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Preservation of Acidithiobacillus caldus: A moderately thermophilic bacterium and the effect on subsequent bioleaching of chalcopyrite
Hydrometallurgy 96 (2009) 333–336
Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / h y d r o m e t
Preservation of Acidithiobacillus caldus: A moderately thermophilic bacterium and the effect on subsequent bioleaching of chalcopyrite Wei-Min Zeng a,b, Hong-Bo Zhou a,b, Min-Xi Wan a,b, Wei-Liang Chao c, Ai-Ling Xu a,b, Xue-Duan Liu a,b, Guan-Zhou Qiu a,b,⁎ a b c
School of Minerals Processing and Bioengineering, Central South University, Changsha, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha, China Department of Microbiology, Soochow University, Tai Bei, Taiwan
a r t i c l e
i n f o
Article history: Received 7 September 2008 Received in revised form 8 November 2008 Accepted 14 November 2008 Available online 25 November 2008 Keywords: Acidithiobacillus caldus Leptospirillum ferriphilum Thermophilic bacterium Culture preservation Bioleaching Chalcopyrite
a b s t r a c t Four bacterial preservation methods: passage culture, sterile sand tube preservation, freezing preservation and freeze-drying preservation, were used to store Acidithiobacillus caldus strain S2, a moderately thermophilic bacterium. Among the four methods, the second is shown to be the best for short term preservation (6 months), and the fourth is the best for long term preservation (15 months). Specifically, the results show that using the second method, 32% cell viability was obtained after 6 months, and the cell survival rate reaches 17% using the fourth method after 15 months. In the bioleaching experiments, A. caldus strain S2 preserved via the second and the fourth methods, along with Leptospirillum ferriphilum strain YSK, were applied to bioleach chalcopyrite. Under the two preservation methods for A. caldus strain S2, the results show that 3.72 g/L and 1.68 g/L copper are extracted in the first 20 days and the maximum biomass delays to the 15th day and to the 18th day, respectively. When the bioleaching time was extended to the 40th day, copper extraction increased significantly to 5.02 g/L and 4.88 g/L, respectively. However, A. caldus strain S2 under no preservation, along with L. ferriphilum strain YSK, extract only 0.54 g/L Cu from the 20th day to the 40th day and finally release 5.14 g/L Cu. Therefore, the preserved culture shows almost the same total copper extraction as the unpreserved one in 40 days. As a result, sterile sand tube preservation and freeze-drying preservation greatly prolong the lag phase of cell growth without much decrease in the bioleaching ability of A. caldus strain S2. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Acidithiobacillus caldus is Gram-negative strictly aerobic bacteria and commonly found in acid mine drainage (AMD) and bioleaching reactors in moderately thermophilic environments (Baker and Banfield, 2003; Johnson and Hallberg, 2003; Zhou et al., 2009). It is considered to be the dominant sulfur-oxidizing bacterium in the bioleaching of sulphide ores at 40 °C–50 °C (Fouchera et al., 2003; Okibe et al., 2003). A. caldus promotes the sulphide-leaching efficiency by removing the build-up of solid sulfur which causes an inhibitory layer on the surface of the minerals (Dopson and Lindstrom, 1999; Gomez et al., 1996; Semenza et al., 2002). A. caldus together with other iron-oxidizing microorganisms such as Leptospirillum ferriphilum used to bioleach chalcopyrite has exhibited good performance. In our previous study of bioleaching chalcopyrite using A. caldus strain S2 and L. ferriphilum strain YSK, 80% copper extraction was obtained after 20 days, which is rather higher than bioleaching by pure culture L. ferriphilum strain YSK (23.6% copper extraction) (Fu et al., 2008). However, the loss of strains and the decrease ⁎ Corresponding author. Key Laboratory of Biometallurgy, Ministry of Education, Changsha 410083, China. Tel.: +86 731 8879212. E-mail address: [email protected] (G.-Z. Qiu).
of their bioleach capability often occur in many research and industrial applications (Yang et al., 2006; Zhang et al., 2000). Furthermore, A. caldus strain S2 that aids in the chalcopyrite bioleaching is not held in any international culture collection, so it is very important to determine the most effective preservation method which would not diminish its bioleaching capability. The methods for storing microorganisms generally include passage culture, sterile sand tube preservation (Li et al., 2006; Lu and Guo, 2007), freezing preservation, and freeze-drying preservation. Freezing preservation and freeze-drying preservations are considered to be the most feasible and effective storage for pure strains and mixed cultures (Morgan et al., 2006; Otero et al., 2007). However, most methods currently employed on freezing and freeze-dying preservations require highly specialized storage systems, substantial technical equipment, and often require very low temperature controlled environments (Hays et al., 2005). For these, they are generally expensive, technically demanding and manpower intensive. Passage culture and sterile sand tube preservations are taken for the short-term storage by many laboratory researchers due to their simple technical equipment demands, simple operation and low preserving cost. However, there are few reports about the preservation of A. caldus in the bioleaching of chalcopyrite. In this study, passage culture, sterile
0304-386X/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.11.003
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sand tube preservation, freezing preservation and freeze-drying preservation were used on A. caldus strain S2. Its characterization for bioleaching of chalcopyrite, along with L. ferriphilum strain YSK, before and after preservation, was then studied to investigate the effect of bacterial preservation on the chalcopyrite bioleaching performance. 2. Materials and methods 2.1. Culture medium and microorganisms A. caldus strain S2 and L. ferriphilum strain YSK (Genbank accession numbers of 16S rRNA genes: DQ256484 and DQ343299, respectively) were isolated and conserved. A. caldus strain S2 was maintained in Starky basal salt medium (pH 2.5) with sulfur as the energy source. Starky-S medium (in g/L) contains: (NH4)2SO4(3), KH2PO4(3), MgSO4·7H2O(0.5), CaCl2·2H2O(0.25), S(10) The medium for bioleaching chalcopyrite(20) with A. caldus and L. ferriphilum included FeSO4·7H2O(10) besides the above Starky-S medium. A. caldus strain S2 after preservation was revived and cultured in a shaking incubator at 180 rpm and 42 °C. 2.2. Cell preservation 2.2.1. Passage culture A. caldus strain S2 growing in stable growth phase was inoculated in 10% amounts into Starky-S medium for 15 days as the first passage culture. After 15 days the cell density clearly began to decrease and then the second passage culture was carried out, as the first time. When the strain was cultured to the 6th day, cell numbers in a 20 µL sample were counted with a hematocyte counter and optical microscope. 2.2.2. Sterile sand tube preservation Cells were harvested and re-suspended to a cell density of about 109 cells/mL by centrifugation (10,000 g, 10 min, and 20 °C) then 1 mL was added into glass tubes containing 20 g sterile sand. The tubes were stored in a dry box at room temperature. Every 3 months one of the tubes was taken out for cell reviving by dumping the contents into 10 mL Starky-S medium, slightly stirring for 20 min, adding 5 mL of clarified liquid into the Starky-S medium and culturing for 6 days. Then the cells were counted under an optical microscope and the results were compared with other preservation methods. 2.2.3. Freezing preservation Cells were harvested and concentrated by centrifugation (10,000 g, 10 min, 4 °C) then re-suspended into 20%w/w mannitol as a cryoprotectant. 500 µL samples of cell re-suspensions were preserved at −20 °C and −72 °C in a refrigerator-freezer, stored for 15 months and revived every 3 months. The frozen samples were revived by thawing for 10 min at 30 °C in a water bath, then inoculated in medium for 6 days — when the cell density was analyzed under optical microscope. 2.2.4. Freeze drying preservation The cell re-suspensions were prepared as above but small aliquots (20 µL) were dispensed into 1.5 mL tubes and then frozen by direct immersion in liquid nitrogen (−196 °C). The frozen samples were dried by a freeze-dryer for a period of 10 h at 4 °C and stored at 4 °C, −20 °C and −72 °C in a refrigerator–freezer. The frozen sample was again revived by thawing for 10 min at 30 °C in a water bath, then inoculated in medium for 6 days when the cell density was analyzed under optical microscope. 2.3. Cell viability Cell viability (V) was measured by cell density counted before preservation (N0) and after different times of storage (Nt). The viability rate (V%) was expressed by the cell survival rate that was calculated as
following: V (%) = 100 × Nt / N0, where N0 and Nt were the cell density cultured after 6 days and every 3 months, respectively. 2.4. Minerals composition The concentrate sample was collected from Meizhou in Guangdong province, China, with a particle size less than 75 μm. The components of the mineral sample were analyzed by XRD. The mineral sample consisted of mainly chalcopyrite (62%), galena (26%) and chalcocite (11%). 2.5. Bioleaching of chalcopyrite along with L. ferriphilum strain YSK Bioleaching experiments were performed in a 500 mL shake flask with 200 mL medium in a shaking incubator at 180 rpm and 42 °C. The initial pH value was adjusted to 2.5 by 1:1 sulfuric acid. A. caldus strain S2 and L. ferriphilum strain YSK (1:1) were inoculated into leaching medium with 10% inoculum amounts. The inoculums used include: the unpreserved A. caldus strain S2 along with L. ferriphilum strain YSK; the preserved strain S2 by sterile sand tube preservation after 3 and 6 months along with strain YSK; and the preserved strain S2 by freeze drying preservation after 3, 6, 9, 12 and 15 months along with strain YSK. Distilled water was added to the flask in order to compensate for evaporation losses. The experiment was performed in two stages: 0– 20 days and 20–40 days at 2% pulp density. The controlled experiments involved the following two cases: control 1, bioleaching of chalcopyrite with pure A. caldus strain S2 unpreserved; control 2, bioleaching of chalcopyrite with pure L. ferriphilum strain YSK. The levels of Cu2+, Fe2+ in solution were analyzed by atomic absorption spectrophotometry and by titration with potassium dichromate, respectively at the end of experiments, while the biomass of leaching system was counted and analyzed everyday under an optical microscope (Olympus, CX21). 3. Results and discussion 3.1. Cell preservation By analyzing the survival rate of the cells preserved, the preservation ways including passage culture, sterile sand tube preservation, freezing preservation and freeze drying preservation were compared to find the feasible way for short-term and long-term storages of strain A. caldus. The cell density of unpreserved A. caldus strain S2 achieved a maximum of 1.2 × 108 cells/mL in 6 days. When growth continues to 15 days, the cell density decreases to about 4.8 × 107 cells/mL and then the cell density decreases rapidly to less than 107 cells/mL. Passage culture has passed through 30 times in 15 months. In the initial several times of passage culture, cell density remains at 1.1– 1.2 × 108 cells/mL. After that, cell density continually decreases to 7.4 × 107 cells/mL at the 30th passage culture. Clearly, after 15 months the viability of strain A. caldus has declined remarkably due to bacterial degeneration (Bai, 2007) which leads the decreasing adaptability of A. caldus strain S2 in the growth medium. The results of sterile sand tube preservation after 3, 6, 9 and 12 months show that the longer the preservation time, the less the cell viability. Especially at the 12th month, few cells can be revived. This demonstrates that sterile sand tube preservation is not suitable for long-term storage; but for short-term storage of A. caldus strain S2 (up to 6 months), cell density reaches 3.8 × 107 cells/mL) and this preservation method is proven to be simple, low-cost and effective. Freezing storage at −20 °C and −72 °C has a very different effect on A. caldus strain S2. Freezing at −20 °C causes a high cell death rate and a majority of cells cannot be revived after storage for 6 months. However, freezing at −72 °C shows a good performance, although even after the first freezing, more than 50% cells have lost activities. Fonseca et al.
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(2001) reported that at very high freezing rates (such as at −72 °C), the bacterial suspension moved to a microcrystalline state, thus reducing the physicochemical and biochemical reaction rates. In contrast, at low freezing rates, damage to the cell structure was generated by the formation of ice crystals and by the dehydration of the cells, due to the high electrolyte concentrations in the medium. Mazur (1970) also explained that the better preservation at −72 °C can be ascribed to lower enzymatic reaction rates at this temperature, corresponding to lower molecular mobility, thus limiting cell damage induced by ice crystals. As a result, it can be seen that a lower storage temperature (−72 °C instead of −20 °C) led to better preservation of cell activity during frozen storage. Freeze drying has been used to preserve microorganisms for decades and it is the preferred method for culture strains worldwide. The products after freeze drying are available for transporting and storage (Morgan et al., 2006). In this experiment, the effect of different storage temperatures on cell viability after freeze-drying was investigated. The results show that freeze-drying preservation has a good performance for storing A. caldus strain S2. Furthermore, storing at −20 °C and −72 °C shows higher cell viability. After preservation for 15 months, the revived cell density achieved 1.2 × 107 cells/mL and 2 × 107 cells/mL, respectively. However, when the cells were preserved at 4 °C after 15 months, the cell density could not be revived. This indicates that after freeze-drying, the storage temperature does play a key role for cell preservation. 3.2. Comparison of cell survival rates For further investigating and analyzing the effects of these preservation methods on storing A. caldus strain S2, the survival rates were calculated as in Section 2.3 as shown in Table 1. Passage culture shows a high 61% survival rate and it seems that bacterial degeneration does not make lethal effect on cell preservation. However, this preservation method needs frequent manual-operation and observation that is easy to cause microbial contamination. Sterile sand tube preservation is considered to be a simple operation and low cost method. In this study, 32% cell viability was obtained after storing A. caldus with sterile sand tube preservation for 6 months so it is the feasible way for storage of A. caldus over a short term. Freezing would cause serial damages on cell structure and further affect the biological activity. It can be seen from Table 1 that the cell viability after freezing largely decreases with all greater than 50% before 3 months. Fortunately, frozen storage at −72 °C after freezedrying shows good cell preservation compared with other methods with up to 17% cell survival rate after 15 months. As a result, freezedrying preservation is proposed for the storage A. caldus over a long term.
335
Fig. 1. Cu2+, Fe2+ and Fe3+ concentrations in the bioleaching of chalcopyrite with L. ferriphilum strain YSK and A. caldus strain S2 preserved by sterile sand tube preservation after 3 and 6 months.
chalcopyrite. Thus, in this experiment, both of L. ferriphilum and preserved A. caldus were used to bioleach chalcopyrite. CuFeS2 þ 2Fe3þ →Cu2þ þ 2Fe2þ þ 0:125S8
ð1Þ
0:125S8 þ 1:5H2 O þ O2 →Hþ þ SO2− 4
ð2Þ
Fe2þ þ Hþ þ O2 →0:5H2 O þ Fe3þ
ð3Þ
Fig. 1 shows the Cu2+, Fe2+ and Fe3+ concentrations in the bioleaching of chalcopyrite after 20 days with L. ferriphilum strain YSK and A. caldus strain S2 preserved by sterile sand tube preservation. Before preservation, the mixed culture could bioleach chalcopyrite with 4.6 g/L Cu, and the copper extraction reaches 82%. It can be seen from Fig. 1 that after preservation, the rate of copper extraction continually decreases and the concentration reaches only 3.7 g/L Cu. The decline of cell viability requires preserved A. caldus strain S2 to grow for a longer time to form enough biomass to bioleach chalcopyrite and further prolong the total bioleaching time. It can be observed that the maximal biomass occurs at the 15th day when strain A. caldus is preserved after 6 months, while the maximal biomass is obtained at the 11th day when using unpreserved A. caldus strain S2 (Fig. 3). On the other hand, preservation would give some damage to cell activity (Fonseca et al., 2001), which may be another reason for the decrease of copper extraction.
3.3. Bioleaching experiments Chalcopyrite is a primary sulphide ore, which is leached by ferric ions according to Eq. (1). Sulfur is then dissolved by the sulfur-oxidizing microorganisms like A. caldus (Eq. (2)) and ferric ions are obtained by the action of ferrous ion-oxidizing microorganisms (Eq. (3)) like L. ferriphilum (Coram and Rawlings, 2002; Sand et al., 2000). Ferrous ion- and sulfuroxidizing microorganisms play important roles in the bioleaching of Table 1 The survival rate (%) of strain A. caldus during several preservation methods Storage time (months)
3
6
9
12
15
Passage culture Sterile sand tube preservation Frozen preservation at − 20 °C Frozen preservation at − 72 °C Freezing drying and frozen preservation at 4 °C Freezing drying and frozen preservation at −20 °C Freezing drying and frozen preservation at −72 °C
92 76 25 41 38 43 48
83 32 10 35 24 30 37
80 7 0 22 12 21 29
66 0 0 12 4 14 23
61 0 0 0 0 10 17
Fig. 2. The Cu2+, Fe2+ and Fe3+ concentrations in the bioleaching of chalcopyrite with L. ferriphilum strain YSK and A. caldus strain S2 preserved by freezing dry preservation at −72 °C after 3, 6, 9, 12 and 15 months.
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viability in less than 6 months. Freeze-drying preservation is considered to be the best way to store A. caldus strain S2 over a long term, since A. caldus can store for 15 months with up to 17% cell viability. During the bioleaching experiments, the rate of copper extraction by preserved L. ferriphilum strain YSK and A. caldus strain S2 decreases significantly in 20 days, compared with that by unpreserved L. ferriphilum strain YSK and A. caldus strain S2. The main reason is that preservation affects the cell activity and prolongs the growth phase time. When the bioleaching time is extended to 40 days, copper extraction significantly increases showing that preservation does not affect its long term ability for bioleaching chalcopyrite. Acknowledgements
Fig. 3. The variations of biomass during the bioleaching of chalcopyrite by L. ferriphilum strain YSK and A. caldus strain S2 unpreserved (■) or preserved by sterile sand tube preservation for 6 months (●) or by freeze drying preservation for 15 months (▲).
The concentrations of Fe2+ and Fe3+ always keep at a low level around 0.62 g/L and 1.04 g/L respectively, after preservation of 6 months, until some hindering factors happen such as the formation of jarosite on the surface of the mineral (Gomez et al., 1999a,b; Sandstorm and Petersson, 1997). Bioleaching of chalcopyrite with L. ferriphilum strain YSK and A. caldus strain S2 preserved by freeze drying preservation at –72 °C is shown in Fig. 2. After 15 months preservation it releases a rather low concentration of Cu2+, Fe2+ and Fe3+, (1.68 g/L, 0.14 g/L and 0.29 g/L, respectively) and the maximum biomass is only 1.6 × 108 cells/mL achieved by the 18th day (Fig. 3). It seriously affects bioleaching of chalcopyrite and leads to a low final copper extraction. After sterile sand tube preservation and freeze drying preservation, bioleaching microorganisms have to take a longer time to adapt the growth environment and reviving. Through prolonging the culture time, microorganisms may recover their growth and bioleaching ability. As a result, an additional experiment for prolonging the bioleaching time to the 40th day was carried out. For the unpreserved culture, the copper concentration increased slightly and finally reached 5.14 g/L Cu (92% extraction). The culture preserved by sterile sand tube preservation after 6 months gave a high copper extraction of 5.02 g/L while the culture preserved by freeze drying preservation after 15 months, finally achieves up to 4.88 g/L Cu. The control experiments showed the lowest extraction of copper with only 0.74 g/L Cu in control 2 and 0.26 g/L Cu in control 1. Compared to the unpreserved culture, the preserved one gives almost the same total copper extraction at the 40th day and do not show any obvious decrease in bioleaching ability. It is notable that with the unpreserved culture, most of the copper is leached from the ore sample before 20 days and it is thought that chalcopyrite passivation seriously affects extraction so that it is unnecessary to prolong the culture time (Sand et al., 2000; Stott et al., 2000). However, in the bioleaching with preserved culture, microorganisms pass through a long growth lag phase as living cell numbers build up again and more time is required. In this experiment, both L. ferriphilum and A. caldus were proved to be necessary for bioleaching of chalcopyrite. 4. Conclusions Four preservation methods: passage culture, sterile sand tube preservation, freezing preservation and freeze drying preservation were used to store A. caldus strain S2, a moderately thermophilic bacterium. It is feasible to preserve A. caldus strain S2 for a short term with sterile sand tube preservation, which has low cost and gives relatively high cell
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Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / h y d r o m e t
Preservation of Acidithiobacillus caldus: A moderately thermophilic bacterium and the effect on subsequent bioleaching of chalcopyrite Wei-Min Zeng a,b, Hong-Bo Zhou a,b, Min-Xi Wan a,b, Wei-Liang Chao c, Ai-Ling Xu a,b, Xue-Duan Liu a,b, Guan-Zhou Qiu a,b,⁎ a b c
School of Minerals Processing and Bioengineering, Central South University, Changsha, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha, China Department of Microbiology, Soochow University, Tai Bei, Taiwan
a r t i c l e
i n f o
Article history: Received 7 September 2008 Received in revised form 8 November 2008 Accepted 14 November 2008 Available online 25 November 2008 Keywords: Acidithiobacillus caldus Leptospirillum ferriphilum Thermophilic bacterium Culture preservation Bioleaching Chalcopyrite
a b s t r a c t Four bacterial preservation methods: passage culture, sterile sand tube preservation, freezing preservation and freeze-drying preservation, were used to store Acidithiobacillus caldus strain S2, a moderately thermophilic bacterium. Among the four methods, the second is shown to be the best for short term preservation (6 months), and the fourth is the best for long term preservation (15 months). Specifically, the results show that using the second method, 32% cell viability was obtained after 6 months, and the cell survival rate reaches 17% using the fourth method after 15 months. In the bioleaching experiments, A. caldus strain S2 preserved via the second and the fourth methods, along with Leptospirillum ferriphilum strain YSK, were applied to bioleach chalcopyrite. Under the two preservation methods for A. caldus strain S2, the results show that 3.72 g/L and 1.68 g/L copper are extracted in the first 20 days and the maximum biomass delays to the 15th day and to the 18th day, respectively. When the bioleaching time was extended to the 40th day, copper extraction increased significantly to 5.02 g/L and 4.88 g/L, respectively. However, A. caldus strain S2 under no preservation, along with L. ferriphilum strain YSK, extract only 0.54 g/L Cu from the 20th day to the 40th day and finally release 5.14 g/L Cu. Therefore, the preserved culture shows almost the same total copper extraction as the unpreserved one in 40 days. As a result, sterile sand tube preservation and freeze-drying preservation greatly prolong the lag phase of cell growth without much decrease in the bioleaching ability of A. caldus strain S2. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Acidithiobacillus caldus is Gram-negative strictly aerobic bacteria and commonly found in acid mine drainage (AMD) and bioleaching reactors in moderately thermophilic environments (Baker and Banfield, 2003; Johnson and Hallberg, 2003; Zhou et al., 2009). It is considered to be the dominant sulfur-oxidizing bacterium in the bioleaching of sulphide ores at 40 °C–50 °C (Fouchera et al., 2003; Okibe et al., 2003). A. caldus promotes the sulphide-leaching efficiency by removing the build-up of solid sulfur which causes an inhibitory layer on the surface of the minerals (Dopson and Lindstrom, 1999; Gomez et al., 1996; Semenza et al., 2002). A. caldus together with other iron-oxidizing microorganisms such as Leptospirillum ferriphilum used to bioleach chalcopyrite has exhibited good performance. In our previous study of bioleaching chalcopyrite using A. caldus strain S2 and L. ferriphilum strain YSK, 80% copper extraction was obtained after 20 days, which is rather higher than bioleaching by pure culture L. ferriphilum strain YSK (23.6% copper extraction) (Fu et al., 2008). However, the loss of strains and the decrease ⁎ Corresponding author. Key Laboratory of Biometallurgy, Ministry of Education, Changsha 410083, China. Tel.: +86 731 8879212. E-mail address: [email protected] (G.-Z. Qiu).
of their bioleach capability often occur in many research and industrial applications (Yang et al., 2006; Zhang et al., 2000). Furthermore, A. caldus strain S2 that aids in the chalcopyrite bioleaching is not held in any international culture collection, so it is very important to determine the most effective preservation method which would not diminish its bioleaching capability. The methods for storing microorganisms generally include passage culture, sterile sand tube preservation (Li et al., 2006; Lu and Guo, 2007), freezing preservation, and freeze-drying preservation. Freezing preservation and freeze-drying preservations are considered to be the most feasible and effective storage for pure strains and mixed cultures (Morgan et al., 2006; Otero et al., 2007). However, most methods currently employed on freezing and freeze-dying preservations require highly specialized storage systems, substantial technical equipment, and often require very low temperature controlled environments (Hays et al., 2005). For these, they are generally expensive, technically demanding and manpower intensive. Passage culture and sterile sand tube preservations are taken for the short-term storage by many laboratory researchers due to their simple technical equipment demands, simple operation and low preserving cost. However, there are few reports about the preservation of A. caldus in the bioleaching of chalcopyrite. In this study, passage culture, sterile
0304-386X/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.11.003
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sand tube preservation, freezing preservation and freeze-drying preservation were used on A. caldus strain S2. Its characterization for bioleaching of chalcopyrite, along with L. ferriphilum strain YSK, before and after preservation, was then studied to investigate the effect of bacterial preservation on the chalcopyrite bioleaching performance. 2. Materials and methods 2.1. Culture medium and microorganisms A. caldus strain S2 and L. ferriphilum strain YSK (Genbank accession numbers of 16S rRNA genes: DQ256484 and DQ343299, respectively) were isolated and conserved. A. caldus strain S2 was maintained in Starky basal salt medium (pH 2.5) with sulfur as the energy source. Starky-S medium (in g/L) contains: (NH4)2SO4(3), KH2PO4(3), MgSO4·7H2O(0.5), CaCl2·2H2O(0.25), S(10) The medium for bioleaching chalcopyrite(20) with A. caldus and L. ferriphilum included FeSO4·7H2O(10) besides the above Starky-S medium. A. caldus strain S2 after preservation was revived and cultured in a shaking incubator at 180 rpm and 42 °C. 2.2. Cell preservation 2.2.1. Passage culture A. caldus strain S2 growing in stable growth phase was inoculated in 10% amounts into Starky-S medium for 15 days as the first passage culture. After 15 days the cell density clearly began to decrease and then the second passage culture was carried out, as the first time. When the strain was cultured to the 6th day, cell numbers in a 20 µL sample were counted with a hematocyte counter and optical microscope. 2.2.2. Sterile sand tube preservation Cells were harvested and re-suspended to a cell density of about 109 cells/mL by centrifugation (10,000 g, 10 min, and 20 °C) then 1 mL was added into glass tubes containing 20 g sterile sand. The tubes were stored in a dry box at room temperature. Every 3 months one of the tubes was taken out for cell reviving by dumping the contents into 10 mL Starky-S medium, slightly stirring for 20 min, adding 5 mL of clarified liquid into the Starky-S medium and culturing for 6 days. Then the cells were counted under an optical microscope and the results were compared with other preservation methods. 2.2.3. Freezing preservation Cells were harvested and concentrated by centrifugation (10,000 g, 10 min, 4 °C) then re-suspended into 20%w/w mannitol as a cryoprotectant. 500 µL samples of cell re-suspensions were preserved at −20 °C and −72 °C in a refrigerator-freezer, stored for 15 months and revived every 3 months. The frozen samples were revived by thawing for 10 min at 30 °C in a water bath, then inoculated in medium for 6 days — when the cell density was analyzed under optical microscope. 2.2.4. Freeze drying preservation The cell re-suspensions were prepared as above but small aliquots (20 µL) were dispensed into 1.5 mL tubes and then frozen by direct immersion in liquid nitrogen (−196 °C). The frozen samples were dried by a freeze-dryer for a period of 10 h at 4 °C and stored at 4 °C, −20 °C and −72 °C in a refrigerator–freezer. The frozen sample was again revived by thawing for 10 min at 30 °C in a water bath, then inoculated in medium for 6 days when the cell density was analyzed under optical microscope. 2.3. Cell viability Cell viability (V) was measured by cell density counted before preservation (N0) and after different times of storage (Nt). The viability rate (V%) was expressed by the cell survival rate that was calculated as
following: V (%) = 100 × Nt / N0, where N0 and Nt were the cell density cultured after 6 days and every 3 months, respectively. 2.4. Minerals composition The concentrate sample was collected from Meizhou in Guangdong province, China, with a particle size less than 75 μm. The components of the mineral sample were analyzed by XRD. The mineral sample consisted of mainly chalcopyrite (62%), galena (26%) and chalcocite (11%). 2.5. Bioleaching of chalcopyrite along with L. ferriphilum strain YSK Bioleaching experiments were performed in a 500 mL shake flask with 200 mL medium in a shaking incubator at 180 rpm and 42 °C. The initial pH value was adjusted to 2.5 by 1:1 sulfuric acid. A. caldus strain S2 and L. ferriphilum strain YSK (1:1) were inoculated into leaching medium with 10% inoculum amounts. The inoculums used include: the unpreserved A. caldus strain S2 along with L. ferriphilum strain YSK; the preserved strain S2 by sterile sand tube preservation after 3 and 6 months along with strain YSK; and the preserved strain S2 by freeze drying preservation after 3, 6, 9, 12 and 15 months along with strain YSK. Distilled water was added to the flask in order to compensate for evaporation losses. The experiment was performed in two stages: 0– 20 days and 20–40 days at 2% pulp density. The controlled experiments involved the following two cases: control 1, bioleaching of chalcopyrite with pure A. caldus strain S2 unpreserved; control 2, bioleaching of chalcopyrite with pure L. ferriphilum strain YSK. The levels of Cu2+, Fe2+ in solution were analyzed by atomic absorption spectrophotometry and by titration with potassium dichromate, respectively at the end of experiments, while the biomass of leaching system was counted and analyzed everyday under an optical microscope (Olympus, CX21). 3. Results and discussion 3.1. Cell preservation By analyzing the survival rate of the cells preserved, the preservation ways including passage culture, sterile sand tube preservation, freezing preservation and freeze drying preservation were compared to find the feasible way for short-term and long-term storages of strain A. caldus. The cell density of unpreserved A. caldus strain S2 achieved a maximum of 1.2 × 108 cells/mL in 6 days. When growth continues to 15 days, the cell density decreases to about 4.8 × 107 cells/mL and then the cell density decreases rapidly to less than 107 cells/mL. Passage culture has passed through 30 times in 15 months. In the initial several times of passage culture, cell density remains at 1.1– 1.2 × 108 cells/mL. After that, cell density continually decreases to 7.4 × 107 cells/mL at the 30th passage culture. Clearly, after 15 months the viability of strain A. caldus has declined remarkably due to bacterial degeneration (Bai, 2007) which leads the decreasing adaptability of A. caldus strain S2 in the growth medium. The results of sterile sand tube preservation after 3, 6, 9 and 12 months show that the longer the preservation time, the less the cell viability. Especially at the 12th month, few cells can be revived. This demonstrates that sterile sand tube preservation is not suitable for long-term storage; but for short-term storage of A. caldus strain S2 (up to 6 months), cell density reaches 3.8 × 107 cells/mL) and this preservation method is proven to be simple, low-cost and effective. Freezing storage at −20 °C and −72 °C has a very different effect on A. caldus strain S2. Freezing at −20 °C causes a high cell death rate and a majority of cells cannot be revived after storage for 6 months. However, freezing at −72 °C shows a good performance, although even after the first freezing, more than 50% cells have lost activities. Fonseca et al.
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(2001) reported that at very high freezing rates (such as at −72 °C), the bacterial suspension moved to a microcrystalline state, thus reducing the physicochemical and biochemical reaction rates. In contrast, at low freezing rates, damage to the cell structure was generated by the formation of ice crystals and by the dehydration of the cells, due to the high electrolyte concentrations in the medium. Mazur (1970) also explained that the better preservation at −72 °C can be ascribed to lower enzymatic reaction rates at this temperature, corresponding to lower molecular mobility, thus limiting cell damage induced by ice crystals. As a result, it can be seen that a lower storage temperature (−72 °C instead of −20 °C) led to better preservation of cell activity during frozen storage. Freeze drying has been used to preserve microorganisms for decades and it is the preferred method for culture strains worldwide. The products after freeze drying are available for transporting and storage (Morgan et al., 2006). In this experiment, the effect of different storage temperatures on cell viability after freeze-drying was investigated. The results show that freeze-drying preservation has a good performance for storing A. caldus strain S2. Furthermore, storing at −20 °C and −72 °C shows higher cell viability. After preservation for 15 months, the revived cell density achieved 1.2 × 107 cells/mL and 2 × 107 cells/mL, respectively. However, when the cells were preserved at 4 °C after 15 months, the cell density could not be revived. This indicates that after freeze-drying, the storage temperature does play a key role for cell preservation. 3.2. Comparison of cell survival rates For further investigating and analyzing the effects of these preservation methods on storing A. caldus strain S2, the survival rates were calculated as in Section 2.3 as shown in Table 1. Passage culture shows a high 61% survival rate and it seems that bacterial degeneration does not make lethal effect on cell preservation. However, this preservation method needs frequent manual-operation and observation that is easy to cause microbial contamination. Sterile sand tube preservation is considered to be a simple operation and low cost method. In this study, 32% cell viability was obtained after storing A. caldus with sterile sand tube preservation for 6 months so it is the feasible way for storage of A. caldus over a short term. Freezing would cause serial damages on cell structure and further affect the biological activity. It can be seen from Table 1 that the cell viability after freezing largely decreases with all greater than 50% before 3 months. Fortunately, frozen storage at −72 °C after freezedrying shows good cell preservation compared with other methods with up to 17% cell survival rate after 15 months. As a result, freezedrying preservation is proposed for the storage A. caldus over a long term.
335
Fig. 1. Cu2+, Fe2+ and Fe3+ concentrations in the bioleaching of chalcopyrite with L. ferriphilum strain YSK and A. caldus strain S2 preserved by sterile sand tube preservation after 3 and 6 months.
chalcopyrite. Thus, in this experiment, both of L. ferriphilum and preserved A. caldus were used to bioleach chalcopyrite. CuFeS2 þ 2Fe3þ →Cu2þ þ 2Fe2þ þ 0:125S8
ð1Þ
0:125S8 þ 1:5H2 O þ O2 →Hþ þ SO2− 4
ð2Þ
Fe2þ þ Hþ þ O2 →0:5H2 O þ Fe3þ
ð3Þ
Fig. 1 shows the Cu2+, Fe2+ and Fe3+ concentrations in the bioleaching of chalcopyrite after 20 days with L. ferriphilum strain YSK and A. caldus strain S2 preserved by sterile sand tube preservation. Before preservation, the mixed culture could bioleach chalcopyrite with 4.6 g/L Cu, and the copper extraction reaches 82%. It can be seen from Fig. 1 that after preservation, the rate of copper extraction continually decreases and the concentration reaches only 3.7 g/L Cu. The decline of cell viability requires preserved A. caldus strain S2 to grow for a longer time to form enough biomass to bioleach chalcopyrite and further prolong the total bioleaching time. It can be observed that the maximal biomass occurs at the 15th day when strain A. caldus is preserved after 6 months, while the maximal biomass is obtained at the 11th day when using unpreserved A. caldus strain S2 (Fig. 3). On the other hand, preservation would give some damage to cell activity (Fonseca et al., 2001), which may be another reason for the decrease of copper extraction.
3.3. Bioleaching experiments Chalcopyrite is a primary sulphide ore, which is leached by ferric ions according to Eq. (1). Sulfur is then dissolved by the sulfur-oxidizing microorganisms like A. caldus (Eq. (2)) and ferric ions are obtained by the action of ferrous ion-oxidizing microorganisms (Eq. (3)) like L. ferriphilum (Coram and Rawlings, 2002; Sand et al., 2000). Ferrous ion- and sulfuroxidizing microorganisms play important roles in the bioleaching of Table 1 The survival rate (%) of strain A. caldus during several preservation methods Storage time (months)
3
6
9
12
15
Passage culture Sterile sand tube preservation Frozen preservation at − 20 °C Frozen preservation at − 72 °C Freezing drying and frozen preservation at 4 °C Freezing drying and frozen preservation at −20 °C Freezing drying and frozen preservation at −72 °C
92 76 25 41 38 43 48
83 32 10 35 24 30 37
80 7 0 22 12 21 29
66 0 0 12 4 14 23
61 0 0 0 0 10 17
Fig. 2. The Cu2+, Fe2+ and Fe3+ concentrations in the bioleaching of chalcopyrite with L. ferriphilum strain YSK and A. caldus strain S2 preserved by freezing dry preservation at −72 °C after 3, 6, 9, 12 and 15 months.
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viability in less than 6 months. Freeze-drying preservation is considered to be the best way to store A. caldus strain S2 over a long term, since A. caldus can store for 15 months with up to 17% cell viability. During the bioleaching experiments, the rate of copper extraction by preserved L. ferriphilum strain YSK and A. caldus strain S2 decreases significantly in 20 days, compared with that by unpreserved L. ferriphilum strain YSK and A. caldus strain S2. The main reason is that preservation affects the cell activity and prolongs the growth phase time. When the bioleaching time is extended to 40 days, copper extraction significantly increases showing that preservation does not affect its long term ability for bioleaching chalcopyrite. Acknowledgements
Fig. 3. The variations of biomass during the bioleaching of chalcopyrite by L. ferriphilum strain YSK and A. caldus strain S2 unpreserved (■) or preserved by sterile sand tube preservation for 6 months (●) or by freeze drying preservation for 15 months (▲).
The concentrations of Fe2+ and Fe3+ always keep at a low level around 0.62 g/L and 1.04 g/L respectively, after preservation of 6 months, until some hindering factors happen such as the formation of jarosite on the surface of the mineral (Gomez et al., 1999a,b; Sandstorm and Petersson, 1997). Bioleaching of chalcopyrite with L. ferriphilum strain YSK and A. caldus strain S2 preserved by freeze drying preservation at –72 °C is shown in Fig. 2. After 15 months preservation it releases a rather low concentration of Cu2+, Fe2+ and Fe3+, (1.68 g/L, 0.14 g/L and 0.29 g/L, respectively) and the maximum biomass is only 1.6 × 108 cells/mL achieved by the 18th day (Fig. 3). It seriously affects bioleaching of chalcopyrite and leads to a low final copper extraction. After sterile sand tube preservation and freeze drying preservation, bioleaching microorganisms have to take a longer time to adapt the growth environment and reviving. Through prolonging the culture time, microorganisms may recover their growth and bioleaching ability. As a result, an additional experiment for prolonging the bioleaching time to the 40th day was carried out. For the unpreserved culture, the copper concentration increased slightly and finally reached 5.14 g/L Cu (92% extraction). The culture preserved by sterile sand tube preservation after 6 months gave a high copper extraction of 5.02 g/L while the culture preserved by freeze drying preservation after 15 months, finally achieves up to 4.88 g/L Cu. The control experiments showed the lowest extraction of copper with only 0.74 g/L Cu in control 2 and 0.26 g/L Cu in control 1. Compared to the unpreserved culture, the preserved one gives almost the same total copper extraction at the 40th day and do not show any obvious decrease in bioleaching ability. It is notable that with the unpreserved culture, most of the copper is leached from the ore sample before 20 days and it is thought that chalcopyrite passivation seriously affects extraction so that it is unnecessary to prolong the culture time (Sand et al., 2000; Stott et al., 2000). However, in the bioleaching with preserved culture, microorganisms pass through a long growth lag phase as living cell numbers build up again and more time is required. In this experiment, both L. ferriphilum and A. caldus were proved to be necessary for bioleaching of chalcopyrite. 4. Conclusions Four preservation methods: passage culture, sterile sand tube preservation, freezing preservation and freeze drying preservation were used to store A. caldus strain S2, a moderately thermophilic bacterium. It is feasible to preserve A. caldus strain S2 for a short term with sterile sand tube preservation, which has low cost and gives relatively high cell
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