A Study of a Composite Biofilm Reactor for the Treatment of Mariculture Wastewater: Performance and Microbial Communities

Abstract

Mariculture wastewater is one of the main sources of saline wastewater. This study used a waterfall aeration biofilm reactor combined with a sequencing batch reactor (WABR-SBR) to treat simulated mariculture sewage. Despite the high inhibition by salinity, the reactor maintained a high removal efficiency for organic matter and ammonium nitrogen. The ammonia nitrogen removal rate was greater than 99%, while that for nitrite, which is extremely toxic to farmed animals, was greater than 80%. Fourier transform infrared spectroscopy and scanning electron microscopy showed that salinity affected the surface structure and composition of biofilms, which became compact and secreted more solute to resist the impact of salinity. High throughput 16S rRNA sequencing revealed that the main phyla in the biofilms were Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Metagenomic annotation of genes further indicated nitrogen metabolism pathways under high salinity. The conclusions of this study can provide a theoretical foundation for the biological treatment of high-salt wastewater and provide a technical reference for further application of the WABR-SBR composite system.

1. Introduction

In recent decades, mariculture has become the fastest-growing segment of the food manufacturing industry due to the decline in global marine capture fishery production [1]. As a significant part of the marine economy, mariculture plays a major role in providing high-quality protein, promoting coastal economic development, and maintaining the ecological balance [2]. China is the world’s largest mariculture, capture fishery, and seafood exporter, accounting for 67% of the world’s marine products production; in 2020, the country’s mariculture production reached a record 21.35 million tonnes [3,4].

According to its salinity, aquaculture water is divided into freshwater, brackish water, and seawater, with salinity levels of <0.5 g/L, 0.5–30 g/L, and >30 g/L, respectively. Of these, brackish water aquaculture is practised mainly in coastal environments, while seawater aquaculture, also known as mariculture, is mainly carried out in the ocean [5]. China has more than 3 million square kilometres of sea area, spanning tropical, subtropical, and temperate regions. These, along with rich marine biological resources, have created unique conditions for the progress of mariculture in China [6]. Presently, mariculture in China accounts for one-third of the total aquaculture, and its range is gradually expanding to the ocean depths [7]. Due to China’s vast ocean area and development potential, some experts predict that the scale and proportion of marine aquaculture will increase significantly in the future [8,9].

The rapid development of aquaculture is also known as the Blue Revolution, and the sustainable growth of aquaculture is particularly important as a response to the growing global population and increasing need for aquatic products [5]. Over the last decade, China has promulgated several regulations and policies on the green development of aquaculture, clarifying the new direction of marine aquaculture, guiding the transformation of traditional mariculture, and promoting its sustainable development [2].

When cultivating marine organisms, a substantial amount of mariculture wastewater is discharged into the sea due to the water circulation between closed and semi-open seawater systems. The tailwater of mariculture is one of the main sources of saline wastewater [10]. The pollutants in mariculture wastewater are primarily soluble organic matter, nitrogen-containing compounds, and suspended solids derived from unused feed residues and aquatic animal waste. Untreated wastewater from mariculture seriously threatens marine ecosystems in estuaries and coasts, causing marine eutrophication and changes in biodiversity in adjacent sea areas [11]. For example, Liu et al. [12] found that in the Yellow Sea, China, no large-scale floating green algae were recorded before 2006; however, from 2007 to 2012, large-scale green tides erupted for six consecutive years. This was mainly due to the large-scale application of fermented chicken manure as feed, after which wastewater was discharged into the sea, resulting in seawater eutrophication. When large numbers of algae occur in eutrophic water bodies, they consume oxygen, resulting in hypoxia and causing the death of aquatic organisms (including plants and animals), imbalance of nutrient structure, and the eventual formation of a feedback loop [13].

To date, the treatments for mariculture tailwaters mainly include physical, chemical, and biological methods. Although the physical methods—mechanical filtration and foam separation techniques—are low in cost and easy to apply, they can only remove suspended particulate matter from the water body. Dissolved pollutants, especially ammonia nitrogen, which is highly toxic to aquatic organisms, cannot be removed. For this reason, physical methods are often used only for the preliminary treatment of wastewater. The chemical methods used for aquaculture wastewater treatment usually involve chemical oxidation. Ozone oxidation [14] is a standard method that quickly decomposes organic matter and reduces inorganic matter without secondary pollution. Additionally, after ozone treatment, the dissolved oxygen (DO) content in the water is high. However, the treatment cost is high compared to other methods, and the residual ozone can also have a toxic effect on living organisms. The electrochemical oxidation method [15] also has limited applications in actual production because of its high cost and equipment requirements. Compared to physical and chemical methods, biochemical treatment technology uses the growth and metabolism of microorganisms to absorb ammonia nitrogen and organic compounds in the water. This biological method is inexpensive, does not produce secondary pollution, and is the most effective treatment for dissolved pollutants. Upflow anaerobic sludge beds [16], moving bed biofilm reactors (MBBR) [17] and sequencing batch reactor-activated sludge processes [18] have been successfully applied to marine aquaculture wastewater treatment.

Researchers have suggested that the waterfall aeration biofilm reactor (WABR) maintains high removal efficiency of chemical oxygen demand (COD) and ammonia nitrogen in the treatment of distributed sewage in rural China [19] and food wastewater with a high carbon-nitrogen ratio [20]. The optimal carbon-nitrogen ratio of the folding aeration reactor has been explored [21]. The WABR has the advantages of low cost, easy maintenance, and efficient treatment performance. However, there is no relevant research on the tolerance and performance of WABR in treating high-salinity wastewater; therefore, this study combines WABR with a sequencing batch reactor (SBR) to treat mariculture wastewater.

The objectives of this study were to: (a) evaluate the COD degradation and denitrification performance of high salinity wastewater treated by a WABR-SBR, (b) explore the morphological changes, surface structure, and functional groups of the biofilm under different salinity conditions, (c) study the changes in microbial community richness and diversity at different salinities, and (d) explain the mechanism of nitrogen metabolism in the reactor under high salinity. The conclusions of our research can provide a theoretical foundation for the biological treatment of high-salt wastewater and provide a technical reference for further application of the WABR-SBR composite system.

2. Materials and Methods

2.1. Reactor Setup and Operating Conditions

A combined reactor (WARB-SBR) with an effective volume of 30 L was used in this study. A structural diagram of the reactor is shown in Figure 1. The main structure of the WARB is composed of six folding aeration inclined plates, each of which has a 5° angle. These are covered with non-woven fabric and flexible fibre filler to provide a suitable substrate for the stable attachment of microorganisms; the flexible fibre filler used in the laboratory was obtained from Shijiazhuang Songyu Environmental Protection Co., Ltd. (Shijiazhuang, China). The WABR was erected in the water tank; then, wastewater was pumped into it and distributed to the top inclined plate by the elevator pump. Gravity caused the wastewater to flow downward through the six-layer inclined plate and then flow into the water tank, restarting the cycle. The bioreactor was started by inoculation with activated sludge from a municipal sewage treatment plant in Guangzhou, China. During the experiment, the operating conditions remained constant: the temperature was 15–20 °C, pH 7.2 ± 0.2, water circulation speed was 7 L/min, and hydraulic retention time (HRT) was 2 days.

2.2. Experimental Water Quality

The experiment was divided into two stages: the initiation period (C) of 30 days and the salinity domestication period (S) of 50 days. The salinity gradient was increased by 0, 10, 20, and 30 g/L during the domestication period, which was divided into four stages (S1, S2, S3, S4). Synthetic simulated wastewater was used throughout the experimental phase, and the organic carbon in the wastewater came from glucose. The experiment used wastewater with a high organic load to promote biofilm formation. After 30 days, the membrane was stable, the reactor exhibited efficient processing performance, and the salinity of the biofilm was domesticated. Salinity and trace elements were provided by sea crystals produced by Zhejiang Blue Sea Crystal Salt Products Co. Ltd. In addition, the following ingredients were added: KH₂PO₄ 10 mg/L and NaHCO₃, 100 mg/L.

2.3. Chemical and Biological Analysis

Wastewater samples were collected every 24 h for a total of 80 days. The treatment performance of the reactor was evaluated, and the chemical oxygen demand (COD), nitrate, and nitrite nitrogen concentrations were analysed according to the Chinese State Environmental Protection Agency (CSEPA) standard methods. Ammonium was measured using Nessler’s reagent spectrophotometry. The dissolved oxygen (DO), temperature, and pH were measured using a portable dissolved oxygen meter with a temperature readout (HQ30d, HACH, Loveland, CO, USA) and a digital pH meter (A221, Thermo, Waltham, MA, USA), respectively. The biofilm was freeze-dried and coated with palladium gold, and its structure was determined using a scanning electron microscope (SEM, Car Zeiss EVO LS10, Zeiss British Company, Cambridge, UK). In addition, the samples were lyophilised to characterise the major functional groups using Fourier transform infrared spectroscopy (FTIR; Nexus Por Euro, MA, USA).

2.4. Microbial Community Determination

At the end of each salinity condition, biofilm samples (surface area of 1 cm²) were taken from the centre of the third layer of the reactor inclined plate and were recorded as S1, S2, S3, and S4, respectively. All collected samples were kept at −80 °C. DNA extraction, PCR amplification of the 16S rRNA gene, and metagenomic analyses were performed by Novogene Company (Tianjin, China).

3. Results and Discussion

3.1. Start-up Performance of Biofilms

The reactor was started under high organic-loading conditions. A stable biofilm was observed on the inclined plate during the 12-day run, while flocculent sludge was produced at the bottom of the tank. Other researchers have reported that there are a large number of denitrifying bacteria in the sludge, which also affect the removal of pollutants [20]. As shown in Figure 1, under the condition that the COD of the influent was 3000 mg/L, the removal rate on the third day of operation reached more than 60%, and the COD of the effluent of the 10th COD was reduced to 300 mg/L. The removal efficiency reached 90% and remained stable, mainly due to the enrichment and degradation of heterotrophic microorganisms. Figure 2 shows that when the influent ammonia nitrogen was 200 mg/L, the ammonia nitrogen removal rate reached 50% on the second day, and on the 12th day of operation, it reached 90% and remained stable. There was a brief accumulation of nitrite during the initiation period, but the treatment effect became stable on the 26th day of operation. There was no nitrite nitrogen and nitrate-nitrogen accumulation in the reactor, indicating that nitrifying bacteria and denitrifying bacteria were effectively enriched and remained active in the system.

3.2. Performance on the Domestication Phase of Biofilm

After 30 days, the film matured, the efficiency of the reactor reached its highest value and remained stable, and there was no accumulation of nitrite and nitrate nitrogen. Simulated marine aquaculture wastewater was pumped into the tank, and biofilms were domesticated sequentially at salinities of 0, 10, 20, and 30 g/L. The average COD removal rates in the four stages of S1, S2, S3, and S4 were 84.8%, 67.6%, 67.7%, and 64%, respectively (Figure 3a). These results suggest that high salinity leads to a decrease in the removal capacity of organic matter because salt has adverse effects on microbial growth and activity. The presence of high salinity is known to lead to the loss of metabolic activities and cell membrane lysis of mixed cultures, giving rise to the release of intracellular components and soluble microbial products, resulting in a reduction in the utilisation of organic matter [22,23]. However, the WABR-SBR maintained some degradation performance after salinity impact, indicating that microorganisms attached to the folding plate could adapt to high salt environments and maintain high organic matter removal efficiency.

In biological denitrification treatment, ammonia nitrogen in sewage is oxidised to nitrite by ammonia-oxidising bacteria (AOB) under aerobic conditions and oxidised to nitrate by nitrifying bacteria (NOB). Then, under hypoxic conditions, denitrifying bacteria are used to reduce nitrate to nitrogen and escape from the sewage. Studies [24] have shown that the impact of salinity on filamentous bacteria and protozoa is much greater than the impact on autotrophic bacteria. Ammonia-oxidising bacteria and NOB are autotrophic bacteria and have ammonium salt oxidation as their energy source. These halophilic bacteria can tolerate salinity changes and survive in high salinity environments while maintaining physiological activity [24]. Therefore, through early domestication and adaptation processes, AOB and NOB can adhere stably to the biofilm, enhancing the ability of the composite system to resist salinity shock and maintain a high ammonia nitrogen removal rate.

The effects of increasing salinity on the ammonia nitrogen removal rate and ammonia nitrogen effluent concentration are shown in Figure 3b. When the salinity was 30 g/L, the ammonia nitrogen in the wastewater was close to complete removal, and the total removal rate was above 99%. The nitrite effluent (Figure 3c) was consistent with the fluctuation of ammonia nitrogen, fluctuating at the beginning of each salinity condition, then quickly recovering and remaining stable; the final effluent was below 0.1 mg/L, with an overall removal rate of over 80%. The effluent is in line with China’s national marine aquaculture wastewater discharge standards and can meet the treatment requirements of marine aquaculture wastewater.

Figure 2 shows the influent and effluent concentrations of nitrate nitrogen. With the increase in salinity, the effluent nitrate-nitrogen concentration increased sharply, reaching a maximum of over 30 mg/L in S3, but then slowly decreased and eventually stabilised at 19.2 mg/L in S4. This result demonstrates that salinity has a strong inhibitory effect on denitrification, in which nitrate is used as an electron acceptor for respiration to obtain energy in the absence of oxygen [25]. In this study, the organic load was low, while the water body maintained a high DO value (Supplementary Materials Table S1) and flocculent sludge formed in start-up stage disappeared; therefore, it was not conducive to the enrichment of denitrifying bacteria, eventually leading to the accumulation of nitrates. However, the reactor also maintained a specific denitrification capacity, indicating that a thicker biofilm on the folded inclined plate can provide an anaerobic environment for denitrifying bacteria. Feng et al. [26] used an MBBR to treat mariculture wastewater, which showed low denitrification efficiency. There may be two reasons for this: one is that high salinity levels are not conducive to denitrification due to denitrifying bacteria being more sensitive to salt than nitrifying bacteria [27]; the other is that the lack of organic matter in the influent limits the efficiency of denitrification, resulting in poor denitrification under oligotrophic stress. Some experiments [26] have used biodegradable polypropylene carriers to supply carbon sources to improve denitrification efficiency and achieved good results under high salinity conditions and oligotrophic stress; the nitrate accumulation was 1.16 ± 0.18 mg/L. This is similar to the effluent in this experiment, these findings also offer the prospect of a system to treat high salinity wastewater by adding an external carbon source.

3.3. FTIR Analysis

Infrared spectroscopy can provide detailed information on the composition and structure of biofilms. Figure 4 shows the FTIR spectra of the biofilms at different salinities. While the position of the infrared spectrum peaks in the biofilm samples showed negligible changes under different salinity conditions, their intensities varied. Six types of peaks were scanned for all four salinity levels. Screening according to the literature [28,29] revealed that the main functional groups detected in the biofilm samples included C-H (3290 cm⁻¹) of proteins, protein-related amide groups α C=O (1637 cm⁻¹), protein-related amide II regions (1544 cm⁻¹), aliphatic CH₂-groups (2922 cm⁻¹), C-O-C (1078 cm⁻¹) of polysaccharides, and unsaturated carbon bonds of polysaccharides (650 cm⁻¹). Overall, the spectroscopic results confirmed that biofilms contained substances such as proteins, polysaccharides, fats, carboxyl groups, and hydroxyl compounds, and that there were many types of proteins, which was in line with the composition characteristics of extracellular polymeric substances (EPS). These substances are products located on the surface of or outside the cell and consist of proteins, lipids, carbohydrates, DNA, and humic acids [30].

Studies have shown that the EPS content of biofilms is affected by environmental factors and operating conditions, and its compositional characteristics are mainly dependent on the wastewater matrix [31]. Wang et al. [32] found that salinity had a greater impact on EPS content than on EPS composition, and Liu and Tay [33] further revealed that high EPS content was conducive to sediment formation and biofilm stability. Lay et al. [32] also found that EPS had high adsorption properties and affinity for cations, thereby forming an adsorption layer on the surface of the membrane, which changed the interface electrochemical potential and further affected the mass transfer of salts in the biofilm. Under S1 conditions, two peaks at 1390 cm⁻¹ and 1229⁻¹ were scanned; the corresponding functional groups were carboxyl or hydroxyl compounds C-H, CH₂-, and protein-related amide III regions, respectively. No peaks were observed at high salinity, which may be due to the stress effect of high salinity on microbial cells. Studies have found that moderately halophilic bacteria could cope with salinity in the environment by producing compatible solutes, with the most common one being proline [34]. However, we did not find proline in our experiments.

3.4. Biofilm Morphology at Different Salinities

Scanning electron microscopy (SEM) was used to compare the morphologies of biofilms at different salinities. Under the S1 condition, the biofilm had a loose and porous mesh structure (Figure 5a), and a large number of bare and buried spherical and rod-like microorganisms were observed. Figure 5b shows the presence of numerous hyphae closely connecting the microorganisms with the membrane structure. When the salinity rose to 10 g/L, a large number of salt crystals were observed on the biofilm, which became compact and showed a decrease in porosity (Figure 5c). In addition, the exposed microorganisms and mycelium were significantly reduced, and the observable rod-like microorganisms appeared shorter and more wrinkled (Figure 5d); this phenomenon became more prominent with an increase in salinity. Through microscopic examination, Peng et al. [24] found that the sludge floc contained a rich variety of protozoa, zoogloeal, and filamentous bacteria. As the salinity increased, the microbial composition changed, and the number of filamentous bacteria and protozoa gradually decreased until they completely disappeared, which was consistent with the results observed in this study [18]. When the salinity reached 20 g/L and 30 g/L, the whole biofilm was dense (Figure 5e,g), which could hinder the mass transfer of salt to a certain extent. Exposed microorganisms were difficult to observe on the biofilm surface. However, a small number of microorganisms, mainly filamentous bacteria, were found embedded in the zoogloea [35]. Some broken cells could be observed on the cell membrane (Figure 5f,h). This effect was due to the high salinity outside the bacterial cytoplasmic membrane creating an osmotic pressure difference and leading to water loss and the bursting of cells [36].

3.5. Microbial Community

3.5.1. Microbial Richness and Diversity

To further understand the effect of salinity on the bacterial communities in the system, 16s rRNA amplicon sequencing analysis was performed on four biofilm samples at different salinities. The values for all samples, shown in

Table 1. Alpha indices of the microbial community on biofilm samples at different salinities.

Sample Name	S1	S2	S3	S4
OTU	558	658	670	643
Shannon	5.363	5.847	5.945	6.033
Simpson	0.923	0.928	0.930	0.943
Chao1	576.676	700.512	710.539	647.737
ACE	581.869	704.361	711.179	659.415
Coverage	0.999	0.998	0.998	0.999

, guaranteed that the given data were sufficient to cover all bacterial species, and the analysis results were reliable. The most abundant operational taxonomic units (OTU) number (670) was obtained in S3, the highest Chao1 and abundance-based coverage estimators (ACE) indices were observed simultaneously, and the highest Simpson and Shannon indices were obtained in S4. The ACE and Chao values represent the richness of the microbial community, while the Shannon and Simpson values indicate the diversity of the microbial community [37]. It was found that the bacterial abundance in the system increased with increasing salinity, which may be due to salinity stimulating the growth of halophilic heterotrophic bacteria. Furthermore, many of the bacteria attached to the biofilm resisted the impact of salinity by proliferating, thus improving the overall richness of the microbial community in medium- and low-salinity environments [17,32]. However, under the S4 conditions, excessive salinity inhibited further proliferation of the microorganisms.

The microbial differences and similarities of the WABR-SBR under salinity at 0–30 g/L were analysed using a Venn diagram (Figure 6). The unique OTU numbers from the biofilm were 65, 56, 52, and 53 at salinities of 0, 10, 20, and 30 mg/L, respectively. The existence of unique OTUs demonstrated that some unique microbes appeared at different salinities, excluding some coexisting microbes.

3.5.2. Composition of the Microbial Community

Figure 7a shows the relative abundance of biofilms at the phylum and genus levels (top 10 in abundance). The four most abundant classes are Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. The relative abundances of Actinobacteria and Proteobacteria for S1–4 were 52.8% and 26.6%, 48.9% and 16.5%, 48.6% and 15.8%, and 45.1% and 33.1%, respectively. It is apparent that salinity has a certain inhibitory effect on Actinobacteria and Proteobacteria, while the dominant population still maintains high abundance. The predominant phyla were consistent with previous reports of high-salinity wastewater treatment using biological methods [38]. It is well known that the filamentous structure of Actinobacteria is much more stable than others, and Actinobacteria can resist sloughing by increasing EPS and adhesion force in high salinity [39]. This is in line with our SEM observation that filamentous bacteria were dominant in the matured biofilm. Proteobacteria are reported to perform a significant role in nitrogen removal [40]. It is worth noting that the Chloroflexi—a group of bacteria that produces energy through photosynthesis—were also present in abundance. Sorokin et al. [41] isolated a nitrite-oxidising bacterium belonging to Chloroflexi from a nitrification bioreactor that used NO₂⁻ and CO₂ as substrates to grow and form as an energy and carbon source to grow under nutrient-rich conditions, participating in the second part of the nitrification reaction (i.e., the oxidation of NO₂⁻). This presents the possibility that reactors can be combined with algae to treat sewage.

As Figure 4 shows, only 8 of the top 10 classes, which included unidentified Bacteria and Enterobacteriaceae, could be identified. The Micropruina, Propioniciclava, and Brooklawnia (all three of which belong to Propioniaceae and Actinobacteria) maintained high abundances in S1, S2, S3, and S4. The Propioniaceae are facultative anaerobes that use glucose and other carbohydrates as metabolic substrates and produce a large amount of propionic and acetic acid and a small amount of gas. The high abundance of this group of bacteria also ensures the stable COD removal performance of the system. The presence and physiological activity of anaerobes proved the presence of an anaerobic environment in the thick biofilm on the collapsible aerated inclined plate. Under S2 and S3 conditions, a high abundance of Bacteroides was observed; however, their number decreased sharply under S4 conditions. Bacteroides exist widely in aquatic environments and are the main bacteria in anaerobic digestion. They can adapt to medium and low salinity and are closely associated with the removal of nutrients [42]. Azoarcus was highly abundant in S4 (9.75%); it can adapt to high salinity and plays a vital part in the simultaneous removal of organic matter and nitrogen in the air inner-loop sequencing batch reactor [43] and the hybrid sequencing batch biofilm reactor [44].

3.5.3. Mechanisms of Nitrogen Removal of the Metagenome at High Salinity

To illustrate the nitrogen removal mechanism in mariculture wastewater treatment, the distribution of functional genes and abundance of enzyme families were classified based on the KEGG database. According to the reference pathway (KO) of nitrogen metabolism (map00910), the nitrogen metabolism pathways are plotted in the Supplementary Information (Figure S1) and can be divided into core (Figure S2) and subordinate (Figure S3) pathways. According to Kanehisa et al. [45], core nitrogen metabolism consists of five modules, including four reduction pathways (M00529 participates in denitrification, M00530 in dissimilatory nitrate reduction, M00531 in assimilatory nitrate reduction, and M00175 in nitrogen fixation), and one oxidation pathway (M00528, which is involved in nitrification). The abundance of the KEGG module classification at different salinities in the nitrogen metabolism pathway can be seen in Figure S4. Modules M00531 and M00528 revealed higher abundance under high salinity conditions, which ensured the efficient ammonia removal capacity of the WARB-SBR. We found that M00530 was most abundant at low salinity (S2), which is supported by the inhibition of denitrification in the high-salinity wastewater in this study; however, M00175 and M00529 showed the opposite trend and were less abundant at low salinities. The higher levels of M00804 in high-salinity sewage ensured a continuous low level of nitrite in the effluent. In short, nitrification-denitrification for nitrogen removal at low salinity was ubiquitous, whereas assimilation was prevalent at high salinity [46].

Combined with key enzyme coding [47], the abundance of key enzymes involved in the primary nitrogen metabolism is presented in Figure 8. As shown in Figure S1, many enzymes participate in the nitrification–denitrification process. These include hydroxylamine dehydrogenase (EC:1.7.2.6) and ammonia monooxygenase (EC:1.14.99.39), which oxidised ammonia nitrogen to nitrite, and nitrate reductase/nitrite oxidoreductase (EC:1.7.7.2; EC:1.7.5.1; EC:1.7.99; EC:1.9.6.1.), which was involved in the conversion between nitrate and nitrite. Moreover, nitrite was reduced to nitric oxide by nitrite reductase (EC:1.7.2.1), nitric oxide reductase (EC:1.7.2.5) was responsible for reducing nitric oxide to nitrous oxide, and nitrogen oxide was eventually reduced to nitrogen by nitrous oxide reductase (EC:1.7.2.4). As the salinity of the wastewater increased, EC:1.7.2.4 and EC:1.7.2.1 showed a marked decline, indicating that salinity inhibited denitrification in the system. In S2 and S3, glutamate dehydrogenase (EC:1.4.1.3 and EC:1.4.1.4) and glutamate-ammonia ligase (EC:6.3.1.2) both increased, followed by a sharp decline in S4, whereas the abundance of ammonia monooxygenase and hydroxylamine dehydrogenase dramatically increased. The results indicated that the removal of ammonia nitrogen under medium and low salinity was mainly through assimilation to form L-glutamine, further forming L-glutamate, which participates in glutamate metabolism but is mainly converted to nitrite at high salinity [46].

To clarify the role of the biofilm in nitrogen metabolism, Figure 9 itemises the proportions of key functional genes in biofilm samples related to the main nitrogen metabolism pathways. The nitrate reductase genes (nirS, norB, and nosZ) and nitrogen fixation genes (nifD, nifH, and nifK) in S1 showed high abundance but were not prominent at high salinity. In S4, the dominant genes were the assimilatory nitrate reduction genes (narB, nasA, and nirA) and the nitrification genes (pmoA-anoA and hao). The pmoA-amoA is involved in encoding ammonia monooxygenase [48], and the hao gene is the key gene that encodes hydroxylamine dehydrogenase [49]. In the biological nitrogen cycle, inorganic nitrogen is fixed, and ammonia nitrogen is formed through a series of reactions. Then, ammonia nitrogen is oxidised to hydroxylamine by ammonia monooxygenase and further oxidised to nitrite by hydroxylamine dehydrogenase [50]. The biofilm provided a habitat for heterotrophic bacteria and strengthened the assimilatory nitrate reduction pathway for protein synthesis at high salinity.

4. Conclusions

This study showed that the WABR-SBR system could effectively treat mariculture wastewater. The effluent quality indexes were stable, and the removal rate of ammonia nitrogen reached 99%. The effluent had no accumulation of nitrite, which is toxic to cultured organisms. High-throughput sequencing was implemented to elucidate the composition and changes in the microbial community structures of the system. Metagenomics was used to analyse functional genes and enzyme families associated with nitrogen metabolism, further explaining the metabolic process. Our results provide new insights into the microbial treatment of mariculture wastewater.