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.

Carbon availability shifts the nitrogen removal pathway and microbial community in biofilm airlift reactor.

This study investigated the response of nitrogen removal performance and microbial community to different carbon composites in biofilm airlift reactors for wastewater treatment. Three reactors were filled with poly (butylene succinate) and bamboo powder composite at the blending ratio of 9:1, 1:1 and 1:9. Increasing the component of bamboo powder in the carrier reduced the carbon availability and had an adverse effect on nitrate removal efficiency. However, bamboo powder improved the ammonia removal rate which mainly through autotrophic nitrification. Three reactors exhibited distinct microbial compositions in both bacterial and fungal diversity. High inclusion of bamboo power decreased the relative abundance of denitrifiers Denitromonas and increased the relative abundance of nitrifiers, including Nitromonas, Nitrospina and Nitrospira. Moreover, correlation network revealed a competitive interaction between the taxa responsible for ammonia removal and nitrate removal processes. Those results indicated the feasibility of steering nitrogen removal pathway through carrier formulation in wastewater treatment.

Integration of Marine Macroalgae (Chaetomorpha maxima) with a Moving Bed Bioreactor for Nutrient Removal from Maricultural Wastewater.

Rather than direct nutrient removal from wastewaters, an alternative approach aimed at nutrient recovery from aquacultural wastewaters could enable sustainable management for aquaculture production. This study demonstrated the feasibility of cultivating marine macroalgae (Chaetomorpha maxima) with a moving bed bioreactor (MBBR-MA), to remove nitrogen and phosphorus in aquaculture wastewater as well as to produce macroalgae biomass. MBBR-MA significantly increased the simultaneous removal of nitrate and phosphate in comparison with only MBBR, resulting in an average total nitrogen (TN) and total phosphorus (TP) removal efficiency of 42.8 ± 5.5% and 83.7 ± 7.7%, respectively, in MBBR-MA while MBBR had no capacity for TN and TP removal. No chemical oxygen demand (COD) removal was detected in both reactors. Phosphorus could be a limiting factor for nitrogen uptake when N : P ratio increased. The recovered nitrogen and phosphorus resulted in a specific growth rate of 3.86%-10.35%/day for C. maxima with an uptake N : P ratio of 6. The presence of macroalgae changed the microbial community in both the biofilter and water by decreasing the relative abundance of Proteobacteria and Nitrospirae and increasing the abundance of Bacteroidetes. These findings indicate that the integration of the macroalgae C. maxima with MBBR could represent an effective wastewater treatment option, especially for marine recirculating aquaculture systems.

Denitrification performance and microbial communities of solid-phase denitrifying reactors using poly (butylene succinate)/bamboo powder composite.

This study explored the denitrification performance of solid-phase denitrification (SPD) systems packed with poly (butylene succinate)/bamboo powder composite to treat synthetic aquaculture wastewater under different salinity conditions (0‰ Vs. 25‰). The results showed composite could achieve the maximum denitrification rates of 0.22 kg (salinity, 0‰) and 0.34 kg NO3--N m-3 d-1 (salinity, 25‰) over 200-day operation. No significant nitrite accumulation and less dissolved organic carbon (DOC) release (<15 mg/L) were found. The morphological and spectroscopic analyses demonstrated the mixture composites degradation. Microbial community analysis showed that Acidovorax, Simplicispira, Denitromonas, SM1A02, Marinicella and Formosa were the dominant genera for denitrifying bacteria, while Aspergillus was the major genus for denitrifying fungus. The co-network analysis also indicated the interactions between bacterial and fungal community played an important role in composite degradation and denitrification. The outcomes provided a potential strategy of DOC control and cost reduction for aquaculture nitrate removal by SPD.

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.

Multi-omics analysis reveals niche and fitness differences in typical denitrification microbial aggregations.

Suspended floc and fixed biofilm are two commonly applied strategies for heterotrophic denitrification in wastewater treatment. These two strategies use different carbon sources and reside within different ecological niches for microbial aggregation, which were hypothesized to show distinct microbial structures and metabolic fitness. We surveyed three floc reactors and three biofilm reactors for denitrification and determined if there were distinct microbial aggregations. Multiple molecular omics approaches were used to determine the microbial community composition, co-occurrence network and metabolic pathways. Proteobacteria was the dominating and most active phylum among all samples. Carbon source played an important role in shaping the microbial community composition while the distribution of functional protein was largely influenced by salinity. We found that the topological network features had different ecological patterns and that the microorganisms in the biofilm reactors had more nodes but less interactions than those in floc reactors. The large niche differences in the biofilm reactors explained the observed high microbial diversity, functional redundancy and resulting high system stability. We also observed a lower proportion of denitrifiers and higher resistance to oxygen and salinity perturbation in the biofilm reactors than the floc reactors. Our findings support our hypothesis that niche differences caused a distinct microbial structure and increased microbial ecology distribution, which has the potential to improve system efficiency and stability.

Performance and microbial community analysis of Combined Denitrification and Biofloc Technology (CDBFT) system treating nitrogen-rich aquaculture wastewater.

This study proposed two novel Combined Denitrification and Biofloc Technology (CDBFT) systems (one under blue LED light (L1) and the other without light (C1), each containing a denitrification (DE) reactor and a biofloc-based reactor) for the enhanced total nitrogen (TN) removal. Long-term operation (110 days) suggested that simultaneous nitrification and denitrification was achieved in both C1 and L1. Significantly higher total nitrogen removal efficiency (TNRE) was observed in L1-CDBFT (92.2%) than C1-CDBFT (87.5%, P < 0.05; after day 14). Further 24-hour nitrogen transformation test showed the boosted nitrate removal of L1-BFT than C1-BFT. High-throughput sequencing analysis revealed that phyla Rotifera and Nematoda which were indispensable for aquatic animal larviculture, were only found in L1-BFT. Nevertheless, CDBFT effluent from both systems was suitable for tilapia culture based on water quality, biofloc characteristics and tilapia survival rates. Overall, this study highlights the significance of developing CDBFT for TN removal especially under lights.

Effect of stock density on the microbial community in biofloc water and Pacific white shrimp (Litopenaeus vannamei) gut microbiota.

Biofloc technology is an efficient approach for intensive shrimp culture. However, the extent to which this process can influence the composition of intestinal microbial community is still unknown. Here, we surveyed the shrimp intestinal bacteria as well as the floc water from three biofloc systems with different stock densities. Our study revealed a similar variation trend in phylum taxonomy level between floc bacteria and gut microbiota. Microbial community varied notably in floc water from different stock densities, while a core genus with dominating relative abundance was detected in gut samples. Extensive variation was discovered in gut microbiota, but still clustered into groups according to stock density. Our results indicated that shrimp intestinal microbiota as well as bacteria aggregated in flocs assembled into distinct communities from different stock densities, and the intestinal communities were more similar with the surrounding environment as the increase of stock density and resulting high floc biomass. The high stock density changed the core gut microbiota by reducing the relative abundance of Paracoccus and increasing that of Nocardioides, which may negatively influence shrimp performance. Therefore, this study helps us to understand further bacteria and host interactions in biofloc system.

Influence of stocking density on growth, digestive enzyme activities, immune responses, antioxidant of Oreochromis niloticus fingerlings in biofloc systems.

A 120-day feeding trial was conducted to evaluate the effect of different stocking densities on growth, the non-specific immunities, antioxidant status and digestive enzyme activities of Oreochromis niloticus fingerlings under a zero-water exchange biofloc system. Tilapias (0.51 ±â€¯0.05 g) were randomly distributed in twelve tanks, each with 300 L water. The experimental design was completely randomized using three replications with four treatments 166 orgs m-3 (LD, low density), 333 orgs m-3 (MD, middle density) and 600 orgs m-3 (HD, high density) with glucose added as biofloc groups, and a clear water group without glucose added as a control 333 orgs m-3. The fish cultured in LD and MD group showed higher final body weight. For the digestive enzymes, the lipase, trypsin, and amylase activities were all depressed in HD group and control group. Regarding the immune and antioxidant abilities, significantly lower values (P < 0.05) of the lysozyme, complement 3, and glutathione were observed for the fish that reared in the control group and HD group. The stress indicator, the cortisol, 5-hydroxytryptamine, and glucose concentrations were also depressed in HD group and control group, meanwhile the alanine aminotransferase, aspartate transaminase and alkaline phosphatase were all higher in HD group and control group. The significant higher survival was observed in the LD and MD group after Vibrio harveyi challenge test. The results of the experiment indicated that the biofloc in situ had the effects of anti-crowding stress.

Poly(butylene succinate)/bamboo powder blends as solid-phase carbon source and biofilm carrier for denitrifying biofilters treating wastewater from recirculating aquaculture system.

In this study, Poly(butylene succinate)/bamboo powder (PBS/BP) was newly applied and tested for 8 months as the carbon source in two moving bed reactors for nitrate removal in real RAS wastewater (fresh/sea water), with the purposes of simultaneous reducing the cost of PBS packing and effluent DOC. Fast start-ups were obtained in both reactors, in which high denitrification rates were observed (0.68 ± 0.03 and 0.83 ± 0.11 kg [Formula: see text]-N m-3 d-1 for fresh and sea water, respectively) with no nitrite and low ammonia accumulation. Reduced DOC concentrations in the effluents were also observed compared to pure PBS. The freezing of PBS/BP showed a further slower release of DOC, which might be beneficial to the life of the PBS/BP for the denitrification process, however, microbial activity, especially in high salinity wastewater, was observed to have declined. Illumina sequencing revealed that the autotrophic genus arcobacter was discovered first time in solid-phase denitrification system with salinity. Redundancy analysis (RDA) was used to reveal the relationships between environmental factors and the microbial community. In overall, PBS/BP blends were proven to be an economically attractive carbon source for nitrate removal in RAS.

The impact of DO and salinity on microbial community in poly(butylene succinate) denitrification reactors for recirculating aquaculture system wastewater treatment.

The interactions between environmental factors and bacterial community shift in solid-phase denitrification are crucial for optimum operation of a reactor and to achieve maximum treatment efficiency. In this study, Illumina high-throughput sequencing was applied to reveal the effects of different operational conditions on bacterial community distribution of three continuous operated poly(butylene succinate) biological denitrification reactors used for recirculating aquaculture system (RAS) wastewater treatment. The results indicated that salinity decreased OTU numbers and diversity while dissolved oxygen (DO) had no obvious influence on OTU numbers. Significant microbial community composition differences were observed among and between three denitrification reactors under varied operation conditions. This result was also demonstrated by cluster analysis (CA) and detrended correspondence analysis (DCA). Hierarchical clustering and redundancy analysis (RDA) was performed to test the relationship between environmental factors and bacterial community compositions and result indicated that salinity, DO and hydraulic retention time (HRT) were the three key factors in microbial community formation. Besides, Simplicispira was detected under all operational conditions, which worth drawing more attention for nitrate removal. Moreover, the abundance of nosZ gene and 16S rRNA were analyzed by real-time PCR, which suggested that salinity decreased the proportion of denitrifiers among whole bacterial community while DO had little influence on marine reactors. This study provides an overview of microbial community shift dynamics in solid-phase denitrification reactors when operation parameters changed and proved the feasibility to apply interval aeration for denitrification process based on microbial level, which may shed light on improving the performance of RAS treatment units.

Simultaneous ammonia and nitrate removal in an airlift reactor using poly(butylene succinate) as carbon source and biofilm carrier.

In this study, an airlift inner-loop sequencing batch reactor using poly(butylene succinate) as the biofilm carrier and carbon source was operated under an alternant aerobic/anoxic strategy for nitrogen removal in recirculating aquaculture system. The average TAN and nitrate removal rates of 47.35±15.62gNH4-Nm(-3)d(-1) and 0.64±0.14kgNO3-Nm(-3)d(-1) were achieved with no obvious nitrite accumulation (0.70±0.76mg/L) and the dissolved organic carbon in effluents was maintained at 148.38±39.06mg/L. Besides, the activities of dissimilatory nitrate reduction to ammonium and sulfate reduction activities were successfully inhibited. The proteome KEGG analysis illustrated that ammonia might be removed through heterotrophic nitrification, while the activities of nitrate and nitrite reductases were enhanced through aeration treatment. The microbial community analysis revealed that denitrifiers of Azoarcus and Simplicispira occupied the dominate abundance which accounted for the high nitrate removal performance. Overall, this study broadened our understanding of simultaneous nitrification and denitrification using biodegradable material as biofilm carrier.

The effects of different seeding ratios on nitrification performance and biofilm formation in marine recirculating aquaculture system biofilter.

Rapid start-up of biofilter is essential for intensive marine recirculating aquaculture system (RAS) production. This study evaluated the nitrifying biofilm formation using mature biofilm as an inoculum to accelerate the process in RAS practice. The effects of inoculation ratios (0-15 %) on the reactor performance and biofilm structure were investigated. Complete nitrification was achieved rapidly in reactors with inoculated mature biofilm (even in 32 days when 15 % seeding ratio was applied). However, the growth of target biofilm on blank carrier was affected by the mature biofilm inoculated through substrate competition. The analysis of extracellular polymeric substance (EPS) and nitrification rates confirmed the divergence of biofilm cultivation among reactors. Besides, three N-acyl-homoserine lactones (AHLs) were found in the process, which might regulate the activities of biofilm. Multivariate analysis based on non-metric multidimensional scaling (nMDS) also indicated the great roles of AHLs and substrate supply which might fundamentally determine varied cultivation performance on target biofilm.

Biological denitrification using poly(butylene succinate) as carbon source and biofilm carrier for recirculating aquaculture system effluent treatment.

Nitrate removal is essential for the sustainable operation of recirculating aquaculture system (RAS). This study evaluated the heterotrophic denitrification using poly(butylene succinate) as carbon source and biofilm carrier for RAS wastewater treatment. The effect of varied operational conditions (influent type, salinity and nitrate loading) on reactor performance and microbial community was investigated. The high denitrification rates of 0.53 ± 0.19 kg NO3(-)-N m(-3) d(-1) (salinity, 0‰) and 0.66 ± 0.12 kg NO3(-)-Nm(-3) d(-1) (salinity, 25‰) were achieved, and nitrite concentration was maintained below 1mg/L. In addition, the existence of salinity exhibited more stable nitrate removal efficiency, but caused adverse effects such as excessive effluent dissolved organic carbon (DOC) and dissimilation nitrate reduce to ammonia (DNRA) activity. The degradation of PBS was further confirmed by SEM and FTIR analysis. Illumina sequencing revealed the abundance and species changes of functional denitrification and degradation microflora which might be the primary cause of varied reactor performance.