In-Situ Biofloc Affects the Core Prokaryotes Community Composition in Gut and Enhances Growth of Nile Tilapia (Oreochromis niloticus).

Biofloc technology is commonly applied in intensive tilapia (Oreochromis niloticus) culture to maintain water quality, supply the fish with extra protein, and improve fish growth. However, the effect of dietary supplementation of processed biofloc on the gut prokaryotic (bacteria and archaea) community composition of tilapia is not well understood. In this study one recirculating aquaculture system was used to test how biofloc, including in-situ biofloc, dietary supplementation of ex-situ live or dead biofloc, influence fish gut prokaryotic community composition and growth performance in comparison to a biofloc-free control treatment. A core gut prokaryotic community was identified among all treatments by analyzing the temporal variations in gut prokaryotes. In-situ produced biofloc significantly increased the prokaryotic diversity in the gut by reducing the relative abundance of dominant Cetobacterium and increasing the relative abundance of potentially beneficial bacteria. The in-situ biofloc delivered a unique prokaryotic community in fish gut, while dietary supplementation of tilapias with 5% and 10% processed biofloc (live or dead) only changed the relative abundance of minor prokaryotic taxa outside the gut core microbiota. The modulatory effect of in-situ biofloc on tilapia gut microbiota was associated with the distinct microbial community in the biofloc water and undisturbed biofloc. The growth-promoting effect on tilapia was only detected in the in-situ biofloc treatment, while dietary supplementation of processed biofloc had no effect on fish growth performance as compared to the control treatment.

Impact of early-life rearing history on gut microbiome succession and performance of Nile tilapia.

BACKGROUND: Fish gut microbial colonisation starts during larval stage and plays an important role in host's growth and health. To what extent first colonisation could influence the gut microbiome succession and growth in later life remains unknown. In this study, Nile tilapia embryos were incubated in two different environments, a flow-through system (FTS) and a biofloc system (BFS); hatched larvae were subsequently cultured in the systems for 14 days of feeding (dof). Fish were then transferred to one common recirculating aquaculture system (RAS1, common garden, 15-62 dof), followed by a growth trial in another RAS (RAS2, growth trial, 63-105 dof). In RAS2, fish were fed with two types of diet, differing in non-starch polysaccharide content. Our aim was to test the effect of rearing environment on the gut microbiome development, nutrient digestibility and growth performance of Nile tilapia during post-larvae stages. RESULTS: Larvae cultured in the BFS showed better growth and different gut microbiome, compared to FTS. After the common garden, the gut microbiome still showed differences in species composition, while body weight was similar. Long-term effects of early life rearing history on fish gut microbiome composition, nutrient digestibility, nitrogen and energy balances were not observed. Still, BFS-reared fish had more gut microbial interactions than FTS-reared fish. A temporal effect was observed in gut microbiome succession during fish development, although a distinct number of core microbiome remained present throughout the experimental period. CONCLUSION: Our results indicated that the legacy effect of first microbial colonisation of the fish gut gradually disappeared during host development, with no differences in gut microbiome composition and growth performance observed in later life after culture in a common environment. However, early life exposure of larvae to biofloc consistently increased the microbial interactions in the gut of juvenile Nile tilapia and might possibly benefit gut health.

Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia.

Sustainable aquafeed production requires fishmeal replacement, leading to an increasing use of plant-derived ingredients. As a consequence, higher levels of antinutritional substances, such as non-starch polysaccharides and phytate, are present in aquafeeds, with negative effects on fish performance, nutrient digestibility and overall gut health. To alleviate these negative effects, providing exogenous digestive enzymes and/or probiotics can be an effective solution. In this study, we tested the effect of dietary supplementation of enzymes (phytase and xylanase) and probiotics (three strains of Bacillus amyloliquefaciens) on nutrient digestion kinetics and volatile fatty acid content along the gut, and the distal gut microbiome diversity in Nile tilapia. Chyme volatile fatty content was increased with probiotic supplementation in the proximal gut, while lactate content, measured for the first time in vivo in fish, decreased with enzymes along the gut. Enzyme supplementation enhanced crude protein, Ca and P digestibility in proximal and middle gut. Enzymes and probiotics supplementation enhanced microbial interactions as shown by network analysis, while increased the abundance of lactic acid bacteria and Bacillus species. Such results suggest that supplementation with exogenous enzymes and probiotics increases nutrient availability, while at the same time benefits gut health and contributes to a more stable microbiome environment.

Integration of Algae to Improve Nitrogenous Waste Management in Recirculating Aquaculture Systems: A Review.

This review investigates the performance and the feasibility of the integration of an algal reactor in recirculating aquaculture systems (RAS). The number of studies related to this topic is limited, despite the apparent benefit of algae that can assimilate part of the inorganic waste in RAS. We identified two major challenges related to algal integration in RAS: first, the practical feasibility for improving nitrogen removal performance by algae in RAS; second, the economic feasibility of integrating an algal reactor in RAS. The main factors that determine high algal nitrogen removal rates are light and hydraulic retention time (HRT). Besides these factors, nitrogen-loading rates and RAS configuration could be important to ensure algal performance in nitrogen removal. Since nitrogen removal rate by algae is determined by HRT, this will affect the size (area or volume) of the algal reactor due to the time required for nutrient uptake by algae and large surface area needed to capture enough light. Constraints related to design, space, light capture, and reactor management could incur additional cost for aquaculture production. However, the increased purification of RAS wastewater could reduce the cost of water discharge in places where this is subject to levees. We believe that an improved understanding of how to manage the algal reactor and technological advancement of culturing algae, such as improved algal reactor design and low-cost artificial light, will increase the practical and economic feasibility of algal integration in RAS, thus improving the potential of mass cultivation of algae in RAS.

Using poly(ß-hydroxybutyrate-ß-hydroxyvalerate) as carbon source in biofloc-systems: Nitrogen dynamics and shift of Oreochromis niloticus gut microbiota.

Inorganic­nitrogen removal is essential for the sustainable operation of aquaculture industry and also influences the health of aquatic animals, which may be accomplished by utilizing biofloc technology. In this paper, we studied the use of three different carbon sources 1) longan seed powder (LP), 2) Poly(ß-hydroxybutyrate-ß-hydroxyvalerate) (PHBV) and 3) synthesized PHBV and LP (PHBVL) in biofloc systems for 90days to investigate the nitrogen dynamics and gut microbiota of Nile tilapia (Oreochromis niloticus). The PHBVL and PHBV groups had higher total inorganic­nitrogen removal efficiencies (70.99±19.45% and 63.54±19.44%) than the LP group (35.02±11.21%), which had an accumulation of nitrate. Meanwhile, the biofloc in PHBVL and PHBV group generally had a higher amino acid composition, particularly for methionine and lysine, but was not reflected in the tilapia muscle. High-throughput sequencing indicated that the different carbohydrates shaped different bacterial community compositions in the fish gut after exposure in the three environments for 90-day. These differences, which resulted in different gut digestive enzyme activities (amylase, lipase and trypsin), and growth performance, which the food conversion ratio in the PHBVL group was lower than LP and PHBV group, the final body weight in PHBVL group was average 4.33% and 3.65% bigger than in LP and PHBV group. Network analysis revealed that the keystone taxa (90.33%) were Proteobacteria, Chloroflexi, Actinobacteria, Planctomycetes, Verrucomicrobia and Bacteroidetes, which relative abundance varied in the fish gut in the three groups. The experiment verified the feasibility and advantage to use biodegradable polymers (BDPs) as carbohydrates for biofloc systems.

Managing the Microbial Community of Marine Fish Larvae: A Holistic Perspective for Larviculture.

The availability of high-quality juveniles is a bottleneck in the farming of many marine fish species. Detrimental larvae-microbe interactions are a main reason for poor viability and quality in larval rearing. In this review, we explore the microbial community of fish larvae from an ecological and eco-physiological perspective, with the aim to develop the knowledge basis for microbial management. The larvae are exposed to a huge number of microbes from external and internal sources in intensive aquaculture, but their relative importance depend on the rearing technology used (especially flow-through vs. recirculating systems) and the retention time of the water in the fish tanks. Generally, focus has been on microbes entering the system, but microbes from growth within the system is normally a substantial part of the microbes encountered by larvae. Culture independent methods have revealed an unexpected high richness of bacterial species associated with larvae, with 100-250 operational taxonomic units associated with one individual. The microbiota of larvae changes rapidly until metamorphosis, most likely due to changes in the selection pressure in the digestive tract caused by changes in host-microbe and microbe-microbe interactions. Even though the microbiota of larvae is distinctly different from the microbiota of the water and the live food, the microbiota of the water strongly affects the microbiota of the larvae. We are in the early phase of understanding larvae-microbe interactions in vivo, but some studies with other animals than fish emphasize that we so far have underestimated the complexity of these interactions. We present examples demonstrating the diversity of these interactions. A large variety of microbial management methods exist, focusing on non-selective reduction of microbes, selective enhancement of microbes, and on improvement of the resistance of larvae against microbes. However, relatively few methods have been studied extensively. We believe that there is a lot to gain by increasing the diversity of approaches for microbial management. As many microbial management methods are perturbations of the microbial community, we argue that ecological theory is needed to foresee and test for longer term consequences in microbe-microbe and microbe-larvae interactions. We finally make some recommendations for future research and development.

Resistance and resilience of small-scale recirculating aquaculture systems (RAS) with or without algae to pH perturbation.

The experimental set-up of this study mimicked recirculating aquaculture systems (RAS) where water quality parameters such as dissolved oxygen, pH, temperature, and turbidity were controlled and wastes produced by fish and feeding were converted to inorganic forms. A key process in the RAS was the conversion of ammonia to nitrite and nitrite to nitrate through nitrification. It was hypothesized that algae inclusion in RAS would improve the ammonia removal from the water; thereby improving RAS water quality and stability. To test this hypothesis, the stability of the microbiota community composition in a freshwater RAS with (RAS+A) or without algae (RAS-A) was challenged by introducing an acute pH drop (from pH 7 to 4 during three hours) to the system. Stigeoclonium nanum, a periphytic freshwater microalga was used in this study. No significant effect of the algae presence was found on the resistance to the acute pH drop on ammonia conversion to nitrite and nitrite conversion to nitrate. Also the resilience of the ammonia conversion to the pH drop disruption was not affected by the addition of algae. This could be due to the low biomass of algae achieved in the RAS. However, with regard to the conversion step of nitrite to nitrate, RAS+A was significantly more resilient than RAS-A. In terms of overall bacterial communities, the composition and predictive function of the bacterial communities was significantly different between RAS+A and RAS-A.

Probiotic legacy effects on gut microbial assembly in tilapia larvae.

The exposure of fish to environmental free-living microbes and its effect on early colonization in the gut have been studied in recent years. However, little is known regarding how the host and environment interact to shape gut communities during early life. Here, we tested whether the early microbial exposure of tilapia larvae affects the gut microbiota at later life stages. The experimental period was divided into three stages: axenic, probiotic and active suspension. Axenic tilapia larvae were reared either under conventional conditions (active suspension systems) or exposed to a single strain probiotic (Bacillus subtilis) added to the water. Microbial characterization by Illumina HiSeq sequencing of 16S rRNA gene amplicons showed the presence of B. subtilis in the gut during the seven days of probiotic application. Although B. subtilis was no longer detected in the guts of fish exposed to the probiotic after day 7, gut microbiota of the exposed tilapia larvae remained significantly different from that of the control treatment. Compared with the control, fish gut microbiota under probiotic treatment was less affected by spatial differences resulting from tank replication, suggesting that the early probiotic contact contributed to the subsequent observation of low inter-individual variation.

The impact of rearing environment on the development of gut microbiota in tilapia larvae.

This study explores the effect of rearing environment on water bacterial communities (BC) and the association with those present in the gut of Nile tilapia larvae (Oreochromis niloticus, Linnaeus) grown in either recirculating or active suspension systems. 454 pyrosequencing of PCR-amplified 16S rRNA gene fragments was applied to characterize the composition of water, feed and gut bacteria communities. Observed changes in water BC over time and differences in water BCs between systems were highly correlated with corresponding water physico-chemical properties. Differences in gut bacterial communities during larval development were correlated with differences in water communities between systems. The correlation of feed BC with those in the gut was minor compared to that between gut and water, reflected by the fact that 4 to 43 times more OTUs were shared between water and gut than between gut and feed BC. Shared OTUs between water and gut suggest a successful transfer of microorganisms from water into the gut, and give insight about the niche and ecological adaptability of water microorganisms inside the gut. These findings suggest that steering of gut microbial communities could be possible through water microbial management derived by the design and functionality of the rearing system.

The colonization dynamics of the gut microbiota in tilapia larvae.

The gut microbiota of fish larvae evolves fast towards a complex community. Both host and environment affect the development of the gut microbiota; however, the relative importance of both is poorly understood. Determining specific changes in gut microbial populations in response to a change in an environmental factor is very complicated. Interactions between factors are difficult to separate and any response could be masked due to high inter-individual variation even for individuals that share a common environment. In this study we characterized and quantified the spatio-temporal variation in the gut microbiota of tilapia larvae, reared in recirculating aquaculture systems (RAS) or active suspension tanks (AS). Our results showed that variation in gut microbiota between replicate tanks was not significantly higher than within tank variation, suggesting that there is no tank effect on water and gut microbiota. However, when individuals were reared in replicate RAS, gut microbiota differed significantly. The highest variation was observed between individuals reared in different types of system (RAS vs. AS). Our data suggest that under experimental conditions in which the roles of deterministic and stochastic factors have not been precisely determined, compositional replication of the microbial communities of an ecosystem is not predictable.

Decomposition of high protein aquaculture feed under variable oxic conditions.

The microbial decomposition of nitrogen-rich organic matter in aquaculture ponds is affected by the oxic-anoxic conditions gradient at the soil-water interface as well as by resuspension practices. To investigate these interactions, the decomposition of a 49% protein fish feed was analyzed in 10 marine lab-scale systems with different exposures to aerobic and anaerobic conditions. The degree of coupling between oxic and anoxic conditions in the system had a strong effect on product accumulation and loss from the culture system. Pure oxic or anoxic conditions proved to be less favorable than mixed aerobic-anaerobic systems with respect to the metabolites accumulated in the system. Short 15-min resuspension events and a continuous alternation of oxic and anoxic conditions at 12-h time intervals proved to be the best options to minimize the accumulation of organic matter in the systems. The correct coupling of aerobic-anaerobic conditions in space and time is a key to maintain a good water quality condition for the cultured animals and also to improve the decomposition and recycling of organic matter, reducing the environmental impact from the effluents.