Recirculating Mariculture System Research Articles

Rhamnolipid-assisted solid-phase denitrification process for enhanced nitrate removal of recirculating mariculture system water

Solid-phase denitrification (SPD) is a promising approach for treating recirculating mariculture systems (RMS) water, but its relatively slow nitrate removal rate presents a challenge. This study, for the first time, investigates the enhanced nitrate removal in the SPD process by rhamnolipid (RL) addition. The enhancement by RL addition (20 and 40 mg L−1) was significant and largely persisted even after RL dosing was stopped. Specifically, the nitrate removal in RL-assisted SPD reactors was enhanced by 1.7–2.6 and 1.9–2.1 times for influent NO3−-N concentrations of 100 and 200 mg L−1, respectively. The enhancement was attributed to the improved biofilm formation, as indicated by the increased contents of biomass and extracellular polymeric substances. Moreover, the abundances of denitrification and biofilm formation functional bacteria (Pseudomonas and Denitromonas) also increased. RL addition increased abundances of functional genes involved in carbon degradation and nitrate removal. The denitrification and assimilatory nitrate reduction pathways achieved nitrate removal together. Therefore, RL-assisted SPD presents a novel strategy for treating nitrate-rich RMS water.

Start-up of solid-phase denitrification process for treatment of nitrate-rich water in recirculating mariculture system: Carbon source selection and nitrate removal mechanism

Efficient nitrate removal from recirculating mariculture system (RMS) water is of significance since high concentration of nitrate would cause chronic health effects on aquatic organisms and eutrophication. Solid-phase denitrification (SPD) is a safer and more sustainable approach than conventional heterotrophic denitrification by dosing liquid carbon sources. Thus, its application for treating nitrate-rich RMS water was investigated in this study. Poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was identified with the best nitrate removal among four kinds of carbon sources. PHBV-filled reactors started with mariculture, municipal and mixing sludges (at the ratio of 1:1) and fed with 200 mg L-1 nitrate-rich RMS water all achieved over 81% nitrate removals with a HRT of 4 days. The dissolved organic carbon concentrations of the reactors were in the range of 3–9 mg L-1. Arcobacter, Halomonas, and Psedomonas were dominant genera responsible for nitrate removal in different reactors. Metagenomic analyses indicate that both denitrification and assimilatory nitrate reduction (ANR) are the main contributors to nitrate removals. Metagenomic results illustrated nirB/D cooperated with nasA may perform ANR pathway, which transformed nitrate to ammonia for biosynthesis. These results indicate that SPD could be a safer alternative for treating nitrate-rich RMS water, and provide new insights into nitrogen metabolism pathways in SPD process.

Multi-Costimulatory Pathways Drive the Antagonistic Pseudoalteromonas piscicida against the Dominant Pathogenic Vibrio harveyi in Mariculture: Insights from Proteomics and Metabolomics.

Vibrio harveyi is the dominant pathogen in mariculture, and biocontrol of this pathogen using antagonistic probiotics is a long-standing biological challenge. Here, Pseudoalteromonas piscicida WCPW15003 as a probiotic effectively antagonized dominant pathogenic V. harveyi in a mariculture, with a growth-of-inhibition ratio of 6.3 h-1. The antagonistic activities of cells and intracellular components of WCPW15003 made a greater contribution to the antagonistic process than did extracellular metabolites and caused the dominance of WCPW15003 during the antagonistic process in vitro. WCPW15003 was safe for the pearl gentian grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus) and, as a consequence of the antagonistic effect on V. harveyi, protected the fish from an immune response in vivo. A comprehensive combined proteomics and metabolomics analysis of antagonistic WCPW15003 and pathogenic V. harveyi in a coculture compared to a monoculture was performed to investigate the antagonistic molecular mechanisms. The results showed that during the antagonistic process, WCPW15003 in a coculture had significantly downregulated metabolic pathways for histidine metabolism, arginine biosynthesis, and phenylalanine metabolism, and upregulated glycerophospholipid metabolism, leading to a competitive advantage against the co-occurring species, V. harveyi. This defined a mechanism by which multi-costimulatory pathways drove P. piscicida WCPW15003 against V. harveyi. IMPORTANCE V. harveyi as a dominant pathogen has become a major hazard in mariculture development and seafood safety, and biocontrol of this pathogen using antagonistic probiotic agents is a long-standing biological challenge. P. piscicida WCPW15003 has promise as a novel, safe, and effective bioagent for specifically inhibiting dominant pathogenic V. harveyi and protects mariculture animals from infection by this pathogen by moderating the host immune response, which is heavily driven by multi-costimulatory pathways in a coculture of WCPW15003 and V. harveyi. This work identified a direction for comprehensively elucidating the molecular mechanism of WCPW15003 antagonism against the dominant pathogen in mariculture using modern molecular biology techniques and provided deep insights into the advantages and potential of this antagonistic probiotic against V. harveyi for the construction of an environmentally friendly, recirculating mariculture system.

Ecosystem Services of Ecosystem Approach to Mariculture: Providing an Unprecedented Opportunity for the Reform of China’s Sustainable Aquaculture

China is the biggest provider of mariculture products, and the industry is still growing rapidly. Increasing scientific evidence indicates that mariculture may provide valuable ecosystem goods and services in China. Here, we performed a systematic literature review of studies with the aim of understanding the ecosystem services of mariculture and a comprehensive review of ecosystem approaches that may improve mariculture ecosystem services and goods in China. We highlight four ecosystem services functions in China, including food supply, nutrient extraction, carbon sequestration, and biodiversity conservation. Given the further reform of China’s mariculture, we outlined several ecosystem approaches including integrated multi-trophic aquaculture (IMTA), carrying capacity assessment and monitoring, marine spatial planning, and waste treatment and recirculating mariculture system. We conclude that the ecosystem services of the ecosystem approach to mariculture provide an unprecedented opportunity for the reform of China’s sustainable aquaculture. Finally, a synthesis of sustainable development of mariculture, along with the five recommendations for future mariculture development in China, is outlined.

Characterization of aerobic denitrification genome sequencing of Vibrio parahaemolyticus strain HA2 from recirculating mariculture system in China

A novel halophilic bacterium capable of heterotrophic nitrification–aerobic denitrification was isolated from the polyethylene fiber of a recirculating aquaculture system and identified Vibrio parahaemolyticus HA2. In this study, the degradation rate of ammonia nitrogen, nitric nitrogen and nitrite nitrogen were 99.06%, 97.35% and 34.91% respectively, and the pH increases furtherly proved existing denitrification during the growth of strain HA2. Genome sequencing showed that the ammonia monooxygenase (AMO), periplasmic nitrate reductase (NAP), nitrite reductase (NIR), nitric oxide reductase (NOR), hydroxylamine oxidase (HAO) and nitrous oxide reductase (N2OR) of strain HA2 were associated with the progress of nitrification and denitrification. Utilization of nitrite and nitrate as well as existence of NAP gene further proved aerobic denitrification ability of strain HA2. The aim is indicating great potential of strain HA2 for future full-scale applications in aquaculture

Proliferation of antibiotic resistance genes in coastal recirculating mariculture system

The abuse of antibiotics has caused the propagation of antibiotic resistance genes (ARGs) in aquaculture systems. Although the recirculating systems have been considered as a promising approach for preventing the coastal water pollution of antibiotics and ARG, rare information is available on the distribution and proliferation of ARGs in the recirculating mariculture system. This study firstly investigated the proliferation of ARGs in coastal recirculating mariculture systems. Ten subtypes of ARGs including tet (tetB, tetG, tetX), sul (sul1, sul2), qnr (qnrA, qnrB, qnrS), and erm (ermF, ermT) were detected. The absolute abundances of the ARGs detected in the mariculture farm were more than 1 × 104 copies/mL. The sulfonamide resistance genes (sul1 and sul2) were the most abundant ARGs with the abundance of 3.5 × 107–6.5 × 1010 copies/mL. No obvious correlation existed between the antibiotics and ARGs. Some bacteria were positively correlated with two or more ARGs to indicate the occurrence of multidrug resistance. The fluidized-bed biofilter for wastewater treatment in the recirculating system was the main breeding ground for ARGs while the UV sterilization process could reduce the ARGs. The highest flux of ARGs (6.5 × 1021 copies/d) indicated that the discharge of feces and residual baits was the main gateway for ARGs in the recirculating mariculture system to enter the environments.

High-throughput sequencing analysis of the microbial community in coastal intensive mariculture systems

The conventional and recirculating mariculture systems are two typical intensive mariculture systems in the coastal zone. This study used high-throughput sequencing method to investigate the structural profiles of microbial communities in conventional and recirculating mariculture farms. The results showed that 13,842 OTUs (operational taxonomic units) were detected at a similarity level of 97%. Conventional and recirculating systems exhibited significant difference in microbialcommunity based on the results of the taxonomy and relative abundance of bacteria. Among the top 10 genera in coastal intensive mariculture systems, the predominant bacteria in the conventional intensive mariculture system were Pseudofulvibacter, Polaribacter, Glaciecola, Crocinitomix, Colwellia, Oleispira, and Balneola, while those in the recirculating systems were Vibrio, Alteromonas, and Pseudoalteromonas. Bacterial communities of different fish ponds suggested that the bacterial groups exhibited fish-specific or water treatment stage-specific features. Potential pathogens such as Vibrio, Arcobacter, Pseudoalteromonas, and Shewanella were readily accumulated as dominant bacteria in the recirculating mariculture system. The detection of similar potential pathogens in both the mariculture systems and the adjacent coastal water indicate that the mariculture farms without recirculating system could be the hotspots of pathogens and have great influences on the surrounding coastal environment. The wastewater treatment units used in recirculating farm could remove bacteria effectively, suggesting that the recirculating mariculture system may be more environmentally friendly than the conventional mariculture system.

Formation of disinfection byproducts in a recirculating mariculture system: emerging concerns

Disinfection is commonly employed in recirculating mariculture systems (RMS) to control animal diseases and improve seawater quality; however, little is known about the occurrence of disinfection byproducts (DBPs) formed in such RMS. Beijing Aquarium is a typical RMS with artificially prepared seawater and mainly adopts a decentralized treatment strategy for different animal tanks, including sand filtration, foam fractionation, and disinfection (O3, UV, and O3/ClO2). This study reveals that the adopted disinfection processes were highly effective in controlling marine heterotrophic bacteria; however, some concerns were raised on the formation of various kinds of DBPs, including secondary oxidants, inorganic oxyanions, and hazardous organic species. Free chlorine and free bromine were generated from ozonation at health-relevant concentrations. High concentrations of BrO3(-) and ClO3(-) were formed in mammal tanks, which exceeded the USEPA-regulated maximum contaminant level (MCL) for drinking water by 19-25 and 52-54 times, respectively. Extremely high concentrations of NO3(-) were detected in mammal tanks, which considerably exceeded the MCL regulated by the Sea Water Quality Standard of China for the mariculture industry (Class II) by about 1100 times. Undoubtedly, the presence of various DBPs poses serious health threats to aquarium animals. To solve these problems, potential control measures for DBPs are proposed.

Colwellia aquaemaris sp. nov., isolated from the Cynoglossus semilaevis culture tank in a recirculating mariculture system.

A Gram-staining-negative, heterotrophic, facultatively anaerobic bacterium, designated S1(T), was isolated from the Cynoglossus semilaevis culture pond in a recirculating mariculture system in Tianjin, China. The taxonomy of strain S1(T) was studied by using a polyphasic approach. Cells of strain S1(T) were non-spore-forming, curved rods, 0.4-0.6 µm wide and 1.2-2.0 µm long, and motile by means of a single polar flagellum. The strain was positive for oxidase and catalase activities. Strain S1(T) was able to grow at 4-30 °C (optimum, 25 °C), at pH 5.5-10.0 (optimum, pH 6.5-7.5) and in the presence of 1-5 % (w/v) NaCl (optimum, 2 %). Strain S1(T) contained Q-8 as the sole respiratory quinone and C16 : 1ω7c/C16 : 1ω6c and C16 : 0 as the predominant cellular fatty acids. The genomic DNA G+C content was 40.1 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequences placed strain S1(T) in the genus Colwellia, and it formed a distinct lineage in the phylogenetic tree together with Colwellia meonggei MA1-3(T), Colwellia aestuarii SMK-10(T), Colwellia polaris 537(T) and Colwellia chukchiensis BCw111(T), with 97.7, 96.1, 95.9 and 95.0 % 16S rRNA gene sequence similarity to these strains, respectively. DNA-DNA relatedness of strain S1(T) to Colwellia meonggei MA1-3(T) was 23.5±3.6 %. On the basis of the phylogenetic and phenotypic evidence, strain S1(T) is considered to represent a novel species of the genus Colwellia, for which the name Colwellia aquaemaris sp. nov. is proposed. The type strain is S1(T) ( = CGMCC 1.12165(T) = JCM 18479(T)).

Bacterially mediated removal of phosphorus and cycling of nitrate and sulfate in the waste stream of a “zero-discharge” recirculating mariculture system

Simultaneous removal of nitrogen and phosphorus by microbial biofilters has been used in a variety of water treatment systems including treatment systems in aquaculture. In this study, phosphorus, nitrate and sulfate cycling in the anaerobic loop of a zero-discharge, recirculating mariculture system was investigated using detailed geochemical measurements in the sludge layer of the digestion basin. High concentrations of nitrate and sulfate, circulating in the overlying water (∼15 mM), were removed by microbial respiration in the sludge resulting in a sulfide accumulation of up to 3 mM. Modelling of the observed S and O isotopic ratios in the surface sludge suggested that, with time, major respiration processes shifted from heterotrophic nitrate and sulfate reduction to autotrophic nitrate reduction. The much higher inorganic P content of the sludge relative to the fish feces is attributed to conversion of organic P to authigenic apatite. This conclusion is supported by: (a) X-ray diffraction analyses, which pointed to an accumulation of a calcium phosphate mineral phase that was different from P phases found in the feces, (b) the calculation that the pore waters of the sludge were highly oversaturated with respect to hydroxyapatite (saturation index = 4.87) and (c) there was a decrease in phosphate (and in the Ca/Na molar ratio) in the pore waters simultaneous with an increase in ammonia showing there had to be an additional P removal process at the same time as the heterotrophic breakdown of organic matter.

Wenyingzhuangia marina gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from a recirculating mariculture system

A Gram-stain-negative, strictly aerobic and heterotrophic bacterial strain, designed strain D1(T), was isolated from a recirculating mariculture system in Tianjin, China. Its taxonomic position was determined using a polyphasic approach. Cells of strain D1(T) were non-flagellated short rods, 0.3-0.5 µm wide and 0.5-1.0 µm long. Growth was observed at 15-30 °C (optimum, 25 °C), at pH 5.5-9.0 (optimum, pH 6.5-7.0) and in the presence of 1-8% (w/v) NaCl (optimum, 2-3 %). Cells contained carotenoid pigments but not flexirubin-type pigments. Strain D1(T) contained MK-6 as the sole menaquinone and phosphatidylethanolamine (PE) as the sole phospholipid and four unidentified lipids. The major cellular fatty acids (>10%) were iso-C15 : 0 (23.2 %), iso-C17 : 0 3-OH (15.2%), C(16 : 1)ω7c/C(16 : 1)ω6c (14.3%), iso-C(15 : 0) 3-OH (13.5%) and iso-C15 : 1 G (10.8%). 16S rRNA gene sequence analyses indicated that strain D1(T) belonged to the family Flavobacteriaceae and showed closest phylogenetic relationship to the genus Lutibacter, with highest sequence similarity to Lutibacter aestuarii MA-My1(T) (92.2%). The DNA G+C content of strain D1(T) was 35.9 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain D1(T) was considered to represent a novel species in a new genus of the family Flavobacteriaceae, for which the name Wenyingzhuangia marina gen. nov., sp. nov. is proposed. The type strain of the type species is D1(T) ( = CGMCC 1.12162(T) = JCM 18494(T)).

Photobacterium aquae sp. nov., isolated from a recirculating mariculture system

A Gram-staining-negative, heterotrophic, facultatively anaerobic bacterium, designated AE6(T), was isolated from a grouper (Epinephelus malabaricas) culture tank in a recirculating mariculture system located in Tianjin, China. Strain AE6(T) was able to grow at 15-40 °C (optimum, 30-35 °C), at pH 5.5-10.0 (optimum, pH 7.0-7.5) and in the presence of 0.5-7% (w/v) NaCl (optimum, 2-3%). It contained Q-8 as the predominant respiratory quinone, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) as the major polar lipids and C(16 : 1)ω7c/C(16 : 1)ω6c (40.4%), C(18 : 1)ω7c (15.5%) and C(16 : 0) (13.5%) as the predominant cellular fatty acids. The genomic DNA G+C content was 47.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain AE6(T) belonged to the genus Photobacterium (94.2-96.8% of 16S rRNA gene sequence similarity) and formed a distinct phylogenetic lineage within the genus and exhibited the highest sequence similarity to Photobacterium aphoticum CECT 7614(T) (96.8%). Multilocus sequence analysis (MLSA) using four loci (gyrB, rpoA, pyrH and recA) also revealed that strain AE6(T) was phylogenetically related to the genus Photobacterium. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, strain AE6(T) is considered to represent a novel species of the genus Photobacterium, for which the name Photobacterium aquae sp. nov. is proposed. The type strain is AE6(T) ( = CGMCC 1.12159(T) = JCM 18480(T)).

Constructed wetland with Salicornia as a biofilter for mariculture effluents

The performance of a constructed wetland (CW) with Salicornia persica (Chenopodiaceae) as a biofilter for effluent water drained from a semi-open recirculating mariculture system was studied in southern Israel. The results demonstrate the effectiveness of N, P and total suspended solid (TSS) removal from mariculture effluent by a CW operating with this plant. The CW was exposed to high (3.3±0.26gNm−2d−1) and low (0.13±0.02gNm−2d−1) nutrient loads (NL) in two hydraulic regimes, surface flow (SF) and subsurface flow (SSF). In addition to the CW being studied as marine fishpond effluent water purifier, the Salicornia yield was evaluated as a marketable agricultural by-product. Eight CW ponds were covered under a 750m2 greenhouse. The surface area of each CW pond was 24.3m2 (13.5m length, 1.8m width and 0.30m average depth) and volume of 7.3m3. The fishponds' effluent drained in a continuous mode into one end of each CW pond and out from the opposite end (plug flow) at a hydraulic load (HL) of 0.5m3h−1, a hydraulic loading rate (HLR) of 0.49md−1, and a hydraulic retention time (HRT) of 1.51d−1. Dissimilation processes such as ammonification, nitrification and denitrification with assimilation by the plants performed as in natural wetlands. Salicornia wet yield was 23±1.6 and 26±4.6kgm−2y−1 in the SF and SSF flow regimes, respectively, at the low nitrogen load, and 20.1±2.4 and 17.4±3.1kgm−2y−1 in the SF and SSF flow regimes, respectively, at the high nutrient load. Total nitrogen (TN) uptake by Salicornia tissue itself excluding the wetland activity (including roots) was 29.1±3.3 and 26.9±3.1gNm−2y−1 in the SF and SSF flows, respectively, at low nitrogen load, and 18.5±3.5 and 13.3±3.3gNm−2y−1 at high N loads in the SF and SSF flow regimes, respectively. The contribution of Salicornia as a nitrogen biofilter at high NL was negligible (0.5%–0.9% of the total dissolved nitrogen [TDN]) compared to the low NL (56%–61.4% of the TDN) in both SF and SSF regimes respectively. Our results show that the SF regime with Salicornia would likely be more efficient in facilities with low NL (e.g., fish hatcheries), whereas the SSF regimes would be more efficient with high NL (e.g., super-intensive fish farms). Using CW systems for effluent treatment requires a relatively extensive area. According to our results, about 10,000m2 of CW with Salicornia are required to remove nitrogen and TSS produced from 900kg of 45% crude protein fish feed (11m2kg−1 of feed) during one year. The income generated from selling the Salicornia as an agricultural crop, together with savings on water treatment and potential fines, contributes to the system's economical viability.

Sulfide-induced nitrate reduction in the sludge of an anaerobic digester of a zero-discharge recirculating mariculture system

The anaerobic digester is a vital component in a zero-discharge mariculture system as therein most of the organic matter is mineralized and nitrogen-containing compounds are converted to gaseous N 2. Although denitrification is a major respiratory process in this nitrate-rich treatment stage, also sulfate respiration takes place and may cause undesirable high sulfide concentrations in the effluent water. To examine the effect of sulfide on nitrate reduction, in situ depth profiles of inorganic nitrogen and sulfur compounds were determined. Additionally, nitrate reduction was examined as a function of ambient sulfide concentrations in sludge collected from different locations in the anaerobic reactor. Depth profiles showed high concentrations of nitrate and low concentrations of sulfide and ammonia in the aqueous layer of the reactor. A sharp decrease of nitrate and an increase in sulfide and ammonia concentrations was measured at the water–sludge interface. Nitrate reduction was highest in this interface zone with rates of up to 8.05 ± 0.57 μmol NO 3 − h −1 g (sludge) −1. Addition of sulfide increased the nitrate reduction rate at all sludge depths, pointing to the important role of autotrophic denitrification in the anaerobic reactor. Dissimilatory nitrate reduction to ammonia (DNRA) was found to be low in all sludge layers but was enhanced when sludge was incubated at high sulfide concentrations. Although nitrate reduction rates increased as a result of sulfide addition to sludge samples, no differences in nitrate reduction rates were observed between the samples incubated with different initial sulfide concentrations. This as opposed to sulfide oxidation rates, which followed Michaelis–Menten enzymatic kinetics. Partial oxidation of sulfide to elemental sulfur instead of a complete oxidation to sulfate, could explain the observed patterns of nitrate reduction and sulfide oxidation in sludge incubated with different initial sulfide concentrations.

Identification of Bacteria Potentially Responsible for Oxic and Anoxic Sulfide Oxidation in Biofilters of a Recirculating Mariculture System

Bacteria presumably involved in oxygen- or nitrate-dependent sulfide oxidation in the biofilters of a recirculating marine aquaculture system were identified using a new application of reverse transcription-PCR denaturing gradient gel electrophoresis (DGGE) analysis termed differential-transcription (DT)-DGGE. Biofilter samples were incubated in various concentrations of sulfide or thiosulfate (0 to 5 mM) with either oxygen or nitrate as the sole electron acceptor. Before and after short-term incubations (10 to 20 h), total DNA and RNA were extracted, and a 550-bp fragment of the 16S rRNA genes was PCR amplified either directly or after reverse transcription. DGGE analysis of DNA showed no significant change of the original microbial consortia upon incubation. In contrast, DGGE of cDNA revealed several phylotypes whose relative band intensities markedly increased or decreased in response to certain incubation conditions, indicating enhanced or suppressed rRNA transcription and thus implying metabolic activity under these conditions. Specifically, species of the gammaproteobacterial genus Thiomicrospira and phylotypes related to symbiotic sulfide oxidizers could be linked to oxygen-dependent sulfide oxidation, while members of the Rhodobacteraceae (genera Roseobacter, Rhodobacter, and Rhodobium) were putatively active in anoxic, nitrate-dependent sulfide oxidation. For all these organisms, the physiology of their closest cultured relatives matches their DT-DGGE-inferred function. In addition, higher band intensities following exposure to 5 mM sulfide and nitrate were observed for Thauera-, Hydrogenophaga-, and Dethiosulfovibrio-like phylotypes. For these genera, nitrate-dependent sulfide oxidation has not been documented previously and therefore DT-DGGE might indicate a higher relative tolerance to high sulfide concentrations than that of other community members. We anticipate that DT-DGGE will be of general use in tracing functionally equivalent yet phylogenetically diverse microbial populations in nature.

An application of membrane inlet mass spectrometry to measure denitrification in a recirculating mariculture system

The detrimental effects of aquaculture waste discharges require development of recirculating and/or pretreatment systems. Denitrification, in combination with nitrification, removes fixed nitrogen from culture water in recirculating systems. Three experiments were conducted to measure denitrification rates in biofilter media (“bioballs”) and sediments from a recirculating mariculture system. In this case, “sediments” refer to solids accumulated at the bottom of the biofilter. Bioballs and sediments were collected from the biofilter attached to a 3.7 m diameter shrimp culture tank. Dissolved dinitrogen and oxygen concentrations were calculated from N 2/Ar and O 2/Ar ratios measured with a membrane inlet mass spectrometer (MIMS) optimized for high precision analysis of dissolved gases in water. Denitrification and oxygen flux were calculated from concentration changes as culture water passed through experimental chambers containing bioballs or sediments. Rates are reported per liter of inflow water flowing through the chambers, rather than per hour, because flow rates and biofilter design vary among aquaculture systems. Mean denitrification rates were 22.5±3.8 and 93.6±2.9 μmol N l −1 ( n=6) in bioball and sediment chambers, respectively. Denitrification removed 1.0±0.4% and 3.9±0.3% of the total nitrogen (less N 2) passing through bioball and sediment chambers, respectively. Ammonium accumulated in sediment chambers at 27.0±5.4 μmol N l −1, but ammonium flux in bioball chambers was not different from zero. Nitrite accumulation rates in bioball and sediment chambers were 20.8±7.8 and 24.0±2.4 μmol N l −1, respectively. The nitrate removal rate from sediment chambers was 115±35.4 μmol N l −1, but nitrate accumulated in bioball chambers at a rate of 76.8±16.6 μmol N l −1. The data showed that sediments accumulated in biofilters were important sites of nitrogen removal in this system. However, ammonium accumulation and nitrate removal in sediment chambers suggest that dissimilatory nitrate reduction to ammonium (DNRA) may be an important process in sediments accumulated in biofilters. The occurrence of DNRA in biofilters would result in less efficient removal of fixed N. MIMS technology has been used in many analytical chemistry applications but recently was identified as a useful tool for measuring denitrification in natural systems. This work is the first known application of MIMS technology to aquaculture issues and offers advantages over other methods to estimate denitrification in aquaculture systems.

A nitrogen budget for a closed, recirculating mariculture system

Nitrogen dynamics were evaluated in a closed, recirculating mariculture system by constructing a mass and isotopic balance of all major nitrogen pools. The experimental system consisted of 12 238-l, closed, independent, recirculating systems, each containing red drum ( Sciaenops ocellatus), a biological filter, water pump and subsurface aeration. The standard system used in our laboratory was compared to two treatments in order to assess their influence on system nitrogen dynamics and fish growth. Treatments included: (1) increased dissolved oxygen concentration; and (2) inoculation with a commercially available probiotic advertised to enhance water quality. Concentrations of total ammonia, nitrite, nitrate, and dissolved organic nitrogen, nitrogen in suspended solids, 5-day biochemical oxygen demand and chemical oxygen demand were measured periodically throughout the experiment. Stable isotope ratio mass spectrometry was utilized to determine δ 15N values for the feed, fish, nitrate, solids associated with the biological filter, suspended solids and settled solids. Final mean values of chemical and isotopic measurements were not significantly different ( P>0.05). Additionally, wet weight gain per fish or the percent dry nitrogen assimilated by the fish were not significantly different. However, survival of fish reared in the oxygen treated tanks was lower (82% survival) compared to the fish in the control (100%) and probiotic (96%) treatments. The isotopic composition of all measured nitrogen pools was enriched in 15N compared to the δ 15N of the feed entering the system. The nitrogen mass balance and isotopic data demonstrate the occurrence of denitrification in recirculating mariculture systems. Release of isotopically light nitrogen to the atmosphere via denitrification resulted in a nitrogen loss of 9–21% of the total nitrogen input.

Simulation model of recirculating mariculture with seaweed biofilter: development and experimental tests of the model

A simulation model was developed for an experimental recirculating mariculture system in Elat, Israel. The system cultures gilthead seabream ( Sparus aurata), using a seaweed biofilter ( Ulva lactuca) to maintain water quality, reduce the flow-through rate of seawater, and reduce the nutrient load in effluents. The model describes the production, transfer, transformations, and losses of inorganic N in the system, in a relatively simple compartment model framework. The model was able to match very closely the experimental data on which it is based. Moreover, the model was able to predict the system's behavior in two additional experiments, which provided independent tests of the model under nonstandard operating conditions (reduced food input and reduced water recirculation rate). The success of the model beyond the range of conditions used in its construction suggests that the model provides a valid mechanistic description of the essential processes affecting inorganic N in the system. Sensitivity analysis of the model indicates that levels of ammonia-N and oxidized N in the system were most strongly affected by the rates of ammonia-N excretion by the fish and ammonia-N uptake by the seaweed, followed by the water inflow and recirculation rates. Sensitivities to internal transformation rate parameters were lower, which might explain the relative seasonal constancy of the system. Simulation experiments indicate that the experimental system could support much higher stocking densities of fish without exceeding tolerable levels of ammonia-N.