Growth and activity of pure and mixed bioleaching strains
on low grade chalcopyrite ore
J.J.Plumb
a,*
,N.J.McSweeney b ,P.D.Franzmann
a
a
CSIRO Land and Water,Underwood Avenue,Floreat,WA 6014,Australia
b
School of Biomedical,Biomolecular and Chemical Sciences,The University of Western Australia,35Stirling Highway,Crawley,WA 6009,Australia
Received 23May 2007;accepted 22September 2007
Available online 7November 2007
Abstract
The kinetics of iron and sulfur oxidation by selected bioleaching strains grown in simple chemically de?ned media have in some instances been studied exhaustively.However,there is very little available information on the growth and leaching kinetics of known biole-aching strains on low grade chalcopyrite ore.Eleven known bioleaching strains were shown to be capable of growing,although at a slow rate,on low grade chalcopyrite ore in pure culture.When the same eleven strains were added as a mixed inoculum and enriched on low grade ore at 28,35,45,55and 65°C,the resultant mixed populations were shown to comprise both iron and sulfur oxidising microor-ganisms.Rates of iron and sulfur oxidation by mixed cultures generated at each temperature varied considerably.Fastest rates of iron and sulfur oxidation were observed at 65°C.Dominant strains in the mixed cultures were determined using PCR-DGGE and included Metallosphaera hakonensis ,Sulfolobus metallicus ,Sulfobacillus thermosul?dooxidans ,Acidimicrobium ferrooxidans ,Acidithiobacillus caldus and Leptospirillum ferriphilum .Further development of methods to accurately quantify microbial growth and activity on low grade ore will likely help us understand the role of bioleaching microorganisms in heap bioleaching of low grade ore.Crown Copyright ó2007Published by Elsevier Ltd.All rights reserved.
Keywords:Archaea;Bacteria;Sul?de ores;Bioleaching;Hydrometallurgy
1.Introduction
There is a paucity of information in the public domain on the ability of bioleaching acidophiles to grow on and leach Cu from low grade chalcopyrite ore.Two pub-lished studies describe qualitatively the bacterial popula-tions associated with operating chalcocite bioheaps,but give no indication of the ability of the individual or mixed microbial populations to grow on or leach Cu from the chalcocite ore (Readett et al.,2003;Hawkes et al.,2004).Another paper describing work done by Rio Tinto as part of their Heap Leach Program at Bingham Canyon,pro-vides a useful quantitative description of the mesophilic and moderately thermophilic bioleaching organisms in the test heap environment,and identi?ed some of the bac-terial population members as strains of Acidithiobacillus ferrooxidans ,Leptospirillum ferrooxidans ,Acidithiobacillus thiooxidans and ‘‘Ferromicrobium acidophilus ’’(Ream et al.,1999).Although the results from the Rio Tinto test work showed quantitatively the distribution of bioleaching microorganisms within the test heap using MPN viable cell counts,a more detailed description of the population struc-ture in the heap and a quantitative assessment of growth rate and leaching kinetics on chalcopyrite ore was not obtained.From this study it was therefore not possible to determine the full extent of microbial diversity of the heap population or which population members were the most successful chalcopyrite bioleaching strains.
Microorganisms can be grouped generally according to the temperature range over which they grow opti-mally.Although not de?ned strictly,the temperature-based groupings are psychrophiles (<15°C),mesophiles (15–40°C),moderate thermophiles (40–60°C),thermophiles
0892-6875/$-see front matter Crown Copyright ó2007Published by Elsevier Ltd.All rights reserved.doi:10.1016/j.mineng.2007.09.007
*
Corresponding author.Tel.:+61893336253;fax:+61893336211.E-mail address:Jason.plumb@csiro.au (J.J.Plumb).
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Minerals Engineering 21(2008)
93–99
(60–80°C)and hyperthermophiles(>80°C).However, these groupings do not re?ect the ability of some strains to grow well at temperatures far removed from their temperature optimum.For example,Sulfobacillus thermo-sul?dooxidans is a moderate thermophile that grows opti-mally between40and60°C.By using the Ratkowsky equation to determine cardinal temperatures it was shown that S.thermosul?dooxidans had a T OPT of51.2°C(Franz-mann et al.,2005).However,S.thermosul?dooxidans grows well in the mesophilic temperature range(<40°C),and has an estimated T MIN of about12°C.Another strain,Lepto-spirillum ferriphilum,is considered a mesophile and was shown to have a T OPT of38.6°C and T MAX of48.5°C. So although L.ferriphilum is a mesophile it is capable of growing at temperatures within the moderate thermophile temperature range.Therefore,depending on the prevailing temperature in the heap environment,whether constant or variable,it is possible that mixed cultures of strains from the di?erent temperature groupings can establish.The response of the mixed culture to changes in operating tem-perature will depend on the makeup of the mixed culture and the ability of population members to grow at the selected temperature.A mixed culture that contains moder-ate thermophiles or thermophiles would be expected to achieve superior bioleaching performance,compared to a mixed culture containing only mesophiles,when operating temperature increases to50°C or greater.
Currently,there is no clear understanding of the mini-mum ore grade required for successful heap bioleaching of chalcopyrite ore.Mine-site operators may be able to dis-cern an ore grade cut-o?beneath which processing of ore becomes uneconomic,however,the relationship between ore grade and growth by bioleaching microorganisms is not known.The Kennecott ore used in this project was a low grade ore with about0.4%Cu content.With respect to the microbiology of bioleaching,the content of mineral sul?des and reduced iron and sulfur in the ore is important. For lithotrophic bioleaching acidophiles,ferrous iron, reduced sulfur species and mineral sul?des act as the energy sources for growth.How di?ering concentrations of these energy sources a?ect the growth of bioleaching acidophiles is not well understood.It may be useful to know the min-imum ore grade required to establish and maintain an active bioleaching population,or whether threshold con-centrations of these energy sources in?uence the success or failure of heap bioleaching or cause selection of speci?c strains.Although other factors associated with heap biole-aching,such as aeration and solution chemistry are impor-tant for microbial growth,by studying the relationship between ore grade and microbial growth,it is hoped a bet-ter understanding of how microorganisms bioleach low grade ore will be obtained.
The objectives of this research were to test the ability of pure and mixed cultures of bacteria and archaea to grow on low grade chalcopyrite ore over a range of tempera-tures,to identify the dominant microorganisms in mixed cultures,to test the ability of mixed cultures to oxidise Fe2+,S0and chalcopyrite concentrate and to evaluate the e?ect of ore grade on the growth kinetics of bioleaching strains.
2.Materials and methods
2.1.Microbial strains and routine culture conditions
Eleven microbial strains were used in this study and included Acidithiobacillus ferrooxidans DSM584,Acidi-thiobacillus thiooxidans DSM14887T,L.ferrooxidans DSM2705T,L.ferriphilum ATCC49881T,Acidimicrobium ferrooxidans DSM10331T,S.thermosul?dooxidans DSM 9293T,Acidithiobacillus caldus DSM8584T,Ferroplasma acidiphilum DSM12658T,Acidianus brierleyi DSM1651T, Sulfolobus metallicus DSM6482T and Metallosphaera hakonensis DSM7519T.Strains were grown as reported previously(Franzmann et al.,2005).M.hakonensis was grown on the same medium used to grow S.metallicus. Each of the11test strains except for M.hakonensis,were tested individually for their ability to grow on the Kenne-cott ore prior to commencement of mixed culture work. M.hakonensis was not included in the pure culture experi-ments due to a delay in receiving this culture.
2.2.Low grade ore
Low grade ore used in these experiments was obtained from the Kennecott Utah Copper operation in North America.The chemical,physical and mineralogical proper-ties of the Kennecott ore were determined using a variety of analytical techniques,including quantitative X-ray di?rac-tion,QEMSCAN,dissolution under controlled but varied conditions with ICP-AES elemental analysis of solutions. The ore contained(wt%):chalcopyrite(CuFeS2)0.5%,pyr-ite(FeS2)0.2%,with other copper sul?des bornite (Cu5FeS4),covellite(CuS)and chalcocite(Cu2S)only pres-ent at<0.05%in the ore.The elemental composition of the ore was(wt%):Cu0.5,Fe3.0,S0.7,S2à0.4,Na1.2,K5.4, Mg2.9,Ca1.4,Al6.7,Si24.9.While for the ore as a whole,this chalcopyrite was encapsulated within gangue mineral particles,in the pulverised ore sample the chalco-pyrite would be exposed to leachate as individual grains. The gangue minerals were moderately acid-consuming, necessitating continued acid addition to maintain the desired test pH.The main acid consumer was biotite,which comprised about20wt%of the ore.
2.3.Growth and activity tests
Erlenmeyer?ask(250mL)cultures containing100mL of9K medium(pH 1.8)supplemented with5%wt:vol Kennecott ore crushed and ground to a particle size of less than75l m were inoculated with10%vol:vol.Flask cul-tures were incubated at close to the T OPT for each strain with shaking at150rpm for7–14days.Two additional subcultures(10%vol:vol)were performed to remove
94J.J.Plumb et al./Minerals Engineering21(2008)93–99
carried-over Fe2+,S0,pyrite or yeast extract from stock cultures of each strain.This meant the Kennecott ore was the only available growth substrate and no organic carbon was provided.Due to acid consumption by the chalcopy-rite ore,the pH in each?ask was monitored twice a week and adjusted to pH1.8using18M H2SO4.Cell numbers were monitored using a Thoma counting chamber and a phase contrast microscope.Culture samples were collected in a microcentrifuge tube,vortexed brie?y and then centri-fuged at low speed(3000rpm for5min)to remove ore par-ticles prior to counting.
Mixed cultures were prepared in duplicate in250mL Erlenmeyer?asks using the growth medium and growth conditions described above.Mixed cultures were incubated at28,35,45,55and65°C.Each mixed culture was inocu-lated with approximately equal numbers of cells from each of the11test strains,except for the28and35°C mixed cultures which were not inoculated with M.hakonensis due to delays in receiving this strain.Given the expected T MIN($30°C)of this thermophilic strain,it seemed unli-kely that M.hakonensis or either of the other two thermo-philes would persist at these temperatures.Mixed cultures were subcultured once a week using a10%v:v inoculum and cell growth was monitored using a Thoma counting chamber and a phase microscope as described above. Cell counts were made immediately prior to subculturing. The pH of each culture was monitored and adjusted as described above.
Subsequent tests were conducted to measure the ability of mixed cultures enriched on low grade ore to oxidise Fe2+and S0and to leach Cu from low grade ore.Tests were performed in250mL Erlenmeyer?asks as described above using9K medium at pH1.8supplemented with either20g Là1ferrous sulfate or4g Là1S0or5%wt:vol of Kennecott ore.Uninoculated control?asks were pre-pared for each treatment.Tests were conducted at the same temperature used to generate each mixed culture with shak-ing at150rpm.
2.4.Microbiological and chemical analyses
Characterisation of microbial population structure in each of the mixed cultures was performed using denatur-ing gradient gel electrophoresis(DGGE)of polymerase chain reaction(PCR)ampli?ed16S rRNA genes.Brie?y, cells were separated from the mineral ore by vortexing at maximum speed for10min followed by low speed centri-fugation for1min at3,000g.Total genomic DNA was extracted from cells in the supernatant as described previ-ously(Plumb et al.,2001)but without the bead beating step.The crude DNA extracts were then puri?ed before ampli?cation using ultraclean PCR clean-up kit(MO BIO Laboratories Inc.)according to the manufacturer’s instructions.PCR of16S rRNA genes was performed using the27F primer(bacteria)or25F primer(archaea) together with the1492R primer(universal)as previously described(Plumb et al.,2002).Ampli?cation of$500bp fragments of the16S rRNA gene,and subsequent analysis of fragments using DGGE was performed as described pre-viously(Hawkes et al.,2006)except that a denaturing gra-dient of30–80%was used.Nucleic acid sequence data obtained from excised bands from the DGGE pro?les were compared with sequences in the publicly accessible Gen-Bank database using the basic local alignment search tool (BLAST;Altschul et al.,1997).
For measurement of Fe2+concentration,cells were removed from1mL of sample by centrifugation at13,000g for8min.Fifty microliters of0.1%(w/v)N-phenylanthrani-lic acid indicator and2mL0.1M H2SO4were added to 500l L of sample supernatant.The resulting solution was titrated against0.005M potassium dichromate.The ferrous iron concentration was calculated from a standard curve of ferrous iron concentration versus the volume of0.005M potassium dichromate required to reach the titration end-point.Measurements were made in triplicate.For measurement of total S and Cu concentration,samples were centrifuged to pellet cells as described above and400l L of the resulting supernatant was diluted in20mL of ddH2O. Total sulfur and Cu concentration were determined using inductively coupled plasma–optical emission spectrometry (UltraTrace Analytical Laboratories,Canningvale,Western Australia).
3.Results and discussion
All test strains grew on low grade Kennecott ore when tested in pure culture after repeated subculturing to remove carried-over substrates from stock cultures(Fe2+,S0, pyrite),albeit mostly at a relatively slow rate(Figs.1and 2).F.acidiphilum showed an initial decrease in cell numbers followed by an increase in cell numbers after four days, indicating cell growth.All strains showed either constant cell numbers or a signi?cant increase in cell numbers throughout the14day incubation period.Cell numbers of
J.J.Plumb et al./Minerals Engineering21(2008)93–9995
Acidithiobacillus ferrooxidans increased relatively rapidly initially before levelling o?.Compared to the other strains tested,the thermophilic strains S.metallicus and Acidianus brierleyi grew particularly well,increasing in number to greater than4·107cells mLà1after170h.Of all the strains tested,S.thermosul?dooxidans and Acidimicrobium ferroox-idans grew least well,perhaps re?ecting the preference of these two strains for mixotrophic or heterotrophic growth when provided with organic carbon.Growth of all strains on Kennecott ore was somewhat unexpected given the low grade of the ore and the possibility of substrate-limiting conditions or non-ideal conditions for growth of each strain (e.g.su?cient concentrations of Fe2+or reduced sulfur compounds for strains capable of oxidising only Fe2+or sulfur).These results provide an indication of the ability of the bioleaching strains to grow on Kennecott ore in pure culture under relatively ideal conditions.The near-optimal aeration and substrate availability due to the thorough mix-ing within the shake?asks would be expected to provide more favourable conditions for growth than a static heap
environment.Also,the?ne particle size of the ore used in these experiments probably promotes growth by providing better accessibility for the microorganisms to the mineral sul?des.In each of the?ask cultures the vast majority of cells appeared planktonic with no signi?cant evidence of cell attachment to the ore particles.
Cell numbers in mixed cultures grown on5%wt:vol Kennecott ore and subcultured repeatedly over several weeks,are shown in Figs.3and4.Cell numbers were greater than107cells mLà1in all of the mixed cultures. The28and35°C mixed cultures maintained good cell numbers after98days incubation and14subcultures. The28°C culture was dominated by rod-shaped cells, whereas the35°C culture contained a more even mix of rods and cocci.Given the considerable length of the incu-bation period(seven days)and the number of transfers,it appears that a well adapted mixed culture capable of grow-ing on the Kennecott ore was established.The45,55and 65°C cultures were monitored over a period of almost three months with weekly subculturing.Cell numbers greater than107cells mLà1were maintained at each of these three temperatures.Some rod-shaped cells were detected in the45°C culture,although the three higher temperature mixed cultures were dominated by cocci typi-cal of thermophilic Archaea.This result was expected for the55and65°C cultures however,it was thought that the rod-shaped bacterial cells would dominate the45°C mixed culture,given that this temperature was very close to the predicted T MIN of the three thermophilic strains. For all cultures grown on Kennecott ore it was noted that without addition of H2SO4to control pH,acid consump-tion by the Kennecott ore resulted in an increase in pH
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up to 5.0.Production of acid via biological oxidation of sulfur was not su?cient to overcome the acid consumption by the ore to maintain a low pH over the course of the seven day incubation period between subcultures.
Cell numbers in mixed cultures grown on Kennecott ore,varied considerably over time (Figs.3and 4).The reason for the variability between subcultures is not completely clear.It is likely that some of the variability was due to inac-curacies involved in performing cell counts using a phase contrast microscope,although repeated tests have shown that cell counts can be done reproducibly within one third of an order of magnitude.Variations between the propor-tions of attached and unattached cells probably contribute to variations in total cell counts although very little cell attachment in shake ?ask cultures was observed.Another possible source of variability is ore heterogeneity which would in?uence the amount of cell growth depending on the quantity and accessibility of mineral sul?de particles.PCR-DGGE analysis of the mixed cultures detected the following bioleaching strains:28°C Acidithiobacillus cal-dus ,35°C Acidithiobacillus caldus ,L.ferriphilum ,and S.thermosul?dooxidans ;45°C Acidithiobacillus caldus ,Acidi-microbium ferrooxidans and S.thermosul?dooxidans ;55°C Acidimicrobium ferrooxidans ,S.thermosul?dooxidans ,M.hakonensis and S.metallicus ;65°C M.hakonensis and S.metallicus .No archaea were detected in the 28,35and 45°C mixed cultures using PCR-DGGE.This was surpris-ing given the apparent abundance of archaea-like cell mor-photypes in the 45°C mixed culture.It is therefore not clear why no archaea were detected in the 45°C mixed culture using PCR-DGGE.
Mixed cultures were tested for their ability to oxidise Fe 2+
and S 0and to leach Cu from the Kennecott ore (Figs.5–7).All mixed cultures oxidised Fe 2+.The 55and 65°C mixed cultures achieved the greatest rate of Fe 2+oxidation with a very rapid initial rate of oxidation.A slow initial rate
of oxidation was observed in the 45,35and 28°C mixed cul-tures,particularly for the two lower temperature cultures.This result suggests that numbers of iron oxidisers were rel-atively low in the lower temperature mixed cultures and therefore achieved a slow initial rate of Fe 2+oxidation.After about three days of incubation the rate of Fe 2+oxida-tion increased signi?cantly.PCR-DGGE characterisation of the microbial populations in the mixed culture did not detect any iron oxidising bacteria in the 28°C culture and detected one iron oxidiser,L.ferriphilum ,in the 35°C cul-ture.PCR-DGGE detected both M.hakonensis and a spe-cies of Sulfolobus ,assumed to be S.metallicus ,in the 55and 65°C mixed cultures and S.thermosul?dooxidans and Acidimicrobium ferrooxidans in the 55°C mixed culture only.All of these species are capable of oxidising Fe 2+.S.thermosul?dooxidans and S.metallicus are noted metal mobilisers,capable of growing on a range of di?erent min-eral sul?des (Golovacheva and Karavaiko,1978;Huber
J.J.Plumb et al./Minerals Engineering 21(2008)93–9997
and Stetter,1991).The rate of Fe2+oxidation in the55and 65°C mixed cultures is considerable given that these tem-peratures are well beneath the T OPT determined for S.met-allicus and M.hakonensis of71and81°C,respectively.In addition,55°C is higher than the T OPT determined for Acid-imicrobium ferrooxidans and S.thermosul?dooxidans of49 and51°C,respectively,although it is less than the T MAX for each species;60and64°C,respectively.The e?ect of temperature on the rate of abiotic Fe2+oxidation is shown in Fig.5,with a signi?cant di?erence between the abiotic oxidation rate at65and28°C.
Data for the oxidation of S0by the mixed cultures were more interesting.All mixed cultures except for the45°C mixed culture showed moderate or high rates of sulfur oxi-dation.The greatest rate of S0oxidation was achieved by the65°C mixed culture.The rate of S0oxidation in the 55°C mixed culture was considerably less than the65°C mixed culture.This result is interesting as PCR-DGGE analysis showed that the microorganisms in the65°C mixed culture were also in the55°C mixed culture.The di?erence in S0oxidation rate by the mixed cultures at these two tem-peratures is di?cult to explain,but it may be due to S.ther-mosul?dooxidans preferring to oxidise Fe2+,with S0being oxidised by only the two thermophilic archaea at sub-opti-mal temperatures.S0oxidation by mixed cultures at45,35 and28°C showed contrasting results.Greater rates of S0 oxidation were achieved by the35and28°C mixed cultures than by the45°C mixed culture.This result is di?cult to explain given that PCR-DGGE detected the sulfur oxidiser Acidithiobacillus caldus in each mixed culture,and that 45°C is close to the T OPT for sulfur oxidation by Acidithio-bacillus caldus of49°C.Acidithiobacillus caldus oxidises S0 more rapidly than other bacterial strains on a S0-rich med-ium(Franzmann et al.,2005).In contrast to Fe2+oxidation, temperature was shown to have very little e?ect on the rate of abiotic S0oxidation,as was expected.
To compare the leaching performance of each of the mixed cultures at their respective temperatures,a brief leach test was performed(Fig.7).The results of the test were fairly unremarkable but show the general e?ect of temperature on the rate of Cu dissolution from chalcopy-rite.The65°C mixed culture achieved the most rapid Cu dissolution,followed by the55°C mixed culture.The abi-otic control test at65°C achieved a low initial rate of Cu dissolution for a period of about10days,before the Cu dissolution rate increased considerably.No cells were detected in the abiotic control tests at each of the two tem-peratures,so it was assumed that the e?ect was indeed abi-otic.It is possible that favourable conditions for leaching were created due to abiotic oxidation of Fe2+released from the Kennecott ore over time,thus creating favourable redox conditions.The signi?cant increase in recovery of Cu under abiotic conditions at65°C may also be due to ore heterogeneity with perhaps a higher initial grade in this sub-sample of ore.No additional measurements were made to provide con?rmation of this.At the end of the20day trial about15%of total Cu was recovered.4.Conclusions
The overall outcomes from this work were mixed.It was demonstrated that all the test strains were capable of growing on Kennecott ore in pure culture.This result was slightly unexpected,given the paucity of previous work describing successful growth of bioleaching strains on low grade chalcopyrite ore,and also the low grade of the Kennecott ore(i.e.low concentration of accessible growth substrate).Both pure and mixed cultures sus-tained growth on Kennecott ore over an extended period of time.PCR-DGGE analysis of mixed cultures showed that successful bioleaching strains under mixed culture conditions were M.hakonensis,S.metallicus,S.thermo-sul?dooxidans,Acidimicrobium ferrooxidans,Acidithioba-cillus caldus and L.ferriphilum.In all mixed cultures generated on Kennecott ore,both iron and sulfur oxidis-ing microorganisms were selected,however subsequent metabolic screening of the mixed cultures showed that mixed cultures may or may not oxidise Fe2+and S0 rapidly.
The experimental results also demonstrated the rela-tively slow growth of strains on Kennecott ore and the inability to achieve high cell densities on Kennecott ore using near-ideal growth conditions for the test strains. Experiments to determine the e?ect of ore grade on micro-bial growth kinetics were not attempted,in part due to an inability to accurately quantify di?erences in growth kinet-ics on low grade ore.Cell counting provided a useful way to monitor the overall growth of bioleaching strains,how-ever,a more convenient and reproducibly quantitative method is required in order to properly understand the role of bioleaching microorganisms in heap bioleaching of low grade ore.The knowledge gained in this preliminary study using near-ideal growth conditions,will lead to an increased understanding of the growth of microorganisms on low grade ores under conditions more typical of a heap bioleaching operation.The future application of respiro-metric methods to provide accurate and reproducible mea-surements of oxygen uptake rate may represent the best approach to furthering this work.
Acknowledgements
This work was conducted as part of AMIRA funded projects P768and P768A‘‘Improving Heap Bioleaching’’. The authors thank AMIRA and the sponsoring companies, Anglo American,BHP-Billiton,Phelps Dodge,Rio Tinto, and WMC for their funding support and permission to publish this work.
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