The hard stuff: Metals in bioremediation

The hard stuff: metals in bioremediation Anne O. Summers University of Georgia, Athens, USA Metals, both toxic and valuable, are found in deposits of variable richness. Human activities have dispersed metals beyond their natural deposits throughout soil, sediments, and freshwater. The idea that biological processes or products could contain toxic metals or enhance recovery of valuable metals has been discussed for many years. Recent advances in microbial biogeochemistry and bioinorganic chemistry indicate that these long-standing aspirations could soon be fulfilled. Current Opinion in Biotechnology 1992, 3:271-276

Introduction Toxic metals have been identified in many United States Environmental Protection Agency [1,2] and US Department of Energy [3] target sites, but commercial applications of metal bioremediation or recovery processes have b e e n rare. This review will consider the difficulties of metal bioremediation and some promising n e w advances in basic science which may lead to n e w developments in metal bioremediation processes. In this context, the term 'metals' will include transition metals that are toxic at any dose (such as Pig and Gd) or that can be toxic in high doses (such as Cu, Zn, Go, Cr), metalloids (such as As, Se, Te, Sb), which are often found as oxoanions, and radionuclides of the nuclear fuel cycle.

Problems of metal bioremediation A number of problems have been encountered in attempts to apply biotechnology to metal remediation and recovery. To begin with, metals are nearly invisible elements in biology and their study has traditionally b e e n left to those specifically interested in metalloenzymes. It has only recently become firmly established that metals play central roles in every aspect of biology, even in the regulation of gene expression. Nonetheless, metals are complex analytical subjects and most modern biochemists know a lot more about sequencing DNA than about speciating mercury or characterizing iron-sulfur centers. Secondly, metals in low doses are relatively idiosyncratic poisons and lack the noxious odors and tastes that often provoke public outrage over organic contaminants. Thus, regulatory guidelines, even o n widespread toxic metal exposure such as that from dental amalgams, are often contradictory. Thirdly,

'remedied' metals are still metals. Whereas biological and chemical processes can change offending organic substrates into innocuous end products, such as CO2, a metal may be changed in valence or in chelation state by these agents but it remains the same metal. Fourthly, metals are chemically promiscuous. Expecting to find the perfect organism (or process) to immobilize permanently a toxic metal in some remote substratum, ignores the very real possibility that what one microbe can precipitate, another microbe (or some other hydrogeological or anthropogenic process) can solubilize. Finally, in situations where remediation involves metal recovery, the recovered metals must either be recycled by the discharger for his own use or sold to someone else, or stored in some way so that they cannot re-enter the biosphere. These problems are not faced in the bioremediation of organics since there are no constraints on releasing carbon dioxide into the biosphere. Even in organic bioremediation, however, it is important to consider h o w metals influence organic bioconversions and how the organic remediation processes modulate the eventual fate of the metals.

Metal bioremediation cycles Metal bioremediation can be envisaged as a set of pathways (Fig. 1). The basic problem is the occurrence of metals in soil, rock or sediment, or in water, at concentrations too dilute to be worth mining (if the metals are valuable) or sufficiently concentrated (if the metals are toxic) to be an environmental concern. Biorecovery is the process employed for metals that are more valuable than toxic, and the h o p e of cheaper and more efficient strategies for their enrichment and re-use (i.e. increased profits) is the driving force for interest in this area. For metals that are toxic but not intrinsically valu-

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Environmental biotechnology able the processes are referred to as bioremediation, and the driving forces are increasingly strict standards for water and air quality, including those set forth by the US Resource Conservation and Recovery Act, and a variety of other federal and state regulations. Meeting these standards will increase the cost of doing business and therefore will add extrinsic value to these toxic metals. This extra value could warrant the scrubbing of metals from a waste stream or site (Fig. 1), and even their re-use in a more contained version of the industrial process that originally led to their distribution into the environment. In bioremediation, another frequently considered scenario is that of 'fixation', meaning the conversion of a metal to a very stable, insoluble form (e.g. a sulfide), so that mobility of the metal in the biosphere is severely, and perhaps permanently, diminished. Processes that enhance, rather than decrease, mobility of metals through aqueous systems or into the air are less frequently considered. Such dissipative processes occur naturally for some metals (e.g. Hg and Se) but deliberate use of such processes is limited b y the lack of any consensus on what is 'dilute enough' not to constitute a hazard.

value, then by concentration of the dilute soluble metal b y solvent extraction or cementation processes, and finally, in the case of solvent extraction, by an electrowinning process to convert the concentrated metal ions into a saleable product [4,5]. Extraction processes cause pollution and electro-winning is energy intensive. Toxic metals in aqueous streams are often precipitated and concentrated b y the addition of lime, limestone, caustic soda, or sulfide and the resulting sludge is either stabilized for subsequent hazardous landfill disposal, or used as a feedstock for mineral concentrators and smelters for metal recovery. Ion exchange resins m a y be u s e d in some applications instead of electro-winning. There is no routine treatment for toxic metals dispersed in soils or sediments other than extensive washing with aqueous solutions of acids or phosphates (which themselves must be disposed of) and subsequent capture of the metals on resins, or b y their concentration into a chemical sludge [6"]. Existing methods can yield final metal concentrations in output streams of I m g l - 1 , but newer regulations require concentrations of less than 1 part per million for m a n y metals.

Existing alternatives for metal remediation or recovery

General strategies for metal bioremediation or recovery

Low grade ores of valuable metals are typically processed first b y chemical extraction (or biological extraction for certain c o p p e r ores) to solubilize the metal

Despite these problems there has b e e n considerable recent progress in the field. Both living cells (typically microbial) and non-living biomaterials can function in

Dilute metals in soil or water

11

I Capture valuable metals

Scrub

I

Make toxic metals less available to biosphere

Fixation~

I Clean and concentrate Sell~ I

l Re-Use ]

Recycle

Precipitate in soil or sediment 1

D issi pation

?f

Very dilute in water or soil

?

Very dilute in air

?

Fig. 1. General scheme of metal remediation and recovery cycles. Profit is the driving force behind the recovery of valuable metals while remediation of toxic metals is driven by regulations. The question marks indicate presently undefined processes.

Metals in bioremediation Summers

metal recovery and remediation. In the discussion that follows, I will distinguish first and second generation processes in both categories (Table 1) based on the extent of biotechnological modification of the agents, regardless of the mechanical devices or structures by which the organisms or biomaterials interact with metals.

First generation processes using living cells First generation processes that involve living cells are naturally occurring processes. Some have b e e n k n o w n for m a n y years but others have been discovered more recently. Technological interventions are minimal and usually involve modifying the physiological behavior of the organisms by nutritional amendments. The classic example of the application of microbe-metal interactions to the biorecovery of ores makes use of naturally occurring communities of chemolithotrophic bacteria, such as Thiobacillus and Sulfolobus (or Acidianus), which release Cu(ID from pyritic ores as a result of iron and sulfur oxidation by the bacteria [4]. The Cu(II) in the leachate is recovered electrolytically. Unfortunately, acid produced microbially from the leaching of u n - i m p o u n d e d c o p p e r pyrite leads to acidification and metal contamination of surface waters. So, although this bacterial process assists c o p p e r recovery from low grade ores, it also creates problems that b e c o m e targets for other bioremediation solutions. A more recent batch variation on this theme is successfully used commercially in South Africa to enhance recovery of refractory gold ores [7"']. Recently, a first generation biotreatment for sulfate- and metal-rich acid mining wastes has b e e n developed by

combining sufficient organic material with the wastewater to foster growth of sulfate-reducing bacteria. Such sulfide-producing microbes have b e e n exploited in artificial wetlands created to remove metals and to raise the p H of acid mine wastes (DM Updegraff et al., abstract, American Chemical Society Geochemical Symposium 1992, 203:174). The offending metals are deposited as fairly insoluble sulfides and it is expected that they will only find their way b a c k into surface waters very slowly, although there are no precise data available to verify this. Several naturally occurring microbial processes that affect selenium have b e e n characterized recently. The dissimilatory reduction of toxic selenium oxyanions to selenium metal results in the immobilization of this element in the soil [8"]. This process is carried out naturally in a mixed anaerobic culture enrichment b y facultative bacteria indigenous to waters loaded with selenate from soil in various regions of the American West. The rate of selenium reduction is sufficient for real-time recovery of water quality by the u n a m e n d e d bacterial process. However, nitrate from agricultural run-off does inhibit the process [9"q. A second group of organisms metabolize selenium in a completely different manner. Aerobic bacteria (possibly halophiles) thriving in briny, agricultural runoff evaporation ponds generate volatile dimethyl selenium. This natural process is greatly stimulated b y the addition of carbon sources and surface-enhancing additives such as glass beads [10"]. Both reduction and methylation of selenium has b e e n observed in a floating microbial mat ecosystem generated in the laboratory [11"]; the removal of selenium from the water column was very efficient, although no applications of this system in the field have b e e n reported.

Table 1. Strategies for metal bioremediation and recovery. Processes

Examples

Agents

Copper ore leaching Metal precipitation in acid mine drainage Selenium precipitation Selenium volatilization

Sulfur oxidizers [4] Sulfate reducers Selenate reducers [9°'] Selenium methylators [10 °']

Enhanced ore leaching or toxic metal deposition

Genetically engineered strains

Biomass chelation/accumulation

BIOCLAIM, BIO-FIX [4,22"']

Engineered biomimetics

Designer ionophores [28,P1]

Employing live cells First generation

Second generation a

Employing non-living biomaterials First generation Second generation aSuch processes have not yet been developed.

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Environmental biotechnology Pure cultures of an iron-reducing strain, GS-15, previously g r o w n anaerobically on Fe(III) and acetate (as an electron donor), can reduce U(VI) to insoluble U(IV) at a rate of about 0 . 6 m m o l e l - l h - 1 , several orders of magnitude faster than its reduction by sulfide, which has previously b e e n thought to be the sole agent of uranium reduction in natural settings [12"]. Alteromonas putrefaciens can also cause rapid U(VI) reduction using H 2 a s an electron donor. Reduction of U(VI) was observed in a u r a n i u m - a m e n d e d Potomac River sludge and w a s a b o u t fourfold faster in non-autoclaved sediment than in autoclaved sediment, which contained considerable natural sulfide as well as Fe(III). There is apparently little potential for re-oxidation of the uranium in a m i x e d ecosystem. These and other first generation processes warrant continued exploration to unearth novel microbial processes and to optimize them for primary or field waste treatment. In situ processes will always be subject to the idiosyncracies of each site, although with a deeper knowledge of such microbial processes more sophisticated consortia and/or nutritional a m e n d m e n t s can b e designed.

Second generation processes using living cells Second generation processes involving the actions of living ceils will e m p l o y organisms genetically optimized for specific applications either in the field or in contained waste-stream processors. At present, there are no examples of such processes but there are a number of possibilities. These include ceils that hyper-accumulate specific metals, cells that release their accumulated metals in response to a simple and benign signal, ceils that generate energy via the oxidation or reduction of toxic metals, and cells that p r o m o t e the release of soil-bound metals to facilitate washing. The intriguing new systems noted above, as well as the burgeoning area of plasmid-determined bacterial resistance to toxic metals [13"'-18",19",20"], indicate that such designer microbes are within reason. Many eubactefia carry genes that render t h e m specifically resistant to such metals as Hg, Cd, Co, Zn, and to oxyanions of arsenic, antimony, and tellurium. Some of these metal-resistance systems function b y mediating efflux of the toxic metal from the cell, while others involve conversion of the metal to a less toxic form [21"]. M1 such systems are remarkable for their high affinity and specificity for metals and the molecular basis of several resistance loci are n o w being w o r k e d out. Expression of metal resistance is regulated in bacteria, offering the possibility of very sensitive bioswitches for metal biosensors and for the recovery or remediation systems themselves.

First generation recovery by non-living biomaterials First generation metal recovery strategies using non-living biomaterials have consisted of non-specific microbial or plant biomass, sometimes treated with a strong base to enhance the affinity of the biomass for met-

als [4,22"]. The capacity of these systems approaches that of existing ion exchange resins and, like these resins, biosorbents generally lack the desired specificity in metal binding. The major ligands are negatively charged (carboxyl or phosphoryl) groups that have a range of unspecified affinities for both hard and soft metals. Captured metals are released either by incineration, or b y washing the biomass with strongs acids and then re-generating its metal ion exchange capacity by treatment with sodium carbonate or a similar caustic solution. In the protonated form, the biomass can b e used to recover certain anions such as chromium. The efficiency of metal release b y acid from the biomass is comparable to release b y ion exchange resins. Biosorbents can b e manufactured for approximately the same cost as existing chemical resins and in formulations with greater mechanical and osmotic stability. The major drawback to the widespread use of biosorbents is that there are few characteristics to differentiate their performance from existing synthetic resins, which are already well established in the water treatment market.

Second generation systems using non-living biomaterials The second generation of non-living metal biorecovery systems could involve ion-specific solid phase resins, designed on the basis of current research into the methods used by living systems to generate highly specific, high-affinity metal-binding sites; examples of some of the m a n y elegant studies include [23",24",25"'-27"',28]. At present there is only one patented example of such a process [P1], employing siderophore analogs for removal of iron and radionuclides from aqueous media [22"]. As w e learn more about modulation of metal affinity and specificity in living systems, resins that can be efficiently loaded or stripped with slight changes of pH, temperature, or counterions, can be developed.

Conclusion Metal bioremediation is an area to which biotechnology (in the strict sense of using genetically engineered organisms or designer biochemicals for novel tasks) has not b e e n applied. Given that metal remediation requires such unusual requirements as active concentration of intrinsically toxic elements, it will definitely benefit from genetic engineering. Molecular genetics and biochemistry can elucidate the chemical bases for the high affinity and specificity which are the hallmarks of biological systems. Such knowledge can then guide the synthesis of biomimetic systems that are more robust, and have a higher intrinsic capacity and specificity, than the present biosorbent tools. Genetic engineering will also allow optimization, over-expression, and/or complete redesign of living cell metal-sequestering or metal-mobilizing systems. The problems of metal ion bioremediation are very different from those of organic bioremediation, but investments in intensive interdisciplinary collaborations between basic and ap-

Metals in bioremediation Summers 275 p l i e d scientists in this e c o n o m i c a l l y i m p o r t a n t a r e a c a n b e e x p e c t e d to y i e l d d r a m a t i c returns in the n e a r future.

Acknowledgments I appreciate critical comments on this article by Corale Brierley, Don Kurtz, and Michael Mooiman. My own work on toxic metal resistant bacteria is funded by the National Institutes of Health and Battelle-Pacific Northwest Laboratories.

References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.

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19.

SLAWSONRM, VAN DYKE MI, LEE H, TREVORSJT: G e r m a n i u m and Silver Resistance, A c c u m u l a t i o n , a n d Toxicity i n M i c r o o r g a n i s m s . P l a s m i d 1992, 27:72-79. Describes a newly developing area. The mechanisms have yet to be worked out. 20. MISRATK: Bacterial R e s i s t a n c e to I n o r g a n i c M e r c u r y •. Salts a n d O r g a n o m e r e u r i a l s . Plasmid 1992, 27:4-16 A review that thoroughly describes the best understood metal resistance system: the biotransformation of mercury compounds. 21. •.

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8. •.

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9. •.

23. ..

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22. •.

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AO Summers, Department of Microbiology and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2605, USA. [email protected]