Scholarly Journals of Biotechnology Vol. 2(4), pp. 69-78, May 2013 Available online at http:// www.scholarly-journals.com/SJB ISSN 2315-6171 © 2018 Scholarly-Journals

Full Length Research Paper Microbial isolation and identification associated with drinking water from various sources in Sokoto Metropolis

Umar, S.1 and Baki, A.S.2

1College of Life Sciences and Technology, Beijing University of Chemical Technology, Beijing, China. 2Department of Microbiology, Faculty of Science, Usmanu Danfodiyo University, Sokoto.

Accepted 33 April, 2013

The study was aimed at isolation and identification of microbes present in drinking water collected a Sokoto metropolis. A total of twenty (20) samples were collected from 4 different areas. Fungi, isolation were identifies using colonial and morphological characteristics. The results obtained indicated that fungal count range from 1.1x104 to 8.0x103cfu/ml. Six (6) species of fungi which include Aspergillus niger, Aspergillus oryzae, Mucor racemosus, Rhizopus stololnifer, Aspergillus flavus and Talaromyce flavus of fungi, isolated from drinking water indicated that Aspergillus niger with (39.28%) had the highest rate of occurrence followed by Aspergillus aryzae (17.85%), Aspergillus flavus (17.85-%), Mucor racemosus (10.71%), Rhizopus stolonifer (7.14%) and Taloromyce flavus (7.14%) which had the least rate of occurrence. It was observed from this study that all the isolated fungi were potential pathogens that can be deleterious to human health.

Key words:- Isolation, microbes, Identification, Aspergillus flavus, Talaromyce flavus.

INTRODUCTION

Biofilm is made up of aggregates of micro organism, such industry. Many problems in drinking water distribution as bacteria, fungi, diatoms, protozoa, algae and any system are microbial including biofilm growth microbial exogenous materials, which are embedded in a hydrated mediated corrosion and persistence of pathogens extracellular matrix and attached to a solid surface (pipe, (Donlan, 2002). Biofilms may be formed on variety of shiphall, teeth, lung, etc). Water is one of the most surfaces, in living tissues, indwelling medical devices, abundant and essential commodities of man occupying industrial or potable water piping system, or normal about 70% of the earth’s surface, yet greater percentage aquatic ecosystems. They are suspected of being the of the world’s population, most especially in developing primary source of microorganisms in water distribution countries live without access to safe water Adriano and systems. In addition to health related importance, Joanna ( 2007). Nigeria for example, is located on microbial growth in destruction systems can cause taste coastal West Africa where water is abundant; most of the and odour problems, which can be contributed by the population lacks adequate and safe drinking water. substances produced by some species of fungi. (Adriano Biofilms playing a major role in drinking and waste water and Joanna, 2007). treatment processes due to their enhanced properties of Understanding the effect of the environmental mineralization, bioaccumulation the organism form conditions on these fungi may help to improve distribution assemblages which are irreversibly associated with a system management strategy. The mature and surface and enclosed in a matrix of extracellular concentration of biodegradable compounds present in polymeric substance (EPS) of their own origin which form treated drinking water as the water temperature are some matrix Donlan (2002). of the factors contributing to biofilm formation in potable Water quality is crucial for both household and food water system. In this study the effect of phosphorus concentration, temperature, light and darkness, on the biofilm forming potential of moral these seature have recently been reviewed Flemming (2008) and Flemming *Corresponding author e-mail: [email protected] and Wingender (2010). For long time, biofilms were Scholarly J. Biotechnol. 70

considered literally as a side issue and they experienced mould and filamentous fungi in drinking water. little awareness, although they were a common sight all 3.To determine the microbiological quality of drinking the time. Their relevance for environmental processes as water. well as in medicine and public hygiene has gained attention only in past few decades, since then, sophisticated methods have been introduced in to biofilm MATERIALS AND METHODS research such as fluorescence microscopy and confocal laser scanning ceroscopy analysis, and most powerful, Sample Collection molecular biology (Flemming, 2008). When cases of illness are registered, epidemiological studies are Twenty (20) samples of water were collected from four conducted in order to demonstrate similarities in heretic different areas within Sokoto metropolis viz: Bado, Arkilla, profiles of strains isolated from clinical and environmental Gidan Dare and Kwakwalawa. Standard method specimens, to track the source of infection. Drinking described by American public Health Association (APHA, water and associated biofilms are often among the prime 1999), was adopted. 300ml of water was poured candidates tested when gastrointestinal diseases and aseptically in to 300ml sterilized bottle. For tap water and different types of infections are recorded. All this has borehole water, the samples were collected by allowing allowed investigating biofilm biology in much greater the water to run to waste for 2 or 3 minutes and then the detail and, thus, taking views of the life of microorganism water was aseptically collected in sterile bottles. Water in the real world. Stewart (2008). Nigeria are focused on from wells were collected in a sterilized bottle fitted with a the occurrence and significance of bacteria with little weight at the base. Water from river was collected by attention to other microorganisms such as fungi. Even in dipping the sterilized bottle into the water. All the collected drinking water pathogenic contamination and disease sample were then labeled accordingly. And were taken to outbreaks may occur demonstrating the imperative the laboratory for analysis. And then seal. Sample requirement for comprehensive water safety plans collected were then transported to the laboratory in an ice implementation, (Szewzyk et al., 2000; wingender and cooler for storage as soon as possible (Adrano and Flemming, 2010). Joana, 2007). Drinking water associated biofilm induce aesthetic problems consisting in colour, odour and taste Sample Processing and Analysis degradation due to chemical compound release and more important, they pose a threat to human and animal Nine milliliters (9mls) of distilled water was poured with health by hosting pathogenic or toxins producing the aid of sterilize syringe into the three test tubes each bacteria, viruses, protozoa, algae, fungi and per sample and autoclaved at 1210c for 15 minutes, one invertebrates. All of them share common feature and take ml (1ml) of each sample was transferred into the already substantial ecological benefits from these structures, the sterilized tubes thereby making it to be homogenized (10- great majority of water related health problem are the 1, 10-2, 10-3) respectively for the whole number of result of microbial contamination (Riley et al., 2011). samples. The last (1ml) of the final tube was discarded. Naturally occurring biofilms contact with drinking water The essence is to reduce the microbial load present in were identified and described as microbial reservoirs for the sample, (Adrano and Joana, 2007): further contamination (Szewzyk et al., 2000; wingender and Flemming, 2010). Fungal infections are becoming Isolation and Identification of fungi more and more important because of increasing numbers of immunesuppresed patient. None less, water borne Colonies growing on the SDA plate, after were fungi are associated with taste and odour problems, subcultured on to another freshly prepared sterile SDA contamination of food and beverage preparation and in a plates for identification test. During identification, a drop variety of health related effects. Opportunistic pathogens, of lacto-phenol blue was placed on a microscope slide. potentially causing superficial or systemic infections, Using sterile inoculating needle, a small portion of fungal allergenic or toxigenic species of fungi (Yeasts and growth was gently removed and placed of lacto-phenol moulds) have been Isolated from drinking water blue and then gently spread it out with two dissecting worldwide. (Nagy and Olson, 2011). This study was needles so that it can easily be identified when viewing aimed at determining the microbiological quality of and covered with a cover glass and then examined drinking water in Sokoto Metropolis and was achieved microscopically. The slide was placed on a stage using with the following objectives; x10 objective lens, the fungal structure were observed and fungi were identified. (Chei, Kalhatkar, et al., (2000). 1.To determine the occurrence, distribution and significance of mould species in selected public Anova drinking water in Sokoto metropolis. 2.To determine and analyzed the biofilms potential of A complete randomized design (CRD) f – test was used Umar and Bello, 71

Table 1 Fungal colony count cfu/ml in the different samples of drinking water.

Sample Fungal colony counts (cfu/ml) A 18 1.8x104 B 15 1.5x104 C 11 1.1x104 D 8 8x103 Key A = Bado; B = Gidan dare;C = Arkilla ;D = Kwakwalawa

Table 2: percentage frequency of occurrence of fungi isolated from drinking water sample collected at Bado.

Isolated organisms Frequency of occurrence Percentage(%) of occurrence of the organisms Apergillus niger 3 50 Aspergillus oryzae 2 33.33 Mucor racemosus 1 16.67 TOTAL 6 100

Table 3: percentage frequency of occurrence of Fungi isolated from Water sample collected at Arkilla.

Isolated organisms Frequency of occurrence Percentage(%) of occurrence of the organisms Aspergillus niger 1 20 Aspergullus oryzae 1 20 Mucor racemosus 2 40 Rhizoipus stolonifer 1 20 TOTAL 5 100

Table 4: percentage frequency of occurrence of Fungi isolated from Gidan Dare Drinking Water.

Isolated organisms frequency of occurrence Percentage(%) of occurrence of the organisms Apergillus flavus 2 33.33 Aspergillus nigers 3 50 Talaromyces flavus 1 16.67 TOTAL 6 100 to compare between the mean colony count of different presented in table 1. The sample D had the highest source of drinking water. Null hypothesis says there is fungal connt of 8.0 x 103cfu/ml and sample C had the leat significant difference between the mean colony counts of count of 1.1 x 104. different sources of drinking water while alternative The fungal isolated and identified in this research are hypothesis says there is no significant difference between presented in table 2 showing Aspergillus niger, the mean colony counts of different source of drinking Aspergillus oryzea and Mucor racemosis respectively. water. If the calculated f – value is greater than tabular The percentage frequency of the fungal isolates in Arkilla value then there is significant difference between the area are shown in table 4.3 and Mucor racemosis had the various sources of drinking water. But if the tabular value highest percentage frequency of occurrence of (40%) and is greater than calculated f – value then there is no Aspergillus niger, Aspergillus oryzea and Stolonifer statistically significant difference between the various rhizypus had the least percentage frequency occourrence sources of drinking water. Therefore, the result is not of (20%). Table 4 represented the percentage frequency significant different. of occunrence of fungi from drinking water analysis which includes three (3) species of fungi. Aspergillus flavus (33.33%), Aspergillus niger (50%) and Talaromyces RESULTS AND DISCUSSION flavus (16.67%). Table 5 indicated the percentage frequency of occurrence of fungi isolated from drinking The mean fungal count of of the fungi isolate are water collectd at kwakwalawa which include Scholarly J. Biotechnol. 72

Table 5: percentage frequency of occurrence of Fungi isolated from sample collected at Kwakwalawa River

Isolated organisms Frequency of occurrence Percentage(%) of occurrence of the organisms Aspergillus oryzae 2 18.18 Aspergillus flavus 3 27.27 Aspergillus niger 4 36.40 Talaromyces flavus 1 9.10 Rhizopus stolonifer 1 9.10 TOTAL 11 100

Table 6: percentage frequency of occurrence of Fungi isolated from Drinking Water sample representing all collection Areas.

Isolated organism Frequency of occurrence Percentage(%) of occurrence of the organisms Aspergillus niger 11 39.28 Aspergillus oryzae 5 17.85 Mucor recemosus 3 10.71 Rhizopus stolonifer 2 7.14 Aspergillus flavus 5 17.85 Talaromyce flavus 2 7.14 Total 28 100

Table 7: Colonia and morphological characterization of fungi isolated from water sample collected at Bado

Fungi isolate Colonial and morphological characterization Aspergillus niger This consists of a compact white or yellow basal/felt/with a dense black conidiophoresor dark conidiophore. Conidia head radiate lending to split into loose column with age conidiophore stipes, smooth-walled, by a fine but often brown colours. Conidian globosse to subglolose phialides born on matulate. Aspergillus oryzae Consisting of a felt of long conidiophores often intermixed with aerial mycelium. Conidial heads radiate, pale greenish yellow, later becoming light to dull brown. Conidiophores hyaline, mostly rough-walled. Mucor rceamosus Colony white, becoming brownish gray with age. Consisting of tall and short sporangiophores branched. The short branch sometimes recurved with encrusted walls

five(5) species of fungi. Aspergillus oryzae (18.18%), Talaromyce flavus and Rhizopus stolonifer. Aspergillus flavus (27.27%), Aspergillus niger (36.40%), In this study the most frequently isolated fungi was Talaromyces flavus (9.10%) and Rhizopus stolonifer genus Aspergillus. These findings are consistent with (9.10%). The percentage frequency of occurrence of works conducted by (Arvanitidou et al., 1990 and 2000) fungi isolate in all area are show in table 6 and and Gunhild et al., (2006), that Aspergillus was the most Aspergillus niger had the highest percentage frequency of commonly isolated generally in water. Aspergillus are occurrence of (39.28%) followed by Aspergillus oryzae known to produce aflatoxins (B1, B2, G1 and G2), the (17.85%), Aspergillus flavus (17.85%), Mucor racemosus most toxic and potent hepatocarcinogenic natural (10.71%)a, Rhizopus stolonifer (7.14%) and Talaromyce compounds ever characterized (Bennett and Klich, 2003). flavus (7.14%) had the least rate of occurrence. These fungi cause a wide range of diseases on humans, The results obtained from this study showed that all the ranging from hypersensitivity reactions to invasive fungi isolated were present in drinking water sample infections associated with angio-invasions, A. niger, A. collected from Sokoto metropolis. Pure cultures obtained oryzae and A. Flavus where found on several occasions from the growth of the samples were subjected to during this study. The finding of A. niger in agreement microscope for identification and fungi identified from 20 with the work conducted by (Hageskal et al., 2006), in sample of drinking water were Aspergillus niger, which he also reported the frequency occurrence of A. Aspergillus oryza, Aspergillus flavus, Mucor recemosus, niger in drinking water. However, the presence of A. Umar and Bello, 73

Table 8: colonial and morphology characterization of fungi isolated from sample collected at Arkilla

Fungi isolation Colonia and morphological characterization Aspergillus niger this consist of a compact white or yellow basal/felt/with a dense black conidiophores. Conidia head radiate lending to spit in to loose column with age condiophore stripes, smooth-walled, by a fine but often brown colours. Conidia globosse to subglolose phialides born on matulate. Aspergillus oryzae consisting of a felt of long conidiophores often intermixed with aerial mycelium. Conidial heads radiate, pale greenish yellow,later becoming light to dull brown. Conidiophores hyaline, mostly rough-walled. Mucor racemosus colony white becoming brownish gray with age. Consisting of tall and short sporangiophores branched. The short branched sometimes recurved with encrusted walls. Rhisopus stolonifer colony whitish becoming graynish-brown due to brownish sporangiophores and brown-black sporangia, sporangiophores tall, solitary or in groups of 2-7 usually 3- 4 from the almost colorless to dark- brown, smooth or slightly rough-walled stolons opposite the branched rhizoid

Table 9: Colonia and morphological characterization of fungi isolated from drinking water collected at Gidan Dare

Fungi isolation Colonia and morphological characterization Aspergillus flavus usually consisting of a dense felt of yellow- green conidiophores. Conidia heads typically radiate, later splitting into several looses columns, yellow-green becoming dark yellow- green.. Aspergillus niger This consist of a compact white or yellow basal/felt/with a dense black conidiophores or dark conidiophores. Conidia head radiate lending to split in to loose column with age conidiophore stipes, smooth-walled, by a fine but often brown color. Conidia globosse to subgolos phialides born on matulate. Talaromyce flavus consisting of a basal felt in which numerous ascomata usually forming a continuous, thick yellow layer. Anamorph scanty, usually not affecting the colony appearance. Reverse ranging from arrange red when young to orange brown in age or purple with the pigment diffusing in the agar.

Table 10: Colonia and morphological characterization of fungi isolated from water sample collected at Kwakwalawa river

Fungi isolate Colonia and morphological characterization Aspergillus oryzae consisting of a felt of long conidiophores often intermixed with aerial mycelium. Conidial heads radiate, pale greenish yellow, latter becoming light to dull brown. Conidiophores hyaline, mostly rough-walled. Aspergillus flavus usually consisting of a dense felt of yellow-green condiophores. Conidial heads typically radiate, later splitting in the several looses columns, yellow- green becoming dark yellow-green. Aspergillus niger this consist of a compact white or yellow basal/felt/with a dense black conidiophore or dark conidiaphores. Conidia head radiate lending to split in to loose column with age conidiophores stripes smooth-walled, by a fine but often brown colours. Conidia globases to subglolose phialides born on matulate. Talaromyuce flavus consist of a basal felt in which numerous ascomata usually forming a continuous, thick yellow layer. Anamoph scanty, usually not affecting the colony appearance. Reverse ranging from orange red when young to orange brown in age or purple red with the pigment diff using the agar. Rhizopus stolonifer colony whitish becoming grwyish-brown due to brownish sporangiophores and brown-black sporangia. Sporangiophores tall, solitary or in groups of 2-7 usually 3-4 from the almost colorless to dark brown, smooth or slightly rough- walled stolons opposite the branched rhizoids. Scholarly J. Biotechnol. 74

Figure 1: Represented the percentage frequency of occurrence of fungi isolated from 5 sample of drinking water collected at Bado which included three (3) species of fungi. Aspergillus nigers (50%), Aspergillus oryzae (33.33%), and Mucor racemosus (16.67%).

Figure 2: Represented the percentage frequency of occurrence of fungi Isolated from 7 water sample collected at Arkilla which include four (4) species of fungi. Aspergillus nigers (20%), Aspergillus oryzae (20%), Mucor racemosus (40%), and Rhizopus stolonifer (20%). Umar and Bello, 75

Figure 3: Indicated percentage frequency of occurrence of fungi isolated from 4 sample collected at Gidan Dare which include three (3) species of fungi. Aspergillus flavus (33.33%), Aspergillus nigers (50%), Talaromyces flavus (16.67%).

Figure 4 Indicated the percentage frequency of occurrence of fungi isolated from 4 water sample collected at Kwakwalawa River which includes five (5) species of fungi. Aspergillus nigers (36.40%), Aspergillus aryzas (18.18%), Aspergillus flavus (27.27%), Talaromyces flavus (9.10%), and Rhizopus stolonifer (9.10%). Scholarly J. Biotechnol. 76

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