脱氢酶活性测定水中活体藻含量的研究
摘要
近年来大量工业废水和生活污水的排入,加速了湖泊水库的富营养化进程,其后果是在气候适宜时导致藻类的大量繁殖代谢。由于藻类致臭、密度低、沉淀效果差,给以湖泊水库为水源的饮用水生产带来诸多危害;同时,藻类还产生毒素,对人类的饮水健康构成了严重威胁,因此,研究解决富营养化水源给水处理技术,特别是藻类的去除,直接关系到人们饮水安全健康,成为亟待解决的问题。衡量藻类去除效果的定量检测方法是杀藻除藻研究的必须条件,简便高效的检测方法将有助于该项研究的普及和深入。目前,常用的藻类检测方法如平板计数法、叶绿素a法等步骤繁琐、检测结果不稳定,误差较大。由于这两种方法本身特有的局限性,广泛适用于各种杀藻除藻技术处理后的水样比较困难,尤其是不能定量测定杀藻处理后活体藻的含量。因此,需要有一种更加快速、稳定、实用的测定水中活体藻类含量的方法。为解决这一问题,本文研究探讨了利用检测藻类脱氢酶活性来间接测定活体藻类含量的方法。
脱氢酶是一类蛋白质,能够激活某些特殊的氢原子,使这些氢原子被适当的受氢体转移而将原来的物质氧化。在氧化过程中,脱氢酶是作用在代谢物上的第一个酶,为生物体提供必不可少的能量和还原当量。生物体的脱氢酶活性(DHA)在很大程度上反映了生物体的活性状态,能直接表示生物细胞对其基质降解能力的强弱。因此,脱氢酶活性检测被广泛应用于污水生化处理、细菌菌落总数检验、水质毒性检验、土壤污染评价等研究与应用领域。
脱氢酶活性可以通过加入人工受氢体TTC(2,3,5-Triphenyltetrazoliumchloride,2,3,5-氯化三苯基四氮唑)的办法进行检测,其最重要的优点是在反应期间使颜色增加和生物学条件下的不可逆性:当微生物细胞内有生物氧化(即脱氢反应)时,TTC便接受氢原子而被还原成红色的TF(Triphenyl Formazane,三苯甲(月朁)),通过红色的TF生成速度来确定脱氢酶活性。
本文提出了通过测定藻细胞脱氢酶活性,测定含藻水样中活体藻的含量的方法。选择使用TTC作为显色剂。通过波长扫描确定了TF的最大吸收波长为487nm。研究了藻类不同pH值环境、发色培养温度、发色培养时间、TTC浓度对藻类脱氢酶酶促反应的影响,确定使用0.8%的TTC溶液,在pH值8.4的环境中,于32±1℃水浴中暗处发色培养1h为最佳反应条件。
为了使脱氢酶的酶促反应快速终止,保证反应速率计算的准确性,比较了几种终止剂甲醛、乙醇、丙酮、浓硫酸对藻类脱氢酶的灭活效果,实验结果表明,甲醛的效果最好,而且不会像浓硫酸那样使藻类和滤膜变色,影响比色分析。所以,研究选择甲醛为终止剂,加入量为1mL。
为了将藻细胞脱氢酶酶促反应中生成的TF萃取出来,克服藻细胞叶绿素的干扰,研究了多种单组份、双组份萃取剂对叶绿素、酶促反应生成的TF的萃取能力,确定以4mL丙酮与5mL石油醚的混合溶剂做萃取剂。
脱氢酶活性需要通过酶促反应生成TF的速度来表征,因此需要使用TF标准曲线来定量。制作标准曲线需要使用还原剂将TTC还原为TF。研究中比较了制作标准曲线时两种还原剂硫化钠和连二亚硫酸钠的稳定性与还原反应速度,发现硫化钠做还原剂生成的TF较稳定。同时筛选比较了硫化钠浓度、还原反应时间对TTC还原反应的影响。确定最佳实验条件为1mL 8%硫化钠溶液作还原剂,还原反应时间5分钟。
依据上述各反应条件研究结果确定藻类脱氢酶活性的最佳检测条件为:在三(羟甲基)氨基甲烷盐酸盐(Tris-HC1)缓冲溶液、pH=8.4的反应环境中,加入1mL0.8%的TTC溶液,水浴温度32±1℃、发色培养1h,使用双组分萃取剂——4mL丙酮和5mL石油醚的混合溶剂进行萃取。
本研究将TTC-脱氢酶活性检测法应用于水体中活体藻类含量的检测之中,水样取10～1000mL,其最低检出浓度为0.004μg TF/mL·h,最佳测定范围为0.014～6.00μg TF/mL·h,在此范围内检测结果有较好的线性关系,回归系数为0.9999。
本方法应用于实际水样的活体藻含量测定,测定结果与水样体积线性正相关,相关系数为0.9873;检测从某植物园映日湖、植物园喷泉、山大南区喷泉、植物园内小河四个不同地点取回的水样,在实验室培养0、15、30、45天后,其脱氢酶活性测定结果与叶绿素a法测定结果进行比较,两者线性正相关,相关系数分别为0.9369、0.9728、0.9855、0.9325。
本方法应用于杀藻处理后的藻类的检测。当藻类细胞没有发生溶裂,细胞形态没有被破坏时,计数法无法辨别藻体死活;而叶绿素a法在叶绿素还未氧化变色或颜色变化不明显时,无法定量测定活体藻含量,显示杀藻、抑藻效果。本方法克服了上述两方法的缺陷,较好地反应出杀藻剂使用前后活体藻脱氢酶活性的变化,尤其对于那些氧化能力适中、能够抑制藻类活性又不使其细胞内物质溶出造成水体二次污染的化学药剂的杀藻实验研究,其灵敏性优于叶绿素a法。
本文从酶作用动力学的角度,对本研究涉及的藻类脱氢酶酶促反应进行了研究探讨,对pH、温度对酶促反应的影响等实验结果进行了酶促反应动力学分析,实验结果基本符合理论解释,为本研究的结果打下了一定的理论基础。
总之,本文将脱氢酶活性检测基本原理应用于淡水中活体藻类含量的测定,并系统研究了该方法的发色培养时间、培养温度、终止剂、萃取剂等反应操作条件。与现有的脱氢酶活性检测、淡水藻类含量测定方法相比,本方法具有下列优点:
(1)通常的脱氢酶活性检测均使用单组分有机溶剂做萃取剂。本文研究确定使用双组分萃取剂,克服了单组分萃取剂易受藻细胞叶绿素干扰的缺点,提高了对藻细胞内脱氢酶促反应生成的TF的萃取效果。
(2)克服了计数法在形态上无法辨别藻体死活、叶绿素a法在叶绿素未变色或变色不明显时无法准确定量测定活体藻含量的缺点,可以更准确定量测定活体藻类含量,解决了一些物理、化学或生物方法杀藻后,水中活体藻含量定量测定的问题。
A large input of nitrogen and phosphorus compounds from municipal and industrial wastes as well as from agriculture promotes development of algae blooms which are the most common indicators of eutrophication in lakes and reservoirs.The excessive growths of algae,especially in some drinking water sources,have caused many problems,such as uncomfortable taste and odour,filters clogging,and algal toxins,which threaten the human health.Therefore,the study of algicides and algae removal has received more attention in recent decades. However,the routine detection methods for algae in fresh water,such as cell number counting and determination of chlorophyll a,have relatively large determining errors,and furthermore,make it difficult or impossible to compare the amount changes of living algae before and after algae control,so that it is hard to assess the effectiveness of algae control technologies.In order to solve this problem,the dehydorgenase activity(DHA) of living algae was researched to determine the amount of living algae in fresh water.
Dehydrogenase is a kind of protein,which can activate some special hydrogen atoms to be transferred by some appropriate hydrogen accepters to oxidate the initial substance.During the oxidation,the dehydrogenase is the first enzyme acting on the metabolizing substances,producing necessary energy and reducing equivalent for Organism.The dehydrogenase activity(DHA) of organism can reflect the activated condition of microbes and the capacity of degrading their substrates.Therefore,DHA detection is widely used in detecting activated sludge, total number of bacterial colony,water toxicity,and soil pollution.
DHA can be detected by using an artificial hydrogen acceptor,TTC (2,3,5-Triphenyl tetrazoliumchloride),which changes from colorless to red triphenyl formazane(TF) when it accepts hydrogen atoms in dehydrogenation. This is an indirect method.Its most important advantage is that the color of TF, which darkens during reaction,is irreversible under such biological conditions. Thus DHA can be observed by the amount of TF produced.
This study proposed a method to determine the amount of living algae in fresh water by measuring the algal DHA.TTC was selected to be the hydrogen acceptor. The max wave length of absorption of TF is 487nm,measured through wave length scanning.The conditions effecting on the enzyme-catalyzed reaction were studied, such as pH,incubating temperature and duration,and the concentration of TTC. The optimized conditions were decided as follows:using 0.8%of TTC,and incubating in pH 8.4 at 32±1℃of water bath in dark for 1 hour.
In order to insure the accuracy of calculation of reaction velocity,the terminating reagents were used to end the dehydrogenation in algal cells.Several terminating reagents were compared on their restraining effect of algal dehydrogenase,such as formaldehyde,ethanol,acetone and sulfuric acid(98%). The results showed that formaldehyde did not change the color of algae and filter membrane to interfere the colorimetry as sulfuric acid did,and performed a better effect of ending the reaction.Therefore,1 mL of formaldehyde was added to be the terminating reagent.
In order to extract TF from algal cells and overcome the interference of chlorophyll,several different extractants were compared with the capacities of extracting chlorophyll and TF.Then a mixed solvent of 4mL acetone and 5mL petroleum ether was decided to be the extractant.
In order to present DHA by the amount of TF,the calibration curve of TF was prepared,when reducing reagent was needed to reduce TTC to TF.Sodium sulfide and sodium hyposulphite were compared on their stability and reacting speed, finding that TF reduced by sodium sulfide was more stable than sodium hyposulphite.Meanwhile,some conditions impacting the reduction of TTC were studied such as the concentration of sodium sulfide and the reduction duration.The optimized conditions were 1 mL of sodium sulfide solution as a reducer,whose concentration was 8%,and the reduction duration was 5 minutes.
According to the above research results of conditions,the detecting conditions of algal DHA were as follows:pH 8.4 of buffer solution of tris(hydroxymethyl) aminomethane hydrochloride,0.8%of TTC,32±1℃of water bath,incubating for 1 hour,then extracted by a mixed solvent of 4mL acetone and 5mL petroleum ether.
This method was used in determining the amount of living algae in fresh water bodies.Its lowest detectable concentration is 0.004μg TF/mL·h when the water sample is 10～1000mL,and its best measuring range is 0.014～6.00μg TF/mL·h with a significant linearity relation and a correlation coefficient of 0.9999.
This method was applied in determining the amount of living algae in water samples.The results had a positive linearity relation with the volumes of water sample,whose correlation coefficient is 0.9873.Four water samples were collected from four places.Their DHA and chlorophyll a were measured respectively after 0, 15,30 and 45 days of culturing in laboratory.Comparing the determining results of DHA and chlorophyll a of each sample,the positive linearity relations were observed with their correlation coefficients of 0.9369,0.9728,0.9855 and 0.9325.
This method was used in determining the living algae after algicide treatment. The cell number counting can not tell if the algae cells are alive or dead when the algae cells are not dissolved and broken and their shape has not changed.And the method of chlorophyll a can not determine quantificationally the amount of living algae when the chlorophyll in algae cells has not been oxidated or the color has not changed obviously.This method overcomes the above disadvantages,and presents well the change of algal DHA before and after algicide treatment.It is more sensitive than the method of chlorophyll a in this respect.
From the point of view of dynamics of enzymic function,the dehydrogenase-catalyzed reaction in algal cells was studied.The impact of pH,and temperature on the reaction were analyzed,using the theory of dynamics of enzyme-catalyzed reaction,and found out that the experimental results basicly followed the theory.This made a theoretical base for this study.
Above all,this study proposed a method to determine the amount of living algae in fresh water by DHA,applying the theory of DHA detection.Its reacting and operating conditions were studied,such as pH,incubating temperature and duration,extractants,and so on.Compared with the existing methods of DHA detection and determination of algae in fresh water,this study has the following advantages:
(1) The single-component solvents were normally used as the extractant in some relevant studies.In this study,a two-component solvent,a mixture of acetone and petroleum ether were decided to be the extractant,to overcome the interference of chlorophyll in algal cells and increase the extracting efficiency of TF.
(2) This method overcomes the disadvantage of cell number counting and chlorophyll a,which can not determine the living algae amount while the cell number counting can not tell if the algae cells are alive or dead from their shapes, and the chlorophyll has not changed color or the color has not changed obviously. It can quantificafionally determine the amount of living algae,and resolve the problem of quantificational determination of living algae in fresh water after some physical,chemical or biological techniques are used to kill or remove the algae.
脱氢酶是一类蛋白质,能够激活某些特殊的氢原子,使这些氢原子被适当的受氢体转移而将原来的物质氧化。在氧化过程中,脱氢酶是作用在代谢物上的第一个酶,为生物体提供必不可少的能量和还原当量。生物体的脱氢酶活性(DHA)在很大程度上反映了生物体的活性状态,能直接表示生物细胞对其基质降解能力的强弱。因此,脱氢酶活性检测被广泛应用于污水生化处理、细菌菌落总数检验、水质毒性检验、土壤污染评价等研究与应用领域。
脱氢酶活性可以通过加入人工受氢体TTC(2,3,5-Triphenyltetrazoliumchloride,2,3,5-氯化三苯基四氮唑)的办法进行检测,其最重要的优点是在反应期间使颜色增加和生物学条件下的不可逆性:当微生物细胞内有生物氧化(即脱氢反应)时,TTC便接受氢原子而被还原成红色的TF(Triphenyl Formazane,三苯甲(月朁)),通过红色的TF生成速度来确定脱氢酶活性。
本文提出了通过测定藻细胞脱氢酶活性,测定含藻水样中活体藻的含量的方法。选择使用TTC作为显色剂。通过波长扫描确定了TF的最大吸收波长为487nm。研究了藻类不同pH值环境、发色培养温度、发色培养时间、TTC浓度对藻类脱氢酶酶促反应的影响,确定使用0.8%的TTC溶液,在pH值8.4的环境中,于32±1℃水浴中暗处发色培养1h为最佳反应条件。
为了使脱氢酶的酶促反应快速终止,保证反应速率计算的准确性,比较了几种终止剂甲醛、乙醇、丙酮、浓硫酸对藻类脱氢酶的灭活效果,实验结果表明,甲醛的效果最好,而且不会像浓硫酸那样使藻类和滤膜变色,影响比色分析。所以,研究选择甲醛为终止剂,加入量为1mL。
为了将藻细胞脱氢酶酶促反应中生成的TF萃取出来,克服藻细胞叶绿素的干扰,研究了多种单组份、双组份萃取剂对叶绿素、酶促反应生成的TF的萃取能力,确定以4mL丙酮与5mL石油醚的混合溶剂做萃取剂。
脱氢酶活性需要通过酶促反应生成TF的速度来表征,因此需要使用TF标准曲线来定量。制作标准曲线需要使用还原剂将TTC还原为TF。研究中比较了制作标准曲线时两种还原剂硫化钠和连二亚硫酸钠的稳定性与还原反应速度,发现硫化钠做还原剂生成的TF较稳定。同时筛选比较了硫化钠浓度、还原反应时间对TTC还原反应的影响。确定最佳实验条件为1mL 8%硫化钠溶液作还原剂,还原反应时间5分钟。
依据上述各反应条件研究结果确定藻类脱氢酶活性的最佳检测条件为:在三(羟甲基)氨基甲烷盐酸盐(Tris-HC1)缓冲溶液、pH=8.4的反应环境中,加入1mL0.8%的TTC溶液,水浴温度32±1℃、发色培养1h,使用双组分萃取剂——4mL丙酮和5mL石油醚的混合溶剂进行萃取。
本研究将TTC-脱氢酶活性检测法应用于水体中活体藻类含量的检测之中,水样取10～1000mL,其最低检出浓度为0.004μg TF/mL·h,最佳测定范围为0.014～6.00μg TF/mL·h,在此范围内检测结果有较好的线性关系,回归系数为0.9999。
本方法应用于实际水样的活体藻含量测定,测定结果与水样体积线性正相关,相关系数为0.9873;检测从某植物园映日湖、植物园喷泉、山大南区喷泉、植物园内小河四个不同地点取回的水样,在实验室培养0、15、30、45天后,其脱氢酶活性测定结果与叶绿素a法测定结果进行比较,两者线性正相关,相关系数分别为0.9369、0.9728、0.9855、0.9325。
本方法应用于杀藻处理后的藻类的检测。当藻类细胞没有发生溶裂,细胞形态没有被破坏时,计数法无法辨别藻体死活;而叶绿素a法在叶绿素还未氧化变色或颜色变化不明显时,无法定量测定活体藻含量,显示杀藻、抑藻效果。本方法克服了上述两方法的缺陷,较好地反应出杀藻剂使用前后活体藻脱氢酶活性的变化,尤其对于那些氧化能力适中、能够抑制藻类活性又不使其细胞内物质溶出造成水体二次污染的化学药剂的杀藻实验研究,其灵敏性优于叶绿素a法。
本文从酶作用动力学的角度,对本研究涉及的藻类脱氢酶酶促反应进行了研究探讨,对pH、温度对酶促反应的影响等实验结果进行了酶促反应动力学分析,实验结果基本符合理论解释,为本研究的结果打下了一定的理论基础。
总之,本文将脱氢酶活性检测基本原理应用于淡水中活体藻类含量的测定,并系统研究了该方法的发色培养时间、培养温度、终止剂、萃取剂等反应操作条件。与现有的脱氢酶活性检测、淡水藻类含量测定方法相比,本方法具有下列优点:
(1)通常的脱氢酶活性检测均使用单组分有机溶剂做萃取剂。本文研究确定使用双组分萃取剂,克服了单组分萃取剂易受藻细胞叶绿素干扰的缺点,提高了对藻细胞内脱氢酶促反应生成的TF的萃取效果。
(2)克服了计数法在形态上无法辨别藻体死活、叶绿素a法在叶绿素未变色或变色不明显时无法准确定量测定活体藻含量的缺点,可以更准确定量测定活体藻类含量,解决了一些物理、化学或生物方法杀藻后,水中活体藻含量定量测定的问题。
A large input of nitrogen and phosphorus compounds from municipal and industrial wastes as well as from agriculture promotes development of algae blooms which are the most common indicators of eutrophication in lakes and reservoirs.The excessive growths of algae,especially in some drinking water sources,have caused many problems,such as uncomfortable taste and odour,filters clogging,and algal toxins,which threaten the human health.Therefore,the study of algicides and algae removal has received more attention in recent decades. However,the routine detection methods for algae in fresh water,such as cell number counting and determination of chlorophyll a,have relatively large determining errors,and furthermore,make it difficult or impossible to compare the amount changes of living algae before and after algae control,so that it is hard to assess the effectiveness of algae control technologies.In order to solve this problem,the dehydorgenase activity(DHA) of living algae was researched to determine the amount of living algae in fresh water.
Dehydrogenase is a kind of protein,which can activate some special hydrogen atoms to be transferred by some appropriate hydrogen accepters to oxidate the initial substance.During the oxidation,the dehydrogenase is the first enzyme acting on the metabolizing substances,producing necessary energy and reducing equivalent for Organism.The dehydrogenase activity(DHA) of organism can reflect the activated condition of microbes and the capacity of degrading their substrates.Therefore,DHA detection is widely used in detecting activated sludge, total number of bacterial colony,water toxicity,and soil pollution.
DHA can be detected by using an artificial hydrogen acceptor,TTC (2,3,5-Triphenyl tetrazoliumchloride),which changes from colorless to red triphenyl formazane(TF) when it accepts hydrogen atoms in dehydrogenation. This is an indirect method.Its most important advantage is that the color of TF, which darkens during reaction,is irreversible under such biological conditions. Thus DHA can be observed by the amount of TF produced.
This study proposed a method to determine the amount of living algae in fresh water by measuring the algal DHA.TTC was selected to be the hydrogen acceptor. The max wave length of absorption of TF is 487nm,measured through wave length scanning.The conditions effecting on the enzyme-catalyzed reaction were studied, such as pH,incubating temperature and duration,and the concentration of TTC. The optimized conditions were decided as follows:using 0.8%of TTC,and incubating in pH 8.4 at 32±1℃of water bath in dark for 1 hour.
In order to insure the accuracy of calculation of reaction velocity,the terminating reagents were used to end the dehydrogenation in algal cells.Several terminating reagents were compared on their restraining effect of algal dehydrogenase,such as formaldehyde,ethanol,acetone and sulfuric acid(98%). The results showed that formaldehyde did not change the color of algae and filter membrane to interfere the colorimetry as sulfuric acid did,and performed a better effect of ending the reaction.Therefore,1 mL of formaldehyde was added to be the terminating reagent.
In order to extract TF from algal cells and overcome the interference of chlorophyll,several different extractants were compared with the capacities of extracting chlorophyll and TF.Then a mixed solvent of 4mL acetone and 5mL petroleum ether was decided to be the extractant.
In order to present DHA by the amount of TF,the calibration curve of TF was prepared,when reducing reagent was needed to reduce TTC to TF.Sodium sulfide and sodium hyposulphite were compared on their stability and reacting speed, finding that TF reduced by sodium sulfide was more stable than sodium hyposulphite.Meanwhile,some conditions impacting the reduction of TTC were studied such as the concentration of sodium sulfide and the reduction duration.The optimized conditions were 1 mL of sodium sulfide solution as a reducer,whose concentration was 8%,and the reduction duration was 5 minutes.
According to the above research results of conditions,the detecting conditions of algal DHA were as follows:pH 8.4 of buffer solution of tris(hydroxymethyl) aminomethane hydrochloride,0.8%of TTC,32±1℃of water bath,incubating for 1 hour,then extracted by a mixed solvent of 4mL acetone and 5mL petroleum ether.
This method was used in determining the amount of living algae in fresh water bodies.Its lowest detectable concentration is 0.004μg TF/mL·h when the water sample is 10～1000mL,and its best measuring range is 0.014～6.00μg TF/mL·h with a significant linearity relation and a correlation coefficient of 0.9999.
This method was applied in determining the amount of living algae in water samples.The results had a positive linearity relation with the volumes of water sample,whose correlation coefficient is 0.9873.Four water samples were collected from four places.Their DHA and chlorophyll a were measured respectively after 0, 15,30 and 45 days of culturing in laboratory.Comparing the determining results of DHA and chlorophyll a of each sample,the positive linearity relations were observed with their correlation coefficients of 0.9369,0.9728,0.9855 and 0.9325.
This method was used in determining the living algae after algicide treatment. The cell number counting can not tell if the algae cells are alive or dead when the algae cells are not dissolved and broken and their shape has not changed.And the method of chlorophyll a can not determine quantificationally the amount of living algae when the chlorophyll in algae cells has not been oxidated or the color has not changed obviously.This method overcomes the above disadvantages,and presents well the change of algal DHA before and after algicide treatment.It is more sensitive than the method of chlorophyll a in this respect.
From the point of view of dynamics of enzymic function,the dehydrogenase-catalyzed reaction in algal cells was studied.The impact of pH,and temperature on the reaction were analyzed,using the theory of dynamics of enzyme-catalyzed reaction,and found out that the experimental results basicly followed the theory.This made a theoretical base for this study.
Above all,this study proposed a method to determine the amount of living algae in fresh water by DHA,applying the theory of DHA detection.Its reacting and operating conditions were studied,such as pH,incubating temperature and duration,extractants,and so on.Compared with the existing methods of DHA detection and determination of algae in fresh water,this study has the following advantages:
(1) The single-component solvents were normally used as the extractant in some relevant studies.In this study,a two-component solvent,a mixture of acetone and petroleum ether were decided to be the extractant,to overcome the interference of chlorophyll in algal cells and increase the extracting efficiency of TF.
(2) This method overcomes the disadvantage of cell number counting and chlorophyll a,which can not determine the living algae amount while the cell number counting can not tell if the algae cells are alive or dead from their shapes, and the chlorophyll has not changed color or the color has not changed obviously. It can quantificafionally determine the amount of living algae,and resolve the problem of quantificational determination of living algae in fresh water after some physical,chemical or biological techniques are used to kill or remove the algae.
引文
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