好氧反硝化细菌YX-6特性及鉴定分析

从对虾池塘筛选得到1株高效的好氧反硝化细菌,命名为YX-6。对该菌生长及反硝化性能间的关系进行研究;同时研究了不同温度、pH、盐度及碳源对该菌生长及反硝化性能的影响。结果表明,该菌反硝化作用主要发生在对数生长期,可将亚硝酸盐氮由10mg/L降至0;该菌最适生长及反硝化温度为30℃;pH值范围为7～9时最适于该菌生长及反硝化性能的发挥。该菌最适盐度范围为0～15;丁二酸钠、乙酸钠为该菌生长及反硝化的最适碳源。通过对YX-6菌株生理生化及16SrRNA分子鉴定,初步鉴定为凝结芽孢杆菌(Bacillus coagulans)。对该菌株亚硝酸还原酶基因进行序列分析,结果表明,该菌含有亚硝酸还原酶nirS基因。     

温室养鳖池底质硝化细菌的分离及其硝化性能的初步研究

通过对温室养鳖池底质硝化细菌的富集，使其浓度达到10^7个／mL，用硅胶平板分离得到硝化细菌纯培养物。经鉴定主要有四种菌：硝化杆菌（Nitrobacter sp.）、硝化球菌（Nitrococcussp.）；亚硝化单胞菌（Nitrosomonas sp.）和亚硝化球菌（Nitrosococcus sp.）。同时，对四种硝化细菌的硝化强度进行了测定，结果依次分别为78.7%、61.8%、74.3%和59.9%。而利用盐酸环丙沙星对硝化细菌硝化性能进行了干扰试验表明，0.4mg/L以上的盐酸环丙沙星，对亚硝化球菌和硝化球菌的硝化强度影响较大；同样浓度的抗生素对亚硝化单胞菌和硝化杆菌的影响相对较小。     

电气石对硝化菌生长和生物膜形成、熟化的影响

在培养基中添加电气石培养硝化菌,研究电气石对硝化菌生长的影响,并在此基础上将电气石添加到普通陶粒(CM)原料中制备了功能性陶粒(FCM),通过测定水中氨氮、亚硝酸盐氮和硝酸盐氮的含量变化,比较功能性陶粒和普通陶粒两种载体上生物膜的生长状况。结果表明:添加电气石的培养基中的亚硝化细菌和硝化细菌数量明显高于未添加电气石的对照组;FCM上的生物膜熟化过程对氨氮的去除率在第14 d趋于稳定,硝酸盐氮含量从第12 d逐渐升高,分别比CM早7 d和6 d,能较早发挥生物硝化功能。     

硝化毛球和底沙对硝化细菌脱氮效果的影响

本实验模拟工厂化养殖模式建立养殖水体净化装置,研究硝化毛球和底沙对硝化细菌净化效果的影响,结果表明:装载硝化毛球、铺设底沙和只投加硝化细菌制剂的三个实验组对养殖水体水质具有一定的净化效果,氨氮、亚硝氮等指标均低于空白组。其中装载硝化毛球的实验组氨氧化细菌、亚硝酸盐氧化细菌可在短时间大量生长繁殖,形成优势,使养殖池氨氮、亚硝酸盐浓度维持在较低水平;铺设底沙的实验组对硝化细菌净化水质效果影响不大。装载硝化毛球的实验组,水质最清澈,无异味,养殖池底部无残渣碎屑,青虾生长状况良好,增重最多。     

一株高效脱氮菌株的分离鉴定及应用潜力分析

为了获得对虾养殖池塘中高效去除亚硝态氮和氨氮的菌株,采用富集培养分离的方法,从养殖水体中筛选得到1株去除亚硝态氮和氨氮的菌株,培养24 h后的去除率分别为96.17%和88.27%,编号为O-11。基于形态学、分子生物学及生理生化鉴定结果,明确了该菌株基本生物学特征以及可能的分类地位。分离菌株在20~30℃时有利于亚硝态氮的去除,而温度为20~35℃时对氨氮的去除效果较好;分离菌株在盐度小于30的环境中对亚硝态氮的去除能力受盐度变化的影响不大;在碱性环境中分离菌株对氨氮的去除能力较高。安全性检验可知,在菌浓度为10~5~10~8 cfu/mL的菌株O-11对凡纳滨对虾(Litopenaeus vannamei)是安全的,且在菌浓度为10~5 cfu/mL时能显著提高对虾的存活率,促进对虾生长。这说明,分离菌株O-11在水产养殖水体中有害氮脱除方面具有潜在的应用价值。     

低温海水硝化细菌富集培养过程及影响因素

多数硝化细菌的适宜温度是28℃左右,低于15℃硝化活性会基本丧失。为解决这一问题,通过构建低温海水硝化细菌富集培养装置,在11~14℃、pH值7.0~7.8、溶解氧4.0~4.5mg/L条件下,经过150d富集培养得到AOB硝化强度为21mg(NH_3-N)/(L·d),NOB硝化强度为93mg(NO_2~--N)/(L·d)的富集培养物。对富集培养物研究表明,当温度为15℃时,pH值为8.0、初始氨氮浓度为30mg/L条件下氨氧化活性较强;当温度为15℃时,pH值为7.0、初始亚硝氮浓度为80mg/L的条件下亚硝酸盐氧化活性较强。     

载体对硝化细菌富集培养过程的影响

分别采用活性污泥和惰性材料MX作为载体富集培养硝化细菌,研究了富集培养过程中生物相结构和性质变化。结果表明,在25～28℃,pH 7.5～8.5,DO 2～5mg/L,氨氮浓度100mg/L条件下,分别经过19d和15d的富集培养,可以得到硝化速率为4.18mg(NH3-N).[g(MLSS).h]-1和10.1mg(NH4+-N).[g(MLSS).h]-1的硝化细菌培养物,以惰性材料MX为载体富集培养的硝化细菌培养物硝化速率明显高于活性污泥为载体。在2种富集培养过程中,硝化细菌培养物的色泽和结构、MLSS、SV30、SVI、硝化强度和硝化速率等均呈现规律性变化且2种培养方法间表现出明显差异。     

海洋硝化菌群的富集驯化和筛选的初步研究

以循环养殖废水为介质,利用不断提高氨氮浓度的方法富集驯化海洋硝化菌群。经过70d的富集驯化,海洋异养细菌数目变化不大,氨氧化细菌和亚硝酸氧化细菌分别增加了43倍和29倍。驯化液中氨氧化能力提高55倍,造成了亚硝酸盐和硝酸盐的积累。利用选择性培养基从驯化液中分离到7株硝化菌,通过菌落形态和菌体形态观察对分离的菌株进行了初步的鉴定。通过硝化能力测试,筛选出硝化能力强的3株细菌。     

海绵中氯丙酸脱氯微生物的分离及其初步研究

以氯丙酸为单一碳源,通过富集培养从大连海域繁茂膜海绵中分离筛选得到139株氯丙酸降解菌。经过初步筛选得到35株降解能力较强的菌株。对这些菌株的耐盐试验表明,分离到的大部分菌株均具有较强的盐耐受能力,多数菌株为中度嗜盐菌。其中部分菌株可以在14.6%以上盐度培养基中生长,可能为极端嗜盐菌。多底物降解试验表明,大多数菌株均能降解多种底物,这些菌株可用于有机氯污染环境生物修复。同时也说明海绵对水体中有机氯污染物具有去除作用。     

一株异养硝化-好氧反硝化菌株的分离鉴定及其对养殖废水的脱氮效果

养殖废水排放严重制约海水养殖业健康发展,为了筛选出适用于养殖废水高效脱氮的菌株,利用BTB培养基从对虾育苗尾水沉淀池塘底泥和海水养殖废水处理池中分离纯化得到5株好氧反硝化菌株。经异养硝化能力试验研究发现5种菌株均具有一定的硝化能力,其中WM2菌株氨氮去除效果显著,去除率达到76.3%,经形态学鉴定、16S rDNA基因序列分析和系统发育树构建分析,鉴定其为革兰氏阴性菌,属于海生杆菌(Marinobacterium)属,海生杆菌属作为脱氮细菌研究在以往的研究中鲜有报道。将菌株WM2接种到实际的半滑舌鳎工厂化养殖废水中,处理48 h后的氨氮去除率达到89.38%、硝态氮去除率为88.77%、亚硝态氮去除率为99.66%,脱氮效果显著。因此本研究筛选出的菌株WM2作为新型异养硝化-好氧反硝化细菌对养殖废水脱氮具有良好的应用潜力。     

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.     

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.     

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.