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Advances in Environmental Biology, 11(8) August 2017, Pages: 25-38

AENSI Journals

Advances in Environmental Biology

ISSN-1995-0756 EISSN-1998-1066

Journal home page: http://www.aensiweb.com/AEB/

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

Study of bacterial diversity in intertidal sediments along the marine ecosystem of Uran, Navi Mumbai

1Prabhakar R. Pawar and 2Abdel Rahman Mohammad Said Al-Tawaha

1Arts, Science and Commerce College, Department of Zoology, Mokhada, Dist. – Palghar, Pin - 401 604, India 2Department of Biological Sciences, Al Hussein Bin Talal University, Ma’an, P.O. Box 20, Jordan 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 19 July 2017; Accepted 29 July 2017; Available online 26 September 2017

ABSTRACT The objective of this study was to assess the bacterial diversity in intertidal sediments along the marine ecosystem of Uran, 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). Intertidal sediments samples were collected during spring low tides for bacterial diversity monthly

from June 2013 to May 2015. Standard microbiological methods like Presumptive test, Confirmed test and Completed tests were adopted for present study. Eleven bacterial species belonging to nine genera, five families, four orders and four classes were isolated from sediment

samples. Potential 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 were isolated. Bacterial load reported in coastal sediment of Uran is attributed to the coastal tourism, coastal

dredging, excavation through mangroves for gas pipelines and erection of electricity poles, Container Freight Stations (CFS), road

construction, inadequate sanitary facilities, open defecation and poverty among local community. An overall result of the study suggests that presence of potential pathogenic bacteria in intertidal sediments of Uran poses a high health risk to the public.

KEYWORDS: Bacterial diversity, Fecal pollution, Jawaharlal Nehru Port, Potential pathogens, Sediments, Uran,

INTRODUCTION

Sediments can also be defined as the material deposited at the bottom of rivers, which are silt and deposits.

Soils and Sediments are home to an extraordinary range of microbial and animal groups. Sediments form a

natural buffer and filter system and often play an important role in the storage and release of nutrients in the

aquatic ecosystems. Sediments are indicators of quality of overlying water and its study is a useful tool in the

assessment of environmental pollution status. Microbial survival in polluted soils depends on intrinsic

biochemical and structural properties, physiological, and genetic adaptation [15].

Marine sediments harbor microbial communities that play a significant role in the decomposition,

mineralization, and recycling of organic carbon. Microbial number and species composition in the soil habitat

differ from place to place depending upon the physical, chemical and biological factors of the particular habitat

[6]. Lipp et al [17] reported that microbial abundances from sediments of continental margins and the open

ocean indicate that margin sites generally have a larger microbial population due to larger amounts of organic

matter.

Sediment is a special realm in aquatic ecosystems. The microbial richness in sediment is much higher than

those of the corresponding water bodies. Sediment receives deposition of microbes and organic matter from the

upper water layer and provides a matrix of complex nutrients and solid surfaces for microbial growth [35]. The

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26 Prabhakar R. Pawar et al, 2017


intertidal zone in marine ecosystems acts as a mixing zone between terrestrial and marine habitats and the

intertidal sediment is thought to have a significantly different bacterial community than marine sediment [31].

Bacteria within coastal and shelf sediments play an important role in global biogeochemical cycles;

however, factors influencing assemblage composition have not been extensively studied [14]. Winter et al., [37]

reported that microbial composition in the oceans is believed to be influenced by combinations of resource

availability, temperature, pressure, and selective loss factors such as grazing and viral lysis.

According to Donovan et al., [8], as sediments act as a reservoir for pathogens, it is important that they, too,

be evaluated to determine if they pose a potential risk to human health. Very large variations have been recorded

in bacteria concentrations in sediments from different sources as well as within a single stream or water body.

Literature reports for values of E. coli concentrations in sediment vary from 1 to 500000 MPN or CFU per gram

of dry weight [22].

Yazhini et al [38] noted that sewage and untreated industrial effluents are being discharged from decades

into the sea which leads to increase in pollution level. Enormous loads of organic, inorganic, sewages and

untreated effluents are in fluxed into the coastal waters due to recreational activities, industrialization and

urbanization. These inputs results in outburst of microbial loads besides spoiling the water quality [7]. The

under-treated effluents from the coastal population and discharges from industrial belt regions often pose an

adverse impact on marine and estuarine species [3].

The recreational safety of water bodies is established through the microbiological examination of water

samples [20]. Swarnakumar et al [33] revealed that regular microbial monitoring in the coastal environment is

an integral and essential part in predicting the microbial pollution of coastal waters.

Maritime activities 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, coastal tourism at Peerwadi coast and large number of Container Freight Stations

[CFS] in the mangroves stretch have affected the water quality and coastal biodiversity of Uran [23, 24, 25, 26,

27, 28].

Objective of the present study is to assess the bacterial diversity in intertidal sediments along the marine

ecosystem of Uran, 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.

Sediment samples from intertidal shorelines were carefully collected by scooping from a portion of the top

5 cm of sediment in a 50 ml sterile bottle, after removing debris and shells. Filled sediment bottles were capped

before they were placed together in a zip-lock polyethylene bag according to sampling station.

Immediately after collection, samples were placed in cooling boxes and immediately transferred to the

laboratory. In the laboratory, all sediment 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 [2].

Laboratory analysis:

For sediment samples, 1 g of finely pulverized sediments were added in a flask containing 99 ml of sterile

buffered peptone water and mixed well for 20 seconds. Serial dilutions of sediment samples were made

following standard methods. Standard microbiological methods described by APHA [2], Cappuccino and

Sherman [5] and Food Safety & Standards Authority of India, Lab Manual 14 [10] were followed for

bacteriological analysis of sediment (Table 1).

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)


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 (HIMEDIA Mumbai M463)

Methyl Red test MR-VP Broth


Voges- Proskauer test MR-VP Broth (HIMEDIA Mumbai M070)

Citrate Utilization test Simmon’s Citrate Agar


Urease test Urea broth (HIMEDIA Mumbai M112)

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:

Table 2: Expression of result for sediment on number of colonies

Number of colonies on petriplates Expression of result

No colonies on petriplates < 1X101 organisms/ml

< 30 colonies in 1:10 dilution 3 X 102 (30 X 10 = 3 X 102 )

> 300 colonies in last dilution > 300 X 10X , Where,

“x” = Number of dilutions used

RESULTS AND DISCUSSION

A total of 11 bacterial species of bacteria were enumerated from three sampling stations by plating

technique. Of the recorded bacterial species, 54.55% belonged to Enterobacteriaceae, 18.18% to

Staphylococcaceae and 9.09% each to Bacillaceae, Micrococcaceae and Pseudomonadaceae. The eleven species



recovered were belonged to 9 genera, 5 families, 4 orders and 4 classes. Based on colony morphology, Gram

staining and biochemical tests, identified bacterial strains were represented by Bacillus, Escherichia, Klebsiella,

Micrococcus, Providencia, Pseudomonas, Salmonella, Shigella and Staphylococcus.

Coliform bacteria from sediment were detected with three basic tests like presumptive, confirmed, and

completed tests performed sequentially on each sediment sample.

A. Presumptive test for total coliforms:

Presumptive test is performed to determine presence of coliform bacteria in a sediment sample and also to

obtain an index indicating the possible number of organisms present in the sample.

Most Probable Number (MPN):

Significantly high MPN Index/100 ml was recorded during monsoon of 2014 at site II (350) and site III (91)

as compared to site I (18). Also during pre-monsoon of 2014, higher MPN Index/100 ml was noted at site III

(110) where as for site I and II, it was 17 and 26 respectively. In post-monsoon, lower MPN Index/100 ml in the

range of 17-26 was recorded from sediment of all study sites (Table 3).

Higher MPN Index recorded during monsoon is attributed to the lower salinity. Ortega et al [2010] reported

that salinity relates to microbial contamination and sites characterized by lower salinities, were also

characterized by higher levels of bacterial indicators. Salinity was most strongly correlated with bacterial

indicators and lower salinity is associating with higher levels of indicator bacteria [13]. High density of

coliforms could be also due to agricultural and urban surface run-off [3, 30].

Table 3: Most Probable Number (MPN) of sediment 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 + - + + + + - - - - - - + - - 4 – 1-1 21

21 II - + - + + - - + - + + - - - - 3 – 2-1 17

III + - + + + + - - - - - - + + - 4 – 1-2 26

2

Pre- monsoon

May 2014

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

51 II - + + + + - + - - + - - - + - 4 – 2- 1 26

III + + - + + + - + - + - - - - + 4 – 3-1 110

3

Monsoon

Aug 2014

I + + - - + - - - + + + + + + + 3 – 2- 5 18

153 II + + + + + + + + + - + + + - + 5 – 4- 4 350

III + + + + + - + - + - - + - + + 5 – 2 -3 91

Standard Plate Count (SPC):

Results on Standard Plate Count (SPC) for coliforms in sediment are in agreement with the MPN Index/100

ml. Exceptionally high SPC count in the range 442-927 CFU/ml was recorded during monsoon of 2014 at all

sites. During pre-monsoon of 2014, slightly higher SPC (324-713 CFU/ml) was observed from all sites. In post-

monsoon of 2013, moderate number of colonies (76-113 CFU/ml) was noted. Mean SPC/ml of sediment

recorded highest value (572 X 104 ) at site I where as site II and III have 413 X 104 and 293 X 104 respectively.

High bacteria count recorded in sediment is attributed to the discharge of untreated effluents,

gastrointestinal inputs and greater from municipal sewage contamination [32]. High density of coliforms at

Sheva creek and Peerwadi coast could be also due to highest population densities at Mora fish landing centre

and is directly related to the municipal sewage pipe outlet from the urban area and port effluents [23] (Table 4).

Table 4: Standard Plate Count (SPC) on Nutrient Agar/MMGA of sediment 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 76 713 927 76X104 713X104 927X104 572X104 426X104 II 90 536 613 90 X104 536X104 613X104 413X104

III 113 324 442 113X104 324X104 473X104 293X104

B

1 ml

from

10-5

10-5

I 46 624 692 46X105 624X105 692X105 454X105

304X105 II 60 311 481 60X105 311X105 481X105 284X105

III 87 133 298 87X105 133X105 298X105 173X105

C 1 ml from

10-6

10-6

I 43 503 476 43X106 503X106 476X106 341X106 204X106 II 59 248 208 59X106 248X106 208X106 172X106

III 73 102 117 73X106 102X106 117X106 98X106



D 1 ml

from 10-7

10-7

I 42 453 331 42X107 453X107 331X107 276X107

158X107 II 56 142 152 56X107 142X107 152X107 117X107

III 62 87 89 62X107 87X107 89X107 80X107

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 sediment 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 [3, 33].

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 5).

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 [33] (Table 6 to

Table 14) (Fig. 2).

pathogenic or potentially pathogenic bacteria isolated from intertidal sediments along Uran coast includes

Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Providencia rettgeri,

Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhimurium, Shigella flexneri, Staphylococcus

aureus and Staphylococcus epidermidis (Table 15). 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 [16].

Ferguson et al [9] reported that a major cause of bacterial pollution of coastal waters is urban runoff in

rivers/channels and storm drains that discharge into the ocean. According to Whitman and Nevers [36], survival

of indicator bacteria in sediment may be enhanced because of protection from UV inactivation and predation,

moisture, buffered temperatures and availability of nutrients originating from algae, debris and plankton. The

phytoplankton and zooplankton provide both nutrients and surfaces for indicator bacteria to survive in the

marine environment [18, 21].

Swarnakumar et al [33] noted that higher densities of pathogenic bacteria in sediments could be due to rich

organic content and degradation and recycling of organic and inorganic materials [19]. Rodrigues et al [30]

documented that high bacterial load in sediments may be due to enhanced survival by low/no exposure to

stressors, such as sunlight and predation, or by increased availability of nutrients.

Ghaderpour et al [11] stated that potentially pathogenic bacteria are get transmitted from ocean to human

either by the pelagic (water) or by benthic (sediment) pathways. Ingestion of suspended pathogenic bacteria or

organic particulates containing these pathogens by fish the grazing of contaminated organic particulates and

sediments on the mangrove roots by prawns and demersal fishes, may eventually reach humans. Consumption of

such contaminated fishery/aquaculture products could lead to food borne illness in humans.

Walsh et al [34] cited that taxonomic richness of microbial flora is highest at the seafloor and declines with

increasing sediment depth. Sources and transmission mechanism of microbial contamination in ocean includes

sewage, sewage sludge, run off/ floods, groundwater and river discharge [12].

The pathogens found in the marine environment are responsible for a broad spectrum of acute and chronic

human diseases, e.g. gastroenteritis, ocular and respiratory infections, hepatitis, myocarditis, meningitis, and

neural paralysis [4]. Abdelzaher et al [1] described that global estimates indicate that each year more than 120

million cases of gastrointestinal disease and 50 million cases of severe respiratory diseases are caused by

swimming and bathing in wastewater-polluted coastal waters.

Results of the present study are in agreement with the work of Ravikumar et al [29] on microbial diversity

in relation to biochemical constituents along Palk Strait, Kalaivani and Sukumaran [15] on bacterial diversity in

marine sediment at different seasons in Karankura, Tamilnadu, India, Bose et al [3] on microbial health in and

around the Lower Stretch of Hooghly Estuary and Chakravarty et al [7] on bacteria of the recreational beach

waters of Visakhapatnam, India.

Coastal environment of Uran receives domestic and industrial wastes Thane Belapur industrial area,

municipal sewage from Navi Mumbai, and effluents with marine debris from Jawaharlal Nehru Port and other

port establishments. Coastal tourism, coastal dredging, excavation through mangroves for gas pipelines and

erection of electricity poles, Container Freight Stations (CFS), road construction, inadequate sanitary facilities,

open defecation and poverty among local community have deteriorated the water quality of Uran coast [23].



An overall result of the study suggests that presence of potential pathogenic bacteria in intertidal sediments

of Uran poses a serious health risk to the public. Outcomes of the study can be used as the baseline for future

research and to develop the management strategy for conservation of the Uran coastal environment.

Table 5: Colony characteristics of bacterial strains isolated from sediment 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 5: 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 6: Indole production test of sediment 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 7: Methyl Red (M. R.) test of sediment of Uran coast


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: Voges - Proskauer test (V. P. test) of sediment of Uran coast


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: Citrate utilization test of sediment 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 10: Hydreogen sulfide test of sediment of Uran coast

Sr.

No.

Season Site Test

tube

Colour of

medium

H2S production

(positive)

H2S production

(negative)

1

Post-monsoon (Oct13 to

Jan 14)

I B Black +

II B Black +

III B Black +

2

Pre-monsoon

(Feb 14 to May

14)

I B Black +

II B Black +

III B Black +

3

Monsoon (Jun 14 to Sept

14)

I B No black colour +

II B Black +

III B Black +

Control I, II, III A No black colour +

Table 11: Urease test of sediment 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 Deep Pink +

2

Pre-monsoon

(Feb 14 to May

14)

I B Pink colour +

II B Pink colour +

III B Pink colour +

3

Monsoon

(Jun 14 to Sept 14)

I B Light Orange +

II B Light Orange +

III B Pink colour +

Control I, II, III A Light Orange +

Table 12: Litmus milk reactions of sediment of Uran coast

Table 12(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 Brick Red - - - -

II B Brick Red - - - -

III B Brick Red - - - -

2

Pre-monsoon

(Feb 14 to

May 14)




3

Monsoon (Jun 14 to

Sept 14)




Control I, II,

III

A Brick Red - - - -



Table 12(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 Deep purple band at the top of

the brownish translucent

medium

- + -

II B Deep purple band at the top of

the brownish translucent

medium

- + -

III B Deep purple band at the top of the brownish translucent

medium

- + -

Control I, II, III

A Brick Red - - -

Table 13: Nitrate reduction test of sediment 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 No red colour - + NO2/NH3/N2

III B No red colour - + NO2/NH3/N2

2

Pre-monsoon

(Feb 14 to May 14)


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

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

3

Monsoon

(Jun 14 to Sept

14)


II B Red colour - - NO3

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

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

Table 14: Biochemical tests of bacterial strains isolated from sediment 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 15: Species diversity of bacteria recorded from sediment 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)


Enterobacteriales Enterobacteriaceae Salmonella enterica (Le Minor & Popoff 1987)


proteobacteria

Enterobacteriales Enterobacteriaceae Salmonella

typhimurium (Lignieres 1900)


proteobacteria

Enterobacteriales Enterobacteriaceae Shigella flexneri

(Castellani & Chalmers 1919)


Pseudomonadales Pseudomonadaceae Pseudomonas aeruginosa (Schröter 1872)

Monsoon_SPC_MC_Site_ III Monsoon_SPC_MMGA_Site_ I

Monsoon_SPC_MMGA_Site_ I Post-monsoon_SPC_NA_Site III



Post-monsoon_SPC_Endo_Site_ III Post-monsoon_SPC_MC_Site_ III

Fig. 2: Colonies of identified bacterial strains

Pre-monsoon_SPC_MC_Site_I Pre-monsoon_SPC_MC_Site_II

Pre-monsoon_SPC_Endo_Site_II

Fig. 2: Continued



Conclusion:

Present study showed that intertidal sediments along the Uran coast are facing severe threat of fecal

contamination. 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 microbial pollution of the coastal sediment. Bacterial load reported in coastal sediment of

Uran is attributed to the coastal tourism, coastal dredging, excavation through mangroves for gas pipelines and

erection of electricity poles, Container Freight Stations (CFS), road construction, inadequate sanitary facilities,

open defecation and poverty among local community. An overall result of the study suggests that presence of

potential pathogenic bacteria in intertidal sediments of Uran poses a high health risk to the public.

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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