Effect of ozone on Vibrio removal in a simulated earthen shrimp pond

The Pacific white shrimp (Litopenaeus vannamei) has been a pivotal player in the global aquaculture industry, particularly in recent years, witnessing both substantial growth and formidable challenges. Originating from experimental cultivation in Thailand in 1998, the Pacific white shrimp has thrived due to its accelerated growth, heightened survival rates, and adaptability to diverse nutrient sources at various pond depths (Liao & Chien 2011; Maliwat et al. 2021). However, the once-prominent status of the Pacific white shrimp faces significant threats, notably from disease outbreaks that have plagued shrimp farms globally.

Disease outbreaks, particularly early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND), have emerged as critical concerns for the Pacific white shrimp industry. The onset of EMS, first identified in China in 2009, has been marked by a range of causative factors, symptoms, and profound effects on shrimp populations (Zorriehzahra & Banaederakhshan 2015; Kumar et al. 2021). In Thailand, the onset of EMS in late 2012 and its continuation into 2013 resulted in a significant decline in white shrimp production, causing output to drop by half to 310,705 tons in 2013. The primary causative agent, Vibrio pathogenic bacteria, proliferates in pond bottom soil, leading to adverse symptoms such as lethargy, diminished feeding, soft shells, and discoloration of the hepatopancreas (Santos et al. 2020). The consequence is a mortality rate ranging from 30 to 100%, depending on variables such as shrimp species, farming conditions, and predisposing factors.

The focus on Vibrio pathogens is justified by their significant impact on shrimp aquaculture and their well-documented association with severe disease outbreaks. Vibrio species, particularly Vibrio parahaemolyticus, have been identified as the primary causative agents of EMS/AHPND, leading to substantial economic losses in the shrimp farming industry. These bacteria are known for their rapid proliferation in aquaculture environments, especially in pond bottom soils where they can persist and cause recurrent infections (Kumar et al. 2021). Unlike other potential pathogens, Vibrio species have a unique ability to thrive in diverse environmental conditions, making them particularly challenging to manage. Their role in major disease outbreaks underscores the critical need to develop effective strategies to control their population in aquaculture systems.

Various treatments have been explored to combat these pathogenic outbreaks, including the use of antibiotics, probiotics, and water treatment chemicals. However, these approaches often come with significant drawbacks, such as the development of antibiotic resistance, negative environmental impacts, and the potential for chemical residues in shrimp products.

In this study, ozone treatment has emerged as a promising alternative due to its potent oxidizing properties and ability to degrade organic matter and microorganisms without leaving harmful residues. The use of ozone in shrimp farming and aquaculture systems has garnered significant attention as a potential solution to control pathogenic bacteria and improve water quality. Previous studies have demonstrated that ozone treatment can effectively reduce the concentration of harmful bacteria and other pathogens in aquaculture environments. Powell et al. (2015) highlighted ozone's strong oxidizing properties, which can deactivate a wide range of microorganisms, including Vibrio spp., known to cause diseases in shrimp. Schroeder et al. (2015) further supported these findings by showing that ozone treatment could lower bacterial loads in water, thereby enhancing the overall health of aquaculture systems. Spiliotopoulou et al. (2018) demonstrated that ozone could improve water clarity and reduce the organic load, which is crucial for maintaining a healthy environment for shrimp. Dien et al. (2022) confirmed that the application of ozone in aquaculture could significantly diminish the presence of pathogenic bacteria in pond bottom sediment, a common reservoir for harmful microorganisms. These studies collectively underscore the potential of ozone treatment to enhance water quality and control bacterial populations, making it a promising alternative to traditional chemical treatments.

The present research aims to address the critical gap in understanding the optimal conditions for ozone application in shrimp aquaculture ponds. Specifically, the study focuses on determining the optimal exposure time and residual ozone concentration (ROC) levels for the effective eradication of Vibrio pathogenic bacteria in pond bottom soil, a primary contributor to EMS or AHPND in Pacific white shrimp. This research seeks to provide a sustainable strategy for managing pond bottoms post-shrimp culture, reducing reliance on harsh chemicals, shortening soil preparation time for shrimp ponds, and minimizing the risk of disease outbreaks. The specific objectives of the study are as follows: (1) to study the effect of the concentration and duration of exposure to ozone on the reduction of the amount of Vibrio pathogens and the rate of removal of inorganic nitrogen from the pond bottom soil and (2) to evaluate the potential of using ozone to eliminate Vibrio pathogens in simulated shrimp ponds in the laboratory.

As the global aquaculture industry navigates the complexities of sustaining shrimp production, this research contributes valuable insights that can inform more environmentally friendly and economically viable practices in shrimp farming. By exploring the intricate interplay between ozone application, Vibrio pathogenic bacteria control, and disease prevention, this study aims to pave the way for a more resilient and sustainable future for the Pacific white shrimp aquaculture sector.

Dissolved ozone was prepared using ozone generators with built-in air pumps (Ebase brand models OZ 1060T, Thailand) capable of producing maximum ozone amounts of 1,000 mg/h. Utilizing the corona discharge method, ozone was generated through dry air passing between two electrodes with different potentials, causing the oxygen bonds to split and form oxygen atoms. These oxygen atoms were then combined to create ozone molecules (Gonçalves 2009). In this study, synthetic saltwater was prepared by using the saltwater originating from salt farming, which was then diluted with tap water to achieve a salinity of 5 ppt. The saltwater was sterilized by autoclaving at 121 °C for 30 min prior to use. Ozone was added to the sterilized saltwater at a flow rate of 7.5 L/min. The ROC was analyzed using the Indigo Colorimetric Method by APHA (2017).

Vibrio pathogens were analyzed using the spread plate on thiosulfate-counting technique citrate-bile-sucrose agar (TCBS) medium (Uchiyama 2000). Soil samples (1 g-wet weight) were added to 9 mL of 0.85% sodium chloride (NaCl) solution, followed by serial dilution to obtain an inoculum ranging from 10⁻¹ to 10⁻⁵. Next, 0.1 mL of the soil solution was pipetted onto the surface of TCBS agar. A sterilized triangular glass rod was then used to evenly spread the soil solution over the agar surface. The plates were incubated in an incubator at 30 °C for 10–12 h, and bacterial colonies growing on the agar surface were counted in triplicate. Colonies in the range of 30–300 were counted from the appropriate dilution (Tagliavia et al. 2019).

This analysis focused on the effect of ozone supplement on total ammonia nitrogen (TAN) removal rates of soil in control tanks (without ozone) and treatment tanks (with an initial ROC of 1.0 mg/L and an exposure time of 30 min). The 10 g of soil was added to 0.5 L of water with a salinity of 5 ppt and 1 mg-N/L of ammonium chloride (NH₄Cl). Oxygen was continuously added to maintain dissolved oxygen (DO) levels of >5 mg/L. Water samples were collected every 1.5 h over a period of 7.5 h to analyze the variations of TAN, nitrite, and nitrate concentrations using a method specified in the Standard Methods for the Examination of Water and Wastewater (APHA 2017). Furthermore, the decrease in TAN concentrations over time was used to calculate TAN removal rates of the soil, comparing between control and treatment tanks.

All experiments and analyses were performed in triplicates. Mean values were reported with standard deviations. Mean value differences were analyzed using one-way analyses of variance followed by a Tukey HSD post hoc test performed in GraphPad Prism (version 7.0) and Duncan's multiple range test followed by Statistical Package for the Social Sciences (SPSS) (version 22; IBM, New York, USA). Differences were considered to have statistical significance at p-values of <0.05.

Soil samples collected from the bottom of the outdoor earthen shrimp pond provided insights into the environmental conditions. Soil has an average pH of 7.52 ± 0.11, aligning with the typical range observed in general shrimp ponds, where the soil pH ranges from 6.73 to 7.39 (Munsiri et al. 1996). Total organic carbon and organic matter contents, which followed the method of Walkley (1947), were 2.27 ± 0.27% and 3.91 ± 0.46%, respectively. These organic carbon contents were comparable to those reported in small (0.04 ha, 0.51 − 2.90%) and large (0.16 ha, 0.27 − 1.77%) outdoor earthen shrimp ponds (Satanwat et al. 2023). The content of total organic carbon >0.5% is normally recommended for allowing benthic productivity, while elevated contents of organic carbon indicate the accumulation of organic matter, such as food residues, shrimp waste, and dead microorganisms. Thus, the typical content should be <3% for shrimp production (Boyd 2003). In addition, high organic content also contributes to the high oxygen consumption rate (OCR) of the soil. The average OCR of the soil samples was observed to be 4.61 ± 1.79 mg-O₂/g-soil/day. The mineral content comprised carbon (0.85 ± 0.02%), hydrogen (0.68 ± 0.08%), nitrogen (0.71 ± 0.04%), and no sulfur. Comparison with general shrimp ponds revealed variations, notably higher nitrogen content, attributed to food input, protein proportion, and shrimp density during rearing. Soil moisture content stood at 15.52%, providing an approximate classification of relatively high sandy composition in shrimp ponds (Brandt et al. 2017). Also, soil mineral content typically ranges from 0.65 to 1.45% carbon, 0.17 to 0.28% nitrogen, and 0.29 to 0.52% sulfur (Munsiri et al. 1996).

In terms of pathogens, the concentration of Vibrio pathogens was 1.0 ± 0.0 × 10³ CFU/mg, which is relatively low compared to the previous study. Prapatsorn (1995) studied the bacterial amounts in the soil at the bottom of shrimp ponds in Chanthaburi province, Thailand. The study reported concentrations of Vibrio spp. before stocking, during aquaculture, and after harvest, were as 3.3 × 10⁵, 4.1 × 10⁶–6.0 × 10⁷, and 2.3 × 10⁸ CFU/mg, respectively. Lower pathogen concentrations in the present study might be attributed to variations in a pond environment. Additionally, it is possible that the samples were collected from dried bottom soil where pathogens were not as abundant as in muddy soil.

Regarding the Vibrio pathogen removal efficiencies, presented as percentages with statistically significant differences marked where applicable, and log inactivation, which allows for the comparison of different treatment conditions, such as varying initial ROCs and exposure times, seen in Figure 3(b). Notably, at a 30-min contact time, initial ROC of 1.0 mg/L showed a significantly higher removal efficiency (p < 0.01). For the Vibrio pathogen removal over a 5-min contact time, the removal efficiency ranged from 57.8 to 69.1% across all experimental sets, with no statistically significant differences observed. Upon extending the contact time to 30 min, experimental sets with initial ROCs of 0.3 and 0.6 mg/L maintained similar effective Vibrio removal efficiencies, ranging from 81.5 to 88.3%. Notably, an initial ROC of 1.0 mg/L exhibited higher elimination efficiency, reaching 98.6%, indicating its ability to almost completely eradicate Vibrio in the soil. Subsequently, at a 60-min exposure time, the removal efficiency showed no statistically significant differences across all experimental sets, achieving a total efficiency of 100% (approximately 5.0 log inactivation). This suggests that, with an extended exposure period, all ROCs were equally effective in completely removing Vibrio pathogens from the soil. In terms of the application of ozone for pathogen disinfection in shrimp cultivation systems, a safe ozone level of 0.06 mg/L is typically recommended, while long-term exposure of 0.1 mg/L can cause soft-shell syndrome (Schroeder et al. 2010). Therefore, instead of injecting ozone directly into the shrimp tank, this study proposed a protocol of supplying ozonated water to treat pathogens in the soil prior to shrimp cultivation. This approach avoided the negative effects of ozone on shrimp and ensured that an ROC was sufficient for minimizing pathogens in the bottom soil.

Appropriate ROC was implemented to eliminate Vibrio pathogens from soil prior to the beginning of the shrimp cultivation. During the soil preparation phase, a comparison was conducted between control sets (untreated soil) and treatment sets (soil treated with ozone), employing an initial ROC of 1.01 ± 0.04 mg/L for an exposure time of 30 min. Initially, both untreated and ozone-treated soils had similar Vibrio pathogen concentrations (2.00 − 2.17 × 10⁵ CFU/mg). However, after exposure to ozone, the treatment sets exhibited an impressive 97.23% eradication efficiency, reducing the pathogen count to 6.00 ± 1.41 × 10³ CFU/mg.

This part of the experiment focuses on the application of ozone-treated soil to control pathogens during the 45-day shrimp cultivation in the simulated outdoor aquaculture tanks. A comparison was conducted between control tanks (untreated soil) and treatment tanks (soil treated with ozone), as follows.

In Figure 5(b), the results depict Vibrio pathogen concentrations in the water during days 0 − 5, with no detection of Vibrio pathogens in the initial stages. This aligns with the low levels of organic matter, food waste, and shrimp waste during the early shrimp cultivation phase, resulting in minimal pathogen growth in the system, consistent with the low Vibrio pathogen analysis results in the soil. During days 10 − 17, Vibrio pathogens in the water were detectable in both control and treatment tanks, with concentrations ranging from 10² − 10³ CFU/mL. Notably, the control tanks experienced a surge in Vibrio pathogens, reaching 5.50 × 10⁴ CFU/mL on day 24, indicating an increase with the prolonged shrimp cultivation period due to the accumulation of more waste in the system and subsequent bacterial proliferation, as observed in the Vibrio pathogen analysis of the soil in the control tanks. In contrast, the treatment tanks effectively controlled the Vibrio pathogen concentrations in water, maintaining a consistent value of 4.25 ± 1.06 × 10³ CFU/mL. Clearly, differences between control and treatment tanks were also observed thereafter, during days 31 − 45.

The increase in Vibrio concentrations between 31 and 45 days is due to the accumulation of organic matter, deteriorating water quality, and optimal conditions for bacterial growth. Ozone treatment, which improves water quality by oxidizing organic matter, disinfecting pathogens, and increasing oxygen levels, can effectively reduce the number of Vibrio pathogens compared to the control. The Vibrio counts in control tanks were reported at 4.00 − 6.00 × 10⁴ CFU/mL, while lower counts were reported in the treatment tanks at 1.05 − 2.50 × 10⁴ CFU/mL. The results indicated that controlling Vibrio pathogens in soil contributed to lower concentrations in the water. Pumkaew et al. (2021) similarly reported that treatment tanks with ozone injection could reduce V. parahaemolyticus cell density from 10⁷ to 10⁵ CFU/m² in the biofilter and V. parahaemolyticus counts from 10⁴ to 10⁰ CFU/mL in the water. In contrast, control tanks without ozone injection showed the remaining V. parahaemolyticus cell density at 10⁷ CFU/m² in the biofilter and V. parahaemolyticus counts at 10⁴ CFU/mL in the water.

Regarding nitrite variations (Figure 6(b)), the initial high concentrations observed during the shrimp cultivation resulted from residual nitrite from the soil acclimation process and might have also stemmed from the TAN oxidation process. Thereafter, nitrite concentrations tended to decrease during the first 15 days of shrimp cultivation, declining from 1.72 to 0.24 mg-N/L (control) and from 1.37 to 0.15 mg-N/L (treatment). Interestingly, the treatment tanks demonstrated slightly lower nitrite concentrations compared to control tanks. This decrease in nitrite concentrations could be attributed to the activity of NOB, possibly indicative of ozone-induced inhibition over prolonged exposure. Powell et al. (2015) mentioned that residual ozone is expected to oxidize nitrite, resulting in a decrease in its concentration. As well, Pumkaew et al. (2021), demonstrated that long-term ozone concentrations at 0.4 mg/L inhibited the efficiency of AOB in nitrification processes (p < 0.05), compared to normal feeding conditions with a 0.3 mg/L ozone concentration. However, even at a ROC of 0.4 mg/L, ammonia treatment remained feasible, with a recorded ammonia treatment rate of 30.29 ± 0.56 mg-N/m²/day, indicating effective control of ammonia levels.

In terms of nitrate concentrations (Figure 6(c)), according to complete nitrification, an increase in nitrate was observed since the first day of shrimp cultivation. The concentrations in both tanks increased from 2.02 to 3.14 mg-N/L at the beginning to 12.36 − 12.45 mg-N/L on the final day of the experiment. Nitrate production results from the oxidation of nitrite through the nitrification process, driven by NOB. Notably, the comparison between the control tanks (untreated soil) and the treatment tanks using residual ozone at 1.01 ± 0.04 mg/L showed no significant differences in nitrate concentration. This underscores the negligible impact of residual ozone on NOB activity in the soil.

Water quality variables during the 45-day shrimp cultivation in the simulated outdoor aquaculture tanks are summarized in Table 1. Adequate DO levels, averaging 6.71 ± 0.63 mg/L (ranging from 6.00 to 8.23 mg/L) for the control tanks and 6.56 ± 0.60 mg/L (ranging from 6.11 to 8.01 mg/L) for the treatment tanks, fall within the suitable range for aquaculture and shrimp propagation. Maintaining suitable DO levels (greater than 5 mg/L) is crucial for shrimp growth and organic matter degradation, while prolonged low DO levels can pose risks to aquatic animals, making them susceptible to bacterial diseases and other health issues (Boyd 1979; Bashir et al. 2020; Anushka & Mishra 2022; Li et al. 2023).

The control tanks exhibited an average pH of 7.68 ± 0.34 (ranging from 7.10 to 8.24), while the treatment tanks recorded an average pH of 7.76 ± 0.44 (ranging from 7.05 to 8.50). Fluctuations in pH might be attributed to the diurnal photosynthesis of phytoplankton due to the presence of white LED lights operating on a 12/12 light-dark cycle daily. The 12-h LED light exposure during the experiment caused a decrease in carbon dioxide (CO₂) concentration in the water, resulting in a higher pH value. Conversely, the decrease in pH at night is a consequence of the respiration process of aquatic organisms, which releases CO₂ and makes the water more acidic. The suitable water pH value for aquaculture should be between 7.5 and 8.5, and it is important to avoid significant pH fluctuations during the day as it can lead to shrimp stress, exhibited growth, weakened immune system, and increased susceptibility to infection (Galdeano et al. 2018; Bashir et al. 2020). Alkalinity levels were within the range of 80.00–176.67 mg/L as CaCO₃ for the control tanks and 70.00–170.00 mg/L for the treatment tanks. The decrease in alkalinity might be attributed to its consumption in treating inorganic nitrogen through nitrification, necessitating the addition of sodium bicarbonate (NaHCO₃). Adequate alkalinity, essential for shrimp survival and growth, aligns with the suitable range of 80–150 mg/L (Limsuwan & Chanratchakul 2004).

Water temperature showed consistent values between the control (29.18 ± 0.55 °C) and treatment (29.21 ± 0.52 °C) tanks. Temperature fluctuations, impactful for shrimp as cold-blooded organisms, were well-managed within the optimal range of 28–32 °C for shrimp growth. A temperature increase of 1 °C can elevate their metabolic rate by 10 times, resulting in higher food and oxygen demands (Landsberg et al. 2009). Additionally, water temperature changes indirectly impact aquaculture systems by influencing the decomposition of organic matter by microorganisms, leading to fluctuation in DO levels (Mugwanya et al. 2022). Salinity values remained consistent in both tanks, with the control tanks having an average salinity of 10.86 ± 0.73 ppt (ranging from 10.00 to 12.00 ppt) and the treatment tanks recording 10.67 ± 0.66 ppt (ranging from 10.00 to 12.00 ppt). These values are within the general range of 0–35 ppt suitable for Pacific white shrimp cultivation (Huang et al. 2024).

Calcium concentrations were within the range of 100.00–240.00 mg/L for both control and treatment tanks. The decline in calcium levels is attributed to its uptake in shrimp shell formation, further exacerbated by the presence of Pogoda Prickly-winkle (Tectarius pagodus). Calcium adjustment and snail removal were undertaken to address this, ensuring adequate calcium levels crucial for shell formation, acid–base balance, blood clotting, muscle contraction, and absorption of vitamin B12. To address this, the calcium value was adjusted by adding calcium chloride (CaCl₂) to 200 mg/L, and snails were removed by scooping them out of the ponds. Calcium is important for shell formation, maintaining acid–base balance within the body, blood clotting, muscle contraction, and absorption of vitamin B12. Calcium is also a key component of the external structure of shrimp. Calcium deficiency can result in thin and soft shrimp shells, leading to slow hardening of the shell after molting (Li & Cheng 2012). Magnesium concentrations were in the range of 300.00–533.33 mg/L (control) and 300.00–500.00 mg/L (treatment). The decline in magnesium levels is attributed to utilization by crustaceans and snails. Magnesium adjustment, involving the addition of magnesium chloride (MgCl₂), ensures balanced magnesium–calcium ratios crucial for shrimp body structure, digestive enzyme stimulation, and energy conversion. To address this, magnesium was adjusted by adding magnesium chloride (MgCl₂) to 500 mg/L. Magnesium plays a crucial role in the body structure of shrimp, constituting approximately 70% of the body structure of shrimp, with the remaining 30% found in tissues and blood. Magnesium stimulates the activity of shrimp digestive enzymes, facilitating the transformation of food into energy for vital functions. Additionally, magnesium can be effective only when it maintains a balanced ratio with calcium, ideally at a ratio of 3 parts magnesium to 1 part calcium. Imbalanced magnesium and calcium levels in the water can lead to issues such as unstable salinity and decreased hard shell formation during molting, along with tense muscles and potential heart failure in shrimp (Fieber & Lutz 2011).

Initially, shrimp had an average weight of 9.41 ± 0.45 and 10.18 ± 0.50 g, and an average length of 10.64 ± 0.20 cm and 10.96 ± 0.20 cm, for control and treatment tanks, respectively. Shrimp were cultivated at 10 shrimp/tank to provide the initial density of 0.94 ± 0.06 kg/m³ (control) and 1.02 ± 0.06 kg/m³ (treatment). Shrimp were fed daily with artificial feed pellets at a rate of 2–5% of their weight. After 45 days of cultivation in the simulated outdoor aquaculture tanks, shrimp had an average final weight of 14.85 ± 0.45 and 15.82 ± 0.77 g, and an average final length of 12.40 ± 0.09 and 13.12 ± 0.43 cm, for control and treatment tanks, respectively. The final densities were 1.49 ± 0.09 kg/m³ (control) and 1.58 ± 0.13 kg/m³ (treatment). The ADG was 0.12 ± 0.03 g/day and 0.13 ± 0.02 g/day, the survival rate was 89.75% ± 11.33 and 89.60 ± 5.43, and the FCR was 1.96 ± 0.25 and 1.96 ± 0.20 for control and treatment tanks, respectively, which were represented in Table 2. These results demonstrated similar growth rates, survival rates, and conversion rates between the control tanks (untreated soil) and the treatment tanks with soil treatment with an initial ROC of 1.01 ± 0.04 mg/L for 30 min before starting the white shrimp culture system. This aligns with the findings of Pumkaew et al. (2021), in which the 40-day white shrimp cultivation in RAS with (0.3 mg/L) and without ozone supplement had ADG of 0.036–0.037 g/day and survival rates of 80.00–81.67%.

This study has investigated the impact of ROC and exposure duration on Vibrio pathogens, soil ammonia treatment rates, and water quality parameters within a simulated shrimp pond system. The experiment unfolded across three distinct phases, each contributing valuable insights.

In the initial phase, the analysis of soil from an outdoor shrimp farming system revealed neutral pH, elevated organic matter, and total organic carbon levels, indicative of organic sediment accumulation from food remnants and shrimp excretion. The relatively high soil oxygen demand suggested active microbial processes. Although Vibrio pathogen concentration in the soil was initially low, likely due to soil drying post-shrimp harvest, this phase laid the foundation for subsequent investigations. The second phase compared soil conditioning between the control set (untreated soil) and the experimental set (ozone-treated soil). Ozone treatment exhibited a notable disinfection efficiency of 97.23%, significantly reducing initial Vibrio pathogen concentrations. The soil ammonia treatment rates in the experimental set displayed a slightly slower reduction, hinting at potential ozone-induced impacts on bacterial activity. However, both sets demonstrated comparable abilities to treat ammonia through nitrification. In the final phase, ozone was introduced to control Vibrio pathogens during a 45-day simulated shrimp culture. The experimental set, benefiting from ozone soil treatment, showcased lower Vibrio pathogen concentrations in both soil and water compared to the control set. Intriguingly, ROC showed no significant influence on ammonia and nitrite concentrations, indicating an unaffected nitrification process. Furthermore, the monitored water quality parameters, including ammonia, nitrite, and nitrate, exhibited similar inorganic nitrogen dynamics between the control and experimental sets.

In summary, the application of an initial ROC of 1.0 mg/L over a 30-min exposure period proved effective in reducing Vibrio pathogens in soil without adverse effects on soil ammonia treatment rates and water quality parameters in the simulated shrimp pond system. These findings endorse the potential utilization of ozone as a viable method for controlling Vibrio pathogens in shrimp aquaculture systems, thereby providing valuable insights for future research endeavors and practical applications in the field of shrimp farming.

This research is funded by Thailand Science Research and Innovation Fund Chulalongkorn University (No. 6641/2566), with special thanks to the Center of Excellence in Marine Biotechnology, Faculty of Science, Chulalongkorn University and the Tha Khai Land Development Project, ‘Song Nam’, the Chaipattana Foundation, Chacheongsao Province, for supporting the research work and being generous with tools, equipment, chemicals, and soil samples, as well as advice on scientific analysis in various fields. The first author (Sitthakarn Sitthi) acknowledges the Second Century Fund (C2F) for a Postdoctoral fellowship, at Chulalongkorn University, Thailand.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.