Assessment of microbial diversity in marine waters along ... · Monitoring the microbiological...

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

AENSI Journals

Advances in Environmental Biology

ISSN-1995-0756 EISSN-1998-1066

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

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

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12 Prabhakar R. Pawar, 2017


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.



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.



Surface water samples were collected seasonally during spring low tides. Samples were collected in heat-

sterilized 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 40C 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 Medium

Presumptive Test Most Probable Number (MPN) Brilliant Green Bile Broth (HIMEDIA Mumbai M121)

Standard Plate Count (SPC) Nutrient agar (NA)

(HIMEDIA Mumbai M001) Mineral Modified Glutamate Agar (MMGA)

(HIMEDIA Mumbai M6431)

Confirmed Test Inoculation on slants and plates of selective &

differential media

Endo agar

(HIMEDIA Mumbai M1077) MacConkeys agar

(HIMEDIA Mumbai M081B)

Completed Tests for

coliform species

identification

Colony morphology -----

Gram staining -----

Motility test Hanging Drop Method

Biochemical Tests

Indole Production test Tryptone water


Methyl Red test MR-VP Broth (HIMEDIA Mumbai M070)

Voges- Proskauer test MR-VP Broth


Citrate Utilization test Simmon’s Citrate Agar (HIMEDIA Mumbai M099)

Urease test Urea broth


H2S Production test SIM agar (HIMEDIA Mumbai M181F)

Nitrate Reduction test Trypticase nitrate broth

(HIMEDIA Mumbai M439S)

Litmus Milk test Litmus milk broth


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).



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 Reading MPN Index/

100 ml

Mean MPN

Index/

100 ml.

10 X 1 X 0.1 X

Tube Tube Tube

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

1

Post-

monsoon Nov 2013

I - + - + + - + - - + + - - - - 3 – 2-1 17

18

II + + - + - - - - + + - - - - + 3 – 2-1 14

III + - + + + + - - - - - - + - - 4 – 1-1 21

2

Pre-

monsoon

May 2014

I + + + + - + + - + - - - - - + 4 – 3-1 33 69

II + + + + + - + - - - + - - - + 5 – 1- 2 63

III + + + + + + + - + - - + - - - 5 – 3-1 110

3

Monsoon

Aug 2014

I + + + + + + + + + + + - + + + 5 – 5- 4 1600

1867 II + + + + + + + + + + + + + + - 5 – 5- 4 1600

III + + + + + + + + + + + + + + + 5 – 5- 5 ≥ 2400



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 post-

monsoon, 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

Petri

Plate

Dilu tion

Dilution Factor

Site

No. of colonies

per plate

No. of colonies

SPC/ml= ────────

Dilution factor

Mean

SPC/ml

Average

SPC/ml

Sample Sample

Post-

mon

Nov 2013

Pre-

mon

May 2014

Mon

Aug

2014

Post-

mon

Nov 2013

Pre-

Mon

May 2014

Mon

Aug

2014

A

1 ml

from

10-4

10-4

I 137 67 583 137X104 67X104 583X104 263X104

222X104 II 65 162 236 65 X104 162X104 236X104 176X104

III 97 113 473 97X104 113X104 473X104 228X104

B

1 ml from

10-5

10-5

I 50 41 434 50X105 41X105 434X105 175X105 155X105 II 54 118 181 54X105 118X105 181X105 118X105

III 72 53 391 72X105 53X105 391X105 172X105

C 1 ml

from 10-6

10-6

I 30 28 387 30X106 28X106 387X106 149X106

116X106 II 41 63 124 41X106 63X106 124X106 76X106

III 43 39 289 43X106 39X106 289X106 124X106

D 1 ml from

10-7

10-7

I 22 17 213 22X107 17X107 213X107 84X107 67X107 II 34 32 96 34X107 32X107 96X107 54X107

III 27 21 138 27X107 21X107 138X107 62X107

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].



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

characteristics

Bacterial species

1 2 3 4 5 6

Size 2-4 mm 3-6 mm 2-5 mm 2 mm ~ 4 mm 2-4 mm

General Surface Form

Irregular Circular Circular Circular Circular Circular

Elevation Umbonate Slightly Raised Umbonate Convex Convex Low convex

Margin Undulate Entire Entire/

Undulate

Entire - Undulate

Texture Rough Smooth Mucoid Smooth - Smooth

Appearance Dull Shiny Shiny - Dull -

Pigmentation White to

creamy or brownish

Colourless

/Cream

White Bright yellow

Non-diffusing

Grey Blue green

Optical Property Opaque Translucent Translucent - Opaque Translucent

Morphology Rod Rod Rod Coccus Straight Rod Rod

Motility Motile Motile Non-motile Non-motile Motile Motile

Gram Staining Gram +ve Gram –ve Gram –ve Gram +ve Gram –ve Gram –ve

Bacillus subtilis

Escherichia coli

Klebsiella pneumoniae

Micrococcus luteus

Providencia rettgeri

Pseudomonas aeruginosa

Table 4: Continued.

Colony characteristics

Bacterial species

7 8 9 10 11

Size 2-4 mm 2-4 mm 2 mm 1-2 mm 1-2 mm

General Surface

Form

Circular Circular Circular Circular Circular

Elevation Convex Raised Convex Raised Convex Convex

Margin Entire - Entire edges Entire Entire

Texture Smooth Smooth Smooth Smooth Smooth

Appearance Shiny Moist - - Shiny

Pigmentation Colourless Grey Colourless White Golden-brown

Optical Property Translucent - Moderately translucent

- Opaque

Morphology Rod Rod Rod Coccus Coccus

Motility Motile Motile Non-Motile Non-motile Non-motile

Gram Staining Gram –ve Gram –ve Gram –ve Gram +ve Gram +ve

Salmonella typhimurium

Salmonella enterica

Shigella flexneri

Staphylococcus epidermidis

Staphylococus aureus

Table 5: Indole production test of water of Uran coast

Sr. No.

Season Site Test tube

A

(Control)

B C D E

1

Post-monsoon

Nov 2013

I - + + + +

II - + + + +

III - + + + +

2

Pre-monsoon

May 2014

I - + + + +

II - + + + +

III - + + + +

3

Monsoon

Aug 2014

I - + + + +

II - + - - -

III - + + + +



Table 6: Methyl Red (M. R.) test of water of Uran coast

Sr. No. Season Site Test tube

A (Control)

B

1

Post-monsoon

Nov 2013

I - +

II - +

III - +

2

Pre-monsoon May 2014

I - +

II - +

III - -

3

Monsoon

Aug 2014

I - -

II - -

III - -

Table 7: Voges - Proskauer test (V. P. test) of water of Uran coast

Sr. No. Season Site Test tube

A

(Control)

B

1

Post-monsoon

Nov 2013

I - +

II - +

III - +

2

Pre-monsoon

May 2014

I - +

II - +

III - +

3

Monsoon

Aug 2014

I - +

II - +

III - +

Table 8: Citrate utilization test of water of Uran coast

Sr.

No.


A

(Control)

B

1

Post-monsoon

Nov 2013

I - +

II - +

III - +

2

Pre-monsoon

May 2014

I - -

II - +

III - +

3

Monsoon Aug 2014

I - +

II - +

III - +

Table 9: Hydreogen sulfide test of water of Uran coast

Sr. No.


Colour of medium

H2S production (positive)

H2S production (negative)

1

Post-monsoon

(Oct13 to Jan 14)

I B Black +

II B +

III B +

2

Pre-monsoon

(Feb 14 to May

14)

I B No black colour +

II B +

III B +

3

Monsoon

(Jun 14 to Sept 14)

I B Black +

II B +

III B +

Control I, II, III A No black colour +

Table 10: Urease test of water of Uran coast

Sr. No.

Season Site Test tube Colour of medium

Urea Hydrolysis (positive)

Urea Hydrolysis (negative)

1

Post-monsoon

(Oct13 to

Jan 14)

I B Light Orange +

II B Light Orange +

III B Light Orange +

2

Pre-monsoon (Feb 14 to May

14)

I B Light Orange +

II B Light Orange +


3

Monsoon

(Jun 14 to Sept

14)

I B Deep Pink +

II B Light Orange +


Control I, II, III A Light Orange +



Table 11: Litmus milk reactions of water of Uran coast

Table 11(a): Lactose fermentation:

Sr.

No.

Season Site Test

tube

Appearance

of medium

Lactose

fermentation

(acid)

Acid

followed

by reduction

Acid, reduction

and curd

Acid, gas

reduction and

curd

1

Post-monsoon (Oct13 to

Jan 14)

I B Orange - - + +

II B Orange - - + +

III B Orange - - + +

2

Pre-monsoon

(Feb 14 to May 14)

I B Orange - - + +

II B Orange - - + +

III B Orange - - + +

3

Monsoon (Jun 14 to

Sept 14)

I B Pink band at the top of the

medium

-

+

-

-

II B Pink band at the top of the

medium

-

+

-

-

III B Pink band at the

top of the medium

-

+

-

-

Control I, II,

III

A Brick Red - - - -

Table 11(b):

Sr.

No.

Season Site Test

tube

Appearance of

medium

Litmus

reduction

Proteolysis Alkaline

reaction

1

Post-monsoon

(Oct13 to

Jan 14)

I B Medium is unchanged - - +

II B Medium is unchanged - - +

III B Medium is unchanged - - +

2

Pre-monsoon

(Feb 14 to May 14)

I B Medium is unchanged - - +

II B Medium is unchanged - - +

III B Medium is unchanged - - +

3

Monsoon

(Jun 14 to

Sept 14)

I B Brownish translucent

medium

- + -

II B Brownish translucent

medium

- + -

III B Brownish translucent

medium

- + -

Control I, II,

III

A Brick Red - - -

Table 12: Nitrate reduction test of water of Uran coast

Sr.

No.

Season

Site Test

tube

Red coloration

with solution A and B

(+) or (-)

Red coloration

with Zinc (+) or (-)

Nitrate

reduction (+) or (-)

End

products

1

Post-monsoon (Oct13 to Jan

14)

I B Red colour - - NO3

II B Red colour - - NO3

III B Red colour - - NO3

2

Pre-monsoon

(Feb 14 to May

14)

I B No Red colour No Red colour + NO2/NH3/N2

II B No Red colour No Red colour + NO2/NH3/N2

III B No Red colour No Red colour + NO2/NH3/N2

3

Monsoon (Jun 14 to Sept

14)

I B Red colour - - NO3

II B No Red colour No Red colour + NO2/NH3/N2

III B No Red colour No Red colour + NO2/NH3/N2

Control I, II, III A Red colour - - NO3

Table 13: Biochemical tests of bacterial strains isolated from water along Uran coast

Sr.

No.

Bacterial species Bio-chemical tests

Indole

production

test

Methyl

Red

test

Voges-

Proskauer

test

Citrate

utilization

test

H2S

production

test

Urease

test

Litmus

milk

test

Nitrate

reduction

test

1 Bacillus subtilis - + + + + - -

2 Escherichia

coli

+ + - - - - + +

3 Klebsiella pneumoniae

- - + + - + + +

4 Micrococcus

luteus

- - + + - + -

5 Providencia rettgeri

+ + - + - +

6 Pseudomonas

aeruginosa

- - - + - - +



7 Salmonella

typhimurium

- + - + + - + +

8 Salmonella enterica

- + - + + - +

9 Shigella flexneri - + - - - - +

10 Staphylococcus

epidermidis

- + - + + +

11 Staphylococus

aureus

- + + + - +

+

Table 14: Species diversity of bacteria recorded from water along Uran coast

Phylum Class Order Family Binomial Name

Actinobacteria Actinobacteria Micrococcales Micrococcaceae Micrococcus luteus (Schroeter

1872)

Firmicutes Bacilli Bacillales Bacillaceae Bacillus subtilis (Ehrenberg 1835)

Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus epidermidis

(Winslow & Winslow 1908)

Firmicutes Coccus Bacillales Staphylococcaceae Staphylococcus aureus (Rosenbach 1884)

Proteobacteria Gamma-

proteobacteria

Enterobacteriales Enterobacteriaceae Escherichia coli (Migula 1895)

Proteobacteria Gamma-proteobacteria

Enterobacteriales Enterobacteriaceae Klebsiella pneumoniae (Schroeter 1886)


proteobacteria

Enterobacteriales Enterobacteriaceae Providencia rettgeri

(Rettger 1904)

Proteobacteria Gamma-proteobacteria

Enterobacteriales Enterobacteriaceae Salmonella enterica (Le Minor & Popoff 1987)


proteobacteria

Enterobacteriales Enterobacteriaceae Salmonella

typhimurium (Lignieres 1900)


proteobacteria

Enterobacteriales Enterobacteriaceae Shigella flexneri

(Castellani & Chalmers 1919)


proteobacteria

Pseudomonadales Pseudomonadaceae Pseudomonas aeruginosa

(Schröter 1872)

Monsoon_SPC_MC_Site I Monsoon_SPC_MC_Site II



Monsoon_SPC_MC_Site III _A Monsoon_SPC_MC_Site III _B

Post-monsoon_SPC_NA_Site II_A Post-monsoon_SPC_NA_Site II_B

Fig. 3: Colonies of identified bacterial strains



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.

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