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。     

养殖水体中水质净化菌的筛选和鉴定

通过富集培养的方法,从养殖水体和淤泥中筛选出4株优良的水质净化菌.各菌株经鉴定分别为假单胞菌属(Pseudomonas)、凝结芽孢杆菌(Bacillus coagulans)、黄色节杆菌(Arthrobacter flavescens)、坚强芽孢杆菌(Bacillus firmus).它们对模拟养殖水体中高达5mg/L的氨态氮和亚硝态氮的降解率均能达到80%和90%以上,其中假单胞菌属还具有反硝化作用.     

一株去除亚硝态氮细菌分离鉴定及特性研究

自对虾养殖后期池水中分离纯化出一株高效去除亚硝态氮的细菌,进行了菌体的亚显微形态观察和16SrDNA同源序列分析,并探讨了起始pH、温度和碳氮比对该菌株去除亚硝态氮效果的影响。结果表明,分离菌株在初始亚硝态氮质量浓度为55.51mg/L的异养硝化培养基中培养12h后,亚硝态氮去除率达到98.69%;根据形态学特征、生理生化特性及16SrDNA序列分析,确定该菌株为脱氮海洋单胞菌,菌株去除亚硝态氮的最适宜条件为:初始pH 9,温度35℃,碳氮比16。     

混料设计优化复合菌剂比例的研究

采用混料设计（Mixture）对复合菌比例进行优化,测定了3株芽孢杆菌（Bacillus spp.）和1株溶藻弧菌（Vibrio alginolyticus）不同比例搭配对细菌生长的光密度（OD）、氨氮和亚硝氮去除率的影响,建立了各搭配比例与试验指标之间的回归方程。使用Design Expert 8.0的优化功能（Optimization）对满足所有期望的响应值进行优化,得到复合菌的最优比例为芽孢杆菌BD6株 5.2%、BZ5株22%、溶藻弧菌VZ5 株72.8%。对该最优比例进行验证试验,得到的结果与预测基本一致。按上述比例搭配的复合菌株在第24 小时即可获得较高的氨氮和亚硝氮去除率,第72 小时的氨氮去除率为98.37%、亚硝氮去除率为93.81%。混料设计和调优软件可用于混合菌中多组分和多目标参数优化,以获得最优的复合菌比例组合。     

两株光合细菌的分离鉴定及其水质净化效果研究

为了筛选能够高效净化水质的光合细菌菌株,采用半固体试管法和双层平板法,从市售光合菌菌液中分离得到2株光合细菌,分别命名为GHJ-1、GHJ-2,对菌株进行了形态学观察、生理生化及16S rDNA鉴定,比较了这2株光合细菌对青鱼(Mylopharyngodon piceus)养殖污水的净化效果。结果表明:经鉴定这两株光合细菌为Rhodovulum sp菌株。菌株GHJ-1、GHJ-2对污水进行15 d的净化处理后,硝酸盐去除率分别为91.4%和96.3%;亚硝酸盐的去除率分别为68.9%和82.7%;氨氮去除率分别为56.8%和67.5%;COD Mn的去除率分别为61.7%和42.4%。     

光合细菌菌剂和沼泽红假单胞菌对实验水体氮磷营养盐和微生物群落的影响

为比较光合细菌菌剂与沼泽红假单胞菌(Rhodopseudomonas palustris)的生理生态特性,分析了不同初始菌量的菌剂PG和菌株PSB-1对实验水体氨氮(NH_4~+-N)、亚硝氮(NO_2~--N)、硝氮(NO_3~--N)和活性磷(PO_4~(3-)-P)的降解效果,通过高通量测序分析了菌剂PG的优势菌组成及实验结束时水体细菌数量和微生物群落组成。结果显示,菌剂PG组对实验水体的PO_4~(3-)-P、NO_3~--N和NO_2~--N有一定的降解作用,其最大降解率分别为40.98%、28.28%和20.12%。菌株PSB-1组仅对实验水体的NO_2~--N和PO_4~(3-)-P有一定的降解效果,其最大降解率分别为14.19%和9.88%。菌剂PG的主要优势菌为红假单胞菌属(Rhodopseudomonas sp.)。实验7 d后实验组水体细菌数量和微生物群落结构发生变化,水体细菌数量增长,形成以异养细菌为优势菌的菌群结构。结果表明光合细菌菌剂PG对水质因子的降解效果优于沼泽红假单胞菌PSB-1,但与报道的高效光合细菌菌株的降解能力存在一定差距。     

益生菌固定化的研究及对氨氮、亚硝氮的降解作用

采用海藻酸钠和聚乙烯醇进行益生菌固定化实验,对固定化小球进行性能评价,并对其净化养殖水的能力进行初步研究。结果表明,海藻酸钠具有机械强度高、传质性能较好、生物毒性较低和固定操作容易的优点;l司定化沼泽红假单胞菌对氨氮去除率可达43．8％、亚硝氮去除率可达39．4％,对养殖水的净化能力优于悬浮态菌。     

抗弧菌光合细菌的分离鉴定及对氨氮和亚硝态氮的降解特性

该研究采用双层平板涂布法和划线法,从不同地区的海洋环境样品中分离纯化光合细菌,以副溶血弧菌(Vibrio parahemolyticus)、创伤弧菌(V.vulnificus)、鳗弧菌(V.anguillarum)为指示菌,采用牛津杯法测定海洋光合细菌菌株的抑菌作用,采用靛酚蓝分光光度法和盐酸萘乙二胺分光光度法测定不同菌株对氨氮(NH_4~+-N)和亚硝态氮(NO_2~--N)的降解作用,筛选出具有抗弧菌并高效降解NH_4~+-N和NO_2~--N复合功能的优良菌株。结果显示,从30个海水、海泥等样品中分离得到3株光合细菌,分离自连云港车牛山岛海水样品的菌株P-3,对3种弧菌均具有较强的抑制作用,其中对鳗弧菌的作用最强,抑菌圈直径为5.3 mm。3株光合细菌均具有一定的降解NH_4~+-N和NO_2~--N作用,菌株P-3的降解作用最强,在含有50 mg·L~(-1) NH_4~+-N和NO_2~--N的培养基中培养4 d,降解率分别为89.68%和94.98%。经形态学观察、生理生化试验和16S rDNA序列分析,确定P-3为沼泽红假单胞菌(Rhodopseudomonas palustris)。     

刺参池塘低温有机污染物降解细菌的分离筛选及其降解特性

利用刺参(Apostichopus japonicus)饵料培养基,在低温(16±1)℃下,从刺参养殖池塘环境中富集驯化分离有机污染物低温降解细菌;根据菌株生长情况、好氧性和生物安全性进行了初步筛选,然后在菌体接种密度1×106 CFU/mL、(16±1)℃、pH 8.0、160 r/min条件下,通过检测菌株对饵料中有机物(COD)和氨态氮(NH4+-N)成分的去除率来最终筛选获得优良低温有机污染物降解细菌,并对其降解特性进行研究。菌株筛选结果表明,从分离的17株低温细菌中共获得3株菌株DR7、DR8和DR11,它们能同时高效降低和去除饵料浸出物中COD和NH4+-N,5 d COD去除率分别为37.7%、50.0%和21.0%,NH4+-N去除率分别为95.0%、98.0%和99.8%。根据菌株的形态特征、生理生化特性以及16S rRNA序列分析,初步鉴定DR7和DR11属于假单胞菌属细菌(Pseudomonas sp.),DR8属于副球菌属细菌(Paracoccus sp.)。菌株降解特性研究结果表明,菌株培养时间对饵料降解有显著影响(P<0.05),在0～6 d内3菌株对饵料中有机物和氨态氮的去除过程明显,其中DR8降解力最强;各组合菌株的饵料降解效果差异显著(P<0.05),但并不完全优于对照单菌株,其中最佳组合为DR8-DR11。研究认为,筛选的3株低温有机污染物降解细菌为良好的菌种资源,在刺参养殖池塘污染环境调控及刺参微生态制剂的研制方面具有较好的潜在应用前景。     

微生物对水产养殖水质调控作用研究

正本实验研究了光合细菌、放线菌、枯草芽孢杆菌三种细菌优化配比成的复合功能菌去除养殖水体有机氮效果。结果表明,光合细菌、放线菌、枯草芽孢杆菌混合培养生态制剂能有效去除养殖水体中的有机氮,对高浓度的氨氮、亚硝酸盐氮的去除率可达99.6%和94%。     

混养锦鲤对微咸水凡纳滨对虾池水质及产量的影响

在6口面积均为0.4 hm2的微咸水池中养殖凡纳滨对虾,密度为7.5×105尾/hm2,其中4口混养锦鲤,密度分别为1500、3000、60001、2 000尾/hm2,研究混养锦鲤对凡纳滨对虾池水质及产量影响。结果表明:混养池水质比较稳定,透明度、NH4+-N和NO2--N波动幅度较小且数值较低,混养池对虾平均产量为3345 kg/hm2,比单养池平均产量2268 kg/hm2高47.5%,其中混养锦鲤3000尾/hm2的池虾产量最高,达到3765 kg/hm2。     

A novel micro‐organism for removing excess ammonia‐N in seawater ponds and the effect of Cobetia amphilecti on the growth and immune parameters of Litopenaeus vannamei

To investigate the feasibility of using micro‐organisms for ammonia‐N removal, six strains were isolated from Chinese white shrimp, Fenneropenaeus chinensis, seawater culture ponds in Dongying (Shandong, China). Of these, strain DY‐01, which exhibited the highest capacity to degrade ammonia‐N, removed 61.7% of the total ammonia‐N (50 mg/L) in 8 hr. An investigation of the factors affecting the removal efficiency indicated the optimum conditions to be 30°C, pH 8.0, and a salinity level of 30 g/L; 16S rDNA gene sequencing and biochemical analysis identified strain DY‐01 as Cobetia amphilecti, which has not previously been reported to degrade ammonia‐N. This strain also boosted the growth of Pacific white shrimp, Litopenaeus vannamei (p < .05), at a concentration of 10⁷ CFU/mL, with no harmful effects on the shrimp's immune system. This study has thus identified a novel aerobic nitrifying bacterium that is potentially an excellent candidate for improving the water quality in mariculture ponds.     

Application of nitrification and denitrification processes in a direct water reuse system for pacific white shrimp farmed in biofloc system

The aim of the present study was to propose a low-cost nitrogen removal system through the nitrification / denitrification process in order to maintain the water quality required for the Pacific white shrimp superintensive cultivation in closed systems without water renewal. The increase in productivity consequently causes the accumulation of organic matter and nitrogenous compounds, especially ammonia nitrogen and nitrite, which in high concentrations can be lethal to aquatic organisms. In addition, the accumulation of solids in the system provides conditions for the emergence of opportunistic pathogens, microalgae booms, and increases the producer's cost of inputs to maintain the equilibrium physicochemical relationships required for shrimp farming. The experimental productive cycle lasted 36 days using Litopenaeus vannamei shrimps with 7.1 g ± 0.56 g and density of 350 shrimps m⁻³. The nitrogen removal efficiency observed during the study period was 71.3 ± 5.3 %, and the shrimp had a survival of 92.9 % and a final weight of 13.1 ± 1.4 g. Thus, we established a system (ammonia and nitrite), capable of managing solids without interaction with the sea, ensuring high biosecurity against exogenous diseases in marine shrimps farms.     

藻—溞系统对凡纳滨对虾养殖水体调控效果的研究

取5000 mL灭菌后的凡纳滨对虾养殖尾水于5000 mL高压灭菌的烧杯中,分为10组,分别为蛋白核小球藻组(C1)、蛋白核小球藻+大型溞组(C2)、衣藻组(C3)、衣藻+大型溞组(C4)、隐藻组(C5)、隐藻+大型溞组(C6)、蛋白核小球藻+衣藻组(C7)、蛋白核小球藻+衣藻+大型溞组(C8)、衣藻+隐藻组(C9)、衣藻+隐藻+大型溞组(C10),每个处理组设3个平行。各藻种原始藻液25 mL,接种初始密度为1×10^( 8) 个/mL,大型溞密度为1个/L。试验烧杯置于25 ℃、3000lx和14L∶10D光暗比的培养箱内常规培养。每间隔3 d的10:00测定溶液的pH、溶解氧、总氮、氨氮、硝态氮、亚硝态氮、总磷和叶绿素a等水质指标,研究不同藻—溞组合系统对凡纳滨对虾养殖水体净化效果及其生长的影响。试验结果显示,各处理组藻类及大型溞均可正常生长,且藻—溞组合系统具有较好的水质净化调控效果,尤其是蛋白核小球藻+衣藻+大型溞组,至试验结束时其硝态氮、亚硝态氮和氨氮的去除率分别为82%、77%和99%;混合藻生长速度要低于单一藻培养,其中蛋白核小球藻组和衣藻组微藻的相对生长速率最高,分别为27.7%和26.9%;且蛋白核小球藻最有利于大型溞的生长繁殖,至试验结束时由5个增至88个,其次是衣藻,增至80个,而隐藻最差,仅增至59个。培养初期,大型溞的扰动作用能够促进藻类的生长,随着大型溞数量的增多,大型溞的牧食压力能够阻止藻密度过密,从而能够长久维持有益藻相的稳定。在本试验研究条件下,接种蛋白核小球藻、衣藻以及大型溞最适于养殖水环境调控,维持藻相稳定。     

Strategies for ammonium and nitrite control in Litopenaeus vannamei nursery systems with bioflocs

In biofloc technology (BFT) rearing systems, nitrogen compounds, specially ammonia and nitrite, have to be controlled by microbial pathways, mainly through the activity of heterotrophic and chemoautotrophic bacteria. The objective of this work was to assess different water preparation strategies (heterotrophic, chemoautotrophic and mature) in BFT system for nursery of Pacific white shrimp (Litopenaeus vannamei). A 35-day study was conducted with post-larvae shrimp (0.08 g) stocked in twelve 300 L tanks at a stocking density of 2000 shrimp m⁻³. The water preparation strategies for shrimp rearing that were evaluated in this study included: i) Heterotrophic treatment, where the water received sugar as a carbon source; ii) Chemoautotroph treatment, where ammonium and nitrite salts were added to the water; and iii) Mature treatment, which was created by the addition of a significant amount of water containing mature biofloc from another established BFT system. In both mature and chemoautotrophic treatments, the nitrification process was able to keep toxic nitrogen compounds (ammonia and nitrite) at low levels without the addition of carbohydrates. In contrast, heterotrophic system showed peaks of ammonia and nitrite during the rearing cycle, and the level of these compounds were found to be higher in this treatment (relative to the mature and chemoautotrophic treatments). The chemoautotrophic system exhibited a lower abundance of bacteria from the family Vibrionaceae in the beginning of the experiment compared to the heterotrophic and mature treatments. The combination of low Vibrionaceae abundance and good water quality resulted in improved growth performance in this treatment. These findings demonstrate the importance of manipulating the environment of BFT systems to induce an enrichment of nitrifying bacteria before stocking shrimp. We have also found that the addition of a carbon source to BFT systems is necessary only in emergency situations, when ammonia spikes need to be controlled.     

Effects of Inorganic and Organic Fertilization on Physicochemical Parameters,Bacterial Concentrations,and Shrimp Growth in Litopenaeus vannamei Cultures with Zero Water Exchange

To identify ways to improve water quality and shrimp production in closed systems, two parallel experiments (one in tanks and one in ponds) were conducted using Pacific white shrimp, Litopenaeus vannamei, cultures. In both experiments, the effects of inorganic (Nutrilake®) and organic (molasses) fertilization on physicochemical parameters, bacterial concentrations, and shrimp performance under zero water exchange were evaluated. Fertilization with both molasses and Nutrilake enhanced the feed conversion rate, as well as shrimp survival and production. In tanks, the shrimp survival and production rates were highest in the molasses treatment, but this effect was not observed in ponds. In ponds, fertilization with Nutrilake increased nitrogen and phosphorus concentrations more than did the controls and molasses treatments toward the end of the experiment. In tanks, fertilization with molasses reduced ammonia concentrations toward the end of the experiment, but the same effect was not observed in ponds. In ponds, fertilization reduced the proportion of Vibrio spp. bacteria, which most likely reduced the incidence of disease from these potentially pathogenic organisms. In both culture systems, fertilization increased the proportion of Bacillus spp., which most likely enhanced food availability.     

三种微生物制剂对有机物废水中氨氮及亚硝酸盐的影响

实验采用复合净水菌、复合芽孢杆菌、光合细菌与空白对照比较了三种微生物制剂对有机物废水中氨氮及亚硝酸盐的处理效果。结果显示：清除氨氮方面：利用微生态制剂微生物制剂净化7d,三种微生物制剂对有机物污染水体中氨氮的清除率均极显著大于对照组（P〈0.01）,处理组之间复合芽孢杆菌的氨氮降解率56.58%,显著大于降解率分别为42.64%和44.02%的复合净水菌和光合细菌组（0.01〈P〈0.05）。清除亚硝酸盐方面：三种微生物制剂对亚硝酸的降解率均极显著大于对照组（P〈0.01）,而复合芽孢杆菌对亚硝酸的降解率68.84%,显著大于复合净水菌47.70%及光合细菌组37.17%（0.01〈P〈0.05）。结果表明：三种微生物制剂对有机物污染的水体中的氨氮及亚硝酸盐均具有降解效果,其中复合芽孢杆菌对于降低水体中氨氮和亚硝酸盐效果最佳。     

Effects of different feed management treatments on water quality for Pacific white shrimp Litopenaeus vannamei

Increasing feeding rates may provide an increase in production, thus nutrients such as nitrogen, phosphorus and organic matter will also increase. These nutrients promote a greater oxygen demand and concentrations of toxic metabolites which can lead to frequent problems with low dissolved oxygen and an abundance of blue‐green algae. Four feed management practices were evaluated among sixteen 0.1 ha ponds culturing Pacific white shrimp (Litopenaeus vannamei). Feeding treatments included hand feeding using the Standard Feeding Protocol (SFP), SFP plus 15% from 8 to 16 weeks, an automatic‐solar timer which fed SFP+15%, and an AQ1 acoustic demand feeder allowing up to 12 kg/day·pond based on shrimps feeding response. Samples were analysed at weeks 0, 4 and 8–16 for the following parameters: chlorophyll a, total ammonia nitrogen, nitrite–nitrogen, nitrate–nitrogen, total nitrogen, total phosphorus, soluble reactive phosphorus, total suspended solids, total suspended volatile solids, turbidity, conductivity, salinity and biological oxygen demand. Samples were collected and shipped overnight to Auburn, Alabama for off‐site analysis. On‐site water quality was also obtained at the farm. The AQ1 acoustic demand feeder produced the most shrimp with a yield of 4,568 kg/ha; however, the AQ1 also had the highest total ammonia nitrogen and nitrite–nitrogen levels late in the growing season. The AQ1 feeder may be a viable, reduced labour and cost alternative for the shrimp commercial industry; however, such technologies must also be matched to the ability of the production system to process nutrients.     

对虾高密度养殖过程中水质的周期变化与分析

通过对对虾高密度养殖池中水质的连续监测，得出养殖水体水质污染状况及一般规律。养殖水体的污染主要是含氮废物的污染，在高密度养殖池养殖后期，养殖水体中氨氮首先达到峰值2.32m/L，随后亚硝酸盐的含量也迅速达到峰值0．773mg／L，在高密度养殖池中活性磷的含量较高，没有显示出明显的磷缺乏现象。