Use of Serratia marcescens MY1112 as Probiotic Bacteria for Tiger Shrimp Culture in the Acid-sulfate Soil Ponds

Probiotic bacteria have been commonly used for disease prevention in aquaculture systems, but sometimes these probiotic bacteria do not work in certain ponds. This experiment aimed to find out if the probiotic bacteria of Serratia marcescens MY1112 could work properly for tiger shrimp culture in acid-sulfate soil ponds. Eight 0.5-ha ponds located in Samataring village of Sinjai regency were used. Two treatments of bacteria probiotic combinations were applied here, namely A) Brevibacillus laterosporus BT951, Serratia marcescens MY1112, and Bacillus licheniformis BM58; and B) Use of Bacillus subtilis BM12 to replace S. marcescens MY1112 in the bacteria combination. The shrimp production and their survival rate in treatment A were significantly better than those of treatment B. However, probiotic bacteria of Serratia marcescens MY1112 could work better in the acid-sulfate soil pond if combined with the dolomite application weekly.


Introduction
Very low pH, high acidity potential, and content of highly-toxic elements are characteristic of acidsulfate soil (ASS) ponds in the Eastern part of South Sulawesi province [1].During pond preparation, the reddish brown color sediment is shown after pond drying, and then it is called acid-sulfate soil or "cat's clay" [2].The ASS ponds have very low productivity if the farmers use them for the shrimp culture during the rainy season because rainwater reacts to the iron pyrite (FeS2) on the pond dike and decreases the pond water pH that causes some mortality of the cultured shrimp.This problem could be solved by the shrimp farmers if the ponds are reclamation (bioremediation) before being used for the shrimp culture.Bioremediation procedures like pond drying, water filling or water submerging, and water flushing should be done several times during the dry season, and finally, pond liming before filling with good quality water for the shrimp culture.Most of the extensive shrimp farmers do not care to do this procedure [1].
At present tiger shrimp and Pacific white-leg shrimp culture in the world are still burdened by vibriosis and white spot syndrome virus (WSSV) [3,5,7,8,11,17].Viral pathogens could cause mortality over 60%, but now bacterial pathogens like Vibrio harveyi and V. parahaemolyticus also cause high mortality in the shrimp culture in China, Vietnam, and Thailand [4].Currently, white-leg shrimp culture has already dominated Indonesian shrimp culture since white-leg shrimp (Litopenaeus vannamei) are more productive than tiger shrimp (Penaeus monodon) [6,18] Many aquaculture researchers have tried to overcome vibriosis and WSSV in tiger shrimp culture.They try to prevent shrimp diseases in eco-friendly ways.Some of them use mangrove herbs to evaluate their capability to protect tiger shrimp from diseases [19].Other researchers use microalgae like Chlorella sp and Porphyridium spp to solve vibriosis [9, 10], while other researchers prevent shrimp vibriosis using of mucus of Nile tilapia (Oreochromis niloticus) [8].
Nowadays, one of the standard operational procedures to prevent cultured shrimp from diseases is using probiotic bacteria [5,7,11,17].Some RICA probiotic bacteria have been applied in a shrimp hatchery and the grow-out ponds [5,7].Bacillus subtilis given in the vannamei shrimp feed enhanced the shrimp growth and their resistance to diseases [14,15].Using New Generating Sequencing (NGS) the genera of Pseudoalteromonas were found in the wastewater treatment plant (WWTP) of the super intensive vannamei shrimp in Takalar Regency previously applied this bacterium in the cultured vannamei shrimp ponds [18].A proteolytic bacterium of Pseudoalteromons ganghwensis isolated from the vanname shrimp pond could be used as a probiotic agent for vanname shrimp culture [13].Until now, the main causative agents of tiger shrimp mortality in Indonesia are Vibrio harveyi, V. parahaemolyticus, and White Spot Syndrome Virus (WSSV) [5, 6, 8-10].However, there were no reports that acid-sulfate soil ponds as the causative agent of mass shrimp mortality in the brackishwater ponds.The extensive shrimp farmers in South Sulawesi only know that they produced about 75 kg/ha/three months with an average survival rate of about 18%.This mainly happened because of the acid-sulfate soil ponds in the Eastern part of South Sulawesi Province.This paper evaluates whether Serratia marcescens strain MY1112 or Bacillus subtilis strain BM12 could be used as the shrimp disease preventive agent in the acid-sulfate soil (ASS) ponds without bioremediation.

Materials
Four RICA probiotic bacteria used in this experiment were RICA1, RICA2 (Serratia marcescens strain MY1112), RICA4 (Bacillus subtilis strain BM12), and RICA5.Each probiotic bacteria was applied alternately every week for about 2.5 L of three-day culture per pond of about 0.5 ha in size.with about 50 cm in water depth.Each probiotic was fermented every week alternately following standard operational procedures [5].In the shrimp pond of about 0.5 ha, 20,000 tiger shrimp postlarvae PL12 were stocked, and then after 20 days of culture (DOC 20), they were fed with commercial pellet feed.The pellet feed given to the shrimp was about 20% of total shrimp biomass on DOC 20, then gradually decreased to about 2% one week before harvesting (three months of culture).

Methods
Eight 0.5-ha shrimp ponds located in Samataring Village of Sinjai Regency, South Sulawesi, Indonesia were used in this experiment to see the best combination of probiotic bacteria applied in the acid-sulfate soil ponds.Two treatments were evaluated here, namely: A) RICA1, Serratia marcescens MY1112 (RICA2), and RICA5; B) RICA1, Bacillus subtilis BM12 (RICA4), and RICA5.
All of the eight selected ponds were considered to be acid-sulfate soil ponds based on the differences in pond bottom pH before oxidizing (pH Fresh) and after oxidizing with hydrogen peroxide (pH Fox).If this value (pH Fresh -pH Fox) is more than 3, then we call it an acid-sulfate soil pond.All of the ponds were prepared by the farmers without any bioremediation.The ponds used here were dried, liming, and fertilized using basic fertilizer before being filled up with the source brackishwater.Each pond of about 0.5 ha in size was then stocked with 20,000 pcs of tiger shrimp postlarvae 12 (Penaeus monodon).After 20 days of stocking, the cultured shrimp were fed with commercial pellet feed.
The pond sediment and the pond water were sampled biweekly to check the number of Total heterotrophic bacteria (TPC) analyzed with tryptic soy agar (TSA) media, while total Vibrio count (TBV) analyzed with thiosulphate citrate bile salt sucrose agar (TCBSA) [5,20,21].After TPC and TBV were counted separately, the TBV/TPC ratio was calculated in percentage of TBV compared to heterotrophic bacteria (TBV/TPC x 100%).Data of these TBV / TPC ratios of each sampling were then tabulated and analyzed graphically.
The shrimp pond water quality parameters (in situ and ex-situ) were checked biweekly.In situ, water quality parameters (water temperature, dissolved oxygen, water pH, salinity, and total alkalinity) were checked directly in the field using a DO-meter YSI model, a pH meter, and an Atago hand refractometer, respectively.While total alkalinity was measured titrimetically.Ex-situ water quality parameters like total organic matter (TOM) were measured titrimetically, while ammonia-nitrogen (NH3-N), nitritenitrogen (NO2-N), and nitrate-nitrogen (NO3-N) were observed using a spectrophotometer in the RIBAFE laboratory [2,22].

Results
The survival rate and production of reared tiger shrimp in treatment A (using probiotic bacteria of RICA 1-2-5) were better than those of treatment B (using probiotic bacteria of RICA 1-4-5) (Table 1).This result means that combination probiotic bacteria using Serratia marcescens strain MY1112 in treatment A had better responses than those using Bacillus subtilis strain BM12 in treatment B. This was reasonable since the probiotic bacteria of Serratia marcescens strain MY1112 was isolated from the mangrove leaf living around the brackishwater pond area, meanwhile, Bacillus subtilis strain BM12 originated from seaweed living in the seawater having relatively high salinity and water pH.There were no differences between treatments A and B on dissolved oxygen (DO) in tiger shrimp ponds, namely 3.78 -6.36 mg/L in range (Table 2).The salinity range of the pond water during the experiment (March to June) was relatively normal for the reared tiger shrimp which was 14 -26 ppt.Pond water pH here was relatively low and quite dangerous for the cultured tiger shrimp in both treatments.This very low pond water pH (5.00 -8.86) was mainly caused by the acid-sulfate soil ponds.Decreasing the total alkalinity of the shrimp pond water to 20.1 mg/L CaCO3 equivalent especially during and after rain was not good at all for the growing phytoplankton and neither for tiger shrimp.This very low total alkalinity (less than 80 mg/L CaCO3 equivalent) was harmful to the cultured tiger shrimp due to unstable pond water pH which possibly enhanced the growth of Vibrio spp and might suppress the reared tiger shrimp.Concentrations of total organic matter (TOM) in the pond waters were relatively low in both treatments (16.3 -66.9 mg/L) (Table 3) since these values could be controlled by the availability of Brevibacillus laterosporus strain BT951 in the pond water.Ammonia-nitrogen (NH3-N) concentrations were sometimes increasing over the normal values (0.45 mg/L) [2], but these values would not be a problem in treatment A which had enough probiotic bacteria of Serratia marcescens MY1112 having a role of changing ammonia-nitrogen to nitrite-nitrogen (NO2-N).Instead of Serratia marcescens MY1112 in treatment B, there was Bacillus subtilis BM12 which functions in changing organic matter to ammonianitrogen but does not control the increase of ammonia-nitrogen.That was why ammonia-nitrogen (NH3-N) in treatment B was relatively high.The concentration of nitrite-nitrogen (NO2-N) and nitrate-nitrogen (NO3-N) during the shrimp culture experiment was mostly low for the cultured tiger shrimp in both treatments [2].This might be controlled by the availability of Bacillus licheniformis BM58 in both treatments.The range of dissolved oxygen during observation (3.78-6.52 mg/L) was quite good for probiotic bacteria growth.However, after rain before the six-week experiment, the pond water pH dropped to 5.00 finally enhancing the Vibrio growth in the pond causing TBV/TPC ratios in the pond sediment and the pond water over 10% (figure 1 and Figure 2), so that some mortality of the shrimp occurred during this time.

Discussions
Probiotic bacteria of Serratia marcescens strain MY1112 (in treatment A) had better effects than probiotic bacteria of Bacillus subtilis strain BM12 (in treatment B).The average survival rate and the shrimp production of treatment A (16.55+4.14% and 93.9+9.4 kg/ha/three months) were significantly better than those of treatment B (5.89+2.22%and 46.9+23.6 kg/ha/three months).These results might be related to the origin of the bacteria, where Serratia marcescens strain MY1112 was originated from the mangrove leaf grown in the pond area with brackishwater condition, while Bacillus subtilis strain BM12 was originated from the seaweed that mostly in the area of higher salinity and higher water pH [6].In other words, Serratia marcescens strain MY1112 could live and function better than Bacillus subtilis strain BM12 in the acid-sulfate soil ponds that mostly have very low water pH.During rainy days just before the sixth week of culture, the coming rain could suddenly drop pond water alkalinity up to 20.1 mg/L CaCO3 equivalent and pond water pH up to 5.0 (Table 2).This condition might become a stressor for the cultured tiger shrimp.Very low pond water alkalinity (much less than 80 mg/L CaCO3 equivalent) could affect the daily variation of the pond water pH so this might be one of the shrimp stressors.The other fact is that the bacteria Serratia marcescens strain MY1112 in the "in vitro" test could grow faster than Bacillus subtilis strain BM12.Based on this condition, the bacteria Serratia marcescens strain MY1112 could inhibit the Vibrio growth in the ponds, so that the TBV/TPC ratio in the pond sediment and the pond water in treatment A could be minimized, while the Bacillus subtilis strain BM12 could not work as good as Serratia marcescens strain MY1112.Figure 1 and Figure 2 show the fluctuation of TBV/TPC ratios in the pond sediment and the pond water of the tiger shrimp that sometimes increased by over 10% after six weeks of culture, which is dangerous for the cultured shrimp [5].The increasing number of Vibrio spp more than 10% of the heterotrophic bacterial count in the pond sediment and the pond water could be dangerous to the cultured tiger shrimp.Concentrations of total organic matter (TOM) in the pond waters were relatively low in both treatments (16.3 -66.9 mg/L) since this organic matter from the shrimp feces and uneaten feed could be minimized by Brevibacillus laterosporus strain BT951 in the pond water.The ammonia-nitrogen (NH3-N) concentrations were sometimes increasing over the normal values (0.45 mg/L) [2], but these values would not be a problem in treatment A which had enough probiotic bacteria of Serratia marcescens MY1112 that change ammonia-nitrogen to nitrite-nitrogen (NO2-N).Instead of Serratia marcescens MY1112 in treatment B, there was Bacillus subtilis BM12 in treatment B which functions as an organic matter demineralizer, but not controlling ammonia-nitrogen in the shrimp pond water.The concentration of nitrite-nitrogen (NO2-N) and nitrate-nitrogen (NO3-N) during the shrimp culture experiment was mostly low for the cultured tiger shrimp in both treatments [2].This might be controlled by the availability of Bacillus licheniformis BM58 in both treatments.
Based on Table 2 the problem here in the acid-sulfate soil pond was very low total alkalinity (sometimes reach to 20.1 mg/L CaCO3 equivalent) and very low pond water pH (5.0), it is recommended that dolomite lime [CaMg(CO3)2] for about 3-5 mg/L should be applied every week and just before or after rain.The range of total alkalinity of the pond water (20 -156 mg/L CaCO3 equivalent) was not good for the growing tiger shrimp, because pond water pH was daily variation and the phytoplankton was relatively hard to grow finally made very clear pond water (not enough phytoplankton in it).During the rainy season, the total alkalinity of the pond water should be maintained at over 80 mg/L CaCO3 equivalent or over 100 mg/L CaCO3 equivalent during the dry season.The decrease of total alkalinity below 80 mg/L CaCO3 equivalent might cause unstable pond water pH and possibly cause the stress of the cultured tiger shrimp.This usually occurs during the rainy season.That is why we still need some dolomite to stabilize the pond water pH during the rainy season.Since total alkalinity is related to the availability of calcium and magnesium in the pond water [2] we need dolomite lime frequently to prevent the decrease of the pond water pH.To minimize dolomite lime in the acid sulfate soil ponds, bioremediation (reclamation) during pond preparation should be done properly in the dry season by drying, water submerging, water flushing, and dolomite liming.

Conclusion
The use of probiotic bacteria of Serratia marcescens MY1112 had better results than the use of Bacillus subtilis BM12 on the survival rate and the production of reared tiger shrimp in extensive acid-sulfate soil ponds of Samataring village of Sinjai regency.TBV/TPC ratio in the pond sediment and the pond water using Serratia marcescens MY1112 was relatively lower than that of using Bacillus subtilis BM12 Without bioremediation, we need dolomite lime application weekly to prevent unstable pond water pH.

Acknowledgments
The authors thank the Ministry of Marine Affairs and Fisheries for the research funding.Their appreciation is also delivered to all researchers and technicians of the Research Institute for Brackishwater Aquaculture and Fisheries Extension office, especially Nurjanna, Rezki Ayu Lestari, Siti Dwi Hadiyanti, drh.Munawarah Syam, Sitti Rohani, Kurniah, Rahmi, and Muhammad Rusli, who helped them in sampling, and analyzing bacteria, soil and water quality parameters, and other activities in the RIBAFE laboratory and the field works.

Figure 1 .
Figure 1.TBV/TPC ratio (%) in the reared tiger shrimp pond sediment applied A) Combination probiotic bacteria of RICA1, Serratia marcescens MY1112, and RICA5 or B) Combination probiotic bacteria of RICA1, Bacillus subtilis BM12, and RICA5 in extensive acid-sulfate soil shrimp ponds in Samataring village of Sinjai regency, Indonesia.

Figure 2 .
Figure 2. TBV/TPC ratio (%) in the reared tiger shrimp pond water applied A) Combination probiotic bacteria of RICA1, Serratia marcescens MY1112, and RICA5 or B) Combination probiotic bacteria of RICA1, Bacillus subtilis BM12, and RICA5 in extensive acid-sulfate soil shrimp ponds in Samataring village of Sinjai regency, Indonesia.

Table 1 .
Tiger shrimp survival rate and its production in extensive acid-sulfate soil ponds of Samataring village of Sinjai Regency, Indonesia (during a rainy season) *) The same superscript at the same column means Not significantly different (P>0.05),N = 4 replications/treatment A) Combination of RICA1, Serratia marcescens MY1112, and RICA5 B) Combination of RICA1, Bacillus subtilis BM12, and RICA5

Table 2 .
Range values of "in situ" water quality parameters in extensive acid-sulfate soil ponds used for tiger shrimp culture in Samataring village of Sinjai regency, Indonesia during the rainy season