Indonesian Aquaculture Journal, 13 (2), 2018, 95-101

Available online at: http://ejournal-balitbang.kkp.go.id/index.php/iaj

USE OF DIFFERENT PROBIOTICS FOR PREVENTION OF VIBRIOSIS DISEASE
ON TIGER SHRIMP LARVAE REARED IN FIBERGLASS TANKS
Nurbaya# and Muharijadi Atmomarsono
Research Institute for Coastal Aquaculture and Fisheries Extension
Jl. Makmur Dg. Sitakka No. 129, Maros 90512, South Sulawesi
(Received 6 March 2018; Final revised 7 June 2018; Accepted 8 June 2018)

ABSTRACT
To counter disease problems caused by vibriosis in shrimp hatchery, this recent study used three different
probiotics to be tested on tiger shrimp (Penaeus monodon) postlarvae. The study arranged four treatments
as follows: A: a combination of three liquid-form probiotics Brevibacillus laterosporus BT951, Bacillus subtilis
BM12, and B. licheniformis BM58; B: a combination of three powder-form probiotics Brevibacillus laterosporus
BT951, Bacillus subtilis BM12, and B. licheniformis BM58; C: a commercial powder probiotic containing
Bacillus subtilis; and D: control (without probiotic), each treatment with three replications. This study was set
up in a completely randomized design experiment using twelve fiberglass tanks filled with 750 L sterile sea
water and stocked with 30,000 nauplii in the Awarange shrimp hatchery of the Research Institute for
Brackishwater Aquaculture and Fisheries Extension Installation in Barru. Variables observed in this study
were the survival rate of the shrimp postlarvae at the end of the experiment, total vibrio count (TBV) and
total plate count of common bacteria (TPC) in the culture water. The results showed that the survival rate
of tiger shrimp applied either in liquid (A: 61.5±4.7%) or powder form (B: 48.6±6.8%), and control (without
probiotic) (D: 51.2±4.4%) were not significantly different (P>0.05). However, survival rates in these three
treatments differed (P<0.05) with that of the commercial probiotic (C: 21.7±9.9%). TBV/TPC ratio in the
tank waters treated with the commercial probiotic (2.26-37.52%) was much higher than that of the liquid
form probiotic (0.86-1.98%), powder form probiotic (1.25-8.37%), and control (1.93-2.84%). Ammonianitrogen in treatment C (1.462-2.989 mg/L) was relatively higher than that of in treatment A (1.595-2.435
mg/L), treatment B (1.644-2.115 mg/L), and treatment D (1.051-1.858 mg/L).
KEYWORDS:

probiotic; survival rate; tiger shrimp postlarvae; vibriosis

INTRODUCTION
Tiger shrimp (Penaeus monodon) aquaculture has
suffered various shrimp diseases in both hatchery and
grow-out systems, worldwide. Shrimp diseases
caused by white spot syndrome virus, yellow head virus,
and vibriosis occurred not only in tiger shrimp culture, but also in whiteleg shrimp culture (Lightner,
2011; Atmomarsono & Susianingsih, 2013). The use
of chemicals and antibiotics to cure shrimp diseases
have been practiced in Thailand and Vietnam (Tran et
al., 2017). Because of their negative impacts on human health, these chemical uses in aquaculture have
now been banned in Indonesia. This has left fewer
alternatives to combat or prevent these diseases oc#

Correspondence: Research Institute for Brackishwater
Aquaculture and Fisheries Extension. Jl. Makmur Dg. Sitakka No.
129, Maros 90512, South Sulawesi, Indonesia
Phone: + 62 411 371544
E-mail: nurbayaeka@gmail.com

currence. One way to solve this problem is using
probiotic to prevent shrimp diseases (Poernomo,
2004; Waston et al., 2008). The main culprit of shrimp
diseases is vibriosis caused by Vibrio harveyi which
could wipe out 100% of the cultured shrimp (Flegel,
2012; Atmomarsono & Nurbaya, 2014). As an opportunistic pathogen, Vibrio harveyi will only become
pathogenic bacteria causing shrimp diseases when
the pond water quality is poor (Atmomarsono &
Susianingsih, 2015). This problem could be minimized
by practicing the best aquaculture practices and applying proper probiotics. Probiotic bacteria are
needed in the culture of larval aquatic organisms including black tiger shrimp to prevent bacterial diseases (Atmomarsono et al., 2009; Uddin et al., 2013).
Probiotic bacteria are non-pathogenic bacteria
usually used to protect the shrimp from pathogenic
microorganisms, inhibit the growth of pathogenic
bacteria, neutralize poor pond water quality such as

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Use f different probiotics for prevention of vibriosis disease ..... (Nurbaya)

organic matter, H2S, NH3-N, and NO2-N, and function
as food (Poernomo, 2004; Wang et al., 2008).
Probiotic bacteria can be isolated from sea sediment,
pond sediment, mangrove leaves, and macroalgae
(Atmomarsono et al., 2009; Susianingsih et al., 2012;
Tampangallo et al., 2013; Atmomarsono & Nurbaya,
2014). There are various types of domestically produced and imported probiotics available in the market. Some of them are useful in the shrimp culture,
but many of them are in effective.
The Research Institute for Brackishwater Aquaculture and Fisheries Extension develops and produces
RICA (Research Institute for Coastal Aquaculture)
probiotics originally isolated from the sea sediment,
brackishwater pond, mangrove, and seaweed. RICA1 (Brevibacillus laterosporus BT951) is useful in controlling organic matter and H2S in the ponds, while
RICA-2 (Serratia marcescens MY1112) is beneficial for
the shrimp growth and controlling ammonia-nitrogen (Atmomarsono et al., 2009). RICA-3
(Pseudoalteromonas sp. Edeep-1 BL542) has an important role in controlling nitrite and pathogenic Vibrio
harveyi (Atmomarsono & Susianingsih, 2013). RICA-4
(Bacillus subtilis BM12) is useful in controlling organic
matter and Vibrio harveyi in pond water. RICA-5 (Bacillus licheniformis BM58) is useful in controlling ammonia and nitrite in pond water (Tampangallo et al., 2013).
A shrimp disease usually occurs when the accumulation of organic matter in the pond waters and sediments can not be controlled properly by the available
natural bacteria. Since each RICA probiotic bacteria
has a different role, the combination use of three
different RICA probiotics might offer a full potential
solution package to control organic wastes, ammonia-nitrogen, nitrite-nitrogen, and pathogenic Vibrio
harveyi in shrimp ponds.
The aim of this study was to determine the best
combination of probiotics in hatchery larval rearing
of tiger shrimp using fiberglass tanks.
MATERIALS AND METHODS
This research was carried out in the shrimp
hatchery of the Research Institute for Brackishwater
Aqua-culture and Fisheries Extension Installation,
Awarange, Barru Regency, South Sulawesi. A Completely Randomized Design (CRD) experiment with
four treatments and three replications was used in
this research. The four treatments applied were: A: a
combination of three liquid-form probiotics RICA-1
(Brevibacillus laterosporus BT951), RICA-4 (Bacillus subtilis
BM12), and RICA-5 (Bacillus licheniformis BM58); B: a
combination of three powder-form probiotics RICA1 (Brevibacillus laterosporus BT951), RICA-4 (Bacillus

96

subtilis BM12), and RICA-5 (Bacillus licheniformis BM58);
C: a commercial powder probiotic containing Bacillus
subtilis; and D: control (without probiotic).
This research used twelve fiberglass tanks of one
m3 in volume filled with 750 L of chlorinated-sterile
sea water of about 30 ppt and continuously aerated.
Each tank was stocked with 30,000 nauplii (40 pcs/L),
fed with Skeletonema sp. 15,000-75,000 cell/mL (started
from the second day, just before Zoea-1 stage), commercial feed (started from Zoea-1 stage), and
brineshrimp 10-20 nauplii Artemia/shrimp larvae
(started from Mysis-3 stage or about day-9). Water
exchange for about 5%-20% was carried out daily after
Mysis-3 stage. The probiotic applications for treatment A and B were as follows RICA-1 probiotic
(Brevibacillus laterosporus BT951) was applied on day-3
(D-3), RICA-4 (Bacillus subtilis BM12) was applied on
D-6, RICA-5 (Bacillus licheniformis BM58) on D-9, and
followed by RICA-1 applied daily from day-10 (PL-1)
to day-21 (PL-12). For treatment C, a commercial
powder probiotic was applied on the same day and
the same amount with treatment B (1 g/m3). All of
the applied probiotics in the shrimp culture water
were estimated to reach about 101 cfu/mL in population. To make this density, 100 mL probiotic in nutrient broth (liquid-form probiotic) was added to 900
mL sterile sea water and stirred for about 24-hours
before used for the shrimp culture water in treatment A. To make powder-form probiotic (treatment
B), 200 mL liquid-form probiotic was added to 400 g
sterile cassava meal, then dried at 40°C in an oven
for about 48-hours before used. The density of the
applied bacteria in each treatment was checked before application.
Water salinity in the tank was maintained about
30 ppt, while water pH and temperature were monitored daily. The concentrations of total organic matter (TOM), ammonia-nitrogen (NH3-N), and nitritenitrogen (NO2-N) in the tank waters were monitored
four times, namely at the stages of Zoea-1 (D-2), PL1 (D-10), PL-4 (D-13), and PL-10 (D-19). Using a modified spreading method (Buller, 2004), the total number of common bacteria (Total Plate Count= TPC) in
the tank water was measured in Tryptic Soy Agar (TSA),
while the total number of Vibrio spp. (Total Bacteria
of Vibrios= TBV) in the tank water was measured in
Thiosulphate Citrate Bile Sucrose Agar (TCBSA). Both TBV
and TPC were monitored in the same days with the
water quality monitoring. The data on TBV and TPC
were then analyzed descriptively. TBV/TPC ratios of
the tank waters were also calculated based on the
percentage of the number of TBV and TPC in each
sample. When TBV/TPC ratio had increased more than

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Indonesian Aquaculture Journal, 13 (2), 2018, 95-101

10%, some pathogenic Vibrio spp. might be harmful
to the shrimp (Atmomarsono & Nurbaya, 2014). The
survival rate and weight of tiger shrimp postlarvae in
each tank were calculated after the shrimp reached
postlarvae 12 (PL-12) and analyzed statistically to see
if any differences among the treatments tested (Steel
& Torrie, 1981).
RESULTS AND DISCUSSION
Table 1 shows that the average survival rate of
tiger shrimp postlarvae (PL-12) in the tank water
treated either liquid-form or powder-form RICA
probiotics were significantly (P<0.05) higher (61.5
± 4.7% and 48.6 ± 6.8%) than that of the commercial
probiotic powder (21.7 ± 9.9%). However, there were
no significant differences (P>0.05) between the average survival rates of tiger shrimp postlarvae in the
control (51.2 ± 4.4%) and those of in treatment A
and B. These results showed that RICA probiotics
either in liquid-form or in powder-form had better
results than the commercial probiotic powder, but
not better than the control (without probiotic). The
shrimp mortality might be caused by the increasing
number of vibrios and the increased level of ammonia-nitrogen concentration in the tank water.
RICA probiotics (treatment A and B) contained
three different bacteria, e.g.: Brevibacillus laterosporus
BT951, Bacillus subtilis BM12, and Bacillus licheniformis
BM58, functioning together to control Vibrio spp.,
total organic matter, ammonia-nitrogen, and nitritenitrogen. Treatment C (commercial probiotic) contained only Bacillus subtilis functioning in controlling
Vibrio spp. and total organic matter. This was the reason why the concentration of ammonia-nitrogen
(1.462-2.989 mg/L) and TBV/TPC ratio in treatment C
(2.26%-37.52%) were relatively higher than those in
the liquid form probiotic (1.595-2.435 mg/L and 0.86%1.98% respectively) and powder form probiotic (1.6442.115 mg/L and 1.25%-8.37% respectively). However,
RICA probiotics (treatment A and B) and control (without probiotic) had not different significantly on the
survival rates of the tiger shrimp postlarvae (Table
1). This might be caused by a low concentration of
probiotic bacteria in the tank water (about 2.3 x 101

cfu/mL) at the initial stocking. This research advises
that the probiotic bacteria should be applied at least
ten times of the current application for future experiment or commercial applications.
The survival rates of tiger shrimp postlarvae resulted in this research were similar to those of Uddin
et al. (2013) results, which were 52% for probiotictreated postlarvae and 35% for untreated postlarvae.
However, based on the individual survived postlarvae,
the study findings (8-25 pcs/L) were relatively lower
than that of Uddin et al. (2013) results (35-52 pcs/L).
This difference might be caused by a higher ammonia-nitrogen concentration in the shrimp culture tank
water in this research after PL-1 stage (2.30-5.08 mg/
L) (Table 2) compared to Uddin et al. (2013) results
(0.9-2.5 mg/L).
It was previously reported by Atmomarsono et al.
(2009) that RICA-1 probiotic consisting of Brevibacillus
laterosporus BT951 could be used as an anti vibriosis,
and was useful in demineralization of total organic
matter. Tampangallo et al. (2013) also reported that
RICA-4 (Bacillus subtilis BM12) functioned as organic
matter demineralizer, and RICA-5 (Bacillus licheniformis
BM58) functioned as ammonia-nitrogen and nitritenitrogen controller in the culture media. Treatment
C consisting of Bacillus subtilis could demineralize total
organic matter, but not ammonia-nitrogen. This was
the reason ammonia-nitrogen in treatment C was
relatively higher than those of the other treatments.
This ammonia-nitrogen could be toxic for tiger shrimp
postlarvae.
According to Chin & Chen (1987) in Boyd (1990)
96-h LC50 and 24-h LC50 for tiger shrimp postlarvae
were only about 1.26 mg/L and 5.71 mg/L, respectively. This means that the concentration of ammonia-nitrogen in the culture media of this present research was relatively dangerous to tiger shrimp
postlarvae. This might be the cause a direct or indirect shrimp mortality through the increase of TBV/
TPC ratio (percentage ratio of total bacteria of vibrios
and total common bacteria) in the culture water during PL-4 stage in the control tank (19.68%) and during
PL-10 stage in the commercial probiotic treated tank

Table 1. Survival rate and average weight of tiger shrimp postlarvae reared in
fiberglass tank with different probiotics
Probiotics
A.
B.
C.
D.

Liquid-formed RICA
Powder-formed RICA
Commercial powder probiotic
Control (without probiotic)

Survival rate (%) Postlarvae weight (mg/pcs)
61.5 ± 4.7 a
48.6 ± 6.8 a
21.7 ± 9.9 b
51.2 ± 4.4 a

1.7 ± 0.2a
2.4 ± 0.5a
2.0 ± 0.5a
1.3 ± 0.6a

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Use f different probiotics for prevention of vibriosis disease ..... (Nurbaya)

Table 2. Ranges of ammonia-nitrogen concentrations in tiger shrimp culture tank water at
different larvae stages treated with different probiotics
Probiotics
A.
B.
C.
D.

Liquid-formed RICA
Powder-formed RICA
Commercial powder probiotic
Control (without probiotic)

Zoea-1
(mg/L)

PL-1
(mg/L)

PL-4
(mg/L)

PL-10
(mg/L)

0.076-0.217
0.038-0.167
0.014-0.271
0.039-0.074

2.332-2.560
2.477-2.660
2.392-2.535
2.298-2.346

4.520-5.083
4.171-4.690
3.966-5.058
4.231-4.835

1.595-2.435
1.644-2.115
1.462-2.989
1.051-1.858

(37.52%). This finding was in line with the results reported by Atmomarsono & Nurbaya (2014) and
Susianingsih et al. (2017) where the increase of TBV/
TPC ratio in the culture water of more than 10% could
be dangerous to the cultured shrimp.
Based on the individual weight size of the
harvested postlarvae, postlarvae in treatment A
(liquid-form RICA probiotic) were relatively more
homogenous than that of the other treatments.
However, the postlarvae in treatment B (powder-form
RICA probiotic) were relatively heavier (2.4±0.5) than
that of the other treatments. The smallest, lightest,
and varying in weight of harvested tiger shrimp
postlarvae were found in the control (1.3± 0.6) (Table
1). These results showed that all kinds of probiotics
were needed to maintain the culture water clean, so
that the cultured tiger shrimp could grow well
homogenously. Besides that, without any addition
probiotics, the natural bacteria were not enough to
demineralize some of organic matter and ammonianitrogen. As a result, the ratio of total bacteria of
vibrios and total common bacteria (TBV/TPC) in the
control tank water during PL-4 stage had increased
over 10%.
In general, vibrio numbers in the cultured shrimp
water in this experiment were not significantly high
(1-6,860 cfu/mL). According to Defoirdt (2007) Vibrio
harveyi could be dangerous when its population in
the cultured shrimp water reached 10 4 cfu/mL.
However, eventhough the vibrio numbers in this
research were still lower than 104 cfu/mL, they might
cause shrimp mortality when TBV/TPC ratio increased
over 10% (Atmomarsono & Nurbaya, 2014; Susianingsih
et al., 2017).
Table 3 shows that nitrite-nitrogen concentrations
in the tiger shrimp culture tank water tended to
increase following the culture period. Fortunately, the
highest concentration of nitrite-nitrogen in this
research (1.843 mg/L) was still considered as a safe

98

level for tiger shrimp postlarvae. According to Chen
& Chin (1988a) in Boyd (1990) the concentration of
nitrite-nitrogen for tiger shrimp postlarvae should
be less than 4.5 mg/L. However, since ammonianitrogen concentration in the culture tank water was
relatively high, it could cause the cultured shrimps to
be more susceptible to pathogenic Vibrio harveyi
especially when TBV/TPC ratio increased over 10% such
as in treatments C and D (Table 5).
In general, a high concentration of ammonianitrogen followed by relatively low concentration of
nitrite-nitrogen indicated that in the culture water
might be not enough nitrification bacteria. In
treatments A and B, RICA-4 and RICA-5 probiotics
(Bacillus subtilis BM12 and Bacillus licheniformis BM58
respectively) could function as nitrification bacteria.
Unfortunately, RICA-4 and RICA-5 probiotics were not
used anymore in this research after tiger shrimp
reached postlarvae one (PL-1). Instead of using RICA4 and RICA-5 probiotics alternately, RICA-1 probiotic
was used everyday from PL-1 to postlarvae 12 stage
following the procedure used for the commercial
probiotic application in treatment C. It is suggested
that for the future experiments, RICA-1, RICA-4, and
RICA-5 probiotics should be used alternately to
prevent the increase of total organic matter, ammonianitrogen, nitrite-nitrogen concentrations, and TBV/
TPC ratio in the shrimp culture water.
Based on Table 4, the ranges of total organic matter
concentrations in the tiger shrimp culture tank water
were quite high (29.4-64.4 mg/L). According to Madeali
et al. (2009) the concentration of total organic matter
in the shrimp culture water should be less than 30
mg/L, otherwise, the total organic matter could
trigger the growth of pathogenic microorganisms
that might be dangerous to the cultured shrimp.
Since total organic matter concentrations in the
shrimp culture water were relatively high in all tanks,

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Indonesian Aquaculture Journal, 13 (2), 2018, 95-101

Table 3. Ranges of nitrite-nitrogen concentrations in tiger shrimp culture tank water at
different larvae stages treated with different probiotics
Zoea-1
(mg/L)

PL-1
(mg/L)

PL-4
(mg/L)

PL-10
(mg/L)

0.001-0.002
0.007-0.033
0.007-0.024
0.007-0.007

0.014-0.043
0.040-0.064
0.023-0.031
0.042-0.054

0.028-0.034
0.053-0.188
0.033-0.042
0.032-0.200

1.385-1.843
0.823-1.469
0.938-1.672
0.360-1.686

Probiotics
A.
B.
C.
D.

Liquid-formed RICA
Powder-formed RICA
Commercial powder probiotic
Control (without probiotic)

Table 4. Ranges of total organic matter concentrations in tiger shrimp culture tank
water at different larvae stages treated with different probiotics
Probiotics
A.
B.
C.
D.

Liquid-formed RICA
Powder-formed RICA
Commercial powder probiotic
Control (without probiotic)

Zoea-1
(mg/L)

PL-1
(mg/L)

PL-4
(mg/L)

PL-10
(mg/L)

29.4-52.6
40.0-51.3
40.0-54.4
46.9-50.7

46.9-57.6
50.0-63.8
50.0-61.3
47.6-54.4

45.7-60.1
48.8-53.2
37.5-63.8
42.5-52.6

50.0-64.4
41.9-57.6
43.2-53.8
45.0-48.2

any kind of probiotic which could demineralize total
organic matter was vitally important. The three
treatments in this experiment had this kind of
probiotic. Unfortunately, only RICA-1 probiotic
(Brevibacillus laterosporus BT951) was used in
treatments A and B after reaching PL-1. Bacillus subtilis
is one of the commonly used probiotic bacteria in
the commercial probiotic products. This could explain
the high level of the general total organic matter
concentration and the high concentration of ammonianitrogen. These two water quality parameters might
trigger the growth of pathogenic vibrios, in TBV/TPC
ratio over 10% in the control (19.68%) and treatment
C (37.52%) (Table 5). This level of TBV/TPC ratios was
relatively dangerous to shrimp (Atmomarsono &

Nurbaya, 2014; Susianingsih et al., 2017). Madeali et
al. (2009) similarly reported that the pathogenicity
of Vibrio harveyi was enhanced by the high
concentration of total organic matter (more than 30
mg/L) in the cultured shrimp water.
During the experiment, other water quality
parameters in the treatment tanks were also
monitored daily. Ranges of water quality parameters
were as follows 30.0-32.4 ppt for water salinity,
30.0°C-32.2°C for water temperature, 7.88-8.22 for
water pH, and 4.45-5.34 mg/L for dissolved oxygen.
These water quality parameters were within the
standard for the cultured tiger shrimp based on the
study of Atmomarsono et al. (2009).

Table 5. Ranges of percentage of total bacteria of vibrios and total plate count
ratio (TBV/TPC) in tiger shrimp culture tank water at different larvae stages
treated with different probiotics
Probiotics
A.
B.
C.
D.
*)

Liquid-formed RICA
Powder-formed RICA
Commercial powder probiotic
Control (without probiotic)

Zoea-1
(mg/L)

PL-1
(mg/L)

PL-4
(mg/L)

PL-10
(mg/L)

0.20-0.40
0.04-3.00
0.20-0.67
0.03-0.33

0.38-1.12
0.51-7.85
0.92-8.03
0.68-3.10

0.31-1.96
1.25-1.75
2.09-5.61

0.86-1.98
1.25-8.37

2.26-37.52
2.36-19.68 1.93-2.84

*)

*)

Bolded values mean lethal level for the cultured shrimp

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Use f different probiotics for prevention of vibriosis disease ..... (Nurbaya)

CONCLUSION
This current study concludes that in comparison
with the control (without probiotic), the application
of liquid-form or powder-form RICA probiotics could
not increase (P>0.05) survival rate of tiger shrimp
postlarvae. Compared with commercial probiotic, the
application of liquid-form or powder-form RICA
probiotics had produced a better survival rate
(P<0.05) of tiger shrimp postlarvae. The survival rate
of tiger shrimp postlarvae might be decreased by the
high concentration of ammonia-nitrogen after PL-1
stage (2.30-5.08 mg/L) and the increase of TBV/TPC
ratio after PL-4 stage (more than 10%). This study
suggests that the three different species of probiotic
bacteria should be used alternately started from zoea1 until PL-12 at ten times concentration compared to
the current research use to demineralize total organic
matter, ammonia-nitrogen, and nitrite-nitrogen.
ACKNOWLEDGEMENTS
The authors would like to thank to all technicians
and researchers of the Research Institute for
Brackishwater Aquaculture and Fisheries Extension
who provided help during the research experiment.
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