1.

INTRODUCTION
Pollution of natural waters with waste water arising from various industries has become a serious
problem globally Textile industries are large industrial consumers of waters as well as producers
of wastewaters with the increased demand for textile products leading to increase in the
generation of textile waste water which makes the textile industry one of the main sources of
severe pollution problems worldwide (Sen & Demirer, 2003; Santos, 2005; Vanlier et al.,
2007). Effluents from the textile factory commonly contain high concentrations of organic and
inorganic chemicals and are characterized by high Chemical Oxygen Demand (COD), Biological
Oxygen Demand (BOD), Total Dissolved Solid (TDS), pH, Total Suspended Solids (TSS)
values, and low dissolved oxygen (DO) value as well as strong color. The major concern with
color is its aesthetic character at the point of discharge with respect to the visibility of the
receiving waters (Solmaz et al., 2006; world bank, 2010; Mansour et al., 2012)The textile
factory in Ethiopia dates back to 1939 in relation with Italian colonialism era, when the first
industrial textile factory was established in Dire-Dawa in the name of Dire-Dawa textile mill.
Since 2010, the Ethiopian government has put effort to improve, support, and expand the textile
&industry, serving the domestic market but mainly with the aim to export and be competitive at
the global market. Ethiopia has potential of building a textile factory with governmental support,
offering low-cost production and raw material and with a growing young population eager for
jobs (Alderin, 2014). The factory is one of the largest employers in Ethiopia, with 35,000 direct
employees (cotton farming (10%) and textile/garment manufacturing (90%)), excluding the
500,000 engaged in the informal hand-loom weaving sector (Federal, 2011). Bahir Dar textile
factory is one of Ethiopia textile factories, manufacturing 100% cotton products, including yarns
and fabrics. It was established in 1961 from the fund of Italian war reparation in the town of
Bahir Dar, 570 km nort west of Addis Ababa, Ethiopia. The factory has production capacity of 17
tons per day of yarns and 5,000 meters wool fabrics. The major factories in Bahir Dar town
including Bahir Dar textile factory, Bahir Dar tanneries, and Bahir Dar Abattoir are built at the
edge of head of Blue Nile River, where most of them dispose their solid and liquid wastes
directly into this river. Despite the extensive work that has been conducted on Bahir Dar
tanneries effluents with regard to physicochemical parameter and aquatic macro invertebrates
(Wondie & Wosnie, 2011) little information is documented about the effect of Bahir Dar textile
factory effluents on head of Blue Nile River.
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Safe drinking water and proper sanitation have constantly been recognized as indispensable
factors to sustain life. Nevertheless, despite remarkable global progress to improve access to
drinking water facilities, currently there are 884 million and an additional 2.5 billion people
lacking improved water sources and sanitation respectively. (United Nations Water Global
Annual Assessment of Sanitation and Drinking Water (GLAAS), 2012). This crisis is further
compounded by factors such as increasing poverty, accelerated population growth and rapid
urbanization coupled with hydrological variability and climate change. These socio-economic
and environmental factors place even further stress on the deteriorating water and sanitation
infrastructure, more so in developing regions, where billions are still at risk of Water, Sanitation
and Hygiene (WASH) related diseases. Despite meeting the Millennium Development Goals
regarding access to potable water, the depletion of existing finite water resources still continue to
be a major problem, with projections that approximately 605 million people will still lack access
to improved drinking water by 2015 (GLAAS, 2012; UNICEF and WHO, 2012). In addition,
lack of access to potable water is estimated to cost countries between 1%7% of their annual
GDP, with slow water and sanitation-related progress further impeding national economic growth
(GLAAS, 2012; UNICEF, WHO, 2012 and WWAP, 2012). This together with the above
named factors serve as the major driving force behind the increased use of wastewater,
surrounding surface water and grey water for various recreational, agricultural and aquaculture
activities (WHO, 2011). Reliable wastewater treatment systems serve as a good indication of the
level of development within a municipality as well as community health, with the degree and
quality of wastewater determining the impact these treatment plants on surrounding water
sources into which it is released of Water.(Department of Water Affairs, 2011 ) Over the last
few years, the quantity of municipal wastewater produced has drastically increased due to the
constant increase in population numbers together with an increased dependence on diminishing
water resources. This coupled with the discharge of inefficiently treated wastewater into
surrounding surface water sources serve as a direct threat, not only to the macro- and micro flora
and fauna present, but also to the provision of good quality water required for all socio-economic
functions (UNICEF and WHO, 2011). Thus the constant monitoring of the operational status of
existing wastewater treatment plants (WWTPs) as well as increasing emphasis on environmental
and water resource health has become key factors in determining the quantity and quality of
wastewater generated by respective municipalities. This review highlights the importance of
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adequate wastewater management as one of the key steps in to protecting and ensuring the
supply of safe drinking water and maintenance of good public health. In addition, it highlights
the inefficiency of traditional wastewater practices as well as consequences of inadequately
treated effluent discharge on the environment and human health.
1.1 Sources of Domestic and Industrial Wastewater
Wastewater is defined as any storm water runoff, as well as industrial, domestic or commercial
sewage or any combination thereof carried by water. The type and volume of wastewater
generated is determined by both, population numbers and the combination of surrounding
domestic, recreational and industrial activities, all of which affect discharge patterns as well as
the chemical status of the treated effluent (CIDWT Decentralized Wastewater Glossary, 2009). In
order to set up an efficient waste management system, proper identification and characterization
of the influent entering a wastewater treatment plant is essential (Mara, 2004). This is based on
the physical, chemical and biological characteristics of the influent; the immediate and
downstream effect on the surrounding environment into which the wastewater will be discharged
as well as the currently laid out environmental and discharge standards. Four main types of
wastewater have been identified, namely domestic, industrial, agricultural and urban. Urban
wastewater is defined as a combination of domestic and industrial wastewater as well as
surrounding sewage infiltration and rain water whilst agricultural wastewater consists of
wastewater generated through processes from surrounding farms, agricultural activities and
sometimes contaminated groundwater (Hamdy et al., 2013). Generally, the focus is mainly on
domestic and industrial sewage as a source of plant influent and contamination, however
agricultural runoff is now becoming increasingly important due to the high quantities of
pesticides and fertilizers being used, ultimately contributing to surface water eutrophication
(Department of Water Affairs, 2011). Domestic wastewater is defined as sewage which
generally consists of black water composed of fecal matter (human and animal wastes) together
with grey water sources composed of various wastewater constituents. These components
originate from a range of household activities (washing and bathing) with each forming
approximately 32.5% and 67.5% of domestic sewage respectively.(Environmental Protection
Administration, Taiwan.2013). Initially, this water is used for drinking, food preparation, hot
water systems, bathing, personal hygiene, washing and gardening and may ultimately form part
3 | Page

of the domestic wastewater being excreted into the environment. (Department of Water Affairs
and Forestry, 1996). Within a household individual domestic wastewater streams all contribute
different amounts to the overall nutrient and element load comprising the discharged effluent.
Industrial wastewater however, is defined as sewage consisting of industrial wastes such as pulp,
paper, petrochemical runoff as well as various chemicals, salts and acids. These sources vary
widely in composition and often require special tertiary treatments in order to comply with
discharge regulations. The composition of industrial wastewater varies based on the type of
surrounding industry together with respective contaminant and pollutant composition with
general classification into inorganic and organic industrial wastewater.(Rosenwinkel et al.,
2005).
1.2 Impact of Improperly Treated Wastewater Effluent
1.2.1 Effect on the Environment, Micro- and Macrofauna
The biggest concern associated with microbial pollution is the risk of human and livestock
related illnesses after exposure to contaminated water sources. Often the discharge of improperly
treated effluent from WWTPs results in the deposition of large amounts of organic matter and
nutrients which have major detrimental effects on the health of these surrounding environments
as well as micro- and macro-fauna present. Excessive nutrient loading can lead to eutrophication
and temporary oxygen deficiencies that ultimately alter the energy relationship and water
balance, disrupting biotic community structure and function. Excessively turbid effluent
discharge can also result in the deposition of sand and grit into the aquatic system, disrupting
sediment characteristics and hindering natural water flows. (Wakelin et al., 2008) In addition,
the overall hydrological and physicochemical environment is often affected due to the discharge
of improperly treated effluent with many of the micro- and macro-fauna within these water
bodies exhibiting distinct physiological tolerance levels. Disturbances to the overall environment
can severely affect those intolerant individuals either in the form of adverse behavioural
characteristics or more severely in the form of death. Often death decreases a large degree of
resource competition and predation within the environment thereby resulting in the proliferation
of tolerant organisms. This ultimately causes an imbalance amongst the group of organisms
present and the overall alterations to the surrounding environment in the form of nutrient
modifications, light and oxygen content, food sources as well as habitat loss (Coetzee, 2003).
4 | Page

Furthermore, the deposition of excessive nutrients leads to profuse plant growth along river
banks which in certain cases may be visually pleasing but can serve as an additional health
hazard due to entanglement and poor visibility. Benthic microbial and algal growth may also
cause rock and wood surfaces to become slippery, posing a threat to human safet (Wakelin et al.,
2008)
1.2.2 Effect on Human Health
Communities situated downstream or near to municipal sewage outfalls or contaminated water
sources are at the highest risk of illness due to increased microbial pathogens and deteriorating
physico-chemical parameters (Wakelin et al., 2008). Often the discharge of extremely turbid
effluent in conjunction with dense algal blooms results in poor visibility within these water
bodies thus creating dangerous situations for recreational users. In addition, water bodies used
for full contact recreational activities may serve as a source of various infectious diseases which
may be contracted either by ingestion of contaminated water or through full body contact
(Department of Water Affairs and Forestry, 1996). However, depending on the type of
waterborne disease and on the physical health of the individual concerned, the person may either
recover completely or suffer permanently from the resultant disease. In addition, a variety of skin
and ear infections may arise as a result of contaminated water coming into contact with broken
skin or penetration of the ear. Furthermore, discharge of improperly treated effluent often results
in an increased number of bacterial, viral and protozoan pathogens which may result in a range
of waterborne related diseases such as giardiasis and gastroenteritis (Okoh et al., 2010). A
number of indirect health hazards such as chemical contaminants, disease-transmitting organisms
such as mosquitos and fresh water snails implicated in malaria and bilharzia, may also arise
depending on the state of the surface water source, leading to additional human health hazards
(Coetzee, 2003).
Waterborne diseases are a significant public health issue, and many originate from contact with
water contaminated with human fecal material (Balarajan et al., 2010; Dufour, 1984; Scott,
2002). An estimated 850 billion gallons of untreated human wastewater and storm water are
discharged into U.S. surface waters each year (U.S.Environmental Protection Agency, 2004).
Because such combine sewer overflow runoff contains raw sewage capable of carrying numerous
human pathogens (e.g., Shigella sonnei, noro viruses, and Cryptosporidium) (CDC, 2002),
5 | Page

solids, debris, and toxic pollutants (i.e., antibiotics, hormones, and caffeine, as well as steroids,
metals, and synthetic organic compounds) (U.S. Environmental Protection Agency, 2004), it is
an important public health concern. There is also now strong evidence that enterococci and other
fecal indicator bacteria in recreational waters themselves contribute to gastrointestinal illness
(Cabelli et al., 1982; U.S. Environmental Protection Agency, 1986) as well as eye, ear, nose,
skin, respiratory, and other infections (Grimes, 1991; Pruss, 1998; U.S. Environmental
Protection Agency, 1994). Ensuring public water quality therefore requires that we develop
improved methods to more accurately identify human fecal pollution. The lack of accurate
methods for identifying sources of fecal pollution has stimulated the recent development of a
number of microbial source tracking (MST) methods. In general terms, these MST methods can
be divided into culture-based and culture-independent techniques (Simpson et al., 2002). Most
culture-based methods for identification of human sources depend on matching panels of
environmental bacterial isolates with known human fecal indicator type strains. A major
limitation of this approach is its requirement for the development of large collections of isolates
from both water and human fecal samples. Thus, MST methods that do not require cultivation,
such as the direct detection of bacterial 16S rRNA gene sequences using PCR, are becoming
more widespread (Bernhard & Field, 2000; Dick et al., 2005). While these assays have now
been used in field applications (Ber & Field, 2000; Dick, 2005; Shanks et al., 2006) a recent
study demonstrates understandable cross-reactivity when highly conserved genomic regions are
targeted (Carson et al., 2005). Since ribosomal genes are not directly involved in microbe-host
interactions, it is possible that other bacterial genetic markers encoding factors related to host
specificity might be better candidates for MST assays. Although a significant number of bacterial
genes have been identified as relevant to host-microbe interactions in the human gut (Hooper &
Gordon, 2001; Hooper at al., 2001), the challenge remains to identify which of these genes are
from bacteria that are truly restricted to this specific niche. We hypothesize that direct
comparisons of the genetic coding capacities of entire human fecal bacterial communities can
identify such factors involved in host-microbe interactions and that these would be the best
targets for PCR assays designed to identify sources of human fecal contamination (Shanks et al.,
2006). We recently developed a nucleic acid analysis method called genome fragment
enrichment (GFE) to identify differences in the genomes of phylogenetically related bacterial
species and to identify differences in total microbial community DNA obtained from different
6 | Page

sources. Here we describe the extension of this approach to address the highly significant
problem of identification of human fecal contamination in water, and we report important
differences encountered relative to other animal sources. A large number of candidate
Waterborne diseases are a significant public health issue, and many originate from contact with
water contaminated with human fecal material (Balarajan et al., 1991). An estimated 850 billion
gallons of untreated human wastewater and storm water are discharged into U.S. surface waters
each year (U.S. Environmental Protection Agency, 2004). Because such combine sewer
overflow runoff contains raw sewage capable of carrying numerous human pathogens (e.g.,
Shigella sonnei, noroviruses, and Cryptosporidium) (CDC, 2002) solids, debris, and toxic
pollutants (i.e., antibiotics, hormones, and caffeine, as well as steroids, metals, and synthetic
organic compounds) , it is an important public health concern. There is also now strong evidence
that enterococci and other fecal indicator bacteria in recreational waters themselves contribute to
gastrointestinal illness, as well as eye, ear, nose, skin, respiratory, and other infections . Ensuring
public water quality therefore requires that we develop improved methods to more accurately
identify human fecal pollution. The lack of accurate methods for identifying sources of fecal
pollution has stimulated the recent development of a number of microbial source tracking (MST)
methods. In general terms, these MST methods can be divided into culture-based and cultureindependent techniques . Most culture-based methods for identification of human sources depend
on matching panels of environmental bacterial isolates with known human fecal indicator type
strains. A major limitation of this approach is its requirement for the development of large
collections of isolates from both water and human fecal samples. Thus, MST methods that do not
require cultivation, such as the direct detection of bacterial 16S rRNA gene sequences using
PCR, are becoming more widespread While these assays have now been used in field
applications a recent study demonstrates understandable cross-reactivity when highly conserved
genomic regions are targeted. Since ribosomal genes are not directly involved in microbe-host
interactions, it is possible that other bacterial genetic markers encoding factors related to host
specificity might be better candidates for MST assays. Although a significant number of bacterial
genes have been identified as relevant to host-microbe interactions in the human gut (Hooper et
al., 2001) the challenge remains to identify which of these genes are from bacteria that are truly
restricted to this specific niche. We hypothesize that direct comparisons of the genetic coding
capacities of entire human fecal bacterial communities can identify such factors involved in host7 | Page

microbe interactions and that these would be the best targets for PCR assays designed to identify
sources of human fecal contamination (Shanks at al., 2006). We recently developed a nucleic
acid analysis method called genome fragment enrichment (GFE) to identify differences in the
genomes of phylogenetically related bacterial species and to identify differences in total
microbial community DNA obtained from different sources. Here we describe the extension of
this approach to address the highly significant problem of identification of human fecal
contamination in water, and we report important differences encountered relative to other animal
sources. A large number of candidate marker sequences are described, with a major characteristic
being that almost half are predicted to encode bacterially secreted or cell surface factors located
at the interface with host cells.
Objectives

Sample collection from polluted water bodies.

Analysis of physico chemical parameter of water sample.
Analysis of microbial diversity of contaminated water and identification by barges

manual.
Identification of bacteria by 16S rRNA sequencing.

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2. REVIEW OF LITERATURE
Shanks. (2006) reported that they used genome fragment enrichment and bioinformatics to
identify several microbial DNA sequences with high potential for use as markers in PCR assays
for detection of human fecal contamination in water. Following competitive solution-phase
hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were
sequenced and were determined to define 289 different genomic DNA regions. These putative
human specific fecal bacteria DNA sequences were then analyzed by dot blot hybridization,
which confirmed that 98% were present in the source human fecal microbial community and
absent from the original pig fecal DNA extract. Comparative sequence analyses of these
sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or
surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26
of the candidate sequences predicted to encode factors involved in interactions with host cells
were then used in the PCR and did not amplify markers in DNA from any additional pig fecal
specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal
sources, with more than half amplifying markers only in specimens from dogs or cats. Four
assays were more specific, detecting markers only in specimens from humans, including those
from 18 different human populations examined. They then demonstrated the potential utility of
these assays by using them to detect human fecal contamination in several impacted watersheds.
Naidu and Olaniran. (2014) reported that more than 1.8 billion people have gained access to
potable water and improved sanitation worldwide. Whilst this represents a vital step towards
improving global health and well-being, accelerated population growth coupled with rapid
urbanization has further strained existing water supplies. Whilst South Africa aims at spending
0.5% of its GDP on improving sanitation, additional factors such as hydrological variability and
growing agricultural needs have further increased dependence on this finite resource. Increasing
pressure on existing wastewater treatment plants has led to the discharge of inadequately treated
effluent, reinforcing the need to improve and adopt more stringent methods for monitoring
discharged effluent and surrounding water sources. This review provides an overview of the
relative efficiencies of the different steps involved in wastewater treatment as well as the
commonly detected microbial indicators with their associated health implications. In addition, it
highlights the need to enforce more stringent measures to ensure compliance of treated effluent
9 | Page

quality to the existing guidelines. The study was conducted in 2013/14 with the objective of
determining the effects of Bahir Dar textile factory effluents on the head of Blue Nile River
water quality. Dissolve oxygen was higher at the upstream site of the river, whereas BOD5, TDS,
and total alkalinity values were higher at wastewater outlet of the factory site. The mean values
of dissolved oxygen, BOD5, and total alkalinity were above maximum permissible limits set by
WHO for drinking water at head of Blue Nile River. The mean value of BOD5 was above
permissible limit of IFC for textile effluents to be discharged to surface water. A total of 836
aquatic macro invertebrate individuals belonging to 21 families were collected. The ShannonWiener Diversity Index, the Hilsenh off family-level biotic index, family richness, and percent
dipterans were calculated. Hilsenh off family-level biotic index and percent dipterans metrics
differed significantly among sampling sites ( < 0.05). Hilsenh off family-level biotic index was
higher at the most downstream site but percent dipterans were higher at site of discharge of
effluent to the head of Blue Nile River. Therefore, there is indication that effluent demands
frequent control and proper treatment before being discharged to the environment.
Kumar et al., reported that dyes residues in textile effluents are hazardous for humans and
animals health. Such pollutants can be degraded into non-harmful molecules using biological
approaches that are considered cheaper and ecologically safer. Isolated 15 bacterial cultures from
soil that could be used in biological system were showed decolonization capacity for Acid Green
dye (33.9% to 94.0%) using thin layer chromatography and broth culture method. The most
promising cultures (AMC3) to decolorize Acid green Dye (94.6%) was re-coded as NSDSUAM
for submitting at IMTECH, Chandigarh for sequencing. The 16SrRNA sequencing suggested that
it can be a variant of Pseudomonas geniculata (99.85% identical similarity) with difference of 2
base pairs to reference strain Pseudomonas geniculata ATCC 19374(T). Thus present study
proposed dye decolorizing efficiency of the isolated strain of Pseudomonas geniculata that was
previously unnoticed. The sequence is deposited in NCBI GenBank with the accession number
KP238100. Rivers are of special significance in Hinduism, not only for its life-sustaining
properties, but also because of its use in rituals and because of the stress given to cleanliness.
Throughout India seven principle holy rivers are nourishing flora and fauna of the region. Of the
seven, the Ganges (Ganga), Yamuna, and Sarasvati are most important. The river Yamuna in
India represents a unique niche for biodiversity. Nowadays, Indian textile Industry is one of the
leading textile industries in the world. However, risks of pollution of water and air have been
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highly increased. Previously, textile effluents have been considered as the most polluting among
all in the industrial sectors by volume and composition of effluent. Typically, textile effluents is
characterized by high values of BOD, COD, color and pH. In-complete use and the washing
operations give the textile effleunts a considerable amount of dyes. That induces persistent color
coupled with organic load leading to disruption of the total ecological/symbiotic balance of the
receiving water stream. Allergic, neurotoxic, carcinogenic effects of these dyes have been
reported by several scientists. Dyes may also affect photosynthetic activity in aquatic habitat
because of reduced light penetration and induce toxicity due to presence of aromatics
compounds, heavy metels, chlorides and other toxic compounds. Humans non-cancer health
hazards are affecting the kidneys, liver, male reproductive organs and developing fetus. Recent
approaches are much focused on the use of microorganisms for the elimination of dyes as it is
comparatively low cost and easier than other conventional processes of decontamination. The
present study was carried out to characterize textile dye degrading bacteria isolated from dye
contaminated soil.
Wang reported that textile industry can be classified into three categories viz., cotton, woolen,

and synthetic fibers depending upon the used raw materials. The cotton textile industry is one of
the oldest industries in China. The textile dyeing industry consumes large quantities of water and
produces large volumes of wastewater from different steps in the dyeing and finishing processes.
Wastewater from printing and dyeing units is often rich in color, containing residues of reactive
dyes and chemicals, such as complex components many aerosols high chroma high COD and
BOD concentration as well as much more hard-degradation materials. The toxic effects of
dyestuffs and other organic compounds, as well as acidic and alkaline contaminants, from
industrial establishments on the general public are widely accepted. At present, the dyes are
mainly aromatic and heterocyclic compounds, with color-display groups and polar groups. The
structure is more complicated and stable, resulting in greater difficulty to degrade the printing
and dyeing wastewater (Shaolan Ding et al., 2010). According to recent statistics, China's
annual sewage has already reached 390 million tons, including 51% of industrial sewage, and it
has been increasing with the rate of 1% every year. Each year about 70 billion tons of wastewater
from textile and dyeing industry are produced and requires proper treatment before being
released into the environment (State Environmental Protection Administration, 1994) .Textile
Printing and dyeing processes include pre-treatment, dyeing and printing, finishing. The main
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pollutants are organic matters which come from the pre-treatment process of pulp, cotton gum,
cellulose, hemicelluloses and alkali, as well as additives and dyes using in dyeing and printing
processes. Pre-treatment wastewater accounts for about 45% of the total, and dyeing/printing
process wastewater accounts for about 50%~55%, while finishing process produces little. In
China, chemical fiber accounts for about 69% of total in which polyester fibers accounts for
more than 80%. Cotton accounts for 80% of the natural fiber production. Therefore, the dyeing
wastewater analysis of production and pollution is based on these two fibers. Pre-treatment of
cotton includes desizing and scouring. The main pollutants are the impurities in the cotton,
cotton gum, hemicelluloses and the slurry, alkali in weaving process. The current average COD
concentration in the pre-treatment is 3000 mg/L. The main pollutants in dyeing/printing are
auxiliaries and the residual dyes. The average concentration of COD is 1000 mg/L and the total
average concentration is 2000 mg/L after mixing. Pre-treatment of polyester fibers mainly
involves in the reduction with alkali. The so-called reduction is treating the polyester fabric with
8% of sodium hydroxide at 90 C for about 45 minutes. Some polyester fabrics will peel off and
decompose into terephthalic acid and ethylene glycol so that a thin polyester fabric will have the
feel of silk. This process can be divided into continuous and batch type. Taking the batch type as
an example, the concentration of COD is up to 20000 mg/L 60000 mg/L. The wastewater from
reduction process may account for only 5% of the volume of wastewater, while COD accounts
for 60% or more in the conventional dyeing and finishing business. The chroma is one pollutant
of the wastewater which causes a lot of concerns. In the dyeing process, the average dyeing rate
is more than 90%. It means that the residual dyeing rate in finishing wastewater is about 10%,
which is the main reason of contamination. According to the different dyes and process, the
chroma is 200 to 500 times higher than before. pH is another factor of the dyeing wastewater.
Before the printing and the dyeing process, pH is another factor the pH of dyeing wastewater
remains between 10 to 11 when treated by alkali at high temperature around 90C in the process
of desizing, scouring and mercerization. Polyester base reduction process mainly uses sodium
hydroxide, and the total pH is also 10 to 11. Therefore, most dyeing water is alkaline and the first
process is to adjust the pH value of the textile dyeing wastewater. The total nitrogen and
ammonia nitrogen come from dyes and raw materials, which is not very high, about 10 mg/L.
But the urea is needed while using batik techniques. Its total nitrogen is 300 mg/L, which is hard
to treat. The phosphorus in the wastewater comes from the phosphor detergents. Considering the
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serious eutrophication of surface water, it needs to be controlled. Some enterprises use trisodium
phosphate so that the concentration of phosphorus will reach 10 mg/L. So, this phosphorus must
be removed in the pre-treatment. In the production process, suspended substance comes from
fiber scrap and undissolve draw materials. It will be removed through the grille, grid, etc. The
suspended solids (SS) in the outflow mainly come from the secondary sedimentation tank, whose
sludge has not been separated completely which will reach 10-100 mg/L as usual. Sulfide mainly
comes from the sulfur, which is a kind of cheap and qualified dye. Due to its toxicity, it has been
forbidden in developed countries.
Cabral, (2010) reported that water is essential to life, but many people do not have access to
clean and safe drinking water and many die of waterborne bacterial infections. In this review a
general characterization of the most important bacterial diseases transmitted through water
cholera, typhoid fever and bacillary dysenteryis presented, focusing on the biology and
ecology of the causal agents and on the diseases characteristics and their life cycles in the
environment. The importance of pathogenic Escherichia coli strains and emerging pathogens in
drinking water-transmitted diseases is also briefly discussed. Microbiological water analysis is
mainly based on the concept of fecal indicator bacteria. The main bacteria present in human and
animal feces (focusing on their behavior in their hosts and in the environment) and the most
important fecal indicator bacteria are presented and discussed (focusing on the advantages and
limitations of their use as markers). Important sources of bacterial fecal pollution of
environmental waters are also briefly indicated. In the last topic it is discussed which indicators
of fecal pollution should be used in current drinking water microbiological analysis. It was
concluded that safe drinking water for all is one of the major challenges of the 21st century and
that microbiological control of drinking water should be the norm everywhere. Routine basic
microbiological analysis of drinking water should be carried out by assaying the presence of
Escherichia coli by culture methods. Whenever financial resources are available, fecal coliform
determinations should be complemented with the quantification of enterococci. More studies are
needed in order to check if ammonia is reliable for a preliminary screening for emergency fecal
pollution outbreaks. Financial resources should be devoted to a better understanding of the
ecology and behavior of human and animal fecal bacteria in environmental waters.

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Environ, (2011) reported that water is a valued natural resource for the existence of all living
organisms.

Management of the quality of this precious resource is, therefore, of special

importance. In this study river water samples were collected and analysed for physicochemical
and bacteriological evaluation of pollution in the Unity Road stream segment of Asa River in
Ilorin, Nigeria. Juvenile samples of Clarias gariepinus fish were also collected from the
experimental Asa River and from the control Asa Dam water and were analysed for comparative
histological investigations and bacterial density in the liver and intestine in order to evaluate the
impact of pollution on the aquatic biota. The water pH was found to range from 6.32 to 6.43
with a mean temperature range of 24.3 to 25.8 C. Other physicochemical parameters monitored
including total suspended solids, total dissolved solids, biochemical oxygen demand and
chemical oxygen demand values exceeded the recommended level for surface water quality.
Results of bacteriological analyses including total heterotrophic count, total coliform and
thermotolerant coli form counts revealed a high level of faecal pollution of the river. Histological
investigations revealed no significant alterations in tissue structure, but a notable comparative
distinction of higher bacterial density in the intestine and liver tissues of Clarias gariepinus from
Asa River than in those.
Zbinden et al. (2014) reported that many bacteria causing systemic invasive infections originate
from the oral cavity by entering the bloodstream. Recently, a novel pathogenic bacterium,
Streptococcus tigurinus, was identified as causative agent of infective endocarditis,
spondylodiscitis and meningitis. In this study, they sought to determine the prevalence of S.
tigurinus in the human oral microbial flora and analyzed its association with periodontal disease
or health. They developed a diagnostic highly sensitive and specific real-time TaqMan PCR
assay for detection of S. tigurinus in clinical samples, based on the 16S rRNA gene. they
analyzed saliva samples and subgingival plaque samples of a periodontally healthy control
group (n = 26) and a periodontitis group (n = 25). Overall, S. tigurinus was detected in 27 (53%)
out of 51 patients. There is no significant difference of the frequency of S. tigurinus detection by
RT-PCR in the saliva and dental plaque samples in the two groups: in the control group, 14
(54%) out of 26 individuals had S. tigurinus either in the saliva samples and/or in the plaque
samples; and in the periodontitis group, 13 (52%) out of 25 patients had S. tigurinus in the
mouth samples, respectively (P = 0.895). The consumption of nicotine was no determining
factor. Although S. tigurinus was a frequently detected species of the human oral microbial flora,
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it was not associated with periodontal disease. Further investigations are required to determine
whether S. tigurinus is a commensal or an opportunistic oral pathogen with a potential for
development of invasive infections.

15 | P a g e

3. MATERIALS AND METHOD

Materials used
Isolation of bacteria

Nutrient agar test

Table 1: composition of Nutrient agar media

Component

Quantity

Peptone
Agar
Beef extract
NaCl
Distilled water

5gm
15gm
3gm
5gm
1000ml

Grams staining
Table: 2 composition of Grams staining

Component
Crystal violet
Grams iodine
Ethyl alcohol
Safranine

Quantity
4-6 drop
2-4 drop
4-6 drop
2-3 drop

Screening of bacteria

Starch hydrolysis test

Table 3: composition of starch hydrolysis test

Component
Starch
Beef extract
Peptone
Agar
Distilled water

16 | P a g e

Quantity
20gm
3gm
5gm
15gm
1000ml

 Biochemical Test

Sugar fermentation test

Table:4 Composition of sugar fermentation test

Component
Peptone
Carbohydrate
Sodium chloride
Phenol red
Distilled water
PH

Quantity
10gm
5gm
5gm
0.01gm
1000ml
7.3

Methyl red /Vogesprokauer(MR/VP)

Table :5 composition of MR/VP

Component
Peptone
Dextrose
Dipotassium
diphosphate
Distilled water
VP reagent 1
VP reagent 2
Methyl red

Quantity
7gm
5gm
5gm
1000ml
600l
400 l
4-6 drop

Indole test
Table:6 composition of Indole test

Component
Tryptone
Distilled water
KOVEX reagent

Casein hydrolysis test

17 | P a g e

Quantity
10gm
1000ml
4-6 drop

Table:7 composition of Casein hydrolysis test

Component
Casein
Beef extract
Peptone
Agar
Distilled water

Quantity
20gm
3gm
5 gm
15 gm
1000ml

Catalase test
Table:8 composition of catalase test

Component
Hydrogen peroxide
Distilled water

Quantity
1ml
2 drop

Citrate utilization test

Table:9 composition of citrate test

Component
Dipotassium
Sodium chloride
Sodium citrate
Magnesium sulphate
Agar
Bromothymol blue
Distilled water

Glassware used

18 | P a g e

Quantity
1.0 gm
5.0 gm
2.0 gm
0.2 gm
15 gm
0.8 gm
1000ml

Table:10 Glassware list

S.N
.
1
2
3
4
5
6
7

Glassware
Test tube
Petriplate
Arymeyer flask
Beaker
Tissue paper
Measuring cylinder
Inoculation loop

S.N
.
8
9
10
11
12
13
14

Glassware
Aluminium foil
Cuvette
Eppendroff tube
Test tube stand
Microtip
L-Spreader
Slide

Broth preparation
Table:11 broth media

Composition
Peptone
Yeast
NaCl
Distilled water

Quantity
5gm
3gm
5gm
1000ml

DNA Isolation from bacteria

Table:12 Composition of DNA isolation

Component
Bacterial culture
Tris-HCL buffer(PH-8.0)
EDTA buffer
TE buffer solution (PH-8.0)
TAE buffer
SDS solution
Phenol chloroform
Propenol

19 | P a g e

Quantity
1ml
10 ml
1 ml
50X(diluted to 1X)
50X(diluted to 1X)
10%
25:24:1
Double volume of upper
transparent layer

Agarose Gel Electrophoresis (0.8% Gel)

Table13: Composition of agar gel preparation

Component
Agarose
TA E buffer
Ethidium bromide
DNA loding dye
Distilled water

Quantity
0.2gm
50X(diluted t0 1X)
3l
5l
25ml

Spectrophotometric Quantification of Genomic DNA

Table 14: composition of spectrophotometric quantification

Component
Template DNA
Distilled water

Quantiy
10l
990l

Polymerase chain reaction(PCR)

Table15:Composition for PCR

Component
Template DNA
PCR buffer
dNTP
Universal forward primer
Universal backward primer
Taq polymerase
Distilled water

20 | P a g e

Quantity
5l
2l
2.5l
1l
1l
1l
7.5l

 Instruments used

Fig1.:(a) Thermo cycler

(d) Electrophoresis Unit

(g) Spectrophotometer

21 | P a g e

(b) Analytical Balance

(e) vortex

(h) Centrifuge

(c) Gel Documentation System

(f) pH meter

(i) Water-bath

Methodology
Sample collection
Water sample was collected from local aria of dollyganj gomti nadi (lucknow).
Serial dilution (Culturing of soil bacteria by dilution method)
Eight test tube were taken and marked as tube 1to 8.
1gm of soil was measured and dissolved in 1 ml of distilled water in 1 tube.
1 ml of solution dissolved in 9 ml of distilled water in 2 tube and then tube was vortex in
order to mix the soil sample. Concentration of first tube.
After that 1 ml of solution and was transferred into next tube containing 9 ml of distilled
water in order 10-1 concentration.
Now step were repeated 8 time in order to prepare 10-8 concentration.
Inoculating agar plates using spread plate techniques

Firstly nutrient agar media was prepared for growth of soil bacteria and Autoclave it at
15

lb/inch2 at 121.5C

Poured in Petri plates and cooled.

Now add 200l of 10-8 dilution sample was added onto the nutrient agar plate and spread
it using the sterilized L-shaped glass rod (spreader).

The plates were incubated at 37C for 24 hours.

Isolation of single bacterial colonies

The principle of this technique is to streak a suspension of bacteria until single cell are
separated on the plate. Each individual cell then grows in isolation to produce a clone of
identical cells known as a Colony. The majority of these cells are genetically identical.
However, during growth, mutation of a single colony can give rise to low level of
mutants cells.

Protocol (Streak plate method) .

22 | P a g e

Flamed a Nichrome loop (3mm across and has 6cm stem). Allowed the loop to cooled or
cooled by immersion in a sterile area of the medium.

Flamed the neck of an overnight broth culture and removed a loop of cells or picked a
colony from overnight agar culture.

Streaked the cell at one side of a well dried agar plate (in a manner to make the first arm
of pentagon).

Flamed the loop and cooled as before.

Streak again, starting from one end to make second arm of pentagon.

Repeated the steps 4 and 5 to complete the pentagon.

Labeled each plate to indicate the strain no, genotype of strain, date of culture and name.

Wrapped the plates.

Incubated the plate at 37C with the medium facing downwards to reduce the chance of
droplets

of

condensation

falling

on

the

medium

surface.

Identification of Unknown Bacteria species

Grams staining
Grams staining technique which is used to differentiate bacteria of the basis of their Cell wall
composition. The bacterial which appeared purple in colour are called as Grams positive while
the bacteria which appear pink in colour are Grams negative in nature. Gram-positive cells have
a thick peptidoglycan cell wall that is able to retain the crystal violet-iodine complex that occurs
during staining, while Gram-negative cells have only a thin layer of peptidoglycan. Thus Grampositive cells do not decolorize with ethanol, and Gram-negative cells do decolorize, this allows
the Gram-negative cells to accept the counter stain safranine. Gram-positive cells will appear
23 | P a g e

blue

to

purple,

while

Gram-negative

cells

will

appear

pink

to

red.

Procedure

Prepared heat-fix smear.

Stained the slide by flooding it with crystal violet for 2-3 minutes

Pour off excess dye and washed gently in tap water.

Added Grams iodine for one minute for 1-2 minutes by washing with iodine, then added
and leaved it on the smear until the minute is over.

Washed with tap water and drained (did not blot).

Washed with 95% alcohol for 30 seconds.

Washed with tap water at the end of 30 seconds to stop decolourization and drained.

Counter stained with 0.25% safranine for 2-4 minutes.

Washed, drained, bloted, and examined under oil at 1 OOX objective.

Indole test
Some bacteria can produce indole from amino acid tryptophan using the enzyme tryptophanase.
Production of indole is detected using Ehrlichs reagent or Kovaxs reagent. Indole reacts with
the aldehyde in the reagent to give a red colour. An alcoholic layer concentrates the red colour as
a ring at the top.Indole test mainly shown by Gram negative bacteria like .E.coli, Bacillus spp.,
Enterobacter spp., Klebsiellaspp., Staphylococcus spp. etc.

24 | P a g e

Procedure

Bacterium to be tested was inoculated in tryptone water, which contains amino acid
stryptophan and incubated overnight at 37C.

Following incubation few drop of Kovaxs reagent were added. Kovacs reagent consists
of para-dimethyl aminobenzaldehyde, isoamyl alcohol and concentrated HCI. Ehrlichs
reagent is more sensitive in detecting idole production in anerobes and non-fermenters.

Red or pink coloured ring formed at the top is taken as positive.

Methyl Red (MR) Test

This is to detect the ability of an organism to produce and maintain stable acid end products from
glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation
that they overcome the buffering action of the system. Methyl Red is a pH indicator, which
remains red in colour at a pH of 4.4 or less. Methyl red test mainly shown by Gram
negative/positive bacteria like Neisseria spp., E.coli, Bacillus pp., Staphylococcus spp. etc.

Procedure

The bacterium to be tested was inoculated into glucose phosphate broth, which contains
glucose and a phosphate buffer and incubated at 37C for 48 hours.

25 | P a g e

Over the 48 hours the mixed-acid producing organism must produce sufficient acid to
overcome the phosphate buffer and remain acid.

The pH of the medium was tested by the addition of 5 drops of MR.

Voges-Proskauer(VP)Test
While MR test is useful in detecting mixed acid producers, VP test detects butylene glycol
producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylene
glycol. In this test two reagents, alpha-naphthol ( VP-I or Barritts reagent-A) and 40% KOH
(VP-lI or Barritts reagent B) are added to test broth after incubation and exposed to atmospheric
oxygen. If acetoin is present; it is oxidized in the presence of air and KOH to diacetyl.Diacetyl
then reacts with guanidine components of peptone in the presence of alphanaphthol to produce
red color. Role of alpha-naphthol is that of a catalyst and a color intensifier. VP test mainly
shown by Gram negative/positive bacteria like Enterobacter spp., Kiebsiella spp., etc.
Procedure

Bacterium to be tested was inoculated into glucose phosphate broth and incubated for at
least48hours.

0.6 ml of alpha-naphthol was added to the test broth and shaken. 0.2 ml of 40% KOH
was added to the broth and shaken.

The tube was allowed to stand for 15 minutes.

The negative tubes were held for one hour, since maximum color development occurs
within one hour after addition of reagents.

Citrate Utilization Test or Simmons citrate agar test

26 | P a g e

This test detects the ability of an organism to utilize citrate as the sole source of carbon and
energy. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator
bromothymol blue. The medium also contains inorganic ammonium salts, which is utilized as
sole source of nitrogen. Utilization of citrate involves the enzyme citritase, which breaks down
citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO 2.
Production of Na2CO3 as well as NH3 from utilization of sodium citrate and ammonium salt
respectively results in alkaline pH. This results in change of mediums color from green to blue.
Citrate utilization test shown by Gram negative & positive bacteria like Neisseria spp., E.coli,
Bacillus spp., Enterobacter spp., Klebsiella spp., Staphylococcus spp. etc.
Procedure

Bacterial colonies were picked up from a straight wire and inoculated into slope of
Simmons citrate agar and incubated over night at 37C.

Sugar fermentation test (Glucose, Sucrose and Mannitol)

Many bacteria ferment various carbohydrates such as sucrose, lactose under anaerobic
conditions. Sugar fermentation media containing phenol red as a pH indicator is used. When the
bacterium is inoculated into the tube, the bacterium which ferments the sugar will result in the
production of acid (Lactic acid, formic acid or acetic acid) that will detect the presence of gas.
Sugar fermentation test showed by E. coli, Proteous vulgaris, Kiebsiella pneumoniae and
Salmonella spp. etc.

Procedure

Use a single carbohydrate for each of medium batch medium prepared.

Keep one tube as un-inoculated or as a control.

27 | P a g e

Incubated at optimum temperature for 24 hours.

If the organism has the ability to ferment sugar the medium changes its color from
yellow to pink.

28 | P a g e

 Casein hydrolysis test

The test determines whether an organism can produce the exoenzymecasesase or caseinase.
Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein is
a large protein that is responsible for the white color of milk. This test is conducted on milk agar
which is a complex media containing casein, peptone and beef extract, if an organism can
produce casein and then there will be a zone of clearing around the growth.

Procedure

Streak a plate of casein agar with the culture to be tested.

Incubate plates at optimum temperature for 24 hours

Starch hydrolysis test

Iodine reagent complexes with starch to form a blue black colour in culture clear whole
surrounding colonies is inactive of their ability to digest the starch in medium. Clear colony
show the positive for their ability to digest the starch.
Procedure

Inoculated a starch plate with the microbe to be tested.

Incubated at optimum temperature for 48 hrs.

Iodine solution added on starch media and observed result.

Catalase activity

29 | P a g e

During aerobic respiration in presence of oxygen, microbes show catalase activity.

Procedure

The bacterium to be tested was inoculated into nutrient agar media and incubated for 24
hours.

Took one drop distilled water on slide and inoculated bacterium on it.

Immediately place 1 drop of 3% hydrogen peroxide on to the organism on the plate.

Observe the organism for immediate formation of bubbles indicating oxygen

production.

Preparation of nutrient broth

Nutrient broth media was prepared for growth of soil bacteria and Autoclave it at 15
lb/inch2 at 121.5C

After cooled poured in two test tubes.

Inoculated bacterial sample 1 and 2.

Incubated at 37C for 24 hours.

DNA Isolation from bacterium

In prokaryotes, DNA is double stranded and circular, and is found throughout the cytoplasm. The
cell membranes must be distrupted in order to release the DNA in extraction buffer. SDS is used
30 | P a g e

to disrupt the call membrane. DNA can be protected from endogenous nucleases by chelating
Mg++ using EDTA.Mg++ ion is considered as a necessary cofactor for most nucleases. Isoamyl
alcohol chloroform is used to denature and separate protein from DNA. Chloroform is also a
protein denaturant which stabilize the rather unstable boundary between an aqueous phase and a
pure phenol layer. The denatured protein forms a layer at the interface between the aqueous and
organic phases which are removed by centrifugation . DNA released from the disrupted cells is
precipitated by cold absolute ethanol or isopropanol.

Procedure

Took 1-2 ml bacterial culture in eppendorf tube.

Centrifuged it at 8000 rpm for 5 minutes.

Discarded the supernatant from the sample and collect the pellet.

Resuspended the pellet in 900 l TE buffer and pellet mixed uniformaly by vertex.

Added 1/10 volume of 10% SDS solution. (90l)

Warmed the tube to 50-60C and incubated for 2 hrs.

After incubation mixed 600l of phenol: chloroform: isoamyal alcohol(25:24:l). White

precipitate is formed.

Kept for 5mm and centrifuged it at 10000 rpm for 10 mm.

Collected upper transparent layer in eppendorf tub.

Kept in refrigerator for 10-15 mm.

Added double volume of chilled propanol to form precipitate.

31 | P a g e

Kept it on 0C for 15 mm for DNA precipitation.

Centrifuged it at 10000 rpm for 10 mm.

Kept the tube in inverted position on filter paper.

Dissolved the dry DNA pellet in 50.l TE buffer for further study.

Agarose Gel Electrophoresis (0.8%)

Preparation of Agarose gel

Dissolved agarose in lx TAE buffer in a conical flask.

Placed the conical flask in an oven and heat until the solution becomes transparent.

Used hand gloves remove the conical flask out from oven & wait until it becomes palm
bearable.

Wiped the gel casting tray & comb with ethanol. Sealed the open sides using cello tape &
set up the apparatus.

Poured the agarose solution in the casting tray. Leaved for solidification for 15-20
minutes.

Preparation of DNA samples

Mixed the DNA sample (pellet) in 50 l of TE buffer in an eppendorf.

32 | P a g e

Loading of DNA samples

Mixed 3 i.tl of gel loading dye with 5 il of DNA sample with the help of a micropipette.

Carefully removed the comb from the solidified gel so that the wells dont break.

Filled the tank with TAE buffer.

Placed the agarose gel in the electrophoresis tank.

The wells will be placed towards cathode.

Loaded the samples in the gel carefully using pipettes.

Run the sample till it travels the half area of the gel.

Removed the gel from the electrophoresis tank and observed it on UV transilluminator.

Spectrophotometric determination:

Switched on Spectrophotometer and entered the type of DNA to be quantified.

Prepared the dilution 10 l ds DNA sample and 990 l ultrapure distilled water.

Now kept the sample in spectrophotometer and took the readings of sample at 260/280
nm wavelengths.

Absorption Ratio = 260/280nm wavelength

A260/280 = 1.7 - 2.0 (useful DNA for PCR) (Best 1.8)
A-260 Absorbance by DNA

33 | P a g e

A-280 Absorbance by Protein

The ratio indicates the purity of the sample. Ratio of 1.8 is considered to be the ideal
concentration. Value less than 1.8 indicates presence of unwanted RNA and more than 1.8
indicates presence of protein.

Polymerase Chain Reaction (PCR)

Amplification of DNA was performed in a total volume of 30l.

The reaction mixture contained 2l of l0x reaction buffer (500Mm KC1, 15Mm MgCl 2.
100Mm Tris. HCI.pH :8.3,0.1%w/v gelatine.

5l of template DNA was added.

Added 1l of each primer (Forward and Reverse primer).The primers were used at a
final concentration of 5pM/l.

Added 1I of 10Mm dNTP mix. The four deoxy ribonucleotide tri phosphate (dNTP,
dTTP, dGTP and dCTP) were used at a concentration of 10Mm each.

To each sample, 1l of Taq polymerase (3U/l) were added.

The remaining volume made up by sterile water.

34 | P a g e

Table 19: PCR reaction mixture composition

Chemical

Stock

Working (l)

Template DNA

PCR Buffer

10x

dNTPs

2.5mM

Forward primer

100 ppm

Backward primer

100 ppm

Taq polymerase

5U

Distilled water

Total

20

Amplification was carried out in a thermal cycler. Initial denaturation at 94C for 3 minutes
followed by 35 cycles, each cycle consisted of denaturation of DNA for 30 seconds at 94C,
annealing of the primers for 30 seconds at 56C and elongation 72C for 1 minutes. The PCR
products were analysed by gel electrophoresis in a 1.2% Agarose in Tris-Borate buffer (pH:8.3).
Table 20: Temperature Cycle of PCR

Sequencing:

35 | P a g e

Steps

Temperature Required

Heating the lid

105C

Initial denaturation

94C for 3minutes

Denaturation

94C for 30seconds

Annealing

56C for 30seconds

Extension

72C for Iminutes

Final extension

72C forminutes

No of cycles

35

The amplified product was send for sequencing and sequencing was done using Sangers Method.
The sequence alignment was further done using bioinformatics tool i.e. BLASTN.

The page for BLAST was opened from www.ncbi.nlm.nih.gov

Fig 2: BLAST Homepage

Blastn was choosen for nucleotide alignment and sequence obtained after sequencing
was either pasted in the given box or browsed from the computer where the file was
saved.

Fig 3: Page of blast where nucleotides query sequence is pasted.

36 | P a g e

The result obtained shows the aligned sequences with your query sequence. The
sequences with maximum identity were selected to construct the phylogenetic tree.

Fig 4: CLUSTALW2 Homepage

To construct the phylogenetic tree CLUSTAL W2 was used. Homepage of CLUSTAL W2

was opened from the URL www.ebi.ac.uk.

37 | P a g e

Fig 5: Page of CLUSTALW2 where nucleotide query sequence is pasted

The selected sequences from BLAST result was pasted or browsed from the computer.
Clicked on submit.
Multiple sequence alignment result was obtained. Using the BLAST phylogenetic tree was
constructed by clicking on Phylogenetic tree on the same page.

38 | P a g e

4. Result and discussion

Sample collection water sample was collected from the local area of dollyganj gomtinadi
(lucknow).

Fig. 6. collected sample of water from Gomti River Lucknow.

Isolation of bacteria
Bacteria isolation was done by serial dilution method of water sample .10 dilution was spread
and lawn of bacteria growth was obtained.

Fig 7.Serial Dilution from soil sample for culture of bacterial colony.

39 | P a g e

 Spreading

Fig8. spreading water sample from Gomti river on Nutrient agar plate for growth of desired bacterial
colony

Streaking

Fig.9 streaking of bacterial colony isolated from water sample on NAM for single colony

Grams staining
Among bacterial specie was isolated from water sample named as bacterial species .The bacterial
species was stained with Grams stain in order to study its morphology and shape.The bacterial
species was found to be Grams negative, and rod and bacterial species Grams positive, cocci.
The probability of Bacterial spp. may be E.coli, Corynebacterium, Bacillum ,Nerisseria,
Pseudomonas, Micrococcus, Staphylococcus, Streptococcus, Enterococcus, Lactococcus and
40 | P a g e

Aerococcus spp. Among bacterial species was isolated from contaminated water sample. The
bacterial spp. was stain in order to study its morphology and shape. The bacterial spp.was found
to be Gram-positive, rod shaped.

Figure 10.Gram staining of Bacteria sample 1(a)Grams positive(b)rod shaped (c)Stain used:Crystal
Violet (1min)& Safranin (30sec) (d) Decolouring Agent:70% ethanol (30sec) (e) Mordant:Grams
Iodine(1min)

Identification of isolated bacterial species by using biochemical

Urease test:
The bacterial sp. were shows the positive result and able to hydrolyse the urea as urease enzyme
was produced by the bacterial species.

Fig 11.Urease test

41 | P a g e

Indole test:
The bacterial spp. was shows positive result for indole test and its mean bacteria was able to
produce tryptophanase enzyme after addition of KOVAX reagent form red coloure ring at top
position.

Figure12:Bacterial species show positive result of Indole test (a) Red color Ring
from,Tryptophanase enzyme was produced (b) Reagent used Kovaxs Reagent;
(c) Media used Tryptone broth

Methyl-Red (MR) & Vogesproskauer(VP) test:

The bacterial spp. was shows positive result of MR test hence the bacterial spp .produce acid by
oxidation of glucose and due to which red colour ring form after addition or methyl red.The
bacterial spp. showed positive result for VP test hence the bacterial species not red color form after
adding of alpha- nephthol (VP-1 or Barretts reagent-A)and 40% KOH (VP-2 or Barretts reagentB).Thus the result positive Vp test

Figure.13: Bacterial species show Positive result for MR test;(a)Red color ring from due to
production of acid after addition of Methyl red (b)Bacteria spec ise, show positive result for VP
test; b) Red color ring form after alpha-naphthol (VP-1 or Barrits reagent A) and 40% KOH

42 | P a g e

Casein hydrolysis test:

The bacterial spp. was not able to hydrolyze the casein as protease enzyme was produce by the
bacterial species. Hence the result for casein hydrolysis test was found to be negative result.

Figure.14: Negative result of Casein hydrolysis test (a)No clear zone around the growth
(b)Media Used:Skim Milk Agar Media

Sugar fermentation test:

Bacterial spp. Acts on Glucose and product an acid and in turn changes pink color of the
indicator to yellow and in thus fermentative in reaction. While some bacterial spp. lacks the
capacity to attack the tryptone and produces a deep blue color (i.e.an alkaline reaction) and is
thus oxidative in reaction. The isolate bacteria species showed positive test for sugar
fermentation test

.
Fig .15: Positive test for Sugar fermentation (b) The colour of media was change from pink to
yellow (c) Reagent used: Phenol Red ;(d)Media used: Glucose fermentation media

43 | P a g e

Catalase activity
During aerobic respiration in presence of oxygen, microbes show catalase activity. Catalase
positive culture will produce bubbles of oxygen within one minute after addition of H 2O2.
Bacterial species showed catalase positive result.

Figure.16: Bacterial species show Positive result for Catalase test

Broth preparation- Bacterial cell isolated from streaking plate and inoculated in broth media for
DNA isolation.

Figure17.:Prepration of nutrient broth

DNA isolation

44 | P a g e

Fig 18:Isolated DNA and reagent used in DNA isolation-1X TE buffer,10% SDS,
phenol:chloroform:isoamyl alcohol,Propanol.

Agarose Gel Electrophoresis(0.8%)

For the confirmation of DNA presence 0.8 % gel was prepared in order to visulaize DNA under
Gel Doc System.

Fig 19: Reagent used for Agarose gel formation-1X TAE buffer and Agarose

45 | P a g e

1
7

2
8

3
9

Figur.20: Agrose Gel Electrophoresis (a)Agrose gel (b)1X TAE Buffer (c)EtBr(d) loading dye Lanel-1kb
DNA Ladder,2,3,4& 5Genomic DNA of target bacteria

Spectrophotometric Quantification
After isolation, quantification of DNA was done using Double Beam Spectrophotometer and the
reading obtained were as follows:
Table24: Result of Quantification of DNA using Spectrophotometer
Sr No

Water samples

Mfactor

Ratio

DNA Conc

Gomti

1.00

1.03

1.2

46 | P a g e

Protein
Conc
2.17

 PCR amplification
The DNA extracted was amplified using PCR, 20l reaction mixture was prepared 10X Taq
DNA Polymerase buffer, 1l dNTPs, 1l primers (Forward and Reverse), 1l Taq DNA
polymerase, 5l template DNA and 9l sterile distilled water.The sample was observed on a
1.2% gel in Gel documentation system.

1
5

Figure.21; PCR(a)PCR Buffer (b) Primer (Forword and Backword) (c) Distilled water (d) Taq
Polymerase (e) ladder Lanel-1kb DNA Ladder, Lane 2,3-PCR Product of 1.5-1.8 kb.
16S rRNAsequencing using Sanger Method:
Sample 1: Water sample (Gomti river)
AGGATGAACGCTGGCGGCGTGCCTAATACATGCTCGAGCGAACGGACGAAACAGAAGCTTGCTTCTCTGATGTTAGCG
GCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATA
ATATTTTGAACCGCATGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGCTGCATTAGCTAG
TTGGTAAGGTAACGGCTTACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACA
CGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGA
GTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGG
TACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTA
TTGGGCGTAAAGCCCGCGCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGG
AAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAAC
ACCAGTGGGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCGGT
AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACT
CCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGAAA
GTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACAACTCTAGAGATAGAGCCTTCCCCTT
CGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGTTA

47 | P a g e

GCGCAACCCTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGTTGACTGCCGGTGAAACCGGAGGAAGGTGGG
GATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAATACAAAGGGCAGCGAAAC
CGCGAGGTCAAGCAAATCCCATAAAGTTGTTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATCGAAGCTGGAAT
CGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGGTCACACCACGAGA
GTTTGTAACACCCGAAGCCGGTGGAGTAACCTTTTAGGAGCTAGCCGTAGGTGGGACAAAACTATGATTGGGGTG

Bioinformatic Analysis:

Fig 22: Result of BLAST

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Fig 23: Sequences with maximum identity obtained after BLAST

Fig 24: Multiple Sequence Alignment using CLUSTALW2

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Fig 25: Phylogenetic tree

The above bioinformatic and phylogenetic analysis helped to identify the genus of the
bacteria. The genus of the bacteria is Staphylococcus sps. which has the maximum
identity with the query sequence.

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5. CONCLUSION
State government should also provide water testing laboratory and some common filtration
plant for improving the drinking water quality. Water is essential to life, but many people do not
have access to clean and safe drinking water and many die of waterborne bacterial infections. In
this review a general characterization of the most important bacterial diseases transmitted
through watercholera, typhoid fever and bacillary dysenteryis presented, focusing on the
biology and ecology of the causal agents and on the diseases characteristics and their life cycles
in the environment. The importance of pathogenic strains and emerging pathogens in drinking
water-transmit diseases also briefly discussed. Safe drinking water and proper sanitation have
constantly been recognized as indispensable factors to sustain life.
The present investigation on strategy in DNA isolation was used to isolate bacterial DNA
directly from the contaminated water .For this study, the water sample selected was from Gomti
river. The purpose of this study was to collect the water from that river and identify the bacteria
present in it. The method followed was to first collect the sample from the location, DNA
isolation then qualitative and Quantitative determination of the isolated DNA using Agarose Gel
Electrophoresis and UV Double Beam Spectrophotometer respectively. Isolated DNA was
further used for PCR amplification. Sequencing was then done of the amplified PCR product.
Genetic relatedness of the identified species was then done using Phylogenetic tree using
bioinformatic tools. The genus of the bacteria is Staphylococcus sps. which has the maximum
identity with the query sequence.

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