Effects of potassium monopersulfate on nitrification activity and bacterial community structure of sponge biocarrier biofilm in Litopenaeus vannamei aquaculture system

ABSTRACT Effects of potassium monopersulfate (KMPS) on the nitrification activity, aquacultural water quality and bacterial community structure of sponge biocarriers with pre-cultured biofilm (SBBF) were analysed through shaking flask experiments and L. vannamei aquaculture experiments. Changes in the ammonia oxidation rate (AOR) and nitrite oxidation rate (NOR) of SBBF under six KMPS concentration treatments (0, 1, 2, 3, 4 and 5 mg/L) were studied. The results showed that the AOR and NOR of SBBF treated with high concentrations of KMPS (3, 4 and 5 mg/L) were significantly lower than those of the control group (CK) (p < 0.05). However, compared with the first dosing of NH4Cl and NaNO2, the inhibition of AOR and NOR by KMPS on AOR and NOR was weakened after the second and third dosing times. That is, AOR and NOR can recover partially or completely over time. The L. vannamei aquaculture experiment was performed using four concentrations of KMPS (0, 2, 4 and 8 mg/L). The results showed that with increasing KMPS dosage, the average and peak concentrations of NH4+-N and NO2–-N in each treatment significantly increased (P < 0.05), and the final body weight of shrimp significantly decreased (P < 0.05). Furthermore the highest dose (8.0 mg/L) of KMPS reduced the survival rate by 9.33% compared to the CK. High-throughput sequencing analysis of the biofilm structure showed that the relative abundances of Nitrospirota, Nitrosomonas and Nitrococcus, which are related to nitrogen cycling, and beneficial bacteria including Firmicutes and Bacilli decreased with the addition of KMPS (p < 0.05). GRAPHICAL ABSTRACT


Introduction
Over the last few years, the world-wide production of Litopenaeus vannamei has increased due to the increasing demand for human consumption [1].The negative impacts of traditional aquaculture are widespread, including environmental hazards from the discharge of farm effluent, waste of water due to the absence of a water recycling system, nutrient pollution and frequent disease outbreaks in aquaculture animals [2].
Recirculating aquaculture systems (RASs) can control environmental conditions and allow for reuse of aquaculture water through filtration and purification, which helps to address some of the impacts of aquaculture systems [3].Compared to pond aquaculture, RAS seems to provide a long-term growth advantage [4].However, in closed aquaculture environments, a RAS can increase the risk of pathogen transmission caused by high-density farming [5].Current disinfection methods for aquaculture water include the use of antibiotics, UV irradiation treatments and ozone to reduce the bacterial load in the water and avoid the proliferation of potentially disease-causing microorganisms [6].These treatments are effective but have some drawbacks, such as gradual accumulation of antibiotics in water and increased resistance of animals and microorganisms to those antibiotics [7], impacts on the growth and repair mechanism of microorganisms due to UV radiation, causing growth cell death or regenerative cell death and the formation of ozone disinfection byproducts and the toxicity of residual cyclohexanone in shrimp limiting ozone wide application of RASs [6][7][8][9].
A possible alternative disinfectant is potassium monopersulfate, which has been widely used in epidemic prevention, medical environment elimination, foreign pathogen defence (COVID-19) and other fields [10][11][12].It is usually applied in the form of potassium monopersulfate triple salt (2KHSO 5 •KHSO 4 •K 2 SO 4 ) (KMPS), whose active ingredient and oxidation potential energy source is KHSO 5 (PMS).On the one hand, KMPS is more likely to be activated to produce sulphate radical anions due to its asymmetrical molecular structure [13].On the other hand, KMPS is traded in the form of powder, which is convenient to store, handle and transport.
The discharge standard of pollutants for aquaculture tailwater implemented in 2021 requires that the total nitrogen concentration in tailwater shall not exceed 5 mg/L during discharge.According to the statistics of the China Marine Ecological Environment Status Bulletin in 2020, inorganic nitrogen pollutants were the main index exceeding the standard of water in aquaculture areas.The inorganic nitrogen in aquaculture tail water mainly includes ammonia nitrogen, nitrite and nitrate, and concentration of ammonia nitrogen and nitrite exceeding their standards, it will cause the outbreak of aquaculture biological diseases and affect the growth and yield of aquaculture individuals.RAS reduces inorganic nitrogen by biofilm filtration, that is, nitrification by microorganisms attached to the substrate [14,15].A substrate suitable for microbial adhesion is the key to the development of biological filtration technology.Sponge biocarriers (SBs) have low cost, high porosity and suitability for stable attachment and growth of microorganisms and thus have received increasing attention from researchers in recent years [15][16][17].SB fillers can effectively enrich native microorganisms in water and remove some eutrophic substances, such as nitrogen and phosphorus, to purify water and are thus widely used in major industrial wastewater treatment and occasionally used in aquaculture [18].KMPS is widely used as a disinfectant and substrate modifier in the field of aquaculture [19].However, the impact of KMPS on the ammonia oxidation activity, nitrite oxidation activity, and microbial community structure composition of the RAS biofilm is currently unclear, and it is worth conducting in-depth research on its interaction effects.Consequently, this study aimed to (i) determine the effects of KMPS on the ammonia oxidation rate (AOR) and nitrite oxidation rate (NOR) of sponge biocarriers with precultured biofilm (SBBF) at different concentrations; (ii) analyse the effects of different doses of KMPS on the water quality and growth of L. vannamei in aquaculture systems; and (iii) compare the structural composition and diversity of SBBF biofilm bacterial communities using high-throughput sequencing methods.

Effect of KMPS on the nitrification activity of SBBF
The following experiments were performed to determine the stabilization of ammonia oxidation activity and nitrite oxidation activity under the regulation of KMPS.The experiments were carried out in six beakers, each with 1000 mL of experimental water and 10% (V/ V) SBBF with initial KMPS (Shanghai Macklin Biochemical Co., Ltd, CAS:70693-62-8) concentrations of 0 mg/L (CK), 1.0 mg/L (Group A), 2.0 mg/L (Group B), 3.0 mg/L (Group C), 4.0 mg/L (Group D) and 5.0 mg/L (Group E).Once KMPS was added, the solutions were treated with a six-joint electric stirrer for 2 h.
Five pieces (10% V/V) of KMPS-treated SBBF were randomly removed from each of the above six beakers and added to six 1000 ml flasks containing 400 ml artificial seawater and 0.1% (V/V) trace element solution.Ammonium chloride (Hushi, CAS:7647-14-5) was added to the flasks to provide an initial concentration of NH 4 + -N of 5 mg/L.Another six 1000 ml flasks were made with the same elements except for ammonium chloride, which was replaced with sodium nitrate (Hushi, CAS:7623-00-0) to make the initial concentration of NO During the experimental period, no water exchange was performed, and KMPS was added every five days.SBBF in 2 mm polyester mesh was immersed in the shrimp culture pond at a rate of 5% (V/V), and the SBBF was cleaned every 15 days to restore adsorption capacity.
Each experimental unit was stocked with 16 shrimp post-larvae (density: 800 shrimp/m 3 ).The temperature was controlled at 24-26°C by a heating rod, and DO was maintained between 7.0 and 8.5 mg/L.Shrimp with an average body length of 1.50 ± 0.10 cm and average weight of 0.1 ± 0.004 g were obtained from the Haida shrimp hatchery (Guangzhou).Feeding was performed 4 times a day, offering commercial bait (Shenzhen Aohua Group Co., LTD) at approximately 4.0% of the total shrimp weight.

Physicochemical analysis of water quality
The water quality of each culture system was monitored in terms of temperature, dissolved oxygen (DO), pH (HQ30D water quality analyser, Hach company, Loveland company, USA) and turbidity (2100Q turbidity analyser, Hach company, USA) in each tank were measured daily during the experiments.NH 4 + -N and NO 2 − -N were measured daily by Nessler's reagent spectrophotometry and N-(1-naphthyl)-ethylenediamine spectrophotometry respectively.NO 3 − -N was determined by the naphthylethylenediamine hydrochloride spectrophotometry method every 3 days [20,21].
Growth performance of L. vannamei At the end of the experiment, body weight (g) and number of individuals were determined.After that, the following variables were calculated: Survival rate (percent) = A f /A i × 100 where A f = final shrimp number, and A i = initial shrimp number.
DNA extraction and high-throughput sequencing method An E.Z.N.A.® soil DNA Kit (Omega Biotek, Norcross, GA, US) was used to extract the total DNA from five pieces biofilm samples from each container were collected on Days 15 and 30 of the experiment.The samples were placed into a beaker, distilled water was added, and biofilm samples were obtained after sonication for 15 min and were denoted as T0_15, T1_15, T2_15, T3_15, T0_30, T1_30, T2_30, and T3_30.The purity and concentration of the biofilm samples were checked by an ultramicro spectrophotometer P360 (Implen GmbH, München, Germany).After quality inspection, PCR amplification was performed [22].The sequence amplification region was 338F-806R, and the amplification primers used were 338F: 5 'ACTCCTACGGGGAGGCAG-CAG 3' and 806R: 5 'GGACTACHVGGGTWTCTAAT 3' The PCR product was extracted from a 2% agarose gel [4].The Illumina MiSeq sequencing platform was used for high-throughput sequencing, and BLAST analysis of all phyla and the top 30 genera in total abundance was performed.

Statistical analysis
Origin and SPSS were used for data processing, and the data are expressed as the mean and standard error (mean ± SE).Error bars indicate the standard error.ANOVA was used to determine significant differences, and P < 0.05 was considered significant.

Results
Variation in NH 4 + -N and NO 2 --N concentrations at different KMPS concentrations Changes in ammonia nitrogen and nitrite nitrogen concentrations in different experimental groups after three doses of ammonium chloride (sodium nitrite) were analysed (Figure 1 and 3).After the first addition of ammonium chloride, NH in Group E was three times longer than that in the CK.Similarly, after the second and third additions of sodium nitrite, the NO 2 --N conversion rates at different concentrations of KMPS were 5.42-5.83mg/L•h −1 and 5.78-6.16mg/L•h −1 , respectively.There was a significant difference in NO 2 --N conversion rates after the third addition of sodium nitrite compared to the first addition, with an extremely significant increase in NO 2 --N conversion rates (p = 0.000) (Figure 3).

Effect of KMPS on the AOR and NOR of SBBF
The AOR in each group increased gradually with increasing ammonium chloride addition times (Figure 4).There were significant differences in AOR levels between treatments at the same ammonium chloride addition times, and within each treatment group, the AOR increased with increasing ammonium chloride addition times.
The AOR was the highest in the CK group, and the lowest in Groups D and E. The change trend of the NOR was similar to that of the AOR and increased gradually with increasing sodium nitrite addition time.After adding sodium nitrite for the first time, the NOR decreased with increasing KMPS concentration to   different degrees.The NOR of the CK was the highest, and those of the D and E groups were the lowest.After adding sodium nitrite for the second and third times, the NOR in each system was higher than that after the first addition.

Effects of KMPS on water quality in L. vannamei aquaculture systems
Table 1 shows the water quality conditions during the aquaculture of L. vannamei.No significant differences in temperature, pH or DO concentration were found between treatments.The turbidity of each system showed an upwards trend, but at a low KMPS concentration, it did not differ significantly between systems.NH 4 + -N and NO 2 --N remained at low levels in the T0 systems (NH 4 + -N < 0.05 mg/L and NO 2 --N < 1.0 mg/L) (Figure 5).The concentrations of NH 4 + -N and NO 2 --N fluctuated with KMPS addition, and the fluctuation degree was T3 > T2 > T1 > T0.The NH 4 + -N content was higher overall in the experimental groups than in T0.NH 4 + -N in T1, T2 and T3 increased sharply after the addition of KMPS and then declined after 3 days, with average decreases of 27.27%, 14.28% and 5.77% in 6 cycles, respectively.The peak value of NH 4 + -N in the experimental groups was higher than that in T0 (P = 0.000), specifically, T3 > T2 > T1 > T0.The concentration of NO 2 --N varied greatly, and the mean NO 2 --N concentrations in the T1, T2 and T3 systems were 1.09 mg/L, 2.14 mg/L and 3.46 mg/L, respectively, showing 2.11fold, 5.11-fold and 8.89-fold increases compared with T0.Similarly, NO 2 --N accumulated immediately after the addition of KMPS and then declined after 4 days, and the peak value of NO 2 --N in the experimental groups was higher than that in T0 (P = 0.000).The NO 3 --N concentrations in all groups tended to rise throughout the experiment, and the order of accumulation was T0 > T1 > T2 > T3, with significant differences among the different systems (P = 0.000).

Effect of KMPS on the growth performance of L. vannamei
The final body weight of shrimp was significantly different among all treatments (Table 2).T0 presented the highest average weight gain at harvest, which was significantly higher than that of T1, T2 and T3.Survival rates on T0, T1 and T2 treatments were 100%, which was significantly higher than that of T3 (P = 0.000).For FCR, significant differences were observed  between T3 and the other treatments, but there were no significant differences between T0, T1 and T2 (P = 0.000).
Effect of KMPS on the bacterial community structure of the SBBF Numbers of operating units (OTUs) and alpha diversity indices of bacterial communities in SBBF biofilm samples from different systems are shown in Table 3.
The depth and coverage rate of each sample were higher than 50000 and 99%, respectively, indicating that the detected sequence results truly reflect the diversity of microbial communities.A total of 1214 OTUs were observed in nine samples, and a low average similarity and low number of shared OTUs among the samples were observed, representing 10.71% of the total reads (Figure 6).The OTU numbers at the initial time (P0_0) were the lowest, followed by the OTU numbers at 15 d and 30 d, and the Chao index and the Ace index were lowest at the initial time.When the samples of the same experimental group on the 30th day were compared with those on the 15th day, the Shannon index increased, while the Simpson index decreased.
The hierarchical clustering of the nine samples is shown in Figure 6.The structural composition of the bacterial community was more different in P0_0 than in the other eight groups of samples.The microbial communities differed significantly under different KMPS concentrations; biofilm samples from different time periods of T0 and T1 clustered together, while those of T2 and T3 were dispersed.This indicates that the similarity of bacterial community composition in the T2 and T3 systems was lower than that in the T0 and T1 systems, and the difference in bacterial community composition in the T2 and T3 culture systems was greater than that in the T0 and T1 systems.
The P0_0 sample was the farthest from the other 8 samples, indicating a significant difference in bacterial community composition between P0 and the other samples (Figure 7(a)).Biofilm samples with the same KMPS treatment concentration often gathered together, with a total variation of PC1 = 31.95%and PC2 = 28.30%,further confirming the interaction between KMPS and biofilm bacterial communities.The four selected environmental factors accounted for a total of 37.57% of the differences in OUT-level community structure, with RDA1 accounting for 23.42% and RDA2 accounting for 14.12% (Figure 7 Figure 8 shows the composition of the microbial community structure at the phylum to genus level.Thirtyfour bacterial phyla and 544 bacterial genera were detected.The diversity of bacteria in the P0 sample was lower than that in the other samples.The dominant phyla among the nine groups were Proteobacteria (38.55-71.60%),Bacteroidetes (7.29-12.01%),Actinobacteriota (0.35-16.01%),Planctomycetes (2.31-22.46%)and Chloroflexi (2.31-13.25%),and additional bacteria included Nitrospirota, Firmicutes and others (Figure 8 (a)).After the addition of KMPS, the relative abundance of Proteobacteria, Actinobacteriota, Nitrospirota and Chloroflexi decreased, while the relative abundance of Bacteroidetes increased.
The top 30 dominant genera in terms of total abundance are presented in the form of heatmap plots (Figure 8(b)).The dominant genera overall in biofilms were norank_f_Rhodobacteraceae (2.46-13.50%),Nitrococcus (1.38-9.16%)and Bacillus (0.27-8.04%).Consistent with the results of the phylum level analysis, the genus diversity in P0 was lower than that in the other samples, but the abundance of bacteria related to nitrification, such as Nitrococcus, Nitrosomonas and Nitratireductor, was more plentiful than that in the other biofilm samples.The abundance of norank_f_Rhodobacteraceae and Flavobacterium increased with increasing KMPS concentrations, whereas the abundance of Bacillus decreased with increasing KMPS concentration.

Discussion
Adding peroxides to the biofilter significantly reduces the removal efficiency of ammonium and nitrite, as peroxides reduce the abundance of ammonia oxidizing bacteria (AOB) and nitrate oxidizing bacteria (NOB).The results showed that KMPS inhibited both ammonia oxidation activity and nitrite oxidation activity of SBBF, and the AOR and NOR of the CK were higher than those of the experimental group.When KMPS concentrations were above 1.0 mg/L, ammonia oxidation activity was inhibited, while KMPS concentrations above 2.0 mg/L inhibited nitrite oxidation activity.Teitge et al. [23] tested the effect of different concentrations of peracetoacetic acid (PAA) on water quality and found that lower concentrations of peroxide had less effect on nitrification, similar to the results of this     AOB, NOB is more sensitive to peroxides.However, the ammonia oxidation process is more susceptible to organic matter.Schatteman et al. [27] studied the effect of phenylhydrazine on the rate of ammonia oxidation and found that 5 μM phenylhydrazine significantly inhibited the ammonia oxidation process, and the accumulation of nitrite decreased by 50% compared to the control group.The AOR was more affected by KMPS in the low concentration groups (1, 2 , and 3 mg/L), while nitrite oxidation activity was more easily inhibited by KMPS in the high concentration groups (4 and 5 mg/L).
With increasing dosing times of ammonium chloride and sodium nitrite, the AOR and NOR increased in each group, which shows that the inhibition of KMPS on the SBBF nitrification process is temporary and recoverable, similar to the results obtained by Chen et al. [28].
Water quality is considered a major limiting factor for shrimp survival, especially in terms of pH and DO, NH 4 + -N and NO 2 --N concentrations [29].Temperature and DO were maintained within the range suitable for shrimp growth [30].The turbidity of all systems increased due to bait residues and defecation but was maintained at a low level.Inorganic nitrogen is an important environmental factor in shrimp culture and has an important effect on the growth, survival and physiological functions of shrimp.In this study, NH 4 + -N and NO 2 --N concentrations were maintained at low levels in the 0.0, 2.0 and 4.0 mg/L KMPS experimental groups, but increased with increasing KMPS concentrations in the 2.0, 4.0 and 8.0 mg/L KMPS experimental groups, suggesting that the continued inhibitory effect of KMPS on nitrifying microorganisms led to the accumulation of NH 4 + -N and NO 2 --N in the water.
The biomass of shrimp is affected by inorganic nitrogen, especially nitrite and nitrate, which negatively affects the growth and survival of the organisms [31,32].As the KMPS concentration increased, the concentrations of NH 4 + -N and NO 2 --N increased and the final mean body weight of shrimp decreased.High concentrations of KMPS (≥ 4.0 mg/L) decreased the survival of L. vannamei, and the survival rate of shrimp in the experimental group with 8.0 mg/L KMPS was decreased by 9.33% compared to the experimental group with 0.0 mg/L KMPS.Meanwhile, the FCR of the group with 8.0 mg/L KMPS was relatively high, indicating that the addition of high-dose KMPS weakened the natural productivity of the culture system, thus reducing the feed conversion efficiency and slowing the growth rate of shrimp.Teitge et al. [23] found lower body weights in PAA-treated L. vannamei than in the control group, which is consistent with the results of this study.
High-throughput sequencing technology was used to analyse bacterial community diversity and abundance based on 16S rDNA amplicon sequencing technology [33].Microbial diversity is a key component in maintaining stable ecological functions, and in aquaculture systems, microbial diversity is closely related to shrimp diseases [34].Similar to other studies on the dominant phyla of shrimp, Proteobacteria, Bacteroidetes, Actinobacteriota and Chloroflexi were the dominant phyla [35,36].The results indicated that the dosing of KMPS reduced the diversity of the bacterial community structure in the experimental groups.As the concentration of KMPS increased, the relative abundances of Chloroflexi, Actinobacteriota and Nitrospirota decreased, and the relative abundance of the phylum Bacteroidetes increased.The relative abundances of Nitrosomonas and Nitrosococcus decreased in the experimental group with the highest KMPS concentration.Proteobacteria is the predominant gastrointestinal bacterial phylum present in the gut of healthy shrimp (L.vannamei) [37]; bacteria of this phylum are gram-negative with extremely diverse shapes and mainly participate in complexes and nitrogen degradation [38,39].Firmicutes breaks down carbohydrates, such as starch and fibre, that are difficult to digest in food, which increases the efficacy of the utilization of energy in the diet and reduces FCR and feed costs.Butyrate-producing bacteria in Firmicutes produce substances beneficial to the health of the host gut.Li et al. [40] found that the relative abundance of Firmicutes was one of the factors promoting rapid growth in transgenic common carp [35,41].Furthermore, Firmicutes also plays key roles in NH 4 + -N, NO 2 --N and NO 3 --N consumption [42].The reduction in ammonia nitrogen in aquatic systems is also closely related to the abundance of Planctomycetes.Planctomycetes contains a large number of anaerobic ammonia-oxidizing bacteria that play an important role in the nitrogen cycle by reducing inorganic nitrogen to produce nitrogen (N 2 ).Anammox is an advantageous process for biological denitrification of wastewater, reducing sludge production and providing significant energy savings, and has been successfully implemented in a variety of practical wastewater treatment applications, such as domestic wastewater, waste leachate and yeast factory wastewater [43,44].However, aquaculture has a lower concentration of inorganic nitrogen and requires sufficient dissolved oxygen, which is not suitable for the survival of anaerobic ammonia oxidation bacteria, making anaerobic ammonia oxidation much less effective.Planctomycetes are widespread in natural and artificial systems and have been shown to play an important role in denitrification systems for agricultural and domestic wastewater [21,45,46], and the lower concentration of inorganic nitrogen in the 30-d biofilm sample of the 2 mg/L KMPS experimental group may be related to denitrification.The reduced abundance of Proteobacteria, Firmicutes and Planctomycetes caused by a high dose of KMPS may further interfere with both the digestive and immune capacity of L. vannamei, leading to a decrease in the shrimp survival rate and an increase in FCR.Actinobacteria are an important part of the degradation of organic matter, mainly macromolecules, such as starch and protein, and play a significant position in the natural nitrogen cycle [47,48].Although the relative abundance of Actinobacteria varies with shrimp habitat and feed composition, studies have shown that Actinobacteria are always one of the dominant phyla in the gastrointestinal tract of the L. vannamei gut [37,49].In this study, a lower abundance of Actinobacteria led to a higher feed conversion rate of shrimp.Nitrospirota are closely related to the biological nitrification process, and their relative abundance directly influences the ammonia/nitrate/nitrite cycling system, which in turn affects water quality and shrimp health [18,50].Gao et al. [51] believed that nitrification performance was significantly correlated with the number of nitrifying bacteria.In this experiment, when the level of KMPS addition was 4 mg/L and 8 mg/L, the abundance of Nitrospirota in the T2_30 and T3_30 samples decreased by 72.99% and 66.67%, respectively, compared with that of T0, leading to the accumulation of NO 2 --N in the water, affecting the survival of L. vannamei, reducing the final shrimp yield, and improving the feed conversion rate, and these findings may be related to the inhibitory effect of KMPS on NOB, leading to a decrease in the NOR.Bacteroidetes are considered one of the richer heterotrophic bacteria in aquatic environments, with the ability to use a wide range of carbohydrates and breakdown organic matter to provide nutrients and energy for the host [52,53].The relative abundance of Bacteroidetes in the 8.0 mg/L KMPS experimental group was 7.83% higher than that in the 0.0 mg/L KMPS experimental group at 30 d.The Flavobacterium family of the phylum Bacteroidetes is pathogenic and may be one of the reasons for the slow growth and reduced survival of shrimp in the 8.0 mg/L KMPS experimental group.
Nitrosomonas is a typical AOB responsible for the nitrification of NH 4 + -N and NO 2 --N [54].Nitrate is the main form of biologically active nitrogen, which is produced through the nitrite oxidation process by nitrite oxidizing bacteria (NOB) such as Nitrococcus and Nitrobacter [55].Nitratireductor are aerobic gram-negative bacteria with the ability to reduce nitrate to nitrite under anoxic conditions, and they also participate in the degradation of organic pollutants [56,57]; in the high concentration KMPS experimental group, the relative abundance of these bacteria decreased.The abundance of Nitrosomonas and Nitrococcus in the 8.0 mg/L KMPS experimental group decreased by 34.02% and 45.22%, respectively, and the decline of water quality may be related to the reduction of AOR and NOR caused by the inhibition of KMPS on AOB and NOB.The existence of Rhodobacteraceae can effectively remove phosphorus [58,59].The relative abundance of norank_f__Rhodobacteraceae in T1 was the highest, followed by that in T2 and T3, and the average concentration of total phosphorus was also the lowest in T3.Bacilli play a role as probiotics, aiding in growth, nutrient absorption and health [60].The relatively high abundance of these bacteria in systems with low doses of KMPS indicates that KMPS can directly influence the presence of beneficial bacteria in the culture system, which could help to explain why growth was reduced when the KMPS dose was increased.
In this study, the effects of KMPS on the nitrification activity and growth performance of Litopenaeus vannamei and the bacterial community structure of sponge biocarriers with precultured biofilms (SBBFs) were analysed through shaking flask experiments and L. vannamei aquaculture experiments.The following conclusions were drawn: KMPS inhibited the ammonia oxidation activity and nitrite oxidation activity of the nitrifying biofilm, and the inhibition increased with increasing of KMPS concentration.The inhibition of nitrifying biofilms by KMPS was temporary and reversible; as the NH 4 Cl and NaNO 2 doses increased, the AOR and NOR of the experimental groups with different concentrations of KMPS increased.
The application of KMPS affects the water quality of L. vannamei cultures.While NH 4 + -N and NO 2 --N concentrations were maintained at low levels in the 0.0, 2.0 and 4.0 mg/L KMPS experimental groups, high KMPS concentrations (≥ 4.0 mg/L) were able to affect the survival of L. vannamei, reduce the final production of shrimp and increase feed conversion rates.High-throughput sequencing of SBBFs showed that the dosing of KMPS reduced the diversity of the bacterial community structure in the experimental groups.As the concentration of KMPS increased, the relative abundances of Chloroflexi, Actinobacteriota and Nitrospirota decreased, and the relative abundance of the phylum Bacteroidetes increased.The relative abundances of Nitrosomonas and Nitrosococcus were decreased in the experimental groups with high KMPS concentrations, leading to a reduction in the AOR and NOR and the deterioration of water quality.

Figure 2 .
Figure 2. Accumulation of NO 2 --N in different groups after sponge biocarriers with precultured biofilms treatment with different concentrations of KMPS.

Figure 3 .
Figure 3. Removal process of NO 2 --N after sponge biocarriers with precultured biofilms treatment with different concentrations of KMPS.

Figure 1 .
Figure 1.Removal process of NH 4 + -N after sponge biocarriers with precultured biofilms treatment with different concentrations of KMPS.

Figure 4 .
Figure 4. Variation in the AOR and NOR in different groups after sponge biocarriers with precultured biofilms treatment with different concentrations of KMPS.

4 +-N and NO 2 -
(b)).NO 3 --N is the main environmental factor, and KMPS concentration is positively correlated with the NH -N contents.

Figure 5 . 2 -
Figure 5.The concentration variations of NH 4 + -N, NO 2 --N and NO 3 --N in L. vannamei aquaculture systems with sponge biocarriers after treatment with different concentrations of KMPS (period 30 d, ↓ indicates the addition of KMPS).

Figure 6 .
Figure 6.Comparison of OTUs (a) and hierarchical clustering (b) of the SBBF biofilm samples treatment with different concentrations of KMPS.The numbers in the ellipse represent the OTUs specific to each sample.

Figure 8 .
Figure 8.The bacterial community in sponge biocarriers samples treatment with different concentrations of KMPS at the phylum (a) and genus (b) levels.
The peak concentration of NO 2 --N accumulation with 4 and 5 mg/L KMPS after the third addition of sodium nitrite decreased by 48.83% and 44.34%, respectively, compared with the second addition.After the first addition of sodium nitrite, the conversion rate of the CK (4.67 mg/L•h −1 ) was significantly higher than that of the other groups (4.27 mg/L•h −1 , 3.99 mg/L•h −1 , 3.32 mg/L•h −1 , 1.59 mg/L•h −1 , and 1.35 mg/L•h −1 ; P = 0.000), and the complete transformation time of NO 2 --N 4 + -N conversion in Groups A, B, C, D and E within 48 h (1.52-28.60%)wassignificantlylowerthan that in the CK (41.53%) (P = 0.000).The time required for NH 4 + -N to decrease to undetectable levels in Groups D and E was almost twice as long as that in the CK, which meant that NH 4 + -N conversion was reduced by 46%.After the second and third addition of ammonium chloride, the total transformation time of NH 4 + -N in each group was reduced by 14.3%, 25.0%, 33.3%, 45.5%, 53.8%, and 53.8%, when compared with those after the first addition.The accumulation of NO 2 --N in the NH 4 +-N removal process of each experimental group is shown in Figure2.The NO 2 --N concentration tends to increase and then decrease.After the first addition of sodium nitrite, NO 2 --N peak concentrations in the CK were significantly lower than those in Group A (P = 0.017) and extremely significantly lower than those in Groups B, C, D, and E (P = 0.000), with 5 mg/L KMPS taking longer to reach peak concentrations and having the highest peak concentration (108 h, 3.19 mg/L).

Table 1 .
Physical and chemical parameters of water in the L. vannamei aquaculture systems treatment with different concentrations of KMPS.
Note: Different superscripts in the same row indicate significant differences (p < 0.05).

Table 2 .
Growth performance of L. vannamei in different experimental treatments during aquaculture treatment with different concentrations of KMPS (30 days).

Table 3 .
OTU numbers and alpha diversity index of the SBBF biofilms treatment with different concentrations of KMPS.