Study on the use of acidophilic iron oxidizing bacteria for dissolving iron from low-Graded chalcopyrite ores

Journal of Science and Technology 54 (4A) (2016) 164-171 STUDY ON THE USE OF ACIDOPHILIC IRON OXIDIZING BACTERIA FOR DISSOLVING IRON FROM LOW-GRADED CHALCOPYRITE ORES Nguyen Van Hung, Dinh Thuy Hang * Institute of Microbiology and Biotechnology, E2 Building, 144 Xuan Thuy, Cau Giay, Hanoi * Email: dthang@vnu.edu.vn; dthangimbt@gmail.com Received: 15 August 2016; Accepted for publication: 6 October 2016 ABSTRACT Biomining is a microbiological-based approach to extract minerals from ores without adding acids and other extraction chemicals. Having biological nature, the process is considered environmental friendly and tends to be applied more widely nowadays, especially for low- graded ores, for which chemical extraction is no longer efficient. In this study, two iron oxidizing bacterial strains Acidithiobacillus sp. IOB1 and Leptospirillum sp. IOB2 originated from mining sites in Vietnam were used in laboratory leaching experiments for the recovering minerals from low-graded chalcopyrite ores. The obtained results showed that both strains could significantly accelerate the leaching process, the highest iron dissolution rate was estimated for 0.83 g kg 1 d 1 within two weeks of incubation with strain IOB2, about 30 % and 50 % respectively higher than leaching with strain IOB1 or control without bacteria, respectively. The iron extraction from ores was also evidenced by EDX analyses comparing the ore particles before and after incubation with the bacteria. Microscopic observation of DAPI-stained ore particles also showed high cell density attached to the particles, instead of freely moving in the liquid extract. Furthermore, FISH analyses using a specific probe for the -Proteobacteria GAM42a revealed that only 50 % of DAPI-stained cells were Acidithiobacillus sp. IOB1 – like in the leaching experiment with this train as starting culture, indicating that other bacterial species have developed and contributed to the metal dissolution here; however very rare positive signals were observed in leaching experiment with strain IOB2 as starting culture or the control without added bacteria. Thus, the acidophilic iron oxidizing strains Acidithiobacillus sp. IOB1 and Leptospirillum sp. IOB2 could serve as potential microbial sources for the development of biologically based approaches to extract minerals from ores, especially for those having low mineral contents. Keywords: Acidophilic iron oxidizing bacteria, bioleaching, low-graded chalcopyrite ores, Acidithiobacillus sp., Leptospirillum sp. 1. INTRODUCTION Application of microbes to extract metals from ores is an economical and environment friendly technique in modern mining industry. The bio-based mining is collectively termed as Study on the use of acidophilic iron oxidizing bacteria for dissolving iron from low-graded 165 biomining or bioleaching, employing microorganisms to leach metals from sulfide minerals via two processes, direct and indirect actions. During the direct bioleaching, minerals susceptible to oxidation undergo direct enzymatic attack by microorganisms. Certain bacteria such as Acidithiobacillus ferrooxidans oxidize ferrous ions to ferric ions and Acidithiobacillus thiooxidans oxidize sulfur presenting in ores to sulfates according to the following reactions 1, 2 : Fe 2+ + 1/4O2 + H2 Fe 3+ + 1/2 H2O S + 3/2O2 H2O + H2SO4 During the indirect action, bacteria produce strong oxidizing agents such as ferric ions and sulfuric acid which react with metals and allow them to be extracted from the ores as proposed in the following reaction 1, 3 : MS + 2Fe 3+ M 2+ + 2Fe 2+ + S An acidic environment is necessary for the indirect bioleaching to keep ferric ions and other metal ions in solution, and this could be reached by the bacterial production of sulfuric acid to the environment 2 . In the mining industry, both the direct and indirect actions are used in conjunction to optimize cost-effectiveness 1 . Most of bioleaching microorganisms are characterized as being autotrophic as well as acidophilic. They are not only different in terms of operating termperature and pH range, but also in terms of ability to extract metals from different ores. For examples, Thiobacillus ferrooxidans extracts iron from pyrite (FeS2) and arsenopyrite (FeAsS) while Thiobacillus thiooxidans extracts sulfur from sulfur ores. Sulfolobus acidocaldarius performs bioleaching on copper and molybdenum from chalcopyrite and molybdenite, while Pseudomonas aeruginosa and Rhizopus arrhizus extract uranium from low quality uranium ores 2 . Nowadays, bioleaching is used widely in mining industry all over the world. Specifically, the technology is applied for approximately 20 % mining and processing of copper (from covellite (CuS), chalcocite (Cu2S), bornite (Cu5FeS4), and chalcopyrite CuFeS2) and many other transition metals from sulfide ores such as pentlandite ((Ni,Fe)9S8), millerite (NiS), sphalerite (ZnS), and galena (PbS) 3 . Industrial-scale bioleaching of refractory gold concentrates is practiced in South Africa, Brazil, Australia, Ghana, Peru, China and Kazakhstan 3, 4 . Nevertheless, there exists a problem in the low efficiency of conventional bioleaching which depends to the large extent on the microbial species 5 . Hence, screening for new high- performance bioleaching microbes as well as improving the performance of known bioleaching species by genetic engineering have been proposed for achieving good leaching rates 6 . This study aimed to demonstrate bioleaching process for recovering minerals from low grade chalcopyrite ores in a laboratory model by using acidophilic iron oxidizing bacterial strains newly isolated in Vietnam. The study would provide scientific basis for the bioleaching technology in Vietnam where metal mines are mostly of low grades and locate dispersedly. 2. MATERIALS AND METHODS 2.1. Acidophilic iron oxidizing bacteria (FOB) sources and cultivation procedure Two strains of acidophilic iron oxidizing bacteria Acidithiobacillus sp. IOB1 and Leptospirillum sp. IOB2 were isolated from wasted sulfidic ores via enrichment step 7 . The Nguyen Van Hung, Dinh Thuy Hang 166 strains were grown in 9K medium (per liter): K2HPO4, 0.5 g, Ca(NO3)2 0.01 g, MgSO4.7H2O 0.5 g, (NH4)2SO4, 3 g, FeSO4.7H2O, 44.22 g, pH was adjusted to 3.3 ± 0.2 by 1N sulfuric acid. The growth conditions were 28 C, rotation shaking at 120 rpm in the dark. 2.2. Leaching experiment Wastes after chemical extraction of chalcopyrite ore (CuFeS2) used in the experiments were provided by Phu Dien Joint Stock Company. The wastes were in the form of grayish powder with particle size of 0.2 – 0.3 mm. The experiments were carried out in plastic vessels constructed with double bottoms, the upper was porous to allowed air to flow through while holding the ore particles with the help of an acid inert, fine-porous net (pore sizes 0.1 mm); the deeper for keeping leaching solution (Fig. 1). Figure 1. Schematic design of the leaching experiments. Each experimental vessel contained 100 g ore waste, immerged in 500 ml 9K medium (pH 5, without Fe 2+ ). As inocula, the strains Acidithiobacillus sp. IOB1 and Leptospirillum sp. IOB2 were added from liquid cultures at a cell number of 10 6 cell ml 1 . Oxygen was supplied from air blowers to keep the concentration of dissolved oxygen in the medium at 3 mg L 1 . Vessels were kept in the dark at room temperature for one month. During the experiments, pH and total content of iron (Fe 2+ and Fe 3+ ) were regularly checked. Since the bacterial growth led to the production of sulfuric acid and drop pH in the leaching solution, a part of this solution was periodically (at different intervals 4 – 8 days, at the time points when pH in the chelating solution dropped to 2 or slightly below) exchanged with fresh 9K medium (pH 5, without Fe 2+ ) to reach pH 3.5 – 4 to promote the bacterial growth. 2.3. Analytical methods 2.3.1. Measurement of iron dissolution Total iron content was measured by using method provided by German standard methods for the examination of water, waste water and sludge (DIN 38406-E1-1). The chemical principle of method lies on the reaction of ferrous iron with O-phenanthroline to produce a complex (at pH in the range 3 – 9) colored in purple, which can be quantified at wave length 510 nm. 2.3.2. EDX analyses Study on the use of acidophilic iron oxidizing bacteria for dissolving iron from low-graded 167 Ore particles in the bioleaching experiments were collected at different time points, washed with ethanol 70 and 100 % then applied on sample holders covered with aluminum foils. Samples were then analyzed for elements under scanning electron microscope Hitachi connected with an energy-dispersive X-ray spectroscopy (EDX) analyzer unit. The changes in iron content in ore particles were observed in at least five distant positions on the sample holder. EDX analyses expressed only semi-quantitative changes in iron content of the ore material. 2.3.3 FISH analyses Ore samples from the leaching experiments were washed in 1 PBS, then fixed in 4 % glutaraldehyde over night (not more than 16 h) at 4 C. The samples were then washed with distilled water for several times, applied onto polycarbonate membrane filters and dried in the air. A piece of membrane filter (1/4 – 1/6 filter sheet) was used in hybridization. Hybridization was performed with γ-proteobacteria specific probe GAM42a (5’- AAACGATGTGGGAAGGC-3’) having cy3 at 5’-end of the oligo-nucleotide probe sequences at stringency of 35 % formamide and 80 % NaCl for 3 hours at 46 C 8 . DAPI counter staining was carried out with 50 l DAPI (0.1 mg ml 1 ) for 3 min in the dark, samples were then washed in 80 % ethanol for few seconds and let to air dry (the samples should be protected from light). Cells were then visualized under fluorescence microscope Zeiss Axio Plus with proper filters for DAPI and Cy3, and images were taken by AxioCam ICc3 (Zeiss). To minimize bleaching effects, a droplet of Citifluor:Vecta shield 1:1 vol:vol (Zeiss) was applied on the top of the samples. 3. RESULTS AND DISCUSSION 3.1. Iron dissolution in the bioleaching experiments Bioleaching experiments (Fig. 1) were carried out with chalcopyrite ore of low metal content (ore wastes after chemical extraction). Growth of the bacterial strains IOB1 and IOB2 used as inoculums in the leaching experiments was recorded via changing of pH (Fig. 2A) of the chelating solution as well as the amount of iron (Fig. 2B) dissolved from the ore particles into the solution. At the beginning of the experiments, pH was set to 4.5 by using diluted sulfuric acid to promote the bacterial growth. At the day fifth, pH dropped to 2, indicating acid production by the bacteria. At this time point, acid production was not observed in control without the bacterial inoculates. Figure 2. Changing in pH (A) and total iron extracted from the ore into the chelating solution (B) during leaching experiments using strains Acidithiobacillus sp. IOB1 and Leptospirillum sp. IOB2 as inocula. A B Nguyen Van Hung, Dinh Thuy Hang 168 During the experiments, pH decreased periodically (within different intervals of 4 – 8 days) to 2 and below due to the bacterial growth (Fig. 2A). Accordingly, at the time points when the pH reached critical values below 2, at which growth of the bacteria significantly slowed down, it was adjusted to 3 – 3.5 by replacing 30 % of the chelating solution with freshly prepared 9K medium. Figure 2 showed that both strains IOB1 and IOB2 started to produce acid and dissolve iron right after the first few days of incubation. In the first two weeks, iron dissolution rate in the presence of strain Leptospirillum sp. IOB2 was higher than that by strain Acidithiobacillus sp. IOB1. Similar effects were also observed in other bioleaching studies 9 . From the data obtained, a maximum rate of 0.83 g kg 1 d 1 could be calculated for the iron dissolution in the presence of strain Leptospirillum sp. IOB2 at the first 10 day of leaching experiments. This rate was 30 % and more than 50 % higher than that occurred in leaching experiment with Acidithiobacillus sp. IOB1 and the control without inocula, respectively. In addition, element analysis by EDX carried out for the ore material after 4 weeks of experiments (Fig. 3) showed that in the presence of Leptospirillum sp. IOB2 the iron content in the ore has decreased most significantly (Fig. 3C) in comparison to the experiment with Acidithiobacillus sp. IOB1 (Fig. 3B) and the control without bacteria (Fig. 3A). Figure 3. Element analyses (EDX) of ore particles in the biochelating experiments. A - Control without added bacteria; B – Acidithiobacillus sp. IOB1 was added; C – Leptospirillum sp. IOB2 was added. It has been shown previously that acidophilic species of the genus Leptospirillum were capable of growing at extremely acidic environments, such as pH 1.5 and below and being stable at high ferric-ferrous iron ratio 10 . With these properties, species of Leptospirillum might better adapt to the environmental conditions inside the ore bulk during chelating processes. Indeed, species of Leptospirillum have been found dominant in commercial bio-oxidation tanks of different ore types 11 . The accumulation of iron extracted from the ore was most significant in the first 20 days of Study on the use of acidophilic iron oxidizing bacteria for dissolving iron from low-graded 169 the experiments, afterward the iron accumulation rate slowed down gradually. It has been speculated that the limitation in air supply and surface attachment of bacterial cells to the ore particles were the main reasons. 3.2. Role of the acidophilic iron oxidizing bacteria in leaching process Mix cultures of acidophilic iron oxidizing bacteria have been proven for higher metal dissolution rate than single strains 2 . It should be noted that the leaching experiments in this study were carried out under unsterile condition, i.e. besides the main bacterial strains that were added as inocula, other species could also develop in the ore bulk. FISH analyses using GAM42a probe, specific for -Proteobacteria (including Acidithiobacillus spp.) revealed that IOB1-related group made just 50 % of total cell count (stained with DAPI) associated with ore particles even in the leaching experiment with IOB1 as inoculum (Fig. 4). This means some other acidophilic iron oxidizing species have been developing and shared significant role in population genetically and, perhaps also functionally. Figure 4. FISH analyze of ore particles from the leaching experiment with Acidithiobacillus sp. IOB1 as starting culture using GAM42a probe specific for the -Proteobacteria. A – DAPI stained cells; B – Cy3-GAM42a hybridized cells. In the leaching experiment with Leptospirillum sp. IOB2, just a few positive signals in the hybridization with GAM42a probe could be observed (data not shown), indicating that Acidithiobacillus sp. IOB1 – like could not compete with Leptospirillum IOB2 as a starting culture for bioleaching process here. In practice, Leptospirillum spp. have been found dominant in commercial bioleaching tanks because they could form strongly attached biofilms and adapt better than Acidithiobacillus spp. to the environment provided in bioleaching tanks 10, 11 . In control without bacterial inocula, much lower cell density was observed and positive signals in hybridization with GAM42a probe were quite rare (data not shown). Apparently, iron dissolution observed in the control must be due to in situ microbial community, however the community development was rather slow, and metabolic activity was not high in comparison to the other leaching experiments where acidophilic iron oxidizers were added as starting cultures. 4. CONCLUSIONS In this study, two strains of acidophilic iron oxidizing bacteria Acidithiobacillus sp. IOB1 and Leptospirillum sp. IOB2 originated from mining sites in Vietnam were used as starting A B Nguyen Van Hung, Dinh Thuy Hang 170 cultures for bioleaching experiments in the laboratory. These strains showed high capability of promoting metal solubilization from low-graded chalcopyrite ore, especially the strain Leptospirillum sp. IOB2. It has been shown clearly that the addition of these bacteria at the beginning of chelating process resulted in 50 % higher iron dissociation from the ore comparing to control without starting culture. Nevertheless, combination of both bacterial groups, i.e. Acidithiobacillus spp. and Leptospirillum spp. would be best for bioleaching. The newly obtained bacterial strains used in this study could have very promising application potential in mining industry in Vietnam, where most of mineral ores are of low grade and locate far apart from each other. Acknowledgements. The research funding from Asia Research Center, VNUH (Grant number CA. 14.12A) is acknowledged. REFERENCES 1. Rohwerder T., Gehrke T., Kinzler K., Sand W. - Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation, Appl. Microbiol. Biotechnol. 63 (2003) 239-248. 2. Rawlings D. E. - Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates, Microb. Cell Factor. 4 (2005) 13-29. 3. Rawlings D. E. - Heavy metal mining using microbes, Annu. Rev. Microbiol. 56 (2002) 65-91. 4. Dopson M., Baker-Austin C., Ram K. P., Bond P. - Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic micro-organisms, Microbiology 149 (2003) 1959-1970. 5. Norris P. R., Burton N. P., Foulis A. M. - Acidophiles in bioreactor mineral processing, Extremophiles 4 (2000) 71-76. 6. Okibe N., Johnson D. B. - Biooxidation of pyrite by defined mixed cultures of moderately thermophilic acidophiles in pH-controlled bioreactors: significance of microbial interactions, Biotech. Bioeng. 87 (2004) 574-583. 7. Nguyen T. H. T., Nguyen V. H., Dinh T. H. – Isolation of the acidophilic iron oxidizing bacteria via modified liquid serial dilution method, J. Biotech. (2016) in press. 8. Manz W., Amann R., Ludwig W., Wagner M. and Schleifer K. H. - Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions, Syst. Appl. Microbiol. 15 (1992) 593-600. 9. Rawlings D. E., Tributsch H., Hansford G. S. - Reasons why 'Leptospirillum'-like species rather than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores, Microbiology 145 (1999) 5-13. 10. Tuffin M., Hector S. B., Deane S. M. and Rawlings D. E. - Resistance determinants of a highly arsennic resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank, Appl. Environ. Microbiol. 72 (2006) 2247 - 2253. Study on the use of acidophilic iron oxidizing bacteria for dissolving iron from low-graded 171 11. Kunnunen P. H. M. and Puhakka J. A. - High-rate ferric sulfate generation by a Leptospirillum ferriphilum-dominated biofilm and the role of jarosite in biomass retention in a fluidized-bed reactor, Biotech. Bioeng. 85 (2004) 697 - 705. TÓM TẮT NGHIÊN CỨU SỬ DỤNG VI KHUẨN OXY HÓA SẮT ƯA AXIT ĐỂ HÒA TÁCH SẮT TỪ QUẶNG CHALCOPYRITE NGHÈO Nguyễn Văn Hưng, Đinh Thúy Hằng* Viện Vi sinh vật và Công nghệ sinh học, Đại học Quốc gia Hà Nội Nhà E2, 144 Xuân Thủy, Cầu Giấy, Hà Nội * Email: dthang@vnu.edu.vn; dthangimbt@gmail.com Tuyển khoáng sinh học là cách tiếp cận thông qua vi sinh vật để hòa tách các khoáng trong quặng mà không cần sử dụng axit hay các hóa chất khác. Có bản chất sinh học, cách tiếp cận này được đánh giá là thân thiện với môi trường và ngày càng được ứng dụng rộng rãi, đặc biệt đối với các loại quặng nghèo không còn thích hợp để áp dụng tuyển khoáng hóa học. Trong nghiên cứu này, hai chủng vi khuẩn oxy hóa sắt ưa axit là Acidithiobacillus sp. IOB1 và Leptospirillum sp. IOB2 có nguồn gốc từ mỏ khoáng sản ở Việt Nam được sử dụng trong thí nghiệm tuyển khoáng ở quy mô phòng thí nghiệm để hòa tách kim loại từ quặng chalcopyrite nghèo. Kết quả cho thấy cả hai chủng đều có khả năng tăng cường quá trình hòa tách kim loại, tốc độ hòa tách cao nhất ở mức 0,3 g kg 1 d 1 có được ở thí nghiệm với chủng Leptospirillum sp. IOB2 trong hai tuần đầu thí nghiệm, cao hơn 30 % và 50 % tương ứng so với thí nghiệm có chủng Acidithiobacillus sp. IOB1 và đối chứng không có vi khuẩn bổ sung ban đầu. Việc hòa tách sắt từ quặng trong các thí nghiệm này còn được minh chứng qua phân tích thành phần nguyên tố trong quặng bằng thiết bị EDX. Quan sát bằng kính hiển vi huỳnh quang mẫu quặng và dịch tuyển khoáng nhuộm DAPI cho thấy phần lớn tế bào sinh trưởng bám dính với các hạt quặng. Lai huỳnh quang tại chỗ với đầu dò GAM42a đặc hiệu cho phân lớp -Proteobacteria (có bao gồm cả các loài Acidithiobacillus) cho thấy chỉ có 50 % lượng tế bào nhuộm DAPI trong thí nghiệm có bổ sung chủng Acidithiobacillus sp. IOB1 có tín hiệu lai với đầu dò này, chứng tỏ ngoài Acidithiobacillus spp. đã có các loài vi khuẩn oxy hóa sắt ưa axit khác cùng phát triển ở đây, đóng vai trò quan trọng trong quần thể về mặt di truyền cũng như về chức năng hòa tách quặng. Rất ít tín hiệu dương tính trong phân tích với đầu dò đặc hiệu được tìm thấy ở thí nghiệm bổ sung chủng Leptospirillum sp. IOB2 hay trong mẫu đối chứng. Như vậy hai chủng vi khuẩn oxy hóa sắt ưa axit Acidithiobacillus sp. IOB1 và Leptospirillum sp. IOB2 có tiềm năng ứng dụng trong việc phát triển công nghệ tuyển khoáng sinh học, đặc biệt hữu hiệu đối với các loại quặng có hàm lượng khoáng thấp. Từ khóa: Acidithiobacillus sp., Leptospirillum sp., quặng chalcopyrite nghèo, tuyển khoáng sinh học, vi khuẩn oxy hóa sắt ưa axit.