Comparing denitrification rates and carbon sources in commercial scale upflow denitrification biological filters in aquaculture

Aerobic biological filtration systems employing nitrifying bacteria to remediate excess ammonia and nitrite concentrations are common components of recirculating aquaculture systems (RAS). However, significant water exchange may still be necessary to reduce nitrate concentrations to acceptable levels unless denitrification systems are included in the RAS design. This study evaluated the design of a full scale denitrification reactor in a commercial culture RAS application. Four carbon sources were evaluated including methanol, acetic acid, molasses and Cerelose™, a hydrolyzed starch, to determine their applicability under commercial culture conditions and to determine if any of these carbon sources encouraged the production of two common “off-flavor” compounds, 2-methyisoborneol (MIB) or geosmin. The denitrification design consisted of a 1.89 m³ covered conical bottom polyethylene tank containing 1.0 m³ media through which water up-flowed at a rate of 10 lpm. A commercial aquaculture system housing 6 metric tonnes of Siberian sturgeon was used to generate nitrate through nitrification in a moving bed biological filter. All four carbon sources were able to effectively reduce nitrate to near zero concentrations from influent concentrations ranging from 11 to 57 mg/l NO₃–N, and the maximum daily denitrification rate was 670–680 g nitrogen removed/m³ media/day, regardless of the carbon source. Although nitrite production was not a problem once the reactors achieved a constant effluent nitrate, ammonia production was a significant problem for units fed molasses and to a less extent Cerelose™. Maximum measured ammonia concentrations in the reactor effluents for methanol, vinegar, Cerelose™ and molasses were 1.62 ± 0.10, 2.83 ± 0.17, 4.55 ± 0.45 and 5.25 ± 1.26 mg/l NH₃–N, respectively. Turbidity production was significantly increased in reactors fed molasses and to a less extent Cerelose™. Concentrations of geosmin and MIB were not significantly increased in any of the denitrification reactors, regardless of carbon source. Because of its very low cost compared to the other sources tested, molasses may be an attractive carbon source for denitrification if issues of ammonia production, turbidity and foaming can be resolved.     

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.     

Rapid start-up of a biological filter in a closed aquaculture system

Seeding of new biological filters with nitrifying bacteria is effective in reducing activation time of the new filters. In a series of tests, seeded filters were activated in an average of 10.7 days, compared with 22.2 days for the unseeded controls. The shorter activation period results from the reduced time required for the nitrifying organisms to increase in numbers to their environmental equilibrium in the system tested.     

Effect of particulate organic carbon on heterotrophic bacterial populations and nitrification efficiency in biological filters

Competition between heterotrophic and nitrifying bacteria is of major practical importance in aquaculture biofilter design and operation. This competition must be understood to minimize the negative impact of heterotrophic bacteria on an aquaculture system. On the other hand, the heterotrophic population is suspected of having a positive effect against pathogenic bacteria. Little information is available on the bacterial communities present within aquaculture systems, except for nitrifying bacteria, but a combination of traditional aquacultural engineering research methods and novel microbiological techniques offers new opportunities for the study of these communities.

The heterotrophic bacterial population activity and the nitrification efficiency of a submerged biological filter were studied for an influent TAN concentration of 2 mg/l and varying C/N ratios. The TAN removal rate was found to be 30% lower at a C/N ratio of 0.5 than at a C/N ratio of 0. For higher C/N ratios the reduction in nitrification efficiency was 50% while the attached bacterial abundance was doubled. Moreover, results confirm that abundance of sheared and attached bacteria are correlated. It is not known to what extent biofilter configuration might influence the relationship between heterotrophic and nitrifying bacteria, and further work will be carried out with moving bed and fluidized filters. A better understanding of the role of the heterotrophic bacteria in RAS will help to optimize any positive “biocontrol” effect and to minimize the microbial degradation of rearing water and the reduction of nitrification rates.     

Application of microbead biological filters

The application of floating microbead filters to aquaculture is reviewed and discussed. The microbead filter is distinctly different from the more commonly used floating bead filters that are used today. Conventional bead filters work in pressured vessels and use a media that is only slightly buoyant. The required mass of beads for the volume required make the media a relatively expensive component of a floating bead filter in contrast to sand or microbead media that is much less expensive on a per volume basis. Microbead filters use polystyrene beads (microbead) that are 1–3 mm in diameter (floating bead filters use media approximately 3 mm in diameter also). Microbead have an overall bulk density of 16 kg/m³ and a specific surface area of 3936 m²/m³ (for 1 mm beads). This material can be obtained commercially in bulk for roughly US$ 4 kg⁻¹ of material. Biological filters that use microbeads for their nitrifying substrate can be thought of as a trickling bio-filter in terms of how the flow distribution and collection mechanics are designed and operated. For design purposes, microbead filters can be assumed to nitrify approximately 1.2 kg of TAN/m³ of media per day for warm water systems with influent ammonia–nitrogen levels from 2 to 3 mg/l. For cool water applications, rates should be assumed to be 50% of warm water rates or use rates similar to those used for fluidized sand beds. Designs and results in several applications are presented. Microbead filters have been used successfully by several commercial growers after being first introduced in the mid 1990s. Effects of capitalization for equipment and buildings upon production costs is discussed and presented in graphical form.     

EVALUATION OP A RECIRCULATING FRESHWATER SALMON REARING FACILITY USING CLINOPTILOLITE FOR AMMONIA REMOVAL

The Seattle Aquarium has reared coho and Chinook fingerlings since 1978 in a 36,000 liter recirculating freshwater system utilizing clinoptilolite (clino) for ammonia removal. The system has effectively produced 140,000 smolts, due in large part to the efficient removal of fish-excreted ammonia by the clino filters. Quantitative studies were performed to assess clino filter loading capacity and to establish effective recharging techniques. The operational characteristics of the clino filters and suggestions for using them in freshwater recirculating culture systems are discussed.     

Ammonia removal in selected aquaculture water reuse biofilters

Four fixed-film biological filters (rotating biological contactor, biodrum, trickling filter, and a submerged anaerobic filter) were tested for the removal of ammonia using a simulated warmwater fish and invertebrate culture water supply. Filter design may be determined based on the results of ammonia removal efficiency over a wide range of hydraulic loads. The rotating biological contactor (RBC) provided the best ammonia removal (over 90%) up to about 0·06 m³ m^?2 day^?1 (1·2 gpd ft^?2). The biodrum removed over 80% of the ammonia to a hydraulic load of 0·05 m³ m^?2 day^?1 (0·9 gpd ft^?2). The trickling filter removed 50% of the ammonia at a hydraulic loading of 0·012 m³ m^?2 day^?1 (0·3 gpd ft^?2).     

ION EXCHANGE SYSTEMS FOR WATER RECIRCULATION

Since the Bozeman Study in 1972, several pilot plants have been investigated to evaluate the application of the ion exchange systems for water recirculation in fish hatcheries. Pioneering work was made at the biological research laboratory of Kramer, Chin and Mayo, Inc., followed by a pilot plant testing at the Oregon Aqua Fish Hatchery in Newport, Oregon. In 1976, two 50-gallon per minute capacity ion exchange bed systems were installed and tested at Dworshak National Fish Hatchery in Orofino, Idaho. Recently, water reuse is accomplished by pumping approximately 120 gallons per minute from the raceway and filtering it through the pressure sand filter and the clinoptilolite filters for the removal of ammonia in the Seattle Aquarium. The paper will discuss the physical and chemical properties of ion exchange materials, pilot plant test results, engineering design criteria and application, and economics as compared to conventional biological ammonia removal systems. In addition, discussion will also be made in the area of pretreatment prior to ion exchange bed and regeneration as well as operational procedure adjustment. Further works needed to be conducted to provide optimum operating conditions will be discussed.     

A quantitative method for detecting ammonia‐oxidizing bacteria in coastal aquaculture systems

There is a need to quantify autotrophic nitrifiers in coastal aquaculture systems for evolving a bioremediation strategy. Autotrophic nitrifiers are extremely slow‐growing organisms, which cannot be detected by traditional methods as they are notoriously difficult to culture. Molecular techniques based on functional genes could be deployed for the detection of nitrifiers. Ammonia monooxygenase (amoA), that catalyses the oxidation of ammonia to hydroxylamine in the rate‐determining step of nitrification is largely unique to ammonia‐oxidizing bacteria (AOB). In the present study, a quantitative real‐time polymerase chain reaction assay targeting amoA was developed to estimate AOB population size in coastal soil, ammonia‐removing bioaugmentors and the solid matrix. To achieve this objective, different set of primers and a dual labelled probe have been designed for SYBR Green and TaqMan real‐time assays. The abundance of AOB ranged from 10⁴ to 10⁶ order of magnitude in the samples. In the present study, biofilm formation of the consortium of nitrifying bacteria onto bagasse has also been quantified. The results demonstrate that the developed method is a rapid and sensitive tool for the quantitative detection of nitrifying bacteria in aquatic and related environment. This helps in making the bioremediation approach for ammonia removal by immobilization of nitrifying bacteria onto the natural substrate.     

不同基质构建海水养殖系统硝化功能的比较分析

为比较不同基质构建海水养殖系统硝化功能的强弱,选取纤维毛球、陶粒、螺旋式生物绳等7种基质,其中陶粒、流化床填料、纤维毛球采取不同放置方式,共建立14个模拟海水养殖系统,比较不同基质硝化功能建立过程以及同种基质不同放置方式对氨氮和亚硝氮的去除效果。结果表明,单位体积珊瑚骨的氨氧化活性和亚硝酸盐氧化活性高于其他载体,在氨氮初始浓度20 mg/L条件下,氨氮和亚硝氮降解至检测不出分别需要3 d和11 d,而纤维毛球、陶粒、螺旋式生物绳、流化床填料、丝带内芯悬浮球、海绵内芯悬浮球硝化系统的建立分别需要18、26、30、25、27和22 d。纤维毛球100目筛绢悬挂、陶粒网兜悬挂、流化床填料100目筛绢悬挂优于其他放置方式,其中纤维毛球100目筛绢悬挂硝化功能建立时间为18 d,效果最优,流化床填料100目筛绢悬挂、陶粒网兜悬挂硝化系统建立分别需要21、24 d。     

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.

     

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。     

Pre-stocking acclimatisation of brown trout Salmo trutta: effects on growth and capture in a fast-flowing river

Abstract  A field experiment was carried out over two consecutive years in a fast-flowing river to evaluate the effects of a 5-day acclimation period on the subsequent growth and capture of stocked juvenile brown trout, Salmo trutta L. Post-stocking growth of both acclimated and non-acclimated fish remained very poor for at least 44 to 50 days. After this slow start, all fish grew significantly but acclimated trout performed 40.1% better than non-acclimated individuals. However, the better growth did not lead to higher capture rates. Capture success post-stocking was low, regardless of acclimation. Flooding, combined with low fitness of the stocked fish in the first weeks was considered the main reason for the unsatisfactory stocking success. It was concluded that in-stream acclimation aids rapid adaptation of stocked trout and thus imparts some growth advantages, although this does not necessarily result in higher yields.     

Organic and inorganic carbon levels in recycled seawater during the culture of tropical prawns Penaeus sp.

Suspended particles and dissolved organic carbon in laboratory recirculation systems were broadly categorised by size filtration and ultraviolet absorption. Their concentrations in recycled seawater increased during the culture of tropical prawns with increase in feeding rate and also with a ‘load’ factor calculated to take account of the volume of water used to culture a known biomass of prawns. In systems operated without periodic water renewal the proportion of organic matter in filterable material and the integrated ultraviolet absorbance (Σ A 250–330 nm) of filtrate increased with time. The increasing absorbance observed, particularly in the higher wavelengths (Σ A 290–330 nm), indicated the accumulation of metabolically derived organic material that was resistant to microbial degradation. Mean numbers of bacteria varied immensely from 7 to 25 000 × 10³ ml^?1 but were lower in foamed than unfoamed systems.The relation between the concentration of dissolved inorganic carbon (a measure of carbonate alkalinity) and pH in the systems varied according to the type of media in the biological filter and the method used to control pH. Results closest to natural seawater were obtained in systems containing filters with limestone media periodically dosed with sodium hydroxide solution. Higher levels of inorganic carbon for a given pH above pH 7·9 were, however, noted when sodium hydroxide was used with plastic media filters, a feature beneficial to heavily loaded systems.     

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.     

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。     

Ammonia production and oxidation during the culture of marine prawns and lobsters in laboratory recirculation systems

Data obtained during the culture of tropical prawns Penaeus sp. and European lobsters Homarus gammarus (L.) in 20 laboratory recirculation systems over a number of years were used to obtain gross estimates of (a) the rates and diurnal patterns of ammonia excretion, (b) food utilization and wastage, and (c) nitrification rates in biological filters. Ammonia excretion in Penaeus monodon decreased with increase in animal size from 0·93 to 0·30 mg N g^?1 day^?1 at 1·6 and 27 g live weight, respectively. Large lobsters (300 g) excreted 0·3 mg N g^?1 day^?1. Peak excretion times were approximately 3, 9 and 15 h after a single morning feed for prawns and after 6 and 12 h for lobsters. Uneaten food solids amounted to 11% (lobsters) and 32% (prawns) of the daily ration and, for 4 g animals, only 69% (lobsters) and 45% (prawns) of the food nitrogen in the daily ration was converted into crustacean flesh. Nitrification rates ranged from 0·03 to 0·43 g N oxidized per m² of filter media surface per day. Some evidence for periodicity in nitrifying activity was also found and is discussed in relation to the published literature.     

不同pH和滤料对硝化细菌消氨效果的影响

为了解在不同pH和滤料条件下硝化细菌对氨氮(NH_4~+-N)和亚硝酸盐氮(NO_2~--N)的去除效果,通过试验,探讨了5.0~10.0等6个pH梯度以及陶环、珊瑚石、生物刷和生物球等4种滤料的消氨效果。在pH 8.0~9.0时,至试验第7天氨氮去除率分别达99.86%、98.95%,明显高于pH 6.0、7.0和10.0组(去除率分别为66.18%、71.43%和70.51%)。在pH 7.0~9.0时,亚硝酸盐氮浓度的增加小于氨氮浓度的下降,特别是在pH 9.0时两者浓度变化差异明显。生物刷、陶环、珊瑚石和生物球分别在试验的第3、4、6、7天,氨氮去除率达100%。陶环组和珊瑚石组,NO_2~--N质量浓度在达到最高值(9.60 mg/L和10.00 mg/L),之后开始逐步下降。生物刷组和生物球组在达到最高值(9.55 mg/L和11.00 mg/L)之后基本维持不变。结果表明:硝化细菌适宜碱性的环境条件(pH 8.0~9.0),水体pH 9.0最有利于硝化细菌对NH_4~+-N和NO_2~--N的去除。不同滤料对硝化细菌去除NH_4~+-N和NO_2~--N有不同的影响。陶环对硝化细菌去除NH_4~+-N和NO_2~--N都有良好效果,生物刷只对去除NH_4~+-N有良好效果,珊瑚石只对去除NO_2~--N有良好效果。多种滤料配合使用有利于产生优势互补的效果。     

Effect of shelter acclimation on the post-release movement and putative predation mortality of hatchery-reared black-spot tuskfish <Emphasis Type="Italic">Choerodon schoenleinii</Emphasis>, determined by acoustic telemetry

In this study, the effect of shelter acclimation on the post-release movement and putative predation mortality of hatchery-reared black-spot tuskfish Choerodon schoenleinii was examined using acoustic telemetry. We acclimated four 1-year-old fish to shelters in cages before release and compared their movements with six nonacclimated fish. Since it was not possible to compare the behavioral pattern between the former and the latter fish due to the short periods the latter fish were available to be monitored, we also compared their movements with those of large nonacclimated fish that were less likely to be preyed upon. Sixty-seven percent of the nonacclimated fish showed atypical movements before the signals ceased to be detected, a pattern that suggested a predation event had occurred, whereas none of the acclimated and large nonacclimated fish showed the atypical movements. In addition, the probability of detection cessation was about 13 times lower in the acclimated than nonacclimated fish. The signal detection patterns suggest that the acclimated fish utilized night-time shelters from the first night after release, while the large nonacclimated fish started to utilize shelters several days after release. Therefore, it is likely that the shelter acclimation enhanced the shelter utilization by tuskfish, possibly decreasing post-release predation mortality.     

一株淡水螺旋藻的盐度驯化及其净化养殖废水作用

为了研究螺旋藻吸收作为海水养殖废水净化手段的可能性,通过2种方法对一株淡水螺旋藻钝顶螺旋藻(Spirulina platensis)进行了海水环境的适应性驯化试验.结果表明,逐级提高海水比例法可以使得淡水螺旋藻较好地适应海水环境.还对驯化成功的螺旋藻进行了生长环境如pH、温度、光照度、COD等分析考查,得出最适宜的生长或操作条件.结果表明,螺旋藻使用的最佳条件为pH 6～11、温度25～40 ℃,最佳使用浓度为91×104 cell/mL左右,加入有机物能够促进螺旋藻的生长繁殖.驯化后的螺旋藻对于实际的养殖废水有较好的净化效果.