Experimental Study of Zn2+ on Growth of Chlorella in Reclaimed Water for Waterscape Recharge

中国环境学会  2011年 06月22日

  Wu shan  Shi jingwei  Hu yueheng
  Civil School of Beijing University of Technology, Beijing, 100124      Water-2004@bjut.edu.cn
  Abstract: From the trace elements the conditions of eutrophication of in the reclaimed water for waterscape recharge were discussed. Through algae growth potential experiments (AGP), the effects of different concentrations of Zn2+ on the growth of algae were studied. The results show that, 0.01μg/L of Zn2+ limited the growth of Chlorella; 1-100μg/L of Zn2+ promoted the growth of Chlorella pyrenoidosa and algae multiplied more quickly with Zn2+ concentration of 50μg/L ;when the concentration of Zn2+ reached 1mg/L ,it has been shown to inhibit the growth of Chlorella; the toxic effects of 100mg/L of Zn2+ have completely led to the death of Chlorella. It was concluded that appropriate Zn2+ concentration might be one of the key factors affecting Chlorella bloom in the reclaimed water.
  Keywords: reclaimed water; Zn2+, Chlorella Pyrenoidosa; chlorophyll; eutrophication
  1.        Intruction

  With the level of urbanization and industrialization increasing, water pollution and water shortages have been increasingly serious. Clean water for the landscape is not feasible, so people have more and more attached importance to reclaimed water for waterscape recharge. However, because the reused water contains relatively high pollutant, so the nutrient salt is rich ,therefore reclaimed water for waterscape recharge is extremely easy to produce water bloom so as to seriously influence water body landscape under outside suitable conditions[1].
  The water blooms which arise in the resurgent water are conditioned by many kinds of environment factor that the trace element is also playing the important role besides major elements N、P. Zn is component of related enzyme with which the algae carries on photosynthesis and metabolism, for example acid phosphatase、alkaline phosphatase[2]. If the algae lack Zn, its growth rate and the photosynthesis speed greatly will reduce. The research of trace element to the algae influence mostly took the red tide algae and superiority algae of the natural water body as the object of study, but reports are few to pelagic algae in the reclaimed water for waterscape. Through the algae growth potential experiment (AGP) and the analysis in the different situation water quality of the resurgent water landscape, the aim of this study is to understand the limit factor of eutrophication in the water body and discuss the influence that the algae grow under the zinc acclimation in the reclaimed water for waterscape from chlorophyll a and algae growth change, thus provide the reference for preventing water bloom in the resurgent water landscape as well as to assist in the development of water quality guidelines, as part of a suite tests for assessing metal bioavailability.
  2.        Material and Methods

  2.1    Materials
  The eukaryon chlorella (Chlorella pyrenoidosa. Fachb-9) was used in this laboratory which was bought from Institute of Hydrobiology, Chinese Academy of Sciences. The chemical reagent is analytical (AR), and the water is distilled.
  2.2    Methods
  2.2.1 Acclimation of Algae
  The freshwater green alga was sterilely cultured in the SE culture medium without soil extracts and was incubated on a 12/12 h light–dark cycle (2000lux, cool white fluorescent lighting) at 25±1℃. After 30d, the alga was acclimated to the medium and grew well to satisfy the experimental request.。
  Table 1   SE medium


SE medium




























A5 solution



Distilled water


  ﹡: A5 solution is obtained by joining the following drugs in the distilled water (100mL)


SE medium


















  2.2.2 Configuration of Zn2+ concentration of SE
  Firstly, 1g/L、1mg/L and 1μg/L Zn2+ of the reserve solution were separately made by the liquid dilution method, using analytically pure ZnSO4·7H2O. Then, SE medium without Zn2+ were made according to Table1, simultaneously different Zn2+ concentration of SE were made by putting 3 kinds of respective reserve solution to SE without Zn2+ according to Table 2. Each experimental group of matching volume 150mL contains 3 parallelisms in the 250mL flask.

  2.2.3 Inoculation
  Take the right amount of algae solution firstly and then centrifuge 10min under 3500r/min, at last abandon the supernate, the precipitation algae cells were left behind. Wash the precipitation with 15mg/L NaHCO3 and then centrifuge 10min under 3500r/min again, so as to remove nourishing substance and other materials in the algae cells. Similarly the cells were washed 2 times by the above method, finally the aerosol became the test vaccination mother liquor. The volume of NaHCO3 rested with the cell density which has been calculated when algae was used to inoculate. After each antiseptic experimental group were carried on the isometric vaccination, the conical flask was sealed with the sterile culture vessel seal membrane, setting in the illumination incubator to culture on a 12/12 h light–dark cycle (3500lux, pH of the experiment solution≤8.5,cool white fluorescent lighting) at 25±1℃. Because the illumination might be non-uniform, every other 4~5h shake nutrient solution and change position of various experimental groups 1 time. The experimental operation carried on under the aseptic condition.

  2.2.4 Endpoint measurements
  Firstly mensurate the entire absorption spectrum of chlorella with the ultraviolet spectrophotometer, determine that its biggest absorption peak is 515nm as optical density. Then start from the third day of inoculation, measure all the samples one time every other 48 hour.  The growth of algae was expressed with OD value which was determined in the wave length 515nm with the 720S spectrophotometer. Take the nutrient solution which was not added algae as the blank comparison. The empirical datum was from the average values of 3 parallel, and both the time of take sample and the determination time were at basic same each time.
  Average specific growth rate[3 was calculated with the following formula].
  K—average specific growth rate;
  T—culture time(d)
  A1—OD515nm at the end of culture time;
  A0—OD515nm at the beginning of culture time
  The chlorophyll[4] a is an important target reflecting the algae biomass. Subsamples for chlorophyll a (3ml) were collected in centrifugal tubes, then chlorophyll a was determined by spectrophotometer colorimetry after 90% methanol extraction.
  The formula as follows: c=13.9×OD665nm 
  Then start to determine chlorophyll from the third day of inoculation, and determine subsamples one time every other 48 hour until at the end of experiment.
  Take subsample (2.5mL) from algae solution at the culture time (25d), the photosynthesis speed and the respiratory rate were determined with oxygen electrode[5]. Net photosynthesis rate was mensurated at 25℃(64μmol photons·m-2·s-1), respiratory rate was determined under at 25℃ too.
  3.        Results

  3.1 Effect of Zn2+ on Chlorella growth
  The results of Zn2+ growth potential tests for chlorella were shown in Figure 1. The curves of each group were basically straight at the experiment previous 7 days except I group, thereby the chlorella was in the lag phase; moreover that the curves of A, B, C, D, E, F, G, H group were greatly close at the previous 7 day are possibly due to surplus Zn2+ in the algae cell which can temporarily satisfy the growth demand of the chlorella, therefore Zn2+ in the medium is not important to influence the growth of algae. But the algae start to grow massively from the 8th day, the different concentration of zinc is obvious gradually to influence the growth of the chlorella except I group, thereby each group of algae enters the exponential phase. The growth movement of B, C, D, E, F, G, H algae cell were similar with the blank group (A group), but the OD value of B, C group is slightly lower than the control group so as to explain 0.01μg/L Zn2+ has limited the chlorella growth; OD of D, E, F group is higher than the control group, consequently 1-100μg/L Zn2+ can promote the chlorella multiplication. Although G, H group of OD had increased, the growth curves were lower than the control group, so the 1mg/L Zn2+ obviously had suppressed the chlorella multiplication. When the concentration of Zn2+ gradually increased, the inhibitory action was getting bigger and bigger. The demonstration that the curve of I group was completely straight in the entire experiment period explained that Zn2+ poisonous effect already caused chlorella stop to grow and die completely within six days when Zn2+ achieved 100mg/L, moreover observed the color of algae solution compared with the control group was obvious whitening.
  Moreover, T-test of A, B, C, D, E, F group algae cell OD value indicated that E had the significant difference explanation with B group with the SPSS data statistics (sig<0.05). Zn2+ concentration for 50μg/L can obviously promote the growth of chlorella under this experimental condition; T-test of A, E, F, G group indicated that Zn2+ of 1mg/L is remarkably poisonous to chlorella (sig<0.05).
  The influences of Zn2+ to chlorophyll a for chlorella were present in Figure 2. When Zn2+ concentration was 0.01-0.1μg/L (B, C group), the chlorophyll a was slightly lower than the control group (A group). The extremely low zinc concentration may limit the algae growth. When Zn2+ concentration was 1-100μg/L (D, E, F group), the chlorophyll-a content is higher than the control group (A group), indicated that the low zinc concentration can promote the chlorella growth; the chlorophyll-a multiplication was remarkable at Zn2+ of 50μg/L (sig<0.05). Although G, H group of chlorophyll-a had increased, the curve was lower than the control group, so the Zn2+ of 1mg/L already suppressed the photosynthesis of chlorella; the chlorophyll-a content of Zn2+10mg/L (H group) increased slowly, demonstrated the zinc of inhibitory action was remarkable (sig<0.05). When the Zn2+ concentration achieved 100mg/L, the chlorophyll a stopped to grow and had the intense inhibitory action to photosynthesis.

  3.2 Average specific growth rate of chlorella
  The statistical results of Average specific growth rate for chlorella were present in Figure 3. when Zn2+ concentration is 0.01-0.1μg/L, the average specific growth rate of chlorella is lower than the blank group; when the Zn2+ concentration is 1-100μg/L, the chlorella OD value is increasing; when the Zn2+ concentration is beyond10mg/L, the specific growth rate of chlorella is lower than the control group; OD for chlorella does not increase nearly at Zn2+100mg/L, explained that the suitable low Zn2+ concentration promotes the growth of chlorella and high Zn2+ suppresses he growth of chlorella. The Zn2+ concentration which is suitable for the growth of chlorella is 1-100μg/L under this experimental condition.

  3.3 Effect of Zn2+ on photosynthesis and respiration of Chlorella
  When Zn2+ concentration is below 100μg/L, the net photosynthesis rate of Chlorella first reduces (0-0.1μg/L), and then gradually increases; when Zn2+ concentration is more than 100μg/L, the net photosynthesis rate of Chlorella reduces gradually only; the net photosynthesis rate of Chlorella is not monitored at 100mg/L of Zn2+. When Zn2+ concentration is beyond 100μg/L, the respiratory rate of chlorella reduces gradually. (Table 3).
  4.        Conclusions

  Zinc is essential element required for the normal functioning of enzyme systems within algae. The zinc plays the vital role in many physiological processes for algae and is component of photosynthesis and related metabolism enzyme. High Zn2+concentration which exceeds those required for optimal growth can urge the nucleic acid to degrade and suppress both NADPH formation in the chloroplast and the algae cell ATP level, however the low zinc concentration may promote the multiplication of algae. The different affinities between different metal ions and algal cells are mainly responsible for the different inhibitions of Chlorella′s growth[3]..
  YanHai[7] found the safety concentration and 96h-EC50 of Zn for Chlorella pyrenoidosa ′s growth were respectively 65μg/L、473μg/L with the standard method of algal bioassay for evaluating the toxicity of toxic chemicals. Zhouhong[8]described the low Zn2+ concentration promoted Selenastrum minutum′s growth, when Zn2+ concentration surpasses those required for optimal growth, this algae's growth is restricted and will indeed die. This phenomenon is also obviously observed in this experiment. In this experimental condition, when zinc concentration is 1-100μg/L, both OD value and chlorophyll a increase and are higher than the blank group along with the culture cycle. This explained that the suitable low zinc concentration has promoted the chlorella′s growth and the most remarkable concentration is 50μg/L which is approximately same with the zinc content in SE culture medium; when zinc concentration is beyond 1mg/L, although the OD value and chlorophyll a has increased, they both are lower than the control group. This indicated that high zinc concentration has the varying degree poisonous effect to the chlorella′s growth. Generally speaking, excessive or too few Zn2+equally will suppress the chlorella cell's growth and division.
  In 2008 28% of the reclaimed water will be used in the urban landscape water body, but because of eutrophication of the reclaimed water for landscape, as well as the hygienic and aesthetic problems which are getting more and more serious, moreover in the summer the green alga of water bloom in the reclaimed water body is different from blue-green alga of water bloom in the natural water body. Average effluent scopes of Zn2+ for three quarter of four reclaimed water treatment plants in Beijing is between 30.1-183.3μg/L. According to this experimental result, if take this kind of resurgent water as supplement of landscape water body, Zn2+ will possibly promote the superiority algae′s growth , but Both biotic and abiotic factors affect the sensitivity of algae to zinc.. Although Zn2+content in the reclaimed water body is suitable, the useful Zn2+ may be very possibly low, which also will limit the algae′s growth, so the existence condition of Zn should be concerned in the water body. Moreover the biological exploitability of zinc to alga as well as the effective absorption of algae to zinc is different. Therefore eutrophication of the resurgent sewage for landscape is conditioned by not only the nutrient salt but also the trace element. Through Zn2+ growth potential tests for chlorella and analyzing water quality of the resurgent sewage for landscape in the different situation, which understand the limit factor and forecast the algae population change in the water body. By strictly controlling Zn2+ content of reclaimed wastewater for landscape , the growth of green alga is in the limit scope as far as possible. Keep the condition within limits where chlorella can be suitable to grow from the very beginning yield, which provides the scientific basis for preventing eutrophication of the reclaimed wastewater in Beijing and discusses theoretically the control factor or inducing factor of algae's growth with the special water quality so as to have a more thorough understanding about the importance and the necessity of the landscape water treatment.
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  Table 3  Effect of Zn2+ on photosynthesis and respiration of Chlorella pyrenoidosa (after 25d)

  Concentration of Zn2+ (μg/L)










Net photosynthesis rate





















Respiration rate





















  Fig.3  Average specific growth rate of Chlorella Pyrenoidosa cell optical density at different Zn2+ concentrations

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