Impact of Ultraviolength Ray on Bacterial Isolates of Well Water

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Published on International Journal of Health, Nursing, & Medicine
Publication Date: September, 2019

Balogun, O. B. & Ayilara-Akande, S. O.
Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji Osun State, Nigeria
Department of Microbiology, Federal University Technology, Oye Ekiti, Nigeria

Journal Full Text PDF: Impact of Ultraviolength Ray on Bacterial Isolates of Well Water (Studied from Ikeji – Arakeji Metropolis).

Wellwater can be a source of drinking water for both rural and urban settlement but can also be source of pathogenic organism which can be threat to human being. The present study was designed to enumerate, isolate and identify microorganisms and physicochemical assessment of well water from Ikeji-Arakeji was also determine, also to investigate the effect of Ultraviolet ray (UV) on the populations, identities of bacteria and also on the physicochemical conditions from selected well water sample and was collected from Ikeji Arakeji Metropolis. Bacterial and physicochemical analysis was done before exposure to UV light which is the control and after exposure to UV light at 254nm. So it’s imperative to carry out bacterial analysis on wellwater. The microbial analysis was carried out by standard methods. The bacteria isolated from waste water samples were Proteus aureus, Staphylococcus aureus, Klebsiella spp, Proteus vulgaris, Bacillus subtilis, Enterobacter aerogenes, Salmonella Typhi, Pseudomonas florescens, Citrobacter spp, Escherichia coli and Micrococcus luteus. The pH of the well sample was neutral after treated with UV light while mean temperature was (25°C) and the mean colour was (18.83 co/pt) highest mean chemical composition value was Total Dissolve solid (TDS) 165 (mg/l).. The bacterial load was dwlindling after the four hours exposure to the Ultraviolent light, Enterobacter aerogenes, Pseudomonas Fluorescens and Salmonella typhi was not inhibited after the 24 hours of exposure. It also had dwindling effect on physicochemical parameters of the wellwater. The presence of pathogens in water for drinking is of public health significance considering the possibility of the presence of other bacteria, that are implicated in gastrointestinal infection water borne diseases. It was observed that there was high population of bacteria in the wellwater sample before exposure to Ultraviolet ray 1.25× 103 the microbial population were observed been reduced to 8.5× 103. It is therefore recommended that wellwater from industries should be treated with ultraviolet ray before drinking especially in rural settlements. This will help reduce microbial population that constitute a serious hazard to public health that are implicated in gastro-intestinal water borne diseases.

Keywords: Microorganisms, Physicochemical parameters, Ultraviolet ray & Bacterial load.

Water is a transparent fluid which forms the world’s streams, lakes, oceans, and rain, and is the major constituent of the fluids of living things (Ashbolt et al., 2001). As a chemical compound a water molecule contains one oxygen and two hydrogen atoms that are connected by covalent bonds (Boboye, 2017). It is the only substance occuring in three phases as solid, liquid and gas on earth surface, it dissolves more substances in greater quantities than any other common liquids. The public health significance of water quality cannot be over emphasized (Aksu and Vur, 2004). Many infectious diseases are transmitted by water through the faecal-oral route. Diseases contacted through drinking water kill about five (5) million children annually and make 1/6th of the world population sick (WHO, 2004).
Wellwater is an excavation or structure created in the ground by digging, driving, boring, or drilling to access ground waters in underground aquifers. Water of good drinking quality is of basic importance to human physiology as well as indispensable to man continued existence. Its role as a medium of water borne disease which constitutes a significant percentage of the diseases that affect human and animals cannot be underestimated (Ballester and Sunyer, 2000). This is the most important concern about the quality of water. Guideline for bacteriological water differs from country to country but they all conform to WHO recommendation (Blatchley et al., 2003).
The standards for drinking water are most stringent than those for recreational waters. Availablity of facilities and financial constraints are the major obstacles in the provision of water of good quality in developing countries and rural areas (Bezuidenhou et al., 2002). In Nigeria, treated pipe borne water is limited to urban areas and there is inadequate treated pipe borne water for rural areas because of frequent epileptic supply. Such services may not even be available in certain areas in Nigeria. Due to this scenario, an increasing number of people in semi urban and urban areas in Nigeria including Ikeji Arakeji Osun State depends on wellwater as their source of water supply. There has been increasing cases of food and water borne diseases in many parts of the country (Nigeria) particularly typhoid fever and cholera (Balogun, 2016). The introduction of water treatment plants and various disinfection processes and frequent bacteriological analysis of water quality ensured the delivery of safe drinking water and this have drastically reduced the occurence of water borne illness (Dada et al., 1990). The occasional occurence of waterborne disease outbreaks, however, points out the continuing importance of strict supervision and control over the quality of public and private water supplies.
The supply of reliable wholesome drinking water that is colourless, odourless, tasteless, and free of pathoogens (W.H.O, 2014) is important in promoting healthy living (Ajayi et al., 2008). Up till date, availabilty of wholesome drinking water in the developing nations remains a critical and urgent problem with immense social and health concern to homes, communities, government and international community (Akhlaq et al., 1990).
Ultraviolet or UV energy is found in the electromagnetic spectrum between visible light and x-rays and can best be described as invisible radiation. In order to kill microorganisms, the ultraviolet rays must actually strike the cell (Bitton, 1994). Ultraviolet energy penetrates the outer cell membrane, passes through the cell body and disrupts its DNA preventing reproduction (Awad et al., 2003; Gordan et al., 1996; Lamikaran, 1999).
The degree of inactivation by ultraviolet radiation is directly related to the UV dose applied to the water (Edema et al., 2001). The dosage, a product of UV light intensity and exposure time is measured in microwatt second per square centimeter. The accompanying table lists dosage requirements to destroy common microorganisms (Blatchley et al., 1993). Most UV units are designed to provide a dosage greater than 30,000 after one year of continuous operation. Notice that UV does not effectively disinfect some organisms (most molds, protozoa, and cysts of Giardia lamblia and Cryptosporidium) since they require a higher dose. The classical use of UV light has occurred in biological safety cabinets in laboratories, although in recent years its used has been extended to inactivation of microorganisms in the food-processing industry, in potable water, and in wastewater) (Ballester and Sunyer, 2000). Ultraviolet light inactivates microorganisms by forming pyrimidine dimers in RNA and DNA, which can interfere with transcription and replication (Reintheler et al., 1987). The germicidal effect of UV light treatment is dependent on microbial exposure, but when used on opaque foods with irregular surfaces, UV light may cause less microbial destruction. Although the opacity and high absorption coefficient of milk has been considered a barrier to the use of UV light as a disinfectant, UV light treatment of milk has been shown to reduce bacterial counts of monocytogenes in goat milk and Staphylococcus. aureus in cow milk (Kreft, et al., 1986). Therefore, the objectives of this study this study will be investigating the bacterial content of well water that serves as the major source of drinking water in Ikeji Arakeji Osun State, a semi urban area in Nigeria, also to determine the eff of UV light on microbial load, identities and physicochemical properties.

All glassware’s used at all stages of analysis were thoroughly washed with detergent and rinsed properly with distilled water to remove all traces of residual washing compounds. The glassware’s were wrapped with aluminum foil paper and sterilized in an autoclave at 121oC for 15 minutes.

For the purpose of this study, water sample was collected from a well in Ikeji-Arakeji village, of Osun state. The sample was collected with a clean sterile bottle water container which had been passed through ultraviolet light screen for twenty four hours. The bottle was fitted with screw cap and neck of the bottle was protected with aluminum foil. The samples were transported to the laboratory and examined within six hours of collection to reduce changes which might occur in the bacteria count of the water.
The well is the dug type and all were covered. The samples was collected by jerking the drawer three to five times into the well and at the sixth time the water was quickly drawn out of the well, the bottle was rinsed with the sample and it was poured into the bottle and immediately covered with the aluminum foil paper and fitted with screw cap.
2.1 Physicochemical Analysis
The physicochemical tests included the determination of temperature, turbidity, odour, colour, total solid, total dissolved solid, dissolved oxygen, biological oxygen demand, total suspended solid, pH, conductivity, sulphate, chloride, nitrate, total acidity, partial acidity, total hardness, phosphate, and chloride content using the methods of FAO (1997).

2.2 Sterilization of materials
The glass wares were thoroughly washed with detergent rinsed thoroughly with distilled water and then allowed to dry. The glass wares were sterilized in the hot air oven at 160°C for one hour. The inoculating loop was also sterilized by flaming. The work bench was disinfected by swabbing with 95% ethanol. All work in the laboratory was done in a sterile environment.

2.3 Preparation of media
The media used in this research work were Nutrient agar, Potato dextrose agar, Mannitol salt agar, MaConkey agar, Peptone water and they were all prepared according to manufacturer’s instructions. The media was dissolved in the adequate amount of distilled water. The media were all homogenized and autoclaved at 121°C for 15 minutes.

2.4 Serial dilution method
Nine (9) ml of distilled water was measured into each of the test tubes and sterilize at 121oC for 15minutes using the autoclave. 1ml from the stock was taken into the first test tube and serial dilution was carried out. Bacteria were isolated using pour plate technique with sterile Nutrient agar and incubated at 37oC. The plates were incubated at 37oC for 24hours. Sub culturing was done repeatedly until pure culture was obtained; colony counting was done by means of a Gallenkamp colony counter.

2.5 Colonial morphology
Colonies of isolates were examined for the forms, sizes, surface, colors, elevations, margins, optical qualities.

2.6 Biochemical characterization
Identification of bacteria was based on morphological characteristics such as edge, size, optical characteristic, biochemical characterization such as Gram staining, Motility test, Sugar fermentation tests, Citrate, Catalase, Indole, Methyl red, Voges-prauskauer, and oxidase e.t.c, which is carryout according to Chessbrough (2006) method.

2.7 Gram’s staining reaction
The gram stain is important in bacteriology examination. It helps in two main groups, possibly showing major evolutionary relationships among organisms. The two groups are designated as Gram positive (blue to purple reaction to stain) and Gram negative (pink to red reaction to stain). Smears of 24 hours old isolates were placed on slides and heat fixed. Crystal violet was first applied and allowed to remain for 1 minute and washed off with water. Grams iodine was added. This stayed for another 1 minute and was later washed off. The smear was decolorized with alcohol, washed with water and counter stained with safranin. Smear was then blot dried with clean filter paper and observed under (x100) oil immersion lens of the microscope. (Chukwura, 2001).

Table 1 shows the morphological characteristic of the isolates which is based on form, size, surface, colour, elevation, margin,texture and optical quality.

TABLE 1: Morphological characterization of Isolates

Table 2 shows the microscopy and biochemical characteristics of the probable bacteria which is based on gram stain result, motility, catalase, oxidase, methylred, vogesproskauer, glucose, lactose citrate urease and indole test.

TABLE 2: Microscopy and biochemical characteristics of probable bacteria

KEYS: Mo, motility; Cat, catalase; Ox, Oxidase; MR, Methyl Red; VP, VogesProskaeur; Glu, Glucose; Lac, Lactose; EMB, Eosin Methylene Blue; Ci, Citrate; Ur, Urea; In, Indole; –R, Gram negative rod; +R, Gram Positive rod; -C, Gram negative Cocci; +C. Gram Positive cocci; – Negative reaction; + Positive reaction; N,
Figure 1: Effects of UV light (254nm) on the bacteria load on waste water from well water

Figure 1: Effects of UV light (254nm) on the bacteria load on waste water from Wellwater

Figure 2: Effects of UV light (254nm) on the bacteria load from well water using Statistical analysis

Table 5 : Effect of UV light on the probable bacteria isolated from well water

Table 1: Shows the physicochemical properties of wellwater

Figure 1: Statistical analysis using individual value plot.

Figure 2 : Physical properties of raw and treated water

Figure 3: Mineral composition of raw and treated water

Eleven bacteria were isolated form well water in Ikeji-Arakeji metropolis, colonial and biochemical characterization was done to know the probable isolates in the study, eleven bacterial genera were routinely isolated in the water sample which includes pathogens and opportunistic pathogens (Pearce, 2007). Members of the Enterobacteriaceae predominated in the bacterial isolated and this includes, the genera Escherichia, Bacillus, Pseudomonas, Klebsiella, Proteus, Staphylococcus, Streptococcus, Micrococcus, Salmonella, Shigella and Enterobacter. Showed the frequency of distribution of the bacterial isolates in the sampling points and it was revealed that the bacterial isolates Escherichia coli, Salmonella spp, Bacillus subtillis and Enterobacter spp. were the most prevalent isolates, while the least prevalent were Shigella spp., Bacillus spp., Salmonella spp. This further confirmed faecal contamination as a major source of water pollution in the rainy season.
The bacteriological assessment of well water reveals the presence of bacterial contaminants and this is in agreement with the findings. high total bacterial count is indicative of the presence of high organic and dissolved salts in the water. The primary sources of these bacteria in water are animal and human wastes (Adetuyi et al., 2017). These sources of bacterial contamination include surface runoff, pasture and other land areas where animal wastes are deposited. Additional sources include seepage or discharge from septic tanks, sewage treatment facilities and natural soil /plant bacteria (EPA, 2002).
Escherichia coli are a taxonomically well-defined member of the family (Meylan et al., 1996). It is abundant in human and animal faeces, it is found in sewage, and all natural waters and soil subject to recent fecal contamination, whether from humans, wild animals, or agricultural activity (Wilson et al., 1993). E.coli is widely preferred as an index of fecal contamination and is widely used as an indicator of treatment effectiveness. The presence of E.coli, as with the presence of thermo tolerant coliform, is significant to water safety (Schlegel, 2002).
Pseudomonas spp are bacteria that are environmentally widespread, with some being opportunistic pathogens (Prescott et al., 2005). Pseudomonas aeruginosa is commonly found in soil, feaces, water, and sewage but cannot be used as an index of fecal contamintion, since it is not invariably present in sewage and feaces and may also multiply in the enriched aquatic environment and on the surface of organic materials in contact with water (Neden et al., 1992).
Proteus spp are widely distributed in nature as saprophytes, being found in decomposing animal matter, sewage, manure soil, and in human and animal feces. They are opportunistic pathogens commonly responsible for urinary and septic infections, often nosocomial. There are three species namely; (Prescott et al., 2005). The isolation of bacterial genera known to be pathogenic in man, calls for a serious concern. Specifically, E. coli, S. aureus B. subtilis, S. pyrogenes, P. aeruginosa, Klebsiella spp, and Enterobacter spp. were isolated during research work
The microorganism where exposed to 24 hours of Ultraviolent ray to check for inhibiting effect on the bacterial load and the bacteria isolated.
There was heavy bacterial load at the beginning of the experiment as time increases there was dwindling effect on the population of bacteria may be because of disinfecting ability of Ultraviolet ray which is consistent to the findings. Some of the microorganisms were seen throughout the experiment such microbes are Escherichia coli, Bacillus subtilis, Salmonella typhi, Proteus spp. The pH of 7.22 of the wellwater sample were in agreement with pH assigned by EPA as the standard pH of which was reduces to 6.5 after application of UV light (Kadam et al., 2007).
The colour of the wellwater samples were not in agreement with the standard limit for colour of drinking water recommended by EPA. The standard colour limit recommended by 15 (colour unit) (EPA, 2002) while the colour of the sample was 18 pt/co but reduce exigently to the standard limit. The low turbidity observed with the surface waters were in agreement with FAO standards on turbidity. High turbidity is often associated with higher levels of disease causing microorganism such as bacteria and other parasites. Rivers may get contaminated from soil runoff thereby increases its turbidity, which is a measure of cloudiness of water (FAO, 1997). Fewer number of disease causing microorganisms may be an indication of lower turbidity value experienced with well samples. At no time can turbidity (Cloudiness of water) go above 5 nephelometric units (NTU) (Boboye et al., 2017). The total dissolved solid of all water samples are in agreement with the environmental protection agency standard of 500mg/l.
Total dissolved solid in drinking water has been associated with natural sources, sewage urban runoff, industrial waste water and chemical used in the water treatment process (though of aesthetic rather than health hazards (Bohrerova et al., 2006).
Biochemical oxygen demand, COD, ammonia, chloride, nitrate were below the detection limit of the techniques, suggesting that organic matter is absent or is present in very low amounts in the water which is similar to the findings of Adetuyi et al. (2017).
Phosphate level in wellwater was above the 3mg/l. This limit should be controlled to avoid eutrophication of the ponds (Boboye et al., 2017). Phosphate may be introduced into the wellwater through through surface run off and could also be from the building materials (Ashbolt et al., 2001).
Sulphate concentration in the wellwater without concrete varied from 8.22 (mg/l) after application of ultraviolet ray and (22 mg/l) without the application of ultraviolet ray so the concrete wellwater significantly higher than that of non concrete wellwater. These values are similar to that of boboye et al. (2017) (68 mg/l) and different from Qin et al. (2012) who reported 0.66-1.09mg/l in his research. He suggested the use of detergent and soaps by residents which got into the water body may be responsible for the high value of sulphate (Schwartz et al., 2010). Electrical conductivity is a useful indicator of mineralization and salinity or total salt in a water sample. The FAO acceptable limit for conductivity in aquaculture is 20-1500 μs/cm (Wolfe, 1990). This limit was not exceeded in the wellwater before and after exposure to ultraviolet ray. The iron content of the water samples used in this study is in agreement with EPA standard of 0.3mg/l (Wilson, et al., 1993). The chloride content or limit recommended by EPA is 250mg/l, this is in agreement with the chloride content of all the water samples analysed. All parameters of physicochemical analysis have been documented as National Secondary Drinking Water Regulation (Balogun et al., 2019), they are non enforceable guidelines regulating contaminants that may cause cosmetic effect (such as taste, odour or colour) in drinking water (EPA, 2002)
Ultraviolet water purifiers destroy harmful microbes, including yeast, bacteria, algae, molds, virus and oocysts near the UV light. UV light deactivates the DNA of bacteria, viruses and other pathogens, which destroys their ability to multiply and cause disease. (Adetuyi et al., 2017)
As UV light penetrates through the cell wall and cytoplasmic membrane, it causes a molecular rearrangement of the microorganism’s DNA, which prevents it from reproducing. Specifically, UV light causes damage to the nucleic acid of microorganisms by forming covalent bonds between certain adjacent bases in the DNA Schwartz et al (2000). The formation of such bonds prevents the DNA from being “unzipped” for replication, and the organism is unable to reproduce (Ajayi, 2008). Klebsiella spp and streptococcus were inhibited at eighteen (18) hours which is in congruent to the findings of Wilson et al. (1993), Citrobacter spp was inhibited at 24 hours which is in correlation to the work done by Kreft (2006). Proteus spp, Enterobacter aerogenes. Pseudomonas Fluorescens and Salmonella typhi. was not inhibited.
Ultraviolet treatment involves the conversion of electrical energy in a low-pressure mercury vapor (USEPA, 1996). Because of the purification properties of the soil, however it can be contaminated. Ground water are found to be contaminated due to improper construction, shallowness, animal wastes, proximity to toilet facilities, sewage, refuse dump sites and various human activities around the well (Bitton, 1994).
The total bacteria counts for the water sample are generally high exceeding the limit of 1.0x 103cfu/ml which is the standard limit of heterotrophic count for drinking water (EPA, 2002). The primary sources of these bacteria in water include animal and human wastes and the two sources of bacterial include pasture, surface run off, leaching of effluents which can lead to oral faecal contamination and other land areas where animal wastes had been deposited. (Balogun et al., 2019).
Although 100% destruction of microorganism cannot be guaranteed, it is possible to achieve 99.9% reduction in certain applications and with proper maintenance and in order for a UV unit to successfully disinfect water some criteria or variables must be considered and also there are dosage required for Ultraviolet to carry out 99.9% destruction of various organisms e.g bacteria recirculating system (Balogun et al., 2016). UV units are most often used in constant flow recirculating systems.

The outcome of this study has shown that there is high incidence of contamination in well water by pathogenic organisms. The water research conducted on is not fit for consumption or usage but treatment with ultraviolet light have denaturing effect on pathogenic microbes isolated from wellwater. Also most of the physicochemical properties except few parameters were reported with lower values than the permissible level, even the few parameter values were essentially reduce below the standard value after application of UV light. There was no significant different between the values of elemental composition that was exposed to UV light compared to those that was not exposed.

I therefore recommend that wells should be dug at least 30meters away from toilet and refuse, dump sites and should be very deep and covered adequately. Also, water around this area should undergo UV treatment before being used for drinking purposes because it assures 99% of destruction of organisms from water without posing any danger to the health of the masses.