African Journal of Biotechnology Vol. 8 (14), pp. 3369-3376, 20 July, 2009
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals

Full Length Research Paper

Effect of Bacillus subtilis on the growth and survival rate of shrimp (Litopenaeus vannamei)
Hadi Zokaei Far1, Che Roos B. Saad1*, Hassan Mohd Daud2, Sharr Azni Harmin1 and Shahram
Shakibazadeh1
1

Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, university Putra Malaysia, 43400 Serdang,
Selangor, Malaysia.

2

Accepted 17 March, 2009

The effect of Bacillus subtilis, isolated from digestive tract of Macrobrachium rosenbergii was investigated on growth and survival rate of Litopenaeus vannamei during 60 days of culture. Sixteen aquaria with four replicates were used for treatments and controls. Treatment groups were consisted of
) shrimp fed diet with B. subtilis (T1), and ii) shrimp fed diet mixed with B. subtilis and commercial probiotic (T2). Control groups were consisted of ) shrimp fed diet with commercial probiotic as positive control, and ii) shrimp fed unaltered diet as negative control. Results showed that B. subtilis was proliferated in digestive tract of treated shrimps, and the number of Vibrio spp. was reduced in digestive tract during the cultural period. Survival rate, 75.5± 4.62 %, and yields of shrimps, 190.00 ±
13.13 g, treated with B. subtilis were significantly greater (P 0.05) was found between aquaria with or without the addition of bacterial supplement for the other parameters; final mean weight, SGR,
FCR, weight gain, and daily gain (Table 1). It seemed that shrimps treated in T1 sacrificed some of the growth performance due to be higher density (P < 0.05) compared to other groups or perhaps it was due to decreasing of survival rate of T2, T3, and T4 during the first two weeks of experiment, because according to the general regulation of aquaculture it was expected that the treatments with lower population densities (T2, T3 and T4) ex-

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b

Mean weight

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80
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Figure 1. The mean yield of shrimp (a), and mean survival rate (%) (b), of L. vannamei by using diets supplemented with B. subtilis (T1), commercial probiotic and B. subtilis (T2), positive control (T3), and negative control (T4).

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40
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M ean survival rate (%)

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Figure 2. Evaluation of survival rate during cultural period for treatment groups (B. subtilis; T1, commercial probiotic and B. subtilis; T2), and control groups (positive control; T3, and negative control; T4).

hibits higher individual growth compared to those with higher population densities (T1). Therefore, by this actual hypothesis, parameters of final mean weight, SGR, FCR, weight gain, daily gain of T2, T3, and T4 should have shown greater results compared to T1, while it is interesting to note that the obtained results were indicated that there is no significant differences (P > 0.05) for these parameters between all the treated and control groups. It is highly recommended that this similarity of growth parameter results which is in contrast with the expectation is due to the T1 efficiency, otherwise, at least the final mean weight of the two treatments (T1 and T4) with 30% differrence in density would be alter.
Bacteriological study
Total bacterial count in digestive tract of T1 (2.9 ± 0.59 ×

10
9
8
7
6
5
4
3
2
1
0

T1
T2
T3
T4

W0

W2

W4

W6

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Week
Figure 3. Log total B. subtilis count (CFU/g) in shrimp digestive tract during 60 days of culture with or without feed supplemented. B. subtilis (T1), commercial probiotic and B. subtilis (T2), positive control (T3), and negative control (T4).

11

10 CFU/g), was significantly higher than the other groups (Figure 8a). This significant difference started from the fourth week of experiment and it was continued until the end of this study. In the beginning of the experiment there were no observed Bacillus spp. in digestive tract of shrimps, but they were appeared in all treated shrimps with different concentration and finally, there were significant differences for B. subtilis counted bet8 ween T1 (6.8 ± 0.05 × 10 CFU/g) and the other treated
4
groups; T2 (4.5 ± 0.23 × 10 CFU/g) and T3 (4.2 ± 0.29 ×
4
10 CFU/g), (Figure 3). No significance differences (P >
0.05) were observed for Vibrio spp. counted between all treated groups and the control groups (Figures 4 and 5).
Total bacterial count in digestive tract of T1 ranged
4
11 from 5.9 × 10 to 2.9 ± 0.59 × 10 CFU/g while this con4 centration for T2, T3 and T4 ranged from 5.9 × 10 to 9.7

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Log total count (CFU/g)

Log total count (CFU/g)

Far et al.

Week

Week

Log total count (CFU/g)

Log total count (CFU/g)

Figure 4. Log total viable bacteria, B. subtilis, Vibrio spp. CFU/g in shrimp digestive tracts during 60 days of culture with different feed diet supplement (a: T1, b: T2).

Week

Week

Figure 5. Log total viable bacteria, B. subtilis, Vibrio spp. CFU/g in shrimp digestive tracts during 60 days of culture with different feed diet supplement (a: T3, b: T4).

8

4

8

4

± 0.18 × 10 , 5.9 × 10 to 5.9 ± 0.16 × 10 and 5.9 × 10
9
to 2.9 ± 0.16 × 10 CFU/g, respectively (Figures 4, 5 and
8a). Mean concentration of Vibrio spp. in digestive tract for all the treatments and control groups ranged from 1.1
4
3
× 10 to 2.8 ± 0.28 × 10 CFU/g (Figures 4 and 5). As indicated in figures, the number of Vibrio spp. was decreasing in digestive tract of all the treated and control groups. Total bacterial counts in rearing waters aquaria were
4
9
4
ranged from 2.8 × 10 to 8.4 ± 0.16 × 10 , 2.8 × 10 to 1.9
9
4
8
4
± 0.31x 10 , 2.8x 10 to 3.9 ± 0.36 × 10 and 2.8 × 10 to
8
4.7 ± 0.24 × 10 CFU/ml for T1, T2, T3 and T4, respecttively (Figures 6 and 7). There were significance differrences for total viable bacterial counted in rearing water

of T1 and the other groups from day 45 to 60 (Figure 8b).
During the fourth and sixth week of culture, Vibrio spp. concentrations in the shrimp rearing water of T3 and T4 were significantly higher than T1 and T2 and in the end of experiment it was significantly higher for T3 (3.1 ± 0.25 ×
4
10 CFU/ml) compared to other groups. The number of
Vibrio spp. decreased in rearing tank waters of all the treated and control groups (Figures 6 and 7). B. subtilis was only found in rearing water of probiotic treatments
3
from day 30 to 60, and ranged from 4.4 ± 0.22 × 10 to
4
3
3
6.2 ± 0.04 × 10 , 1.5 ± 0.21 × 10 to 5.6 ± 0.15 × 10 and
3
3
3.5 ± 0.29 × 10 to 3.3 ± 0.24 × 10 for T1, T2 and T3, respectively (Figures 6 and 7). Low CFU number of B. subtilis in the rearing water tanks of T4 was counted (>30

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Log bacterial count (CFU/ml)

Afr. J. Biotechnol.

Log bacterial count(CFU/ml)

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Week

Week

Log bacterial count (CFU/ml)

Log bacterial count (CFU/ml)

Figure 6. Log total bacteria, B. subtilis, Vibrio spp. CFU/ml in shrimp rearing water during 60 days of culture with different feed diet supplement (a: T1, b: T2).

Week

Figure 7. Log total bacteria, B. subtilis, Vibrio spp. CFU/ml in shrimp rearing water during 60 days of culture with different feed diet supplement (a: T3, b: T4).

CFU/plate) (Figure 7b).
DISCUSSION
It is important to provide healthy shrimp with higher production and probiotics has a great deal of potential
(Gomez-Gil et al., 2000). Moriarty (1995, 1998) added
Bacillus spp. as probiotic in the penaeid shrimp ponds; the result of this study shows increasing survival rate and decreasing of luminous Vibrio densities in the pond water. In P. monodon, Bacillus used as a probiotic was able to colonize both the culture water and the shrimp digestive tract; the Bacillus also was able to replace
Vibrio spp. in the gut of the shrimp, thereby increasing shrimp survival (Rengpipat et al., 1998a). Similar results

were obtained in the present study. Significant differrences for survival rate (P < 0.05) in this study were observed in shrimps treated with B. subtilis (T1) compared to the other groups. Also significant differences (P
< 0.05) for shrimp yield between T1 and the other experimental groups could be a great deal to determine the positive effect of bacteria B. subtilis on growth rate of treated shrimp (T1), however, there were no observed significant differences (P > 0.05) for growth parameters according to the results (Table 1). Therefore in competition on increasing growth between all the experimental groups, T1 had the greatest growth performance by considering the significant differences of survival rate.
According to results of this study it is highly recommended that constantly application of B. subtilis to shrimp culture with the optimal dose had influenced them to

Far et al.

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LL o g totall countt (CFU/ml) og to ta c o u n (C F U / m l )

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Figure 8. Log total viable bacterial count in (a) shrimp digestive tract (CFU/g), and (b) shrimp rearing water
(CFU/ml), during 60 days of culture with or without feed supplemented. B. subtilis (T1), commercial probiotic and
B. subtilis (T2), positive control (T3), and negative control (T4).

achieve the optimal growth rate and the greatest survival rate compared to the other experimental groups, and finally the greatest shrimp yield was obtained, which is the most important concern in the shrimp culture industry.
Effects of commercial probiotic on aquaculture has been investigated by researchers, and some of these research have not shown any positive effects on growth parameters or survival rate or any promising result on the cultural condition. For instance, Shariff et al. (2001) found that treatment of P. monodon with a commercial Bacillus probiotic did not significantly increase survival. Dennis et al. (2000) used a commercial bacteria supplement to culture on L. vannamei in outdoor tanks with no water exchanging. This study did not improve shrimp survival rate, mean final weight and FCR, or the quality of the shrimp tank water. Samocha et al. (1998a,b) tested a commercial probiotic with P. setiferus, and no promising data for growth parameters or water quality was obtained.
Commercial probiotic as positive control, applied in the present study, did not improved growth performance of the related shrimp (T2 and T3) compared to T4, while it was expected that commercial probiotic used in this study should had shown a great promotion on L. vannamei culture due to be high density of bacteria inside [L.
6
6 acidophilus (10 CFU/ml) B. subtilis (10 CFU/ml)
6
L.plantarum (10 CFU/ml)]. It is difficult to directly assess different studies using probiotics, because the efficacy of a probiotic application depended on many factors
(Gomez-Gil et al., 2000) such as species composition, application level, frequency of application and environmental conditions. This indicates that the quantity of probiotics was only one of the factors promoting the growth or survival rate of shrimp larvae (Zhou, 2009).
Bacillus bacteria are able to out-compete other bacteria for nutrients and space and can exclude other bacteria through the production of antibiotics (Moriarty, 1998;
Verschuere et al., 2000). Many different antibiotic compounds are produced naturally by a range of Bacillus

species, and it appears that other bacteria would be unlikely to have resistance genes to all of the antibiotics produced by the Bacillus probionts (Moriarty, 1998).
Bacillus administration also has been shown to increase shrimp survival by enhancing resistance to pathogens by activating both cellular and humoral immune defenses in shrimp (Rengpipat et al., 2000).
Result of total viable bacteria and B. subtilis bacteria counted in digestive tract and rearing water showed that there were significance differences (P < 0.05) between bacterial counted in T1 and the other groups. It seemed
B. subtilis was proliferated in digestive tract of shrimp in
T1. B. subtilis has been shown to produce a wide variety of antibacterial and antifungal compounds in culture media (Alexander, 1977; Katz and Demain, 1977; Korzybski et al., 1978). It produce novel antibiotics such as difficidin and oxydifficidin that have activity against a wide spectrum of aerobic and anaerobic bacteria (Zimmerman et al., 1987) as well as more common antibiotics such as bacitracin, bacillin, and bacillomycin B (Parry et al.,
1983). It is expected that gram-negative bacteria were replaced with Bacillus probiont (Austin et al., 1995).
Rengpipat et al. (1998) showed that Bacillus S11 is harmless for shrimp and culture system, while providing greater survival during normal culture period and following disease challenge by luminescent bacterial challenges. This suggests that probiotic treatment is an effective alternative for enhancing shrimp health.
The findings of this study show the presence of viable
B. subtilis in digestive tract and rearing water of shrimp fed with it (Figures 4, 5, 6, and 7). B. subtilis was colonized in rearing water while water quality was not affected by feed additives. However, as indicated in figures, there was a competitive exclusion between B. subtilis and
Vibrio spp. in rearing water and digestive tract during the cultural period (Figures 4, 5, 6, and 7). It is clear that B. subtilis almost eliminated Vibrio spp. from digestive tract by action of competitive exclusion. Perhaps B. subtilis

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proliferated in digestive tract had promoted the wellbeing of shrimps or contributed to the direct or indirect protecttion of shrimp against harmful bacteria and provided the well growth performance and also the great survival rate.
However using of probiotics in aquaculture is still on perspective but this study suggests the using of B. subtilis as probiotic in culture with L. vannamei, while there is still need to consider all parameters which might affect and lead to loss of this bacteria during the cultural period.
ACKNOWLEDGEMENTS
The authors would sincerely like to express their thanks to Mr. Jasni MD. Yusoff for his technical help and providing many valuable suggestions and comments during the experimental period.
REFERENCES
Alexander M (1997). Introduction to soil microbiology. John Wiley and
Sons, Inc. New York. p. 418.
Anderson IG, Shamsudin MN, Nash G (1989). A preliminary study on the aerobic heterotrophic bacteria flora in giant fresh water prawn,
Macrobrachium rosenbergii, hatcheries in Malaysia. J. Aquacult. 81:
213-223.
Austin B, Stuckey LE, Robertson PAW, Effendi I, Griffith DRW (1995). A probiotic strain of Vibrio alginolyticus effective in reducing disease caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalli. J. Fish Dis. 18: 93-96.
Avakh KM (2006). Assessment of putative bacteria as probiotics in juvenile freshwater prawn, Macrobrachium rosenbergii. Unpublished doctoral dissertation, University of Putra, Malaysia.
Boyd CE, Tucker CS (1998). Pond Aquaculture Water Quality
Management. Kluwer Academic Publishers, USA.
McIntosh D, Samocha TM, Jones ER, Lawrencea AL, McKee DA,
Horowitz S, Horowitzc A (2000). The effect of a commercial bacterial supplement on the high-density culturing of Litopenaeus vannamei with a low-protein diet in an outdoor tank system and no water exchange. Aquacult. Eng. 21: 215-227.
Felix N, Sudharsan M (2004). Effect of glycine betaine, a feed attractant affecting growth and feed conversion of juvenile freshwater prawn
Macrobrachium rosenbergii. J. Aquacult. Nutr. 10: 193-197.
Fuller R (1989). A review: probiotics in man and animals. J. Appl.
Bacteriol. 66: 365-378.
Gildberg A, Mikkelsen H (1998). Effects of supplementing the feed to atlantic cod (Gadus morhua) fry with lactic acid bacteria and immunostimulating peptides during a challenge trail with Vibrio anguillarum.
J. Aquacult. 167: 103-113.
Gomez-Gil B, Roque A, Turnbull JF (2000). The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms.
Aquacult. 191: 259-270.
Kamarudin MS, Jones DA, Vay L, Abidin AZ (1994). Ontogenetic changes in digestive enzyme activity during larval development of
Macrobrachium rosenbergii. J. Aquacult. 123: 323-333.
Katz E, Demain AC (1977). The peptide antibiotics of Bacillus:
Chemistry, biogenesis, and possible functions. Bacteriol. Rev. 41:
449-474.
Korzybski T, Kowszyk-Gindifer Z, Kurylowicz W (1978). Antibiotic isolated from the genus Bacillus (Bcillaceae). In: Antibiotic - origin,
Nature and properties. Am. Soc. Microbiol. Washington, DC. 3: 15291661.
Moriarty DJW (1998). Control of luminous Vibrio species in penaeid aquaculture pond. Aquacult. 164: 351-358.

Moriarty DJW, Body AGC (1995). Modifying microbial ecology in ponds: the key to sustainable aquaculture, p. 1-10. In Proceedings of Fish
Asia ’95 Conference: 2nd Asian Aquaculture and Fisheries Exhibition and Conference. RAI Exhibitions, Singapore, Singapore.
Moriarty DJW, Body AGC (1995). Modifying microbial ecology in ponds: the key to sustainable aquaculture. pp. 1-10.
Parker RB (1974). Probiotics, the other half of the antibiotics story. J.
Anim. Nutr. Health. 29: 4-8.
Parry JM, Turnbull PCB, Gibson JR (1983). A colour atlas of Bacillus species. Wolf Medical Publication p.186. Lt., London.
Rengpipat S, Phianphak W, Piyatiratitivorakul S, Menasaveta P (1998).
Effects of a probiotic bacterium in black tiger shrimp Penaeus monodon survival and growth. J. Aquacult. 167: 301-313.
Rengpipat S, Rukpratanporn S, Piyatiratitivorakul S, Menasaveta
(2000). Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). J. Aquacult. 191:
271-288.
Robertson PAW, O'Dowd C, Burrells C, Williams P, Austin B (2000).
Use of Carnobacterium sp. as a probiotic for Alantic salmon, salmo salar L. and rainbow trout Oncorhynchus myksis, Walbaum. J.
Aquacult. 185: 235-243.
Samocha TM, Lawrence AL, Horowitz A, Horowitz S (1998). The use of commercial probiotics in the production of marine shrimp under no water exchange. The Second International Conference on
Recirculating Aquacult. July 1998, Virginia Polytechnic Institute, pp.
373-375.
Samocha TM, Lawrence AL, Horowitz A, Horowitz S (1998).
Commercial bacterial supplement-its potential use in the production of marine shrimp under no water exchange. In: Jory DE (Ed.),
Proceedings of the First Latin American Shrimp Culture Congress &
Exhibition, October 1998, Panama City, Panama.
Shariff M, Yusoff FM, Devaraja TN, Srinivasa Rao SP (2001). The effectiveness of a commercial microbial product in poorly prepared tiger shrimp, Penaeus monodon (Fabricius), ponds. Aquacult. Res.
32: 181-187.
Sugita H, Hirose Y, Matsuo N, Deguchi Y (1998). Production of the antibacterial substance by Bacillus sp. strain NM12, an intestinal bacterium of Japanese coastal fish. J. Aquacult. 165: 269-280.
Sugita H, Tanaami H, Kobashi T, Deguchi Y (1981). Bacterial flora of coastal bivalves. Bull Jpn. Soc. Sci. Fish, 47: 655-661.
Uma A, Abraham TJ, Sundararaj V (1999). Effect of a probiotic bacterium, Lactobacillus plantarum on disease resistance of Penaeus indicus larvae. Indian J. Fish. 46(4): 367-373.
Venkat HK, Sahu NP, kamal K Jain (2004). Effect of feeding
Lactobacillus-based probiotic on the gut microflora, growth and survival of postlarva of Macrobrachium rosenbergii (de Man). J.
Aquacult. Res. 35: 501-507.
Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000). Probiotic bacteria as biological control agents in aquaculture. J. Microbiol. Mol.
Biol. Rev. 64: 655-671.
Wang YB, Xu ZR, Mei-Sheng X (2005). The effectiveness of commercial probiotics in northern white shrimp Penaeus vannamei ponds. Fish. Sci. 71: 1036-1041.
Zimmerman SB, Schwartz CD, Monaghan RL, Pleak BA, Wiessberger
B, Gilfillan EC, Mochales S, Hernandez S, Currie SA, Tejera E,
Stapley EO (1987). Dfficidin and oxydifficidin: Novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. J. Antibiotics,
40(12): 1677-1681.
Zhou X, Wang Y, Li W (2009). Effect of probiotic on larvae shrimp
(Penaeus vannamei) based on water quality, survival rate and digestive enzyme activities. Aquaculture, 287: 349-353.
Ziaei-Nejad S, Habibi Rezaei M, Azari Takami Gh, Lovett LD,
Mirvaghefi AR, Shakouri M (2006). The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquacult.
252: 516-524.