Mierob Eeol (1985) 11:139-148

MICROBIAL ECOLOGY 9 1985 Springer-Verlag

Copper Toxicity to Cyanobacteria and its Dependence on Extracellular Ligand Concentration and Degradation Wilson F. Jardim and H.W. Pearson The University of Liverpool, Department of Botany, Liverpool L69 3BX, UK Abstract. Copper toxicity to the cyanobacterium Plectonema boryanum (UTEX 594) has been investigated in the presence of citrate and nitrilotriaeetic acid (NTA) using a copper-saturated culture medium. The coppercitrate complex was biodegradable, and toxicity was dependent on the free ligand concentration. The ratio of citrate to copper also affected the extent o f metal toxicity. N T A was not degraded by P. boryanum. Ligand degradation in the cyanobacterial culture increased the ionic copper concentration and caused a concomitant reduction in growth. The ecological implications of these findings are discussed.

Introduction

Recently, the differentiation of the metal forms has become the principal aim when monitoring copper toxicity to phytoplankton, because the ionic species seems to be predominantly responsible for the deleterious effects [9, 23, 24]. In the environment, the amelioration of the toxic effects of ionic copper can be due to its complexation by naturally occurring ligands. Hart and Davis [ 14] suggested that organic copper complexes were the dominant species in the Yarra River, Australia, and Blutstein and Shaw [5] found that in lake water samples, about 50% of the apparent complexing capacity was attributable to soluble organic compounds. The importance of organic compounds in controlling metal toxicity within the aquatic environment has also been suggested by other workers [21, 24]. In laboratory toxicity tests, organic compounds have been used to complex copper and detoxify the metal present in solution. These include synthetic organic chelators such as EDTA and N T A [2, 9, 18], amino-acids [3, 11], and buffers [1, 23]. However, little attention has been focused on the assimilation of the metal, the ligand, and the metal-complex itself by aquatic organisms in such toxicity experiments [ 15]. Since at least some of the compounds used as complexing agents can be degraded by the organisms during growth, the results of bioassay tests may be difficult to interpret. Bollman and Robinson [7] have studied the assimilation of 14C-carboxylic acids by blue-green algae and their ability to compete with heterotrophie bacterial populations for such substrates, and Butler and Capindale [8] have calculated the rate of assimilation of citric and adipic acids by the cyanobacterium

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W . F . Jardim and H. W. Pearson

Anacystis nidulans. Recently, Kilpi et al. [17] showed that catechol, methylcatechol, and chlorocatechol could be degraded by the Pseudomonas sp. HV3 via an ortho and meta fission pathway. The degradation o f organic chelators by microbes has also been shown to occur in natural waters and sewage samples investigated [10, 22]. In the experiments described here, changes in the ionic copper concentration in the culture medium during cyanobacterial growth in the presence of citrate or N T A has been investigated. A copper-saturated culture medium (SCM) was used to facilitate measurement of ionic and soluble copper concentrations.

Materials and Methods

Growth The cyanobactefium Plectonema boryanum (UTEX 594--The University o f Texas Culture Collection) was grown in M1 culture medium [16] under continuous light (62 t~E m-2s -~) at 24 (_+2)* C. A 5 liter Pyrex flask containing 4 liters of culture medium was stirred magnetically using Teflon coated bars. The pH was buffered at either 7.20 or 7.00 using 5 m M HEPES (N-2-hydroxylethyl Piperazine-N'-2-EthanesulphonieAcid). Growth was monitored by optical density measurements at 540 nm using a PYE UNICAM SP8-100 UV/Visible spectrophotometer. Although growth in the relatively nutrient-poor M1 culture medium was suboptimal compared with growth in conventional laboratory media for cyanobacteria, its use minimized copper complexation by the medium [ 16] and still supported a trebling o f the biomass concentration within 6 days.

Saturated Culture Medium (SCM) At a fixed buffered pH, the MI culture medium was saturated by the addition of a known amount o f copper. When an excess o f ligand is then added to the system, it will not only solubilize any copper precipitate but also decrease the ionic concentration of the metal in solution. I f the ligand is then consumed (or degraded by any physical or chemical means), the ionic copper concentration will change markedly near the point ofequimolar concentration for the metal and ligand (assuming a 1: l complex stoichiometry). Also, the system will again become saturated with respect to copper, and the drop in the soluble metal concentration can be followed as a function o f the ligand uptake.

Experimental Conditions A, B, and C Three different sets o f conditions were used in this experiment. Under experimental condition A, the nominal total copper concentration (Cut) was 5 x l0 -s M and the ligand concentration as citrate (Cit) was 1 x 10 -4 M. This gave an initial ratio ofligand : metal o f 2:1. When free ofligand, the soluble copper concentration (Cu~o0 in the SCM was 2 x 10 -5 M. On addition of citrate, (CU~o3 became equal to (Cut). Thus, the ligand was able to solubilize 3 x 10 -~ M o f the copper originally precipitated. Copper-free culture medium acted as a control. It should also be noted that HEPES complexes copper and increases solubility in the basic M l medium [16]. Under experimental condition B,(Cut) = 5 x 10-5 M and (Cit) = 2.5 x 10 -4 M, giving a ratio of citrate:copper o f 5:1. This ratio was used to give a higher free ligand concentration than in condition A. Finally, under condition C, the total copper concentration was (Cut) = 5 x 10 -5 M, but the ligand used was NTA at a nominal concentration o f (NTA) = 6 x 10 -5 M.

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Citrate Determinations The Boehringer Mannheim citric acid UV analysis kit was used. In this assay, citrate is first converted to oxaloacetate and acetate by the enzyme citrate lyase. The stoichiometric amount of nicotinamide-adenine-dinucleotide (NADH) oxidized by the product of the citrate conversion is determined by measuring changes in the NADH concentration at 340 nm [6]. Citrate standard solutions were made up in either culture medium or deionized water, but no differences in absorbanee readings were detectable.

Ionic Copper Determinations The ionic copper concentration was monitored using an Orion 92-29A cupric electrode with a Russel SRI AG/AgCIreference electrode. The potential was measured using a Thuflby 1503 digital multimeter with input impedance > 1GfL Standard ionic metal buffer solutions [4] and samples were measured at the same ionic strength (I = 0.02 M). Polycarbonate beakers were used in the titrations.

Soluble Copper Determinations Sample aliquots of 25 ml were filtered through GF/C filter pads (Whatman). The filtrate was acidified with one drop of cone HNO3 and analyzed for copper using a Varian AA 1275 flame Atomic Absorption Spectrophotometer.

Results The results obtained u n d e r experimental conditions A are plotted in Figures 1 and 2. Plectonema showed m a x i m u m rates o f growth between days 2 and 4 in b o t h the control a n d the copper-citrate m e d i u m , although there was a difference in the a m o u n t o f b i o m a s s p r o d u c e d in the 2 cultures (Fig. 1). At day 4, the optical density value o f the control was 0.157 (+0.003), whereas in the coppercitrate m e d i u m it was 0.121 (+0.006). The rates o f growth o f the copper-citrate cultures were approximately 72% o f the controls between days 2 and 3 and 53% between days 3 a n d 4. T h e citrate disappeared f r o m the m e d i u m at 2 different rates. Until day 2, the rate was approximately 0.3/~mol h -1 in both algal cultures. C o n c o m i t a n t with an increase in algal biomass, between days 2 a n d 4, the uptake o f citrate increased to nearly 1.25 # m o l h -1 in the copper-containing m e d i u m a n d 1.65 ~mol h-~ in the controls. The effect o f ligand disappearance on the speciation o f copper in SCM can be seen by c o m p a r i n g Figures 1 and 2. T h e ionic copper concentrations o f 1.6 x 10 -s M r e m a i n e d a b o u t the same until day 2, but as the citrate was c o n s u m e d , the ionic c o p p e r increased, so that on d a y 3 (near the e q u i m o l a r concentration) it was 6.0 x 10 -s M. At d a y 4 the highest value o f 8.5 x 10 -7 M was reached. This was a b o u t 50 times higher than the initial ionic c o p p e r concentration. The consequence o f this increase was to reduce algal growth c o m p a r e d with that in the copper-free treatment (Fig. 1). T h e variation in the soluble copper concentration is also plotted in Figure

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2. Since the m e d i u m was initially saturated wtih copper, the o b s e r v e d decrease in the soluble c o n c e n t r a t i o n o f c o p p e r as the ligand d i s a p p e a r e d w o u l d be expected. T h e initial value o f (Cuso0 = 5.2 ( + 0 . 1 5 ) x l0 -s M r e m a i n e d a b o u t the s a m e until d a y 2, b u t after 4 d a y s d r o p p e d to its lowest value o f 2.0 ( + 0 . 1 ) x 10 -5 M as a consequence o f citrate degradation. H o w e v e r , w h e n after 6 days additional citrate was a d d e d to the m e d i u m to restore the original concentration, the 94% r e c o v e r y in the soluble c o p p e r concentration indicated that the c o p p e r was still present in the culture m e d i u m . T h e results o b t a i n e d u n d e r e x p e r i m e n t a l condition B are plotted in Figure 3. In contrast to the results o b t a i n e d u n d e r condition A, the copper-citrate culture m e d i u m s u p p o r t e d m i c r o b i a l growth as good or better t h a n the controls. At d a y 6, the optical density values were 0.157 ( + 0 . 0 2 ) a n d 0.133 ( + 0 . 0 5 ) for the copper-citrate and the control cultures respectively. Citrate degradation was not detected until day 2, but thereafter, b o t h the control and the c o p p e r - c o n t a i n i n g cultures s h o w e d similar rates o f uptake. During the last 3 days o f the experiment, the average rate o f d i s a p p e a r a n c e was 1.20/zmol citrate h -1. T h e ionic c o p p e r concentration decreased during the course o f the e x p e r i m e n t in response to a slight increase in the p H f r o m 7.24 to 7.38 by d a y 6. As the

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ratio o f ligand : copper was 5: l instead o f 2:1, ligand utilization does not cause an increase in Cu E+(as in treatment A) since the citrate concentration remained above the equimolar level with copper throughout the experiment. Figure 4 shows the variation in the ionic copper concentration, pH, and optical density in a Plectonema culture growing in SCM containing NTA. The experiment lasted 20 days under condition C. Growth became more rapid from day 6 onwards, and reached stationary phase after 16 days. The pH variation followed the same pattern, rising from 7.03-7.34 after 20 days of algal growth. The variation in the ionic copper concentration in SCM indicates that no ligand degradation occurred during the 20 days. The initial ionic metal concentration was 4.4 (+0.4) x 10 -9 M at the first day and remained about the same until day 6. As the pH of the medium increased slightly during the growth, there was a predicted drop in (Cu2+). During the stationary growth phase there was no appreciable change in pH, and (Cu 2§ remained near its lowest value o f 1.3 x 10 -9 M. The decrease in the ionic copper concentration in SCM under condition C (as a result o f the increase in pH) indicated that the cyanobacterium was unable to metabolize NTA. As the free ligand concentration was 1 x 10 -5 M, any small drop in the N T A concentration would be reflected by an increase in

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(Cu2+). The soluble copper concentration (4.9 _+ 0.1 x 10 -5) in the m e d i u m also r e m a i n e d the same t h r o u g h o u t the experiment.

Discussion W h e n establishing correlations between metal concentration and toxicity to microorganisms, the use o f strong complexing c o m p o u n d s such as metal buffers has been widely used. This is because they p r o v i d e a m e a n s o f calculating low ionic metal concentrations in the m e d i u m [1, 9, 24]. T h e disappearance o f citrate from algal culture m e d i a has been pointed out before by Nielsen and K a m p - N i e l s e n [ 19], and Butler and Capindale [8] found that Anabaena variabilis assimilated citrate in b o t h the light and the dark. In the light, the rate o f uptake was a p p r o x i m a t e l y 0 . I 0 / ~ m o l s citrate mg -t dry weight h -1. In this work, it has been shown that care must be taken when choosing complexing c o m p o u n d s to be used in metal toxicity tests involving m i c r o o r ganisms, since i f the c o m p o u n d s are degraded, conditions will alter markedly during the course o f the experiment.

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T h e results obtained u n d e r experimental conditions A and B show how contradictory conclusions could be reached about the ability o f citrate to ameliorate copper toxicity. U n d e r condition A, the disappearance o f citrate in the m e d i u m resulted in a 50-fold increase in the ionic copper concentration. In contrast, u n d e r experimental condition B with a high ratio o f ligand: copper, the ionic c o p p e r concentration in the m e d i u m did not increase because an excess o f free ligand r e m a i n e d to buffer the cupric ions. Therefore, as might be predicted, no toxic effects were discernible in the copper-containing culture. I f on the other h a n d a nonmetabolizable ligand is used to ameliorate copper toxicity, and assuming that no abiotic degradation occurs, then the ionic concentration o f the metal would tend to remain the same or even to decrease if the p H increases as a consequence o f algal growth. T h e results in this paper could also partially explain inconsistent results obtained during copper toxicity tests where organic ligands have been used to complex the metal. F o r example, G u y and Kean [ 13] indicated that while c o p p e r c o m p l e x e d by N T A and E D T A was not toxic to Selenastrum, c o p p e r complexes f o r m e d with citrate and ethylenediamine were. T h e degradation o f m e t a l - N T A complexes in the e n v i r o n m e n t is a controversial m a t t e r [25]. As pointed out by Chau and Shiomi [10], some h e a v y m e t a l - N T A complexes are very resistant to degradation. W a r r e n and Malec

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[26] studied the degradation of NTA and amino and imino-acids in natural water samples. They found that following a period o f acclimatization, degradation occurred within 3 days. Acclimatization is supposed to be the key factor in NTA degradation by microorganisms present in sewage [22]. Nevertheless, in this work, an attempt to acclimatize Plectonema to NTA-containing medium for more than 10 generations (30 days) did not promote any ligand degradation during the subsequent 20 day experiment. Preliminary studies had shown that after growing in citrate-free culture medium for some time, Plectonema seemed to metabolize the ligand more slowly than cultures that had been maintained on citrate-containing medium. An acclimatization period could be observed as an increased lag phase, the length of which could be correlated with the size of the original inoculum. However, once in exponential growth, acclimatized and nonacclimatized cultures showed the same rates of citrate utilization. According to Raboy et al. [20], heterotrophic growth ofPlectonema boryanum on glucose involved both adaptation and enrichment of a fast-growing genotype. Three possible mechanisms could explain the apparent utilization of citrate in the copper-complexed form: (1) diffusion of the complex into the microbial cells; (2) cleavage of the complex at the cell surface followed by an uptake o f the free ligand; and (3) utilization of free (or weakly bound) citrate present in the medium with a continuous shift in the equilibria involved. As citrate is lipid-soluble [7, 8], the diffusion of the complex across the cell membrane would seem feasible. However, since after 6 days the addition of more citrate to the medium to replace that consumed (condition A) caused the total copper concentration to return to 94% of its original concentration, either the copper remains outside the cell, or if copper does enter the cells as a coppercitrate complex, there is a mechanism for its expulsion. In the Plectonema cultures, an optical density value o f 0.130 absorbance units at 540 nm corresponded to a biomass of approximately 30 mg dry weight cell 1'. Assuming a steady rate o f citrate uptake to be 1.25 gmol 1-1 h -1, between days 2 and 4 this algal biomass would have had to exclude nearly 4 mg of copper from the cells, i.e., 13% of its own dry weight. For the mechanism for citrate assimilation proposed in (2) to take place, cleavage of the copper-complex would have to occur at specific cell receptor sites with high affinities for copper. According to Gavis [ 12], in phytoplankton, such receptor sites on cell surfaces were able to bind copper very strongly, with conditional stability constants (K') ranging from 8.6 x 108-5.7 x I0 zo. However, sophisticated kinetic experiments not attempted in this study would be necessary to investigate mechanisms (2) and (3) in any detail, and the possibility of more than one mechanism operating at the same time cannot be ruled out. The bioavailability o f citrate (and thus presumably other organic ligands), even when strongly complexed by metals such as copper, has ecological implications. Consumption o f the ligand by heterotrophs may increase metal toxicity by increasing the ionic concentration, and this can actually coincide with a decrease in the soluble metal concentration, as evidenced by the results under conditions A. Predicting such an event in the environment may also be

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complicated by the realization that any increase in ionic metal concentration will, in part, be dependent on the concentration of the ligand, and thus the relative speeds of its degradation and production. Furthermore, even though the organism under study may be incapable of metabolizing the metal complex, toxicity could be increased by the activity of associated microorganisms which can. These findings also serve to emphasize the care needed and the caution that must be taken in interpreting the results from bioassays. Acknowledgment. Supported by the Brazilian Government (CNPq) scholarship 200 110/80.

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20. Raboy B, Parian E, Shilo M (1976) Heterotrophic capacities of Plectonema boryanum. Arch Mierobiol 110:77-85 21. Ramamoorthy S, Kushner DJ (1975) Heavy metal binding components of river water. J Fish Res Bd Can 32:1755-1776 22. Rossin AC, Perry R, Lester JN (1982) The removal of NTA and its effects on metal removal during biological sewage treatment. Part 1. Adsorption and acclimatization. Environ Pollut (A) 29:271-302 23. Sunda W, Guillard RRL (1976) The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J Mar Res 34:511-529 24. Sunda W, Lewis JAM (1978) Effect of complexafion by natural organic ligands on the toxicity of copper to a unicellular alga, Monochystis lutheri. Limnol Oceanogr 23:870-876 25. Thorn NS (1971) Nitrilotriacetie acid: a literature survey. Water Res 5:391-399 26. Warren CB, Malec EJ (1972) Biodegradation of nitrilotriacetic acid and related imino and amino acids in river waters. Science 176:277-279