**4. Discussion**

*4.1. TAN Removal Pathways in Different Systems*

4.1.1. Nitrification in Different Systems

In previous studies, apparent nitrification was observed in heterotrophic systems, where heterotrophic assimilation was promoted [7,33]. This agrees well with the findings of the present study, in which the nitrification rate was relatively high in the BFT treatments during early culture but was not significantly different from that in the CW treatment. Several water quality variables demonstrated nitrification in the BFT systems. NO2 −-N is the product of the first step in the nitrification process. The accumulation of NO2 −-N and a decrease in TAN were observed during the early days of culture, suggesting that the first nitrification step (NH4 <sup>+</sup> + 1.5 O2 → NO2 <sup>−</sup> + 2H<sup>+</sup> + H2O) progressed at a greater rate than the second step (NO2 <sup>−</sup> + 0.5 O2 → NO3 −). Schveitzer, et al. [33] made a similar observation in BFT systems and arrived at the same conclusion. A dramatic increase in NO3 −-N was observed from days 19 to 29, indicating complete oxidation of nitrite to nitrate during the period. Nitrite increased to a high level in the middle of the experiment and Emilie, et al. [34] also observed this trend after 20 d. However, nitrification in the BFT systems became inhibited as the experiment progressed. The nitrification rates in the BFT systems decreased and almost reached zero at the end of the study. Previous studies have demonstrated that heterotrophic bacteria efficiently compete with nitrifying bacteria for nutrients and space resulting in inhibited nitrification in *L. vannamei* and *Oreochromis mossambicus* culture systems [7,8,12].

#### 4.1.2. Heterotrophic Bacteria Assimilation

The heightened ammonia concentrations in BFT systems during week 1 of this study may indicate that heterotrophic bacterial communities require considerable time to develop. The water had not received added carbohydrates to stimulate the growth of heterotrophic bacteria before it was added to the culture tanks. The addition of carbohydrates began after the shrimp were placed into the tanks, and the bacterial community required time to become functional in the systems [6]. Gerardi [35] reported that the heterotrophic bacterial community takes 14 days to reach a stable stage. In this study, although the average TAN concentration was not significantly different among the treatments, the dominant TAN removal pathway may have been different. The low TAN concentration in the CW system was maintained by water exchange and nitrification. This was supported by the significantly higher nitrification rate in the CW system during the experiment period. TAN in the BFT systems was mainly removed through heterotrophic bacterial assimilation, and nitrification was greatly inhibited after 14 d.

#### *4.2. Microbial Composition*

Previous studies have shown that different carbon sources can affect the structure and function of bacterial communities in aquaculture systems [8,10,36,37]. In this study, Proteobacteria and Bacteroidetes were the dominant phyla of bacteria in all three systems. This finding agrees with Cardona, et al. [34] who reported that these two phyla represent more than 90% of the total bacteria present in BFT systems. Proteobacteria are widely dispersed in the aquatic environment and play an important role in nutrient cycling and the mineralization of organic compounds [34,38]. Bacteroidetes are an important part of the heterotrophic bacterial community of many water bodies [39]. Other phyla with different abundance among treatments, including Cyanobacteria, Planctomycetes, Chloroflexi, and Verrucomicrobia were found in *L. vannamei* culture systems [40]. Significant growth and proliferation of Cyanobacteria were observed in the P-BF system in this study, and this was reported by Miranda-Baeza, et al. [41] in BFT systems with added molasses. This phylum has developed a variety of ecological and physiological adaptation strategies to grow under poor conditions with high organic matter loads [41]. The phylum Planctomycetes, which was abundant in the M-BF system, performs anaerobic ammonium oxidation (i.e., oxidizing ammonium with nitrite as the electron acceptor to yield dinitrogen gas) to maintain good water quality [36,42].

*Leucothrix* was the most abundant genus in the M-BF system, and this was consistent with our previous studies [43]. One study reported that *Leucothrix* forms filaments and is usually found as an epiphyte on algae and invertebrates [43]. Species of this genus promote high mortality rates in shrimp [44,45], and this may be the reason for the decreased survival rate in the M-BF system. In addition, *Vibrio*, which are also bacterial pathogens to aquatic organisms, increased significantly in the CW system compared to the BFT systems [46,47]. Inhibition of a population of pathogenic *Vibrio* was reported in BFT systems [46].

#### *4.3. The Correlation between Carbon Sources, Microbial Communities and Environmental Factors*

According to the RDA, a close correlation was observed between bacterial composition and the water quality parameters. This result agrees with the findings of previous studies, which reported that bacterial community structure in the aquatic environment is affected by the abiotic environment [48]. Zhang, et al. [49] revealed that abiotic environmental factors, such as TAN and TN, have large impacts on bacterial populations in *L. vannamei* culture ponds, and Li, et al. [50] reported that TAN and Chl strongly affect the bacterial composition in the sediment of intertidal regions used for mariculture. Many of the environmental factors changed in the culture systems after adding the carbon sources. (Table 1). Different bacteria perform different ecological functions and the changing ecology and microbiota co-affected shrimp culture [51].

The changes in the functional profiles involved in the bacterial community provide valuable information from a functional perspective [31]. FAPROTAX is a gene functional annotation tool based on 16S rRNA sequencing that is appropriate for environmental samples. The results of the functional predictions indicated that biofilms formation and stress tolerance increased in the M-BF system. Biofilm contributes to the metabolism of the nitrogenous compounds generated within biofloc culture systems [52]. This finding indicates that adding molasses changed the environment to a more stressful condition but improved nitrogen metabolism in another aspect. The functional comparison using BugBase showed that adding PHBV produced a less stressful environment for bacteria compared to that of molasses. Our previous study also showed that shrimp are subject to be stressed in BFT systems where molasses serves as the carbon source other than PHBV [53].

### *4.4. Practical Value*

Carbon sources are often applied in aquaculture systems to maintain a high C/N ratio and control the concentrations of nitrogenous metabolites in the system [54]. The carbon sources used are often by-products of the food processing and animal feed industries that are inexpensive and locally available [54]. Water exchange is greatly reduced in those systems to favor heterotrophic nitrogen assimilation pathways [33,55–57]. In the present study, the water exchange rate in the BFT systems was only one-fifth of that applied to the CW system, but the average TAN concentrations were not significantly different among the three systems. This agrees with the findings of Hargreaves [12] and Avnimelech [58], who demonstrated the usefulness of BFT in reducing water use while maintaining adequate water quality.

The growth rate of shrimp was better in the BFT systems than in the CW system, suggesting the benefits of BFT systems for improving the growth rate, decreasing the FCR of shrimp, and reducing feed costs. Avnimelech [59] also observed that the growth rate of overwintering tilapia fingerlings in BFT systems was significantly higher than in the control. Shrimp may utilize bioflocs as a source of protein and additional feed for growth [4,60]. The mechanism for the promoting effect of biofloc on the growth of aquatic animals is unclear. Some researchers have proposed that biofloc contains fungal proteins, and their amino acids facilitate absorption by aquatic animals. In addition, biofloc is a rich source of proteins and lipids continuously available in situ for consumption by shrimp [54].

Water-soluble carbon sources, such as molasses, must be added many times to a culture system, which increases the difficulty of system management. Such carbon sources are also limited by excessive DOC and color problems [61]. In the present study, water from the M-BF system was more turbid than that from the CW and P-BF systems. The biofloc in the P-BF system was almost in the PVC tube, which caused low biofloc volume in this system. Moreover, PHBV was added as a carbon source only once, so this was not a major management effort. Boley, et al. [61] demonstrated that solid carbon sources are better alternatives to water-soluble carbon sources because they reduce supervision and management efforts. Adding PHBV had a similar function for improving water quality and producing less stress compared with the molasses-added treatments. Taken together, our results suggest that insoluble biodegradable polymers could be good alternatives for improving water quality and promoting production in intensive aquaculture systems.

#### **5. Conclusions**

The carbon sources-added group maintained water quality with reduced water exchange compared to the control. The dominant ammonia removal pathways changed after adding a carbon source, where the TAN concentration was mainly removed through heterotrophic bacterial assimilation, and nitrification was greatly inhibited after day 14. Moreover, this study confirmed the effects of adding carbon sources on bacterial community composition in culture tanks, and *Leucothrix* was closely related to molasses treated systems. Results from the present study also indicated that the environment of the molasses-added systems tended to be more stressful to shrimp as the relative abundance of several functions, such as biofilms formation and stress tolerance increased in the treatment. The relatively better shrimp performance and less stressful culture environment in the PHBV-added systems suggest that PHBV may be a better alternative as a carbon source in BFT systems.

**Author Contributions:** Conceptualization, methodology, and writing—original draft preparation, L.L. and Y.X.; writing—review and editing, L.L.; visualization, Y.X.; data curation, project administration, L.L., S.D. and X.T.; supervision, L.L., S.D., Q.G. and X.T.; data curation, project administration, Y.X.; funding acquisition, L.L., X.T. and S.D.; investigation, Y.X.; validation and resources, L.L. and X.T.; formal analysis: Y.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key R&D Program of China (No. 2020YFD0900200 and No. 2017YFE0122100) and the Natural Science Foundation of Shandong Province, China (ZR2020MC194).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data from this study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
