1株亚硝酸盐降解菌的筛选、鉴定、降解条件及效果

从养殖水体中筛选出1株对哑硝酸盐具有高效降解能力、增殖速度快、稳定和安全的优良菌株,经细菌学常规榆测和16S rRNA序列分析确定为巨大芽孢杆菌(Bacillus megaterium,),并命名为SZ-3.对SZ-3菌株降解条件的单因素实验发现,其最佳降解条件是:pH 7.0,温度33℃,降解所需的最低葡萄糖含量为0.01 mg/L,在24 h内对亚硝酸盐的降解率达69.58%.在哑硝酸盐质量浓度为5 mg/L的100 L污染水体养殖实验中,添加1 000 mL,含量为1×108 CFU/mL的SZ-3培养菌液离心后的纯菌体,在28℃的水温条件下经96 h,SZ-3菌株对亚硝酸盐的降解率可达91.78%.表明该菌株对污染水体中的亚硝酸盐具有较强的降解效果.本研究旨在为研发高效实用的降解亚硝酸盐微生态活性菌奠定基础.     

铁离子对固定化和游离态硝化细菌活性的影响

采用聚乙二醇—海藻酸钠—氯化钙方法包埋淡水型和海水型硝化细菌,比较分析铁离子对固定化和游离态硝化细菌中氨氧化菌和亚硝酸盐氧化菌活性的影响。结果表明,Fe~(3+)对游离态硝化细菌的影响大于固定化硝化细菌,当Fe~(3+)浓度为1mg·L~(-1)时固定化淡水硝化细菌氨氧化活性最高,游离态淡水型氨氧化活性受Fe~(3+)影响较大,120h后明显降低,但对亚硝酸盐氧化活性影响均较小。在不同Fe~(3+)浓度条件下,固定化海水硝化细菌氨氧化活性和亚硝酸盐氧化活性与不加Fe~(3+)组基本相同,而游离态海水型氨氧化和亚硝酸盐氧化活性均随Fe~(3+)浓度增大而略有降低。     

固定化光合细菌净化养殖水质研究

采用海藻酸钠进行光合细菌固定化试验,比较了固定化菌和悬浮态菌的生长和生理特性,观察了其在载体中的分布,并对其净化养殖水的能力进行了初步研究.结果表明,固定化大大提高了光合细菌的生长速率;粒径为3.5mm,菌初始密度为0.12mg/L为最佳固定化条件.在生长初期,细菌在载体内的分布不均匀,后期趋于一致.固定化光合细菌对养殖水的净化能力大大优于悬浮态菌.     

一株亚硝酸盐降解菌的分离鉴定及其降解特性

为获得高效降解亚硝酸盐菌,从鄱阳湖水域筛选出1株新型反硝化细菌,命名为X10。本实验对菌株进行16S rDNA基因序列分析,构建系统发育树并结合生化指标对该菌株进行鉴定;研究了亚硝酸盐浓度、pH值、温度、接种量对其生长及脱氮能力的影响,以及菌株对养殖水体中亚硝酸盐氮的降解能力。结果显示,X10鉴定为木糖氧化无色杆菌;温度30 ℃、pH 7.0、接种量3%,经48 h培养后菌株对筛选培养基中亚硝酸盐(浓度300 mg/L)的降解率最高,达99.8%;将该菌液(2×10⁸ CFU/mL)按1%的接种量加入人工养殖池塘水中(亚硝酸盐质量浓度为0.45 mg/L,pH为6.7),28 ℃水温下经96 h后亚硝酸盐降解率达82.6%;安全性评价实验显示,X10菌落周围无溶血现象,并且高浓度菌液(5×10⁸ CFU/mL)对斑马鱼无致死作用。研究表明X10在水产养殖中具有较好的应用前景。     

复合硝化菌制剂对水质改良的应用效果

室内静态水体中0.25mg/L复合硝化菌制剂使用后,7d内氨氮平均降解率为34.84%,亚硝酸盐氮的平均降解率为19.05%。0.5mg/L组氨氮平均降解率为45.05%,亚硝酸盐的平均降解率为41.79%。1.0mg/L组的氨氮平均降解率为55.26%,亚硝酸盐氮平均降解率为51.20%。氨氮和亚硝酸盐氮的最大的降解峰值出现6d之间。而养殖池塘中,0.5mg/L复合硝化菌制剂后,5d内氨氮的降解率为13.61%～28.03%,7d内亚硝酸盐氮的降解率为9.30%～25.58%。0.2mg/L复合硝化菌制剂使用后,6d内氨氮的降解为23.40%～34.75%,7d内亚硝酸盐氮的降解率为16.33%～36.13%。试验结果表明,复合硝化菌制剂在养殖池塘中使用后,有降解速度快、降解能力强、维持时间长等特点,适宜于作为净化和调控养殖水质的渔用微生物制剂使用。     

不同载体固定化海洋微生物酯酶ETM-b的性能比较

分别采用海藻酸钠包埋法、明胶包埋交联法、壳聚糖吸附交联法制备固定化海洋芽孢杆菌酯酶ETM-b,并对其固定化条件进行了研究。结果发现,壳聚糖制备的固定化酶效果最好,壳聚糖2%、戊二醛浓度1%、小球与酶液4∶3(g/ml)时制备的固定化酶的活性回收最高,达到66%。壳聚糖制备的固定化酶使用10次,相对活性保留70%,具有良好的操作稳定性。固定化酶在非水介质中具有转化α-乙酸萘酯(α-Naphthyl acetate)的能力,在异辛烷、正辛烷、正己烷中活性表现最高。     

光合细菌的固定化及对养殖水体的净化作用

为了解固定化光合细菌对养殖水体的净化作用,分别采用海藻酸钠、PVA/海藻酸钠和NaCS微胶囊3种不同材料对光合细菌进行包埋,并比较了包埋后的光合细菌对养殖水体的净化效果。结果表明:以3种不同材料包埋的光合细菌,对养殖水体中氨氮、亚硝酸盐和COD的去除效果比对照组显著。以海藻酸钠—CaCl2法包埋的光合细菌小球,对养殖水体中氨氮、亚硝酸盐和COD的去除效果较好,但抗冲击力和机械性能较差;而以PVA/海藻酸钠—饱和硼酸法制备的光合细菌小球,传质性稍差;NaCS-PDMDAAC微胶囊,因其膜较薄,故抗冲击力较差,但其中空的纯液态环境为细胞的生长提供了更大的空间,尤其是在水处理10 d后,被包埋的细胞对氨氮、亚硝酸盐和COD的去除效果明显。     

亚硝酸盐氮对金鱼鱼种的急性毒性效应

采用常规生物急性毒性试验方法,研究了亚硝酸盐氮对体长(6.50±0.50) cm、体质量(9.12±0.45) g金鱼的急性毒性效应.试验结果表明:金鱼对亚硝酸盐氮的耐受性随接触时间的增加而明显降低;亚硝酸盐氮对金鱼24、48、72、96 h半致死质量浓度分别为1328.726、784.352、731.890、341.470 mg/L,95%可信区间依次为(1070.889～1648.393)、(578.031～1064.193)、(580.943～922.001)、(283.320～411.504) mg/L,安全质量浓度为34.15 mg/L.     

非离子氨氮和亚硝酸盐氮对暗纹东方鲀稚鱼的急性毒性试验

在水温21~23℃,pH 8.2~8.5,溶解氧6.00~7.50mg/L的条件下,采用半静水法研究了非离子氨氮和亚硝酸盐氮对全长(1.6±0.2)cm、体质量为(0.11±0.05)g的暗纹东方鲀稚鱼的急性毒性效应。试验结果表明,暗纹东方鲀稚鱼受到非离子氨氮和亚硝酸盐氮胁迫后,先后出现鱼体体色变白、扭曲、侧游、失去平衡、昏迷等中毒症状。随着非离子氨氮和亚硝酸盐氮质量浓度的提高和胁迫时间的延长,暗纹东方鲀稚鱼死亡率逐渐升高,存在明显的剂量效应和时间效应关系。非离子氨氮和亚硝酸盐氮对暗纹东方鲀稚鱼96h半致死质量浓度分别为0.46mg/L(95%置信限0.34~0.64mg/L)和290.12mg/L(95%置信限255.16~329.87mg/L),安全质量浓度分别为0.046 mg/L和29.01mg/L。非离子氨氮和亚硝酸盐氮对暗纹东方鲀稚鱼具有一定毒性,且非离子氨氮毒性大于亚硝酸盐氮毒性。     

甘蔗渣载体填料在海水曝气生物滤池中的应用

为研究甘蔗渣作为载体填料用于海水曝气生物滤池中的可行性,在海水曝气生物滤池中培养生物膜,并以此为基础构建海水养殖排放水处理系统。通过监测水体总氨氮(TAN)、亚硝酸盐氮(NO2--N)等水质指标浓度变化,水体游离细菌与载体附着细菌密度变化,评价甘蔗渣载体生物滤池的降解效果。结果显示,以甘蔗渣为载体的生物滤池挂膜所需时间为26 d,挂膜完成后甘蔗渣附着可培养总菌和芽孢杆菌密度分别为3×108cfu/g和7.8×107cfu/g。在处理养殖水体时,生物滤池中水体氨氮和亚硝酸盐氮浓度分别控制在0.2 mg/L和0.05 mg/L以下,同时,水体中芽孢杆菌数量由3.3×103cfu/L增加至7×104cfu/L,弧菌数量由4.9×103cfu/L下降至3.1×101cfu/L。研究表明,以甘蔗渣为载体的海水曝气生物滤池能快速有效地完成挂膜,并在海水养殖排放水处理中取得较好效果。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.