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Effects of Nitrogen Forms on the Production ofCyanobacterial Toxin Microcystin-LR by an IsolatedMicrocystis aeruginosaHai Yan a , Gang Pan a b , Hua Zou a , Lirong Song c & Mingming Zhang aa State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, Chinab Qingdao University of Science and Technology, Qingdao, Chinac Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

Available online: 06 Feb 2007

To cite this article: Hai Yan, Gang Pan, Hua Zou, Lirong Song & Mingming Zhang (2005): Effects of Nitrogen Forms onthe Production of Cyanobacterial Toxin Microcystin-LR by an Isolated Microcystis aeruginosa , Journal of EnvironmentalScience and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 39:11-12, 2993-3003

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JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH

Part A—Toxic/Hazardous Substances & Environmental Engineering

Vol. A39, Nos. 11–12, pp. 2993–3003, 2004

Effects of Nitrogen Forms on the Production of

Cyanobacterial Toxin Microcystin-LR by an Isolated

Microcystis aeruginosa

Hai Yan,1 Gang Pan,1,2,* Hua Zou,1 Lirong Song,3 and Mingming Zhang1

1State Key Laboratory of Environmental Aquatic Chemistry, Research Center

for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, China2Qingdao University of Science and Technology, Qingdao, China

3Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

ABSTRACT

A cyanobacterial strain, which produced high content of microcystin-LR

(MC-LR) but no microcystin-RR (MC-RR), was isolated from the hypertrophic

Dianchi Lake in China and identified as Microcystis aeruginosa DC-1. Effects of

nitrogen containing chemicals and trace elements on the growth and the

production of MC-LR by this strain were studied. In the presence of bicine,

compared with urea and ammonium, nitrate greatly promoted the growth and the

production of MC-LR. However, leucine and arginine, which were the

constitutional components in the molecular structure of MC-LR or RR, inhibited

the production of MC-LR. Iron and silicon up to 10mg/L had little effects on the

growth of M. aeruginosa DC-1, but the production of MC-LR was apparently

enhanced. Under all conditions studied here, only MC-LR but no RR was

detected within the cells of M. aeruginosa DC-1. Thus, chemical forms of

nitrogen, rather than the usually concerned the total nitrogen, and trace elements

played important roles in the production of MC toxins during cyanobacterial

blooms.

*Correspondence: Gang Pan, Research Center for Eco-environmental Sciences, Chinese

Academy of Sciences, P.O. Box 2871, Beijing 100085, China; E-mail: gpan@mail.rcees.ac.cn.

2993

DOI: 10.1081/LESA-200034799 1093-4529 (Print); 1532-4117 (Online)

Copyright & 2004 by Marcel Dekker, Inc. www.dekker.com

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Key Words: Microcystin-LR; Microcystis aeruginosa DC-1; Nitrogen; Iron;

Silicon.

INTRODUCTION

Anthropogenic discharge of nitrogen and phosphorus in natural waters has ledto the occurrence of harmful cyanobacterial bloom, which is becoming a seriousenvironmental problem worldwide. Microcystins (MC) are produced by variouscyanobacteria such as Microcystis, Anabaena, Oscillatoria, and Nostoc species,[1–3]

which inhibit eukaryotic protein phosphatases and cause excessive phosphorylationof cytoskeletal filaments and liver failure of animals.[4] To understand the conditionsof MC production by harmful cyanobacterial bloom is of fundamental significancefor the control of this environmental pollution.

Environmental factors may play an important role in the production of MCsince some cyanobacterial species could show different toxicity under differentlaboratory conditions.[5–7] It was reported that the production of MC by Oscillatoriaagardhii and M. aeruginosa correlated to high level of nitrogen and low level ofphosphorus.[8–10] However, Utkilen and Gjlme[11] found that the production of MCby M. aeruginosa was stimulated by iron, but not nitrate and phosphate. Orr andJones[12] suggested that the production of MC was controlled by the rate of celldivision rather than the metabolic pathways. So far, most attention has been focusedon the effects of total nitrogen and phosphorus on the growth of cyanobacteria,[9,10]

but the roles of different nitrogen chemical forms and trace elements on the growthof cyanobacteria and especially the production of MC were less understood.

MC-LR and MC-RR are the two most commonly found toxins among manycyanobacterial blooms worldwide.[13,14] Tillett et al.[15] described the completebiosynthesis pathway of MC-LR. It was reported that the content of MC-RR in theisolated strains of M. viridis and M. aeruginosa from Dianchi Lake were higher thanthat of MC-LR.[16,17] Park et al.[18] observed that the content of MC-RR in collectedcyanobacterial cells from Lake Suwa in Japan was much higher than that ofMC-LR. Here a MC-LR rather than MC-RR producing strain M. aeruginosa DC-1was isolated from the Dianchi Lake of China. The effects of different nitrogencontaining compounds and trace elements on the growth and the production ofMC-LR were investigated. Results indicated that chemical forms of nitrate and traceelements of iron and silicon played important roles in promoting the growth andproduction of MC-LR by cyanobacteria, which may be important for theunderstanding and practical controlling of harmful cyanobacterial blooms.

MATERIALS AND METHODS

Cyanobacterium

A MC-LR producing cyanobacterial strain used in this article was isolatedfrom the hypertrophic Dianchi Lake of China using the method recommended

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by Shirai.[19] and identified as a unicellular Microcystis aeruginosa and coded asstrain DC-1 by Institute of Hydrobiology Research, Chinese Academy of Sciences.

Culture Medium

According to the culture medium reported,[20] the basic medium for the cultureof M. aeruginosa DC-1 consisted of 100mg of NaHCO3, 70mg of MgSO4

7H2O, 20mg of CaCl2, 70mg of KCl, 50mg of NaCl, 100mg ofb-Sodiumglycerophosphate, 5mg of Na2EDTA, 0.5mg of FeCl3 6H2O, 5mg ofMnCl2 4H2O, 5mg of CoCl2 6H2O, 0.8mg of Na2MoO4 2H2O, 20mg of H3BO3,and 1000mL of distilled water. Different nitrogen compounds were used andprepared as initial 60mg/L of total nitrogen in all experiments. When two nitrogensources were used, each nitrogen form (such as nitrate, ammonia, urea, or bicine(N,N-bis (2-hydroxyethyl) glycine)) was set to 30mg/L. In the presence of nitrate andbicine, bicine nitrogen was 30mg/L and the third nitrogen of leucine or arginine wasset to 15mg/L. FeCl3 6H2O or Na2SiO39H2O was used to prepare 1.0 g/L of iron orsilicon solution that was diluted with the medium using nitrate and bicine as nitrogensources, respectively. Initial pH of the medium was adjusted to 8.60 using 1.0N HCland NaOH solutions. The medium and all experimental utensils were sterilized at124�C for 20min.

Culture Conditions

M. aeruginosa DC-1 grew in a 100mL flask containing 45mL inoculum in aclimatic chamber at 24�C with the light intensity of 15 mmolm�2 s�1 in 12:12 h light-dark circle. A hemocytometer was used to count the number of cyanobacterial cellsunder a microscope and to calculate the density of cyanobacterial cells. The initialdensity of cells was set to 6.35� 106 cells/mL and samples in the bottle for eachcondition were taken for assay every two days during the period of 10 days’experiment. The data presented here were the average values of three parallelsamples with the standard deviation.

Dry Weight Concentration of M. aeruginosa DC-1 Cells

Cells in the culture solution were harvested with a centrifuge tube at 12,000 rpmfor 10min. Different densities of cells were prepared using 0.5% NaCl solution.50mL of cyanobacterial solution was filtered through a 0.22 mm membrane, whichwas previously dried at 103�C for 2 h and then weighed. The wet cells togetherwith the membrane were also dried at 103�C for 2 h and then weighed. Thedry weight of cells in 50mL was obtained from the difference between theabove-mentioned two weights. A linear relationship, Dry weight concentrationof cells (g/L)¼�0.697� 10�3

þ 0.008� 10�6�Density of cells (cells/mL)

(R2¼ 0.9989), was obtained, which was used to calculate dry weight concentration

of cells with density of cells in the following experiments.

Effects of Nitrogen Forms on the Production of Cyanobacterial Toxin MC-LR 2995

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Determination of MC within the Cells

Both MC-LR and MC-RR bought from Sigma (95% purity) were analyzedby HPLC (Shimadzu-10A) with a Waters mBondapak C18 column (300� 3.9mm)and a Diode Array Detector at 239 nm. The mobile phase was 40% (v/v) acetonitrile-water solution containing 0.03% (v/v) of trifluoroacetic acid. The flow-rate was1.0mL/min and the injection amount was 20 mL.

Various ratios of methanol solutions were tested to extract MC fromM. aeruginosa DC-1, in which 40% of methanol-water was found to be the optimalsolution to extract MC and used throughout the extracting procedures.

To measure the content of MC within the cyanobacterial cells, 40mL ofcyanobacterial culture solution was centrifuged at 12,000 rpm for 10min and thenthe supernatant was discarded. 5mL of 40% methanol–water solution was added tocell pellet to extract MC within cyanobacterial cells. The extraction solution wasvibrated using a ultrasonic equipment for 1 h and then centrifuged at 12,000 rpm for10min. The supernatant was filtered through a 0.22 mm membrane and measured forMC-LR on HPLC. The MC-LR content in the cells was calculated from the ratiobetween the concentration of MC-LR in the extraction solution (mg/L) and the dryconcentration of cells (g/L).

RESULTS AND DISCUSSION

Analysis of MC-LR and MC-RR

Figure 1 was the HPLC profile of standard solution of 10mg/L of MC-RR andMC-LR. Peaks of MC-RR and MC-LR appeared at 3.5 and 8.6min, respectively.The wavelength of 239 nm was the maximum absorbance of MC-RR and LR (Fig. 1),which agreed with literatures[21,22] and was used for the detection of MC on HPLC inthis experiment. Good linear relationships between the peak heights and thestandard concentrations of MC-LR or RR were obtained at the wavelength of239 nm (Fig. 2).

Extraction of MC

The extraction of MC in the cells of M. aeruginosa DC-1 (40 g-dry weight ofcells/L) was investigated by using different concentration of methanol solutions.Figure 3 indicated that the extraction efficiency of MC-LR within cells using 40%methanol was slightly higher than those using other methanol solutions and wastherefore used to extract MC-LR within cells in the following experiments. Solventsreported in the extraction of MC varied greatly and it was difficult to determinewhich is the most appropriate because the range of MC content differs greatly indifferent samples. Lawton and Edwards[23] recommended 50–80% methanol as theoptimal extraction solution.

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Effects of Nitrogen Compounds on the Growth

of M. aeruginosa DC-1

It was shown that M. aeruginosa DC-1 could grow in mixed nitrogen sourcescontaining bicine and another nitrogen compound nitrate, ammonia or urea,

Figure 2. Calibration curves for the measurement of MC-LR and RR by HPLC.

Figure 1. The HPLC profiles for standard MC-RR (RT¼ 3.5min) and MC-LR

(RT¼ 8.6min). Concentrations of standard MC-LR and RR are 10mg/L, respectively.

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in which nitrate supported the most rapid and stable growth (Fig. 4). At the presenceof bicine and nitrate, the growth of M. aeruginosa DC-1 were slightly promoted byleucine but heavily inhibited by arginine (Fig. 5). Rao et al.[24] found that MAmedium[20] containing bicine and nitrate as nitrogen sources supported the bestgrowth of M. aeruginosa among four medium tested. Our results confirmed thatthe nitrogen chemicals of bicine and nitrate were important in the rapid growth ofM. aeruginosa DC-1. The reason that leucine can promote the growth while arginineinhibited the growth of M. aeruginosa DC-1 may be related to the fact thatM. aeruginosa can uptake more leucine than arginine.[25] Normal physiologicalmetabolism of this cyanobacterium might be interfered by arginine, which wasresponsible for the severe inhibition of growth.

Effects of Trace Elements on the Growth

of M. aeruginosa DC-1

Figures 6 and 7 indicated that the growth of M. aeruginosa DC-1 could hardlybe affected by iron and silicon at an initial concentration of 10mg/L, respectively.This may be due to the fact that iron is a necessary element in the photosynthesis ofmicroalgae and silicon is another necessary element for the growth of M. aeruginosaDC-1.

Effects of Nitrogen Compounds on the Production of MC-LR

Figure 4 showed that in the presence of bicine, nitrate promoted the highestproduction of MC-LR than those of urea and ammonium, and the maximum

Figure 3. Extraction of MC-LR from M. aeruginosa DC-1 using different concentrations of

methanol. The dry weight concentration of cells in extracting solution was 4.0 g/L.

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Figure 5. Effects of leucine and arginine on the growth and the production of MC-LR of

M. aeruginosa DC-1. Solid symbol: cell density; blank symbol: content of MC-LR in cells.

Figure 4. Effects of nitrogen compounds on the growth and the production of MC-LR of

M. aeruginosa DC-1. Solid symbol: cell density; blank symbol: content of MC-LR in cells.

Effects of Nitrogen Forms on the Production of Cyanobacterial Toxin MC-LR 2999

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MC-LR content could reach 3.5mg/g at day 8. At the presence of bicine and nitrate,leucine and arginine, which are the constitutional components in the molecularstructures of MC-LR or RR, did not promote the production of MC-LR, althoughthe growth was slightly increased when leucine was added (Fig. 5). Although thegrowth of M. aeruginosa DC-1 was hardly affected, the production of MC-LR wasmuch stimulated by iron and silicon (Figs. 6 and 7). MC-producing cyanobacterialstrains always contain the mcy genes but nontoxic-producing strains may or may notcontain mcy genes,[26] which indicate that experimental factors may play importantroles on the expression of the mcy genes. In the presence of bicine, nitrate supportedmore rapid growth and higher content of MC-LR within the cells of M. aeruginosa

Figure 7. Effects of silicon on the growth and the production of MC-LR of M. aeruginosa

DC-1. Solid symbol: cell density; blank symbol: content of MC-LR in cells.

Figure 6. Effects of iron on the growth and the production of MC-LR of M. aeruginosa

DC-1. Solid symbol: cell density; blank symbol: content of MC-LR in cells.

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DC-1 (Fig. 4), which is in agreement with the findings that the production of MC was

controlled by environmental effects on the rate of cell division of M. aeruginosa.[12]

Our results on the contrasting roles of leucine in the promotion of growth and the

inhibition of MC-LR production (Fig. 5) indicated that the mechanism of MC

producing was not solely correlated to the growth of cyanobacterium.It was proposed that the MC toxins are an intracellular chelator that inactivates

free cellular Fe2þ and the production of MC are controlled by the amount of free

Fe2þ present.[11] The binding of copper and zinc to MC were also confirmed by

Humble et al.[27] Here we proposed that two main contrasting factors were

responsible for the change of MC-LR content in our experiments. With the increase

in initial iron concentration, more Fe3þ was converted to Fe2þ that was accumulated

and uptaken by M. aeruginosa DC-1, which might consume more MC-LR to chelate

Fe2þ and cause the decrease in the content of MC-LR within cells at day 2 (Fig. 6).

However in order to reduce the stress of Fe2þ afterward, the production MC-LR was

stimulated and the maximum content of MC-LR in cells was obtained after 2 d when

initial iron concentration was set to 5mg/L (Fig. 6). However the mechanism of the

promotion of MC-LR production by silicon needed further studies.

CONCLUSION

A MC-LR producing unicellular cyanobacterium of Microcystis aeruginosa

DC-1 was isolated from Dianchi Lake in China, and the effects of nitrogen

containing compounds and trace elements on the growth and the production of MC

of this cyanobacterium were studied. Some important facts can be concluded as

follow: (1) Only MC-LR rather than MC-RR was produced by this isolated

cyanobacterial strain under all conditions tested. (2) In the presence of bicine, nitrate

promoted the most growth and production of MC-LR than those of urea and

ammonium. (3) In the presence of bicine and nitrate, the growth of DC-1 was slightly

promoted by leucine but inhibited by arginine. The productions of MC-LR were all

inhibited when leucine and arginine were added, respectively. (4) The growth of

M. aeruginosa DC-1 were not affected by iron or silicon up to 10mg/L, however the

production of MC-LR was obviously inhibited in the lag growth phase but

promoted in exponential growth phase. (5) The content of MC-LR within the

cyanobacterial cells was lowered in the lag and late exponential growth phases but

peaked in the early exponential growth phase.

ACKNOWLEDGMENTS

The research was funded by the Chinese National Key Project for Basic

Research on the Processes of Lake Eutrophication and the Mechanism of

Cyanobacterial Blooming (2002CB412308), Chinese NNSF grant 20177029 and

Chinese ‘‘863 program’’ (2002AA601011).

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