Isolation and Characterization of Novel Metal Accumulating Extracellular Protease Secreting Bacteria from Marine Coastal Region of Digha in West Bengal, India

Problem Statement: The objective of the present piece of work was to explore the existing biodiversity of protease secreting microbes in a marine ecosystem, the site selected being the coastal region of West Bengal (Digha), India. Approach: The culture dependent method was used for isolation and characterization of novel microbes from the point of biotechnological application. The isolate obtained was characterized on basis of its morphological, biochemical, physiological properties and finally its molecular identity was revealed through 16S rDNA analysis. The complete characterization would ease the application of this isolate for various purposes which would require the understanding of the detailed properties. Results: The isolation was done on milk medium while further characterization was done in Luria Bertani broth as well as Carbon Minimal Salt medium. The isolate was found to be DNase & protease positive, spore forming, Gram-positive bacilli and exhibited growth in wide range of substrates, temperature [20C-40C] & pH [5.5-10]. The Scanning Electron Micrograph revealed a unique pililike bridge between every 3-4 bacterial cells, which indicates the characteristic stacking pattern of growth found in certain groups of bacteria. Moreover it was found to grow in presence of a wide range of metals namely iron, lead, mercury, zinc, aluminum, copper, nickel, cadmium and silver. Energy dispersive X Ray Fluorescence analysis conducted for two of the metals, chromium and copper indicated the intracellular accumulation of these metals. Conclusion: This property provides potential for its application in remediation as well as recovery of heavy metals. The isolate with this property can be a potential candidate to carry out bioaugmentation.


INTRODUCTION
Microbes account for more than 90% of ocean biomass. The marine microbial group including bacteria, archaea, lower eukaryotes & their associated viruses are found to be diverse as their origin vary-from the ice-covered polar regions of the Arctic & Antarctic to the boiling hypothermal vents in the depth of the sea. Still much of the microbial life remains unknown, because of the fact that culture dependent identification could not reveal the entire existing microbial diversity. Marine microbes are reported to play important roles in the earth system from being the major primary producers in the ocean and acting as sources of natural products like enzymes and antibiotics.
One of the important contribution of the microbes in aquatic system is in the geochemical cycle of earth and in some way they explain the evolution of living organisms from inorganic matter [1] . The microbes inhabiting the aquatic environment do have importance in the population dynamics and thereby contributing to the functional aspect operating in the ecosystem [2] . The diverse group of microbes have applications in various fields like production of enzymes, drugs and various other commercial products [3][4][5][6] . Taking into account these importance, the primary goal would be to explore the marine environment to look for novel microbes which can have various applications.
Today microbes do have importance in the management and sustenance of environment [7] . Many reports present the role of microbes in detoxification of contaminated sites, may be by their interaction with heavy metals or degradation of chemicals like halogenated, aromatic compounds [8][9][10][11][12][13][14][15] . Not only marine environment but microbes from different ecosystems were reported to play significant role in maintaining the ecosystem as well as played varied roles to decontaminate the environment [16] .
The present study reports the characterization of one of the isolate from a marine site of West Bengal, India. The basic objective was to screen out potential microbes, to characterize them and to exploit their properties in various applications.

MATERIALS AND METHODS
Isolation of pure strain: Milk media (10% milk, 0.3% yeast extract and 1.5 % agar) was used as a selective medium for isolation of protease secreting microbes, the source being the water collected from coast of West Bengal (Digha). From a mixed population of microbes isolated by spread plate technique, pure isolate was obtained by repeated streak-plate method on Luria Bertani agar medium (LB medium) (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar).

Characterization of the bacteria Morphological characterization:
Morphological characterization of the isolate was based on the light and electron microscopy. Other than simple and gram staining, the detailed characterization was conducted through various other staining procedures for detection of endospore, capsule, flagella as per standard procedure. The bright field microscopy was conducted at 100X magnification on a Zeiss Axiostar Plus microscope. Scanning Electron Microscopy (SEM) revealed the ultrastructure of the cell. The bacterial cell fixation and preparation technique for SEM was followed as reported earlier [17] .

Biochemical characterization:
Biochemical characterization included the test for presence of enzymes namely protease, DNase, lipase, lecithinase, oxidase & catalase. The specific ready made medium (Himedia laboratories) were used for the enzymatic assay. The detailed procedure of enzymatic assay and result assessment was as reported earlier [17] .

Physiological characterization:
Physiological characterization includes determination of optimum pH, temperature for growth, efficiency in substrate utilization, growth profile under enriched condition as well as sensitivity for different antibiotics.
• pH profile: pH is a limiting factor, which governs bacterial growth. To determine the pH optima, LB medium meant for growth of the isolate was adjusted to different pH ranging from 2-13 and was seeded with 1% inoculum. Post overnight growth • Light sensitivity: To determine the lightsensitivity, two sets of LB media were inoculated with 1% culture, one of the set was incubated in presence of luminescent light and remaining set was incubated in the dark. The temperature and incubation conditions for both were 37°C at constant shaking of 150 rpm for overnight. Thereafter growth was checked in terms of OD at 660 nm in Beckman UV-Vis spectrophotometer.
• Growth pattern: To determine the growth pattern, 1% of inoculum from overnight grown parent culture was seeded into sterile 100 mL LB broth. OD at 660 nm was checked in UV-visible spectrophotometer at a gap of 30 min until the culture reaches stationary phase.
• Antibiotic sensitivity: Antibiotic sensitivity was checked for 18 different antibiotics. The different antibiotics used and the detailed procedure for sensitivity assay was followed as reported by others [17] Metal sensitivity: The tolerance was checked for eleven different heavy metals The metals used in the study and detailed procedure to determine the tolerance profile, in terms of MIC was as per the protocol reported elsewhere [17] . The quantitation of metal accumulation was determined by Energy Dispersive X Ray Fluorescence analysis. The protocol for sample preparation and details of the analytical procedure was reported earlier [17,18] .

Pathogenicity detection by real-time PCR:
The detection for the presence of pathogenic gene was done using TaqMan kit (Applied biosystems.) for Staphylococcus aureus and Pseudomonas aeruginosa.

Molecular characterization:
The molecular characterization was done on the basis of 16S rDNA sequence analysis. The steps for the characterization included the genomic DNA isolation followed by PCR amplification of universal 16S rDNA gene. The amplified fragment was then cloned into pGEMT easy vector and transformed into E coli DH5α host. The positive transformants were screened out and sent for sequencing. The details about genomic DNA isolation, the primer sequence, PCR conditions, transformation procedure was as reported by Ray Chaudhuri and Thakur as well as Ray Chaudhuri et al. [19,20] .
The sequence obtained was subjected to nucleotide-nucleotide BLAST, a tool for checking percentage of similarity with the existing database. The sequence being novel was submitted to GenBank.
All the experiments were repeated at least thrice and the data represented are the mean.

Characterization of the isolate:
The preliminary characterization was based on morphological and biochemical properties. The isolate was bacilli as demonstrated by light microscopy as well as electron microscopy ( Fig. 1, 3). As depicted from staining results presented in Table 1, the isolate was found to be Gram-positive in nature and possessed endospores (Fig.  2). Other structural features like absence of capsule and flagella were clearly put forth by the staining procedures.
The biochemical nature of the isolate indicates presence of enzymes like DNase and protease.

Physiological characterization:
The physiological properties of the isolate were concluded in terms of pH, temperature and substrate.
A wide variety of substrates were found to support the growth of the isolate which indicates the metabolic efficiency of the isolate (Fig. 4). The isolate was found to grow within a pH range of 5-10 with optimum growth at pH 7 (Fig. 5). The temperature range found suitable for growth was between 20-45°C, optimum being at 35°C (Fig. 6). These physiological conditions are reported as the optimum for most of the functions in a living organism.
Growth pattern: The isolate follows a typical sigmoidal pattern of growth curve. An initial lag pase of two hours succeded by five hours of log phase followed by a stationary phase (Fig. 7). Metal sensitivity: The isolate was found to demonstrate tolerance for metals shown in Fig. 8. The minimum tolerance was for metals like mercury, cadmium and silver. The maximum tolerance was demonstrated for iron followed by lead. Moreover the EDXRF analysis done for two of the metals among these namely chromium and copper indicates intracellular accumulation of these metals ( Table 2).  Figure 9 depicts the amplification plots obtained using the probes for specific genes for the two strains (Staphylococcus aureus and Pseudomonas aeruginosa) mentioned above. As observed from Fig.  9, the internal positive control shows high copy number with respect Table 2: Table representing

DISCUSSION
The presence of enzyme like DNase indicates the possible defense mechanism in bacteria which participate in host prey interaction. This in the long run enables the existence and adaptability of the organism. The usability of substrates like glucose, lactose, sucrose, fumarate malonate and glycerol indicate towards the aerobic metabolic pathway because these substrates could be easily assimilated and directly enters the Tricarboxylic Acid cycle (TCA). Ethanol not giving substantial growth means that glyoxylate pathway was not supported.
The pH and temperature profile of an isolate would indicate the adaptability of the isolate to different habitats. These factors do have an effect on survivability since they have roles in enzymatic function as well as overall metabolic efficiency. The isolate being able to withstand wide range of pH and temperature was capable of surviving in adverse condition.
An idea of the entire growth cycle would facilitate the complete characterzation of the isolate and further enable designing for practical applications.
The metal tolerance data would definitely facilitate their application in remediation of heavy metals. These metals are commonly found in the mine effluents, small scale battery industries, tanneries etc. These tailings could be removed by the application of this isolate. The EDXRF data indicates the potential for the removal of these toxic metals from the site and moreover certain processes can be followed for the recovery of economically important metals from the microbes.
The analysis of partial sequence with 99% similarity identifies the isolate as Bacillus sp.

CONCLUSION
This preliminary characterization of the isolate regarding its morphological, biochemical and physiological characters as well as the molecular identity would definitely lead to the application of the isolate for various purposes. The property of the isolate for heavy metal uptake would explain the role of this group of microbes in the geochemical mechanism under the water level and it would also facilitate the application of marine microbes for leaching and removal of the toxic metal dumped into the aquatic ecosystem. The reason for non amplification of pathogenic gene in both tests could also be due to its molecular identity which was different from those used for the test. Another important fact regarding this microbe was that in spite of its marine origin it was able to grow and propagate under non halophilic condition. Moreover the protease secreting property of these isolates would open up the exploitation of the marine microbes for production of enzymes. Thus the future prospect lies in the application of this isolate for purposes like heavy metal remediation and commercial product development involving enzyme.