Received January 2003; accepted February 2003nopr.niscair.res.in/bitstream/123456789/11337/1/IJBT 2(3) 451-464.pdf · chemolithotrophs, They include mesophilic Thiobacillus thiooxidans,

Indian Journal of BiotechnologyVol 2, July 2003, pp 451-464

Role of Acidothermophilic Autotrophs in Bioleaching of Mineral Sulphide Ores

Valentina V Umrania*Microbiology Department, MVM Science and Home Science College, Kalawad Road, Rajkot 360 002, India

Received 13 January 2003; accepted 20 February 2003

Living organisms synthesize a wide array of enzymes, which catalyze a myriad of reactions both inside andoutside the cell. The acidothermophilic iron-oxidizing bacteria represent a group of obligately autotrophicchemolithotrophs, They include mesophilic Thiobacillus thiooxidans, Leptospirillum ferrooxidans and thermophilicbacteria such as Sulfolobus and Acidianus species. Several studies have shown the importance and feasibility ofmicrobiological prospecting for sulphide ore deposits. Acidothermophilic autotrophic bacteria are now considered asan ideal source to exploit more unusual commercial applications of Geo-biotechnology, especially for metal andmining industry. The mining of copper, uranium, molybdenum, zinc, silver, gold, etc, from their sulphide ores issuccessfully possible with these microbes. The predominant characteristic of chemolithotrophs is their ability tosurvive and flourish in a completely inorganic aqueous environment with a supply of oxidizable substrate and CO2,

A number of different species have now been isolated from high temperature regions and their potential for the rapidleaching of some ores. The ability of such isolates to tolerate high concentration of toxic heavy metals makes themexcellent tools for accumulation and/or for biochemical transformation of metals. The outline of such applicationsare described in the present review.

Keywords: acidothermophiles, bioleaching, mineral sulphide ores, bio-oxidation, chemolithotrophs

IntroductionMicroorganisms play a predominant role in the

solubilization, transport and deposition of metals andminerals in the environment. A better understandingof these processes has allowed scientists to furthercharacterize bacterial leaching of metals from oresand to propose innovative microbe based technologiesfor metal reclamation. Microbial technology presentsan economic alternative for mining and waste watertreatment at a time when high grade mineral resourcesare depleting, energy costs are increasing and adverseenvironmental effects are becoming more apparent asa result of few limitations of conventionaltechnologies.

The recovery of metals from their ores has been anobject of man's activities for centuries. The role ofbacteria in dissolving metallic sulphides from theirores has been known since Roman times. The practiceof percolating acidified water through heaps of lowgrade ores to remove the metal sulphide formed bybacterial activity within the dump ore was carried outin Anglesey in the 16th century and in Spain in the 18th

century. But this process, known as bacteriallyassisted leaching, has only been developed on a large-

*Tel: +91-281-2461104,2577842; Fax: +91-281-2461984Email: [email protected]

scale in this century principally in the USA~ Chile andRomania for copper recovery. Extraction of uraniumis currently being carried out using this technique inCanada. Further development of this technology maylead to the recovery of metals from undergroundregions inaccessible to man by conventionaltechniques and to the extraction of metals from theash and slag left after the burning of coal containingsignificant amount of metallic ores.

The use of acidophilic, chemolithotrophic iron- andsulphur-oxidizing microbes in processes to recovermetals from certain types of copper, uranium andgold-bearing minerals or mineral concentrates is nowwell established. During these processes insolublemetal sulphides are oxidized to soluble metalsulphates. Mineral decomposition is believed to bemostly due to chemical attack by ferric iron, with themain role of the microorganisms being to reoxidizethe resultant ferrous iron back to ferric iron. Currentlyoperating industrial biomining processes have usedbacteria that grow optimally from ambient to 50°C,but thermophilic microbes have been isolated thathave the potential to enable mineral biooxidation tobe carried out at temperatures of 80°C or higher. Thedevelopment of higher-temperature processes willextend the variety of minerals that can becommercially processed (Rawlings, 2002).

452 INDIAN J BIOTECHNOL, JULY 2003

The interaction between microorganisms andminerals (or metals) is extremely varied but mainlymicrobiological processes related to three differenttypes are of importance in the mineral biotechnology(Karavaiko & Groudev, 1985).

1. Oxidation of sulphide minerals, elementalsulphur, ferrous iron and some other metals intheir reduced valency forms by chemolithotrophicbacteria.

2. Formation by heterotrophic microorganisms oforganic (organic acids, polysaccharides, etc.) andinorganic (peroxides, etc.) compounds causingdegradation of the mineral structures bysolubilization of their compounds to the relevantions or as complexes and chelates. In some cases,the degradation is connected with an enzymaticoxidation or reduction of individual chemicalelements.

3. Formation by heterotrophic microorganisms andalgae of large amounts of biomass or of somemetabolites (mainly organic compounds but alsosome inorganic compounds such as hydrogensulphides), which are capable of accumulating orprecipitating metal ions from solutions.

The processes of the first type are the most specificand at the same time are largely used for the practicalpurposes. The need of bacterial leaching is felt in theare beneficiation processes mainly due to the reasonssuch as:

1. The high-grade mineral resources are depletingcontinuously and new ores are not being locatedso frequently.

2. The conventional pyrometallurgical or otherprocesses are becoming more and more costly.

3. The mining industries generally create a lot ofpollution, which add to environmental imbalances(Brierley, 1982).

The principal benefits of bacterial leaching are lowoperating costs and mitigation of air pollution.

Chemistry of the Leaching ProcessesThe role of T. ferrooxidans and thermophilic

bacteria in leaching is complex and not preciselydefined. Some researches supports the concept thattheir function may be "indirect", whereby themicrobes generate ferric iron, which oxidizes the

mineral. Other investigations indicate "direct"leaching in which the microbes contact and adhere tothe mineral surface, oxidizing the mineral without theuse of ferric iron oxidant.

The fundamental reaction for indirect leaching isthe microbial oxidation of ferrous iron (equation 1) inacidic condition for the purpose of energy generation:

The ferric sulphate thus generated serves to oxidizeminerals such as chalcopyrite (equation 2), chalcocite(3), covellite (4), and uraninite (5):

(2)

(3)

(4)(5)

The resulting soluble metal sulphates are recoveredby solvent extraction, ion exchange, or by othermethods. The iron, now reduced to the ferrous state, isreoxidized by the microorganisms accroding toequation (1). The sulphur, which is often present as anend product of the metal solubilization (equations 2, 3and 4) may also be oxidized to produce sulphuricacid.

The "direct" mechanism of metal leaching takesplace without ferrous iron as an oxidant. Pyrite maybe oxidized directly by the microbes as per equation(6).

... (6)

This results in the solubilization of iron. The iron issubsequently oxidized accroding to equation 1 and theferric iron then participates in the "indirect" leachingprocess. Copper-containing minerals can also beleached by the "direct" process (equations 7 and 8).

2CuFeS2 +811202~ 2CUS04 + Fe2(S04h + H20(7)

The "direct" leaching mechanism has not beenconclusively demonstrated for iron-containing

UMRANIA: BIOLEACHING OF MINERAL SULPHIDE ORES

minerals such as chalcopyrite (equation 7). Becausesolubilized iron facilitates the "indirect" mechanisms,even minerals without iron, such as chalcocite(equation 8) are oxidized in part by the "indirect"process. The presence of any iron as a contaminantwill initiate an "indirect" leaching reaction.

Pyrrhotite (Fel_xS; or FeS in a simplified formula)is another common iron sulphide in sulphidemineralizations, but its chemical and microbiologicaloxidation is not well characterized. The oxidation ofthis mineral has been reported to be relatively fasterthan that of pyrite (Ahonen et aI, 1986) and itsmicrobiological oxidation at elevated temperatureshas also been reported (Norris 3l. Parrott, 1986), butstrictly comparable experimental data relative to othersulphide minerals are not available. Pyrrhotiteoxidation is an acid-demanding reaction that alsoproduces major amounts of elemental sulphur as a by-product. Ferric iron can again act as a chemicaloxidant and is regenerated via bacterial oxidation:

The microbiological leaching of chalcopyrite is arelatively slow reaction by· comparison withbiological leaching rates of secondary Cu-oxide andCu-sulphide minerals. Complete oxidation ofchalcopyrite can be represented by equation (7).

Iron plays a central catalytic role in the chemicaland biological oxidation of mineral sulphide minetailings and its oxidation state is the main factor thatdetermines the prevailing redox potential. After irongets oxidized to the ferric oxidation state, it tends tohydrolyze in solution and this reaction leads to a netincrease in acid formation in the environment. Theferric hydroxide can further interact with varioussulphates to form Fe(III) hydroxy-sulphate andoxyhydroxide complexes.

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Thermophiles in LeachingBeck (1967) observed temperature of 60 to 80°C in

leach dump environments and concluded that one mayhave "to consider the role of microbial activity inleaching operation where these temperatures occur."Murr and Briereley (1978) observed increase intemperature during copper leaching. In pyriteleaching, significant differences occurred with T.ferrooxidans and A. brierleyi; the thermophileextracted about 1.3 times more iron over anequivalent period of time (Ngubane & Baecker,1988).

A wider temperature range can be very importantfor organisms in various ways, since it makes anorganism more versatile with regard to changes in theenvironment (Wiegle, 1990). As thermophiles canfunction at elevated temperatures, the kinetics ofbiological and most of the chemical reactions areinfluenced favourably with increases in temperature.The presence and importance of thermophiles incommercial leaching operation has been described.Both these factors are important in leachingoperations. Faster reaction rates reduce the time forwhich ores and waste must be processed to extractmetal values. These thermophilic acidophiles possessthe ability to bioleach over a wide range oftemperatures. They are potentially well suited toindustrial leaching applications where considerabletemperature fluctuations limit the growth of othernon-thermophilic bioleaching microorganisms.(Plumb et aI, 2002).

Sulfolobus and Related OrganismsA microorganism that grows at high temperature

and low pH was isolated first from an acid thermalregion of Yellowstone National Park by Brierley in1966 and further characterized by Brierley andBrierley in 1973. The genus Sulfolobus was firstdescribed by Brock et al (1972). These organismsgrow in a temperature range of 50 to 80°C underacidic conditions. As these organisms oxidize sulphuras well as ferrous iron they become excellentcandidates for use in microbial leaching. The isolationof Sulfolobus species from hot acid springs and soilsaround the world has been reported in the book"Thermophiles" (Brock, 1978).

Weiss (1974) speculated that survival of theorganism in extreme environments may depend on theunique features of their cell envelops. Several of thepep tides of Sulfolobus are unique (Langworthy et aI,

454 INDIAN J BIOTECHNOL, JULY 2003

1974) and only inositol-containing phsopholipids arepresent. He proposed that thermophily is related to thelong chain of isopranols, and acidophily is correlatedwith ether lipids. Millonig et al (1975) characterizedtwo strains of acidophilic thermophiles isolated by DeRosa et aZ (1975) from volcanic hot springs nearNaples, Italy. Using the transmission electronmicroscope, these organisms and Sulfolobus (Brock etaZ, 1972; Brierley & Brierley, 1973) were comparedand characterized. The presence of pilli was noted inthese organisms. Shivvers and Brock (1973) alsoreported on sulphur oxidation by S. acidocaldarius.De Rosa et al (1975) studied six microbial strains,designated MT, similar to Sulfolobus (Brock et ai,1972). These bacteria isolated from pools with atemperature range of 74 to 89°C and a pH of 1.4 to2.6 were cultured in spring water amended with 0.1%yeast extract.

A number of strains of spherical bacteria, lackingcell walls and resembling S. acidocaldarius have beenshown to be able to grow at temperatures up to about80°C using sulphur and iron oxidation for energy.Such bacteria are able to leach recalcitrant mineralssuch as chalcopyrite and molybdenite at 60°C moreefficiently than mesophilic bacteria (Brierley & Murr,1973; Brierley, 1974, 1975, 1977, 1978; Brierley &Brierley, 1978). Zilling et aZ in 1980 establishedtaxonomic relationship among several strains ofSulfoZobus and proposed three species of SulfoZobus:S. acidocaldarius, S. brierleyi and S. solfataricus. Theisolation of Sulfolobus from metal leachingenvironments was not reported until 1983. Marsh andNorris (1983) found these microbes in sample of adrainage channel (pH 1.5, 37°C) emanating from coalpile at the Birch Coppice Colliery, Warwickshire,UK.

The mechanisms for CO2 assimilation by S.brierZeyi were determined by Kandler and Stetter(1981). The autotrophic CO2 fixation occurs viareductive carboxylic acid pathway. The Calvin-Benson cycle is apparently not present in Sulfolobusspecies. In the last decade, comparative microbialleaching studies by Marsh and Norris (1983), Kargiand Robinson (1985), Brierley and Brierley (1986),Norris and Parrot (1986) have shown that appreciablyfaster rates of leaching can be obtained withthermophilic Sulfolobus than T. ferrooxidansoperating at 30 to 37°C.

In 1989, Gertrud et al, obtained and proposed threenovel strains of spherical thermoacidophilic metal-

mobilizing archaebacteria from a Solfataric field inItaly. These new isolates grew aerobically on singlesulphidic ores like pyrite, chalcopyrite and sphaleriteand on combinations of them (ore mixture G6 andG IN). Growth was also obtained on the syntheticsulphides CdS, SnS, and ZnS and on So. Arsenopyrite,bornite, cinabar, chalcocite, covellite or galena andthe synthetic sulphides CuS, FeS, MoS2 and Sb2S3 didnot serve as substrates. During growth on So,sulphuric acid was formed by the isolate TH-2. Thepresence of yeast extract (0.005%) did not change theproduction rate of sulphate significantly. The newisolates were able to grow on complex organicsubstrates such as beef extract, cas amino acids,peptone, tryptone and yeast extract but growth wasnot obtained on sugars.

The effects of organic substances on growth andinorganic substance oxidation by Sulfobacillusthermosulfooxidans and Asporogenes species werestudied by Vartanyan et al (1990). A new genus-Sulfurococcus, thermoacidophilic archaebacteria,which oxidizing sulphur, ferrous iron and sulphideminerals have been reported by Karavaiko et al(1993). In the same year, Concetta and Teresareported about chemolithotrophic, sulphur-oxidizingbacteria from a marine shallow hydrothermal vent ofvolcano (Italy). Fatty acid composition of the lipids inthermoacidophilic bacteria of the genus Sulfobacillusand carbon metabolism in S. thermosulfidooxidansstrain 1269 has been reported by Zakharchut et al(1994). Konishi et al (1995) studied bioleaching ofpyrite by acidophilic, thermophilic, Acidianusbrierleyi. They reported that the specific growth rateon pyrite for A. brierleyi was about four times morethan that of the mesophilic T. ferrooxidans.

Another lob-shaped thermophilic isolate Sulfolobushakonensis was obtained by Takayanagi et al (1996)from a geothermal area in Hakone, Japan. The isolatewas found to be aerobic, facultative chemolithotrophgrew on SO and reduced sulphur compounds optimallyat pH 3.0. In the same year, Williams et al reported anew strain of aerobic thermophilic bacteria from hotsprings in Portugal and were identified as Thermusoshimai. A study on the enhanced stability ofcarboxypeptidase from S. solfataricus at high pressurewas carried out by Bee et al (1996). In the same year,Wright et al did work on cloning of a potentialcytochrome P450 from the S. solfataricus. Masullo etal also in the same year did work on purification andcharacterization of NADH oxidase from two species

UMRANIA: BIOLEACHING OF MINERAL SULPHIDE ORES

of Sulfolobus, S.acidocaldarius and S. solfataricus. Astudy was carried out by Ianniciello et al in 1996 onthe expression of thermostable elongation factor I-a inE. coli from S. solfataricus. Immunochemicaldetection of ADP-ribosylating enzymes in thisorganism was also carried out (Faraone-Mennella etal, 1996). A study on a see Y homologous gene in thecrearchaean S. acidocaldarius was carried out byKath and Schaefer (1996). Santin et at in the sameyear studied enzymatic synthesis of 2-~-D-galactopyranosyloxy-ethyl methacrylate (GaIEMA)by this thermophilic archaeon. Knapp et at (1996) didthermal unfolding of the DNA-binding proteins Sso7dfrom the same hyperthermophile.

Rod-shaped Thermophilic Thiobacillus likeMicrobes

The facultative, thermophilic, iron-oxidizingmicrobes catalyze important reactions for leachingcertain metals from low-grade ores and mine wastes.Several reports have been published, which dealspecifically with mineral leaching by facultative,thermophilic, iron-oxidizing microbes. Le Roux andWakerely (1988) were the first to report leachingusing facultative, thermophilic bacterium, TH-l,growing at 50°C were more effective than T.ferrooxidans in leaching nickel from volarite(Ni2FeS4)'

Observation of rod-shaped organisms, which grewat temperatures up to 55°C was made by Kaplan(1956), Schwartz and Schwartz (1965), Brierley(1966), and Schoen and Ehrlich (1968). In anecological study of hot, acid soils, Fliermans andBrock (1972) observed the presence of Thiobacillus attemperatures of 55°C. In a study of bacterial survivalat high temperatures and low pH, Weiss (1973)observed rod-shaped bacteria at concentration of 107

to 108 cells / ml in environment at pH 2 to 3 and at 75to 80°C. Mosser et al (1974) noted that some hotsprings in Yellowstone National Park containthermophilic Thiobacilli. Bohlool (1975) observedrod-shaped bacteria in New Zealand hot springsranging from 43 to 84°C. Sulfolobus and the rod-shaped bacteria coexist in some springs, but generallywhere both organisms are found, the rodspredominate. Le Roux et al (1977) isolated severalthermophilic, rod-shaped bacteria on ferrous iron andthiosulfate media from hot springs in South WestIceland where temperature ranged from 58 to 86°Cand a pH of 4.1 to 8.9. However, the moderate, iron-

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oxidizing, thermophilic bacteria are recognizablylarger than T. ferrooxidans; cell sizes range from 1.6to 4.2 mm in length and about Imm in diameter(Brierley, 1978). Research by Noguchi et al (1977) onthe acidostability of T. ferrooxidans spheroplasts maybe applicable to the stability of acidophilicthermopiles. Spheroplasts lack the peptidoglycanlayer but remain acid stable.

Thiobacillus TH-2 isolated from a test leach facilityrequired organic supplement for the growth. The samewas found to be true for Thiobacillus TH-3 isolatedfrom a copper leach dump. T. thermosulfidooxidanswas found to be heterotrophic growing on sugars(Golovacheva & Kavavaiko, 1977). Marsh and Norris(1983) demonstrated chemolithoautotrophic growth ofseveral strains of facultative thermophiles usingferrous iron and minerals sulphides. The data provideincreasing evidence for the ability and potential foruse of facultative thermophiles in mineral leaching.

Sugio et al (1985, 1987) carried out a systematicstudy of the sulphur-oxidizing systems of-varieus T.ferrooxidans and T. thiooxidans strains, i.e. involvinga large number of strains, three different substratesand three different activities. They obtained resultswhich indicated the existence of variability amongdifferent strains not only in their ability to grow oncertain substrates but also in their response todifferent substrate, by changing Fe2+ and SOoxidativeactivities. This variability in adaptation was inagreement with the possible mechanism as describedearlier by Holmes et al (1988). In 1996, Shooner et alobtained and proposed a novel species of facultativelyautotrophic, moderately thermophilic bacterium, T.thermosulfatus from sewage sludge samples enrichedwith elemental sulphur. This isolate showedproduction of various intermediates during growth onthiosulphate. These included tetrathionate, trithionate,and sulphate. Robertson et al (2002) isolated sevenFe2+ oxidising acidophilic bacterial strains at 50°Cfrom a pyritic coal from Collie, Australia and from aFe2

+ oxidising fIuidised bed reactor running at 60°C,which was originally inoculated with the Collie coal.The 16S rRNA gene of five of the strains waspartially sequenced. The strains isolated from thereactor were closely related (99% similarity in genesequence) to Sulfobacillus thermosulfidooxidans andthe strains isolated directly from the coal had a 97%gene sequence similarity to Sulfobacillusyellowstonensis. Thiobacillus like acidothermophilicorganisms could solubilize pyrrhoitite in the presence

456 INDIAN J BlOTECHNOL, JULY 2003

of 14 heavy metals at pH 2.5 and 60°C after theiradaptation with all the toxic heavy metals in naturalindustrial soils (Umrania et al, 1998).

Factors Affecting BioleachingFollowing mineral sulphide dissociation, the

chemical oxidation of the reduced valence state ionicspecies is greatly enhanced by the catalytic activity ofthe ubiquitous mixed communities ofmicroorganisms. A major contributor to the microbialconsortia is the chemoautotrophic bacterium, T.ferrooxidans, the other sulphur- and iron oxidizingthermophilic bacteria include Sulfolobus species aswell as Th-1, Th-2 and Th-3, Acidianus brierley(Brierley & Brierley, 1973, 1978; Le Roux et al,1977). The degradation of these mineral materials is adynamic process involving a succession of microbialpopulations, which develop according to theprevailing environmental conditions. They, in turn,are controlled by a complex array of physico-chemical conditions, which are generally site-specific.

Temperature ProfileThere have been few attempts to measure heat

profiles in mine tailings. Temperature distributionshave been measured in waste rock dumps to evaluateheat source distributors as indicators of mineralsulphide oxidation sites. Harries and Ritchie (1983)showed that surface temperature changes affectedtemperature distributions in a waste rock dump downto a depth of approximately 6 m. Fluctuations inambient temperature are more likely to affect airflowthrough waste materials thus influencing the oxygenlevel. The oxidation of pyrite, a common componentof the minerological assemblages associated withmine tailings and waste materials, is an exothermicreaction (Harries & Ritchie, 1983). Indeed,temperature can increase up to 56 to 59°C wherepurely chemical reactions involving sulphides, copperbearing waste and copper ore have been studied (Murr& Berry, 1976; Murr & Brierley, 1978). Even highertemperatures in excess of 80°C have been observed inlow-grade copper dumps in Bulgaria (Groudev et al,1978) and in the USA (Beck, 1967).

Thus, it is now known that groups of specializedthermophilic chemolithotrophic bacteria maycontribute to the biological oxidation of tailings andwastes at significantly higher temperatures than thosefavoured by the mesophilic Thiobacilli.

As reported by Brierley and Le Roux (1977),ferrous iron oxidation occurred 50% at 30°C, 97% at

40°C and 94% at 50°C after five days of incubation.The apparent greater decrease of ferric iron at 50°Cwas attributed to its decreased solubility at the highertemperature, shorter incubation periods used at 55 and60°C assuming that the rate of any biologicaloxidation would be greater.

Growth FactorsBrierley and Le Roux (1977) suggested the

requirement of yeast extract by moderatethermophiles, which made it difficult to use thesebacteria in metal leaching from cost point of view.But supplementing the media with partially reducedsulphur compounds (e.g. NiS, FeS, S203, etc.)supported the growth of microbes without addition ofyeast extract. However, reduced sulphur source wasnot required by all moderate thermophiles. In thepresence of yeast extract and Fe+2,TH-1 incorporated1% carbon from CO2, while T. ferrooxidans derived78% of its carbon from CO2, suggesting that moderatethermophiles do not fix CO2 (Brierley et al, 1978),which was proved wrong by incorporating 14C02 andcomparing CO2 fixation of TH-3 and T. ferrooxidans,which was almost same (Schacklett, 1983). May be,moderate thermophiles require higher concentrationof CO2, as it was shown by him by increasing CO2concentration from - 0.03% v/v to 1 to 10% v/v.

As mentioned earlier, thermophilic Thiobacilli TH-1 is an acidophilic chemolithotrophic heterotrophgrowing on media containing ferrous iron or pyritewhen supplemented with yeast extract or glutathione(Brierley et al, 1978). Virtually, CO2 fixation is nottaking place during growth on iron. The growth ratewas maximum with 0.005 to 0.02% (w/v) yeastextract and it was reduced with lower or higher yeastextract concentration. Growth was completelyinhibited by 0.1% (w/v) glucose in the presence of0.02% (w/v) yeast extract. FeS04 -grown or pyrite-grown inoculum of the thermophile were reported togrow rapidly at 50°C when transferred to elementalsulphur, copper sulphide, pyrite, chalcopyrite, or acopper concentrate when yeast extract was alsoprovided. However, serial subculture of the organismon sulphur resulted in successively less growth witheach transfer and eventual failure to grow.

A similar thermophile (Brierley & Lockwood,1977) could grow rather slowly at pH 2.6-3.0 whenmedium employed with cysteine and cystine (100mg/l) in the absence of yeast extract. For continuedsuccessful subculture in a cysteine supplemented Fe

UMRANIA: BIOLEACHING OF MINERAL SULPHIDE ORES

medium, TH-3 appeared to require a further growthfactor, which could be supplied by a trace amount(e.g. 0.5 mg/l) of yeast extract. At the lower yeastextract concentration only TH-3 was able to maintainthe initially high rate of ferrous iron oxidation (Norriset al, 1978).

By itself, yeast extract will serve as a sole energysource for these organisms but in manometric studies,oxygen was not used by the bacteria when yeastextract was the only substrate. Most investigatorshave given this need for yeast : extract only asuperficial examination, but Shivvers and Brock(1973) reported that supplementing inorganicsubstrates with yeast extract has a complex effect onchemoautotrophic metabolism. They proposed thatyeast extract affects both carbon assimilation andenergy generation. Sulphur oxidation was greatlyinhibited by yeast extract, but because of increasedcellular production, the total sulphur oxidized wasonly reduced by approximately one third. Possibly,enzymes responsible for inorganic substrate oxidationwere repressed and CO2 assimilation was likewisesuppressed.

The thermophilic Thiobacillus, isolated by Le Rouxet al (1977) has been studied by Brierley and Le Roux(1977). Acidothermophilic Thiobacillus grew onferrous iron at 30 to 50°C, but 0.02% yeast extractwas required. Growth did not occur at highertemperatures. Oxygen uptake was not enhanced whenthe bacterium oxidized iron in the presence of yeastextract, and oxygen uptake increased with increasingiron concentration to 81 mM (4.9 g/l). The growth ofacidothermophilic Thiobacillus on pyrite requiresyeast extract, and growth was observed at 40 to 55°C;pyrite oxidation occurred from pH 1.1 to 2.6.Increasing pyrite concentrations enhanced oxygenuptake. The author measured growth on pyrite by pHdecline, which infers oxidation of the sulphidemoiety; iron dissolution was not measured, so it wasnot known whether the iron moiety was biogenicallyoxidized. When the organism was supplied withferrous iron, pyrite, and yeast extract as substrates,ferrous iron was not entirely oxidized. Brierley and LeRoux (1977) suggested that this might be due to themicrobe having a two-enzyme system; one for pyriteoxidation and the second for ferrous iron oxidation.The authors suggested that these systems functionindependently. Restriction profiles of chromosomalDNA were studied by Kondrat'eva et al (2002) indifferent Acidithiobacillus ferrooxidans strains grown

457

on medium with Fe2+ and further adapted to anotheroxidation substrate (So, FeS2, or sulphide oreconcentrates ).

pH ProfileThe oxidative dissolution of a sulphide mineral is

commonly associated with an increasing acidificationof the surrounding medium. This alone provides aheavy selection pressure on the development of themicrobial succession since a range of pH conditionsare encountered. Variations in pH also affect thedevelopment and viability of microbial populationsthrough the availability of electron donors such asferrous iron, the oxidation of which is sensitive to pH(Kelly & Tuovinen, 1988). The minerologicalcomposition of the tailings or waste materialinfluences both mineral sulphide dissociation andbacterial establishment. Highly silicaceous orcarbonaceous gangue associations consume acid;thereby displacing conditions beyond the pH rangesuitable to many leaching organisms. The chemicalstability of iron and its compounds is very sensitive toconditions of pH and eH and will to a large degreedetermine the types of biological populations thatdevelop. Iron oxidation is usually rapid and issensitive to both pH and oxygen concentration,particularly at pH values above 3.5 (Ackman &Kleinmann, 1984). Thus, it could be said proved thatthe growth of autotrophic iron bacteria are largelydependent on the establishment of appropriate acidicenvironments.

Agitation ProfileThe availability of oxygen in leach dump is

undoubtedly one factor, which controls bacterialmetal extraction. CO2 solubility is low in acidsolutions and therefore may be a limiting factor ingrowth of chemolithotrophic acidophilic bacteria(Brierley, 1978). These aerobic bacteria requireadequate supply of oxygen, which can be achieved inthe laboratory by aeration or shaking.

Ore CharacteristicsThe effect of particle size on leaching has been

extensively studied for chalcopyrite and sphalerite.The size of the particles to be leached is critical.Torma et al (1970) found highest zinc extraction rate(17.6 mm/hr) with the finest particle size of sphaleriteconcentrate. If the total available surface area of theparticle is increased, the rate of metal extraction isincreased to a point. Pinches et al (1976) also

458 INDIAN J BlOTECHNOL, JULY 2003

concluded that the most important factor affecting theextraction of copper from a concentrate was the sizeof mineral particles. They also discovered as didTorma (1976) that regrinding allows additionalcopper to be solubilized from chalcopyriteconcentrate. They found that the rates increaselinearly with decreasing particle size but it was lessdependent when the particle size was very small.They were working with particle sizes ca. 4 to 20 urn.When the surface area was increased, either bydecreasing the particle size or by increasing the solidconcentration, each had similar effects on the leachrate. Therefore, surface area concentration is a realvariable in leach rates. These findings correspond tothe studies on sphalerite by Torma et al (1970) and onarsenopyrite concentrates by Pinches (1975). It wasshown that as the extraction of metal increasesexponentially, the growth of bacteria also increasesexponentially. It was suggested that as the particlesurface area decreases because of bacterial attachmentand reaction products, leaching decreases. It wasfurther suggested that particle-particle collision resultsin bacterial attrition and reduce the effective numberof bacteria taking place in the reaction. These studiesstrongly indicate that mineral particle size anddistribution influence the bacterial growth rate andhence leaching of metal sulphide mineral. Brierley(1977) reported greatest copper extraction from thesmallest seive sizes (-16+48 mesh; 1.00 mm -300urn), of low grade porphyry ores with chalcopyrite.These results were similar to the results obtained byEhrlich and Fox (1967) that the particles with greatersurface areas leached faster. Examination of theleaching of chalcopyrite by Sulfolobus BC atincreasing pulp densities was carried out by Le Rouxand Wakerley (1988). They also found markedinfluence of pulp density on the rate of leaching.Maximum leaching rate of approximately 50 mgCu/lIhr was attained at about 15% w/v pulp densityand good bacterial growth occurred up to 25% w/vpulp density.

Considerable potentiality of Sulfolobus was alsoreported by Norris & Parrot (1986). A Sulfolobusstrain has been shown to be very effective at solubili-zation of nickel from pyrrhotite and pentlanditeconcentrates. Soluble nickel reached about 0.7 g Ni/lwith a medium initially containing 1% concentratepulp density increased to about 10% pulp density. TheSulfolobus solubilized iron at a much greater rate thanT. ferrooxidans.

The effect of pulp density (w/v) on bioleachingculture capacity with respect to this copperconcentrate was studied by Rubioa and Garda Frutos(2002). The results of the batch tests show that it ispossible, operating at 10% of pulp density to attaincopper extraction of 94% in 10 days and, at higherpulp densities (20%), to attain good copper extraction(80%) in only 14 days. In the same way, the culturehas been amply tested with different chalcopyriticores and copper concentrates.

Bioleaching Activity on Natural OresIn bioleaching, chemical and minerological

characteristics of an ore is important and hence mustbe established for each ore (Tuovinen, 1990).Bioleaching becomes a method of choice when CuS,FeS, PbS, ZnS, etc. occur together and are to berecovered quantitatively. Pyrite is the most abundantsulphide mineral and is associated with other sulphideminerals, such as those of copper, nickel and zinc orwith uranium ores. Pyrite and arsenopyrite may alsooccur in the same mineralizations, as in precious-metal-containing sulphidic ore materials (Lawrence &Marchant, 1988).

The first successful leaching of molybdenum by amicroorganism was accomplished using Sulfolobus(Brierley & Murr, 1973). The mineral molybdeniteMoS2 is particularly refractory to leaching. A secondproblem with leaching of molybdenite is toxicity ofthe resultant soluble molybdenum to the leachingorganism. Although, mesophilic T. ferrooxidans isinhibited by 5 to 90 mg Moll (Tuovinen et al, 1971),Sulfolobus has a remarkable resistance to this metal.Molybdenite serves as an energy source for growth ofSulfolobus (Brierley, 1974) respiration, using sulphuras an energy source, occurred at 2000 mg Moll; cellgrowth was inhibited near a concentration of 750 mgMoll.

Sulfolobus was examined for possible extraction ofcopper from copper sulphite minerals. Preliminarystudies (Brierley & Murr, 1973) indicated that themicrobe could oxidize a chalcopyrite (CuFeS2)concentrate with copper solubilization occurring atrate of 10 to 16 mg Cu/l/day over a thirty day periodat 60°C. Another study of copper leaching (Wyckoff& Davidsons, 1977) suggested that microbesresembling Sulfolobus were more effective than"Sulphur bacteria" (presumably T. ferrooxidans) inleaching chalocopyrite.

UMRANIA: BIOLEACHING OF MINERAL SULPHIDE ORES

In a more detailed study, S. acidocaldarius wasused for copper leaching from porphyry copper orewith chalocopyrite as the primary mineral (Brierley,1977). The ore sample, obtained from Duval SierritaCorporation, AZ, possessed a mineralization ofprimarily chalcopyrite, with some digenite (CU9S5)and covellite (CuS). The ore assayed 0.31% Cu,0.05% Mo, 0.02% Zn, 5.7% Fe, 0.01 % Pb, and 0.01 %Ni. The amount of copper leached by S.acidocaldarius was 38% at an average rate of21mg/l/day; only 4% of the copper was leached fromthe control column at an average rate of 1.9 mg/l/day.Similar leach results obtained using a chalocopyriteore obtained from the Pinto valley Mines, citiesService Corporation AZ by Brierley in 1980.Sulfolobus appears to be advantageous in extractingcopper from the refractory mineral, chalcopyrite. T.ferrooxidans and Sulfolobus were compared forcopper leaching from various copper containingminerals including chalcocite, covellite andchalcopyrite (Brierley et al 1978). Marsh et al (1983)made a comparative study of Sulfolobus species withregard to their ability to oxidize minerals. The rateand extent of mineral dissolution were found to bestrain dependent. A study by Acevedo et al (1983),involving the leaching of Chilean copper ores,suggested that the facultative thermopile TH-3 wasthe most effective microbe for solubilizing copper.Mineral concentrate dissolution at high temperatureswas carried out by Norris and Parrot (1986).

Thermophilic microbial treatment of precious metalores was carried out by Hutchins et al (1988) andreported considerable improvement in the economicsof gold recovery. Barrett et al (1988) isolated andcharacterized a moderately thermophilic mixedculture of autotrophic bacteria and their application tothe oxidation of refractory gold concentrates andreported their results in Perth International GoldConference. Ngubane and Baecker (1988) alsoreported successful utilization of thermophilic pyriteleaching by Sulfolobus brierleyi. Lawrence andMarchant (1988) made the comparison of mesophilicand thermophilic oxidation systems for the treatmentof refractory gold ores and concentrates. As reportedby Brierley (1990) Sulfolobus and Acidianus speciescan be used for solubilization of copper, molybdenumand nickel as well as other metals, which occur insulphide minerals (such as pyrite, arsenopyrite).

From an economic point of view, microbial metalextraction would become more attractive if the metal

459

could not be easily extracted by conventionalmethods, or if these were too costly. Bioleachingrecovery system for cobalt, nickel, molybdenum,gold, etc could be attractive because of the value ofthese metals. The main disadvantage of theseprocesses is linked with their relatively slow leachingrates than conventional pyrometallurgical processes,because of the slow growth rates of microbes. It canbe further optimized with faster growing and morespecifically their metal solubilizing ability formaximum conversion of insoluble metal sulphides.

A simple membrane dialysis bioreactor wasdeveloped for a large-scale axenic culture ofSymbiobacterium thermophilum, a symbioticthermophile that requires co-cultivation with anassociating thermophilic Bacillus strain S for normalgrowth (Veda et al, 2002). A new type ofmicrofiltration (MF) bioreactor has been developedfor improving efficiency of the production ofextremophilic enzymes (Schiraldi et al, 2001). In spiteof the difficulties in cultivating hyperthermophiles,they achieved, in 300 hrs of fermentation, more than38 gll dry weight of Sulfolobus solfataricus using aMF technique, and demonstrated that the activity ofalcohol dehydrogenase (ADH), as the reporterenzyme, was not affected by cell density. Howeverhyperthermophile cultivation is difficult to scale upbecause of evaporation and the very low growth rate.Thus, to achieve high productivity they cultivated, inthe MF bioreactor, recombinant mesophilic hostsengineered for the production of two thermophilicenzymes, namely, trehalosyldextrin-forming enzyme(SsTDFE) and trehalose-forming enzyme (SsTFE)from Sulfolbbus solfataricus. An electric water heaterhas been modified by Worthington et al (2003) for thelarge-scale cultivation of aerobic acidophilichyperthermophiles to enable recovery of secretedproteins. Critical changes include thermostatreplacement, redesign of the temperature controlcircuit, and removal of the cathodic anticorrosionsystem. These alterations provided accuratetemperature and pH control. The bioreactor was usedto cultivate selected strains of the archaeon Sulfolobussolfataricus and other species within this genus.

The published literature is also available on a fewbacterial strains resembling T. ferrooxidans andpossessing thermophilic, acidophilic or/and iron-oxidizing characteristics. They include Sulfolobus BC,Sulfolobus solfataricus, S. acidocaldarius, S.hakonensis, Acidianus brierleyi, TH-l, TH-2, TH-3,

460 INDIAN J BIOTECHNOL, JULY 2003

T. thermosulfatus, Thermus oshimai, etc. (Brock et al,1972; Brierley & Brierley, 1973; Le Roux et ai, 1977;Zilling et al, 1980; Hutchins et ai, 1988; Shooner etal, 1996~ Williams et ai, 1996; Takayanagi et al,1996; Urnrania, 1998). These types of microbes arenow in use for recovery of metal from waste ores andthe leaching of iron pyrite from coal. Sulfolobus likeorganisms capable of covellite solubilization wereisolated from industrial soil samples, Rajkot, India byUrnrania & Joshi (2002). The isolate JV Cu-12acidothermophilic bacterium could solubilize copperup to 91% within 5 days.

As suggested by Petersen and Dixon (2002)thermophiles have been shown to be the onlymicroorganisms to leach chalcopyrite successfully.Heap leaching may be a feasible alternative toconventional bioreactors, provided a high temperatureenvironment can be maintained within the heapwithout external heating. In their study thermophilicheap leaching of a chalcopyrite concentrate coatedonto inert support rocks was studied in sets of smallheated columns. The temperature was graduallyincreased to 70°C, while successively introducingvarious mesophile and thermophile cultures.Individual columns were dismantled afterprogressively longer leach periods and the residualconcentrates analyzed. Copper extractions in excessof 90% were achieved within 100 days.Electrochemical techniques were conducted to clarifythe role of solution potential and temperature under avariety of experimental conditions similar to thosefound during the mesophilic and thermophilicbiooxidation of chalcopyrite (CuFeS2) (Tshilombo etal,2002).

Metal ToleranceMetal tolerance among microorganisms is well

known with several areas .now receiving intensiveattention at the molecular and genetical level. Variousmicrobial mechanisms are implicated for survival inthe presence of potentially toxic concentrations ofmetal species. A given organism often relies directlyand/or indirectly on several survival strategies. Manyof such organic and inorganic metal species can beaccumulated by microbial cells as a result of physico-chemical mechanisms and transport systems ofvarying specificity, independent of, or directly andindirectly dependent on metabolism. The pervasivenature of metals in the environment has resulted in thewidespread appearance of metal resistance inmicroorganisms (Duncan et ai, 1994).

Microbial metal resistance is heterogeneous in boththeir genetic and biochemcial bases and may bechromosomally-, plasmid- or transposon encodedwith one or more genes being involved (Duncan et ai,1994). At the biochemical level microorganismsdemonstrate a diversity in the type of resistancemechanisms they have evolved. This includes sixdifferent fundamental types. These differentmechanisms may occur singly or in variouscombinations to produce resistance. The fivemechanisms generally proposed are illustrated asfollows:

1 Exclusion of the metal by a permeability barrier2 Exclusion by active export of metal from the cell3 Intracellular physical sequestration of the metal

by binding proteins to prevent it from damagingmetal sensitive cell materials

4 Extracellular sequestration5 Extracellular detoxification where the metal is

chemically modified to render it less active.

In addition to the five general mechanisms thespecific reduction in metal sensitivity of cellulartargets for metal damage provides a sixth mechanismof resistance. .The specific maintenance of a metalsensitive cell component may be achieved in thefollowing four ways:

1 By mutation altering the component to decreaseits sensitivity, without unduly affecting its normalrole

2 By increasing the amount of the affected cellcomponent, if inactivation is not total

3 By repair of the component, in general onlyfeasible for DNA

4 By bypassing it, either through utilizing aplasmid-encoded metal resistant form of thecomponent to bypass the metal sensitivechromosomal component, analogous to thecommon mechanism of trimethoprim resistance(Amyes & Gemell, 1992), or through increasingactivity in an alternative (shunt) pathway that isrelatively metal-resistant.

Metal toxicity could reduce industrial applicationof bioleaching processes (Norris & Kelly, 1978). Anotable tolerance to heavy metals is found in commonleaching bacterium, T. ferrooxidans (lngledew, 1986;Boscecker, 1987) and this property is clearlyexhibited during iron oxidation (Agat~, 1982),

UMRANIA: BIOLEACHING OF MINERAL SULPHIDE ORES

Bioleaching process is affected by the toxicity ofmetal like Ag, Co, Cu, Hg, Ni and Zn (Garcia & DaSilva, 1991). Metals like mercury and silver are verytoxic to the bacteria even at low concentration(Mahapatra & Mishra, 1984; Garcia & Da Silva,1991). According to Kazutami et al (1975) silver ionhad the most harmful effect on growth and the ironoxidizing activity of T. ferrooxidans even at 10-3 Mconcentration.

Silver is frequently associated with base metalsulphide such as Cu, Pb and Zn (Gupta & Ehrlich,1989). Available literature reports inhibition oforganism at Ag concentration as low as 0.005 mM(Norris & Kelly, 1978). Royez al (1981) have alsoreported 0.1 ppm Ag concentration inhibitory for thegrowth of T. ferrooxidans culture. Tuovinen (1990)observed a lag phase longer than 3 weeks for the fourstrains of T. ferrooxidans tested by them.

T. ferrooxidans can tolerate Aluminium, 0.37 M;Zinc, 0.15 M; Cobalt, 0.17 M; Manganese, 0.18 M;Copper, 0.16 M; Chromium, 0.10 M; Uranium, 0.001M; Mercury, 0.05 M; Silver 10-9 M_1O-5 M; andMolybdenum, 0.03 M; while oxides of Selenium,Tellurium and Arsenic are inhibitory (Brierley, 1978).

On one hand, a very few reports are available onthe metal tolerance by moderate thermophilicchemolithtrophic bacteria and on the other hand, theimportance of thermophiles in metal recoveryprocesses at mine sites are getting more applicability.The heavy metal sensitivity restricts the application ofthermoacidophiles in metal extraction processes. Theability of acidothermophilic bacteria to oxidizeinorganic substrate makes them potential microbes foruse in the leaching of metallic sulphides and reduceorganic load.

The leaching of molybdenite (MoS2) by Sulfolobuswas first reported in 1973 by Brierley and Murr andfurther described thoroughly by Brierley in 1974. Theleaching of molybdenite by the chemoautotrophicthiobacilli is limited because of their inability totolerate high concentration of soluble molybdenum.But metal tolerant Sulfolobus can be developed whichcan tolerate 2000 ppm (=21 mM) hexavalentmolybdenum, thus toxicity of Mo is not a problemwith this Sulfolobus. A molybdenite concentrate isleached at a maximum rate of 6.6 mg Mosolublized/l/day; this rate was maximized bysupplementing the medium with 0.02% yeast extractand 1% iron (II) sulphate. The tolerance to highconcentration of soluble molybdenum is unique to this

461

organism. Several copper sulphide ores andconcentrates were leached in stationary batch reactorsusing Sulfolobus. The first evaluation of the potentialof A. brierleyi for bioleaching of metals indicated thatthis microbe could grow on the common coppersulphide minerals chalcocite and chalcopyrite.(Brierley & Brierley, 1978).

Further studies considered the use of extremethermophiles for copper leaching. Sulfolobus BCdeveloped tolerance for copper from a limit of about 3g Cull to about 27 g Cull during progressiveacclimatization (Le Roux & Wakerley, 1988).Comparison of Sulfolobus BC with T. ferrooxidansfor copper leaching ability from the chalcopyriteconcentrate demonstrated the thermophile to be muchmore effective. Copper leaching by the Sulfolobusoccurred at an overall rate of about 11.5 mg Cull/hrwith 83% copper extraction at the end of the test. TheT. ferrooxidans leached the copper at a rate of 2.5 mgCull/hr for 19% copper extraction (Le Roux &Wakerley, 1988).

Sulfolobus are not only utilized for molybdenite,chalcopyrite and chalcocite but also for preciousheavy metal recovery. These thermophiles viz.Acidianus, Sulfolobus and thermophilic eubacteriahave been evaluated for microbial pretreatment of theore to degrade the encapsulating sulphide matrix. Thisencapsulated sulphide matrix will facilitate contactbetween the cyanide and the precious metal (Hutchins "et al, 1988; Barrett et al, 1988).

The influence of metallic ions on the activity ofSulfolobus BC has been examined (Mier et al, 1996).The maximum tolerance to increasing quantities ofmetal in cultures grown on copper concentrate wasdetermined after a period of progressive adaptation. Incase of silver, mercury, ruthenium and molybdenum,only adapted organisms led to an increase in theoxidation rate of ferrous iron and sulphur.

Thus, the use of thermophilic bacteria for microbialpretreatment of precious metal ores can offereconomic advantages over common bioleachingprocesses in terms of both increased reaction rates anda lower requirement for cooling. Additional work isrequired to evaluate the relative merits of the differentgroups of leaching bacteria as applied to preciousmetal ores.

AcknowledgementThe author gratefully acknowledge Dr A D Agate,

former Director, Agharkar Research Institute, Pune,

462 INDIAN J BIOTECHNOL, JULY 2003

Dr S R Dave, Head, Microbiology Department,Gujarat University, Ahmedabad and Dr J M Dave,Retd Principal, Gujarat Agriculture University fortheir encouragement and guidance. She is indebted toDr Neepa Pandhi, Microbiology Department, M NVirani Science College, Rajkot for her help in readingthis manuscript.

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