The influence of biofloc system on Vibrio composition, the growth and the gut microvilli performance of the Pacific white shrimp P enaeus vannamei

Biofloc technology has been shown to have a positive impact on shrimp culture by controlling pathogenic Vibrio . This study aimed to analyze the effect of biofloc on the Vibrio composition in water and shrimp gut, as well as the growth performance and microvilli of Pacific white shrimp ( Penaeus vannamei ). Shrimp post-larvae measuring 0.66 ± 0.02 g were reared in a glass aquarium (working volume 22 L) of 6 units with a density of 150 shrimp m -3 for 28 days. The treatments included rearing in the biofloc system with C/N ratio 10, and without a biofloc system as a control. The results showed that Vibrio was highly prevalent in the control gut (4.13%) and biofloc water (3.77%), but only a few were found in the control water (0.16%) and biofloc-treated gut (0.11%). V . hepatarius (1.10%) and V . nereis (1.06%) were found to dominate the Vibrio bacterial community in the biofloc system maintenance media, while Vibrio sp. Hep-1b-8 (2.26%) and V . parahaemolyticus (0.80%) known as a pathogenic bacteria dominated the control shrimp gut. The biofloc system significantly increased the digestive enzyme activity, growth performance, and microvilli length in the shrimp gut. In conclusion, the application of a biofloc system in shrimp culture can affect the composition and abundance of bacterial communities in both the culture environment and the shrimp gut, and improve growth performance with higher digestive enzyme activity and longer microvilli in the gut


INTRODUCTION
Global aquaculture production increased by 4.5% between 2010 and 2020, with Pacific white shrimp (Penaeus vannamei) being one of its primary commodities, with a production of 5.81 million tons in 2020 (FAO, 2022).According to KKP (2020), Indonesia was one of the world's major shrimp producers, with 1.053.206tons produced in 2019 and contributing more than $2 billion USD to worldwide exports (FAO, 2022).Shrimp production has the highest economic value in Indonesia among all aquaculture commodities, followed by tilapia (Oreochromis sp.) and catfish (Clarias sp.).The potential for expanding the white shrimp culture industry is still considerable, given the broad target market, which includes Japan, the United States, the United Kingdom, and other countries that import shrimp (FAO, 2020).
Despite a significant increase in production of almost 120% in 2020 compared to 2010, the intensification of white shrimp culture remains a challenge due to the decrease in water quality caused by excessive feed and shrimp metabolism waste.This leads to instability in the microbial composition of the water, which increases the threat of bacterial diseases caused by various Vibrio, such as V. harveyi, V. alginolyticus, V. parahaemolyticus, and others, commonly known as vibriosis (Valente & Wan, 2021).Vibrio are naturally occurring as opportunistic pathogens in the marine and estuarine environment that can infect white shrimp under poor environmental and low shrimp immunity, causing various abnormalities such as gill necrosis, lethargy, loss of appetite, and mass mortality reaching up to 100% in acute conditions (Raja et al., 2017;Abdel-latif et al., 2022).
The dominance of Vibrio in the digestive tract of white shrimp is also suspected to be one of the agents causing white feces disease (WFD) (Kurniawinata et al., 2022;Subash et al., 2023).Apart from vibriosis, infections caused by V. parahaemolyticus strains containing the pirA and pirB toxins have been reported to cause acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS).These infections have gained global attention due to their widespread occurrence, starting with the first EMS outbreak in China in 2009 and rapidly spreading to other countries such as Vietnam (2010), Malaysia (2011), Thailand (2012), Mexico (2013), and others.AHPND has also resulted in significant losses for the aquaculture industry worldwide (Kumar et al., 2020).Shinn et al. (2018) estimated the economic losses caused by AHPND to be approximately $4 billion USD in the Asian region between 2009 and 2018, with a total more than $44 billion USD worldwide (Tang & Bondad-reantaso, 2019).Besides probiotics as a solution to prevent vibriosis disease (Wang et al., 2018;Kewcharoen & Srisapoome, 2019;Torpee et al., 2021), the application of biofloc technology is also known to have a positive impact on shrimp culture activity.Biofloc technology converts nitrogen from feed and metabolic waste into microbe biomass by adding organic carbon sources, this process creates biofloc, which is an aggregate of heterotrophic bacterial biomass, phytoplankton, fungi, and other organisms (Avnimelech, 2009).
According to previous studies, biofloc has been shown to enhance the immune response of white shrimp (Ekasari et al., 2014;Panigrahi et al., 2018) and function as a biocontrol agent for pathogenic bacteria.Biofloc can produce extracellular compounds such as bromophenol, poly-βhydroxybutyrate, carotenoids, chlorophyll, amino acids, and other compounds that can decrease the virulence factors of bacteria, modulate gut microbiota structure, and enhance the shrimp gut microvilli height (Crab et al., 2010;Silva et al., 2016;Qiao et al., 2020).Additionally, our previous research showed that biofloc can lower the density and bacterial biofilms activity, increase the immune response, and enhance the resistance of white shrimp against V. parahaemolyticus infection (Gustilatov et al., 2022).According to Tepaamorndech et al. (2020) and Schveitzer et al. (2020), biofloc can initiate a shift in bacterial dominance in the aquaculture environment and affect the diversity of microbiota in the shrimp gut.
The change in microbiota composition is related to the modulation of various functions, including the suppression and competition with populations of pathogenic bacteria and the enhancement of shrimp health with an appropriate environmental and gut microbiota composition (Xiong, 2018).Suitable microbiota conditions in the environment and gut, along with minimal pathogens, are essential in preventing disease outbreaks and maintaining good shrimp growth performance.Based on the research findings, it indicates the benefits of biofloc application as a biocontrol agent for pathogenic bacteria and modulation of the environmental microbiota and gut microbiota composition of white shrimp.The role of biofloc in influencing the microbiota especially Vibrio composition as a major in shrimp in the environment and gut, and its relationship with growth performance and microvilli of white shrimp, needs further investigation.Therefore, this study aims to analyze the application of biofloc in influencing the Vibrio composition of water and gut microbiota, as well as the growth performance and microvilli of white shrimp.

Experimental Design and Biofloc Preparation
In this study, a completely randomized design (CRD) with two treatments and three replications was used.The control treatment involved rearing shrimp without a biofloc system, while the biofloc treatment involved rearing shrimp with a biofloc system.The biofloc used for the initial shrimp maintenance (biofloc inoculants) came from a biofloc in shrimp culture container, where molasses was used as the source of organic carbon.During the shrimp culture process, molasses was added once a day, two hours after the morning feeding, with a C:N ratio of 10.
To determine the amount of carbon needed, the De Schryver et al. (2008) carbon requirements scheme was used, assuming a 40% protein content of the feed, 16% nitrogen in protein, around 85% nitrogen excretion, and approximately 38% carbon content in molasses.Based on these values, approximately 14.3 grams of molasses was required for every 10 grams of feed given.Biofloc inoculants were added to the biofloc treatment aquarium by adding 25% inoculant to 75% seawater.

Shrimp Maintenance
The experiment involved rearing post-larvae 10 white shrimp to an average weight of 0.66 g before moving the treated group to aquariums with an initial density of 150 individuals/m 3 (33 shrimp per aquarium).The shrimp were fed four times a day, at 7:00, 11:00, 15:00, and 19:00, at an 8% feeding rate.During maintenance, molasses was given to the shrimp aquarium once per day, two hours after the morning meal.The treatment had an estimated C:N ratio of 10, whereas the control group received no molasses.The white shrimp were grown for 28 days.Every three days, water quality measures such as dissolved oxygen, pH, and salinity were also monitored, while total ammonia nitrogen, nitrite, nitrate, and total suspended solids were observed at the beginning, middle, and end of the cultivation at the Environmental Laboratory, Department of Aquaculture, Bogor Agricultural University, following APHA (2005) standards.

Parameters of observation Microbiota composition in water and shrimp gut
At the end of the maintenance period, a 1 L sterile sample bottle was used to collect water samples, which were then filtered using a 0.22 µm millipore filter paper (Merck, Germany) under a vacuum.The shrimp intestines were then dissected and aseptically collected.The filter paper and intestine samples were separately placed in 2 mL microtubes and treated with DNA Shield (Zymo, US) for DNA preservation.The samples were stored in a -80°C freezer for subsequent DNA extraction.Sequencing preparation included DNA extraction from the water and shrimp intestine samples, PCR amplification, quantification and mixing of PCR products, purification of PCR products, and library preparation.

NGS (Next Generation
Sequencing) technology was used to analyze the genomic DNA (Kurniawinata et al., 2021).The PCR amplification of the 16S rRNA gene in the V3-V4 region was performed using the 515F-806R primers (5'-GTGCCAGCMGCCGC-GGTAA-3' and 5'-GGACTACHVGGGTWTCTAAT-3'), which were linked to barcodes.Equal amounts of PCR products from each sample were collected, end-repaired, A-tailed, and then ligated with Illumina adapters.The resulting library was sequenced on the Illumina paired-end platform to generate 250bp paired-end raw reads.The quantified library will be collected and sequenced on the Illumina platform, according to the effective library concentration and the required amount of data.The data obtained were analyzed using Microsoft Excel 2013 and tabulated using Origin 2022 software.

Shrimp growth performance
The shrimp biomass was measured at the beginning and end of the maintenance period.Sampling was done before feeding when the shrimp gut was empty.Specific growth rate (SGR), feed conversion ratio (FCR), and survival rate (SR) parameters were calculated using the equation according to Li et al. (2021):

Statistical Analyses
The obtained data were tabulated using Microsoft Excel 2013 and Origin 2022 software.The data analysis of growth performance, digestive enzyme activity, and microvilli length in the gut was conducted using analysis of variance (ANOVA) with SPSS version 20.If a significant difference was found, a further test using the Tukey test with a 95% confidence interval was performed.The diversity analysis of microbiota in the culture media and shrimp digestive tract, and gut histology was done descriptively.

Water and shrimp gut bacterial composition and Vibrio abundance
The study investigated the impact of biofloc on the bacterial composition of rearing water and shrimp gut samples.A heatmap displaying the 25 most prevalent genera for each sample treatment was produced (Figure 1), with the color of the heatmap indicating the relative abundance of the genus (darker red indicates higher relative abundance).The findings indicated that the biofloc treatment resulted in differences in the dominant genus composition compared to the control.The control water was dominated by Flavilitoribacter, Phaeodactylibacter, Lewinella, Kriegella, and Rubinisphaera genera, with relative abundance of 7.31%, 6.39%, 4.72%, 2.13%, and 1.76% respectively.

Digestive enzyme
The measurement of enzyme activity in the digestive tract of shrimp includes the activity of amylase, protease, and lipase enzymes.Sampling of the shrimp digestive tract was done at the end of the treatment and in an empty gut condition.The shrimp digestive tract was collected using aseptic techniques and transferred to a sample bottle with 0.5 g.After that, the samples were stored in a deep freezer at -80°C for later analysis of digestive enzyme activity, which followed method by Muttharasi et al. (2021).

Shrimp gut histology and microvilli height
Histopathology of the gut was observed at the end of the rearing period.The gut tissue was aseptically collected, fixed in Bouin's solution for 24 hours, and then cut into 1×1 cm pieces with a thickness of 3-5 mm.The subsequent steps involved dehydration, clearing, embedding, blocking paraffn, , sectioning, and staining (Munaeni et al., 2020).Microvilli length in the gut was then observed using a microscope (Olympus CX31) with IndomicroView software.

Water and shrimp gut Vibrio composition and relative abundance
The analysis of Vibrio relative abundance in the shrimp gut and shrimp rearing water revealed that Vibrio was prevalent in the biofloc water and control gut, but not in the control water and biofloc gut samples (Figure 3).Moreover, the dominant Vibrio species in the biofloc water sample were V. hepatarius (1.10%) and V. nereis (1.06%), whereas in the control gut the dominant species were Vibrio sp.Hep-1b-8 (2.26%) and V. parahaemolyticus (0.80%)

Shrimp digestive enzyme activity and growth performance
The results of the examination on the shrimp digestive enzymes activity following 28 days of maintenance in the biofloc system revealed a significant increase in the activity of lipase and protease, compared to the control treatment (P<0.05) (Table 1).Although there was no significant difference compared to the control treatment, amylase activity exhibited a similar pattern.Furthermore, growth performance indicators, as shown by the SGR and FCR revealed significantly better results in the biofloc system for shrimp rearing compared to the control treatment (P<0.05).Finally, no significant difference was found in the SR parameter.

Shrimp gut histology and microvilli height
The biofloc system is able to elongate the microvilli of the shrimp gut, thereby increasing its surface area.This is demonstrated in the histological image of the shrimp gut in Figure 4, which reveals denser and longer microvilli.Additionally, Figure 5 displays data indicating that the length of the microvilli was significantly higher in the biofloc treatment at 76.47 µm compared to the control treatment, which measured 29.76 µm (P<0.05).

Water quality parameter
The water quality observations are presented in Table 2.At day 14 of the rearing period, the biofloc treatment exhibited significantly lower levels of nitrite compared to the control treatment (P <0.05).Additionally, on day 28, the nitrate parameter of the biofloc treatment also showed significantly lower levels.Though not significantly different, a similar trend was observed in the TAN parameter overall.

Discussion
This study was conducted to analyze the diversity of microbiota composition, particularly the composition and relative abundance of Vibrio, in both maintenance media and white shrimp gut reared in a biofloc system using high-throughput/ next-generation sequencing (NGS) technology.NGS is a type of DNA sequencing technology that allows for the analysis of the whole genome, all exons within known genes (whole exome), or only selected genes' exons (target panel) on a large scale (Alfaro et al., 2019), thus it can be used to identify and analyze the abundance of microbiota in maintenance media and shrimp gut.One approach in aquaculture activities to convert nitrogen waste into microbial biomass is through the application of biofloc system.The controlled addition of carbohydrates with the appropriate C:N ratio will promote the growth of heterotrophic bacteria, and the production of bacterial protein will consume nitrogen (Avnimelech, 2009;Padeniya et al., 2022).According to Tepaamorndech et al. (2020), the biofloc system is capable of initiating changes in bacterial dominance in both the culture media and the gut microbiota of shrimp.
The report from Wei et al. (2016), also showed that differences in carbon sources in the biofloc system can produce different diversity and abundance of microbiota, and ultimately exhibit different responses to the presence of pathogenic bacteria, especially Vibrio, in the aquaculture environment.Before bacteria can thrive and dominate the aquaculture environment, they may have been normal microflora bacteria and also come from the cultivated shrimp.Subsequently, bacteria must be able to compete for nutrients and sites to grow and reproduce with other microorganisms (Ferreira et al., 2020).The observation of the microbiota composition in the shrimp culture media in this study revealed differences in composition between the biofloc system and the control treatment.In the biofloc treatment, Vibrio also showed dominance and became one of the bacteria composing the floc, which is consistent with Tepaamorndech et al. (2020), showing the dominance of Vibrio in biofloc.
Furthermore, the bacteria that participated in dominating the biofloc were Hoflea, Aliiroseovarius, Ruegeria, and Novipirellula.Hoflea is a marine bacteria that functions as a neutrophilic iron-oxidizing bacteria (Sorokina et al., 2012), while Ruegeria is known to have the ability to degrade organophosphate-containing substances using phosphotriesterase enzymes and acrylate degradation (Hussaan et al., 2021;Bullock et al., 2017).In contrast to the control treatment, the bacteria that dominated the microbiota in the shrimp culture media were from the genus Flavilitoribacter, Phaeodactylibacter, Lewinella, Kriegella, and Rubinisphaera.Biofloc can also influence the composition of the microbiota in the digestive tract of shrimp.Based on the observations, the abundance of Vibrio in the biofloc treatment was lower compared to the control treatment, while the genera that dominated in the biofloc treatment were Sedimentitalea, Aliiroseovarius, Ruegeria, Roseovarius, and Sulfitobacter.
Sedimentitalea is known as a type of bacteria found in the digestive tract of Japanese flying squid (Kim et al., 2016) and also in marine sediment (Sun et al., 2010).This may be correlated to the biofloc treatment, where the water sample showed higher abundance values of Sedimentitalea compared to the control treatment.The same trend was also shown by Aliiroseovarius and Ruegeria, where both types of bacteria dominated the water and shrimp gut, indicating that bacteria that dominate the biofloc culture can also dominate the shrimp digestive tract.Furthermore, Sulfitobacter was also found to dominate the shrimp digestive tract in the biofloc treatment.
This bacterium is known to have several functions in the marine environment, including organic sulfur cycling, production of sodiumchannel blocking toxins, host chemical defense, and marine oil biodegradation.Consequently, secondary metabolites of Sulfitobacter may play important roles in marine ecosystems (Long et al., 2011).In contrast to the biofloc treatment, the shrimp gut in the control treatment was dominated by Klebsiella, Vibrio, Kriegella, Streptomyces, and Maribacter bacteria, where Klebsiella and Vibrio are known to exhibit pathogenic characteristics in aquatic animals (Vaneci-Silva et al., 2022;Valente & Wan, 2021).Vibrio dominance in the shrimp gut also correlates with diseases such as Vibriosis and WFD, further leading to decreased survival rates, growth performance, and increased feed conversion ratio (Kurniawinata et al., 2022).
This was confirmed by the relative abundance of Vibrio (Figure 2), which was lower in the gut of shrimp in the biofloc treatment compared to the control treatment.This proves that the biofloc system is capable of influencing the dominance and abundance of microbiota compositions both in the water and in the shrimp gut, as well as reducing the potential dominance of pathogenic bacteria especially Vibrio.According to Asplund et al. (2011), most Vibrio strains are harmless, although some species have the ability to cause diseases, such as V. parahaemolyticus, which can cause acute hepatopancreatic necrosis disease (AHPND) in white shrimp (Han et al., 2015).In the control treatment, Vibrio sp.Hep-1b-8 and V. parahaemolyticus were found to dominate the shrimp intestine (Figure 3), and these are suspected to be potentially pathogenic bacteria.
On the other hand, Vibrio species that dominate the biofloc media are V. hepatarius and V. nereis.Based on research, V. hepatarius does not exhibit pathogenic activity and has even been reported to have probiotic action in white shrimp against V. parahaemolyticus infection (Ramirez et al., 2022).Therefore, it appears that biofloc has the ability to minimize the dominance of pathogenic bacteria in the environment and the digestive tract of shrimp.Biofloc has a sufficiently complete nutrient composition including protein, carbon, ash content, fatty acids, minerals, and other nutrients, making it useful as a natural feed that is always available in the culture media (Rajkumar et al., 2015, Toledo et al., 2016).In addition, biofloc can enhance digestive enzyme activity, leading to better utilization of feed for growth support (Wang et al., 2016).This is supported by the biofloc aggregate components, which consist of microbes that produce extracellular products that act as a supplement to endogenous digestive enzymes to break down proteins and carbohydrates in the animal's intestinal tract.This can facilitate digestion, absorption, and utilization of feed (Najdegerami et al., 2016;Zafar et al., 2022).This can be seen in the higher overall digestive enzyme activity observed in the biofloc treatment compared to the control.With a higher nutrient intake and better digestive enzyme activity, shrimp growth is improved and the feed conversion ratio (FCR) is lower in the biofloc treatment.One of the extracellular products of biofloc is polyβ-hydroxybutyrate (PHB), which can reach a concentration of 50-200 mg g -1 suspended solids in biofloc (Ruan et al., 2011;Klanian et al., 2020).
This compound has various functions, such as controlling Vibrio bacteria (Hoseinifar et al., 2016;Liu et al., 2010), serving as a carbon source and influencing the microbiota community (Qiao et al., 2020), and providing energy for cells in the intestinal tissue (Duan et al., 2017).According to Ringø et al. (2016), PHB particles are partially degraded into β-hydroxybutyrate in the shrimp gut, and the release of this fatty acid provides the shrimp with energy, resulting in an improved gut epithelium.This energy further supports longer and denser microvilli growth, increasing the surface area and optimizing nutrient absorption.This is also supported by the research of Duan et al. (2017) andSahin et al. (2021), which demonstrate the effect of PHB on increasing the length and density of fish intestinal microvilli.
Finally, due to the nitrogen uptake for bacterial growth, the concentration of ammonia in water decreases rapidly (Padeniya et al., 2022).This is evident from the observation of water quality at the end of maintenance which showed significantly lower nitrite and nitrate values in the biofloc treatment compared to the control treatment.This is largely influenced by the ability of the microorganisms that compose the biofloc to reduce the levels of nitrite and nitrate.
of the shrimp at the end of the maintenance period (g) W0 = Average weight of the shrimp at the beginning of the maintenance period (g) t = duration of the maintenance period (day) FCI = Amount of feed consumed (g) Ft = Final shrimp biomass (g) F0 = Initial shrimp biomass (g) Fd = Observed dead biomass (g) Nt = Number of shrimp at the end of the maintenance period N0 = Number of shrimp at the beginning of the maintenance period

Figure 1 .
Figure 1.Relative abundance heat-map of the top 25 genera in rearing water and shrimp gut.

Figure 2 .
Figure 2. The Vibrio relative abundance in rearing water and shrimp gut.

Figure 3 .
Figure 3. Vibrio composition and relative abundance heat-map in rearing water and shrimp gut.

Table 1 .
Digestive enzyme activity and growth performance of shrimp reared in the biofloc system.*within the same column indicate significantly higher value (P< 0.05).Each treatment has three replications.SGR: specific growth rate, FCR: feed conversion ratio, SR: survival rate.

Table 2 .
Water quality in the white shrimp rearing medium.