Microbial resistance to heavy metals - Environmental Science

May 30, 2012 - Publication Date: December 1983. Copyright © 1983 American Chemical Society. ACS Legacy Archive. Cite this:Environ. Sci. Technol. 1983...
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Microbial resistance to heavy metals Some microorganisms have developed "strategies" for combating effects of toxic inorganics, and several may prove useful for their removal from wastewater

John M. Wood Gray Freshwater Biological Institute University of Minnesota Navarre, Minn. 55392 Hong-Kang Wang Beijing Agricultural University People's Republic of China

It is clear that living organisms have influenced steady-state levels of elements in the atmosphere, in the oceans, and at the surface of the Earth throughout geological time (/). Both organic compounds and some inorganic complexes have been formed through biological activities that began about four billion years ago. Before examining some of the processes involved in element uptake by living cells, one should consider some of the selection principles involved in the chemistry of life. Several fundamental questions must be asked: • Which elements are essential for the growth and cell division of microorganisms, plants, and animals? • Why were these elements selected during the evolution of microorganisms more than four billion years ago? • What is the role of the geosphere in determining the uptake of essential elements? • What is the role of the biosphere in the selection of these elements? Obviously, the uptake of elements and their use by living cells depend on the chemical and physical properties of each element. Of the more than 100 elements in the periodic table, 30 have been found to be required for microbial 582A

Environ. Sci. Technol., Vol. 17, No. 12, 1983

life, although not all of these elements are necessary for the growth and cell division of every microbial species. Therefore, in addition to the bulk elements—carbon, nitrogen, hydrogen, and oxygen—26 others are required in

intermediate to trace amounts. An overabundance of any of these elements can cause buildup to an intracellularly toxic level, which often results in death. The reason for the selection of these

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Some mechanisms of microbial resistance to heavy metals 30 elements in the evolution of micro­ organisms appears to have been de­ termined by two factors: their abun­ dance in the Earth's crust and their solubility in water under strictly an­ aerobic conditions. Table 1 presents a list of elements in order of their crustal abundances. Twenty-two of the 26 el­ ements are found to be essential for life in higher organisms. Nonessential el­ ements are generally of low abundance in the Earth's crust, and therefore should not be effective in competing with essential elements in cells through their specific transport systems. The effect of solubility properties of elements under strictly anaerobic conditions is best illustrated by the biological use of iron and the rejection of aluminum. Under anaerobic con­ ditions, iron will be present in com­ plexes of lower oxidation state, [Fe(II) salts], which would be water-soluble and available for transport into prim­ itive anaerobic bacteria. By contrast, aluminum will not be soluble under those same conditions, unless the pH is extremely low. The same is true for the possible solubilization of lead, tin, and antimony. Metal ion selection Williams has determined a number of chemical parameters that must be considered for the uptake of metal ions by living cells (2). They are charge, ionic radius, preference for the coor­ dination of metals to certain organic ligands, and coordination geometry and coordination numbers for reten­ tion. Spin pairing between metal ions for more stability and the degree of covalence of metal-ligand interactions, available concentrations of metal ions in the aqueous environment, kinetic controls pertinent to metal ion trans­ port and binding, and chemical reac­ tivities of metal ions in solution also are major factors. The principles for the uptake of es­ sential metal ions by primitive micro­ organisms can now be set forth. First ofall, the availability of metal ions for transport into cells is restricted by their natural abundance and solubility in water. Solubility is profoundly in­ fluenced by pH, temperature, standard reduction potential (E°), the presence of competing anions and cations, and the presence of surface-active sub­ stances, such as particulates and macromolecules including proteins,

During the 200 years following the beginning of industrialization, huge changes in the distribution of elements at the surface of the Earth have oc­ curred. Microorganisms are adapting to these changes by evolving strate­ gies to maintain low intracellular con­ centrations of toxic pollutants. An understanding of the biochemi­ cal basis for resistance to metal ion toxicity is emerging, but it is compli­ cated by the different resistance mechanisms. Several strategies for resistance to metal ion toxicity have been identified: • The development of energydriven efflux pumps that keep toxic element levels low in the interior of the cell. Such mechanisms have been described for Cd(ll) and As(V). • Oxidation (e.g., AsOf~ to AsO*-) or reduction (e.g., Hg 2+ to Hg°), which can enzymatically and intracellularly convert a more toxic form of an ele­ ment to a less toxic form. • Biosynthesis of intracellular polymers that serve as traps for the removal of metal ions from solution. Such traps have been described for cadmium, calcium, nickel, and copper. • The binding of metal ions to cell surfaces. • The precipitation of insoluble metal complexes (e.g., metal sulfides and metal oxides) at cell surfaces. • Biomethylation and transport through cell membranes by diffusioncontrolled processes. Each of the mechanisms for resis­ tance to toxicity requires inputs of cellular energy and as such represents a nonequilibrium component for the distribution of elements at the Earth's surface.

humic acids, clays, and the like. Both the pH and the E° can vary widely from outside the living cell to inside that cell. For example, many essential transition metal ions such as iron, copper, cobalt, chromium, and nickel occur in higher oxidation states outside the cell, but in lower oxidation states inside the cell. The pH can vary widely outside the cell, but is usually between 7.0 and 7.2 inside the cell. Such changes in oxidation state and pH affect the chemical reactivity of chemical species; for instance, they determine whether inorganic com­

plexes function as nucleophiles or electrophiles. Most metal ions function as Lewis acids (electron acceptors), but depending on pH, oxidation state, and complexation, metal complexes also can function as bases. This is especially true for thiol-containing complexes. Changes in oxidation state can profoundly affect steric factors in ad­ dition to coordination geometry and coordination number. Moreover, changes in pH and E° can have an ef­ fect on the charge of the inorganic complex, and such alterations in charge can greatly influence the transport of metals that are known to be involved in the most primitive metabolic pathways. Outside the cell, chemical properties can be used to predict interactions between chemical species, often in quite complex situations. Pearson has summarized the order for complexa­ tion of inorganic ions on the basis of his theory of "hard" and "soft" acids and bases (3). Table 2 outlines ligand preferences for a number of trace ele­ ments, both essential and nonessential. It should be noted here that many of the more reactive metals are soft acids preferring coordination to bases found in living systems, such as thiolate groups, which are present in sulfurcontaining amino acids. For example, the stability for Cu(II) is as follows: S = > CN- > C03= > OH- > Ρ04Ξ > NH 3 > S0 4 = > I" > Br- > CI" > F-. In living cells, the important tran­ sition metals bind best to sulfur-rich bases, followed by nitrogen-oxygen bases, oxygen bases alone, and finally by coordination to water molecules alone. Such bases are readily available to metal ions in living cells because of fast exchange of protons in the pH range from 7.0 to 7.2 However, the coordination number becomes a very important factor for the kinetics of binding and stability. Some metal ions, once coordinatecovalently saturated in the interior of biological macromolecules, are diffi­ cult to replace with other competing metal ions. This is demonstrated clearly by the specific selection of metals in those metalloproteins con­ taining such metals as iron, copper, zinc, cobalt, nickel, manganese, and molybdenum. Williams has extended Pearson's Environ. Sci. Technol., Vol. 17, No. 12, 1983

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TABLE 1

Crustal abundance of elements a a 0 , Si, A l , a Fe, Na, Ca, Mg, K, H, Mn, P, S, C , V, CI, Cr, Zn, Ni Cu, Co, N, P b , S n , a a a a Br, B e , As, F, Mo, W, T l , I, S b , C d , Se; all the rest are less than 0.1 /ug/g. a

Element has no known biological function. Note: Range of concentration 0 = 46.6% to Se, which is 0.1 /ug/g.

TABLE 2

Classification of hard and soft acids and bases a Hard acceptor H+, N a + , K+, B e 2 + , M g 2 + , C a 2 + , M n 2 + , A l 3 + , C&+, Co3+, Fe3+, As3+

Intermediate Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+

Hard donor H 2 0 , O H " , F", Cr,

PO3~,

sor. co|_, o2~

Soft acceptor C u + , A g + , A u + , TI+, H g 2 + , CH 3 Hg+

Intermediate

Soft donor

B r - , N 0 2 ~ , SO§~

S H - , S 2 - , RS", C N " , S C N - , CO, R 2 SRSH

« Source: Pearson, 1968; Forstner and Wittman, 1979.

ideas by recognizing those factors im­ portant to the transport and parti­ tioning of metals in cells (2, 4). For example, the failure of a cell to trans­ port and use sufficient essential metal could arise from the following: • low availability, • excessive competition from other metal ions with similar chemical properties (e.g., Co(II) and Ni(II), Ca(II) and Cd(II), P 0 4 s and As0 4 =, Li + and Na + , etc.), • inadequate synthesis of carrier molecules by the cell, • excessive excretion of metal ions by the cell, or • failure of the energy-driven up­ take systems. Excessive element uptake can occur through the reversal of the above five factors. It is important to recognize that living cells are not at equilibrium with the external environment, and therefore a kinetic approach to metal ion transport, binding, toxicity, and resistance to toxicity is much more meaningful than a thermodynamic approach. Transport in aerobic bacteria The research of J. B. Neilands and his group at the University of Cali­ fornia at Berkeley has led to a clearer understanding of the molecular biol­ ogy of iron transport in certain bacte­ ria (5, 6). Ligands are excreted into the external environment to form ex­ tremely stable complexes with Fe(III). These ligands then bind to a specific 584A

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receptor site at the cell surface. This mechanism provides a very elaborate yet efficient system for the uptake of iron, which involves the investment of considerable energy by the cell. Figure 1 illustrates those parameters to be considered in both the transport and the control of intracellular con­ centrations of essential trace elements. Consider a general trace metal M and ligands Li through L 4 which can coordinate to M. The free external concentration of M is determined by environmental factors and so are the stability con­ stants for ML ι (where Li is a variety of ligands including, for example, humic acids). The internal concen­ trations are determined by: • selective carrier ligands, L2, • ligands, L 3 , that can remove M from solution at the cell surface, • concentration gradients that are established between the exterior and the interior of the cell through binding to L2 or by energy-coupled channels and pumps, and • internal concentrations of M that also can be controlled by removal of M from solution by special biomolecules, i.e., L 4 (e.g., the removal of Cd by the protein metallothionen due to non­ specific binding). It is apparent that uptake is con­ trolled by metabolic activity; of crucial importance is the specific design of L2 for transport of each specific metal ion (for example, the transport of iron into bacteria and fungi by siderophores,

small chelating agents excreted by microbes for binding and transporting essential metal ions) (2,3). L2 must be able to compete effectively with ex­ ternal ligand Li; therefore, stability constants are of critical importance to element transport. Also ML 2 must have an affinity for cell membranes by reacting at a specific membrane re­ ceptor site. This is well established for iron transport in unicellular organisms containing no nucleus, known as prokaryotes (5). Therefore, there will be a KD for each element in this membrane interaction. Krj is an equi­ librium constant for the interaction of a metal complex with the surface of a microbial cell. Competition between similar elements, such as Co(II) and Ni(II), must be taken into account, and the kinetics for ML2 interactions outside the cell, at the membrane, and inside the cell are very important in determining intracellular concentra­ tions of M. It is clear from these concepts that the accumulation of a trace metal by a cell is not at equilibrium, because metabolic activity is responsible for the synthesis of L2, L3, and L4 as well as energy-coupled channels, pH gradi­ ents, and redox potential differences. Clearly, uptake requires the invest­ ment of cellular energy. Therefore the thermodynamics of trace-metal ion transport depends on the following parameters: • log Kaq—stability constants for ligand-metal ion interactions in aqueous solution, • ΔρΗ—internal vs. external pH, • ΔΕ0—internal vs. external redox potentials, • free (M)—outside the cell, • free (L,) through (L4)—inside the cell, outside the cell, and in the cell membranes. M and L1-L4 are expressed in molar­ ities or molalities. The problem becomes even more complicated if one considers these principles for uptake in terms of ki­ netics. For example, one must under­ stand the rates of input from the envi­ ronment, the rates for transport through cell membranes, the rates for cellular exclusion or inclusion on cel­ lular traps, and the rates of exit from the cell. Outside the cell, the availability of a trace metal ion is determined by its abundance and solubility in aqueous solution. Inside the cell, redox poten­ tials and pH gradients are determined by different environmental situations. High pH and high E° favor higher oxidation states for trace metals, and it follows that low solubility and

availability result. Transfer through membranes requires a combination with a carrier or the presence of special channels in membranes, such as Ca 2 + channels and the like. Carrier molecules can be specific small molecules or proteins. Nevertheless, there is still a great lack of understanding of the selectivity principles involved with trace metal ion transport and of those mechanisms that energize inward transport. Essentiality vs. toxicity The biochemical basis for resistance to toxicity is complicated by the great variety of reactions at the molecular and cellular levels, even in closely related organisms and tissues. Several pathways for resistance to the effects of toxicants have been identified. Williams has pointed out that metal ion interactions in biology can be divided into three classes: ions in fast exchange with biological ligands, ions in intermediary exchange with biological ligands, and ions in slow exchange with biological ligands {4). Examples of those elements in fast exchange include the alkali metals Na + and K + , the alkaline earth metals Ca 2 + and Mg 2+ , and H + . Those that sometimes can be in intermediary exchange are Fe 2+ and Mn 2+ , while examples of those in slow exchange are generally in the active sites of metalloenzymes (Fe3+, Zn 2+ , Ni2+, Cu 2 + ). Metal-to-metal interactions and covalency predominate in the slow-exchange metals, and this provides the basis for stability. However, competition for those metal ions in fast exchange is often severe. Living cells have membranes that act as initial barriers to metal ion uptake. In prokaryotes, the external cell membrane represents the only barrier, but in eukaryotes (nucleus-containing cells of advanced organisms), there are many membranous organelles that can partition metal ions by a variety of mechanisms. A comparative study of metal ion resistance between blue-green algae (prokaryotes) and green algae (eukaryotes) demonstrates how important membranes are to metal ion uptake and toxicity. The green algae are much more resistant to high concentrations of toxic metal ions such as Cu 2 + and Ni 2 + than are the blue-green algae (7, 8). The external cell membranes of prokaryotes carefully select those ions in fast exchange, as exemplified by the rejection of N a + and Ca 2 + and selection of K + and Mg 2+ (9). In eukaryotes, spatial partitioning of metals occurs, even for those in slow ex-

FIGURE 1

Transport and control of intracellular and essential trace elements

Outside cell ML,

ML3 ·* E° high pH variable

change, because metal-binding macromolecules can be partitioned in different organelles and in tissues of different cell lines (e.g., metallothionenbinding Zn 2+ , Cd2+, Cu2+, and Hg2+ in the kidney cortex). The regulation of metal ion transport is presented in a general way in Figure 2, which attempts to show how once the cell buffering capacity for essential metal ions is exceeded, toxicity becomes evident. Toxic effects become evident at much lower concentrations for nonessential metals. In the natural environment, nonessential metals seldom reach concentrations in excess of l Mg/g (Table l). Examples of exceptional circumstances in which higher metal concentrations occur in nature include active volcanic regions such as deep sea vents, hot springs, and volcanic lakes. However, over the 200 years following the beginning of industrialization, huge changes in the distribution and solubilization of metal ions at the Earth's surface have occurred. This is attributable to the application of new chemistry in industrial society. Activities ranging from mining to modern agriculture pose risks to health and welfare and challenge those regulatory systems for the transport of metal ions through cell membranes that took billions of years to evolve. Microorganisms that have short generation times, and consequently increased ev-

Cell membrane

olution rates, have adapted themselves to deal with high concentrations of metal ions. Microbial resistance to metal ions Over the past two centuries, vast quantities of energy have been used to extract and process elements at the surface of the Earth. Microorganisms are adapting to these changes by evolving strategies to maintain low intracellular concentrations of toxic pollutants. This adaptation to resist toxic substances has arisen in two distinct patterns. Some microorganisms have inherited the ability to resist high concentrations of toxic elements through their evolution under extreme environmental conditions {10). Other microorganisms have acquired a transferred resistance to the polluted environment relatively recently, and certainly since the start of the Industrial Revolution. Such bacteria have achieved resistance through the acquisition of extrachromosomal DNA molecules (plasmids)

ill). Whether primitive or recent, each of the mechanisms for resistance to toxicity relies on inputs of cellular energy and thus represents a nonequilibrium component for the distribution of elements at the Earth's surface. Therefore, kinetic aspects of these processes are especially important to the field of environmental health and toxicity. Rates of uptake of all elements by cells Environ. Sci. Technol., V o l . 17, No. 12, 1983

585A

586A

Environ. S c i . Technol., V o l . 17, No. 12, 1983

Metabolic activity vs. concentration for essential and nonessential elements i

i

>* ΙΛΙ

Biomethylation Recent analyses of oil shale show that primitive organisms actively synthesize organometals and organometalloids. Therefore, biomethyla­ tion must have given certain micro­ organisms selective advantages for the elimination of heavy metals such as mercury and tin and of metalloids such as arsenic and selenium. The synthesis of less polar organometallic com­ pounds from polar inorganic ions has certain advantages for cellular elimi­ nation by diffusion-controlled pro­ cesses (12,13). The microbial synthesis of organo­ metallic compounds from inorganic precursors is well understood in both the terrestrial environment and in the sea. Mechanisms for B12-dependent synthesis of metal alkyls (requiring the presence of vitamin B12) have been discovered for the metals Hg, Pb, Tl, Pd, Pt, Au, Sn, and Cr and for the metalloids As and Se (14-18). Also, pathways for the synthesis of organoarsenic compounds have been shown to occur by a mechanism involving Sadenosylmethionine as the methylating coenzyme (19). To date, two different mechanisms have been determined for methyl transfer from methyl B12 to heavy metals: electrophilic attack by the at­ tacking metals on the Co-C bond of methyl B] 2 , and methyl-radical transfer to an ion pair between the at­ tacking metal ion and the corrinmacrocycle (the portion of the vitamin Bi 2 molecule that complexes its central cobalt atom). Metal ions that displace the methyl group by electrophilic at­ tack are Hg(II), Pb(IV), Tl(III), and Pd(II). Examples of free radical transfer are Pt(II)/Pt(IV), Sn(II), Cr(II), and Au(III). The latter mechanism provides experimental support for Kochi's ideas on charge transfer complex formation and elec­ tron transfer (20). The ecological significance of B]2-dependent biomethylation is best illustrated by B12-dependent and Bi2-independent strains of Clostridi­ um cochlearium. The B|2-dependent strain is capable of methylating Hg(II) salts to CH 3 Hg + , whereas the Bi 2 independent is incapable of catalyzing this reaction. Both strains transport Hg(II) into cells at the same rate, but the Bi2-independent strain is inhibited by at least a 40-fold lower concentra­

FIGURE 2

oil act

determine whether they live or die. However, it should be recognized that resistance mechanisms in higher as well as lower organisms can be quite variable.

.a to Φ

2

Λ\

""\^ /f

A \ V χ

\\

\

A

/

\

\

[Element concentration]

FIGURE 3

The mercury cycle3

a

S h o w i n g reactions catalyzed by bacteria, chemical disproportionation by Η2Ξ, and the photochemistry of organomercury compounds

tion of Hg(II) than the Bi2-dependent strain. This result clearly demonstrates that the dependent strain of C. coch­ learium uses biomethylation as a mechanism for detoxification, which gives the organism a clear advantage in mercury-contaminated systems. This biomethylation capability was shown to be plasmid mediated (21 ) . Once methylmercury is released from the microbial system, it enters food chains as a consequence of its rapid diffusion rate. In the estuarine environment, the reduction of sulfate by Desulfovibrio species to produce

hydrogen sulfide is important in re­ ducing CH3Hg + concentrations by S=-catalyzed disproportionation to volatile (CH 3 ) 2 Hg and insoluble HgS. It should be mentioned 'that there is overwhelming evidence to support the notion that membrane transport of methylmercury is diffusion controlled. Fluorescence techniques and highresolution nuclear magnetic resonance spectroscopy show that diffusion is the key to CH 3 Hg+ uptake (13). Also, a field study of the uptake of CH 3 Hg+ by Mediterranean tuna perfectly fits the diffusion model for biota in tuna

food chains (22). An updated view of the mercury cycle is presented in Fig­ ure 3 (23). Some details of the individual re­ actions of this mercury cycle will il­ lustrate the molecular biology and biochemistry of the following equilib­ ria (23): CH 3 Hg+ *± Hg 2 + *± Hg" Microorganisms have been isolated that catalyze these reactions both forr ward to Hg° and back to CH 3 Hg + . The enzymes carrying out the for­ ward reactions are coded by DNA on bacterial plasmids and transposons— the latter is a small amount of DNA transferable between closely related microorganisms—and not by normal chromosomal genes (24-26). There­ fore, it is not too surprising that mer­ curic and organomercurial strains of bacteria have been isolated from a variety of ecosystems such as soil, water, and marine sediments (27-32). The enzymology of methylmercury hydrolysis and mercuric ion reduction is now understood in some detail (24-26). In fact, the sequence of the active site of mercuric ion reductase is now determined and has been found to be identical to that of glutathione re­ ductase (33, 34). Clearly, the reduction of Hg 2 + to Hg°, which is volatile, represents a very effective detoxification mecha­ nism. Much less is known about the reverse reaction, that is, the oxidation of Hg° to Hg 2 + . However, an enzyme critical to the oxygen cycle (catalase) will carry out this reaction. Microbial methylation of the mercuric ion is also widespread (32,35,36). Biomethylation has been shown to occur in sedi­ ments and in human feces (37). The important role played by sulfide in the biological cycle for mercury is presented in Figure 3. Hydrogen sul­ fide is extremely effective at volatil­ ization and precipitation of mercury through disproportionation chemistry in the aqueous environment. The same is true for the volatilization and pre­ cipitation of lead compounds. This chemistry is important to the mobili­ zation of metals from the aquatic en­ vironment into the atmosphere. Such reactions occur only in polluted lakes, rivers, coastal zones, estuaries, and salt marshes where Desulfovibrio species have access to sulfate in anaerobic ecosystems (Figure 3). The dispro­ portionation of organometals by H 2 S is outlined below: 2 CH 3 Hg+ + H 2 S -* (CH 3 ) 2 Hg + HgS

Synechococcus. This nickel-tolerant microorganism was grown in I0~s M nickel sulfate; note dense intracellular granules (magnification 62 200X) 2 (CH 3 ) 3 Pb+ + H 2 S — (CH 3 ) 4 Pb + (CH 3 ) 2 PbS Once in the atmosphere, volatile organometallics such as dimethylmercury are unstable, since metal-carbon bonds are susceptible to homolytic cleavage by light. Intracellular traps for ion removal The biosynthesis of intracellular traps for the removal of metal ions from solution represents a temporary measure adopted by cells to prevent metals from reaching toxic levels. This temporary measure precedes mecha­ nisms for the expulsion of these metals from the cell by vacuoles. However, such temporary traps can be very ef­ fective; one example is the biosynthesis of metallothionen and the removal of cadmium (2) or copper (38) by this sulfhydryl-containing protein. This strategy adopted for the bio­ synthesis of intracellular traps fits quite closely the predicted partitioning for elements in organic or inorganic matrices. For instance, Na, K, Mg, Ca, Al, P, Si, and Β prefer to react with an oxygen-donor matrix, but Cu, Zn, Fe, Ni, Co, Mo, Cd, and Hg prefer a nitrogen- and sulfur-donor matrix. A recent example that we discovered in our laboratory came about through the selection of nickel-tolerant mutants of the cyanobacterium Synechococcus. Mutants that would tolerate up to 20 Χ ΙΟ - 5 Μ nickel sulfate were se­ lected (39) and were found to synthe­ size large quantities of an intracellular polymer (40). Nickel analysis and electron microscopy of sections of these nickel mutants showed that this polymer effectively removes nickel from solution by providing an intra­

cellular mechanism to prevent nickel toxicity. The photograph above is an electron micrograph of a nickel mutant of Synechococcus showing the pres­ ence of large granules. We failed to isolate similar mutants of Synechococcus showing tolerance to ΙΟ - 5 Μ copper sulfate. However, we found that our nickel-tolerant mutants also were resistant to copper. This was presumably caused by the stronger coordination of copper to the granules. Mutants with intracellular trapping mechanisms tend to bioconcentrate the toxic metal intracellular^ to ap­ proximately 200 times over the exter­ nal concentration. However, while this strategy works quite well for some or­ ganisms, it does not compare favorably with those organisms that bind or precipitate metals extracellularly. In­ tracellular concentrations of metal ions can be controlled by deposition on a solid surface, as in the crystallization of calcium salts in blood platelets or the removal of copper and nickel by intracellular granules. The concentration of free metal ions also can be controlled by the biosyn­ thesis of ligands in the form of small molecules with high stability constants. For example, the removal of iron by siderophores fits this category. Energy may be expended by the cell to pump the metal ion out of the cell (for ex­ ample, by the sodium-potassium ATPase pump). The cell may synthe­ size ligands that bind metals strongly at the cell surface or use the activities of surface-bound enzymes to precipi­ tate metals extracellularly. Efflux mechanisms The resistance of microbial cells to the toxic elements arsenic, antimony, Environ. Sci. Technol., Vol. 17, No. 12, 1983

587A

and cadmium has been shown to occur through the evolution of cellular exclusion mechanisms. In Staphylococcus aureus and in Escherichia coli resistance to arsenate, arsenite, and antimony(III) salts has been shown to be induced by an operonlike system (41). An operon is a DN A region that codes for several enzymes in a reaction pathway. Each of the three ions will induce resistance to ions of the other two elements. Arsenate is transported through cell membranes in competition with the transport of phosphate ions, but arsenite is transported through a phosphate-independent mechanism (42). The genetics of arsenate efflux from resistant cells is quite well understood at the genetic level and is the subject of two recent reviews (//, 24). The toxicity of arsenic and antimony salts resides in the reactivity of these compounds with sulfhydryl groups (43). Sulfhydrylcontaining enzymes are inactivated by arsenicals and antimonials. In the case of cadmium, resistance has been shown to be mediated by a plasmid. Resistant cells of S. aureus have a very efficient chemosmotic efflux system specific for Cd 2+ ions (24). Two separate plasmid genes have been shown to be responsible for Cd 2+ resistance. These genes also prevent Zn 2 + toxicity. The cad-A gene codes for proteins that are involved in the efflux of Cd 2+ on the inside and for two H + from the outside of the cell. The cad-B gene is believed to be responsible for the synthesis of a Cd2+-binding protein, which may be similar to metallothionen (24, 44). Arsenate, cadmium ions, and antimony ions are accidentally taken up by normal transport systems designed for

the transport of ions such as phosphate and zinc. Therefore, the uptake of these elements is nonselective. However, resistance to toxicity by these microorganisms resides in their ability to remove these elements selectively by energy-driven efflux systems.

surface must be important, because only surface-active substances such as humic acids could compete effectively for nickel binding (63). Table 3 shows the ability of these seven strains of nickel-tolerant algae to bioconcentrate nickel simply through ion exchange.

Surface binding of metal ions Microorganisms, including the algae, synthesize extracellular ligands that complex metals and prevent their cellular uptake. The research groups of François Morel and Pamela Stokes, at MIT and the University of Toronto, respectively, have carried out extensive work on the complexation of copper with a variety of extracellular substances (45-49). Considerable literature also exists on the toxicity of nickel and copper to algae (50-57). Cyanobacteria and brown and green algae all bioconcentrate nickel (57-62). Recently, we used the isotope nickel-63 in a study of nickel binding by seven different strains of nickeltolerant algae (63). The cyanobacteria were found to be more sensitive to nickel toxicity than the green algae, which points out differences in transport mechanisms for prokaryotes and for eukaryotes. Both cyanobacteria and green algae could concentrate nickel primarily at the cell surface to 3000 times over the concentration in the culture medium. Both nickel binding and nickel toxicity were shown to be very pH dependent. The optimum pH for binding was between 8 and 8.5. Binding was shown to be rather specific with charge, ionic radius, and coordination geometry being the predominant factors. The only significant competing cation for Ni(II) binding was Co(II). The orientation of ligands at the cell

Precipitation at the cell surface

The precipitation of insoluble metal complexes occurs through the activities of membrane-associated sulfate reductases (63) or through the biosynthesis of oxidizing agents such as oxygen or hydrogen peroxide (23). The reduction of sulfate to sulfide and the diffusion of 0 2 and H2O2 through the cell membrane provide highly reactive means by which metals can be complexed and precipitated. This process depends on the metabolic activity of the cell and is closely tied to heavymetal resistance. Several bacteria have been found that precipitate silver as Ag2S at the cell surface. Also, certain fungi are very efficient at recovering uranium (64). However, the most interesting organisms are certain strains of green algae that grow in acidic conditions and at high temperatures (1). Cyanidium caldarium is such an organism; it has the remarkable ability to grow in media containing either 1 Ν H2SO4 or 1 Ν HC1. A strain of C. caldarium has been adapted to grow at 45 °C in acid mine water from a copper-nickel mine. This thermophilic alga grows extremely well in sulfuric acid over the pH range from 0 to 4 and removes toxic metal ions from solution by their precipita­ tion at the cell surface as metal sul­ fides. Batch cultures of C. caldarium have been grown that remove very high concentrations of metals from solu-

TABLE 3

Cfa for 63 Ni (II) 6 by different strains of algae at different pH conditions pH Cf Algae

Scenedesmus ATCC 11460 Scenedesmus B-4 Synechococcus ATCC 17146 Synechococcus Nic7 OscillatoriaUTEX 1270 Chlamydomonas UTEX 89 Euglena UTEX 753 63 a r , _ M9 of Ni(ll) removed per gram of alga

5

4

NS NS NS NS NS 27 NS

C

NS 90 NS NS 29 38 NS

6

Environ. Sci. Technol., Vol. 17, No. 12, 1983

2

7.9 X 10 2.8 X 102 4.2 X 102 2.2 X 102 9.5 X 102 5.4 X 102 NS

μ® of 63Ni(tl) in culture medium ' [63Ni(ll)] = 0.02 Mg/mL (0.34 μΜ), incubated for 6 h at 20 °C in the light. : NS = no significant uptake at the 9S % confidence level.

588A

7

9

8 3

1.8 X 10 6.6 X 102 3.0 X 103 4.4 X 102 1.1 X 103 NS 17

2.2 1.0 3.3 5.5

3

X 10 X 103 X 10a X 102 11 NS 6.9 X 102

2.0 X 103 4.0 X 102 3.1 X 103 NS NS NS NS

tion—68% of the iron, 50% of the copper, 41% of the nickel, 53% of the aluminum, and 76% of the chro­ mium. Since C. caldarium is both ther­ mophilic and acidophilic, it is easily grown free of other contaminating autotrophs without having to use sterilization procedures for the acid mine water medium. This alga has great promise as a biological agent for the recovery of metals and for cleaning metal-polluted wastewaters. In the early 1950s, Allen (64) was the first to report the isolation of C. caldarium from acidic hot springs in California. This was subsequently confirmed by Seckbach et al. (65). Since that time, substantial research has been done on the microbiology, life cycle, structure, and ecology of this unusual alga (/ ). This organism has an optimum growth temperature of 45 °C and grows very well in temperatures ranging from 35 °C to 55 °C. C. caldarium adapts to fit a whole range of temperature and pH without any apparent selection of specialized strains for specific ecosystems. Cul­ tures grow faster when provided with 5% CO2, and excellent cell yields are obtained under strictly autotrophic conditions (7). However, more than double the cell yield is obtained if cultures are provided with 1% of a soluble carbon source such as glucose. Therefore, C. caldarium grows heterotrophically in the dark on a variety of organic substrates, including mono­ saccharides, disaccharides, mannitol, glycerol, ethanol, succinate, glutamate, lactate, and acetate. The.organism is easily maintained on acid media with viabilities of stock cultures lasting longer than one year (66). A culture of C. caldarium isolated from the Waimangu Caldron Outlet, North Island, New Zealand, was slowly adapted to growth in acid mine water by adding 10% increment in­ creases of mine water to a culture grown under the conditions described by Allen. Using this procedure, one can select strains of C. caldarium that grow very well on a culture medium consisting of unfiltered acid mine water provided with 5% C 0 2 . Table 4 presents data on the ele­ mental composition of acid mine water at pH 2.1 and also shows the removal efficiencies for stationary-phase cul­ tures of C. caldarium in the presence and absence of glucose plus ammo­ nium sulfate. The photograph above shows a section of C. caldarium ex­ amined under the electron microscope; microcrystals of metal sulfides are found to adhere to the external cell

Precipitation. Inorganic complexes precipitate on cell surface o/Cyanidium caldarium grown in acid mine water (pH 2.1 with 5% CO2 at 45 °C; magnification 31 700X) TABLE 4

Removal of metal ions by precipitation from acid mine water a> b

Elementce

Acid mine water (PH 2.1) (pH (ppm)

Culture supernatant + 1 % (NH 4 ) 2 8Ο4 + 5% C0 2 (ppm)

Removed

Ca Mg Fe Cu AI Cr Ma Ni Ρ

342 456 632 119 329 1.31 28.4 4.32 26.0

219 228 205 60.6 155 0.31 13.2 2.55 13.5

36 50 68 50 53 76 54 41 50

%

Culture supernatant + 5 % CO2 (ppm) 277 342 386 95 245 0.38 20.9 2.69 19.1

! %

Removed 19 25 39 20 25 71 26 28 29

ι

" By Cyanidlum caldarium. b Cells grown for 1 wk from 1 L inoculum in In 8 L acid mine water. 0 Analyzed by plasma emission spectroscopy.

membrane. Cells contain approxi­ mately 20% metal on the basis of dry weight. Clearly, toxic metals such as copper, nickel, and chromium are prevented from entering the cell through an ex­ tracellular precipitation mechanism. This result suggested to us that C. caldarium possesses a membraneassociated sulfate reductase system. Heterotrophic cultures, such as one oi this alga, which are allowed to attain anaerobic conditions in the dark pro­ duce hydrogen sulfide gas quite effi­ ciently. Therefore, sulfide precipitation of metals can be regarded as a cellular detoxification mechanism. Of special interest are the removal efficiencies of chromium and nickel, which are present in very low concen­ trations in the acid mine water. These metals are known to have potential as carcinogens, and their selective re­ moval from wastewaters is highly de­

sirable. Organisms such as C. caldar­ ium may well be effective in treating polluted waters so that effluents can meet federal standards for such toxic elements. Genetic engineering The examples previously given suggest that microorganisms can be selected from extreme environmental circumstances or can be manipulated genetically to recover toxic elements from industrial wastewaters. This is certainly true for those organisms with resistance mechanisms coded by the cell genome. However, a word of cau­ tion should be expressed with regard to the uses of plasmids to engineer special organisms genetically for this purpose. It is more than likely that the toxic conditions found in industrial waste­ waters will cause plasmid losses from organisms, even though they can be shown to function extremely well in the Environ. Sci. Technol., Vol. 17, No. 12, 1983

589A

laboratory. Also, at ambient pH and temperature, organisms introduced into industrial wastewaters must compete for nutrients with those or­ ganisms already established in this special environment. The use of biotechnology looks much more promising for those organisms—such as C. caldarium—that grow under extreme conditions of pH and tem­ perature and that use light as the major energy source. Acknowledgment We wish to acknowledge the research of postgraduate colleagues Y.-T. Fanchiang and F. K. Gleason. Francis Engle and Steve Michurski also are thanked for their technical assistance. We wish to dedicate this paper to R.J.P. Williams, F.R.S., for his encouragement, insight, and contribu­ tions to our knowledge of this subject. Some of the research reported in this paper was supported by grants from the National Institutes of Health AM 18101 and by a grant from Atlantic Richfield Company. Before publication, this article was read and commented on for suitability as an ES& Τ feature by Bruce E. Rittmann of the University of Illinois, Urbana, 111. 61801, and Fumio Matsumura of Michi­ gan State University, East Lansing, Mich. 48824. References (1) Lovelock, J. E. "Gaea. Λ New Look at Life on Earth"; Oxford University Press: Oxford, U.K., 1979. (2) Williams R.J.P. Phil. Trans. Roy. Soc. Lond. Ser. B. 1981, 57, 294. (3) Pearson, R. J. Chem. Educ. 1968, 45, 643. (4) Williams, R.J.P. "Structural Aspects of Metal Toxicity"; Dahlem Konferenzen on Changing Biogeochemical Cycles of Metals and Human Health; Berlin, F.R.G., March 20-25, 1983. (5) Neilands, J. B. Chem. Scr. 1983, 21, 123. (6) Neilands, J. B. "Trace Metals in Health and Disease"; Kharasch, N., Ed.; Raven Press: New York, N.Y., 1979; p. 27. (7) Wood, J. M.; Wang, Hong-Kang. Environ. Sci. Techno/., in press. (8) Wang, Hong-Kang; Wood, J. M. Environ. Sci. Technol., in press. (9) Williams, R.J.P. Pure Appl. Chem., in press. (10) Brock, T. D. "Thermophilic Micro­ organisms and Life at High Temperatures"; Springer-Vcrlag: Heidelburg, F.R.G., 1978. (11) Silver, S. In "Biomincralization and Bio­ logical Metal Accumulation"; Westbroek, P.; deJong, E. W., Eds.; D. Reidel Publishing Company: Amsterdam, The Netherlands, 1983; p. 439. (12) Wood, J. M. Nalurwissenschaflen 1975, 62, 357. (13) Wood, J. M.; Cheh, Α.; Dizikes, L. J.; Ridley, W. P.; Rackow, S.; Lakowicz, J. R. Fed.Proc. 1978, 37, 16. (14) Ridley, W. P.; Dizikes, L. J.; Wood, J. M. Science 1977,197. 329. (15) Ridley, W. P.; Dizikes, L. J.; Cheh Α.; Wood, J. M. Environ. Health Perspec. 1977, /9,43. (16) Wood, J. M. Q. Rev. Biophys. 1978, 9(2) 467. (17) Wood, J. M.; Fanchiang, Y.-T.; Ridley, W. P. ACS Monograph No. 82; Brinckman, F. E.; Bellama, J. M., Eds.; 1978; p. 52. 590A

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John M. Wood (/.) is a professor of bio­ chemistry and ecology at the Gray Freshwater Biological Institute, University of Minnesota. In 1972, he was the recipient of the Synthetic Organic Chemical Man­ ufacturers Association award for his work on the biomethylation of mercury. That same year, he received a Guggenheim Fellowship and spent a year at Oxford University, U.K. In 1981, he received the E. J. Zimmerman Award from the ACS for research in environmental science. In 1982 he participated in a Nobel sympo­ sium on inorganic biochemistry. A mem­ ber of the editorial board o/Science, Wood received his BSc and PhD in biochemistry from the University of Leeds, U.K. He is the author of more than 100 papers in the chemical and biochemical literature. Hong-Kang Wang (r.) is an associate professor of chemistry at the Beijing Ag­ ricultural University in the People's Re­ public of China. His expertise is in the area of heavy-metal pollution of soils and sediments, and he was instrumental in drawing up the guidelines for the use of sludge fertilizers, with special reference to heavy metals, for crop production in China. He is a standing member of The Chinese Agriculture and Environmental Protection Society and is deputy editorin-chief of the Chinese Agriculture and Environment Protection Journal. He has been a visiting professor at the Gray Freshwater Biological Institute, where he worked on the uses of algae for the re­ moval of heavy metals from polluted wastewater.