw Hill, London. Pp. 135 -140

Richardson, M. D. and Richardson, R.(2015). Aspergillus and aspergillosis.In Molecular Biology of Food andWater Borne Mycotoxigenic andMycotic Fungi; Paterson, R. R. M.,Lima, N., Eds. ; Food MicrobiologySeries; CRC Press: Boca Rotan, FL,USA, pp. 151–164.

Skaar, I. and Hageskal, G. (2015). Fungiin drinking water. In: Molecular Biol-ogy of Food and Water Borne Myco-toxigenic and Mycotic Fungi;Paterson, R. R. M., Lima, N., Eds. ;FooMicrobiology Series; CRC Press:Boca Rotan, FL, USA, pp. 597–606

Samson, R. A. ; Hoekstra, E. S. and Fris-vad, J. C. (2004) Introduction to Foodand Airborne Fungi, 7th ed. ; Cen-traalbureau voor Schimmelcultures:Utrecht, The Netherlands.

Sokari, T. G., Yubedee, A. G., andLugbe P. B. (1996). Garri as aneffective medium for certainMycological studies. Niger DeltaBiologia. 1(1): 86-89.

Warris, A., Gaustad, P; Meis, J. F. G. M.Voss, A., Verweij, P. E. andAbrahamsen, T. G. (2001). Recoveryof filamentous fungi from water in apaediatric bone marrow transplan-tation unit. Journal of HospitalInfections 47: 143–148.

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ISOLATION AND CHARACTERISATION OF AQUATIC FUNGI …