metal speciation and microbial growth

8/3/2019 Metal Speciation and Microbial Growth

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Journal of General Microbiology (1991), 137, 725-734. Printed in Great Britain

Review ArticleMetal speciation and microbial growth-the hard (and soft) factsMARTIN . HUGHES~*nd ROBERT . POOLE*Department ojChe mist ry and Microbial Physiology Research Group,2Kings College London, C ampden Hill R oad,London W8 7 A H , U KMicro-organisms require certain metallic elements forgrowth and function (Hughes & Poole, 1989). Theseinclude, for example, the bulk elements potassium andmagnesium, and trace elements such as manga nese, iron,copper, zinc and molybdenum. Essential metals areshown in Fig. 1, the Periodic Table. The need for theseessential metals is, or should be, acknowledged in thecomposition of growth media. However, higher concen-trations of these metals, and the heavy metals such assilver, cadmium, mercury, tin and lead (Fig. l ) , may betoxic. In any study of the biological effects of eitheressential or toxic metals it is imperativ e tha t full accountbe taken of the speciation or chemical form(s) of themetal ion in the growth medium, in the organismsenvironment, and in experimental media such asbuffers; the bioavailability, and hence reactivity, of themetal is critically dependent upon its speciation (Bern-hard et al., 1986; Morrison et al., 1989). Unfortunately itappears that little attention is sometimes given to thesematters, and that this has led to some invalid andmisleading conclusions in the m icrobiological literature .In this review it is intended to survey the factors thatcontrol the speciation of metal ions in media, tosummarize methods for establishing the speciation ofmetal ions in solution and to suggest some simpleguidelines for estimating the bioava ilability of me tal ionsin media. Some comm only-used media will be assessed inthe light of these comm ents and atten tion draw n to errorsthat have resulted from a failure to recognize theimportance of metal speciation.A recent and topical example will serve to illustrate theimportance of speciation effects and the ease with w hichthey are overlooked. It has been suggested in both thescientific and popular press that levels of atmosphericcarbon dioxide could be reduced by sowing the oceanwith tons of iron filings to boost growth of m arine algae(Davies, 1990; Martin et al., 1990). Little mention hasbeen made of the problems of maintaining the iron in aform which is bioavailable. Significantly, Hutn er (1972)cites much earlier work, which demonstrated that the

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fertility of the Sargasso Sea is limited only by theamounts of chelating agents present, which in turndetermines the amount of iron which is bioavailable;merely adding ED TA to samples of the water significant-ly enhanced algal growth.Chemical speciation: factors that affect the availabilityof metal ions in growth mediaAn essential metal is usually added to the growth-medium as a simple metal salt, often a s the aquacomplex[M(H20)6]Xnwhere Mn+= metal ion, X- = anion, andthe coordination number is 6). However, the metal ionmay well be present in the complete growth medium inseveral different forms depending upon the chemicalproperties and concentrations of the anions a nd chelat-ing agents present in solution and on the pH value.Analysis for the concentration of the metal ion in themedium by techniques such as atomic absorption oremission spectroscopy will give the total concentrationand will not reveal the way in which the metal ion isdistributed in its various species. These individualspecies may be coordination complexes differing in theidentity of the ligands in the coordination sphere, theoverall charge (positive, neutral or negative), theoxidation state of the metal, lipophilicity, and affinitytowards b inding sites on cell surfaces. Only one of thesespecies, perhaps representing a minor fraction of thetotal metal ion concentration, may be responsible for thebiological effect exerted by the m etal. Ideally it would bevaluable to know the concentration a nd reactivity of allspecies of the metal ion present in the medium. Somefactors that may determine bioavailability are sum mar-ized below prior to a fuller discussion.Charge on th e complexNeutral complexes are m ore likely than charged ones tobe transported across biological membranes by non-specific routes, and so will exert toxic effects more

0001-6692O 1991 SG M


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726 M . N . Hughes and R . K . Poole

Li Be B C N 0 F N eNa Mg A l Si P S C1 ArK Ca & Ti V Cr Mn Fe Co Ni Cu Za & G e A s Se Br Kr-b Sr Y Zr N b M o T c &I R h A g Cd In Sn Sb Te I XeCs Ba La Hf Ta W R e 0 s I r pt &I H g TI Pb Bi P o At Rn

Fig. 1. The Periodic Table of the E lements showing metals and m etalloids of interest to inorganic microbiologists. Essential metals ormetalloids are shown in bold, toxic metals are italicized and metals used as drugs, antimicrobial agents and probes are underlined.Hydrogen, helium, the lanthanides and the actinides are omitted.

readily. For example, at neutral pH values the non- Precipitationcharged aquamonocarbonato complexes of Cu(I1) andZn(I1) are more toxic than the charged aquacom plexesformed at lower pH values and the anionic biscarbonatocomplexes formed at higher pH values (Stumm &Bilinski, 1972).Complexa onThis effect is probably the most significant overall inassessing the bioa vailability of me tal ions. The bioa vaila-bility or toxicity of a metal ion will vary considerablywith the chemical constitution of the medium and hencethe availability of ligands. Many studies on metaltoxicity are of little comparative value because they hav ebeen carried out using a rich medium or one whichcontains a specific ligand that strongly complexes themetal ion under study. The toxicity of the metal ion willbe greatly reduced under these conditions as it will bestrongly complexed. This explains why Tetrahymenapyrifbrmis can tolerate a 100-fold higher concentration ofCu(I1) in a rich organic medium than in a defined one(Nilsson, 1981), why addition of yeast extract to culturesof Aerobacter aerogenes (Klebsiella pneumoniae) protectsthe bacteria from the usual toxic effects of Cu(I1)(MacLeod et al., 1967), and why yeast extract canalleviate the toxic effects of Ag(1) on the oxidation ofFe(11) by Thiobacillus jerrooxidans (Tuovinen et al.,1985). Bird et al. (1985) have also drawn attentio n to theeffect of growth medium on the chemical speciation ofCu(II), and the implication for toxicity studies.Inorganic anions can also affect the bioavailability ofmetal ions through com plexation, possibly by their effecton the charge on the complex, as described above for thecarbonate ligand. Examples of the effects brought aboutby such changes of speciation include the protectiveeffect of high concentrations of N aC l(2 -4 M) n the toxiceffects of Cd C lz by forming a nionic ch loro complexes(Onishi et al., 1984) and the enhanced uptake ofgermanium by algae at high pH (Yanagimoto et al., 1983)probably due to the formation of anionic hydroxoger-manium species.

Th is is particularly releva nt to toxicity studies, as salts ofthe heavy elements are often of low solubility. Phos-phates in particular are readily precipitated out ofsolution. The insolubility of cadmium and other phos-phates and even of silver chloride does not seem alwaysto have been appreciated ; inely divided precipitates arenot easily detected in the turbid media of growingcultures! This has led to claims for organisms withremarkably high tolerances to cadmium or silver, whenin fact the concentration of th e toxic cation in solutionhas been negligible. Trace essential metal ions may alsobe precipitated or co-precipitated as phosphates.A further example of precipitation is associated withmetal ions such as Cu(I1) and Fe(III), which althoughusually present as aqua cations in acidic aqueoussolution, may also be precipitated as hydroxides oroxides at higher pH values. Certain o rganisms can utilizeprecipitates as a source of metals, either by abstractingthe metal ion directly or by producing ligands thatcomplex and solubilize the metal ion. F or example, th esiderophores solubilize iron(II1) hydroxide. However,problems of precipitation may be overcome by designinga medium either with replacements for anions that causeprecipitation or with appropriate complexing ligands.Thus, phosphate may be replaced by glycerophosphate,while addition of citrate will ensure that Cu(I1) andFe(II1) remain soluble at around p H 7. Note, however,that the bioavailability of these metal ions is thendecreased compared to that of aqua complexes.

Changes in speciation during growthSpeciation of metal ions may change during growth ofthe organism. Exam ples in th e literature include precipi-tation of metal ions as oxalates or phosphates, precipita-tion resulting from a chan ge in pH , an d complexation bya ligand synthesized by the cell. In one case, thespeciation of Cu(I1) changed during growth of Pseudo-rnonas testosteroni from a 1 :3 C u :N H3 complex to a 1 :2


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Me tal ions in microbiological media 727

species due to the decrease in the medium of theconcentration of the am mo nium ion as it was utilized bythe organism (A. Ahm ad, M . N . Hughes, A. M . Nobaran d R. K . Poole, unpublished work).Loss of metal species by binding to surfacesA further general complication should be noted. Certainmetal ions (for example Sn(I1) species) are readily lostfrom solution by adsorption to the wall of the reactionvessel. This phenomenon is only significant whencarrying out experiments at micromolar concentrationsof metal species. It may be alleviated by using plasticvessels.Problems of precipitation: quantitative aspectsSome anions present in growth media may cause theprecipitation of metal ions, particularly the heavy metalions. Inspection of tables of solubilities of inorganiccompounds (for example, W east, 1978) shows that m anycomm on metal phosphates are insoluble in water (such asthose of Zn(II), Fe(II), and Cd(I1)) or only slightlysoluble in water (Fe(II1)). However, the solubility datafor sparingly soluble compounds must be considered interms of the effects of common ions and pH uponsolubility.Solubility product and the common ion eflectFor sparingly soluble salts, those whose solubility is lessthan 0.01 M, he product of the molar concentrations ofthe ions is constant at a fixed temp erature . This cons tant,the 'solubility product', may be calculated from thesolubility of the compound in aqueous solution. Forexample, the solubility of silver chloride is 1-0 5x M:thus the solubility product is 1.1 x 10-lo M? .

MX e + X- Kspc M+l x [X-1MTY pM4++ qXp- K s p$ Mq+]Px CXP-14Precipitation of the sparingly soluble salt takes placewhen the product of the concentrations of the ionsexceeds the value of the solubility product. In growthmedia, the concentration of the anion may be muchgreater than that of the metal ion due to the presence ofanother electrolyte containing the anion . Th e product ofthe concentrations of the ions M + an d X - in solution m aythen exceed the solubility product, so that precipitationof the solid takes place readily. This common ion eBect isillustrated by the decreased solubility of silver chlorid e inthe presence of add ed chloride. T he solubility product ofAgCl is 1.1 x 10-lo M ~ .n the presence of 1 mM chlorideion, the maxim um co ncentration of Ag+ in solution is1.1 x M in the ab-, compared with 1-05x

sence of added chloride.Ksp= 1.1 x = [Ag+]x [ I x

The solubility of AgCl has decreased by a factor of 100due to the presence of only 1 mM-NaCl.Several other factors affect solubilities. Complexformation will decrease the concentration of the metalion and soallow more solid to pass in to solution. Increasein acidity may also increase solubility if the anioninvolved in precipitation is the anion of a weak acid.Protonation of the anion leads to a decrease in itsconcentration, which allows further dissolution of solid.This factor is important for the salts of acids such asphosphoric acid and arsenic acid.Some studies on the interactions of micro-organismswith silver will now be discussed to illustrate theuncertainties that can arise when precipitation is notfully considered. Silver ions, Ag +, are well known to beextremely toxic to micro-organisms (Trevors, 1987),although this toxicity is alleviated thro ugh co mplexing ofAg+ to cellular and medium compon ents, by precipita-tion of silver compou nds and by reduction to the metal(Belly & Kyd d, 1982). Of the 300 p.p.m . of silver nitrateadded to nutrient broth, only 3 p.p.b. of Ag+ wasdetected (Belly & Kydd, 1982). Pooley (1 982) has shownthat Thiobacillus ferrooxidans catalysing the bio-oxida-tion of sulphidic silver ores accumulated silver sulphideparticles on cell surfaces. Significantly, Ag,S is highlyinsoluble, with a solubility product of 1.6 x M 3.Ehrlich (1987), in an investigation of the bioleaching ofsilver from a mixed sulphide ore, prepared an inoculumof T. jerrooxidans 'by cultivation in 9K mediumcontaining M Ag N0 3'. The 9K m edium (Silverman& Lundgren , 1959) contain s 0.1 g KC1 l- ' , an d is 1.34 mMin chloride. From the solubility product of AgCl it maybe calculated that the maximum concentration of Ag+under these conditions is 8.21 x M, mplying thatthe added Ag+ would have been precipitated. Thismedium contains amm onium sulphate at 3 g l - ' , giving aconcentration of ammonium ion of 0.0454 M . Howeverthe pK, for N H,+ s 9-26. At the pH of the medium (2.0)the concentration of N H 3 will be 2- 5 x M, nd willbe insufficient to solubilize the A gCl via comp lexation.Sugio et al. (1981) reported that silver-ion-resistant T .jerrooxidans grew in 9K medium at 5 x M-AgNO,.From their analysis, they claimed that the silver waspresent as insoluble species equivalent to 4.6 x lo-4 Mand soluble species at 2.78 x M. It is clearlyunrealistic to claim that the organism is resistant to5 x M silver! It should be noted that the amount ofsoluble Ag+ in the 9K medium can be calculated as8.21 x lo-* M ,which is very different from the reportedvalue for soluble silver. There may be problems withanalysis. Their suggestion that reduced glutathione


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(GSH ) was involved in the resistance m echanism may beincorrect, as the GSH could function by complexing theAg+ and removing it from the cell wall. A medium forstudying the effect of silver ions on Escherichia coli ha sbeen described by Ghandour e t a l . (1988).Hydrolysis and precipitation of cationsSpeciation of metal cations in aqueous solution changeswith change of pH, and may lead to precipitation ofhydroxides or oxides, unless ligands are present tocomplex the cation. In aqueous solution, divalent metalions undergo hydrolysis to give hydroxo complexes,followed by dimerization through the formation of 0x0bridges and finally giving precipitates.

M(H,O);+ e (H,O),(OH)+ + H +M(H,O),(OH)+ M(H,O),(OH), + H +2M(H,O),(OH)+ (H,O)SMOM(H20);+ + H 2 0

At higher pH values these precipitates will redissolvegiving anionic hydroxometallate complexes (e.g.Zn(OH)i-) (Baes & Mesmer, 1976).The pKa values of the aqua cations of Ni(II), Zn(II),Cu(I1) and Cd(I1) are 9.9, 9-0, 8.0 and 9.9 respectively.Precipitation usually begins to occur about 2-3 pH unitsbelow the pKa value of the aqua complex, provided thatthe con centration of m etal ion is sufficient. As the pH ofsolutions of the divalent transition metal ions is raisedprecipitation occurs in the order of the Lewis acidity ofthe metal ion, taking place first for the smallest cation,namely Cu(I1). The stronger the Lewis acid the moreacidic is the coordinated water molecule. Precipitationeffects are thus more important for the aqua cationsM(H 20) i+ uch as Fe3+and A13+, and will occur at lowerpH values. Precipitation of Fe(II1) begins at pH values2-3.The complexing power of ligands and the likelyspeciationof metal ionsIt is possible to formulate some general conclusionsabout the speciation of metal ions in growth med ia froma consideration of the chemical properties and concen-trations of the metal ions and ligands present. T he abilityof a metal ion to complex a ligand depends upon itspolarizing power, that is upon the charge/radius ratio ofthe cation. A cation of high polarizing pow er is seen bythe ligand a s a centre of high density of positive c harge,and strong interaction takes place between the me tal ionand the ligand. Such a metal ion is described a s a strongLewis acid. Ionic size decreases from left to right acrossthe Periodic Table (Fig. 1). This means th at the alkalimetal cations Na+ and K + interact very weakly withligands, and can be disregarded from a speciation point

of view. The doubly-charged cations M g2+ and C a2 +interact more strongly with ligands than do the c ationsNa+ and K+, but weakly compared to the transitionmetal ions. In general, for small ligands, M g2+ bindsmore strongly than does Ca2+,but this can be reversed.The ionic radii of the transition metal ions decrease insize down to Cuz+,with an increase to Zn2+.Thus, inbroad terms, the strength of interaction between divalenttransition metal ions and ligands increases from left toright across the first transition series, Mn(I1) < Fe(I1) Zn(I1). Cations such asFe 3+ bind more strongly still, in view of their grea terpolarizing power (i.e. Lewis acidity).The binding of some of the toxic metal ions to ligandscannot be compared straightforwardly with that of theessential metal ions in terms of polarizing power. Thecations K + and T1+ have similar ionic radii a nd th e samecharge, but T1+ often interacts about a thousand timesmore strongly with ligands than does K +. Thi s situationarises because TI+ has mo re electrons tha n does K + and istherefore polarized more easily. The thallium cation isdescribed as soft while the p otassium cation is hard.Ha rd cations are usually small, of high charge an d cannotbe polarized. Table 1 classifies metal ions of biologicalinterest as hard, soft or intermediate and also includesinformation on simple ligands (Pearson, 1963). It can beseen that oxygen-donor ligands are hard and sulphur-donors are soft. As a general rule, hard ligands prefer tobind to hard metals an d soft ligands prefer to bind to softmetal ions. But, if both hard and soft metal ions arepresent, then the soft metal ions will often win thecompetition for binding ligands, and will displaceessential metal ions from their sites. Inspection of Tab le1 shows that essential metal ions are hard or borderlinehard, while the comm on toxic metals, with the exceptionof aluminium, are soft. The information in Table 1provides a useful indication of likely combinations ofmetals and ligands. These conclusions are summ arized inTable 2, where the preferences of metal ions for donor

Table 1. Hard and soft acids and basesAcids Bases

Hard Borderline Soft Hard Borderline SoftNa+K +Rb+Mgz+Caz+Mnz+Fe3+~ 1 3 +Co3+

Fez+ Cu+Coz+ Pb2+NiZ+ CdZ+Cuz+ Hg2+Zn2+ T1+Ag+M I )Sn(I1)

HZO Pyridine CN-ROH RSH-COT RZSNH3 szoj-RNHZPorphyrinc1-Poi-so:-


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Metal ions in microbiological media 729

Table 2. Preferences of metal ionsforligand donor groups

Na A1 V Pb Co HgK Fe Cr Ga NiMn T1 c uZna Fe CdMo PbMg

atoms are shown. A knowledge of the polarizing powerof the cations will allow further conclusions to be drawn,as this will show, for example, which of the divalenttransition metal ions present in a medium will wincompetitions for binding sites (this will normally beCu(II), the strongest Lewis acid).The interaction of metal ions and ligands can bedescribed quantitatively by the use of formation con-stants. Indeed it is possible for the detailed speciation ofmetal ions in media to be calculated using formationconstant data.Consider the reversible interaction of a metal ion witha monodentate ligand (a ligand with only one donoratom). A series of stepwise equilibria exist for theformation of metal-ligand complexes of stoicheiometry1 : 1 , 1 :2 , 1 : 3 up to 1 :N, where N is the maximumcoordination number of the metal ion. Each of thesesteps can be described by an equilibrium constant (theformation constant), as shown below (charges and watermolecules are omitted for simplicity).

M + L + M L K1 = MLI/[MI[LlM L + L e ML;!ML2 + L +ML3 K2 = [ML,I/[MLI[LlK3 = [ML,l/[ML2I[LlM L , N - , , + L e M L N KN = [MLNI/[ML(N-

These equilibria can also be described in terms of overallformation constants, the two types of formation constantbeing related by P N = K, x K 2 x K3 x .. KN.M + L e M L P1 = [MLI/[MI[LlM + 2L +ML2M + 3L +ML3 P 2 = [ML21/[M1[L12P 3 = ML31[M1[L13

Compilations of formation constants for a wide range ofmetals and ligands are available (Sillen& Martell, 1971).These are usually determined at fixed temperature andionic strengths. The greater the value of the formationconstant, the higher is the affinity of the metal ion for theligand. Inspection of formation constant data will showwhich ligand out of those available in the medium willcomplex the metal. Alternatively, the data will showwhich metal ion will bind a particular ligand. Theseconclusions will depend upon the concentrations of metaland ligand, as a high concentration of a weakly bindingligand may be able to complex a metal ion in the presenceof a low concentration of a strongly binding ligand.Under such conditions calculations should be carriedout. Selected formation constants are shown in Table 3for metal ions of microbiological interest. It is clear thatCu(I1) binds most strongly of all the divalent transitionmetal ions : it complexes glycine with approximately athousand-fold greater affinity than does Zn(I1). How-ever, the data alsoshow that Cu(I1) cannot compete with

Table 3. Formation constantsfor metals and ligands of microbiological interestValues are taken from Sillen & Martell (1971).

log formation constant ( K , )

GlycineCysteineAspartateHistidine*GlutamateCitratePolyphospha teEDTAMESNTAGlycylglycine8-H ydroxy-quinolineThioglycollicacid

3-44 1.38 3.44 4.3 5.23 5.77 8.62 5.52 3.5 4.8 9.122.43 1.60 3.74 5.94 7.12 8.57 5-841.9 2.05 3.3 4.6 5.06 5.90 7.85 5.453.6 3.3 3.7 4.4 11.4 5.0 5.9 5-0 8.0 6.53.0 3.2 5*5* 3.0 6*5 * 3.0 5.5 6.0* 1.691.7 1.0 9.12 11.0 14.0 13.9 25.1 16.2 18.6 18.8 16.3 5.84 16 16.6 18.30.8 0-7 0.72.1 1.0 7.0 6.4 7.4 8.8 15.9 10.6 12.8 10.5 4.4 11.80.8 0.8 1.7 5.812.0 13.2 13.5 15.0 26.3 13.9 15.2 25.4 17.1 10.6

4.11 6.2 9.33 19.2 9.86 12.27.74 9.3 13.9 15.9 18.3 12.9 1 1 . 1

10.9 14.4 15 15~ ~~* Values of p2 .


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730 M . N . Hughes and R.K. Poole

Fe(III), the conclusion reached earlier on the basis ofpolarizing power. The data also illustrate how therelative affinity of Mg2+ and Ca2+ or ligands can varydepending upon the nature of the ligand. Formationconstants for complexes of the divalent transition metalcations with nitrogen donor ligands increase in the seriesMn(I1) < Fe(I1) < Co(I1) < Ni(I1) < Cu(1I) > Zn(I1).This sequence, discussed earlier, is the Irving-Williamsseries.

Metal ions compete with protons for sites on ligands.Ligands are often weak acids H,L and complex as theanionic species. An increase in acidity will cause theligand anion to be protonated and so lead to an increasein the concentration of the free metal ion. This effect willdepend upon the pK, value of the ligand, and socompetition for a metal ion by two ligands at a fixed pHvalue could well be determined by their pK, values.Complexation will be favoured by lower pK, values.An important measurement in the context of specia-tion is the concentration of free metal ion (i.e. uncom-plexed, usually defined as pM= -log[M]). It is arelatively simple matter to calculate this when only oneligand is present: this would allow, for example, acomparison of the concentrations of free metal ion in twomedia containing different ligands.

When the metal ion is in excess and the ligand bindsstrongly, then [M,] =[M,] - [L,], where f = free andt = total. If the ligand is in excess and is not protonatedunder the pH conditions of the experiment, thenL,=ML + L, [ML] - [M,] and the calculation is alsostraightforward.

If protonation of the ligand has to be considered, thecalculation is more complex, as L, = ML + L + HL +H 2 L + . . . H,L (charges not included). An additionalfactor has to be introduced to allow for the concentrationof protonated ligand species, which will be unable tocombine with the metal. This can be calculated at anypH value knowing the pK, values for the ligand.

The effect of additional ligands in the medium mustalso be considered. If the formation constants for theircomplexation with a metal ion are similar, then theligands will compete for the metal, which will bedistributed among the two ligands. Competition betweentwo metals for a particular ligand cannot be assessedreadily from formation constant data if other ligands arepresent. One of the two metal ions may be bound to amuch greater extent than the other by a second ligand; itsfree concentration is effectively reduced and it loses thecompetition which it may have been expected to win on asimple comparison of formation constants (Ringbom,1963). Such complications emphasize the difficulties of

calculating the speciation of a metal ion in complexmedia and hence the need for physical methods to studyspeciation.

Methods fordetermining speciationThe measurement of the concentrations of individualchemical species is a much more difficult exercise thanthe determination of total metal concentration. Concen-trations of some metal species may be very low (perhapsdown to nanomolar), and the nature of the samples maychange with time. In extreme cases, metal-ligandsystems may take days to come to equilibrium. Further-more, the metabolic activity of the micro-organism mayresult in the modification of the distribution of species,with the production of volatile species or with new metalcomplexes being formed by complexation with ligandssecreted by the cell. Ideally measurements of speciationby instrumental methods should be related to assays forbiological activity, whether it be to study the toxicity oressential functions of a metal.

Experimental methods for determination of chemicalspeciation must involve either techniques that allow thesimultaneous analysis of a number of species or the use ofa chromatographic procedure to separate species fol-lowed by analysis of fractions (Bernhard et al., 1986;Duffield & Williams, 1989). Such analysis of fractionscould involve atomic absorption analysis for the metal,which will not distinguish between the fractions in achemical sense, or techniques that can distinguishbetween different species and characterize them asunique entities. It is possible that some species may notbe detected in these procedures. It is essential, therefore,that the total concentration of the metal in the sample ofmedium be determined, and that a mass balance shouldbe attempted with the sum of the individual species.Failure to achieve a balance indicates that some specieshave not been detected. Unfortunately, determination ofthe total metal content itself may be unreliable, whetherdue to interferences or to poor technique. Othercomplications could result from changes in speciationwith time. Many metal complexes are labile, with themetal-ligand bonds breaking and reforming rapidly.Ideally the metal-ligand bond should not be brokenduring the time needed for analysis or under theconditions of the determination. Organometallic com-pounds (with a metal-carbon bond) are a particularproblem.

It is clear that determination of speciation can be aresearch exercise in its own right, although determina-tion of oxidation state speciation is usually straightfor-ward, and other methods may give information onsomecomponents of the system fairly readily. However, it isunrealistic to assume that major speciation studies can be


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carried out routinely, and it is therefore important thatresearchers are able to make general judgements on thebioavailability of metal ions in the media used in aparticular investigation.Some experimental approaches to th e study of metalion speciation are discussed in the following paragraphs.

In situ methodsThese methods avoid disturbing the reaction system, butare not always particularly sensitive. Often one speciesonly will be analysed for. A number of spectroscopicmethods are useful. For transition metal com plexes, thed-d absorptions in the electronic spectrum will giveinformation about the geometry of the species and som einformation about the ligand environment around themetal centre (Hughes & Poole, 1989), as the wavelengthof the absorption is sensitive to the ligands (thespectrochemical series). Th e major problem with the useof d-d spectroscop y is the lack of intensity of the d -dabsorptions, molar absorptivities lying in the range 10-100 M- m-l. Charge -transfer bands, usually in the UV,are also sensitive to the ligand and are much moreintense. ESR techniques are particularly valuable fordistinguishing paramagnetic species but will also giveinformation about the ligands coordinated to theparamagnetic metal. Resonance Raman and FTIRspectroscopy yield information about metal speciation.The R ama n techniqu e is the m ore useful as it can be usedmore readily for aqueous solutions, since the watermolecule does not absorb strongly in the Ramanspectrum. Raman spectra of complexes at concentra-tions in the range 10-4-10-6 M can be determined underfavourable cond itions.Electrochemical method s (Lund , 1986) also allow thedetermination of metal species in media. Ion-selectiveelectrodes may be used to determine the conc entration offree metal ion in solution, with a detection limit aroundloW6 . A copper-selective electrode has been used todetermine the conce ntration of free copper in studies onthe toxicity of copper towards algae (Sunda & Lewis,1978). Toxicity was related to the concen tration of freecopper and not to the total copper concentration. Thisdoes not exclude, however, toxic effects also being d ue tospecies such as Cu(OH)+ whose concentration will bedirectly proportional to tha t of the free copper a t a fixedpH value. To distinguish between these two possibilities,experiments will also have to be carried ou t at a differentpH value (Guy & Ross-Kean, 1980). Zevenhuizen et al.(1979) have used a Cu(I1)-specific electrode to constructa useful graph illustrating th e dep endency of pC u on thetotal Cu(I1) added to solutions of potential complexingagents. In 0.5% phosphate buffer at pH 7, copper(I1) iscomplexed and m aintained in a soluble complex a t metal

concentrations up to 2 p.p.m. A t higher concentrationsof Cu (II), insoluble copper(I1) pho sphate is precipitated.The addition of sodium potassium tartrate allowedadded Cu(I1) to remain in solution up to 63 p.p.m.Polarographic (usually differential pulse polaro-graphy) and voltammetric techniques give the concen-tration of free metal ion and of complexes which areeasily reduced. Differential pulse polarography hasdetection limits around lo- M. The polarogram allowsthe determination of the concentration of the speciesbeing reduced, w hile the half-wave potential is chara c-teristic of the species, as the redox potential of thecomplex will vary with the ligands present. Strippingvoltammetry is a particularly valuable method for thestudy of metal speciation. It has better sensitivity anddetection limits. One approac h to studying the bindingproperties of a medium would be to titrate the medium

with a solution of the metal a nd record voltammogramsafter each addition. T his would allow the estimation ofthe metal-complexing properties of the medium as aresponse would be obtain ed only when free metal ions oreasily reducible complexes were present in solution(Lund, 1986). Th e binding of m etal ions to microb ial cellsurfaces can also be determined by these techniquessince added metal ions would not be detected until metal-binding sites on cell surfaces were saturated and freemetal ions were present in solution (I. Savvaidis, M. N .Hughes & R. R. Poole, unpublished work).

Separation and analysisThese approaches largely involve the use of ionchromatography coupled to some type of detectionsystem, such as UV-visible spectroscopy, conductivityand other electrochemical methods, atomic absorption oremission spectroscopy, and mass spectrometry. Krull(1986) has critically reviewed these methods.Computer simulationIn theory, speciation of metal ions in microbiologicalmedia can be characterized by computer simulationstudies, based upon formation constant data for themetals and ligands present in the medium. Suchstrategies have been applied to more complex biologicalfluids, such as hum an blood plasma (May & Williams,1981). These calculations app ly to systems in thermodyn -amic equilibria and do not take into account any slowreactions taking place in the medium or microbialtransformations of metallic species.Questionsto be asked about growth mediaI . LigandsAre strongly-complexing ligands present in the me dium ?


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If in doubt look up tables of formation constants.Compare concentrations of the ligand with that of themetal. Is there sufficient ligand to complex all the metal?Will neutral complexes be formed? Is the ligand likely tocomplex strongly enough to make the metal unavailableto the cell? Will the ligands be attacked by the organismsor undergo chemical decomposition under the growthconditions? EDTA appears to be resistant to microbialattack (Pirt, 1975) but will undergo photochemicaldecomposition over extended times, and should not beused in media for growing algae under intenseillumination.

chelating agents, as such, are omitted. Hence the mediaare buffered, in respect to transitional metals, only by theweak chelation lent by histidine and other amino acids.Pirt (1975) has drawn attention to the requirement thatmetal buffers (i.e. chelating agents) in culture mediumshould not be metabolized. In his formulation of achemically defined medium designed to yield biomass [ofAerobacter aerogenes (Klebsiella pneurnoniae)] at up to10 g dry biomass 1- in aerobic culture, the amount ofEDTA included is 1 mol EDTA per mol magnesium plusmetals other than alkali metal ions. Some other recipesfor bacteriological media described at length and innumber by Guirard & Snell(l981) omit chelating agentsas such, and must rely on the presence of caseinhydrolysate, yeast extract (itself a source of metals), Trisbuffer, phosphates and other components for metal ionchelation. Sometimes it is recommended that trace

2. Bufer sWill buffers complex the metal ion? These may well bepresent at concentrations higher than other potentialligands.3 . PrecipitationAre the metals likely to precipitate at pH 6 upwards ashydroxides and/or oxides? Is there the need for achelating agent such as EDTA to maintain solubility? Ifso, note the effects on the bioavailability of the metal.Consider selecting a ligand of lower affinity for the metalion. The metal must be bound strongly enough to remainsoluble but weakly enough to avoid dramatic effects onits bioavailability.4. AnionsAre anions present that may precipitate metal ions oraffect speciation? Phosphate, sulphate, arsenate andchloride could all cause precipitation of cations (andcause co-precipitation of other essential trace elements)or affect bioavailability (e.g. Zn(II), Cd(I1)). If theinclusion of inorganic phosphate in the medium givesprecipitates, glycerophosphate should be considered asan alternative phosphate source (e.g. Poole et al., 1989).Glycerophosphate is hydrolysed during autoclaving andso must be filter-sterilized.5 . Antagonistic relationshipsAre there possibilities of antagonistic relationshipsbetween cations or between anions in the growthmedium? Literature examples include sulphate andmolybdate, phosphate and arsenate, and Ag+ and Cuz+(Hughes & Poole, 1989; Ghandour et al., 1988). The softtoxic cations often exert toxic effects by competingsuccessfully with essential elements.A look at growth mediaHutners (1972) oft-cited review of microbial nutritionmade the important point that inspection of cataloguesof commercial media for cell cultures indicates aprevailing inadequacy in respect to trace elements, . .Trace elements beside iron are seldom included and

element concentrates be prepared in acid to ensure thesolubility of metal ions, but what happens when such asolution is added to the bulk of the medium at pH 7?A defined medium for fission yeast (Edinburghminimal medium; Mitchison, 1970) relies on citrate formetal chelation, but a number of other recipes for yeastmedia contain no identifiable chelator. Thus the com-mercially available (Difco) W ckerhams medium (seeCampbell, 1988), a defined medium for yeast describedby Light & Garland (1971) used for metal-limitationstudies in a chemostat, and a more recent description ofGO minimal medium for budding yeast (Rickwood etal., 1988) include no specific chelator. The relatively lowpH of media for fungi would be expected to alleviate, inpart, metal limitation.In our experience, several media and their constituenttrace element solutions described in the literature cannotbe prepared or autoclaved without formation of a visibleprecipitate, which must indicate the insolubility of atleast a part of the metal ion complement. Which fractionof the trace element solution should be used? Undersuch circumstances, especially when an aim is toreproduce the growth conditions described by others, itmay be prudent to adopt such unsatisfactory media,since improving the medium by adding a chelator maymarkedly influence the growth pattern and physiology ofthe culture. But why should less attention be paid to pFeor pCu than to pH?Apart from solubilizing metal ions, chelating agents inmedia widen the margin between adequacy of supply ofmetal ions and their toxicity in unchelated media.Hutner (1972) cites the example of potential coppertoxicity from its presence in peptones, sea water andcommercial grade chemicals. Chelators can also be usedto advantage when collecting aqueous samples formicrobiological analysis by preventing injury to theorganisms from contaminating metals (Domek et al.,


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1984). On the other h and, chelators themselves ca n betoxic. For example, the growth of Legionella pneumophilain a defined medium is very sensitive to inhibition byEDTA, citrate or 2,2-bipyridyl, but inhibition isreversed by the addition of com pounds of various metals(Reeves et al., 1988).Careful consideration of the composition of themedium is also necessary in studies on metal toxicity tomicrobes (Hughes & Poole, 1989). Com plex ingredientsof growth med ia, such as yeast extract, proteose peptoneand am ino acids, complex metal ions effectively (Rama-moorthy & Kush ner, 1975), explaining the very differentmetal toxicities expressed in different media. Forexample, in the design of a medium selective forEscherichia coli strains containing the relatively zinc-(and azide-) resistant bd-type cytochrome ox idase, up t o10 mM-ZnSO, was required in a rich, meat extract ag ar(where the Zn 2+ was strongly complexed), or in aphosphate-rich defined medium (where the Zn2+ wa sprecipitated as phosphate), whereas in a medium low ininorganic phosph ate 0-2mM-ZnSO, was adequate. Simi-lar results were observed for other m etal ions (Poole et el.,1989). Environmental constituents such as clays andsediment also modify metal toxicity when they arepresent, by adsorbing the metal species (see Gadd &Griffiths, 1978), as will dead cells.However, it should not be thought that whollyinorganic media are ideal for toxicity studies ! Theircomposition has to be considered carefully. Indee d, tracemetals may be removed from growth media in metal-limitation studies by precipitation as insoluble hydrox-ides, phosphates, sulphides or carbonates (Pirt, 1975;Hughes & Poole, 1989). Thus, Fe(II1) hydroxide isprecipitated in Tris/succinate growth medium, pH 8, asTris does not complex Fe(II1) strongly enough to pre ventthis occurring. This explains why Neilands (1980)recommends this medium for growth of enteric ba cteriain severely iron-deficient conditions. Similarly, it is no tsurprising that phosphate protects against the toxiceffects of a range of metals, carbonate against lead,fluoride against aluminium, and chloride against cad-mium. T he lethal effects of cobalt and zinc are decreasedin media prepared with hard water. The presence ofMg2+protects against the toxicity of Ni2+,as shown inthe enhanced toxicity of Ni2 + n lake water com pared tosea water.The complexing properties of buffers in m edium (Tris,citrate and phosphate) has been commented upon.Other, so-called Good buffers (Good et al . , 1986) oftenexhibit negligible metal-binding properties : examplesinclude the sulphonic acids MES, pKa = 6.15; PIPES,pK, = 6.80; TES, pKa = 7.5; and HEPES, pKa = 7.55(Calbiochem, 198 1). However, quantitativ e forma tionconstant data for the interaction of these buffers with all

the metal ions likely to be present in microbiologicalmedia (whether toxic or essential) are not readilyavailable. Use of a buffer that interacts strongly with ametal cation m ay not only affect the bioavailability of thecation but may also result in poor control of pH. If asignificant amount of the buffer is complexed withcations the hydrogen ion s will be unab le to compe te withmetal ions for sites on the buffer and the pH will fall.This in turn may lead to decreased growth rates.Post-scriptIt is hoped that this mini-review m ay m ake some smallcontribution to the arrival of that turning point in thedevelopmen t of inorganic microbiology when attentionturns from consideration of the total concentration ofmetal in a biological situation to an apprec iation of metalspeciation.

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