| 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. |
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