1.

INTRODUCTION
Water is one of the most vital resources essential for sustaining life on Earth. Access to clean and safe
drinking water is a fundamental human right and crucial for maintaining public health, agriculture,
and environmental balance. However, due to rapid industrialization, urbanization, and population
growth, water quality is declining at an alarming rate.
Contaminated water is a primary source of waterborne diseases such as cholera, dysentery, typhoid,
and hepatitis. Different water sources – rivers, lakes, ponds, wells, tap water, borewell water, and
even rainwater – harbour unique microbial communities and chemical compositions. The quality of
these sources is influenced by several factors including environmental conditions, human activities,
and natural geochemical processes.
Physicochemical parameters such as pH, turbidity, total dissolved solids (TDS), dissolved oxygen (DO),
hardness, and chloride content serve as key indicators of water quality. Similarly, microbial
populations, including coliforms, E. coli, Pseudomonas, and fungi, are essential markers of
contamination and potability.
Waste management is a serious problem worldwide. In India, waste management regulations are
formulated for sustainable development, so municipalities and other agencies are authorized to
create an environment to minimize waste generation in society. To regulate the rise of waste
generation, different legislations concerning the way of disposal and the way to trot out generated
waste were enacted and acquired underneath the umbrella of the surroundings Protection Act (EPA),
1986. However, different types of waste are generated in society, and the control of specific types of
waste requires separate regulations and laws, and requires special attention. In fact, cities and
countries usually produce waste as a by-product. With rapid urbanization since the 1990s, India has
faced major challenges in managing and disposing of waste, which is mainly generated in
metropolitan areas and large cities. It is estimated that in 2018, 377 million urban residents in India
generated 62 mil- lion tons of municipal solid waste. Among these huge solid wastes, only 43 million
tons of wastes were collected, and 11.9 million tons were recycled, which are now part of the solid
processing. It is considered a basic and mandatory service for all communities in India to keep the
city clean. Unfortunately, most of the municipalities of India dump solid waste on a daily basis at an
area inside or outside town haphazardly in a very random manner. This is a serious problem of solid
waste management in India where waste disposal and management are handled.
This study aims to isolate and identify microbial populations from different water resources and
evaluate their quality using standard physicochemical and microbiological parameters. The results
will be compared against WHO and BIS standards to determine the potability and contamination
status of each source.
drinking water, certain microbes may evade the treatment and infiltrate the distribution Water is
thought to provide a medium for the dissemination and growth of microorganisms linked to humans.
A vital human right, consumers may experience health effects if their drinking water is contaminated
by opportunistic pathogenic environmental microorganisms. Therefore, it is important to safeguard
human health by avoiding microbial contamination of drinking water.

For drinking and other household uses, untreated surface water from rivers, dams, and streams is
used directly in rural areas. Because they are not protected, these unprotected water sources are
susceptible to contamination from agricultural runoff, sewage effluents, and animal excrement,
making them unfit for human consumption. To detect faecal pollution in water and the presence of
these pathogens, faecal coliforms Aeromonas and Pseudomonas are utilized as indicators that may
have negative effects on consumers' health, particularly those who have impaired immune systems.
It has been noted that antibiotic use in human therapy and agricultural processes has increased
alarmingly. This extensive use of antibiotics in both human and animal medicine has led to the
emergence of antibiotic-resistant bacteria, which has an impact on the management of illnesses. As a
result, antibiotic resistance has grown to be a significant public health concern, and it is generally
known that it exists in drinking water, surface water, and wastewater. The risk brought on by the
pathogenicity of bacteria is made worse by their resistance to drugs. The presence of multidrug-
resistant organisms may rise as a result of the biological treatment procedures used in wastewater
treatment facilities, which may lead to a selective increase in antibiotic-resistant bacteria. Despite
chlorination's reduction of germs in network. Additionally, it has been previously observed that
microbes exhibit drug resistance.
A detailed investigation into all major water sources will provide insights for environmental
monitoring, public health protection, and sustainable water resource management.
Water is a natural resource and is essential to sustain life. Accessibility and availability of fresh clean
water does not only play a crucial role in economic development and social welfare, but also it is
essential element in health, food production and poverty reduction. Water helps to maintain the
moisture of internal organs of the body; maintains normal volume and consistency of fluids such as
blood and lymph; regulates body temperature; removes poisons or toxins from the body through,
urine sweat and breathing; and is essential for regulating the normal structure and function of the
skin. Around 700 million people suffer from lack of access to clean and safe water and 2.2 million
people die from water-borne diseases every year globally. Infants are the most vulnerable targets of
these diseases.3 The problem is even severe in developing countries where generally the drinking
water is untreated. Bacteria constitute one of the major contaminants of water and they have been
reported to persist even in the extreme environmental conditions and oligotrophic conditions.
Diseases related to contamination of drinking water constitute a major burden on public health. The
principal risk to the health is from ingestion of water contaminated with faeces containing pathogens
that cause infectious diseases such as cholera and other diarrhoea diseases, dysenteries and enteric
fevers. As a result, water related diseases continue to be one of the major health problems globally.
It is estimated that globally 80% of all illnesses are linked to use of unsafe and microbiologically poor
water quality. Increase in antibiotic resistance level is now a global problem. Infections with
antibiotic resistant bacteria make the therapeutic options for infection treatment, extremely difficult
or virtually impossible in some instances. Antibiotic resistance is not only found in pathogenic
bacteria but also in environmental organisms inhabiting terrestrial and aquatic habitats. Higher
numbers of resistant bacteria occur in polluted habitats compared with unpolluted habitats,
indicating that humans have contributed substantially to the increased proportion of resistant
bacteria occurring in the environment. Antibiotics exert a selection in favour of resistant bacteria by
killing or inhibiting growth of susceptible bacteria; resistant bacteria can adapt to environmental
conditions and serve as vectors for the spread of antibiotic resistance. The main risk for public health
is that resistance genes are transferred from environmental bacteria to human pathogen. In general
terms, the greatest microbial risks are associated with ingestion of water that is contaminated with
human or animal faeces. Wastewater discharges in fresh waters and costal seawaters are the major
source of faecal microorganisms, including pathogens. Acute microbial diarrheal diseases are a major
public health problem in developing countries. People affected by diarrheal diseases are those with
the lowest financial resources and poorest hygienic facilities. Children under five, primarily in Asian
and African countries, are the most affected by microbial diseases transmitted through water.
Microbial water-borne diseases also affect developed countries. In the USA, it has been estimated
that each year 560,000 people suffer from severe water-borne diseases, and 7.1 million suffer from a
mild to moderate infections, resulting in estimated 12,000 deaths a year. 11 In 2015, reports revealed
that around 319 million people in sub– Saharan African, characterized by the shortage of clean and
safe water for drinking. Antimicrobial agents’ resistance has been recognized as an emerging
worldwide problem, in both human and veterinary medicine, and drug abuse is considered the most
important factor for the emergence, selection and dissemination of antimicrobial agent resistant
bacteria.
The traditional waterborne enteric pathogens are Shigella spp. (four species causing dysentery),
Salmonella enterica (subsp. enterica ser. Typhi, causing typhoid), and Vibrio cholerae (serogroups O1
and O139, causing cholera). These pathogens have been largely controlled by water
treatment/disinfection, and as a result, they are rarely a problem via drinking water in developed
regions. However, Shigella sonnei, along with closely related shiga toxin and verotoxin-producing E.
coli persist in the sewage of developed nations due to person-to-person and foodborne transmission
(Ashbolt et al., 2015).
A genus of rod-shaped, gram-negative, non-spore-forming enterobacteria, mostly motile, is called
salmonella. They are facultative anaerobes and chemo organotrophs, deriving their energy from
organic substances through oxidation and reduction reactions. Growing most species on media
containing ferrous sulfate allows one to easily detect hydrogen sulfide that they create. There are
two phases that most isolates go through: a motile phase (I) and a nonmotile phase (II). It is possible
to transition non-motile cultures into the motile phase after primary culture. Closely linked to the
Escherichia genus, salmonella can be found in both warm- and coldblooded animals, including
humans, as well as in food-borne illnesses including typhoid fever and paratyphoid fever (Oludairo et
al., 2022; Stella et al., 2018).
Shigella are non-motile, facultative anaerobic, gram-negative rods that do not generate spores.
Shigella's pathogenicity, physiology (inability to digest lactose or decarboxylate lysine), and serology
set it apart from the closely related Escherichia coli bacterium. The most frequent cause of bacillary
dysentery is Shigella. This illness is not the same as enterotoxigenic diarrhea caused by Escherichia
coli, which results in excessive amounts of watery diarrhea. The genus contains four sero-groups: A.
with twelve serotypes of S. dysenteriae, B. with six serotypes of S. flexneri, C. with eighteen
serotypes of S. boydii, and D with one serotype of S. sonnei (Strockbine et al., 2015).
Although E. coli is considered to be a more accurate indication of faecal pollution, it still has a
number of drawbacks that should be taken into account before relying solely on the findings of E. coli
testing for faecal contamination (Rana et al., 2024; Hossain et al., 2024; Li et al., 2021). It has even
been demonstrated that E.
Drinking water and wastewater (hereafter “water ”) services require significant energy, consuming
about 3% of total U.S. electricity, though the proportion varies regionally and is expected to increase
in the future with scarcer water supplies and stricter treatment standards [ 5 , 6 , 15 , 24 ]. In
developing countries, the share of electricity consumed by water services may be even greater. The
energy demands of individual water systems vary by location, size, water source, climate, and
treatment process [ 4 , 18 , 19 , 26 ]. In recent years especially, the water sector has turned to energy
management as a means to meet rising energy prices and operate more sustainably, creating new
opportunities for synergy in the energy–water nexus.
Thanks to water storage and built-in redundancy, water systems have some operational flexibility and
can help balance loads in the electric grid during demand response (DR) events. As a subset of
demand-side management (DSM) strategies, DR events are initiated when power sup- pliers cannot
keep up with instantaneous demand and need to temporarily shed or shift demand in order to regain
some capacity. During DR events, large industrial users may volunteer to pause their operations, for
which the power company later compensates them, effectively buying back unused power at critical
times. Reviews by Schäfer et al. [16] and Zohrabian et al. [27] showed that the water sector can offer
significant DR benefits to power providers and that there has been some success to date. But
Zohrabian et al. [27] observed that while the water sector has progressed in its energy management
practices, it still lags behind other industries in DR.
DR solutions are increasingly necessary as energy systems transition from steady fossil fuels to
cleaner but intermittent renewables and consequently must be more nimble than ever in balancing
supply and demand [25] . The water sector can support this transition by participating in DR
programs, but several barriers remain. Here, the author describes a few challenges and
recommendations not fully developed elsewhere, based on both available literature and personal
experience in energy management programs with 10 power companies and over 60 water utilities in
the United States.
About 70% of the earth’s surface is covered with water either as freshwater, brackish water or as
saline water (Sen, 2019). Water, an important earth resource, is very vital to humans, plants and
animals. Man utilises this indispensable natural resource for various activities including domestic,
industrial, agricultural and or recreational purposes. To the ecosystem, water serves as a habitat for
aquatic organisms such as fish, crocodiles, snails, frogs, bacteria, fungi, protozoans, algae and viruses
and these organisms interact with each other and form the aquatic ecosystem (Sen, 2019).
Potable water is crucial to human existence. Unfortunately, some people struggle to have access to
safe water. The physical and chemical properties of water have great implications on its microbial
quality and the occurrence of antibiotic-resistant bacteria (ARB). Most often, when the physical
(colour, odour, temperature etc.) and chemical (pH, electrical conductivity, heavy metals, COD, DO
etc.) parameters of water are above permissible limits, the microbial load of the water will be high
with elevated ARB levels (Ho et al., 2021). The microbial quality of water largely determines its
usefulness, especially for domestic usage. Globally, bacterial contamination of water brings about
serious public health threats (Hile et al., 2023). Bacteria are one of the major pathogens responsible
for waterborne diseases, and they have been implicated in many gastrointestinal outbreaks
worldwide (Delgado-Gardea et al., 2016). Worldwide as a result of waterborne diseases, 525,000
children (< 5) come down with diarrhoea on a yearly basis with 117,000 deaths recorded in Nigeria
alone (WHO, 2017; UNICEF, 2018).
However, despite the continuous efforts to improve water quality worldwide, waterborne disease
outbreaks are still reported (Hile et al., 2023). Bacteria associated with waterborne diseases include
Escherichia coli, Legionella spp., Pseudomonas aeruginosa, Aeromonas and Mycobacterium spp.
(Stec et al., 2022). Other waterborne pathogens are usually obligate pathogens; they multiply only in
an infected host.
They include Campylobacter, Salmonella, Shigella, Escherichia coli, Acinetobacter spp., Clostridium
spp., Bacillus anthracis and Helicobacter pylori. (Delgado-Gardea et al., 2016). When in the human
hosts, these waterborne pathogens are usually eliminated using antibiotics.
For decades, antibiotics are the widely known effective antimicrobials used for treatment of
infectious diseases while curbing the rate of morbidity and mortality (Chukwu et al., 2020). However,
the misuse of antibiotics has led to the development of antimicrobial resistance. Several sources of
antibiotic contamination into water bodies have been identified which include: surface runoff,
hospital discharge, sewage and wastewater treatment effluents, municipal waste and animal
wastewater effluents (Guo et al., 2018).
The deposition of antibiotic residues into the environment leads to the development of antimicrobial
resistance in some bacteria. These bacterial species are referred to as antibiotic-resistant bacteria
(ARB) and they contain antibiotic resistance genes (ARGs) which are transferred horizontally or
vertically to other bacteria (Peterson & Kaur, 2018). Related studies have also revealed that heavy
metals do combine with antibiotics in compound pollution (Zhou et al., 2022). The synergistic
selection pressure between heavy metals and antibiotics increases the population of co-resistant
microorganisms in the environment, impairs microbial communities and the expression of genes in
water bodies causing changes in the structure and diversity of natural microbial communities (Wang
et al., 2023).
Heavy metals, even at very low concentrations, can alter the bacterial efflux pump system
expression, thereby promoting cross-resistance to antibiotics and contributing to the development of
multidrug-resistant bacterial species (Xu et al., 2022; Chukwu et al., 2023). Bacterial species become
vulnerable to antibiotics due to persistent and continuous exposure to antibiotics; this increases
bacterial selection pressure which in turn enhances the increased abundance of resistant bacterial
strains (Danner et al., 2019). Antimicrobial resistance (AMR) has become a serious global issue,
gaining top priority on the public health agenda as it leads to a reduction in treatment options for
bacterial infections thereby reducing clinical efficacy while increasing treatment costs and mortality
(Oladipo et al., 2019; Achi et al., 2021). These water bodies, which once considered as divine source
of water are now increasingly being abused and severely polluted. Our fresh water bodies are
contaminated with different kinds of pollutants resulting from increasing human population,
urbanization and industrialization. In the context of international regulations, the contamination of
water bodies by organic micro pollutants is the subject of constant interest and is always under
investigation. Although the provision of safe drinking water has been one of humanity’s most
successful public health interventions. It is a defining aspect of a developing country’s ignorance of
the risks and inappropriate training of the staff and managers working on drinking water systems,
which still results in unnecessary waterborne disease outbreaks in affluent communities. Disposal of
domestic wastes in lakes is causing undesirable changes in physio-chemical and biological
characteristics of these waters.[1] Microbes including bacteria, viruses and protozoa are the common
cause of diseases in present aquatic ecosystems. Many major human diseases such as typhoid fever,
cholera and other diarrheal diseases, poliomyelitis and viral hepatitis A and E are water borne. These
pathogens reach water sources through faecal and sewage pollution. The aquatic systems are mostly
dominated by bacteria and fungi and in the natural environments micro-organisms have very specific
roles with regard to the recycling of materials and purification of water. Salmonella, Acinetobacter,
Chromobacterium, Alcaligens, Flavobacterium, Staphylococcus aureus, Pseudomonas aeruginosa,
Clostridium botulinum, Vibrio cholerae and Escherichia coli are the main human pathogens
responsible for water contamination.[2] The presence of coliform a rod shaped, gram negative
bacteria in the water indicates the faces of human beings and other warm-blooded animals in large
numbers and can be easily detected in high dilutions. This confirms the presence of E. coli in the
water bodies giving a definite proof of faecal pollution thus not being suitable for bathing and
drinking. Whereas waste water reuse has been extensively implemented in some European and
African countries, yet in South Africa, only a few waste water reuse schemes have been documented
and there is limited implementation of this alternative in communities.
The intrusion of biological agents into water systems can pose serious public health risks as these
agents cannot be easily detected and can remain hidden until a widespread contamination exists.
Alarming increases in the consumption of antibiotics through human therapy and agricultural
processes have been reported and this extensive usage in both human and animal medicine has
resulted in the development of antibiotic-resistant bacteria which affect the treatment of infections.
Antibiotic resistance has therefore become a major public health issue and its presence in waste
water, surface water, and drinking water is well documented. The hazard associated with the
pathogenicity of microbes is aggravated by its ability to resist destruction by antibiotics. Biological
treatment processes in the waste water treatment plants may result in a selective increase of
antibiotic-resistant bacteria and therefore increase the occurrence of multidrug-resistant organisms
Although microorganisms in drinking water are reduced by chlorination, they may survive the
treatment process and enter the distribution system. Moreover, the presence of antibiotic resistance
in microorganisms has been previously reported [24–26]. Considering the fact that the public health
of a community may be related to the quality of treated waste water supplied and that public health
can be protected by reducing the pathogenic microorganisms in drinking water, the present study
was designed to isolate environmental bacteria from surface and drinking water in Mafikeng and
identify the Pseudomonas and Aeromonas species using polymerase chain reaction (PCR). A further
objective was to characterize the isolates using their antibiotic resistance profiles.

2. REVIEW OF LITERATURE
A significant amount of research has been conducted worldwide to evaluate the quality of water
resources. Kumar et al. (2021) demonstrated that river water near industrial zones showed high
microbial contamination, exceeding WHO permissible limits for coliform counts. Singh and Verma
(2019) reported that well water in rural India often exceeded BIS standards for hardness and
microbial content due to poor sanitation practices. Patel et al. (2020) observed significant fungal
diversity in pond water influenced by agricultural runoff and organic matter accumulation. WHO
(2017) guidelines emphasize the importance of maintaining pH between 6.5–8.5, turbidity below 5
NTU, and zero coliform count per 100 ml of drinking water. APHA (2017) outlined standard
methodologies for physicochemical and microbiological water testing. The combination of microbial
and physicochemical analyses provides a comprehensive assessment of water safety, highlighting the
need for integrated studies covering multiple water sources. Certain microorganisms including
bacteria, viruses and parasites are well known water contaminants of which several may lead to
waterborne disease and epidemics. Role of water in spreading communicable diseases is evident due
to combined source of water, which is drinking as well as bathing. Contaminated water with faecal
coliform severely affects the performance of humans. Contamination of water sources is counted
when the man’s action is adding or causing the addition of pollutants thus by altering its physical,
chemical and biological characteristics to such an extent that it’s utility for any reasonable purpose or
its environmental value is demonstrably depreciated. An estimation of bacterial production is a
crucial step in understanding quantitatively the function and contribution of bacteria in material
cycling within given aquatic habits. Wild and domestic animals seeking drinking water can also
contaminate the water through direct defecation and urination. [2] Various studies have evolved,
wherein contamination of various water bodies across the country with different microorganisms is
notified time and again.

3. AIM AND OBJECTIVES

Aim:
To isolate and identify microbial populations from various water resources and evaluate their water
quality using physicochemical and microbiological analyses.

Objectives:
To collect water samples from multiple sources including river, lake, pond, well, tap water, and
borewell.
To isolate and identify bacterial and fungal species present in the samples.
To analyze physicochemical parameters such as pH, turbidity, TDS, DO, hardness, and chloride.
To compare water quality parameters with WHO and BIS drinking water standards.
To determine the contamination status and potability of each water source.
To correlate microbial diversity with environmental conditions and physicochemical parameters.
4. MATERIALS AND METHODOLOGY
Materials:
Sterile water sample collection bottles (500 ml)
Nutrient Agar, MacConkey Agar, Potato Dextrose Agar
pH meter, turbidity meter, TDS meter
Titration kits for hardness and chloride
Incubator, microscope, biochemical test kits

Methodology:
4.1 Sample Collection:
Water samples will be collected in sterile containers from river, lake, pond, well, tap water, and
borewell.
Samples will be stored in an icebox and transported to the laboratory for processing within 6 hours.
4.2 Microbial Isolation:
Serial dilution and spread plate techniques will be used for bacterial and fungal isolation.
Nutrient Agar and MacConkey Agar will be used for bacterial growth; PDA for fungi.
Plates will be incubated at 37°C for bacteria (24–48 hrs) and 28°C for fungi (48–72 hrs).
4.3 Identification:
Bacterial isolates will be identified using Gram staining and biochemical tests (IMViC, catalase,
oxidase).
Fungal species will be identified based on colony morphology and microscopic examination.
4.4 Physicochemical Analysis:

pH – digital pH meter
Turbidity – nephelometric method
TDS – TDS meter
DO – Winkler’s method
Total Hardness – EDTA titration method
Chloride – Argentometric titration
4.5 Data Analysis:
Microbial load expressed as CFU/ml will be statistically analyzed.
Water quality parameters will be compared with WHO and BIS standards.
Correlation between physicochemical and microbiological data will be evaluated.
5. EXPECTED OUTCOME
Isolation and identification of dominant bacterial and fungal species from all water sources.
Comprehensive evaluation of physicochemical and microbial quality of each source.
Assessment of potability based on WHO and BIS standards.
Establishment of correlations between microbial populations and environmental factors.

6. SIGNIFICANCE OF THE STUDY

Provides critical data for environmental monitoring and public health safety.
Helps in identifying contamination sources and risk factors in water resources.
Supports sustainable water resource management and policy development.
Contributes to academic research in microbiology, environmental science, and public health.

7. REFERENCES
APHA. (2017). Standard Methods for the Examination of Water and Wastewater (23rd ed.). American
Public Health Association.
Kumar, R., Sharma, P., & Gupta, A. (2021). Assessment of microbial contamination in river water.
Journal of Environmental Microbiology, 45(2), 112–120.
Patel, S., Mehta, V., & Roy, R. (2020). Fungal diversity in pond ecosystems and their impact on water
quality. International Journal of Water Research, 12(3), 221–230.
Singh, K., & Verma, P. (2019). Evaluation of physicochemical and microbial parameters of well water.
Indian Journal of Environmental Protection, 39(5), 380–388.

World Health Organization. (2017). Guidelines for Drinking-Water Quality (4th ed.). WHO Press.