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Extracellular DNA enhances the adsorption of Sulfobacillus thermosulfidooxidans strain ST on chalcopyrite surface
Hydrometallurgy 176 (2018) 97–103
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Extracellular DNA enhances the adsorption of Sulfobacillus thermosulfidooxidans strain ST on chalcopyrite surface
T
Runlan Yua,b, Chunwei Houa, Ajuan Liua, Tangjian Penga, Mingchen Xiaa, Xueling Wua,b, Li Shena,b, Yuandong Liua,b, Jiaokun Lia,b, Fei Yange, Guanzhou Qiua,b, Miao Chenc,d, ⁎ Weimin Zenga,b,c, a
School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha 410083, China c CSIRO Mineral Resources, Clayton, Victoria, Australia d Centre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University, Melbourne 3000, Australia e School of Public Health Management, Central South University, Changsha, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Adsorption Sulfobacillus thermosulfidooxidans strain ST Extracellular DNA Chalcopyrite In situ observation and monitoring
Extracellular polymeric substances (EPS) and their key components play an important role in bioleaching. Herein, the amount of extracellular DNA (eDNA) secreted by Sulfobacillus thermosulfidooxidans strain ST during bioleaching of chalcopyrite concentrate was investigated through traditional extraction method and confocal laser scanning microscope (CLSM) analysis. DNase I was added into the bioleaching system to degrade eDNA, and then the effects of eDNA deficiency on bacterial adsorption, copper extraction and the synthesis of other key components of EPS were evaluated. The results of adsorption experiment indicated that the adsorption time was prolonged and the equilibrium adsorption quantity was decreased when the eDNA was absent. The bioleaching experiment showed that eDNA in the EPS was favorable to the copper extraction, which increased by 18.5% compared with that of the eDNA-deficient group. Furthermore, it was found that the removal of eDNA decreased the formation of other key EPS components like polysaccharides and proteins. In situ observation and monitoring of eDNA on the chalcopyrite surface by CLSM were carried out. The results showed that the loss of eDNA attenuated the fluorescence intensity of each component of EPS, which was consistent with the above results. These studies suggested that eDNA in the EPS could enhance the adsorption of bacteria on the surface of chalcopyrite and further increase the copper extraction.
1. Introduction Bioleaching technology can be applied to dissolve the metallic ions from low grade ores into the leaching solution by the mineral-leaching microorganisms (mainly archaea, bacteria and fungi), and it can provide advantages of low operating cost, operational simplicity and environmental friendliness compared with the physical and chemical treatment methods (Zhou et al., 2015). The process of bioleaching, includes the adsorption of bacteria onto mineral surface, the interface biochemical reactions, the formation of intermediate species, and desorption of bacteria from mineral surface. Adsorption of microorganism on mineral surface is an initial and important step of bioleaching. It will influence the physicochemical properties of the mineral surface, such as hydrophobicity, oxidation-reduction potential, dissolution and precipitation of surface elements, and thus remarkably affect the metal
⁎
extraction rate. In the bioleaching process, microorganisms secrete extracellular polymeric substances (EPS) and, thus, a biofilm layer is formed on the minerals surface. Biofilm plays a unique and essential role during the interface reactions among microorganisms, mineral and solution, which has become a hot research topic in the field of bioleaching (Zeng et al., 2010a; Sand and Gehrke, 2006). EPS in the biofilm are generally composed of polysaccharides, proteins, fatty acids, nucleic acids and some inorganic substances. The contents of extracellular polysaccharides and proteins are very high, being easy to be extracted and purified. Therefore these two major components have been widely studied. However, the content of extracellular DNA (eDNA) is low, and it is very difficult to be extracted and analyzed, thus the reports about the characterization and functions of eDNA are relatively few. The presence of eDNA in the extracellular matrix was firstly
Corresponding author. E-mail address: [email protected] (W. Zeng).
https://doi.org/10.1016/j.hydromet.2018.01.018 Received 26 April 2017; Received in revised form 4 January 2018; Accepted 22 January 2018 0304-386X/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Short-term adsorption experiment
described in 1956 (Catlin, 1956), and this report identified eDNA as a critical component of extracellular matrix that participated in several processes related to bacterial colonization, intercellular contact, initial bacterial adhesion and surface aggregation (Spoering and Gilmore, 2006; Bakkali, 2013; Jakubovics et al., 2013). Other studies have suggested that eDNA may be a functional factor of oncogenes (Bandich et al., 1965). In addition, when studying the process of human immune responses that involve in T and B lymphocytes, it was found that eDNA presented in the biofilm could transmit information between these cells types (Anker et al., 1980). Furthermore, it has been found that the eDNA and chromosome DNA were homologous (Wu and Xi, 2009; Qin et al., 2007). The release of eDNA involves a set of complex mechanisms. It is widely accepted that eDNA is mainly released from cell lysis (Steinmoen et al., 2002; Vorkapic et al., 2016). However, additional mechanisms involving active eDNA secretion have been proposed (Farah et al., 2005; Antonova and Hammer, 2015) and even an active transport via vesicles for creating a biofilm matrix has been reported (Lewenza, 2013). Although eDNA plays a significant role in biofilm development and, be considered an important EPS composition, its analysis is still challenging due to the difficulties of its extraction from the extracellular matrix without any contamination from genomic DNA released by cell lysis during the extraction process. Till now a variety of methods have been used for the in situ analysis of eDNA, such as laser scanning confocal microscopy (CLSM), environmental scanning electron microscopy (ESEM), atomic force microscopy (AFM) and fluorescence microscopy (EFM). Furthermore, DNase I was usually used to degrade eDNA and then the comparison experiments with and without eDNA were carried out to investigate the function of eDNA. Several studies have suggested that removal of bacterial eDNA had an effect on the adsorption of bacteria and the formation and stability of biofilm (Das, 2010; Sena-Vélez et al., 2016; Tetz and Tetz, 2010; Gilan and Sivan, 2013; Özdemir et al., 2015). However, the functions of eDNA during bioleaching and its characterization have not been reported so far. In this paper, a typical acidophile Sulfobacillus thermosulfidooxidans strain ST was used to bioleach chalcopyrite concentrate. DNase I was added to degrade eDNA in the adsorption experiment and bioleaching experiment. The effects of eDNA deficiency on the bacterial adsorption, the production of extracellular proteins and polysaccharides, and the copper extraction were investigated. These would be beneficial to clarify the adsorption and bioleaching mechanism of microorganism.
Bacterial suspension (1 mL) with a known cell density was added into 30 mL sterilized medium, and then 1.5 g chalcopyrite powder was added. The initial bacterial cell density was 3.2 × 108 cells/mL, pH 1.6. The medium was incubated at 45 °C in a shaking incubator at 170 r/ min. These experiments were divided into four groups, each group was performed in triplicate. Among them, the first group was inoculated with untreated cells; the second group was inoculated with untreated cells and adding DNase I (Deoxyribonuclease I from bovine pancreas; Sigma) at a concentration of 50 μg/mL (Under the bioleaching conditions, DNase I retains about 40% of its maximal activity ensuring complete eDNA degradation in our experiments); the third group was inoculated with EPS-deficient cells (The free cells in logarithmic phase were collected and suspended with the sterile 9 K medium, and EPS was removed through sonication method associated with centrifugation to obtain EPS-deficient cells (Comte et al., 2006; Yu et al., 2011)); the fourth group was inoculated with EPS-deficient cells and adding DNase I at a concentration of 50 μg/mL. The complete content of shaking flasks was withdrawn every half an hour to count the number of free cells. The amount of attached bacterial cells can be calculated by the differences in bacterial cells number before and after adsorption, assuming that (1) any decrease in unattached bacterial cells was caused by bacterial adhesion to mineral surface (Wang et al., 2012), and that (2) the total biomass of bacteria was basically unchanged within 24 h of incubation (Tan et al., 2011; Tan and Chen, 2012). 2.4. Bioleaching experiments Bioleaching experiments were carried out in 50-mL shake flasks containing 30 mL medium, and the inoculation cell density was 8 × 106 cells/mL, and other conditions were similar to those mentioned above. In order to visualize the effect of eDNA in the bioleaching process, bacterial cultures of one group were treated with DNase I at a concentration of 50 μg/mL. Each group was performed in triplicate. DNase I was added every two days to ensure that there was no eDNA in the whole leaching process (Although activity of the enzyme slightly decreased as a function of time, the DNase I activity was still detectable after 2 days of incubation. Nevertheless, in order to guarantee efficient remove of eDNA, cultures were supplement with additional aliquots of DNase I within this time period.). No additional treatment was added to the other groups. The complete content of shaking flasks was withdrawn every day to analyze the redox potential, pH and the concentrations of Cu2+, Fe2+ and Fe3+ in solution. The copper concentration in the solution was measured using the BCO (bisoxaldihydrazone) light-intensity method. The total iron in solution was determined by inductively coupled plasma- atomic emission spectrometer (ICP-AES). Phenanthroline spectrophotometric method was used for determination of ferrous iron concentration, and the ferric ion concentration was the difference between total iron and ferrous iron concentrations. The pH and redox potential were measured with the E201F pH electrode and a platinum electrode with an Ag/AgCl reference electrode, respectively. The free cells in leaching solution were counted by optical microscopy. The amount of attached cells was analyzed using a previously reported method (Zeng et al., 2010b). All the tests were carried out in triplicate.
2. Materials and methods 2.1. Bacterial strain and culture conditions The bacterial strain used in this study was Sulfobacillus thermosulfidooxidans strain ST, which was isolated from an acid hot spring in Tengchong, Yunnan (southwestern China) (Guo et al., 2014). The 9 K basal salt medium (Silverman and Lundgren, 1959) was used for culturing and it had the following compounds: 3.0 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H2O, 0.5 g/L K2HPO4, 0.1 g/L KCl, 0.01 g/L Ca (NO3)2. Chalcopyrite concentrate (50 g/L) was added as an energy source. The strain was incubated at 45 °C in this medium supplement with 0.02% yeast extract and at an initial pH of 1.6.
2.5. Extraction of EPS from bioleaching residue of chalcopyrite 2.2. Mineral components The extraction of EPS from the mineral surface was carried out as reported previously (Zhang et al., 2010; Yu et al., 2017) in 50-mL shake flasks containing 30 mL medium. One group was treated with DNase I at a concentration of 50 μg/mL, and the other group was not treated, each group was performed in triplicate. The ore residue was analyzed on the 5th, 10th, 15th, 20th day of the incubation. In order to harvest the EPS from the adsorbed bacteria, the leaching slurry was settled for
The mineral sample was collected from Meizhou in Guangdong province, China. The mineral sample was chalcopyrite concentrate which mainly consisted of CuFeS2 (88.1%), CuS2 (4.19%), FeS2 (3.7%) and SiO2 (2.7%). The main elements of the sample were as followed: 29.19% Cu and 28.76% Fe. Mineral powders used in the experiments had a size distribution of less than 74 μm (95% of grains). 98
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increased until adsorption equilibrium was reached. This indicated that the adsorption of bacteria on the chalcopyrite is a rapid process (Wang et al., 2012; Gu et al., 2014). Without treatment, a maximal adsorption of 5.7 × 109 cells was achieved. However, when DNase I was added into the medium, the adsorption equilibrium time increased to 2 h, and the amount of adsorbed cells decreased to 3.8 × 109 cells, which indicated that absence of eDNA affected the adsorption capacity of bacterial cells. On the other hand, the EPS-deficient cells reached the adsorption equilibrium at 2.5 h, and the adsorption percentage was only 27% (2.7 × 109 cells). When the DNase I was added into the medium, there was no significant change in the amount of adsorbed cells and equilibrium time. The results were compared with the original bacterium, which showed that the adsorption capacity decreased significantly. This indicated that other EPS components such as extracellular proteins and polysaccharides also played an important role in the bacterial adsorption. Furthermore, it can be seen that the adding of DNase I had no obvious effect on the bacterium itself from the point of view of adsorption, but only degraded eDNA and then reduced the adsorption capacity of bacterial cells. Some studies have also confirmed that addition of DNase I had no effect on the survival and growth of bacterial cells (Sena-Vélez et al., 2016). Therefore, it can be concluded that the absence of eDNA would seriously decrease the adsorption of bacterial cells on the mineral surface. Adsorption kinetics of Sulfobacillus thermosulfidooxidans strain ST on the chalcopyrite surface was measured (Fig. 1A). Classic McKay secondary rate equation (shown as Eq. (1)) (Ho and Mckay, 1999) was used to analyze the adsorption kinetic process.
2 h, then the supernatant was decanted. The residual ore was centrifuged at 2500 ×g for 2 min at 4 °C. The supernatant was decanted and the obtained slag was re-suspended with 10 mL of sterile distilled water (SDW). The same operation was performed for twice, aiming to remove the free cells. Then 1.5 g of glass beads with a diameter of 0.2 mm were added into the re-suspension and shaken on a vortexer (250 r/min) for 10 min. Then it was centrifuged at 2500 ×g for 2 min, the supernatant was collected, and the cell number was counted by an optical microscope. If there were remaining bacteria, the above steps were repeated until no bacteria were observed under the microscope. The collected fractions containing the detached cells were centrifuged at 15000 ×g for 10 min at 4 °C and the resulting supernatant was defined as EPS extract of the bacteria originally attached to the mineral surface. The extracts were filtered through 0.2-μm filters and then the soluble fractions were stored at −20 °C in aliquots until analyses were performed (Zeng et al., 2010a; Gehrke et al., 1998). Possible contamination with non-EPS components of bacterial cells (cell wall and membrane fragments as well as cytoplasmic compounds from disrupted cells) was checked by measuring the ratio of proteins and polysaccharides in EPS extracts (Bo et al., 1996). In addition, the amount of DNA was used as criterion to rule out contamination of EPS extracts by disrupted cells, as the eDNA value does normally not exceed 2 to 15% of total EPS (Liao et al., 2001). 2.6. Chemical analysis of EPS components and contents The total quantity of EPS can be represented by the sum of proteins, polysaccharides and DNA contents. Quantitative estimation of protein content in EPS was measured by bicinchoninic acid (BCA) spectrophotometry method (Zhang et al., 2015). And it was similar to the Lowry assay, since it also depends on the conversion of Cu2+ to Cu+ under alkaline conditions. The Cu+ was then detected by reaction with BCA. The polysaccharide content in EPS was determined by phenolsulfuric acid method using glucose as a standard (Yao et al., 2010). The eDNA in EPS needed to be purified by the cetyltrimethylammonium bromide (CTAB)-DNA precipitation method (Wu and Xi, 2009; Corinaldesi, 2005) and then quantified by the spectrophotometer NanoDrop®ND-1000.
t 1 1 = + t qt kq e2 qe
(1) 9
In the formula qe is equilibrium adsorption amount, 10 cells; qt is the adsorption amount at t hours, 109 cells; K is rate constant, 109 cells/ h. The kinetic diagram was obtained through Formula (1) and shown in Fig. 1B. According to Fig. 1B and the fitting Eq. (1), the value of the equilibrium adsorption qe was calculated as 5.85 × 109 cells (line 1). The fitted rate equation agreed well with experimental data (5.74 × 109 cells), the error was less than 2%, indicating that the adsorption reaction of original cells can be approximately described by McKay second reaction model. Nonetheless, the fitted rate equations of treated cells were not in good agreement with experimental data (Fig. 1B, lines 2, 3, 4), and the error was between 32% and 57%. This suggested that the adsorption kinetics of treated cells no longer agreed with the Classic McKay secondary rate equation. It seemed that the adsorption characteristics of treated cells have changed, which was consistent with Fig. 1A. The adsorption of bacterial cells on the surface of minerals mainly involved three kinds of forces: van der Waals interactions, electrostatic interactions and acid-base interaction (Das et al., 2011; Das et al., 2014). The existence of eDNA would benefit the adsorption of cell, this is mainly due to the presence of eDNA and other components of EPS on bacterial cell surfaces would promote the surface hydrophobicity, meanwhile the mineral surface is also hydrophobic. In addition, eDNA can combine with some metal ions, creating a positive zeta potential, while the mineral surface generally has a negative charge. Therefore, the electrostatic interaction enhances the adsorbing of bacterial cells onto the mineral surface (Pogliani and Donati, 1999).
2.7. CLSM analysis of EPS on the chalcopyrite surface during bioleaching In order to compare the content of EPS before and after the addition of DNase I, the EPS on the surface of slag were stained by specific fluorescent dyes, and the results were observed by CLSM. The slag should be cleaned with SDW to remove the free cells on the surface. Propidium iodide (PI) can be used to stain eDNA (Qin et al., 2007; Okshevsky and Meyer, 2014) and it cannot penetrate living cells membranes (Zrelli et al., 2013; Fuxman Bass et al., 2010). Fluorescent probes FITC-WGA (wheat germ agglutinin) (Neu et al., 2001; Yu et al., 2017) and FITC-D 70 (fluorescein isothiocyanate) were also performed to visualize extracellular polysaccharides and proteins. 3. Result and discussion 3.1. Effect of eDNA on the adsorption behavior of Sulfobacillus thermosulfidooxidans strain ST In order to study the adsorption behavior of bacterial cells on the surface of chalcopyrite, the adsorption experiment of Sulfobacillus thermosulfidooxidans strain ST was carried out. The effect of eDNA on adsorption capacity of bacterial cells was studied. DNase I was added into the medium to degrade eDNA and then the comparison experiment with and without eDNA was carried out to investigate the function of eDNA on bacterial adsorption. Fig. 1A shows the adsorption curve of bacterial cells before and after the addition of DNase I. It shows that with prolonged incubation the amount of attached cells gradually
3.2. Bioleaching of chalcopyrite concentrate by Sulfobacillus thermosulfidooxidans strain ST Variations in the number of free and attached cells during bioleaching were shown in Fig. 2. Without adding DNase I, the attached cells had a relatively shorter lag phase, and then increased quickly after 2 days. The number of attached cells could reach the maximum at the 99
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Fig. 1. Adsorption behavior of Sulfobacillus thermosulfidooxidans strain ST on the chalcopyrite surface. (A) Adsorption curve of Sulfobacillus thermosulfidooxidans strain ST within in 3 h under different conditions; (B) Fitting curve according to the secondary rate equation.
Fig. 2. The variations of the free cells number in the solution and the attached cells number in the ore residue during bioleaching of chalcopyrite concentrate by the original and treated cells, respectively. The treated cells (DNase I addition) means its eDNA in the EPS was degraded by DNase I during the whole process of bioleaching.
Fig. 3. The variations of copper extraction during bioleaching of chalcopyrite concentrate by the original and treated cells respectively. The treated cells (DNase I addition) means its eDNA in the EPS was degraded by DNase I during the whole process of bioleaching.
11th day (1.1 × 1010 cells). After that, the number of attached cells kept stable till the 18th day and then began to decrease. Meanwhile, the number of free cells began to grow from the 4th day and reached the highest at the 14th day, which was 3.1 × 1010 cells. However, when DNase I was added into the bioleaching system, the number of attached cells began to increase quickly only from the 6th day, and reached the maximum at the 13th day (0.8 × 1010 cells). The number of free cells began to increase after the 7th day and the maximum number was 2.6 × 1010 cells at the 16th day. The adaptation period was prolonged compared with the group without addition of DNase I. It can also be seen that after the number of attached cells achieved an equilibrium, the number of free cells was still increasing. But the number of adsorbed and free bacterial cells decreased compared with the group without addition of DNase I. These results indicated that the addition of DNase I would lead to eDNA deficiency, which affected the adsorption capacity of bacteria, and then inhibited the release of energy sources from mineral. The reduction of energy source would further affect the growth of free cells in the bioleaching solution. Alternatively, after adding DNase I, part of the attached cells will detach from the mineral/biofilm and become free cells, but the number of these cells was difficult to be calculated.
Fig. 3 shows copper extraction as a function of time during bioleaching of chalcopyrite concentrate. In the early stage of bioleaching, although the microbes were still in the adaptation period, a few copper and iron were leached. This was due to that CuS2 in the concentrate was easily leached by the acid in the medium. As bioleaching continued, copper extraction steadily increased until the 18th day, achieving 10.6 g/L, and the copper extraction percentage reached 82%. However, in the experiment with the addition of DNase I, a maximal copper extraction of 8.2 g/L was achieved at the 21th day. The iron was also an important parameter in bioleaching of chalcopyrite concentrate. In the whole bioleaching process, the concentration of ferrous ion increased first and then decreased (Fig. 4). And the highest ferrous iron concentration (2.2 g/L) was achieved at the 9th day. Meanwhile, the concentration of ferric ions gradually increased until reaching stability. In the experiment with the addition of DNase I, the ferrous iron concentration reached the maximum (1.5 g/L) at the 11th day, followed by a rapid decrease, and reached the plateau at the 17th day. During this process, the concentration of ferric ion increased gradually, and the maximal concentration of 1.2 g/L was achieved at the end of bioleaching. 100
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Fig. 5. The variation of the contents of each major EPS component as a function of time during bioleaching of chalcopyrite by Sulfobacillus thermosulfidooxidans strain ST in the presence and absence of DNase I. EPS is given in mg and refers to the amount extracted from the total ore residue.
Fig. 4. The variations of ferrous and ferric ion concentrations during bioleaching of chalcopyrite by the original and treated Sulfobacillus thermosulfidooxidans strain ST, respectively.
It can be seen from Figs. 3 and 4 that the high concentration of total iron would be beneficial to copper extraction. Iron in solution at the beginning was mainly ferrous iron and the presence of ferric iron was more notable only in the end. At the end of bioleaching, ferrous iron was continually oxidized to ferric iron, therefore the concentration of ferric iron increased gradually till the end. When the leaching rate of copper decreased, the total iron began to keep stable. Because of the large amount of acid produced by oxidation of reduced sulfur species, the pH of the medium was reduced to 1.1 and the redox potential value increased to 590 mV, and this would inhibit the copper extraction (Hiroyoshi et al., 2008; Okamoto et al., 2005). Furthermore, jarosite was found when the components of ore residue were analyzed by XRD. Therefore, the formation of passivation layer was also an inducement resulting in the decrease of copper leaching rate (Zeng et al., 2010b). The addition of DNase I decreased the extraction of copper and ferrous iron. This was due to the absence of eDNA, which affected the number of attached cells (Fig. 2). It further indicated that the attached cells were a more important factor for the dissolution of chalcopyrite concentrate compared with the free cells in the bioleaching solution (Zeng et al., 2010b; Fowler and Crundwell, 1998).
Original
DNase I addition
3.3. Extraction and characterization of EPS components during bioleaching of chalcopyrite concentrate During the extraction process of EPS from the mineral surface, the ratio of proteins and polysaccharides in EPS was below 5.0 and the ratio of eDNA in the EPS was below 15%. According to the research of Bo et al. (1996), the ratio between proteins and polysaccharides was found in the range 0.2 to 5.0. In addition, relevant studies showed that the ratio of eDNA in the total EPS is below 15% (Liao et al., 2001; He et al., 2014). It shows that the applied method for EPS extraction did not lead to noticeable contamination with intracellular components. Fig. 5 shows the variation of the contents of major EPS components like extracellular proteins, polysaccharides and eDNA at different bioleaching time points. They can also be detected by. CLSM, which combined with fluorescent probes for staining extracellular proteins, extracellular polysaccharides and eDNA, respectively (Fig. 6). These EPS components were asymmetrically covered on the mineral surface. It was reported that the cells preferred to attach on the cracks/defects and formed a monolayer biofilm on chalcopyrite concentrate surface (Zhang et al., 2014). Only a small amount of EPS was
Fig. 6. The CLSM observation of EPS components after bioleaching of 12 days by the Sulfobacillus thermosulfidooxidans strain ST in the presence and absence of DNase I. DNase I treatment (A2, B2, C2), no DNase I treatment (A1, B1, C1). (A: extracellular protein; B: extracellular polysaccharide; C: extracellular DNA).
produced at the first 5 days, this was mainly due to that the bacteria were still in the adaptation period (Fig. 2). After that, as the increase of energy sources released from mineral, the amount of attached cells increased, which resulted in the increase of EPS on the surface of minerals. At the final stage of bioleaching, the total EPS of attached cells 101
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remained stable at 53.2 mg. This indicated that once the EPS were produced, it was difficult to remove or degrade them in the bioleaching system. The extracellular proteins and extracellular polysaccharides were the major components in the EPS (Figs. 5 and 6). The contents of extracellular polysaccharides and extracellular proteins could reach the highest value at the 10th day, which were 26.1 mg and 34.8 mg, respectively. Then the content was maintained at a steady level, which was related to the stability of biofilm. However, the content of eDNA was much lower than that of other components. Especially in the initial stage of bioleaching the content of eDNA was the lowest, but with the extension of bioleaching time, its content increased gradually and reached 3.8 mg at the 20th day in the experiment without the addition of DNase I. It can be seen from Figs. 2 and 5 that at the later stage of bioleaching, the attached cell number did not increase, but the amount of eDNA increased gradually. This may be due to that the harmful environment (high metal ion concentration, extreme low pH) at the later stage of bioleaching would increase the cell lysis and then release DNA into the solution (Steinmoen et al., 2002; Vorkapic et al., 2016). During the bioleaching with addition of DNase I, the total EPS and the content of each component were reduced obviously (Fig. 5). The fluorescence intensity of all the EPS components decreased significantly (Fig. 6). Especially for eDNA, its fluorescence intensity was even disappeared (or under detection limit). This was because the absence of eDNA reduced the adsorption capacity of bacterial cell, therefore, the amount of bacterial cells adsorbed on the surface of the mineral was reduced, which in turn leading to a decrease of EPS. Furthermore, eDNA as a bridge in EPS was reported to combine other extracellular substances and then keep the biofilm structure stable (Cruz et al., 2012; Kerchove and Elimelech, 2008). Therefore, the deficiency of eDNA would partly inhibit the formation of EPS/biofilm on the mineral surface during bioleaching. This further proved that the addition of DNase I not only degraded eDNA formed on the mineral surface, but also inhibited the formation of other EPS components.
475–481. Antonova, E.S., Hammer, B.K., 2015. Genetics of natural competence in vibrio cholerae and other vibrios. Microbiol. Spectr. 3 (3). Bakkali, M., 2013. Could DNA uptake be a side effect of bacterial adhesion and twitching motility? Arch. Microbiol. 195 (4), 279–289. Bandich, A., Wilcox, T., Borenfreund, E., 1965. Circulating DNA as a possible factor in oncogenesis. Science 148 (3668), 374–376. Bo, F., Palmgren, R., Keiding, K., Nielsen, P.H., 1996. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 30 (8), 1749–1758. Catlin, B.W., 1956. Extracellular deoxyribonucleic acid of bacteria and a deoxyribonuclease inhibitor. Science 124 (3219), 441–442. Comte, S., Guibaud, G., Baudu, M., 2006. Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS: part II. Consequences of EPS extraction methods on Pb2+ and Cd2+ complexation. Enzym. Microb. Technol. 38 (2), 237–245. Corinaldesi, C., 2005. Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Appl. Environ. Microbiol. 71 (1), 46–50. Cruz, L.F., Cobine, P.A., Fuente, L.D.L., 2012. Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility. Appl. Environ. Microbiol. 78 (5), 1321–1331. Das, T., 2010. Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl. Environ. Microbiol. 76 (76), 3405–3408. Das, T., Krom, B.P., Van, d.M.H.C., Busscher, H.J., Sharma, P.K., 2011. DNA-mediated bacterial aggregation is dictated by acid–base interactions. Soft Matter 7 (6), 2927–2935. Das, T., Sehar, S., Koop, L., Wong, Y.K., Ahmed, S., Siddiqui, K.S., Manefield, M., 2014. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation. PLoS One 9 (3), e91935. Farah, C., Vera, M., Morin, D., Haras, D., Jerez, C.A., Guiliani, N., 2005. Evidence for a functional Quorum-Sensing type AI-1 system in the extremophilic bacterium Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 71 (11), 7033–7040. Fowler, T.A., Crundwell, F.K., 1998. Leaching of zinc sulfide by Thiobacillus ferrooxidans: experiments with a controlled redox potential indicate no direct bacterial mechanism. Appl. Environ. Microbiol. 64 (10), 3570–3575. Fuxman Bass, J.I., Russo, D.M., Gabelloni, M.L., Geffer, J.R., Giordano, M., Catalano, M., Zorreguieta, A., Trevani, A.S., 2010. Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms. J. Immunol. 184 (11), 6386. Gehrke, T., Telegdi, J., Thierry, D., Sand, W., 1998. Importance of extracellular polymeric substances from Thiobacillus ferrooxidans for bioleaching. Appl. Environ. Microbiol. 64 (7), 2743–2747. Gilan, I., Sivan, A., 2013. Extracellular DNA plays an important structural role in the biofilm of the plastic degrading actinomycete. Adv. Microbiol. 3 (8), 543–551. Gu, G.H., Hu, K.T., Li, S.K., 2014. Surface characterization of chalcopyrite interacting with Leptospirillum ferriphilum. Trans. Nonferrous Metals Soc. China 24 (6), 1898–1904. Guo, X., Yin, H.Q., Liang, Y.L., Hu, Q., Zhou, X.S., Xiao, Y.H., Ma, L.Y., Zhang, X., Qiu, G.Z., Liu, X.D., 2014. Comparative genome analysis reveals metabolic versatility and environmental adaptations of Sulfobacillus thermosulfidooxidans strain ST. PLoS One 9 (6), e99417. He, Z.G., Yang, Y.P., Zhou, S., Hu, Y.H., Zhong, H., 2014. Effect of pyrite, elemental sulfur and ferrous ions on EPS production by metal sulfide bioleaching microbes. Trans. Nonferrous Metals Soc. China 24 (4), 1171–1178. Hiroyoshi, N., Kitagawa, H., Tsunekawa, M., 2008. Effect of solution composition on the optimum redox potential for chalcopyrite leaching in sulfuric acid solutions. Hydrometallurgy 91 (1), 144–149. Ho, Y.S., Mckay, G., 1999. Pseudo-second order model for sorption processes. Process Biochem. 34 (5), 451–465. Jakubovics, N.S., Shields, R.C., Rajarajan, N., Burgess, J.G., 2013. Life after death: the critical role of extracellular DNA in microbial biofilms. Lett. Appl. Microbiol. 57 (6), 467–475. Kerchove, A.J., Elimelech, M., 2008. Calcium and magnesium cations enhance the adhesion of motile and nonmotile pseudomonas aeruginosa on alginate films. Langmuir ACS J. Surf Colloids 24 (7), 3392. Lewenza, S., 2013. Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa. Front. Microbiol. 4 (1), 21. Liao, B.Q., Allen, D.G., Droppo, I.G., Leppard, G.G., Liss, S.N., Allen, D.G., 2001. Surface properties of sludge and their role in bioflocculation and settleability. Water Res. 35 (2), 339. Neu, T., Swerhone, G.D., Lawrence, J.R., 2001. Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology 147 (Pt 2), 299–313. Okamoto, H., Nakayama, R., Kuroiwa, S., Hiroyoshi, N., Tsunekawa, M., 2005. Normalized redox potential used to assess chalcopyrite column leaching. J. MMIJ 121 (6), 246–254. Okshevsky, M., Meyer, R.L., 2014. Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms. J. Microbiol. Methods 105, 102–104. Özdemir, C., Karaca, B., Akçelik, N., 2015. The role of extracellular DNA in Salmonella biofilms: is it in intimate relationship with matrix or initial adhesion? In: Conference: 25th ECCMID Conference, At Copenhagen, Denmark. Pogliani, C., Donati, E., 1999. The role of exopolymers in the bioleaching of a non-ferrous metal sulphide. J. Ind. Microbiol. Biotechnol. 22 (2), 88–92. Qin, Z., Ou, Y., Yang, L., Zhu, Y., Tolkernielsen, T., Molin, S., Qu, D., 2007. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology 153 (Pt 7), 2083. Sand, W., Gehrke, T., 2006. Extracellular polymeric substances mediate bioleaching/
4. Conclusion Bacterial adsorption on mineral surface is required for the production of extracellular DNA. The adsorption kinetics of bacterial cells on the surface of chalcopyrite can be described by the McKay level two rate equation. The equilibrium adsorption quantity calculated by this equation was in good agreement with the experimental data. The content of eDNA in EPS was relatively low, but eDNA played an important role in the adsorption process on the mineral surface. The lack of eDNA would decrease the formation of other EPS components. In situ detection combined with traditional extraction and analysis methods, can systematically study the formation and content of eDNA in different stages of bioleaching. Furthermore, the method about “specific enzymatic lysis” (like using DNase I to degrade eDNA) would be also a good way to investigate the function of other EPS components. This paper would provide some theoretic evidence for elucidating the function of eDNA in bioleaching process, and be also conducive to the further development of biohydrometallurgy theory. Acknowledgements This work was supported by the National Natural Science Foundation of China (nos. 31470230, 51320105006, 51604308), the Fundamental Research Funds for the Central University of Central South University (2017zzts378). References Anker, P., Jachertz, D., Stroun, M., Brögger, R., Lederrey, C., Henri, J., Maurice, P.A., 1980. The role of extracellular DNA in the transfer of information from T to B human lymphocytes in the course of an immune response. Int. J. Immunogenet. 7 (6),
102
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1686–1690. Yu, R.L., Ou, Y., Tan, J.X., Wu, F.D., Sun, J., Lei, M., 2011. Effect of EPS on adhesion of Acidithiobacillus ferrooxidans on chalcopyrite and pyrite mineral surfaces. Trans. Nonferrous Metals Soc. China 21 (2), 407–412. Yu, R.L., Liu, A.J., Liu, Y.N., Yu, Z.J., Peng, T.J., Wu, X.L., Shen, L., Liu, Y.D., Li, J.K., Liu, X.D., Qiu, G.Z., Chen, M., Zeng, W.M., 2017. Evolution of Sulfobacillus thermosulfidooxidans secreting alginate during bioleaching of chalcopyrite concentrate. J. Appl. Microbiol. 122 (6). Zeng, W.M., Qiu, G.Z., Zhou, H.B., Liu, X.D., Chen, M., Chao, W.L., Zhang, C.G., Peng, J.H., 2010a. Characterization of extracellular polymeric substances extracted during the bioleaching of chalcopyrite concentrate. Hydrometallurgy 100 (3–4), 177–180. Zeng, W., Qiu, G., Zhou, H., Peng, J., Chen, J.M., Tan, S.N., Chao, W., Liu, X., Zhang, Y., 2010b. Community structure and dynamics of the free and attached microorganisms during moderately thermophilic bioleaching of chalcopyrite concentrate. Bioresour. Technol. 101 (18), 7079–7086. Zhang, D., Pan, X., Mostofa, K.M., Chen, X., Mu, G., Wu, F., Liu, J., Song, W., Yang, J., Liu, Y., Fu, Q., 2010. Complexation between Hg (II) and biofilm extracellular polymeric substances: an application of fluorescence spectroscopy. J. Hazard. Mater. 175 (1–3), 359–365. Zhang, R., Bellenberg, S., Castro, L., Thomas, R.N., Wolfgang, S., Mario, V., 2014. Colonization and biofilm formation of the extremely acidophilic archaeon Ferroplasma acidiphilum. Hydrometallurgy 150, 245–252. Zhang, W., Cao, B., Wang, D., Zhang, W., 2015. Variations in distribution and composition of extracellular polymeric substances (EPS) of biological sludge under potassium ferrate conditioning: effects of pH and ferrate dosage. Biochem. Eng. J. 106, 37–47. Zhou, H.B., Mao, F., Wang, Y.G., 2015. Acidophilic microorganisms and bioleaching technology. Bull. Mineral. Petrol. Geochem. 34 (2), 269–276. Zrelli, K., Galy, O., Latourlambert, P., Kirwan, L., Ghigo, J.M., Beloin, C., Henry, N., 2013. Bacterial biofilm mechanical properties persist upon antibiotic treatment and survive cell death. New J. Phys. 15 (12), 5026.
biocorrosion via interfacial processes involving iron (III) ions and acidophilic bacteria. Res. Microbiol. 157 (1), 49–56. Sena-Vélez, M., Redondo, C., Graham, J.H., Cubero, J., 2016. Presence of extracellular DNA during biofilm formation by Xanthomonas citri subsp. citri strains with different host range. PLoS One 11 (6), e156695. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium ferrobacillus ferrooxidans: I. An improved medium and a harvesting procedure for securing high cell yields. J. Bacteriol. 77 (5), 642–647. Spoering, A.L., Gilmore, M.S., 2006. Quorum sensing and DNA release in bacterial biofilms. Curr. Opin. Microbiol. 9 (2), 133–137. Steinmoen, H., Knutsen, E., Håvarstein, L.S., Steinmoen, H., Knutsen, E., Havarstein, L.S., 2002. Induction of natural competence in streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proc. Natl. Acad. Sci. 99 (11), 7681–7686. Tan, S.N., Chen, M., 2012. Early stage adsorption behaviour of Acidithiobacillus ferrooxidans on minerals I: an experimental approach. Hydrometallurgy 119-120 (5), 87–94. Tan, S.N., Burgar, I., Chen, M., 2011. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. 102 (102), 9143–9147. Tetz, V.V., Tetz, G.V., 2010. Effect of extracellular DNA destruction by DNase I on characteristics of forming biofilms. DNA Cell Biol. 29 (8), 399. Vorkapic, D., Pressler, K., Schild, S., 2016. Multifaceted roles of extracellular DNA in bacterial physiology. Curr. Genet. 62 (1), 71–79. Wang, Z.H., Xie, X.H., Liu, J.S., 2012. Experimental measurements of short-term adsorption of Acidithiobacillus ferrooxidans onto chalcopyrite. Trans. Nonferrous Metals Soc. China 22 (2), 442–446. Wu, J., Xi, C., 2009. Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. Appl. Environ. Microbiol. 75 (16), 5390–5395. Yao, F.U., Yang, H.Y., Fang, Y.J., Zhang, C.Z., 2010. Determination of polysaccharide content in extracellular polymers of leaching bacteria. J. Cent. South Univ. 41 (5),
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Extracellular DNA enhances the adsorption of Sulfobacillus thermosulfidooxidans strain ST on chalcopyrite surface
T
Runlan Yua,b, Chunwei Houa, Ajuan Liua, Tangjian Penga, Mingchen Xiaa, Xueling Wua,b, Li Shena,b, Yuandong Liua,b, Jiaokun Lia,b, Fei Yange, Guanzhou Qiua,b, Miao Chenc,d, ⁎ Weimin Zenga,b,c, a
School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China Key Laboratory of Biometallurgy, Ministry of Education, Changsha 410083, China c CSIRO Mineral Resources, Clayton, Victoria, Australia d Centre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University, Melbourne 3000, Australia e School of Public Health Management, Central South University, Changsha, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Adsorption Sulfobacillus thermosulfidooxidans strain ST Extracellular DNA Chalcopyrite In situ observation and monitoring
Extracellular polymeric substances (EPS) and their key components play an important role in bioleaching. Herein, the amount of extracellular DNA (eDNA) secreted by Sulfobacillus thermosulfidooxidans strain ST during bioleaching of chalcopyrite concentrate was investigated through traditional extraction method and confocal laser scanning microscope (CLSM) analysis. DNase I was added into the bioleaching system to degrade eDNA, and then the effects of eDNA deficiency on bacterial adsorption, copper extraction and the synthesis of other key components of EPS were evaluated. The results of adsorption experiment indicated that the adsorption time was prolonged and the equilibrium adsorption quantity was decreased when the eDNA was absent. The bioleaching experiment showed that eDNA in the EPS was favorable to the copper extraction, which increased by 18.5% compared with that of the eDNA-deficient group. Furthermore, it was found that the removal of eDNA decreased the formation of other key EPS components like polysaccharides and proteins. In situ observation and monitoring of eDNA on the chalcopyrite surface by CLSM were carried out. The results showed that the loss of eDNA attenuated the fluorescence intensity of each component of EPS, which was consistent with the above results. These studies suggested that eDNA in the EPS could enhance the adsorption of bacteria on the surface of chalcopyrite and further increase the copper extraction.
1. Introduction Bioleaching technology can be applied to dissolve the metallic ions from low grade ores into the leaching solution by the mineral-leaching microorganisms (mainly archaea, bacteria and fungi), and it can provide advantages of low operating cost, operational simplicity and environmental friendliness compared with the physical and chemical treatment methods (Zhou et al., 2015). The process of bioleaching, includes the adsorption of bacteria onto mineral surface, the interface biochemical reactions, the formation of intermediate species, and desorption of bacteria from mineral surface. Adsorption of microorganism on mineral surface is an initial and important step of bioleaching. It will influence the physicochemical properties of the mineral surface, such as hydrophobicity, oxidation-reduction potential, dissolution and precipitation of surface elements, and thus remarkably affect the metal
⁎
extraction rate. In the bioleaching process, microorganisms secrete extracellular polymeric substances (EPS) and, thus, a biofilm layer is formed on the minerals surface. Biofilm plays a unique and essential role during the interface reactions among microorganisms, mineral and solution, which has become a hot research topic in the field of bioleaching (Zeng et al., 2010a; Sand and Gehrke, 2006). EPS in the biofilm are generally composed of polysaccharides, proteins, fatty acids, nucleic acids and some inorganic substances. The contents of extracellular polysaccharides and proteins are very high, being easy to be extracted and purified. Therefore these two major components have been widely studied. However, the content of extracellular DNA (eDNA) is low, and it is very difficult to be extracted and analyzed, thus the reports about the characterization and functions of eDNA are relatively few. The presence of eDNA in the extracellular matrix was firstly
Corresponding author. E-mail address: [email protected] (W. Zeng).
https://doi.org/10.1016/j.hydromet.2018.01.018 Received 26 April 2017; Received in revised form 4 January 2018; Accepted 22 January 2018 0304-386X/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Short-term adsorption experiment
described in 1956 (Catlin, 1956), and this report identified eDNA as a critical component of extracellular matrix that participated in several processes related to bacterial colonization, intercellular contact, initial bacterial adhesion and surface aggregation (Spoering and Gilmore, 2006; Bakkali, 2013; Jakubovics et al., 2013). Other studies have suggested that eDNA may be a functional factor of oncogenes (Bandich et al., 1965). In addition, when studying the process of human immune responses that involve in T and B lymphocytes, it was found that eDNA presented in the biofilm could transmit information between these cells types (Anker et al., 1980). Furthermore, it has been found that the eDNA and chromosome DNA were homologous (Wu and Xi, 2009; Qin et al., 2007). The release of eDNA involves a set of complex mechanisms. It is widely accepted that eDNA is mainly released from cell lysis (Steinmoen et al., 2002; Vorkapic et al., 2016). However, additional mechanisms involving active eDNA secretion have been proposed (Farah et al., 2005; Antonova and Hammer, 2015) and even an active transport via vesicles for creating a biofilm matrix has been reported (Lewenza, 2013). Although eDNA plays a significant role in biofilm development and, be considered an important EPS composition, its analysis is still challenging due to the difficulties of its extraction from the extracellular matrix without any contamination from genomic DNA released by cell lysis during the extraction process. Till now a variety of methods have been used for the in situ analysis of eDNA, such as laser scanning confocal microscopy (CLSM), environmental scanning electron microscopy (ESEM), atomic force microscopy (AFM) and fluorescence microscopy (EFM). Furthermore, DNase I was usually used to degrade eDNA and then the comparison experiments with and without eDNA were carried out to investigate the function of eDNA. Several studies have suggested that removal of bacterial eDNA had an effect on the adsorption of bacteria and the formation and stability of biofilm (Das, 2010; Sena-Vélez et al., 2016; Tetz and Tetz, 2010; Gilan and Sivan, 2013; Özdemir et al., 2015). However, the functions of eDNA during bioleaching and its characterization have not been reported so far. In this paper, a typical acidophile Sulfobacillus thermosulfidooxidans strain ST was used to bioleach chalcopyrite concentrate. DNase I was added to degrade eDNA in the adsorption experiment and bioleaching experiment. The effects of eDNA deficiency on the bacterial adsorption, the production of extracellular proteins and polysaccharides, and the copper extraction were investigated. These would be beneficial to clarify the adsorption and bioleaching mechanism of microorganism.
Bacterial suspension (1 mL) with a known cell density was added into 30 mL sterilized medium, and then 1.5 g chalcopyrite powder was added. The initial bacterial cell density was 3.2 × 108 cells/mL, pH 1.6. The medium was incubated at 45 °C in a shaking incubator at 170 r/ min. These experiments were divided into four groups, each group was performed in triplicate. Among them, the first group was inoculated with untreated cells; the second group was inoculated with untreated cells and adding DNase I (Deoxyribonuclease I from bovine pancreas; Sigma) at a concentration of 50 μg/mL (Under the bioleaching conditions, DNase I retains about 40% of its maximal activity ensuring complete eDNA degradation in our experiments); the third group was inoculated with EPS-deficient cells (The free cells in logarithmic phase were collected and suspended with the sterile 9 K medium, and EPS was removed through sonication method associated with centrifugation to obtain EPS-deficient cells (Comte et al., 2006; Yu et al., 2011)); the fourth group was inoculated with EPS-deficient cells and adding DNase I at a concentration of 50 μg/mL. The complete content of shaking flasks was withdrawn every half an hour to count the number of free cells. The amount of attached bacterial cells can be calculated by the differences in bacterial cells number before and after adsorption, assuming that (1) any decrease in unattached bacterial cells was caused by bacterial adhesion to mineral surface (Wang et al., 2012), and that (2) the total biomass of bacteria was basically unchanged within 24 h of incubation (Tan et al., 2011; Tan and Chen, 2012). 2.4. Bioleaching experiments Bioleaching experiments were carried out in 50-mL shake flasks containing 30 mL medium, and the inoculation cell density was 8 × 106 cells/mL, and other conditions were similar to those mentioned above. In order to visualize the effect of eDNA in the bioleaching process, bacterial cultures of one group were treated with DNase I at a concentration of 50 μg/mL. Each group was performed in triplicate. DNase I was added every two days to ensure that there was no eDNA in the whole leaching process (Although activity of the enzyme slightly decreased as a function of time, the DNase I activity was still detectable after 2 days of incubation. Nevertheless, in order to guarantee efficient remove of eDNA, cultures were supplement with additional aliquots of DNase I within this time period.). No additional treatment was added to the other groups. The complete content of shaking flasks was withdrawn every day to analyze the redox potential, pH and the concentrations of Cu2+, Fe2+ and Fe3+ in solution. The copper concentration in the solution was measured using the BCO (bisoxaldihydrazone) light-intensity method. The total iron in solution was determined by inductively coupled plasma- atomic emission spectrometer (ICP-AES). Phenanthroline spectrophotometric method was used for determination of ferrous iron concentration, and the ferric ion concentration was the difference between total iron and ferrous iron concentrations. The pH and redox potential were measured with the E201F pH electrode and a platinum electrode with an Ag/AgCl reference electrode, respectively. The free cells in leaching solution were counted by optical microscopy. The amount of attached cells was analyzed using a previously reported method (Zeng et al., 2010b). All the tests were carried out in triplicate.
2. Materials and methods 2.1. Bacterial strain and culture conditions The bacterial strain used in this study was Sulfobacillus thermosulfidooxidans strain ST, which was isolated from an acid hot spring in Tengchong, Yunnan (southwestern China) (Guo et al., 2014). The 9 K basal salt medium (Silverman and Lundgren, 1959) was used for culturing and it had the following compounds: 3.0 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H2O, 0.5 g/L K2HPO4, 0.1 g/L KCl, 0.01 g/L Ca (NO3)2. Chalcopyrite concentrate (50 g/L) was added as an energy source. The strain was incubated at 45 °C in this medium supplement with 0.02% yeast extract and at an initial pH of 1.6.
2.5. Extraction of EPS from bioleaching residue of chalcopyrite 2.2. Mineral components The extraction of EPS from the mineral surface was carried out as reported previously (Zhang et al., 2010; Yu et al., 2017) in 50-mL shake flasks containing 30 mL medium. One group was treated with DNase I at a concentration of 50 μg/mL, and the other group was not treated, each group was performed in triplicate. The ore residue was analyzed on the 5th, 10th, 15th, 20th day of the incubation. In order to harvest the EPS from the adsorbed bacteria, the leaching slurry was settled for
The mineral sample was collected from Meizhou in Guangdong province, China. The mineral sample was chalcopyrite concentrate which mainly consisted of CuFeS2 (88.1%), CuS2 (4.19%), FeS2 (3.7%) and SiO2 (2.7%). The main elements of the sample were as followed: 29.19% Cu and 28.76% Fe. Mineral powders used in the experiments had a size distribution of less than 74 μm (95% of grains). 98
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increased until adsorption equilibrium was reached. This indicated that the adsorption of bacteria on the chalcopyrite is a rapid process (Wang et al., 2012; Gu et al., 2014). Without treatment, a maximal adsorption of 5.7 × 109 cells was achieved. However, when DNase I was added into the medium, the adsorption equilibrium time increased to 2 h, and the amount of adsorbed cells decreased to 3.8 × 109 cells, which indicated that absence of eDNA affected the adsorption capacity of bacterial cells. On the other hand, the EPS-deficient cells reached the adsorption equilibrium at 2.5 h, and the adsorption percentage was only 27% (2.7 × 109 cells). When the DNase I was added into the medium, there was no significant change in the amount of adsorbed cells and equilibrium time. The results were compared with the original bacterium, which showed that the adsorption capacity decreased significantly. This indicated that other EPS components such as extracellular proteins and polysaccharides also played an important role in the bacterial adsorption. Furthermore, it can be seen that the adding of DNase I had no obvious effect on the bacterium itself from the point of view of adsorption, but only degraded eDNA and then reduced the adsorption capacity of bacterial cells. Some studies have also confirmed that addition of DNase I had no effect on the survival and growth of bacterial cells (Sena-Vélez et al., 2016). Therefore, it can be concluded that the absence of eDNA would seriously decrease the adsorption of bacterial cells on the mineral surface. Adsorption kinetics of Sulfobacillus thermosulfidooxidans strain ST on the chalcopyrite surface was measured (Fig. 1A). Classic McKay secondary rate equation (shown as Eq. (1)) (Ho and Mckay, 1999) was used to analyze the adsorption kinetic process.
2 h, then the supernatant was decanted. The residual ore was centrifuged at 2500 ×g for 2 min at 4 °C. The supernatant was decanted and the obtained slag was re-suspended with 10 mL of sterile distilled water (SDW). The same operation was performed for twice, aiming to remove the free cells. Then 1.5 g of glass beads with a diameter of 0.2 mm were added into the re-suspension and shaken on a vortexer (250 r/min) for 10 min. Then it was centrifuged at 2500 ×g for 2 min, the supernatant was collected, and the cell number was counted by an optical microscope. If there were remaining bacteria, the above steps were repeated until no bacteria were observed under the microscope. The collected fractions containing the detached cells were centrifuged at 15000 ×g for 10 min at 4 °C and the resulting supernatant was defined as EPS extract of the bacteria originally attached to the mineral surface. The extracts were filtered through 0.2-μm filters and then the soluble fractions were stored at −20 °C in aliquots until analyses were performed (Zeng et al., 2010a; Gehrke et al., 1998). Possible contamination with non-EPS components of bacterial cells (cell wall and membrane fragments as well as cytoplasmic compounds from disrupted cells) was checked by measuring the ratio of proteins and polysaccharides in EPS extracts (Bo et al., 1996). In addition, the amount of DNA was used as criterion to rule out contamination of EPS extracts by disrupted cells, as the eDNA value does normally not exceed 2 to 15% of total EPS (Liao et al., 2001). 2.6. Chemical analysis of EPS components and contents The total quantity of EPS can be represented by the sum of proteins, polysaccharides and DNA contents. Quantitative estimation of protein content in EPS was measured by bicinchoninic acid (BCA) spectrophotometry method (Zhang et al., 2015). And it was similar to the Lowry assay, since it also depends on the conversion of Cu2+ to Cu+ under alkaline conditions. The Cu+ was then detected by reaction with BCA. The polysaccharide content in EPS was determined by phenolsulfuric acid method using glucose as a standard (Yao et al., 2010). The eDNA in EPS needed to be purified by the cetyltrimethylammonium bromide (CTAB)-DNA precipitation method (Wu and Xi, 2009; Corinaldesi, 2005) and then quantified by the spectrophotometer NanoDrop®ND-1000.
t 1 1 = + t qt kq e2 qe
(1) 9
In the formula qe is equilibrium adsorption amount, 10 cells; qt is the adsorption amount at t hours, 109 cells; K is rate constant, 109 cells/ h. The kinetic diagram was obtained through Formula (1) and shown in Fig. 1B. According to Fig. 1B and the fitting Eq. (1), the value of the equilibrium adsorption qe was calculated as 5.85 × 109 cells (line 1). The fitted rate equation agreed well with experimental data (5.74 × 109 cells), the error was less than 2%, indicating that the adsorption reaction of original cells can be approximately described by McKay second reaction model. Nonetheless, the fitted rate equations of treated cells were not in good agreement with experimental data (Fig. 1B, lines 2, 3, 4), and the error was between 32% and 57%. This suggested that the adsorption kinetics of treated cells no longer agreed with the Classic McKay secondary rate equation. It seemed that the adsorption characteristics of treated cells have changed, which was consistent with Fig. 1A. The adsorption of bacterial cells on the surface of minerals mainly involved three kinds of forces: van der Waals interactions, electrostatic interactions and acid-base interaction (Das et al., 2011; Das et al., 2014). The existence of eDNA would benefit the adsorption of cell, this is mainly due to the presence of eDNA and other components of EPS on bacterial cell surfaces would promote the surface hydrophobicity, meanwhile the mineral surface is also hydrophobic. In addition, eDNA can combine with some metal ions, creating a positive zeta potential, while the mineral surface generally has a negative charge. Therefore, the electrostatic interaction enhances the adsorbing of bacterial cells onto the mineral surface (Pogliani and Donati, 1999).
2.7. CLSM analysis of EPS on the chalcopyrite surface during bioleaching In order to compare the content of EPS before and after the addition of DNase I, the EPS on the surface of slag were stained by specific fluorescent dyes, and the results were observed by CLSM. The slag should be cleaned with SDW to remove the free cells on the surface. Propidium iodide (PI) can be used to stain eDNA (Qin et al., 2007; Okshevsky and Meyer, 2014) and it cannot penetrate living cells membranes (Zrelli et al., 2013; Fuxman Bass et al., 2010). Fluorescent probes FITC-WGA (wheat germ agglutinin) (Neu et al., 2001; Yu et al., 2017) and FITC-D 70 (fluorescein isothiocyanate) were also performed to visualize extracellular polysaccharides and proteins. 3. Result and discussion 3.1. Effect of eDNA on the adsorption behavior of Sulfobacillus thermosulfidooxidans strain ST In order to study the adsorption behavior of bacterial cells on the surface of chalcopyrite, the adsorption experiment of Sulfobacillus thermosulfidooxidans strain ST was carried out. The effect of eDNA on adsorption capacity of bacterial cells was studied. DNase I was added into the medium to degrade eDNA and then the comparison experiment with and without eDNA was carried out to investigate the function of eDNA on bacterial adsorption. Fig. 1A shows the adsorption curve of bacterial cells before and after the addition of DNase I. It shows that with prolonged incubation the amount of attached cells gradually
3.2. Bioleaching of chalcopyrite concentrate by Sulfobacillus thermosulfidooxidans strain ST Variations in the number of free and attached cells during bioleaching were shown in Fig. 2. Without adding DNase I, the attached cells had a relatively shorter lag phase, and then increased quickly after 2 days. The number of attached cells could reach the maximum at the 99
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Fig. 1. Adsorption behavior of Sulfobacillus thermosulfidooxidans strain ST on the chalcopyrite surface. (A) Adsorption curve of Sulfobacillus thermosulfidooxidans strain ST within in 3 h under different conditions; (B) Fitting curve according to the secondary rate equation.
Fig. 2. The variations of the free cells number in the solution and the attached cells number in the ore residue during bioleaching of chalcopyrite concentrate by the original and treated cells, respectively. The treated cells (DNase I addition) means its eDNA in the EPS was degraded by DNase I during the whole process of bioleaching.
Fig. 3. The variations of copper extraction during bioleaching of chalcopyrite concentrate by the original and treated cells respectively. The treated cells (DNase I addition) means its eDNA in the EPS was degraded by DNase I during the whole process of bioleaching.
11th day (1.1 × 1010 cells). After that, the number of attached cells kept stable till the 18th day and then began to decrease. Meanwhile, the number of free cells began to grow from the 4th day and reached the highest at the 14th day, which was 3.1 × 1010 cells. However, when DNase I was added into the bioleaching system, the number of attached cells began to increase quickly only from the 6th day, and reached the maximum at the 13th day (0.8 × 1010 cells). The number of free cells began to increase after the 7th day and the maximum number was 2.6 × 1010 cells at the 16th day. The adaptation period was prolonged compared with the group without addition of DNase I. It can also be seen that after the number of attached cells achieved an equilibrium, the number of free cells was still increasing. But the number of adsorbed and free bacterial cells decreased compared with the group without addition of DNase I. These results indicated that the addition of DNase I would lead to eDNA deficiency, which affected the adsorption capacity of bacteria, and then inhibited the release of energy sources from mineral. The reduction of energy source would further affect the growth of free cells in the bioleaching solution. Alternatively, after adding DNase I, part of the attached cells will detach from the mineral/biofilm and become free cells, but the number of these cells was difficult to be calculated.
Fig. 3 shows copper extraction as a function of time during bioleaching of chalcopyrite concentrate. In the early stage of bioleaching, although the microbes were still in the adaptation period, a few copper and iron were leached. This was due to that CuS2 in the concentrate was easily leached by the acid in the medium. As bioleaching continued, copper extraction steadily increased until the 18th day, achieving 10.6 g/L, and the copper extraction percentage reached 82%. However, in the experiment with the addition of DNase I, a maximal copper extraction of 8.2 g/L was achieved at the 21th day. The iron was also an important parameter in bioleaching of chalcopyrite concentrate. In the whole bioleaching process, the concentration of ferrous ion increased first and then decreased (Fig. 4). And the highest ferrous iron concentration (2.2 g/L) was achieved at the 9th day. Meanwhile, the concentration of ferric ions gradually increased until reaching stability. In the experiment with the addition of DNase I, the ferrous iron concentration reached the maximum (1.5 g/L) at the 11th day, followed by a rapid decrease, and reached the plateau at the 17th day. During this process, the concentration of ferric ion increased gradually, and the maximal concentration of 1.2 g/L was achieved at the end of bioleaching. 100
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Fig. 5. The variation of the contents of each major EPS component as a function of time during bioleaching of chalcopyrite by Sulfobacillus thermosulfidooxidans strain ST in the presence and absence of DNase I. EPS is given in mg and refers to the amount extracted from the total ore residue.
Fig. 4. The variations of ferrous and ferric ion concentrations during bioleaching of chalcopyrite by the original and treated Sulfobacillus thermosulfidooxidans strain ST, respectively.
It can be seen from Figs. 3 and 4 that the high concentration of total iron would be beneficial to copper extraction. Iron in solution at the beginning was mainly ferrous iron and the presence of ferric iron was more notable only in the end. At the end of bioleaching, ferrous iron was continually oxidized to ferric iron, therefore the concentration of ferric iron increased gradually till the end. When the leaching rate of copper decreased, the total iron began to keep stable. Because of the large amount of acid produced by oxidation of reduced sulfur species, the pH of the medium was reduced to 1.1 and the redox potential value increased to 590 mV, and this would inhibit the copper extraction (Hiroyoshi et al., 2008; Okamoto et al., 2005). Furthermore, jarosite was found when the components of ore residue were analyzed by XRD. Therefore, the formation of passivation layer was also an inducement resulting in the decrease of copper leaching rate (Zeng et al., 2010b). The addition of DNase I decreased the extraction of copper and ferrous iron. This was due to the absence of eDNA, which affected the number of attached cells (Fig. 2). It further indicated that the attached cells were a more important factor for the dissolution of chalcopyrite concentrate compared with the free cells in the bioleaching solution (Zeng et al., 2010b; Fowler and Crundwell, 1998).
Original
DNase I addition
3.3. Extraction and characterization of EPS components during bioleaching of chalcopyrite concentrate During the extraction process of EPS from the mineral surface, the ratio of proteins and polysaccharides in EPS was below 5.0 and the ratio of eDNA in the EPS was below 15%. According to the research of Bo et al. (1996), the ratio between proteins and polysaccharides was found in the range 0.2 to 5.0. In addition, relevant studies showed that the ratio of eDNA in the total EPS is below 15% (Liao et al., 2001; He et al., 2014). It shows that the applied method for EPS extraction did not lead to noticeable contamination with intracellular components. Fig. 5 shows the variation of the contents of major EPS components like extracellular proteins, polysaccharides and eDNA at different bioleaching time points. They can also be detected by. CLSM, which combined with fluorescent probes for staining extracellular proteins, extracellular polysaccharides and eDNA, respectively (Fig. 6). These EPS components were asymmetrically covered on the mineral surface. It was reported that the cells preferred to attach on the cracks/defects and formed a monolayer biofilm on chalcopyrite concentrate surface (Zhang et al., 2014). Only a small amount of EPS was
Fig. 6. The CLSM observation of EPS components after bioleaching of 12 days by the Sulfobacillus thermosulfidooxidans strain ST in the presence and absence of DNase I. DNase I treatment (A2, B2, C2), no DNase I treatment (A1, B1, C1). (A: extracellular protein; B: extracellular polysaccharide; C: extracellular DNA).
produced at the first 5 days, this was mainly due to that the bacteria were still in the adaptation period (Fig. 2). After that, as the increase of energy sources released from mineral, the amount of attached cells increased, which resulted in the increase of EPS on the surface of minerals. At the final stage of bioleaching, the total EPS of attached cells 101
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remained stable at 53.2 mg. This indicated that once the EPS were produced, it was difficult to remove or degrade them in the bioleaching system. The extracellular proteins and extracellular polysaccharides were the major components in the EPS (Figs. 5 and 6). The contents of extracellular polysaccharides and extracellular proteins could reach the highest value at the 10th day, which were 26.1 mg and 34.8 mg, respectively. Then the content was maintained at a steady level, which was related to the stability of biofilm. However, the content of eDNA was much lower than that of other components. Especially in the initial stage of bioleaching the content of eDNA was the lowest, but with the extension of bioleaching time, its content increased gradually and reached 3.8 mg at the 20th day in the experiment without the addition of DNase I. It can be seen from Figs. 2 and 5 that at the later stage of bioleaching, the attached cell number did not increase, but the amount of eDNA increased gradually. This may be due to that the harmful environment (high metal ion concentration, extreme low pH) at the later stage of bioleaching would increase the cell lysis and then release DNA into the solution (Steinmoen et al., 2002; Vorkapic et al., 2016). During the bioleaching with addition of DNase I, the total EPS and the content of each component were reduced obviously (Fig. 5). The fluorescence intensity of all the EPS components decreased significantly (Fig. 6). Especially for eDNA, its fluorescence intensity was even disappeared (or under detection limit). This was because the absence of eDNA reduced the adsorption capacity of bacterial cell, therefore, the amount of bacterial cells adsorbed on the surface of the mineral was reduced, which in turn leading to a decrease of EPS. Furthermore, eDNA as a bridge in EPS was reported to combine other extracellular substances and then keep the biofilm structure stable (Cruz et al., 2012; Kerchove and Elimelech, 2008). Therefore, the deficiency of eDNA would partly inhibit the formation of EPS/biofilm on the mineral surface during bioleaching. This further proved that the addition of DNase I not only degraded eDNA formed on the mineral surface, but also inhibited the formation of other EPS components.
475–481. Antonova, E.S., Hammer, B.K., 2015. Genetics of natural competence in vibrio cholerae and other vibrios. Microbiol. Spectr. 3 (3). Bakkali, M., 2013. Could DNA uptake be a side effect of bacterial adhesion and twitching motility? Arch. Microbiol. 195 (4), 279–289. Bandich, A., Wilcox, T., Borenfreund, E., 1965. Circulating DNA as a possible factor in oncogenesis. Science 148 (3668), 374–376. Bo, F., Palmgren, R., Keiding, K., Nielsen, P.H., 1996. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 30 (8), 1749–1758. Catlin, B.W., 1956. Extracellular deoxyribonucleic acid of bacteria and a deoxyribonuclease inhibitor. Science 124 (3219), 441–442. Comte, S., Guibaud, G., Baudu, M., 2006. Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS: part II. Consequences of EPS extraction methods on Pb2+ and Cd2+ complexation. Enzym. Microb. Technol. 38 (2), 237–245. Corinaldesi, C., 2005. Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Appl. Environ. Microbiol. 71 (1), 46–50. Cruz, L.F., Cobine, P.A., Fuente, L.D.L., 2012. Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility. Appl. Environ. Microbiol. 78 (5), 1321–1331. Das, T., 2010. Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl. Environ. Microbiol. 76 (76), 3405–3408. Das, T., Krom, B.P., Van, d.M.H.C., Busscher, H.J., Sharma, P.K., 2011. DNA-mediated bacterial aggregation is dictated by acid–base interactions. Soft Matter 7 (6), 2927–2935. Das, T., Sehar, S., Koop, L., Wong, Y.K., Ahmed, S., Siddiqui, K.S., Manefield, M., 2014. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation. PLoS One 9 (3), e91935. Farah, C., Vera, M., Morin, D., Haras, D., Jerez, C.A., Guiliani, N., 2005. Evidence for a functional Quorum-Sensing type AI-1 system in the extremophilic bacterium Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 71 (11), 7033–7040. Fowler, T.A., Crundwell, F.K., 1998. Leaching of zinc sulfide by Thiobacillus ferrooxidans: experiments with a controlled redox potential indicate no direct bacterial mechanism. Appl. Environ. Microbiol. 64 (10), 3570–3575. Fuxman Bass, J.I., Russo, D.M., Gabelloni, M.L., Geffer, J.R., Giordano, M., Catalano, M., Zorreguieta, A., Trevani, A.S., 2010. Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms. J. Immunol. 184 (11), 6386. Gehrke, T., Telegdi, J., Thierry, D., Sand, W., 1998. Importance of extracellular polymeric substances from Thiobacillus ferrooxidans for bioleaching. Appl. Environ. Microbiol. 64 (7), 2743–2747. Gilan, I., Sivan, A., 2013. Extracellular DNA plays an important structural role in the biofilm of the plastic degrading actinomycete. Adv. Microbiol. 3 (8), 543–551. Gu, G.H., Hu, K.T., Li, S.K., 2014. Surface characterization of chalcopyrite interacting with Leptospirillum ferriphilum. Trans. Nonferrous Metals Soc. China 24 (6), 1898–1904. Guo, X., Yin, H.Q., Liang, Y.L., Hu, Q., Zhou, X.S., Xiao, Y.H., Ma, L.Y., Zhang, X., Qiu, G.Z., Liu, X.D., 2014. Comparative genome analysis reveals metabolic versatility and environmental adaptations of Sulfobacillus thermosulfidooxidans strain ST. PLoS One 9 (6), e99417. He, Z.G., Yang, Y.P., Zhou, S., Hu, Y.H., Zhong, H., 2014. Effect of pyrite, elemental sulfur and ferrous ions on EPS production by metal sulfide bioleaching microbes. Trans. Nonferrous Metals Soc. China 24 (4), 1171–1178. Hiroyoshi, N., Kitagawa, H., Tsunekawa, M., 2008. Effect of solution composition on the optimum redox potential for chalcopyrite leaching in sulfuric acid solutions. Hydrometallurgy 91 (1), 144–149. Ho, Y.S., Mckay, G., 1999. Pseudo-second order model for sorption processes. Process Biochem. 34 (5), 451–465. Jakubovics, N.S., Shields, R.C., Rajarajan, N., Burgess, J.G., 2013. Life after death: the critical role of extracellular DNA in microbial biofilms. Lett. Appl. Microbiol. 57 (6), 467–475. Kerchove, A.J., Elimelech, M., 2008. Calcium and magnesium cations enhance the adhesion of motile and nonmotile pseudomonas aeruginosa on alginate films. Langmuir ACS J. Surf Colloids 24 (7), 3392. Lewenza, S., 2013. Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa. Front. Microbiol. 4 (1), 21. Liao, B.Q., Allen, D.G., Droppo, I.G., Leppard, G.G., Liss, S.N., Allen, D.G., 2001. Surface properties of sludge and their role in bioflocculation and settleability. Water Res. 35 (2), 339. Neu, T., Swerhone, G.D., Lawrence, J.R., 2001. Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology 147 (Pt 2), 299–313. Okamoto, H., Nakayama, R., Kuroiwa, S., Hiroyoshi, N., Tsunekawa, M., 2005. Normalized redox potential used to assess chalcopyrite column leaching. J. MMIJ 121 (6), 246–254. Okshevsky, M., Meyer, R.L., 2014. Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms. J. Microbiol. Methods 105, 102–104. Özdemir, C., Karaca, B., Akçelik, N., 2015. The role of extracellular DNA in Salmonella biofilms: is it in intimate relationship with matrix or initial adhesion? In: Conference: 25th ECCMID Conference, At Copenhagen, Denmark. Pogliani, C., Donati, E., 1999. The role of exopolymers in the bioleaching of a non-ferrous metal sulphide. J. Ind. Microbiol. Biotechnol. 22 (2), 88–92. Qin, Z., Ou, Y., Yang, L., Zhu, Y., Tolkernielsen, T., Molin, S., Qu, D., 2007. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology 153 (Pt 7), 2083. Sand, W., Gehrke, T., 2006. Extracellular polymeric substances mediate bioleaching/
4. Conclusion Bacterial adsorption on mineral surface is required for the production of extracellular DNA. The adsorption kinetics of bacterial cells on the surface of chalcopyrite can be described by the McKay level two rate equation. The equilibrium adsorption quantity calculated by this equation was in good agreement with the experimental data. The content of eDNA in EPS was relatively low, but eDNA played an important role in the adsorption process on the mineral surface. The lack of eDNA would decrease the formation of other EPS components. In situ detection combined with traditional extraction and analysis methods, can systematically study the formation and content of eDNA in different stages of bioleaching. Furthermore, the method about “specific enzymatic lysis” (like using DNase I to degrade eDNA) would be also a good way to investigate the function of other EPS components. This paper would provide some theoretic evidence for elucidating the function of eDNA in bioleaching process, and be also conducive to the further development of biohydrometallurgy theory. Acknowledgements This work was supported by the National Natural Science Foundation of China (nos. 31470230, 51320105006, 51604308), the Fundamental Research Funds for the Central University of Central South University (2017zzts378). References Anker, P., Jachertz, D., Stroun, M., Brögger, R., Lederrey, C., Henri, J., Maurice, P.A., 1980. The role of extracellular DNA in the transfer of information from T to B human lymphocytes in the course of an immune response. Int. J. Immunogenet. 7 (6),
102
Hydrometallurgy 176 (2018) 97–103
R. Yu et al.
1686–1690. Yu, R.L., Ou, Y., Tan, J.X., Wu, F.D., Sun, J., Lei, M., 2011. Effect of EPS on adhesion of Acidithiobacillus ferrooxidans on chalcopyrite and pyrite mineral surfaces. Trans. Nonferrous Metals Soc. China 21 (2), 407–412. Yu, R.L., Liu, A.J., Liu, Y.N., Yu, Z.J., Peng, T.J., Wu, X.L., Shen, L., Liu, Y.D., Li, J.K., Liu, X.D., Qiu, G.Z., Chen, M., Zeng, W.M., 2017. Evolution of Sulfobacillus thermosulfidooxidans secreting alginate during bioleaching of chalcopyrite concentrate. J. Appl. Microbiol. 122 (6). Zeng, W.M., Qiu, G.Z., Zhou, H.B., Liu, X.D., Chen, M., Chao, W.L., Zhang, C.G., Peng, J.H., 2010a. Characterization of extracellular polymeric substances extracted during the bioleaching of chalcopyrite concentrate. Hydrometallurgy 100 (3–4), 177–180. Zeng, W., Qiu, G., Zhou, H., Peng, J., Chen, J.M., Tan, S.N., Chao, W., Liu, X., Zhang, Y., 2010b. Community structure and dynamics of the free and attached microorganisms during moderately thermophilic bioleaching of chalcopyrite concentrate. Bioresour. Technol. 101 (18), 7079–7086. Zhang, D., Pan, X., Mostofa, K.M., Chen, X., Mu, G., Wu, F., Liu, J., Song, W., Yang, J., Liu, Y., Fu, Q., 2010. Complexation between Hg (II) and biofilm extracellular polymeric substances: an application of fluorescence spectroscopy. J. Hazard. Mater. 175 (1–3), 359–365. Zhang, R., Bellenberg, S., Castro, L., Thomas, R.N., Wolfgang, S., Mario, V., 2014. Colonization and biofilm formation of the extremely acidophilic archaeon Ferroplasma acidiphilum. Hydrometallurgy 150, 245–252. Zhang, W., Cao, B., Wang, D., Zhang, W., 2015. Variations in distribution and composition of extracellular polymeric substances (EPS) of biological sludge under potassium ferrate conditioning: effects of pH and ferrate dosage. Biochem. Eng. J. 106, 37–47. Zhou, H.B., Mao, F., Wang, Y.G., 2015. Acidophilic microorganisms and bioleaching technology. Bull. Mineral. Petrol. Geochem. 34 (2), 269–276. Zrelli, K., Galy, O., Latourlambert, P., Kirwan, L., Ghigo, J.M., Beloin, C., Henry, N., 2013. Bacterial biofilm mechanical properties persist upon antibiotic treatment and survive cell death. New J. Phys. 15 (12), 5026.
biocorrosion via interfacial processes involving iron (III) ions and acidophilic bacteria. Res. Microbiol. 157 (1), 49–56. Sena-Vélez, M., Redondo, C., Graham, J.H., Cubero, J., 2016. Presence of extracellular DNA during biofilm formation by Xanthomonas citri subsp. citri strains with different host range. PLoS One 11 (6), e156695. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium ferrobacillus ferrooxidans: I. An improved medium and a harvesting procedure for securing high cell yields. J. Bacteriol. 77 (5), 642–647. Spoering, A.L., Gilmore, M.S., 2006. Quorum sensing and DNA release in bacterial biofilms. Curr. Opin. Microbiol. 9 (2), 133–137. Steinmoen, H., Knutsen, E., Håvarstein, L.S., Steinmoen, H., Knutsen, E., Havarstein, L.S., 2002. Induction of natural competence in streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proc. Natl. Acad. Sci. 99 (11), 7681–7686. Tan, S.N., Chen, M., 2012. Early stage adsorption behaviour of Acidithiobacillus ferrooxidans on minerals I: an experimental approach. Hydrometallurgy 119-120 (5), 87–94. Tan, S.N., Burgar, I., Chen, M., 2011. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. 102 (102), 9143–9147. Tetz, V.V., Tetz, G.V., 2010. Effect of extracellular DNA destruction by DNase I on characteristics of forming biofilms. DNA Cell Biol. 29 (8), 399. Vorkapic, D., Pressler, K., Schild, S., 2016. Multifaceted roles of extracellular DNA in bacterial physiology. Curr. Genet. 62 (1), 71–79. Wang, Z.H., Xie, X.H., Liu, J.S., 2012. Experimental measurements of short-term adsorption of Acidithiobacillus ferrooxidans onto chalcopyrite. Trans. Nonferrous Metals Soc. China 22 (2), 442–446. Wu, J., Xi, C., 2009. Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. Appl. Environ. Microbiol. 75 (16), 5390–5395. Yao, F.U., Yang, H.Y., Fang, Y.J., Zhang, C.Z., 2010. Determination of polysaccharide content in extracellular polymers of leaching bacteria. J. Cent. South Univ. 41 (5),
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