Analysis of Microbial and Physicochemical Properties of Pond Water Samples Collected from Different Locations of Agra (U.P.) India

With two-thirds of the Earth's surface covered by water and the human body consisting of 75 percent water, it is evident that water plays a crucial role in sustaining life on our planet. Presently, our drinking water falls short of being pure, containing bacteria, viruses, inorganic minerals (resulting in hard water), and a chemical concoction that is unacceptable, if not potentially harmful, for human consumption. It is well-known that freshwater fish and their aquatic environments can harbor pathogenic bacteria, particularly those belonging to the coliform group (Leung, Huang, and Pancorbo 1992; Pulella, Fernandes, Flick, Libey, Smith, and Coale, 1998; Ramos and Lyon 2000). The presence of coliform bacteria in water can be attributed to fecal contamination, stemming from the discharge of faeces by humans, all warm-blooded aquatic creatures, and some reptiles. Coliform bacteria encompass members of the Enterobacteriaceae family, such as Escherichia coli, Enterobacter aerogenes, Salmonella, and Klebsiella. The faecal indicator bacterium, E. coli, is widely recognized as a bioindicator of faecal contamination in drinking water.

Investigation of pond water and sediment samples reveal a close correlation between nitrifying microorganisms (including ammonifying bacteria, nitrite bacteria, nitrobacteria, and denitrifying bacteria) and various physicochemical factors. Ammonifying bacteria are primarily associated with dissolved oxygen, denitrifying bacteria show a negative correlation with phosphorus, nitrite bacteria are strongly linked with nitrites, and nitrobacteria exhibit an inverse correlation with ammoniac nitrogen.

In terms of human health, water plays a crucial role, and the quality of the supplied water is vital in determining the well-being of individuals and entire communities. Ensuring safe water quality is a major concern for public health, as the health and prosperity of humanity are intricately connected to the quality of water used (Sharma et al., 2005; Venkateswaran et al., 2011). Freshwater is a limited resource essential for agriculture, industry, and human existence. Without a sufficient quantity and quality of freshwater, sustainable development would be unattainable (Kumar, 1997).

The analysis of microbial and physicochemical properties of pond water samples is crucial for understanding the overall water quality and its suitability for human consumption. In the existing literature, concerns about the safety of potable water have been highlighted due to the presence of microscopic organisms, infections, and inorganic minerals. This study focuses on assessing the microbial parameters in pond water samples collected from diverse locations in Agra, Uttar Pradesh, India.

Microbial analyses of water environments have been conducted using the Serial Dilution method to isolate bacteria from collected pond samples. Quantification of the coliform group is essential to gauge the extent of waterborne microorganisms. Additionally, physicochemical parameters such as pH, dissolved oxygen (DO), biochemical oxygen demand (BOD), and chemical oxygen demand (COD) play a pivotal role in determining water quality. Results of studies of various physico-chemical parameters have shown that the level of pollution of river rises as sewage and industrial waste is dumped into it (Mishra KD, 1991). BOD and COD are the factors used as the pollution indicator of a water body. Amongst the two, BOD is widely considered the more important factor than COD while analysing the performance of wastewater/STP (Sharma, P., Sood, S., and Mishra, S. K., 2020). The literature also emphasizes the significance of considering microbial parameters in assessing overall water health

The findings from past studies underscore the prevalence of higher bacterial colonies in pond water samples, pointing to potential concerns regarding water safety. The presence of elevated levels of free CO2 is identified as a potential factor contributing to low pH values in river water samples (Tiwari, S. K., Gupta, A. K., and Asthana, A. K. L., 2020), which in turn may impact bacterial counts. Understanding these relationships is essential for comprehensively evaluating the suitability of pond water for drinking purposes.

Research analysis shows that parameters such as total coliform, temperature and TDS are on rise on yearly basis. On the other hand, parameters like DO and pH do not show similar pattern. It also suggests that factors like poor sewage, improper management of wastewater and high tourist flow are the major cause of poor water quality (Sharma et al., 2020).

This literature review integrates findings from prior studies, establishing a foundation for the current research on the microbial and physicochemical analysis of pond water samples in different locations of Agra, Uttar Pradesh, India.

Collection of Samples

In our study, samples were collected from three ponds of Agra to conduct the practical. sampling was performed according to Standard Methods for the Examination of Water and Waste water 20^th edition (APHA, 1999).

Sample Preparation

Each sample comprised of 1 ml pond water suspended in 99 ml of distilled water ina flask.

ISOLATION

Serial dilution

In this procedure, a small measured weight is mixed with large volume of sterile water called diluents or dilution blank. Serial dilutions are later prepared by transferring a known volume of the dilution to second dilution blank and so on. 

Identification

Bacteria were identified by various staining techniques i.e., simple staining, gram’s staining, negative staining, and endospore staining.

Bacteriological analysis:

Bacteriological analysis of drinking water samples was done by Multi-tube Fermentation Method (UNICEF, 2002). The Most Probable Number (MPN) of bacteria present in water sample has been estimated from the specially developed statistical table. Confirmatory tests on selective culture medium were also performed.

Physicochemical analysis

The parameters like pH, Dissolved Oxygen (DO), Biological Oxygen Demand (BOD), Chemical Oxygen Demand(COD)were analyzed using standard procedures [14].

Microbial analysis

The microbial analyses of bacteria were measured by standard plate count (SPC)using standard nutrient. Microbial analysis of pond water samples was studied within 24 hours of collection. The number of bacterial colonies was counted by colony counter. All estimations were carried out using three replicates.

Identification Results

Three samples of Pond water were collected from different regions of Agra and cultured on Nutrient Agar and then tested through various morphological and biochemical testes for identification of microorganisms.

Biochemical Tests Results:

Amylase test

A typical positive starch hydrolysis reaction is shown by Bacillus subtilis by the production of exoenzyme amylase which has diffused into the medium surrounding the growth.

Mac-Conkey agar test

Lactose aging strains develop as Red or Pink and might be encompassed by a zone of corrosive encouraged bile. The red shading is because of generation of corrosive from lactose, assimilation of nonpartisan red and an ensuing shading change of the colour when the pH of medium falls beneath 6.8.

Mannitol salt agar test

Due to the presence of S.aureus the colour of the medium turns to yellowish whereasS. epidermidisdoes not change the colour of the medium.

Bacteriological examination of water by multiple-tube fermentation test or multiple –tube test (MPN index)

The test is performed sequentially in three stages:-

1. Presumptive
2. Confirmed and,
3. Completed.

Lactose stock tubes are immunized with various water volumes in the possible test. Tubes that are sure for gas creation are vaccinated into splendid green lactose bile juices in the affirmed test and positive tubes are utilized to compute the most likely number (MPN) of coliforms in the water test taking after the measurable table. The finished test including the immunization of EMB agar plate, supplement agar incline and splendid green lactose bile soup and planning of a gram the slide from supplement agar inclination is utilized to set up that coliforms microscopic organisms are available in the example. The finished procedure including the affirmed and finished test requires no less than 4 days of hatching and exchanges.

(i) Presumptive test

The aging tubes were inspected for the creation of corrosive (yellow shading) and gas after 24 hours of hatching. Creation of corrosive (shading change) and gas takes place after 24 hours. Brooding demonstrates a positive test for coliforms microbes. In the event the gas creates in tubes after 48 hours brooding, the possible test is suspicious and if there is no gas created after 48 hours hatching, it appears, negative possible test. (i.e. coliforms are truant). Record the quantity of tubes demonstrating the positive hypothetical test are held and utilized for affirmed test.

(ii) Confirmed test

All the vaccinated bile stock tubes were analyzed for the gas generation. On the off chance that no gas is delivered in any of the vaccinated tube, the nonappearance of coliforms in the water test is affirmed demonstrating that the water test is not polluted with the sewage water. Creation of gas as the splendid green lactose bile juices tubes after 48 hours of hatching shows a positive affirmed test for coliforms microscopic organisms in the water test. Record number of tubes demonstrating positive affirmed test and ascertain the most likely number (MPN) esteem from the MPN table by applying the mix of positive and negative result saw in the 15 immunized lactose maturation tubes. For instance, if gas shows up in all the 5 tubes marked 10 and 1 tube named 1 the MPN table would be roughly 31 in 100 ml of water test.

Table 2: Confirmed test of Coliform Bacteria present in 100 ml Water sample for various combination of Positive results when five 10 ml portions, five 1 ml Portion and five 0.1 ml portions are used

(iii) Completed test

Lactose stock maturation tubes were watched for the generation of gas. Gram recolor the creature found on the inclination and watch the slide for the positive or negative Gram response and cell morphology. Generation of gas from lactose, vicinity of Gram negative, non-spore shaping bar come about a positive finished test and affirm the location of coliforms microbes in the water test showing the faecal defilement of water, along these lines that water considered as not consumable. Then again, if no remote enteric pointer microscopic organisms are identified in a particular volume (100 ml) of water, it is viewed as consumable or suitable for human utilization.

Eosin Methylene Blue test

Acid is produced by the gram negative bacteria responsible for fermentation of lactose. When this acid acts on the dyes, the colonies show dark purple colour. In EMB agar, the colonies of E.coli show characteristic green soft lustre. Non-fermenters of lactose show light lavender colour or are colourless.

Least coliform populace (13 MPN/100 mL) was identified in S3: Pond Water Test (Farah), though, a most extreme coliform populace was found in S1: Pond Water Test (Arsena) (1600 MPN/100 mL). Then again, water test gathered from Repura discovered MPN List of 240 MPN/100 ml demonstrating coliform defilement. Faecal coliform microscopic organisms just disconnected from three diverse Pond water tests were broke down for recognition of E. coli. Present study result demonstrates that all pond water tests were discovered present with E. coli sullying.

Physico-chemical analysis of drinking water

Three pond water samples were collected from different locations of Agra and were analyzed for pH, Temperature, EC, TDS, Dissolved Oxygen and Biological Oxygen Demand (BOD).

Total 3 pond water samples S1, S2 and S3 (*S1: Pond Water Sample, Arsena; *S2: Pond Water Sample, Repura and *S3: Pond Water Sample, Farah) were analyzed for Physico-chemical parameter (pH, Electrical Conductivity and Total suspended solids) analysis of pond water.

Table 3: Physico-chemical parameters of Pond Water Samples

Fig. 1: Graphical representation of physiological parameters of pond water sample showing pH, Temperature, EC and TDS values

pH

pH of tube pond water tests was discovered 8. 0. The pH values broke down utilizing pH meter was observed to be pretty much comparative for every specimen, where the pH values ran from 6-8. Test 1 and 2 was found to have the most elevated pH esteem (8) and the least for Test 3 (7). The pH values for Tests 1, 2 and 3 was observed to be between 7-8 amid the study period, which is pretty much like the outcomes got by Mishra et. al., 2008. The pH was inside of the reasonable reach 6 to 8 as recommended by WHO. As indicated by Medera et. al., 1982, the pH of most normal waters ranges from 6.5 - 8.5 while deviation from the unbiased 7.0 is as a consequence of the CO2/bicarbonate/carbonate harmony.

Electrical Conductivity (EC)

Pond water samples S1, S2 and S3 collected from (*S1: Pond Water Sample (Arsena); *S2: Pond Water Sample (Repura); *S3: Pond Water Sample (Farah), EC values were found to range from 1.1 (Ω/cm) to 3.2 (Ω/cm). Pond water sample collected from Farah (S3), Agra showed lowest EC value 1.1 (Ω/cm) and Pond water sample collected from Arsena (S1) and Repura showed result with highest EC value 3.0 and 3.2 (Ω/cm). EC has closed relationship with total dissolved solids, thus high EC value indicate that there as high concentration of dissolved solids in water. Water with high EC value has salty taste.

Dissolved Oxygen Determination:

Dissolved oxygen observed was high in Pond water samples S1, S2 and S3 collected. Pond Water Sample, Arsena S1 showed 40 mg/L, Pond Water Sample (Repura) S2 showed 26 mg/l and Pond Water Sample (Farah) S3 showed 48 mg/l. This is a measure of the amount of gaseous oxygen dissolved in aqueous solution that plays a vital role in the biology of cultured organisms (Dhawan and Karu, 2002). These values agree with those of Saloom and Duncan, 2005.

The total amount of sodium thiosulfate solution (titrate) used and the dissolved oxygen content of water (mg/litre) was calculated by applying the equations:-

D.O (in mg/litre)= (8*x 1000 X N / V) X v

Where;

           V= Volume of sample taken (ml)

           v =Volume of titrate used 

           N=Normality of the titrate

           * = 8 is the constant since 1 ml of 0.025 sodium thiosulphate solution is equivalent to 0.2 mg oxygen.

Table 4: Determination of Dissolved Oxygen of pond water samples

Biological Oxygen Demand Determination:

BOD and COD are considered as essential and important parameters to decide about the quality of the drinking water (Mirza et. al., 2006). Highest amount of BOD was observed in Sample 3 (12 mg/L) while Samples 1, 2 the lowest (10 mg/L and 6.5 mg/l). Chemical oxygen demand determines the oxygen required for chemical oxidation of organic matter. COD values convey the amount of dissolved oxidisable organic matter including the non-biodegradable matters present in it (Mahananda et. al., 2010). Sample 3 had the highest amount (12 mg/L) while Sample 2 had the lowest (6.5 mg/L), which indicated that Sample 2 is highly polluted.BOD of the water in mg /litre calculated by applying the formula

BOD (in mg l^-1)= D₁ - D₂

Where;  

             D1= Initial DO in sample (mg I ^-1)         

             D 2=DO after 3 days incubation (mg I^-1).

Results of the microbial analysis of the water samples showed that bacterial colonies found were higher. In general, high levels of free CO₂ might be the reason for low pH values obtained in the river water samples, which may consequently affect the bacterial counts. Groundwater is found to be contaminated due to improper construction, shallowness, animal wastes, proximity to toilet facilities, sewage, refuse dump sites, and various human activities around the well (Bitton, 1994). The presumed reason for contamination of pond water accounts for the microbial load of well water close to refuse disposal site which have high microbial count than the one far away from refuse disposal site (Shittu, 2008).

In the present study, it has been found that these 3 pond waters were polluted by human and animal wastes. Therefore, conservation of these ponds is important to protect them from further contamination and for the human needs.

Starch hydrolysis test of Pond Water sample S1, S2, S3 [(S1: Pond Water Sample (Arsena); S2: Pond Water Sample (Repura) and *S3: Pond Water Sample (Farah)] showed growth of Bacillus subtilis and Bacillus cereus)

E.coli showed growth on MacConkey Agar ofPond Water sample; S1: Pond Water Sample (Arsena). Staphylococcus aureus showed growth on Mannitol Salt Agar from sample S1.

Al-Harbi (2003) observed that Escherichia coli present in swine feces may be responsible for the contamination of the water, of the sediment and of the digestive system of tilapia in fish ponds. In the present study, the thermo tolerant coliforms reached fish ponds water and went through the intestinal excretions of mammal’s animals, as in the case of sheep, geese, herons and other birds found around the fish pond studied.

The bacterial group, together with the growths, has been generally viewed as mineralizers, in charge of the transformation of natural matter into inorganic matter, in this manner taking up a focal part for supplement come back to the amphibian and physical biological communities. Natural decay happens in similarity with microbial movement and thickness, which are controlled by the extent of carbon, nitrogen and phosphorous, and, thus, affected by manure presentation in the amphibian framework.

The number of microscopic organisms found in fish ponds was more noteworthy in the stormy period, relating to abnormal state of fish generation, when out streaming waters might convey a lot of allochthonous materials to the fish ponds furthermore the high temperatures might support the quick propagation of aggregate coliforms.

The water from animals reproducing ranch wells might be a potential wellspring of Escherichia coli. In the present study, the surroundings of the source to estate territories and to the dairy cattle and goat reproducing ranch areas might have cultivated contact with lingering water and source sullying by coliforms, principally amid the stormy period. Until 14 years back, swine reproducing effluents streamed out specifically into the fish Pond 3, which has most likely added to a more prominent coliform inflow and, thusly, defiled consequent fish ponds considerably further.

In the present study, microorganism focuses have additionally exhibited an expanding prosperous pattern toward the gushing in the fish ponds, considering as wealthy the source water, and as profluent the water from the last fish pond, as these are persistent stream frameworks. The E/A relationship of the entire framework was brought up in all parameters considered, with the relationship found for thermo tolerant coliforms in the stormy and dry periods being 29.18 and 4.00, separately, exhibiting a high thermo tolerant coliform augmentation speed inside of the framework, particularly for the blustery period.

The fish ponds concentrated on might be viewed as biodegradable (Valente et. al., 1997), as in all focuses, with the exception of the source and the principal fish pond in the stormy period, the COD/Body mean proportion was lower than 5. The variety among fish pond was more prominent than the variety between the periods, a reality which is identified with the administration utilized and to the sort of ceaseless water stream framework, with no past treatment. In fish pond, the high Body and COD values discovered show a high natural compound fixation. The higher Body fixations in the F3a, F5a, F5b and F6 fish pond might likewise be identified with the natural strong accessibility in the aforementioned fish pond, while the vicinity of coliforms decided a microbiological tainting potential.

Table 5: Determination of Biological Oxygen Demand of pond water samples

Fig. 3: Graphical representation of BOD of pond water sample of S1, S2 and S3

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