Microbial community dynamics during a demonstration-scale bioheap leaching operation

Hydrometallurgy 125–126 (2012) 34–41 Contents lists available at SciVerse ScienceDirect Hydrometallurgy journal homepage: www.elsevier.com/locate/hydromet Microbial community dynamics during a demonstration-scale bioheap leaching operation Anna-Kaisa Halinen a,⁎, Nelli J. Beecroft a, 1, Kirsi Määttä a, Pauliina Nurmi a, Katja Laukkanen b, Anna H. Kaksonen a, 2, Marja Riekkola-Vanhanen b, Jaakko A. Puhakka a a b Department of Chemistry and Bioengineering, Tampere University of Technology, BO Pox 541, FIN-33101 Tampere, Finland Talvivaara Mining Company Plc. Ahventie 4.B.47, FIN-02170 Espoo, Finland a r t i c l e i n f o Article history: Received 6 February 2012 Received in revised form 23 April 2012 Accepted 3 May 2012 Available online 9 May 2012 Keywords: Bioleach communities Complex sulfide ore Demonstration-scale bioheap Mesophiles Thermophiles a b s t r a c t In the present work the microbial community of a low grade nickel ore demonstration-scale bioheap was examined under varying weather (outside air temperature between + 30 and − 39 °C) and operational conditions over a period of three years in Talvivaara, Finland. After the start-up of heap irrigation, oxidation of pyrrhotite and pyrite increased the heap temperature up to 90 °C. Leach liquor temperatures varied between 60 and 15 °C over the operation period, affecting the progress of sulfide ore oxidation. The microbial communities were profiled by polymerase chain reaction (PCR) — denaturing gradient gel electrophoresis (DGGE) followed by partial sequencing of 16S rRNA gene. Large temperature gradients prevailed resulting in the simultaneous presence of active mesophilic and thermophilic iron- and/or sulfur-oxidisers in the heap. As mineral oxidation progressed microbial diversity decreased and Acidithiobacillus ferrooxidans became increasingly dominant. The number of bacteria in the leach liquors was in the range of 105–10 7 cells mL − 1. After one year of bioheap operation several ore samples were drilled from the heap and A. ferrooxidans, Acidithiobacillus caldus, an uncultured bacterium clone H70 related organism, Ferrimicrobium acidiphilum and a bacterium related to Sulfobacillus thermosulfidooxidans were found. Cell counts from the ore samples varied between 10 5 and 10 7 cells g− 1 ore sample. The archaeal species present in leach liquors were novel and related to uncultivated species. During the secondary leaching phase the leaching community remained steady. A. ferrooxidans dominated, and an uncultured bacterium clone H70related organism and Leptospirillum ferrooxidans were present. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Heap bioleaching of low-grade sulfide ores has become an important process for metal recovery (for reviews, see: Rawlings, 2002; Watling, 2008). During the last twenty years the process has been optimized successfully including ore crushing, agglomeration, aeration, leach liquor distribution and stacking stages (Brierley and Brierley, 2001). Leach liquor pH can be adjusted before irrigation. Temperatures are affected by the composition and concentration of the sulfidic minerals because of exothermic oxidation reactions. Aeration and irrigation rates affect evaporation and heat dissipation (Ehrlich, 2001; Rawlings, 2002; Watling, 2006). Microorganisms present in bioheaps are mainly ferrous iron- and sulfur-oxidizing chemolithotrophs, although some heterotrophs have been reported (Hallberg and Johnson, 2001). The regeneration of ferric iron (Fe3 +) and proton release (H+) are essential for metal sulfide ⁎ Corresponding author. Tel.: + 358 50 346 3935. E-mail address: anna-kaisa.halinen@tut.fi (A-K. Halinen). 1 Present address: Division of Microbial Sciences, University of Surrey, Guildford, GU2 7XH, UK. 2 Present address: CSIRO Land and Water, Underwood Avenue, Floreat Park, WA 6014, Australia. 0304-386X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2012.05.001 oxidation and dissolution of valuable metals. As sulfuric acid is produced by the oxidation of sulfur, these organisms generate an acidic growth environment. Many of the chemolithotrophic acidophiles are sensitive to organic matter and thus heterotrophic acidophiles detoxify the bioleaching environment. A small fraction of the bioleaching microorganisms is found in the leach liquor, while most of the microorganisms adhere to the mineral surfaces (Rohwerder et al., 2003; Crundwell, 1996). Studies of microbial communities inhabiting commercial reactor based, bioleaching processes have been successfully carried out in recent years (Pradhan et al., 2008). However, microorganisms inhabiting industrial bioheaps and dumps have gained less attention (Demergasso et al., 2005). The aim of the present work was to study the microbial community structures and their dynamics during a demonstration-scale complex sulfide ore (17 000 tons) bioheap leaching operation. Spatial and temporal changes in microbial communities were monitored and included strong fluctuations. 1.1 . Talvivaara ore deposit Talvivaara complex multi-metal black schist ore deposit is located in central-eastern Finland with 1550 million tons of classified 35 A-K. Halinen et al. / Hydrometallurgy 125–126 (2012) 34–41 Heap 1 Heap 2 increased the heap temperature up to 90 °C. Leach liquor temperatures remained always at above 15 °C over the operation period, even during the boreal winter. 2.2. Inoculation of Heap 1 Manhole 1 Pond 1 PPV=175 m3 Manhole 2 Pond 2 V=136 m3 Irrigation 1 Irrigation 2 PPon Fig. 1. Diagram of the sampling points of the Talvivaara bioheaps with the direction of the liquid flow marked with arrows. Each heap had its own liquid circulation. The amount of the ore of Heap 1 was 10 255 tons and for Heap 26 703 tons, respectively. resources (Talvivaara, 2012). The mineral composition of the sulfides used in the demonstration-scale bioheaps was 61.2% pyrrhotite [(Fe1 − x)(S2), where X = 0.7–0.9], 24.3% pyrite (FeS), 5% pentlandite [(Fe,Ni,Co)9S8], 6.5% alabandite (MnS), 2.4% chalcopyrite (CuFeS2) and 1% sphalerite [(Zn,Fe)S]. Valuable metal contents were as follows: 0.27% Ni, 0.56% Zn, 0.14% Cu and 0.02% Co (for detailed description see Riekkola-Vanhanen, 2007). Prior to the bioheap demonstration, laboratory scale studies had demonstrated the amenability of Talvivaara ore to bioleaching (e.g. Puhakka and Tuovinen, 1986a, b, c; Riekkola-Vanhanen and Heimala, 1999; Wakeman et al., 2008; Halinen et al., 2009a, b). After a year of bioleaching 65% of nickel and 60% of zinc were leached. After 48 months, 99% of nickel and zinc were leached. 2. Materials and methods 2.1. Design and start-up of the demonstration heaps During summer 2005, a 17 000 ton demonstration plant was constructed at the Talvivaara mine site (Fig. 1). A representative ore sample was mined, crushed to 80% —8 mm, agglomerated and stacked in a two-part heap (8 m high, 30 × 120 m). Heap 1 was agglomerated with sulfuric acid solution (pH 1.8) including inoculum (described below). Heap 2 was agglomerated with sulfuric acid solution only. Irrigation of the heaps was started in August 2005. The irrigation flow rate was at the beginning 10 L m − 2 h − 1 on Heap 1 and 20 L m − 2 h − 1 on Heap 2. It was decreased later to 5 L m − 2 h − 1 on both heaps. Leach liquors were collected by subsurface drains below the heaps and directed to manholes. From the manholes liquors flowed to pregnant leach solution (PLS) ponds and back to irrigation (Fig. 1). The operational volumes of ponds 1 and 2 were 175 m 3 and 136 m 3, respectively. Ten percent side stream was removed continuously for metal recovery and replaced with well water. After the start-up of irrigation, the oxidation of pyrrhotite and pyrite Primary heap construction MH1, MH2, P1, P2 12/2005-6/2006 August 2005 9/2005 MP, MH1, MH2 12/2005, 1/2006 +Archaea The iron and sulfur-oxidizing enrichment culture was originally enriched from mine site water samples on Fe 2 +, S 0 and Talvivaara ore powder at pH 1.8 (Halinen et al., 2009a). The enrichment culture was grown in laboratory to the volume of 4.5 m 3 (Geological Survey of Finland (GTK), Outokumpu). It was transported to the mine site and pumped into a microbial pond (MP) with initial water volume of 40 m 3. Most of the water used originated from on-site drilled well (temperature 5 °C). Liquid pH in the pond was adjusted to 1.8 with sulfuric acid and pulp concentration (w v − 1) was set to 1% prior to inoculation. After the inoculation ammonium sulfate concentration was increased stepwise to 0.4 g L − 1 using 25% (v v − 1) stock solution and 500 kg of elemental sulfur was added. No liquid heating or cover was used. The volume of 40 m 3 was increased to 150 m 3 with well water. Inoculation of Heap 1 was accomplished during agglomeration and by irrigating the heap by acidic microbial solution, total inoculum volume being 99 m 3. Heap 2 was not inoculated. 2.3. Secondary bioheaps On February 2007 after 18 months of operation, the heaps were reclaimed and restacked to the secondary bioheap. Irrigation rate was 2 L m − 2 h − 1. No aeration was provided. Bioleaching of copper and cobalt was continued (data not shown). Minor amounts of nickel and zinc were bioleached, probably from the parts that were not reached during the primary phase. 2.4. Sampling First samples for microbiological analyses were taken from the microbial pond (MP), where the inoculum was grown, and from the manholes (MH 1 and 2) that collected the irrigation and rain water that percolated trough the heaps. Next samples were taken after 3 months of bioleaching. Samples (50 mL) from manholes and ponds (P 1 and 2) were collected thereafter every month. In July 2006 pond samples were changed to irrigation samples (IR 1 and 2). Sampling was continued when primary bioheaps were reclaimed to the secondary bioheaps. Fig. 1 shows the sampling points and the sampling and analysis program was as presented in Fig. 2. 2.5. Cell counts Total cell counts were estimated from the samples with 4′, 6-diamidino-2-phenylindole (DAPI) staining technique using epifluorescence microscopy. Microbes were detached from the ore samples according to methods described in Halinen et al. (2009a). 15 g of the ore sample was mixed with 40 mL of sterile Zwittergentwashing solution (0.38 g L − 1 ethylene glycol tetraacetic acid, 3.35 − 4 g L − 1 Zwittergent, 3.73 g L − 1 KCl, pH adjusted to 2.5 with 2 M HCl). The mixture was shaken and sonicated 5 × 1 min in order to detach microorganisms from ore particles. Thereafter, the sample was allowed to settle for about 30 min to prevent the small ore MH1, MH2, IR1, IR2 8/2006, 11/2006-1/2007 8/2006 +Ore samples Heaps are reclaimed to secondary bioheaps 2/2007 End of phase two November 2008 2, 4, 6, 7, 10 and 12/2007 Samples from the secondary bioheaps MH1, MH2, IR1, IR2 Fig. 2. Timescale of sampling. MP = microbial pond, MH1 = manhole 1, MH2 = manhole 2, P1 = pond 1, P2 = pond 2, IR1 = irrigation 1, IR2 = irrigation 2. 36 A-K. Halinen et al. / Hydrometallurgy 125–126 (2012) 34–41 1,0E+08 Total cell count (cells ml-1) PRIMARY HEAPS 1,0E+07 1,0E+06 SECONDARY HEAPS 1,0E+05 MH2 P1 P2 IR1 IR2 A ug O -05 ct N -05 ov Ja 05 nM 06 ar A -06 pr Ju 06 nA 06 ug Se -06 p N -06 ov D -06 ec Fe 06 bA 07 pr M -07 ay Ju 07 lSe 07 pO 07 ct D 07 ec -0 7 1,0E+04 MH1 Date Fig. 3. The cell counts (DAPI) from leach liquors during the bioleaching. MH1 = manhole 1, MH2 = manhole 2, P1 = pond 1, P2 = pond 2, IR1 = irrigation 1, IR2 = irrigation 2. particles from interfering with DAPI staining. Microbial numbers were counted from supernatant to account for attached cells. 2.6. Microbial community analyses The microbial communities were investigated during the bioleaching from the leach liquors. Samples were sent to Tampere University of Technology and 15 mL duplicates of every sample were filtered on a 0.2 μm pore size polycarbonate filter prior to concentrating the microorganisms for DNA extraction (Cyclopore Track Etched Membrane, Whatman). The filters were rinsed with 5 mL of 0.9% (wt vol − 1) NaCl at pH 1.8 for 1 min to remove the excess metals and then neutralized with 5 mL of 40 mM Na-EDTA in phosphate buffered saline (PBS; 130 mM NaCl, 5 mM Na2HPO4, and 5 mM NaH2PO4 adjusted to pH 7.2) for 1 min. The filters were stored at −20 °C prior to nucleic acid extraction. The microbial communities were investigated by polymerase chain reaction (PCR) — denaturing MP MH1 MH2 MH1 MH2 P1 P2 MH1 MH2a P1 P2 std 9/05 9/05 9/05 12/05 12/05 12/05 12/05 1/06 1/06 1/06 1/06 std gradient gel electrophoresis (DGGE) followed by partial sequencing of 16S rRNA gene as described previously (Halinen et al., 2009a). In October 2006 several ore samples were obtained by drilling from the heaps. The first samples were drilled from a part of Heap 1 where the temperature was 80–90 °C at the depths of 1–2 m and 3–4 m. Next samples were taken from Heap 1, from the depth of 1–2 m and 4–5 m in area where the temperature was 65–75 °C. Last samples were drilled from Heap 2 from area where the temperature was 20–35 °C, from the depth of 0–1 m and 4–5 m (Fig. 5). Characteristic leach liquor at the time when ore samples were drilled from Heap 1 (PLS1) were as follows: temperature (T) 46.5 °C, dissolved oxygen (DO) 2.2 mg L − 1, pH 2.75, redox 331 mV (Pt electrode against an Ag 0/AgCl reference), soluble Fe 2 + 12.6 g L − 1, soluble Fetot 13.7 g L − 1 and from the Heap 2: T 35.1 °C, DO 2.1 2 mg L − 1, pH 2.67, redox 392 mV, soluble Fe 2 + 6.2 g L − 1, soluble Fetot 7.4 g L − 1. 3. Results and discussion 3.1. Bioleach conditions in heaps Ferrous iron concentrations were high during the first half year of bioleaching, being mainly between 20 and 35 g L − 1 and between 10 and 20 g − 1 L − 1 in Heaps 1 and 2, respectively. After 6 months ferrous iron concentrations started to decrease steadily, being between 10 and 20 g − 1 L − 1 in both heaps. Ferric iron concentration remained low in both heaps (data not shown). Leach liquor pH values of both heaps were quite similar varying during the 10 months between 3.5 and 3.0 and after that between 3.0 and 2.5. The pH was maintained by continuous adjustment by dosing sulfuric acid. Redox potentials varied between 200 and 400 mV in both heaps increasing toward 400 mV as leaching progressed. At the beginning (autumn 2005) leach liquor temperatures were between 25 and 55 °C in both heaps. During the first year, temperatures varied between 20 and 50 °C in Heap 1 and between 20 and 40 °C in Heap 2. In the second winter leach liquor temperatures were between 20 and 40 °C in Heap 1 and between 15 and 25 °C in Heap 2. Temperatures inside the heaps varied greatly, being between 15 and 90 °C during the first year. In the second winter temperatures inside the heaps started to drop being still 80 °C in the hottest zone. 3.2. Cell counts Total cell counts (DAPI staining) in leach liquors from the primary and secondary phase were as presented in Fig. 3. Liquid volumes a b b c Sample 1 7.75E+05 T=80-90 ºC depth 1-2 m Sample 3 2.49E+06 T=65-75 ºC depth 1-2 m Sample 5 1.82E+07 T=20-35 ºC depth 0-1 m d l e f g h i m n Heap 1 Heap 2 8m j k 120 m Fig. 4. Bacterial DGGE profiles of partial 16S rRNA gene fragments retrieved from leach liquors after 4 months of bioleaching. Samples were from microbial pond (MP), manhole 1 (MH1), manhole 2 (MH2), pond 1 (P1), pond 2 (P2). Identity of DGGE bands: a) and c) Acidithiobacillus ferrooxidans, b) A. caldus, d) A. thiooxidans, e) Leptospirillum ferrooxidans, f) Thiomonas arsenivorans, g) Alicyclobacillus acidocaldarius, h) and m) Alicyclobacillus tolerans i), Ferrimicrobium acidiphilum, j and k) Sulfobacillus thermosulfidooxidans, l) A. caldus, n) an uncultured Firmicutes bacterium clone H70 related bacterium that is related to Moorella sp. Please see Table 1 that presents the percentages of 16S rRNA gene similarities and data base designations. Sample 2 9.13E+06 T=80-90 ºC depth 3-4 m Sample 4 1.48E+06 T=65-75 ºC depth 4-5 m Sample 6 9.74E+06 T=20-35 ºC depth 4-5 m Fig. 5. The average cell counts from the drilled ore samples from Talvivaara bioheaps. The locations and temperatures of the sample sites are shown in a side view of the heaps. Table 1 Bacteria present in demonstration-scale bioheaps for complex sulfide ore during the primary leaching phase. MP = microbial pond, MH1 = manhole 1, MH2 = manhole 2, P1 = pond 1, P2 = pond 2, IR1 = irrigation line 1, IR2 = irrigation line 2. + corresponds weak band on DGGE and ++ corresponds strong band on DGGE, empty cell = not detected. Time (months) Sample\ species Acidithiobacillus ferrooxidans AP310 (99%, DQ355183) A. caldus MTH-04 (99–96%, AY427958) A. thiooxidans ORCS8 (100%, AY830900) 8/2005 9/2005 12/2005 1/2006 2/2006 3/2006 4/2006 5/2006 6/2006 8/2006 11/2006 12/2006 1/2007 9/2005 12/2005 1/2006 2/2006 3/2006 4/2006 5/2006 6/2006 8/2006 11/2006 12/2006 1/2007 12/2005 1/2006 2/2006 3/2006 4/2006 5/2006 6/2007 12/2005 1/2006 2/2006 3/2006 4/2006 5/2006 6/2007 8/2006 11/2006 12/2006 1/2007 8/2006 11/2006 12/2006 1/2007 0 1 4 5 6 7 8 9 10 12 15 16 17 1 4 5 6 7 8 9 10 12 15 16 17 4 5 6 7 8 9 10 4 5 6 7 8 9 10 12 15 16 17 12 15 16 17 MP MH 1 ++ + + ++ Thiomonas arsenivorans (100%, AY950676) MH 2 P1 P2 IR1 IR2 + + ++ + + + + + + + + + + ++ ++ + ++ ++ ++ ++ + + Leptospirillum ferrooxidans DSM 2705 (98%, X86776) Alicyclobacillus acidocaldarius (99%, AB059665) Alicyclobacillus tolerans (100%, AF137502) Ferrimicrobium acidiphilum T23 (100%, AF251436) + ++ + ++ + + + ++ ++ ++ + + + + ++ + + + + + ++ + + + + ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++ + ++ ++ ++ + ++ ++ ++ Bacterium related to clone H70 (91%, DQ328625) + + + + ++ ++ ++ + ++ ++ ++ ++ + ++ ++ + ++ ++ ++ ++ ++ ++ ++ Sulfobacillus thermosulfidooxidans N19-50-01 (100%, EU499919) + ++ + ++ + + + + + + ++ ++ + ++ + ++ + + + + + + + + ++ ++ A-K. Halinen et al. / Hydrometallurgy 125–126 (2012) 34–41 Date + ++ + + + + + + + ++ ++ ++ + + + + + ++ + ++ + + + + + 37 38 A-K. Halinen et al. / Hydrometallurgy 125–126 (2012) 34–41 Table 2 Archaea present in demonstration-scale bioheaps for complex sulfide ore during the primary leaching phase. MH1 = manhole 1, MH2 = manhole 2, P1 = pond 1, P2 = pond 2. + corresponds weak band on DGGE and ++ corresponds strong band on DGGE, empty cell = not detected. Date Time (months) Species\sample 12/2005 1/2006 12/2005 1/2006 12/2005 1/2006 12/2005 1/2006 4 5 4 5 4 5 4 5 MH1 Uncultured crenarchaeote clone JG36-GR-88 (100%, AJ535129) Thermoplasma acidiphilum strain 122-1B2 (93%, NR_028235) Uncultured archaeon SAGMA-X (99%, AB050229) ++ MH2 ++ P1 ++ ++ ++ ++ P2 ++ ++ increased drastically during the first four months resulting in dilution of leach liquors. During the primary phase cell counts were higher in Heap 2 leach liquors than in Heap 1 even though Heap 2 was not inoculated. The high ferrous iron concentrations in Heap 1 may have affected the growth of microorganisms. At the end of the primary bioleach phase, cell counts were quite similar in all samples being between 3.2 × 10 6 mL − 1 and 7.2 × 10 6 cells mL − 1. Cell counts increased most in manhole 1 being 1.5 × 10 5 mL − 1 at the beginning and 5.2 × 10 6 cells mL − 1 in the end on primary phase. In the secondary phase, cell counts varied between 1.8 × 10 6 and 6.0 × 10 7 cells mL − 1, being higher than in primary phase, likely resulting from the stabilized growth conditions. All cell counts decreased toward the end of the secondary phase. After restacking of the ore, the oxygen supply likely improved and decreased again toward the end. The total cell count in the drilled ore samples averaged 10 6 cells ore g − 1. Highest cell counts (1.8 × 107 cell ore g − 1) were detected from Heap 2 near the surface where the temperature varied between 20 and 35 °C (Fig. 5). A rough estimate based on liquid and solid sample analysis and associated volumes gives a total of 2 × 10 16 cells in the heap, while the liquid phase estimate is 3 × 1014 cells. This shows that more than 98% microorganisms were attached. 3.3. Microbial community characteristics 3.3.1. Start-up phase Duplicate DGGE profiles after 4 months of bioleaching were as shown in Fig. 4. The enrichment culture used in Heap 1 inoculation contained Acidithiobacillus ferrooxidans (99% 16S rRNA gene sequence similarity), Acidithiobacillus thiooxidans (100%), Leptospirillum. ferrooxidans (98%), Sulfobacillus thermotolerans (99%) and a species related to Acidithiobacillus caldus (96%) (Halinen et al., 2009a). When the inoculums was grown in the microbial pond (MP) A. ferrooxidans (100%), A. caldus (99%), A. thiooxidans (100%), L. ferrooxidans (98%), Alicyclobacillus acidocaldarius (99%), Alicyclobacillus tolerans (100%) and Ferrimicrobium acidiphilum (100%) were present. Several acidophilic microorganisms were also detected at the beginning from the manhole samples. A. ferrooxidans (99%), A. caldus (96%), A. acidocaldarius (99%), Thiomonas arsenivorans (100%) and S. thermosulfidooxidans (100%) were present (Fig. 4). During the first six months the microbial communities of the leach liquors were diverse and dominated by A. ferrooxidans (99%). S. thermosulfidooxidans (100%) and a bacterium related to Firmicutes clone H70 (91%) were also detected frequently. The DGGE band of the novel bacterium related to clone H70 was cut out, DNA isolated, PCR amplified and sequenced and submitted to GenBank (accession JQ941953). Table 3 Bacteria present in demonstration-scale bioheaps for complex sulfide ore during the secondary leaching phase. MH1 = manhole 1, MH2 = manhole 2, IR1 = irrigation line 1, IR2 = irrigation line 2. + corresponds weak band on DGGE and ++ corresponds strong band on DGGE, empty cell = not detected. Date Time (months) Sample\species Acidithiobacillus ferrooxidans AP310 (100%, DQ355183) Bacterium related to clone H70 (91%, DQ328625) Leptospirillum ferrooxidans DSM 2705 (100%, X86776) 2/2007 4/2007 6/2007 7/2007 10/2007 12/2007 2/2007 4/2007 6/2007 7/2007 10/2007 12/2007 2/2007 4/2007 6/2007 7/2007 10/2007 12/2007 2/2007 4/2007 6/2007 7/2007 10/2007 12/2007 0 2 4 5 8 9 0 2 4 5 8 9 0 2 4 5 8 9 0 2 4 5 8 9 MH1 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ + + + ++ + MH2 IR1 IR2 ++ + + + + + + + + + ++ + + ++ + + + + A-K. Halinen et al. / Hydrometallurgy 125–126 (2012) 34–41 3.3.2. Temporal dynamics of microbial communities The bacterial community composition was monitored over time from manholes (MH) and ponds (P) that collected the leach liquor. Bacterial species detected throughout the primary leaching phase were as presented in Table 1. After 6 months of bioheap operation L. ferrooxidans (100%) was first observed and it was present thereafter in nearly all samples. The microbial diversity in both heaps varied and decreased with time, with A. ferrooxidans remaining as the dominant bacterium and the bacterium related to clone H70 (91%) being present. Archaea were analyzed after 133 and 163 days of bioleaching from leach liquors of primary heaps and three uncultured species were found (Table 2). One species was related to an uncultured Crenarchaeote (100%) retrieved from uranium mining waste from the pond 1 on both days. From the same sample a species related to other uncultured archaeon SAGMA-X (99%), also found in deep South African gold mines, was detected. This species is a crenarchaeotic phylotype (Takai et al., 2001). In Pond 2 on day 133 one species related to a clone with the nearest known species of Thermoplasma acidophilum (93%) was detected. This species was also present on day 163 from samples MH1, MH2 and P2. At the secondary leaching phase the leaching community remained steady. A. ferrooxidans dominated and the bacterium related to clone H70 and L. ferrooxidans were present (Table 3). 3.3.3. Microbial communities on mineral surfaces Several ore samples were drilled from the primary bioheaps in October 2006 and their microbial communities were as shown in Table 4. A. ferrooxidans (99%) was present in nearly all samples. The bacterium related to clone H70 (90%) and A. caldus was detected from the areas of wide temperature variation. S. thermosulfidooxidans (99%) was found from the high temperature zones of the heap. F. acidiphilum (99%) was present in the areas where temperature varied between 20 and 35 °C. 3.3.4. Mesophiles present in demonstration scale bioheaps Temperatures of the leach liquors were mainly between 20 and 50 °C. Microorganisms were therefore mainly mesophilic and moderately thermophilic. Genus Acidithiobacillus (formerly Thiobacillus) includes e.g., A. ferrooxidans, A. thiooxidans and A. caldus that were detected at the primary phase. A. ferrooxidans was the dominating micro-organisms during the operation time. A. ferrooxidans grows optimally at 30–35 °C and at temperature range of 10–37 °C (Kelly and Table 4 Bacteria present in the drilled ore samples from the demonstration-scale bioheaps for complex sulfide ore during the primary leaching phase. First sample was drilled in a part of the Heap 1 where temperature was 80–90 °C in the depth of 1–2 m. Second was taken in the same place in the depth of 4–5 m. Next samples were taken from Heap 1 in the area where temperature was 65–75 °C. Last samples were drilled from the Heap 2 from the area were temperature was 20–35 °C in the depths of 0–1 m and 4–5 m, respectively. Species\sample 80–90 °C 65–75 °C 20–35 °C 1– 2m 3– 4m 1– 2m 0– 1m 4– 5m + + + + Acidithiobacillus ferrooxidans + AP310 (99%, DQ355183) A. caldus related bacterium (95%, AY427958) + Bacterium related to clone H70 (90%, DQ328625) Sulfobacillus thermosulfidooxidans + strain YN22 (99%, DQ650351) Ferrimicrobium acidiphilum T23 (99%, AF251436) 4– 5m + + + + + + + + + + 39 Wood, 2000). A. ferrooxidans was also detected from the ore samples from the high temperature area (80–90 °C), even though the temperature greatly exceeded their growth temperature. A. ferrooxidans is able to oxidize reduced inorganic sulfur compounds (RISCs), ferrous iron molecular hydrogen, formic acid and some other metal ions (Rohwerder et al., 2003). It can also grow anaerobically with H2 or S 0 as an electron donor and S 0 or Fe 3 + as an electron acceptor (Ohmura et al., 2002). A. thiooxidans grows in a temperature range of 10–37 °C and has optimum temperature at 28–30 °C. A. caldus has a growth rate that exceeds that of A. thiooxidans at temperatures over 30 °C (Norris et al., 1986). It has been reported as the dominant sulfur-oxidizing bacterium in bioleaching reactors at temperatures between 40 and 50 °C (Dopson and Lindström, 1999; Okibe et al., 2003). A. caldus has a growth temperature range of 32–52 °C and optimum temperature of 45 °C (Kelly and Wood, 2000). A. thiooxidans and A. caldus that are incapable of pyrite degradation utilize the sulfide moiety of the mineral when it is first released by the action of iron-oxidizing bacteria like A. ferrooxidans. L. ferrooxidans was detected in leach liquors for the first time after 6 months of bioleaching even though it was present in the inoculum. Competition between A. ferrooxidans and Leptospirillum species has been reviewed (Rawlings et al., 1999; Coram and Rawlings, 2002). Leptospirillum species dominate in environments with greater concentrations of ferric iron. The environments classified by high ferrous iron concentration (>5 g L − 1) seem to select for A. ferrooxidans (Pizarro et al., 1996). A. ferrooxidans and Sulfobacillus spp. are able to oxidize both ferrous iron and RISCs and might exploit the leaching environment more effectively than A. caldus or L. ferrooxidans that are specialized to oxidize iron or sulfur oxidizers. However, growth ranges and other factors may have overriding effects. The growth temperature range of L. ferrooxidans is wider (b10–45 °C) compared to that of A. ferrooxidans (Johnson, 2001). Other mesophiles detected were F. acidiphilum and Thiobacillus arsenivorans. F. acidiphilum has been found in several acidophilic environments (Johnson et al., 2009). It is mesophilic iron-oxidizing obligate heterotroph and grows below 37 °C with the optimum pH 2. T. arsenivorans has been originally isolated from a disused mine site by growth using arsenite [As(III)] as energy source. Optimum growth occurred at temperatures between 20 and 30 °C, and at pH between 4.0 and 7.5 (Battaglia-Brunet et al., 2006). 3.3.5. Thermophiles present in demonstration scale bioheaps At the beginning of bioleaching, temperatures of the Talvivaara demonstration-scale heaps were high and thermophilic and thermotolerant microorganisms were present in leach liquors. S. thermosulfidooxidans was detected from leach liquors several times during the first six months of bioleaching. It was also present in ore samples where temperatures were between 65 °C and 90 °C. The genus Sulfobacillus includes Gram-positive rods that obtain energy by oxidizing both ferrous iron and elemental sulfur (Norris et al., 1996). S. thermosulfidooxidans grows optimally at 50 °C and at pH 1.9–2.4 (Brandl, 2001; Robbins, 2000). The optimum temperature of S. thermotolerans is 40 °C and growth range 20–60 °C (Bogdanova et al., 2006). The ability to form endospores is advantageous for survival of bacteria during low temperature periods in a heap operated in boreal conditions, where high seasonal variation in temperature occurs. Sulfobacillus species and their occurrence in acidic and bioleaching environments have been reviewed by Watling et al. (2008). The genus Alicyclobacillus was also detected at the beginning of the bioleaching. It was reclassified from genus Bacillus by Wisotzkey et al. (1992). They are heterotrophic, aerobic or facultative aerobic, gram-positive or gram variable, spore-forming bacteria and grow at temperatures between 25 and 70 °C and pH values of 2.5 to 6.0. 40 A-K. Halinen et al. / Hydrometallurgy 125–126 (2012) 34–41 The novel bacterium that was related to uncultured Firmicutes clone H70 (91%) was present during the whole bioleaching time, except in the enrichment culture. Clone H70 was detected in acidic hot springs (T = 55 °C) in North America (Wilson et al., 2008). The use of extremely thermophilic archaea (e.g., Sulfolobus and Acidianus) in bioleaching processes is getting more attention. The role of archaea in the biomining community has been considered to rather scavenge the organic material than leach minerals (Johnson, 1998, 2001) but their activity in mineral sulfide oxidation of the bioheap cannot be ruled out. The archaeal species present in the Talvivaara demonstration-scale bioheap leach liquors were related to uncultivated species. 4. Conclusion The following conclusions can be drawn from the microbial characterization in demonstration-scale bioheap leaching of a complex sulfide ore: 1. The temperature conditions and profiles varied over a wide range (15–90 °C) during the 30 months of operation of the bioheaps. 2. The temperature increased due to exothermic biologically catalyzed oxidation of the sulfidic materials and especially that of pyrrhotite. 3. The total numbers of microbial cells in the heap were estimated to be approximately 2 × 10 16 with over 98% of cells being on the ore surfaces. 4. When the enrichment culture was grown in the microbial pond A. ferrooxidans, A. caldus, A. thiooxidans, L. ferrooxidans, A. acidocaldarius, A. tolerans and F. acidiphilum were present. 5. During the first six months the microbial communities of the leach liquors were diverse and dominated by A. ferrooxidans (99%). An uncultured bacterium related to Firmicutes clone H70 (91%) and S. thermosulfidooxidans (100%) were also detected frequently. 6. L. ferrooxidans was first observed after 6 months of bioheap operation and in all subsequent samples. The microbial diversity in both heaps varied and decreased with time, A. ferrooxidans remaining the dominating bacterium. 7. At the secondary leaching phase the leaching community remained steady. A. ferrooxidans dominated and the bacterium related to an uncultured clone H70 and L. ferrooxidans were present. In conclusion the multi-metal, low-grade nickel bioheap harbored a diverse microbial community that underwent spatial and temporal changes during leaching. It should be pointed out that DNA based community profiling indicates the presence of a microorganism but not it's activity in a given sample. Microorganisms having different growth temperatures were considered beneficial to the bioheap leaching. Acknowledgment This work was funded by the Talvivaara Project Ltd and the National Agency for Innovation and Technology (TEKES), Finland. The authors wish to thank Eila Kuronen and Sin Man Kwan for technical assistance. References Battaglia-Brunet, F., Joulian, C., Garrido, F., Dictor, M.C., Morin, D., Coupland, K., Johnson, D.B., Hallberg, K.B., Baranger, P., 2006. Oxidation of arsenite by Thiomonas strains and characterization of Thiomonas arsenivorans sp. nov. 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