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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Minerals Engineering 24 (2011) 870–875 Contents lists available at ScienceDirect Minerals Engineering journal homepage: www.elsevier.com/locate/mineng Effects of ultraviolet irradiation on bacteria mutation and bioleaching of low-grade copper tailings Yingbo Dong, Hai Lin ⇑, Han Wang, Xiaolan Mo, Kaibin Fu, Hongwei Wen School of Civil and Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China Key Laboratory of the Ministry of Education of China for High-Efficient Mining and Safety of Metal Mines, Beijing 100083, China a r t i c l e i n f o a b s t r a c t Article history: This paper studies the effects of ultraviolet irradiation on the mutation of bacteria (i.e., Acidithiobacillus ferrooxidans LD-1) and on the bioleaching system for low-grade copper tailings. It is shown that ultravi- olet irradiation can lead to an obvious LD-1 mutation, can improve bio-activity and bioleaching capabil- ity. The mutant LD-1 bacteria, after ultraviolet irradiation for 30 min, had the best oxidation activity, the Received 20 December 2010 Accepted 23 March 2011 Available online 16 April 2011 2 + oxidation rate of ferrous ion (Fe ) in mutant bacteria culturing medium can reach 100% after 24 h (18 h less than that of the original bacteria under the same leaching conditions). After 30 days, copper extrac- tion with mutant bacteria is increased by about 17%, compared with original bacteria, and by 70% com- pared to chemical leaching. These results show that the effects of mutant bacteria after ultraviolet irradiation are better than those of the original bacteria and much better than those of chemical leaching. The improved bioleaching rate of the mutant bacteria is attributed to the faster propagation rate and capabilities for stronger sulfuric acid generation. This study shows that mutant bacteria from ultraviolet irradiation have great potential for improving copper recovery from low-grade copper tailings. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: Low-grade copper tailing Bioleaching Ultraviolet irradiation Bacteria mutation Copper leaching 1 . Introduction Recently, conventional mineral processing methods have been Qiu et al., 2008), there are still some challenges in producing excel- lent strains for industrial application. For example, high efficiency strains are extremely scarce, and the oxidation ability of bacteria or the ability to resist adverse environments is inadequate. In addi- tion, both the domestication of strains and genetic engineering have some disadvantages, such as the former is time-consuming and inefficient, genetic engineering is complex and still in the early stages of development. Therefore, mutation breeding has become the most popular method for enhancing bioleaching activity of strains. Induced UV mutation is the simplest and effective physical mutation method. The effective ultraviolet wavelength is mainly around 255 nm which is the same with the DNA absorption spec- trum of general bacteria. Hence, UV mutation has a strong bacteri- cidal effect. In this study, copper tailing from Hubei province, China, was used to investigate UV mutation of LD-1 bacteria. Ultraviolet Irradi- ation can change the chemical structure of DNA and increase muta- tion frequency. The effects of mutagen on the oxidation activity of mutant LD-1 bacteria and the bioleaching capability of the bacteria on copper tailings are the main factors studied in this work. used to recover valuable metals in tailings, but these methods have limitations. Compared to conventional mineral processing methods, microbial leaching technology is a more effective way to treat low-grade tailings, due to many advantages, such as low cost, low investment, fast processing, simple equipment, wide range of applications, easy management and environment friendly, etc. Bioleaching technology has been widely used in the leaching of low-grade primary ores (Plumb et al., 2008; Farah et al., 2005), but only a few studies on bioleaching of tailings have been done (Wang and Li, 2004). However, compared with the pri- mary ore, the nature of tailings is different. Therefore, selecting and breeding the strain for leaching tailings is becoming more and more important (Wen, 2008). Low mineral recovery could cause the tailings to be stockpiled and discharged even when they still contain rich useful minerals (Donto et al., 2007; Fourier and Blight, 2001). Tailings therefore have great potential as valuable secondary resources. The main bacteria breeding methods include domestication, mutagenesis and genetic engineering (Akcil, 2004). Although much research on breeding of leaching bacteria has been done all over the world (Roel et al., 1987; Brierley, 2008; Gericke et al., 2001; 2 . Materials and methods 2 .1. Microbial strain and culture conditions ⇑ Corresponding author. Tel.: +86 138 01283734; fax: 86 010 62333603 E-mail address: linhai@ces.ustb.edu.cn (H. Lin). Acidithiobacillus ferrooxidans LD-1, isolated from acid mine water (pH = 4.5) from the Daye copper mine in Hubei Province, 0 892-6875/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mineng.2011.03.020 Author's personal copy Y. Dong et al. / Minerals Engineering 24 (2011) 870–875 871 was obtained by various stages of domestication. The stages were conducted to gradually simulate the tailing environment. The opti- mal cultivating conditions of LD-1 bacteria were set at 30 °C, the initial pH value at 2.0, and rotation speed at 160 rpm. The culture medium, optimized 9 k medium, consisted of the following com- to an optimized 9 k medium, in which the cell density was ad- 5 À1 justed to 5 Â 10 cell mL after inoculation. The oxidation proper- ties of mutant bacteria were analyzed. The mutant bacteria with the best oxidation activity were used in the tailings bioleaching experiments. 4 ) 2 SO 4 2.0 g/L, K 2 HPO 2 O 44.2 g/L. The medium, without medium m filter and was added 4 4 2 pounds: (NH 0.25 g/L, MgSO Á7H O 0.25 g/ L, KCl 0.1 g/L, and FeSO FeSO , was autoclaved at 112 °C for 30 min. The FeSO 4 Á7H 2 .4. Bioleaching experiments 4 4 was separately sterilized through a 0.2 l Bioleaching experiments were carried out in 250 mL flasks con- taining 90 mL sulfuric acid solution (pH = 2) and 10 mL of inocula. aseptically to the iron free medium. 7 À1 The initial density of the inoculated solution was 5 Â 10 cell mL . 2.2. Low-grade tailings The mineral concentration was 5% (wt/vol). Flasks were main- tained at 30 °C and shaken at 160 rpm. The number of viable bac- teria, pH value, redox potential, and copper ion concentration in the leaching solution were determined at certain intervals. The loss of water due to evaporation was compensated by distilled water in order that the solution was maintained at 100 mL in the flasks. Low-grade tailings were collected from the abandoned reservoir of the copper mine at Daye. The copper-bearing mineral compo- nents of the tailings were chalcopyrite and trace amounts of copper blue. The chemical analysis of the tailings is shown in Table 1. X- ray diffraction (XRD) patterns are shown in Fig. 1. The main gangue minerals were dolomite, calcite, and quartz. The content of copper- bearing minerals was low. The particle size of samples was 2 .5. Analytical methods À250 lm (over 85%). Copper-bearing minerals were finely dissem- Ferrous iron was determined by titration with potassium dichromate (K CrO ). The concentration of dissolved copper ion 2 7 inated and intergrown with particles of gangue. The particle size of copper minerals was generally about 0.005 mm ꢀ0.1 mm. in the leaching solution was analyzed by atomic absorption spec- trometry (AAS). The pH value and redox potential were measured by a S20 seven easy pH/Eh process controller. The bacterial number was determined by blood cell counting chambers under a ZBM- 300E biological microscope. The lethality rate was determined by calculating the bacterial number. The leached residues were fil- tered and were dried using a freeze drier. The morphological fea- ture and chemical composition of the leached residues were analyzed by SEM and XRD. The oxidation property of the bacteria was expressed by the oxidation rate of ferrous ions. Fe2 oxidation rate was calculated using the equation: 2.3. Mutations The bacteria culture in the logarithmic growth phase was cen- trifuged for about 20 min at 5000 rpm, and the solid cells were ob- tained by removing the supernatant. The solid cells were then washed three times with acidified distilled water (pH = 2). Finally, the cells were suspended in basal salts medium without energy sources and the density of the cells was adjusted to + 1 Â 108 cell mLÀ1 . 1 0 mL of bacteria solution were added to a petri-dish and irra- CFen2þ diated with UV light (power of 15 W, wavelength of 254 nm, dis- tance of 30 cm) for 10, 30, 60, and 120 mins. The numbers of mutant bacteria with different irradiation times were counted un- der a microscope, so the bacteria lethality rates could be calcu- lated. Meanwhile, the different mutant bacteria were introduced Fe2þ oxidation rate % ¼ 1 À CFe02þ Â 100% ð1Þ where CFen2þ is the concentration of Fe2+ in the medium after an n-hr 2 + culture, CFe02þ is the initial concentration of Fe in the medium. Table 1 Chemical analysis of copper tailings. Elements Cu Fe 9.40 Na 1.78 2 O 3 S SiO 45.39 TiO 0.28 2 Al 2 O 3 MgO 6.46 P Zn Content (%) 0.20 5.32 CaO 18.38 8.98 Mn 0.02 Mo K 2 O 2 O 2 Content (%) 3.43 0.22 0.11 0.03 Fig. 1. XRD pattern of the copper tailings. Author's personal copy 872 Y. Dong et al. / Minerals Engineering 24 (2011) 870–875 3 . Results and discussion 2.6 2 2 2 2 2 .5 .4 .3 .2 .1 2 3 .1. The bacteria lethality rates 0 1 min 0min UV is the most common physical mutagen. Ultraviolet irradia- 30min 0min 120min tion can increase the energy of the inner election in the atom and can make the inner election into the active molecule. The en- ergy of UV is about 3–5 ER. Even though the energy is very weak and the penetrability of UV cannot cause ionization, the UV can change the structure of DNA; for example, DNA strand breakage, cross linking of intramolecular and intermolecular in the DNA, cross linking of nucleic acid and protein, hydration of cytosine and uralic, formation of pyrimidine dimmers (Pullman and Pull- man, 1963; Rohwerder et al., 2003). The main effect of UV muta- tion is to format thymine dimmers which can change the biological activity of DNA and cause bacterial mutation even after death (Yang et al., 2010). Excessive UV irradiation can cause loss of a large segment of DNA cells or can make the cross linked DNA unopened and prevent replication and transcription of DNA. In other words, excessive UV can induce cell death. The amount of mutagen can influence the bacteria lethality directly. Large amounts of mutagen cause high lethality (about 90–99%). The few- er the positive mutant strains, the more negative mutant strains are in the survival cells. However, high efficiency strains may be selected from a few positive mutant strains. Small amounts of mutagen can cause a lethality of 50–80%. While more positive mu- tant strains are in the survival cells, a few high efficiency strains may also be there (Beggs, 2002). For this test work, the relationship between irradiation time and lethality rate is shown in Fig. 2. The results show that the lethality rate increased with UV irra- diation time. When the irradiation time was 10 min, the lethality rate of bacteria was about 17%, indicating that the lethality rate of bacteria was low in small amounts of mutagen. When the irra- diation time was 120 min, the lethality rate reached 85%. The main cause of UV mutation is that UV irradiation can change the DNA, as base pairs of DNA chains can absorb UV strongly. The sensitivity of the pyrimidine is almost 100 times greater than that of the purine. The double bond of thymine changes into a single bond under UV light, and then the two newly formed single bands of carbon atoms connect together, so a circular band is formed between two thy- mines, which were thymine dimmers (Xu et al., 2010). When the irradiation time was longer, the amounts of newly formed thymine dimmers increased, causing a large range of genetic variation and a high lethality of bacteria. 6 0 6 12 18 24 30 Time/h 36 42 Fig. 3. Evolution of pH vs. time by bacteria with different mutation time. 6 5 5 4 10 60 10 60 0min 10min 30min 60min 120min 410 360 0 6 12 18 24 30 36 42 Time/h Fig. 4. Evolution of Eh vs. time by bacteria with different mutation time. ium under the same conditions, at 30 °C, initial pH value of 2.0, rotation speed 160 rpm. The initial density of inoculated solution 5 À1 was 5 Â 10 cell mL . The pH and Eh of the culture solution, oxi- 2+ dation rate of Fe , and growth curves were determined at various intervals. The pH values and the Eh of the culture solution are shown in Figs. 3 and 4, respectively. The pH value is a very important factor in the culture of bacteria. A change of pH will affect bacteria growth and oxidation of Fe . The results in Fig. 3 show that the pH increased at first and subsequently decreased, the reason being that H is consumed in the bacteria metabolism. In the first culti- vation stage, the main reaction was oxidation of Fe in the med- ium; this reaction can be accelerated by the involvement of bacteria, which leads to the pH increasing rapidly (Meruane and Vargas, 2003). As culture time progressed, Fe was oxidized to 2+ + 2+ 2+ 3+ 3+ 3+ Fe and the concentration of Fe in the medium increased. Fe was involved in the hydrolysis reaction (Eq. (2)) and led to the for- mation of H , and so the pH decreased slightly in the late cultiva- + tion stage. 3 .2. The culture of mutant bacteria Fe þ þ 3H 3 O ! FeðOHÞ3 þ 3Hþ ð2Þ 2 The stability of the DNA structure may be changed by mutation. Fig. 4 shows the evolution of Eh in the culture solution. The change of [Fe ]/ [Fe ] or the concentration of Fe in medium re- flects the change of Eh. Oxidation of Fe can provide the necessary energy for bacterial growth, so that the concentration of Fe and Eh in solution increases gradually. When Fe was oxidized com- pletely, the Eh reached its highest value. The results in Figs. 3 and 4 demonstrate that the trends of pH and Eh in the culture sys- tem of the original bacteria both lag behind that of the mutant bac- teria. The time for the pH to reach its maximum in the culture system of the original bacteria lags 18 h behind that of the mutant bacteria and the time for the Eh to reach a stable state lags by at least 10 h. After 36 h, the Eh is close to a stable state, between The mutation site may be in a metastable state, which increases the probability of reverse mutation or inhibition of gene mutation. The mutant bacteria were cultured and observed over a long period to ensure the stability of mutants. After four generations of culture, the different mutant bacteria were cultured in optimized 9 k med- 3 + 2+ 3+ 2 + 3+ 2 + 100 8 6 4 2 0 0 0 0 0 5 90 mV and 600 mV, in the culture system of mutant strains. Thus, the oxidation activity of mutant bacteria is improved and the growth rate of mutant bacteria is accelerated. 0 20 40 60 80 100 120 Figs. 5 and 6 show the Fe +oxidation rate and growth curve of the original strain and the mutant bacteria, respectively. The re- sults show that the oxidation activity and growth rate of mutant 2 Irradiation time/min Fig. 2. Effect of irradiation time on lethality rate. Author's personal copy Y. Dong et al. / Minerals Engineering 24 (2011) 870–875 873 1 00 9 .5 8 8 6 4 2 0 0 0 0 0 8 0min 1 3 0min 0min original bacteria mutant bacteria 60min 1 20min 7.5 0 5 10 15 20 25 30 0 6 12 18 24 30 36 42 Time/h Leaching time/d Fig. 5. Fe2+oxidation rate vs. time by bacteria with different mutation time. Fig. 7. Growth curves of the original bacteria and mutant bacteria in leaching system. 9 8 7 6 5 .5 .5 .5 .5 .5 3 5 0 1 3 6 1 min 28 0min 0min 0min 20min 2 1 1 4 7 0 original bacteria mutant bacteria acid leaching 0 6 12 18 24 30 36 42 Time/h 0 5 10 15 20 25 30 35 Leaching time/d Fig. 6. Growth curves of different bacteria. Fig. 8. The copper extractions under the different conditions. bacteria are both better than those of the original bacteria, which 2 + are in concert with the trend of pH and Eh. After 42 h, the Fe oxi- dation rate of the four mutants reached 100% and that of original bacteria only about 65%. These indicate that the oxidation activities of the four mutants are improved greatly. Fig. 6 shows that the mu- tants grow faster than the original bacteria. The mutant bacteria with 30 min irradiation have the best oxidation activity and growth rate. The results show that UV irradiation for 30 min is effective for mutation. So, the mutant bacteria with 30 min irradi- ation were selected as the experimental bacteria in the tailing leaching experiments. minor role in initial leaching. The alkaline gangue was almost com- pletely dissolved after 5 days and the pH of the leaching system tended to stay around 2.0. This pH value was fitting for the growth of leaching bacteria, so acids were not added to the leaching sys- tem. After 30 days, copper extractions reached 18.5% (acid system), 2 6.4% (original bacteria system), and 30.8% (mutant bacteria sys- tem), respectively. These results indicate that bioleaching of tail- ings had the advantage over acid leaching; copper extraction of mutant bacteria was increased by about 17% compared with that of original bacteria. UV mutation can improve the performance of the bacteria leaching of tailings greatly. 3.3. Leaching experiment by mutant strain 3 .4. SEM analysis of leached residues The effects of original and mutant bacteria, after 30 min irradi- ation, and acid on tailings leaching were compared in this experi- ment. Some inorganic elements in the tailings can replace the nutrients in the medium and the experimental results show that inorganic basal medium had little improvement on leaching effi- ciency of tailings. So the inorganic basic medium was not needed in the leaching experiment. Since tailings contained much alkaline gangue, the pH of the leaching system increased with dissolution of alkaline gangue in the early leaching stage. Therefore, 5 mL of SEM images of the tailing surface and leached residues are shown in Figs. 9 and 10, respectively. SEM analysis of the leached residues and tailings reveal that the surface of the leached resi- dues (Fig. 10) is rougher than that of the tailings (Fig. 9). The min- eral particles of the leached residues are covered with a polyporous layer, and etched pits were also present, especially in the area of defect, crack and dislocation of mineral particle. This may be because the attachment of bacteria to the mineral particles was selective and the bacteria preferred to attach to the area of high charge surface such as defect surfaces. EDS anal- ysis showed that the weight proportion and atom number pro- portion of sulfur in the leached residues were much higher than those in the tailings sample, but the weight proportion and atom number proportion of Cu, Mg, Ca, Si, Al and Fe in leached residues were lower than those in the tailings sample. This indicated that the presence of the mutant strain enriched the elemental sulfur on the mineral surface during the bioleaching process (Yu et al., 2008). Cu and Fe oxidized fast and dissolved to an ionic state with the bacterial action. Meanwhile, the main elements of gangue minerals in tailings dissolved in the leaching solution in the leaching process. 1 0% sulfuric acid was added to the leaching system in the first five days by timing and quantization. Comparison of the growth curves of original and mutant bacte- ria in the leaching system is shown in Fig. 7. The results show that mutant bacteria grow faster than original bacteria in the process of tailings leaching. The growth concentration of mutant bacteria is 0 2 .6 orders of magnitude higher than that of original bacteria after 0 days leaching. Comparison of copper extraction of original bac- teria, mutant bacteria and acid treatment is shown in Fig. 8. All these three treatment results are enhanced greatly after 5 days leaching, being 16.8%, 18.6% and 15%, respectively. These indices demonstrate that the leaching effect of bacteria was only a little better than that of acid. The possible reasons could be as follows: high pH value in initial leaching was detrimental to bacteria growth; sulfuric acid was needed regularly during the leaching period; acids played a leading role while bacteria played a more In the bioleaching process, Acidithiobacillus ferrooxidans LD-1 caused the oxidation of chalcopyrite and pyrite, and led to the for- mation of copper iron and ferric iron (Gleisner et al., 2006). Ferric Author's personal copy 874 Y. Dong et al. / Minerals Engineering 24 (2011) 870–875 Fig. 9. SEM image and EDS patterns of copper tailings (Â300). Fig. 10. SEM image and EDS patterns of the leach residue (Â300). iron is a very effective oxidant and leaching agent of minerals in leaching systems (Rohwerder et al., 2003; Komnitsas and Pooley, other main elements in the gangue mineral decreased by dif- ferent degrees, but the weight proportion and atomic num- ber proportion of sulfur increased. 1 991). Ferric iron can oxidize chalcopyrite to copper ions, ferrous 0 ions and elemental sulfur in solution (Vilca ez et al., 2008; Stott et al., 2000). The direct oxidation of A. ferrooxidans LD-1 on chalco- pyrite played a dominant role while ferric iron played a more min- or role in the leaching system (Bhattacharya et al., 1990). References Akcil, A., 2004. Potential bioleaching developments towards commercial reality: Turkish metal mining’s future. Minerals Engineering 17 (3), 477–480. Beggs, C.B., 2002. A quantitative method for evaluating the photoreactivation of ultraviolet damaged microorganisms. 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