Antagonistic Bacillus cereus TC-1 Isolated from Solar Salt Work in Southern India

Microbes from extreme environments have attracted considerable attention in recent years. This is primarily due to the secret that they hold about the molecular evolution of life and stability of the macromolecules1. They are often under extreme conditions of e.g., pressure, temperature, salinity, and depletion of micronutrients, with survival and proliferation often depending on the ability to produce biologically active compounds. The diversity of marine environments has exerted a driving force on bacteria selection leading to new adaptive strategies and the synthesis of new metabolites2. Microbial secondary metabolites have been recognized as a major source of compounds endowed with ingenious structures and potent biological activities3.


Introduction
Microbes from extreme environments have attracted considerable attention in recent years. This is primarily due to the secret that they hold about the molecular evolution of life and stability of the macromolecules 1 . They are often under extreme conditions of e.g., pressure, temperature, salinity, and depletion of micronutrients, with survival and proliferation often depending on the ability to produce biologically active compounds. The diversity of marine environments has exerted a driving force on bacteria selection leading to new adaptive strategies and the synthesis of new metabolites 2 . Microbial secondary metabolites have been recognized as a major source of compounds endowed with ingenious structures and potent biological activities 3 .
The genus Bacillus constitutes a diverse group of rod-shaped, Gram-positive bacteria, characterized by their ability to produce a robust spore. Most Bacillus species are not harmful to mammalians, including humans and are commercially important as producers of a high and diverse amount of secondary metabolites (antibiotics, bio insecticides, fine chemicals and enzymes) 4,5 . The genus Bacillus has been in use in the biotechnology industry for a very long time with a number of new cultures exhibiting a variety of benefits to humans. Members of the Bacillus genus are often considered microbial factories for the production of a vast array of biologically active molecules potentially inhibitory for phytopathogen growth, such as kanosamine or zwittermycin A from B. cereus 6 .
Microorganisms represent the most common candidates as sources of new enzymes because of their broad biochemical diversity, feasibility of mass culture and ease of genetic manipulation. Microbial alkaline proteases dominate the worldwide enzyme market, with a two third share of the detergent industry 7,8 . Nowadays increasing emphasis is being laid on extremophiles for the presence of such enzymes, mainly due to the mechanisms and strategies that help them to function under stressful growth conditions 9 .
Antagonistic activities between micro-organisms have been widely reported 10 and in many instances the inhibition is due to the production of bacteriocins and other Extra Cellular Products (ECP). Bacteria showing antagonistic activity have potential application as bio control agents. Sugita et al 11 isolated a strain of Bacillus sp. that was antagonistic to 63% of the bacterial isolates from fish intestine. Some bacteria have been observed to be antagonistic to even viruses 12 and such bacteria have potential use in biocontrol of viral diseases 13 . The present study focuses isolation, identification, biochemical characterization and antagonistic studies of the halophilic Bacillus cereus from the crystallizer pond of the solar salt works.

Materials and Methods
Water samples were collected from the condensed ponds (C-I,II & III) from the solar salt works of Thamaraikulam, Kanyakuari, Tamilnadu, India. The physicochemical parameters of the samples were studied following the standard methods by Bhaskaran 14 and given in the Table 1.

Antagonistic Bacillus cereus TC-1 Isolated from Solar Salt Work in Southern India
Saline water samples were decimally diluted and spread in the specific media Bacillus cereus Agar Base (Himedia, Mumbai, India) and incubated the plates at 37 0 C for 24 hours. A total number of 3 tentative isolates picked based on morphological characteristics and checked by microscopy (gram staining and spore staining) tested for motility and for presumptive identification.
The presumptive isolates subjected to biochemical characterization were based on sugar fermentation pattern in basal broth medium as per the standard method. The Bacillus isolates were tested using 15 carbohydrate discs (Himedia, Mumbai) for their ability to ferment different sugars. Isolates were also tested for catalase, indole, gelatin hydrolysis and lactic acid production.
Genomic DNA isolated from B. cereus TC-1 strain and 100 ng was PCR amplified using 16S rRNA universal primers. The PCR product was cloned in to pTZ57R vector and transformed to DH 5 á following the method of Sambrook et al 15 . The transformants were sequenced by using ABI 3700 automated DNA sequencer. Sequences were compared with other 16S rRNAs obtained from Genbank using the BLAST program. Phylogenetic tree was constructed by clustal algorithm using Gene Bee software values were determined.
To optimize the B. cereus TC-1 growth in different NaCl concentration, Nutrient Broth was enriched with 1, 2, 3, 4 and 5 % NaCl and studied the growth curve. The inoculated cultures were incubated at 37 0 C in a shaker at 100 rpm and bacterial growth was monitored at 0, 12, 36, 48, 60, 72, 84 and 86 h after inoculation.
Alkaline protease assay was done in the skim milk agar base and the activity was estimated by Hagihara 16 . The enzyme (0.5 ml) was added to 3.0 ml casein (0.6% w/v in 20 mM borax NaOH buffer, pH 10) and the reaction mixture was incubated at 37 0 C for 10 min before the addition of 3.2 ml of TCA mixture (0.11 M trichloroacetic acid, 0.22 M sodium acetate, 0.33 M acetic acid). The terminated reaction mixture was incubated for 30 min at room temperature. The precipitates were removed by filtration through Whatman no. 1 filter paper and the absorbance of the filtrate was measured at 280 nm. One unit of alkaline protease activity was defined as the amount of enzyme liberating 1 lg of tyrosine per minute under assay conditions. Enzyme units were measured using tyrosine (0-100 mg) as standard.
A lawn culture of the pathogenic Vibrio harveyi, V. parahaemolyticus, V. anguillarum, V. alginolyticus and V. vulnificus were prepared by pouring 2 ml of each young culture over Muller-Hinton Agar media plates, separately. Air dried the plate by keeping it in the incubator at 30 0 C for 15 minutes. Three millimeter diameter wells were punched in the plates using a sterile gel puncher. Thirty microlitres of an 18 hour culture of B. cereus TC-1 (16-18 hours in nutrient broth, supplemented with 5 % sodium chloride) was pipette into the wells and plates were incubated for 24 hrs. Zone of inhibition around the wells was recorded.
One way and two way Analysis of Variance (ANOVA) were carried out using the software PASW statistics data editor and Ky plot respectively. Means were compared at 0.05 % for One Way ANOVA and 0.001 % level.

Results and Discussion
Based on the morphological, physiological, biochemical and genetic identification, the rod shaped bacteria was confirmed as Bacillus cereus and submitted to the NCBI gene bank, accession number is GU939623.1. Cluster algorithm of Gene Bee analysis revealed that, B. cereus TC-1 was highly similar that the B. thuringiensis strain (100%) (GeneBank acc. No: AM292032.1) followed by the Bacillus sp. (92%) (GeneBank acc. No: AY853168.1) and the least similarity, 72% of the B. thuringiensis strain (AY138289.1) (Figs 1 &2). Many types of bacterial species have been isolated from various salt environments such as soils and salterns including the Gram-negative halophilic like species of the genera Vibrio, Alteromonas, Acinetobacter, Marinomonas, Pseudomonas 17 and the genera Marinococcus,  NaCl played an important role for the growth of B. cereus TC-1. The bacterial cells belongs to 2% NaCl growth media reached the stationary phase at 24 hours with less number of bacterial counts. The concentration between 4-6% responsible for more number and reached the stationary phase around 48 hours and significantly increased (P<0.05). Also two way ANOVA revealed that the growth rate was varied significantly among the different groups (F= 4.4333; 32. 0493; P<=0.001). The concentrations such as 12 and 15 had very slow growth with less numbers of cells and this result revealed that the NaCl concentration 4 -6 was the optimum (Fig 3). Recently Patel et al 19 , isolated and identified the haloalkaliphilic, gram positive, aerobic, coccoid Bacillus pseudofirmus-Po2 by 16s rRNA sequencing analysis from the seawater sample in Gujarat, India. They also screened and optimized the production of alkaline protease using NaCl, nitrogen sources and metal ions etc. Figure 4a shows the positive alkaline protease activity produced by B. cereus TC-1 in the skimmed milk agar base. The clear zone indicated the hydrolysis of casein as a result of alkaline protease production. The lowest alkaline protease production was observed in 1 % NaCl of 92.24 U/ml. This was significantly (P<0.05) increased to 130.55 and 170. 85 respectively in the 2 and 3 % NaCl enriched growth media. Further the production was decreased the 4 to 6 % NaCl enriched growth media (Fig  4b). Patel et al 19 , optimized the growth of haloalkaliphilic Bacillus pseudofirmus-Po2 at 10 %( w/v) NaCl and declared that the halotolerant nature of the strain Po2. Similar trends were also evident in Salinicoccus alkaliphilus sp. nov., a moderately halophilic and alkaliphilic coccus isolated form Baer Soda Lake in Mongolia, which could grow over a wide range of NaCl, 0-25% (w/v) with optimum at 10% (w/v) 20 . The present study, alkaline protease production also higher in 4 (130.55U/ ml) and 6 % (170.85U/ml) NaCl enriched growth media and it reflected that due to the halophilic nature of the bacterial strain. The lower and higher concentrations are failed to attain more enzyme production. Po2 produced protease in the range of 5-20% (w/ v) NaCl, optimally (162-170 U/ml) at 10% (w/v) NaCl 19 . Similar results have also been reflected by the haloalkalophilic archaeon, Natronococcus occultus in which protease secretion was optimum at 1-2 M NaCl 21 . However, in the case of the archaebacterium Halobacterium mediterranei, a much higher salt requirement (25%, w/v) for serine protease secretion was reported 22 .
In vitro antagonistic activity of Bacillus cereus TC-1 against the five pathogenic Vibrio sp was tabulated in Table 2   Bacillus is an interesting genus to investigate for antimicrobial activity since Bacillus species production a diverse array of antimicrobial peptides representing several different basic chemical structures 23 , with a distinct diversity in their inhibitory activities against a variety of micro organisms 24 25 . Pseudoalteromonas sp. A1-J11 isolated from coastal seawater of Kagoshima Bay, Kagoshima Prefecture, Japan was found to produce anti-Vibrio substances extracellularly 26 . Our previous study, the Bacillus cereus TC-1 isolated from coconut retting water effectively suppressed the pathogenic Vibrio harveyi and Aeromonas hyrophila by in vitro and in vivo level. They are significantly decreased the bacterial loads in the culture tanks 27,28 . Bacillus strains are one of the most recognized beneficial bacteria used against bacterial or viral disease in shrimp aquaculture; they release antibacterial substances 29 . There is less evidence that Bacillus strains exert harmful effects on shrimp or the environment 30 . The B. cereus TC-1 isolated from solar salt works having higher antagonistic activities against the pathogenic Vibrio sp. Further works are need to in vivo treatments against the aquatic pathogens in laboratory as well as field trails and characterize the virulence factors at molecular level.