Chemosphere 85 (2011) 1325–1330

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Physiological and biochemical responses of Microcystis aeruginosa to phosphite

Juan Zhang, Jinju Geng ⇑, Hongqiang Ren ⇑, Jun Luo, Aiqian Zhang, Xiaorong WangState Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 April 2011Received in revised form 20 July 2011Accepted 22 July 2011Available online 9 September 2011

Keywords:Reduced phosphorusPhosphitePhosphateMicrocystis aeruginosaP cycle

0045-6535/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2011.07.049

⇑ Corresponding authors. Tel.: +86 25 89680360.E-mail address: jjgeng@nju.edu.cn (J. Geng).

Phosphorus (P) is a key biological element and limiting nutrient in aquatic environments. Phosphate (+5)is traditionally associated with the P nutrient supply. However, phosphite (+3) has recently generated agreat deal of interest, because of the possibility that it is a P source based on recognition of its vital role inthe original life of the early earth. This study investigated whether phosphite can be an alternative Psource for Microcystis aeruginosa PCC 7806, one of the predominant bloom species in freshwater systems.The results indicated that M. aeruginosa could not utilize phosphite as a sole P-nutrient directly for cellgrowth at any concentration, but that phosphite could boost cell numbers and chlorophyll a (Chl-a) con-tent as long as phosphate was provided simultaneously. Specifically, Chl-a production increased sharplywhen 5.44 mg P L�1 phosphite was added to 0.54 mg P L�1 phosphate medium. Analysis of the maximumyield of PSII indicated that phosphite may stimulate the photosynthesis process of cells in phosphate–phosphite medium. In addition, phosphite failed to support cell growth, even though it more readily per-meated the cells in P-deficient medium than in P-sufficient medium. Alkaline phosphatase activity (APA)analysis indicated that, unlike organic P, phosphite inhibits the response of cells to deficient P status,especially under P-deprived conditions.

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1. Introduction

Phosphorus (P) is a necessary nutrient for life on earth. Phosphateminerals, which comprise the majority of inorganic P, have low sol-ubility and mobility, which limits their availability (Benitez-Nelson,2000). The concentration of P can be used to predict the total bio-mass of phytoplankton (Zhang et al., 2007). It has been assumed thatP occurs exclusively as phosphate (Geng et al., 2005a; Han et al.,2011). However, recent evidence suggests that other less oxidizedforms of P (namely reduced P, with an oxidation state lower than+5) that are more soluble and active play a critical role in P bioavail-ability (Metcalf and Wolfe, 1998; Morton et al., 2003). It has evenbeen suggested that the P geochemistry in the early earth was con-trolled by reduced forms of P such as phosphite (H2PO�3 and/orHPO2�

3 , +3) (Pasek, 2008). Many microorganisms can use phosphiteand hypophosphite (H2PO�2 , +1) as alternative P sources, and thereis genetic evidence that this capability is ancient (White and Metcalf,2007). Moreover, application of phosphite fertilizer in agriculture isincreasing worldwide, despite the dispute over whether it can beused as a P source (Rickard, 2000; McDonald et al., 2001a; Thaoand Yamakawa, 2009). Phosphite has been sold as commercial prod-ucts known as biostimulants or nutri-phosphite in the American andEuropean market since 1998, and claims that these products boostplant growth have been made; however no studies have confirmed

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these reports or that phosphite was actually involved in P metabo-lism. In addition to nutrient function, phosphite also serves as a fun-gicide in agricultural field. Phosphite compounds have beenrecognized as excellent fungicides for the control of many importantplant diseases caused by Oomycetes, particularly Phytophthora sp.(Thao and Yamakawa, 2009). Nevertheless, few studies have inves-tigated the existence and role of reduced P in the biogeochemical Pcycle in lake ecosystems.

P is an important nutrient for algal growth, and its deficiency cangreatly influence the production of Chl-a and the rate of photosyn-thesis (Shen and Song, 2007; Wu et al., 2009). The role of P in eutro-phication has been at the forefront of hydro-biological researchduring the last few decades (Correll, 1998; Geng et al., 2005b).However, the effects of reduced P on eutrophication have not beenconsidered in past studies, in part because these previous effortshave neglected the existence of reduced P in the environment. Re-duced forms of P are often misclassified as organic P by traditionalanalysis methods (Morton et al., 2003), even though they areactually potential contributors to measured dissolved reactive Pconcentrations (Hanrahan et al., 2005). To date, ion chromatographymethods have been widely employed to identify phosphite andhypophosphite in the environment (McDowell et al., 2004). Pechet al. (2009) confirmed the presence of 0.06 ± 0.02 lM phosphiteand 0.05 ± 0.01 lM phosphate in a geothermal pool. We recentlymeasured 0–0.05 mg P L�1 of phosphite in bottom water collectedfrom Lake Taihu, China (unpublished data). Reduced P may be intro-duced to the environment from many different sources, including

1326 J. Zhang et al. / Chemosphere 85 (2011) 1325–1330

phosphate ore, corroding metals such as iron, and industrial prod-ucts. The primary product of schreibersite oxidation by water isphosphite, with >50% of the total aqueous P occurring in this form(Pasek, 2008). Moreover, reduced P products are routinely used inenvironmental applications, including fertilizers, fungicides, insec-ticides, herbicides, rodenticides, fumigants, flame retardants, andchemical intermediates (Morton et al., 2005).

Approximately 1% of bacterial species are capable of using re-duced P compounds such as phosphite and hypophosphite as thesole P sources (Pasek, 2008). Additionally, some bacteria can oxi-dize hypophosphite to phosphite. Phosphine gas (PH3, �3) isdetectable globally in the atmosphere and as part of the atmo-spheric link of the P cycle on earth (Han et al., 2011); accordingly,PH3 has been attributed to anaerobic metabolism and can ulti-mately be converted to phosphate after complex oxidationthrough hypophosphite and phosphite. PH3 had a positive rela-tionship with the biomass of algae and Chl-a in Lake Taihu (Genget al., 2005b; Niu et al., 2003). However, no studies have beenconducted to investigate the effects of phosphite on algal growthand photosynthesis to date. Therefore, it is necessary to study thealgal bioavailability of phosphite in lakes and its possible link toeutrophication.

Given the importance of P cycling in lake ecosystems, additionalstudies are needed to clarify the relationship between reduced Pand lake eutrophication. In this study, the bioavailability of phos-phite by M. aeruginosa, one of the predominant species involvedin algal blooms in freshwater lakes of China (Cembella et al.,1984; Fujimoto et al., 1997; Wu et al., 2009), was investigated.The purpose of this study was to determine if phosphite will boostthe cell growth and photosynthesis of M. aeruginosa.

2. Materials and methods

2.1. Algae and culture conditions

M. aeruginosa (PCC 7806) obtained from the Institute of Hydrobi-ology of Chinese Academy was investigated in modified-P BG-11medium at 25 ± 1 �C under illumination (2400 lx) using cool whitefluorescent lights with a 12-h dark:12-h light cycle. The basal P-freeBG-11 medium consisted of 150 g L�1 NaNO3, 20 g L�1 Na2CO3,75 g L�1 MgSO4�7H2O, 36 g L�1 CaCl2�2H2O, 1 g L�1EDTA (disodiumsalt), 6 g L�1 citric acid, 6 g L�1 (NH4)3Fe(C6H5O7)2, 35 g L�1 ofKNO3, 2.86 g L�1 H3BO3, 1.81 g L�1 MnCl2�4H2O, 0.22 g L�1 ZnSO4�7H2O, 0.39 g L�1 NaMoO4�2H2O, 0.079 g L�1 CuSO4�5H2O, and0.049 g L�1 Co(NO3)2�6H2O in distilled water. All components wereautoclaved separately and mixed upon cooling. All P substrates wereprepared based on P-free BG-11 medium using a single filter-steril-ized (0.22 lm) P source (Na2 (HPO3) �5H2O and NaH2PO4�H2O) toform different P media.

2.2. Sample collection and analysis

Before the experiment, cells were cultured in phosphate med-ium until they reached their exponential growth stage, after whichthey were transferred to P-free BG-11 medium for about 1 week toexhaust the accumulated P in the cells. The P-starved cells werethen re-inoculated into different P media for 2 or 3 weeks (depend-ing on the growth status).

All the experiments were conducted in triplicate. At regularintervals, samples were taken and cell numbers were counted ona compound microscope equipped with a hemocytometer. Eachsample was measured at least three times, with a maximum devi-ation of about 20%. Chl-a was extracted with 90% acetone extrac-tion and measured as described by the standard methods (StateEnvironmental Protection Administration of China, 2002).

A chlorophyll fluorescence monitoring system (PAM 210; Walz,Efeltrich, Germany) was applied to monitor the photosyntheticperformance in algae as described by Schreiber and Bilger (1987).Dark-adapted minimal fluorescence (Fo) with all PSII reaction cen-ters open was determined by measuring the modulated light(0.15 lmol m�2 s�1 PAR), which was sufficiently low not to induceany significant variable fluorescence. The dark adapted maximalfluorescence (Fm) with all PSII reaction centers closed was deter-mined by 0.8 s saturating pulse 8000 lmol m�2 s�1 PAR. The vari-able fluorescence, Fv, was calculated as the difference between Fo

and Fm. After being dark-adapted for 15 min, the optimal quantumyield of PS II photochemistry was measured and calculated fromthe Fv/Fm ratio based on the method described by Ting and Owens(1992). The detection limit of the fluorometer was approximately106 cells of M. aeruginosa per mL.

Phosphate was measured using the molybdenum-antimonyspectrophotometric method (State Environmental ProtectionAdministration of China, 2002). Because phosphite is unable toreact with molybdenum-antimony regent, its levels can bedetermined in vitro after oxidation into phosphate with persul-fate solution (Morton et al., 2003). Cells in the P medium werecentrifuged at 12,000 g for 10 min at 4 �C, washed three timeswith P-free medium and then digested with 5% persulfate for30 min.

The alkaline phosphatase activity (APA) was assayed accordingto the method described by Berman (1970). Briefly, a 2 mL cellsample (unfiltered) was mixed with 1 mL of freshly prepared p-nitrophenyl phosphate, disodium hexahydrate (p-NPP) at10�3 mol L�1 and 2 mL of Tris-Cl (pH = 8.6), after which it wasincubated under dark culture conditions for 24 h and then mea-sured at 410 nm by UV-1100. Reactions were monitored by contin-uously following the production of p-nitrophenol.

3. Results and discussion

3.1. Phosphite stability in medium

Before the experiment, the stability of phosphite in the mediumwas investigated. The percentage of oxidation of phosphite in BG-11 medium was examined in freshly prepared and after 3 weeks ofstorage medium under the culture conditions. As shown in Table 1,phosphite was stable in medium during the 3 weeks of storage un-der the cultured conditions. In phosphite medium with phosphiteconcentrations ranging from 0 to 500 mg P L�1, the oxidation per-centage of the phosphite ranged from 0% to 1.48%.

If the phosphite is unstable and easily oxidized to phosphate,the generated phosphate rather than the phosphite itself will serveas the P source. The results indicated that the phosphite was stablein BG-11 medium, at least during the experimental period. Otherstudies have also shown that phosphite solution is stable, even un-der aerobic conditions, and that it will not be readily oxidizedwithout strong oxidants (McDowell et al., 2004). 31P nuclear mag-netic resonance (NMR) revealed that phosphite in morpholinepro-panesulfonic acid (MOPs) medium had no detectable oxidationproducts after 2 weeks under aerobic conditions when the Pconcentration ranged from 250 to 1000 lM (Metcalf and Wolfe,1998).

3.2. Phosphite as the sole P source for growth of M. aeruginosa

P-starved M. aeruginosa were inoculated into medium contain-ing four levels of phosphite ranging from 0.5 to 500 mg P L�1. Theresults revealed that 500 mg P L�1 phosphite caused a decrease inthe cell number and even death after 16 d, whereas the cellnumber of cultures grown on medium containing 0–50 mg P L�1

Table 1Phosphite stability in medium before inoculation.

Phosphite(mg P L�1) inmedium

Oxidation of phosphite (%)in freshly preparedmedium

Oxidation of phosphite (%) inmedium after 3 weeks ofstorage

0 0 00.54 1.48 ± 1.36 1.29 ± 0.285.44 0.46 ± 0.13 0.44 ± 0.0350 0.18 ± 0.00 0.18 ± 0.03500 0.13 ± 0.01 0.14 ± 0.00

J. Zhang et al. / Chemosphere 85 (2011) 1325–1330 1327

phosphite fluctuated within a small range (Fig. 1). A wide range ofphosphite concentrations was set to determine if an appropriatephosphite level to accelerate the M. aeruginosa growth exists, andto judge the toxicity of phosphite to M. aeruginosa. Some studieshave reported that phosphite was toxic to all first crops (Thaoand Yamakawa, 2009). These results suggest that phosphite isnot a suitable P source for the growth of M. aeruginosa. Phosphiteappears to be inert and is not toxic to M. aeruginosa until its con-centration reaches 500 mg P L�1.

Many microorganisms such as Escherichia coli and Pseudomonasfluorescens have been found to utilize phosphite as the sole Psource since the late 1950s (Casida, 1960; Malacinski and Kon-etzka, 1966; White and Metcalf, 2007). In addition, Desulfotignumphosphitoxidans was found to use phosphite as the sole electron do-nor in energy metabolism (Schink and Friedrich, 2000). Despite M.aeruginosa being one of the oldest species on earth (Pasek, 2008),its ability to utilize phosphite as the sole P source likely disap-peared during evolution. As a result, M. aeruginosa could not utilizephosphite as a sole P-nutrient directly at any concentration. Like M.aeruginosa, most plants do not appear to be able to use phosphiteas a P source (Thao and Yamakawa, 2009). Furthermore, the resultsshow that, even if phosphite serves as a fungicide, phosphite atconcentrations as high as 50 mg P L�1 fails to damage cells untilthe concentration reaches 500 mg P L�1.

A high phosphite concentration was also tested to determine ifminor oxidation of phosphite into phosphate was sufficient to sup-port M. aeruginosa growth. Based on Table 1, the addition of500 mg P L�1 phosphite would add 0.65 mg P L�1 phosphate, whichis an adequate P level for the growth of M. aeruginosa (Fig. 2a),while 500 mg P�1 phosphite caused the death of M. aeruginosa.Phosphite has been shown to prevent the utilization of phosphateby M. aeruginosa. Phosphite was also found to prevent the acclima-tion of plants and yeast to phosphate deficiency by specificallysuppressing the expression of phosphate-starvation induciblegenes (Varadarajan et al., 2002).

11.09 1.16

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Fig. 1. Cell numbers of M. aeruginosa under different phosphite concentrations.

3.3. Combination of phosphite and phosphate as the P sources forgrowth of M. aeruginosa

Some studies have suggested that combinations of phosphiteand phosphate are more effective than either phosphite or phos-phate alone in plant assimilation (Förster et al., 1998; Young,2004; Lovatt and Mikkelsen, 2006). A recent study indicated thatthe effect of phosphite on the growth of hydroponic lettuce wasdependent on the phosphate status of the plants, and that thoseinsufficiently fertilized with phosphate may become vulnerableto even low phosphite concentrations (Thao et al., 2009). Generally,phosphite and phosphate can be detected simultaneously in theaquatic environment. Therefore, cell growth may be enhanced ifcells are provided with some phosphate in the phosphite medium.

Preliminary tests on phosphate as the sole P source for M. aeru-ginosa growth suggest that four phosphate supply statuses couldbe defined according to cell growth. P-deficient in phosphate med-ium containing less than 0.02 mg P L�1, P-insufficient in phosphatemedium less than 0.54 mg P L�1, P-sufficient in phosphate mediumcontaining 0.54 mg P L�1, and P-excess in phosphate medium con-taining 5.44 mg P L�1 (Fig. 2a). These findings are consistent withthe response of M. aeruginosa PCC7806 to low inorganic phosphate(Wang et al., 2010).

As shown in Fig. 2a, in phosphate control groups, the cell num-bers primarily depend on the phosphate concentration. In phos-phate–phosphite groups, when phosphate existed in the medium,phosphite appeared to bring a slight increase in cell numbers(Fig. 2b).

3.4. Combination of phosphite and phosphate as the P sources forphotosynthesis of M. aeruginosa

Chl-a is also an indication of cell growth status and photosyn-thetic ability. As shown in Fig. 3, the Chl-a level increased as thephosphate concentration increased from 0 to 5.44 mg P L�1 inphosphate control medium or in phosphate–phosphite medium.Nevertheless, phosphite affected the Chl-a content in phosphate–phosphate medium. When phosphate (0.01 or 0.10 mg P L�1) wasdeficient in phosphate–phosphite medium, phosphite led to aslight increase in the Chl-a level. When phosphate (0.54 mg P L�1)was sufficient in the phosphate–phosphite medium, phosphitewould sharply increase the Chl-a content (P < 0.05, n = 27). How-ever, under excess phosphate conditions (5.44 mg P L�1) in thephosphate–phosphite medium, phosphite led to a decrease inChl-a content (P < 0.05, n = 27).

Chlorophyll fluorescence parameters were applied to monitorthe photosynthetic response of M. aeruginosa under these condi-tions. The activity of PSII measured as the maximum quantumyield (Fv/Fm) was examined at 7 and 21 d after inoculation(Fig. 4). The maximum yield of photosynthesis in all P mediumon day 7 was much greater than that on day 21. Additionally, pho-tosynthesis was higher in mixed phosphate–phosphite mediumthan in sole phosphate medium at both time points, despite therebeing no obvious trend.

The Fv/Fm value indicated that PSII function and oxygen evolu-tion are the two of the most sensitive photosynthetic parameters.Kromkamp and Peene (1999) discovered that low Fv/Fm valuescoincided with low phosphate and silicate concentrations. Fv/Fm

may be used as an indicator of P limitation deficiency for theunicellular strains (Shen and Song, 2007).

It is widely accepted that Chl-a production is associated withphosphate concentration and total P (TP) concentrations in lakes(Dillon and Rigler, 1974; Hendrickson et al., 2004; Wu et al.,2009). However, the role that phosphite plays in the productionof Chl-a is unclear. As shown in Fig. 3, phosphite only increased sig-nificantly in Chl-a concentrations when cells were under sufficient,

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mg P L-1 phosphate and 5.44 mg P L-1 phosphite

mg P L-1 phosphate and 5.44 mg P L-1 phosphite

mg P L-1 phosphate and 5.44 mg P L-1 phosphite

5.44 mg P L-1 phosphite

Fig. 2. Cell numbers of M. aeruginosa in phosphate (a) and phosphite–phosphate medium (b) during 3 weeks of culture.

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Fig. 3. Chl-a contents in different P medium after 3 weeks of culture.

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phosphate

Fig. 4. Maximum yield of photosynthesis (Fv/Fm) in different P media.

1328 J. Zhang et al. / Chemosphere 85 (2011) 1325–1330

but not excessive phosphate conditions. In the fertilizer market,many phosphite fertilizer products have been reported to improvephotosynthesis, respiration, cellular strength and enhance the en-ergy generating Krebs cycle. Indeed, Chl-a fluorescence is an effec-tive method for assessing the physiological state of higher plantsand algae under various stress conditions (Baker, 2008). Fv/Fm isthe maximum quantum efficiency of PSII, and the Fv/Fm of cells inphosphate–phosphite medium was slightly higher than in eithersole phosphite or phosphate medium, which means that phosphitecan contribute to the increase in Chl-a concentrations, especiallywhen cells are under sufficient phosphate supply. Another study

also concluded that the application of phosphite to the Hass avo-cado plant (Persea americana Mill) enhanced photosynthetic activ-ity (Cervera et al., 2007).

3.5. Change of TP and APA in vitro

The TP and APA in vitro were determined on the day 21. Asshown in Fig. 5, TP in the sole phosphite medium was much higherthan in the P-free medium, which indicates that phosphite can beeasily absorbed in the sole phosphite medium. Phosphite can bewell assimilated by the leaves and roots of the plants, but will

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Fig. 5. Concentrations of total P and APA in vitro in different P media.

J. Zhang et al. / Chemosphere 85 (2011) 1325–1330 1329

not provide P as a nutrient (Thao et al., 2009). However, althoughthe TP in the sole phosphite medium was the same as in the phos-phate–phosphite medium (Fig. 5), the Chl-a content in the phos-phate–phosphite medium was much higher than in the solephosphite medium (Fig. 3). These findings indicate that the in-creased phosphite in cells did not enhance the cell growth, but in-stead increased the Chl-a concentration. When applied withphosphite fertilizer, phosphite likely just remains in or is translo-cated in plants, where it has an adverse effect on growth (Ticconiet al., 2001). Phosphite could also be assimilated and concentratedby yeast cultured with 0.1 mmol L�1 phosphite, especially under P-deprivation conditions (McDonald et al., 2001b).

The electron acceptors for the oxidation of phosphite are pro-tons derived from water. Thus, alkaline phosphatase is a phos-phite-dependent, hydrogen evolving hydrogenase as well as aphosphomonoesterase (White and Metcalf, 2007). Besides, alkalinephosphatases play an important role in supplying P during Pdepletion (Shen and Song, 2007). In phosphite–phosphate mediumand sole phosphate medium, the APA concentration remains lowand almost constant; this suggests that P is sufficient. In the P-freemedium, P-free medium stimulated higher APA, which is a re-sponse of the cells to P deficiency. In phosphite medium, theAPA value decreases because of the false P supply, which attenu-ates the expression of the normal signal of P deficiency. APA canbe an indicator of P limitation of algal growth. APA is located inthe membranes and can directly decompose dissolved organicphosphate (DOP) as a P nutrient for algal growth when P is defi-cient (Huang and Hong, 1999; Huang et al., 2005). The measure-ment of APA in vitro showed that it depends on the status of Psupply in vitro (Fig 5). Specifically, the expression of APA was ex-tremely high in P-free medium, then partly restrained by5.44 mg P L�1 phosphite and nearly completely inhibited by0.54 mg P L�1 phosphate. These findings suggest that phosphitemay inhibit the expression of APA, especially under P-deprivedconditions. Phosphite has been reported to intensify the deleteri-ous effect of P deficiency by tricking P-deprived plant cells intosensing that they are P sufficient, when in fact their cellular Plevels are extremely low (McDonald et al., 2001b; Ticconi et al.,2001; Varadarajan et al., 2002). This response mechanism of M.aeruginosa to phosphite is different from that of many other micro-organisms, some of which can utilize phosphite through thehydrolysis of phosphite by BPA (a phosphomonoesterase in bacte-ria), which is a similar mechanism as that employed for theutilization of organic P (Yang and Metcalf, 2004). At the molecularlevel, APA expression is largely restrained in the presence ofphosphite by tricking cells to sense that they are in an environ-ment containing sufficient P.

4. Conclusions

Phosphite at levels of 0–500 mg P L�1 was stable in mediumduring 3 weeks of storage under the cultured conditions with anoxidation percentage of 0–1.48%. M. aeruginosa could not utilizephosphite as a sole P-nutrient directly at any concentration. Phos-phite could boost cell numbers and Chl-a content of M. aeruginosaas long as appropriate levels of phosphate were provided simulta-neously. Increased phosphite in cells failed to enhance cell growth,but did increase the Chl-a concentration. Chl-a contents increasedsharply when 5.44 mg P L�1 phosphite was added to 0.54 mg P L�1

phosphate medium by stimulating the photosynthesis process.Sole phosphite can easily be absorbed, but does not support thegrowth of M. aeruginosa. Phosphite inhibits the response of cellsto deficient P status, especially under P-deprived conditions.

Acknowledgments

This work was supported by the National Basic Research Pro-gram of China (No. 2008CB418003), the National Science Founda-tion of China (No. 21077051), the Jiangsu Natural ScienceFoundation (No. BK2008276) and the International Foundation ofScience (No. A/4425-1). We also thank Dr. Yang Yu from NanjingInstitute of Geography and Limnology, Chinese Academy of Sci-ences for his help with the measurement of the chlorophyllfluorescence.

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