Int. J. Aquat. Biol. (2022) 10(3): 201-208 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2022 Iranian Society of Ichthyology Original Article Evaluation of Pseudomonas stutzeri AM1 and Pseudomonas oleovorans ST1.1 isolated from shrimp pond sediments as probiotics for whiteleg shrimp, Litopenaeus vannamei culture Thi Cam Tu Phan, Ngoc Ut Vu, Thi Tuyet Ngan Pham, Hung Hai Vu, Truong Giang Huynh College of Aquaculture and Fisheries, Can Tho University, Campus II, 3/2 street, Ninh Kieu District, Can Tho City, Vietnam. s Article history: Received 26 March 2022 Accepted 27 May 2022 Available online 2 5 June 2022 Keywords: Probiotics Pseudomonas Litopenaeus vannamei Water quality Growth performance Abstract: This study aimed to isolate the probiotic potential of nitrifying bacterial strains and to evaluate their effects on water quality and growth performance of the whiteleg shrimp, Litopenaeus vannamei. Based on an initial screening of 100 isolates identified from sediment samples, 12 strains could remove nitrogen compounds and two strains (Pseudomonas stutzeri AM1 and P. oleovorans ST1.1) showed highly efficient nitrogen removal ability. Within 96 h, total ammonia nitrogen (TAN) removal efficiency in the two strains was 28.0-31.6% and 21.5-24.9%, respectively. The water addition of 103 CFUmL-1 of P. stutzeri AM1 (T1) and P. oleovorans ST1.1 (T2) effectively reduced TAN, nitrite, nitrate, and total sulfide and increased the survival rate and biomass of shrimp. However, no significant differences were found between the control (T0) and treatment groups (T1 and T2) in the final weight, weight gain and specific growth rate of shrimp. Overall, P. stutzeri AM1 (T1) and P. oleovorans ST1.1 used as water supplements improved water quality and the survival rate of whiteleg shrimp. Introduction Shrimp farming is one of the fastest-growing aquaculture sectors in many countries worldwide. In Vietnam, the whiteleg shrimp, Litopenaeus vannamei is a hugely popular farmed shrimp; in 2020, this species's farming area and production reached 113,418 ha and 900,000 tons, respectively, increasing by 12% over the same period in 2019 (MARD, 2021). In recent years, environmental problems caused by poor water quality are one of the threats to shrimp production (Wang et al., 2018). Through several studies, probiotics have demonstrated significant potential as therapeutic options for improving water quality (Cai et al., 2019; Nimrat et al., 2019; Kim et al., 2021). Probiotics in aquaculture can be administered through water additives or feed supplements during the rearing of aquatic species (Zhou et al., 2009; Leyva-Madrigal *Correspondence: Truong Giang Huynh DOI: http//doi.org/10.22034/ijab.v10i3.1580 E-mail: htgiang@ctu.edu.vn et al., 2011). A variety of bacteria (Bacillus, Lactobacillus, Lactococcus, Entercoccus, Clsotridium, Aeromonas, and Pseudomonas), yeasts, and unicellular algae have been studied for use as probiotics in aquaculture (Irianto and Austin, 2002; Vijayan et al., 2005). Members of the genus Pseudomonas are commonly found in aquatic environments, including shrimp culture ponds (Preetha et al., 2015). The members of this genus are Gram-negative rod with polar flagella providing motility, oxidase-positive, and catalase-positive, and it is obligate respiratory (Jurat-Fuentes and Jackson, 2012). Based on the results of Pseudomonas application in several studies, these species have been proven to control pathogens, enhance growth performance, immune system function, and improve environmental conditions in the culture systems favorable to the 202 Phan et al./ Pseudomonas stutzeri and P. oleovorans as probiotics for whiteleg shrimp species being cultured (Preetha et al., 2015; Hai et al., 2009). In addition, Dou et al. (2021) suggested that Pseudomonas species possess great potential to remove nitrogen from wastewater based on aerobic autotrophic nitrification and anaerobic heterotrophic denitrification processes. For example, Jin et al. (2015) reported that Pseudomonas sp. ADN-42, isolated from soil, has good nitrogen removal performance. Similar results were found in P. stutzeri (Wen et al., 2010), P. putida Y-9 (Xu et al., 2017), and P. putida ZN1 (Zhang et al., 2018). The present study aimed to isolate and identify efficacious probiotic Pseudomonas spp. from shrimp pond sediments and examine their efficacy to remove nitrogen and enhance growth performance, survival, and water quality in whiteleg shrimp in vitro and in vivo conditions. Two strains, Pseudomonas stutzeri AM1 and P. oleovorans ST1.1, obtained in this study, exhibits huge potential as a probiotic candidate, as they effectively improved shrimp survival and water quality. The present study's findings can be applied to enhance health status and water quality in the shrimp culture industry. Materials and Methods Isolation of candidate probiotic bacteria: Sediment samples were collected from extensive shrimp ponds in Soc Trang, Tra Vinh, and Kien Giang provinces, and Pseudomonas spp. were isolated from the collected samples using AOB and NOB media (Chankaew et al., 2017). Pseudomonas isolates were screened by applying the nitrogen removal tests (Yang et al., 2011). They were identified using 16S rRNA sequencing according to the manufacturer’s instructions after PCR amplification using a 27F-CM primer (5′ - AGAGT TTGATCMTGG CTCAG - 3′) and a 1492R primer (5′ - TACGGYTACCTTGTT ACGACTT - 3′) (Frank et al., 2008). The purified products were sequenced by the Nam Khoa Biotek company, Ho Chi Minh City, Vietnam. Sequences were analyzed through http://www.ncbi.nlm.nih .gov/blast by comparing them with bacterial 16S rRNA sequences in Genbank to identify species. Experimental design Experiment 1: In vitro evaluation of nitrogen removal efficiency of P. stutzeri AM1 and P. oleovorans ST1.1: Wastewater was collected from shrimp pond and analyzed for initial concentrations of TAN, NO2 --N, and NO3 --N. A single colony of strains AM1 and ST1.1 were cultivated in a Luria Bertani broth medium and incubated with shaking until the stationary phase. The bacterial cells were harvested by centrifuging at 3000 rpm for 10 min at 4℃ and washed twice with 0.9% sterile NaCl solution. Then, the cells were re- suspended in sterile saline (0.9% NaCl) and adjusted to achieve a bacterial cell density of 1×108 CFU mL- 1 used for experiments (Lami et al., 2020). The two-candidate probiotic bacteria were tested on the Duran bottle scale for nitrogen removal efficiency. Pseudomonas stutzeri AM1 (T1, T2, and T3) and P. oleovorans ST1.1 (T4, T5, and T6) at different concentrations (103, 104, and 105 CFUmL- 1) were inoculated into 200 mL of wastewater in 250 mL Duran bottle shaken at 150 rpm, 30ºC for four days. Each treatment was run in triplicates. Water parameters, including TAN, NO2 --N, NO3 --N, pH, and temperature, were measured daily to calculate the nitrogen removal efficiency using the formula of Nitrogen removal (%) = ((Ci -Ct) / Ci) × 100, Where Ci is the initial concentration of nitrogen and Ct is the concentration of nitrogen after sampling time. Experiment 2: Evaluation of P. stutzeri AM1 and P. oleovorans ST11 on water quality and growth performance of whiteleg shrimp: Based on experiment 1, P. stutzeri AM1 and P. oleovorans ST1.1 at a concentration of 103 CFUmL-1 were used for experiment 2. This experiment was carried out in the wet lab of the College of Aquaculture and Fisheries, Can Tho University. Shrimp were fed a commercial diet (40% crude protein, 6% crude lipids, and 4% ash) for 7 days. After acclimatization, 100 shrimp (0.66±0.01 g) were randomly stocked into each composite tank, with a capacity of 500 L. The groups were given three treatments: the control group (T0) was not supplemented with probiotics, 203 Int. J. Aquat. Biol. (2022) 10(3): 201-208 and the two other experimental groups were cultured in water supplemented every three days with probiotics (103 CFUmL-1) containing P. stutzeri AM1 (T1) and P. oleovorans ST1.1 (T2), respectively. Each group consisted of three replicates. The commercial feed was supplied to the shrimp three times daily at 3-5% body weight. Experiments were performed with a 50% water exchange twice a week and lasted for 30 days. Each tank was supplied with constant aeration during the experimental period to maintain the dissolved oxygen concentration (>4 mgL-1), and the salinity was maintained at 20 ppt. The water temperature and pH were 28.8-30.0℃ and 7.8-8.3, respectively. Total alkalinity, TAN, NO2 --N, NO3 —N, and total sulfide (S2⁻) were analyzed two times a week following the procedures of the standard methods (APHA, 2017). At the end of the experiment, the final weight (FW), weight gain (WG), daily weight gain (DWG), specific growth rate (SGR), survival rate (SR), and shrimp biomass (B) for all groups were measured and calculated following the method described by Niu et al. (2015): WG (g) = Final weight - Initial weight DWG (g day-1) = [Final weight - initial weight]/days of culture SGR (% day-1) = 100 × (Ln final mean weight - Ln initial mean weight)/days of culture SR (%) = 100 × [number of final shrimp / number of initial shrimp] B (kg.m-3) = [final weight × final population]/volume of water Statistical analysis: Statistical analysis was carried out using SPSS ver. 22 (SPSS Inc. Chicago, IL, USA), and the results were presented as mean ± standard error (SE). All data were subjected to one- way analysis of variance (ANOVA), and Tukey’s multiple comparisons test was then used to identify significant differences between treatments. All statistical significance tests were at the P<0.05. Results Isolation of probiotic bacteria: Hundreds of microbial strains were isolated from the samples collected from the sediment of shrimp ponds. Six strains, CN2.1, CN6.1, CN8.1, ST1.1, TB7.2, and TV3.1 showed a high ammonium removal ability (AOB), whereas strains AM1, CN6.2, CN7.1, TB3.2, TB7.1, and TV4.1 demonstrated high nitrite removal efficiencies (NOB) (Table 1). The ST1.1 and AM1 strains had the highest nitrogen removal ability among the twelve bacterial strains and were selected for further study. After BLAST analysis, partial 16S rRNA gene sequences analysis revealed 99% homology of isolates ST1.1 and AM1 with P. oleovorans and P. stutzeri, respectively. The nitrogen removal performance of strains AM1 and ST1.1: Temperature and pH ranged 28.8- 29.5ºC and 7.5-7.8, respectively. The aerobic inorganic nitrogen removal capacity of the strains ST11 and AM1 was shown in Figure 1. In both strains ST1.1 and AM1, the removal efficiencies of TAN, NO2 --N, and NO3 - -N during 96 h were 21.5- 31.6, 3.5-14.4, and 8.1-19.2%, respectively. Although TAN, NO2 --N, and NO3 --N were reduced in all treatments after 24 and 48h, no significant differences were found between treatments (P>0.05). After 96 h of incubation, TAN in the treatments supplemented with strains ST1.1 and AM1 at different concentrations (T1, T2, T3, T4, T5, and T6) was significantly reduced than the control treatment. There were no significant differences AOB Strains Ammonia removal (%) NOB Strains Nitrite removal (%) CN2.1 69 AM1 76.6 CN6.1 66.3 CN6.2 74.7 CN8.1 69.8 CN7.1 74.7 ST1.1 73.2 TB3.2 72.1 TB7.2 64.6 TB7.1 70.5 TV3.1 62.1 TV4.2 71.3 Table 1. Nitrogen removal performance of the isolated nitrifying bacteria. 204 Phan et al./ Pseudomonas stutzeri and P. oleovorans as probiotics for whiteleg shrimp between T1, T2, T3, T4, T5, and T6 (P>0.05). The higher removal efficiency and faster removal rate of NH4 +-N indicated that strains ST1.1 and AM1 possessed a higher potential for heterotrophic nitrification. Effects of P. stutzeri AM1 and P. oleovorans ST1.1 on water quality parameters: Water temperature, pH, DO, and alkalinity in each group were in a Figure 1. Nitrogen removal performances of strains AM1 and ST11 during the experimental period. a a ab b b0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 1 3 6 9 12 15 18 21 24 27 30 T A N ( m g L -1 ) Days of culture T0 T1 T2 A a a a b b b 0 1 2 3 4 5 6 7 8 1 3 6 9 12 15 18 21 24 27 30 N O 2 - - N ( m g L -1 ) Days of culture T0 T1 T2 B a a ab b b 0 1 2 3 4 5 6 7 1 3 6 9 12 15 18 21 24 27 30 N O 3 - - N ( m g L -1 ) Days of culture T0 T1 T2 C 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 1 3 6 9 12 15 18 21 24 27 30 S 2 - (m g L -1 ) Days of culture T0 T1 T2 D Figure 2. Concentrations of TAN, NO2--N, and NO3- -N in different treatments during the experimental period. 205 Int. J. Aquat. Biol. (2022) 10(3): 201-208 proper range for shrimp growth and survival from the beginning until the end of the experiment, and there were no significant differences between groups (Table 2). TAN in all groups reached the highest concentrations on day 9 of the experiment, and significant differences were found among treatments. TAN in the control group was significantly higher than the T1 and T2 (P<0.05). The TAN concentrations in all groups decreased later on day 12 and remained quite stable until the end of the experiment (Fig. 2A). The concentrations of NO2 --N in all groups gradually increased during the experimental period, and reached a peak at the end of the experiment. NO2 —N concentrations in two experimental groups inoculated with probiotics (T1 and T2) were significantly lower than those of the control group on days 24, 27, and 30, but no significant difference was found between the two probiotic treated groups (P>0.05) (Fig. 2B). The concentrations of NO3 --N in all treatments varied from 10 to 8 mg L-1. NO3 --N in the T1 and T2 were significantly lower than those in the control group on days 21 and 24 (P<0.05). However, there were no significant differences among all treatments on the other sampling days (P>0.05) (Fig. 2C). Total sulfide (S2-) in all treatments gradually increased from the beginning of the experiment until day 15 and decreased sharply later. No significant differences in total sulfide were found between treatments (Fig. 2D). Effects of P. stutzeri AM1 and P. oleovorans ST1.1 on growth performance of the whiteleg shrimp: Table 3 shows the effects of inoculating P. stutzeri AM1 and P. oleovorans ST1.1 into the experimental tanks’ water on the survival and growth performance of whiteleg shrimp. The results showed that the experimental group inoculated P. stutzeri AM1 (T1) showed a significantly higher survival rate and biomass compared with the control group (P<0.05). However, no significant differences were found in these parameters between T2 and the control groups. Similarly, there were no significant differences in the FW, WG, and SGR between all groups (P>0.05). Discussion The role of probiotics in controlling the colonization Table 2. Water temperature, pH, DO, and alkalinity during the experimental period. Parameters Treatments T0 T1 T2 pH 7.87±0.05a 7.79±0.07a 7.8±0.08a Temperature (℃) 28.1±0.3a 28.1±0.3a 28.1±0.3a DO (mg L-1) 4.2±0.07a 4.21±0.08a 4.19±0.06a Alkalinity (mgCaCO3 L -1) 137.8±4.0a 137.5±4.1a 137.4±4.0a Values shown are mean ± SE. Mean values within a row followed by the same letters show that there is no significant difference among the groups (P>0.05). Table 3. Growth performance and survival rate of the whiteleg shrimp. Parameters Treatments T0 T1 T2 Initial weight (g) 0.66±0.01a 0.67±0.01a 0.65±0.01a Final weight (g) 5.28±0.09a 5.54±0.09a 5.45±0.14a WG (g) 4.62±0.08a 4.87±0.08a 4.8±0.13a DWG (g day-1) 0.154±0.003a 0.162±0.003a 0.16±0.004a SGR (% day-1) 6.92±0.04a 7.06±0.06a 7.07±0.05a Survival rate (%) 74.3±1.8b 88.7±3.5a 78.3±2.2ab Biomass (kg m-3) 0.98±0.01b 1.23±0.03a 1.07±0.04b Values shown are mean ± SE. Mean values within a row followed by the same letters show that there is no significant difference among the groups (P>0.05). 206 Phan et al./ Pseudomonas stutzeri and P. oleovorans as probiotics for whiteleg shrimp and overgrowth of pathogens has been documented in many studies (Moriarty, 1999). In addition, the application of probiotics in aquaculture also enhances innate immunity (Chauhan and Singh, 2019), prevents microbial diseases (Meidong et al., 2018), promotes growth and improves water quality (Ma et al., 2009; Kolndadacha et al., 2011). Zhang et al. (2011) and Tran et al. (2019) reported that several Pseudomonas species could remove nitrogen. In the present study, two bacterial isolates, AM1 and ST1.1 showed the highest efficiency in removing nitrogen. According to 16S rRNA sequencing, these candidates were identified as P. stutzeri and P. oleovorans. Based on in vitro results, the concentration of NH4 +-N, NO2 --N, and NO3 --N decreased significantly after 96 h incubation, suggesting that strains AM1 and ST11 have a strong ability to oxidize and assimilate nitrogen in the water. Similar results were also reported with strains of P. putida Y-9 (Xu et al., 2017), Pseudomonas sp. (Tran et al., 2019), and P. fluorescens NB14 (Duo et al., 2021). Yang et al. (2017) reported that the removal efficiency of total nitrogen was directly related to bacterial growth. In addition, the results of this study indicated that strain AM1 has a strong ability to remove nitrogen compounds than ST11. In water-supplemented conditions, the effects of P. stutzeri and P. oleovorans application had no impact on shrimp growth parameters (final weigh, WG, DWG, and SGR), but the survival rate and biomass of shrimp in the treatment supplemented with P. stutzeri AM1 (T1) were significantly greater than those in control one. These results suggest that the application of P. stutzeri AM1 via water addition improved the survival of whiteleg shrimp. This observation is similar to Balcázar et al. (2007) report, where the application of P. aestumarina SLV22 at 105 CFU g-1 diet for 28 days enhanced the survival rate of whitelegs shrimp. Ngo and Ravi (2009) also reported that Penaeus latisulcatus shrimp fed P. synxantha and P. aeruginosa (2106 CFU kg-1 diet) diets for 84 days had a significantly higher survival rate. Some previous studies demonstrated that the increased survival of shrimp supplemented with probiotics (Lactobacillus plantarum, Bacillus sp., Pediococcus acidilactici, P. fluorescens) might be related to the decreased abundance of Vibrio spp. in the digestive tract (Lone et al., 1999; Wang et al., 2007; Castex et al., 2008; Vanmaele et al., 2015). In addition, Borges et al. (2008) reported that probiotics promoted the health of animals through improved water quality. In our study, water quality parameters, including pH, temperature, DO and alkalinity were within suitable ranges for the culture of whiteleg shrimp (Law 1988; Wybanet al., 1995; Nonwachaiet al., 2011; Boyd, 2007) and were not significantly different between the treatments. However, the TAN, NO2 --N, NO3 --N, and total S2- concentrations were lower when the probiotic (P. stutzeri or P. oleovorans) was applied compared with no probiotic application. This could be suggested that P. stutzeri and P. oleovorans played an important role in improving water quality. In conclusion, based on the in vivo and in vitro experiments, the two candidate probiotics P. stutzeri AM1 and P. oleovorans ST11 isolated from the shrimp pond sediment, had the clear ability to remove nitrogen compounds in wastewater from shrimp culture. Additionally, these isolates also effectively enhanced the survival rate of shrimp and water quality, which is very important for application in shrimp farms. 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