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产土霉异味蓝藻的生态学调查及其生理学特性的研究
Alternative TitleEcological investigation and physiological characteristics of earthy-musty odor-producing cyanobacteria
张婷
Subtype博士
Thesis Advisor宋立荣
2008-06-15
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword蓝藻 异味 土霉味 2-甲基异茨醇 土腥素 伪鱼腥藻 库氏鞘丝藻
Abstract2-甲基异茨醇 (2-Methylisoborneol, 2-MIB) 和土腥素 (Geosmin) 是饮用水工业和水产养殖业中最常见的两种土霉味化合物,是造成水体及水产品异味问题的主要原因。蓝藻通常被认为是富营养化水体中 2-MIB 和 Geosmin 最主要的生物来源。自上世纪八十年代以来,我国众多水体富营养化趋势日益加重,常伴随着蓝藻水华的频繁暴发,由此引发的蓝藻毒素和蓝藻异味物质的污染严重影响了我国水环境的安全。本文系统调查了湖北省熊河水库土霉味化合物的周年时空变化,分析了土霉异味的生物来源,分别研究了产 2-MIB 蓝藻伪鱼腥藻 (Pseudanabaena sp.) 和产 Geosmin 蓝藻库氏鞘丝藻 (Lyngbya kuetzingii) 异味产生的特性及其相关影响因子。主要研究内容和结果如下: 1) 采用微波蒸馏-顶空固相微萃取-气质联用测定了鱼体中常见的两种土霉味化合物 2-MIB 和 Geosmin。结果表明:微波蒸馏 6 min、载气流量 70 mL•min-1 为微波蒸馏萃取的最佳条件。所得馏分再进行顶空固相微萃取,并于 GC-MS 中分析检测。基于此检测方法,2-MIB 和 Geosmin 的检出限均达到 0.1 µg•kg-1,且其在 1~20 µg•kg-1 的范围内线性关系良好,相关系数 R 分别达到 0.987、0.995,故该方法可用于分析水产品中痕量的 (ppb 级) 土霉味化合物。 2) 熊河水库属于富营养化水体 (Ⅲ~Ⅳ 类水质),水体及鱼体中均存在土霉味化合物 2-MIB 和 Geosmin,相关分析结果显示,其主要生物来源分别为卷曲鱼腥藻 (Anabaena circinalis) 和伪鱼腥藻 (Pseudanabaena sp.)。Geosmin 和 2-MIB 在 3 个样点的空间分布有所不同:Geosmin 在 3 个样点的浓度水平表现为 A 点 > B 点 > C 点,而 2-MIB 则为 B 点 > A 点 > C 点。此外,鱼的种类不同,其对异味化合物的积累程度也差异显著。经检测,Geosmin 在浮游植物食性的鲢鱼肌肉内的含量高于杂食性的鲤鱼。 3) 采用毛细管分离和一系列纯化方法,从熊河水库中获得伪鱼腥藻纯培养物,并建立了稳定的培养体系。通过对野外与室内培养条件下藻丝的形态学观察,结合 16S rRNA 基因序列分析技术,确定所分离的藻株为伪鱼腥藻 Pseudanabaena sp.。经 SPME-GC-MS 检测显示伪鱼腥藻 (Pseudanabaena sp.) 可产生土霉味化合物 2-MIB。 4) 在实验室培养条件下,研究了从熊河水库分离的藻株伪鱼腥藻 (Pseudanabaena sp.) 的生长特性、胞外 2-MIB 产量和产率 (即单位生物量内的产量) 随培养时间的变化以及胞外 2-MIB 产量与生物量的关系。结果表明,伪鱼腥藻 (Pseudanabaena sp.) 的比生长速率 µ 达到 0.255 ± 0.011 d-1,但对数期持续的时间较短。在整个培养过程中,伪鱼腥藻 (Pseudanabaena sp.) 在迟缓期、对数期以及稳定生长后期 2-MIB 胞外产量增加。胞外 2-MIB 产率 在迟缓期、稳定生长后期和衰亡期呈增长趋势,对数期反而降低。伪鱼腥藻 (Pseudanabaena sp.) 2-MIB 的胞外产量还与细胞数存在着明显的正相关关系。 5) 比较了温度和光照强度对伪鱼腥藻 (Pseudanabaena sp.) 生长和产 2-MIB 能力的影响。结果表明,伪鱼腥藻 (Pseudanabaena sp.) 对光照强度非常敏感,最适生长的光照强度为 25 μmol•m-2•s-1,但对温度的敏感程度不及光强,藻细胞在 20、25 和 30 °C 下均表现出良好的生长状态。2-MIB 产量随温度的升高逐渐增大,并在 35 °C 下达到峰值;过高或过低的光照强度均能降低 2-MIB 产量,最高均值出现在 40 μmol•m-2•s-1 条件下。而 2-MIB 产率则在不同温度和光照强度下具有相似的变化规律:在生长状态较好的情况下,2-MIB 产率相对处于最低水平;相反,在生长状态较差的情况下,2-MIB 产率反而较高;2-MIB 产率与细胞数呈负相关关系。但温度和光照对 2-MIB 由胞内向胞外释放的作用效果存在较大的差异:温度对 2-MIB 释放的贡献较小,而增大光强可促进胞内 2-MIB 向胞外释放。 6) 在实验室培养条件下,研究了产 Geosmin 蓝藻——库氏鞘丝藻 (L. kuetzingii) 的生长特性及产 Geosmin 能力。结果显示,库氏鞘丝藻 (L. kuetzingii) 的比生长速率 µ 为 0.130 ± 0.008 d-1。溶解态 Geosmin 产量先随培养时间的延长而增加,达到其最大值后则随培养时间的延长而降低。溶解态 Geosmin 产率在对数期最低,在静止生长后期出现了一个小小的增幅。此外,在对数生长期,胞内 Geosmin 比率较高;在稳定生长后期,胞外 Geosmin 比率较高。 7) 以 BG11 为基础培养基,研究了不同环境因子和营养盐对库氏鞘丝藻 (L. kuetzingii) 生长和 Geosmin 产量的影响。结果表明,库氏鞘丝藻 (L. kuetzingii) 最适生长光强为 20 µmol•m–2•s–1,最适生长温度为 25 °C;但 Geosmin 产量和产率的最大值均出现在 10 µmol•m–2•s–1、10 °C 条件下。Chl a 含量随 PO4-P、NO3-N 浓度增大而增加,相反,限制生长的低磷 (0.2 mg PO4-P•L-1)、无磷、低氮 (2.47 mg NO3-N•L-1) 以及无氮条件反而显著促进 Geosmin 的产生。过高浓度 (0.980 g•L-1) 或完全缺失 Ca2+ 均抑制藻细胞的生长,在所设四种 Ca2+ 浓度下,Ca2+ 浓度越高,Geosmin 产量越大。库氏鞘丝藻 (L. kuetzingii) 的 Cu2+ 毒性阈值可能为 0.4 mg•L-1。当 Cu2+ 浓度大于 0.4 mg•L-1 时,蓝藻细胞和异味化合物均可被完全除去;当 Cu2+ 浓度小于或等于 0.4 mg•L-1 时,Chl a 含量和 Geosmin 产量均与 Cu2+ 浓度呈负相关关系,相关系数 R 分别为 -0.974 (P < 0.01)、-0.860 (P < 0.05)。溶解态 Geosmin 在库氏鞘丝藻 (L. kuetzingii) 胞外和胞内分布情况的结果显示,在最适生长条件 (20 µmol•m–2•s–1, 25 °C; BG11 培养基) 下,胞外 Geosmin 产量随着生长逐渐增大,并在静止期达到最大值;而胞内溶解态 Geosmin 含量则在对数生长晚期就达到其最大值。然而,在不适宜生长的条件 (e.g., 10 °C, 10 µmol•m–2•s–1, 0 & 0.2 mg PO4-P•L-1, 0 & 2.47 mg NO3-N•L-1, 0.980 g Ca2+•L-1) 下,释放到培养基中的 Geosmin 含量较低,主要积累于细胞内。
Other AbstractTwo major lipophilic compounds, 2-methylisoborneol (2-MIB) and geosmin, are primarily responsible for imparting earthy-musty off-flavors in aquaculture and drinking water industries. Cyanobacteria have been attributed to the main biological sources of the two odorous compounds in eutrophic aquatic systems. As China is a developing country, the off-flavor problem will be an austere aquatic environmental problem for the future, due to environmental protection lagging behind the rapid economic growth. Thus making a study of cyanobacteria-producing off-flavors will provide insight into scientific significance and attempt to meet the urgent demands to our country. Based on the above reasons, the present dissertation established an instrumental method for the analysis of off-flavors in fish, investigated annual dynamics and biological origins of earthy-musty odor in Xionghe Reservoir, and studied the physiological characteristics and odorous compounds production in Pseudanabaena sp. and Lyngbya kuetzingii. The main results are summarized as follows: 1) 2-MIB and geosmin were determined by microwave mediated distillation with headspace solid-phase microextraction and gas chromatography-mass spectrum (MWDE-SPME-GC-MS). It was investigated and discussed the effects of the key parameters, microwave processing time and carrier gas flow rate, in microwave mediated distillation on extracts. It’s concluded that both 6 min of microwave time and 70 mL•min-1 of nitrogen flow were the optimized condition. On the optimized condition, odorous compounds could be completely distilled from fish flesh. Then the odorous compounds were absorbed in the fiber under HS-SPME. Finally, they were desorbed at 250 °C and determined by GC-MS. The limits of detection for 2-MIB and geosmin in fish were both 0.1 µg•kg-1. There were good linear correlation for the two odorous compounds in the range of 1~20 µg•kg-1, and the calibration coefficients were 0.987 and 0.995, respectively. Therefore, trace levels of off-flavor at ppb in fish could be quantified by this method with satisfactory result. 2) To find out the doubted odorous compounds and their origins, the annual variation of physicochemical parameters, algae and odorous compounds at three sites in Xionghe Reservoir were investigated. During May 2007 to Apr 2008, the algal composition, algal cell number, and earthy-musty odorous compounds were identified monthly and monitored in Site A, B and C. Meanwhile, physicochemical parameters such as total nitrogen (TN), total phosphorus (TP), dissolved oxygen (DO), pH, transparency, water temperature and chlorophyll a (chl a) were determined. Two earthy-musty odorous compounds, 2-MIB and geosmin existed in the reservoir were identified by using GC-MS. During the episodes, a great deal of Anabaena circinalis was present on the surface of water body. The highest concentration of geosmin reached 2.7 µg•L-1 while no 2-MIB was detected in July 2007, which showed that geosmin was mainly responsible for the off-flavor episodes in summer. The biological origin of 2-MIB may be related to Pseudanabaena sp., a MIB-producing cyanobacteria, isolated from sediment in Xionghe Reservoir. The concentrations of 2-MIB and geosmin were significantly correlated with the biomass of Pseudanabaena sp. and Anabaena circinalis (P < 0.01), respectively. 3) A filamentous, nonheterocystous cyanobacterium was isolated from Xionghe Reservoir with Pasteur-type pipette and a series of purification. Based on phenotypic analysis and molecular phylogenetic analyses dealt with the sequence of the 16S rRNA gene, this strain was named Pseudanabaena sp., which could produce 2-MIB detected by GC-MS. 4) It was investigated that the effect of culture time on the growth, extracellular 2-MIB production and production rate, and the relationship between extracellular 2-MIB production and cell numbers. The results showed that the specific growth rate (µ) of Pseudanabaena sp. was up to 0.255 ± 0.011 d-1. Extracellular 2-MIB production was increasing during the lag, exponential and late-stationary phase. Extracellular 2-MIB production rate (2-MIB production per unit cell number) was increasing during the lag, late-stationary and decline phase, while decreased at exponential phase. There was a significantly positive correlation between extracellular 2-MIB production and cell numbers. 5) The cell number, 2-MIB production and production rate of Pseudanabaena sp. response to temperature were compared with that response to light intensity. Pseudanabaena sp. was more sensitive to light intensity than to temperature. 25 μmol•m-2•s-1 was the optimum light intensity to the growth, while Pseudanabaena sp. could grow well among 20、25 and 30 °C. 2-MIB production increased as temperature increasing, and reached the peak value at 35 °C. Both high and low light intensities could decrease the 2-MIB production, and the peak value was only observed at 40 μmol•m-2•s-1. Increasing light intensity could stimulate the release of 2-MIB into culture medium, while temperature did not play a part in the release. However, the variety of 2-MIB production rate under different temperatures was the same as that at different light intensities. 2-MIB production rate was negatively correlated with cell number. 6) The characteristics of the growth, geosmin production and production rate of L. kuetzingii were studied in the laboratory. The specific growth rate (µ) of L. kuetzingii was only 0.130 ± 0.008 d-1. Dissolved geosmin production increased as the growth progressed, when reached its maximum value, and then declined. Geosmin production rate (geosmin production per unit chl a concentration) decreased rapidly at the exponential phase, while small amplitude was occurred at late-stationary phase. The ratio of intracellular dissolved geosmin to total geosmin was relatively high at exponential phase. Otherwise, the ratio of extracellular geosmin was high at late-stationary phase. 7) The effects of light intensity, temperature, phosphate-P, nitrate-N, and the micronutrients calcium and copper on the growth, geosmin production and production rate by L. kuetzingii were determined. Of the 3 temperatures—10, 25, and 35 °C—tested, 10 °C yielded the maximal geosmin production and production rate, but optimal chl a production was observed at 25 °C. In the studies on light intensity, the maximal geosmin production and producton rate were observed at 10 µmol•m–2•s–1, while 20 µmol•m–2•s–1 yielded the optimal chl a production. Chl a synthesis by L. kuetzingii was positively correlated to PO4-P and NO3-N concentrations. The production and production rateof geosmin were remarkably promoted by 0 and 0.2 mg PO4-P•L-1, whereas 0 and 2.47 mg NO3-N•L-1 maximized the production rate with a significant difference (P < 0.01). Geosmin production increased with an increase in the Ca2+ concentration in the culture medium, while chl a synthesis was inhibited at 0.980 g Ca2+•L-1. It was suggested that more geosmin was synthesized with lower chl a demand. The Cu2+ toxicity threshold of L. kuetzingii was 0.4 mg•L-1. Chl a and geosmin were not detected when the Cu2+ concentration in the growth media was higher than 0.4 mg Cu2+•L-1. Chl a and geosmin production were negatively correlated with Cu2+ ion concentrations lower than 0.4 mg Cu2+•L-1 (R = –0.974, P < 0.01; and R = –0.860, P < 0.05, respectively). Meanwhile, the relative amounts of extra- and intracellular dissolved geosmin were investigated. Under optimum growth conditions (20 µmol•m–2•s–1, 25 °C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed, and reached the maximum in the stationary phase, while the intracellular dissolved geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under unfavorable growth conditions (e.g., 10 °C, 10 µmol•m–2•s–1, 0 & 0.2 mg PO4-P•L-1, 0 & 2.47 mg NO3-N•L-1, 0.980 g Ca2+•L-1), less geosmin was released into the culture medium, and mainly accumulated in cells.
Pages165
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12304
Collection学位论文
Recommended Citation
GB/T 7714
张婷. 产土霉异味蓝藻的生态学调查及其生理学特性的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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