IYIICROBIAL
`€COLOGV
`
`301
`
`Contents
`
`279 M. M. Roper and K. C. Marshall
`Effects of a Ciay Mineral on Microbial Predation and Parasitism
`of Escherichia coil
`
`291
`
`303
`
`319
`
`331
`
`345
`
`351
`
`361
`
`3?3
`
`381
`
`J. D. Buck
`Comparison of in Siru and in Vitro Survival of Candida albicans
`in Seawater
`
`G. M. Gadd and A. J. Griffiths
`Microorganisms and Heavy Metal Toxicity
`
`S. G. Berk. A. L. Mills. D. L. Hendricks. and Fl. Fl. Colwell
`Effects of Inge-st:ng Mercury—Containing Bacteria on Mercury
`Tolerance and Growth Rates oi Ciliates
`
`C. I. Maytield and W. E. Inniss
`Interactions Between Freshwater Bacteria ann
`Ankistrodesmus braunni in Batch and Continuous Culture
`
`D. C. Coleman. C. V. Cole, H. W. Hunt, and D. A. Klein
`Trophic Interactions in Soils as They Affect Energy and Nutrient
`Dynamics. I. Introduction
`
`M. A. Herzberg. D.A. Klein. and D. C. Coleman
`Tiophic Interactions in Soils as They Afiect Energy and Nulltefll
`Dynamics. II. Ptiysiotogical Responses of Selected
`Rhizosphere Bacteria
`
`H. V. Anderson. E. T. Elliott. J. F. McClellan. D. C. Coleman,
`C.V. Cole, and H. W. Hunt
`Trophic Interactions in Soils as They Affect Energy and Nutrient
`Dynamics. lll. Biotic Interactions of Bacteria. Amoebae and
`Nematodes
`
`D. C. Coleman, H. V. Anderson. C. V. Cole. E.T. Elliott,
`L. Woods, and M. K. Campion
`Trophic Interactions in Soils as They Affect Energy and Nutrient
`Dynamics. IV. Flows of Metabolic and Biomass Carbon
`
`C. V. Cole, E.T. Elliott, H. W. Hunt. and D.C. Coleman
`Trophic interactions in Soils as They Affect Energy and Nutrient
`Dynamics. V. Phosphorus Transformations
`Index to Volume 4
`
`Indexed in Current Contents
`
`MCBEBU "{4)279'337 (1973)
`ISSN: 0095-3628
`
`Springer-Verlag New York Heidelberg Berlin
`\lOLUlYl€4/l‘lUlYlB€R4/1977-78
`
`301
`301
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`ARGENTUM
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`MICROBIFILECOLOGV
`
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`tSS.\l: ltlJ‘).‘i-_’it).‘_t»t
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`302
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`

`l\-Iierobial
`
`I-‘.eolog_\- -l:3t}3—3]'r‘t]lJ7t<t
`
`Microorganisms and Heavy Metal Toxicity
`
`Geoffrey M. Gatltl and Alan J. Gritliths
`Department of t\licr'obio|ogy. L.li1ivc|'sit_\' (‘allege C'artlilT. Newport Roatl. Cartlili. Sntitlt Wales.
`Cl-‘Z IT.-\
`
`Abstract. The environmental and microbiological factors that can influence
`heavy metal toxicity are discussetl with a view to untlerstanding the mecha-
`nisms of microbial metal tolerance.
`lt
`is apparent that metal toxicity can be
`heavily intiuencetl by environmental conditions. Binding of metals to organic
`materials. precipitation. cornplexation. and ionic interactions are all impor-
`tant phenomena that must be COI'lSlL'l€l't‘.‘Ll carefully in laboratory and field
`studies.
`it is also obvious that microbes possess a range of tolerance mecha-
`nisms. most t'eaturing some kintl of detoxification. Many of these detoxifica-
`tion mechanisms occur widely in the microbial worltl and are not only specilic
`to microbes growing in metal-contaminatetl environments.
`
`Introduction
`
`The heavy metals constitute a group ot’ about 40 elements with :1 density greater
`than live (80). A Feature ol‘ heavy metal phy_~.io|ogy is that even though many of
`them are essential for growth. they are also reported to have comprehensively
`toxic etiects on cells. mainly as a result oi" their ability to denature protein
`molecules. There are. however. many reports in the literature of microbial
`resistance to heavy metals. The phenomenon of Inicrobial resistance is of some
`limtlamental importance and is particularly relevant to microbial ecology. espe-
`cially in connection with the roles of microbes in polluted ecosystems and in the
`reclamation ofrnetal-contarninatetl natural habitats. It is also important to under-
`stand the mechanisms of microbial tolerance because of the extensive use of
`
`is the
`It
`some metals and metal compountls as fungicides antl ttisinfectants.
`purpose of this review to examine the nature of the interactions between
`microbes antl heavy metals and to attempt to clarify the processes. both environ-
`mental antl microbial. underlying resistance or tolerance.
`
`tlt}95—3h2ti-'78-"tt(ltI<l—t}3tl3 ‘iltlltlti
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`
`lSn\-'i1'onn1ental Influence on Toxicity
`
`Bf.l'.‘(ll:".F.[£.,’ to .-L“ttt'ft’tut:ttt’n!ul ('rm.\'tt'tttwtt_s
`
`One of the Inost important factors that determines the biological availability of a
`metal in a system is its binding to other environmental constituents. If a metal is
`wholly or partially removed by binding. a decrease or complete disappearance of
`toxic effects tnay result.
`[11 the soil. metals can be bound strongly by organic materials such as humic
`and fulvic acids and proteins. Humic acids are especially important and it has
`been stated that practically every aspect of the chemistry of heavy metals in
`soils, sediments. and natural waters is related in some way to the formation of
`complexes with humic substances t" |3..'33. I00). ln some cases metal availability is
`limited by l'Il[1Lllt‘Ig to humic acids to such an extent that deliciency symptoms
`may result in plants growing in such soils I301. The hound metal is often difficalt
`to I'emove. and even in ve|'y sandy soils extraction can require vigorous proce-
`dures (46).
`
`Clay particles can also bind metal cations. and some metals such as zinc may
`enter the crystal
`lattice and become unavailable to organisms l4(1.l I8}. Clay
`particles can reduce the toxicity of certain m.eta|s toward microoI'ganisn1s.
`Experiments with cadmittm have shown that the clay minerals. montmorillonite
`and kaolinite. protected ceI'tain bacteria. actinomycetes. and filamentous fungi
`from the inhibitory effects of cadmium. This protective ability of the clays was
`correlated with their cationvexchange capacity tCF.Ct as it appeared that
`the
`greater the CEC. the greater the amottnt of cadmium absorbed (8,9).
`In aquatic habitats.
`tnetals such as zinc and copper can be bound and
`removed from the water by organic sediments. which effectively reduces the
`total metal ion concentration in soltttion.
`It has also been reported that certain
`oxitlized sediments can bind up to 96“? of added zinc tll.] I4}. Certain waters.
`especially those in moorland areas. contain considerable amounts of humic
`substances and. as in the soil environment. a variet_v of metals can be bound
`including zinc. cobalt. and mercury (13.83). Because of such binding in aquatic
`systems. it has been shown. that toxic effects of certain metals on microbes can
`be decreased (70).
`
`In certain polluted aquatic habitats. metals such as mercury can be trapped
`and hotmd by petroleum. and since many oil-degrading microbes are active at the
`oil—water interface. such removal is of obvious ecological significance in that it
`may enable the growth of metal-sensitive organisms.
`In one study of an oil-
`polluted maI'ine habitat. it was found that the concentration of mercury in the oil
`was 4000 times |1ig|1et' than in the sediment and 3tltl.t)0(l times l1igheI'tl1:1t1 in the
`water samples. Many of the oil-degrading organisms isolated were found to be
`Inerctiry resistant. but
`the extent
`to which the mercury removal
`influenced
`resistant behavior was not determined tl 12).
`Compounds which can chelate metals. for example. citrate. cysteine. gluta-
`mate. and EDTA. can also have a significant effect on micI'obial responses when
`included in growth media. Toxic effects of copper on /-l.t’l't'JtlJ(.‘t{':'t’."
`t.'m'u;m:v.Iv.\-
`were prevented by the addition of yeast extract and cysteine. and this was
`attributed to the ability of these compounds to bind copper {E36}. Similarly. toxic
`
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`

`l\'licroorg'.misms and I-lca\'y Metal Toxicity
`
`3tl:'\
`
`ellects oicopper ascoI'bate to .S‘crrr.'.'iu .lHrl'."(’(‘.\'t'{’H.\' weI'e relieved by the addition
`ofcopper-chelating agents (1 I9}. Citrate and EDTA can reduce toxicity cut‘ some
`metals to A. m'i'n;.:ciii-.s'. In the presence of citrate this organism could grow in
`200 ppm cadmium. but ifglncose was substituted for the citrate an "infinite lag"
`resulted (82). A study of mei'cuI'y toxicity using the protozoan Tm.-'ril'i_\‘iiiciiri
`_.v:yi‘.{',t'i.~Hiii.i' revealed that toxic levels in a complex medium containing proteose
`peptone and liver digest were about 40 times higher than those observed in a
`simpler medium (44). Copper toxicit_y to Ara.-."mwic.' c_w"iii:t'ric':i has also been
`shuw|1 [u be 1‘edttCCLl by the addition Di‘ b‘D'liA {.75}.
`in Inedia without complexing agents. toxicity Inay be pronounced. This is the
`case with ("iiirii-viii: pi'i'mnido.m where a copper concentration as low as 5 pg 1 "
`was toxic (99). These atIthoi's tnade the interesting suggestion that copper is not
`ordinarily present as the ionic form in nattIt'al waters but is usually complexed
`with organic materials such as polypeptides.
`In brewery systems it has been commonly found that metals do not have the
`same effect on fermentation in simple and complex media 06.113].
`111 general.
`termentation is relatively unaffected by metal additions when tested in complex
`Inedia. For example. in tnalt wort and molasses. a brewing yeast was unai'fecled
`by 30 to 40 ppm of copper. but the same yeast. when grown in a simpler sugar
`and mineral salts medium. was completely inhibited by l to 2 ppm copper t
`I I3).
`in activated sludge. metals can be adsorbed by organic matter and a plant
`may be able to withstand quite high additions of metals without serious loss o|'
`activity. lt1 one study. for example. it was found that protozoa were unaliected
`by copper concentrations up to 5 ppm and the reduction in overall e|'ficieacy was
`only 4"}? even at concentrations oi" 25 ppm t69].
`It should be mentioned that although binding to environmental constituents
`usually reduces [u_\it,;i[y_ in sol1‘IC cases toxic action still results even when there
`are no free metallic ions. This was found to occur in certain complex media with
`mercury. Although there were no free mercury ions until the total concentration
`was [60 ppm. a total concentration of It} ppm was found to inhibit the growth of‘
`many aquatic bacteria t'i'4.84). It was suggested that either the ions exerted their
`toxicity and entered the cell as organic complexes or bacterial cells competed
`successfully with the growth media for the bound ions (84).
`Of couI'se. in some cases metal complexes are more toxic than the free metal.
`This was shown for .S'm_nl'i_\'l'omc-:'ii.x i‘.‘:'H't’H.\' using 8-OH~quino|ine (l0"‘ M] and
`ferrous ions t l{}'”"‘ M). When these substances were applied separately. no toxic
`action resulted but a mixture completely inhibited growth £3). Although certain
`metal complexes are more toxic than the free metal. they are o|'ten volatile and
`may disappear from an environment. ’1"liis is the case with methylated derivatives
`of niercury.
`
`pH
`
`pH can have a considerable effect on the availability and thus the toxicity of
`heavy metals in a given enviI'onment.
`in general. at an acid pH metals exist as
`free ionic cations. but at an alkaline pH the ionic cations precipitate as insoluble
`hydroxides or o,\'idcs. Most heavy metal hydroxides are insoluble. Copper. at
`
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`

`filth
`
`C}. M. Gtltltl and .-\.
`
`.|. Cxrilliths
`
`about I ppm. disappears from solution at any Eh when the pH is greater than (1.
`and at an Eh below +200 mV when the pH is less than 6. Zinc precipitates as zinc
`hydroxide. ZI1i()H)g. above pH 5. and above pH 8.5 it forms zincate ion which
`can be precipitated by calcium ions {E7}. The pH at which precipitation occurs
`varies among different metals and among oxidation states of the same element.
`Some metals. for example. coppeI'. have more than one valence state and the
`oxidized slate is favored by high pH. The hydroxides ofthese oxidized states are
`less soluble than those of reduced states and precipitate at low pH values [I9].
`Thus low pH generally incI'eases the availability of metal ions. whereas high pH
`decreases availability. This has been illustrated in soils. where in very acid
`conditions toxicity due to an abundance of iron. Inanganese. copper. and zinc
`can be removed by adding lime which raises the pH ( I946). The inlluence of pH
`on availability is also illustrated by a study ofthe toxicity ofeopper complexes to
`C‘t.'mt'."tt'r.'
`tm'l.-'.\-. Complexes with amino acids were less toxic at pH values of 7
`than at lower pH values. It was suggested that at the lower pH. the stability ofthe
`complex was lessened. releasing free copper ions (7).
`One aspect of pH and metal toxicity that should be mentioned is the occur-
`rence of metal-polluted mine streams which are often very acidic t'_'{L.‘-3.54.85}.
`The low pH can arise from I'tIn-in fro in acid soils and also front the mict'obio|ogi-
`cal oxidation of sulfide-containing minerals by lhiobacilli. for example. I'cle:tsit'Ig
`sulfuric acid. The low pH can then release other metals such as lead. manganese.
`iron. and zinc into solution (H4). The biology of such acid mine streams has
`received some study and it is evident that in spite of metal toxicity. there is still
`much microbiological life in the form of algae. bacteria. yeasts. protozoa. and
`fungi tl4.1(a.39.| 14}. Some bacteria are very tolerant indeed. such as the thioba—
`cilli which can tolerate high concentrations of copper and zinc tlll7'.l I4) and a
`P.s'c'mt’.«nnmtn.s' species which is highly tolerant of copper. manganese. and cobalt
`{(18}.
`It
`is not clear. however. whether the low pH is of any advantage to these
`organisms in reducing toxicity. or whether they are just extreme examples
`surviving by means of other tolerance mechanisms.
`
`hm .f.’.'fc‘t'(t:'!ff)t'l.\'
`
`ions can be markedly affected by the
`The biological activity of heavy metal
`presence of other ions. Cations such as magnesium and calcium can often reduce
`heavy metal
`inhibition. Toxic effects of nickel. cobalt. cadmium. zinc. and
`manganese to .l;‘.\'t-i'm'it'lit':t (‘tiff were decreased in media with a high magnesium
`content. The toxicity of nickel and cobalt to A. t'l(’t"{J‘L’{’lt(’.\'. A.\';:ct'_cillit.t' tlf_L’:’t'. and
`C‘.
`ttrt':‘l.\- was also diminished by Inagnesium. For all four organisms. using
`radioisotopes of nickel and cobalt. it was found that the high magnesium levels
`reduced the amounts of nickel and cobalt taken up by the cells (ll. Inhibitory
`amounts of manganese. iron. cobalt. nickel. and copper to Bm'iHn.t'
`lit‘.-'tc*tt.{t‘E:t'nii.s'
`could likewise be antagonized by the addition of magnesium to the medium.
`although toxic concentrations ofzinc and cadmium were less effectively reduced
`(421. Similarly. calcium and magnesitim have been shown to reduce the toxicity
`ofcadmium toward .4.
`.ttl_:..’(’." {fit}. It has also been found that the iron concentra-
`tion in a medium had a detoxifying effect on copper to the alga C‘ltlm't*l.-‘rt
`
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`

`l\-licroorganisins and He-.n-'_\' Metal 'I'oxicit_\-'
`
`3ll'I'
`
`p_\‘iwioi.:fu.m. At the iI'on concentration tised in algal growth media. copper may
`be adsorbed to the negatively char_e.ed micelles of ferric hydroxide (981.
`Anions are able to reduce metal toxicity by precipitation. The hydroxyl ion
`has already been mentioned with 1'egard to pH. Besides this. phosphate. thiosu|—
`fate. carbonate. and bicarbonate ions can form pI'ecipitales with heavy metals
`depending on their concentrations and the pH of the solution. The addition oi‘
`such anions to growth media often redttces metal toxicity (89).
`Sulfide. from hydrogen sulfide. can also prevent toxicity in niany cases by
`precipitating the metal as an insoluble sullide. Organisms that grow in oi‘ produce
`high sulfide concentrations. e.g.. i)c.rrrif!Ea-i'lii-in r.-‘es-iil_fiii'i’(':m.t'. have been shown
`to be unaffected by lai‘ge additions of heavy metals H05). This meclianisrn of
`tolerance will be discussed later.
`
`Sometimes toxicity ofa metal is increased when other ions are present. In the
`case of the alga C'l.=lom"ln i'iii',erii'i'.s. an asymmetric t'espi1‘alory response occurs
`when lluoride and copper ions are applied jointly: respiration is completely
`inhibited by a mixture. but individually the ions have little effect (451.
`Mixtures of heavy metals often exert a more pronounced effect on microorga-
`nisms. e.g.. a mixture of copper and silver ions on algae {H7}. but this can be
`accounted for by simple additive effects tllr-ll. Synergistic effects of metals on
`microbial growth and survival have. however. not received much attention.
`
`l\’IL“L'lI1II1iSt11S of l\*Iierobial Resistance
`
`I'I_\‘ili'rJ§_:('ii
`
`.S'.ulficl'(’ Pi'm."Ht'IioiI
`
`Microbial hydrogen sulfide production often has significant effects on metal
`toxicity since most heavy metals form insoluble sulfides with H33. Consequently.
`H3S—produeing organisms often exhibit tolerance to heavy metals.
`In yeasts. metal tolerance has often been linked with H33 production. and the
`importance of such H3S~pro¢.|ucing yeasts in nature has often been documented
`(28). Copper- and meI‘ctIry-tolerant strains ofSm'('liui-.«:iii_\':‘es t‘c*i‘c*i-i_s'im’ produce
`more H35 than do their nontolerant parent strains. the metals being precipitated
`as insoluble sulfides (5832). Colonies of copper—to|erant strains appear black or
`dark brown in the presence of copper and contain much copper sulfide (Si.
`Electron micrographs have shown that the copper sulfide was chiefly deposited
`in and around the cell wall (3.457). Similar precipitation. thought to be copper
`sulfide, has also been observed in the fungus Perla -.‘ni‘llaim'i' (Bil.
`Bacteria that are capable of H38 production may exhibit tolerant behavior.
`The sulfate reducer l).~.i'iil_;‘in'ii'2i'io cic.t'irlfiri-it-riii.r produces H33. grows in high
`sullide concentmtions. and may be unaffected by the addition of high concentra-
`tions of heavy metals (105). Likewise. in anaerobic digesters sulfide reduces the
`toxicity of most heavy metals. the H33 again resulting from bacterial reduction of
`sulfates (62).
`
`It has been noted that in some cases sulfide-producing organisms can protect
`sensitive organisms from the toxic effects of metals. When I). tr.-’e.-.'.mf,liri‘i'r‘riits was
`grown in mixed culture with a melal—sensitive slI'ain of P.wia:imiimui.r rl(’l’ll;.,’l'-
`.’lrJ.\‘(l. the latter organism could tolerate higher concentrations of mercurials than
`
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`

`308
`
`C}.
`
`.\-l. Ciadtl and A.
`
`.|. Ciriliilhs
`
`it could in pure eLIltuI'e. Results indicated the H._.S produced by the sulfate
`t'etluceI' protected the pseutlotnonad (I0). 5'.
`tHt.f‘£’t.'.\' was also futlnti to exhibit a
`higher tolerance to mercurials when grown with E. roll. The protective effect of
`the if. en.-‘I’ was partly clue to H._.S protluetion and the extracellular protluction of
`gltltathionc HUI).
`
`.-“’t‘mt"trt't.='mt of (J;j::rtrii'c' ('c:tit;:::iiitt:’.\-
`
`As previously mentionetl. binding or chelation ofa metal by organic substances
`present in the microbial environment can InaI'keL'l|y affect metal toxicity. In some
`cases the microot'ganisms themselves are capable of protlueing such substances
`which may reduce toxicity. Citric acid, which can be protlueetl by many yeasts
`and fungi. can reatlily chelate metal ions such as copper and may protect a fungus
`from copper poisoning (87). A. it.",L:.:»r may be protectetl from the toxic effects of
`lead in this way tl2U). Oxalic acid production has been linketl with coppeI'
`tolerance o|' certain woou.l—rotting fungi. The fungi Cu:-rnH‘tr.i'
`,:mlu.m-i‘s.
`.S‘c'i-pith:
`i’mulir_\-omits. and t'-’m'i:.- nmnn‘:'ul:.- all produce copper oxalate crystals when
`grown on synthetic media containing copper sulfate (87). Oxalie acitl is itselfa
`toxic substance. but metals such as copper and iron. when tJtJI‘l‘l]'.li€Ni3t.i Wllh It.
`remove its toxicity while losing their own. This has been observed with the oxalic
`llCtLi-]‘)|‘()L'il.ICiI1g fungus £'iid'n.rh.='c.- ,mmt.s'i‘r.w't-:.- {E9}.
`Some meI'cury-resistant mutants of Sm'n"i:.u't=:in_~.’c-ex r-w'cw'.s'.-'m= were found to
`have a reqt1i1'ement for methionine. Evidence suggested that
`this compound.
`itself an eflieient chelating agent. was used by the yeast to produce a “simple.
`Llifftlsible substance" which acted as a Lletoxifyiltg agent
`toward n'tercuI'ials
`(93.94).
`
`Intraeellttlat‘ organic substances can also tletertnine metal tolerance. This was
`fouml to be the case with mercuI'y-tole:‘a1tt A. i.=t'_uc*r where a pool of intracellular
`sulfliytlryl eotnpoumls complexed mercury and alleviated its toxic et’t‘ects tot.
`Such sullltytlryl compotttuls have also been observed in eoppe:'—resistant yeasts
`t56.72l.
`
`Upmftv mm’ .-"u‘(‘tiiitm’::tt’wt
`
`Microorganisms possess mechanisms by which metal cations can be taken up
`and accumulated from theirenvironment. Although the amounts oftnetal cations‘
`needed for growth reqttirements are generally small. stteh uptake meeltanisnts
`can still operate at higher concentrations and can inlluence metal toxicity. towartl
`both intlivitlaal aectlmalating or_aanisnts and the nticrobial community. In gen-
`eral. i|'a metal is wholly or partly removed from it system by microbes. toxicity
`may be I‘etl1.teetl. This kincl of detoxification is similar to that which occurs if a
`metal is removed by environntental constituents.
`There appear to be two main types of metal uptake by organisms. The first
`involves noitspeeitic binding. of the metal to cell sttrfaees. slime layers. extracel-
`lular matrices. ete.. whereas the second involves ntetabolism-dependent intracel-
`lulat' uptake.
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`l\licI'oorgi|ni~an.~ and l‘lC'.t\'_\' Metal 'I'osiciI_\'
`
`Ml‘)
`
`The Iirst type can be important since most heavy metals can be adsorbed onto
`the stir|'ace ot‘ rnicrobial cells. both living and dead. and. in fact. the addition or‘
`dead bacterial cells to copper~inhibited laboratory cultures of bacteria is ellective
`in I'educing toxicity (39). With metals such as copper. cadmium. and zinc.
`complexation is possible with polygalacturonic acid. an important constituent of
`the ottter layers of bacterial cells. The Inetal can be I‘ecovered from sttch
`complexes and the polymer regenerated (49). ln yeasts. metabolisnrintlependent
`sttrfitce binding is often to anionic groups of two species. polyphosphate and
`carhoxyl. and such binding is rapid and reversible 09.88}. Isolated cell walls of
`.5‘. u:’l'et'l'.~'inc have been shown to bind their own weight of mercury to “high-
`at'linity" sites (Tl). In the fungus i'\='miv;.s'um_t-port:
`i'ir.s'iii_,’i'r.tii. surface binding of
`zinc to negatively charged groups on the ltyphal stn'i"ace was rapid. reversible.
`and tentperature independent [8I]\ as was the binding of zinc to C".
`.*m'li's (31).
`Similar binding ofcobalt by .-'\-’t'nms-_.«mi'u (‘l':‘t.\‘.\‘(t was also rapid and accounted for
`30¢?’
`to 405-’? of total metal
`ttptalte (l[()l. Surface binding of metals may be
`especially important in sliIne—protlucing organisms or those organisms that grow
`in an extracellular matrix. the extracellular material acting as an “impermeable
`barrier." For example. zoogleoal bacteria. which are comtnon in aquatic liabi-
`tats. can survive and grow in the presence of high concentrations of heavy metal
`ions. the metals being adsorbed and precipitated within the extracellular matrix
`(37). Such organisms are el'|'ective in removing toxic ions from solution and are
`thus of great ecological signilicance in that
`they may allow more sensitive
`organisms to survive in a mixed community. However.
`they may present a
`hazard. especially if eaten by other organisms.
`The second type of Inetal uptake. Inetabolism-dependent transport. has been
`studied in various algae and yeasts ll{1.3|.3'-_’.38.75.76lt bacteria ll8.25.7hl. and
`fungi (81.1 10). A detailed account of the physiology of metal uptake will not be
`given here.
`It
`is. however.
`important to note that
`in most of the organisms
`studied. the amount of metal bound by surfaces is insignilicant when compared
`to the amounts that can be taken up by energyvrequiring processes t l8.3l.?5.8ll.
`It should also be mentioned that
`Inost studies of metal uptake have been
`concerned with low mieronutrient concentrations as opposed to higher concen-
`trations where.
`in order to survive. an organism may have to express some
`mechanism of tolerance.
`
`At higher concentrations. intracellular pI'ecipitation of the metal may occur
`after uptake. This itself can be a means of detoxification since the metal
`is
`compattmentalized and may be converted to another more innocuous form. For
`example. certain yeasts are capable of precipitating thallium within the mito-
`chondria as thallium oxide. The oxide may subsequently be discharged front the
`mitochondria and excreted from the protoplast. This is termed oxidative detoxifi-
`cation t63.o5). "Copper containing particles" have also been observed in C".
`i.*.'i‘i"i'.s' after growth in high copper concentrations (55). In another study. copper
`was used as a "stain" for electron microscopy since it was found that this metal
`attaches to the nucleoli and chromosomes of yeast (64).
`lo the algae S('('.'t‘(’cll{'.\‘-
`aura‘ r.'t‘iirr'li:i‘iiii.- and .S't'tvieu'i:awiit.«_\- m'trau'ti's, precipitation of copper has been
`observed within vacuoles and nuclei when grown at higher cencentrations. At
`lower copper concentrations. electron-dense bodies containing copper were
`concentrated only in the nucleus (33). Precipitation of mercury in electron-dense
`
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`

`3|!)
`
`('3. M. (Judd and ./\.
`
`.l. Grillillts
`
`bodies has also been noted in the hyphae ofthe fungus ("i‘ir_\'.i'os,-mi-iii:i ptH'H'h'H'l'.’l'H
`(ll5l. Likewise. electron-dense bodies. presumed to contain zinc. have been
`observed in the fungus New'o.m.=uspoi-.«i 1‘fl.\'f!i_'fi:’('!(l' after growth in a medium
`containing zinc (SI). There is also evidence for the intracellular deposition of
`iron. as ferrous stilfide. within the sulfate-reducing bacteria .tJc.ml_;‘E:i'ihi'io and
`D(’.\'.‘tUi'J.’i.'!lttl{‘Hflt!ll (52). Crystals of a copper compound thought to be sulfide
`have also been observed in the mycelium of the fungus Porici
`i'uii'i'riiiii.i (8?).
`Decreased uptake or impermeability to a metal may be a means of resistance.
`Decreased uptake is the case with cadmium and .5".
`t’l':'.F."(’H.\' where a resistant
`strain takes up less cadmium than the sensitive parent strain (2l.23.60.l08t.
`There is evidence that the genes for such cadmium resistance are tocated on
`cxtrachromosornal R-factors (plasmids) which are discussed later.
`lmperineability is one explanation of tolerance for those fungi capable of
`growth in high copper concentrations. Pmii:'illii.rui and ;l.s-,im'_Lrillii.s- species have
`been found which can survive in saturated copper sulfate ((2.97).
`
`.-".'I:*n:.d Tm:i.\jfiii'iitritioii
`
`The biological transformation of certain heavy Inetals is an important process
`that can occur in many habitats and be carried out by a wide variety of
`microorganisms. chiefly bacteria and fungi. Metals cannot be broken down into
`other products but may. as a result of biological action. undergo changes in
`valence andior conversion into organotnetallic compounds. Both processes catt
`be considered to be detoxification mechanisms since volatilizittion and I'emoval
`
`of the metal may result.
`Transformatiotts involving changes of valency have been chiefly studied with
`ntercury. Several types of bacteria and yeast have been shown to effect
`the
`I'eduction of cationic mercury tHg”'"l to the elemental state (I-lg") (l?.S9.6Tr'.9l}.
`This usually results in the mercury being volatilized from the mediunt. Bacterial
`mcrcttry resistance is closely linked with this volatilizing ability tl{l9}. The
`oxidation of elemental mercury to its cationic foI'm can also be mediated by
`microbes.
`l3acteria shown to have this ability include E. ruli. P.s'ciit{oiiit:iiri.i'
`_fl!tt)l'(’.\'('t’lt.\'. J”. m’i'H,::i'iin.s.rt. Citi'tH'::.'('!:*t' $11.. Br(('iffti.\'
`.\‘t.t.-"m't'.r'.\'. and B. mc*{.:ritlt('t'-
`lam (47'l.
`
`"Transformation of certain metals into organotnetallic compounds by methyl-
`ation is also an important detoxiiication mechanism. Metals that have been
`shown to undergo tnethylation include mercury (4350.86). lead (34.1 16]. cad-
`mium. and tin (48). Methylation can be al'l'ected by the environmental factors
`mentioned in previous sections and also by the numbers and species of microbes
`present in a particular habitat. Methylation can be catalyzed by a wide variety of
`microorganisms: both aerobic and anaerobic bacteria (48.|(l6.l I 1.] I6). yeasts.
`and fungi (24.34.87.l|l}. Although products of ntethylation may be more toxic
`than the free metal. they at'e often volatile and can be released into the atmo—
`sphere. This is the case with mercury and its methylated derivatives. methyl and
`dimethyl mercury (86).
`Organometallic compounds can also undergo microbiological and chemical
`degradation which may result in the metal being Ieliberated. again usually in at
`
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`

`Microorgatiislns and Heavy Metal To.\icit_v
`
`3ll
`
`volatile form. This degt'adation can also be carried out by many kinds of
`microbes t5l.74.9l.96.l06). Thus concentt'ations of metals and organometallic
`compounds in natural habitats may be reduced by microbial action.
`Metal transformations have been shown to occur in a wide variety of habitats.
`e.g.. lake and river sediments. soil. river water. and activated sludge. and in each
`case the microbial composition has been significantly dit'l'erent (90). As already
`mentioned. a wide vat'iety of microbes can be involved in metal transi'orm-ation.
`and the fact that a specific. transforming flora does not exist further etnphasizes
`that
`the ability to transform is a widely occurring phenomenon and is
`the
`pt'opet'ty of diverse and ttbiqttitous organisms from all kinds of habitats. Since the
`ability to transform and thus detoxify certain metals is a widely occurring
`phenomenon. it follows that metal resistance resulting from this ability will also
`be common.
`
`Gt*m‘rf('ttl'l_\‘ .{)t*tt*l‘!.'liu<*t¢" i‘.-'.-'c‘tc.'l Rt'.t'i.\‘.'dHt't*
`
`Bacterial resistance to some heavy metals can be controlled by genes on extra-
`chromosomal t'esistance (R) factors or plasmids which can also control antibiotic
`resistance (7192). Plasmids have chielly been studied with regard to the transl'er
`of antibiotic resistance. but it
`is now evident that drug and metal resistance are
`closely connected and often occur together in clinical isolates. For example. in a
`study ttsing clinical isolates ot'P. m*ri.g:iiio.rt.-.
`it was found that most ofthe tneta|-
`resistant isolates ex