Responses of the toxic cyanobacterium Microcystis aeruginosa to iron and humic substances

Plant Physiology and Biochemistry 45 (2007) 365e370 www.elsevier.com/locate/plaphy

Research article

Responses of the toxic cyanobacterium Microcystis aeruginosa to iron and humic substances Alicja Kosakowska a,*, Marcin N˛edzi b, Janusz Pempkowiak a a

Institute of Oceanology, Polish Academy of Sciences, Powstan´co´w Warszawy 55, 81-712 Sopot, Poland b Institute of Oceanography, University of Gdan´sk, Piłsudskiego 46, 81-378 Gdynia, Poland Available online 21 March 2007

Abstract Iron is an essential element to marine biota. Different types of dissolved organic matter (DOM), such as humic substances have impacts on the marine coastal waters iron chemistry. The aim of the study was to examine how the presence of humic substances (both aquatic and sedimentary) may affect iron bioavailability to the bloom-forming cyanobacterium Microcystis aeruginosa Kutzing incubated on standard and modified mineral BG-11 media. The final iron concentrations in the growth media ranged from 0.1 to 100 mM. The results demonstrate that both the growth rate and the concentration of chlorophyll a in cultures of M. aeruginosa are limited by insufficient (<10 mM) Fe concentrations. The addition of aquatic humic substances in the presence of iron in concentrations <0.1 mM increased the optical density 25-fold, and the production of chlorophyll a 15-fold as compared with the cultures exposed to iron only at the same concentration. Sedimentary humic acids in the presence of iron at a concentration of 10 mM reduced the growth and production of chlorophyll a by 50% as compared to the cultures exposed to iron only at the same concentration. Possible mechanisms of humic substances e metal ion e alga interactions are discussed. It is suggested that aquatic humic substances could be of great importance in the formation of cyanobacteria blooms. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Cyanobacteria; Growth; Iron; DOM; Complexes; Fulvic acids; Humic acids

1. Introduction In open-ocean surface water iron is present in picomolar (1012 M) concentrations [2]. In the Baltic Sea, dissolved iron concentrations are below the detection limit of the analytical procedure applied, i.e., <108 M [30]. Despite its low concentration, iron is an important factor in marine ecology and biogeochemistry because it limits primary production in several parts of the ocean in the so-called HNLC waters [6]. Iron is an essential element for cyanobacteria and algae owing to its importance in numerous metabolic processes such as respiration, photosynthetic transport, chlorophyll

Abbreviations: HS, humic substances; HA, humic acids; FA, fulvic acids. * Corresponding author. Fax: þ48 58 551 2130. E-mail address: [email protected] (A. Kosakowska). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.03.024

synthesis, nitrate reduction, nitrogen fixation and detoxification of reactive oxygen species [20,22]. In the context of phytoplankton growth limitation, iron has also been reported from coastal waters [15], including estuaries [40], coral reefs [9] and lakes [16], in spite of the relatively high dissolved iron concentrations in such waters. Only a small fraction of dissolved iron(III) occurs in a free hydrated (Fe3þ) or inorganically complexed form; the remaining 99% is strongly complexed by organic ligands [4,21]. This organic complexation prevents iron(III) from forming insoluble oxyhydroxides, thereby maintaining elevated dissolved iron concentrations in seawater. Little is known about the specific nature of these ligands; nevertheless, iron has been reported to complex with two main types of organic substances: those with a high metal-binding affinity (high K) and specificity (siderophores or porphyrins; [15,38]), and those with a lower affinity but present in high concentrations (e.g. humic substances and

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polysaccharides; [33]). In the open ocean and offshore coastal waters the first type is predominant, but in coastal and nearshore areas the second type may also be important. Therefore, high concentrations of dissolved iron in coastal waters by no means guarantee that iron is abundantly available to phytoplankton there. Iron bioavailability is related to many factors, including the presence of organic substances that may interact with (Fe3þ) in the water. A major fraction of DOM in natural waters (30e60% of total dissolved organic carbon) is composed of humic substances (HS), which consist mainly of fulvic acids (FA), but may also comprise humic acids (HA). In Baltic seawater, concentrations of humic substances range from 2 to 5 mg dm3 [29]. The presence of humic substance has been shown to positively affect the growth rate and biomass production of dinoflagellates [8,10] and to favor their growth over diatom species in culture [12,26]. Furthermore, growth and production of Gymnodinium catenatum in culture is stimulated by addition of aquatic HS and natural DOM (isolated from Huon River, south-east Tasmania) as well as by water soil extract [1,8]. Stimulation of G. catenatum growth in the presence of organic matter (DOM and HS) may therefore have been partly due to increased N (and P) availability [8]. Graneli and Moreira [13] also showed a positive correlation between the increase in discharge of HS into rivers draining the Swedish west coast and the natural incidence of dinoflagellate blooms over the last decade. Interestingly, runoff from forested areas produced a greater stimulatory response on Alexandrium growth rates than did runoff from agricultural areas [8,13]. The presence of humic substances has been shown to stimulate photosynthesis in the green alga, Pseudokirchneriella subcapitata [21] and Chlorella vulgaris [11,31] by reducing the toxicity of heavy metals and inhibit the growth of cyanobacterium Microcystis aeruginosa [16] as well as Anabaena circinalis, by reducing the concentration of biologically available Fe [36]. However, other studies have found negative or no effects of DOM on algal growth. For example, Devol and co-workers [7] found that humic and fulvic acid (isolated from lake sediments) added to natural phytoplankton assemblages from Lago Jacaretinga, Central Amazon, Brazil did not change phytoplankton nutrient uptake kinetics or final chlorophyll yields. Many investigators have shown that humic substances can enhance vascular plant growth when present in the nutrient or soil solution at low concentrations. Comprehensive reviews summarizing these observations have been published by Chen and co-workers [4,5]. These investigators reported optimum plant-growth enhancement when humic substances in concentrations of 25e150 mg dm3 were added to the nutrient solution. The mechanism suggested by a number of researchers is the enhanced solubility of microelements in solution and therefore also an enhanced nutrient uptake and plant growth. Plant parameters affected are: root and shoot weight, root initiation, seedling emergence and growth, rhizosphere microbial population, nutrient uptake and flowering. These effects

were found for both HAs and FAs as well as for compostderived HS. In most experiments the activity of HAs was found to be similar to that of FAs, although other results indicated higher activity for low molecular weight products. DOM is also a substrate for microorganisms. In soils, DOM may be the most important carbon source since soil microorganisms require an aquatic environment for all uptake mechanisms [5]. The aim of this study was to examine how the presence of humic substances (both aquatic and sedimentary) may affect iron bioavailability. The bloom-forming cyanobacterium Microcystis aeruginosa was used as the test organism. Experiments were carried out in the laboratory under iron-deficient and iron-sufficient conditions. Humic substances added to the growth media were isolated from both lake water (aquatic humic substances) and marine bottom sediments (sedimentary humic substances). 2. Material and methods 2.1. Organism A xenic culture of the planktonic cyanobacterium Microcystis aeruginosa (PCC 7820 Cluster 1) was used in the experiments. The strain was obtained from the Pasteur Culture Collection of Cyanobacteria. It is a common, bloom-forming alga found primarily in nutrient-enriched freshwater and estuaries of low salinity. The species is colonial, which means that single cells can aggregate to form groups that tend to float near the water surface. Colony sizes vary from a few to hundreds of cells. 2.2. Ferric(III) chloride solutions The stock solution of ferric(III) chloride (Titrisol, Merck, 1 g Fe3þ in 100 cm3) was diluted with deionised water to obtain a 1 mg/cm3 standard solution. This solution was added to the growth media preparation and used in as standard absorption atomic spectroscopy (AAS) measurements. 2.3. Humic substance solutions Humic substances were isolated from filtered (Whatman GF/F) and acidified (pH 2) lake water and from marine sediments by sorption on Amberlite XAD-2 (Serva), desorption with 0.5 M NH3 H2O, and finally, concentration in a rotary evaporator and lyophilisation [29,30]. The aqueous solutions of humic substances were prepared by dissolving their lyophilisates in 10 mM NaHCO3. The final humic substances concentration in modified BG-11 medium was 4 mg dm3. 2.3.1. Humic substances-characteristics The isolates were characterized by measuring UV/VIS absorption spectra after dissolving in 5 mmol dm3 NaHCO3, IR spectra (KBr pellets), and elemental composition (C,H,O,N,S). The results are presented in (Table 1).

A. Kosakowska et al. / Plant Physiology and Biochemistry 45 (2007) 365e370 Table 1 The characteristics of humic substances used in the experiments

and 750 nm [18,24,35]. Optical density (OD) was measured as absorbance at 680 nm.

Humic substances origin Bottom sediments

Lake water

UVa e A280/A400 VISb e A400/A600 IRc e 2950 cm1 1710 cm1 1650 cm1 1440 cm1

4.1 6.1 þþ þ þ þ

4.4 6.7 þ þþ þþ þ

Elemental composition [%] eC eO eH eS eN

46.30 33.20 7.12 1.49 3.12

47.12 35.17 8.20 1.13 2.70

a

UV e ratio of absorbances at 280 and 400 nm. VIS e ratio of absorbances at 400 and 660 nm. c IR e occurrence of absorption bonds at given value-numbers (þ e present, þþ e strong). b

2.4. Incubation medium The cyanobacterium was incubated on both standard and modified mineral BG-11 medium [34]. In the latter, iron in the form of ferric ammonium citrate, along with the chelating agents EDTA and citric acid, were excluded from the original medium. The pH of the medium was adjusted to 7.5 with 1 M KOH. The medium was sterilised at 121  C for 30 min. To reduce iron contamination, glassware and polyethylene equipment were pre-treated with 6N HCl for 24 h, then rinsed with deionised water. 2.5. Cultivation conditions M. aeruginosa cells were incubated on Fe-deficient and Fesufficient growth media respectively containing <0.1 mM Fe and from 1 to 100 mM Fe. The inoculum of 750 mg chl a 1 cm3 was added at the beginning. The cultures were incubated for 12 days at 25  0.1  C under continuous illumination at an intensity of 6 mmol photons m2 s1. All experimental variants were run in triplicate. The controls were cyanobacteria cultures grown in standard BG-11 medium. The iron concentration in the control cultures was the same as in the original BG-11 medium, i.e., 10 mM. 2.6. Measurements Iron concentrations in the cultivation medium were determined by AAS (Video 11E, Thermo Jarell Ash) in the Marine Biogeochemistry Laboratory of the Sopot Institute according to procedures described elsewhere [29]. The optical density and chlorophyll a concentration were used to track cyanobacterial growth. Chlorophyll was measured following standard methods after filtration (GF/F), extraction with acetone, and absorption measurements at 680

2.7. Statistics Means and standard deviations were calculated for all the experiments. Dixon’s Q test ( p ¼ 0.05) was performed to eliminate outliers, and Student’s t-test ( p ¼ 0.05) was used to evaluate the statistical significance of differences between means [39]. 3. Results Fig. 1 shows the results of experiments on the influence of different iron(III) concentrations on cell growth and the chlorophyll a content in a population of Microcystis aeruginosa. The data indicate that 10 mM Fe was the threshold concentration required to support M. aeruginosa growth. The experiments showed that the addition of iron at a concentration of 100 mM to the growth medium increased the cell density and chlorophyll a content in the test organism cultures by 50e 70% in comparison to the control sample. These results demonstrate that the chlorophyll a concentration in cultures and growth of M. aeruginosa is limited by insufficient Fe concentrations, implying that the synthesis of chlorophyll a is strongly inhibited by Fe depletion. Our results are consistent with the results of previous studies. Underscoring the importance of iron in phytoplankton metabolism, several studies have demonstrated that Fe depletion reduces numerous metabolic processes in marine, brackish and freshwater cyanobacteria and algae [20,22,27]. For example, iron stress reduced the growth rate of a Microcystis aeruginosa population isolated from algal blooms in Lake Kasumigaura [16], of Nodularia spumigena isolated from algal blooms in the Gulf of Gdan´sk [28], and also of Anabaena variabilis [37] and Anabaena circinalis NIES-41

200

Optical density and chlorophyll-a [% of control]

Parameter

367

180 160

Optical density chlorophyll-a

140 120 100 80 60 40 20 0

0.01

0.5

1

10

100

iron concentration [ µM ] Fig. 1. The effect of iron(III) concentration in a depleted growth medium, devoid of chelating agents, on the growth rate and concentration of chlorophyll a in a population of Microcystis aeruginosa.

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[36]. In contrast, iron stress stimulates akinete formation in Anabaena cylindrica [14]. Fig. 2 presents the results of experiments on the influence of humic substances on populations of Microcystis aeruginosa under both iron-deficient and iron-sufficient conditions. Aquatic humic substances (AHS) (a low-molecular weight fraction of humic substances), added at a concentration of 4 mg dm3 to media containing Fe(III) at concentrations from <0.1 mM to 10 mM, stimulated M. aeruginosa growth and chlorophyll a production. The addition of AHS in the presence of iron at concentrations <0.1 mM increased the optical density 25-fold, and the production of chlorophyll a 15-fold as compared with the cultures exposed to iron only at the same concentration. The addition of AHS in the presence of iron at concentrations of 10 mM doubled the growth rate and production of chlorophyll a in comparison to the cultures exposed to iron only at the same concentration. Several factors may be responsible for this increase. One of them could be the enhanced availability of iron(III)-AHS complexes as compared to iron(III). This would imply that in the culture medium, and possibly in other aqueous solutions, e.g., seawater, depleted of organic complexing agents, Fe(III) is in a form (a colloidal form?) that is biologically unavailable to algae. Fig. 3 presents the results of experiments on the influence of sedimentary humic substances (SHA) on the population of M. aeruginosa under both iron-deficient and iron-sufficient conditions. Exposure to SHS in the presence of iron at concentrations <0.1 mM had no statistically significant effects on the growth rate or production of chlorophyll a by the test organism. Sedimentary humic substances consisting of humic acids e a high-molecular weight fraction of humic substances e at concentrations of 4 mg dm3 significantly inhibited chlorophyll a production and the number of cells in the cyanobacterium population under iron-sufficient conditions. Humic acids in the presence of iron at concentrations of 10 mM reduced the growth and production of chlorophyll

Cell density and chlorophyll-a content [% of control]

120 100

full medium BG-11

Optical density chlorophyll-a

100

Cell density and chlorophyll-a content [% of control]

368

Optical density chlorophyll-a

80

60

40

20

0 0.1µM Fe

0.1µM Fe 10µM Fe 10 µMFe (BG11)10µMFe + humic acids +humic acids +humic acids

Fig. 3. The effect of iron(III) concentration in a growth medium consisting of sedimentary humic substances (SHS) on the growth rate and concentration of chlorophyll a in a population of Microcystis aeruginosa.

a by 50% as compared to the cultures exposed to iron only at the same concentration. In the full BG-11 medium (with iron and chelating agents) a statistically significant reduction in cyanobacteria growth was observed in the presence of humic acids. The chlorophyll a production was lowered by 20% in relation to the control. It is difficult to explain the inhibition of cyanobacterium by sedimentary fulvic acids. One possibility might be a formation of unbioavailable complexes with humic acids. However, the measured properties (Table 1), if anything, contradict the possibility. The larger UV/VIS absorption indexes indicate more aromatic structures in the aquatic HS than in the sedimentary ones. This combined with larger concentration of oxygen in the former suggests more oxygen containing functional groups in the aquatic HS. The conclusion is supported by IR spectra indicating the presence of carboxyl groups (1710 cm1 bond) and aromatic rings (1650 cm1) in the aquatic as opposed to sedimentary HS. In the consequence aquatic HS should exhibit more pronounced affinity to iron than the sedimentary ones. Another possibility would be the presence of specific organic substances inhibiting growth of M. aeruginosa in sedimentary HS It has been reported that sedimentary humic acids act as agents inhibiting growth of bacteria, although at much larger concentrations [23].

80

4. Discussion 60 40 20 0

0.1µM Fe

0.1µMFe +fulvic acids

10µM Fe

10 µMFe 10µMFe +fulvic acids +fulvic acids

Fig. 2. The effect of iron(III) concentration in a growth medium consisting of aquatic humic substances (AHS) on the growth rate and concentration of chlorophyll a in a population Microcystis aeruginosa.

The present study clearly shows that AHS additives at concentrations of 4 mg dm3 are stimulatory to Microcystis aeruginosa PCC in the presence of iron concentrations from 0.1 mM to 10 mM. Imai et al. [16], however, reported that M. aeruginosa isolated from algal blooms in Lake Kasumigaura was inhibited when the fulvic acid concentration was 2 mg dm3. Growth inhibition was probably due to a deficiency of iron resulting from Fe complexation by AHS. Humic substances inhibit the growth of M. aeruginosa and A. circinalis by reducing

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the concentration of biologically available Fe [16,36]. However, several other possible mechanisms involving DOM may have a negative effect on algae growth, e.g., decreased availability of other trace metals and the possible negative effect of phenolic compounds on DOM [23,36]. These seemingly contradictory results indicate that the origin and properties of fulvic acids are important factors modifying their affinity to iron and their influence on iron transport to cyanobacterial cells. Humic substances have also been reported to stimulate algal growth. The presence of humic substances has been shown to stimulate photosynthesis in the green alga Pseudokirchneriella subcapitata and growth in Chlorella vulgaris A1e76 by reducing the toxicity of heavy metals [21,31] and to enhance phytoplankton growth by increasing Fe availability [11]. The detoxifying activity of humic substances has been examined in detail. Several experiments have demonstrated that the toxicity of heavy metals, e.g., Cd and Zn, towards Pseudokirchneriella subcapitata was significantly reduced in the presence of humic acids, though not in the presence of the Suwannee River AHS. Complexation is not the only mechanism by which DOM can influence the bioavailability of metals in natural waters. It has been amply documented that DOM can also be adsorbed on biotic surfaces, e.g., algal surfaces, and can block surface sites where toxic metals must be adsorbed in order to be taken up by the cell ([3] and references therein; [19]). The results suggested that AHS reduced heavy metal toxicity in different ways: by reducing the quantity of free metal ions, and by being adsorbed on algal surfaces, thus shielding the cells from free ions [21]. Humic and fulvic acids possess several functional groups capable of complexing micronutrients. The most important of these functional groups are the carboxyl (COOH) and hydroxyl (OH) groups. Some researchers attributed the stimulative effects of HS to higher uptake of nutrients. Others, however, suggested that plants growth promoted by a hormone-like activity of HS. Yet, investigators have been unable to prove that plant growth regulators are present in HS preparations. A small fraction of lower molecular weight components of HS can be taken up by plants and are considered to increase cell membrane permeability and to exhibit hormone-like activity. The most widely accepted mechanism for the stimulation of algal growth by DOM is its potential for altering the free ion concentration (‘bioavailability’) of trace metals. These compounds are natural ligands and have the ability to form metal-organic complexes of varying strength [25], which may serve to enhance phytoplankton growth by increasing the availability of some metals (e.g. iron; [17]) while concomitantly decreasing the availability of others (e.g. copper; [17,21,31]). The relatively high degree of the phytoplankton growth stimulation by the so-called low molecular weight humic and fulvic acids fractions may be associated with a direct cell sensitization response, an indirect chelation response, or

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both. A small fraction of lower molecular weight components of HS and their metal- complexes can penetrate the plant cell and the degree of penetration depends on the molecular size of the various entities [32]. The apparently low molecular weight humic fractions have a high number of carboxyl groups associated with their metal holding capacity. It is also possible that the differences in elemental composition of lower and higher molecular weight fractions are associated with differences in their biological properties. On the other hand, the growth inhibition by high concentrations of humic additives may cause ‘over-chelation’, since excess of chelators may make essential ions unavailable for algae [8]. 5. Conclusions Our study has demonstrated that humic substances stimulate or inhibit biomass production in cultures of the cyanobacterium Microcystis aeruginosa. The magnitude of these effects depends on the composition of the phytoplankton community and on the source and properties of the humic substances. Molecular weight appears to be important here, since different results were obtained for lowmolecular-weight aquatic humic substances and sedimentary humic acids, the high-molecular-weight fraction of humic substances. The results indicate elevated iron bioavailability and the possibility of M. aeruginosa blooms in the presence of aquatic humic substances. It is possible, therefore, that aquatic humic substances could be of great importance in the formation of toxic cyanobacteria blooms, especially in eutrophic environments. Acknowledgements This work was carried out within the framework of the Institute of Oceanology’s statutory activities, grant No II.3.2005e2006. We would like to express our gratitude to Prof. Egil T. Gjessing for providing the lacustrine humic substances. References [1] S.I. Blackburn, G.M. Hallegraeff, C.J. Bolch, Vegetative reproduction and sexual life cycle of the toxic dinoflagellate Gymnodinium catenatum from Tasmania, Aust. J. Phycol. 25 (1989) 577e590. [2] P.W. Boyd, A.J. Watson, C.S. Law, E.R. Abraham, T. Trull, R. Murdoch, D.C.E. Bakker, A.R. Bowie, K.O. Buessler, H. Chang, M. Charette, P. Croot, K. Downing, R. Frew, M. Gall, M. Hadfield, J. Hall, M. Harvey, G. Jameson, J. LaRoche, M. Liddicoat, R. Ling, M.T. Maldonado, R.M. McKay, S. Nodder, S. Pickmere, S. Rintoul, K. Safi, P. Sutton, R. Strzepek, K. Tanneberger, S. Turner, A. Waite, J. Zeldis, A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization, Nature 407 (2000) 695e702. [3] P.G.C. Campbell, M.R. Twiss, K.J. Wilkinson, Accumulation of natural organic matter on the surfaces of living cells: implications for the interaction of toxic solutes with aquatic biota, Can. J. Fish. Aquat. Sci. 54 (1997) 2543e2554.

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