Low Cost Medium for Spore Production of Bacillus KKU02 and KKU03 and the Effects of the Produced Spores on Growth of Giant Freshwater Prawn (Macrobrachium rosenbergii de Man)

INTRODUCTION

Giant freshwater prawn (Macrobrachium rosenbergii de Man) is commonly found in the nature among Southeast Asian countries. This prawn is used to be caught along the major rivers in Thailand and is an alternative source of protein, especially for the North-eastern area of Thailand where the productivity of agricultural crop is low. Because of its good taste and texture, the freshwater shrimp has become a very popular food which, consequently, resulting in an over fishing and destroying of its natural habitat. The natural catching, thus, has been reduced dramatically. The production of post larvae in hatcheries with potential for pond culture and number of prawn farms, thus, has significantly increased. The fast growing of shrimp farming and the continuous use of the farming land led to the neglect of good husbandry and environmental management. Under these damaging environmental conditions, shrimps were stressed and weakened and, thus, could cause the development of shrimp diseases. Thus, the major problem for the shrimp farming industry is the shrimp disease. Vaccines and antibiotics are the important disease control measures for the shrimp farming. Vaccines alone, however, could not be used in controlling all shrimp diseases. Thus it is not an economical measure for shrimp protection. Antibiotic and chemotherapeutics treatments are, hence, the important measures for disease controlling in aquaculture. Bacterial resistance to antibiotics development has been well documented. The fear of the spread of this resistance to human pathogens has led to the banning of several antibiotics as so-called growth promoters in animal husbandry within the European Union and the year 2006 is the date proposed for a complete ban of antibiotics in animal feed within Europe (Cartman and La Ragione, 2004; Hong et al., 2005). This concern has also been raised in the aquaculture industry and has led to suggestions for other disease control measures. The use of probiotic is an alternative measure and many species have been used and produced (Westerdahl et al., 1991; Smith and Davey, 1993; Gatesoupe, 1994; Austin et al., 1995; Bly et al., 1997; Gram et al., 1999; Sanders et al., 2003; Hong et al., 2005; Cutting, 2011). However, the fear of antibiotic resistant gene transfer among the bacterial species is another concern. Thus, new isolates of probiotic which could not transfer antibiotic resistance gene are still needed.

Most probiotic, generally found normally in the Gastrointestinal Tract (GIT) of humans and animals, are supplied as live supplements in feed which must have the ability to survive passage through the intestinal tract. However, microorganisms which are not normally found in the GIT, such as the spore forming bacteria, are alternative interesting probiotic sources. One disadvantage of using live non-spore forming bacteria is the stability of the probiotic product. If the spore form is used, thus it can be stored longer on the shelf (Hong et al., 2005; Cutting, 2011).

Bacillus KKU02 and Bacillus KKU03 have been isolated from the intestine of the giant freshwater prawns. It was found that these 2 isolates of Bacillus showed a high potential for using as prawn probiotic (Deeseenthum et al., 2007). However, when these 2 isolates were applied in the field by the farmers, their efficiencies were declined because of the reduction of their viabilities. The advantage of the Bacillus sp. was they could form spores which could survive in some stress conditions, such as heat and dry conditions. In order to use the spore for testing the probiotic efficiency, the mass production of spore need to be examined. Thus, the low cost medium for spore production and the effects of the produced spores of Bacillus KKU02 and Bacillus KKU03 as probiotic on growth of giant freshwater prawn after feeding with the probiotic were the aims of these studies.

MATERIALS AND METHODS

Low cost media formulation for Bacillus KKU02 and KKU03 spores production
Microorganism: Two Bacillus sp. isolated from the intestine of the giant freshwater prawn which were Bacillus KKU02 and Bacillus KKU03 as reported earlier (Deeseenthum et al., 2007), were used in this study.

Media formula for spore production: Four cheap agricultural substrates which were sweet potato (Impomoea batatasil), cassava root (Manihot esculenta), rice (Oryza sativa) and sticky rice (Oryza sativa var. glutinosa), were added to the tested media 200 g L^-1 as carbon source, comparison to Nutrient Broth (NB). Each agricultural substrate medium was boiled for a given time to obtain the aqueous extract which was then supplemented with 20.0 g L^-1 dextrose and use as spore production medium. Control medium was consisted of 20.0 g L^-1 dextrose as a sole carbon source. The initial pH of the medium was adjusted to 7.0 prior to sterilization at 121°C for 15 min. The sterilized culture medium (150 mL in 250 mL Erlenmeyer flasks) was inoculated with 1.0% of 12 h culture of Bacillus KKU02 and KKU03 and cultivated at 37°C on a rotary shaker (150 rpm). The obtained optimum carbon source medium was further examined for the mineral salt supplementation by adding the aqueous extract medium with MgSO₄ 0.1 g L^-1 and (NH₄)₂SO₄ 2.0 g L^-1 which were used in the previous cell production medium (Deeseenthum et al., 2007). In order to obtain the optimum concentration of the carbon source, the concentrations of the obtained carbon source medium were then further varied to 50, 100 and 200 g L^-1. The culture condition was the same as described earlier.

Analysis: Spore concentration was determined by viable plate count technique. Samples were taken every 4-6 h and boiled at 80°C for 10 min for eliminating of the vegetative cells. The remaining spore sample was then used for viable plate counting.

Giant freshwater prawn production using Bacillus KKU02 and Bacillus KKU03 in spore form as probiotic
Preparation of experimental animals: Post larvae of giant freshwater prawns (PL-15) were obtained from a hatchery located in Supanburi province, Thailand. The prawns were acclimatized in the tanks for 60 days and fed with only the commercial feed. Two hundred giant freshwater prawns of uniform size (12.0-14.0 g) were kept in each tank. The prawns were reared with probiotic bacteria in six feed treatments until reach to 90 days.

Experimental conditions: The experiment was conducted for 150 days at the Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand. The experiment was divided into six triplicates experimental groups in 18 concrete tanks (1.50x2.00x1.00 m³ at 0.5 m water height). The total water in each tank was maintained at 1,500 L and aeration was continuously provided. About 50% of water was replaced with fresh water once a week. The details of the experimental groups were as follows:

Treatment 1:	Commercial diet+spore of Bacillus KKU03
Treatment 2:	Commercial diet+spore of Bacillus KKU02
Treatment 3:	Commercial diet+spores of Bacillus KKU02 and KKU03
Treatment 4:	Commercial diet+vegetative cells of Bacillus KKU02 and KKU03 (Deeseenthum et al., 2007)
Treatment 5:	Commercial diet (control)
Treatment 6:	Commercial diet+commercial probiotic (reference control)

The commercial feed used was purchased from Charoen Pokphand Company, Samutsakhon, Thailand (CP No. 9041). The feed was autoclaved at 110°C for 28 min before mixing with the cultured spore for eliminating of the contamination.

Bacterial strains and feeding regime: The Bacillus KKU03 and Bacillus KKU02 were grown in cooked cassava chip aqueous extract medium as described earlier for 48 h at 37°C on a shaker at 150 rpm. The final spore concentration of about 10⁷ spore mL^-1 was mixed with the feed at the ratio of 200 mL to 1 kg feed (the expected concentration in the feed was 2x10⁶ spore g^-1). After acclimatization, the prawns were fed twice daily, at 08.00 am and 06.00 pm. The daily feeding rate was about 10% of total body weight.

Analysis of samples: Ten randomly collected live prawns from each tank were measured for lengths and weights once every 3 weeks. Water quality was checked weekly for pH, dissolved oxygen and temperature. The amount of ammonium, total hardness and total alkalinity were also determined by test kits (HACH^®) obtained from HACH Company, USA.

Statistical analysis: One-way analysis of variance (ANOVA) was used to determine any significant differences among the treatment groups. The comparison was done by using Randomized Complete Block Design (RCBD) test between the six treatments.

Challenge test of the probiotic treated giant freshwater prawns
Experimental conditions: After rearing shrimps in six feed treatments (prawn production section) until reach 120 days, 30 shrimps from each treatment (triplicate of 10 shrimps per tank) were transferred into a glass container (15”x18”x15” at 6” of water height) and challenge with Aeromonas hydrophila which was cultured and maintained using NB at 37°C, 200 rpm for 24 h. The bacterial suspension with a final concentration of 10⁵-10⁸ CFU mL^-1 (300 mL) was added to each tank. The number of survival shrimps was recorded daily until 0% survival was reached in any treatment.

RESULTS

Low cost medium formulation for spore productions of Bacillus KKU02 and KKU03: Four abundant agricultural substrates which were sweet potato (Impomoea batatasil), cassava root (Manihot esculenta), rice (O. sativa Linn.) and sticky rice (O. sativa var. glutinosa), were added to the tested media as carbon sources in comparison to Nutrient Broth (NB). The results were shown in Fig. 1. It was found that all substrates could support the spore productions of Bacillus KKU02 and Bacillus KKU03 better than without adding any agricultural substrates (the control group).


Fig. 1(a-b):	Spore production of Bacillus (a) KKU02 and (b) KKU03 when using different agricultural substrates as carbon sources

It could be seen that only cassava root and sweet potato supplementation gave an equal or better spore concentration than the nutrient broth. However, when consider the price of cassava and sweet potato, the cooked cassava root was the optimum carbon source for Bacillus KKU02 and Bacillus KKU03 spore productions which showed the highest concentration of Bacillus KKU02 and Bacillus KKU03 spores at 8.32 and 1.35x 10⁸ spores mL^-1, respectively.

Supplementation of cooked cassava medium with mineral salts, 0.1 g L^-1 MgSO₄ and 2.0 g L^-1 (NH₄)₂SO₄, resulted in 1.75 times increase of spore productions in both Bacillus strains (Fig. 2). The highest concentrations of Bacillus KKU02 and Bacillus KKU03 spores were 1.62x10⁸ and 6.61x10⁷ spore mL^-1, respectively.

Various cassava concentrations (50, 100 and 200 g L^-1) for spore production were studied in order to obtain the optimum cassava concentration for spore production.


Fig. 2(a-b):	Spore production of Bacillus (a) KKU02 and (b) KKU03 ) when supplementation with mineral salts in the cassava medium

The optimum cassava concentration for spore production of both Bacillus strains was 100 g L^-1 which showed the highest spore concentrations of Bacillus KKU02 and Bacillus KKU03 at 1.78x10⁸ and 1.48x10⁸ spores mL^-1, respectively (Fig. 3).

Giant freshwater prawn production using Bacillus KKU02 and Bacillus KKU03 in spore form as probiotic: Water quality during shrimp cultivation was also concerned in this study. The range of water quality parameters during experimental period in each prawn culture tank were shown in Table 1. All of the measured parameters which were pH, temperature, %DO, the amount of ammonium, hardness and alkalinity were in the ranges of 7.02-8.72, 23-30°C, 4.0-8.5%, 0-0.25, 120-250 and 80-180 ppm, respectively. These results were in the acceptable ranges suggested by Armstrong et al. (1976), New (1990) and Boyd and Zimmerman (2000).

The rearing prawns with the probiotic in six feed treatments for 90 days showed significant differences (p≥0.05) of body weight and length gain between T1, T2, T3, T4, T6 and the control group (T5) during probiotic feeding, as shown in Fig. 4.


Fig. 3(a-b):	Spore production of Bacillus (a) KKU02 and (b) KKU03 at various cassava concentrations

Table 1:	Physical and chemical water quality parameters ranges during shrimp cultivation with 6 feed treatments of pure and mixed cultures of Bacillus KKU 02 and KKU03


Fig. 4(a-b):	(a) Body weight and (b) Length of giant freshwater prawn after rearing for 90 days in six feed treatments

The prawns fed with mixed culture of spore form (T3) exhibited the highest body weight and length at 59.75 g and 13.50 cm, respectively. On the other hand, the body weight and length of the probiotic treatment groups T1, T2, T4 and T6 were not significantly different from each other (p≥0.05).

Challenge test of the treated giant freshwater prawn: After 3 days of post challenge with A. hydrophila, 50% of shrimps in the control group (T5 without any probiotic supplementation) were dead while more than 70% of shrimps in all probiotic treatment groups still survive, as shown in Fig. 5.


Fig. 5:	Percentage of survival in challenged prawn after challenging prawns with A. hydrophila for 7 days compared to the control group

All shrimps in the control treatment (T5) could not survive after 7 days of the challenge. In contrast, the survival rate of prawn in mixed culture treatments both spore and vegetative cell, could survived more than 45% at day 7 of the challenge which were comparable or even better than the commercial probiotic. The lower percentages of shrimp survival in T1 and T2 after 3 days of the challenge were because some shrimps were dead during molting.

DISCUSSION

Low cost medium formulation for spore productions of Bacillus KKU02 and KKU03: Bacillus spores have been reported as probiotic uses both in human and animal from a lot of scientists (Hoa et al., 2001; La Ragione et al., 2001; Casula and Cutting, 2002; La Ragione and Woodward, 2003; Hong et al., 2005; D’Arienzo et al., 2006; Hong et al., 2008; Cutting, 2011). Bacillus KKU02 and KKU03 were reported earlier in using as probiotic in giant freshwater shrimp (Deeseenthum et al., 2007). In order to extend the shelf life of these 2 Bacillus isolates, their spore forms were investigated. In order to use spores as probiotic for feeding shrimp, mass production of spore must be considered, including cheap medium formulation for spore production. Thailand has a lot of cheap agricultural products which could be used for bacteria cultivation. Four abundant agricultural products which were sweet potato, cassava root, rice and sticky rice, were tested in order to obtain the best substrate for spore productions of Bacillus KKU02 and KKU03. Fortunately, cassava which was the cheapest agricultural substrate, was the optimum substrate for our Bacillus spore productions. This could be seen from the obtained spore concentrations which were comparable to those obtained from the Nutrient broth. The control group which contained only dextrose, showed the lowest spore number, possibly because of the limitation of carbon source.

Some minerals, such as calcium magnesium and manganese, have been reported in enhancing spore formulation of some Bacillus sp. (Amaha et al., 1956; Curran, 1958, Kolodziej and Slepecky, 1964; Cote and Gherna, 1999; Monteiro et al., 2005; Dechmahitkul et al., 2007; Omer, 2010). In our previous studies, MgSO₄ and (NH₄)₂SO₄ were used as mineral and nitrogen sources in culturing Bacillus KKU 02 and KKU 03 (Deeseenthum et al., 2007). Thus, these 2 compounds were tested by adding in the cassava medium for spore production. The results showed that the spore concentration was increased 1.7 times, compared to the medium without supplementation.

The concentration of carbon source was also important in spore production. Kang et al. (1992) reported that when glucose higher than 200 g L^-1, Bacillus thuringienesis could not produce its spore. The concentration of cassava in the spore production medium was, thus, examined. The results showed that reducing the cassava to 100 g L^-1 could still get the spore concentration equivalent to when 200 g L^-1 were used.

Thus the optimum conditions for spore production of Bacillus KKU02 and KKU03 were cassava 100 g L^-1, dextrose 20 g L^-1, MgSO₄ 0.1 g L^-1 and (NH₄)₂SO₄ 2.0 g L^-1.

Giant freshwater prawn production using spore of Bacillus KKU02 and Bacillus KKU03 as probiotic: Water qualities, such as pH, %DO, the amount of ammonium, hardness and alkalinity, had some effects on shrimp growth (Armstrong et al., 1976; New, 1990; Boyd and Zimmerman, 2000). Water in the culture tanks was changed once a week in order to make sure that water had no effect on the death and could be used for culturing shrimps. The results showed that water quality during shrimp cultivation was in the acceptable range. Thus, growth performance of the cultured shrimps was affected by the studied Bacillus probiotic.

After feeding shrimps with probiotic, both in vegetative cells and spore forms of Bacillus KKU02 and KKU03 it was found that the weight of the shrimps was significantly higher than feeding only the commercial feed, as shown in Fig. 4, although the shrimp length in all treatments, except in T3, was not significantly different. In addition, the mixed cultures, both live cells and spore forms, exhibited the better results than using pure spore cultures and the commercial probiotic. These results were similar to our previous report when live cells were used (Deeseenthum et al., 2007). This was possibly because the mixed probiotic Bacillus sp. enhanced nutrients utilization in shrimps, as these 2 isolates of Bacillus could produce amylase and protease (Deeseenthum et al., 2007). Moreover, these results also indicated that the spore forms of these two isolates of Bacillus gave the same results when the vegetative cells were used. Thus, the spore of these 2 Bacillus isolates could also be used as probitics. The pure Bacillus spore of both stains, in addition, showed the equivalent obtaining weight to the commercial probiotic treatment. These results indicated that our Bacillus stains had a high potential for commercialization.

Challenge test of the treated giant freshwater prawn after raring for 90 days: The survival of probiotic fed shrimps was enhanced after challenging with A. hydrophila, especially in the mixed culture, confirming the advantages of probiotic use. This was possibly because of the immune stimulation or pathogen growth inhibition by the probiotic Bacillus sp. which was reported by a number of investigators (Sakai et al., 1995; Itami et al., 1998; Moriarty, 1998; Rengpipat et al., 2000; Bachere, 2003; Vaseeharan and Ramasamy, 2003; Balcazar et al., 2006; Pandiyan et al., 2013). The low survival of shrimps in the pure spore cultures of Bacillus KKU02 (T1) and KKU03 (T2) after 3 days of challenge was possibly because the shrimps accidentally molted during the challenge test. The molting shrimps were easily attacked by the healthy shrimps and easily to be infected by the pathogen. The mixed culture, both live (T3) and spore (T4) forms, showed the highest percentage of survival which was better than the commercial probiotic (T6). These results confirmed that our mixed Bacillus culture had a high potential in commercial use. However, more aspects in using these two isolates of Bacillus, such as the safety and production cost, have to be studied further.

ACKNOWLEDGMENTS

The authors would like to thank the Office of the Higher Education Commission, Thailand for supporting by grant fund under the program “Strategic Scholarships for Frontier Research Network for the Join Ph.D. Program Thai Doctoral degree” for this research, Graduate School, Khon Kaen University and Agricultural Biotechnology Research Center for Sustainable Economy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand, for the financial support of this research. Thank you to the Department of Biotechnology, Faculty of Technology, Khon Kaen University and the Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen, Thailand and the Department of Environmental Science, Faculty of Science, Udon Thani Rajabhaj University, Udon Thani, Thailand for providing facilities for this research.