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.     

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.     

Improved performance of an intensive rotifer culture system by using a nitrifying inoculum (ABIL)

Abstract A dense nitrifying culture (ABIL) has been examined for its capacity to stimulate rotifer growth in a labscale culture system. The nitrifiers were applied in different ways. When ABIL was added directly to rotifer batch cultures, it gave rise to significantly higher population densities (factor 1.5–2.5 higher, P  < 0.05). The nitrifiers were subsequently examined for their capacity to enhance the start-up of bioreactors, commonly installed in aquaculture rearing tanks. Of the different carrier materials used in these bioreactors, i.e. CaCO₃, gravel and a PVC matrix (Bionet), CaCO₃ gave by far the best results. In a third set of experiments, effectively nitrifying bioreactor systems were connected to rotifer culture tanks and operated over a period of up to 10 days. It was demonstrated that the ABIL inoculated CaCO₃-based bioreactor allowed excellent rotifer growth reaching rotifer densities up to 5500 rotifers per mL. Moreover, a new system in which the ABIL culture was recirculated through hollow fibres was developed and demonstrated to be effective for supporting rotifer growth up to 3500 rotifers per mL. Overall, the use of the dense nitrifying culture either in seed batch cultures, conventional bioreactors or hollow fibre bioreactor systems in support of rotifer cultures was demonstrated to be effective for improving the water quality and the rotifer growth.     

硝化细菌分子生态学研究进展

硝化细菌在促进水域生态系统的氮循环、保持健康水产养殖环境方面发挥着巨大作用。本文分析了硝化细菌分子生态学研究的意义,介绍了硝化细菌的主要种类及其系统进化关系,综述了硝化细菌分子生态学研究进展,并对中国的研究现状进行了分析。结合作者的工作实践,认为中国应加强对水产养殖环境硝化细菌分子生态学研究,为养殖环境的污染防治和微生态制剂的研制等提供理论依据。     

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.     

Short‐term effect of the inoculation of probiotics in mature bioflocs: Water quality parameters and abundance of heterotrophic and ammonia‐oxidizing bacteria

The aim of this study was to evaluate the short‐term effect of probiotic inoculation on the abundance of heterotrophic and ammonia‐oxidizing bacteria in mature biofloc, as well as on total suspended solids (TSS), chlorophyll‐a and nitrogenous compounds in water. A completely randomized design consisting of five treatments (three commercial probiotics, one native consortia and one control) was performed. At the beginning of the experiment (day 1), each treatment was inoculated with the respective probiotic: PondPlus^® (PP), Efinol^® PT (EF) and Epicin^® ponds (EP), native consortia UE, whereas the control was not inoculated. Water parameters and bacterial abundance were evaluated at 0, 2, 4, 6 and 8 days. The addition of probiotics, either native or commercial, did not show any significant effect on the TSS, Chl‐a and colony‐forming unit (CFU) of heterotrophic bacteria when they were added to the systems containing mature biofloc. A significant increase in ammonium oxidizing bacteria was registered with the probiotics PP and EP, although the levels of total ammonia nitrogen, NO₃‐N and NO₂‐N were statistically similar among all treatments. Modifications on most of the parameters measured were associated with the factor of time, rather than the inclusion of probiotics. Results suggest that the bacterial conglomerates in mature stage contain well‐established bacterial communities that are difficult to be affected by the addition of probiotics.     

Suppression of Saprolegnia infections in rainbow trout (Oncorhynchus mykiss) eggs using protective bacteria and ultraviolet irradiation of the hatchery water

Since formalin is widely used in prevention of Saprolegnia infections in salmonid fish hatcheries, there is a need for more environmentally safe treatment methods. Therefore, we screened 360 bacterial isolates against their ability to antagonize the growth of Saprolegnia parasitica hyphae in vitro, and best strains were selected according to their antagonistic properties and colonization capability on rainbow trout egg surface. Protective bacterial cultures of Pseudomonas sp. M162, Pseudomonas sp. M174 and Janthinobacterium M169 were tested for prevention of Saprolegnia sp. infections during incubation trials of rainbow trout (Oncorhynchus mykiss) eggs with UV irradiated (400 mWs cm^?2) and non‐treated inlet water. UV irradiation of inlet water significantly decreased mortality during the incubation. Lowest mortalities were observed in protective culture treated groups incubated with UV‐irradiated inlet water. UV irradiation increased the dominance of the main bacterial colonizers and variation in the bacterial species diversity between the experimental units.     

Infection of Tilapia Oreochromis sp. by Vibrio vulnificus in Freshwater and Low-salinity Environments

The first isolation of Vibrio vulnificus in southern Taiwan from hybrid tilapia Oreochromis sp. raised in freshwater and brackish water environments is documented in this report. The infection was only found in fish in ponds where the salinity was less than 10 ppt. Tilapia raised in water of higher salinities in the same region were not affected. Grossly visible signs of infection included dark coloration, lethargy, and external hemorrhage and ulceration of the skin. The most prominent internal signs of infection included splenomegaly and severe hemorrhagic lesions in the liver. Septicemia was documented in moribund fish. All bacterial isolates from moribund fish were tested by polymerase chain reaction, using V. vulnificus hemolysin/cytolysin gene‐specific primers. Sequence data from the 16S ribosomal RNA gene suggest that these isolates were V. vulnificus. The isolates were indole and mannitol positive, characteristics shared by human clinical isolates and isolates from freshwater European eel, Anguilla anguilla. The isolates from tilapia were unique in that they were negative for ornithine decarboxylase and citrate.     

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.     

Identification of conditions underlying production of geosmin and 2-methylisoborneol in a recirculating system

Geosmin and 2-methylisoborneol (MIB) are semi-volatile terpenoid compounds produced as secondary metabolites by benthic and planktonic cyanobacteria, several genera of fungi, and various actinomycetes. These off-flavor compounds pose a heavy economic burden in the aquaculture industry rendering fish unmarketable unless purified by purging with large quantities of clean water. In the present study, the presence of off-flavor compounds was examined in a recirculating aquaculture system (RAS) for tilapia culture. In this zero-discharge system, where water from the fish basins is recirculated through parallel aerobic (drum filter and a trickling filter) and anaerobic treatment loops (sedimentation/digestion basin), concentrations of geosmin and, in particular, MIB were highest in the aerobic treatment loop. Lowest concentrations were detected in the anaerobic treatment loop. This latter finding pointed toward a possible reduction of these compounds in this basin. Two bacterial strains of the streptomycetes family were isolated from the aerobic, organic-rich, drum filter and the nitrifying trickling filter. In vitro tests with these isolates, closely related to Streptomyces roseoflavus and Streptomyces thermocarboxydus, revealed that MIB production exceeded geosmin production under all conditions tested and was significantly higher under aerobic than under anoxic conditions. Under the latter conditions, with nitrate as an electron donor, the S. roseoflavus-like isolate was capable of denitrification. Based on the results obtained in this study, it was concluded that aerobic, organic-rich conditions stimulate the growth of actinomycetes and subsequent production of geosmin and MIB in the system. The observed reduction of these compounds in the anaerobic water treatment component may serve in designing treatment steps aimed at alleviating the problem of geosmin and MIB accumulation in recirculating systems.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.     

Pyocyanin as a safe aquaculture drug for the control of vibriosis in shrimp recirculating aquaculture system (RAS)

Diseases are one of the most critical limiting factors in aquaculture. Recirculating aquaculture systems (RAS) are one of the most functionally viable sustainable aquaculture production systems adopted world over. In the event of any eventuality caused by bacterial pathogens, antibiotics or other antibacterial agents cannot be applied due to the vulnerability of biological filters which form an integral part of the RAS. Because of this, newer drugs are required for the effective control of diseases in RAS which would not interfere with the activity of nitrifying bacteria used in the biological filters. The antagonistic activity of pyocyanin, a bioactive compound produced by Pseudomonas aeruginosa, against vibrios is well established. The purpose of this study was to prove the effectiveness of pyocyanin as an aquaculture drug for application in RAS by employing a pilot-scale shrimp culture under RAS. It was noted that at the concentration of 5 mg L^?1, pyocyanin could significantly bring down the population of Vibrio spp. in RAS without affecting noticeably the other natural heterotrophic bacteria. Also, pyocyanin at this concentration did not inhibit the activity of nitrifying bacterial consortia used in the SBSBR (stringed bed suspended bioreactor) of RAS. The reared shrimp (Penaeus monodon) showed 100% survival after the application of pyocyanin, besides exhibiting normal health signs. Pyocyanin was non-toxic to the shrimp hybrid cell line (PmLyO-Sf9) at the concentration required for its application in disease management (IC₅₀?=?419.26 mg L^?1). The present study has demonstrated that pyocyanin is effective as an environment-friendly and safe aquaculture drug for the application in RAS-based shrimp culture to control Vibrio spp. without impeding nitrification established through the deployment of nitrifying bioreactors.

     

Seasonal dynamics of microbial community in an aquaculture system for Nile tilapia (Oreochromis niloticus)

Studies on bacterial abundance and diversity will improve the understanding of the microbial ecology to optimize aquaculture production, water quality, disease control and environmental impact of effluents. We comparatively evaluated an aquaculture system in dry and rainy seasons by Fluorescent in situ Hybridization (FISH) and culture‐dependent methodology. Overall, a negative correlation between seasonality in bacterial and flagellates abundance was observed. Alpha‐, beta‐, gamma‐proteobacteria and Cytophaga‐Flavobacter were observed with seasonal variation. Putative pathogenic bacteria such as coagulase‐negative Staphylococcus, non‐fermenter Gram‐negative rods (Pseudomonas sp. and Burkholderia sp.), and Enterobacteriaceae were also detected in this study with significant seasonal variation. Focusing on medically important bacteria, our data show that microbial diversity in the environment associated with aquaculture, as it is practiced, may be altered in growth ponds used for fish cultivation. As an ecological consequence, potentially pathogenic bacteria might be released in high concentrations to the downstream environments posing potential threats to human and animal health.     

Discharge management in fresh and brackish water RAS: Combined phosphorus removal by organic flocculants and nitrogen removal in woodchip reactors

The current study combined P and N removal using organic flocculant chemicals and woodchip bioreactors in both freshwater and brackish water (7 ppm) recirculating aquaculture systems (RAS). The use of carbon (C) containing flocculant chemicals in the process was hypothesized to further stimulate C-demanding N removal (denitrification) in bioreactors. The trial of combined P and N removal consisted of four treatments: freshwater and brackish water RAS with and without the addition of supernatant from flocculation process to the woodchip reactor. Duplicate woodchip reactors were used per treatment and the trial was run for six weeks. 56% and 49% of P was removed from fresh and brackish sludge water, respectively. The nitrate-N (NO₃-N) removal rate was improved in the treatment when supernatant from flocculation process was used together with RAS discharge water when compared against the control. In brackish water RAS, the improvement was more pronounced (from 6.6–16.5 g NO₃-N m⁻³ d^-1) than in freshwater RAS (from 5.1–6.5 NO₃-N m⁻³ d^-1). In the freshwater bioreactors using supernatant, N was largely discharged as a nitrite-N (NO₂-N). High NO₂-N concentrations in freshwater reactors allude to incomplete denitrification reactions taking place. The results suggest that the organic flocculants did provide an additional C source for denitrification, which improved the N-removal process. However, in freshwater RAS this might have been partly due to untargeted processes such as DNRA (dissimilatory nitrate reduction to ammonium), and/or insufficient denitrification reactions taking place (excessive NO₂-N production).     

Diversity of bacteria in sewage treatment plant used as fish culture pond in southern Ghana

Bacterial populations from different stages of sewage treatment ponds in Ghana have been found to consist essentially of the same bacterial species, but the predominant species varied with each system. Subtyping of the bacterial strains recovered from the different ponds, and comparing the species present was a valuable tool in determining correlations between the flora of the different environments. The results indicated that the diversity among the bacteria populations from the sewage treatment ponds was generally high, with mean diversity above 0.95 in each pond. Twenty‐five species of bacteria were identified as associated with the fish culture systems in this study. The identified bacteria included one genus of spiral and curved bacteria, Campylobacter sp., one genus of Gram‐negative aerobic rod, Pseudomonas sp., 16 genera of Gram‐negative facultative anaerobic rods, Actinobacillus sp., Aeromonas sp., Citrobacter sp., Edwardsiella sp., Enterobacter sp., Escherichia sp., Flavobacterium sp., Hafnia sp., Klebsiella sp., Pasteurella sp., Proteus sp., Salmonella sp., Serratia sp., Shigella sp., Vibrio sp. and Yersinia sp., one Gram‐negative anaerobic bacterium, Bacteroides sp., three Gram‐positive cocci, Micrococcus sp., Staphylococcus sp. and Streptococcus sp., two endospore‐forming rods, Bacillus sp. and Clostridium sp., and one Actinomycete, Corynebacterium sp.     

硝化细菌对海参养殖系统水质的净化效果

氨和亚硝酸盐对海洋生物有强烈的毒害作用,是海水养殖系统的主要污染物。本文研究硝化细菌制剂对海参养殖系统水质的净化效果。结果表明:硝化细菌对养殖系统水质有明显的净化效果。投加菌剂的实验组氨氮和亚硝酸盐氮出现峰值的时间和对照组相比明显缩短,表明投加硝化细菌制剂后,养殖系统内的氨氧化细菌、亚硝酸盐氧化细菌可在短时间内形成优势,促进了氨和亚硝酸盐的进一步转化。对照组氨氧化细菌和亚硝酸盐氧化细菌需要较长的时间才形成优势,从而导致氨氮和亚硝酸盐氮的积累。观察实验过程中海参的生长情况发现,实验组海参生长状况良好,而对照组中海参在19d时全部死亡。     

硝化细菌浓度及滤料对养殖水中氨氮处理效果的影响

分别研究了不同硝化细菌浓度(0、20、60、120 mL/100 L)和不同微生物滤料(珊瑚石、锅炉煤渣、牡蛎壳)对养殖水中氨氮处理效果的影响。结果显示,添加硝化细菌后,水体中的氨氮浓度呈现下降趋势,在8～12 h出现极低值后,开始上升,但上升速度较慢;随着水体中硝化细菌添加量的增加,水体中的氨氮浓度下降速度加快;水体中亚硝酸氮浓度呈现先上升后下降的趋势,并在4～6 h出现极高值,然后迅速下降,且硝化细菌添加量越高,下降速度越快。硝化细菌对以珊瑚石和锅炉煤渣为滤料的养殖水体中氨氮和亚硝酸氮的处理效果显著优于牡蛎壳,但珊瑚石和锅炉煤渣之间无显著差异。综合试验结果,应急水质处理时,硝化细菌菌剂的添加量以一次60 mL/100 L(或以活菌计数为1.2×109个/100 L)、间隔24 h添加1次为宜;经过脱硫筛选之后的锅炉煤渣可以作为循环水养殖用滤料。     

A study on bacteria associated with the intestinal tract of farmed yellow seahorse,Hippocampus kuda (Bleeker, 1852): characterization and extracellular enzymes

The microenvironment of bacteria associated with the gastrointestinal tract of an animal influences the host in many ways, including the metabolism of several nutrients. Isolation, molecular characterization and enzymatic activities of culturable bacteria associated with the gastrointestinal tract of hatchery‐reared oceanic yellow seahorse, Hippocampus kuda, were investigated in the present study. 16S rRNA sequencing of bacterial isolates yielded 10 different bacterial genera belonging to two major groups: proteobacteria (62.9%) and firmicutes (37.03%). The predominant microbial flora belonged to the genera Vibrio spp., Enterovibrio sp. and Bacillus spp. Burkholderia cenocepacia, hitherto unreported from the gastrointestinal tract of marine fish, was retrieved from the seahorse gut, albeit in small numbers. Enzymatic studies indicated that the bacterial isolates possess the ability to degrade lipids, cellulose, xylan, starch and proteins. Lipase activity was exhibited by a majority of the isolates (70.4%), indicating that the bacterial flora associated with the intestinal tract of seahorses is capable of digesting foods rich in lipids. The possible implications for the role of these bacteria in the digestion and physiological processes are discussed. To our knowledge, this is the first time that the characterization and enzymatic activities of bacteria associated with the intestinal tract of farmed seahorses have been reported.     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.