Isolation and Characterization of Microorganisms from Mangrove Soil of CBD Belapur Creek, Navi Mumbai, India

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Research article ISSN 0976 – 4402

Isolation and characterization of microorganisms from mangrove soil of CBD Belapur creek , Navi Mumbai ,MS India

Bhat M.R, Shewade Leena

Department of Biotechnology and Bioinformatics, Pd. Dr. D. Y. Patil University, Plot no. 50, Sector-15,CBD Belapur, Navi Mumbai,Kokan Bhavan 400614, MS, India.

manisbhat@gmail.com doi:10.6088/ijes. 2013030600046

ABSTRACT

In the present study, mangrove soil samples from five different stations of CBD Belapur (19° 1′25.33″ N, 73° 2′27.65″E) Navi-Mumbai, MS, India, were collected for the isolation of micro-organisms. Among the soil samples screened, all gram positive isolates were obtained. Out of all the isolates, fourteen distinct isolates were selected and characterized. On the basis of biochemical tests, the isolates were assigned to the genera Bacillus. All the isolates demonstrated very strong L-asparginase activity, whereas few were positive for Protease and Amylase activity. Isolates were observed to grow at pH ranging from 7-8 and had temperature optima of 28⁰C and few had 55⁰C. Most of the organisms could tolerate about 300ppm of heavy metals in the form of AlCl3, MnSO4 and ZnSO4; upto 1000ppm of K2CrO4 and only about 10 ppm of HgCl2. In addition, a few of these isolates could tolerate more than 8% NaCl. Antibiotic efficacy of the isolates was studied, in which most of the organisms were sensitive to antibiotics tested except Penicillin G.

Keywords: Asparaginase, biochemical characterization, mangrove, heavy metal, saline ecology.

1. Introduction

Mangroves are the coastal wetland forests generally found near the intertidal regions of estuaries between creeks, lagoons, marshes etc. Mangroves provide a unique ecological site to different microbes. Because of richness in carbon and other nutrients mangrove ecosystem harbors diverse microbial communities which can adapt themselves in the extreme conditions there. Microorganisms forms integral part of the mangrove ecosystem. They help in recycling and transformation of various nutrients and thus make the mangrove ecosystem more productive. The microorganisms of mangrove are essential in the productivity, conservation, and rehabilitation of mangrove ecosystems (Gina Holguin · Patricia Vazquez · Yoav Bashan , 2001) Role of bacteria in promotion of growth of halotolerant plants have been studied (Y.

Bashan - M. Moreno - E. Troyo , 2000). Culturable bacterial distribution in sediments of mangroves reported from arid zone in 2005 (Barbara Gonzalez et. al, 2005). Molecular diversity of N2 fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (Ana L. et. al, 2007). Mangrove ecosystem shows diversity of microbes such as bacteria, fungi, actinomycetes etc. Bacteria includes various types like nitrogen fixing bacteria, phosphate solublising bacteria, Sulphate reducing bacteria, photosynthetic anoxygenic bacteria , methanogenic bacteria, enzyme producing bacteria.(K.Sahoo and N.K.Dhal, 2009). Actinomycetes isolated from mangrove habitats are a potentially rich source for the discovery of anti-infection and anti-tumor compounds, and

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new nitrogen-fixing bacteria from the rhizosphere of mangrove trees, their isolation, identification and in vitro interaction with rhizosphere Staphylococcus species have been studied (Gina Holguin et.al 1992).Evaluation of Synergism between Phyllobacterium sp.

(N2-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere as plant growth promoters have been reported (Adriana Rojas et. al 2001) Antibiotic resistance in bacteria from shrimp farming in mangrove areas of south was studied(Le T.X et. al , 2005).Characterization of marine bacteria and the activityof their enzyme systems involved in degradation of the algal storage glucan laminarin have been studied (Anne-Carlijn et.al,2006). Diversity,antimicrobial activity and enzymes production have increased the scope of finding industrially important marine actinomycetes from the Bay of Bengal was excecuted (Subramani Ramesh and Narayanasamy Mathivanan, 2009).

Asparaginase producing marine actinomycetes from mangrove have been isolated and characterized (P. Dhevagi and E. Poorani, 2006). Various bacterial strains have been isolated and studied for their enzymatic activity (Z. Mudryak and B. Podroraska, 2006). Useful extracellular enzyme activity of bacteria isolated from Bhitarkanika mangrove ecosystem of Orissa coast have been reported (Gupta, N , Das, S. and Basak, U.C , 2007). Antimicrobial activity of actinomycetes of Kerala mangrove have been reported. Two of the strains of actinomycetes showed broad spectrum activity (Mohan Remya and Ramasamy Vijayakumar, 2008). Various heavy metals have been detected in mangrove microbial communities due to domestic and industrial effluents. The accumulated heavy metals may enter into the food chain and can cause deleterious effects to human beings, animals and to plants as well.

Phytoremediation and phytoextraction are the possible ways to deal with heavy metal accumulation (Alexander A, 2000). Characterization and evaluation of stress and heavy metal tolerance of some predominant Gram negative halotolerant bacteria have been reported from mangrove soils of Bhitarkanika, Orissa, India. The isolates tolerated 600 - 1000 ppm K2CrO4 but only up to 10 - 20 ppm CdNO3( Mishra R. R. et.al, 2009). Navi Mumbai is the world’s largest planned city, just 35 kms away from main Mumbai city. Navi Mumbai is spread in the horizons of 344 square km, having coastal stretch of 34.2 kms along Thane and Panvel cities. This CBD Belapur (19° 1′25.33″N, 73° 2′27.65″E) coastal stretch remains largely unexplored for its microbial diversity. Hence, in the current study the microbial diversity study of CBD Belapur mangroves was undertaken.

2. Material and methods

2.1 Site description and sample collection

Soil samples were collected in sterile Petri plates from five different mangrove locations of C.B.D. Belapur and Nerul region, India. The five different stations were Diwale village seashore, Palm beach-Uran junction, Nerul signal on Palm beach road, Belapur creek and near Nerul lake. Soil was collected from 4cm deep from each of these stations using a sterile spatula. The soil sample was collected in St. plastic bags from 1-2 cm below the pneumatophores, it was then brought to the laboratories and stored at 4^oC till further processing.

2.2 Isolation of microorganisms

Suspensions of all the five soil samples were prepared using physiological saline. Spread plate technique on Nutrient Agar plates was used for the isolation of microorganisms (Dubey and Maheshwari, 2005)

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2.3 Identification

Serial tenfold dilutions of mangrove soil samples were spread on St. Nutrient agar and incubated at R.T. for 24 hrs. Total 42 isolates were obtained out of which only 14 which were morphologically dominant were selected for further study. Of the 14 isolates, all 06 isolates were gram positive. Their gram nature was confirmed by gram staining. All the isolates were assigned a specific number i.e. MS (Mangrove soil) 1,2,3,4…. All the isolates were identified by carrying out IMViC tests. With the help of Bergey’s manual all the isolates were identified.

(Table no.1and 2) (Dubey and Maheshwari, 2005)

Isolates were checked for presence of enzymes like protease, amylase, L- asparaginase and Glutaminase qualitatively by standard methods.(Gulati et.al. 1997 and S. Anil Kumar, 2000) 2.4 Optimization of cultural conditions

Optimum temperature, pH, NaCl concentration required for optimum growth was checked for all isolates by standard method. Various temperatures, pH NaCl concentrations were used during optimization. (Dubey and Maheshwari, 2005)

2.5 Study of Antibiotic efficacy of the isolate

It was carried out with Combi I and IV discs (Hi media) on St. Muller and Hinton agar by swab spread technique. (Dubey and Maheshwari, 2005)

2.6 Optimization of heavy metal tolerance

An effort was made to study the effects of varying concentrations of different heavy metals on the growth of the soil isolates. The heavy metals used for the purpose were aluminium(AlCl3), chromium(KCrO4), manganese(MnSO4), zinc(ZnSO4) and mercury(HgCl2). It was carried out by standard method. (Dubey and Maheshwari, 2005)

Table 1 (a) and (b): Biochemical characteristics

Biochemical test / Isolate

MS 1 MS 2 MS 3 MS 4 MS

5

MS 6 Starch

hydrolysis

+ + + + + +

Citrate test + - + - - +

Indol test + + + + + +

M.R. + - + - - +

V.P. + - + - - +

Nitrate reduction

+ + + + + +

Catalase test + + + + + +

Glucose + + + + + +

Lactose - - - -

Arabinose + + + + + +

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. Biochemical

test / Isolate MS 7

MS 8

MS 9

MS 10

MS 11

MS 12

MS 13

MS 14 Starch

hydrolysis

+ + + + + + + +

Citrate test - + - + + + + +

Indol test + + + + + + + +

M.R. - + - + + + + +

V.P. - + - + + + + +

Nitrate reduction

+ + + + + + + +

Catalase test + + + + + + + +

Glucose + + + + + + + +

Lactose - - - -

Arabinose + + + + + + + +

Spores + + + + + + + +

Gram staining

+ baccilli

Motility - - - -

Table 2: Optimization of cultural characteristics for isolates Isolate No/

Optimization

MS 2,4,5,8,14 MS 1,6,13 MS 3,7,10,11 MS 9,12

Temperature 28 – 30⁰C 28 – 30⁰C , also Grows at 55⁰C

28 – 30⁰C 28 – 30⁰C,

pH 7-8 7-8 7-8 7-8

% NaCl Con.

6.5% 8% Did not

tolerate 6.5%

Nacl

8%

Probable Sp. B. macerans, B.

brevis

B. circulans, B.

circulans

B.

licheniformis

B. coagulans B. subtilis

Spores + + + + + +

Gram staining

+ bacilli

+ baccilli

Motility - - - -

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Table 3: Antibiotic Susceptibility test

Anti-biotic MS 1

MS 2

MS 3

MS 4

MS 5

MS 6

MS 7

MS 8

MS 9

MS 10

MS 11

MS 12

MS 13

MS 14

Cephalo- ridin – 30mcg

S S S S S S S S S S S S S S

Cefa- droxil-

2mcg

Cefpodo- xime – 25

mcg

Erythro- mycin- 15

mcg

Genta- mycin -10

mcg

Ofloxacin - 1mcg

Penicillin- G -10 U

R S R R S R R S S R S S R S

Vanco- mycin - 30mcg

Table 4: Antibiotic Susceptibility test

Sr No.

Anti-biotic M S 1

MS 2

MS 3

MS 4

MS 5

MS 6

MS 7

MS 8

MS 9

MS 10

MS 11

MS 12

MS 13

MS 14

1. Ampi- cillin -10 mcg

2. Cephalo- thin-30 mcg

3. Chloram- phenicol - 30 mcg

4. Clinda- mycin -2 mcg

5. Oxacillin- 1mcg

Antibiotic efficacy: R= Resistant; S= sensitive , mcg - micrograms

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Table 5: Heavy metal tolerance Isolates/Metals ZnSo

4 K

2CrO

4 MnSo

4 Hgcl

2 AlCl

2

MS 1 300 1000 300 10 300

MS 2 300 1000 300 10 500

MS 3 300 1000 700 10 300

MS 4 300 1000 500 10 300

MS 5 200 1000 500 10 300

MS 6 200 1000 500 10 300

MS 7 200 1000 700 10 300

MS 8 200 1000 500 10 300

MS 9 500 1000 500 10 300

MS 10 100 1000 500 10 300

MS 11 500 1000 500 10 300

MS 12 300 1000 300 10 300

MS 13 300 1000 300 10 300

MS 14 300 500 300 10 300

Table 6: Qualitative enzyme testing

Enzyme Test Observation Isolates

Protease NA + milk Zone of ppt around

colonies

MS 2,5,11,12,14 Amylase 1% stach agar – 06

% KI

Clear zone around colonies

Positive test by all isolates

L-Asparginase St. Zobell marine bagar + 0.5%

L-aspargine + 8% NaCl + 2 drops of phenol red indicator

Plate turns pink with pink zone around colonies

L-Glutaminase St. Zobell marine bagar + 0.5%

L-glutamine + 8% NaCl + 2 drops of phenol red indicator

Plate turns pink with pink zone around colonies

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3. Result and conclusion

Here an attempt was made for Isolation and identification of microorganisms from mangroves soil of CBD Belapur region. All isolates exhibited good growth on nutrient agar.

Out of total 42 isolates obtained only 14 isolates were selected for further study, as these isolates were morphologically distinct and consistent in growth characteristics on nutrient agar. In general, all isolates formed colonies which were moist, small, with regular margin and were translucent. All isolates were found to be gram positive bacilli. Based on morphological and biochemical characteristics all isolates were identified with the help of IMViC tests and Bergey’s manual. All isolates were Lactose negative. Isolates MS 2,4,5,8,14 were found to be gram positive bacilli and were citrate , MR , VP negative and positive for all other tests like Glucose , Arabinose , sorbitol (Table No.1). Hence all these isolates could be B. macerans, B. brevis B. circulans, B. circulans as they grew at 30⁰C and at 6.5% Nacl concentration(Table No.2). Isolates MS 1,6,13 were found to be gram positive bacilli and were positive for all biochemical tests carried out (Table No.1). Theses isolates grew at 55⁰C and at 8% Nacl concentration (Table No.2).

Hence all these isolates could beB. licheniformis. MS 3,7,10,11 and 9 ,12 were found to be gram positive bacilli and were positive for all biochemical tests carried out (Table No.1).

These isolates grew at 30⁰C and Isolate No. MS 3,7,10,11 did not tolerated 6.5% NaCl concentration and Isolate No. MS 9,12 tolerated 6.5% NaCl concentration (Table No.2).

Hence these Isolates could beB. coagulans and B. subtilisrespectively. Extracellular enzyme analysis was as per Table no. 6. All the gram positive isolates were found to be producing amylase and protease enzyme. In the amylase qualitative assay isolates exhibited clear, shining zones around colonies after flooding the plate with Iodine solution. Similarly these isolates showed zone of precipitation on nutrient agar plates confirming positive protease test.

All isolates produced pink coloration around colonies on Zobell marine agar supplemented with aspargine and glutamine. All the isolates were found to be producing L-asparginase and glutaminases. (Table no. 6).

Antibiotic susceptibility assay reveled that all isolates were sensitive to the antibiotics used.

This might be due to that the bacteria had no intrinsic resistance to several antibiotics, as they were not exposed to those antibiotics and hence no resistance developed. Some isolates showed resistance to Penicillin G. (Table no.3 and 4). All the isolates were tolerant towards five different heavy metals used. MIC values were varying as per the isolates and heavy metal (Table no.5). MIC values for Mercuric Chloride were very less i. e. 10 ppm (micro gram) for all isolates. Some isolates exhibited relatively high MIC values which may be due to their stress tolerance characters.

4. Conclusion

Mangrove regions are unique swampy regions with water region being alkaline in nature and sediment or soil region having a neutral to slightly acidic pH. Since mangrove environment is prevalent to stress conditions such as salt stress, microorganisms growing under such stress conditions could have a potential for bioremediations programmes. The soil isolates were halo-tolerant and could tolerate relatively high concentrations of heavy metals. Mangroves are saline coastal ecosystem rich in Carbon and other nutrients. They harbor large numbers of population of unique bacteria. The present study reveals the mixed population of bacteria of CBD Belapur mangroves. Further studies on these organisms and more evaluation of their

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