**1. Introduction**

*Penaeus vannamei* has become the most dominant species in shrimp farming worldwide [1]. The high demand in the market has led to the expansion of shrimp production and the widespread availability of high stocking density culture patterns. However, with the increasing intensification of shrimp farming, feed residues and shrimp metabolic products led to the accumulation of nitrogen pollutants, including ammonia, nitrite and nitrate nitrogen, in cultured water [2,3]. This problem is of increasing concern in intensive farming practices. The continued increase of nitrogen pollution in culture systems not only leads to

**Citation:** Wang, M.; Liu, Y.; Luo, K.; Li, T.; Liu, Q.; Tian, X. Effects of *Bacillus pumilus* BP-171 and Carbon Sources on the Growth Performance of Shrimp, Water Quality and Bacterial Community in *Penaeus vannamei* Culture System. *Water* **2022**, *14*, 4037. https://doi.org/10.3390/ w14244037

Academic Editor: Christophe Piscart

Received: 30 October 2022 Accepted: 8 December 2022 Published: 10 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the deterioration of water quality in the culture system but also affects the normal physiological functions and immune performance of shrimp and eventually possibly causes frequent diseases in shrimp culture [4–6]. Therefore, it is urgent to develop healthy farming modes and good water quality management technologies for the sustainable development of aquaculture.

Previous studies have shown that the concentration of harmful nitrogen such as ammonia, nitrite and nitrate in culture water could be effectively controlled by adding appropriate probiotics [7–9], ultimately reducing the morbidity of farmed species and increasing the production of farmed animals [10–12]. Probiotics can not only reduce the concentration of hazardous nitrogenous substances accumulated in water through the process of nitrification, denitrification and assimilation but also inhibit the growth of pathogenic microorganisms by competing for physical space and nutrients and secreting bacteriocins and lysozyme [13–15], and thus are widely used in aquaculture.

The addition of a carbon source at an appropriate dosage to the culture water can increase the carbon-to-nitrogen ratio (C/N ratio) and promote the proliferation of heterotrophic bacteria so that ammonia, nitrite and nitrate nitrogen can be removed by the assimilation and denitrification of heterotrophic bacteria [2,16,17]. Molasses is currently one of the most commonly used carbon sources in shrimp aquaculture [18]. However, molasses as an added carbon source is highly soluble and can lead to the rapid growth of heterotrophs and consumption of oxygen, which can easily lead to a rapid decrease in dissolved oxygen, affect the stability of water quality, and eventually threaten the survival of aquatic animals [18,19]. For example, Pérez-Fuentes et al. [18] found that dissolved oxygen decreased significantly from 3.2 mg·L−<sup>1</sup> to 1–1.5 mg·L−<sup>1</sup> when the concentration of added molasses exceeded 0.12 g·L−1, which may lead to mortalities of aquatic animals. PHBV (poly-3-hydroxybutyrate-co-3-hydroxyvalerate) produced by bacteria has many excellent properties, such as thermoplastic, biodegradable, and can exist as a solid material, and has been used as a carbon source for denitrifiers in wastewater treatment systems with good results [20–22]. Compared with conventional carbon sources such as molasses, PHBV is characterized by slow carbon release and easy control, which means the concentration of dissolved organic carbon in water that can be used by heterotrophic bacteria will keep appropriate and stable, so it is a continuous carbon source after application in water and can be used as a biofilm carrier for bacteria [20,22]. However, to our knowledge, the practical application of PHBV as a carbon source in shrimp culture has rarely been reported [3,20].

Biological denitrification is considered one of the most effective, environmentally friendly, and inexpensive biotechnologies for reducing nitrogen levels in aquaculture wastewater [23]. As more heterotrophic nitrifying-aerobic denitrifying bacteria have been isolated, interest in their use for the effective removal of nitrogen accumulated in aquaculture systems has increased in recent years [24–27]. Studies have shown that heterotrophic nitrifying bacteria can convert nitrogen-containing compounds into NH2OH, NO2 −-N or NO3 −-N, etc., by nitrification while using carbon sources for their growth [20,28]. Meanwhile, some bacteria also have simultaneous aerobic denitrification, which can convert NO2 −-N or NO3 −-N into gasses such as NO, N2O and N2 [20,28]. Therefore, denitrification treatment with heterotrophic nitrifying-aerobic denitrifying bacteria has the advantages of high economic benefits and environmental friendliness and has gradually become a research focus in recent years [24,26,27]. Unlike the traditional methods of nitrogen removal by autotrophic nitrification and anaerobic denitrification, heterotrophic nitrifying-aerobic denitrifying bacteria can not only avoid the manipulation of separation of aerobic and anaerobic zones but also have the advantage of rapid growth and high denitrification efficiency [24,29]. Heterotrophic nitrifying and aerobic denitrifying microorganisms such as *Halomonas* spp., *Pseudomonas* spp., *Alcaligenes* spp., *Bacillus* spp. and other genera have been isolated successively [26,27]. However, the relevant research was conducted only on the laboratory scale, with few reports on the pilot scale and above, and most of them are based on biofortification, i.e., aerobic denitrifying microorganisms are exogenously added to the bioreactor in the form of microbial agents to improve the denitrification efficiency of

the reactor [24,25,29]. A strain of *Bacillus pumilus* BP-171 with the ability of heterotrophic nitrification-aerobic denitrification was isolated from a shrimp culture pond and showed good denitrification ability under laboratory conditions [29]. In addition, Li et al. [20] found that BP-171 significantly reduced nitrite concentration when added to a *P. vannamei* culture system with two other probiotic strains and PHBV simultaneously.

In this study, a shrimp culture experiment was set up to determine the possible effects of different treatments by adding a single BP-171, the combination of BP-171 and PHBV, and the combination of BP-171 and molasses on shrimp growth performance, water quality, and water microbiota in a *P. vannamei* culture system to understand the mechanisms of action of *B. pumilus* BP-171 and provide necessary information for its potential application in shrimp culture practice.

#### **2. Materials and Methods**

#### *2.1. Experimental Animals*

The juvenile *P. vannamei* were purchased from Qingdao Zhengda Agricultural Development Co., Ltd. (Qingdao, China). Before the culture experiment, shrimp were allowed to acclimate under experimental conditions for two weeks. During acclimation, the water temperature was controlled at 25 ± 0.5 ◦C and salinity at 29.0 ± 1.0‰. Water was exchanged once daily at a 10% exchange rate and continuously aerated. *P. vannamei* was fed three times a day (7:00, 12:00 and 18:00). At the end of acclimation, healthy *P. vannamei* of similar size were used for the culture experiment. The feed was obtained from Guangdong Yuehai Feeds Group; the main components and nutrient contents of the feed are shown in Supplementary Table S1.

#### *2.2. Experimental Strain and Carbon Sources*

*Bacillus pumilus* BP-171 was obtained from the Microbial Culture Collection, Lab of Aquaculture Ecology, Ocean University of China, a heterotrophic nitrifying-aerobic denitrifying strain [29]. PHBV was purchased from Ningbo Tianan Biological Material Co., Ltd. (Ningbo, China). It is white and cylindrical, with a height of 4 mm and an inner diameter of 1 mm. PHBV was activated in seawater with sufficient aeration for 10 d before the experiment [22]. Molasses (23.7% of total organic carbon content) was purchased from Jinan Pengduo Trading Co., Ltd. (Jinan, China).

## *2.3. Experimental Design*

The experiments were performed in 12 white polyethylene tanks with a volume of 500 L. Four experimental groups were designed, i.e., the probiotic group B applied with a single *B. pumilus* BP-171, the group BP added with the strain BP-171 and PHBV, the group BM with the strain BP-171 and molasses, and the control group DZ without the probiotic and carbon source. Three replicates were set up for each treatment group, and each replicate was randomly stocked with 70 shrimp, and the average weight of the shrimp was 6.06 ± 0.02 g.

The viable bacteria of *B. pumilus* BP-171 were regularly added to the water every seven days. The final concentration of probiotic bacteria in the water of each treatment group was designed as 1 × 107 cfu·L<sup>−</sup>1.

PHBV particles were placed in a PVC pipe with an inner diameter of 10 cm and a height of 35 cm, and the ends of the PVC pipe were covered with suitable sieves to prevent the PHBV particles from leaking. For aeration, an air stone was placed in the PVC pipe to allow the continuous release of the carbon source into the water with water currents. After assembly, the entire device containing 500 g of PHBV was placed in the corresponding tanks [20].

The molasses was applied with reference to the formula of Avnimelech [30] as follows:

$$
\Delta \text{N} = \text{(Feed} \times \text{N\%)} \times \text{\%N execution}
$$

$$
\Delta \text{CH} = \Delta \text{N} \times \text{[C/N]} \text{mic/(\%C} \times \text{E)}
$$

ΔN is the amount of nitrogen required to produce new bacteria. Feed is the amount of feed fed, and N% is the percentage of nitrogen in the feed, %N excretion is the percentage of feed nitrogen converted to ammonia in the culture system and is approximately 50%. ΔCH is the amount of molasses added. [C/N] mic is the C/N ratio of the heterotrophic bacteria themselves, %C is the carbon content of the added carbohydrate, and E is the efficiency of assimilation by heterotrophic bacteria, approximately 0.4.

An appropriate amount of molasses was diluted with seawater and poured evenly into the experimental tanks twice daily.

#### *2.4. Experimental Management*

The strain of *B. pumilus* BP-171 was inoculated into 2216E liquid medium and cultured at 160 r/min, (28.0 ± 1.0) ◦C to logarithmic phase, and the concentration of viable bacteria in the fermentation broth was 1 × 109 cfu·mL<sup>−</sup>1.

During the experimental period, *P. vannamei* was fed 5% of the total weight of shrimp three times daily (7:00, 12:00, and 18:00). Uneaten feed particles and feces were collected for 1 h after feeding, dried at 60 ◦C, and weighed. Using a portable dissolved oxygen meter (YSI 550A, Fisher Scientific, Hanover Park, IL, USA), a salinity meter (YSI EC300A, Fisher Scientific, Hanover Park, IL, USA), and a pH meter (YSI pH100A, Fisher Scientific, Hanover Park, IL, USA), temperature (25–28 ◦C), salinity (28–31‰), pH (7.8–8.0), and dissolved oxygen (>5 mg·L<sup>−</sup>1) were measured daily.

The feeding trial was conducted in workshop 16 of Qingdao Ruizi Group Co. (Qingdao, China) and lasted for 30 days.

#### *2.5. Sample Collection and Measurement*

#### 2.5.1. Growth Performance of Shrimp

The shrimp were counted and weighed for each treatment group at the beginning and end of the experiment, respectively. The survival rate, feed efficiency rate, and specific growth rate of *P. vannamei* were calculated as follows.

Survival rate (SR) = (Nt/N0) × 100%;

Feed efficiency rate (FER) = (Wt − W0)/Wf × 100%;

Specific growth rate (SGR) = (lnWt − lnW0)/T × 100%.

Nt is the number of alive shrimp on the day the feeding trial ended, and N0 is the number of shrimp put in the tank when the feeding trial started. Wt is the final wet weight of shrimp when the feeding trial ended, and W0 is the initial wet weight when the feeding trial started. T represented the days from the start to the end of the experiment.

#### 2.5.2. Water Quality Parameters

The water sample of 500 mL was collected every seven days. The parameters of ammonia, nitrite, nitrate, total nitrogen, soluble reactive phosphate, and total phosphorus were determined using an automatic chemical analyzer (Clever Chem 380G, DeChem-Tech. GmbH, Germany) according to the instructions. Water samples for DOC (Dissolved organic carbon) and TOC (Total organic carbon) were analyzed by a multi-2100s TOC analyzer (Analytik Jena). Besides, the average values at different time points of the above parameter were calculated to compare the difference of corresponding parameters among different treatments.

#### 2.5.3. DNA Extraction, Amplification, Purification, and Sequencing

Bacterial samples were collected on Day 30 when the feeding trial ended. A 1L water sample was filtered through a filter membrane with a pore size of 0.22 μm, then the bacterial samples were stored in a −80 ◦C refrigerator. Total genomic DNA was extracted from the water sample using the E.Z.N.A.® Water DNA Kit (Omega, GA, USA), and PCR amplification was performed using primers 338F (5 -ACTCCTACGGGAGGCAGCA-3 ) and 806R (5 -GGACTACHVGGGTWTCTAAT-3 ) specific for the V3 and V4 regions of the 16S rRNA gene. PCR products were then recovered to generate sequencing libraries, and the constructed libraries were sequenced at high throughput using the Illumina HiSeq platform. Raw reads were processed by splicing, filtering, and removal of chimeras to obtain effective reads. The sequences were clustered to obtain operational taxonomic units (OTUs) using UPARSE software (version 7.0) [31] with sequenced reads at a 97.0% similarity level. OTUs were taxonomically annotated using the Silva database (http://www.arb-silva.de/ (accessed on 1 July 2022)).

## *2.6. Statistical Analysis*

Mothur software (version 1.30.2) was used to analyze the diversity of sample sequences, including alpha diversity such as ACE index, Chao1 index, Shannon index, Simpson index, and Good-coverage as well as beta diversity such as PCA (Principal Component Analysis), PCoA (Principal Co-ordinates analysis), and PLS-DA (Partial Least Squares Discriminant Analysis). Chao index and ACE index were used to estimate the number of OTU, the total number of species, reflecting the species richness of α diversity in a community, but the algorithms are different. Both the Shannon index and Simpson index were used to estimate the α diversity of the bacterial community in samples. They consider not only the richness of species in the community but also the evenness of species. However, the algorithms of the two are different. In addition, the higher the Shannon value is, the higher α-diversity is. However, the higher the Simpson value is, the lower α-diversity is. Analysis and visualization of OTU-based Venn diagrams were performed using the VennDiagram package in R (v3.3.1). Based on the species composition of each treatment group at each taxonomic level, bar graphs were generated using the ggplot2 package in R (v.3.3.1), which can be used to visualize the dominant species of each group at a given taxonomic level and the relative abundance of each dominant species. Using the stats package in R (v.3.3.1) and the scipy package in Python, hypothesis tests were performed among species in the different groups based on the species abundance data of bacterial community using the one-way test ANOVA or the Kruskal-Wallis H test to evaluate the significance level of differences in species abundance and obtain species with significant differences between groups.

RDA analysis (redundancy analysis) was performed using the vegan package in R (v.3.3.1), and the significance of RDA analysis was determined by permutest analysis similar to ANOVA. Spearman correlation coefficients between environmental factors and selected species were calculated using the vegan package in R (v.3.3.1) for correlation analysis, and Spearman correlation significance tests were performed using the corrplot package in R (v.3.3.1). Ecological networks were created based on CoNet software in Cytoscape (v.3.8.2) and visualized using Cytoscape (Faust et al., 2016). The KEGG Module database was used to link bacterial taxa to gene sets with particular metabolic capacities and other phenotypic traits. The Shapiro-Wilk test was used to test the data for normal distribution (*p* > 0.05), and Levene's test was used to test for chi-square (*p* > 0.05). One-way analysis of variance (ANOVA) and Duncan's multiple comparison method in IBM SPSS Statistics 24.0 software were used to analyze the significance of differences between groups. The Kruskal-Wallis test was used for analysis when the data were not normally distributed or when there was unequal overall variance. *p* < 0.05 indicated significant differences.

#### **3. Results**

#### *3.1. Growth Performance of Shrimp*

The growth performance of shrimp is shown in Table 1. The survival rate of shrimp in groups B, BP, and BM was significantly higher than that in the control (*p* < 0.05), and there was no significant difference among groups B, BP, and BM (*p* > 0.05). The final body weight and specific growth rate (SGR) of shrimp were significantly higher in groups BP and BM than in groups B and the control (*p* < 0.05), and they were highest in group BP (*p* < 0.05). The gross weight of shrimp in the BP group was the highest and significantly higher than

that of the other groups (*p* < 0.05), while that of groups B and BM was significantly higher than that of the control group (*p* < 0.05). The feed efficiency rate of shrimp in the BP group was significantly higher than that in other groups (*p* < 0.05), while no significant difference was found between other groups and the control (*p* > 0.05).

**Table 1.** Gross weight, survival rate, specific growth rate, and the feed efficiency rate of *P. vannamei* (Mean ± S.E.).


Note: 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. Data are expressed as mean ± standard error, *n* = 3. Values in the same row with different superscripts are significantly different among treatments (*p* < 0.05).

#### *3.2. Water Quality Parameters*

3.2.1. Changes in Ammonia, Nitrite, Nitrate, and Total Nitrogen

The average values of the temperature, dissolved oxygen, and pH in the water changed from 25.6 ± 0.3 ◦C, 7.21 ± 0.51 mg/L, and 7.46 ± 0.21 ◦C to 25.9 ± 0.3 mg/L, 7.43 ± 0.17 and 7.62 ± 0.19 during the period of the experiment, and no significant differences were found among the groups (*p* > 0.05). The salinity of the water was (30.0 ± 1.0) ‰ during the feeding experiment.

The concentrations and changes of nitrogen in water during the feeding experiment are shown in Figure 1. The concentration of total ammonia nitrogen (TAN) in groups B, BP, and BM was significantly lower than that in the control (*p* < 0.05), and the concentration in group BM was significantly higher than that in groups B and BP. The lowest concentration occurred in the B group and was reduced by 70.22% compared with the control (*p* < 0.05). The concentrations of TAN in the control increased during the experimental period (Figure 1A). In contrast, the TAN concentration in the BM group peaked at about Day 18, whereas the concentration in the B and BP groups leveled off after Day 6 and was significantly lower than that in the control group during the experiment (*p* < 0.05).

Similarly, the concentration of nitrite nitrogen in the B, BP, and BM groups was significantly lower than that in the control (*p* < 0.05). The concentration in the B group was significantly lower than that in the other groups (*p* < 0.05) and reduced by 76.88% compared to the control. In addition, the nitrite-nitrogen concentration in the control group increased from Day 1 to Day 24, stabilized after Day 24, and was significantly higher than all treatment groups (Figure 1B). In comparison, the concentration in the treatment groups leveled off after Day 6 and was significantly lower than that in the control during the experiment (*p* < 0.05).

The concentration of nitrate nitrogen in groups B, BP, and BM was significantly lower than that in the control group (*p* < 0.05). The concentration in the BP group was significantly lower than that in the other groups (*p* < 0.05) and reduced by 26.24% compared to the control. Moreover, the nitrate-nitrogen concentration in water showed an increasing trend in all treatment groups until Day 18 and stabilized after Day 18 in groups B and DZ (Figure 1C). However, concentration in groups BP and BM continued to increase until the end of the experiment. From Day 18 to 30, the nitrate-nitrogen concentration was significantly higher in the control group than in groups B and BP.

**Figure 1.** Changes of Ammonia nitrogen (**A**), Nitrite nitrogen (**B**), Nitrate nitrogen (**C**), and Total nitrogen (**D**) in the water of 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 on the same day are significantly different among treatments in each figure (*p* < 0.05).

The concentration of total nitrogen was significantly lower in groups B, BP, and BM than in the control group (*p* < 0.05). The concentration in group BP was significantly lower than in the other groups (*p* < 0.05) and was reduced by 40.02% compared to the control. The concentration of total nitrogen in the control group showed an increasing trend (Figure 1D). The values in the treatment groups leveled off between Day 6 and 30 and were significantly lower than that in the control group (*p* < 0.05).

#### 3.2.2. Changes in Soluble Reactive Phosphorus (SRP) and Total Phosphorus (TP)

The concentrations of SRP and TP are presented in Figure 2. There was no significant difference in SRP and TP concentrations among all the groups during the experiment, with an overall increasing trend.

#### 3.2.3. Changes of Dissolved Organic Carbon (DOC) and Total Organic Carbon (TOC)

The concentrations and changes of organic carbon are presented in Figure 3, respectively. The concentrations of DOC and TOC in the groups without the addition of carbon sources (DZ and B) were significantly lower than those in the groups with the addition of carbon sources (BP and BM) (*p* < 0.05). The DOC and TOC concentrations in the DZ and B groups showed a trend of stabilization, while the concentrations in the culture system with carbon source addition increased until Day 18. After Day 18, the concentrations of DOC and TOC in the BP group increased slowly and stabilized gradually, while the concentrations in the BM group decreased rapidly. The average concentrations of DOC and TOC between the B group and the control group showed no significant difference (*p* > 0.05).

**Figure 2.** Changes of SPR (**A**) and TP (**B**) in the water of 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 on the same day are significantly different among treatments in each figure (*p* < 0.05).

**Figure 3.** Changes of DOC (**A**) and TOC (**B**) in the water of 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 on the same day are significantly different among treatments in each figure (*p* < 0.05).
