Aquaculture Water Quality Improvement by Mixed

Bacillus and Its Effects on Microbial Community

Structure
Xue Li 
Liaoning University
Tianie Wang 
Liaoning University
Baorong Fu 
(

fbr2021@163.com
)
Liaoning University
Xiyan Mu 
Liaoning University

Research Article

Keywords: Nitrogen, Phosphorus, Mixed Bacillus, Water quality, Degradation, Microbial community
structure

Posted Date: December 13th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-1057159/v1

License:


This work is licensed under a Creative Commons Attribution 4.0 International
License.
 
Read Full License

Page 1/19
Abstract
Nutrients nitrogen and phosphorus pollution in aquaculture is one of the greatest challenges threatening
the survival of aquatic organisms, which requires efficient and sustainable remediation approach.
Microbial remediation, especially the application of probiotics, has recently gained popularity in
improving the water quality and maintaining the health condition of aquatic animals. In the present study,
two groups of mixed Bacillus (Bacillus megaterium and Bacillus subtilis (A0+BS) and Bacillus
megaterium and Bacillus coagulans (A0+BC)) were applied to aquaculture system of Crucian carp to
improve the treatment of nitrogenous and phosphorus compounds. The effects of mixed Bacillus on
water quality, and the structure and function of microbial communities in aquaculture water were
investigated. Our results showed that the improvement effect of mixed Bacillus A0+BS on water quality
was better than that of A0+BC, and the NH4+-N, NO2--N, NO3--N and total phosphorus (TP) concentrations
were reduced by 46.3%, 76.3%, 35.6%, and 80.3%, respectively. In addition, both groups of mixed Bacillus
increased the diversity of bacterial community and decreased the diversity of fungal community.
Microbial community analysis showed that mixed Bacillus A0+BS increased the relative abundance of
bacteria related with nitrogen and phosphorus removal, e.g., Proteobacteria, Actinobacteria, Comamonas,
and Stenotrophomonas, but decreased the relative abundance of pathogenic bacteria (Acinetobacter and
Pseudomonas) and fungi (Epicoccum and Fusarium). The redundancy analysis showed that NH4+-N,
NO2--N, and TP were primary environmental factors affecting the microbial community in aquaculture
water. PICRUST analysis indicated that all functional pathways in the treatment groups were up-
regulated, and all pathways in A0+BS group were richer than those in other groups. These results
indicated that mixed Bacillus A0+BS addition produced good results in reducing nitrogenous and
phosphorus compounds and shaped a favorable microbial community structure to further improve water
quality.

Highlights
Mixed Bacillus A0+BS was directly applied in aquaculture as free bacteria.
Mixed Bacillus A0+BS shaped a favorable microbial community structure.
Water quality was improved by the mixed Bacillus A0+BS addition.
All functional pathways were enriched after adding mixed Bacillus especially A0+BS in aquaculture.

1. Introduction
In recent decades, the eutrophication of rivers, lakes, reservoirs and coastal waters has become one of the
most important water pollution problems in the world due to the excessive discharge of nitrogen and
phosphorus (Wang et al., 2019). The rapid development of intensive aquaculture and the discharge of
aquaculture water cause the high concentration of nutrients nitrogen and phosphorus in surface water
bodies, which makes algal blooms occur frequently (Conley et al., 2009). The release of nitrogen-
containing metabolites and algal toxins change the balance of micro-ecology, resulting in the
Page 2/19
proliferation of a large number of pathogenic microorganisms (Lieke et al., 2019; Zokaeifar et al., 2014).
These proliferating pathogenic microorganisms are very easy to cause infectious diseases of fish and
shrimps during the process of aquaculture, and cause damage to the aquaculture water and the
surrounding environment (Shakya et al., 2013; Wang et al., 2018b). Hence, exploring the cost-effective
and non-toxic water quality improvement technology has given rise to major global concern.

Currently, a number of physical, chemical, and biological methods are used in the process of aquaculture
wastewater (Yin et al., 2018). These include water exchange, biofilters (Jahangiri et al., 2018), ozone
oxidation (Schroeder et al., 2011), and biofloc technology (Liu et al., 2019). However, most of these
methods have disadvantages such as time-consuming, high cost, and toxic by-products (Mook et al.,
2012; Hlordzi et al., 2020). In addition, the use of antibiotics is common practice in aquaculture to control
fish and shrimp diseases (Banerjee et al., 2017). However, this has resulted in an increase in antibiotic
resistant strains in aquaculture environment, which can be transmitted to human through food chain
(Hoseinifar et al., 2017; Zhou et al., 2018). To avoid the above adverse situation, bioremediation by
probiotics has become high in aquaculture (Hoseinifar et al., 2018). Various microorganisms have been
successfully applied to reduce toxic pollutants generated in the aquatic system with simple, stable and
relatively low operational cost (Verschuere et al., 2000; Lu et al., 2012). Probiotics as a kind of safe
additive are used in aquaculture for purifying water quality, inhibiting potential pathogenic
microorganisms, improving feed utilization, and enhancing the health of the host (Olmos et al., 2011;
Selim et al., 2015; Yang et al., 2011). This efficiently reduced the dependent of aquaculture on antibiotics.

Bacillus species have been widely used in aquaculture based on the characteristic of high survival rate,
inhibition of pathogens, and immunity regulation of aquatic organisms (Xu et al., 2013). Many
researchers have reported that Bacillus species play significant roles in removing nitrogenous and
phosphorus compounds and maintaining the balance of the microbial community structure (Choi et al.,
2002; Soltani et al., 2019). Bacillus species can remove the different forms of nitrogen from aquaculture
wastewater through ammonification (Hui et al., 2019), nitrification (Rout et al., 2017), and denitrification
(Verbaendert et al., 2011) as well as nitrogen fixation. Literature has proven that Bacillus can reduce
phosphate ions to the acceptable range in aquaculture (Thurlow et al., 2019). However, with the
increasing deterioration of aquaculture water and the complexity of pollution factors, the single bacterial
species has certain limitations in terms of nitrogen and phosphorus removal, disease control. Compared
with a single strain, the synergistic interaction between mixed Bacillus could enhance the nutrients
removal rate, and such system were more resistant to environmental condition (John et al., 2020).
However, the current application of mixed Bacillus in aquaculture water mainly focuses on the removal
effect of different combinations of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, and
Bacillus lateralis on the nitrogenous and phosphorus compounds (Elsabagh et al., 2018; Kuebutornye et
al., 2019). Bacillus megaterium is widely used for the removal of nitrogenous and phosphorus
compounds (Zhao et al., 2021). Gao et al. (2018) found that Bacillus megaterium could keep a high nitrite
removal rate when the temperature was between 25°C and 40°C and the pH was between 7.0 and 9.0.
Therefore, it is necessary to further study the combined effect of Bacillus megaterium and other

Page 3/19
functional strains. Meanwhile, the potential interaction mechanism between exogenous Bacillus and
microbial community is still unclear.

In addition, the method of adding Bacillus was mainly through mixing with feed and then adding it to
aquaculture water in most of the previous studies (Cai et al., 2019). Nayak et al. (2020) pointed out that
direct addition of Bacillus species into the rearing water as water additive could rapidly degrade the
unspent feed and organic excreta. In this study, using the Bacillus megaterium isolated from eutrophic
water by our research group and other functional Bacillus (Bacillus subtilis, Bacillus coagulans) from
Liaoning Huaxing Biological Technology Co. Ltd, based on the diffusion efficiency of Bacillus in
aquaculture water, the beneficial effects of mixed Bacillus species on water quality and micro-ecosystem
in Crucian aquaculture were ascertained by directly adding mixed Bacillus (Bacillus megaterium +
Bacillus subtilis, Bacillus megaterium + Bacillus coagulans). The changes in microbial populations, the
structure and function of microbial community in aquaculture water had been explored by Illumina-HiSeq
sequencing. Our results would help to understand the degradation mechanism of nitrogen and
phosphorus in water by mixed Bacillus and to provide a theoretical basis for microbial combined action
in water purification.

2. Material And Method

2.1. Strains
Bacillus megaterium (A0) was previously isolated from eutrophication aquaculture pond located at
Shenyang (Liaoning Province, China). It had the effect of removing nutrients nitrogen and phosphorus,
and no fish death or other abnormal phenomenon was apparent (unpublished data). Bacillus subtilis (BS)
and Bacillus coagulans (BC) were purchased from Liaoning Huaxing Biological Technology Co. Ltd. The
two Bacillus species can improve the water quality and regulate the balance of micro-ecosystem in
aquaculture water.

2.2. Mixed Bacillus culture

Stock cultures of the single Bacillus were maintained in 60% glycerol and stored at -80 ℃. The solid
medium that was used to culture the single Bacillus contained beef extract 3.0 g/L, tryptone 10.0 g/L,
NaCl 5.0 g/L, pH 7.0. The solid medium was sterilized in an autoclave at 115 ℃ for 30 min. The Bacillus
mixture in this study comprised two Bacillus species, namely mixed Bacillus A0+BS and mixed Bacillus
A0+BC. A co-culture experiment about mixed Bacillus was carried out in a 250 mL flask containing
proliferation medium (glucose 8 g/L, yeast extract 12.5 g/L, KH2PO4 3 g/L, MnSO4·H2O 0.18 g/L,
MgSO4·7H2O 0.125 g/L, pH 7.0), and the medium was sterilized in an autoclave at 115 ℃ for 30 min
before use. These flasks were shaken in an incubator shaker (LRH-70, Shanghai, China) at 160 rpm, 30
℃ for 24h. The grown cell cultures were made into pellets by centrifuging at 4000g, 4 ℃ for 10 min. The
pelleted bacteria were re-suspended and washed three times in sterile saline. The density of the cell
suspension was calculated by using a spectrophotometer (2600 UV/VIS, Shanghai, China), and the

Page 4/19
relationship between the density of the suspension and the colony forming unit (CFU) was determined
using the expansion plate technique (Zokaeifar et al., 2014). The final concentration of cell suspension
was altered to 2.0×108 CFU/mL.

2.3. Batch experiments

Crucian carp, water and sediment were all obtained from aquaculture pond located at Yuhong district
Aquaculture Plant (YAP, Northeast of Shenyang, Liaoning Province, China). The water quality parameter
was with a pH of 7.2, dissolved oxygen (DO) of 7.65 mg/L, ammonia nitrogen (NH4+-N) of 1.34 mg/L,
nitrate nitrogen (NO3−-N) of 0.18 mg/L, nitrite-nitrogen (NO2−-N) of 0.41 mg/L, and TP of 0.204 mg/L.

On the basis of pre-experiment, 180 health Crucian carp (average weight 60 ± 3 g) were used to study.
Fishes were divided among 9 aquariums (L × W × H, 1.00 m × 0.50 m × 0.60 m) for the ensuing outdoor
experiments, with 20 fishes per aquarium. 10cm-sediment collected from fish ponds were put into each
tank to simulate aquaculture pond and each tank was filled with 180 L of pond water. All tanks were
aerated continuously. Water temperature was 14 ~ 24℃ and pH was 7.1~7.4 during experiment. Fishes
were fed the bait twice a day.

Nine tanks were randomly divided into three groups, and each group had three replicate tanks: (1) control
group (CK); (2) treatment group: Bacillus megaterium and Bacillus subtilis group (A0+BS); (3) treatment
group: Bacillus megaterium and Bacillus coagulans group (A0+BC), with the mixed Bacillus added to the
water at a final concentration of 6.0×105 CFU/mL (Nai and Fotedar, 2010; Zokaeifar et al., 2014),
respectively. According to the effect time of the mixed Bacillus, the experimental period was designed to
be 15 days.
2.4. Water quality analysis
Samples collected from the four locations of each tank were mixed before storage in polyethylene plastic
bottles (500 mL) for all the tests once every two days. The collected water sample were filtered and then
analyzed for NH4+-N, NO3−-N, NO2−-N, and TP following these methods as described by Chinese EPA
(2002). NH4+-N levels were determined by salicylic acid spectrophotometry method. NO3−-N levels were
determined by UV spectrophotometry method. NO2−-N levels were determined by N-(1-naphthyl)-ethylene
diamine dihydrochloride spectrophotometric method. TP levels were determined by ammonium
molybdate spectrophotometric method.

2.5. DNA extraction and high-throughput sequencing

Tank water samples were collected in sterile tubes and then were filtered through a 0.2 µm pore size
polycarbonate filter. Total DNA was extracted from the filter using the E.Z.N.A. Soil DNA Kits (Omega Bio-
Tek, USA) according to the manufacturer's protocol.

All DNA samples were sent to Sangon Bio-tech Co., Ltd. (Shanghai, China). The V3-V4 region of the
bacterial 16S rRNA genes was amplified by using specific primers: 341F (5′-CCTACGGGNGGCWGCAG-3′)

Page 5/19
and 805R (5′-GACTACHVGGGTATCTAATCC-3′). The ITS1-ITS2 region of fungi was amplified by using
specific primers: ITS1F (5′-CCCTACACGACGCTCTTCCGATCTN-3′) and ITS2R (5′-
GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3′). The PCR conditions were as follows: 1 cycle at 94 ℃
for 3 min, followed by 30 amplification cycles (94 ℃ for 30s, 45 ℃ for 20s, and 65 ℃ for 20s), 20
amplification cycles (94 ℃ for 20s, 55 ℃ for 20s, and 72 ℃ for 30s), l cycle at 72 ℃ for 5 min. The PCR
products were detected by gel electrophoresis using 2% agarose gel. High-throughput sequencing of 16S
rRNA and ITS gene was carried out using an Illumina MiSeq.

2.6. Statistical analysis

Correlation and statistical differences were analyzed using SPSS software (Vision 19.0). Differences were
considered significantly at P < 0.05. Cutadapt Version 1.2.1 was used to remove primer adapters to
ensure the quality of information analysis. Pear Version 0.9.6 was used for sequence splicing. Prinseq
Version 0.20.4 was used for sequence filtering. Mothur Version 1.30.1 was used to remove the chimera
sequence. Finally a high-quality series for analysis was acquired. The operational taxonomic units
(OTUs) were clustered by UPARSE at the 97% similarity level. QIIME Version 1.8.0 software was used to
perform Alpha diversity analysis on OTUs, including Shannon Index and Chao Index. The RDP classifier
Bayesian algorithm was used to perform taxonomic analysis on the 97% similarity level OTU
representative sequence, and the community composition of each sample bacterial phylum and genus
level was counted. The KEGG (Kyoto Encyclopedia of Genes and Genomes) microbial metabolic function
was obtained by standardizing the OTU abundance table with PICRUST software. Redundancy analysis
(RDA) was used to estimate the relationship among dominant microbial communities and water quality
indicators using CANOCO 5.0 at a level of phylum and genus.

3. Results
3.1. Improvement of water quality
As shown in Fig. 1A, the NH4+-N concentration of the three groups increased within the first 5 days and
decreased rapidly from the 5th to the 15th days, decreasing by 24.4% (CK), 46.3% (A0+BS) and 32.1%
(A0+BC), respectively. The NH4+-N concentration in the A0+BS group was lower than the other two
groups.

As shown in Fig. 1B, the NO2−-N concentration in the CK group maintained a relative stable level from the
first 9 days and decreased slowly during the 9th to 15th days. In the treatment groups, the NO2−-N
concentration maintained a relative stable level during the first 11 days and decreased from the 11th to
15th days, decreasing by 76.3% (A0+BS), 65.8% (A0+BC), respectively. The NO2−-N concentration in the
A0+BS group was lower than those of other groups.

As shown in Fig. 1C, the NO3−-N concentration of the three groups continued to increase and reached the
highest level at the end of the experiment. The NO3−-N concentration in the A0+BS group was lower than
Page 6/19
those of other groups.

As shown in Fig. 1D, the TP concentration of the three groups increased within the first 3 days, decreased
from 3rd to the 11th days, and maintained a relative stable level from 11th to 15th days. The
concentration of TP in A0+BS was lower than those of other groups.
3.2. Diversity analysis of the microbial community
A total of 250292 bacterial and fungal high-quality sequences were obtained from the three groups after
removing low quality sequences. The coverage of effective sequences was greater than 83% for all
groups, indicating that the analysis of both the bacterial and fungal sequence were rational. In all groups,
the bacterial OTUs numbers were higher than that of fungi, among which the CK group was significantly
higher than other groups in bacterial (Fig. 2A) and fungal (Fig. 2D) groups (P < 0.05).

Shannon was used to estimate the microbial diversity. Chao was used to describe the total number of
species. Alpha diversity of the microbial communities of different groups showed the bacterial
communities had a higher community diversity and total number of species than that of fungi, indicating
that bacteria was an important part of the indigenous microbial population. Shannon index of bacterial
communities from the A0+BS group was the highest compared with other groups and significantly higher
than CK group (P < 0.05) (Fig. 2B). There were significant differences in Shannon index between fungal
communities (P < 0.05), and the fungal community diversity of CK group was the highest compared with
treatment groups (Fig. 2E). Chao index of bacterial communities from the CK group was significantly
higher than A0+BC group (P < 0.05), and the species richness of CK group was the highest (Fig. 2C). No
significant differences in Chao index between fungal communities (P > 0.05), and the species richness of
A0+BS group was the highest (Fig. 2F).

3.3. The composition analysis of the microbial community

at the phylum and genus level
At the phylum level, the bacterial community structure had high diversity in CK group, and its dominant
bacteria mainly included Firmicutes (57.8%), Proteobacteria (25.3%), Bacteroidetes (8.2%), and Chloroflexi
(3.7%). The composition of dominant bacteria in A0+BS and A0+BC groups was basically the same,
including Proteobacteria (89.1%, 90.4%), Bacteroidetes (4.8%, 5.7%), and Actintobacteria (3.9%, 2.2%),
among which the proportion of Proteobacteria was the largest in every treatment group (Fig. 3A).

At the genus level, 23 genera had relative abundance greater than 2%. The dominant bacteria in CK group
were mainly Lysinibacillus (27.6%), Solibacillus (9.5%), and Haematobacter (5.8%). The dominant
bacteria in A0+BS group were Comamonas, Caulobacter, Stenotrophomonas, Acidovorax,
Pseudoxanthomonas, Methylobacterium, Bosea, Dokdonella, and Thermomonas, among which
Comamonas (8.1%), Caulobacter (8%) and Stenotrophomonas (7.2%) accounted for the larger proportion.
The dominant bacteria in A0+BC group were mainly Comamonas, Acinetobacter, Stenotrophomonas, and
Pseudomonas, among which Comamonas (27%), Acinetobacter (14%), and Stenotrophomonas (9.5%)
accounted for the larger proportions (Fig. 3B).
Page 7/19
Fungi covered 36 genera in all groups, of which 12 genera had relative abundance greater than 1%. The
dominant fungi in CK group were mainly Cladosporium (21%), Epicoccum (21.3%), and Fusarium (5.4%).
The dominant fungi in A0+BC group were mainly Cladosporium (48.12%), Unclassified (29.47%), and
Guehomyces (10.79%). The dominant fungi in A0+BS group were mainly Unclassified (92.63%) (Fig. 3C).
3.4. Relationships between the environmental factors and
bacterial microbial community
The CANOCO software was used to analyze the correlation between bacterial community (dominant
phyla and genus) changes and environmental factors including NH4+-N, NO2−-N, NO3−-N, and TP
(Fig. 3D). The results of the RDA between bacterial community and environmental factors showed that
the RD-1 and RD-2 axes explained 91.92 % and 7.96 % of the total variance, respectively. TP, NH4+-N, and
NO2−-N were the main factors affecting bacterial community structure. At the phylum level, the
abundance of Firmicutes and Bacteroidetes positively correlated with NH4+-N, NO2−-N, and NO3−-N. The
correlation of Proteobacteria with water quality was similar to Actinobacteria, with negative correlation
with nitrogenous compounds and TP. At the genus level, the abundance of pathogenic genera
Acinetobacter negatively correlated with NH4+-N, NO2−-N, NO3−-N, and TP.

3.5. Functional profile of the microbial community

The potential functional characteristic of the microbial community was carried out using KEGG analysis
(Fig. 4). The predicted sequences from the three groups (CK, A0+BS, and A0+BC) were mainly clustered
into four categories (metabolism, environmental information processing, genetic information processing,
and cellular processes) related to the effect of mixed Bacillus on microbial function, among which,
metabolism accounted for the largest proportion. Metabolism comprised 12 pathways, and each of the
other three categories comprised less than 4 pathways. Almost all functional pathways were more
abundant in the treatment groups than CK and the A0+BS group was more enriched than A0+BC group.
Among others, the pathways of amino acid metabolism, carbohydrate metabolism, energy metabolism,
and xenobiotics biodegradation and metabolism were more abundant in the cluster of metabolism. The
pathway of replication and repair was the most enriched in the cluster of genetic information processing.
The pathway of membrane transport was the most abundant among all pathways.

4. Discussion
Bioremediation is an eco-friendly method to achieve the sustainable development of aquaculture (Nayak.,
2020). Nowadays, probiotics were successfully used for maintaining water quality parameters and
promoting the growth, health and disease resistance of various fish and shellfish species in aquaculture
(Gomez-Gil et al., 2000; Verschuere et al., 2000). In aquaculture, common probiotics include Alteromonas,
Bacillus, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Photorbodobacterium, and et al
(Dawood et al., 2018; Wang et al., 2018a). Bacillus species had been proven to possess better probiotic
properties and widely used in different types of aquaculture practices (Ghosh et al., 2016). In an

Page 8/19
aquaculture environment with severe eutrophication, mixed Bacillus could be advantageous over single
Bacillus because of the co-operative interactions between the co-cultivated Bacillus that enhanced the
removal efficiency of nutrients (Ren et al., 2021; Kesarcodi-Watson et al., 2012). However, the effects of
mixed Bacillus on nutrients nitrogen and phosphorus, and aquatic microorganism are still unknown in
aquaculture.

The Chao index indicated species richness and was sensitive to the change of rare species (Wang et al.,
2018b), and the Shannon index indicated species richness and evenness in the sample. These results
showed that the addition of mixed Bacillus A0+BS increased the diversity of bacterial community in
aquaculture water, and the addition of mixed Bacillus A0+BC caused the loss of rare species.
Simultaneously, highly diverse populations improved the stability of the water ecosystem. Similarly, Wu et
al. (2014) had reported that the addition of Bacillus subtilis in shrimp culture water increased the diversity
of bacterial community.

In addition, bacterium far outnumbered fungi in terms of total number and diversity of species (Fig. 2).
Similar results had been found in most previous studies (Bahram et al., 2020). In A0+BS group, the Chao
and Simpson index of fungi increased, indicating that the species richness of fungi increased and the
diversity decreased. Due to the poor coverage of ITS database and the existence of many highly novel
fungal systems (Zhang et al., 2021b), it was difficult to make further discussion. In conclusion, the
addition of mixed Bacillus increased the bacterial diversity and decreased the fungal diversity.

The addition of mixed Bacillus caused microbial community shifts and changed the status of nitrogen
and phosphorus. High-throughput sequencing showed that mixed Bacillus could not colonize in the new
environment and thus could not become dominant species, indicating that mixed Bacillus might have an
indirect effect on indigenous bacteria. A similar result was reported by Skjermoetal et al. (2015). Previous
studies have proven that carbon sources caused shifts in microbial community structure and function in
aquaculture waters (Zhao et al., 2018; Wang et al., 2020). Mixed Bacillus was cultivated on a medium rich
in carbon sources and used in liquid form finally. In this study, Proteobacteria and Actinomyces increased,
whereas, Bacteroides and Firmicutes decreased in the treatment groups compared to CK group, a
possible reason that mixed Bacillus addition showed potential carbon source effects and consequently
affects microbial communities. Previous studies found that the dominance of Proteobacteria and
Actinomyces in various types of aquaculture systems (Jiang et al., 2020). Tang et al. (2014) found that
the dominant phyla was Actinobacteria, Proteobacteria, and Bacteroidetes in freshwater aquaculture
system farming of L. vannamei. Duarte et al. (2019) also reported that Proteobacteria and Actinomyces
were dominated in semi-intensive aquaculture system for sea bass (Dicentrarchus labrax). In most
freshwater environments, Proteobacteria can use nitrogen and phosphorus pollutants in water and
participate in a variety of metabolic pathways (Chen et al., 2017; Shapleigh, 2011). Actinobacteria might
transform elements such as nitrogen and phosphorus and inhibit the growth and reproduction of
pathogenic bacteria (Wang et al., 2018a). Taken together, after mixed Bacillus A0+BS addition, the
increase in the abundance of Proteobacteria and Actinobacteria played an important role in improving
nutrients nitrogen and phosphorus concentration in aquaculture water. Bacteroidetes and Firmicutes
Page 9/19
could serve as sentinels of changes to trophic state for monitoring wastewater impacts (Velmurugan et
al., 2011; He et al., 2016). In this study, the decrease in the abundance of Bacteroidetes and Firmicutes
after mixed Bacillus A0+BS application indicated that aquaculture water eutrophication was mitigated.

At the level of genus classification, the microbial community in aquaculture water was relatively scattered
and highly diverse. Comamonas and Stenotrophomonas dominated in aquaculture water with mixed
Bacillus addition. Comamonas is a heterotrophic nitrifying-aerobic denitrifying bacteria (Su et al., 2019).
Stenotrophomonas is a heterotrophic nitrifying bacterium, which can assimilate and nitrify ammonia
nitrogen (Zeng et al., 2020). Qiao et al. (2020) had pointed out that heterotrophic nitrifying-aerobic
denitrifying bacteria had greater advantages in denitrification and removal of organic matters, compared
with traditional deammonia bacteria. Interestingly, some nitrogen removal bacteria (e.g., Dokdonella,
Acidovorax, and pseudoxanthomonas) appeared in aquaculture water after mixed Bacillus A0+BS
addition. Among which, Dokdonella is a common denitrification bacteria in sewage treatment, which has
nitrification and denitrification functions (Schuster et al., 2008). Pseudoxanthomonas belongs to
denitrifying bacteria and is the key microorganism for nitrogen removal (Schreier et al., 2010). Acidovorax
can directly reduce nitrate to nitrogen without any accumulation of denitrifying intermediates (Wang and
Chu, 2016; Gentile et al., 2007). In this study, the relative abundance of Dokdonella and Acidovorax was
negatively correlated with nitrogenous compounds, further supporting this hypothesis. These results
suggested that nitrification and denitrification existed simultaneously in aquaculture water after mixed
Bacillus A0+BS addition, which accelerated the removal of nitrogenous compounds.

Acinetobacter and Pseudomonas, two well-known pathogenic bacterium causing infections in fish (Xia et
al., 2008; Sihag et al., 2012), were more enriched in the treatment A0+BC compared to the A0+BS.
Previous studies indicated that Actinobacteria could control pathogen in aquaculture through produce
antibiotics (Goodfellow and Fiedler.,2010). Thus, in our study, the increase in the abundance of
Actinobacteria might have been a contributing factor to the decrease in the abundance of Acinetobacter
and Pseudomonas in the treatment A0+BS. These results implied that mixed Bacillus A0+BS addition
could effectively inhibit pathogenic bacteria.

Fungi played important roles in the decomposition of organic matters in aquaculture water (Grossart and
Rojas-Jimenez, 2016). It was found that the abundance of Epicoccum and Fusarium decreased in this
study, indicating that the mixed Bacillus and its associated bacteria reduced nutrients, through
decomposition and assimilation, that promoting the growth of these fungi. Moreover, Bacillus species
could suppress the growth of some fungi by producing lipopeptides (Zhao et al., 2010). However, little
information was available about the increased abundance of Unclassied genera after the mixed Bacillus
addition in aquaculture water. Thus, it would be worth exploring the functions of Unclassied genera in the
future study, especially with respect to its identification of such fungi and interaction with Bacillus.

The NH4+-N and NO2−-N concentrations showed an overall decreasing tendency, whereas, the NO3−-N
concentration showed an overall increasing tendency in aquaculture water during the entirety of this test,
a possible reason that microorganisms in aquaculture water converted NH4+-N to NO2−-N, and NO2−-N to

Page 10/19
NO3−-N by nitrification process. The NH4+-N, NO3−-N, and NO2−-N concentrations in the treatment were
lower than those of the CK. This was likely because the mixed Bacillus addition caused shifts in bacterial
community structure and function, and the dominated bacteria could be responsible, through nitrification-
denitrification and assimilation, for the removal of nitrogenous compounds in aquaculture water. In
addition, mixed Bacillus A0+BS was more effective in removing nitrogenous compounds compared with
the mixed Bacillus A0+BC. Phosphate produced by microorganisms decomposing organic matter can be
absorbed and utilized by phosphorus accumulating bacteria (Chun et al., 2018; Li et al., 2020). Previous
studies have already demonstrated that TP could be reduced in catfish ponds with B. velezensis (Thurlow
et al., 2019). In our study, there was no improvement in TP removal after mixed Bacillus A0+BC addition,
whereas, mixed Bacillus A0+BS had higher performance in the accumulation and utilization of
phosphorus. These results might suggest suggesting that the synergic effect of Bacillus subtilis and
Bacillus megaterium played important roles in the removal TP. In addition, higher abundance of dominant
bacteria associated with the removal of nitrogenous and phosphorus compounds appeared in the
treatment A0+BS. Previous studies indicated that adding microbial agents every 7 days could maintain
good water quality (Liang et al., 2015; Zhang et al., 2013). Our results showed that the mixed Bacillus
A0+BS directly applied in aquaculture water as free bacteria every 13 days was a safety and effective
measure to improve water quality.

In addition to the influence of foreign bacteria, the microbial community structure in aquaculture water
might also be affected by water physicochemical characteristics (Hou et al., 2017; Ruprecht et al., 2021).
Zhang et al. (2016) found that NH4+-N and TN were major factors that shaped water microbial
communities in shrimp cultural ecosystems. Zhang et al. (2021a) showed that TN was the primary
environmental factor that shaped water microbial community divergence in Heihe river. In our study,
NH4+-N, NO2−-N, and TP were primary environmental factors. Therefore, creating favorable environmental
conditions for the growth and reproduction of functional bacteria, may be beneficial to improve the
removal effect of mixed Bacillus on nutrients nitrogen and phosphorus (Li et al., 2021).

According to PICRUST analysis and prediction, all functional pathways were up-regulated after mixed
Bacillus addition, and exhibited a higher functional abundance under the mixed Bacillus A0+BS
application, which might result in accelerated nitrogen cycles in aquaculture ecosystem (Arias-Andres et
al., 2018; Cornejo-D'Ottone et al., 2020). Membrane transport is one of the most important biological
processes (Cornejo-Granados et al., 2017). In our study, the pathway of membrane transport was the
most enriched in all functional pathways. Metabolism was the main category of taxonomic function
(Raiyani and Singh., 2020; Lu et al., 2020). The enriched amino acid metabolism, carbohydrate
metabolism, and energy metabolism explained the aquaculture water quality improvement achieved after
mixed Bacillus A0+BS addition. In addition, most of the secondary metabolites produced by
microorganisms through metabolism were allelochemicals, which would affect the competitive
relationship between microorganisms (Gomes et al., 2017; Song et al., 2017). Thus, in our study, different
allelochemicals produced by mixed Bacillus might be another reason for the differences in the microbial
community structure between the groups. These results showed that mixed Bacillus A0+BS promoted

Page 11/19
microbial growth and reproduction and shaped the microbial community structure and function, thereby
accelerating the conversion and utilization of nitrogenous and phosphorus compounds in aquaculture.

5. Conclusion
Mixed Bacillus directly used as free bacteria was applied to aquaculture and showed a relatively long
action time in this test without secondary pollution. The mixed Bacillus A0+BS effectively decreased the
nutrients nitrogen and phosphorus concentrations. Also, the addition of mixed Bacillus A0+BS shaped a
favorable microbial community structure, increased probiotics related to the removal of nitrogen and
phosphorus compounds and reduced the pathogenic bacteria in aquaculture. The functional pathways
were up-regulated under the mixed Bacillus A0+BS application, which promoted the conversion and
utilization of nitrogenous and phosphorus compounds in aquaculture ecosystem. In summary, the mixed
Bacillus A0+BS addition increased microbial diversity, inhibited pathogenic bacteria, and up-regulated
microbial functional pathways, thereby improving water quality. Therefore, the mixed Bacillus A0+BS
could be applied to aquaculture as a safer, more effective, and eco-friendly method to improve the water
quality and induce a stable microbial community structure.

Declarations
Availability of date and materials

All data generated or analyzed during this study are available within the article and its supplementary
materials.

Competing interests 

The authors declare no competing interests.

Funding 

This work was supported by Major science and technology project of water pollution control and
management in China (2015ZX07202-012) and the Central Public-interest Scientific Institution Basal
Research Fund (2020TD11).

Author contribution 

XL carried out all conceptualization, data curation, writing-original draft, writing-review and editing. TW
provided technical and theoretical support. BF (corresponding author) was responsible for this study and
provided funding acquisition and project administration. XM (corresponding author) help to guard the
manuscript and provide project administration.

Ethical approval and Consent to participate

Page 12/19
Not applicable.

Consent for publication 

Not applicable.

References
1. Arias-Andres M, Kettner MT, Miki T, Grossart HP (2018) Microplastics: new substrates for
heterotrophic activity contribute to altering organic matter cycles in aquatic ecosystems. Sci Total
Environ 635: 1152-1159.
2. Banerjee G, Ray AK (2017) The advancement of probiotics research and its application in fish
farming industries. Res Vet Sci 115: 66-77.
3. Bahram M, Netherway T, Frioux C, Ferretti P, Coelho LP, Geisen S, Bork P, Hildebrand F (2020)
Metagenomic assessment of the global distribution of bacteria and fungi. Environ Microbiol 23: 316-
316.
4. Cai Y, Yuan W, Wang S, Guo W, Li A, Wu Y, Chen X, Ren Z, Zhou Y (2019) In vitro screening of putative
probiotics and their dual beneficial effects: To white shrimp (Litopenaeus vannamei) postlarvae and
to the rearing water. Aquaculture 498: 61-71.
5. Chen Y, Liu Y, Wang X (2017) Spatiotemporal variation of bacterial and archaeal communities in
sediments of a drinking reservoir, Beijing, China. Appl Microbiol Biotechnol 101 (8): 3379-3391.
6. Chinese EPA (2002) Methods for the Examination of Water and Wastewater, fourth ed. China
Environmental Science Press, Beijing (in Chinese).
7. Choi YS, Hong SW, Kim SJ, Chung IH (2002) Development of a biological process for livestock
wastewater treatment using a technique for predominant outgrowth of Bacillus species. Water Sci
Technol 45: 71-78.
8. Chun SJ, Cui Y, Ahn CY, Oh HM (2018) Improving water quality using settleable microalga Ettlia sp.
and the bacterial community in freshwater recirculating aquaculture system of Danio rerio. Water Res
135: 112-121.
9. Conley DJ, Paerl HW, Howarth RW, Boesch DF, Seitzinger SP, Havens KE, Lancelot C, Likens GE (2009)
Controlling eutrophication: nitrogen and phosphorus. Science 323: 1014-1015.
10. Cornejo-D’Ottone M, Molina V, Pavez J, Silva N (2020) Greenhouse gas cycling by the plastisphere:
the sleeper issue of plastic pollution. Chemosphere 246: 125709.
11. Cornejo-Granados F, Lopez-Zavala AA, Gallardo-Becerra L, Mendoza-Vargas A, Sánchez F, Vichido
R, Brieba LG, Viana MT, Sotelo-Mundo RR, Ochoa-Leyva A (2017) Microbiome of Pacific Whiteleg
shrimp reveals differential bacterial community composition between Wild, Aquacultured and
AHPND/EMS outbreak conditions. Sci Rep 7: 11783.
12. Dawood MAO, Koshio S, Mohamed MAbdel‐Daim, Doan HV (2018) Probiotic application for
sustainable aquaculture. Rev Aquac 11 (3): 907-924.
Page 13/19
13. Duarte LN, Coelho F, Cleary D, Bonifácio D, Martins P, Gomes N (2019) Bacterial and microeukaryotic
plankton communities in a semi-intensive aquaculture system of sea bass (Dicentrarchus labrax): a
seasonal survey. Aquaculture 503: 59-69.
14. Elsabagh M, Mohamed R, Moustafa EM, Hamza A, Farrag F, Decamp O, Dawood MAO, Eltholth M
(2018) Assessing the impact of Bacillus strains mixture probiotic on water quality, growth
performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquac
Nutr 24: 1613-1622.
15. Gao J, Gao D, Liu H, Cai J, Zhang J, Qi Z (2018) Biopotentiality of High Efficient Aerobic Denitrifier
Bacillusmegaterium S379 for Intensive Aquaculture Water Quality Management. J. Environ Manage
222: 104-111.
16. Gentile ME, Jessup CM, Nyman JL, Criddle CS (2007) Correlation of Functional Instability and
Community Dynamics in Denitrifying Dispersed-Growth Reactors. Appl Environ Microbiol 73 (3): 680-
690.
17. Ghosh AK, Bir J, Azad MAK, Hasanuzzaman AFM, Islam MS, Huq KA (2016) Impact of commercial
probiotics application on growth and production of giant fresh water prawn (Macrobrachium
Rosenbergii De Man, 1879). Aquac Rep 4: 112-117.
18. Gomes MP, Garcia QS, Barreto L.C, Pimenta LPS, Matheus MT, Figueredo CC (2017) Allelopathy: an
overview from micro to macroscopic organisms, from cells to environments, and the perspectives in
a climate-changing world. Biologia 72: 113-129. 
19. Gomez-Gil B, Roque A, Turnbull JF (2000) The use and selection of probiotic bacteria for use in the
culture of larval aquatic organisms. Aquaculture 191: 259-270.
20. Goodfellow M, Fiedler HP (2010) A guide to successful bioprospecting: informed by actinobacterial
systematics. Antonie van Leeuwenhoek 98: 119-142.
21. Grossart H, Rojas-Jimenez K (2016) Aquatic fungi: targeting the forgotten in microbial ecology. Curr
Opt Microbiol 31: 140-145.
22. He T, Guan W, Luan Z, Xie S (2016) Spatiotemporal variation of bacterial and archaeal communities
in a pilot-scale constructed wetland for surface water treatment. Appl Microbiol Biotechnol 100:
1479-1488.
23. Hlordzi V, Kuebutornye F KA, Afriyie G, Abarike ED, Lu Y, Chi S, Anokyewaa MA (2020) The use of
Bacillus species in maintenance of water quality in aquaculture: A review. Aquac Rep 18: 10053.
24. Hoseinifar SH, Dadar M, Ringoe E (2017) Modulation of nutrient digestibility and digestive enzyme
activities in aquatic animals: the functional feed additives scenario. Aquac Res 48 (8): 3987-4000.
25. Hoseinifar SH, Sun Y, Wang A, Zhou Z (2018) Probiotics as means of diseases control in aquaculture,
a review of current knowledge and future perspectives. Front Microbiol 9: 2429.
26. Hou D, Huang Z, Zeng S, Liu J, Wei D, Deng X, Weng S, He Z, He J (2017) Environmental Factors
Shape Water Microbial Community Structure and Function in Shrimp Cultural Enclosure Ecosystems.
Front Microbiol 8: 2359.

Page 14/19
27. Hui C, Wei R, Jiang H, Zhao Y, Xu L (2019) Characterization of the ammonification, the relevant
protease production and activity in a high-efficiency ammonifier Bacillus amyloliquefaciens DT. Int
Biodeterior Biodegrad 142: 11-17.
28. Jahangiri L, Esteban M (2018) Administration of Probiotics in the Water in Finfish Aquaculture
Systems: A Review. Fishes 3: 33.
29. Jiang W, Ren W, Li L, Dong S, Tian X (2020) Light and carbon sources addition alter microbial
community in biofloc-based Litopenaeus vannamei culture systems. Aquaculture 515: 734572
30. John EM, Krishnapriya K, Sankar TV (2020) Treatment of ammonia and nitrite in aquaculture
wastewater by an assembled bacterial consortium. Aquaculture 526: 735390.
31. Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L (2012) Performance of single and multi-strain
probiotics during hatchery production of Green shell TM mussel larvae, Perna canaliculus.
Aquaculture 354: 56-63.
32. Kuebutornye F, Abarike ED, Lu Y (2019) A review on the application of Bacillus as probiotics in
aquaculture. Fish Shellfish Immunol 87: 820-828.
33. Li B, Jing F, Wu D, Xiao B, Hu Z (2020) Simultaneous removal of nitrogen and phosphorus by a novel
aerobic denitrifying phosphorus-accumulating bacterium, Pseudomonas stutzeri ADP-19. Bioresour
Technol 321: 124445.
34. Li C, Pan G, Wang X, Qiang X, Qiang T (2021) The effects of non-metallic organic tanning agents on
the microbial community structure in wastewater. J Clean Prod 279: 123553.
35. Liang Q, Zhang X, Lee KH, Wang Y, Yu K, Shen W, Fu L, Shu M, Li W (2015) Nitrogen removal and
water microbiota in grass carp culture following supplementation with Bacillus licheniformis BSK-4.
World J Microbiol Biotechnol 31 (11): 1711-1718.
36. Lieke T, Meinelt T, Hoseinifar SH, Pan B, Straus DL, Steinberg CEW (2019) Sustainable aquaculture
requires environmental-friendly treatment strategies for fish diseases. Rev Aquac12: 943-965.
37. Liu H, Li H, Wei H, Zhu X, Han D, Jin J, Yang Y, Xie S (2019) Biofloc formation improves water quality
and fish yield in a freshwater pond aquaculture system. Aquaculture 506: 256-269.
38. Lu L, Tan H, Luo G, Liang W (2012) The effects of Bacillus subtilis on nitrogen recycling from
aquaculture solid waste using heterotrophic nitrogen assimilation in sequencing batch reactors.
Bioresour Technol 124: 180-185.
39. Lu T, Xu N, Zhang Q, Zhang Z, Debognies A, Zhou Z, Sun L, Qian H (2020) Understanding the
influence of glyphosate on the structure and function of freshwater microbial community in a
microcosm. Environ Pollut 26: 114012.
40. Mook WT, Chakrabarti MH, Aroua MK, Khan GMA, Ali BS, Islam MS, Abu Hassan MA (2012) Removal
of total ammonia nitrogen (TAN), nitrate and total organic carbon (TOC) from aquaculture
wastewater using electrochemical technology: A review. Desalination 285: 1-13. 
41. Nai VH, Fotedar R (2010) A review of probiotics in shrimp aquaculture. J Appl Aquacult 22: 251-266.

Page 15/19
42. Nayak SK (2020) Multifaceted applications of probiotic Bacillus species in aquaculture with special
reference to Bacillus subtilis. Rev Aquac 13 (2): 862-906.
43. Olmos J, Ochoa L, Paniagua-Michel J, Contreras R (2011). Functional feed assessment on
Litopenaeus vannamei using 100% fish meal replacement by soybean meal, high levels of complex
carbohydrates and Bacillus probiotic strains. Mar Drugs 9 (6): 1119-1132.
44. Qiao Z, Sun R, Wu Y, Hu S, Liu X, Chan J, Mi X (2020) Characteristics and metabolic pathway of
sediment biomass for heterotrophic nitrification and aerobic denitrification in aquatic ecosystems.
Environ Res 191: 110069.
45. Raiyani NM, Singh SP (2020) Taxonomic and functional profiling of the microbial communities of
Arabian Sea: A metagenomics approach. Genomics 112 (6): 4361-4369.
46. Ren W, Wu H, Guo C, Xue B, Long H, Zhang X, Cai X, Huang A, Xie Z (2021) Multi-Strain Tropical
Bacillus spp. as a Potential Probiotic Biocontrol Agent for Large-Scale Enhancement of Mariculture
Water Quality. Front Microbiol 12: 699378.
47. Rout PR, Bhunia P, Dash RR (2017) Simultaneous removal of nitrogen and phosphorous from
domestic wastewater using Bacillus cereus GS-5 strain exhibiting heterotrophic nitrification, aerobic
denitrification and denitrifying phosphorous removal. Bioresour Technol 244: 484-495.
48. Ruprecht JE, Birrer SC, Dafforn KA, Mitrovic SM, Crane SL, Johnston EL, Wemheuer F, Navarro A,
Harrison AJ, Turner IL, Glamore WC (2021) Wastewater effluents cause microbial community shifts
and change trophic status. Water Res 200: 117206.
49. Schreier HJ, Mirzoyan N, Saito K (2010) Microbial diversity of biological filters in recirculating
aquaculture systems. Curr. Opin. Biotechnol 21: 318-325.
50. Schroeder J, Croot P, Von-Dewitz B, Walle U, Hanel R (2011) Potential and limitations of ozone for the
removal of ammonia, nitrite, and yellow substances in marine recirculating aquaculture systems.
Aquacult Eng 45 (1): 35-41.
51. Schuster SC (2008) Next-generation sequencing transforms today's biology. Nat Methods 50 (1): 16-
18.
52. Selim KM, Reda RM (2015) Improvement of immunity and disease resistance in the Nile tilapia,
Oreochromis niloticus, by dietary supplementation with Bacillus amyloliquefaciens. Fish Shellfish
Immunol 44: 496-503.
53. Shakya M, Gottel N, Castro H, Yang ZK, Gunter L, Labbé J, Muchero W, Bonito G, Vilgalys R, Tuskan G,
Podar M, Schadt CW (2013) A multifactor analysis of fungal and bacterial community structure in
the root microbiome of mature Populus deltoides trees. PloS One 8 (10): e76382.
54. Shapleigh JP (2011) Oxygen control of nitrogen oxide respiration, focusing on α-proteobacteria.
Biochem Soc Trans 39 (1): 179-183.
55. Sihag RC, Sharma P (2012) Probiotics: the new ecofriendly alternative measures of disease control
for sustainable aquaculture. J Fish Aquat Sci 7: 72-103.
56. Skjermo J, Bakke I, Dahle SW, Vadstein O (2015) Probiotic strains introduced through live feed and
rearing water have low colonizing success in developing Atlantic cod larvae. Aquaculture 438: 17-23.
Page 16/19
57. Soltani M, Ghosh K, Hoseinifar SH, Kumar V, Lymbery AJ, Roy S, Ringø E (2019) Genus Bacillus,
promising probiotics in aquaculture: aquatic animal origin, bio-active components, bioremediation
and efficacy in fish and shellfish. Rev. Fish. Sci. Aquac 27 (3): 331-379.
58. Song H, Lavoie M, Fan XJ, Tan HN, Liu GF, Xu PF, Fu ZW, Paerl HW, Qian HF (2017) Allelopathic
interactions of linoleic acid and nitric oxide increase the competitive ability of Microcystis
aeruginosa. ISME J 11: 1865-1876.
59. Su JF, Yang S, Huang TL, Li M, Liu JR, Yao YX (2019) Enhancement of the denitrification in low C/N
condition and its mechanism by a novel isolated Comamonas sp. YSF15. Environ Pollut 256:
113294.
60. Tang YY, Tao PY, Tan JG, Mu, HZ, Peng L, Yang DD, Tong SL, Chen LM (2014) Identification of
bacterial community composition in freshwater aquaculture system farming of Litopenaeus
vannamei reveals distinct temperature-driven patterns. Int J Mol Sci 15 (8): 13663-13680.
61. Thurlow CM, Williams MA, Carrias A, Ran C, Newman M, Tweedie J, Allison E, Jescovitch LN, Wilson
AE, Terhune JS, Liles MR (2019) Bacillus velezensis AP193 exerts probiotic effects in channel catfish
(Ictalurus punctatus) and reduces aquaculture pond eutrophication. Aquaculture 503: 347-356.
62. Velmurugan N, Kalpana D, Cho JY, Lee GH, Park SH, Lee YS (2011) Phylogenetic analysis of
culturable marine bacteria in sediments from South Korean Yellow Sea. Microbiology 80: 261-272.
63. Verbaendert I, Boon N, De Vos P, Heylen K (2011) Denitrification is a common feature among
members of the genus Bacillus. Syst Appl Microbiol 34: 385-391.
64. Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic bacteria as biological control
agents in aquaculture. Microbiol Mol Biol Rev 64: 655-671.
65. Wang A, Ran C, Wang Y, Zhang Z, Ding Q, Yang Y, Zhou Z (2018a) Use of probiotics in aquaculture of
China - a review of the past decade. Fish Shellfish Immunol 86: 734-755. 
66. Wang J, Chu L (2016) Biological nitrate removal from water and wastewater by solid-phase
denitrification process. Biotechnol Adv 34 (6): 1103-1112.
67. Wang R, Zhang Y, Xia W, Xin W, Guo C, Bowker J, Chen Y (2018b) Effects of aquaculture on lakes in
the Central Yangtze River Basin, China, I. water quality. N Am J Aquacult 80: 369-378.
68. Wang X, Chen Z, Shen J, Kang J, Zhang X, Li J, Zhao X (2020) Effect of carbon source on pollutant
removal and microbial community dynamics in treatment of swine wastewater containing antibiotics
by aerobic granular sludge. Chemosphere 260: 127544.
69. Wang X, Daigger G, de Vries W, Kroeze C, Yang M, Ren N-Q, Liu J, Butler D (2019) Impact hotspots of
reduced nutrient discharge shift across the globe with population and dietary changes. Nat Commun
10 (1): 2627
70. Wu D, Zhao S, Peng N, Xu C, Wang J, Liang Y (2014) Effects of a probiotic (Bacillus subtilis FY99-01)
on the bacterial community structure and composition of shrimp (Litopenaeus vannamei, Boone)
culture water assessed by denaturing gradient gel electrophoresis and high-throughput sequencing.
Aquac Res 47 (3): 857-869.

Page 17/19
71. Xia L, Xiong D, Gu Z, Xu Z, Chen C, Xie J, Xu P (2008) Recovery of Acinetobacter baumannii from
diseased channel catfish (Ictalurus punctatus) in China. Aquaculture 284 (1-4): 285-288. 
72. Xu D, Wang Y, Sun L, Liu H, Li J (2013) Inhibitory activity of a novel antibacterial peptide AMPNT-6
from Bacillus subtilis against Vibrioparahaemolyticus in shrimp. Food Control 30: 58-61.
73. Yang X, Wang S, Zhang D, Zhou L (2011) Isolation and nitrogen removal characteristics of an aerobic
heterotrophic nitrifying-denitrifying bacterium, Bacillus subtilis A1. Bioresour Technol 102 (2): 854-
862.
74. Yin H, Yang C, Jia Y, Chen H, Gu X (2018) Dual removal of phosphate and ammonium from high
concentrations of aquaculture wastewaters using an efficient two-stage infiltration system. Sci Total
Envir 635: 936-946.
75. Zeng X, Huang JJ, Hua B, Champagne P (2020) Nitrogen removal bacterial strains, MSNA-1 and
MSD4, with wide ranges of salinity and pH resistances. Bioresour Technol 310: 123309.
76. Zhang H, Sun Z, Liu B, Xuan Y, Jiang M, Pan Y, Gong C (2016) Dynamic changes of microbial
communities in Litopenaeus vannamei cultures and the effects of environmental factors.
Aquaculture 455: 97-108. 
77. Zhang Q, Jian S, Li K, Wu Z, Guan H, Hao J, Wang S, Lin Y, Wang G, Li A (2021a) Community
structure of bacterioplankton and its relationship with environmental factors in the upper reaches of
the Heihe River in Qinghai Plateau. Environ Microbiol 23 (2): 1210-1221.
78. Zhang X, Fu L, Deng B, Liang Q, Zheng J, Sun J, Zhu H, Peng L, Wang Y, Shen W, Li W (2013) Bacillus
subtilis SC02 supplementation causes alterations of the microbial diversity in grass carp water.
World J Microbiol Biotechnol 29 (9): 1645-1653. 
79. Zhang Z, Deng Q, Cao X, Zhou Y, Song C (2021b) Patterns of Sediment Fungal Community
Dependent on Farming Practices in Aquaculture Ponds. Front Microbiol 12: 542064.
80. Zhao J, Li Y, Chen X, Li Y (2018) Effects of carbon sources on sludge performance and microbial
community for 4-chlorophenol wastewater treatment in sequencing batch reactors. Bioresour
Technol 255: 22-28.
81. Zhao Y, Mao X, Zhang M, Yang W, Di HJ, Ma L, Li B (2021) The application of Bacillus Megaterium
alters soil microbial community composition, bioavailability of soil phosphorus and potassium, and
cucumber growth in the plastic shed system of North China. Agric Ecosyst Environ 307: 107236.
82. Zhao Z, Wang Q, Wang K, Brian K, Liu C, Gu Y (2010) Study of the antifungal activity of Bacillus
vallismortis ZZ185 in vitro and identification of its antifungal components. Bioresour Technol 101
(1): 292-297.
83. Zhou H, Wang X, Li Z, Kuang Y, Mao D, Luo Y (2018) Occurrence and Distribution of Urban Dust-
Associated Bacterial Antibiotic Resistance in Northern China.Environ Sci Technol Lett 5 (2): 50-55.
84. Zokaeifar H, Babaei N, Saad CR, Kamarudin MS, Sijam K, Balcazar JL (2014) Administration of
Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune
response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus
vannamei. Fish Shellfish Immunol 36: 68-74.
Page 18/19
Figures

Figure 1

Variations in the nitrogenous compounds and total phosphorus in aquaculture water. (A) NH4+-N, (B)
NO2--N, (C) NO3--N, (D) TP.

Figure 2

The bacterial and fungal community diversity in aquaculture water from CK, A0+BC, A0+BS group. (A)
OTUs, (B) Shannon Index and(C) Chao Index belong to the bacterial diversity. (D) OTUs, (E) Shannon
Index and(F) Chao Index belong to the fungal diversity. Different letters indicate significant differences
between communities (P< 0.05).

Figure 3

Relative abundance of bacteria in phylum (A) and genus (B) levels and fungus in genus (C) level in
aquaculture water. Redundancy analyses(D) of main bacteria phyla, genera and water quality in
aquaculture water.

Figure 4

Variation of metabolism function profiles of bacterial community between the three groups analyzed by
PICRUST.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.

GraphicalAbstract.png

Page 19/19