Assessment of microbial diversity in marine waters ...

Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

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Advances in Environmental Biology ISSN-1995-0756

EISSN-1998-1066

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Assessment of microbial diversity in marine waters along the Uran coastal area of Navi Mumbai, Maharashtra, India Prabhakar R. Pawar Arts, Science and Commerce College, Department of Zoology, Mokhada, Dist. – Palghar, Pin - 401 604, India. Address For Correspondence: Prabhakar R. Pawar, Arts, Science and Commerce College, Department of Zoology, Mokhada, Dist. – Palghar, Pin - 401 604, India E-mail: [email protected] This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Received 13 May 2017; Accepted 27 July 2017; Available online 26 September 2017

ABSTRACT The objective of this study was to evaluate the diversity of total coliforms, fecal coliforms and pathogenic bacteria from three study sites (Sheva creek, Peerwadi coast and Dharamtar creek) along Uran coastal area of Navi Mumbai. Uran is located along the eastern shore of Mumbai harbor opposite to Coloba and is included in the planned metropolis of Navi Mumbai and its port, the Jawaharlal Nehru Port (JNP). Surface water samples were collected during spring low tides for microbial diversity monthly from June 2013 to May 2015. Standard microbiological methods like Presumptive test (Most Probable Number and Standard Plate Count), Confirmed test (Inoculation on slants and plates) and Completed tests (Colony morphology, Gram staining and Biochemical tests) were adopted for present study. A total of 11 bacterial species belonging to 9 genera, 5 families, 4 orders and 4 classes were isolated from marine water samples. Identified bacterial strains were represented by Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Providencia rettgeri, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus and Staphylococcus epidermidis. Of the recorded bacterial species, 54.55% belonged to Enterobacteriaceae, 18.18% to Staphylococcaceae and 9.09% each to Bacillaceae, Micrococcaceae and Pseudomonadaceae. Heavy microbial load reported in marine waters of Uran is correlated to the disposal of untreated sewage and waste water, lack of sanitary facilities, human settlement in the vicinity of the coast, poverty among local community and discharge of untreated effluents from Jawaharlal Nehru Port (JNP) and other port related establishments. Presence of potential pathogenic bacteria could pose a high health risk to the local community due to contamination of fishery and aquaculture products.

KEYWORDS: Contamination, Coliforms, E. coli, Fecal pollution, Microbial diversity, Jawaharlal Nehru Port, Uran INTRODUCTION The study of marine bacterial diversity is important in order to understand the community structure and pattern of distribution. In the marine environment, 90% of bacteria are Gram-negative with different characteristics and are better adapted for survival in the marine environment. Bacteria maintain the pristine nature of the environment and also serve as biological mediators through their involvement in the biogeochemical processes like breakdown of organic matter, mineral cycle and sedimentation. Marine microbes are a potential source for commercially important bioactive compounds and also show bioremediation capability [6]. The sources of coastal water contamination are point discharges (discharge of treated and untreated sewage from shoreline outfalls), and non-point discharges (runoff from naturally vegetated areas, the storm water runoff from urban, commercial, and industrial lands and malfunctioning or poorly sited septic systems) introduce significant amounts of pathogens in coastal waters. Water-borne pathogen contamination in water resources and

Copyright © 2017 by authors and American-Eurasian Network for Scientific Information (AENSI Publication).

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

related diseases are a major water quality concern throughout the world. Gastrointestinal diseases caused by various bacteria, viruses, and protozoa have been the causes of many outbreaks [17]. Monitoring the microbiological quality of water relies largely on examination of indicator bacteria such as coliforms, Escherichia coli, and Pseudomonas aeruginosa. E. coli is a member of the faecal coliform group and is a more specific indicator of faecal pollution than other faecal coliforms. E. coli are the predominant member of the human colonic normal flora with its only natural habitat is the large intestine of warm-blooded animals. With few exceptions, E. coli generally does not survive well outside of the intestinal tract and its presence in environmental samples, indicates recent faecal contamination [8, 15, 28]. Among the leading sources of chemical and biological contamination of waters and associated beaches are sewer systems, septic tanks, storm water runoff, industrial wastes, wastewater injection wells, cesspits, animal wastes, commercial and private boat wastes, and human recreation. One of the primary concerns in public health is the risk that humans using the marine environment for recreational activities will encounter microbial pathogens [2, 12]. Population explosion and inadequate infrastructure to properly treat and dispose the sewage, lack of sanitary condition, poverty, overexploitation of natural water has resulted in the discharge of considerable quantities of untreated waste into the natural waters. This organic pollution is especially severe in the coastal waters due to the large density of inhabitants in coastal areas [3, 13]. Assessment of water contamination requires both quantitative and qualitative analysis to pinpoint the location and the identity of water pollution. Effective method for monitoring water constituents includes microbiological techniques, including culturing and plating of water borne-microbes [7]. Due to onset of Jawaharlal Nehru Port (JNP, an international port), and port associated establishments like Oil and Natural Gas Commission (ONGC), Liquid Petroleum Gas Distillation Plant, Grindwell Norton Ltd., Gas Turbine Power Station (GTPS), Bharat Petroleum Corporation Limited (BPCL) Gas Bottling Plant, DP World, and large number of Container Freight Stations [CFS] in the mangroves stretch of coastal ecosystem of Uran, it is under considerable stress [18, 19]. Uran coast is a famous tourist spot in Navi Mumbai and large number of tourists visits the Peerwadi coast on weekends. These activities affect the water quality and coastal marine diversity from Uran coast resulting in coastal pollution due to anthropogenic inputs [20, 21, 22, 23]. Though published literature is available on the water quality of coastal ecosystem of in and around Mumbai, no detailed reports are available on the microbial diversity from coast of Mumbai and Navi Mumbai; hence the present study is undertaken. Objective of the present study is to evaluate the microbial diversity in relation to the impacts of pollution in marine waters along Uran coast, Navi Mumbai with respect to E. coli, total coliforms and fecal coliforms. MATERIALS AND METHODS Study Area: Uran (18º 50'5'' to 18º50'20'' N, 72º57'5'' to 72º57'15'' E) with the population of 28,620 is located along the eastern shore of Mumbai harbor opposite to Coloba. Uran is bounded by Mumbai harbor to the northwest, Thane creek to the north, Dharamtar creek and Karanja creek to the south, and the Arabian Sea to the west. Uran is included in the planned metropolis of Navi Mumbai and its port, the Jawaharlal Nehru Port (JNPT) (Fig. 1). The Uran coast is a tide-dominated and the tides are semidiuranal. The average tide amplitude is 2.28 m. The flood period lasts for about 6–7 h and the ebb period lasts for about 5 h. The average annual precipitation is about 3884 mm of which about 80% is received during July to September. The temperature range is 12–36ºC, whereas the relative humidity remains between 61% and 86% and is highest in the month of August.

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Fig. 1: Map showing the location of three study sites namely the Sheva creek, Peerwadi coast and the Dharamtar creek along Uran coast Study Location: For the present study, three sampling sites, namely Sheva creek, site I (18º 50' 20'' N, 72º 57' 5'' E), Peerwadi coast, site II (18º 50' 10'' N, 72º 57' 1'' E) and Dharamtar creek, site III (18º 48' 3'' N, 72º 58' 31'' E) separated approximately by 10 km were selected. These sites were selected on the basis of their strategic locations for Jawaharlal Nehru Port, industries, port related infrastructural facilities and different anthropogenic activities along the entire coastal area. Sheva creek is characterized by extensive mud flats with sparse mangrove vegetation and less rocky stretches. Jawaharlal Nehru Port (JNP) and other port related establishments are located in the stretch of the creek. Gharapuri Island (Elephanta caves), a famous tourist spot is present on the north side of the creek. Intertidal region of Peerwadi coast has major portion of rocky substratum. Dharamtar creek is with rocky and coral substratum towards the Dronagiri Mountain whereas remaining part of the creek is dominated by the marshy areas and mud flats. Towards the Revas and Karanja side, the Dharamtar creek has mangrove associated habitats due to presence of dense and natural mangrove habitat. Sheva creek and Dharamtar creek are considered as high anthropogenic pressure zones. Field Sampling: The present study was carried out for a period of two years, i.e., from June 2013 to May 2015. At each site, three sampling stations separated approximately by 1 km were set up for sample collection.

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Surface water samples were collected seasonally during spring low tides. Samples were collected in heatsterilized glass bottles of 250 ml capacity with ground glass stopper. To neutralize the bactericidal effect of chlorine in water, 0.25 ml of fresh 1.8 % aqueous solution of sodium thiosulphate was added in sample bottles. Sample bottle were filled by holding it at its lower part and submerge it to a depth of about 30 cm with mouth facing slightly upward. Bottles were stoppered immediately and were wrapped in a Kraft paper. Water samples were properly labeled with full details of the source, time and date of collection. Immediately after collection, samples were placed in cooling boxes and immediately transferred to the laboratory. In the laboratory, all water samples were stored in a 40C freezer until microbiological analyses were carried out within 24 h of sampling and stored at 4 0C according to the standard method [1]. Laboratory analysis: Serial dilutions of water samples were made following standard methods. Standard microbiological methods described by APHA [1], Cappuccino and Sherman [5] and Food Safety & Standards Authority of India, Lab Manual 14 [9] were followed for bacteriological analysis of water. Enumeration for Escherichia coli, total coliforms, fecal coliforms and coliform species identification was made using presumptive test, confirmed test and completed tests by methods based on lactose fermentation. Table 1: Standard methods adopted for microbiological studies of water Test Technique Presumptive Test Most Probable Number (MPN) Standard Plate Count (SPC)

Confirmed Test

Inoculation on slants and plates of selective & differential media

Completed Tests for coliform species identification

Colony morphology Gram staining Motility test Biochemical Tests Indole Production test Methyl Red test Voges- Proskauer test Citrate Utilization test Urease test H2S Production test Nitrate Reduction test Litmus Milk test

Medium Brilliant Green Bile Broth (HIMEDIA Mumbai M121) Nutrient agar (NA) (HIMEDIA Mumbai M001) Mineral Modified Glutamate (HIMEDIA Mumbai M6431) Endo agar (HIMEDIA Mumbai M1077) MacConkeys agar (HIMEDIA Mumbai M081B) --------Hanging Drop Method

Agar

(MMGA)

Tryptone water (HIMEDIA Mumbai M463) MR-VP Broth (HIMEDIA Mumbai M070) MR-VP Broth (HIMEDIA Mumbai M070) Simmon’s Citrate Agar (HIMEDIA Mumbai M099) Urea broth (HIMEDIA Mumbai M112) SIM agar (HIMEDIA Mumbai M181F) Trypticase nitrate broth (HIMEDIA Mumbai M439S) Litmus milk broth (HIMEDIA Mumbai M609)

Expression of result: Colonies were counted using Quebec colony counter and the results were expressed as number of microorganisms per ml of original culture is calculated by multiplying the number of colonies counted by the dilution factor: Number of cells per ml = number of colonies X dilution factor RESULTS AND DISCUSSION A total of 11 bacterial species belonging to 9 genera, 5 families, 4 orders and 4 classes were isolated from marine water samples from Sheva creek, Peerwadi coast and the Dharamtar creek along Uran coast. Identified bacterial strains were represented by Bacillus, Escherichia, Klebsiella, Micrococcus, Providencia, Pseudomonas, Salmonella, Shigella and Staphylococcus. Of the recorded bacterial species, 54.55% belonged to Enterobacteriaceae, 18.18% to Staphylococcaceae and 9.09% each to Bacillaceae, Micrococcaceae and Pseudomonadaceae (Fig. 2).

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Fig. 2: Percentage representation of families of bacteria from water of Uran coast To detect coliform bacteria (indicators of fecal contamination) in water, the three basic tests like presumptive, confirmed, and completed tests are performed sequentially on each water sample. A. Presumptive test for total coliforms: Presumptive test is performed to determine presence of coliform bacteria in a water sample and also to obtain an index indicating the possible number of organisms present in the sample. 

Most Probable Number (MPN): The data on Presumptive test by Most Probable Number (MPN) of the water indicates that at all sites; the MPN Index/100 ml of water sample is highest during monsoon. In monsoon, at site I and II, the MPN Index/100 ml is 1600 where as at site III, it was ≥ 2400. In pre-monsoon, the MPN Index/100 ml at all sites is in the range of 33 to 110 while in post-monsoon, it was within the range of 14 to 21 (Table 2). Significantly high MPN Index/100 ml recorded at all sites is correlated to the storm water runoff which is a significant source of pollution to the sea, which can include bacteria, viruses and sediment. It is also attributed to the agricultural, urban surface run-off, waste water discharges and from domestic and wild animals. Stream flow-River flow is the primary transport media of fecal coliform bacteria to ocean [4, 16]. Table 2: Most Probable Number (MPN) of water of Uran coast Sr. No. Season Site Gas 10 X 1X Tube Tube 1 2 3 4 5 1 2 PostI - + - + + - + 1 monsoon II + + - + - - Nov 2013 III + - + + + + I + + + + - + + 2 PreII + + + + + - + monsoon III + + + + + + + May 2014 I + + + + + + + 3 Monsoon II + + + + + + + Aug 2014 III + + + + + + +

3 -

4 + + +

5 + + -

0.1 X Tube 1 2 + - - - + - +

+ + +

+ + +

+ + +

+ + +

+ +

3 + -

4 -

+ + +

+ + +

Reading

MPN Index/ 100 ml

5 + + + -

3 – 2-1 3 – 2-1 4 – 1-1 4 – 3-1 5 – 1- 2 5 – 3-1

17 14 21 33 63 110

+ +

5 – 5- 4 5 – 5- 4 5 – 5- 5

1600 1600 ≥ 2400

Mean MPN Index/ 100 ml.

18 69

1867

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24



Standard Plate Count (SPC): Results on Standard Plate Count (SPC) for coliforms in water are in agreement with the MPN Index/100 ml. Highest bacterial density was recorded during monsoon at all sites followed by pre-monsoon and post-monsoon. Bacterial count in the range of 236-583 CFU/ml is recorded during monsoon whereas in pre-monsoon and postmonsoon, they are in the range of 67-162 CFU/ml and 65-137 CFU/ml respectively. High densities of bacteria recorded from marine waters of Uran is correlated to the discharge of untreated effluents, gastrointestinal inputs and greater from municipal sewage contamination [11, 25] (Table 3). Table 3: Standard Plate Count (SPC) on Nutrient Agar/MMGA of water of Uran coast No. of colonies Petri No. of colonies SPC/ml= ──────── Plate Dilu Dilution Sit per plate Dilution factor tion Factor e Sample Sample PostPreMon PostPremon mon Aug mon Mon Nov May 2014 Nov May 2013 2014 2013 2014 1 ml I 137 67 583 137X104 67X104 A from 10-4 II 65 162 236 65 X104 162X104 4 10-4 III 97 113 473 97X10 113X104 1 ml I 50 41 434 50X105 41X105 B from 10-5 II 54 118 181 54X105 118X105 10-5 III 72 53 391 72X105 53X105 C 1 ml I 30 28 387 30X106 28X106 -6 6 from 10 II 41 63 124 41X10 63X106 10-6 III 43 39 289 43X106 39X106 7 D 1 ml I 22 17 213 22X10 17X107 from 10-7 II 34 32 96 34X107 32X107 10-7 III 27 21 138 27X107 21X107

Mean SPC/ml

Average SPC/ml

Mon Aug 2014 583X104 236X104 473X104 434X105 181X105 391X105 387X106 124X106 289X106 213X107 96X107 138X107

263X104 176X104 228X104 175X105 118X105 172X105 149X106 76X106 124X106 84X107 54X107 62X107

222X104

155X105

116X106

67X107

B. Confirmed test for total coliforms: (Inoculation on Slants and Plates of Selective & Differential media) Confirmed test is performed to confirm the presence of coliform bacteria in a water sample for which the presumptive test was positive. To distinguish among morphologically and biochemically related groups of organisms, selective and differential media (Endo agar and MacConkeys agar) be streaked from a positive lactose broth tube obtained from the presumptive test. Results of inoculation on slants and plates of Endo agar and MacConkeys agar shows circular and irregular colonies with size ranging from 1 - 6 mm. Pigmentation of the colonies vary from creamy, blue green, grey or pinkish red. C. Completed test for coliform species identification: Completed test is performed to confirm the presence of coliform bacteria. The isolated coliform colonies appeared on Endo agar and MacConkeys agar were picked from the confirmatory test plate and examined for colony morphology, Gram staining and motility test (Table 4). Species identification and differentiation of enteric coliforms of the family Enterobacteriaceae was made on the basis of their biochemical properties and enzymatic reactions in the presence of specific substrate from the results of biochemical tests like Indole Production test, Methyl Red test, Voges- Proskauer test, Citrate Utilization test, Urease test, H2S Production test, Nitrate Reduction test and Litmus Milk test (Table 5 to Table 13) (Fig. 3). Eleven species of bacteria representing 9 genera, 5 families and 4 orders were identified from the water samples of Uran coast. These include Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Providencia rettgeri, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus and Staphylococcus epidermidis (Table 14). Significantly high density of bacteria recorded during monsoon could be due to increased land runoff along with reduced intensity of sunlight due to overcast conditions monsoon [13]. It is also correlated to the increased turbidity from land runoff causing reduction in penetration of sunlight resulting in extended survival of indicator bacteria [27]. Engelbrecht and Tredoux [8] reported that a 100 faecal coliform count represents a 60-E. coli count, while 2000 faecal coliforms represent 1200 E. coli. Geissler et al. [10] documented that E. coli is generally considered the most reliable since its presence directly relates to faecal contamination with its implied threat of the presence of enteric diseases. According to Tallon et al [29], E. coli is a member of thermotolerant and is an indigenous member of the intestinal flora of healthy humans and warm-blooded animals. In present study, detection of E. coli at all the three study sites indicates fecal pollution of recent origin. Higher density of coliforms recorded in present study is attributed to discharge of wastewater, especially sewage disposal with fecal material contaminated by human and warm-blood animals; runoff after rain and hurricane and direct contamination by wild animals [14].

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Results of the characteristic features of the bacterial strains by biochemical tests are in agreement with the work of Ramesh Babu et al [24] on microbial status and characteristic features from polluted coastal habitats at Visakhapatnam, India, Saravanan et al [26] on microbial diversity in marine ecosystem of Parangipettai, Tamil Nadu, India and Yazhini et al [30] on microbial contamination of sea water at Puducherry sea shore, India. The Uran coast acts as a sink receiving large daily volume of domestic and industrial wastes and effluents from Asia’s largest industrialized zone namely Thane Belapur industrial area, Navi Mumbai Municipal Corporation and effluents from Jawaharlal Nehru Port [19]. Maritime activities of JNP and local dredging activities have promoted the changes in physico-chemical parameters, and inorganic nutrients in the seawater surrounding the Jawaharlal Nehru Port [18]. Anthropogenic impact of these activities has affected the water quality and diversity of macrobenthos from coastline of Uran [21, 22, 23]. Since no reports are available on the microbial pollution from Uran coastal area, results of the study can be used as the baseline for future research. It will further helpful to develop the management strategy for the conservation of coastal marine ecosystem. Table 4: Colony characteristics of bacterial strains isolated from water along Uran coast Colony Bacterial species characteristics 1 2 3 4 Size 2-4 mm 3-6 mm 2-5 mm 2 mm General Surface Irregular Circular Circular Circular Form Elevation Umbonate Slightly Raised Umbonate Convex Margin Undulate Entire Entire/ Entire Undulate Texture Rough Smooth Mucoid Smooth Appearance Dull Shiny Shiny Pigmentation White to Colourless White Bright yellow creamy or /Cream Non-diffusing brownish Optical Property Opaque Translucent Translucent Morphology Rod Rod Rod Coccus Motility Motile Motile Non-motile Non-motile Gram Staining Gram +ve Gram –ve Gram –ve Gram +ve Bacillus Escherichia Klebsiella Micrococcus subtilis coli pneumoniae luteus Table 4: Continued. Colony characteristics Size General Surface Form Elevation Margin Texture Appearance Pigmentation Optical Property Morphology Motility Gram Staining

5 ~ 4 mm Circular

6 2-4 mm Circular

Convex -

Low convex Undulate

Dull Grey

Smooth Blue green

Opaque Straight Rod Motile Gram –ve Providencia rettgeri

Translucent Rod Motile Gram –ve Pseudomonas aeruginosa

Bacterial species 7 2-4 mm Circular

8 2-4 mm Circular

9 2 mm Circular

10 1-2 mm Circular

11 1-2 mm Circular

Convex Entire Smooth Shiny Colourless Translucent

Raised Smooth Moist Grey -

Raised Convex Entire Smooth White -

Convex Entire Smooth Shiny Golden-brown Opaque

Rod Motile Gram –ve Salmonella typhimurium

Rod Motile Gram –ve Salmonella enterica

Convex Entire edges Smooth Colourless Moderately translucent Rod Non-Motile Gram –ve Shigella flexneri

Coccus Non-motile Gram +ve Staphylococcus epidermidis

Coccus Non-motile Gram +ve Staphylococus aureus

Table 5: Indole production test of water of Uran coast Sr. Season Site No.

1

Post-monsoon Nov 2013

2

Pre-monsoon May 2014

3

Monsoon Aug 2014

I II III I II III I II III

Test tube A (Control) -

B

C

D

E

+ + + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Table 6: Methyl Red (M. R.) test of water of Uran coast Sr. No. Season Site

1

Post-monsoon Nov 2013

2

Pre-monsoon May 2014

3

Monsoon Aug 2014

Test tube A (Control) -

I II III I II III I II III

Table 7: Voges - Proskauer test (V. P. test) of water of Uran coast Sr. No. Season Site

1

Post-monsoon Nov 2013

2

Pre-monsoon May 2014

3

Monsoon Aug 2014

I II III I II III I II III

Table 8: Citrate utilization test of water of Uran coast Sr. Season No.

1

Post-monsoon Nov 2013

2

Pre-monsoon May 2014

3

Monsoon Aug 2014

Test tube B B B B B B B B B A

+ + + + + -

Test tube A (Control) -

B + + + + + + + + +

Test tube A (Control) -

I II III I II III I II III

Table 9: Hydreogen sulfide test of water of Uran coast Sr. Season Site Test No. tube Post-monsoon I B 1 (Oct13 to II B Jan 14) III B Pre-monsoon I B 2 (Feb 14 to May II B 14) III B Monsoon I B 3 (Jun 14 to Sept II B 14) III B Control I, II, III A Table 10: Urease test of water of Uran coast Sr. Season Site No. Post-monsoon I 1 (Oct13 to II Jan 14) III Pre-monsoon I 2 (Feb 14 to May II 14) III Monsoon I 3 (Jun 14 to Sept II 14) III Control I, II, III

Site

B

Colour of medium Black

B + + + + + + + +

H2S production (positive) + + +

H2S production (negative)

No black colour

+ + +

Black

+ + +

No black colour

Colour of medium Light Orange Light Orange Light Orange Light Orange Light Orange Light Orange Deep Pink Light Orange Light Orange Light Orange

+

Urea Hydrolysis (positive)

Urea Hydrolysis (negative) + + + + + +

+ + + +

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Table 11: Litmus milk reactions of water of Uran coast Table 11(a): Lactose fermentation: Sr. Season Site Test Appearance No. tube of medium

1

2

3

Post-monsoon (Oct13 to Jan 14) Pre-monsoon (Feb 14 to May 14) Monsoon (Jun 14 to Sept 14)

I II III I II III I

II

III

Control

Table 11(b): Sr. Season No. Post-monsoon (Oct13 to 1 Jan 14) Pre-monsoon 2 (Feb 14 to May 14) Monsoon (Jun 14 to 3 Sept 14)

Control

B B B B B B B

Orange Orange Orange Orange Orange Orange Pink band at the top of the medium Pink band at the top of the medium Pink band at the top of the medium Brick Red

B

B

I, II, A III

Site I II III I II III I

Test tube B B B B B B B

II

B

III

B

I, III

II, A

Lactose fermentation (acid) -

Acid followed by reduction -

Acid, reduction and curd + + + + + +

Acid, gas reduction and curd + + + + + +

-

+

-

-

-

+

-

-

-

+

-

-

-

-

-

-

Appearance of medium Medium is unchanged Medium is unchanged Medium is unchanged Medium is unchanged Medium is unchanged Medium is unchanged Brownish translucent medium Brownish translucent medium Brownish translucent medium Brick Red

Table 12: Nitrate reduction test of water of Uran coast Sr. Site Test Red coloration No. Season tube with solution A and B (+) or (-) Post-monsoon I B Red colour (Oct13 to Jan II B Red colour 1 14) III B Red colour Pre-monsoon I B No Red colour (Feb 14 to May II B No Red colour 2 14) III B No Red colour Monsoon I B Red colour (Jun 14 to Sept II B No Red colour 3 14) III B No Red colour Control I, II, III A Red colour

Litmus reduction -

Proteolysis +

Alkaline reaction + + + + + + -

-

+

-

-

+

-

-

-

-

Red coloration with Zinc (+) or (-)

Nitrate reduction (+) or (-)

End products

No Red colour No Red colour No Red colour No Red colour No Red colour -

+ + + + + -

NO3 NO3 NO3 NO2/NH3/N2 NO2/NH3/N2 NO2/NH3/N2 NO3 NO2/NH3/N2 NO2/NH3/N2 NO3

Table 13: Biochemical tests of bacterial strains isolated from water along Uran coast Sr. Bacterial species Bio-chemical tests No. Indole Methyl VogesCitrate production Red Proskauer utilization test test test test 1 Bacillus subtilis + + + 2 Escherichia + + coli 3 Klebsiella + + pneumoniae 4 Micrococcus + + luteus 5 Providencia + + + rettgeri 6 Pseudomonas + aeruginosa

H2S production test + -

Urease test

Litmus milk test

-

+

Nitrate reduction test +

-

+

+

+

-

+

-

+

-

-

-

+

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

7 8 9 10 11

Salmonella typhimurium Salmonella enterica Shigella flexneri Staphylococcus epidermidis Staphylococus aureus

-

+

-

+

+

-

-

+

-

+

+

-

+

-

+ -

+

-

+

+

+ +

-

+

+

+

-

+

+

Table 14: Species diversity of bacteria recorded from water along Uran coast Phylum Class Order Family Actinobacteria Actinobacteria Micrococcales Micrococcaceae Firmicutes

Bacilli

Bacillales

Bacillaceae

Firmicutes

Bacilli

Bacillales

Staphylococcaceae

Firmicutes

Coccus

Bacillales

Staphylococcaceae

Proteobacteria

Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria

Enterobacteriales

Enterobacteriaceae

Enterobacteriales

Enterobacteriaceae

Enterobacteriales

Enterobacteriaceae

Enterobacteriales

Enterobacteriaceae

Enterobacteriales

Enterobacteriaceae

Gammaproteobacteria Gammaproteobacteria

Enterobacteriales

Enterobacteriaceae

Pseudomonadales

Pseudomonadaceae

Proteobacteria Proteobacteria Proteobacteria Proteobacteria

Proteobacteria Proteobacteria

Monsoon_SPC_MC_Site I

+

+

Binomial Name Micrococcus luteus (Schroeter 1872) Bacillus subtilis (Ehrenberg 1835) Staphylococcus epidermidis (Winslow & Winslow 1908) Staphylococcus aureus (Rosenbach 1884) Escherichia coli (Migula 1895) Klebsiella pneumoniae (Schroeter 1886) Providencia rettgeri (Rettger 1904) Salmonella enterica (Le Minor & Popoff 1987) Salmonella typhimurium (Lignieres 1900) Shigella flexneri (Castellani & Chalmers 1919) Pseudomonas aeruginosa (Schröter 1872)

Monsoon_SPC_MC_Site II

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Monsoon_SPC_MC_Site III _A

Post-monsoon_SPC_NA_Site II_A Fig. 3: Colonies of identified bacterial strains

Monsoon_SPC_MC_Site III _B

Post-monsoon_SPC_NA_Site II_B

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Prabhakar R. Pawar, 2017 Advances in Environmental Biology, 11(8) August 2017, Pages: 11-24

Pre-monsoon_SPC_Endo_Site_ III

Pre-monsoon_SPC_MC_Site_ I

Fig. 3: Continued. Conclusion: Results of the present study showed that marine waters along the Uran coastal area is undergoing severe sewage pollution. Presence of pathogenic or potentially pathogenic bacterial strains like Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Providencia rettgeri, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus and Staphylococcus epidermidis indicates the fecal contamination in coastal water. Heavy microbial load reported in marine waters of Uran is correlated to the disposal of untreated sewage and waste water, lack of sanitary facilities, human settlement in the vicinity of the coast, poverty among local community and discharge of untreated effluents from Jawaharlal Nehru Port (JNP) and other port related establishments. Presence of potential pathogenic bacteria could pose a high health risk to the local community due to contamination of fishery and aquaculture products. ACKNOWLEDGEMENTS Financial support provided by University Grants Commission, New Delhi [File No: 42–546/2013 (SR) dated 22nd Mar 2013] is gratefully acknowledged. The author is thankful to The Principal, Veer Wajekar Arts, Science and Commerce College, Mahalan Vibhag, Phunde (Uran), Navi Mumbai 400 702 for providing necessary facilities for the present study. Special thanks to Dr. Rahul B. Patil for providing healthy cooperation during field visits for photography of the study sites. Thanks are due to Mr. Sanket S. Shirgaonkar, who worked as a Project Fellow for the present study. Thanks to Dr. Atul G. Babar for the graphic design of the study area and the distribution maps. REFERENCES [1] American Public Health Association (APHA). 2005. Standard methods for the examination of water and wastewater. Vol. 21. American Public Health Association; Washington, D.C. [2] Ashbolt, Nicholas J., Willie O.K. Grabow and Mario Snozzi, 2001. Indicators of microbial water quality. World Health Organization (WHO). Water Quality: Guidelines, Standards and Health. Ed. by Lorna Fewtrell and Jamie Bartram. Pub. by IWA Publishing, London, UK. ISBN: 1 900222 28 0. [3] Banoo, Sabnam., Rabi Narayan Kar and Chitta Ranjan Panda, 2014. Seasonal variation and distribution of sewage pollution indicator and human pathogenic bacteria along Odisha coast. Indian J Mar Sci., 43(5): 859-869. [4] Bose, Rahul., Hare Krishna Jana, Sufia Zaman and Abhijit Mitra, 2014. Study of the Microbial Health in and Around the Lower Stretch of Hooghly Estuary. J Marine Sci Res Development, S11: 004. doi:10.4172/2155-9910.S11-004. [5] Cappuccino, James G., and Natalie Sherman, 2004. Microbiology: A Laboratory Manual. PEARSON Education, Inc. 7th Edition. pp: 528.

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