Veterinary Microbiology 159 (2012) 443450

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Potential Bacillus probiotics enhance bacterial numbers, water quality

and growth during early development of white shrimp (Litopenaeus
vannamei)
Subuntith Nimrat a,*, Sunisa Suksawat b, Traimat Boonthai c, Verapong Vuthiphandchai d
a
Department of Microbiology and Environmental Science Program, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
b
Environmental Science Program, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
c
Biological Science Program, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
d
Department of Aquatic Science, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand

A R T I C L E I N F O A B S T R A C T

Article history: Epidemics of epizootics and occurrence of multiresistant antibiotics of pathogenic bacteria
Received 15 January 2012 in aquaculture have put forward a development of effective probiotics for the sustainable
Received in revised form 18 April 2012 culture. This study examined the effectiveness of forms of mixed Bacillus probiotics
Accepted 20 April 2012 (probiotic A and probiotic B) and mode of probiotic administration on growth, bacterial
numbers and water quality during rearing of white shrimp (Litopenaeus vannamei) in two
Keywords: separated experiments: (1) larval stages and (2) postlarval (PL) stages. Forms of Bacillus
Bacillus probiotics and modes of probiotic administration did not affect growth and survival of
Probiotics
larval to PL shrimp. The compositions of Bacillus species in probiotic A and probiotic B did
Litopenaeus vannamei
not affect growth and survival of larvae. However, postlarvae treated with probiotic B
Growth
Water quality
exhibited higher (P < 0.05) growth than probiotic A and controls, indicating Bacillus
probiotic composition affects the growth of PL shrimp. Total heterotrophic bacteria and
Bacillus numbers in larval and PL shrimp or culture water of the treated groups were higher
(P < 0.05) than in controls. Levels of pH, ammonia and nitrite of the treated shrimp were
signicantly decreased, compared to the controls. Microencapsulated Bacillus probiotic
was effective for rearing of PL L. vannamei. This investigation showed that administration
of mixed Bacillus probiotics signicantly improved growth and survival of PL shrimp,
increased benecial bacteria in shrimp and culture water and enhanced water quality for
the levels of pH, ammonia and nitrite of culture water.
2012 Elsevier B.V. All rights reserved.

1. Introduction
White shrimp (Litopenaeus vannamei) are commercially
cultivated in Thailand since 2001 to replace Penaeus
monodon destroyed by viral disease. Production of white
shrimp reached nearly 400,000 tons in 2006 and repre-
sented over 98% of total shrimp production in Thailand
(Wyban, 2007). Marine shrimp aquaculture in Thailand has
suffered also from opportunistic and pathogenic bacteria
* Corresponding author. Tel.: +66 38 103 120; fax: +66 38 393 490.
E-mail address: subunti@buu.ac.th (S. Nimrat).
such as Vibrio sp. and viruses (Flegel, 2006). Excessive use
of antimicrobial agents for disease control has resulted in
evolution of multi-antibiotic resistance of pathogenic
bacteria and subsequent prohibition of chloramphenicol
and the group of nitrofurans in shrimp culture because of
their potential harmful effects on human health (Serrano,
2005). Therefore, benecial bacteria called probiotics
provide an alternative approach to chemotherapeutic
agents to sustainable aquaculture as well as being
environment-friendly (Balcazar et al., 2006; Utiswannakul
et al., 2011).
Probiotics provides advantages to their hosts including
enhancement of digestive enzymatic activity, activation of

0378-1135/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2012.04.029
444 S. Nimrat et al. / Veterinary Microbiology 159 (2012) 443450
immune defense, inhibitory activity against pathogen,
promotion of growth and survival, improvement of water
quality and biodegradation of pond bottom soil containing
organic sludge (Balcazar et al., 2006; Nimrat et al., 2008;
Cerezuela et al., 2011; Utiswannakul et al., 2011). Foremost
among probiotics in aquaculture are photosynthetic
bacteria, yeast, Lactobacillus, Vibrio and Pseudomonas,
especially Bacillus are also applied in aquaculture (Sahu
et al., 2008; Utiswannakul et al., 2011).
There are various problems related with probiotic
products containing Bacillus species available in Thailand
and other countries such as high cost and ineffectiveness
(Nimrat and Vuthiphandchai, 2011). Recently, Nimrat et al.
(2011) developed an effectively novel probiotic for rearing
of L. vannamei through microencapsulated or freeze-dried
forms of microbial probiotics: bacteria, yeast and micro-
algae. Species composition of bacterial probiotics remains
to be investigated on their effect of growth, survival and
microbiological aspects in order to increase efcacy and
reduce complication of production processes. The purpose
of this study was to investigate the efcacy of species
composition of Bacillus as microencapsulated and freeze-
dried forms on growth, bacterial numbers and water
quality during rearing of L. vannamei larvae and postlarvae.
2. Materials and methods
2.1. Experimental shrimp
Vibrio harveyi-free L. vannamei nauplii were obtained
from a private hatchery located in Chon Buri Province,
Thailand, and transported to the hatchery of Department of
Aquatic Science, Burapha University. Nauplii were held at
100 per liter in 500-l ber glass tanks containing 30%
seawater disinfected with 0.01 g l 1 of chlorine. Each day
prior to the beginning of experiments, nauplii were fed
microalgae, Chaetoceros, at 106 cells ml 1 and 10% of
rearing seawater was exchanged. Weight and total length
of shrimp larvae were measured on a digital balance
(Mettler Toledo, AT 200, Greifensee, Switzerland) and
vernier caliper, respectively, before experiments.
2.2. Probiotic administration and cultivation
Two probiotic products in this study consisted of Bacillus
strains (probiotic A and probiotic B). Bacteria in both products
were isolated from P. monodon intestine and cultured
environments in Chon Buri Province, Thailand in 2005 using
spread plating method. Bacillus probiotics was chosen on a
basis of biodegradation capacity of nutrients (protein, lipid
and starch) and in vitro inhibitory ability of pathogenic V.
harveyi isolated from infected shrimp during vibriosis
outbreak at Chanthaburi Province, Thailand (Nimrat et al.,
2008). Two probiotic products were effective for P. monodon
culture by reducing Vibrio and improving growth perfor-
mance (Boonthai et al., 2011). Species identication of Bacillus
was determined by morphological and biochemical char-
acteristics and genetic identication based on 16S rRNA gene
sequencing. Probiotic A contained Bacillus thuringiensis BUU
001, Bacillus megaterium BUU 002, Bacillus polymyxa BUU 003,
Bacillus licheniformis BUU 004 and Bacillus subtilis BUU 005
while Probiotic B consisted of B. subtilis BUU 006, B. polymyxa
BUU 007, B. megaterium BUU 009, Bacillus circulans BUU 010
and Bacillus pumilus BUU 012. Each strain of probiotic was
grown in Trypticase Soy Broth (TSB; Difco, Detroit, MI, USA)
and stored in TSB with 300 ml l 1 glycerol at 20 8C.
Each Bacillus strain grown on Trypticase Soy Agar
(Difco, Detroit, MI, USA) was separately inoculated into a
500-ml ask containing 200 ml of TSB and shaken at
200 rpm, 30 8C for 24 h. Bacterial cell suspensions were
harvested by centrifugation at 8228  g, 4 8C for 5 min
(Nimrat et al., 2008, 2011). The cell pellets were collected
and washed three-time with sterilized 1 g l 1 Peptone
Water, pH 7.0. Then, they were separately resuspended in
the Peptone Water and spectrophotomecally adjusted to
1.5 at an O.D.580 (approximately 1010 CFU ml 1) for
subsequent production of freeze-dried probiotics and
microencapsulated probiotics.
2.3. Preparation of freeze-dried probiotics
Bacterial suspensions of each species were separately
freeze-dried according to Boonthai et al. (2011). Briey,
cell suspensions were mixed with 200 g l 1 skim milk and
freeze-dried in the freeze dryer (Flexi-DryTM, Stone Ridge,
NY, USA) at 40 8C for 60 h. Enumeration of total counts of
viable freeze-dried bacteria was determined by the spread
plate technique. Freeze-dried bacteria were diluted serially
with 1 g l 1 Peptone Water and 0.1 ml of each dilution was
pipetted aseptically onto Plate Count Agar (PCA; Difco,
Detroit, MI, USA) and incubated at 30 8C, 2448 h. Freeze-
dried powder was kept in sterilized containers.
2.4. Production of microencapsulated probiotics
All strains of bacteria probiotics were separately
microencapsulated using the protocol developed by
Nimrat et al. (2011). A 25-ml cell suspension of bacteria
probiotics (approximately 1010 CFU ml 1) was homoge-
nized in 100 ml of 30 g l 1 sodium alginate solution (Fluka,
Steinheim, Germany). The alginate-cell mixture was
dispensed slowly down the side of a beaker containing
600-ml corn oil supplemented with 2 ml l 1 Tween 80 and
magnetically stirred at 200 rpm for 15 min. A calcium
chloride solution (100 ml of 0.1 mol l 1; Carlo Erba
Reagenti, Rodano, Italy) was added quickly and gently
along the side of a beaker until the oilwater emulsion was
broken. Microencapsulated beads were formed in the
aqueous phase within 5 min and removed by centrifuging
at 350  g, 4 8C for 5 min. Microencapsulated-bead suspen-
sions were ltered through Whatman No. 4 lter paper
under vacuum and the adhered beads were eluted with
1 g l 1 Peptone Water and again ltered through a 55-mm
net. Spherical microencapsulated beads with 38.81  11.10
to 40.60  11.24 mm in diameter were collected and re-
suspended in 1 g l 1 Peptone Water and kept at 4 8C in the
Peptone Water until use.
2.5. Artemia enrichment
Bioenrichment of Artemia was performed following the
protocol of Nimrat et al. (2011). Artemia franciscana (Crytal,
 S. Nimrat et al. / Veterinary Microbiology 159 (2012) 443450
Ocean Star International, Snowville, Utah, USA) cysts were
decapsulated and hatched in a tank containing 100 l of
sterilized seawater over 36 h. Seawater was maintained at
30% salinity and 2830 8C until harvest of nauplii. Thirty-
six-hour-old Artemia were enriched separately with ten
strains of microencapsulated probiotics for 6 h, an appro-
priate period for enrichment of Artemia with probiotics, at
a concentration of 109 CFU ml 1. Mild aeration passed
through a 0.45-mm membrane lter (Sartorius, Bedford,
MA, USA) was supplied into the enriching medium to
maintain oxygen level and mix thoroughly the micro-
encapsulated probiotics beads in the medium. Enriched
Artemia were carefully harvested for shrimp larvae
feeding.
2.6. Experimental setup
Shrimp were fed probiotic-enriched Artemia or admi-
nistered with microencapsulated or freeze-dried probio-
tics in the culture water of the two experiments. Animal
ethics have been approved prior to beginning of the
experiments.
2.6.1. Experiment 1
This experiment evaluated the effect of Bacillus
probiotics on larval stages. The experimental design was
completely randomized with 7 treatments in triplicate.
Each treatment was randomly assigned to triplicate glass
tanks (width  length  height = 0.2 m  0.4 m  0.25 m)
wrapped with black plastic to reduce light intensity. Each
tank was stocked with zoea stage 3 of shrimp (n = 1000;
100 zoea l 1). Water (10 l) was ltered through the cotton
lter at about 1 l min 1. An equal amount of each probiotic
species (109 CFU g 1) in forms of freeze-dried Bacillus (FB) or
microencapsulated Bacillus (MB) (T1T2 for probiotics A and
T4T5 for probiotics B) was introduced daily into culture
water to maintain a nal concentration of 109 CFU ml 1.
Shrimp larvae were fed with enriched Artemia (T3 and T6) or
nonenriched Artemia (T1T2, T4T5 and control) at a rate of
34 nauplii per shrimp larvae four-times daily at 6:00,
12:00, 18:00 and 24:00 h. Culture water was not discharged
during the experiment and Chaetoceros was added daily into
culture water at a xed 106 cells ml 1 in all treatments. The
salinity and temperature of culture water were maintained
at a range of 3031% and 3132 8C, respectively. Shrimp
survivals were recorded daily. At the 4th day after beginning
of the experiment, larval shrimp were randomly measured
for weight and length and bacterial numbers were
enumerated.
2.6.2. Experiment 2
The effect of Bacillus probiotic on postlarval (PL) stages
was examined in Experiment 2. Mysis 3 obtained from the
Experiment 1 were reared until reaching PL 1 stage. PL 1
shrimp were reared in the same glass tanks at a density of
500 per tank (50 postlarvae l 1). The experiment was
divided into 7 treatments as previously described. The
treatments and control were repeated in triplicate. One to
fourteen-day-old and fteen to twenty two-day-old PL
shrimp were fed nonenriched Artemia at 810 and 1520
nauplii per postlarvae, respectively, six-time daily at 6:00,
445
10:00, 14:00, 18:00, 22:00 and 02:00 h, except T3 and T6
using enriched Artemia previously described. A small
amount (10%) of culture water in all tanks was exchanged
daily during the experiment. Chaetoceros was added in the
culture water and salinity and temperature were also
maintained as previously described. Postlarvae at the
beginning of experiment and after rearing for 14 and 22
days were examined for growth, survival and bacterial
enumeration.
2.7. Measurement of shrimp growth and survival
Shrimp at zoea 3 and 4-day-old larvae of Experiment 1
and PL 1, 14-day-old and 22-day-old PL shrimp of
Experiment 2 in the treated and control groups were
assessed in triplicate for weight, length, growth rate and
survival rate. Weight and length were measured for 100
shrimp from each tank on a digital balance (Mettler Toledo,
AT 200, Greifensee, Switzerland) and vernier caliper,
respectively. Shrimp growth and survival rate were
calculated according to Nimrat et al. (2011).
2.8. Evaluation of bacterial numbers
The enumeration of bacteria was performed in tripli-
cate in shrimp at the same interval of growth and survival
determination. At each sampling, twenty shrimp larvae or
postlarvae were sampled from each tank, immersed
individually in 0.05 g l 1 formalin solution for 5 min to
eliminate the external bacteria and washed thoroughly
using sterilized water for 1 min to remove the disinfectant.
Whole shrimp were separately homogenized in 1 g l 1
Peptone Water and diluted serially by 10 fold-dilution
method with the same buffer solution. One hundred
milliliter of each dilution was plated onto PCA supple-
mented with 10 g l 1 NaCl for determination of total
heterotrophic bacteria (THB) and total Bacillus numbers by
spread plate standard method (Boonthai et al., 2011;
Nimrat et al., 2011). Water samples (50 ml per tank) were
collected from each of the four edges and the center of each
tank and evaluated for THB and total Bacillus numbers as
previously described. All petri dishes were incubated at
30 8C, 2448 h (Nimrat et al., 2008, 2011). All colonies of
Bacillus were identied using Gram staining, spore
staining, catalase test and other selected biochemical tests
described by Krieg and Holt (1984). All bacterial colonies
and Bacillus colonies were calculated as a colony forming
unit of THB and total Bacillus numbers, respectively.
2.9. Water quality analysis
Dissolved oxygen and pH were monitored in triplicate
daily at 05.00 and 14.00 h. using a regularly calibrated
Multi-Probe Model (Horiba, W-22XD, Kyoto, Japan).
Salinity and temperature were measured in triplicate by
salinometer (Atago, S/Mill, Kyoto, Japan) and thermo-
meter, respectively. Ammonia-nitrogen and phosphate
were measured (American Water Works Association and
Water Pollution Control Federation, 1980) also along with
nitrite-nitrogen (Strickland and Parsons, 1972) and
nitrate-nitrogen (AOAC, 2002).
446 S. Nimrat et al. / Veterinary Microbiology 159 (2012) 443450

2.10. Statistical analysis
Data were expressed as mean  standard deviation
(S.D.). A one-way analysis of variance (ANOVA) was applied
to compare treated groups and the control at a signicance
level of P < 0.05. Duncans New Multiple Range Test (DMRT)
was used to identify signicant differences of parameters in
the ANOVA (Duncan, 1955). All statistics were performed
using SPSS for Windows version 11.5 (SPSS, Chicago, USA).
3. Results
3.1. Growth and survival of larval and PL shrimp
In experiment 1, initial weight and length of zoea 3 did
not differ (P > 0.05) among treated and control groups. At
the end of experiment 1, percentages of weight and length
gain of larval shrimp in treated groups (T1T6) ranged
from 72.0  9.0 to 79.7  8.7% and 89.6  6.9 to 92.4  7.8%,
respectively, which were not signicantly different (P > 0.05)
from those of the controls. Survival rates of the treated groups
(70.9  1.5 to 72.5  0.7%) were not signicantly different
(P > 0.05) compared to that of the control (71.1  2.2%) (data
not shown).
In experiment 2, initial weight and length of PL 1 did not
differ (P > 0.05) among treated and control groups (Table
1). Postlarval shrimp in treatments with mixed freeze-
dried Bacillus (FB; T1 and T4), microencapsulated Bacillus
(MB; T2 and T5) or enriched Artemia (T3 and T6) had
signicantly higher (P < 0.05) survivals, percentages of
weight and length gain, average daily growth (ADG) and
specic growth rate (SGR) than those of controls (Table 1).
However, there was no signicant difference (P > 0.05) in
growth and survival in PL shrimp treated with the same
probiotic group (probiotic A or probiotic B).
Compositions of Bacillus species in probiotic B (T4T6)
had a profound effect on the percentages of weight and
length gain, ADG and SGR of PL shrimp, which were higher
(P < 0.05) than those of Bacillus in probiotic A (T1T3)
(Table 1). Specic growth rate (SGR) in the treatments with
probiotic B (T4T6) had average values between 19.5  0.0
and 19.6  0.1%, which were higher (P < 0.05) than those of
probiotic A (T1T3; 19.3  0.1 to 19.4  0.1%) and the control
(18.6  0.1%). However, survival rate of 22-day-old PL shrimp
in the treatments with probiotic A (80.7  0.6 to 81.1  0.8%)
and probiotic B (83.9  1.5 to 84.7  3.5%) were not different
(P < 0.05), but were higher (P < 0.05) than those of the
control (70.8  0.8%).
3.2. Bacterial number in shrimp larvae and postlarvae and
culture water
In experiment 1, numbers of THB, Bacillus and ratios of
Bacillus/THB number in zoea 3, 4-day-old larvae and
culture water in treatments with Bacillus probiotic
supplementation (T1T6) were signicantly higher
(P < 0.05) than those of controls (Table 2). However,
numbers of THB and Bacillus and ratios of Bacillus/THB
number in the culture waters of the treatments with
inoculation of Bacillus probiotics into culture water (T1T2
and T4T5) were signicantly higher (P < 0.05) than those
of treatments with enriched Artemia (T3 and T6) (Table 2).
No Bacillus were detected in zoea 3, 4-day-old larvae or in
the culture water of controls.
In experiment 2, numbers of THB, Bacillus and ratios of
Bacillus/THB number in treated shrimp (T1T6) at stages of
PL 1, 14-day-old and 22-day-old PL shrimp were sig-
nicantly higher (P < 0.05) than those of controls although
differences (P < 0.05) were not signicant in numbers
among treated groups (Table 3). Ratios of Bacillus/THB
numbers in the culture water among all treatment groups
(T1T6) were not different (P > 0.05), except those of PL 1
inoculated with Bacillus probiotics (T1T2 and T4T5),
which were signicantly higher (P < 0.05) than those of
enriched Artemia (T3 and T6) and control. Bacillus were not
detected in PL 1, 14-day-old and 22-day-old PL shrimp as
well as in the culture water of controls.
3.3. Water quality
There were no difference (P > 0.05) in temperature,
salinity, DO, nitrate and phosphate in the culture water of
the treated groups and controls. However, application of
either microencapsulated or freeze-dried probiotics via
enriched Artemia or water additive had a notable inuence
on pH, ammonia and nitrite values. In both experiments,
pH, ammonia and nitrite levels at 05:00 and 14:00 h for all
treatment groups ranged between 7.7  0.3 to 8.3  0.2,
0.32  0.01 to 0.38  0.02 mg l 1 and 0.05  0.00 to
0.07  0.01 mg l 1, respectively, which were signicantly

Table 1
Growth and survival of treated and untreated PL shrimp.

Treatment Survival Initial Final Length Initial Final Weight ADG SGR (%)
1
rate (%) length length gain (%) weight weight gain (%) (mg day )
(mm) (mm) (mg) (mg)

Control 70.8  0.8b 4.5  0.1a 19.9  0.8c 347.8  16.9c 0.2  0.0a 20.1  0.7c 8,852.7  447.3c 0.8  0.0c 18.6  0.1c
T1 80.7  0.6a 4.4  0.1a 22.2  0.5b 398.5  10.4b 0.2  0.0a 24.4  0.8b 10,710.4  345.8b 1.0  0.0b 19.4  0.0b
T2 80.9  1.0a 4.5  0.1a 21.9  0.6b 393.2  13.9b 0.2  0.0a 24.3  0.8b 10,658.9  335.4b 1.0  0.1b 19.4  0.1b
T3 81.1  0.8a 4.5  0.1a 22.2  0.6b 396.1  15.8b 0.2  0.0a 24.6  0.8b 10,795.6  318.0b 1.0  0.0b 19.3  0.1b
T4 83.9  1.5a 4.5  0.0a 23.7  0.7a 431.8  12.5a 0.2  0.0a 25.9  0.4a 10,987.0  248.8a 1.1  0.0a 19.6  0.1a
T5 84.3  3.1a 4.5  0.1a 23.6  0.7a 431.3  12.6a 0.2  0.0a 25.6  0.6a 11,256.7  462.7a 1.1  0.0a 19.6  0.1a
T6 84.7  3.5a 4.5  0.0a 23.5  0.7a 432.3  12.8a 0.2  0.0a 25.4  1.1a 11,032.7  283.7a 1.1  0.0a 19.5  0.0a

Data were expressed as mean  S.D. Means with different superscript indicate signicant difference (P < 0.05). T1: addition of mixed freeze-dried Bacillus (FB) of
probiotic A, T2: addition of mixed microencapsulated Bacillus (MB) of probiotic A, T3: addition of artemia enriched with mixed MB of probiotic A, T4: addition of
mixed FB of probiotic B, T5: addition of mixed MB of probiotic B, T6: addition of artemia enriched with mixed MB of probiotic B and control: no addition of
probiotics, ADG: average daily weight gain, SRG: specic growth rate.
 S. Nimrat et al. / Veterinary Microbiology 159 (2012) 443450 447

Data were expressed as mean  S.D. Means with different superscript at the same shrimp stage indicate signicant difference (P < 0.05). T1: addition of mixed freeze-dried Bacillus (FB) of probiotic A, T2: addition of mixed
microencapsulated Bacillus (MB) of probiotic A, T3: addition of artemia enriched with mixed MB of probiotic A, T4: addition of mixed FB of probiotic B, T5: addition of mixed MB of probiotic B, T6: addition of artemia
different (P < 0.05) than those of controls (8.2  0.0 to

Ratio (%) (Bacillus

THB number 1)
8.5  0.1, 0.45  0.00 to 0.49  0.00 mg l 1 and 0.09  0.02
to 0.10  0.02 mg l 1, respectively) (data not shown). How-

0.4  0.1(b)

0.5  0.1(b)

0.9  0.1(b)

1.0  0.2(b)
13.3  1.3(a)
14.3  3.8(a)

15.2  4.1(a)

10.9  1.3(a)
10.5  1.5(a)

11.4  2.9(a)
10.2  1.6(a)
14.8  0.4(a)
ever, all values of treatment groups at 05:00 and 14:00 h in
0(c) both experiments were within acceptable ranges for rearing

0(c)
of penaeid shrimp (Boyd and Fast, 1992).

4. Discussion

4.3  1.2  102(b)

6.3  1.1  102(b)

6.7  1.6  102(b)

2.8  0.6  102(b)
8.2  1.6  104(a)

6.9  1.2  104(a)

7.9  1.3  104(a)

7.0  1.6  104(a)

8.7  1.0  104(a)

9.2  0.9  104(a)

1.3  0.6  105(a)

8.0  1.0  104(a)
Bacillus number

4.1. Growth and survival characteristics

(CFU ml 1)

Bacillus microencapsulated and freeze-dried probiotics

0(c)

0(c)
signicantly improved growth and survival of PL shrimp, but
not for larval shrimp. This may be associated with the
efcacy of probiotics that is dependent on duration of
exposure (Ziaei-Nejad et al., 2006). Development of larval
7.4  1.1  104(b)

6.0  1.1  104(b)

9.7  1.1  104(b)

8.8  0.9  104(b)
5.3  1.3  105(a)
6.2  1.3  105(a)

6.0  1.3  105(a)

4.9  1.2  105(a)

8.7  1.1  105(a)

2.3  1.2  106(a)

9.2  1.1  105(a)

7.9  0.1  105(a)
4.2  0.9  104(c)
3.7  0.0  104(c)
Culture water

stages required a short developmental period (4 days),

THB number
(CFU ml 1)

compared to that of PL stages (22 days). Similar growth and

survival of PL shrimp in the present study indicated that the
forms of Bacillus probiotics and modes of probiotic
administration did not affect growth and survival of PL
shrimp. Signicant differences in growth and survival of PL
shrimp over controls were in accordance with other
Ratio (%) (Bacillus

investigations using Bacillus as probiotics (Far et al., 2009;

THB number 1)

Boonthai et al., 2011; Nimrat et al., 2011; Utiswannakul

26.8  2.3(a)
27.5  3.1(a)

32.8  3.5(a)

25.8  3.6(a)
0.8  0.1(a)

0.8  0.1(a)
0.4  0.1(a)

21.7  2.0(a)

23.0  4.6(a)
0.2  0.0(a)
0.5  0.0(a)
0.6  0.0(a)

et al., 2011). Signicantly higher survival rate of treated PL

shrimp compared to the control in the present study was
0(b)

0(b)

probably associated with stress resistance to environmental

uctuation after probiotic administration (Liu et al., 2010).
Substantial enhancement of growth and survival rates
in PL shrimp in the present study may be attributed to
5.4  0.8  103(a)
3.9  0.7  103(a)

6.2  0.9  103(a)

5.9  0.9  103(a)
4.8  0.9  103(a)

7.8  1.0  105(a)

9.0  1.7  105(a)
2.3  0.1  106(a)
8.7  0.9  105(a)
1.5  0.1  106(a)
8.9  0.2  105(a)
4.0  0.7  103(a)
Bacillus number
(CFU larvae 1)

increased numbers of Bacillus probiotics in the digestive

tract of treated shrimp. Allochthonous Bacillus adapted to
digestive tract environments may form a symbiotic
0(b)

0(b)

association essential to improve physiological response

of the host. After colonization, Bacillus generally use a
THB and Bacillus numbers of treated and untreated larval shrimp and culture water.

variety of nutrients for their growth and simultaneously

release relevant digestive enzymes and other necessary
)
1

growth factors that facilitate nutrient assimilation in their

THB number (CFU larvae

enriched with mixed MB of probiotic B and control: no addition of probiotics.

hosts resulting in prevention of intestinal disorders and

higher growth and survival (Wang, 2007; Sahu et al., 2008;
5.7  1.5  105(b)
2.1  0.1  104(b)

3.0  1.1  105(a)

3.7  1.1  105(a)

3.4  1.6  106(a)

4.5  1.3  106(a)

3.9  1.1  106(a)

4.7  1.1  106(a)
2.0  1.0  105(a)
4.2  0.9  105(a)

1.6  0.9  105(a)

4.5  0.8  105(a)

6.0  0.8  106(a)

5.3  1.0  106(a)

Lara-Flores, 2011). Probiotic B demonstrated a greater

Larval shrimp

improvement in growth of PL shrimp than probiotic A. This

can be explained by different bacterial strains of the two
probiotic groups and interactions between bacterial
strains in each probiotic group (Luis-Villasenor et al.,
2011). Therefore, further study is necessary to investigate
the mechanism of mixed Bacillus species of each probiotic
for culture of L. vannamei shrimp.
Treatments

Control

Control

4.2. Bacterial number

T1
T2
T3
T4
T5
T6

T1
T2
T3
T4
T5
T6

Similar numbers of THB, Bacillus and ratios of Bacillus/

THB number among the treated groups for each stage from
Zoea 3 (at beginning

zoea 3 to PL shrimp (22-day-old) indicated that form and

of Experiment 1)

mode of probiotic administration did not affect bacterial

Duration (days)

numbers during this ontogeny. Gradual increases in

Bacillus numbers with probiotic treatments during rearing
from zoea 3 to PL shrimp (22-day-old) suggested their
Table 2

colonization in the digestive tract. This was supported by the

4

elevated ratios of Bacillus to THB numbers. Bacillus probiotics

 448
Table 3
THB and Bacillus numbers of treated and untreated PL shrimp and culture water.

Duration (days) Treatments Postlarval shrimp Culture water

1
THB number Bacillus number Ratio (%) (Bacillus THB number (CFU ml ) Bacillus number Ratio (%) (Bacillus
1
(CFU postlarvae ) (CFU postlarvae 1) THB number 1) (CFU ml 1) THB number 1)

PL 1 (at beginning of Experiment 2) Control 3.1  1.0  106(b) 0(b) 0(b) 6.3  1.8  104(b) 0(c) 0(c)

S. Nimrat et al. / Veterinary Microbiology 159 (2012) 443450

T1 7.5  1.0  106(a) 2.1  0.6  106(a) 53.7  5.5(a) 5.9  1.8  105(a) 8.3  1.1  104(a) 16.4  1.8(a)
T2 8.3  0.9  106(a) 4.3  1.3  106(a) 62.0  6.1(a) 7.5  1.9  105(a) 1.1  0.5  105(a) 18.4  2.0(a)
T3 6.7  0.6  106(a) 1.9  0.8  106(a) 51.5  4.3(a) 4.2  0.9  104(b) 9.7  1.4  102(b) 5.2  0.6(b)
T4 7.7  0.8  106(a) 3.8  1.1  106(a) 55.9  5.3(a) 6.0  1.8  105(a) 9.0  1.0  104(a) 16.5  2.8(a)
T5 8.9  1.2  106(a) 4.0  1.0  106(a) 58.3  2.7(a) 4.7  1.1  105(a) 7.7  1.1  104(a) 17.2  3.6(a)
T6 8.1  0.8  106(a) 3.6  0.8  106(a) 54.4  3.9(a) 6.3  1.8  104(b) 7.8  1.3  102(b) 4.5  0.8(b)

14 Control 2.7  1.2  106(b) 0(b) 0(b) 2.1  1.3  106(b) 0(b) 0(b)
T1 9.9  0.9  106(a) 7.1  0.9  106(a) 82.0  6.2(a) 3.3  1.6  107(a) 9.2  1.2  106(a) 42.3  2.5(a)
T2 1.2  0.5  107(a) 6.6  1.7  106(a) 80.2  5.2(a) 6.0  1.4  107(a) 8.8  0.8  106(a) 54.7  4.5(a)
T3 9.3  0.1  106(a) 5.0  1.2  106(a) 74.7  4.2(a) 4.3  0.4  107(a) 7.2  1.0  106(a) 37.8  3.5(a)
T4 8.7  1.2  106(a) 4.9  0.9  106(a) 76.8  3.8(a) 5.4  1.7  107(a) 6.7  1.9  106(a) 43.0  6.5(a)
T5 3.4  1.1  107(a) 9.7  1.0  106(a) 73.1  5.1(a) 3.9  1.3  107(a) 7.7  1.2  106(a) 47.8  4.6(a)
T6 2.0  0.7  107(a) 8.4  1.1  106(a) 73.8  4.5(a) 3.8  0.1  107(a) 6.3  0.8  106(a) 34.3  3.9(a)

22 Control 1.9  1.0  106(b) 0(b) 0(b) 4.7  1.2  106(b) 0(b) 0(b)
T1 2.3  0.8  107(a) 7.3  0.8  106(a) 76.0  7.2(a) 6.1  1.6  107(a) 7.0  1.2  106(a) 49.9  2.8(a)
T2 9.3  0.9  106(a) 6.1  1.2  106(a) 85.7  3.2(a) 5.4  1.6  107(a) 4.1  1.3  106(a) 51.9  0.6(a)
T3 1.0  0.2  107(a) 7.8  0.9  106(a) 81.1  5.6(a) 7.0  1.5  107(a) 7.7  0.7  106(a) 45.0  1.6(a)
T4 2.1  0.7  107(a) 6.9  1.1  106(a) 76.6  4.3(a) 7.3  1.5  107(a) 7.3  1.0  106(a) 50.8  1.2(a)
T5 1.9  0.7  107(a) 8.7  1.3  106(a) 80.2  6.4(a) 5.5  1.1  107(a) 6.5  1.7  106(a) 50.6  3.6(a)
T6 2.7  0.9  107(a) 9.2  0.9  106(a) 79.6  7.8(a) 5.8  1.3  107(a) 5.4  0.5  106(a) 42.5  2.3(a)

Data were expressed as mean  S.D. Means with different superscript at the same shrimp stage indicate signicant difference (P < 0.05). T1: addition of mixed freeze-dried Bacillus (FB) of probiotic A, T2: addition of mixed
microencapsulated Bacillus (MB) of probiotic A, T3: addition of artemia enriched with mixed MB of probiotic A, T4: addition of mixed FB of probiotic B, T5: addition of mixed MB of probiotic B, T6: addition of artemia
enriched with mixed MB of probiotic B and control: no addition of probiotics.
 S. Nimrat et al. / Veterinary Microbiology 159 (2012) 443450
in this study may replace other bacteria by functioning as a
protective barrier through competitive exclusion for nutri-
ents and habitat, thereby becoming a dominant constitute of
intestinal ora (Balcazar et al., 2006; Utiswannakul et al.,
2011). Ziaei-Nejad et al. (2006) revealed Bacillus numbers at
104105 CFU larva 1 (61.593.0% of total bacterial ora)
when F. indicus larvae were inoculated with a commercial
probiotic product containing 106 CFU ml 1 of Bacillus via
culture water or enriched Artemia. The present study also
supported recent studies carried out in shrimp by Boonthai
et al. (2011) and Nimrat et al. (2011), who pointed out that
mixed Bacillus probiotics were capable of propagating in
digestive tracts of P. monodon and L. vannamei without
affecting THB number when Bacillus probiotics were used as
food or water additives.
4.3. Water quality parameters
Addition of Bacillus probiotics in different form and
mode of application to shrimp larval and PL stages was
associated with signicantly reduced levels of ammonia,
nitrite and pH. Bacillus probiotics A and B inhabited and
grew in water, which was supported by increased ratios of
Bacillus/THB numbers during ontogenetic stages. There
have been several reports of Bacillus genera mineralizing
nitrogenous wastes via nitrication and/or denitrication
resulting in reduced ammonia and nitrite in accord with
the present study (Kim et al., 2005; Wang et al., 2005;
Lakshmanan and Soundarapandian, 2008; Nimrat et al.,
2011). Consequently, nitrication that occurs simulta-
neously with the addition of Bacillus probiotics also
discharges hydrogen ion leading to a reduction of pH
(Camargo and Alonso, 2006).
The present study indicated that microencapsulated
Bacillus probiotics are also as effective as freeze-dried
Bacillus probiotics for enhancing growth and survival of L.
vannamei postlarvae as well as improving pH, ammonia
and nitrite during rearing of PL shrimp. However, the
concept of bacterial probiotic administration needs further
research to assess the bacterial compositions based on
molecular technology and to elucidate the action mechan-
isms of the mixed Bacillus probiotics in order to have better
understanding of bacterial interactions for application in
shrimp cultivation.
5. Conclusion
Addition of Bacillus probiotics as microencapsulated or
freeze-dried forms directly into water or by enrichment of
Artemia are equally effective in improvement of growth,
survival and benecial bacteria of PL shrimp including
some water quality parameters, especially pH, ammonia
and nitrite as compared to controls. Species composition of
Bacillus probiotics B signicantly enhanced the growth and
survival of PL shrimp.
Acknowledgements
This study was partially funded by Thailand Research
Fund (TRF) (grant no. DBG 5380033) and Environmental
449
Science Program, Faculty of Science, Burapha University.
The authors also express their gratitude to Dr. F.W.H.
Beamish for reading manuscript and our colleagues for
many valuable help. We also gratefully acknowledge
Department of Microbiology and Department of Aquatic
Science, Faculty of Science, Burapha University for
providing experimental equipments and facilities.
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