Microbial Diversity in Water and Biofilm Samples from Well Sources in Ilorin Metropolis , Nigeria

The aim of the study was to investigate the microbial populations of water and biofilm samples from seven hand dug wells in Ilorin, Nigeria, which are for private and public use. Physicochemical parameters such as pH, turbidity, total dissolved solids, temperature, conductivity, dissolved oxygen and biological oxygen demand were determined. Biofilm growths were induced from water samples obtained from selected wells. Twenty bacteria isolated were identified using morphological and biochemical characteristics as well as molecular methods and includes Shigella sonnei, Shigella dysenteriae, Micrococcus luteus, Bacillus sphaericus, Salmonella enteritidis, Proteus mirabilis strain IK-MB4-518F, Bacillus licheniformis strain RH104, Bacillus subtilis, Erwinia sp., Proteus vulgaris, Yersina sp., Bacillus cereus, Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa strain GS1, Pseudomonas aeruginosa strain 218B, Staphylococcus epidermidis, Vibrio sp., Escherichia coli and Bacillus pasteurii. Total bacterial count ranged from 1.8×104-1.53×106 cfu/ml. Water samples from these wells are therefore not potable and some of the isolates are potential health hazards to human population.


Introduction
Water is popularly known to occupy about 70% of the earth's surface and its significance in the ecosystem especially as it relates to all human activities cannot be overemphasized.Water is a critical issue for the survival of all living organisms.Many organisms including the great majority of higher plants and most mammals need to have access to freshwater.As plentiful as this resource is in nature, it is however regarded as scarce because the percentage of the whole that is potable, accessible and available for use is very limited (Agbabiaka and Oyeyiola, 2012).
The adequacy of safe water supply is indispensable for consumption, cooking, personal hygienic requirements and the reduction of risks from water related diseases.On a daily basis, each individual should have access to at least 20 litres of safe water for consumption and hygiene (Ersel, 2015).
The presence and survival of pathogenic and opportunistic organisms, especially those of faecal origin pose more threat to health due to the ability of certain bacterial species (including the pathogens) to produce extracellular polymeric substances (EPS) that initiates the development of the biofilm community, thereby increasing their survival rate and virulence (Dessie, 2014).
As access to potable water is limited, hence individuals and communities resort to other less expensive means of obtaining adequate water by their own standards to meet their daily needs.The most common means especially in Nigeria are wide-diameter tube wells.
One of the important sources of well contamination are leachate from various sources considering the rate of population growth, extents of community development and methods of waste managements found around these wells.Researches on landfill leachates have proved the presence of large numbers of pathogenic and opportunistic bacteria, many species belonging to genera such as Enterobacter, Escherichia, Klebsiella, Salmonella, Serratia, Proteus, Pseudomonas and Staphylococcus (Holt et al., 2000;Adeyemi et al., 2007;Flores-Tena et al., 2007), which find their way into surrounding underground waters and ruin their quality (Fatta et al., 1999).This was further explained by Oshode et al., (2008), who confirmed the presence of these organisms which are not only pathogenic and opportunistic but also capable of toxin production which increases the virulence potential of such organisms.Efuntoye et al. (2011), showed that some of these organisms exhibit antimicrobial resistance to certain antibiotics.
There is therefore a need to examine dug-wells on a case by case basis in order to determine their potential to be hazardous to human and animal health.

Dissolved oxygen
Determination of dissolved oxygen was achieved using Winkler titration method.Each water sample from the sampling points was halved.One of the halved samples was placed in the dark (for BOD analysis).The first halved sample was now taken and 2 ml manganese chloride(aq) (4g MnCl2 dissolved in 10 ml distilled water) added to the sample which was followed with the addition of 2 ml alkaline iodide solution (3.3g NaOH = 2g KI dissolved in 10 ml distilled water).The sample was then shaken.Two ml of concentrated hydrochloric acid was added and shaken.Iodine formed is directly proportional to the dissolved oxygen.Fifty ml of the above solution was titrated with 0.0125M sodium trioxosulphate (II) solution using starch as indicator.The end-point was reached when the blueblack colour disappeared.The samples were collected under water to ensure there were no trapped air bubbles.
Biological Oxygen Demand at day 5 (BOD5) One N acid/1 N alkali was added to each water sample to adjust the pH to 7. The sample was then transferred into BOD bottles gently so that bubbles did not come out.One ml of allylthiourea was added to each bottle to avoid nitrification.Dissolved oxygen was then measured using the steps described for dissolved oxygen.The other BOD bottle was incubated at 27 °C for 5 days in a BOD incubator.The amount of oxygen was measured as done earlier BOD5 (mg/l) = D1 -D2 Where BOD = Biological Oxygen Demand; D1 = initial dissolved oxygen (mg/l) in the first halved sample (mg/l); D2 = dissolved oxygen (mg/l) in the second halved sample after 5 days of incubation (Dubey and Maheshwari, 2004).

Sanitary surveillance i.
The nature of the surrounding community and the level of development surrounding each well i.e. student area, market layout, agricultural layout or others.ii.
The adequacy of the well structure i.e. whether or not it is well protected, casted to its depths iii.
The hygienic conditions obtainable around the wells i.e. presence of a dump site, distance from toilets, septic tanks etc.

Microbial analysis of water quality Enumeration of total coliform bacteria
Total bacterial count was done using the standard plate count methods for the examination using 10 -4 dilution factor as described by Fawole and Oso (2001) and incubated at 37 °C for 24 h.The plates were observed for growth after 24 h and the number of discrete colonies on

Sampling areas
Samples were obtained from well 1 (Tanke Bubu), well 2 (Tanke Ajanaku), well 3 (Tanke Ajanaku), well 4 (Tanke Bubu), well 5 (Akerebiata), well 6 (Ipata) and well 7 (Oja Oba) communities within Ilorin metropolis, Kwara State, Nigeria.Three of these wells were private and four were major public wells selected based on population density, nature of the area, absence of and/or unreliable sources of adequate public drinking water distribution systems (DWDS), frequent use of such wells.

Collection of water and biofilm samples
Water samples were collected from the wells in sterile sample bottles using the water fetcher, screwed properly and transferred to the laboratory in coolers.
Two periodic biofilm samples from the 3 rd and 10 th day after immersion were taken from each of these wells similar to the work done by Huang et al. (2009).Two filter membranes were withdrawn aseptically on the 3 rd and 10 th day after the setup had been immersed for analysis in the laboratory.

Physicochemical analysis of water samples
The physicochemical parameters measured were pH, turbidity, temperature, conductivity, dissolved oxygen and biological oxygen demand.pH Potable digital pH meter was standardized with buffer of pH 4, 7 and 9 before being used.The glass electrode was rinsed with distilled water before it was dipped into the samples.The pH was then read when digital reading stabilized (Kanase et al., 2016).

Turbidity
Turbidity of the water samples was determined using a HACH 2100N Turbidimeter.A clean sample bottle of turbidimeter was filled with 25 ml of each sample, covered with light shield and turbidity read in Nephelometric turbidity units (NTU) (Kanase et al., 2016).

Total dissolved solids
The total dissolved solids (TDS) of water samples was measured with a TDS meter in parts per million (ppm) (Kanase et al., 2016).

Temperature
Temperature was determined using a mercury-bulb thermometer dipped into each water sample and left for about 5-10 minutes and the thermometer read and recorded in °C (Kanase et al., 2016).

Conductivity
The conductivity of water samples was measured with a conductivity meter calibrated in ms/cm (Kanase et al., 2016).
plates were observed and recorded in cfu/ml.TCBS was used for the detection of the presence of Vibro sp., by inoculating a 24 h old sample culture grown in peptone water (Mohammadi-Aragh, 2016).

Enumeration of faecal coliform bacteria
One hundred ml of the sample was filtered using a sterile Millipore apparatus and the filter membrane was aseptically transferred unto sterile EMB plate which was incubated at 44.5 °C for 48 h.The plates were observed for growth after 48 h and the numbers of discrete colonies on plates were observed and recorded in cfu/ml (Fawole and Oso, 2001).

Enumeration of biofilm cell formation units (CFU)
The method described by Huang et al. (2009) was adopted with few modifications.Organisms present in the water source were impacted on membrane filter and made to develop a biofilm community.The setup was made up of perforated weighted bottles, perforated Petri dish, Whatman filter paper with minimal organic matter content and high durability measuring 7 cm in diameter and a string was used.The apparatus was disinfected using ethanol and sterilized under UV-light 3 days before it was sunk in the well to a depth of at least one feet below the water surface.For each well under investigation four filter membranes were sunk, a pair for each periodic sample was retrieved on the 3 rd and 10 th day after the apparatus was immersed.
Upon withdrawal, the filter membranes were aseptically transferred into glass flasks containing 50 ml sterile distilled water using sterile forceps.The flasks were mounted on a shaker device for thirty minutes to allow for re-suspension of the organisms in solution successively diluted to 10 -3 after which 1 ml of each of the suspension was drawn and plated unto freshly prepared MacConkey agar, Eosin methylene blue agar, Salmonella-Shigella agar, Pseudomonas Selective Agar, Thiosulfate Citrate Bile Salts Sucrose and Nutrient agar and incubated appropriately for 24-48 hours after which grown colonies were counted (Beściak and Surmacz-Górska, 2011).
Sequencing was done at the International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria.Sequence comparison was done using the BLASTN tool on the National Centre for Biotechnology Information (NCBI) website (www.ncbi.Nlm.nih.gov:80/BLASTN/).

Physicochemical characteristics pH of water sample
The pH of the samples ranged from 6.65 ± 0.15 -7.85 ± 0.05.well 1 sample 2 had the highest pH of 7.85 ± 0.05 while well 4 sample 1 the lowest value of 6.65 ± 0.15 (Fig. 1).

Turbidity of water samples
The lowest values were recorded in samples 3 well 3 and sample 5 well 6 (1 ± 0.00) while the highest was recorded in sample 3 well 7 (23.5 ± 0.5) recorded in Nephelometric Turbidity Unit (NTU).This corresponded to the periods when there was rainfall (Fig. 2).

Total dissolved solids of water sample
The TDS was lowest in well 1 sample 3 141.5 ± 2.5 while the highest was recorded in well 5 sample 5 1421.5 ± 1.5 parts per million (ppm) (Fig. 3).

Temperature
Temperature values expressed in °C ranged from 27.75 -28.5 °C throughout the sample, the highest value of 28.5 °C was obtained in well 7 sample 1 and sample 4, while the lowest was found in well 1 sample 1 and well 5 sample 4 (Table 1).

Conductivity of water samples
The conductivity was measured in ms/cm which ranged from 0.195 ± 0.005-1.755± 0.005.The lowest value was found in the 3 rd sample of well 1 while the highest was found in the 3 rd sample of well 5.These values were obtained around the neutral pH for those samples (Fig. 4).

Dissolved oxygen
The dissolved oxygen (DO) in mg/L values fluctuated with the amount of water present in the wells at the time of withdrawal.The lowest recorded was well 1 sample 5 (35 ± 1.0) while the highest recorded was in well 3 sample 1 and well 5 sample 5 (85 ± 1.0) (Fig. 5).

Biological oxygen demand at day 5
The values of Biological oxygen demand at day 5 (BOD5) were recorded in mg/L.The lowest values were recorded in wells 4 and 6 of sample 3 (3 ± 1.0) while the highest was in well 5 sample 2 (Fig. 6).

Sanitary surveillance
The age, facilities and nature of well structures obtainable at the different sites varied as well as their sanitary conditions (Table 2).

Microbial quality of water samples Total coliform counts
The mean of total coliform counts for water samples is shown in Table 3.
The lowest total coliform count of 1.8 × 10 4 cfu/100ml was recorded in well 5 Sample 2 while the highest counts of 1.53 × 10 6 cfu/100ml was recorded in well 3 Sample 2.

Total faecal coliform counts
The mean of total faecal coliform counts is shown in Table 4.The lowest total faecal coliform count of 2.0 x 10 3 cfu/ml occurred in wells 1, 2, 5, 6 and 7, respectively while the highest count occurred in wells 6 and 7 of sample 1 collection (Table 5).
In biofilm cell forming unit, the lowest count of 1.7 x 10 4 occurred in well 1 of sample 2 at day 10 while the highest of 1.0 x 10 5 occurred in well 6 of sample 1 at day 3.

Molecular identification of isolates
Four isolates identified by molecular method were Pseudomonas aeruginosa XXX which shows 91% similarity with strain GSI, Pseudomonas aeruginosa XXX which shows 94% similarity with strain 218B, Proteus mirabilis XXX which shows 95% similarity with strain IK-MB4-518F and Bacillus licheniformis which shows 94% similarity with strain RH104 using 16S ribosomal RNA gene, partial sequence deposited with Genebank.

Table 1 .
Temperature of water samplesThe structure of the well is not adequate, as it has a large mouth opening without a coverlid and the wall cast has broken at its depths.For this reason it hasn't been cleaned out in a long time.The well is sited downhill to a dump site, drainage system and close to a bathroom.This well has a high water table year round, an adequate structure, as well as a neat and plastered environment, however, the lid is broken.It is however located close to a place of worship where abolition is done constantly.It is also close to an improper drainage system in the backyard of another building.It was cleaned out in the course of this research work.This well has an adequate structure, proper coverlid as well as a neat and plastered environment.It is however located close to a place of worship where abolition is done constantly.It is not cleaned out regularly because of its water depths and slippery nature of its wall.It is not sited close to any visible source of contamination.

Table 6 .
Bacterial frequency distribution among sampling sites