Effects of Oyster Shell Addition on Shrimp Aquaculture and the Dynamic Succession of Surface Biofilm Microbial Communities

doi:10.21203/rs.3.rs-4867248/v1

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Effects of Oyster Shell Addition on Shrimp Aquaculture and the Dynamic Succession of Surface Biofilm Microbial Communities

https://doi.org/10.21203/rs.3.rs-4867248/v1

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In the context of Litopenaeus vannamei aquaculture, the incorporation of oyster shells has proven beneficial for enhancing water quality and the growth conditions of the shrimp. Nonetheless, the specific effects of in-situ water treatment using oyster shells on water quality and shrimp growth, along with the composition and succession dynamics of the microbial community within oyster shell biofilms, have yet to be thoroughly investigated. This study established control, low-concentration, and high-concentration oyster shell addition groups to emulate the in-situ water treatment environment with oyster shells, with the objective of elucidating the impacts of oyster shell addition on the aquaculture setting. The results showed that the addition of oyster shells exerted positive influences on shrimp length, weight, and survival rate, without significant disparities between the high and low concentration groups. Additionally, oyster shell addition resulted in elevated phosphate levels, diminished nitrite levels, and increased nitrate concentrations. Within the biofilm, the relative abundances of Ruegeria, Tenacibaculum, BD2-11_terrestrial_group, and Kapabacteriales exhibited significant declines over time, whereas the relative abundance of Nitrospira demonstrated a marked increase, ultimately emerging as the predominant bacterium in the biofilm during the latter stages of the experiment. Nitrospira also exhibited a notably higher relative abundance in the microbial community of the experimental water group relative to the control group. This research offers valuable insights for the application of oyster shells in shrimp farming and contributes to the theoretical underpinnings necessary for advancing our understanding of the mechanisms through which oyster shell biofilms enhance water quality and foster shrimp health.

Litopenaeus vannamei

oyster shells

biofilm

microbial community

The Pacific White Shrimp (Litopenaeus vannamei), celebrated for its rapid growth, robust survival rate, and formidable adaptability, has emerged as a pivotal industry in global marine aquaculture, commanding substantial economic value(Liao & Chien, 2011). The aquaculture of the Litopenaeus vannamei is significantly impacted by a range of factors, encompassing water quality conditions, environmental variables, aquaculture infrastructure, seed stock quality, and dietary composition. As a tropical shrimp species highly sensitive to water quality, the growth trajectory, health status, and survival rates of the Litopenaeus vannamei are intricately tied to key water parameters such as dissolved oxygen, pH levels, ammonia nitrogen, nitrite, and salinity(Villarreal & Juarez, 2022). Dissolved oxygen is an essential element for the shrimp's survival, insufficient levels can retard growth and may even lead to asphyxiation(Simbeye & Yang, 2014). Fluctuations in pH beyond normal ranges can disturb the shrimp's osmotic equilibrium, compromising their immune function and thereby weakening their disease resistance(Mustapha & Scientific, 2020). Maintaining an optimal salinity range can mitigate the osmotic pressure regulation demands on the shrimp, thereby enhancing their environmental adaptability(Z. Li et al., 2024). Ammonia nitrogen and nitrite are prevalent water contaminants; at elevated concentrations, they pose a toxic threat to the shrimp(Valencia-Castañeda et al., 2018). Prolonged exposure to these substances can hinder shrimp growth, impair immune function, and potentially culminate in widespread mortality(M. Huang et al., 2020).

Oyster shells play a significant role in enhancing aquaculture water quality during shrimp cultivation(X. Huang et al., 2020; Jones, Preston, & Dennison, 2002). Abundant in calcium carbonate, these shells are instrumental in maintaining optimal pH levels within the water, thereby supporting the molting and new shell formation processes in shrimp(Shen YuChun, Xiong BangXi, Wang Hui, & Ye FuLiang, 2007). Moreover, oyster shells exhibit a high adsorption capacity, effectively binding and sequestering harmful substances such as ammonia nitrogen, nitrite, and organic pollutants(An et al., 2024; Patil, Lee, Hwang, Joo, & Kim, 2024). This capability significantly diminishes the concentration of these detrimental elements in the water, thereby reducing the associated risks to shrimp health. Additionally, oyster shells provide an ideal substrate for microbial colonization, promoting the establishment of beneficial microbial films within the aquatic environment.

Microbial films are characterized by their diverse composition, encompassing bacteria, algae, protozoa, and fungi, which together create a dynamic ecosystem on the surfaces of oyster shells(Ivanov, Stabnikova, Sihanonth, & Menasveta, 2006). These microorganisms play a crucial role in the cycling of organic matter and nutrients within the aquatic environment(Geng et al., 2024). Specifically, Nitrosomonas bacteria catalyze the oxidation of ammonia nitrogen in the water to nitrite, a process followed by Nitrobacter bacteria which convert nitrite into nitrate(Daims et al., 2015; Van Kessel et al., 2015). Algae then utilize these nitrate and phosphate compounds as essential nutrients for their growth and reproduction. Additionally, the microorganisms present in the microbial film can be integrated into the food chain, ultimately affecting the microecological balance within the intestines of the shrimp(Zeng, He, & Huang, 2024).

Although the positive role of oyster shells in shrimp aquaculture has gained increasing recognition, the regulatory efficacy of oyster shells in shrimp farming still warrants further validation. Moreover, the mechanisms through which the microbial film on oyster shells influences water quality and the intestinal environment of shrimp remain to be fully understood. This study is designed to explore these aspects by setting up groups with different concentrations of oyster shell additions and monitoring the shrimp cultivation outcomes. Specifically, the research aims to: 1) assess whether there are differences in shrimp cultivation performance among groups with varying concentrations of oyster shells; 2) evaluate the differential effects of different oyster shell concentrations on water quality; and 3) determine whether the microbial film on oyster shells at different concentrations has varying impacts on the shrimp.

Throughout the cultivation process, there was a general upward trend in the levels of chemical oxygen demand (COD), suspended substance, phosphorus, nitrite, ammonium, and nitrate (Fig 1B, Supplementary Table1, Table2). Significant inter-group differences in COD emerged starting from the 7th day, escalating to extremely significant variations among groups from the 49th day onwards. However, a consistent trend of continuous inter-group differences over time was not observed. Similarly, no consistent inter-group differences were noted in suspended substance. Ammonium levels rose significantly and rapidly from the 7th day, with the control group exhibiting consistently higher concentrations than the experimental groups. By the 28th day, the ammonium content in the control group experienced a sharp decline, dropping below that of the experimental groups. Nitrite concentrations in the control group were initially lower than those in the experimental groups until the 14th day, after which they remained consistently higher from the 35th day onwards. Starting from the 7th day, nitrate levels in the control group were persistently lower than those in the experimental groups, while no consistent significant differences were discerned over time between the low-concentration and high-concentration groups. Phosphorus concentrations were consistently lower in the control group compared to the experimental groups, with significant differences becoming evident from the 7th day and extremely significant differences from the 21st day onwards.

On the 70th day of the cultivation period, a statistical analysis was conducted on the body length, weight, and survival rate of the shrimp in each group. The findings revealed that the control group had significantly lower values in terms of body length, weight, and survival rate when compared to the experimental groups (Fig 1C, Supplementary Table3). Within the experimental groups, a significant difference was observed in weight, with the high-concentration group exhibiting a higher average weight than the low-concentration group. However, no significant differences were detected in body length and survival rate between the low and high concentration groups.

The correlation analysis between the growth indices of shrimp and the hydrochemical factors revealed that there was a significant negative correlation between the concentration of nitrite and the body length of the shrimp, while a significant positive correlation was observed between phosphate and body length (Fig 1D, Supplementary Table4).

On the 70th day, there were significant differences in the beta diversity of bacterial communities in the water body as a whole, but no significant differences between the control and experimental groups over time. In the shrimp intestines, there were significant differences in the beta diversity of bacterial communities as a whole, with significant differences between the control and experimental groups, but no significant differences between the high and low concentration groups (Fig2C, Supplementary Table5). Multiple indicators of alpha diversity in the low concentration group showed significant differences (Fig2D, Supplementary Table6, Table7).

In the biofilm, Ruegeria, Tenacibaculum, Nitrospira, BD2-11_terrestrial_group, and Kapabacteriales were the dominant bacteria on day 0 (Fig 3A, Supplementary Table8). Among these, Ruegeria exhibited significant differences over time with an ANOVA F-value of 6.103 and P < 0.001, Tenacibaculum showed an F-value of 5.11 and P = 0.001, BD2-11_terrestrial_group had an F-value of 8.170 and P < 0.001, and Kapabacteriales had an F-value of 2.444 and P = 0.05. These bacteria demonstrated a significant decrease in relative abundance over time. In contrast, Nitrospira showed a significant increase in relative abundance over time with an F-value of 5.951 and P < 0.001. Beginning on day 56, Nitrospira became the most abundant dominant bacterium in the biofilm, reaching relative abundances of 19.6% and 44% in the low and high concentration groups, respectively, by day 70. Additionally, Nitrospira was also the dominant bacterium in the water, with concentrations as high as 12.3% and 7.1% in the experimental groups, and only 0.1% in the treatment groups (Fig 3B, Supplementary Table9). Nitrospira was not the dominant bacterium in the shrimp gut. Burkholderia-Caballeronia-Paraburkholderia, Ruegeria, PeM15, and Mycobacterium were dominant in both water and the shrimp gut, with significant inter-group differences observed for Burkholderia-Caballeronia-Paraburkholderia, Ruegeria, and PeM15 in the gut.

Through one-way ANOVA and random forest analysis, we identified nine key bacterial genera, WPS-2, Bauldia, SWB02, KI89A_clade, Nitrosomonas, Woeseia, PS1_clade, BD2-11_terrestrial_group, and Truepera, in water that exhibited significant inter-group differences (Fig4A, 4B, Supplementary Table9, Table10). Among these, WPS-2 and Bauldia were significantly positively correlated with Chemical Oxygen Demand and Suspended Substance, SWB02 and KI89A_clade were significantly positively correlated with phosphate, and Nitrosomonas was significantly positively correlated with nitrate and phosphorus (Supplementary Table12). WPS-2, Bauldia, and KI89A_clade were significantly positively correlated with shrimp weight, and KI89A_clade was also significantly positively correlated with survival rate(Supplementary Table13).

Tenacibaculum, Kriegella, KI89A_clade, Iamia, JG30-KF-CM45, Fulvivirga, Maribacter, Ruegeria, Mycobacterium, BIrii41, Aureimarina, Ilumatobacter, Halocynthiibacter, SWB02, Nitrosomonas, A4b, Thioclava, and Gracilibacteria were identified as bacteria with significant inter-group differences in the gut (Fig 4A, 5A, Supplementary Table9, Table11). Among these, Tenacibaculum was significantly positively correlated with nitrate and phosphorus. Kriegella, KI89A_clade, Iamia, JG30-KF-CM45, Fulvivirga, Maribacter, and Ruegeria were all significantly positively correlated with Chemical Oxygen Demand and Suspended Substance, while BIrii41 and Mycobacterium were significantly positively correlated with nitrate (Fig 5B, Supplementary Table14). Tenacibaculum was significantly positively correlated with shrimp length and survival rate, while Ilumatobacter was significantly positively correlated with shrimp weight and survival rate (Fig 5C, Supplementary Table15).

The addition of oyster shells could improve the growth indicators of shrimp

The body length, weight, and survival rate of shrimp were significantly lower in the control group compared to the experimental group, thereby confirming that the addition of oyster shells not only enhanced survival rates but also fostered superior growth conditions for individual shrimp. Despite identical feed inputs between the control and experimental groups, the latter demonstrated higher survival rates, with each shrimp receiving, on average, less feed than those in the control group. This finding indicates that the incorporation of oyster shells also improved feed utilization efficiency.

In comparing the high-concentration and low-concentration oyster shell addition groups, it was observed that no significant differences in body length, weight and survival rate. The increased concentration of oyster shell addition did not result in superior biological enhancement, indicating that an addition rate of 4‰ may have already reached the functional limit for oyster shell enhancement within the aquaculture system, or other factors may be constraining the biological enhancement potential of oyster shells. The excessive addition of oyster shells could potentially disturb the water's acid-base equilibrium and elevate pH levels. However, an addition of 16‰ oyster shells did not constitute an excessive level, suggesting that the threshold for detrimental effects has not yet been reached.

The addition of oyster shells could improve the water quality in aquaculture

The monitoring of hydrochemical indicators throughout the aquaculture process demonstrated that oyster shells had a marked impact on enhancing water quality. Phosphate, among the hydrochemical factors, correlated positively with shrimp body length, weight, and survival rate, with concentrations notably lower in the control group than in the experimental group. As a vital water buffer, phosphate stabilizes pH levels and bolsters the shrimp's immune system and resilience, thereby fostering robust growth(Nathanailides et al., 2023; Vijuksungsith et al., 2023). In contrast, nitrite displayed a negative correlation with these same parameters, with concentrations surpassing those of the control group after 35 days. The recognized adverse effects of nitrite on shrimp aquaculture are well-documented(Gross, Abutbul, & Zilberg, 2004; Tomasso, 2012). The initial lower nitrite levels in the control group could be attributed to the high water exchange rates, as ammonia nitrogen derived from feed serves as a primary source of nitrite. As feed input escalated during the aquaculture cycle, ammonia nitrogen levels increased; however, the control group's daily 10% water exchange rate effectively decelerated ammonia nitrogen accumulation, thereby mitigating nitrite buildup. Conversely, the experimental group, with a daily 2% water exchange rate, witnessed more rapid nitrite accumulation relative to the control group. During the later stages of the culture period, the bacterial composition of the biofilm on the oyster shells underwent changes, with Nitrospira becoming overwhelmingly dominant in terms of abundance from day 56, speeding up the process of converting nitrite to nitrate. Consequently, this resulted in a higher concentration of nitrate in the experimental group than in the control group.The incorporation of oyster shells not only augmented phosphate levels but also curbed nitrite concentrations, substantiating the biological enhancement capabilities of oyster shells.

Nitrospira, Ruegferia, and Tenacibaculum were the dominant bacteria in the biofilm

In the bacterial community of the biofilm, prior to the emergence of Nitrospira as the dominant bacteria, Ruegeria and Tenacibaculum were the predominant species within the biofilm. Ruegeria, an aerobic chemoheterotrophic bacterium, was noted for its high capacity for polysaccharide metabolism, which enabled it to contribute to the degradation of algal cell walls(Huo et al., 2011; Yin et al., 2023). Research had demonstrated that Tenacibaculum could rapidly establish biofilms on polystyrene surfaces, exhibiting hydrophobic characteristics that were likely associated with biofilm formation. Additionally, Tenacibaculum maritimum, recognized as a fish pathogen, was known to possess the ability to form biofilms(Avendaño-Herrera, Toranzo, & Magariños, 2006; Levipan et al., 2019).

Regardless of whether in the control or experimental groups, the concentrations of ammonia and nitrite in the water were notably high. However, Nitrospira was not the dominant bacterial genus within the bacterial communities of the control sample waters. It is hypothesized that Nitrospira in both high and low concentration groups originated from biofilms. Microorganisms on the surfaces of oyster shells interconnected through an extracellular polymeric substance matrix, forming a stable community structure. This structure not only provided physical protection for the microorganisms but also conferred advantages in resource acquisition and substance exchange. The favorable microenvironment within the biofilm promoted the growth and activity of Nitrospira(Vijayan et al., 2021; Zhou et al., 2023). The significant enrichment of Nitrospira within the biofilms led to increased accumulation of this genus in water, which, through water and feed, influenced the distribution of Nitrospira within the gut (F = 6.253, P = 0.020); however, it did not dominate the gut microbiota.

The relative abundance of Nitrospira in biofilm of the high concentration group was higher than that in the biofilm of the low concentration group. However, in water, the abundance of Nitrospira was higher in the low concentration group. From day 63 onwards, although there were still significant overall differences in nitrite levels, there were no significant differences between the high and low concentration groups, the treatment group and the high concentration group, or the control group and the low concentration group. This suggests that the nitrite concentration might have constrained the biofilm improvement ability in the high concentration group. The addition of oyster shells at both 4‰ and 16‰ had already provided sufficient Nitrospira to the aquaculture system.

In both water and gut environments, besides Nitrospira, Ruegeria, and Tenacibaculum, Burkholderia-Caballeronia-Paraburkholderia were identified as important differential dominant bacteria. These bacteria, belonging to the Burkholderia genus, were capable of assisting shrimp in digesting food, inhibiting pathogens, and even participating in the shrimp's immune system(Kaur, Selvakumar, & Ganeshamurthy, 2017; Suárez-Moreno et al., 2012). They also played roles in nitrogen fixation and aiding plant nutrient absorption(Sessitsch et al., 2005). Their dominance was likely related to their diverse biological functions.

Tenacibaculum, KI89A_clade, SWB02, and Nitrosomonaswere the inter-group differential bacteria in water and gut

Through the application of random forest analysis and one-way ANOVA, we identified significant inter-group differences in the relative abundance of Tenacibaculum within the shrimp gut. This abundance was notably correlated with shrimp growth indicators, floating particulate matter, and chemical oxygen demand. The relative abundance of Tenacibaculum in biofilms exhibited a gradual decline. We hypothesized that this phenomenon is attributable to the critical role of Tenacibaculum in the formation and structural stability of biofilms(Levipan et al., 2019). During the initial stages of biofilm formation, Tenacibaculum, which aids in establishing a stable biofilm structure, enjoys a competitive advantage in the ecological niche. However, as the biofilm structure matures and the concentration of nitrite increases, Nitrospira, with its nitrogen-fixing capabilities, gains a competitive edge, leading to a decrease in the relative abundance of Tenacibaculum(Palomo, Dechesne, Pedersen, & Smets, 2022). Throughout the aquaculture process, Tenacibaculum continually permeated from the biofilm into the water and the shrimp gut, resulting in a positive correlation between Tenacibaculum abundance and shrimp growth.

KI89A_clade, SWB02, and Nitrosomonas were significantly positively correlated with phosphate, while Nitrosomonas was also significantly positively correlated with nitrate in water. KI89A_clade is a bacterial group whose relationship with shrimp growth remains unclear, although studies have suggested it might be involved in nutrient cycling, particularly in denitrification and nitrogen fixation processes(S. Y. Q. Y. X. Li, Wei, & Ba, 2023; Ruprecht et al., 2021). SWB02 was found to enhance ammonia-oxidizing capacity and produce large quantities of extracellular polymeric substances (EPS). These substances can form granules in ammonia-oxidizing systems, aiding in biofilm formation and potentially influencing the structure and function of microbial communities in biotreatment processes(Han, Yang, Fan, & Jin, 2024). Nitrosomonas, an important group of nitrifying bacteria, plays a key role in the first stage of nitrification by oxidizing ammonia (NH4+) to nitrite (NO2-)(Arp, Sayavedra-Soto, & Hommes, 2002). However, due to the rapid conversion of nitrite to nitrate by Nitrospira, Nitrosomonas showed a significant correlation with nitrate concentration. These processes may have indirect interactions with phosphate, which is a limiting factor for algae growth, and algae are a crucial component of primary producers in the aquatic environment(Lobus, 2022).

The addition of oyster shells had a positive impact on shrimp aquaculture, although no significant differences were observed between groups with varying concentrations of oyster shell addition. In the biofilm on the oyster shells' surface, Nitrospira, Ruegferia, and Tenacibaculum were the dominant species. Over time, the abundance of Tenacibaculum decreased, while Nitrospira increased. This significant proliferation of Nitrospira in the biofilm led to an increase in Nitrospira in the water of the experimental groups, accelerating the conversion of nitrite to nitrate. Consequently, this resulted in higher survival rates and increased body length of the shrimp. Bacteria such as KI89A_clade, SWB02, Nitrosomonas, and Tenacibaculum were affected by the amount of oyster shell added, showing significant differences among the three groups in both water and shrimp intestines. These bacteria have positive implications for the aquaculture system, but their mechanisms of action require further investigation.

In the present study, the concentration gradient of oyster shell addition was not sufficiently comprehensive, with no significant differences observed between the 4‰ and 16‰ oyster shell additions. It is necessary to establish a more scientifically rigorous addition gradient to further investigate the effects of varying concentrations of oyster shell addition on shrimp. Additionally, the microbial communities in the water and intestines were not sampled at distinct time points, leaving the question of whether the dynamic changes in bacterial concentrations within the biofilm would be mirrored in the water and intestines, thereby inducing dynamic shifts in water and intestinal bacteria, unsubstantiated by adequate data. In future research, we will continue to explore the intricate dynamic interplay among microbial communities during the oyster shell addition process.

Biofilm preparation, water environment monitoring, and sample collection

This experiment was conducted at Zhejiang Mariculture Research Institute in Wenzhou, Zhejiang Province, China. Twelve culture tanks, each with an approximate volume of 800L (diameter 1 m, height 1 m), were selected for the aquaculture experiment system. Each experimental tank had a water volume of about 700L and was stocked with 140 shrimp (body length: 4.054 ± 0.326 cm, body weight: 0.367 ± 0.086 g), resulting in a stocking density of 200 shrimp/m³. The 12 culture systems were divided into three groups, each consisting of four replicates: the control group, the high oyster shell concentration group, and the low oyster shell concentration group. The experimental groups were supplemented with oyster shells at concentrations of 4‰ (approximately 2.8L) and 16‰ (approximately 11.2L) of the culture water volume, respectively (shell length: 4.34 ± 0.89 cm; width: 2.73 ± 0.71 cm). The bottom of the oyster shells was aerated to form an upward flow through the shells, while no oyster shells were added to the control group. During the experiment, each tank was continuously aerated to increase oxygen levels, and an equal amount of feed was provided three times daily (8:00, 12:00, and 16:00). Starting from the 15th day, water was exchanged daily, with the control group having a 10% water change and the oyster shell addition groups having a 2% water change.

Every 7 days, 1L water samples were collected from each experimental tank for the determination of physicochemical parameters of the water. Every 14 days, biofilm samples from the oyster shells were collected by randomly selecting three shells of consistent size from each experimental tank. The oyster shell samples were rinsed with sterile physiological saline and the biofilm was separated using an SB-5200DT ultrasonic cleaner (40kHz, 15min). The suspension was then filtered through a 0.2-µm polycarbonate membrane (Millipore Type GTTP, USA), and the samples collected on the filter membrane represented the biofilm.

At each sampling time point, the concentrations of ammonia nitrogen, nitrite, nitrate, and active phosphate in the water were measured. The measurement of the physicochemical parameters of the water was conducted in accordance with the "Specifications for Oceanographic Survey" (GB 17378.4–2007): ammonia nitrogen was determined using the indophenol blue spectrophotometric method; nitrite was measured using the sulfanilamide and hydrochloric acid naphthylethylenediamine diazo method; active phosphate was determined using the ascorbic acid reduction phosphomolybdenum blue method; and chlorophyll-a was measured using the acetone method.

At the beginning of the experiment (D0) and at the end (D70), the body length and body weight of 10 individual shrimp were measured. At the end of the experiment (D70), shrimp yield was recorded, and survival rate was calculated.

DNA extraction and bacterial 16S rRNA gene sequencing

Genomic DNA was extracted using the Power Soil® DNA Isolation Kit (MOBIO, USA) and the QIAamp® DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturers' instructions. DNA concentration and quality levels were measured using an ND-2000 spectrophotometer (NanoDrop Technologies, Delaware, USA). The V4 region of the 16S rRNA gene was amplified using primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA-3') and 806R-B (5'-GGACTACNVGGGTWTCTAAT-3'). The PCR products were purified using a PCR Fragment Purification Kit (TaKaRa, Japan), quantified with an Agilent 2100 Bioanalyzer (Agilent, USA), and sequenced on the MiSeq platform (Illumina, USA).

Processing of Illumina sequencing data

For the 16S rRNA data, we used the QIIME2 command line platform version 2024.2 with default parameters for quality control, filtering, chimera detection, de-noising, detection of sequence variants and taxonomic classification.Taxonomy was assigned to representative sequences using the feature-classifier plugin with the Naive Bayes SILVA132 515F/806R classifier available on the QIIME2 website (https://docs.qiime2.org/2024.2/data-resources/) based on 97% sequence identity(Hall & Beiko, 2018; Quast et al., 2012). After assigning taxonomic information, OTUs related to Archaea, Chloroplast, Unclassified (not affiliated with Bacteria), and singleton OTUs were removed from the dataset. To correct for unequal sequencing depth, the OTU table was diluted at the level of 10,000 sequences per sample to calculate alpha- and beta-diversity estimates, as well as Bray-Curtis dissimilarity between samples.

Statistical analyses

Pearson correlation coefficient was used to calculate the correlation between parameter variables. One-way ANOVA and T-test were used to determine whether there were significant differences between groups. PCA clustering based on Bray-Curtis (BC) distance was used to analyze microbial community clustering, and analysis of similarity (ANOSIM) was used to calculate whether there were significant differences between communities(Granato, Santos, Escher, Ferreira, & Maggio, 2018). Bacteria with a relative abundance greater than 1% were used to plot the bacterial community composition abundance chart. The intersection of bacteria with high contributions predicted by the random forest and bacteria with significant differences identified by one-way ANOVA was taken to obtain the differential bacteria between groups(Rigatti, 2017).

Author Contribution

Xiaolin Huang designed and guided the entire experiment. Xianke Huang and Xiaolin Huang wrote the main manuscript text , Xianke Huang and Shuangshuang Teng prepared all figures and tables. Xiang Zhang, Ming Li, Yilong Cai and Ran Chen conducted the experiment.

Acknowledgement

Thanks to everyone who contributed to the experiment。Thanks to Zhejiang Provincial Science and Technology Project of China.

Data Availability

submission: SUB14648373

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Effects of Oyster Shell Addition on Shrimp Aquaculture and the Dynamic Succession of Surface Biofilm Microbial Communities

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