MICROBIAL
`€COL OGY
`
`rii1111TEJ2_11riTIOI1riL JOUKI1ril
`
`Contents
`
`279 M. M. Roper and K. C. Marshall
`Effects of a Clay Mineral on Microbial Predation and Parasitism
`of Escherichia coli
`
`291
`
`J. D. Buck
`Comparison of in Situ and in Vitro Survival of Candida albicans
`in Seawater
`
`303 G. M. Gadd and A. J. Gri ffiths
`Microorganisms and Heavy Metal Toxicity
`
`319 S. G. Berk, A. L. Mill s, D. L. Hendricks, and R. R. Colwell
`Effects of Ingesting Mercury-Containing Bacteria on Mercury
`Tolerance and Growth Rates of Ciliates
`
`331 C. I. Mayfield and W. E. Inniss
`Interactions Between Freshwater Bc>cteria and
`Ankistrodesmus braunni in Batch and Continuous Culture
`
`345 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
`
`351 M. A. Herzberg, D. A. Klein, and D. C. Coleman
`Trophic Interactions in Soils as They Affect Energy and Nutnent
`Dynamics. II. Physiological Responses of Selected
`Rhizosphere Bacteria
`
`361 R. 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 . Ill. Biotic Interactions of Bacteria, Amoebae, and
`Nematodes
`
`373 D. C. Coleman , R. 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
`
`381 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(4)279-387 (1978)
`ISSN : 0095-3628
`
`Il l Springer-Verlag New York Heidelberg Berlin
`\JOLUM€ 4/NUMBE:R 4/1977-78
`
`

`
`MICROBIAL E:COLOGV
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`

`
`Microbial Ecolog y 4:303- 317 ( 1978)
`
`MICROBIAL E:COLOGY
`
`Microorganisms and Heavy Metal Toxicity
`
`Geoffrey M. Gadd and Alan J. Griffiths
`Department of· Microbiology , University College Cardiff, Newport Road , Cardiff, South Wales ,
`CF2 JTA
`
`Abstract. The environmental and microbiological factors that can influence
`heavy metal toxicity are discussed with a view to understanding the mecha(cid:173)
`nisms of microbial metal tolerance. It is apparent that metal toxicity can be
`heavily influenced by environmental conditions. Binding of metals to organic
`materials, precipitation , complexation, and ionic interactions are all impor(cid:173)
`tant phenomena that must be considered carefully in laboratory and field
`studies. It is also obvious that microbes possess a range of tolerance mecha(cid:173)
`nisms, most featuring some kind of detoxification. Many of these detoxifica(cid:173)
`tion mechanisms occur widely in the microbial world and are not only specific
`to microbes growing in metal-contaminated environments.
`
`Introduction
`
`The heavy metals constitute a group of about 40 elements with a density greater
`than five (80). A feature of heavy metal physiology is that even though many of
`them are essential for growth , they are also reported to have comprehensively
`toxic effects on cells, mainly as a result of their ability to denature protein
`molecules. There are , however, many reports in the literature of microbial
`resistance to heavy metals. The phenomenon of microbial resistance is of some
`fundamental importance and is particularly relevant to microbial ecology, espe(cid:173)
`cially in connection with the roles of microbes in polluted ecosystems and in the
`reclamation of metal-contaminated natural habitats. It is also important to under(cid:173)
`stand the mechanisms of microbial tolerance because of the extensive use of
`some metals and metal compounds as fungicides and disinfectants. It is the
`purpose of this review to examine the nature of the interactions between
`microbes and heavy metals and to attempt to clarify the processes , both environ(cid:173)
`mental and microbial, underlying resistance or tolerance.
`
`0095-3628/78/0004-0303 $03.00
`© 1978 Springer-Verlag New York Inc.
`
`303
`
`

`
`304
`
`G. M. Gaclcl and A. J. Griffiths
`
`Environmental Influence on Toxicity
`
`Binding to En viro n/7/ento/ Co nstitu ents
`
`One of the mo st importa nt factors that determines the biological ava il ab ilit y of a
`metal in a sys tem is its binding to other e nvironm e nta l co nstituents. If a metal is
`wholly or parti a ll y re move d by binding, a decrease or co mplete disappearance of
`to xic effects may result.
`In the soil , me tals ca n be bound stro ngly by organic materials suc h as humic
`a nd fulvic ac id s and prote ins. Humic ac ids are especiall y important a nd it has
`bee n stated that practica ll y every aspect of th e chemistry of heavy metal s in
`soils, sed ime nts , and nat ura l waters is related in so me way to the formation of
`co mplexes with humi c substances (13,23 , 100) . In so me cases me tal ava ilab ility is
`limited by binding to humic acids to suc h a n extent th at deficiency sy mptoms
`may res ult in plants grow ing in such soil s (30). The bound meta l is ofte n difficult
`to remove. and even in ve ry sa nel y so il s ext racti o n ca n require vigo ro us proce(cid:173)
`dures (46).
`Clay particles ca n also bind metal catio ns , a nd so me metals such as zinc may
`enter the c rysta l lattice a nd beco me un ava ilable to o rgani sms ( 46 , 118). Clay
`particles ca n reduce the toxicity of ce tta in metal s toward mi croo rganisms.
`Expe rime nts with cad mium have shown that the clay minerals, mo ntmorillonite
`a nd kaolinite , protected ce rta in bacteri a, ac tinomycetes , and fil a me ntous fungi
`from the inhibitory effects of cadmium . This protective ab ility of th e clays was
`correlated with their cat ion-exc hange capacit y (CEC) as it appea red that the
`greater the CEC, the greate r the amount of cadmium abso rbed (8,9).
`In aquat ic habitats , metals suc h as z inc a nd cop pe r can be bound a nd
`re moved from the water by organic sedime nts, which effe ctively red uces the
`total me ta l ion co nce ntrat ion in solution . It has a lso been reported that certain
`oxidized sediments ca n bind up to 96% of added z inc (11 , 114). Ce rta in waters ,
`especiall y those in moo rla nd a reas , co ntain conside rab le a mounts of humic
`subst ances a nd , as in the soil e nvironme nt, a variety of meta ls ca n be bound
`including zinc , cobalt, and mercury ( 13 ,83). Beca use of such binding in aqu atic
`sys tems , it has been shown that toxic effects of ce rt ain metal s on microbes can
`be decreased (70) .
`In ce rta in polluted aq uatic habitats , meta ls such as merc ury ca n be trapped
`a nd bound by petroleum , a nd since ma ny oil-clegracling microbes are active at the
`oil-water inte rface , suc h re mova l is of obvious ecological signifi ca nce in th at it
`may enable the growth of metal-se nsitive organisms. In one st ud y of an oil(cid:173)
`polluted marine habitat, it was found that the co nce ntration of me rc ury in the oil
`was 4000 times higher than in the sedime nt a nd 300 ,000 times higher than in the
`water sa mpl es. Many of the oil-degrading organisms isolated were found to be
`mercury resistant , but th e ex te nt to w hich the me rc ury re mo va l influenced
`re sistant behavior was not determined (11 2).
`Compounds which ca n chelate meta ls, for example , citrate , cys tei ne, gluta(cid:173)
`mate , a nd EDT A, can also have a significa nt effect on mi crobial responses when
`included in growt h med ia. Toxic effects of co ppe r on Aerobacter aero ge nes
`were prevented by the add ition of yeas t extract a nd cysteine , a nd thi s was
`attributed to the ability of these compounds to bind cop pe r (66) . Simil arly , toxic
`
`

`
`Mic roo rga ni sms and Heav y Meta l Tox ic it y
`
`305
`
`effects of copper ascorbate to Serratia nw rcescens we re re lieved b y the additi o n
`of co pper-che lating agents (119). Citrate a nd E DTA ca n reduce tox ic ity o f so me
`me tals to A. aerogenes. In the prese nce of c itrate thi s o rgani sm co uld grow in
`200 pp m cadmium , but if gluco se was sub stituted fo r the citrate a n " infini te lag"
`resul ted (82). A stud y of me rc ury tox ic ity using the pro tozoa n Te tm liy m ena
`pyr(fo nnis revealed th at tox ic leve ls in a co mpl ex medium co nta ining pro teose
`pepto ne a nd li ve r di gest we re abo ut 40 tim es hi ghe r th a n th ose obse rved in a
`sim ple r medium (44 ). Coppe r tox icit y to A nabaena cvlin drica has a lso bee n
`show n to be redu ced by the addi tio n o f E DTA (35) .
`ln medi a w itho ut co m plex ing age nt s, toxicit y may be pro no un ced . T his is the
`case w ith Chlo re/la pyrenoidosa w he re a co pper co nce ntra tio n as low as 5 J.Lg 1- 1
`was tox ic (99). These a uth ors made th e interesting suggestio n that coppe r is no t
`o rdin arily prese nt as the ionic fo rm in natural waters but is us ua ll y co mplexed
`with o rga nic mate ri a ls such as polype ptides .
`In brewe ry syste ms it has bee n co mmo nl y fo und th at metals do not have the
`sa me effect on fe rm entatio n in simple and co mplex media (36 , 113). In ge ne ra l,
`fe rme ntati o n is re lati ve ly un affected by metal addi tio ns w he n tested in co mpl ex
`med ia . Fo r exa mple , in ma lt wort and mo lasses , a brew ing yeas t was unaffected
`by 30 to 40 pp m of co pper , but the same yeast , whe n grown in a simple r suga r
`a nd mine ra l salts med ium , was co mpl etely inhibited by 1 to 2 ppm co ppe r (113) .
`In act ivated sludge, me tals ca n be ad sorbe d by o rganic matte r a nd a pla nt
`may be a bl e to withsta nd qu ite high additi ons of meta ls witho ut seri o us loss o f
`acti vit y. ln o ne stud y, fo r exa mpl e , it was fo und that pro tozoa we re un affected
`by co pper co ncentra tio ns up to 5 ppm and th e reduc ti on in ove rall efficie ncy was
`o nl y 4% eve n at co nce ntratio ns of 25 ppm (69).
`It s hould be me nti o ned that altho ugh binding to e nvironme nt al constitue nts
`us uall y reduces tox icity, in so me cases toxic actio n s till res ult s eve n w he n th ere
`are no free me tallic io ns . T his was found to occ ur in ce rtain co mpl ex medi a w ith
`merc ury. Although the re we re no free me rc ury io ns un til the total co nce ntrati o n
`was 160 ppm , a total co nce ntra ti on of 10 ppm was found to inhibi t the growt h of
`ma ny aqu ati c bac te ri a (74,84) . It was sugges te d that e ithe r the io ns exe rted the ir
`tox icit y a nd e ntered the cell as orga nic co mpl exes o r bacte ri a l cell s co mpeted
`successfull y w ith the gro wth me di a fo r the bo un d io ns (84).
`Of co urse, in so me cases metal co mpl exes are more tox ic th a n the free metal.
`Thi s was show n fo r S tap liv lococcus aure us using S-OH-quin oline (1 0-" M) and
`fe rro us io ns (1 0- '1 M) . Wh en these sub sta nces we re applied se parate ly, no toxic
`acti o n res ulted but a mi xture co mpl etely inhibi ted grow th (2). Although ce tta in
`meta l complexes are mo re tox ic tha n the free me tal, they a re ofte n volatile a nd
`may di sappea r from a n enviro nm ent. T his is the case w ith me thylated de riv atives
`of me rc ury.
`
`pH
`
`pH ca n have a co nside ra ble effect o n the ava ilability a nd thu s the tox icity of
`heavy metals in a give n e nviro nme nt. In ge ne ra l, at a n acid pH me tals ex ist as
`free io nic catio ns, bu t at a n a lkaline pH the ionic ca tio ns precipitate as insolu ble
`hydrox ides o r ox ides. Mos t heavy metal hyd rox ides are insoluble. Co ppe r, at
`
`

`
`306
`
`G. M. Gadd and A. J. Griffiths
`
`about 1 ppm , disappears from solution at any Eh when the pH is greater than 6,
`and at an Eh below +200 mY when the pH is less th an 6. Zinc precipitates as zinc
`hydroxide , Zn(OHh, above pH 5, and above pH 8.5 it forms zincate ion which
`can be precipitated by calcium ions (27). The pH at which precipitation occurs
`va ries among different metals and among oxidation states of the same element.
`Some metals, for example , copper, have more than one valence state and the
`oxidized state is favored by high pH. The hydroxides of these oxidized states are
`less soluble than those of reduced states and precipitate at low pH values (19).
`Thus low pH generally increases the ava ilability of me tal ions , whereas high pH
`decreases availability . This has been illu strated in so il s, where in very acid
`conditions to xicity due to a n abundance of iron , manganese , copper, a nd zinc
`can be removed by adding lime which raises the pH (19,46). The influence of pH
`on availability is also illustrated by a study of the toxicit y of copper complexe.s to
`Ca ndida uti/is . Complexes with amino acids were less toxic at pH values of 7
`th an at lower pH values. It was suggested th at at the lower pH , the stability of the
`complex was lesse ned , releasi ng free copper ions (7).
`One aspect of pH and metal toxicity that should be mentioned is the occur(cid:173)
`re nce of metal-polluted mine streams which are often very acidic (20,53,54,85) .
`The low pH can arise from run-in from ac id soils and also from the microbiologi(cid:173)
`cal oxidation of sulfide-containing mine ral s by Lhiobacilli , for example , releasing
`sulfuric acid. The low pH can then release other metals such as lead , ma nganese ,
`iron , and zinc into solution (114). The biology of such ac id 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 (14,26,39, 114). Some bacteria are very tolerant indeed , such as the thioba(cid:173)
`cilli which can tolerate high concentrations of copper a nd zinc (107, 114) and a
`Pseudomonas species which is highly tolerant of copper, ma nganese, a nd cobalt
`(68). It is not clear, however, whether the low pH is of any advantage to these
`organisms in reducing toxicity , or whether they a re just extreme examples
`surviving by means of other tolerance mechanisms .
`
`Ion Interoctions
`
`The biological activity of heavy metal ions can be ma rkedly affected by the
`prese nce of other ions. Cations such as magnesium and calcium can often reduce
`heavy metal inhibition. Toxic effects of nickel , cobalt , cadmium, zinc, and
`ma nganese to Escherichia coli were dec reased in media with a high magnesium
`content. The toxicity of nickel and cobalt to A. aerogenes , Aspergillus niger, and
`C. uti/is was also diminished by magnesium. For all four organisms, using
`radioisotopes of nickel and cobalt , it was found that the high magnesium levels
`reduced the a mounts of nickel a nd cobalt taken up by the cells (l) . Inhibitory
`amounts of ma nga nese , iron, cobalt, nickel, a nd copper to Ba cillus licheniformis
`could likewise be antagonized by the addition of magnesium to the medium ,
`although toxic concentrations of zinc and cadmium were less effectively reduced
`(42) . Similarl y, calcium and magnesium have been shown to reduce the toxicity
`of cadmium toward A. niger (61). It has also been found that the iron concentra(cid:173)
`tion in a medium had a detoxifying effect on copper to the alga Chlorella
`
`

`
`Mi c roorgani s m s and Heav y Metal To xicity
`
`307
`
`pvrenoidosa . At the iron concentration used in algal growt h media , copper may
`be adsorbed to the negatively charged micelles of ferric hydrox ide (98).
`Ani ons are ab le to red uce metal toxicity by precipitation. T he hydroxy l ion
`has already been mentioned with regard to pH. Besides thi s, phosphate , thiosul(cid:173)
`fate , carbo nate , and bicarbonate ions can form precipitates wi th heavy me tals
`depending on their co ncentrat ions a nd the pH of the solution . T he add ition of
`such a nio ns to grow th media ofte n reduces metal toxicity (89).
`Sulfide , from hyd rogen sulfide , can also prevent to xicit y in ma ny cases by
`precipitating the metal as an insoluble sulfide . Organisms that grow in or produce
`high sulfide concentrations , e.g ., Desu!f'ovibrio desu(fitri cans , have been shown
`to be un affected by large add itions of heavy metals (105). This mechanism of
`tolerance will be di scussed later.
`Sometimes toxicity of a metal is inc reased when othe r io ns are present. In the
`case of the alga Cl!lorella Fulg aris , a n asy mmetric respiratory respo nse occurs
`when ftuorid e and copper io ns are applied jointly ; respi ration is completely
`inhibited by a mixture , but individually the ions have little effect (45).
`Mixtures of heavy metal s often exe rt a more pronounced effect on microorga(cid:173)
`ni sms , e.g., a mixture of copper and silver ions on algae (117) , but this can be
`accounted for by simple additive effects (114). Synergistic effects of metals on
`microbial growt h and survival have , howeve r, not received much attention.
`
`Mechanisms of Microbial Resistance
`
`Hv dro ge n Su(fide Production
`
`Microbial hydroge n su lfide production often has significant effects on metal
`toxicity si nce most heavy metals form insoluble sulfid es with H 2S. Consequently,
`H 2S-producing orga nisms often exhjbit tol era nce to heavy metals.
`In yeas ts, metal tolerance has often bee n linked with H 2S prod uction , a nd the
`importance of such H 2S-prod ucing yeas ts in nature has often been documented
`(28). Copper- and mercury-tolerant stra ins of Saccharomvces cere l'isia e produce
`more H 2S tha n do th ei r nontole rant parent st rains, the me tals being precipitated
`as insoluble sulfides (58, 72). Colonies of copper-tolerant strains appea r black or
`dark brown in the presence of copper a nd conta in muc h copper sulfide (5).
`E lectron microg rap hs have shown that the coppe r sulfid e was c hiefly deposited
`in and a round the cell wall (3,4,57) . Similar precipitation, thought to be copper
`sulfid e , has also been observed in the fungus Poria Faillantii (87).
`Bacteria that are capable of H 2S production may exhibit tolerant behavior.
`T he sulfate reducer Desu(f'o vibrio destt(furicans produces H 2S , grows in high
`sulfide concentrations , a nd may be un affected by the additi on of high co ncentra(cid:173)
`tions of heavy metals (105). Likewise, in a naerobic digesters sulfide reduces the
`to xicity of most heavy metals, the H 2S aga in resulting from bacterial reduction of
`sulfates (62).
`It has been noted that in so me cases sulfide-prod ucing organisms can protect
`se nsitive organisms fro m the toxic effects of metals. When D. desu(furicans was
`grown in mixed culture with a metal-sensitive stra in of Pseudomonas aem gi(cid:173)
`nosa, the latter organi sm could tol e rate higher concentrations of me rcuri als than
`
`

`
`308
`
`G. M. Gadd a nd A . J. G riffith s
`
`it could in pure culture . Re sult s indica ted the H 2S produced by the sulfate
`reducer protected the pse udomonad (10). S. aureus was a lso found to exhibit a
`higher tolera nce to mercurial s when grown with E. coli. The protective effect of
`the E . coli was pa rtl y du e to H 2S production a nd the extracellular production of
`g luta thione ( 101) .
`
`Production of Organic Compounds
`
`As previously mentioned , binding or chelation of a metal by organic substances
`present in the microbial e nvironme nt can markedly affect metal toxicity. In some
`cases the microorga nism s themse lves are c a pable of producing such substances
`which may reduce toxic ity . Citric acid , which c a n be produce d by ma ny yeasts
`a nd fungi, can readily che late meta l ions s uch as copper and may protect a fungus
`from copper poi so ning (87). A. niger may be protected from the toxic effects of
`lead in this way (120) . Oxa lic acid production has been linked with copper
`tolera nce of certain wood-rotting fungi . The fungi Co rra l/us palustris , Serpula
`laclu y mans, a nd Poria monticula a ll produce copper oxalate crystals when
`grown on synthetic media containing copper sulfa te (87) . Oxa lic acid is itself a
`toxic substa nce , but metals such as copper a nd iron , when complexed wtth It ,
`remove its toxicity while losing their own. Thi s has bee n observed with the oxa lic
`acid-producing fungus Endothia parasitica (29) .
`Some me rcury-res ista nt muta nts of Saccharomyces cerel'isiae were found to
`have a requirement for methionine . Evidence sugges ted tha t this compound ,
`itself a n efficient chelating agent , was used by the yeast to produce a "s imple ,
`diffusible substa nce " which acted as a detoxifying agent towa rd mercuri a ls
`(93,94).
`Intrace llular organic substances can also determine metal tolerance . This was
`found to be the case with me rcury-tolera nt A. niger where a pool of intrace llul a r
`sultl1ydryl compounds complexed mercury a nd a lleviated its toxic etlects (6).
`Such sulfuydryl compounds have also been observed in co pper-resi sta nt yeasts
`(56, 72) .
`
`Uptake and Acc111nulation
`
`Microorganism s possess mechanisms by which metal cations can be taken up
`a nd accumulated from their environment. Although the amounts of metal cations
`needed for growth requirements a re generally s mall , such uptake mechanis ms
`can still operate a t higher concentrations a nd can influ e nce metal toxicity , tow a rd
`both individu a l accumul ating orga ni sm s and the microbi a l community. In gen(cid:173)
`eral, if a metal is wholly or partly removed from a system by microbes , toxicity
`may be reduced . This kind of deto xification is s imil ar to that which occurs if a
`meta l is removed by environmental constituents.
`There appear to be two main types of me tal upt ake by organisms. The first
`involves non s pecific binding of the metal to ceU surfaces, slime laye rs, extracel(cid:173)
`lular matrices, etc. , whereas the second involves meta bolism-dependent intracel(cid:173)
`lular uptake.
`
`I
`l~
`
`-
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`

`
`Microorgani s m s a nd H eav y Me ta l Toxicit y
`
`309
`
`The first type can be important since mo st heavy metals can be adso rbed onto
`the sUiface of microbial cells, both living a nd dead, and, in fact, the addition of
`de ad bacterial cells to copper-inhibited laboratory cultures of bacteria is effective
`in reducing toxicity (39) . With metals such as copper, cadmium, and zinc ,
`complexation is possible with polygalact uronic acid, a n important constituent of
`the outer laye rs of bacteri al cells. The metal can be recovered from such
`complexes a nd the polymer regenerated (49). In yeasts, metabolism-independent
`sUiface binding is often to anionic groups of two species, polyphosphate and
`carboxyl , and such binding is rapid and reversible (79,88). Isolated cell waiJs of
`S. cereFisiae have been shown to bind their own weight of mercury to " high(cid:173)
`affinity " sites (71). In the fungus Neo cosnwspora vasil(["ecta , sUiface binding of
`zinc to negatively charged groups on the hypha! surface was rapid , reversible ,
`and temperature independent (81), as was the binding of zinc to C. uti/is (31).
`Similar binding of cobalt by Ne urospow eras sa was also rapid and acco unted for
`30% to 40% of total metal uptake (II 0). Surface binding of metals may be
`especiaiJy important in slime-producing organisms or those organisms that grow
`in an extracellular matrix , the extracellular material acting as an " impermeable
`barrier. " For example, zoogleoal bacteri a , which are common in aquatic habi(cid:173)
`tats, can survive and grow in the presence of high concentrations of heavy metal
`ions , the metals being adso rbed and precipitated within the extracellular matrix
`(37). Such organisms are effective in re moving toxic ions from solution and are
`thus of great ecological significance 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 metal uptake , metabolism-depe ndent tra nsport , has been
`studied in various algae a nd yeas ts (16,31 ,32,38,75 ,76) , bacteria (18 ,25 ,76) , and
`fungi (81 ,110) . 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 insignificant when compared
`to the amounts that can be taken up by energy-requiring processes (18 ,31 ,75 ,81).
`It should also be mentioned that most studies of metal uptak e have been
`concerned with low micronutrient concentrations as opposed to higher concen(cid:173)
`trations where, in order to survive , an organism may have to express some
`mechanism of tolerance .
`At higher concentrations, intracellular precipitat ion of the metal may occur
`after uptake. This itself can be a mea ns of detoxification since the metal is
`compartmentalized and may be converted to another more innocuous form. For
`example, certain yeasts are capable of precipitating thallium within the mito(cid:173)
`chondria as thaiJium oxide. The oxide may subsequently be disch arged from the
`mitochondria and excreted from the protoplast. This is termed oxidat ive detoxifi(cid:173)
`cation (63,65). "Copper containing particles" have also been observed in C.
`111 ilis after growth in high copper concentrations (55). In another study , copper
`was used as a "stain" for electron microsco py since it was found that this metal
`attaches to the nucleoli and chromosomes of yeast (64). In the algae Scenedes(cid:173)
`nuts acut(formis and Scenedesm us acumitis, 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
`co ncentrated only in the nucleus (33). Precipitation of mercury in electron-dense
`
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`310
`
`G. M. Gadd a nd A. J . Griffiths
`
`bodies has also been noted in the hyph ae of the fungus Chrysospomm pannomm
`(115). Likewise, electron-dense bodies, pre sumed to co nt ain z inc, have bee n
`observed in the fungus Neocos mosporo vasil({ecta after growth in a medium
`co ntaining zinc (8 1). There is also ev ide nce for the intracellular deposition of
`iro n, as fer rous sulfid e , within the sul fate-reducing bacteria Desu(fovib rio a nd
`Desu(fotomaculum (52). Crys tals of a copper compound thought to be sulfide
`have a lso been observed in the myce lium of the fungus Poria Faillantii (87).
`Dec reased uptake or impermeability to a metal may be a mea ns of resistance.
`Decreased uptake is the case with cad mium a nd S. aurei/S w he re a resistant
`strain takes up less cadmium than the se nsiti ve pa re nt stra in (2 1 ,22, 60 ,108).
`There is evide nce that the genes for suc h cadmium resistance are located o n
`extrachromo somal R-factors (pl as mid s) which are di scussed later.
`Impe rmeability is o ne ex planat io n of tolerance for those f ungi ca pable of
`growt h in high co pper conce ntrations. Penicillium a nd Aspergillus species have
`been found w hich ca n survive in sat urated cop per sulfate (12 ,97).
`
`!Vl eta/ Tran sjomzmion
`
`T he biologica l transform ation of certain heavy metals is an importa nt process
`that ca n occur in many habitats a nd be ca rried o ut by a wide variet y of
`microorga ni sms, chiefly bacteria a nd f ungi. Metals ca nnot be broken clown into
`other products but may, as a res ult of biologica l action , und e rgo changes in
`valence and/o r conversio n into organometallic co mpound s. Both processes can
`be co nside red to be detoxification mec ha ni sms since vo lat ilization a nd removal
`of the metal may re sult.
`Transformatio ns in vo lving cha nges of valency have been chiefly studied w ith
`mercury . Several types of bac teria and yeas t have been shown to effect the
`red uctio n of cationic mercury (Hg2+) to the elemental state (H g0
`) (17 ,59 ,67 ,91) .
`This usua ll y result s in the me rc ury bei ng volatili zed from the medium . Bacte ria l
`merc ury resista nce is c losely linked w ith thi s volatili zing abilit y (I 09). The
`oxidation of elemental mercury to its cationic form ca n also be mediated by
`microbes. Bacteria show n to have thi s ability include E . coli, Pseudo monas
`fl uoresce ns, P. aem ginosa, Citroba cter sp., Ba cillus subtilis, a nd B . megmher(cid:173)
`ium (47).
`Transformation of certain metal s into organometallic co mpounds by methyl(cid:173)
`ation is also a n importa nt detoxification mec ha nism. Metals that have been
`shown to unde rgo me th ylation inc lude mercury ( 43 ,50,86) , lead (34, 116), cacl(cid:173)
`mi um , and tin ( 48) . Methyl ation can be affected by the environm e ntal facto rs
`me ntioned in previous sectio ns a nd also by the numbe rs a nd species of microbes
`present in a particular habitat. Methylation can be catalyzed by a w ide va riet y of
`microorga nisms: both ae robic and a naerobic bacte ri a ( 48 , 106, 111 , 116), yeasts ,
`a nd fungi (24 ,34,87 , 111 ). Although products of met hylation may be more toxic
`than the free metal, they a re often volatile and ca n be released into the atmo(cid:173)
`sp here. This is the case with mercury a nd its meth ylated deriva tives , met hyl and
`dimethyl mercury (86).
`Organometallic co mpo und s can also undergo mi cro biological a nd c hemical
`degradation w hich may re sult in the metal being reliberated , aga in usually in a
`
`

`
`Microorgani sms and Heavy Metal To xicit y
`
`311
`
`volatile form . This degradation can also be carried out by many kinds of
`microbes (51 , 74,91 ,96, 106) . Thus concentrations of metals a nd 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 differe nt (90) . As already
`mentioned , a wide variety of microbes can be involved in metal transformation ,
`and the fact th at a specific, transfo rming flora does not exist further emphasizes
`that the ability to transfo rm is a widely occurring phenomenon and is the
`property of diverse and ubiquitou s organisms from all kinds of habitats. Since the
`abiLity to tra nsfo rm and thus detoxify certain metals is a widely occurring
`phenomenon , it follow s that metal resistance resulting fr