EFFECT OF DIFFERENT PROBIOTIC BACTERIA ON SURVIVAL RATE , GROWTH , AND PRODUCTION OF WHITELEG SHRIMP IN TRADITIONAL-PLUS TECHNOLOGY

Instead of culturing tiger shrimp that is frequently burdened by mass mortality, whiteleg shrimp (Litopenaeus vannamei) is then considered as an alternative commodity in Indonesian brackishwater ponds. To prevent the whiteleg shrimp from diseases, different probiotic bacteria were tested in completely randomized design experiment using nine 250-m2 experimental ponds stocked with 10 PLs of whiteleg shrimp fry/m2. Three treatments were applied, namely A) alternate use of probiotic bacteria RICA-1, RICA-2, RICA-3; B) alternate use of probiotic bacteria RICA-4, RICA-5, RICA-3, and C) control (without probiotic bacteria); each with three replications. After 11-week application, the results showed that the best survival rate of whiteleg shrimp was achieved by treatment B (98.83%) and the best production was achieved by treatment A (23.52 kg/250 m2). However, there were no significant differences (P>0.05) among the three treatments tested for the shrimp survival rate. The whiteleg shrimp production in treatment A and B were signicantly better (P<0.05) than that in treatment C (control). These high shrimp production in treatment A and B were mainly caused by the capability of the applied probiotics in controlling some water quality variables and Vibrio numbers.


INTRODUCTION
As an alternative commodity, white whiteleg shrimp (Litopenaeus vannamei) is potentially developed in Indonesia brackishwater ponds.Compared to tiger shrimp, whiteleg shrimp grows faster, can live in the whole water column, more productive, and relatively resistant to unbalance water quality.Whiteleg shrimp can be stocked in smaller size (PL-7), higher stocking density with relatively higher survival rate than tiger shrimp (Anonim, 2003;Poernomo, 2004).
High stocking density of the shrimp fry makes new problem in water quality deterioration that enhances shrimp diseases and finally decreasing brackishwater pond productivity.Vibriosis is the main cause of high mortality in larval stages of penaeid shrimp in the hatchery.Generally, vibriosis in the shrimp is mainly caused by one or some strains of pathogenic vibrios like Vibrio alginolyticus, V. harveyi, V. parahaemolyticus, V. damsela, V. vulnificus, V. penaecida, and V. fischeri (Lightner et al., 1992;Pasharawipas et al., 1998;Ruangpan, 1998).
To solve shrimp disease problems, some researches had been done in many ways, but so far the results were still questionable, like using bacterin, water reservoir and biofilter organisms for wastewater treatment (Muliani et al., 1998;Atmomarsono, 2004;Madeali et al., 2004).In addition, eradication of vibriosis using antibiotics also makes vibrios resistance to certain antibiotic.Thus, the use of probiotic bacteria in the cultured shrimp is considered as the best method for shrimp disease prevention in brackishwater ponds.
Different kinds of probiotics, its doses, and how the farmers apply are important to achieve the best results.Since 2006 BPPBAP (Balai Penelitian dan Pengembangan Budidaya Air Payau) has developed some probiotic bacteria isolated from brackishwater ponds, mangrove leaves, sea sediment, and macroalgae that are currently called as RICA (Research Institute for Coastal Aquaculture) probiotics.Specifically, RICA-1 is used for controlling total oganic matter and H 2 S in the shrimp pond water (Muliani et al., 2006;Atmomarsono et al., 2009).RICA-2 and RICA-4 are used for balancing nitrite and nitrate in the shrimp pond water (Muliani et al., 2004) and enhancing the shrimp growth (Atmomarsono et al., 2009;Tampangallo et al., 2013).RICA-3 is Pseudoalteromonas sp.Edeep-1 that has role for decreasing nitrite and controlling Vibrio spp. in the shrimp pond water (Muliani et al., 2003;2005;Susianingsih et al., 2012a;2012b;Atmomarsono & Susianingsih, 2013).RICA-5 is Bacillus licheniformis that has role for controlling ammonia and nitrite in the pond water (Tampangallo et al., 2013).However, to enhance all the above processes, additional oxygen is needed, since the above bacteria are considered as aerobic bacteria which need oxygen for their growth.
The use of different kinds of probiotic bacteria either in different combination or different species alternately used may affect to the growth, production, and survival rate of the cultured shrimp.Thus, this experiment is aimed at finding out the best alternate use of the RICA probiotics for survival rate, growth, and production of the whiteleg shrimp (Litopenaeus vannamei) in brackishwater ponds with traditional-plus technology using supercharge blower as oxygen supplier.

MATERIALS AND METHODS
This research was conducted at the Marana Experimental Ponds of the Research and Development Institute for Coastal Aquaculture (RICA), Maros using nine 250-m 2 ponds aerated with supercharge blower.
Each pond was stocked with 10 pcs of PL-8 whiteleg shrimp fry/m 2 that is negative WSSV (tested with PCR machine and IQ2000 test kit).Completely Randomized Design (CRD) with three treatments and three replications each were applied in this experiment.Three treatments tested here are A) alternate use of probiotic bacteria RICA-1, RICA-2, and RICA-3; B) alternate use of probiotic bacteria RICA-4, RICA-5, and RICA-3; and C) control (without probiotic bacteria).Whiteleg shrimp was fed with pellet decreasingly from 50% at beginning to 2% at the end of the culture period of total biomass/day.Before being used, probiotic bacteria was cultured using 1,000 g of rice bran; 400 g of fish meal; 500 g of molasses; and 100 g of yeast in 20 L of brackishwater pond water (Poernomo, 2004).All probiotic bacteria were applied twice a week about 10 mg/L of 3-d fermented culture of bacteria.Alternate use of bacteria for treatment A and B were done every two weeks until the harvest time (11 weeks).This means that either RICA-1 probiotic in treatment A or RICA-4 probiotic in treatment B were firstly applied four times, then followed by four time applications of either RICA-2 probiotic in treatment A or RICA-5 probiotic in treatment B, then followed by four time applications of RICA-3 probiotic in treatment A and B, then followed again by application of the first, second, and third probiotic sequently.
Survival rate, growth, and production of whiteleg shrimp were observed after 11 weeks.Data were then analyzed using analysis of variances (ANOVA) and followed by Least Significant Different method (Steel & Torrie, 1981).

Survival Rate, Growth, and Production of Whiteleg Shrimp
Survival rate, growth, and production of whiteleg shrimp after 11 weeks of culture can be seen in Table 1 below.The growth of the whiteleg shrimp in each treatment was shown by its size.
There were no significant differences (P>0.05) for survival rate of the whiteleg shrimp after 11 weeks of culture in this experiment.However, survival rate of the shrimp in treatment B (alternate use of probiotic RICA-4, RICA-5, and RICA-3) was relatively higher than those in treatment A and C. Whiteleg shrimp production in treatment A (alternate use of probiotic RICA-1, RICA-2, and RICA-3) and treatment B (alternate use of probiotic RICA-4, RICA-5, and RICA-3) were significantly better (P<0.05)than that in control.According to Suprapto (2005), probiotic application in intensive shrimp ponds increases survival rate.In this experiment, application of the RICA probiotics alternately in treatment A or B could increase the shrimp production significantly (P<0.05),but not significantly increased the shrimp survival rate (P>0.05).The alternate use of probiotic bacteria could minimize the organic waste in the pond water culture media (Figure 1), and the vibrio numbers (Table 2) as well, so that the whiteleg shrimp in these probiotic-treated ponds could live in better water media than the shrimp in control pond.Verschuera et al. (2000) mentioned that application of probiotic bacteria in the culture media could be used as complement feed for the shrimp or contribute in digestive system in protecting the shrimp from pathogenic bacteria, because probiotic bacteria could release antibacteria like bacteriozyme, lysozyme, protease, siderophore, hydrogen peroxide, and organic acid.

Total Vibrio Count (TBV)
Total number of Vibrio spp.(TBV) in the whiteleg shrimp culture water media during experiment is shown in Table 2.At the beginning of the experiment, total vibrio count in treatment A and B were much higher than that in control pond water media.Fortunately, total number of Vibrio spp. in the pond water of treatment A and B were decreasing much during the culture period of whiteleg shrimp.TBV in these two treatments using RICA probiotics were only 1.92 x 10 2 cfu/mL and 1.25 x 10 2 cfu/mL for the second sampling respectively for treatment A and B, while in control was quite high (1.87 x 10 3 cfu/mL).TBV in treatment A and B for the third sampling were also lower than that in control (Table 2).This result meant that RICA probiotics used in treatment A and B had significant role in controlling vibrio numbers in the shrimp culture water media.Consequently, the whiteleg shrimps in treatment A and B could grow faster than in treatment C (control).Finally the whiteleg shrimp production in treatment A and B were significantly (P<0.05)better than that in control.RICA probiotics consist of three different species of bacteria that firstly selected by their capability of controlling Vibrio spp.So that, it is normal when the number of Vibrio spp. in treatment A and B were lower than that in control.According to    (1993; 2010) total number of Vibrio spp.(TBV) in control pond water for the second sampling (1.87 x 10 3 cfu/mL) is relatively dangerous to the reared shrimp for long time period especially if pond water quality is getting worse like increasing of total organic matter, ammonia-nitrogen (NH 3 -N), and nitritenitrogen (NO 2 -N).It was previously reported by Atmomarsono et al. (1993), that the Vibrio spp.numbers in the reared shrimp pond water should not be over 1 x 10 3 cfu/mL for long time period.
The above result was match with that of reported by Atmomarsono et al. (2009), that appplication of probiotic bacteria that is originated from the sea sediment (RICA-3 = Pseudoalteromonas sp.Edeep-1) could control Vibrio spp. in the culture water media.The use of probiotic bacteria originated from the sea was also reported by Vaseeharan & Ramasamy (2003) using Bacillus subtilis to control pathogenic Vibrio harveyi.Schulze et al. (2006) reported, that application of Pseudoalteromonas undina that is originated from the sea could alleviate the growth of pathogenic virus and bacteria in fish and shrimp culture ponds.Muliani et al. (2009) also reported, that application of bacteria isolate BT951 (RICA-1) which is originated from brackishwater pond (Brevibacillus laterosporus) also could control the growth of Vibrio spp. in the culture pond water.
In general, the number of this Vibrio spp.count (about 10 2 cfu/mL) is still safe for the cultured shrimp as reported by Olafsen et al. (1993in Buller, 2004).Oyster will be sick if the Vibrio spp.count in their hemolymph increase to 10 5 cfu/mL.Moriarty (1997) and Defoirdt (2007) mentioned, that if the Vibrio spp.count in the hemolymph reach 10 5 cfu/mL, then it is called as "quorum sensing", that is the number of bacteria in the hemolymph that possibly causes the diseases of aquatic animals.

Water Quality
In natural condition, water quality variables like water pH, temperature, total organic matter (TOM), ammonia-nitrogen (NH 3 -N), nitrite-nitrogen (NO 2 -N), and nitrate-nitrogen (NO 3 -N) are very important.Organic matter on the pond bottom soil can be used as indicator of the soil fertility.Organic matter in the pond water is also needed by living organisms, but if the concentration of total organic matter is too much, it will enhance the growth of pathogenic organisms like parasite, bacteria, and viruses.So that the amount of organic matter should be maintained at the minimum level as well as ammonia-nitrogen and nitritenitrogen which are considered as toxic substances for the cultured shrimp and fish.
Fluctuation of several water quality variables like total organic matter (TOM), ammonia-nitrogen, nitrite-nitrogen (NO 2 -N), and nitrate-nitrogen (NO 3 -N) are described through below figures.

Total Organic Matter (TOM)
Figure 1 below shows the fluctuation of total organic matter concentration in the cultured shrimp pond water during experiment.
Naturally, total organic matter (TOM) in the body waters consists of three forms, namely soluble, suspended, and coloidal organic matter.Total organic matter in the pond waters during experiment ranged from 63.99-69.45mg/L.Generally, concentrations of total organic matter in treatment C (control) were relatively higher than those in treatment A (alternate use of probiotic RICA-1, RICA-2, and RICA-3) and B (alternate use of probiotic RICA-4, RICA-5, and RICA-3) with the average concentration was 69.27 mg/L, followed by treatment B (66.64 mg/L) and treatment A (65.65 mg/L).However, these concentrations of total organic matter for the whole pond waters were slightly over than the optimal range for whiteleg shrimp culture (30-60 mg/L).
Feeding for whiteleg shrimp with stocking density of 10 pcs of PL-8 shrimp fry/m 2 will not only increase the shrimp body weight, but also increase total organic matter in the pond water and sediment.Reported by Madeali et al. (2004), that increasing of total organic matter in the shrimp pond water up to 30 mg/L could enhance the virulence of Vibrio spp.This might be protected by adding some probiotic bacteria in the pond waters like in treatment A and B. In treatment A, the use of probiotic RICA-1 (Brevibacillus laterosporus) could demineralize total organic matter and ammonia-nitrogen as well.This probiotic RICA-1 also could alleviate the growth of Vibrio spp. in the pond water.This condition made the concentration of total organic matter in this treatment was relatively lower than in treatment B and C (control).However, the use of pobiotic RICA-4 (Bacillus subtilis) that is originated from seaweed (macroalgae) in treatment B also could control the increase of total organic matter in the pond water.
The high concentration of total organic matter in treatment C might be caused by not sufficient natu-ral demineralizing bacteria in the pond water.This could enhance the growth of Vibrio spp.(Table 2).Other result reported by Atmomarsono & Susianingsih (2013), that the alternate use of probiotic bacteria was better than the use of probiotic bacteria combination simultaneously.

Ammonia-Nitrogen (NH 3 -N)
Ammonia-nitrogen is one of the toxic substances in the shrimp pond water that is more harmfull to the cultured shrimp when the water pH and salinity increase.So that the concentration of ammonia-nitrogen must be minimized during the culture period.Fluctuation of the average concentration of ammonia-nitrogen (NH 3 -N) in the vanname shrimp pond waters during experiment could be seen in Figure 2.
The above Figure 2 shows that ammonia-nitrogen (NH 3 -N) concentration in the shrimp pond water during experiment in treatment A (0.0946-0.0989 mg/ L) and treatment B (0.1272-0.1608 mg/L) were much lower than in treatment C (0.1844-0.2818 mg/L).The optimum range of ammonia-nitrogen concentration in the pond water of L. vannamei culture by Adiwijaya et al. (2003) is 0.05-0.1 mg/L.Based on this reference, ammonia-nitrogen concentrations in the pond water of treatment A and B were still in good range, while that of in treatment C for the third sampling was quite high.This might cause stress to the whiteleg shrimp, then finally could not grow well (Table 1).Relatively lower concentration of ammonia-nitrogen in treatment A and B up to the third sampling might be caused by the application of probiotic RICA-2 (Serratia marcescens) in treatment A and RICA-5 (Bacillus licheniformis) in treatment B which are capable of ammonium elimination in the pond water.While the concentration of ammonia-nitrogen in the control pond water (C) was relatively high, because it could not be minimized by natural bacteria in the pond water.

Nitrite-Nitrogen (NO 2 -N)
Nitrite-nitrogen (NO 2 -N) is also considered as one of the toxic substances in the cultured shrimp pond water.So that the concentration of this substance in the culture pond water must be controlled strongly.In natural condition the available nitrite-nitrogen in the pond water will be processed to nitrate-nitrogen that is less toxic substance only if nitrification bacteria like Nitrobacter is available enough and under aerobic condition (enough dissolved oxygen).Otherwise this process will not happen that finally makes harmfull to the cultured shrimp.Concentration of nitrite-nitrogen (NO 2 -N) in the whiteleg shrimp pond waters during experiment could be seen in Figure 3.
The above figure shows that the use of probiotic bacteria seemed to be not effective to decrease concentration of nitrite-nitrogen (NO 2 -N) in the whiteleg shrimp pond waters.Concentration of nitrite-nitrogen in treatment A ranged from 0.0013-0.0217mg/L, in treatment B ranged from 0.0011-0.0201mg/L, and in treatment C ranged from 0.0028-0.0251mg/L.However, the above concentrations of nitrite-nitrogen for the overall pond waters were still in good range for the cultred shrimp that is much less than 4.5 mg/L as suggested by (Chen & Chin, 1998ain Boyd, 1990).
Nitrite-nitrogen is considered as one the immunostressors for the cultured shrimp, so that the con-centration in the pond waters must be minimized as small as possible.Usually this nitrite-nitrogen is affected by concentration of total ammonium nitrogen (TAN) in the pond waters and the available demineralized bacteria naturally.In that case, the additional probiotic bacteria which has ability of neutralizing nitrite-nitrogen will help this process.Adding probiotic bacteria RICA-3 (Pseudoalteromonas sp.Edeep-1) in treatment A and B could not only protect the growth of the pathogenic Vibrio spp., but also neutralize nitrite-nitrogen to become nitrate-nitrogen which is not toxic to the whiteleg shrimp.

Nitrate-Nitrogen (NO 3 -N)
Fluctuation of nitrate-nitrogen (NO 3 -N) concentration in the whiteleg shrimp pond waters during experiment could be seen in Figure 4.
Unlike nitrite-nitrogen and ammonia-nitrogen, nitrate-nitrogen is considered as less toxic substance in the pond waters.Nitrate-nitrogen can be used directly by phytoplankton and other algae in the pond waters if ortho-phoshate and other micro nutrients are available in the waters.So that the high concentration of nitrate-nitrogen is not dangerous to the reared whiteleg shrimp.However, to prevent the pond waters from the phytoplankton bloom, concentration of nitrate-nitrogen should be no more than 1.0 mg/L (Wedemeyer, 1996).So that, if the nitrate-nitrogen concentration of the pond water increases as well as nitrite-nitrogen and ammonia-nitrogen, some molasses or other carbon source should be applied periodically.In this case Atmomarsono (2004) mentioned that C:N:P ratio for the normal growth of usefull phytoplankton should be about 106:16:1.So that in in- tensive shrimp pond which uses quite a lot of artificial feed (pellet), some uneaten feed will be demineralized to ammonium and nitrite that finally increases the amount of total nitrogen in the pond water.In this case, some farmers apply molasses or other carbon sources to stabilize the C:N ratio in the pond water.

CONCLUSION
The high survival rate, growth, and production of whiteleg shrimp in this experiment might be related to the use of probiotic bacteria which control total organic matter, ammonia-nitrogen, and the growth of Vibrio spp. in the pond waters.

Figure 1 .
Figure 1.Monthly fluctuation of total organic matter in the culture pond water during experiment on alternately use of RICA probiotics for whiteleg shrimp in traditional-plus technology.A = Alternate use of probiotic RICA-1, RICA-2, and RICA-3; B = Alternate use of probiotic RICA-4, RICA-5, and RICA-3; C = Control (without probiotic bacteria)

Figure 2 .
Figure 2. Monthly fluctuation of ammonia-nitrogen (NH 3 -N) concentration in the whiteleg shrimp pond water during experiment on alternate use of the RICA probiotic bacteria at the Marana Experimental Pond Station.A = Alternate use of probiotic RICA-1, RICA-2, and RICA-3; B = Alternate use of probiotic RICA-4, RICA-5, and RICA-3; C = Control (without probiotic bacteria)

Figure 3 .
Figure 3. Monthly fluctuation of nitrite-nitrogen (NO 2 -N) concentration in the whiteleg shrimp pond water during experiment on alternate use of the RICA probiotic bacteria at the Marana Experimental Pond Station.A = Alternate use of probiotic RICA-1, RICA-2, and RICA-3; B = Alternate use of probiotic RICA-4, RICA-5, and RICA-3; C = Control (without probiotic bacteria)

Table 1 .
Atmomarsono et Survival rate, size, and production of whiteleg shrimp reared in traditional plus pond aerated with supercharge blower after 11 weeks of culture period applied with alternate use of different probiotics