In recent years, affected by overfishing and environmental pollution in the natural environment, human beings began to rely on aquaculture to maintain the continuous supply of protein sources. Aquaculture industry has developed rapidly in the world, profoundly changing the global food consumption pattern, and bringing huge economic profits to fishery operators (Bohnes et al., 2022). In particularly, the intensive aquaculture mode developed rapidly, but its high input and high-yield aquaculture mode made the content of ammonia nitrogen, nitrite nitrogen, total nitrogen, total phosphorus, organic matter and other substances in the aquaculture water exceed the standard, and then it caused a large number of pathogenic microorganisms to multiply. It could further cause the eutrophication of the aquaculture water body to become increasingly serious (Becke et al., 2019), which not only puts great pressure on the ecological environment but also endangers the normal development and health of cultured animals, and this has seriously damaged the economic benefits of fishery practitioners (Ahmad et al., 2022).

As an important environmental factor in the aquaculture water body, ammonia nitrogen is an important indicator of water quality monitoring. It exists in the water body in the form of ionic ammonia and non-ionic ammonia (Camargo and Alonso, 2006). With the rapid development of high-density intensive aquaculture, excessive ammonia nitrogen in the aquaculture water body has become a common environmental problem. The main source of ammonia nitrogen in aquaculture water is usually from the feed and additives of aquatic animal (Zhou and Boyd, 2015; Boyd, 2018). During the breeding process, nitrogen containing organic substances in residual bait, residues, excreta and the carcasses of aquatic animal are released into the water body (Zhou and Boyd, 2015). It is worth noting that this kind of situation is more likely to occur in the high-density breeding mode (Anh et al., 2010), resulting in the accumulation of ammonia nitrogen. Studies have shown that non-ionic ammonia is the main form of toxicity that causes physiological stress on aquatic animals. Its toxicity is about 300 to 400 times that of ionic ammonia (Xu et al., 2021). Therefore, non-ionic ammonium is a pollutant which could pose great threat to the health of aquatic organisms (Zheng et al., 2021; Guo et al., 2022). Under ammonia nitrogen stress, fish will have acute and chronic toxic effects, and then significant oxidative stress and severe inflammation could be detected in fish. Studies have shown that the increase of non-ionic ammonia will inhibit the excretion of ammonia nitrogen in fish (Xu et al., 2021). In addition, because of good fat solubility of non-ionic ammonia, it is easy to diffuse into the body through the cell membrane, causing damage to important organs such as gill, reducing the oxygen carrying function of hemoglobin which could transport oxygen. As a result of tissue hypoxia, elevated blood CO₂ concentration and pH value would lead fish dyspnea and decreasing feeding rate, inhibiting its growth and development (Jia et al., 2017; Liu et al., 2022). Ammonia nitrogen stimulates a series of toxic reactions, such as hypoxia convulsion, agitation or unresponsiveness of fish until death, which seriously damages the health status of breeding animals and the economic benefits of aquaculture practitioners (Camargo and Alonso, 2006). Therefore, there is a need to focus on the treatment of ammonia nitrogen and other pollutants when dealing with aquaculture water.

Studies have showed that different aquatic organisms have different tolerance to ammonia nitrogen. For example, bluegill is more sensitive while channel catfish has better resistance (Huff et al., 2013a). Nitrogen forms are diverse and can be transformed into each other, so nitrogen cycle generally exists in the material cycle system composed of the earth’s air, water and soil environment (Canfield et al., 2010). As far as the aquaculture system is concerned, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and other nitrogen forms will transform and migrate mutually in these environments in the body of water, sediment and aquatic organisms (Zerkle and Mikhail, 2017; Rose et al., 2021). Their chemical processes are extremely complex and changeable, which could affected by physical, chemical, biological and other factors, and each process interacts and influences each other. It is generally believed that nitric acid nitrogen is non-toxic to aquatic organisms, ammonia nitrogen is toxic, and the intermediate product nitrite nitrogen is toxic, while nitrogen is stable and non-toxic which cannot be directly used by aquatic organisms, and does not participate in the nitrogen transformation process in water (Lou et al., 2016; Kim et al., 2019). It has always been a hot research field for researchers on how to transform toxic nitrogen in the aquaculture water into non-toxic nitrogen or low toxic nitrogen forms (Bucking, 2017). There have been many studies on how to prepare the ammonia nitrogen concentration in aquaculture water, among which the microbial action is the most widely used because of its high efficiency, green and low cost, such as adding microbial agents (Yang et al., 2019; Praveen et al., 2021). Microbial treatment refers to the transformation of ammonia nitrogen through the chemical reaction in the process of microbial growth and metabolism. Adjusting the composition of the microbial community in the water body to realize the biotechnology of using different microorganisms to transform nitrogen to regulate aquaculture water quality has become quite important in the field of aquaculture (Niu et al., 2021). For example, under the action of nitrifying bacteria, the nitrification reaction could take place, which converts the highly toxic ammonia nitrogen into less toxic nitrite or basically non-toxic nitrate. Some products are converted into nitrogen through the denitrification process, thus effectively reducing the concentration of ammonia nitrogen (Yasuda et al., 2017; Holmes et al., 2019). In recent years, there are many researches on nitrogen removal in wastewater. According to the principle of nitrate removal, it can be divided into physical method, chemical method and biological method. It is widely used in industrial wastewater, domestic sewage and aquaculture water. Compared to other sewage treatment, in terms of aquaculture water treatment, its demand for treatment methods is more economic, safe and simple. Autotrophic denitrification is a new biological denitrification technology to remove nitrate in water., Kurt first used hydrogen to carry out hydrogen autotrophic denitrification in a conical reactor (Kurt et al., 1987). The principle is that hydrogen is used as an electronic donor by autotrophic microorganisms to reduce the oxidizing pollutant NO₃ ^- in water to N₂ (Karanasios et al., 2010). Compared with other physical and chemical methods such as adsorption method, chemical precipitation method and active metal reduction method, it has the characteristics of lower cost such as no addition of potentially toxic chemical reagents, harmless to cultured animals, simple operation and easy maintenance (Broman et al., 2017; Zha et al., 2020). It has achieved remarkable results in many water treatment fields such as high ammonia wastewater treatment and drinking water treatment, and most of these applications tested by the hydrogen nutrition process have achieved high efficiency and high denitrification rate (Karanasios et al., 2010; Shinoda et al., 2020). Considering that the aquaculture mode is generally medium and large intensive farms and individual farmers, it is more suitable for the aquaculture water treatment mode.

This study innovatively used the method of filling hydrogen into the aquaculture water body to demonstrate whether the removal of nitrogen and phosphorus were detected and the different pattern of microbial community composition and structure in the water body. And this work would provide the theoretical basis and technical support for removing ammonia nitrogen in aquaculture water.

Considering that the aquaculture mode is generally medium and large intensive farms and individual farmers, it is more suitable for the aquaculture water treatment mode (Li et al., 2021). Relying only on the system of self-regulation can’t guarantee the stability and harmless of the nitrogen cycle in the breeding environment, it is necessary to use external factors to control and promote the transformation direction of the nitrogen form (Akaboci et al., 2018). Nitrification and denitrification process are the most important nitrogen absorption process and mechanism in aquaculture water body (Zhang et al., 2019; Martínez-Espinosa et al., 2020). The present study showed, the ammonia nitrogen content in the water body in Group H was gradually decreased from the initial concentration in the process of hydrogen injection. However, the content of ammonia nitrogen in Group D gradually increased. After 7 days’ treatment, the ammonia nitrogen content in Group H was significantly lower than that in Group D (P <0.05). This suggests that the ammonia nitrogen concentration was significantly reduced in the progression of nitrogen cycle in water. Compared with Group D with 2.4 mg/L ammonia nitrogen, the concentration of ammonia nitrogen in Group H is at a safer level of 1.4 mg/L. For the sensitive aquaculture animals, with a very positive significance, this indicates that water may be more conducive to the survival and development of breeding animals after hydrogen filling treatment. For example, previous study showed that under the environmental conditions of gradient ammonia nitrogen concentration, the three common cyprinid tissues such as liver and gills have a higher health level at low ammonia nitrogen concentration (Wang et al., 2017). In our research here, the results of physical and chemical analysis showed that during the 7-days’ experiment, the ammonia nitrogen content in Group D increased significantly, while the ammonia nitrogen content in Group H decreased significantly.

The microbial diversity of the fish aquaculture water body is closely related to the stability of the water quality environment (Woese, 1994). When the diversity of water microbial community increases, the probability of aquatic biological diseases can be reduced (Woese, 1994; Niu et al., 2019). This study showed that the microbial Sobs index, ACE index, Chao1 index and Shannon-Wiener index of Group H were significantly higher than Group D (P <0.05), while the Simpson index of Group H was significantly lower than Group D p (P <0.05), which showed that a diversity of the microbial community increased significantly, which was conducive to ensuring the circulation of carbon, nitrogen, phosphorus and other elements in the water body and increasing the stability of the water environment (Fuhrman and Jed, 2009). At present, the effect of hydrogen injection in large-scale aquaculture water bodies on microbial communities is not studied. Noting that the study of microbial community changes in large reservoirs by some researchers (Lymperopoulou et al., 2012), it is suggested that the microbial community composition will change with water quality in environments with different spatial sizes, and the influence of hydrogen injection in aquaculture water bodies of large spatial scale could be investigated next.

The microbial community structure of the aquaculture water body is very complex and changeable, which not only affects the water environmental indicators, but also may be closely related to the gut microbial community of the aquatic organisms (Jin et al., 2017). Proteobacteria and bacteroidetes are dominant species of bacteria widely distributed in freshwater bodies. (Henne et al., 2012; Vaz-Moreira et al., 2013; Vaz-Moreira et al., 2017).In Group H of this study, proteobacteria replaced actinomycetes as the first dominant species compared with Group D. At the species level, Dechloromonas_sp._An3-5 and norank_o_Saccharimonadales abundances of Group H were significantly higher than these of Group D (P <0.05). Dechloromonas which is commonly used in the denitrification treatment system, is a denitrifier which belongs to the family Proteobacteria. And Dechloromonas always has participated in the denitrification process of all the enzyme line, which involved in the metabolism of nitrogen such as reducing NO₃ ^- to N₂ (Coates et al., 2001; Pieper and Santos, 2004; Yang et al., 2021). In addition, some bacteria in Proteobacteria, Firmicutes, Bacteroidetes, and Desulphuria all have the function of denitrification (He et al., 2016; Wu et al., 2020; Wang et al., 2021). Not all denitrifiers could undergo complete denitrification, and these microorganisms acted together on the denitrification chain, indicating positive implications for NO₂ ^- and NO₃ ^- in many wastewater treatment systems. Norank_o_Saccharimonadales is related to the metabolism of sugar, and it is the main microorganism of the hydrolysis and fermentation of macromolecular organic matter in the system (Liu et al., 2020). It is closely related to the degradation of organic matter in the water body, which is conducive to the improvement of water quality. Our results also suggest that the above two bacteria might be related to hydrogen autotrophic denitrifying bacteria. The groups of bacteria with nitrification and denitrification in Group H, such as Proteus, Desulfurization bacteria, abundance of Dechloromonas_sp._An3-5 and norank_o_Saccharimonadales was increased, which shows that there are huge differences in the nitrogen cycle process between Group D and Group H. Ammonia nitrogen changes to nitrite nitrogen and nitrate nitrogen which was with lower toxicity, and then obvious nitrification and denitrification occur in the water. As nitrate is the highest valence compound of nitrogen element, it can be reduced to low valence nitrogen. In this experiment, the process is accelerated by filling the reducing gas hydrogen. At this stage, microorganisms with nitrification and denitrification functions could become the dominant bacteria in the system, which might also imply that some strains belong to hydrogen autotrophic denitrifying bacteria. And we believe that it requires further experiments to explore the denitrifying microbial population suitable for aquaculture water.

In conclusion, our findings have demonstrated that 7-days’ hydrogen treatment could effectively reduce ammonia nitrogen concentration in aquaculture water. Meanwhile, hydrogen significantly changed the microbial structures and distributions in the water bodies. The abundance of bacteria such as Proteobacteria, Firmicutes, Bacteroidetes, and Desulphuria increased clearly in Group H, which means nitrification and denitrification occurred in the effect of hydrogen injection. These findings suggested that hydrogen could significantly improve aquaculture water health, reduce the production of ammonia nitrogen, which means hydrogen could be used as a functional external method to protect the aquaculture environment.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA915221.

JH and LW: conceptualization.SN, JH, LH, ML and XL: methodology. SN,WZ and JY: formal analysis and writing –original draft. LH, ML, YL and XL: data curation. SN,JH, and LW: writing – review and editing. SN,WZ and JY: visualization. LW: supervision. All authors contributed to the article and approved the submitted version.