The Impact of the Mechanism of Biocarriers on Bacterial–Microbial Symbiosis for Mariculture Wastewater Treatment: Performance and Microbial Community Evolution

Abstract

Mariculture wastewater is an intractable wastewater, owing to its high salinity inhibiting microbial metabolism. The biocarrier bacterial–microbial consortium (BBM) and bacterial–microbial consortium (BM) were developed to investigate the mechanism of pollutant degradation and microbial community evolution. The BBM exhibited excellent mariculture wastewater treatment, with the highest removal for TOC (91.78%), NH₄⁺-N (79.33%) and PO₄³⁻-P (61.27%). Biocarriers accelerated anaerobic region formation, with the levels of denitrifying bacteria accumulation improving nitrogen degradation in the BBM. Moreover, the biocarrier enhanced the production of soluble microbial products (SMPs) (11.53 mg/L) and extracellular polymeric substances (EPSs) (370.88 mg/L), which accelerated the formation of bacterial and microalgal flocs in the BBM. The fluorescence excitation–emission matrix (EEM) results demonstrated that the addition of biocarriers successfully decreased the production of aromatic-like components in anoxic and aerobic supernatants. Additionally, the biocarrier shifted the bacterial community constitutions significantly. Biocarriers provided an anoxic microenvironment, which enhanced enrichments of Rhodobacteraceae (66%) and Ruegeria (70%), with a satisfying denitrification in the BBM. This study provided a novel biocarrier addition to the BBM system for actual mariculture wastewater treatment.

1. Introduction

Notable amounts of mariculture wastewater are being produced due to the increasing demand for seafood and the reduction in wild fishery resources [1]. As a typical salinity wastewater, it contains complex components such as suspended solids, nutrients and dissolved organic carbon from feed residues and excretions [2]. Discharge of mariculture wastewater results in a severe threat to the coastal environment, such as in marine eutrophication or biodiversity reduction. Moreover, mariculture wastewater has been generally considered as a kind of intractable wastewater owing to high salinity’s impact on microbial metabolism [3]. There are currently various methods to treat such wastewater, such as advanced oxidation processes, reverse osmosis and ion exchange. However, there are problems of a high operating cost and secondary pollutants [4]. Hence, conventional biological methods are more suitable for the removal of contaminants from mariculture wastewater. However, the pollutant removal efficiency of these technologies (e.g., activated sludge wastewater treatment process) cannot meet the requirements of discharge or recycling. Hence, an alternative technology for mariculture wastewater treatment is urgently needed [5].

The bacterial–microbial consortium (BM) provides a sustainable and cost-effective means of biological nutrient removal as well as microalgae bioenergy accumulation in the wastewater treatment process. The BM has been increasingly used for high-salinity wastewater treatment due to its unique features of an excellent nutrient removal efficiency, rapid growth and a high adaptability to surrounding environments [6]. A previous study indicated that a higher ammonium reduction (100 ± 18 mg/(L·d)) was attained in the BM than that in the microalgae system (44 ± 16 mg/(L·d)) [7]. However, the high stress of salinity inhibits the microbial metabolism in the BM dramatically. The activities of functional microorganisms—including denitrification bacteria and phosphate-accumulating organisms—are reduced, resulting in unsatisfactory nutrient removal. Moreover, the high stress of salinity shifted the structure of the polymers that hold the sludge together, resulting in the sludge floc breaking up. It produced sludge particles, leading to a decline in the efficiency of sludge filtering [8]. Soluble microbial product (SMP) and extracellular polymeric substance (EPS) were released throughout the procedure. Interestingly, biocarrier enhanced the chemical oxygen demand (COD) removal rate and color removal rate, with values 2% and 8% greater than that in the conventional bacterial-microbial MBR [9]. In the MBBR, the addition of biocarrier significantly enhanced the biodegradation efficiency of organic matter. The removal rates of NH₄⁺-N and TN increased by 8.7% and 12.5%, respectively, which improved the nitrification and denitrification processes [10]. A previous study reported that total nitrogen removal was about 22.9% higher than that of the control group with the addition of biocarrier in the MBR. This result may be related to the addition of biocarrier, which changed the microbial community structure and led to the formation of anoxic zones. The relative abundance of denitrification and nitrogen metabolism sequences increased [11]. Biocarriers significantly impacted the characteristics of SMPs and EPSs in mariculture wastewater treatment. Biocarriers reduced the production of aromatic-like components in anoxic and aerobic supernatants and caused peak shifts towards shorter wavelengths of SMPs in the aerobic phase [12]. The addition of biocarriers resulted in the enrichment of various functional microorganisms, including Candidatus, Cloacibacterium and Flavobacterium [13]. In addition, the BM has shown significant economic advantages in the treatment of mariculture wastewater. Microalgae photosynthesis replaces traditional aeration and reduces electricity consumption [14]. Moreover, microalgae can use CO₂ as a feedstock for photosynthesis, producing high-value compounds while reducing CO₂. Microalgae have a lot of high-value compounds in their cells that can produce large amounts of biomass and lipids [15]. These substances can be converted into biofuels, biohydrogen, biobatteries and other high-value substances in a clean and sustainable way that has the potential to replace traditional fossil fuels and be environmentally friendly [16]. Hence, the above studies demonstrated that biocarrier can be applied to wastewater treatment and has achieved good results, but the treatment efficiency of organic matter and microbial community transformation after adding biocarrier need to be further investigated.

Therefore, the biocarrier bacterial–microbial consortium (BBM) and BM were developed to explore the mechanism of pollutant degradation and microbial community evolution for mariculture wastewater treatment. The aims of this study were to (1) investigate the mechanism of the biocarrier on the degradation of pollutants in the mariculture wastewater treatment; (2) to explore the effects of biocarrier addition on the characteristics of SMPs and EPSs; (3) to analyze the biocarrier’s effect on the mass balance under the high stress of salinity; and (4) to discuss the biocarrier’s effect on the microbial community structure shift in the BBM. This paper presents new insights into the mechanism by which the BBM promotes the secretion of EPSs, the evolution of microbial community structure, and the enhancement of organic matter removal efficiency. These studies can provide a theoretical basis for the future large-scale application of the BBM treatment of mariculture wastewater.

2. Materials and Methods

2.1. Microorganism and Synthetic Mariculture Wastewater

Chlorella pyrenoidosa (C. pyrenoidosa) was obtained from the Institute of Oceanology, Chinese Academy of Sciences (Qingdao, China) (FACHB-9), and it was maintained in autoclaved BG-11 medium and stored at 4 °C. Lighting parameters were as follows: cool white LED light; a light intensity of 3000 lux; a light/dark cycle of 12 h/12 h. Activated sludge seed was collected from a local sewage treatment plant located in Weihai and inoculated in a 20 L capacity reactor, and the impurities were removed in the process [5]. C. pyrenoidosa and activated sludge were mixed to produce biomass. The biomass was acclimated in an operation using synthetic mariculture wastewater for a month to obtain loose flocs. The pre-cultivated flocs were inoculated in the BBM with an initial concentration of approximately 2000 mg/L.

Synthetic wastewater was added with the following composition: 100 mg/L NH₄Cl, 17.5 mg/L KH₂PO₄, 250 mg/L NaHCO₃, 332 mg/L CH₃COOH, 8.4 mg/L EDTAFeNa, 0.005 mg/L vitamin B12, 20 mg/L Na₂SiO₃⋅9H₂O, 1 mg/L vitamin B1, 36 mg/L CaCl₂⋅2H₂O, 75 mg/L MgSO₄⋅7H₂O, 1 mg/L Na₂EDTA and A5 (1.86 mg/L MnCl2⋅4H₂O, 2.86 mg/L H₃BO₃, 0.22 mg/L Na₂MoO₄⋅2H₂O, 0.05 mg/L Co(NO₃)₂⋅6H₂O, 0.08 mg/L CuSO₄⋅5H₂O, 0.021 mg/L ZnSO₄⋅7H₂O) [17]. The final concentrations of the essential components were 250 mg COD/L, 26 mg N/L and 4 mg P/L in synthetic mariculture wastewater.

2.2. Experimental Setup and Operation

A novel bioreactor, namely a double internal circulating bioreactor, was constructed to cultivate the bacterial–microbial consortium directly in continuous flow mode, as shown in Figure 1. The BBM was a double internal circulating bioreactor using 1 cm diameter round high-density polyethylene packing as biocarriers. Biocarriers flows with the fluid throughout the reactor. The filling rate was 5% of the effective volume of the reactor. The BM was used as the control group without adding biocarriers. This configuration achieved an internal hydrodynamic circulation. Aeration was provided at the bottom of both reactors by gas diffusers (Xiangjin, China) and monitored by an airflow meter (Xiangjin, China). The floc of bacteria and algae were made by fluid flowing upwards in the aeration chamber and downwards in the mixing chamber. The configuration of both bioreactors is shown in Figure 1. Both reactors were set up with a working volume of 15 L. The light intensity was 3000 lx.

2.3. Analytical Methods

The organic and nutrient removal performance of the BBM and the BM was monitored in terms of TOC, NH₄⁺, TP. A total organic carbon analyzer was used to monitor TOC (TOC-L CPH CN200V, Shimadzu, Japan) [18]. The content of chlorophyll a was detected by acetone extraction. Biomass was measured by dry-weight method. The Lowry method [19] and phenol-sulphuric method [20] were used to detect protein and carbohydrate, respectively. Excitation–emission matrix spectroscopy (EEM, FP6500, JASCO, Japan) was used to characterize major components of SMPs and EPSs (F-2700 Hitachi Ltd., Japan). Measurements of EEM fluorescence spectra were carried out repeatedly. To investigate the bacterial community constitution evolution in the BBM and BM, samples were collected at the end of the experiment. The 16S rRNA gene levels Miseq Illumina were used to analyze the bacteria community composition [5].

3. Results and Discussion

3.1. Mariculture Wastewater Treatment Performance

As shown in Figure 2a, the BBM obtained the highest removal of TOC (91.78%). The microalgae provided sufficiently high dissolved oxygen (DO) to heterotrophic bacteria (DO > 7.3 mg/L), enhancing COD removal [21]. A previous study had demonstrated that the BBM had promising performance in removing organic matter. Compared to the BM, the addition of biocarrier provided a suitable environment. The biocarriers improved the hydraulic characteristics by prolonging the actual hydraulic retention time (HRT) and reduced the dead zone, which could benefit mass transfer [22]. The cylindrical biocarrier had a large specific surface area. Aerobic microorganisms multiplied on the surface of the biocarrier and formed a biofilm [23,24]. Organic matter was decomposed rapidly. Gao et al. added modified basalt fiber (MBF) biocarrier. The COD removal efficiency was 93.4%, which may be the result of the effective diffusion and degradation of oxygen and pollutants in the bio-nest [25]. These findings aligned with the previous study’s results, which showed that COD was efficiently utilized, with efficient removal in the biofilm membrane bioreactors coupled with pre-anoxic tanks for treating mariculture wastewater [26]. Additionally, the addition of biocarriers could change the microbial environment inside the system and increase the abundance of beneficial bacteria such as Alphaproteobacteria. Hence, the addition of the biocarrier was beneficial to the removal of organics in the BBM.

As shown in Figure 2b, the BBM and BM system all maintained a stable high removal efficiency of nitrogen of 90% to 100% throughout the whole operation. NH₄⁺-N was mainly removed through the effect of microalgal assimilation and bacterial dissimilation between microalgae and bacteria [27]. Microalgae preferred to take up NH₄⁺-N due to its smaller energy requirement. NH₄⁺-N could be directly transported into microalgal cells, which could be converted to amino acids in the body by glutamine synthetase–glutamate synthase for vital activities and reproduction. NH₄⁺-N was firstly transformed into NO₂⁻-N and NO₃⁻-N by NH₄⁺-N-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), then would be converted to nitrogen through denitrification [28]. The BBM had a higher nitrogen removal efficiency compared to activated sludge. This conclusion was also verified in the previous study, where the removal efficiency of NH₄⁺-N nitrogen in the BBM was about 24% higher than that of activated sludge system [29]. This was because the conversion of ammonium by nitrifying bacteria reduced the toxicity of free NH₄⁺-N on microalgae growth. CO₂ from bacteria were shown to be a carbon source for microalgal growth. In turn, in some cases, microalgae extracellular polysaccharides or amino acids fed bacteria as well [30]. Microalgae and their associated bacteria were involved in a complex nutritional exchange that benefits both the bacteria and the microalgae [31]. The EPSs secreted by bacteria and microalgae could be attached to the surface of the biocarrier, which acted as a diffusional barrier between microbial cells. Biocarriers might protect microorganisms from changes in the surrounding environment through the attachment of biomass [32]. In addition, microbial community succession occurred in the biofilm, driven by the increased microbial diversity in the biofilm of biocarriers. This increased the abundance of beneficial bacteria such as Proteobacteria, which includes AOB, NOB, denitrifying bacteria (Betaproteobacteria) and other bacteria dedicated to denitrification (Rhodobacter) [33]. The biocarriers immobilized specific bacteria in the BBM, which encouraged the enrichment of nitrifiers and denitrifiers. Therefore, the BBM had achieved an excellent nitrogen removal performance [12].

Figure 2c shows that the influent TP concentration varied in the low range of 2.21~2.66 mg/L (average 2.36 mg/L). The removal efficiency of the effluent TP concentration in the stabilized stage of the BBM was above 85%. The removal of phosphorus in the BBM and BM system included the growth assimilation of microalgae and the aerobic phosphorus absorption of activated sludge. In this process, aeration was set and O₂ was produced by promoting the growth of microalgae. Therefore, it was difficult to form anaerobic conditions. It was possible that the removal of TP was mainly due to the assimilation of microalgae [34]. At the initial stage, the removal efficiency of TP showed a decreasing trend, which may be due to the reduction in the phosphorus-accumulating organisms (PAOs) in the two reactors. Polyphosphate-accumulating organisms enriched from activated sludge could store net phosphate as intracellular polyphosphate via alternating anaerobic and aerobic conditions [35]. During the intermediate and late stage, the increasing removal efficiency of TP may be attributed to the adaptation of the bacteria and microalgae consortium in the BBM and BM to the environment, and the enhancement of microbial assimilation and excessive phosphorus absorption capacity in the system. The BBM exhibited a higher TP removal performance than the BM. The biocarriers facilitated the maintenance of nutrient and oxygen levels in the colonies attached to the biocarrier and enabled the growth of a thin biofilm onto the surface despite the turbulence [22]. The performance of the BBM was more stable than that in the BM. In another study, it was demonstrated that thin biofilms had high bacterial activity and provided excellent contaminant removal properties [24,36]. Moreover, biocarriers adopted a cylinder shape with a cross inside and enhanced the attachment of microorganisms. It had large pores inside, which facilitated water and bubble transport. It increased the active area for bacterial growth and metabolic activity, and allowed for a great and fast mass transference [37]. Therefore, bacteria and microalgae attached to the biocarrier can further strengthen the adsorption of pollutants and improve the assimilation efficiency.

3.2. The Accumulation of Chlorophyll a and Biomass Production

As shown in Figure 3, the variation trend of mixed liquor suspended solid (MLSS) production was similar to the chlorophyll a accumulation. The BBM and BM both showed a trend of rising firstly and then decreasing. However, the biocarriers stabilized the regularity of the fluidization of the system, which promoted the secretion of EPS by microorganisms and increased the chlorophyll a accumulation. At the initial and intermediate stage, activated sludge and microalgae were in the growth phase, with sufficient resource availability. The growth of MLSS and chlorophyll a content increased significantly. In the system, microalgae could fix CO₂ during autotrophic growth while secreting organic carbon, mainly as polysaccharides and proteins, which can be used as a carbon source by the heterotrophs. The synergistic support of bacteria and microalgae enhanced the consortium growth, as previously reported [38]. After that, the biomass and chlorophyll a content of the two reactors decreased, which might be due to the continuous increase in biomass, resulting in the poor fluidization state of the reactor system. The study by Qian et al. confirmed this statement [39]. Overall, the growth in the MLSS and chlorophyll a content of the BBM was higher than that in the BM. Hence, biocarriers strengthened the regularity of fluidization in the BBM.

3.3. The Production of SMP and EPS

Figure 4 depicts the contents of protein and polysaccharide in SMPs and EPSs for the BBM and BM. The protein concentration decreased while polysaccharide content increased. In addition, the BBM system had a substantially greater polysaccharide content than the BM system, with a maximum concentration of almost 30 mg/L (Figure 4). Research has demonstrated that SMPs may indicate the activity of the denitrification bacteria in metabolism. The decrease in SMPs indicates a decline in the metabolic activity of denitrification bacteria [40]. This demonstrates that the BBM system outperformed the BM system in terms of of nitrogen removal and denitrification ability. This might also be related to the formation of anaerobic areas inside the bacterial–microbial system attached to the biocarriers. The results show a tendency of first increasing, then decreasing, and then increasing for the EPS concentration in the two systems. The protein and polysaccharide contents were more than 300 mg/L at day 9. Compared to the BM system, the BBM system features an increased EPS content, primarily a higher polysaccharide content. In the bacterial–microbial consortium system, bacteria might find additional growth sites and sticky parts, while microalgae were utilized as a biocarrier [28]. Bacterial-produced materials were more inclined to become attached to microalgae and form EPSs. One characteristic of biocarriers was a large specific surface area, which can adsorb more bacteria and microalgae. This could be a consequence of the BBM system having a higher EPS content than that of the BM system.

In the BBM system, microorganisms produced EPSs as a metabolic byproduct. They were frequently utilized as an adhesive to unite microbial cells to form functional microbial communities. The change in the EPS content indicated a shift in the composition of the microbial community in the reactor. During the operation of the reactor, the fluctuation of EPS content and PN/PS ratio in the BBM system was more obvious than that in the BM system. Bacteria and microalgae must secrete more EPSs to resist these changes than before, and protected bacteria from hydraulic shear forces and toxic chemicals. This was an ordinary method whereby bacteria and microalgae defend themselves when under stress from the outside world. Qiu et al. also reported that biomass resists changes in conditions inside bioreactors by secreting more EPSs [41]. The stability of biofilms was significantly influenced by EPSs. Bacteria and biocarriers might connect through the assistance of EPSs. The addition of biocarriers regulated the hydraulic flow conditions inside the system. For the elimination of phosphate and nitrogen, a stable ecosystem formed by the biofilm on the biocarriers was crucial.

3.4. Fluorescence Excitation–Emission Matrix Analysis of System

Three-dimensional EEM spectroscopy was performed to characterize the components of SMPs and EPSs in the bacteria and microbial substances extracted from the two systems. The substance status of SMPs and EPSs in the BBM and BM systems at the end of the reaction had been shown through the EEM, as demonstrated in Figure 5. The SMP peaks were similar in the two systems. Peak A (Ex = 350~375 nm, Em = 415~435 nm) was humic acid. Tryptophan protein substance was discovered at Peak B (Ex = 265~285 nm, Em = 310~330 nm). Peak C (Ex = 260~275 nm, Em = 430~450 nm) was marine humic acid [17]. The little change in the peak positions of the two systems suggests their structure is similar. However, the peak intensity of SMPs in the BBM sample was high. This was consistent with the findings of the SMP assessment of content in the two systems. From the variation in the peak strength of the substances in the two systems, humic acid content was high in SMPs. Humic acid, as a kind of polyelectrolyte complex, was an important substance in activated-sludge EPSs. The strong hydrophobicity made it easy to attach to the biocarriers. This might also be the reason for the high content of humic acid in the BBM system. Two typical fluorescence peaks were detected in EPS samples. Peak B (Ex = 265~285 nm, Em = 310~330 nm) was the tryptophan protein substance. Peak D (Ex = 220~235 nm, Em = 300~320 nm) was aromatic protein. In fact, these two peaks were identified as corresponding to protein-like substances. Previous studies had shown that aromatic proteins can induce microbial aggregation through hydrophobicity. The peak strength of the BBM was obviously higher than that of the BM, which had a better bioflocculation capability [42]. Consequently, the addition of biocarriers accelerated bacterial–microbial floc formation, demonstrating a satisfying mariculture wastewater treatment.

3.5. Microbial Community Composition

The microbial community constitution evolution between BBM and BM is depicted in Figure 6. Figure 6a displays the distribution of dominant bacterial community shifts at the phylum level. The addition of biocarriers resulted in a significant shift in the microbial community constitution. In the BBM, Proteobacteria (51%) was the dominant bacteria, and Bacteroidota (73%) and Chloroflexi (63%) were the predominant bacteria in the BM. Actinobacteriota maintained the absolute advantage in the raw sludge, reaching 83%. This demonstrated that Actinobacteriota abundance was greatly decreased and that the addition of C. pyrenoidosa had a significant influence on the primary bacterial community structure. Moreover, a huge accumulation of Proteobacteria was obtained in the BBM, which was about 14% higher than that in the BM. The structure of microbial communities within the microalgae system could be altered by biocarriers. The anoxic microenvironment formed in the biocarriers provided an excellent habitat for conventional heterotrophic denitrifying bacteria improving denitrification in the BBM. The previous study demonstrated that Proteobacteria were crucial for the degradation of organic matter and the removal of nutrients [43]. In addition, nitrite-oxidizing bacteria (NOB), NH₄⁺-N-oxidizing bacteria (AOB), denitrifying bacteria and other bacteria dedicated to nitrogen removal were all included in the category of Proteobacteria [44]. Furthermore, C. pyrenoidosa had an intense preference for Protobacteria presence, which may be extremely helpful in the growth and multiplication of these microorganisms [45]. This explained the reason for the increase in Proteobacteria predominance in the BBM and BM systems. During the culture process, the abundance of Actinobacteria in BBM and BM decreased from 83% to 5% and 12%, respectively, indicating that the current culture conditions were not conducive to Actinobacteria. It is possible that the inhibitory effect of C. pyrenoidosa on actinomycetes exacerbated this effect [46]. In general, the diversity of bacteria in the community composition was similar with what is usually found in wastewater treatment systems [47]. This could account for the excellent nutrient and organic matter removal efficiency in the microalgal–bacteria system.

The distribution of bacteria at the class and genus level is depicted in Figure 6b and Figure 6c, respectively. The research results indicate that the predominant classes of the BBM and BM were Alphaproteobacteria (57%, 27%), Bacteroidetes (26%, 73%) and Gammaproteobacteria (23%, 76%), respectively. Alphaproteobacteria occupied the highest proportion and played an important role in the removal of organic carbon and participated in various metabolic reactions in the system (Photosynthesis, NH₄⁺-N oxidation and nitrogen fixation) [48,49]. As an important denitrifying bacterium, Gammaproteobacteria ensured optimal denitrification performance in BM systems. Furthermore, it was frequently discovered that these bacteria predominate in the microbial community in traditional wastewater treatment systems. These bacteria secreted a large amount of SMP, which contributed to the formation of bacteria and microalgae floc, promoted the precipitation of bacteria and microalgae and improved the quality of effluent [50]. But according to our research, Gammaproteobacteria produced more SMPs in the BBM system, in spite of having a low abundance in the system. It might be due to the addition of biocarriers promoting the secretion of this substance. Furthermore, bacteria that could still survive in large quantities under conditions of high salinity include Gammaproteobacteria and Alphaproteobacteria [51]. The widespread presence under high-salinity conditions was one of the conditions that gave the bacteria the ability to remove nitrogen and phosphorus [52,53]. Rhodobacteraceae (66%) were aerobic denitrifying bacteria with a high NH₄⁺-N removal efficiency. This explained the reason why the system continued to have excellent nitrogen removal abilities despite severe salt stress [54]. Bacteroidia are heterotrophic bacteria that perform various kinds of metabolic functions in the process of the degradation of organic matter. However, in the biocarrier-enhanced reaction system, the abundance of these bacteria was changed dramatically. It might be that the addition of biocarriers was not conducive to the growth of some bacteria and had a negative impact on the species.

As illustrated in Figure 6c, the species at the genus level in the BBM and BM were Rhodobacteraceae (66%, 31%), Ruegeria (70%, 30%), Actinomarinales (28%, 72%) and Flavobacteriaceae (26%, 74%). The abundance of Rhodobacteraceae was significantly increased by the addition of biocarriers. The enrichment of Rhodobacteraceae accelerated the formation of biofilm [55]. Microlunatus was one of the reported phosphorus-accumulating bacteria. The addition of biocarriers increased the abundance of Microlunatus. In the BBM system, Microlunatus played a pivotal role in phosphorus removal. This conclusion had been confirmed by previous studies; that is, that the assimilation of microalgae was advantageous in the removal of phosphorus from wastewater [56]. Therefore, the addition of biocarriers resulted in a significant bacterial community shift, and the enrichment of the above functional bacteria enhanced the mariculture wastewater treatment and microalgal growth in the BBM.

3.6. Mass Balance Flow of Carbon and Nitrogen

In the BBM and BM system, bacteria and microalgae could develop a beneficial symbiotic interaction. It was possible to establish synthetic reactions between bacteria and microalgae with specified formulas [57]. Organic matter was oxidized to simple byproducts, while biomass was produced as waste products. The molecular formulas of bacteria and microalgae were CH_1.4N_0.2O_0.4P_0.017 and CH_1.78N_0.12O_0.36P_0.01, respectively. Prior research had demonstrated that bacteria and microalgae have a mass distribution of roughly 5:3, based on the balance of nitrogen [58]. Acetate was the carbon source for the bacterial assimilation processes in (1) and (2), respectively. The following formula illustrates the synthetic reaction of the bacteria and microalgae [57]:

Bacteria assimilate with acetate as a carbon source:

$$\mathrm{C}{\mathrm{H}}_3\mathrm{C}\mathrm{O}{\mathrm{O}}^{-}+0.88{\mathrm{O}}_2+0.22\mathrm{N}{\mathrm{H}}_4^{+}+0.019{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4^{-}+0.8{\mathrm{H}}^{+}=0.91\mathrm{C}{\mathrm{O}}_2+1.6{\mathrm{H}}_2\mathrm{O}+1.1\mathrm{C}{\mathrm{H}}_{1.4}{\mathrm{N}}_{0.2}{\mathrm{O}}_{0.4}{\mathrm{P}}_{0.017}$$

(1)

Microalgae assimilate with CO₂ as a carbon source:

$${\mathrm{CO}}_2+0.12{\mathrm{NH}}_4^{+}+0.01{\mathrm{H}}_2{\mathrm{P}\mathrm{O}}_4^{-}+0.69{\mathrm{H}}_2\mathrm{O}=1.19{\mathrm{O}}_2+0.11{\mathrm{H}}^{+}+{\mathrm{CH}}_{1.78}{\mathrm{N}}_{0.12}{\mathrm{O}}_{0.36}{\mathrm{P}}_{0.01 }$$

(2)

Consistent with previous studies, bacterial metabolism predominantly absorbed carbon and nitrogen contaminants [59]. Furthermore, certain studies had estimated that the growth proportion of bacterial–microbial systems is 0.7 g/g COD of volatile suspended solids (VSSs) [60]. It demonstrated that the system can convert approximately 70 percent of COD to VSSs. Furthermore, 10% to 20% of the organic carbon was released from throughout the system with the wastewater. Bacterial digestion converted about 20–30% of organic carbon to carbon dioxide [61]. About 30–40% of the removed nitrogen could be biodegraded through processes including nitrification and denitrification to produce nitrate, nitrite and nitrogen under the influence of light. The extra nitrogen was subsequently transformed through biological assimilation to biomass [62,63]. As shown in

Table 1. Mass balance of carbon and nitrogen in BBM and BM.

Organic Matter	BBM	BM
Influent Carbon	100	100
Influent Nitrogen	100	100
Effluent C	11.38	13.34
Effluent N	20.67	29.50

, bacteria and biocarriers were closely combined to form an anaerobic area for denitrification and conversion into nitrogen in the BBM system. The nitrogen removal performance was improved (79.33%). The increased abundance of Proteobacteria in the system also further supports this opinion. The addition of biocarriers also changed the microbial environment in the system and increased the abundance of Alphaproteobacteria. It played an important role in the removal of organic carbon. In consequence, the BBM effluent had enhanced mariculture wastewater treatment efficiency and contained less nitrogen.

3.7. Impact of Mechanism of Biocarriers on Bacterial–Microbial Symbiosis for Pollutant Removal

Figure 7 outlines the major biotransformations of organic and inorganic nitrogen in the system. Bacteria played a major role due to their ability to remove nutrients and biotransforming pollutants. The addition of biocarriers could have provided anaerobic conditions for denitrification and have improved the efficiency of pollutant removal [64]. Nitrogen sources in the wastewater mainly existed in the form of inorganic nitrogen such as NH₄⁺-N, NO₃⁻-N and nitrite. They were transformed into each other through various ways, and finally discharged out of the system in the form of N₂ under anaerobic conditions formed by biocarriers. Bacteria and microalgae could both eliminate NH₄⁺-N from the body through metabolism. NH₄⁺-N could penetrate the cell membrane and enter the intracellular environment of microalgal cells. It was further transformed into an important component of proteins and nucleic acids. However, excess NH₄⁺-N would promote the release of free NH₄⁺-N [65]. This could have a negative effect on the photosynthesis process of microalgae cells. The nitrogen-functional bacteria in the system had NH₄⁺-N-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria (AMX) and denitrifying bacteria (DNF). AOB oxidized NH₄⁺-N to NO₂⁻-N during the aerobic phase of the system. NOB converted some of the NO₂⁻-N to NO₃⁻-N. Nitrogen denitrification could be performed because biocarriers provide anoxic zones. Compared with the BM, denitrification was easy to achieve in the BBM system, and it improved the efficiency of nitrogen removal. DNF denitrified NO₃⁻-N to NO₂⁻-N, and AMX subsequently transformed it to N₂. Furthermore, AOB oxidized nitrogen sources that were previously converted to NO₂⁻-N and which do not entirely reoxidize to NO₃⁻-N. Some of the NO₂⁻-N was directly denitrified to N₂ by AMX. Meanwhile, anaerobic ammoxidation could directly convert NH₄⁺-N into N₂ under anaerobic conditions provided by biocarriers. The process also involved several enzymatic reactions [66]. In addition, nitrifying bacteria and denitrifying bacteria could help microalgae alleviate NH₄⁺-N stress, which strengthened the synergistic relationship between bacteria and microalgae. Therefore, the bacterial–microbial system with the addition of biocarriers could improve the removal efficiency of pollutants.

4. Conclusions

The impact of the biocarrier on the BBM for mariculture wastewater treatment and the shift in the bacterial community was investigated. The mariculture wastewater treatment performance of the BBM was superior to that of the BM, with the highest removals for nitrogen (79.33%) and phosphorus (90.28%). The biocarrier provided an anoxia, contributing to the denitrification with a satisfying nitrogen removal. The regularity of the reaction system fluidization was boosted by biocarriers in the BBM, which benefited the accumulation of chlorophyll a and MLSS. Moreover, the BBM obtained the highest polysaccharide (413.39 mg/L) accumulation. The adhesion of polysaccharide accelerated the bacterial–microbial floc formation, improving the biological flocculation in the BBM. Additionally, biocarriers resulted in a significant bacterial community shift, and the enrichment of Rhodobacteraceae and Ruegeria promoted the pollutant degradation in the BBM. The novel BBM has potential for actual mariculture wastewater treatment. However, there is a lack of research on the treatment of mariculture wastewater by the BBM and BM at the genetic level. The mechanism of nitrogen and phosphorus degradation in high-salinity wastewater can be understood more clearly through the genetic analysis of the nitrogen and phosphorus transformation pathway. It provides a theoretical basis for the future practical application of the BBM.