*3.3. The Microbial Diversity, Community Structure, and Function*

#### 3.3.1. Microbial Diversity

The bacterial diversity indices are shown in Table 2. The coverage indices of all groups were above 0.99, indicating that the sequencing data were adequate to represent the bacterial community structure. The Shannon, Simpson, Chao, and Ace indices of bacterial communities of all groups did not show significant differences, indicating that the addition of probiotics and carbon sources did not change the abundance and diversity of bacterial communities in the culture system.

As shown in Figure 4, the similarity analysis among groups using partial least squares discriminant analysis (PLS-DA) showed that the samples of group BP were separated from those of groups BM, DZ, and B along the COMP1 axis. Meanwhile, the samples of group B were separated from those of groups BM and DZ along the COMP2 axis. In addition, the samples within the same group clustered together while the samples from different groups moved away from each other.


**Table 2.** α-diversity indices of bacterial communities in shrimp culture systems.

Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition. Data are expressed as mean ± standard error, *n* = 3.

**Figure 4.** PLS-DA analysis based on OTU level of different *P. vannamei* culture systems. Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP−171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition.

## 3.3.2. Bacterial Community Composition

There were 501, 605, 626, and 565 OTUs in the DZ, B, BP, and BM groups, respectively (Figure 5). In addition, the unique OTUs in the DZ, B, BP, and BM groups were 34, 86, 144, and 66, respectively. The group with the lowest number of specific OTUs was the control group, while the most were found in the BP group.

The predominant phyla were *Proteobacteria*, *Bacteroidetes*, *Actinobacteria*, and *Verrucomicrobia* (Figure 6A). The relative abundance of the phylum *Bacteroidetes* was significantly lower in the BP group than in the control group (*p* < 0.05). However, the relative abundance of the phylum *Proteobacteria* was distinctly higher compared with the control (*p* < 0.05). In addition, the relative abundance of the phylum *Verrucomicrobia* in the B group was observably higher than that in the other groups (*p* < 0.05).

Additionally, the classes *Alphaproteobacteria*, *Flavobacteriia*, *Actinobacteria*, and *Verrucomicrobiae* dominated each group (Figure 6B). The relative abundance of *Flavobacteria* was significantly lower in the BP group than in the other groups (*p* < 0.05). Compared to the control, the relative abundance of *Alphaproteobacteria* was significantly increased in the BP group (*p* < 0.05). Besides, the relative abundance of *Verrucomicrobiae* was remarkably higher in the B group than in the other groups (*p* < 0.05).

**Figure 5.** Venn diagram based on OTU level for different *P. vannamei* culture systems. Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition.

**Figure 6.** Bacterial composition at different levels of phylum (**A**), class (**B**), order (**C**), family (**D**), and genus level (**E**) in different culture systems. Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition.

At the order level, *Rhodobacterales*, *Flavobacteriales*, *Propionibacteriales*, *Rhizobiales*, and *Verrucomicrobiales* were the dominant bacterial taxa in each treatment (Figure 6C). The relative abundance of *Flavobacteriales* was significantly lower in the BP group than in the other groups (*p* < 0.05). In contrast, the abundance of *Rhizobiales* and *Rhodobacterales*, which belong to the bacterial taxonomic class *Alphaproteobacteria* of the phylum *Proteobacteria*, was significantly increased in the BP group compared with the control (*p* < 0.05). In addition, the relative abundance of *Verrucomicrobiales* was prominently higher in the B group than in the other groups (*p* < 0.05).

At the family level, *Rhodobacteraceae*, *Flavobacteriaceae*, *Propionibacteriaceae*, *Phyllobacteriaceae*, and *Verrucomicrobiaceae* were relatively abundant and dominant in each treatment (Figure 6D). The relative abundance of *Flavobacteriaceae* in the BP group was notably lower than that in the other groups (*p* < 0.05), while the abundance of *Phyllobacteriaceae* and *Rhodobacteraceae* in the BP group was significantly higher than that in the control group (*p* < 0.05). Furthermore, *Verrucomicrobiaceae* in the B group was more abundant than in the other groups (*p* < 0.05).

At the genus level, the unclassified *Rhodobacteraceae*, *Donghicola*, *Ruegeria*, *Tessaracoccus*, *Oricola*, and *Marivita* dominated the B, BP, and BM groups (Figure 6E). As depicted in Figure 7, the relative abundance of *Oricola* spp., *Donghicola* spp., and *Marivita* spp. was significantly higher in the BP group than in the other groups (*p* < 0.05). Compared with the control, the relative abundance of *Bacillus* spp. in groups B, BP, and BM was significantly increased, and the relative abundance of *Bacillus* spp. in the BP group was significantly higher than that in groups B and BM (*p* < 0.05).

**Figure 7.** The relative abundance of *Bacillus* spp. (**A**), *Donghicola* spp. (**B**), *Marivita* spp. (**C**) and *Oricola* spp. (**D**) in different groups. Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition. Values with different superscripts are significantly different among treatments in each figure (*p* < 0.05).

3.3.3. The Correlation of Water Quality Indexes and Microbial Community Structure

Redundancy analysis (RDA) revealed the effects of environmental variables (TAN, NO2 −-N, NO3 −-N, TN, SRP, TP, DOC, and TOC) on microbial community structure. It can be seen that TAN, NO2 −-N, NO3 −-N, and TN had strong positive impacts on the

distribution of flora at the phylum level on the first typical axis, and the above four environmental factors had synergistic effects on community structure (Figure 8A). In contrast, DOC and TOC had strong negative effects on the distribution of flora at the phylum level on the first typical axis, and the two environmental factors had synergistic effects on community structure. In addition, nitrate nitrogen was the most influential variable that had a significant effect on bacterial community structure (r<sup>2</sup> = 0.538, *p* = 0.043).

**Figure 8.** The Redundancy Analysis on phylum (**A**) and genus (**B**) level and the correlation heatmap analysis between bacterial taxa and water quality parameters on phylum (**C**) and genus (**D**). Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition.

Similar to the phylum level, TAN, NO2 −-N, NO3 −-N, and TN had strong positive effects on the distribution of flora at the genus level on the second typical axis, and the four environmental factors had synergistic effects on community structure (Figure 8B). In addition, DOC and TOC had strong negative effects on the distribution of flora at the genus level on the second typical axis, and the two environmental factors also had synergistic effects on community structure. Moreover, nitrate nitrogen had the greatest effect on bacterial community structure at the genus level (r<sup>2</sup> = 0.409, *p* = 0.038).

The correlation between environmental variables and bacteria was explored by calculating Spearman coefficients of water quality factors and bacterial taxa at phylum and genus levels, respectively. At the phylum level, *Proteobacteria* showed a significant positive correlation with TOC (*p* < 0.05), while it showed a significant negative correlation with nitrate nitrogen and total nitrogen, respectively (*p* < 0.05) (Figure 8C). In addition, the phylum *Firmicutes* was significantly negatively correlated with ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total nitrogen, respectively (*p* < 0.05). Besides, the phylum Bacteroidetes had a significant negative correlation with TOC (*p* < 0.05) but had a significant positive correlation with total nitrogen (*p* < 0.05).

At the genus level, *Bacillus* spp. showed a significant positive correlation (*p* < 0.05) with DOC and TOC but had a significant negative correlation with nitrate nitrogen and total nitrogen (*p* < 0.05) (Figure 8D). Additionally, *Oricola* spp. was positively correlated with TOC (*p* < 0.05) while negatively correlated with total nitrogen (*p* < 0.05). The *unclassified\_f\_\_Flavobacteriaceae* spp. and *Demequina* spp. both had a significant negative correlation with TOC, while they had a significant positive correlation with nitrate nitrogen and total nitrogen (*p* < 0.05).

#### 3.3.4. Ecological Network Analysis

Ecological network analysis showed that there were more nodes in groups B, BP, and BM than in the control group (Table 3). However, no significant difference was found in negative or positive interactions among different groups. There were more functional modules in the groups to which carbon sources were added (BP and BM) than in the control and B groups, with the highest number of modules in the BP group (Figure 9). The number of functional modules in the B, BP, BM and DZ groups was 7, 26, 16, and 7, respectively.

**Table 3.** Topological properties of the networks.


Note: group B, a single *B. pumilus* BP-171, was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition. Data are expressed as mean ± standard error, *n* = 3.

**Figure 9.** Ecological Network based on OTUs of the bacterial community in the different culture systems. Note: In the ecological network diagram, different nodes represent bacteria from different OTUs, and the line between two nodes indicates that there is some interaction between bacteria from two OTUs, and the red line represents a positive relationship between bacteria from two OTUs, the green line represents a negative relationship. The group B, a single *B. pumilus* BP-171 was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition.

#### 3.3.5. Predictive Functions of Microbiota in the Water

As shown in Figure 10, compared to the control, eight functional modules (Cationic antimicrobial peptide resistance, photosystem II, coenzyme M biosynthesis, bacilysin biosynthesis, kanosamine biosynthesis, cytochrome aa3-600 menaquinol oxidase, photosystem I and lysine biosynthesis) were significantly improved in the B group (*p* < 0.05). In the BP group, 12 functional modules (ketone body biosynthesis, pentose phosphate pathway, ethylmalonyl pathway, entner-Doudoroff pathway, hydroxypropionate-hydroxybutylate cycle, urea cycle, cobalamin biosynthesis, D-Galacturonate degradation, purine degradation, tyrosine biosynthesis, and molybdenum cofactor biosynthesis and nicotinate degradation) were significantly higher than the control (*p* < 0.05). However, 19 functional modules (tetrahydrobiopterin biosynthesis, C4-dicarboxylic acid cycle, nodulation, N-glycan precursor biosynthesis, tetrahydrofolate biosynthesis, threonine biosynthesis, biotin biosynthesis, biotin biosynthesis of BioW pathway, methionine degradation, assimilatory sulfate reduction, abscisic acid biosynthesis, tetrahydrofolate biosynthesis, ascorbate biosynthesis, dTDP-L-rhamnose biosynthesis, pyrimidine ribonucleotide biosynthesis, and coenzyme A biosynthesis) were significantly decreased (*p* < 0.05). In the BM group, three functional modules (coenzyme M biosynthesis, N-glycan biosynthesis, and assimilatory nitrate reduction) were enriched (*p* < 0.05), whereas one functional module (D-Glucuronate degradation) was significantly decreased (*p* < 0.05).

**Figure 10.** Differences of predicted functions based on the KEGG Module database using STAMP. Note: Only data with significant differences (*p* < 0.05) between groups are shown. (**A**) DZ and B group, (**B**) DZ and BP group, (**C**) DZ and BM group. The group B, a single *B. pumilus* BP-171 was added; the group BP, *B. pumilus* BP-171, and PHBV were added; the group BM, *B. pumilus* BP-171, and molasses were added; the group DZ, the control without any probiotics and carbon sources addition.

#### **4. Discussion**

#### *4.1. Effects of Addition of B. pumilus 171 and Carbon Sources on the Growth Performance of Shrimp*

Many previous studies have shown that the addition of probiotics to culture water can promote the growth of aquatic animals by improving FER and regulating the balance of aquatic flora, and reducing toxic substances such as ammonia and nitrite [8,9,32,33]. The results from the present study were consistent with the above findings. Higher survival rate and gross weight of shrimp were observed in the group with the single addition of *B. pumilus* BP-171 compared to the control. In addition to probiotics, carbon sources with the property of improving growth performance were also observed [34–39]. In this study, the shrimp in the group to which both *B. pumilus* BP-171 and carbon sources were added showed better growth performance than the group to which only one probiotic was added, and the group using PHBV as a carbon source showed the best performance. The above results suggest that *B. pumilus* BP-171 promoted the growth performance of shrimp, and a suitable carbon source such as PHBV could further enhance the growth-promoting function of probiotics.

#### *4.2. Effects of the Addition of B. pumilus 171 and Carbon Sources on the Water Quality*

Some probiotics, such as *Bacillus* spp. could be used to improve water quality by reducing the concentration of ammonia and nitrite in the culture system [7–9]. For example, Lee et al. [40] found that total NH4 <sup>+</sup> concentration was significantly lower when *Bacillus* spp. were added to the culture system. Decreased nitrite concentration was observed when *B. subtilis* FY99-01 was used in the culture system of *P. vannamei* [32]. Barman et al. [23] also found that *B. cereus* PB45 could consume nitrite in the culture pond effluent. The addition of carbon sources could also promote the removal of nitrogen from water by increasing the C/N ratio [2,16,17]. However, to our knowledge, the effects of combining probiotics and carbon sources on aquaculture water quality have rarely been reported. In this study, *B. pumilus* BP-171, a heterotrophic nitrifying-aerobic denitrifying strain isolated from shrimp ponds [29], was tested in a shrimp culture system. This strain can not only convert ammonia nitrogen into bacterial biomass by heterotrophic assimilation but also convert nitrite and nitrate nitrogen into gaseous nitrogen by denitrification [29]. This study showed that the concentration of ammonia nitrogen was reduced by more than 60% in the B and BP groups compared to the control, while the concentration of nitrite nitrogen was reduced by more than 69% in the B and BP groups. The removal rates of ammonia and nitrite nitrogen in the B group reached 70.22% and 76.88%, respectively. The average concentrations of nitrate nitrogen in the B and BP groups were reduced by more than 17%, while the concentrations of total nitrogen in the B and BP groups were reduced by more than 35%. The nitrate and total nitrogen removal rates in group BP were 26.24% and 40.02%, respectively. In conclusion, the addition of *B. pumilus* BP-171 alone could reduce the concentrations of ammonia and nitrite in the culture system, while the simultaneous addition of *B. pumilus* BP-171 and PHBV could reduce the concentrations of nitrate and total nitrogen. In addition, PHBV was better than molasses both as a solid carbon source and as a biofilm carrier when used together with *B. pumilus* BP-171.

#### *4.3. Effects of Addition of B. pumilus 171 and Carbon Sources on the Microbial Diversity and Microbiota Compositions in Water*

*Bacillus* spp. is a type of common probiotic used as a water quality improver in aquaculture systems [7–9,41]. BP-171 is a strain of heterotrophic nitrifying-aerobic denitrifying bacteria isolated from shrimp environments with high nitrogen removal capacity [29]. In this study, a single *B. pumilus* BP-171 and various combinations of *B. pumilus* BP-171 with PHBV and molasses were added to the shrimp culture system. Interestingly, no significant difference in the Ace, Chao, Shannon, and Simpson indices of microbiota was observed between the groups in this study, which was in agreement with the results of Kokkuar et al. [3]. However, the addition of *B. pumilus* BP-171 and carbon sources altered

the microbiota composition in the water. The number of OTUs and unique OTUs in the B, BP, and BM groups were all higher than that in the control, and that in the BP group was the highest. In addition, the microbial composition at various taxonomic levels differed distinctly in the different groups.

The abundance of the phylum *Verrucomicrobia*, the class *Verrucomicrobiae*, the order *Verrucomicrobiales*, and the family *Verrucomicrobiaceae* was significantly higher in group B, to which only *B. pumilus* BP-171 was added, than in the other groups. Although the role of *Verrucomicrobia* in aquaculture has rarely been reported, some studies have shown that they were widely distributed in drinking water, freshwater lakes, and marine sediments [42]. In addition, some *Verrucomicrobia* taxa isolated from seawater have been shown to be strictly aerobic chemoheterotrophs that use mono- or disaccharides as carbon and energy sources and can convert nitrate nitrogen to nitrite nitrogen [43,44]. Besides, some *Verrucomicrobia* taxa, which can utilize a variety of organic and inorganic gas molecules such as methane, carbon dioxide, ammonia, and nitrogen gas, were involved in the natural carbon and nitrogen cycles [45]. Thus, *Verrucomicrobia* might involve in the conversion of nitrogen in the culture system, leading to the low concentration of ammonia and nitrite nitrogen in the B group.

The relative abundance of the phylum *Proteobacteria* in the BP group was significantly increased compared with the control when *B. pumilus* BP-171 and PHBV were added simultaneously. Previous studies have shown that the phylum *Proteobacteria* was widely distributed in various regions of the marine environments [46], and many microorganisms involved in nitrogen removal belong to this phylum, including nitrifying and denitrifying bacteria [47]. In this study, it was demonstrated that the abundance of phylum *Proteobacteria* had a negative correlation with the concentration of nitrate and total nitrogen, which might be one origin of a lower concentration of nitrate and total nitrogen in the BP group. In addition, the relative abundance of the class *Alphaproteobacteria* in the BP group reached 75.01%, which was strikingly higher than in other groups. *Alphaproteobacteria* have been shown to have excellent denitrification ability [47,48]. Moreover, the abundance of several dominant bacteria of various taxonomic levels belonging to the class *Alphaproteobacteria* was significantly higher in the BP group than in the control. For example, the order *Rhodobacterales* and *Rhizobiales*, the family *Erythrobacteriaceae* and *Rhizobacteriaceae*, and the genus *Donghicola*, *Oricola*, and *Marivita* as well as *Bacillus*, whose relative abundance was significantly higher in the BP group than in the control. *Rhodobacterales* is considered to be the most abundant denitrifying bacterium widely distributed in the environment [49]. Hu et al. [50] found that the family *Rhodobacteraceae*, as one of the core taxa in shrimp culture ponds, removed nitrite nitrogen from the system mainly by denitrification. Besides, the genus taxa of *Donghicola* and *Marivita*, which were isolated from seawater and are both Gram-negative and aerobic, belong to the class *Rhodobacteraceae*, but their role in bacterial communities has hardly been studied [51]. The order *Rhizobiales*, a type of heterotrophic bacteria with denitrification character, was found to be the second most abundant functional bacterium in ammonia-oxidizing anaerobic systems with a relative abundance of 18.2% [52]. The family *Phyllobacteriaceae* was a group of aerobic bacteria that can utilize various forms of nitrogen for reproduction and was found in marine environments [53,54]. In addition, Zheng et al. [55] identified the most abundant transporter proteins involved in the transport and uptake of carbohydrates from a strain of *Oricola* sp. based on metagenomic and metaproteomic analysis. Among them, three proteins involved in ammonia assimilation and a large number of genes involved in the uptake and metabolism of inorganic nitrogen were also observed in this strain [55]. These results suggest that *Oricola* sp. might be able to utilize carbon sources in the environment and participate in the conversion and removal of nitrogen.

Many studies have shown that *Bacillus* plays an important role in nitrogen cycling via nitrification [56] and denitrification [57]. *B. pumilus* BP-171 was periodically added into different shrimp culture systems in this study. Although an increase in the relative abundance of *Bacillus* spp. compared to other taxa in the microbial community was not observed, the relative abundance of *Bacillus* spp. in groups B, BP, and BM was significantly higher than in the control. Of course, due to methodological limitations, it could not be determined whether the *Bacillus* spp. was the strain BP-171. The results of the present study showed that the microbial composition shifted distinctly at different taxonomic levels when *B. pumilus* BP-171 and different combinations of the strain BP-171 with PHBV and molasses were added. It was also found that the relative abundance of *Oricola* spp. was positively correlated with TOC concentration, while the relative abundance of *Bacillus* spp. was positively correlated with the concentration of TOC and DOC, indicating that the addition of carbon source promoted the proliferation of *Oricola* spp. and *Bacillus* spp. Furthermore, the relative abundance of *Bacillus* spp. showed a significant negative correlation between the concentration of nitrate nitrogen and total nitrogen, while the relative abundance of *Oricola* spp. showed a significant negative correlation with total nitrogen concentration, suggesting an increase in the abundance of *Bacillus* spp. and *Oricola* spp. promoted the conversion and removal of nitrogen. The above correlations between the relative abundance of bacteria, the concentration of TOC and DOC, and the concentration of nitrate and total nitrogen might partially explain the higher removal rate of nitrate nitrogen and total nitrogen in the BP group.

#### *4.4. Effects of Addition of B. pumilus 171 and Carbon Sources on the Ecological Network and Function of the Microbial Community*

The complicated ecological network consisted of negative interactions and positive interactions of interspecies in the bacterial community, which sustained the stability of the bacterial ecosystem in water [58,59]. In general, the cooperative network involving mutualism or synergy bacteria can be efficient but not stable [60]. Negative interactions such as competition can weaken the efficiency of the cooperating network but enhance its stability [60]. In this study, a higher ratio of negative to positive interactions was observed in the ecological network of the BP group, suggesting that the addition of PHBV might strengthen the stability of the shrimp culture ecosystem. Moreover, each module was considered a functional unit, performing an identifiable task [58,61]. In the present study, the largest number of modules was observed in the BP group, which indicated that the addition of PHBV could alter the bacteria in the water to perform more biological functions.

The functional modules in the KEGG Module database represent cellular and organismal level functions, and these modules generally contain various molecular level functions stored in the KO (KEGG Orthology) database [62]. The bacterial community in water can affect the growth of aquatic animals in various ways, such as the inhibition of pathogenic bacteria and the secretion of nutrients [9,11,13,15]. The antimicrobial effect of organic acids has been demonstrated [63–65]. Hydroxybutyrate can exert its inhibitory effect against pathogenic *Vibrio* bacteria [66,67]. In this study, the function prediction analysis showed that 8, 12, and 3 functional modules were significantly enhanced in groups B, BP, and BM, respectively. The hydroxypropionate-hydroxybutylate cycle, ethylmalonyl pathway, and the metabolic activity of organic acids, such as fumarate, were significantly enhanced in the BP group. In addition, previous studies have shown that urea dissolved free amino acids, as well as inorganic nitrogen together sustained the nitrogen demand of bacteria for growth in natural water [68,69]. In the present study, the urea cycle, metabolic activities of nutrient substances such as tyrosine, pyruvate, glyceraldehyde-3P, ribose 5P, and cobalamin, as well as molybdenum cofactor were also remarkably enhanced. Just as previous research has shown that many invertebrates like shrimps have demonstrated the ability to take up a variety of organic compounds, including amino acids, even against the concentration gradient [70–74]. Therefore, the overall promotion of numerous metabolic functions of the microbial community in the water might be partially responsible for the improvement in shrimp growth performance.

#### **5. Conclusions**

In summary, probiotics and various combinations of probiotics with different carbon resources had differential impacts on the growth performance of shrimp, water quality, and bacterial community in the *P. vannamei* culture system. The addition of BP-171 and carbon sources could promote the growth of shrimp to varying degrees and improve the yield of farmed shrimp, with the best in the group of simultaneous addition of BP-171 and PHBV. The single addition of BP-171 could effectively reduce the concentration of ammonia and nitrite nitrogen in the culture system, and the simultaneous addition of BP-171 and PHBV could effectively improve the removal rates of nitrate and total nitrogen. In addition, the addition of BP-171 and carbon sources did not change the abundance and diversity of the bacterial community in the shrimp culture system but altered the structure and function of the bacterial community and enhanced the stability of the community's ecological network.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w14244037/s1, Table S1: Composition of the basic feed; Figure S1: Analysis of rarefaction curves.

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

**Funding:** This study was funded by the National Key Research and Development Program of China (2020YFD0900201, 2019YFD0900403), and the Joint Fund of Think Tank for Biomanufacturing Industry of Qingdao (QDSWZK202111).

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

**Acknowledgments:** We thank all the students whom participated the field works and laboratory analysis.

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

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