铜绿微囊藻水华模拟研究及微囊藻毒素测定方法的改进
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摘要
随着水体富营养化程度日益加剧,蓝藻水华的发生越来越频繁,由蓝藻毒素引起各种动物甚至人类中毒的事件也随之增多,对人类社会、经济和环境造成不良影响。微囊藻毒素成为最受人们关注的问题之一,也是防治水华危害的重点。本论文采用实验室小试和小型模拟装置的方法,研究了在铜绿微囊藻生长过程中,不同环境因子对藻类增殖的特征、氮磷在细胞内外份额的变化、群体形态变化等的影响,并考察了铜绿微囊藻的产毒特性。同时在进行测定微囊藻毒素时,提出了一种微囊藻毒素富集提取的新方法。
研究结果表明,铜绿微囊藻最适生长条件为:温度为29℃,光照强度为4000Lx,pH为9.0,Zn~(2+)浓度为100nmol/L,Fe~(3+)浓度为10000nmol/L,总磷浓度为7.0mg/L,氮磷比为30:1。根据Monod方程和Droop方程,磷酸盐影响铜绿微囊藻生长的比增长率最大值μ_(max)=0.173/d,半饱和常数K_s=0.275mg/L,细胞内磷份额Q_0 =3.803μg/mg(dw);不同氮磷比影响铜绿微囊藻生长的比增长率最大值为0.173/d,半饱和常数为4.990mg/L,细胞内氮份额62.137μg/mg(dw)。
实验室小型模拟装置条件下,通过营养盐对微囊藻生长影响的过程分析结果表明,藻的生长最终发展为磷限制。但随着外源性磷的输入,藻体不再受磷限制,当藻体生物量达到一定量之后,藻的生长可能转变为光限制。试验表明,水体的外加营养盐浓度的逐步提高是对铜绿微囊藻生长的一种外加刺激,将促进铜绿微囊藻的生长。此外,微囊藻对磷营养的摄取存在过度摄取行为,以应对环境中磷的缺乏及为爆发性增殖做准备。在微囊藻培养过程中,水体pH、DO表现出了昼夜变化。
以产毒铜绿微囊藻为研究对象,主要通过对不同的有机相萃取液和沸水浴的处理时间的优化选择,提出了一种新的前处理方法用于高效液相色谱(HPLC)法分析测定微囊藻毒素环境样品。结果表明,有机相萃取液选用CCl_4,沸水浴时间为15min,可以取得较好的提取效果。该方法的MC-RR和MC-LR的回收率分别达到94.6%和95.3%,与传统提取方法相比MC-RR和MC-LR的相对误差为3%和9.3%。
通过对铜绿微囊藻生长与产毒关系的研究结果可知,铜绿微囊藻的产毒能力与所处的生长阶段有关。铜绿微囊藻在对数生长期,产毒量明显增高。铜绿微囊藻通过分泌微囊藻毒素,抑制其他水生物种的生长,这也是铜绿微囊藻在自然水体中能够获得竞争优势,暴发微囊藻水华的原因之一。
The serious eutrophication of water body results in frequent breaking out of cyanobacterial blooms, and some genera of cyanobacteria can produce microcystins, which can cause health problems in animals and humans. The effect of microcystins on society,economy and environment has become one of the problems people concerned mostly, as well as the key in the control of cyanobacterial blooms. The sutdies were established with M. aeruginosa, one of the most deleterious species of harmful algal blooms. The environmental factors such as temperature, illumination, pH, zinc, iron, nitrogen and phosphorus were studied to determine their effect on the growth and microcystins production of M. aeruginosa. And in this paper, a new rapid method for toxin extraction was provided based on boiling water bath/liquid-liquid extraction.
The results indicate that: M. aeruginosa grew well on the condition that the temperature was 29℃, illumination was 4000Lx, pH was 9.0, Zn~(2+) concentration was 100nmol/L, Fe~(3+) concentration was 10000nmol/L, P concentration was 7.0mg/L, N/P was 30:1. The effects of P concentration on the growth rate of M. aeruginosa under simulative equipment can be described using the Monod equation and Droop equation. The max growth rateμ_(max)=0.173/d. The P half saturation constant for algae growth K_s=0.275mg/L. The P subsistence quota of algae Q_0=3.803μg/mg(dw). The P half saturation constant for algae growth was 4.990mg/L. The N subsistence quota of algae was 62.137μg/mg(dw). The max growth rate was 0.173/d.
The results about the effects of nutrients on the growth of M. aeruginosa and environment factors in simulative equipment of experiment were as flows: The last development of M. aeruginosa growth was phosphorus limitation. With imput of extragenous phosphorus, M. aeruginosa was not limited by phosphorus. When the biomass reached a certain value, the growth of M. aeruginosa translated to light limitation. The growth of M. aeruginosa could be improved by increasing the phosphorus concentration gradually as additional stimuli. M. aeruginosa was luxury phosphorus uptake to cope with phosphorus deficiency when the water environment was breakout of algal blooms. Diurnal changes in the simulative equipment of pH and DO were displayed with the growth of M. aeruginosa.
The adsorption and extraction of MCs in M. aeruginosa was studied from the selection of different organic solvents and different time of boiling water bath. The results showed that the extraction was best when used CCl_4 as organic solvent and selected 15min as the time of boiling water bath. Comparing with the traditional extraction methods ,the comparative error of MC-RR and MC-LR were 3% and 9.3%, and the recovery of MC-RR and MC-LR were 94.6% and 95.3%.
Effects of growth on M. aeruginosa production of MCs were as flows: The ability of M. aeruginosa to product toxin was related to the developmental stage. The concentration of MCs were significantly higher in the increased logarithmic phase. The M. aeruginosa achieved advantage of competition which exude MCs to inhibit the growth of other species so as to breakout of water bloom.
研究结果表明,铜绿微囊藻最适生长条件为:温度为29℃,光照强度为4000Lx,pH为9.0,Zn~(2+)浓度为100nmol/L,Fe~(3+)浓度为10000nmol/L,总磷浓度为7.0mg/L,氮磷比为30:1。根据Monod方程和Droop方程,磷酸盐影响铜绿微囊藻生长的比增长率最大值μ_(max)=0.173/d,半饱和常数K_s=0.275mg/L,细胞内磷份额Q_0 =3.803μg/mg(dw);不同氮磷比影响铜绿微囊藻生长的比增长率最大值为0.173/d,半饱和常数为4.990mg/L,细胞内氮份额62.137μg/mg(dw)。
实验室小型模拟装置条件下,通过营养盐对微囊藻生长影响的过程分析结果表明,藻的生长最终发展为磷限制。但随着外源性磷的输入,藻体不再受磷限制,当藻体生物量达到一定量之后,藻的生长可能转变为光限制。试验表明,水体的外加营养盐浓度的逐步提高是对铜绿微囊藻生长的一种外加刺激,将促进铜绿微囊藻的生长。此外,微囊藻对磷营养的摄取存在过度摄取行为,以应对环境中磷的缺乏及为爆发性增殖做准备。在微囊藻培养过程中,水体pH、DO表现出了昼夜变化。
以产毒铜绿微囊藻为研究对象,主要通过对不同的有机相萃取液和沸水浴的处理时间的优化选择,提出了一种新的前处理方法用于高效液相色谱(HPLC)法分析测定微囊藻毒素环境样品。结果表明,有机相萃取液选用CCl_4,沸水浴时间为15min,可以取得较好的提取效果。该方法的MC-RR和MC-LR的回收率分别达到94.6%和95.3%,与传统提取方法相比MC-RR和MC-LR的相对误差为3%和9.3%。
通过对铜绿微囊藻生长与产毒关系的研究结果可知,铜绿微囊藻的产毒能力与所处的生长阶段有关。铜绿微囊藻在对数生长期,产毒量明显增高。铜绿微囊藻通过分泌微囊藻毒素,抑制其他水生物种的生长,这也是铜绿微囊藻在自然水体中能够获得竞争优势,暴发微囊藻水华的原因之一。
The serious eutrophication of water body results in frequent breaking out of cyanobacterial blooms, and some genera of cyanobacteria can produce microcystins, which can cause health problems in animals and humans. The effect of microcystins on society,economy and environment has become one of the problems people concerned mostly, as well as the key in the control of cyanobacterial blooms. The sutdies were established with M. aeruginosa, one of the most deleterious species of harmful algal blooms. The environmental factors such as temperature, illumination, pH, zinc, iron, nitrogen and phosphorus were studied to determine their effect on the growth and microcystins production of M. aeruginosa. And in this paper, a new rapid method for toxin extraction was provided based on boiling water bath/liquid-liquid extraction.
The results indicate that: M. aeruginosa grew well on the condition that the temperature was 29℃, illumination was 4000Lx, pH was 9.0, Zn~(2+) concentration was 100nmol/L, Fe~(3+) concentration was 10000nmol/L, P concentration was 7.0mg/L, N/P was 30:1. The effects of P concentration on the growth rate of M. aeruginosa under simulative equipment can be described using the Monod equation and Droop equation. The max growth rateμ_(max)=0.173/d. The P half saturation constant for algae growth K_s=0.275mg/L. The P subsistence quota of algae Q_0=3.803μg/mg(dw). The P half saturation constant for algae growth was 4.990mg/L. The N subsistence quota of algae was 62.137μg/mg(dw). The max growth rate was 0.173/d.
The results about the effects of nutrients on the growth of M. aeruginosa and environment factors in simulative equipment of experiment were as flows: The last development of M. aeruginosa growth was phosphorus limitation. With imput of extragenous phosphorus, M. aeruginosa was not limited by phosphorus. When the biomass reached a certain value, the growth of M. aeruginosa translated to light limitation. The growth of M. aeruginosa could be improved by increasing the phosphorus concentration gradually as additional stimuli. M. aeruginosa was luxury phosphorus uptake to cope with phosphorus deficiency when the water environment was breakout of algal blooms. Diurnal changes in the simulative equipment of pH and DO were displayed with the growth of M. aeruginosa.
The adsorption and extraction of MCs in M. aeruginosa was studied from the selection of different organic solvents and different time of boiling water bath. The results showed that the extraction was best when used CCl_4 as organic solvent and selected 15min as the time of boiling water bath. Comparing with the traditional extraction methods ,the comparative error of MC-RR and MC-LR were 3% and 9.3%, and the recovery of MC-RR and MC-LR were 94.6% and 95.3%.
Effects of growth on M. aeruginosa production of MCs were as flows: The ability of M. aeruginosa to product toxin was related to the developmental stage. The concentration of MCs were significantly higher in the increased logarithmic phase. The M. aeruginosa achieved advantage of competition which exude MCs to inhibit the growth of other species so as to breakout of water bloom.
引文
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