To explore the nitrogen removal performance and mechanism of a strain of Bacillus mojavensis, C3, isolated from the sediment of the Yellow Sea cold water mass, we investigated its nitrogen removal performance and mechanism. The results showed that C3 effectively removed NH₄⁺-N, NO₂^--N and NO₃^--N from sole nitrogen sources media with the initial inorganic nitrogen concentration of 21 mg/L, with the removal rate of 85.95%, 75.89% and 96.29%, respectively, and the maximum nitrogen removal rate, 0.36 mg/(L∙h), 0.36 mg/(L∙h), and 0.50 mg/(L∙h), correspondingly. Nitrogen balance analysis revealed that inorganic nitrogen in the system was removed through assimilation and heterotrophic nitrification-aerobic denitrification (HN-AD). Among these, nitrogen assimilation dominated the strain's inorganic nitrogen metabolism transformation while nitrogen released into the atmosphere through heterotrophic nitrification-aerobic denitrification varied between 13.29% and 16.83% of the total. The results of ¹⁵N isotope tracing showed that it only produced ¹⁵N₂O but not ¹⁵N₂ when strain C3 used ¹⁵NH₄⁺-N, ¹⁵NO₂^--N and ¹⁵NO₃^--N as the sole nitrogen source, indicating that N₂O was the primary gaseous product of denitrification of C3. Further exploration of the nitrification pathway using ammonia oxidation inhibitors revealed that adding different concentrations of allylthiourea (ATU) had no significant effect on ammonia removal rate or nitrogen loss, suggesting that C3 may possess a novel ammonia monooxygenase with a unique enzymatic structure, being capable of converting NH₄⁺-N to NH₂OH. Whole-genome sequencing analysis revealed that nitrogen utilization during assimilation mainly occurs through the glutamate dehydrogenase (GDH) pathway (extracellular NO₃^-/NO₂^-/NH₄⁺→intracellular NH₄⁺→Glutamate→Bacterial protein) and the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway (extracellular NO₃^-/NO₂^-/NH₄⁺→intracellular NH₄⁺→Glutamine→Glutamate→Bacterial protein). The results indicated that C3 achieves nitrogen dissimilation through the heterotrophic nitrification-aerobic denitrification (HN-AD) pathway which includes heterotrophic nitrification (NH₄⁺→NH₂OH→NO→N₂O) and aerobic denitrification (NO₃^-→NO₂^-→NO→N₂O). These findings provided important insights into the nitrogen transformation metabolism mechanism of B. mojavensis and offered a scientific basis for the potential application of C3 in the treatment of wastewater from marine aquaculture.

Bacillus subtilis is one of the most commonly used probiotics in aquaculture, but there are limited studies on the optimal forms of addition form and the differences in effects of B. subtilis in feed. In this study, we evaluated the growth, immunity and disease resistance of Litopenaeus vannamei fed diets supplemented with different forms of B. subtilis BSXE-2102, including live cells (WB), heat-killed whole cells (WK), fermentation broth (WL), fermentation supernatant (WM) and sonication-killed cell-free extracts (WN) at a concentration of 1×10¹¹ CFU/kg, five replicates each treatment, 42-day cultivation. Commercial feed was used as the control (DZ). The results showed that the survival rate each diet ranged from 85.33% to 94.67%, with WB showed the highest survival rate, significantly higher than that of WL and DZ (P < 0.05). The minced shrimp body weight and specific growth rate of WN were significantly higher than those of DZ (P < 0.05), and feed efficiency and digestibility were also significantly higher in WB, WK and WN groups compared to DZ (P < 0.05). The T-NOS and SOD activities in the serum of shrimp from all treatment groups were significantly higher than those of DZ (P < 0.05), but other antioxidant indicators varied across treatments. WB demonstrated significantly higher POD, LZM activity and PO content than DZ (P < 0.05), with the highest LZM activity recorded. WK had significantly higher ACP activity and T-AOC than DZ (P < 0.05). WL, WM and WN had significantly higher AKP, ACP, POD activity, PO content and T-AOC than DZ (P < 0.05), with the WN group showing significantly higher T-NOS activity and T-AOC than the others (P < 0.05). Compared to DZ, the expression levels of genes SOD, Relish, Toll, Imd, TOR, eIF4E1α and eIF4E2 in the hepatopancreas of shrimp in all treatment groups were significantly upregulated (P < 0.05). Among these, WN had the highest expression levels of SOD, Toll, TOR and eIF4E2 while WB had significantly higher expression of LZM and LGBP than others (P < 0.05). The challenge test of Vibrio parahaemolyticus revealed that the cumulative mortality of WN was significantly lower than that of DZ (P < 0.05). Our findings suggested that adding B. subtilis BSXE-2102 live cells and sonication-killed cell-free extracts to L. vannamei feed can significantly improve its growth performance, immunity and disease resistance. Sonication-killed cell-free extracts may serve as a potential postbiotic to enhance the growth performance, immunity and disease resistance of L. vannamei.

Polysaccharide β-glucan and algal polysaccharides are two prevalent feed additives, yet comparisons of their effects are scarcely reported. In this investigation, we explored the impacts of feeds supplemented with β-glucan and fucoidan, respectively, on the growth, non-specific immunity and resistance to Vibrio parahaemolyticus infection of juvenile shrimp, Litopenaeus vannamei. β-Glucan from Saccharomyces cerevisiae (brewer′s yeast) (YG) and Euglena gracilis (MG) and fucoidan from Macrocystis pyrifera (AP) were supplemented at a concentration of 0.65 g/kg feed, and a commercial feed (DZ) served as the control, five replicates each treatment. The shrimp individuals, initial mean body weight (1.26±0.02) g, were fed with these diets for 42 days. And then challenged with V. parahaemolyticus for 14 days. Results indicated that shrimp survival rate exceeds 90% among all diets, and that of YG, MG and AP significantly higher than that of control (P < 0.05). YG exhibited significantly higher final weight and specific growth rate than control (P < 0.05). Feed efficiency and digestibility were notably superior among all diets (P < 0.05). Compared to the control, β-glucan and fucoidan supplementation significantly elevated the activity of alkaline phosphatase (AKP), acid phosphatase (ACP), total nitric oxide synthase (T-NOS), lysozyme (LZM), peroxidase (POD) and superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and phenoloxidase (PO) content in shrimp serum. YG and MG demonstrated the most pronounced effects, which was followed by AP. Relative expression abundances of LGBP, SOD, HSP70 and proPO in shrimp hepatopancreas was significantly higher among all diets than those of control (P < 0.05), and β-glucan supplementation caused higher expression of LGBP, SOD and HSP70 than fucoidan supplementation (P < 0.05). Expression abundance of Imd, Relish and Toll in hepatopancreas was significantly upregulated among all diets (P < 0.05), and such upregulation was similar among each other. Along the mTOR signaling pathway, β-glucan supplementation significantly increased the expression of TOR, eIF4E2, eIF4E1α and 4EBP in hepatopancreas compared to control (P < 0.05) while fucoidan supplementation significantly upregulated the expression of TOR and eIF4E2 (P < 0.05). V. parahaemolyticus challenging revealed that β-glucan supplementation varied in improving shrimp survival rate among its sources with yeast-coming β-glucan exhibited the most favorable effect (P < 0.05). Our findings imply that appropriate incorporation of β-glucan and fucoidan into feed can enhance the growth performance, immunity and disease resistance of against L. vannamei with the benefit of yeast-coming β-glucan the most comprehensive.

An experiment was conducted to study the effect of adding three Bacillus species to feed on the growth performance, non-specific immunity and disease resistance of Penaeus vannamei. Shrimp was fed with the basal diet supplemented with Bacillus subtilis BSXE-1601 (BD group), B.cereus BCXE-01 (CD group) and B.velezensis 26.3 (BV group) at a density of 1×10⁹ CFU·kg^-1 in the basal diet. The basal diet without bacterial additives was used as the control group. The experiment lasted for 6 weeks. After the feeding trial, a 14 d Vibrio parahaemolyticus challenge experiment was conducted. Results showed that the final weight, specific growth rate and feed efficiency of shrimp in BD and BV were significantly improved as compared to the control (P < 0.05), especially in BV. The activities of peroxidase (POD), superoxide dismutase (SOD), acid phosphatase (ACP) and phenol oxidase (PO) in the serum of shrimp of three treatment groups were significantly higher than those of the control group (P < 0.05). The activities of ACP, AKP and SOD in the serum of BV were significantly higher than those of BD (P < 0.05). Compared with the control, the relative expression level of proPO, LZM, SOD, HSP70, Imd, Toll, Relish, TOR, 4E-BP, eIF4E1α and eIF4E2 in the hepatopancreas of shrimp of BD was significantly higher than that of the control (P < 0.05). The relative expression level of proPO、LGBP、Imd、Toll、4E-BP and eIF4E2 in the hepatopancreas of shrimp of CD group was significantly higher than that of the control (P < 0.05). The relative expression level of proPO, LZM, SOD, LGBP, HSP70, Imd, Toll, TOR, 4E-BP and eIF4E1α in BV was significantly higher than that of the control (P < 0.05). The relative expression level of SOD, LGBP, Toll and 4E-BP in BV was significantly higher than that of CD and BD (P < 0.05). The challenge test of V. parahaemolyticus revealed that the cumulative mortality of shrimp in three treatment groups was significantly lower than that in the control (P < 0.05). In conclusion, the effects of adding B. subtilis BSXE-1601 and B. velezensis 26.3 in the feed markedly enhanced the growth performance of P. vannamei. Two Bacillus species elicited a superior immune response compared to B.cereus BCXE-01. Three Bacillus species significantly enhanced the resistance against V.parahaemolyticus of P.vannamei.

In order to investigate the microbial community characteristics of Takifugu rubripes pond culture systems, 16S rRNA gene high-throughput sequencing technology was used in this study. The community structure and diversity of gut microorganisms, water microorganisms and sediment microorganisms in T. rubripes single culture system, the bi-species polyculture system of T. rubripes with Mercenaria mercenaria, the tri-species polyculture system of T. rubripes with Penaeus japonicus and M. mercenaria, and the traditional bi-species polyculture system of T. rubripes with Penaeus japonicus were compared. The results showed that Campilobacteria, Proteobacteria, Spirochaetota and Firmicutes are the dominant bacteria in the intestinal microbes of T. rubripes. Campylobacter may be the unique dominant bacteria in the gut of T. rubripes. Desulfobacterota, Bacteroidota, Firmicutes and Chloroflexi are the dominant bacteria in the sediment. Proteobacteria, Cyanobacteria, Bacteroides and Actinobacteriota are the dominant bacteria in the water. Although there was no significant difference in microbial α diversity in the gut of T. rubripes in different systems, the PCoA analysis showed that there were significant differences in microbial community structure among gut, sediment and water of T. rubripes in different culture systems. The similarity between gut microbes and environmental microbes each culture system was low, indicating that the intestinal microbial composition of T. rubripes may be less affected by environmental microorganisms. Redundancy analysis showed that the community structure and diversity of environmental microorganisms were affected by sediment and water environmental factors, respectively. Among them, salinity, ammonia nitrogen, nitrous nitrogen, nitrate nitrogen and total nitrogen were significantly correlated with the community structure of water flora (P<0.05) while nitrous nitrogen, nitrate nitrogen and total nitrogen were significantly correlated with the community structure of sediment flora (P<0.05).