International Journal of Advance Engineering and Research Development

Scientific Journal of Impact Factor (SJIF): 4.72 International Journal of Advance Engineering and Research Development Volume 4, Issue 11, November -2017 e-issn (O): 2348-4470 p-issn (P): 2348-6406 Bacterial diversity of mangrove rhizospheresoil - Gulf of Khambhat, Gujarat,India Jitendra Kumar Singh 1, Bhawana Pathak 2 1, 2 School of Environment and Sustainable Development, Central University of Gujarat, Sector 30, Gandhinagar 382030, Gujarat, India Abstract - In the present investigation of the microbial population as well as the diversity of bacteria of the unique mangrove forest ecosystem of Gulf of Khambhat (GoK) region Gujarat, India were analyzed. The maximum water and soil bacterial colonies were presented in monsoon seasons were compare with pre-monsoon and post-monsoon seasons.total ten different bacterial strains were isolated from mangrove rhizosphere soil of four different mangrove forest location of the gulf of Khambhat during a period of 2014-2015. Bacterial isolation was done by the soil dilution method incubated at 37 C for 24 hours. The most dominant mangrove species of study sites are Avicennia marina. Determination of bacterial diversity in the mangrove rhizosphere soil by culture method showed the predominant bacterial genera namely Salinicoccus kekensis, Kocuria flava, Arthrobacter sp., Kocuria sp., Arthrobacter sp., Bacillus endophyticus, Agrococcus lahaulensis, Georgenia satyanarayanai, Planococcus rifietoensis and Virgibacillus dokdonensis sp. present in the Gulf of Khambhat mangrove ecosystem. Key words - Mangroves forest;mangrove soil;rhizosphere;bacterial diversity I. INTRODUCTION Mangroves are an assemblage of salt tolerant woody plants (i.e., halophytes) which grow in intertidal coastal zone of brackish to saline shorelines and estuaries in the tropical and sub tropical regions [1], [2]. Mangrove ecosystems support a greatdiversity of life forms including flora and fauna. They are most productive and play a dynamic role as a most important primary producer within coastal and estuarine environment.in mangrove rhizosphere soil, the microorganisms play a major role in the productivity, environment recovery and conservation, where they participate in the biogeochemical cycles and provide nutritional sources to the plant and animals [3], [4]. The free living bacteria were reported to have significant role in the carbon, sulfur, nitrogen and phosphorous cycles in mangrove forest [5] and participants in formation of detritus in the mangrove ecosystems [6].The bacteria from mangrove soil possess unique biotechnological potentials like nitrogen fixation, phosphate solubilization and sulfur oxidation as well as cellulose degradation [7]. The bacterial diversity decreased with increasing depth of mangrove sediments and its community structure is significantly correlated with sediment characteristics [8]. The mangrove rhizosphere soil are the homeland of microbes because the more availability of nutrient sources [9]. The microbial diversity (bacteria, fungi, actinomycetes, viruses and many more) from mangrove ecosystems has been studied worldwide. The potential microbes are also existing in plenty in the rhizosphere of mangroves. These microbes have potential for bioremediation applications, plant growth promotion and enzyme production under salt stress conditions [10]. Mangrove bacterial communities mainly consist of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospira, and Planctomycetes and others [11]. Mangrove habitats are ecologically and economically important ecosystems that are under severe pressure. Conservation strategies for mangroves should consider the ecosystem as a biological entity, which includes all the environmental parameters and ecological processes that maintain productive mangroves[9]. II. MATERIAL AND METHODS A. Collection of soil samples The soil samples were collected from four different mangrove forest location of gulf of Khambhat(21 00 N - 22 18 N and 72 15 E -72 45 E) in the West coast of India during a period of 2014-2015. @IJAERD-2017, All rights Reserved 857

B. Enumeration of Microbial Population The number of bacterial colony-forming units (CFU) was counted by serial dilution and pour plate method. Adding 1 ml of water sample in 9 ml or 1 gram of soil sample was mixed with 10 ml of 0.8% NaCl (saline) solution of distilled water was serially diluted in sterilized distilled water to get a concentration range from 10-1 to 10-9. After serial dilutions, a volume of 0.1 ml (100 µl) from each dilution was transferred aseptically to petri plate containing nutrient agar media (ZMA) and spread uniformly. Then plates were incubated in the incubator having temperature range of 37±1 C. The bacterial colonies were counted after 24h of incubation. Plates with 20-300 CFU s were used to calculate number of CFUs/mL of the original sample.number of CFUs/ml can be calculated using the following formula- No. of CFUs/mL = Number of CFU Volume plated (in ml) Total dilution used C. Isolation and purification of bacteria The respective coastal mangrove soil sample were ten-fold serially diluted in saline solution (0.9% NaCl) and spread plated on zobell marine nutrient agar plates with pour plate method. The plates were incubated at 37 0 C temperature in incubator for 24 hours to 3 days. After incubation period the plates were observed for the growth of different types of bacterial colonies. The colony's showing discrete natures were picked up and streaked on selective agar plates for purification. All these different types of bacterial colony were purified mechanically by streaking on zobell marine agar media containing 5.0g Peptic digest of animal tissue; 1g Yeast extract; 1g Ferric citrate; 19.45g Sodium chloride; 8.8g Magnesium chloride; 3.24g Sodium sulphate; 1.8g Calcium chloride; 0.55g Potassium chloride; 0.16g Sodium bicarbonate; 0.08g Potassium bromide; 0.034g Strontium chloride; 0.022g Boric acid; 0.004g Sodium silicate; 0.0024g Sodium fluorate; 0.0016g Ammonium nitrate; 0.008g Disodium phosphate and 15g Agar per liter and further sub cultured to obtain pure culture. The all plate of pure bacterial isolates were maintained at 4ºC in refrigerator for further used. D. Identification All pure bacterial strainsisolated from mangrove rhizosphere soil was identified as per the Bergey s manual of determinative bacteriology, based on morphological, physiological, biochemical tests and finally their molecular identification was done through 16S rdna gene sequencing analysis using most established and popular Sanger dideoxy sequencing methods [12]. III. RESULTS AND DISCUSSION The main aim of this study was to identify the bacterial diversity of mangrove rhizosphere soil and assess the sessional variation of the bacterial population of coastal water and sediment at four different location of Khambhat mangrove ecosystem Gujarat, India. The present study rhizosphere soil of mangrove habitat supports for the presence of higher population of gram positive compare with gram negative bacteria. The morphologically, isolated 10 bacterial isolates (MRB1 to MRB10) were observed round, oval, circular, translucent, irregular and the colour was also indicated as Pink red, yellow, light Yellow, Cream to yellow, whitish, pale yellow, Brown, Milky white (Table-1). The bacterial colonies was negative to most of the physiological biochemical tests used in this study. The mangrove forest habitat supports the occurrence of a total ten rhizosphere soil bacterial strain was identified as Salinicoccus kekensis, Kocuria flava, Arthrobacter sp., Kocuria sp., Arthrobacter sp., Bacillus endophyticus, Agrococcus lahaulensis, Georgenia satyanarayanai, Planococcus rifietoensis and Virgibacillus dokdonensis sp. based on morphological, physiological, biochemical and molecular characteristics. Out of ten bacterial species, 9 species occurred at location of Purna estuaries, whereas 5, 4 and 5 species occurred at location of Ghogha coast, Dahej coast and Dumas beach respectively (Table 2). The range of microorganisms are known to occur in the mangrove forest ecosystem, although these differ as to biogeography and their location of habitats with many factors such as ph, temperature, salinity, availability of nutrients, @IJAERD-2017, All rights Reserved 858

depth, verities of substrate, level of contamination and anthropogenic activities effecting microbial communities composition and species occurrences [4], [13]. Isolate No. Table 1. Morphological characterisation and biochemical tests of isolated bacteria Gram staining Colour Motility Indol MR VP Catalase Amylase Urease Oxidase Non MRB1 +vecocci Pink red - - - - - + - motile Pale Non MRB2 +vecoccoid yellow motile Cream to MRB3 +ve Rod Motile + - - + - - + yellowish +ve Rod Light Non MRB4 - - - + + - - Coccus yellow motile Bright Non MRB5 +vecoccoid orange motile +ve MRB6 Brown Motile - - + + + + - Rod Light Non MRB7 +ve Rod yellow motile +ve Rod Greenyellow motile Non MRB8 - - + + + + - Coccus MRB9 +vecocci Orange Motile - - - + - - + Gramvariable Motile - - - + + - + Milky MRB10 white Rod Table 2. Presence of predominant bacterial species in four location of gulf of Khambhat mangrove ecosystem and their taxonomy Isolate No. Identified bacterial Species Ghogha coast Dahej coast Dumas beach Purna estuaries MRB1 Salinicoccuskekensis + - MRB2 Kocuria flava + + MRB3 Arthrobacter sp. + + + MRB4 Arthrobacter sp. + + + MRB5 Kocuria sp. + + + + MRB6 Bacillus endophyticus + + + MRB7 Agrococcus lahaulensis + + + MRB8 Georgenia satyanarayanai + + MRB9 Planococcus rifietoensis + MRB10 Virgibacillus dokdonensis sp. + Total bacterial counts in mangrove coastal water and mangrove soils ranged from 2.2 x 10 4 to 7.1 x 10 5 CFU/ml and 6.6 x 10 6 to 1.93 x 10 8 CFU/g respectively in the gulf of Khambhat mangrove ecosystem (Figure 1 a & b). @IJAERD-2017, All rights Reserved 859

Figure 1. Seasonal variation of bacterial population in (a) coastal water and (b) coastal soil at four different mangrove forest locations during three seasons (2014 to 2015) Among the four location, water and soil sample from the Purna mangroves recorded the highest bacterial counts 7.1 x 10 5 CFU/ml and 1.93 x 10 8 CFU/g respectively. Ghogha coast recorded the lowest bacterial count of 2.2 x 10 4 CFU/ml in water sample during monsoon season and 6.6 x 10 6 CFU/g in soil sample of Dahej coast during pre-monsoon season. The total bacterial count (TBC) in sediments and water samples found highest during monsoon as compare to pre-monsoon and post-monsoon season was also observed by Borade et.al. at Tapi estuary along the Gujarat coast [14].The high salinity in coastal water as well as mangrove soil may have restricted the growth of microbial populations. IV. CONCLUSIONS In conclusion, Mangrove rhizosphere soils are the home to wide range of marine coastal bacterial genera. Total ten bacterial strains isolated from mangrove rhizosphere soil of gulf of Khambhat region. Among the bacterial genera, Arthrobacter, Kocuria and Bacillus were most dominant in the mangrove rhizosphere soils.information of different bacterial genera from the mangrove soil habitat of gulf of Khambhat will not only help to assess the microbial diversity, but also will provide a better description of this complex system, explore the microorganisms for various biotechnological applications and the development of conservation strategies. Extensive exploration, isolation, screening and identification are suggested in search of new leads for ecological remediation applications.further studies on the microbial ecology in mangroves are required for a better understanding of the functional role of the bacteria in these environments. REFERENCES [1] Aheto, D. W., OwusuAduomih, A., &Obodai, E. A. (2011). Structural parameters and above ground biomass of mangrove tree species. Annals of Biological Research, 2 (3), 504 514. [2] Heumann, B. W. (2011). An Object Based Classification of Mangroves Using a Hybrid Decision Tree Support Vector Machine Approach. Remote Sensing, 3, 2440 2460. [3] Holguin, G., Vazquez, P., & Bashan, Y. (2001). The role of sediment microorganisms in the productivity, conservation, and rehabilitation of mangrove ecosystems: an overview. Biology and fertility of soils, 33(4), 265-278. [4] Andreote, F. D., Jiménez, D. J., Chaves, D., Dias, A. C. F., Luvizotto, D. M., Dini-Andreote, F.,...& de Melo, I. S. (2012). The microbiome of Brazilian mangrove sediments as revealed by metagenomics. PloS one, 7(6), e38600. [5] Rojas, A., Holguin, G., Glick, B. R., & Bashan, Y. (2001). Synergism between Phyllobacterium sp. (N2-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere. FEMS Microbiology Ecology, 35(2), 181-187. @IJAERD-2017, All rights Reserved 860

[6] Maria, G. L., & Sridhar, K. R. (2002). Richness and diversity of filamentous fungi on woody litter of mangroves along the west coast of India. Current Science, 1573-1580. [7] Thatoi, H. N., Behera, B. C., Dangar, T. K., & Mishra, R. R. (2012). Microbial biodiversity in mangrove soil of Bhitarakanika, Odisha, India. Int. J. Environ. Biol, 2(2), 50-58. [8] Mendes, L. W., & Tsai, S. M. (2014). Variations of bacterial community structure and composition in mangrove sediment at different depths in Southeastern Brazil. Diversity, 6(4), 827-843. [9] Saseeswari, A., Kanimozhi, G., &Panneerselvam, A. (2016). Bacterial diversity of mangrove soil in Karankadu from East Coast of Tamil Nadu, India. Int. J. Curr. Microbiol. App Sci, 5(4), 750-756. [10] Arora, S., Vanza, M. J., Mehta, R., Bhuva, C., & Patel, P. N. (2014). Halophilic microbes for bio-remediation of salt affected soils. African Journal of Microbiology Research, 8(33), 3070-3078. [11] Ullah, R., Yasir, M., Khan, I., Bibi, F., Sohrab, S. S., Al-Ansari, A.,...&Azhar, E. I. (2017). Comparative bacterial community analysis in relatively pristine and anthropogenically influenced mangrove ecosystems on the Red Sea. Canadian Journal of Microbiology, 63(8), 649-660. [12] Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the national academy of sciences, 74(12), 5463-5467. [13] Mendes, L. W., Taketani, R. G., Navarrete, A. A., & Tsai, S. M. (2012). Shifts in phylogenetic diversity of archaeal communities in mangrove sediments at different sites and depths in southeastern Brazil. Research in microbiology, 163(5), 366-377. [14] Borade, S., Dhawde, R., Maloo, A., Gajbhiye, S. N., &Dastager, S. G. (2014). Occurrence and seasonal variation in distribution of fecal indicator bacteria in Tapi estuary along the West coast of India. @IJAERD-2017, All rights Reserved 861