Studies on the microbial diversity of salted fishes under aerobic conditions

A total of 93 isolates of halophilic and moderately halophilic bacteria were isolated from salted fishes, Scoliodon sp. and Thrissina thryssa obtained from retail fish outlets in Cochin, Kerala, India. The viable count of the bacteria in Scoliodon sp. and Thrissina thryssa ranged from 10-10 per g. The morphological, biochemical, and physiological investigations were done to characterize the isolates, which showed good growth in medium containing 3-10% (w/v) NaCl at 37°C. In Scoliodon sp. the maximum of 87.5% isolates were Gram-positive cocci and in Thrissina thryssa 95.5% of the isolates were Gram-positive rods. The optimum conditions such as temperature, pH, and salts (NaCl) were determined. The utilization of organic compounds such as fructose, lactose, glucose, sucrose, arabinose, trehalose, maltose, salicin, mannose, cellobiose, rhamnose, dulcitol, xylose, raffinose, sorbitol, adonitol, inulin, galactose, inositol, mannitol, hydrogen sulfide, nitrate, citrate utilization, MR-VP, triple sugar iron, as well as the hydrolysis of organic compounds such as casein, gelatin, aesculin, starch, Tween 20, and Tween 80 were also investigated. Moreover, an alkalophilic bacterial strain Bacillus halodurans (B. halodurans) was isolated from Thrissina thryssa and confirmed by 16S rDNA analysis. These studies revealed that salted fishes offer an optimal environment for the viable, diverse, potentially and industrially important bacterial community.


Introduction
Salting and drying has been used for a long time as a method of fish preservation. 1roduction of canned salted fishes is a traditional activity in the fishing industry in the countries around the Mediterranean Sea.Salting is used for the preservation of fishes from spoilage owing to tissue autolysis and microbial action. 2 Microbial ecology of salted fish products is influenced markedly by the water activity of the product.The high salt and low moisture contents of the salted fishes have contributed to the extreme halophilic bacteria, which have been the most studied. 3,4A broad range of bacteria inhabit salted fishes; among these moderate halophilic bacteria are the dominating flora.Moderately halophilic bacteria are a heterogeneous group of microorganisms characterized by growth over a wide range of salt concentrations (0.5-2.5 M NaCl). 5Moderate halophilic bacteria were also recovered from seafoods. 6,7To date, several studies have reported the microbial ecology of hypersaline waters. 8,9ew studies have been done regarding the moderately halophilic and halotolerant bacteria in salted fish products. 10While the microbiology of salted fish has not been studied extensively in the recent past, many different species of moderately halophilic bacteria have been isolated from various habitats such as saline lakes, 11 hypersaline soils, 12 and solar salterns. 13To our knowledge no studies relating to the bacterial profile of salted Scoliodon sp. and Thrissina thryssa have been done.The aim of this work was to characterize the microbial diversity with the main emphasis on the Bacillus halodurans (B.halodurans) strain.

Sampling and culture conditions
Six samples each of dried salted fishes like Scoliodon sp. and Thrissina thryssa were obtained from retail fish shops in Cochin, Kerala, India.Salinity of the samples was checked using a salinometer.Ten-gram samples were excised aseptically from each fish and homogenized in 90 mL of sterile saline water with a Mortar and Pestle and serially diluted up to 10 -4 .Then 0.5 mL of each dilution was used to spread in the growth media.Enumeration and isolation of the bacteria were done on salt agar, high-salt casein agar, and nutrient agar with 3% NaCl.The plates were incubated at room temperature for 3-7 days in sealed polythene bags.

Identification of Bacillus halodurans
Identification and biochemical characterization of B. halodurans using the conventional method was carried out as described by. 19

Genomic DNA isolation for 16S rDNA analysis
Genomic DNA extraction from B. halodurans was performed as described by. 201.5 mL of the late exponential phase bacterial cells were transferred into a microfuge tube and centrifuged at 10,000 rpm for 5 min at 4°C.The supernatant was discarded.The clean cells were suspended in 200 mL of lysis buffer and incubated at 37°C for 30 min.Then, 30 mL of 10% SDS and 10 mL of proteinase K (20 mg/mL) were added.The tube contents were gently mixed and incubated at 37°C for 1 h.An equal volume of phenol:chloroform was added and the tube was centrifuged at 12,000 rpm for N o n -c o m m e r c i a l u s e o n l y 5 min at 4°C.The supernatant was transferred to a new microfuge tube.Twice the volume of cold absolute ethanol was added to the supernatant and gently mixed to precipitate the DNA.The tube was then centrifuged at 12,000 rpm for 5 min at 4°C.The supernatant was discarded.The precipitated DNA was washed with 500 mL of 70% ethanol, and dried at 37°C for 30 min.The DNA pellet was resuspended with 50 mL of TE buffer and kept overnight at 4°C to dissolve the precipitated DNA.

Polymerase chain reaction amplification of 16S rDNA
The 16S rDNA of halophilic bacterium isolate was amplified by PCR using primers 27f and 1525r (Table 1).The PCR was performed using a PTC-150 Mini cycler (MJ Research, Waltham, MA, USA) with a primary heating step for 2 min at 95°C, followed by 30 cycles of denaturation for 20 sec at 95°C, annealing for 60 sec at 55°C, and extension for 2 min at 72°C, then followed by a final extension step for 7 min at 72°C.Each 25 mL reaction mixture contained 2 mL of genomic DNA, 14.25 mL of MilliQ water, 2.5 mL of 10× buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl), 1.5 mL of MgCl 2 (25 mM), 2.5 mL of dNTP's mixture (dATP, dCTP, dGTP, dTTP at 10 mM concentration), 1.0 mL of each primer (20.0 pmoles/mL), and 0.25 mL of Taq DNA polymerase (MBI Fermentas, USA).The PCR-amplified product was analyzed on 1% agarose gel containing ethidium bromide (0.5 mg/mL) and 1 kb DNA molecular weight marker (MBI Fermentas), and documented using a gel documentation system (Alpha Imager 1220, Alpha Innotech Corporation, San Leandro, CA, USA).

Sequencing and in silico analysis of polymerase chain reaction amplicon
The PCR amplicon was purified by the MinElute Gel purification Kit (Qiagen, Hilden, Germany) and was sequenced on an ABI PRISM 377 genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA).The nucleotide sequences obtained were compared against database sequences using BLAST 21 provided by NCBI (http://www.ncbi.nlm.nih.gov) and were aligned and clustered using the CLUSTAL-X version 1.81 program. 22

Isolation of microorganisms
About 75% of the isolates obtained from both the Scoliodon sp. and Thrissina thryssa samples were grown optimally in a 3-10% NaCl media and were thus considered as moderate halophiles. 23Scoliodon sp. and Thrissina thryssa were analyzed for the growth of bacteria by inoculating the sample on salted agar media, (5% NaCl), nutrient agar (3% NaCl), and highsalt casein media (25% NaCl).The counts observed for the samples in two different media are given in Table 2. Nutrient agar with 3% NaCl had an elevated count in both the samples analyzed and Scoliodon sp. had a higher count than Thrissina thryssa.A total number of 93 isolates were randomly selected for characterization.The source of the isolates are given in Table 3.

Pigmentation pattern
All isolates were observed for their colony color.Of the 93 isolates, 21.5% were whitish, 27.9% were yellowish, 34.4% were creamy, 13.9% were pinkish, and 1% were orange in color.

Growth range and optimal growth conditions
Of the 93 isolates, 80.6% were grown in 10% NaCl, 77.4% in 2% NaCl, 75.2% in 0.5% NaCl, 83.8% in 5% NaCl, and 10.75% in 20% NaCl.The optimum NaCl range for growth was 2-10% among the 93 isolates.pH range for growth also varied among the isolates; 95.6% of the 93 isolates were found to have good growth at pH 8, 91.3% at pH 9, and 60.21% at pH 10.The optimum range of pH was found to be 7-9.The optimal growth temperature for the 93 isolates was determined to be 30°C.The isolates thus have a wide growth range, both in salt concentration and pH.

Isolation and identification of Bacillus halodurans
Morphological, physiological, and biochemical characterization of B. halodurans were found to be: a Gram-positive, spore-forming rod; motile; oxidase positive; catalase negative; and utilized glucose, fructose, galactose, sucrose, lactose, and arabinose.Hydrolysis of Tween 40, 60, casein, gelatin, and starch is obtained but the hydrolysis of Tween 20 and reduction of nitrate were not observed.The isolate also exhibited good growth on nutrient agar plates with 15% NaCl and at pH10.0.Moreover, good growth was observed in the temperature range of 15-55°C.These properties support the previous report for the identification of B. halodurans. 19Furthermore, the B. halodurans isolate was confirmed by 16S rDNA analysis.The sequence of the 16S rDNA gene 1502 bp (Figure 1) of our isolate showed 100% identity to that of the previously reported B. halodurans C-125 (BA000004) 24 and B. halodurans MS-2-5 (AB359904). 25Thus, the bacterial strain was identified and confirmed as B. halodurans, on the basis of these results.The 16S rDNA sequence generated in this study was submitted to GenBank and has been given the accession no.GU367604.

Discussion
Several species of moderately halophilic eubacteria, obtained from diverse natural saline habitats, have been isolated and described in recent years. 4Microbial diversity of salted Scoliodon sp. and Thrissina thryssa in this study was the first in saline ecosystems to be studied in detail from a microbiological standpoint.In view of the great commercial importance of the halophilic and moderate halophilic bacteria, it is surprising how little research has been done to study the microbiology of salted fishes.Our primary interest was in characterizing the microorganisms with the potential value of biotechnological interest.Presently there are only nominal reports of the isolation of bacteria from salted foods. 10In our study, we isolated 147 bacterial strains, of which 93 of both halophilic and moderately halophilic bacteria were characterized in detail.The majority of the Gram-positive cocci (87.5%) were found in Scoliodon sp.whereas the maximum numbers of Gram-positive rods (95.5%) were observed in Thrissina thryssa.These results agree with those reported by other authors in salted cod 10 and other cured food commodities such as smoked catfish, 26 fermented sausages, 27 and cured meats. 28ram-positive cocci are the dominant bacterial survivors in salted fishes. 29Staphylococcus aureus and Staphylococcus xylosus have been found in green salted and dried salted products, respectively. 30All the isolates in this study were found to use various organic compounds including sugars as substrates and should be considered chemoorganotrophs.For the first time, we have isolated an industrially import -a n t alkalophilic B. halodurans strain from Thrissina thryssa.Previous studies reported the isolation and identification of Marinococcus and Halobacillus strains from other saline environments, including athalassohaline and thalassohaline lakes and marine waters. 31,32In our study, about 75% of the isolates from both salted fish samples revealed optimal growth in 3-10% NaCl media and were thus considered as moderate halophiles. 33ecent research on different hypersaline habitats focused on the screening of new bacteria with potential applications in biotechnology. 34he potential industrial use of these microorganisms has been highlighted for the production of compatible solutes, biopolymers, and bioremediation processes, 33,34 prompting us to screen our collection of halophiles for molecules of industrial interest.Moreover, extensive studies on these bacteria will certainly offer different potential applications in various pharmacochemical industries. 35,36

Conclusions
Based on the results, it can be concluded that the identification and characterization of halophilic and moderately halophilic bacteria presenting in salted Scoliodon sp. and Thrissina thryssa have opened several possibilities for future research in the field of industrial biotechnology and microbial spoilage aspects.