Novel strains of Bacillus subtilis for improving water quality and controlling pathogens in shrimp aquaculture

Karthik Masagounder, Michelle Dargatz, Evonik Operations GmbH, Germany, Sarah He, Evonik (China) Co., Ltd., China

Whiteleg shrimp production has doubled in the past decade, growing from 2.6 million metric tons (mMT) to 4.97 mMT between 2010 and 2018. It had the largest share (53%) of total crustacean production (FAO, 2020) over this period too. This significant increase in production is partly because of the intensification of shrimp farming, which is gradually shifting from semi-intensive to intensive and superintensive farming. As farming practices intensify, stocking density has gone up from 50 shrimp/m2 to 200-400 shrimp/m2. Unfortunately, the rapid growth and intensification of shrimp farming create tremendous pressure on the water quality and nutrient load of the surrounding environment.

Clean water is an essential resource for the sustainable growth of shrimp farming. With stocking density continuing to increase, there is an escalating challenge to maintain the ideal water quality parameters needed for healthy shrimp. Shrimp feed contains 35-45% crude protein, of which only about 35% is retained in the body. The rest is largely catabolized, contributing to nitrogen waste.

One way to improve feed protein utilization is to formulate feed following the ideal protein concept. This means meeting the exact amino acid requirements of shrimp with no deficiency and no, or little, excess. In practical diets – formulated using ingredients such as fishmeal, soybean meal and wheat products – except for the first limiting amino acid, methionine (Met), the other amino acids are provided in excess and are inevitably catabolized, contributing to nitrogen excretion.

With the use of supplemental amino acids such as AQUAVI® Met-Met (DL-Methionyl DL-Methionine), Biolys® (Lysine Sulfate) and ThreAMINO® (L-Threonine) in diet formulation, we can reduce the inclusion of protein-rich ingredients, and minimize the excess levels of dietary protein or amino acid levels – still meet the requirements for all the limiting amino acids. This will increase the protein utilization to 40-45% (Nunes et al., 2019; Masagounder et al., 2021). However, while this approach can reduce the nitrogen load, there is still a lot going into the culture water which needs to be removed.

Ammonia is the principal nitrogen waste product excreted by shrimp. Ammonia from the hemolymph (blood) of shrimp is largely excreted into the water across the gills via diffusion process in the form of un-ionized ammonia.

Assuming 35-45% protein retention, this means, for 10 g shrimp stocked at 100/m2 in a 1 hectare (ha) pond (water depth 1 m) and fed at 3% body weight per day with a feed containing 35% crude protein, about 11,000-13,000 g ammonia per ha or 1.1-1.3 parts per million (ppm) NH3 is added into the pond every day. In other words, for every 100 kg feed, about 0.4 ppm of NH3 is added into the pond water. If this is not removed, this will quickly increase the ammonia concentration in water. As the concentration of ammonia in the water increases, the diffusion gradient

of NH3 from hemolymph into the water decreases – leading to accumulation of NH3 in the blood causing serious physiological stress and illness to the shrimp. The exact LC50 of total ammonia nitrogen (TAN) on shrimp depends on various parameters such as salinity, pH, species and age and varies, 30-110 mg/L over 48 h. Although ammonia is the principal nitrogenous waste from shrimp, we also find nitrite accumulation in the culture water over time. Nitrite is an intermediate product in the nitrification or de-nitrification cycle. In the nitrification process, ammonia is oxidized by highly aerobic, chemoautotrophic bacteria. During denitrification, heterotrophic bacteria use nitrate in the absence of oxygen (anaerobic process) and nitrate is reduced into nitrite and eventually into a gaseous form of nitrogen. Both ammonia (mainly un-ionized form) and nitrite are toxic to shrimp. As a consequence, the toxicity of NH3-N and NO2-N negatively impacts excretion, respiration, osmoregulation, immunity, antioxidant defense, molting, growth, feed utilization and eventually survival of the animal (Zhao et al., 2020).

A multi-strain Bacillus subtilis-based product for water application

Probiotics are increasingly used in aquaculture to control disease, improve water quality, and enhance the health status of fish and shrimp. Bacillus-based probiotics with species including B. subtilis (Liu et al., 2010; Maia et al., 2016), B. licheniformis (Zhang et al., 2011), B. amyloliquefaciens (Xie et al., 2013) and B. pumilus (Nimrat et al., 2012) are commonly used in shrimp aquaculture for both water and feed application. Gram-positive spore-forming bacteria from the species Bacillus subtilis preferably use glutamine as a nitrogen source but in absence of such, it can incorporate inorganic nitrogen sources, such as ammonia into organic biomolecules. B. subtilis has the genetic features to take up ammonia, nitrite, or nitrate from the environment, reduce nitrate and nitrite to ammonia and feed this into glutamine synthesis. The efficiency of this assimilation of inorganic nitrogen varies among strains as the activities of the required enzymes are usually under strict control. By applying efficient ammonia and nitrite assimilating B. subtilis strains to polluted water, reduction of toxic ammonia and nitrite can be archived by simple growth and biomass formation of the added bacteria.

With this background, Evonik developed a new product called AQUAVI® Pro-Pond (Pro-Pond).

Figure 1. Ammonia and nitrite removal of the three specific B. subtilis strains included in Pro-Pond under 0, 10 (DSM 3351 and 3352), 15 (CCTC M 2020356) and 30 ppt salinity after 48 hours.

Pro-Pond is composed of three B. subtilis strains that were selected based on their ammonia and nitrite removal capabilities under a wide range of salinities. To ensure product stability, Pro-Pond contains the strains in the form of endospores with a total concentration of 1.5×1010 CFU/g.

Ammonia and nitrite removal capacities under salinities ranging from 0 to 30 ppt for the single product strains are depicted in Figure 1. The efficacy of the product containing the combined spores of the three strains was tested under high ammonia concentrations after 24 and 48 hours and is illustrated in Figure 2.

Figure 2. Ammonia and nitrite removal of Pro-Pond after 24 and 48 hours.

Figure 3. Trend of total ammonia level over the whole culture period in the Pro-Pond treated ponds (purple, 0.25 ppm) versus commercial control ponds treated with multiple products (C1, C2, C3). Solid dots indicate the days when the pond was treated with its respective product. Bigger solid grey dots in C3 indicate the days when two different products were applied on the same day. Purple arrows in the top panel indicate drop in NH3 level following the Pro-Pond treatment.

Evaluation in a semi-intensive shrimp farm in China

To evaluate the efficacy of AQUAVI® Pro-Pond on the nitrogen removal efficacy, a commercial pond trial was conducted in collaboration with Prof. Dr. Shijun Chen, South China Agricultural University at the Guangdong province in China between November 2020 and March 2021.

Three earthen ponds at a stocking density of ~80 shrimp/m2 were allotted to the Pro-Pond treatment. The average culture period of the three ponds lasted for 108 days (97, 112, and 115 days for the three ponds). Three other earthen ponds (average area 3,557 m2 or 5.3 mu) were assigned to commercial control and the average culture period lasted 102 days. The three ponds (average area 2,668 m2 or 4 mu) allotted for Pro-Pond group were treated with Pro-Pond at 11.3 times per pond on an average (34 times in total for the three ponds) at a dose of 0.25 ppm or 2.5 kg per ha during each application over the whole culture period (108 days).

On the other hand, the three commercial control ponds were treated with four different commercial products at 10.7 times per pond on an average (32 times in total for the three ponds) over the 102 day culture period. These four commercial products were based on probiotics (Bacillus, nitrifying bacteria, Enterococcus faecalis), yeast and/or enzyme mix. Product selection, dose and application frequency were decided by the farmer based on his experience and the recommendation from the respective product supplier. Ammonia nitrogen and nitrogen were recorded daily for each pond until harvest. Given the differences in product application in the commercial control ponds, the average of Pro-Pond treated ponds were compared with individual commercial control pond on the ammonia level over the culture period (Fig. 3). Pro-Pond was able to keep the ammonia and nitrite levels down (Fig. 3, 4). When the ammonia level increased, the product was applied and we could see that in the following one to two days, the NH3 level went down. Overall, Pro-Pond was marginally better than the commercial treatment on controlling the NH3-N level (0.45 mg/l versus 0.53 mg/l; p-value = 0.10, t-test). There were no differences between the two groups on the nitrite level. Although multiple products were applied in the control ponds by the farmer, Bacillus subtilis alone was sufficient to control nitrogen waste in the commercial ponds. At the end of culture, the average harvest yield of shrimp was 505 kg/mu for the control group and 535 kg/mu for the B. subtilis group (6% higher).

Results demonstrate that Pro-Pond can be used to control nitrogen waste and improve shrimp production.

Potential on controlling common shrimp and fish pathogens

A co-incubation of the Pro-Pond product strains and relevant aquaculture pathogens via antagonism assay was applied to determine the inhibitory potential. The inhibitory zone/halo formed in bacterial lawn of the pathogen after 24 hours was determined and classified in: a negative sign (-) means no inhibition, a positive sign (+) means mild inhibition, double positive sign (++)

Figure 4. Overall concentration of NH3-N and NO2-N in the Control and Pro-Pond groups. NH3-N level in the Pro-Pond treated ponds was marginally lower (-15%) than that in the control ponds which were treated with four different products (p-value = 0.10, t-test).

Figure 5. Final yield of shrimp (kg/mu) for the commercial control and Pro-Pond treated groups.

Table 1. In vitro inhibition profiles of Pro-Pond strains towards different pathogens relevant for aquaculture.

DSM 33351 DSM 33352 CCTCC M 2020356

Vibrio parahaemolyticus Vibrio penaecida Vibrio haarveyi Vibrio anguilarum Vibrio alginolyticus Aeromonas hydrophila Not tested

Streptococcus agalactiae

+++ +++

Not tested

means medium inhibition, and finally triple positive sign (+++) means strong inhibition. The combination of strains yields a broad pathogen inhibition profile is illustrated in Table 1.

Overall, our study results demonstrate that AQUAVI Pro-Pond® developed with three unique strains of B. subtilis bacteria can be used for water application to effectively remove the nitrogenous wastes (NH3 N, NO2-N and NO3-N) from the culture water and potentially keep the pathogen load under control in commercial shrimp farms.

References are available on request.

More information:

Karthik Masagounder, Head of Aqua Research Evonik Operations E: animal-nutrition@evonik.com

Michelle Dargatz

Scientist Evonik Operations

Sarah He

TSM and R&D Manager, Aqua Evonik (China) Co. Ltd.

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