牙鲆海水循环养殖系统生物膜上3种细菌的数量与代谢活性

从牙鲆(Paralichthys olivaceus)海水循环养殖系统中移动床生物滤器载体生物膜上获取异养细菌、氨化细菌和硝化细菌样本.异养细菌用平板涂布法计数,其代谢活性以培养8 h的氧吸收速率表示.氨化细菌、氨氧化细菌(AOB)和亚硝酸氧化细菌(NOB)用MPN法计数,其代谢活性分别以培养24h NH+<4>-N浓度的增加、NH+<4>-N浓度的减少和NO-<2>-N浓度的减少表示.结果表明,生物膜成熟之后,4种生理类群细菌的数量和代谢活性基本保持稳定.异养细菌数量为10-7～108CFU·m-2(载体),氨化细菌、AOB和NOB数量分别为107～108MPN·m-2(载体)、105～106MPN·m-2(载体)和105～106MPN·m-2(载体).分批培养测得异养细菌的氧吸收速率、氨化细菌的氨化速率、AOB的氨氧化速率和NOB的亚硝酸氧化速率分别是0.591～0.738g(O2)·m-2·d-1、0.081～0.135 g(Nh+4-N)·m-2.d-1、0.017～0.031 g(Nh+4-N)·m-2·d-1和0.020～0.038 g(NO-2N)·m-2·d-1.异养细菌的氧吸收速率、氨化细菌的氨化速率和AOB的氨氧化速率与异养细菌、氨化细菌和AOB的数量呈正相关.移动床生物滤器生物膜的平均氨和亚硝酸盐去除速率为(0.121±0.076)g(NH+4-N)·m-2·d-1和(0.139±0.181)g(NO-2-N)·m-2·d-1.同时测定了底物浓度和pH对氨化细菌、AOB和NOB代谢活性的影响.结果显示,氨化细菌的氨化速率、AOB的氨氧化速率和NOB的哑硝酸氧化速率与底物浓度(蛋门胨、NH+4-N和NO-2-N)呈线性相关,pH为8.0时3种生理类群细菌的代谢活性较pH6.5或pH9.5时高.[中国水产科学,2009,16(1):97-103]     

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。     

冰冻对硝化细菌制剂活性的影响

在实验室模拟条件下,研究冰冻对淡水型硝化细菌和海水型硝化细菌制剂中氨氧化细菌和亚硝酸盐氧化细菌活性的影响。结果表明,冰冻对淡水型硝化细菌和海水型硝化细菌制剂活性均有较为明显的抑制作用,且10d处理组的抑制作用高于5d处理组,对淡水型硝化细菌制剂活性的抑制作用大于海水型硝化细菌制剂。液体硝化细菌制剂在冬季运输和保存过程中要采取保温措施,以避免由于冰冻导致的菌剂活性降低。     

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。     

硝化细菌对海水水族箱硝化功能建立过程的影响

氨和亚硝酸盐对海水观赏鱼具有很强的毒害作用,是海水水族箱的主要去除目标。研究考察投加硝化细菌对海水水族箱硝化功能建立的影响。结果表明,投加硝化细菌制剂可以明显缩短硝化功能建立的时间。投加菌剂的实验组水族箱可在9 d时间将40 mg/L氨氮降低到检测不出,亚硝酸氮在第七天出现峰值(37.4 mg/L),亚硝酸氮在第十五天降低到检测不出。不投加菌剂的对照组将40 mg/L氨氮降低到检测不出需要25 d,亚硝酸氮在第二十五天出现峰值(36.6 mg/L),亚硝酸氮在第四十三天降低到检测不出。即实验组完成硝化功能建立需要15 d,而对照组则需要43 d。投加硝化细菌制剂后,海水水族箱内氨氧化细菌、亚硝酸盐硝化细菌可在短时间内形成优势,使氨氮、亚硝酸氮维持在较低浓度水平,缩短硝化系统建立的时间;在不投加菌剂的情况下,氨氧化细菌虽然可在一定时间内形成优势,使氨氮浓度降低,但由于亚硝酸氧化细菌生长更为缓慢,水族箱中亚硝酸积累问题严重。     

刺参养殖池塘底泥理化指标和细菌数量变化的检测

通过检测刺参养殖池塘底泥的6项理化指标和8类细菌数量,研究了山东省莱州刺参养殖池塘底质的周年变化规律。实验结果显示,底泥的pH值和氧化还原电位变化趋势相同,变化范围分别为(7.15±0.40)~(7.80±0.21)和(42.8±85.5)~(-351.0±61.5)mV。硫化物的变化趋势与pH值和氧化还原电位相反,其含量变化范围为(47.66±47.01)~(496.12±418.57)μg/g(干重)。总氮、总磷和有机碳含量变化范围分别为(457.42±103.40)~(865.83±187.85)、(166.83±17.12)~(241.32±27.21)μg/g和0.27%±0.08%~0.37%±0.10%。异养细菌、弧菌和芽孢杆菌数量的变化范围分别为(3.27±5.55)×105~(6.41±8.48)×106、(5.39±4.32)×103~(1.62±2.01)×105和(1.31±1.57)×104~(7.57±1.66)×104 CFU/g。氨化细菌、反硝化细菌和亚硝化细菌数量的变化范围分别为(6.80±1.16)×105~(1.31±1.64)×107个/g、(2.67±2.48)×103~(5.11±5.88)×104个/g和(8.08±8.99)×10~(1.45±1.73)×103个/g,氨化细菌和反硝化细菌在1年内的变化趋势相同。硫还原细菌和硫氧化细菌数量的变化范围分别为(5.62±5.53)×102~(7.22±5.52)×104个/g和(6.50±5.17)×101~(1.00±1.16)×104个/g,两种细菌数量在一年内的变化趋势相反。研究表明,底泥的氧化还原电位与硫氧化细菌数量呈显著正相关(P0.05),硫化物含量分别与芽孢杆菌、反硝化细菌、氨化细菌及硫还原细菌的数量呈极显著正相关(P0.01),有机碳含量与硫还原细菌的数量呈显著正相关(P0.05)。刺参养殖池塘底泥的多项理化指标和细菌数量之间显著相关,关系复杂。     

基于珊瑚骨基质的凡纳滨对虾内循环养殖系统的构建与运行效果

采用珊瑚骨作为生物膜载体,利用海水素配制人工海水,构建盐度为15‰(海水)和5‰(淡水)的两个凡纳滨对虾内循环养殖系统,通过添加硝化细菌菌剂和氮源,分别用8 d和13 d建立硝化功能。按照500尾/m~3密度投入虾苗后,海水系统和淡水系统分别运行97 d和83 d。在运行期间淡水和海水系统养殖水体氨氮浓度始终维持在较低水平,平均浓度分别为(0.015±0.008) mg/L和(0.014±0.008) mg/L;在海水系统运行前60 d,亚硝氮浓度维持在较低水平,在60～90 d,亚硝氮浓度呈缓慢上升趋势,在90 d后,海水系统亚硝氮浓度开始快速增加,最终达到3.43 mg/L;淡水系统在运行前40 d亚硝氮浓度维持在较低水平,40 d后开始小幅上升,运行至70 d后,亚硝氮浓度开始快速增加,最终达到0.52 mg/L。最终海水系统和淡水系统凡纳滨对虾存活率分别为51.5%和48.5%。     

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。     

海水氨氧化细菌菌种保存方法的研究

将筛选得到的—株海水氨氧化细菌,振荡培养至氨氧化细菌对数增长期末期,去除珊瑚砂后对菌液进行分装保存,以研究其在室温、4℃、-18℃及-80℃的保存条件下存活率的变化.试验结果表明,该氨氧化细菌菌液不宜于-18℃保存,室温下保存不宜超过3个月,4℃和-80℃,保存不宜超过6个月.10％的甘油对该氨氧化细菌有抗冻保护作用....     

硝化细菌制剂对淡水水族箱水质的净化效果

研究自制硝化细菌制剂对水族箱水质的净化效果,试验结果表明:硝化细菌制剂对水族箱水质具有明显的净化效果,实验组水族箱水质氨氮、亚硝氮、COD等指标明显低于对照组。投加硝化细菌制剂后,水族箱内氨氧化细菌、亚硝酸盐氧化细菌可在短时间内形成优势,使氨氮、亚硝氮维持在较低浓度水平;在不投加菌剂的情况下,氨氧化细菌虽然可在一定时间内形成优势,使氨氮浓度降低,但由于亚硝酸氧化细菌生长缓慢,水族箱中亚硝酸积累问题严重。     

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.     

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.     

A case study of biofilter activation and microbial nitrification in a marine recirculation aquaculture system for rearing Atlantic salmon (Salmo salar L.)

Marine recirculation aquaculture system (RAS) is a prominent technology within fish farming. However, the nitrifying bacteria in the biofilter have low growth rates, which can make the biofilter activation a long and delicate process with periods of low nitrification rates and variations in water quality. More knowledge on the microbial development in biofilters is therefore needed in order to understand the rearing conditions that favour optimal activation of the biofilters. In this case study, we investigated the activation of two biofilters in a marine RAS for Atlantic salmon post‐smolt associated with either high or low stocking densities of fish by monitoring the microbial communities and chemical composition. The results showed that the microbial communities in both biofilters were similar during the first rearing cycle, despite variations in the water quality. Nitrifying bacteria were established in both biofilters; however, the biofilter associated with low stocking density had the highest relative abundance of ammonia‐oxidizing Nitrosococcus (1.0%) and nitrite‐oxidizing Nitrospira (2.1%) at the end of the first rearing cycle, while the relative abundance of ammonia‐oxidizing Nitrosomonas (2.3%–2.9%) was similar in both biofilters. Our study showed that low fish stocking density during the first rearing cycle provided low and steady concentrations of ammonium, nitrite and organic load, which can stimulate rapid development of a nitrifying population in new marine RAS biofilters.     

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.     

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。     

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。     

非离子氨胁迫对淡水和海水养殖凡纳滨对虾呼吸代谢酶活力影响的比较

为了探讨非离子氨胁迫对淡水和海水两种养殖条件的凡纳滨对虾呼吸代谢酶活力的影响,在实验室条件下研究了非离子氨胁迫(0.1 mg/L和0.5 mg/L)后,两种养殖条件凡纳滨对虾己糖激酶(HK)、丙酮酸激酶(PK)、乳酸脱氢酶(LDH)和琥珀酸脱氢酶(SDH)活力的变化规律,并将两种养殖条件对虾的相关指标进行了比较。结果显示:(1)非离子氨胁迫后,两种养殖条件凡纳滨对虾鳃HK活力变化显著,而肌肉HK活力变化则不显著。(2)非离子氨胁迫后,两种养殖条件凡纳滨对虾鳃PK活力先升高,后逐渐恢复到正常水平;淡水养殖对虾肌肉PK活力则显著升高,而海水养殖对虾肌肉PK活力变化则不显著。(3)0.1 mg/L非离子氨胁迫后,两种养殖条件对虾鳃和肌肉LDH活力变化均不显著,而0.5 mg/L非离子氨对两种养殖条件对虾鳃和肌肉的LDH活力均具有显著影响。(4)非离子氨胁迫后,两种养殖条件对虾鳃和肌肉SDH活力均显著降低。研究表明,非离子氨胁迫对淡水养殖凡纳滨对虾呼吸代谢酶活力具有显著影响;非离子氨胁迫后,凡纳滨对虾有氧代谢迅速减弱,而无氧代谢在胁迫初期略有升高,随后减弱,推测非离子氨胁迫可能使对虾机体主要供能物质发生改变。