High salinity impedes efficient nitrogen removal from mariculture wastewater, which inhibits the colonization and nitrogen removal capabilities of nitrogen-removing microbes. This study aimed to isolate and characterize a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial strain. We evaluated 30 bacterial strains isolated from Portunus trituberculatus aquaculture ponds, among which Pseudomonas aeruginosa SH3 exhibited superior nitrogen removal efficiencies (99 % of NH₄⁺-N, 71 % of NO₂^--N, and 85 % fof NO₃^--N at a salinity of 30 ‰) than the other strains. Single-factor experiments demonstrated that SH3 effectively removed either NH₄⁺-N or NO₂^--N across various C/N ratios (10-20), pH levels (7-9), salinity levels (15-35 ‰), and temperatures (25-35 °C), highlighting its promising nitrogen removal capabilities under conditions suitable for mariculture. Genomic analysis showed that SH3 removes NH₄⁺-N through ammonia assimilation and nitrification and converts NO₂^--N and NO₃^--N via denitrification and assimilatory nitrate reduction. Bioaugmentation with SH3 reduced the startup period by 14 d, addressing a common challenge of prolonged startup times in a moving-bed biofilm reactor used for nitrogen removal in marine recirculating aquaculture systems. Meanwhile, bioaugmentation maintained minimal fluctuations in nitrogen levels throughout the operational period, resulting in consistently low concentrations of NO₂^--N and NH₄⁺-N, both below 1 mg/L. Therefore, strain SH3 exhibits robust nitrogen removal capabilities, demonstrating its practicality and reliability in mariculture wastewater treatment along with providing robust data support for industrial-scale applications.

Anesthesia serves as an effective method to mitigate the stress response in aquatic animals during aquaculture and product transportation. In this study, we assessed the anesthetic efficacy of clove oil, tricaine methane-sulfonate (MS-222), ethanol, and magnesium chloride by anesthesia duration, recovery time, 24-hour survival rate, and the behavior of mud crabs (Scylla paramamosain). Additionally, the optimal anesthetic concentration for varying body weights of mud crabs was also investigated. The results revealed that clove oil emerged as the optimal anesthetic for mud crabs, with a 24-hour survival rate surpassing those observed in MS-222 and magnesium chloride treatments. Ethanol caused amputation and hyperactivity in mud crabs. Regression analyses between the optimal anesthetic concentration of clove oil and the weight categories of 0.03–27.50 g and 27.50–399.73 g for mud crabs yielded the following equations: y = 0.0036 x³ − 0.1629 x² + 1.7314 x + 4.085 (R² = 0.7115) and y = 0.0437 x + 2.9461 (R² = 0.9549). Clove oil exhibited no significant impact on serum cortisol, glucose, lactate content, aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, or superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in mud crabs across different treatment groups. Anesthesia induced by clove oil in mud crabs resulted in an increase in inhibitory neurotransmitters such as glycine. However, the recovery from anesthesia was associated with elevated levels of the excitatory neurotransmitters L-aspartic acid and glutamate. In conclusion, clove oil proves to be a safe and optimal anesthetic agent for mud crabs, exerting no physiological stress on the species.

High concentrations of nitrite in marine aquaculture wastewater not only pose a threat to the survival and immune systems of aquatic organisms but also contribute to eutrophication, thereby impacting the balance of coastal ecosystems. Compared to traditional physical and chemical methods, utilizing microorganism-mediated biological denitrification is a cost-effective and efficient solution. However, the osmotic pressure changes and salt-induced enzyme precipitation in high-salinity seawater aquaculture environments may inhibit the growth and metabolism of freshwater bacterial strains, making it more suitable to select salt-tolerant marine microorganisms for treating nitrite in marine aquaculture wastewater. In this study, a salt-tolerant nitrite-degrading bacterium, designated as DM6, was isolated from the seawater (salinity of 25–30‰) of Portunus trituberculatus cultivation. The molecular identification of strain DM6 was conducted using 16S rRNA gene sequencing technology. The impacts of various environmental factors on the nitrite degradation performance of strain DM6 were investigated through single-factor and orthogonal experiments, with the selected conditions considered to be the key factors affecting the denitrification efficiency of microorganisms in actual wastewater treatment. PCR amplification of key genes involved in the nitrite metabolism pathway of strain DM6 was conducted, including denitrification pathway-related genes narG, narH, narI, nirS, and norB, as well as assimilation pathway-related genes nasC, nasD, nasE, glnA, gltB, gltD, gdhB, and gdhA. The findings indicated that strain DM6 is classified as Pseudomonas aeruginosa and exhibits efficient nitrite degradation even under a salinity of 35‰. The optimal nitrite degradation efficiency of DM6 was observed when using sodium citrate as the carbon source, a C/N ratio of 20, a salinity of 13‰, pH 8.0, and a temperature of 35 °C. Under these conditions, DM6 could completely degrade an initial nitrite concentration of 156.33 ± 1.17 mg/L within 36 h. Additionally, the successful amplification of key genes involved in the nitrite denitrification and assimilation pathways suggests that strain DM6 may possess both denitrification and assimilation pathways for nitrite degradation simultaneously. Compared to freshwater strains, strain DM6 demonstrates higher salt tolerance and exhibits strong nitrite degradation capability even at high concentrations. However, it may be more suitable for application in the treatment of wastewater from marine aquaculture systems during summer, high-temperature, or moderately alkaline conditions.

With the development of the aquatic products processing industry, 6–8 million tons of shrimp and crab shell waste are produced globally annually, but, due to the lack of high-value conversion technology, crab shells are often discarded in large quantities as a by-product of processing. Pseudomonas-based microbial cell factories are capable of biosynthesis of high-value products using a wide range of substrates; however, there is currently no reliable fermentation model for producing high-value chemicals using crab shell waste by Pseudomonas strains. In this study, we first explored the culture conditions of shell fermentation using KT2440 through single-factor and orthogonal experiments, and the optimized fermentation parameters obtained are given as follows: a temperature of 30 °C, fermentation time of 42 h, substrate solid–liquid ratio of 7%, and rotational speed of 200 rpm. After optimization, the maximum cell growth was increased by 64.39% from 350.67 × 10⁸ CFU/mL to 576.44 × 10⁸ CFU/mL. Combined with engineering modification, two engineered strains, KT_+IV and KT_+lasBT, expressing exogenous proteases, were obtained, and the maximum growth was increased from 316.44 × 10⁸ CFU/mL to 1268.44 × 10⁸ CFU/mL and 616.89 × 10⁸ CFU/mL, which were 300.84% and 94.94% higher, respectively. In addition, the engineered strain KT_+NtrcT-D55E, which regulates nitrogen metabolism, was obtained, and the accumulation of intracellular polyhydroxy fatty acid esters (PHA) was increased from 20.00 mg/L to 78.58 mg/L, which was a significant increase of 292.93% relative to the control group. This study provides a theoretical basis and technical support for the high-value utilization of shrimp and crab shell resources and the development of environmentally friendly bioproducts.

With the development of the aquatic products processing industry, 6-8 million tons of shrimp and crab shell waste are produced globally annually, but due to the lack of high-value conversion technology, crab shells are often discarded in large quantities as a by-product of processing. Pseudomonas-based microbial cell factories are capable of biosynthesis of high-value products using a wide range of substrates; however, there is currently no reliable fermentation model for producing high-value chemicals using crab shell waste by Pseudomonas strains. In this study, we first explored the culture conditions of shell fermentation using KT2440 through single-factor and orthogonal experiments, and the optimized fermentation parameters obtained were: with the temperature of 30°C, fermentation time of 42 h, substrate solid-liquid ratio of 7%, and rotational speed of 200 rpm. After optimization, the maximum cell growth was increased by 64.39%, from 350.67 × 10 8 CFU/mL to 576.44 × 10 8 CFU/mL. Combined with engineering modification, two engineered strains KT+IV and KT+lasBT expressing exogenous proteases were obtained, with the maximum growth increasing by 300.84% and 94.94%, respectively. Additionally, an engineered strain KT+NtrcT-D55E regulating nitrogen metabolism was obtained, significantly increasing the accumulation of intracellular polyhydroxyalkanoates (PHA) by 292.93%, relative to the control group. This study provides a theoretical basis and technical support for the high-value utilization of shrimp and crab shell resources and the development of environmentally friendly bioproducts.