Variability of the nitrifying bacteria in the biofilm and water column of a recirculating aquaculture system for tilapia (Oreochromis niloticus) production

The aim of this study was to evaluate variability of nitrifying bacterial community in the biofilm and in the water of a recirculating aquaculture systems (RAS) in a tilapia farming in order to determine if nitrification process is dependent, or not, of nitrifying bacteria abundance. Biofilm and water samples were collected periodically for 30 days and analysed with the fluorescent in situ hybridization (FISH) technique, used to quantify ammonia‐oxidizing bacteria (AOB) and nitrite‐oxidizing bacteria (NOB). Ammonia presented the peak in the first week, while the nitrite's maximum was recorded in the second week. Nitrate increased steadily, indicating nitrification activity. Total bacterial abundance in biofilm increased continuously, while in water, it did not change significantly. In the biofilm, number of AOB was high at beginning, decreased after few days and increased again following augment of ammonia. Number of NOB also showed an increase in abundance in biofilm following the increment of nitrite and nitrate. In water, AOB and NOB did not show major variability. Relative abundance of nitrifying bacteria represented more than 30% of total bacteria in biofilm at beginning of the experiment. Their contribution decreased to >3% in last days. It indicates that nitrifying bacteria are biofilm colonizers, and that their activity seems to be directly related to the concentration of nitrogen compounds. However, contribution of nitrifying bacteria did not vary much along the time. We may conclude that the biofilm‐nitrifying bacteria plays major role in nitrification process in RAS and that the activity of these organisms is dependent of their abundance in response to the concentration of nitrogen compounds.     

循环海水养殖系统硝化滤器中氨氧化微生物分析

研究循环水养殖硝化滤器载体上附着生物膜的微生物群落结构可以为提高其处理速率和效率,并为特异性工程菌构建提供依据。采用改良的AFLP方法分析了循环水养殖硝化滤器载体上附着的氨氧化细菌16S rRNA基因和氨单加氧酶amoA基因片段及其系统发育情况。结果表明:分析16S rRNA基因得到的序列片段比分析amoA基因片段得到了更多信息,准确度较高,可作为分析循环水养殖硝化滤器氨氧化菌群组成的有效方法。克隆测序所得序列与网上公布数据比对,可见存在于循环水养殖硝化滤器载体上的氨氧化细菌与Nitrosomonas cryotolerans、Nitrosomonas oligotropha、Nitrosospira tenuis、Nitrosomonas marina相似度达100%,与Nitrosomonas aestuarii相似度为87%。大部分属于亚硝化单胞菌属(Nitrosomonas),仅少数序列属于亚硝化螺菌属(Nitrosospira)。采用16S rRNA基因和amoA片段分析方法得到的附着于封闭循环海水养殖硝化滤器载体上的氨氧化细菌主要为变形菌(Proteobacteria)的β-亚类的亚硝化单胞菌属(Nitrosomonas)和少量的亚硝化螺菌属(Nitrosospira)氨氧化细菌,以及一定数量的γ-亚类氨氧化细菌。     

Application of the consortia of nitrifying archaea and bacteria for fish transportation may be beneficial for fish trading and aquaculture

The growing popularity of the aquarium trade is greatly increasing the demand for many ornamental fish. While shipping technology has made the worldwide transportation of ornamental fish possible, a significant portion of the fish caught for the aquarium trade perish in transport before being sold to hobbyists. One of the major causes of fish death in transport is ammonia building up to toxic levels in the shipping bags. In order to solve this problem, we investigated the effectiveness of using nitrifying consortia in reducing the ammonia build‐up in marine fish bags during transport. A pre‐activated nitrifying consortium was effective in safely maintaining low ammonia levels during a three‐day experiment. We found that both ammonium chloride and urea can activate nitrifying consortia. Activation of nitrifiers by urea is not only novel but also beneficial due to being less harmful to fish in comparison with ammonia. We also discovered that unexpectedly one nitrifying consortium examined mainly contained ammonia‐oxidizing archaea. The confirmation of the concept of the use of activated nitrifying consortia and the usefulness of nitrifying archaea for fish transportation may be beneficial for the fish trading and aquaculture.     

A novel micro‐organism for removing excess ammonia‐N in seawater ponds and the effect of Cobetia amphilecti on the growth and immune parameters of Litopenaeus vannamei

To investigate the feasibility of using micro‐organisms for ammonia‐N removal, six strains were isolated from Chinese white shrimp, Fenneropenaeus chinensis, seawater culture ponds in Dongying (Shandong, China). Of these, strain DY‐01, which exhibited the highest capacity to degrade ammonia‐N, removed 61.7% of the total ammonia‐N (50 mg/L) in 8 hr. An investigation of the factors affecting the removal efficiency indicated the optimum conditions to be 30°C, pH 8.0, and a salinity level of 30 g/L; 16S rDNA gene sequencing and biochemical analysis identified strain DY‐01 as Cobetia amphilecti, which has not previously been reported to degrade ammonia‐N. This strain also boosted the growth of Pacific white shrimp, Litopenaeus vannamei (p < .05), at a concentration of 10⁷ CFU/mL, with no harmful effects on the shrimp's immune system. This study has thus identified a novel aerobic nitrifying bacterium that is potentially an excellent candidate for improving the water quality in mariculture ponds.     

TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan‐based real‐time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L^?1) for 10 days and challenged with WSSV by feeding WSSV‐positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post‐infection. The TaqMan real‐time PCR assay showed a highly dynamic detection limit that spanned over 6 log₁₀ concentrations of DNA and high reproducibility (standard deviation 0.33–1.42) and small correlation of variability (CV) (1.89–3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L^?1 of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L^?1 of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L^?1 (TAN) had increased susceptibility to WSSV.     

Genetic diversity of nitrate reducing bacteria in marine and brackish water nitrifying bacterial consortia generated for activating nitrifying bioreactors in recirculating aquaculture systems

Nitrate reducing potency of 88 bacterial isolates segregated from marine and brackish water nitrifying bacterial consortia (NBC), generated for activation of nitrifying bioreactors, was confirmed by determining the nitrate reducing capability under aerobic conditions as maintained in nitrifying bioreactors. All the isolates had the potential to be used as bio‐augmentors for activating nitrate dissimilation in recirculating aquaculture system. The existence of nitrate reducers with nitrifiers in NBC and in the reactor configuration negates the requirement of integrating anoxic denitrifying system for effective removal of NO₃^?‐N. Phylogenetic analyses of representative isolates from each cluster of the dendrograms generated based on phenotypic characterization and amplified ribosomal DNA restriction analysis revealed profound diversity of nitrate reducing bacterial flora in the NBC. They were composed of Streptomyces enissocaesilis, Marinobacter sp., Pseudomonas sp., Microbacterium oxydans, Pelagibacterium halotolerans and Alcanivorax dieselolei from marine NBC and Streptomyces tendae, Nesterenkonia sp., Bacillus cereus, Microbacterium oxydans and Brevibacterium sp. from brackish water NBC. The diversity indices of the consortia were calculated using Mega 5.0, primer 7 and VITCOMIC softwares. Marine NBC exhibited higher Shannon wiener diversity and mean population diversity than brackish water NBC. The study delineated higher species richness and diversity in marine NBC than in its brackish water counterpart, a possible reflection of the higher biodiversity of marine systems, and hence, the former is more promising to be used as start‐up cultures for the activation of nitrifying bioreactors after appropriate acclimatization to the desired salinity.     

Development of a Model to Simulate Nitrogen Dynamics in an Integrated Shrimp–Macroalgae Culture System with Zero Water Exchange

The effluents of traditional shrimp monoculture cause pollution and promote eutrophication and hypernutrification of the receiving coastal ecosystems. Integrated aquaculture and a recirculating aquaculture system (RAS) have been proposed as an alternative to address these problems. In this study, we developed a dynamic model to simulate the concentration of total ammonia nitrogen (TAN), nitrite, and nitrate in an integrated culture of whiteleg shrimp, Litopenaeus vannamei, and seaweed, Gracilaria vermiculophylla, in a recirculating and zero water exchange system, and the effect of nitrifying and heterotrophic bacteria was also included. The experiments demonstrated that a dynamic model can explain the concentrations of dissolved inorganic nitrogen and variations in these concentrations over time in the integrated culture. The results also suggest that nitrifying and heterotrophic bacteria play an important role in the transformation of dissolved nitrogenous compounds; therefore, these bacteria should be considered within the dynamics of nitrogen in integrated systems with low water exchange.     

Development of real‐time and quantitative QCM immunosensor for the rapid diagnosis of Aeromonas hydrophila infection

Since bacterial infection cause a significant economic loss in fish farms, it is necessary to develop rapid diagnostic tools. Interests on label‐free biosensors have been raised for the rapid detection of aquatic pathogenic bacteria but have not been extensively studied yet. Here we report a quartz crystal microbalance (QCM) immunosensor system for the rapid and simple detection of Aeromonas hydrophila, a pathogen for fish and human, in comparison with a conventional indirect ELISA method. In QCM immunosensor system, an antibody against A. hydrophila was covalently cross‐linked to the gold surface of sensor chip and bacterial attachment was monitored as real‐time frequency shifts within 5 min. The frequency shifts were very positively related to the amounts of bacterial cells between 6.25 and 100 μg corresponding to 6 × 10⁶ to 10⁸ CFU with a high specificity. The QCM immunosensor was also able to detect bacterial cells in fish tissue extract in a dose‐dependent manner. Indirect ELISA also showed the dose‐dependent reaction and the amplified signal may allow a lower detection limit. However, QCM immunosensor system showed a more linear and reliable standard curve with R² value of almost 1 (0.9999). Moreover, detection of the bacteria was much quicker and simpler.     

丙酮酸钠对硝化细菌制剂活性的影响

分别研究丙酮酸钠对淡水型和海水型硝化细菌制剂中氨氧化细菌(AOB)和亚硝酸盐氧化细菌(NOB)活性的影响。结果表明,丙酮酸钠能明显提高AOB活性,缩短其对环境的适应时间。在丙酮酸钠浓度为7mmol/L时,12h海水型硝化细菌和淡水型硝化细菌对氨氮去除率分别为54.7%和63.4%,是对照组的8.82倍和2.98倍。丙酮酸钠对NOB活性影响与AOB存在不同,其中,丙酮酸钠对海水型硝化细菌NOB有抑制作用,并随其浓度升高而增大,但对淡水型硝化细菌NOB活性影响较小。     

A case study of biofilter activation and microbial nitrification in a marine recirculation aquaculture system for rearing Atlantic salmon (Salmo salar L.)

Marine recirculation aquaculture system (RAS) is a prominent technology within fish farming. However, the nitrifying bacteria in the biofilter have low growth rates, which can make the biofilter activation a long and delicate process with periods of low nitrification rates and variations in water quality. More knowledge on the microbial development in biofilters is therefore needed in order to understand the rearing conditions that favour optimal activation of the biofilters. In this case study, we investigated the activation of two biofilters in a marine RAS for Atlantic salmon post‐smolt associated with either high or low stocking densities of fish by monitoring the microbial communities and chemical composition. The results showed that the microbial communities in both biofilters were similar during the first rearing cycle, despite variations in the water quality. Nitrifying bacteria were established in both biofilters; however, the biofilter associated with low stocking density had the highest relative abundance of ammonia‐oxidizing Nitrosococcus (1.0%) and nitrite‐oxidizing Nitrospira (2.1%) at the end of the first rearing cycle, while the relative abundance of ammonia‐oxidizing Nitrosomonas (2.3%–2.9%) was similar in both biofilters. Our study showed that low fish stocking density during the first rearing cycle provided low and steady concentrations of ammonium, nitrite and organic load, which can stimulate rapid development of a nitrifying population in new marine RAS biofilters.     

Abundance of sulphur‐oxidizing bacteria in coastal aquaculture using soxB gene analyses

Molecular techniques based on sequencing of metagenomic clone libraries provide an insight into the diversity of microbial populations. Using nucleic acid‐based methods, the diversity of soxB genes was examined to detect and characterize sulphur‐oxidizing bacteria in Indian coastal aquaculture environments. Gene‐specific degenerate primers were used to amplify various fragments (710, 753, 483–503, 280 and 239 bp) of soxB genes. Metagenomic clone libraries were constructed for 753, 483–503 and 239 bp fragments of soxB genes. The abundance of soxB revealed the presence of sulphur‐oxidizing organisms. Amino acids in parts of the soxB‐encoded proteins were aligned to known conserved amino acid residues. The level of conservation ranged from 23% to 30%. A phylogenetic tree constructed from aligned amino acid sequences of SoxB revealed different clusters associated with the branches of phototrophic α‐ and γ‐proteobacteria. In general, soxB is widespread among the various phylogenetic groups, although this does not necessarily mean that the organism can use sulphur compounds. Our results suggest that the chemolithoautotrophy based on sulphur oxidation in coastal aquaculture is primarily sustained by the presence of sulphur oxidizers, which involve the soxB gene. This study aids identification of the phylogenetic characteristics related to sulphur bioremediation in poorly characterized coastal aquaculture environments.     

Changes to the histological gill structure and haemolymph composition of early blue swimmer crab Portunus pelagicus juveniles during elevated ammonia‐N exposure and the post‐exposure recovery

It is yet unclear whether sub‐lethal ammonia‐N levels cause irreparable damage to aquatic crustaceans, or if recovery is possible, the potential factors involved. The aim was to investigate the effect of 0.706 and 2.798 mmol L^?1 ammonia‐N exposure on the haemolymph osmolality, Na⁺, K⁺, Ca²⁺, pH, ammonia‐N, total haemocyte counts (THC) and gill histopathology of Portunus pelagicus juveniles at 0, 3, 6, 12, 24 and 48 h respectively. Following 48 h, crabs were transferred to pristine seawater allowing a recovery period up to 96 h and similarly measured. In addition moribund crabs, induced from lethal ammonia‐N levels of 7.036 and 10.518 mmol L^?1, were measured for haemolymph osmolality/ions and pH levels. The results demonstrate that despite severe gill damage within 6‐ and 1 h of 0.706 and 2.798 mmol L^?1 ammonia‐N exposure, respectively, no significant change (P>0.05) in the haemolymph osmolality, Na⁺, K⁺, Ca²⁺ or pH levels occurred or by ammonia‐N‐induced morbidity. Although the gills can completely recover within 24 and 48 h post exposure to 0.706 and 2.798 mmol L^?1 ammonia‐N, respectively, likely facilitated by significant haemocyte increases (P<0.05) within the haemolymph and gill lamellae, dependent factors were the previous ammonia‐N concentration and recovery duration while individual variability was also noticed.     

Impact of neem (Azadirachta indica A. Juss) seed cake upon selective nutrient cycling bacterial population in fish culture

With an objective to examine neem seed cake as bactericide, three treatments—10, 20, and 30 mg L^?1—were applied in outdoor experimental Labeo rohita culture tanks for a period of 120 days. Nitrifying, ammonifying, and denitrifying bacteria declined directly with increasing dosage. Ammonifying and phosphate solubilizing bacteria recovered within a month, nitrifying and denitrifying bacteria after 45 days. A positive relationship was established between soil available-N and ammonifying, nitrifying, and denitrifying bacteria in 10 mg L^?1 (R² = 0.68–0.83). Phosphate solubilizing bacteria responded directly with dosage and contributed significantly to the orthophosphate pool (R² = 0.66–0.78).     

Immunohistochemical and Taqman real-time PCR detection of mycobacterial infections in fish

Real‐time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase β subunit (rpoB) gene and polyclonal anti‐Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus, M. avium ssp. avium, M. bohemicum, M. chelonae ssp. chelonae, M. farcinogenes, M. flavescens, M. fortuitum ssp. fortuitum, M. gastri, M. gordonae, M. immunogenicum, M. malmoense, M. marinum, M. montefiorense, M. phlei, M. phocaicum, M. pseudoshottsii, M. salmoniphilum, M. senegalense, M. shottsii, M. smegmatis, M. szulgi and M. wolinskyi. A detection limit equivalent to 10² cfu g^?1 was registered for M. salmoniphilum‐infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10² cfu g^?1 tissue. Both assays were found to be more sensitive than Ziehl–Neelsen (ZN) staining, where the detection limit was below 8 × 10³ cfu g^?1 tissue. Although specificity testing of the real‐time PCR against a panel of non‐Mycobacterium spp. revealed a degree of cross‐reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross‐reactions were identified (by either real‐time PCR or IHC) on testing of formalin‐fixed paraffin‐embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.     

Enhancement of Hsp70 synthesis protects common carp, Cyprinus carpio L., against lethal ammonia toxicity

Exposure to TEX‐OE^®, a patented extract of the prickly pear cactus (Opuntia ficus indica) containing chaperone‐stimulating factor, was shown to protect common carp, Cyprinus carpio L., fingerlings against acute ammonia stress. Survival was enhanced twofold from 50% to 95% after exposure to 5.92 mg L^?1 NH₃, a level determined in the ammonia challenge bioassay as the 1‐h LD50 concentration for this species. Survival of TEX‐OE^®‐pre‐exposed fish was enhanced by 20% over non‐exposed controls during lethal ammonia challenge (14.21 mg L^?1 NH₃). Increase in the levels of gill and muscle Hsp70 was evident in TEX‐OE^®‐pre‐exposed fish but not in the unexposed controls, indicating that application of TEX‐OE^® accelerated carp endogenous Hsp70 synthesis during ammonia perturbation. Protection against ammonia was correlated with Hsp70 accretion.     

Taxonomic and functional profiling of nitrifying biofilms in freshwater,brackish and marine RAS biofilters

In recirculating aquaculture systems (RAS), the crucial step of eliminating toxic N compounds like ammonia and nitrite is mediated via nitrifying microorganisms and takes place in biofilters. In this study, analyses of microorganisms colonizing biocarriers of nine moving-bed biofilters of three different RAS operated with freshwater, brackish or marine process water uncovered site specific communities. Illumina-based amplicon sequencing of the V4-region of the 16S rRNA gene revealed a high microbial diversity with 1000–2500 species-level operational taxonomic units (OTUs) in all biofilters with the highest diversity in the brackish RAS. Proteobacteria, Bacteriodetes, Plantomycetes, Chloroflexi and Nitrospirae represented the most abundant phyla. 76 out of 674 known genera occurred in all nine biofilters and were defined as core-taxa, including nitrifying bacteria (Nitrosomonas and Nitrospira) as well as members of the (heterotrophic) genera Planctomyces, Blastopirellula, Nannocystis and Lewinella. Nitrifying communities composed of different, closely related and so far uncultured members of Nitrosomonas and Nitrospira were identified, strongly indicating that several potentially novel ammonia and nitrite oxidizing species are present in RAS biofilters. Relatives of known comammox Nitrospira were detected in the brackish biofilters, revealing 94–99 % identity of the 16S rRNA gene sequence to Ns. inopinata. Salinity tolerance tests with biocarriers derived from biofilters of the three distinct RAS showed an unexpected broad physiological flexibility with regard to salinity. Nitrification performance of freshwater nitrifiers was drastically reduced with increasing salinity and nearly completely inhibited at 15 PSU, while the brackish and marine nitrifiers showed a high resistance and maintained nitrification activity in a broad range of salt concentrations. This data can help to improve the nitrification process in RAS with changing salinity of the process water.     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

Detection of Neoparamoeba perurans by duplex quantitative Taqman real-time PCR in formalin-fixed, paraffin-embedded Atlantic salmonid gill tissues

The development and the application of a quantitative duplex real‐time PCR for the detection of Neoparamoeba perurans and the elongation factor α 1 gene (ELF) of Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), are described. A set of primers and probe was designed to amplify a 139‐bp fragment specific to the N. perurans 18S rRNA gene. The test was shown to be very sensitive, being able to detect as little as 13.4 DNA copies per μL corresponding to 0.15 fg of template DNA. In addition, the reaction that detected N. perurans was found to have a high degree of repeatability and reproducibility, to have a linear dynamic range (R² = 0.999) extending over 5 log₁₀ dilutions and to have a high efficiency (104%). The assay was applied to DNA samples extracted from 48 formalin‐fixed, paraffin‐embedded (FFPE) salmon gill tissues showing varying degrees of gill histopathology and amoebic gill disease (AGD)‐type histopathology ranging from absent to severe (each scored 0–3). Neoparamoeba perurans DNA was detected in all the blocks where AGD‐type histopathology was diagnosed microscopically and in 43.6% of the blocks showing signs of gill pathology. The association between parasitic load and gill histopathology and AGD‐type histopathology severity was also investigated. This study also describes the development and the application of a second real‐time PCR for the generic detection of Neoparamoeba spp., Page, 1987. A set of primers and probe conserved among the Neoparamoeba spp. was designed to amplify a 150‐bp fragment within the 18S rRNA gene. Applied to N. perurans‐negative gill tissues, the method was used to exclude the presence of other Neoparamoeba spp. in those blocks where gill pathology was observed microscopically.     

Development of a quantitative real-time PCR for the detection of Tenacibaculum maritimum and its application to field samples

The development and the application of a quantitative real‐time PCR for the detection of Tenacibaculum maritimum are described. A set of primers and probe was designed to amplify a 155‐bp fragment specific to the T. maritimum 16S rRNA gene. The test was shown to be very sensitive, able to detect as little as 4.8 DNA copies number μL^?1. In addition, the assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R² = 0.999) extending over 6 log₁₀ dilutions and a high efficiency (100%). The assay was applied to DNA samples extracted from 48 formalin‐fixed paraffin‐embedded (FFPE) Atlantic salmon, Salmo salar, gill tissues showing varying degrees of gill pathology (scored 0–3) and from 26 jellyfish samples belonging to the species Phialella quadrata and Muggiaea atlantica. For each sample, the bacterial load was normalised against the level of the salmonid elongation factor alpha 1 (ELF) detected by a second real‐time PCR using previously published primers and probe. Tenacibaculum maritimum DNA was detected in 89% of the blocks with no signs of gill disease as well as in 95% of the blocks with mild‐to‐severe gill pathology. Association between bacterial load and gill pathology severity was investigated. T. maritimum DNA was detected at low level in four of the 26 jellyfish tested.     

Activation of a Nitrifying Biofilter for High Density Fish Culture

When a reuse water system operates at high fish loadings and recirculation levels, a readily perofrming biofilter is of utmost importance. The present work aims at reducing the colonization time of nitrifying bacteria with the intention of creating a fully operational trickling biofilter upon the introduction of a substantial fish load into the system. A technique is developed by which the future fish biomass is simulated continuous injection of ammonium salts (25.5 ± 2.6 g H-NH₄ ⁺/m³ of iofilter media (432 to 1411 ky fish/liter per minute of makeup water) with a sustaining water quality. With a pH mainained between 9.0 and 8.5, a hydraulic loading rate of 130 m³/m²/d. inorganic nutrients and an inoculum of active nitrifiers, fully reliable biofilters were obtained within 9 days. The role of alkalinity, inoculum, and mineral nutrients is also discussed.