thermophilic archaeal community succession and function change associated with the leaching rate in...

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Thermophilic archaeal community succession and function change associated with the leaching rate in bioleaching of chalcopyrite Wei Zhu, Jin-lan Xia , Yi Yang, Zhen-yuan Nie, An-an Peng, Hong-chang Liu, Guan-zhou Qiu School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China Key Lab of Biometallurgy of Ministry of Education of China, Central South University, Changsha 410083, China highlights " A first study on community succession and function associated with chalcopyrite leaching rate. " Sulfolobus metallicus was most sensitive to the environmental change. " Acidianus brierleyi showed the best adaptability and highest sulfur oxidation ability. " The bioleaching rate correlated closely with the consortium function embodied by soxB gene. article info Article history: Received 22 November 2012 Received in revised form 24 January 2013 Accepted 25 January 2013 Available online 7 February 2013 Keywords: Community succession Sulfur oxidation soxB gene Bioleaching Thermophilic archaea abstract The community succession and function change of thermophilic archaea Acidianus brierleyi, Metallosph- aera sedula, Acidianus manzaensis and Sulfolobus metallicus were studied by denaturing gradient gel elec- trophoresis (DGGE) analysis of amplifying 16S rRNA genes fragments and real-time qPCR analysis of amplifying sulfur-oxidizing soxB gene associated with chalcopyrite bioleaching rate at different temper- atures and initial pH values. The analysis results of the community succession indicated that temperature and initial pH value had a significant effect on the consortium, and S. metallicus was most sensitive to the environmental change, A. brierleyi showed the best adaptability and sulfur oxidation ability and predom- inated in various leaching systems. Meanwhile, the leaching rate of chalcopyrite closely related to the consortium function embodied by soxB gene, which could prove a desirable way for revealing microbial sulfur oxidation difference and tracking the function change of the consortium, and for optimizing the leaching parameters and improving the recovery of valuable metals. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The mesophilic microorganisms have been widely used in the hydrometallurgical field in the past several decades (Rawlings, 2002). However, increasing number of researchers have been inter- ested in using moderately and/or extremely thermophilic organ- isms to improve the dissolution rate of valuable metals (Konishi et al., 2001; Qin et al., 2013; Wang et al., 2012), due to exothermic reaction during bioleaching that results in temperature increasing to 50 °C or greater in the pregnant leach solution (Riekkola-Vanha- nen, 2007). As reviewed in the literatures (Konishi et al., 2001; Schnell and Rawlings, 1997), the thermophiles, including Acidianus brierleyi, Acidianus manzaensis, Metallosphaera sedula and Sulfolobus metallicus, have inherent advantages for industrial applications of metal extraction from various sulfide minerals when the temperature rises over 60 °C. In our previous work, the result was also confirmed (Zhu et al., 2011). Previous studies had mainly focused on the aspects of leaching behavior and leaching mechanism of thermophilic archaea, while few information about their community succession and function change in the bioleaching of sulfide minerals had been revealed (Qiu et al., 2008). Various kinds of microbe shows distinct meta- bolic activity at different conditions, the environmental factors exercise great impact on the structure and function of microbial community (Li et al., 2011). Moreover, the current methods of characterizing the microbial metabolic activity, such as pH value, sulfate ion concentration and cell concentration are often affected by many factors, and it is hard to accurately reflect the role of a specific species of microbe. However, this information is very important for optimizing the leaching parameters and improving the recovery of valuable metals, therefore, it is necessary to find a desirable method to efficiently characterize the role of various microbes at different conditions during bioleaching processes. In some species of bacteria, sulfur oxidation is catalyzed by sul- fur-oxidizing (Sox) enzyme systems (Chen et al., 2007). Sox is a well-characterized multi-enzyme system for thiosulfate oxidation, and is capable of oxidizing various reduced sulfur compounds 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.135 Corresponding author. Tel.: +86 731 88836944; fax: +86 731 88710804. E-mail addresses: [email protected], [email protected] (J.-l. Xia). Bioresource Technology 133 (2013) 405–413 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

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Bioresource Technology 133 (2013) 405–413

Contents lists available at SciVerse ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Thermophilic archaeal community succession and function changeassociated with the leaching rate in bioleaching of chalcopyrite

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.01.135

⇑ Corresponding author. Tel.: +86 731 88836944; fax: +86 731 88710804.E-mail addresses: [email protected], [email protected] (J.-l. Xia).

Wei Zhu, Jin-lan Xia ⇑, Yi Yang, Zhen-yuan Nie, An-an Peng, Hong-chang Liu, Guan-zhou QiuSchool of Minerals Processing and Bioengineering, Central South University, Changsha 410083, ChinaKey Lab of Biometallurgy of Ministry of Education of China, Central South University, Changsha 410083, China

h i g h l i g h t s

" A first study on community succession and function associated with chalcopyrite leaching rate." Sulfolobus metallicus was most sensitive to the environmental change." Acidianus brierleyi showed the best adaptability and highest sulfur oxidation ability." The bioleaching rate correlated closely with the consortium function embodied by soxB gene.

a r t i c l e i n f o

Article history:Received 22 November 2012Received in revised form 24 January 2013Accepted 25 January 2013Available online 7 February 2013

Keywords:Community successionSulfur oxidationsoxB geneBioleachingThermophilic archaea

a b s t r a c t

The community succession and function change of thermophilic archaea Acidianus brierleyi, Metallosph-aera sedula, Acidianus manzaensis and Sulfolobus metallicus were studied by denaturing gradient gel elec-trophoresis (DGGE) analysis of amplifying 16S rRNA genes fragments and real-time qPCR analysis ofamplifying sulfur-oxidizing soxB gene associated with chalcopyrite bioleaching rate at different temper-atures and initial pH values. The analysis results of the community succession indicated that temperatureand initial pH value had a significant effect on the consortium, and S. metallicus was most sensitive to theenvironmental change, A. brierleyi showed the best adaptability and sulfur oxidation ability and predom-inated in various leaching systems. Meanwhile, the leaching rate of chalcopyrite closely related to theconsortium function embodied by soxB gene, which could prove a desirable way for revealing microbialsulfur oxidation difference and tracking the function change of the consortium, and for optimizing theleaching parameters and improving the recovery of valuable metals.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction Previous studies had mainly focused on the aspects of leaching

The mesophilic microorganisms have been widely used in thehydrometallurgical field in the past several decades (Rawlings,2002). However, increasing number of researchers have been inter-ested in using moderately and/or extremely thermophilic organ-isms to improve the dissolution rate of valuable metals (Konishiet al., 2001; Qin et al., 2013; Wang et al., 2012), due to exothermicreaction during bioleaching that results in temperature increasingto 50 �C or greater in the pregnant leach solution (Riekkola-Vanha-nen, 2007). As reviewed in the literatures (Konishi et al., 2001;Schnell and Rawlings, 1997), the thermophiles, including Acidianusbrierleyi, Acidianus manzaensis, Metallosphaera sedula and Sulfolobusmetallicus, have inherent advantages for industrial applications ofmetal extraction from various sulfide minerals when thetemperature rises over 60 �C. In our previous work, the resultwas also confirmed (Zhu et al., 2011).

behavior and leaching mechanism of thermophilic archaea, whilefew information about their community succession and functionchange in the bioleaching of sulfide minerals had been revealed(Qiu et al., 2008). Various kinds of microbe shows distinct meta-bolic activity at different conditions, the environmental factorsexercise great impact on the structure and function of microbialcommunity (Li et al., 2011). Moreover, the current methods ofcharacterizing the microbial metabolic activity, such as pH value,sulfate ion concentration and cell concentration are often affectedby many factors, and it is hard to accurately reflect the role of aspecific species of microbe. However, this information is veryimportant for optimizing the leaching parameters and improvingthe recovery of valuable metals, therefore, it is necessary to finda desirable method to efficiently characterize the role of variousmicrobes at different conditions during bioleaching processes.

In some species of bacteria, sulfur oxidation is catalyzed by sul-fur-oxidizing (Sox) enzyme systems (Chen et al., 2007). Sox is awell-characterized multi-enzyme system for thiosulfate oxidation,and is capable of oxidizing various reduced sulfur compounds

406 W. Zhu et al. / Bioresource Technology 133 (2013) 405–413

(hydrogen sulfide, elemental sulfur, thiosulfate, and sulfite) to sul-fate (Harada et al., 2009). As one of the most important member ofSox multi-enzyme system, SoxB is not only essential for thiosulfateoxidation but also responsible for sulfide oxidation (Azai et al.,2009), and its detection in a sulfur-oxidizing microbe might beused as an indicator for the presence of components of the Sox en-zyme system and a first indication for evaluating some microbialfunction in sulfide oxidation (Pandey et al., 2009). The Sox en-zymes of mesophilic and neutrophilic bacteria have been foundand well characterized. But little information is available fromthe acidophilic bacteria and archaea that widely used in bioleach-ing (Chen et al., 2007). The partial soxB gene has been detected incloning of the terminal oxidase genes of M. sedula (Auernik et al.,2008; Kappler et al., 2005). Interestingly, we have found in our pre-vious research that there was an identical related sulfur-oxidizingsoxB gene existed in thermopiles A. brierleyi, A. manzaensis, M. sedu-la and S. metallicus, and confirmed soxB gene of four thermophilicarchaea could oxidize sulfur with similar physiological basis.

At present, many techniques have been used in the analysis ofmicrobial community structure (Johnson, 2001), of which thedenaturing gradient gel electrophoresis (DGGE) technique is a fin-gerprint identification technology that widely applied in dynamicanalysis of microbial community diversity, but it cannot providethe cell growth profiles and information of functional gene expres-sion (Xing and Ren, 2006). Real-time qPCR is a reliable technologyto study the microbial community in bioleaching environments.Though real-time qPCR cannot distinguish some microorganismswith very high similarity, it is a useful tool to assist microbial func-tion analysis, and to further understand microbial leaching capac-ity (Chen et al., 2009). Therefore, by combining the DGGE methodand real-time qPCR technique that monitor the changes of commu-nity succession and function of the consortium, further under-standing to the contributions of each specific species to thebioleaching, and the critical role of functional gene in bioleachingof chalcopyrite could be obtained.

In this paper, a predefined thermophilic archea consortium con-sisting of A. brierleyi, M. sedula, A. manzaensis and S. metallicus wasused to leach chalcopyrite. DGGE method was used to investigatethe thermophilic archaeal community succession, and real-timeqPCR technique was applied to monitor the expression level of soxBgene, which was used to characterize the function change of theconsortium during bioleaching of chalcopyrite. Finally, the rela-tionship between the function change of the consortium and theleaching rate of chalcopyrite was analyzed and the effect of envi-ronmental factors was studied.

2. Methods

2.1. Microorganisms and culture media

Four thermophilic archea used in this work were A. brierleyi(JCM 8954), M. sedula (YN 23), A. manzaensis (YN 25) and S. metal-licus (YN 24), which were conserved by the Key Laboratory of Bio-metallurgy, Ministry of Education of China, Central SouthUniversity, China. The basal medium used in this study contained3.0 g/L (NH4)2SO4, 0.5 g/L K2HPO4, 0.5 g/L MgSO4�7H2O, 0.1 g/LKCl, 0.01 g/L Ca(NO3)2 with 0.2 g/L yeast extracts.

2.2. Mineral samples

The mineral samples were collected from Dexing in Jiangxiprovince, China. The mineral consisted of chalcopyrite (80%), pyrite(5%), quartz (5%) and others (5%). The main contents of mineralwere (mass fraction): Cu 32.02%, Fe 30.90% and S 22.65%. The min-eral was grinded to fine powder with the size less than 75 lm.

2.3. Bioleaching experiments

Bioleaching experiments were carried out with the consortiumin 250 mL Erlenmeyer flasks at 65 �C with initial pH (1.0, 1.5, 2.0)and with initial pH 1.5 at temperature (55, 65 �C) on a rotary sha-ker at 180 rpm. The flasks contained 100 mL of basal medium sup-plemented with 10 g/L chalcopyrite as the sole energy source. Theinitial cell density was approximately 1 � 108 cells/mL. The initialpH of the culture was adjusted with diluted sulfuric acid. All exper-iments were performed in triplicate at the same conditions and theabiotic control was also run. Evaporated water was compensatedby additional distilled water. Samples were taken at regular inter-vals to analyze the cell density, copper concentration, communitysuccession and function change of the consortium during bioleach-ing experiments.

The cell density was determined by direct counting with a Neu-bauer chamber counter. Copper concentrations in solution weredetermined by atomic absorption spectrophotometry. The commu-nity succession and function change of the consortium were ana-lyzed with the methods described in Sections 2.4 and 2.5.

2.4. Community structure analysis

2.4.1. Preparation of DNAIn order to sample with sufficient representation, the free and

attached microorganisms of three parallel flasks were used to ex-tract the DNA every 2 days. The separation method of attachedmicroorganisms was described by Zeng et al. (2010). The leachingsolution of samples was allowed to settle for 1.5 h and the super-natant was decanted. Then the pellets were re-suspended with10 mL Milli Q water in a 50 mL centrifuge tube. After this, 1 g ofglass beads with a diameter of 0.2 mm was added into the tube.The tube was pressed on a vortexer for 8 min of vigorous vortexing.Then about 36 mL Milli Q water was added into the tube and thevortexing continued for 2 min. After this, the 50 mL mixture wascentrifuged at 2000g for 2 min to separate the ore residue and solu-tion. About 0.1 mL of the supernatant was used to count the cellnumber under the optical microscope and the others were trans-ferred to a 250 mL flask. The ore residue was used again, under-went vigorous vortexing for 10 min and centrifugation at 2000gfor 2 min, followed by cell collection and counting. After this, theremaining ore residue was then washed repeatedly until no bacte-ria remained. Solutions washed from the pellets were mixed to-gether with the supernatant, and centrifuged at 10000g for10 min at 4 �C to collect the cell.

Total DNA was extracted from the cell pellet of previous collec-tion by washing twice in 10 mM Tris containing 10 mM EDTA pH8.5. Subsequent steps for DNA extraction were described previ-ously (Zhou et al., 2007). The crude DNA was purified by usingWizard plus SV Minipreps DNA purification system (Promega Cor-poration, USA) and stored at �20 �C.

2.4.2. Primers and PCR amplification for DGGEThe 16S rRNA gene fragments were amplified with primers

ARCH 344F-GC (50-ACGGGGTGCAGGCGCGA-30) and ARCH 915R(50-GTGCTCCC CCGCCAATTCCT-30), which are for universal primersfor conserved archaeal 16S rRNA (Casamayor et al., 2002). A 40-bpGC-rich sequence (GC-clamp: 50-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-30) was attached to the 5P end of primer344F-GC to prevent complete melting of the DNA fragments duringthe DGGE analysis. The 16S rRNA gene fragments (around 600 bp)suitable for subsequent denaturing gradient gel electrophoresis(DGGE) analyses were obtained by PCR as previously described(Karr et al., 2005). PCR amplification reactions were performedwith the reagents supplied with T-Gradient ThermoBlock (Biome-tra, Germany). A hot start PCR was performed at 95 �C for 5 min

Table 1Primers used for real-time qPCR.

Species/GI Primers Primer sequences Amplicon length (bp)

Forward primer (50–30) Reverse primer (50–30)

GI:42794901 soxB TAGTGGAAGTGCCCCACAAC AGTCTTGGCTTCTGGCTCTG 213A. brierleyi 16S rRNA TAGGAGGCTTTTCCCCACTT GGAGTACCTCCGACCTTTCC 221M. sedula 16S rRNA ACGGCGGTGATACTTACAGG GACACCTAGCCTGCATCGTT 221S. metallicus 16S rRNA TGGGGCTTTTCTACGCTCTA TCTAGGAGTACCCCCGACCT 232A. manzaensis 16S rRNA AGAGGGCTTTTCCCTACTGC GCCCCTACTCTGGGAGTACC 223

Fig. 1. DGGE fingerprint of archaeal communities in bioleaching of chalcopyrite: (a) with initial pH 1.5 at 55 �C, (b) with initial pH 1.5 at 65 �C, (c) with initial pH 1.0 at 65 �Cand (d) with initial pH 2.0 at 65 �C.

W. Zhu et al. / Bioresource Technology 133 (2013) 405–413 407

and a touchdown PCR was performed as follows. The annealingtemperature was initially set at 71 �C and was then decreased by0.5 �C every cycle until it was 61 �C; then 15 additional cycles werecarried out at 61 �C. Denaturing was carried out at 94 �C for 1 min,primer annealing was performed by using the scheme describedabove for 1 min, and primer extension was performed at 72 �Cfor 1 min. The final extension step was 10 min at 72 �C.

2.4.3. DGGE electrophoresis and their fingerprinting patterns analysisDGGE electrophoresis and their fingerprinting patterns were

analyzed according to the procedure described by He et al.(2010). The cells of four microorganisms used in the study werefirst collected separately for DGGE–PCR amplification, and then

four PCR products were mixed to work as marker. DGGE electro-phoresis was performed using a model DGGE-1 2001 electrophore-sis system (C.B.S Scientific Company Inc., CA.) with a denaturinggradient of 55–75% in a 6% polyacrylamide gel, following the man-ufacturer’s instructions. The gel was made from 10 mL of 0%-dena-turing solution (2 mL of 50� TAE, 20 mL of 30% acrylamide/bis-acrylamide (37.5:1), and 80 mL of water) and 10 mL of 100%-dena-turing solution (2 mL of 50� TAE, 20 mL of 30% polyacrylamidesolution, 80 mL of formamide, and 42 g of urea) using a GM-40Gradient Maker according to the manufacturer’s instructions. Priorto cast, 80 lL of ammonium persulfate (APS) and 6 lL of TEMEDwere added to each solution. PCR products were mixed with 1/3volume of 10� sucrose loading buffer, and DNA fragments were

Fig. 2. Analysis of the relative DGGE band abundance with initial pH 1.5: (a) at55 �C and (b) 65 �C.

408 W. Zhu et al. / Bioresource Technology 133 (2013) 405–413

separated for 5.5 h at 200 V and 60 �C. The gel was stained for20 min with ethidium bromide or silver nitrate and documentedusing a UV transillumination and VisiDoc-It imaging systems(UVP).

DGGE fingerprinting patterns were analyzed by the softwareQuantityone-1-D (Version 4.6.2) and Matlab statistical computingpackage (version 7.1). The former is a powerful, flexible softwarepackage for imaging and analyzing 1-D electrophoresis gels. Theprincipal components analysis (PCA), showing every band patternas one plot and relative changes in community structure, are pre-sented by the distance between the plots.

2.5. Community succession and function analysis

2.5.1. RNA and DNA extractionThe extraction and purification of total RNA were similarly de-

scribed by Galleguillos et al. (2008). The cells were collected by fil-tering through a 0.2 lm pore size membrane and samples werestored at �80 �C. To preserve the RNA during the extraction,1 mL of RNA protect solution (QIAGEN) was added to each filter.RNA was purified using RNeasy kit (QIAGEN) from the filters. TheRNA samples were treated with DNase RQ-1 (Promega) for 1 h at37 �C and then stored at �80 �C. The quality of total extractedRNA was confirmed by 1% agarose gel electrophoresis andquantified using a Thermo Scientific NanoDrop� ND-1000 spectro-photometer and then served as the templates in reverse transcrip-tion reactions to synthesize cDNA with First Strand cDNA SynthesisKits (Fermentas RevertAid™, K1622).

DNA was extracted from the cells collected by filtering 10 mL ofpure cultures of four strains through a 0.2 lm pore size membrane

as described previously (Zhou et al., 2007), and was used to con-struct standard curves.

2.5.2. Detection of PCR primers specificityResults from our previously study indicated that soxB gene ex-

isted in thermophiles of A. brierleyi, A. manzaensis, M. sedula andS. metallicus, and soxB gene of four thermophilic archaea could oxi-dize sulfur with similar physiological basis during bioleaching ofchalcopyrite. Therefore the soxB gene primer was designed forreal-time qPCR, and 16S rRNAs of the four species were chosenas control genes for real-time qPCR analysis. All primers were de-signed by Primer Premier 3.0 on-line design system (http://frod-o.wi.mit.edu/primer3/) and listed in Table 1. The specificfragments were amplified and checked as described by Zhanget al. (2009) and Zeng et al. (2010), respectively.

2.5.3. Quantitative real-time PCRConventional PCR was performed using the ABI Veriti™ Ther-

mal Cycler (Life Tec. Co., USA). The PCR products were diluted seri-ally from 109 to 103 copies/lL by real-time PCR to constructstandard curves. Real-time qPCR was carried out with an iCycleriQ Real-time PCR Detection System (Bio-Rad Laboratories Inc., Her-cules, USA) using 12.5 lL SYBR� Green qPCR Master Mix (ToyoboCo., Ltd., Osaka, Japan), 1 lL of the corresponding primers, 1 lL oftemplate cDNA, and double-distilled water added to a total of25 lL. The amplification program consisted of 1 cycle of 95 �C for3 min, and then 40 cycles of 95 �C for 15 s, 58 �C for 30 s, and72 �C for 30 s. The detailed procedures of real-time qPCR were asdescribed by Zhang et al. (2009). All tests were conducted in trip-licate and were also designed the negative controls.

3. Results and discussion

3.1. Analysis of DGGE fingerprinting patterns

3.1.1. Thermophilic archaeal community structures at differenttemperatures

Fig. 1a shows the DGGE fingerprinting patterns of communitystructures with initial pH 1.5 at 55 �C during bioleaching. Four dif-ferent thermophilic archaea were detected in the system. A. brier-leyi was found to be the predominant microorganism, and A.manzaensis and M. sedula had approximately the same proportion,while the proportion of S. metallicus was small in the whole pro-cess. The result mentioned above was also confirmed by the anal-ysis of the relative fingerprint abundance with Quantityone-1-D(Fig. 2a). The proportion of species A. brierleyi in the bioleachingsystem was about 50%, which was the predominant microorganismin the whole process. On the other hand, the proportion of speciesS. metallicus was very low in the community. In addition, the pro-portions of species A. manzaensis and M. sedula were relatively sta-ble (Zhu et al., 2011), however, the former was in a constantincrease while the latter was in a gradually decrease.

Fig. 1b shows the DGGE fingerprinting patterns of the commu-nity structure with initial pH 1.5 at 65 �C during bioleaching ofchalcopyrite. From Fig. 1b, only M. sedula, A. brierleyi and S. metal-licus were detected and A. manzaensis was not detected in thebioleaching system on day 2. During that period, M. sedula and A.brierleyi grew very fast, while S. metallicus showed a relatively poorgrowth (Zhu et al., 2011). Since day 4, four archaea were detectedwith S. metallicus keeping a rapid growth. Meanwhile A. manzaensisalso kept a growth trend, but its dominance was far inferior to thatof S. metallicus. Compared with the increasing proportion of S.metallicus and A. manzaensis, the proportion of M. sedula and A.brierleyi began to decrease, however, it still accounted for aconsiderable proportion until the end of bioleaching. The result

Fig. 3. PCA analysis of DGGE fingerprint of archaeal communities in bioleaching of chalcopyrite: (a) with initial pH 1.5 at 55 �C, (b) with initial pH 1.5 at 65 �C, (c) with initialpH 1.0 at 65 �C, and (d) with initial pH 2.0 at 65 �C.

W. Zhu et al. / Bioresource Technology 133 (2013) 405–413 409

mentioned above was also confirmed by the analysis of the relativefingerprinting abundance (Fig. 2b). S. metallicus played a predomi-nant role during the whole process, which accounted for 21.5% ofthe original community on day 2, and increased to 32.9% on day10, followed by a decrease by 5.5% on day 14, then stabilized atapproximately 30% from day 16. A. brierleyi and M. sedula were alsothe major species in the whole bioleaching process, which ac-counted for approximately 20–50% of the community. The propor-tion of the former decreased in the early stage, followed by a trendof increasing and decreasing in the later stage while that of M.sedula was continuously decreasing to the end of bioleaching.

Comparing thermophilic archaeal community succession dur-ing bioleaching of chalcopyrite at different temperatures, it wasfound that thermophilic archaeal community structures varieddramatically. The results indicated that the change in the commu-nity was related to the influence of systems at different tempera-tures (Wen et al., 2009), i.e. the response of the communitystructures to change in operating temperature would depend onthe makeup of the mixed culture and the ability of populationmembers to grow at the selected temperature (Plumb et al., 2008).

In order to better understand the changes of thermophilicarchaeal community with initial pH 1.5 at different temperatures,the principal component analysis (PCA) of community structurewas performed (Fig. 3a and b). The results showed that the com-munity succession could be divided into three stages while the

performance of individual species was quite different during thewhole bioleaching process at different temperatures. From thebeginning to day 6, the spots on PCA graph were close together,which meant community structures of those spots were similar.In this stage A. brierleyi and M. sedula were the most predominantmicroorganism in two bioleaching systems. Simultaneously S. met-allicus accounted for a small proportion at temperature 55 �C whileA. manzaensis changed a great deal at temperature 65 �C. From day8 to day 14, the spots were clustered into another group, in whichA. brierleyi and M. sedula were still the most predominant microor-ganism, and A. manzaensis showed an upward trend in two biole-aching systems. From day 16 to the end of bioleaching, the spotswith the similar community structures were also clustered obvi-ously. In this stage, A. brierleyi remained as predominant species,while A. manzaensis replaced M. sedula to be the second mostabundant microorganism and S. metallicus still accounted a smallproportion at 55 �C. In contrast, M. sedula, A. brierleyi and A. manza-ensis had a similar abundance and the proportion of S. metallicus in-creased with time at 65 �C till four species changed to anapproximately equal proportion in the last stage at 65 �C. In addi-tion, the trend of cluster of community at 65 �C was more obviousthan that at 55 �C. Meanwhile, the sample on day 8 indicated anoticeable transitional characteristic between two communityclusters owing to acclimating the varying systems caused by envi-ronmental factors (He et al., 2010).

Fig. 4. Analysis of the relative DGGE band abundance at 65 �C with initial pH: (a)1.0, (b) 2.0.

410 W. Zhu et al. / Bioresource Technology 133 (2013) 405–413

3.1.2. Thermophilic archaeal community structures at different initialpHs

Fig. 1c shows the DGGE fingerprinting patterns of thermophilicarchaeal community structures with initial pH 1.0 at 65 �C. As itcan be seen, the predominant microorganisms were M. sedulaand A. brierleyi during the whole process. While the proportion ofA. manzaensis was decreasing and S. metallicus was not detectedin the early stage. The relative fingerprint abundance supportedthat M. sedula and A. brierleyi were the predominant microorgan-isms and their proportions fluctuated between 18.5–44.2% and35.2–49.5%, respectively. The proportion of A. manzaensis was18.5–28% in the early stage, and then decreased to 9.1% later. S.metallicus was detected with a very low proportion, which was lessthan 10% during the whole process (Fig. 4a).

A comparison of Fig. 1c and d shows a similar community suc-cession between them, with some difference mainly on S. metalli-cus that it was in a weak growth in the early stage of thebioleaching process, but its proportion in community structuregradually increased to coincidence with M. sedula, which was justnext to the predominant microorganism of A. brierleyi by the laststage of bioleaching (Fig. 4b).

Fig. 3b–d shows the principal component analysis of archaealcommunity structure during bioleaching of chalcopyrite at differ-ent initial pH values. The analysis results indicated that the clusterof community was not clear at different initial pH values comparedwith that at different temperatures. The samples were far apartwith each other, which revealed a noticeable transition betweentwo community clusters during bioleaching at different initial pHvalues, indicating that the community structures were quite sensi-tive to the effect of pH value. The results were consistent with theliterature that the microbial community succession were stronglycontrolled by pH value (Liu et al., 2010).

It was noted that, among these species, S. metallicus wasmost profoundly influenced by environmental factors. It was the

predominant microorganism at initial pH 1.5 and 65 �C, it was,however, obviously less in the other conditions. The reason maybe associated with its poor adaptability, which needs to be furtherstudied. A. brierleyi was always one major microorganism in allbioleaching systems. That may be defined by its iron and sulfuroxidizing abilities. The oxidation of chalcopyrite could release fer-rous iron, elemental sulfur and other reductive sulfur compoundsthat can be used as the energy substance for the microbial growth(Sand and Gehrke, 2006). Vilcáez et al. (2008) indicated that as aniron and sulfur-oxidizing archaeon, A. brierleyi has the weakestiron-oxidizing ability among the four species in this study, but arelatively stronger sulfur oxidation ability. In the meantime, theproportions of M. sedula and A. manzaensis varied with time dueto their own characteristics during bioleaching of chalcopyrite atdifferent conditions, which was consistent with the report ofWen et al. (2009).

3.2. The relationship between the function of the consortium and theleaching rate of chalcopyrite

DGGE gave us the succession of community during bioleachingof chalcopyrite, however, it cannot provide the information offunction change of the consortium during bioleaching of chalcopy-rite (Xing and Ren, 2006). To further understand the function ofmicrobial consortia and the comprehensive and accurate informa-tion about bioleaching environments, the expression level of soxBgene in thermophilic archaea community and its relationship tothe leaching rate were studied by real-time qPCR.

3.2.1. The effect of temperatureFig. 5a shows the relationship between soxB gene expression

and cell densities of thermophilic archaea at different tempera-tures. At the beginning, trends of the expression of soxB gene werebasically coincident with that of cell densities in the solution as thecommunity structure at the early stage was stable (Fig. 2). How-ever, the curve of the expression of soxB gene began to deviatefrom that of cell density from day 6. According to the previous re-searches, the main cause of such a deviation could be attributed todifferent sulfur oxidation activities of these thermophilic archaea,which was a result of the variation of expression of the relatedfunctional genes. At the late stage, however, there were no signif-icant different variations between the expressions of soxB gene andthe cell densities, and they were both tended to be descendant dueto the total biomass reduced. The above results indicated, besidesthe factor of the total cell density, the species of the predominantmicroorganism and their sulfur oxidation activities were also theimportant parameters during bioleaching process.

Fig. 5b shows the relationship between soxB gene expression le-vel and chalcopyrite leaching rate at different temperatures. Theresults showed that there was positive correlation between thesoxB gene expression quantity and the leaching rate of chalcopy-rite. In the first stage, the leaching rate of chalcopyrite increasedrapidly with sharp up-regulation of soxB gene expression. Subse-quently, two distinctly peaks appeared independently in time forthe change curves in Fig. 5b. At 65 �C, the first peak occurred atday 4 when the expression quantity of soxB gene reached to a rel-atively high value corresponding to a larger accumulate rate ofcopper concentration. The second peak occurred at day 12 whenthe expression quantity of soxB gene was in a high level with anincreasing of copper concentration. A similar phenomenon also ap-peared at days 6 and 14 during the bioleaching process at 55 �C.According to the above results, it was discovered that expressionlevel of soxB gene could be better embodied the function changeof the consortium.

Fig. 5. Relationships between soxB gene expression level and cell density (a), and between soxB gene expression level and chalcopyrite leaching rate with thermophilicarchaea at different temperatures (b).

W. Zhu et al. / Bioresource Technology 133 (2013) 405–413 411

3.2.2. The impact of pHFig. 6a shows the relationship between soxB gene expression

and cell densities at different initial pH values. As it can be seen,there were some significant inconsistencies between the expres-sion of soxB gene and the cell densities, but it was worthwhile tonote that there were similar growth trends and different cell den-sities with different initial pH values. The cell density at initial pH1.0 was higher than that at initial pH 1.5 and 2.0. However, theexpression quantity of soxB gene at initial pH 1.0 was less than thatat initial pH 2.0. And interestingly, the highest expression quantityof soxB gene occurred at the system with the moderate cell densityat initial pH 1.5.

Fig. 6b shows the relationship between soxB gene expression le-vel and chalcopyrite leaching rate at different pH values. It indi-cated that the copper extraction was not obviously differentduring bioleaching process between pH 1.0, 1.5, 2.0, and the ulti-mate copper concentrations in the solution were 3.0, 3.09 and3.06 g/L respectively. It may be due to the precipitation behavior

of copper combined with other intermediate substances whichleaded to the copper concentration reduced and maintained at acertain level (Bigham et al., 2010). The precipitant varied was re-lated with the change of pH, temperature, the precipitation rateand their chemical compositions (Gramp et al., 2008). In this study,during the first 4 days, the copper concentration in the solution atinitial pH 1.5 was lower than that at initial pH 2.0. The reason maybe that the higher pH value was easier to promote the occurrenceof the metal ion co-precipitation (Klauber, 2008), which was con-sistent with the case that some yellow precipitants appeared atthe stage (Zhu et al., 2011).

The aforementioned analysis results indicated that temperatureand initial pH value had a significant effect on function change ofthe consortium and the leaching rate of chalcopyrite, in whichthe impact temperature was more obvious. Just as shown inFig. 5b, the copper concentration in the solution at temperature55 �C was lower than that at temperature 65 �C, and their ultimatevalues were 2.32 and 3.09 g/L respectively. The impact tempera-

Fig. 6. Relationships between soxB gene expression level and cell density (a), and between soxB gene expression level and chalcopyrite leaching rates with thermophilicarchaea at different pH values (b).

412 W. Zhu et al. / Bioresource Technology 133 (2013) 405–413

ture may be mainly mediated by two pathways: one was the totalbiomass, community structure and sulfur-oxidizing activity of spe-cies, which resulted in different function of the consortium andchange of the leaching rate of chalcopyrite. Another was the impactof chemical reaction dynamics during bioleaching of chalcopyrite,which also could contribute to the results mentioned above. Inmost cases, the chemical reaction rate accelerated with the tem-perature increasing in bioleaching system (Rawlings et al., 2003).In present research work, the leaching rate of chalcopyrite at65 �C outnumbered that at 55 �C, which was in agreement withthe literature report (Rawlings et al., 2003). Currently, people havedifferent viewpoints on the effect of different pH values in biole-aching system. Some people have believed that the lower pH value(1.0) could promote the copper leaching rate (Liu et al., 2010),while others considered that the higher pH value (pH 2.0) wasfavorable to the copper leaching rate (Cordoba et al., 2009). In pres-ent research work, changes of the leaching rate of chalcopyrite atdifferent pH values were not obviously different that the reasonswere as above-mentioned.

4. Conclusions

The analysis results of the community succession of thermo-philes A. brierleyi, M. sedula, A. manzaensis and S. metallicus indi-cated that S. metallicus was most sensitive to the environmentalchange, A. brierleyi showed the best adaptability and sulfur oxida-tion ability and predominated in various leaching systems. The re-sults also showed that temperature and initial pH value had asignificant effect on the community succession. Meanwhile, theleaching rate of chalcopyrite closely related to the consortiumfunction embodied by soxB gene, which could prove a desirableway for revealing microbial sulfur oxidation differences and track-ing the function change of the consortium.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (50974140; 51274257), the Ph.D. ProgramsFoundation of Ministry of Education of China (20090162110054),

W. Zhu et al. / Bioresource Technology 133 (2013) 405–413 413

and the Opening Foundation for Precious Instruments of CentralSouth University, Changsha, China (2012-18).

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