Abstract: In study to obtain a safe Bacillus probiotic for Nile tilapia (Oreochromis niloticus) cultivation, hemolytic activity on blood agar medium was used in isolation of Bacillus probiotic species from gastro-intestinal tract of Nile tilapia. One hundred and three isolates of Bacillus sp. which showed no hemolytic activity were obtained from 2 sampling sites of the Nile Tilapia net-cage culture farms. Among these 103 isolates, however, there was only 1 isolate, named as Bacillus UBRU4 which showed the inhibitory effect on Aeromonas hydrophila growth. The results of physiological and biochemical test and molecular identification (99.90% identity) showed that Bacillus UBRU4 was similar to Bacillus brevis. This was possibly the first report of isolation of Bacillus brevis in aquaculture. The optimum pH and temperature for Bacillus UBRU4 growth on Tryptic soy broth were 6.5 and 37°C, respectively. The maximum cell numbers of Bacillus UBRU4 in modified broth culture medium was obtained when using the medium contained 30 g L-1 of Nile tilapia commercial feed and 20 g L-1 of molasses. The bioactive compound production of Bacillus UBRU4 showed the growth associated characteristic. Partial purified bioactive compounds by 80% saturated ammonium sulfate could increase the activity to 6,400 AU mL-1. The specific activity of the bioactive compound was increased from 1,298 to 5,807 AU mg-1. These results suggested that the Bacillus UBRU4, thus, could possibly be used as high potential probiotic in Nile tilapia feed.
INTRODUCTION
Disease is a major problem for the fish farming industry which currently is the fastest growing food-protein producing sector, with an annual increase of approximately 9% (Deeseenthum et al., 2007). Tilapia (Oreochromis sp.) is a group of fish which is growing importance in the aquaculture industry. Nile tilapia is the most commonly cultured species among Tilapias in many countries around the world (Mehrim, 2009), including Thailand. Only in Ubon Ratchatani province, there are more than 2,000 net-cage cultures. Disease outbreak is an important problem for Nile tilapia farmers and antibiotics are used as effective agents to control disease and growth promoters in domestic animals. Nevertheless, the continuous use of antibiotics has contributed to the occurrence of antibiotic-resistant bacteria population (Rahman et al., 2009) and to an increase in more virulent pathogens. The fear for the spread of this resistance to human pathogens has recently led to the banning of several antibiotics as so-called growth promoters in animal husbandry within the European Union (Naviner et al., 2006; Deeseenthum et al., 2007). Probiotics are viable cell preparation which must have the ability to survive passage through the intestinal tract which has beneficial effects on health of the host by improving its intestinal balance via producing nutrients, enhancing immune response (Song et al., 2006). Probiotics have been used in aquaculture as a means of disease control, digestion aids and immune booster or even in some cases replacing the use of antimicrobial compounds (Irianto and Austin, 2002; EL-Haroun, 2007; Mehrim, 2009). In order to make Nile tilapia products more acceptable to consumers, it is necessary to find the effective probiotics to prevent diseases and improving growth performance with a particular attention to Bacillus spp. because bacilli are spore-forming bacteria and can grow rapidly. However, some Bacillus spp. could be pathogenic bacteria also, such as B. cereus, B. anthracis. Hemolytic activity on blood agar plate is generally used for identifying pathogenic microorganism and rarely use for screening a useful bacteria. Thus, the aims of the present work were isolation and screening of the high potential bacilli probiotic from the gut of Nile tilapias by using blood agar plate. Bioactive compound production, optimizations of the cultivation condition and the modification of the cheap medium for producing of the selected Bacillus sp. were also reported.
MATERIALS AND METHODS
The isolation and screening for the safe Bacillus probiotic from Nile tilapia’s intestinal tract took almost 2 years (from February 2009-November 2010) to obtain the targeted Bacillus probiotic. This was because most of the isolated Bacillus showed the hemolytic activity or no antagonistic activity.
Isolation and screening of the bacilli probiotics from the intestinal tract of Nile tilapia: The Nile tilapia net-cages culture farms located in Moon River at Ban Kudua, Jaramae sub-districts and Moon-noi River at Ban Pakmoonnoi, Bungkasael sub-districts, Maung district, Ubon Ratchatani province, Thailand were the two study sites of this work. The bacilli probiotics from the intestinal microbiota in Nile tilapia were screened over 4 months with regular sampling of 90 fishes in total from the two study sites. Five randomly live Nile tilapia from each site were collected and transferred to the laboratory within 1 h for microbiological analyses. The collected fish were anesthetized and cleaned with alcohol to remove any external microbial contamination. Their gastrointestinal tracts were then dissected, grinded and boiled at 100°C for 5 min in order to isolate for bacilli group on blood agar medium by cross-streak technique. Colonies of spore forming bacteria without clear-zone on blood agar were considered as the isolated bacilli and all isolates were purified by spread plate technique 10 times for further use. All isolated bacilli were screened for the ability of growth inhibition against Aeromonas hydrophila by spotted on lawn technique on tryptic soy agar plates. The plates were swabbed with 24 h cultured A. hydrophila and were spotted on lawn with 24 h cultured bacilli. All plates were incubated at 37°C for 24 h. If clear inhibitory zones formed around, the spots were considered as antagonistic against the growth of A. hydrophila.
Identification of the isolated bacilli probiotics from the intestinal tract of Nile tilapia: Bacillus isolates were characterized by biochemical test and molecular biology assays at the genus or species level. The selected Bacillus were determined on the criteria of Gram reaction, spore shape and position, susceptibility to penicillin, streptomycin and kanamycin, catalase activity, motility, starch digestion, nitrate reaction, acid and gas formation from glucose, growth at temperature of 10 and 45°C, growth in medium with 4, 6.5% salt and 0.2% bile salt. The nucleotide sequencing, 16S rDNA (Full sequence; 1,400 bp), was also used for Bacillus identification by the service at BIOTECH Culture Collection, BIOTECH Central Research Unit, 113, Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Prathumthani, Thailand.
Optimization of cultivation conditions and culture medium for Bacillus cells production: The selected Bacillus was cultured in nutrient broth at 37°C for 24 h and used as inoculum. The initial pH of nutrient broth was adjusted to 4, 5, 6 and 7 prior to sterilize at 121°C for 15 min. All conditions of culture medium were inoculated with 108 CFU mL-1 of inoculum and incubated at 30 and 37°C for 24 h on rotary shaker (150 rpm). Samples were withdrawn during the cultivation process for cell growth determination by total plate count method. For cheap medium optimization, the isolated Bacillus was cultured in modified liquid medium which was contained of molasses at the concentrations of 20 g L-1. The effect of varying Nile tilapia commercial feed CP No. 895 (Charoenpokapan Company, Samutsakhon, Thailand) at the concentrations of 10, 20, 30 and 40 g L-1 was then examined after incubation at 37°C for 24 h. Samples were withdrawn during the cultivation process for cell growth measurement by total plate count method.
Bioactive compound production of the selected Bacillus sp. probiotic: The selected Bacillus sp. was cultured in 1 l Erlenmeyer flasks containing 500 mL nutrient broth and incubated at 37°C in incubation shaker (150 rpm). Samples were withdrawn during the cultivation process for cell growth, protein concentration and antibacterial activity determinations by total plate count method, Lowry method (Waterborg and Matthews, 1994) and two-fold-dilution method (De Carvalho et al., 2006), respectively. The activity of the partial purified bioactive compound, obtained from 80% saturated ammonium sulfate precipitation, was also examined by two-fold-dilution technique. The partially purified substance from the selected Bacillus sp. was serially diluted into 50 mM sodium acetate buffer (pH 5.5) and tested against Aeromonas hydrophila. The antibacterial activity (AU mL-1) was calculated by the following formula: The highest dilution which still showed the positive resultx1,000/sample volume.
RESULTS
Isolation and screening of the bacilli probiotics from the intestinal tract of Nile tilapia: One hundred and sixty randomly live Nile tilapias from two net-cage culture sites were collected for 8 months. The samples were transferred to the laboratory within 1 h for microbiological determination. One hundred and three isolates of bacilli without clear-zone on blood agar (no hemolytic effect) were obtained from both study sites. However, there was only 1 from 103 isolated Bacillus exhibited both no hemolytic activity on blood agar medium (Fig. 1a) and the inhibitory activity against the growth of A. hydrophila by spot on lawn technique (5-6 mm of inhibition zone) (Fig. 1b) and swab paper disc method (5-7 mm of inhibition zone). This isolated Bacillus was found from Nile tilapia of site I and it was designed as Bacillus sp. UBRU4.
Identification of the bacilli probiotic from the intestinal tract of Nile tilapia: The results of Physiological property and Biochemical test of Bacillus sp. UBRU4 were shown in Table 1 and 2. Colony morphology of Bacillus sp. UBRU4 was evaluated after complete expression of the phenotypes on LBY agar, the resulting colony was described in terms of their most distinct features (Table 1). The full phenotype typically became manifest after 1 week of cultivation (2 days at 37°C, followed by 5 days at room temperature). The developed phenotype was exhibited whitish or pale yellow color, circular form, convex elevation, convex elevation in the center, glistening surface, rough, translucent colonies, 3 to 4 mm in diameter and undulated irregular edges (Table 1). Micro-morphology of Bacillus sp. UBRU4 was 1x1.5-2.5 μm of cell size, bacilli shape, gram positive, spore forming bacteria in swollen sporangium which were ellipsoidal shape, central or subterminal endospore position.
It was exhibited that the Biochemical properties of Bacillus sp. UBRU4 (Table 2) were resistance to the antibiotic penicillin and kanamycin but susceptible to streptomycin, catalase positive, motility positive, starch digestion negative, nitrate reducing positive, acid and gas productions from glucose positive, growth at 10 but not at 45°C, growth in medium with 4 salt but not at 6.5% salt, growth in medium with 0.2% bile salt.
| Table 1: | Physiological property of Bacillus sp. UBRU4 |
| Table 2: | Biochemical characteristics of Bacillus sp. UBRU4 |
| Note: Bacillus sp. UBRU4 was tested for susceptibility to some antibiotics (inhibition zone in mm), Gram straining, productions of catalase, amylase and nitrate reductase, motility, formation of acid and gas from glucose, the ability to grow at different temperatures and salts; +was positive result (with activity) and -was negative result (no activity) | |
| Fig. 1(a-b): | Bacillus sp. UBRU4 colonies on Blood Agar (a) and inhibition zone of Bacillus sp. UBRU4 against the growth of A. hydrophilla by spot on lawn method (b) |
| Fig. 2: | Nucleotide sequence of Bacillus sp. UBRU4 by 16S rDNA sequence (full sequence; 1,400 base pairs) |
When comparing these results with those reported by Gordon (1989), it was found that Bacillus sp. UBRU4 was similarly to Bacillus brevis. This result was coincided with the result of molecular biology assays which indicated that the nucleotide sequencing (16S rDNA) of Bacillus sp. UBRU4 (Fig. 2) was similar to B. brevis (99.90% similarity).
Optimization of cultivation conditions and culture medium for Bacillus cell production: In order to obtain the high cell concentration, the effects of pH and temperature on Bacillus sp. UBRU4 growth were studied. It was observed that the highest growth of Bacillus sp. UBRU4 occurred at pH 6.5 and 37°C which the highest cell number was 3.2+0.13x109 CFU mL-1. However, to facilitate the farmer to produce the probiotics by themselves, the modified low cost culture medium for Bacillus sp. UBRU4 cell production was examined. The selected carbon source used in this study was molasses which was a byproduct from sugar cane industry. The nitrogen source was the Nile tilapia commercial feed which was easily for the farmer to use. The results showed that the highest growth of B. brevis UBRU4 occurred at 3% commercial feed which the obtained cell numbers were 5.7±8.6x08 CFU mL-1
Bioactive compound production of the selected Bacillus sp. probiotic: Cell growth, protein content and bioactive compound production of Bacillus sp. UBRU4 were showed in Fig. 3.
| Table 3: | Activity and specific activity of the partial purified bioactive compound of Bacillus sp. UBRU4 |
It was found that the bioactive compound production of Bacillus sp. UBRU4 was growth associated. The activity of the bioactive compound of Bacillus sp. UBRU4 reached the maximum (1600 AU mL-1) at 8 h of cultivation and stayed constant until 17 h of cultivation before declining at the stationary growth phase. Partial purification of bioactive compound by ammonium sulfate precipitation could increase the activity up to 6, 400 AU mL-1, which the specific activity was increased from 1,298 to 5,807 AU mg-1, as shown in Table 3.
DISCUSSION
Probiotics, live microorganisms which may serve as dietary supplements to enhance the growth and health of the host, have received some attentions in aquaculture as a means of diseases control, digestion aids, immune booster, supplementing or replacing the use of antimicrobial compounds (Irianto and Austin, 2002; EL-Haroun, 2007; Abdelhamid et al., 2009; Denev et al., 2009). The probiotic, in addition, may prevent or complete with the potential pathogens in colonizing the gut by production of antimicrobial compounds, or by out-competing them for nutrients or mucosal space (Robertson et al., 2000; Denev et al., 2009; Fernandez et al., 2011). Some of probiotics which are naturally occurring micro-organisms, are able to promote the growth and survival of aquaculture animals by inhibiting the activity of other bacteria that flourish in hatchery cultures.
| Fig. 3: | Cell growth, protein content and bioactive compound activity during batch cultivation of Bacillus sp. UBRU4 |
The Bacillus genus has not been reported as pathogens of the aquatic organisms (Moriarty, 1998). On the other hand, it is often antagonistic against some fresh water fish pathogenic bacteria (Vijayabaskar and Somasundaram, 2008). In addition, Bacillus species could be able to produce antibiotics, amino acids and enzymes (Sanders et al., 2003; EL-Haroun, 2007). Consequently, Bacillus probiotics may have positive nutritional effects on fish (Bagheri et al., 2008). Moreover, these Bacillus species could be able to survive through the pelletization process (Mehrim, 2009). Its application, thus, has been promoted and more widely accepted within the aquaculture industry (Gullian et al., 2004).
This present study, found that all isolates of bacilli from the gastro-intestinal tracts of Nile tilapia, except the Bacillus sp. UBRU4, showed hemolytic activity at days 7-14 of post-cultured on Blood agar medium. Not only exhibited the negative hemolytic activity, the Bacillus UBRU4 also showed the inhibitory activity against the growth of A. hydrophila. This result was agreed with Sanders et al. (2003) who reported that some of different antibiotics exhibiting antagonism against a broad spectrum of microbes have been identified from the Bacillus genus. Using hemolytic activity and antagonistic activity against the pathogenic growth in screening of the safe Bacillus probiotic in this study is one of a few reports at the present (Duc et al., 2004; Rahman et al., 2009; Thirabunyanon et al., 2009). This indicated that Bacillus sp. UBRU4 could possibly be safe for using as probiotic in Nile tilapia culture. Sanders et al. (2003) reported that normally Bacillus species were allochthonous microbes to the host intestinal tract and they were not normal colonizing inhabitants of the host intestinal tract. So in order to find the suspected probiotics, the screening of probiotic Bacillus from Nile tilapia’s gut was spent a long time. This was corresponded to Al-Harbi and Uddin (2004) who reported that the intestine bacteria flora of hybrid tilapia (O. niloticusxO. aures) cultured in Saudi Arabia had some influences from the seasonal quantitative and qualitative and revealed that Bacillus spp. was present in some seasons of the year.
The physiological, biochemical and Molecular characters of the suspected probiotic bacterial which was Bacillus sp. UBRU4, was identified as Bacillus brevis by modifying the method of Gordon (1989) and 16S rDNA (Full sequence; 1,400 bp). Using B. brevis as probiotic in aquaculture in our study, if not the first, is possibly one of the pioneer works.
Clear zone colony of Bacillus sp. UBRU4 showed the antagonism against A. hydrophila and indicated the production of antimicrobial which was considered to be a pathogen-inhibiting mechanism. This result was agreed with Sanders et al. (2003) who reported that dozens of different peptide antibiotics exhibiting antagonism against a broad spectrum of microbes had been identified from the Bacillus genus. Berditsch et al. (2007) reported that the production of antibiotic substance was involved in destroying pathogens. It was consistent with the previous studies that B. brevis could produce antibiotics substances, gramicidin and tyrocidine and these two antibiotics were peptide antibiotics. Moreover, these peptides exerted a wide range of antimicrobial effects, including activity against gram positive and gram negative bacteria, viruses, fungi and single-cell pathogenic eukaryotes. This was similar to the report of Robertson et al. (2000) that a strain of Carnobacterium sp., isolated from the intestine of Atlantic salmon, was evaluated for potential use as a probiotic for salmonids. In vitro studies of this Carnobacterium sp. demonstrated antagonism against A. hydrophila, A. salmonicida, Flavobacterium psychrophilum, Photobacterium damselae subsp. Piscicida, Streptococcus miller, Vibrio anguillarum and V. ordalii. The original B. brevis strain which could produce antibiotic (Gramicidin S) was isolated from soil and was described as B. brevis var. G. B. Initially, B. brevis var. G. B. was delivered to the National Centre of Type Cultures (London, United Kingdom), from which it was obtained by the American Type Culture Collection (ATCC, Manassas, VA) where it was deposited as B. brevis ATCC 9999 (Berditsch et al., 2007). B. brevis Nagano caused complete inhibition of Botrytis cinerea germination. This spore concentration contained Gramicidin S (Edwards and Seddon, 2001). Bacillus species have antagonistic effect on other microorganisms and antibiotics have been recognized as the only means of effective microbial growth control (Kuta et al., 2009).
Optimum pH and incubation temperature for Bacillus sp. UBRU4 growth on Tryptic soy broth were 6.5 and 37°C, respectively. The maximum cell growth in modified broth culture medium was obtained when 30 g L-1 of Nile tilapia commercial feed, without urea adding and 20 g L-1 of Molasses were used. Dechmahitkul et al. (2007) reported that the use of Molasses caused a foaming problem in the fermentor culturation of Bacillus subtilis and defatted soybean meal was suitable for substitution. The modified solid medium for Bacillus sp. UBRU4, Grinded soybean, was performed and Bacillus sp. UBRU4 showed the highest live cell at 48 h (3.6x1010 CFU g-1) of accelerated storage condition (37°C) and then was decreased to 4.5x102 CFU g-1 at 192 h. Because of the potential of antibiotic producing, Bacillus sp. UBRU4 has got Feed-back inhibition activity which was exhibited short shelf-life in accelerated storage condition in Grinded soybean modified medium. The optimum modified medium of Bacillus sp. UBRU4 should be solid media and kept under 4°C in order to maintain alive cell and reduce the effect of antibiotic activity from itself in the surrounding liquid part from the medium.
The production of bioactive compound which produced by the Bacillus sp. UBRU4 was found to be growth association which was similar to other bacteriocins (Mojgani and Amirnia, 2007; Kumari et al., 2008; Sharma et al., 2010). It was well established that Gramicidin and Tyrocidine were produced by B. brevis (Foster and Woodruff, 2010). Thus, the bioactive compound of Bacillus sp. UBRU4 could possibly be one or both of these bacteriocins. The decline of the activity during stationary growth phase was possibly due to the hydrolysis of the protease from the dead cell lysate. The precipitation of the bioactive compound by ammonium sulfate could increase the activity up to 4 folds. These confirmed that the bioactive compound produced by Bacillus sp. UBRU4 was a bioactive peptide or bacteriocin (Bali et al., 2011).
CONCLUSION
Bacillus UBRU4 was only one isolate of Bacillus probiotic specie which was isolated from gastrointestinal tract of Nile tilapia by hemolytic and antagonistic activities. The physiological and biochemical test and molecular identification results indicated that this Bacillus UBRU4 was similar to B. brevis. This is possibly the first report of B. brevis isolation from aquaculture. The modified broth medium contained 3% of Nile tilapia commercial feed and 2% of molasses was the cheap medium for cultivation of this isolated Bacillus sp. probiotics which the farmers could prepare easily by themselves. The produced bioactive compound or bacteriocins of this safe probiotic Bacillus UBRU4 exhibited the growth associated character. The Bacillus UBRU4, thus, could possibly be safe and used as high potential probiotic in Nile tilapia cultivation.
ACKNOWLEDGMENTS
This study was financial supported by the National Research Council of Thailand (NRCT). Thanks to the Fermentation Research Centre for Value Added Agricultural Products and the Department of Biotechnology, Faculty of Technology, Khon Kaen University and the Department of Microbiology, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Ratchathani, Thailand, for supporting the facilities in this research.