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Bioleaching of copper sulfides using mixed microorganisms and its community structure succession in the presence of seawater
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Bioleaching of copper sulfides using mixed microorganisms and its community structure succession in the presence of seawater ⁎
Wei Chena,b, Shenghua Yina,b, , Aixiang Wua,b, Leiming Wangb, Xun Chenb a b
Key Laboratory of Ministry of Education for High-Efficient Mining and Safety of Metal, University of Science and Technology Beijing, Beijing 100083, China School of Civil and Resource Engineering, University of Science and Technology Beijing, Beijing 100083, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Seawater Low-grade copper sulfide ore Bioleaching Bacteria diversity and dynamics Attached bacteria
The bacterial diversity and dynamics in the leaching solution were analyzed during bioleaching of low-grade copper sulfide ore in the presence of seawater in this study. The results indicated a promoting response of appropriate-proportion seawater to bioleaching with improved copper recoveries. A maximum of 84.70% copper recovery was obtained in the presence of 20.00% seawater in contrast to only 72.49% in its absence. The experiments verified that seawater owned a great influence on Attached bacteria and bacterial species. 16S rDNA analysis illustrated that bacterial species decreased distinctly in the presence of seawater. Little difference between blank sample (no seawater) and sample adding 20.00% seawater was indicated by beta diversity index. Bacteria (including Acidithiobacillus ferrooxidans, Sphingomonas leidyi and Lactobacillus acetotolerans) were influenced significantly after adding seawater. Acidithiobacillus ferrooxidans accounted for the highest proportion of the community whether seawater was added or not during bioleaching.
1. Introduction There has been a continuous decline in mined ore grades and easyto-recovery metal resources throughout the world over the past few decades, and thus advisable exploitation of low-grade reserves to meet the ever increasing metal demands is extensively carried out (Davis-
Belmar et al., 2014; Liu et al., 2019; Jia et al., 2018). Chalcopyrite is a kind of sulfide ores bearing copper, which hold the most abundant copper mineral in the world (Giuseppe et al., 2019; Owusu et al., 2014). While compared to copper oxide ores and other copper minerals, copper sulfide ores own characteristics of refractory and recalcitrance (Panda et al., 2015a). Bioleaching is a hydrometallurgical technique
⁎ Corresponding author at: Key Laboratory of Ministry of Education for High-Efficient Mining and Safety of Metal, University of Science and Technology Beijing, Beijing 100083, China. E-mail address: [email protected] (S. Yin).
https://doi.org/10.1016/j.biortech.2019.122453 Received 15 October 2019; Received in revised form 17 November 2019; Accepted 18 November 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Wei Chen, et al., Bioresource Technology, https://doi.org/10.1016/j.biortech.2019.122453
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analysis result showed that the Cu grade was at 0.75%, including 0.04% free copper oxide, 0.06% primary copper sulfide, 0.64% secondary copper sulfide and 0.01% combined copper oxide. The mineralogical analysis of the ore sample indicated that silica (SiO2) was the major mineral phase, which was up to 91.00%. And other main compositions of low-grade copper sulfide ores were Cu − 0.75%, Fe − 1.62%, S − 1.10%, CaO − 0.30%, MgO − 0.04% and Al2O3 − 5.19%.
that is suitable to process these low-grade ores compared to other conventional mineral processing techniques (Fagan et al., 2014; Hong and Valix, 2014; Petersen, 2016), and it has been used in industrial production over past few years (Brierley and Brierley, 2013; Xia et al., 2018). Although bioleaching is an effective promisingly method for extracting refractory copper sulfide ores with advantages of environment friendly and perfect economic benefits, problems including low leaching rate, long bioleaching routine and low efficiency still need to be finished off (Bevilaqua et al., 2013; Klauber, 2008). To solve the problems mentioned above, many efforts have been conducted to improve copper recovery by adding certain additives (Johnson et al., 2008; Muñoz et al., 2007). Liang et al. (2012) investigated the effect of activated carbon on the refractory copper sulfide ores bioleaching and found that the bioleaching efficiency was enhanced in the presence of activated carbon. The effects of metal ions like Ag+ as catalysts on copper extraction during bioleaching were addressed by Xia et al. (2018) and Hu et al. (2002). And two different mostly accepted catalytic reactions by Ag+ during bioleaching of refractory copper sulfide ores were presented. Besides, recent studies on bioleaching of mostly concentrates have shown promising results of metal recovery using cellulose. Yin et al. (2019) investigated the effect of cellulose (rice straw) on the copper recovery during copper sulfide ores bioleaching. In addition, the effect of cellulose (waste newspapers) was presented by Panda et al. (2015b) as well. There are abundant resources in ocean with an average of 35.70 million tons minerals per cubic kilometer of seawater (Lao et al., 2019). Among the more than 100 elements have been identified in the world, 80.00% can be found in seawater (Atzori et al., 2019). Thus extracting metals from seawater is going to be more and more promising. Based on the significant role of seawater, the application of seawater has been more and more extensive in recent years (Yang et al., 2018a). Many metals full of economical value were extracted from seawater directly, such as uranium and magnesium (Li et al., 2019; Luo et al., 2019). Yang et al. (2018b) studied the lithium metal extraction from seawater, which proposed a new method for the shortage of metal resources. The floatability of molybdenite and chalcopyrite in artificial seawater was explored by Suyantara et al. (2018), which provided a reference for copper extraction using seawater. Gong et al. (2019) analyzed the advantages and disadvantages of seawater as circulating cooling water for coastal nuclear power stations, which provided security for nuclear energy by using seawater. Besides, the bacterial community inside seawater or after adding seawater has been studied, which provided a basis for extracting metal resources from seawater in the presence of bacteria (Chen et al., 2019; Kumar et al., 2019). There is no enough researches showing copper extraction using seawater, thus it is still worth studying a better way to recover copper from seawater. In this study, seawater that acted as a potential promoter during bioleaching of low-grade copper sulfide ores was proposed for the first time. And some significant leachable factors, including Cu2+ concentration, Fe3+ concentration, oxidation-reduction potential (Eh), pH value and bacteria concentration, were monitored. The microbial community structure inside the leaching solution during bioleaching was also analyzed by 16S rDNA (Hao et al., 2017). Thus, the results showed in this paper should provide more promising utilization of seawater during and extraction of copper.
2.2. Pretreatment of seawater Seawater used in this study was originated from Guangdong Province, China. For the sake of avoiding the influence of unidentified bacteria inside seawater on bioleaching, high-temperature heating was applied to seawater prior to low-grade copper sulfide ore bioleaching experiments. The preprocessing (high-temperature heating) was performed at a vertical mode steam sterilizers for 30 min at a temperature of 120 ℃. Then the seawater after heating was placed in a laminar flow as cooling. The seawater obtained after cooling was used during the bioleaching experiments and further analytical studies. The specific compositions of seawater were Cl− − 17.87 g/L, Na+ − 9.58 g/L, Mg2+ − 1.23 g/L, Ca2+ − 0.38 g/L and K+ − 0.35 g/L. 2.3. Bacteria and culture The bacteria were collected from acidic waste mine water of a copper mine, Fujian Province, China. Bacteria have been gone through repeatedly laboratory-scale culture experiments in 9 K medium to enrichment, purification and domestication before bioleaching experiments. The components of 9 K medium were showed in Feng et al. (2014), which contained (NH4)2SO4 − 3.00 g/L, MgSO4·7H2O − 0.50 g/L, KCl − 0.10 g/L, K2HPO4- 0.50 g/L, Ca(NO3)2–0.01 g/L and FeSO4·7H2O − 44.20 g/L. Bacteria were inoculated to flasks in the presence of 9 K media, and pH value was adjusted to 2.00 by dilute H2SO4. Then flasks were placed at a temperature of 30 ℃ and 120 r/min shaker during laboratory-scale culture experiments. Thus the bacteria concentration was up to 9.50 × 107 cells/mL at the beginning of the bioleaching experiments.
2.4. Bioleaching experiments In order to study the effect of seawater on low-grade copper sulfide ore bioleaching, five bioleaching experiments were designed firstly. Bioleaching experiments were performed using 250 mL flasks, and the proportion of seawater to leaching solution was varied in those experiments (0.00%, 20.00%, 40.00%, 60.00% and 80.00%). Table 1 showed the summary of experiment schemes. Due to the ore samples were rich in Fe, which can provide enough energy for the bacteria growth. The culture medium here called 0 K medium (i.e. 9 K media without FeSO4·7H2O) containing (NH4)2SO4 − 3.00 g/L, MgSO4·7H2O − 0.50 g/L, KCl − 0.10 g/L, K2HPO4- 0.50 g/L and Ca(NO3)2 – 0.01 g/ L was employed in the bioleaching experiments in this study. The experiments were carried out in 250 mL flasks under the same initial condition of 20.00 mL bacteria (9.50 × 107 cells/mL) were inoculated, and the pH value of the leaching solution was adjusted to 2.00 by adding dilute H2SO4. Then the flasks were placed at a temperature of 30 ℃ and 120 r/min shaker. Moreover, characteristics including pH value, Cu2+ concentration, bacteria concentration and Oxidation-reduction potential (Eh value) were monitored. Normal system mentioned subsequently was referred to experimental samples that are in the presence of both Attached bacteria and free bacteria. Experimental samples after removing Attached bacteria and free bacteria during bioleaching herein were referred to as Attached-bacteria-removed system and Free-bacteria-removed system throughout the text, respectively. All the experiments in this work were conducted in triplicate.
2. Materials and methods 2.1. Feature of ore samples Low-grade copper sulfide ores applied in this study were collected from a copper Mine, Fujian Province, China. The chemical composition analysis result of the low-grade copper sulfide ores was obtained by selective dissolved experiments through Chinese Academy of Sciences. The mineralogical analysis of the ore sample was initially carried out through Atomic Absorption Spectroscopy. The chemical composition 2
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Table 1 Bioleaching experiment schemes. Experimental samples
Seawater (%/mL)
Ores (g)
Medium
TA-0 TA-1 TA-2 TA-3 TA-4 TB-0 TB-1 TB-2 TB-3 TB-4 TB-5
0.00/0.00 20.00/30.00 40.00/60.00 60.00/90.00 80.00/120.00 20.00/40.00 20.00/40.00 20.00/40.00 20.00/40.00 20.00/40.00 20.00/40.00
20.00 20.00 20.00 20.00 20.00 15.00 15.00 15.00 15.00 15.00 15.00
0 k 130.00 mL + Bacteria 20.00 mL 0 k 100.00 mL + Bacteria 20.00 mL 0 k 70.00 mL + Bacteria 20.00 mL 0 k 40.00 mL + Bacteria 20.00 mL 0 k 10.00 mL + Bacteria 20.00 mL – Attached and Free bacteria Removed Attached bacteria Free bacteria and add Attached bacteria at day 9 Removed Free bacteria Attached bacteria and remove Free bacteria at day 9
of H+. Reactions mentioned above all leaded to an increase in pH value. However, S0 contained in low-grade copper sulfide ores was oxidized into dilute sulfuric acid subsequently, which yielded a large amount of H+. Furthermore, formation of precipitation (formed by hydrolysis of Fe3+) produced H+ as well. Both processes mentioned above contributed to the following decrease in pH value. Although variation of pH value during low-grade copper sulfide ore bioleaching was similar regardless of whether seawater was present or not, the minimum pH value of each sample differed ranging from 2.03 to 2.15 after adding seawater. Fig. 1(b) showed that the pH value of Samples TA-0 to TA-4 decreased significantly with leaching time. The decrease of Samples TA-1 and TA-2 was observed to be more striking. For instance, the pH value of Sample TA-1 decreased by 0.38 (from 2.41 to 2.03), while that of Sample TA-4 only decreased by 0.27 (from 2.42 to 2.15). According to Fig. 1(b), Samples TA-0 to TA-4 reached their maximum pH value at day 5, day 3, day 4, day 5 and day 6, respectively. Compared to Sample TA-0 in the absence of seawater, Samples TA-1 and TA-2 in the presence of seawater reached peak pH value more quickly, which indicated adding appropriate-proportion seawater accelerated the increase of pH value. Meanwhile, lower pH values were found in Samples TA-1 and TA-2, where higher Cu2+ concentrations were produced (Fig. 1(a)). Based on previous study that decrease of initial pH value accelerated the dissolution of chalcopyrite (Vilcáez et al., 2009), the decline in pH value was likely responsible for improving copper extraction of low-grade copper sulfide ores in this study. The peak Fe3+ concentration of Samples TA-0 to TA-4 was 84.70 mg/L, 95.90 mg/L, 87.10 mg/L, 78.80 mg/L and 69.30 mg/L, separately. The peak Fe3+ concentration of Samples TA-1 and TA-2 increased by 11.20 mg/L and 2.40 mg/L, respectively, compared to that of Sample TA-0. Thus, it could be deducted that adding appropriateproportion seawater could promote the release of Fe3+. However, there was relatively low Fe3+ concentration of Samples TA-3 and TA-4 according to Fig. 1(c), especially in Sample TA-4. Previous study (Vilcáez et al., 2009) has found that the oxidation process of Fe2+ by dissolved oxygen in the presence of H+ decreased with increasing pH value. There was high pH value of Samples TA-3 and TA-4 (Fig. 1(b)), which explained why the two samples owned low Fe3+ concentration. Fig. 1(d) showed clearly that the Eh value increased remarkably at the initial stage of bioleaching, and then stayed stable in fluctuation. The increase in the Eh value during bioleaching mainly resulted from the oxidation of Fe2+ to Fe3+ by the dissolved oxygen in the presence of H+ (Hiroyoshi et al., 2001). The maximum Eh value of Samples TA-0 to TA-4 was 587 mV, 589 mV, 569 mV, 563 mV and 546 mV, respectively. Samples TA-0 and TA-1 owned the highest Eh value.
2.5. Analysis In this study, concentrations such as Cu2+, Cl−, Ca2+ were detected by ICP Optical Spectrometer (OPTIMA 7000DV, America). Bacteria concentration was measured by optical microscope (Carl Zeiss Axio Lab A1, Germany). Oxidation-reduction potential (Eh value) and pH value were tested by a pH meter (PHS-3E, Inesa, China). The bacterial diversity and dynamics were clarified by 16S rDNA. Residue ores and aggregations were measured through X-ray Diffractionmeter (Smartlab, RIGAKU, Japan). 3. Results and discussion 3.1. Effect of seawater on low-grade copper sulfide ore bioleaching The variations of key chemical parameters (including pH, Eh, Fe3+ concentration, Fe2+ concentration, bacteria concentration and Cu2+ concentration) during low-grade copper sulfide ore bioleaching in the presence of seawater were shown in Fig. 1(a)–(f), separately. 3.1.1. Effect of seawater on copper recovery The effect of different proportion of seawater on low-grade copper sulfide ore bioleaching for copper recovery was shown in Fig. 1(a). Fig. 1(a) indicated clearly that compared to a peak Cu2+ concentration (543.70 mg/L) of Sample TA-0 without adding seawater, that of Samples TA-1 and TA-2 increased significantly. The peak Cu2+ concentration of Samples TA-1 and TA-2 reached 635.30 mg/L and 576.40 mg/L, respectively. However, Samples TA-3 and TA-4 in the presence of seawater only yielded a maximum Cu2+ concentration of 522.60 mg/L and 501.10 mg/L, separately. There was an apparent lower peak Cu2+ concentration of Samples TA-3 and TA-4, which dropped 21.10 mg/L and 42.60 mg/L in comparison with that of Sample TA-0. The maximum copper recovery of Samples TA-0 to TA-4 was 72.49%, 84.70%, 76.85%, 69.68% and 66.81%, respectively. It could be found that with increasing proportion of seawater, the maximum copper recovery decreased gradually ranging from 84.70% to 66.81%. The maximum (635.30 mg/L) and minimum (501.10 mg/L) Cu2+ concentration in the presence of seawater increased by 16.85% and decreased by 7.84% respectively compared with that of Sample TA-0 (543.70 mg/L) in the absence of seawater, which illustrated that appropriate-proportion seawater could improve bioleaching. 3.1.2. Effect of seawater on key chemical parameters As was shown in Fig. 1(b), the pH value increased at first, and then dropped. The peak pH value of Samples TA-0 to TA-4 was 2.43, 2.41, 2.42, 2.41 and 2.42, respectively. The pH value rose during early stage of low-grade copper sulfide ore bioleaching could be resulted from the consumption of H+. Because there were massive oxides (e.g., MgO, CaO and Al2O3) inside bioleaching system, and they could react with H+. In addition, the processes (e.g., oxidation process of Fe2+ to Fe3+) of extracting Cu2+ from low-grade copper sulfide ores also consumed a lot
E = E0 +
RT [electron acceptor] ln [elector donor] nF
(1)
According to Eq. (1), when the factors (including R, T, F, n and E0) are the same, high electron acceptor proportion and low electron donor proportion during bioleaching can yield a higher Eh value. Since Fe3+ can be reduced to act as electron acceptor. The high Eh value of 3
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Fig. 1. Effect of seawater on the low-grade copper sulfide bioleaching performance. (a) Variation of Cu2+ concentration. (b) Variation of pH value. (c) Variation of Ferric concentration. (d) Variation of Eh value. (e) Variation of bacteria concentration. (f) Variation of Ferrous concentration.
Samples TA-0 and TA-1, especially in Sample TA-1, could be verified by the high Fe3+ concentration inside leaching solution.
3.2. Effect of seawater on bacteria during bioleaching 3.2.1. Effect of seawater on bacteria concentration Previous Section 3.1 indicated a significantly promoting effect with improved copper recovery from low-grade copper sulfide ores in the presence of appropriate-proportion seawater. And the effect of seawater 4
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value. Each value inside Fig. 2(c) represented the difference coefficient between two samples. The smaller the difference coefficient value was, the smaller the difference of species diversity between two samples was. The value between Sample TA-0 in the absence of seawater and Samples TA-1 to TA-4 was 0.540, 0.610, 0.646 and 0.696, separately. It could be concluded that the difference became more notable with the increasing proportion of seawater, and adding high proportion of seawater into bioleaching system caused greater difference than adding low proportion seawater. Especially, the difference coefficient value of low proportion of seawater between Sample TA-0 and Sample TA-1 was small, only 0.540. However, that between Sample TA-0 and Sample TA4 adding high proportion of seawater was up to 0.696. Meanwhile, in the bioleaching system in the presence of seawater, the difference coefficient value between Sample TA-1 and Sample TA-2, Sample TA-3 and Sample TA-4 were both relatively low, only 0.457 and 0.475. While that between Sample TA-1 and Sample TA-4 was up to 0.569. Thus, seawater effected bioleaching a lot, and adding different proportion of seawater exhibited diverse influence on bacteria as well. The dominant OTU (OTU 01, OTU 02, OTU 03, OTU 05, OTU 06, OTU 07 and OTU 12) of Sample TA-0 that detected during bioleaching were classified into seven different genus mainly: Acidithiobacillales, Lactobacillales, Ferroplasma, Stenotrophomonas, Thermoplasmatales, Pseudomonadales and Delftia. And the number of the same dominant OTU of Samples TA-1 to TA-3 in comparison with Sample TA-0 was 3 (OTU 01, OTU 02, OTU 05), 2 (OTU 01, OTU 03) and 1 (OTU 01), respectively. Contrary to Samples TA-1 to TA-3, no same dominant OTU was found between Sample TA-4 and Sample TA-0, however. The results of main OTU in each sample showed good accordance with difference coefficient value between samples (Fig. 2(c)). Table 3 indicated the variations of different species in the leaching solution determined by16S rDNA at day 7 and day 14 during low-grade copper sulfide ore bioleaching after adding different proportion of seawater. Acidithiobacillus ferrooxidans dominated the species at both day 7 and day 14 no matter what the proportion of seawater was added, accounting for more than 16.50% of the total population in the leaching solution. As was shown in Table 3, the proportion of Acidithiobacillus ferrooxidans of Sample TA-0 was 18.62% at day 7. The proportion of Acidithiobacillus ferrooxidans of Samples TA-1 and TA-2 was up to 27.18% and 21.25%, respectively. While that in Samples TA-3 and TA-4 was only 18.43% and 17.39%, which indicated that seawater played a vital role in proportion variation of Acidithiobacillus ferrooxidans. This could be due to adding excessive seawater leaded to a failure of oxygen level in bioleaching system (Table 2). However, Acidithiobacillus ferrooxidans was an aerobic bacterium, and lack of air could hinder its growth. Compared to Acidithiobacillus ferrooxidans proportion of Samples TA-0 to TA-3 at day 7, that at day 14 increased significantly up to 25.87%, 32.38%, 27.74% and 23.65%, respectively. While there was an obvious decreasing tendency showed in Sample TA-4 (from17.39 % to 16.82%). Such finding verified the addition of excessive proportion of seawater seriously hindered the growth of Acidithiobacillus ferrooxidans during this low-grade copper sulfide ore bioleaching. Acidithiobacillus ferrooxidans were main leaching bacteria according to previous studies (Yin et al., 2019). The higher proportion of Acidithiobacillus ferrooxidans were, the better copper recovery was obtained in this study. Such result indicated the important role of Acidithiobacillus ferrooxidans on the lowgrade copper sulfide ore bioleaching. Table 3 showed clearly that the proportion of Sphingomonas leidyi increased distinctly after adding seawater, which was widespread present in the ocean. The proportion of Sphingomonas leidyi was only 1.48% in Sample TA-0 at day 7. In contrast, the proportion of Sphingomonas leidyi in Samples TA-1 to TA-4 was up to 3.47%, 3.69%, 4.47% and 4.98%, respectively. Contrary to a decline in Sphingomonas leidyi proportion in Sample TA-0 (from 1.48% to 0.95%), the proportion of Sphingomonas leidyi in Samples TA-1 to TA-4 increased a lot, up to 4.43%, 4.83%, 5.69% and 5.76% after a 14-day bioleaching. It was then concluded that the increasing proportion of Sphingomonas leidyi was as
on bacteria was discussed as follows. Fig. 1(e) showed clearly that the bacteria concentration during bioleaching decreased firstly, then increased rapidly, and stayed stable in fluctuation at last. The decrease in bacteria concentration of Samples TA-0 to TA-4 at first was because it took some time for bacteria to adapt to the new environment (Yin et al., 2019). Due to the fact that the oxidation process of Fe2+ to Fe3+ could provide energy resource for bacteria, the bacteria concentration increased rapidly subsequently with the continuous release of Fe2+ from low-grade copper sulfide ores. However, with continuous consumption of nutrients and environment deterioration, the bacteria concentration tended to stay stable at last. As was shown in Fig. 1(e), the maximum bacteria concentration of Samples TA-0 to TA-4 was 8.53 × 107 cells/mL, 8.59 × 107 cells/mL, 8.11 × 107 cells/mL, 6.48 × 107 cells/mL and 5.12 × 107 cells/mL, respectively. Compared to Sample TA-0 with no seawater was added, the peak bacteria concentration of Samples TA-2 to TA-4 in the presence of a relative high proportion of seawater decreased by 0.42 × 107 cells/mL, 2.05 × 107 cells/mL and 3.41 × 107 cells/mL, separately. However, the maximum bacteria concentration of Sample TA-1 in the presence of low-proportion seawater reached 8.59 × 107 cells/mL, only increased by 0.06 × 107 cells/mL in comparison with that of Sample TA-0. Thus it could be concluded that adding high proportion of seawater had significantly negative impact on bacteria concentration in this study. 3.2.2. Effect of seawater on bacteria community succession According to previous studies (Yin et al., 2019) that bacteria played an important role during bioleaching process. And by comparing Sample TA-0 without seawater was added with Samples TA-1 to TA-4 in the presence of seawater, it was found that high proportion of seawater hindered bacteria growth (mentioned in previous Section 3.2.1). In view of the significant role of bacteria during bioleaching, further analysis was applied. Bacteria inside Samples TA-0 to TA-4 were detected by 16S rDNA (Wang et al., 2018). And the results were shown in Fig. 2(a)–(c). In this work, each OTU (Operational Taxonomic Units) corresponded to a 16S rDNA sequence. That is to say, each OTU represented a bacterial species. Fig. 2(a) revealed that 753 OTUs were identified in total of the whole five bioleaching samples. And the amount of OTU inside Samples TA-0 to TA-4 was 552 OTUs, 531 OTUs, 488 OTUs, 324 OTUs and 290 OTUs, respectively. Among all the OTUs, 138 OTUs were shared by the five samples. Sample TA-0 owned the most unique OTU, up to 151. Compared with Sample TA-0, unique OTU of Samples TA-1 to TA-4 decreased obviously, only at 74 OTUs, 48 OTUs, 34 OTUs and 19 OTUs, separately. Variation of OTU inside each sample showed a great difference. The number of OTU inside Samples TA-1 to TA-4 in the presence of seawater decreased dramatically compared to Sample TA-0 without seawater was added. And in the bioleaching system contained seawater, with the increasing proportion of seawater, the number of OTU decreased remarkably (Samples TA-1 to TA-4). Fig. 2(b) indicated the top 35 OTUs of the five samples to show a specific difference among them. The abundance of OTU 01 to OTU 03, OTU 05 to OTU 07 and OTU 12 were much higher inside Sample TA-0 in the absence of seawater. While in the bioleaching system containing seawater, OTU 01 and OTU 03 to OTU 05 played the dominant role inside Sample TA-1. The most abundant OTU inside Sample TA-2 were OTU 01, OTU 03, OTU 32 and OTU 34. OTU 01 and OTU 19 showed an important role inside Sample TA-3. However, OTU 15 to OTU 17 and OTU 19 took a significant proportion inside Sample TA-4. The differences among Samples TA-0 to TA-4 indicated that the distribution of OTU changed a lot after adding seawater. Namely, the bacterial species inside the bioleaching system changed greatly, and the community structures were successfully differentiated by adding seawater. Based on 16S rDNA test results, the heatmap of beta diversity index of the bacteria was showed in Fig. 2(c), which measured the similarities and the differences among the five samples by giving specific numerical 5
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Fig. 2. Results of bacteria detection by 16S rDNA. (a) VENN map of OTU distribution. (b) Heatmap analysis of top 35 OTUs. (c) Heatmap analysis of beta diversity index.
solution (Table 2). The proportion of Lactobacillus acetotolerans of Samples TA-0 to TA-4 all increased distinctly, thus it could be concluded that Lactobacillus acetotolerans showed little influence on this low-grade copper sulfide ore bioleaching.
Table 2 Dissolved oxygen content in leaching solution. Experimental samples
Dissolved oxygen (mg/L) Day 1
TA-0 TA-1 TA-2 TA-3 TA-4
8.03 7.14 6.26 5.32 4.38
± ± ± ± ±
Day 7 0.21 0.14 0.11 0.12 0.09
6.38 5.83 5.01 4.17 3.42
± ± ± ± ±
Day 14 0.14 0.10 0.16 0.06 0.12
5.46 5.02 3.84 3.29 2.71
± ± ± ± ±
3.3. Effect of seawater on Free bacteria and Attached bacteria
0.16 0.13 0.14 0.07 0.06
Along with the optimum proportion of seawater has been verified (20.00%), experiments aimed at exploring the relationship between seawater and bacteria (Free bacteria and Attached bacteria) were conducted further. The detailed experiment schemes were shown in Table 1. Fig. 1(a) indicated clearly that the maximum Cu2+ concentration of Samples TB-0 to TB-5 were 90.70 mg/L, 554.40 mg/L, 329.50 mg/L, 329.10 mg/L, 339.30 mg/L and 523.60 mg/L, respectively. Compared to a peak Cu2+ concentration (90.70 mg/L) of Sample TB-0 in the absence of bacteria, that of Samples TB-1 to TB-5 in the presence of bacteria showed a great increase, in which the peak Cu2+ concentration ranged from 329.10 mg/L to 554.40 mg/L. In addition, the maximum Cu2+ concentration of Samples TB-2 to TB-4 were 329.50 mg/L, 329.10 mg/L and 339.30 mg/L, separately. An increase of 238.80 mg/L, 238.40 mg/L and 248.60 mg/L showed in comparison with that of Sample TB-0. The results in this study verified that bacteria (including Free bacteria and Attached bacteria) played a vital role in bioleaching. In attached-bacteria-removed system after a 9-day bioleaching, Cu2+ concentration of Sample TB-2 decreased by 239.10 mg/L compared to normal system (Fig. 1(a)). That of Sample TB-4 in free-bacteria-removed system inclined by 187.20 mg/L compared to normal
a result of adding seawater. And the possible reason for such significant difference was believed as follows that adding seawater could provide a more suitable environment for Sphingomonas leidyi to grow. A high proportion of Sphingomonas leidyi was found in both Sample TA-1 with the highest copper recovery and Sample TA-4 with the lowest copper recovery, however. That is to say, no evidence showed Sphingomonas leidyi could promote this low-grade copper sulfide ore bioleaching. The proportion of Lactobacillus acetotolerans in Samples TA-0 to TA-4 at day 7 was 0.14%, 0.89%, 0.96%, 1.06% and 1.83%, separately. And that in Samples TA-0 to TA-4 at day 14 was 0.26%, 0.97%, 1.27%, 1.75% and 2.67%, respectively. The proportion of Lactobacillus acetotolerans in each sample increased with the increasing proportion of seawater, which illustrated an improvement of seawater on Lactobacillus acetotolerans. The possible reason was as follows, Lactobacillus acetotolerans was a bacterium that needed a small amount of air or no air to grow, and adding seawater reduced the dissolved oxygen (D.O.) of leaching 6
Bioresource Technology xxx (xxxx) xxxx 16.82 ± 0.08 1.27 ± 0.02 5.76 ± 0.14 0.14 ± 0.05 0.94 ± 0.02 2.67 ± 0.10 3.46 ± 0.15 1.86 ± 0.06 17.39 ± 0.13 2.36 ± 0.04 4.98 ± 0.01 0.67 ± 0.03 0.79 ± 0.01 1.83 ± 0.03 2.17 ± 0.11 1.52 ± 0.01 23.65 ± 0.15 0.58 ± 0.01 5.69 ± 0.04 0.69 ± 0.02 0.02 ± 0.00 1.75 ± 0.02 0.93 ± 0.01 0.22 ± 0.01 18.43 ± 0.27 1.96 ± 0.01 4.47 ± 0.00 0.84 ± 0.02 1.39 ± 0.02 1.06 ± 0.05 1.85 ± 0.04 2.68 ± 0.11 27.74 ± 1.61 1.63 ± 0.12 4.83 ± 0.01 2.47 ± 0.07 1.74 ± 0.03 1.27 ± 0.03 0.17 ± 0.00 1.84 ± 0.07 21.25 ± 0.83 1.26 ± 0.02 3.69 ± 0.03 2.18 ± 0.03 1.18 ± 0.02 0.96 ± 0.01 0.21 ± 0.01 0.37 ± 0.03 27.18 ± 2.13 1.79 ± 0.06 3.47 ± 0.13 2.31 ± 0.04 2.51 ± 0.12 0.89 ± 0.01 2.52 ± 0.16 2.41 ± 0.11 25.87 ± 1.69 3.68 ± 0.03 0.95 ± 0.05 1.83 ± 0.07 2.57 ± 0.31 0.26 ± 0.03 2.91 ± 0.12 1.46 ± 0.13
32.38 ± 1.69 2.03 ± 0.05 4.43 ± 0.02 3.04 ± 0.01 1.93 ± 0.13 0.97 ± 0.00 2.69 ± 0.04 3.18 ± 0.62
Day 7 Day 7 Day 7 Day 7 Day 14
TA-1
system, however. There was a more significant decline in Cu2+ concentration after removing Attached bacteria compared to removing Free bacteria, which indicated that Attached bacteria played a more vital role in the first 9-day bioleaching. It was said that the ore surface was initially adsorbed by Attached bacteria and then EPS (Extracellular Polymeric Substances) was produced to concentrate Fe3+ to attack copper sulfide ores (Feng et al., 2016). The Fe2+ was gradually produced along with the dissolution of Cu2+, and Fe2+ concentration in free-bacteria-removed system was also higher than that in attachedbacteria-removed system (Fig. 1(f)). The results in this study showed accordance with previous studies (Bevilaqua et al., 2013). Meanwhile, the Cu2+ concentration of Sample TB-0 between day 9 and day 15 in normal system was 35.20 mg/L. And that of Sample TB-2 in attachedbacteria-removed system was 49.40 mg/L. The value was only 7.30 mg/ L in free-bacteria-removed system (Fig. 1(a)). The results showed an important effect of Free bacteria on bioleaching from day 9 to day 15. According to previous researches that the passivation layer formed by hydrolysis of Fe3+ (Eqs. (5)–(9)) on the ore surface would inhibit the Attached bacteria to adsorp ore for releasing copper (Yin et al., 2019). At the middle-end bioleaching, pH value dropped heavily (contributed by Fe3+ hydrolysis). And some component like jarosite was also tested by XRD from residue low-grade copper sulfide ores after bioleaching experiments in this study. As was shown in Fig. 1(a), Samples TB-3 and TB-5 respectively yielded a maximum Cu2+ concentration of 329.10 mg/L and 523.60 mg/L from low-grade copper sulfide ores after a half-month bioleaching process. The Cu2+ concentration of Samples TB-3 and TB-5 after a 9-day bioleaching was 266.60 mg/L and 509.70 mg/L, respectively. And the Cu2+ concentration of Samples TB-3 and TB-5 between day 9 and day 15 was 62.50 mg/L and 13.90 mg/L, separately. Compared to the first 9-day Cu2+ concentration of Sample TB-2 (280.10 mg/L), that of Sample TB-3 (266.60 mg/L) showed a little drop only 13.50 mg/L. And difference between the first 9-day Cu2+ concentration of Sample TB-5 (509.70 mg/L) and Sample TB-1 (519.20 mg/L) was small. In summary, seawater has no significant effect on Attached bacteria and Free bacteria in the first 9-day bioleaching in this study. However, Cu2+ concentration between day 9 and day 15 of Sample TB-3 (62.50 mg/L) increased a lot in comparison with that in Sample TB-1 (35.20 mg/L). And Cu2+ concentration between day 9 and day 15 in Sample TB-5 (13.90 mg/L) increased by 6.60 mg/L only compared to that of Sample TB-4 (7.30 mg/L). The result distinctly demonstrated that the effect of seawater on enhanced copper recovery from low-grade copper sulfide ores was mainly realized by Attached bacteria during middle-final stage bioleaching. And the possible reason was as follows. With the bioleaching proceeding, Fe3+ inside leaching solution hydrolyzed to form precipitation and covered the ore surface, which hindered the contact between Attached bacteria and ore and then impeded bioleaching. It seemed that the retardation of precipitation released after adding seawater in this study.
18.62 ± 0.94 2.62 ± 0.21 1.48 ± 0.03 1.96 ± 0.01 1.69 ± 0.00 0.14 ± 0.00 1.14 ± 0.03 0.32 ± 0.04
According to the preliminary experiments above, analytical studies (e.g., XRD test) were applied to understand the enhanced copper dissolution phenomenon better. The effects of different proportion of seawater on the copper releasing during the low-grade copper sulfide ore bioleaching were analyzed after analytical tests.
Acidithiobacillus ferrooxidans Delftia tsuruhatensis Sphingomonas leidyi Leptospirillum ferriphilum Brevundimonas vesicularis Lactobacillus acetotolerans Ralstonia insidiosa Moraxella osloensis
Day 7
TA-0
Proportion (%)
3.4. Analytical insights into effect of seawater on bioleaching
Species
Table 3 Proportion of different species in leaching solution tested by 16S rDNA.
Day 14
TA-2
Day 14
TA-3
Day 14
TA-4
Day 14
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3.4.1. Detection of dissolved oxygen There was an abundant consumption of the oxygen resulted from bacteria and bioleaching reactions, the D.O. in each experiment sample declined distinctly finally compared to that at first, which could be seen from Table 2. And D.O. in leaching solution decreased in varying degrees in the presence of seawater at the beginning, especially Sample TA-4. Sample TA-0 in the absence of bacteria and seawater kept the highest D.O., however. The D.O. in Samples TA-0 to TA-4 at first were 7
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3.4.3. Bacteria separation analysis In this study, no additional compounds except seawater were added into the leaching solution of each sample before the bioleaching experiments. Surprisingly, a little peptidoglycan was found in Sample TA1 only in comparison with Sample TA-0 and Samples TA-2 to TA-4. It was believed that the appearance of peptidoglycan in Sample TA-1 was the result of metallization of bacteria on ores or cell metabolite from other microorganisms. At the same time, quite small yellow aggregations were found clearly in Sample TA-1, and XRD test results showed that main phases of the aggregations were KFe3(SO4)2(OH)6, (NH)4Fe3(SO4)2(OH)6 and peptidoglycan. And no quite small yellow aggregations could be found by eyes in Sample TA-0 and Samples TA-2 to TA-4, however. In order to obtain bacteria, glass beads were added into the plastic tube and mixed with leaching solution fully. Bacteria inside leaching solution were subsequently separated by centrifuge separation (TG16WS, Xiangli, China). Leaching solution containing bacteria was centrifuged at 600×g for ten minutes for separating supernatant and mineral. Then the bacteria were extracted from supernatant. The test results showed that major components of bacteria were peptidoglycan, phospholipid, lipoprotein and glycoprotein mainly. In comparison with the XRD test results of small yellow aggregations in Sample TA-1, the same peptidoglycan was found. In the light of the facts that there was no additional compounds except seawater were added into the leaching solution, thus the possible reasons of seawater improving bioleaching were as follows. According to previous Section 3.2, bacteria concentration and community succession were influenced greatly during bioleaching in the presence of appropriate proportion of seawater. Bacteria may die due to the concentration difference between inside and outside the cell wall after adding seawater, and then suspended in leaching solution after death. Formation of precipitation like KFe3(SO4)2(OH)6 and (NH)4Fe3(SO4)2(OH)6 (Eqs. (5)–(9)) could attach to the dead bacteria rather than ore surface or living bacteria. Meanwhile, hydrolysis of Fe3+ occurred at any location in the leaching solution. Suspending of dead bacteria in leaching solution made a better covering place for precipitation than ores. Thus, the obstructive effect of precipitation on ores and bacteria was reduced.
8.03 mg/L, 7.14 mg/L, 6.26 mg/L, 5.32 mg/L and 4.38 mg/L, respectively. Due to heating process of seawater before bioleaching experiment reduced the D.O. in seawater, Samples TA-1 to TA-4 in the presence of seawater showed a lower D.O. at the beginning. The D.O. in each sample after a half-month bioleaching was 5.46 mg/L, 5.02 mg/L, 3.84 mg/L, 3.29 mg/L and 2.71 mg/L, separately. The D.O. declined obviously because of reactions showed in Eqs. (2)–(5). Therefore, an extreme low D.O. of 3.29 mg/L and 2.71 mg/L showed in Samples TA-3 and TA-4, which was in response to the relatively low bacteria concentration and copper recovery. CuFeS2 + 4Fe3+ + 2H2O + 3O2 → Cu2+ + 5Fe2+ + 4H+ + 2SO42− (2) CuS + 4Fe3+ + 2H2O + O2 → Cu2+ + 4Fe2+ + 4H+ + SO42− (3) Cu2S + 6Fe3+ + 2H2O + O2 → 2Cu2+ + 6Fe2+ + 4H+ + SO42−(4)
3.4.2. XRD analysis The XRD analysis of residue ores in Samples TA-0 to TA-4 were conducted respectively after bioleaching experiments, which showed clearly that silica (SiO2) was the major mineral phase in those samples. XRD analysis results of residue ores in Samples TA-0 to TA-4 after bioleaching showed great variations in mineral phases whether in the presence of or in the absence of seawater. Four main phases were detected by XRD analysis in Sample TA-0 in the absence of seawater: SiO2, (NH)4Fe3(SO4)2(OH)6, 4Cu2S·CuS and Cu2S. In bioleaching system in the presence of seawater, there were four phases (SiO2, (NH)4Fe3(SO4)2(OH)6, peptidoglycan and 4Cu2S·CuS) in Sample TA-1 mainly. And SiO2, (NH)4Fe3(SO4)2(OH)6, 4Cu2S·CuS and Cu2S owned the most proportion in Sample TA-2. However, the same phases including SiO2, KFe3(SO4)2(OH)6, (NH)4Fe3(SO4)2(OH)6, 4Cu2S·CuS and Cu2S were found significantly in Samples TA-3 and TA4. It could be identified clearly that the same phases including SiO2, (NH)4Fe3(SO4)2(OH)6 and 4Cu2S·CuS were obtained in all experimental samples. There was no KFe3(SO4)2(OH)6 was found in Samples TA-0 and TA-2, where no or low proportion of seawater were added. While in Samples TA-3 and TA-4 treated with high proportion of seawater, a relatively high proportion of KFe3(SO4)2(OH)6 were noticed, which may cause a great obstacle to bioleaching according to previous studies (Bevilaqua et al., 2013). The results in Fig. 1(a) showed that Cu2+ concentration of Samples TA-3 and TA-4 in the presence of high proportion of seawater was less than that of Samples TA-0 to TA-2. It was therefore clear that significant effect of seawater on bioleaching might be related to precipitation like KFe3(SO4)2(OH)6. And the possible reasons of the effects of seawater on bioleaching were as follows. 4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O
(5)
H2SO3 + 2Fe3+ + H2O → 2Fe2+ + 4H+ + SO42−
(6)
[Fe(H2O)6]3+ + H2O → [FeOH(H2O)5]2+ + H3O+ 2+
[FeOH(H2O)5] 3+
+ H2O → [Fe(OH)2(H2O)4]
+
+
+ 2[Fe(OH)2(H2O)4] [Fe(H2O)6] PFe3(SO4)2(OH)6↓ + 2H3O+ + 10H2O
+
4. Conclusions The data in this study represented the firstly detailed investigation of the response of bacterial community to seawater in low-grade copper sulfide ore bioleaching. High copper recovery (84.70%) was obtained after adding 20.00% seawater. Seawater had significant effects on bacterial community structure and little difference showed between blank sample (no seawater) and sample adding 20.00% seawater. Bacterial richness decreased with increasing proportion of seawater. Low proportion of seawater was beneficial to copper extraction and dominant bioleaching bacteria (Acidithiobacillus ferrooxidans). Such study should provide a good reference for low-grade copper sulfide ore bioleaching and better application of seawater.
(7) +
+ H3O
2SO42−
(8) +
+
P
→ (9)
CRediT authorship contribution statement Wei Chen: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing review & editing, Supervision, Project administration, Funding acquisition. Shenghua Yin: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Aixiang Wu: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Leiming Wang: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original
A large amount of Fe2+ inside leaching solution of each sample was oxidized to Fe3+ under acid condition. High Fe3+ concentrations promoted the hydrolysis reactions between H2O and Fe3+, and the reaction processes were shown in Eqs. (5)–(9). The following ions including Ca2+, Mg2+, SO42−, CO32–, Na+, Cl− and K+ were contained in seawater mainly (Suyantara et al., 2018). However, K+ and SO42− were both significant components of precipitation indicated in Eq. (9). Based on the results showed in previous Section 3.3.1, adding high proportion of seawater accelerated the formation of precipitation like KFe3(SO4)2(OH)6, which thus decreased bacteria activity. 8
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draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Xun Chen: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.
185–197. Hu, Y., Qiu, G., Wang, Jun, Wang, D., 2002. The effect of silver-bearing catalysts on bioleaching of chalcopyrite. Hydrometallurgy 64, 81–88. Hong, Y., Valix, M., 2014. Bioleaching of electronic waste using acidophilic sulfur oxidising bacteria. J. Cleaner Prod. 65, 465–472. Jia, Y., Sun, H.Y., Tan, Q.Y., Gao, H.S., Feng, X.L., Ruan, R.M., 2018. Linking leach chemistry and microbiology of low-grade copper ore bioleaching at different temperatures. Int. J. Miner. Metall. Mater. 25 (3), 271–279. Johnson, D.B., Okibe, N., Wakeman, K., Yajie, L., 2008. Effect of temperature on the bioleaching of chalcopyrite concentrates containing different concentrations of silver. Hydrometallurgy 94 (1–4), 42–47. Klauber, C., 2008. A critical review of the surface chemistry of acidic ferric sulphate dissolution of chalcopyrite with regards to hindered dissolution. Int. J. Miner. Process. 86 (1–4), 1–17. Kumar, R., Mishra, A., Jha, B., 2019. Bacterial community structure and functional diversity in subsurface seawater from the western coastal ecosystem of the Arabian Sea, India. Gene 701, 55–64. Liang, C.L., Xia, J.L., Nie, Z.Y., Yang, Y., Ma, C.Y., 2012. Effect of seawater on sulfur speciation of chalcopyrite bioleached by the extreme thermophile Acidianus manzaensis. Bioresour. Technol. 110, 462–467. Lao, Q., Su, Q., Liu, G., Shen, Y., Chen, F., Lei, X., Qing, S., Wei, C., Zhang, C., Gao, J., 2019. Spatial distribution of and historical changes in heavy metals in the surface seawater and sediments of the Beibu Gulf, China. Mar. Pollut. Bull. 146, 427–434. Li, P., Wang, J., Wang, Y., Liang, J., He, B., Pan, D., Fan, Q., Wang, X., 2019. Photoconversion of U(VI) by TiO2: an efficient strategy for seawater uranium extraction. Chem. Eng. J. 365, 231–241. Liu, W.J., Jiang, H., Yu, H.Q., 2019. Emerging applications of biochar-based materials for energy storage and conversion. Energy Environ. Sci. 12, 1751–1779. Luo, W., Xiao, G., Tian, F., Richardson, J.J., Wang, Y., Zhou, J., Guo, J., Liao, X., Shi, B., 2019. Engineering robust metal-phenolic network membranes for uranium extraction from seawater. Energy Environ. Sci. 12, 607–614. Muñoz, J.A., Dreisinger, D.B., Cooper, W.C., Young, S.K., 2007. Silver-catalyzed bioleaching of low-grade copper ores Part I: shake flasks tests. Hydrometallurgy 88, 3–18. Owusu, C., Brito e Abreu, S., Skinner, W., Addai-Mensah, J., Zanin, M., 2014. The influence of pyrite content on the flotation of chalcopyrite/pyrite mixtures. Miner. Eng. 55, 87–95. Panda, S., Akcil, A., Pradhan, N., Deveci, H., 2015a. Current scenario of chalcopyrite bioleaching: a review on the recent advances to its heap-leach technology. Bioresour. Technol. 196, 694–706. Panda, S., Biswal, A., Mishra, S., Panda, P.K., Pradhan, N., Mohapatra, U., Sukla, L.B., Mishra, B.K., Akcil, A., 2015b. Reductive dissolution by waste newspaper for enhanced meso-acidophilic bioleaching of copper from low grade chalcopyrite: a new concept of biohydrometallurgy. Hydrometallurgy 153, 98–105. Petersen, J., 2016. Heap leaching as a key technology for recovery of values from lowgrade ores-A brief overview. Hydrometallurgy 165, 206–212. Suyantara, G.P.W., Hirajima, T., Miki, H., Sasaki, K., 2018. Floatability of molybdenite and chalcopyrite in artificial seawater. Miner. Eng. 115, 117–130. Vilcáez, J., Yamada, R., Inoue, C., 2009. Effect of ph reduction and ferric ion addition on the leaching of chalcopyrite at thermophilic temperatures. Hydrometallurgy 96 (1–2), 62–71. Wang, Y., Li, K., Chen, X., Zhou, H., 2018. Responses of microbial community to pH stress in bioleaching of low grade copper sulfide. Bioresour. Technol. 249, 146–153. Xia, J., Song, J., Liu, H., Nie, Z., Shen, L., Yuan, P., Ma, C., Zheng, L., Zhao, Y., 2018. Study on catalytic mechanism of silver ions in bioleaching of chalcopyrite by SR-XRD and XANES. Hydrometallurgy 180, 26–35. Yang, S., Han, Z., Pan, X., Liu, B., Zheng, D., 2018a. Nitrogen oxide removal from simulated flue gas by UV-irradiated electrolyzed seawater: efficiency optimization and pH-dependent mechanisms. Chem. Eng. J. 354, 653–662. Yang, S., Zhang, F., Ding, H., He, P., Zhou, H., 2018b. Lithium metal extraction from seawater. Joule 2 (9), 1648–1651. Yin, S., Chen, W., Chen, X., Wang, L., 2019. Bacterial-mediated recovery of copper from low-grade copper sulfide using acid-processed rice straw. Bioresour. Technol. https:// doi.org/10.1016/j.biortech.2019.121605.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Science Foundation for Excellent Young Scholars of China (51722401), the Key Project of National Natural Science Foundation of China (51734001) and the Fundamental Research Funds for the Central Universities (FRF-TP-18003C1). References Atzori, G., Mancuso, S., Masi, E., 2019. Seawater potential use in soilless culture: a review. Sci. Hortic. 249, 199–207. Bevilaqua, D., Lahti, H., Suegama, Patrícia H., Garcia, O., Benedetti, A.V., Puhakka, J.A., Tuovinen, O.H., 2013. Effect of na-chloride on the bioleaching of a chalcopyrite concentrate in shake flasks and stirred tank bioreactors. Hydrometallurgy 138 (113), 1–13. Brierley, C., Brierley, J., 2013. Progress in bioleaching-part B: applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 97, 7543–7552. Chen, L., Tsui, M.M.P., Lam, J.C.W., Hu, C., Wang, Q., Zhou, B., Lam, P.K.S., 2019. Variation in microbial community structure in surface seawater from pearl river delta: discerning the influencing factors. Sci. Total Environ. 660, 136–144. Davis-Belmar, C.S., Cautivo, D., Demergasso, C., Rautenbach, G., 2014. Bioleaching of copper secondary sulfide ore in the presence of chloride by means of inoculation with chloride-tolerant microbial culture. Hydrometallurgy 150, 308–312. Fagan, M.A., Ngoma, I.E., Chiume, R.A., Minnaar, S., Sederman, A.J., Johns, M.L., Harrison, S.T.L., 2014. MRI and gravimetric studies of hydrology in drip irrigated heaps and its effect on the propagation of bioleaching micro-organisms. Hydrometallurgy 150, 210–221. Feng, S., Yang, H., Zhan, X., Wang, W., 2014. Novel integration strategy for enhancing chalcopyrite bioleaching by acidithiobacillus sp. in a 7-L fermenter. Bioresour. Technol. 161, 371–378. Feng, S., Yang, H., Wang, W., 2016. Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages. Bioresour. Technol. 200, 186–193. Giuseppe, G., Kazumasa, T., Tatsuya, K., Chiharu, T., 2019. Mechanochemical activation of chalcopyrite: relationship between activation mechanism and leaching enhancement. Miner. Eng. 131, 280–285. Gong, Y., Ma, F., Xue, Y., Jiao, C., Yang, Z., 2019. Failure analysis on leaked titanium tubes of seawater heat exchangers in recirculating cooling water system of coastal nuclear power plant. Eng. Fail. Anal. 101, 172–179. Hao, X.D., Liang, Y.L., Yin, H.Q., Liu, H.W., Zeng, W.M., Liu, X.D., 2017. Thin-layer heap bioleaching of copper flotation tailings containing high levels of fine grains and microbial community succession analysis. Int. J. Miner. Metall. Mater. 24 (4), 360–368. https://doi.org/10.1007/s12613-017-1415-4. Hiroyoshi, N., Miki, H., Hirajima, T., Tsunekawa, M., 2001. Enhancement of chalcopyrite leaching by ferrous ions in acidic ferric sulfate solutions. Hydrometallurgy 60 (3),
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Bioleaching of copper sulfides using mixed microorganisms and its community structure succession in the presence of seawater ⁎
Wei Chena,b, Shenghua Yina,b, , Aixiang Wua,b, Leiming Wangb, Xun Chenb a b
Key Laboratory of Ministry of Education for High-Efficient Mining and Safety of Metal, University of Science and Technology Beijing, Beijing 100083, China School of Civil and Resource Engineering, University of Science and Technology Beijing, Beijing 100083, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Seawater Low-grade copper sulfide ore Bioleaching Bacteria diversity and dynamics Attached bacteria
The bacterial diversity and dynamics in the leaching solution were analyzed during bioleaching of low-grade copper sulfide ore in the presence of seawater in this study. The results indicated a promoting response of appropriate-proportion seawater to bioleaching with improved copper recoveries. A maximum of 84.70% copper recovery was obtained in the presence of 20.00% seawater in contrast to only 72.49% in its absence. The experiments verified that seawater owned a great influence on Attached bacteria and bacterial species. 16S rDNA analysis illustrated that bacterial species decreased distinctly in the presence of seawater. Little difference between blank sample (no seawater) and sample adding 20.00% seawater was indicated by beta diversity index. Bacteria (including Acidithiobacillus ferrooxidans, Sphingomonas leidyi and Lactobacillus acetotolerans) were influenced significantly after adding seawater. Acidithiobacillus ferrooxidans accounted for the highest proportion of the community whether seawater was added or not during bioleaching.
1. Introduction There has been a continuous decline in mined ore grades and easyto-recovery metal resources throughout the world over the past few decades, and thus advisable exploitation of low-grade reserves to meet the ever increasing metal demands is extensively carried out (Davis-
Belmar et al., 2014; Liu et al., 2019; Jia et al., 2018). Chalcopyrite is a kind of sulfide ores bearing copper, which hold the most abundant copper mineral in the world (Giuseppe et al., 2019; Owusu et al., 2014). While compared to copper oxide ores and other copper minerals, copper sulfide ores own characteristics of refractory and recalcitrance (Panda et al., 2015a). Bioleaching is a hydrometallurgical technique
⁎ Corresponding author at: Key Laboratory of Ministry of Education for High-Efficient Mining and Safety of Metal, University of Science and Technology Beijing, Beijing 100083, China. E-mail address: [email protected] (S. Yin).
https://doi.org/10.1016/j.biortech.2019.122453 Received 15 October 2019; Received in revised form 17 November 2019; Accepted 18 November 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Wei Chen, et al., Bioresource Technology, https://doi.org/10.1016/j.biortech.2019.122453
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analysis result showed that the Cu grade was at 0.75%, including 0.04% free copper oxide, 0.06% primary copper sulfide, 0.64% secondary copper sulfide and 0.01% combined copper oxide. The mineralogical analysis of the ore sample indicated that silica (SiO2) was the major mineral phase, which was up to 91.00%. And other main compositions of low-grade copper sulfide ores were Cu − 0.75%, Fe − 1.62%, S − 1.10%, CaO − 0.30%, MgO − 0.04% and Al2O3 − 5.19%.
that is suitable to process these low-grade ores compared to other conventional mineral processing techniques (Fagan et al., 2014; Hong and Valix, 2014; Petersen, 2016), and it has been used in industrial production over past few years (Brierley and Brierley, 2013; Xia et al., 2018). Although bioleaching is an effective promisingly method for extracting refractory copper sulfide ores with advantages of environment friendly and perfect economic benefits, problems including low leaching rate, long bioleaching routine and low efficiency still need to be finished off (Bevilaqua et al., 2013; Klauber, 2008). To solve the problems mentioned above, many efforts have been conducted to improve copper recovery by adding certain additives (Johnson et al., 2008; Muñoz et al., 2007). Liang et al. (2012) investigated the effect of activated carbon on the refractory copper sulfide ores bioleaching and found that the bioleaching efficiency was enhanced in the presence of activated carbon. The effects of metal ions like Ag+ as catalysts on copper extraction during bioleaching were addressed by Xia et al. (2018) and Hu et al. (2002). And two different mostly accepted catalytic reactions by Ag+ during bioleaching of refractory copper sulfide ores were presented. Besides, recent studies on bioleaching of mostly concentrates have shown promising results of metal recovery using cellulose. Yin et al. (2019) investigated the effect of cellulose (rice straw) on the copper recovery during copper sulfide ores bioleaching. In addition, the effect of cellulose (waste newspapers) was presented by Panda et al. (2015b) as well. There are abundant resources in ocean with an average of 35.70 million tons minerals per cubic kilometer of seawater (Lao et al., 2019). Among the more than 100 elements have been identified in the world, 80.00% can be found in seawater (Atzori et al., 2019). Thus extracting metals from seawater is going to be more and more promising. Based on the significant role of seawater, the application of seawater has been more and more extensive in recent years (Yang et al., 2018a). Many metals full of economical value were extracted from seawater directly, such as uranium and magnesium (Li et al., 2019; Luo et al., 2019). Yang et al. (2018b) studied the lithium metal extraction from seawater, which proposed a new method for the shortage of metal resources. The floatability of molybdenite and chalcopyrite in artificial seawater was explored by Suyantara et al. (2018), which provided a reference for copper extraction using seawater. Gong et al. (2019) analyzed the advantages and disadvantages of seawater as circulating cooling water for coastal nuclear power stations, which provided security for nuclear energy by using seawater. Besides, the bacterial community inside seawater or after adding seawater has been studied, which provided a basis for extracting metal resources from seawater in the presence of bacteria (Chen et al., 2019; Kumar et al., 2019). There is no enough researches showing copper extraction using seawater, thus it is still worth studying a better way to recover copper from seawater. In this study, seawater that acted as a potential promoter during bioleaching of low-grade copper sulfide ores was proposed for the first time. And some significant leachable factors, including Cu2+ concentration, Fe3+ concentration, oxidation-reduction potential (Eh), pH value and bacteria concentration, were monitored. The microbial community structure inside the leaching solution during bioleaching was also analyzed by 16S rDNA (Hao et al., 2017). Thus, the results showed in this paper should provide more promising utilization of seawater during and extraction of copper.
2.2. Pretreatment of seawater Seawater used in this study was originated from Guangdong Province, China. For the sake of avoiding the influence of unidentified bacteria inside seawater on bioleaching, high-temperature heating was applied to seawater prior to low-grade copper sulfide ore bioleaching experiments. The preprocessing (high-temperature heating) was performed at a vertical mode steam sterilizers for 30 min at a temperature of 120 ℃. Then the seawater after heating was placed in a laminar flow as cooling. The seawater obtained after cooling was used during the bioleaching experiments and further analytical studies. The specific compositions of seawater were Cl− − 17.87 g/L, Na+ − 9.58 g/L, Mg2+ − 1.23 g/L, Ca2+ − 0.38 g/L and K+ − 0.35 g/L. 2.3. Bacteria and culture The bacteria were collected from acidic waste mine water of a copper mine, Fujian Province, China. Bacteria have been gone through repeatedly laboratory-scale culture experiments in 9 K medium to enrichment, purification and domestication before bioleaching experiments. The components of 9 K medium were showed in Feng et al. (2014), which contained (NH4)2SO4 − 3.00 g/L, MgSO4·7H2O − 0.50 g/L, KCl − 0.10 g/L, K2HPO4- 0.50 g/L, Ca(NO3)2–0.01 g/L and FeSO4·7H2O − 44.20 g/L. Bacteria were inoculated to flasks in the presence of 9 K media, and pH value was adjusted to 2.00 by dilute H2SO4. Then flasks were placed at a temperature of 30 ℃ and 120 r/min shaker during laboratory-scale culture experiments. Thus the bacteria concentration was up to 9.50 × 107 cells/mL at the beginning of the bioleaching experiments.
2.4. Bioleaching experiments In order to study the effect of seawater on low-grade copper sulfide ore bioleaching, five bioleaching experiments were designed firstly. Bioleaching experiments were performed using 250 mL flasks, and the proportion of seawater to leaching solution was varied in those experiments (0.00%, 20.00%, 40.00%, 60.00% and 80.00%). Table 1 showed the summary of experiment schemes. Due to the ore samples were rich in Fe, which can provide enough energy for the bacteria growth. The culture medium here called 0 K medium (i.e. 9 K media without FeSO4·7H2O) containing (NH4)2SO4 − 3.00 g/L, MgSO4·7H2O − 0.50 g/L, KCl − 0.10 g/L, K2HPO4- 0.50 g/L and Ca(NO3)2 – 0.01 g/ L was employed in the bioleaching experiments in this study. The experiments were carried out in 250 mL flasks under the same initial condition of 20.00 mL bacteria (9.50 × 107 cells/mL) were inoculated, and the pH value of the leaching solution was adjusted to 2.00 by adding dilute H2SO4. Then the flasks were placed at a temperature of 30 ℃ and 120 r/min shaker. Moreover, characteristics including pH value, Cu2+ concentration, bacteria concentration and Oxidation-reduction potential (Eh value) were monitored. Normal system mentioned subsequently was referred to experimental samples that are in the presence of both Attached bacteria and free bacteria. Experimental samples after removing Attached bacteria and free bacteria during bioleaching herein were referred to as Attached-bacteria-removed system and Free-bacteria-removed system throughout the text, respectively. All the experiments in this work were conducted in triplicate.
2. Materials and methods 2.1. Feature of ore samples Low-grade copper sulfide ores applied in this study were collected from a copper Mine, Fujian Province, China. The chemical composition analysis result of the low-grade copper sulfide ores was obtained by selective dissolved experiments through Chinese Academy of Sciences. The mineralogical analysis of the ore sample was initially carried out through Atomic Absorption Spectroscopy. The chemical composition 2
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Table 1 Bioleaching experiment schemes. Experimental samples
Seawater (%/mL)
Ores (g)
Medium
TA-0 TA-1 TA-2 TA-3 TA-4 TB-0 TB-1 TB-2 TB-3 TB-4 TB-5
0.00/0.00 20.00/30.00 40.00/60.00 60.00/90.00 80.00/120.00 20.00/40.00 20.00/40.00 20.00/40.00 20.00/40.00 20.00/40.00 20.00/40.00
20.00 20.00 20.00 20.00 20.00 15.00 15.00 15.00 15.00 15.00 15.00
0 k 130.00 mL + Bacteria 20.00 mL 0 k 100.00 mL + Bacteria 20.00 mL 0 k 70.00 mL + Bacteria 20.00 mL 0 k 40.00 mL + Bacteria 20.00 mL 0 k 10.00 mL + Bacteria 20.00 mL – Attached and Free bacteria Removed Attached bacteria Free bacteria and add Attached bacteria at day 9 Removed Free bacteria Attached bacteria and remove Free bacteria at day 9
of H+. Reactions mentioned above all leaded to an increase in pH value. However, S0 contained in low-grade copper sulfide ores was oxidized into dilute sulfuric acid subsequently, which yielded a large amount of H+. Furthermore, formation of precipitation (formed by hydrolysis of Fe3+) produced H+ as well. Both processes mentioned above contributed to the following decrease in pH value. Although variation of pH value during low-grade copper sulfide ore bioleaching was similar regardless of whether seawater was present or not, the minimum pH value of each sample differed ranging from 2.03 to 2.15 after adding seawater. Fig. 1(b) showed that the pH value of Samples TA-0 to TA-4 decreased significantly with leaching time. The decrease of Samples TA-1 and TA-2 was observed to be more striking. For instance, the pH value of Sample TA-1 decreased by 0.38 (from 2.41 to 2.03), while that of Sample TA-4 only decreased by 0.27 (from 2.42 to 2.15). According to Fig. 1(b), Samples TA-0 to TA-4 reached their maximum pH value at day 5, day 3, day 4, day 5 and day 6, respectively. Compared to Sample TA-0 in the absence of seawater, Samples TA-1 and TA-2 in the presence of seawater reached peak pH value more quickly, which indicated adding appropriate-proportion seawater accelerated the increase of pH value. Meanwhile, lower pH values were found in Samples TA-1 and TA-2, where higher Cu2+ concentrations were produced (Fig. 1(a)). Based on previous study that decrease of initial pH value accelerated the dissolution of chalcopyrite (Vilcáez et al., 2009), the decline in pH value was likely responsible for improving copper extraction of low-grade copper sulfide ores in this study. The peak Fe3+ concentration of Samples TA-0 to TA-4 was 84.70 mg/L, 95.90 mg/L, 87.10 mg/L, 78.80 mg/L and 69.30 mg/L, separately. The peak Fe3+ concentration of Samples TA-1 and TA-2 increased by 11.20 mg/L and 2.40 mg/L, respectively, compared to that of Sample TA-0. Thus, it could be deducted that adding appropriateproportion seawater could promote the release of Fe3+. However, there was relatively low Fe3+ concentration of Samples TA-3 and TA-4 according to Fig. 1(c), especially in Sample TA-4. Previous study (Vilcáez et al., 2009) has found that the oxidation process of Fe2+ by dissolved oxygen in the presence of H+ decreased with increasing pH value. There was high pH value of Samples TA-3 and TA-4 (Fig. 1(b)), which explained why the two samples owned low Fe3+ concentration. Fig. 1(d) showed clearly that the Eh value increased remarkably at the initial stage of bioleaching, and then stayed stable in fluctuation. The increase in the Eh value during bioleaching mainly resulted from the oxidation of Fe2+ to Fe3+ by the dissolved oxygen in the presence of H+ (Hiroyoshi et al., 2001). The maximum Eh value of Samples TA-0 to TA-4 was 587 mV, 589 mV, 569 mV, 563 mV and 546 mV, respectively. Samples TA-0 and TA-1 owned the highest Eh value.
2.5. Analysis In this study, concentrations such as Cu2+, Cl−, Ca2+ were detected by ICP Optical Spectrometer (OPTIMA 7000DV, America). Bacteria concentration was measured by optical microscope (Carl Zeiss Axio Lab A1, Germany). Oxidation-reduction potential (Eh value) and pH value were tested by a pH meter (PHS-3E, Inesa, China). The bacterial diversity and dynamics were clarified by 16S rDNA. Residue ores and aggregations were measured through X-ray Diffractionmeter (Smartlab, RIGAKU, Japan). 3. Results and discussion 3.1. Effect of seawater on low-grade copper sulfide ore bioleaching The variations of key chemical parameters (including pH, Eh, Fe3+ concentration, Fe2+ concentration, bacteria concentration and Cu2+ concentration) during low-grade copper sulfide ore bioleaching in the presence of seawater were shown in Fig. 1(a)–(f), separately. 3.1.1. Effect of seawater on copper recovery The effect of different proportion of seawater on low-grade copper sulfide ore bioleaching for copper recovery was shown in Fig. 1(a). Fig. 1(a) indicated clearly that compared to a peak Cu2+ concentration (543.70 mg/L) of Sample TA-0 without adding seawater, that of Samples TA-1 and TA-2 increased significantly. The peak Cu2+ concentration of Samples TA-1 and TA-2 reached 635.30 mg/L and 576.40 mg/L, respectively. However, Samples TA-3 and TA-4 in the presence of seawater only yielded a maximum Cu2+ concentration of 522.60 mg/L and 501.10 mg/L, separately. There was an apparent lower peak Cu2+ concentration of Samples TA-3 and TA-4, which dropped 21.10 mg/L and 42.60 mg/L in comparison with that of Sample TA-0. The maximum copper recovery of Samples TA-0 to TA-4 was 72.49%, 84.70%, 76.85%, 69.68% and 66.81%, respectively. It could be found that with increasing proportion of seawater, the maximum copper recovery decreased gradually ranging from 84.70% to 66.81%. The maximum (635.30 mg/L) and minimum (501.10 mg/L) Cu2+ concentration in the presence of seawater increased by 16.85% and decreased by 7.84% respectively compared with that of Sample TA-0 (543.70 mg/L) in the absence of seawater, which illustrated that appropriate-proportion seawater could improve bioleaching. 3.1.2. Effect of seawater on key chemical parameters As was shown in Fig. 1(b), the pH value increased at first, and then dropped. The peak pH value of Samples TA-0 to TA-4 was 2.43, 2.41, 2.42, 2.41 and 2.42, respectively. The pH value rose during early stage of low-grade copper sulfide ore bioleaching could be resulted from the consumption of H+. Because there were massive oxides (e.g., MgO, CaO and Al2O3) inside bioleaching system, and they could react with H+. In addition, the processes (e.g., oxidation process of Fe2+ to Fe3+) of extracting Cu2+ from low-grade copper sulfide ores also consumed a lot
E = E0 +
RT [electron acceptor] ln [elector donor] nF
(1)
According to Eq. (1), when the factors (including R, T, F, n and E0) are the same, high electron acceptor proportion and low electron donor proportion during bioleaching can yield a higher Eh value. Since Fe3+ can be reduced to act as electron acceptor. The high Eh value of 3
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Fig. 1. Effect of seawater on the low-grade copper sulfide bioleaching performance. (a) Variation of Cu2+ concentration. (b) Variation of pH value. (c) Variation of Ferric concentration. (d) Variation of Eh value. (e) Variation of bacteria concentration. (f) Variation of Ferrous concentration.
Samples TA-0 and TA-1, especially in Sample TA-1, could be verified by the high Fe3+ concentration inside leaching solution.
3.2. Effect of seawater on bacteria during bioleaching 3.2.1. Effect of seawater on bacteria concentration Previous Section 3.1 indicated a significantly promoting effect with improved copper recovery from low-grade copper sulfide ores in the presence of appropriate-proportion seawater. And the effect of seawater 4
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value. Each value inside Fig. 2(c) represented the difference coefficient between two samples. The smaller the difference coefficient value was, the smaller the difference of species diversity between two samples was. The value between Sample TA-0 in the absence of seawater and Samples TA-1 to TA-4 was 0.540, 0.610, 0.646 and 0.696, separately. It could be concluded that the difference became more notable with the increasing proportion of seawater, and adding high proportion of seawater into bioleaching system caused greater difference than adding low proportion seawater. Especially, the difference coefficient value of low proportion of seawater between Sample TA-0 and Sample TA-1 was small, only 0.540. However, that between Sample TA-0 and Sample TA4 adding high proportion of seawater was up to 0.696. Meanwhile, in the bioleaching system in the presence of seawater, the difference coefficient value between Sample TA-1 and Sample TA-2, Sample TA-3 and Sample TA-4 were both relatively low, only 0.457 and 0.475. While that between Sample TA-1 and Sample TA-4 was up to 0.569. Thus, seawater effected bioleaching a lot, and adding different proportion of seawater exhibited diverse influence on bacteria as well. The dominant OTU (OTU 01, OTU 02, OTU 03, OTU 05, OTU 06, OTU 07 and OTU 12) of Sample TA-0 that detected during bioleaching were classified into seven different genus mainly: Acidithiobacillales, Lactobacillales, Ferroplasma, Stenotrophomonas, Thermoplasmatales, Pseudomonadales and Delftia. And the number of the same dominant OTU of Samples TA-1 to TA-3 in comparison with Sample TA-0 was 3 (OTU 01, OTU 02, OTU 05), 2 (OTU 01, OTU 03) and 1 (OTU 01), respectively. Contrary to Samples TA-1 to TA-3, no same dominant OTU was found between Sample TA-4 and Sample TA-0, however. The results of main OTU in each sample showed good accordance with difference coefficient value between samples (Fig. 2(c)). Table 3 indicated the variations of different species in the leaching solution determined by16S rDNA at day 7 and day 14 during low-grade copper sulfide ore bioleaching after adding different proportion of seawater. Acidithiobacillus ferrooxidans dominated the species at both day 7 and day 14 no matter what the proportion of seawater was added, accounting for more than 16.50% of the total population in the leaching solution. As was shown in Table 3, the proportion of Acidithiobacillus ferrooxidans of Sample TA-0 was 18.62% at day 7. The proportion of Acidithiobacillus ferrooxidans of Samples TA-1 and TA-2 was up to 27.18% and 21.25%, respectively. While that in Samples TA-3 and TA-4 was only 18.43% and 17.39%, which indicated that seawater played a vital role in proportion variation of Acidithiobacillus ferrooxidans. This could be due to adding excessive seawater leaded to a failure of oxygen level in bioleaching system (Table 2). However, Acidithiobacillus ferrooxidans was an aerobic bacterium, and lack of air could hinder its growth. Compared to Acidithiobacillus ferrooxidans proportion of Samples TA-0 to TA-3 at day 7, that at day 14 increased significantly up to 25.87%, 32.38%, 27.74% and 23.65%, respectively. While there was an obvious decreasing tendency showed in Sample TA-4 (from17.39 % to 16.82%). Such finding verified the addition of excessive proportion of seawater seriously hindered the growth of Acidithiobacillus ferrooxidans during this low-grade copper sulfide ore bioleaching. Acidithiobacillus ferrooxidans were main leaching bacteria according to previous studies (Yin et al., 2019). The higher proportion of Acidithiobacillus ferrooxidans were, the better copper recovery was obtained in this study. Such result indicated the important role of Acidithiobacillus ferrooxidans on the lowgrade copper sulfide ore bioleaching. Table 3 showed clearly that the proportion of Sphingomonas leidyi increased distinctly after adding seawater, which was widespread present in the ocean. The proportion of Sphingomonas leidyi was only 1.48% in Sample TA-0 at day 7. In contrast, the proportion of Sphingomonas leidyi in Samples TA-1 to TA-4 was up to 3.47%, 3.69%, 4.47% and 4.98%, respectively. Contrary to a decline in Sphingomonas leidyi proportion in Sample TA-0 (from 1.48% to 0.95%), the proportion of Sphingomonas leidyi in Samples TA-1 to TA-4 increased a lot, up to 4.43%, 4.83%, 5.69% and 5.76% after a 14-day bioleaching. It was then concluded that the increasing proportion of Sphingomonas leidyi was as
on bacteria was discussed as follows. Fig. 1(e) showed clearly that the bacteria concentration during bioleaching decreased firstly, then increased rapidly, and stayed stable in fluctuation at last. The decrease in bacteria concentration of Samples TA-0 to TA-4 at first was because it took some time for bacteria to adapt to the new environment (Yin et al., 2019). Due to the fact that the oxidation process of Fe2+ to Fe3+ could provide energy resource for bacteria, the bacteria concentration increased rapidly subsequently with the continuous release of Fe2+ from low-grade copper sulfide ores. However, with continuous consumption of nutrients and environment deterioration, the bacteria concentration tended to stay stable at last. As was shown in Fig. 1(e), the maximum bacteria concentration of Samples TA-0 to TA-4 was 8.53 × 107 cells/mL, 8.59 × 107 cells/mL, 8.11 × 107 cells/mL, 6.48 × 107 cells/mL and 5.12 × 107 cells/mL, respectively. Compared to Sample TA-0 with no seawater was added, the peak bacteria concentration of Samples TA-2 to TA-4 in the presence of a relative high proportion of seawater decreased by 0.42 × 107 cells/mL, 2.05 × 107 cells/mL and 3.41 × 107 cells/mL, separately. However, the maximum bacteria concentration of Sample TA-1 in the presence of low-proportion seawater reached 8.59 × 107 cells/mL, only increased by 0.06 × 107 cells/mL in comparison with that of Sample TA-0. Thus it could be concluded that adding high proportion of seawater had significantly negative impact on bacteria concentration in this study. 3.2.2. Effect of seawater on bacteria community succession According to previous studies (Yin et al., 2019) that bacteria played an important role during bioleaching process. And by comparing Sample TA-0 without seawater was added with Samples TA-1 to TA-4 in the presence of seawater, it was found that high proportion of seawater hindered bacteria growth (mentioned in previous Section 3.2.1). In view of the significant role of bacteria during bioleaching, further analysis was applied. Bacteria inside Samples TA-0 to TA-4 were detected by 16S rDNA (Wang et al., 2018). And the results were shown in Fig. 2(a)–(c). In this work, each OTU (Operational Taxonomic Units) corresponded to a 16S rDNA sequence. That is to say, each OTU represented a bacterial species. Fig. 2(a) revealed that 753 OTUs were identified in total of the whole five bioleaching samples. And the amount of OTU inside Samples TA-0 to TA-4 was 552 OTUs, 531 OTUs, 488 OTUs, 324 OTUs and 290 OTUs, respectively. Among all the OTUs, 138 OTUs were shared by the five samples. Sample TA-0 owned the most unique OTU, up to 151. Compared with Sample TA-0, unique OTU of Samples TA-1 to TA-4 decreased obviously, only at 74 OTUs, 48 OTUs, 34 OTUs and 19 OTUs, separately. Variation of OTU inside each sample showed a great difference. The number of OTU inside Samples TA-1 to TA-4 in the presence of seawater decreased dramatically compared to Sample TA-0 without seawater was added. And in the bioleaching system contained seawater, with the increasing proportion of seawater, the number of OTU decreased remarkably (Samples TA-1 to TA-4). Fig. 2(b) indicated the top 35 OTUs of the five samples to show a specific difference among them. The abundance of OTU 01 to OTU 03, OTU 05 to OTU 07 and OTU 12 were much higher inside Sample TA-0 in the absence of seawater. While in the bioleaching system containing seawater, OTU 01 and OTU 03 to OTU 05 played the dominant role inside Sample TA-1. The most abundant OTU inside Sample TA-2 were OTU 01, OTU 03, OTU 32 and OTU 34. OTU 01 and OTU 19 showed an important role inside Sample TA-3. However, OTU 15 to OTU 17 and OTU 19 took a significant proportion inside Sample TA-4. The differences among Samples TA-0 to TA-4 indicated that the distribution of OTU changed a lot after adding seawater. Namely, the bacterial species inside the bioleaching system changed greatly, and the community structures were successfully differentiated by adding seawater. Based on 16S rDNA test results, the heatmap of beta diversity index of the bacteria was showed in Fig. 2(c), which measured the similarities and the differences among the five samples by giving specific numerical 5
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Fig. 2. Results of bacteria detection by 16S rDNA. (a) VENN map of OTU distribution. (b) Heatmap analysis of top 35 OTUs. (c) Heatmap analysis of beta diversity index.
solution (Table 2). The proportion of Lactobacillus acetotolerans of Samples TA-0 to TA-4 all increased distinctly, thus it could be concluded that Lactobacillus acetotolerans showed little influence on this low-grade copper sulfide ore bioleaching.
Table 2 Dissolved oxygen content in leaching solution. Experimental samples
Dissolved oxygen (mg/L) Day 1
TA-0 TA-1 TA-2 TA-3 TA-4
8.03 7.14 6.26 5.32 4.38
± ± ± ± ±
Day 7 0.21 0.14 0.11 0.12 0.09
6.38 5.83 5.01 4.17 3.42
± ± ± ± ±
Day 14 0.14 0.10 0.16 0.06 0.12
5.46 5.02 3.84 3.29 2.71
± ± ± ± ±
3.3. Effect of seawater on Free bacteria and Attached bacteria
0.16 0.13 0.14 0.07 0.06
Along with the optimum proportion of seawater has been verified (20.00%), experiments aimed at exploring the relationship between seawater and bacteria (Free bacteria and Attached bacteria) were conducted further. The detailed experiment schemes were shown in Table 1. Fig. 1(a) indicated clearly that the maximum Cu2+ concentration of Samples TB-0 to TB-5 were 90.70 mg/L, 554.40 mg/L, 329.50 mg/L, 329.10 mg/L, 339.30 mg/L and 523.60 mg/L, respectively. Compared to a peak Cu2+ concentration (90.70 mg/L) of Sample TB-0 in the absence of bacteria, that of Samples TB-1 to TB-5 in the presence of bacteria showed a great increase, in which the peak Cu2+ concentration ranged from 329.10 mg/L to 554.40 mg/L. In addition, the maximum Cu2+ concentration of Samples TB-2 to TB-4 were 329.50 mg/L, 329.10 mg/L and 339.30 mg/L, separately. An increase of 238.80 mg/L, 238.40 mg/L and 248.60 mg/L showed in comparison with that of Sample TB-0. The results in this study verified that bacteria (including Free bacteria and Attached bacteria) played a vital role in bioleaching. In attached-bacteria-removed system after a 9-day bioleaching, Cu2+ concentration of Sample TB-2 decreased by 239.10 mg/L compared to normal system (Fig. 1(a)). That of Sample TB-4 in free-bacteria-removed system inclined by 187.20 mg/L compared to normal
a result of adding seawater. And the possible reason for such significant difference was believed as follows that adding seawater could provide a more suitable environment for Sphingomonas leidyi to grow. A high proportion of Sphingomonas leidyi was found in both Sample TA-1 with the highest copper recovery and Sample TA-4 with the lowest copper recovery, however. That is to say, no evidence showed Sphingomonas leidyi could promote this low-grade copper sulfide ore bioleaching. The proportion of Lactobacillus acetotolerans in Samples TA-0 to TA-4 at day 7 was 0.14%, 0.89%, 0.96%, 1.06% and 1.83%, separately. And that in Samples TA-0 to TA-4 at day 14 was 0.26%, 0.97%, 1.27%, 1.75% and 2.67%, respectively. The proportion of Lactobacillus acetotolerans in each sample increased with the increasing proportion of seawater, which illustrated an improvement of seawater on Lactobacillus acetotolerans. The possible reason was as follows, Lactobacillus acetotolerans was a bacterium that needed a small amount of air or no air to grow, and adding seawater reduced the dissolved oxygen (D.O.) of leaching 6
Bioresource Technology xxx (xxxx) xxxx 16.82 ± 0.08 1.27 ± 0.02 5.76 ± 0.14 0.14 ± 0.05 0.94 ± 0.02 2.67 ± 0.10 3.46 ± 0.15 1.86 ± 0.06 17.39 ± 0.13 2.36 ± 0.04 4.98 ± 0.01 0.67 ± 0.03 0.79 ± 0.01 1.83 ± 0.03 2.17 ± 0.11 1.52 ± 0.01 23.65 ± 0.15 0.58 ± 0.01 5.69 ± 0.04 0.69 ± 0.02 0.02 ± 0.00 1.75 ± 0.02 0.93 ± 0.01 0.22 ± 0.01 18.43 ± 0.27 1.96 ± 0.01 4.47 ± 0.00 0.84 ± 0.02 1.39 ± 0.02 1.06 ± 0.05 1.85 ± 0.04 2.68 ± 0.11 27.74 ± 1.61 1.63 ± 0.12 4.83 ± 0.01 2.47 ± 0.07 1.74 ± 0.03 1.27 ± 0.03 0.17 ± 0.00 1.84 ± 0.07 21.25 ± 0.83 1.26 ± 0.02 3.69 ± 0.03 2.18 ± 0.03 1.18 ± 0.02 0.96 ± 0.01 0.21 ± 0.01 0.37 ± 0.03 27.18 ± 2.13 1.79 ± 0.06 3.47 ± 0.13 2.31 ± 0.04 2.51 ± 0.12 0.89 ± 0.01 2.52 ± 0.16 2.41 ± 0.11 25.87 ± 1.69 3.68 ± 0.03 0.95 ± 0.05 1.83 ± 0.07 2.57 ± 0.31 0.26 ± 0.03 2.91 ± 0.12 1.46 ± 0.13
32.38 ± 1.69 2.03 ± 0.05 4.43 ± 0.02 3.04 ± 0.01 1.93 ± 0.13 0.97 ± 0.00 2.69 ± 0.04 3.18 ± 0.62
Day 7 Day 7 Day 7 Day 7 Day 14
TA-1
system, however. There was a more significant decline in Cu2+ concentration after removing Attached bacteria compared to removing Free bacteria, which indicated that Attached bacteria played a more vital role in the first 9-day bioleaching. It was said that the ore surface was initially adsorbed by Attached bacteria and then EPS (Extracellular Polymeric Substances) was produced to concentrate Fe3+ to attack copper sulfide ores (Feng et al., 2016). The Fe2+ was gradually produced along with the dissolution of Cu2+, and Fe2+ concentration in free-bacteria-removed system was also higher than that in attachedbacteria-removed system (Fig. 1(f)). The results in this study showed accordance with previous studies (Bevilaqua et al., 2013). Meanwhile, the Cu2+ concentration of Sample TB-0 between day 9 and day 15 in normal system was 35.20 mg/L. And that of Sample TB-2 in attachedbacteria-removed system was 49.40 mg/L. The value was only 7.30 mg/ L in free-bacteria-removed system (Fig. 1(a)). The results showed an important effect of Free bacteria on bioleaching from day 9 to day 15. According to previous researches that the passivation layer formed by hydrolysis of Fe3+ (Eqs. (5)–(9)) on the ore surface would inhibit the Attached bacteria to adsorp ore for releasing copper (Yin et al., 2019). At the middle-end bioleaching, pH value dropped heavily (contributed by Fe3+ hydrolysis). And some component like jarosite was also tested by XRD from residue low-grade copper sulfide ores after bioleaching experiments in this study. As was shown in Fig. 1(a), Samples TB-3 and TB-5 respectively yielded a maximum Cu2+ concentration of 329.10 mg/L and 523.60 mg/L from low-grade copper sulfide ores after a half-month bioleaching process. The Cu2+ concentration of Samples TB-3 and TB-5 after a 9-day bioleaching was 266.60 mg/L and 509.70 mg/L, respectively. And the Cu2+ concentration of Samples TB-3 and TB-5 between day 9 and day 15 was 62.50 mg/L and 13.90 mg/L, separately. Compared to the first 9-day Cu2+ concentration of Sample TB-2 (280.10 mg/L), that of Sample TB-3 (266.60 mg/L) showed a little drop only 13.50 mg/L. And difference between the first 9-day Cu2+ concentration of Sample TB-5 (509.70 mg/L) and Sample TB-1 (519.20 mg/L) was small. In summary, seawater has no significant effect on Attached bacteria and Free bacteria in the first 9-day bioleaching in this study. However, Cu2+ concentration between day 9 and day 15 of Sample TB-3 (62.50 mg/L) increased a lot in comparison with that in Sample TB-1 (35.20 mg/L). And Cu2+ concentration between day 9 and day 15 in Sample TB-5 (13.90 mg/L) increased by 6.60 mg/L only compared to that of Sample TB-4 (7.30 mg/L). The result distinctly demonstrated that the effect of seawater on enhanced copper recovery from low-grade copper sulfide ores was mainly realized by Attached bacteria during middle-final stage bioleaching. And the possible reason was as follows. With the bioleaching proceeding, Fe3+ inside leaching solution hydrolyzed to form precipitation and covered the ore surface, which hindered the contact between Attached bacteria and ore and then impeded bioleaching. It seemed that the retardation of precipitation released after adding seawater in this study.
18.62 ± 0.94 2.62 ± 0.21 1.48 ± 0.03 1.96 ± 0.01 1.69 ± 0.00 0.14 ± 0.00 1.14 ± 0.03 0.32 ± 0.04
According to the preliminary experiments above, analytical studies (e.g., XRD test) were applied to understand the enhanced copper dissolution phenomenon better. The effects of different proportion of seawater on the copper releasing during the low-grade copper sulfide ore bioleaching were analyzed after analytical tests.
Acidithiobacillus ferrooxidans Delftia tsuruhatensis Sphingomonas leidyi Leptospirillum ferriphilum Brevundimonas vesicularis Lactobacillus acetotolerans Ralstonia insidiosa Moraxella osloensis
Day 7
TA-0
Proportion (%)
3.4. Analytical insights into effect of seawater on bioleaching
Species
Table 3 Proportion of different species in leaching solution tested by 16S rDNA.
Day 14
TA-2
Day 14
TA-3
Day 14
TA-4
Day 14
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3.4.1. Detection of dissolved oxygen There was an abundant consumption of the oxygen resulted from bacteria and bioleaching reactions, the D.O. in each experiment sample declined distinctly finally compared to that at first, which could be seen from Table 2. And D.O. in leaching solution decreased in varying degrees in the presence of seawater at the beginning, especially Sample TA-4. Sample TA-0 in the absence of bacteria and seawater kept the highest D.O., however. The D.O. in Samples TA-0 to TA-4 at first were 7
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3.4.3. Bacteria separation analysis In this study, no additional compounds except seawater were added into the leaching solution of each sample before the bioleaching experiments. Surprisingly, a little peptidoglycan was found in Sample TA1 only in comparison with Sample TA-0 and Samples TA-2 to TA-4. It was believed that the appearance of peptidoglycan in Sample TA-1 was the result of metallization of bacteria on ores or cell metabolite from other microorganisms. At the same time, quite small yellow aggregations were found clearly in Sample TA-1, and XRD test results showed that main phases of the aggregations were KFe3(SO4)2(OH)6, (NH)4Fe3(SO4)2(OH)6 and peptidoglycan. And no quite small yellow aggregations could be found by eyes in Sample TA-0 and Samples TA-2 to TA-4, however. In order to obtain bacteria, glass beads were added into the plastic tube and mixed with leaching solution fully. Bacteria inside leaching solution were subsequently separated by centrifuge separation (TG16WS, Xiangli, China). Leaching solution containing bacteria was centrifuged at 600×g for ten minutes for separating supernatant and mineral. Then the bacteria were extracted from supernatant. The test results showed that major components of bacteria were peptidoglycan, phospholipid, lipoprotein and glycoprotein mainly. In comparison with the XRD test results of small yellow aggregations in Sample TA-1, the same peptidoglycan was found. In the light of the facts that there was no additional compounds except seawater were added into the leaching solution, thus the possible reasons of seawater improving bioleaching were as follows. According to previous Section 3.2, bacteria concentration and community succession were influenced greatly during bioleaching in the presence of appropriate proportion of seawater. Bacteria may die due to the concentration difference between inside and outside the cell wall after adding seawater, and then suspended in leaching solution after death. Formation of precipitation like KFe3(SO4)2(OH)6 and (NH)4Fe3(SO4)2(OH)6 (Eqs. (5)–(9)) could attach to the dead bacteria rather than ore surface or living bacteria. Meanwhile, hydrolysis of Fe3+ occurred at any location in the leaching solution. Suspending of dead bacteria in leaching solution made a better covering place for precipitation than ores. Thus, the obstructive effect of precipitation on ores and bacteria was reduced.
8.03 mg/L, 7.14 mg/L, 6.26 mg/L, 5.32 mg/L and 4.38 mg/L, respectively. Due to heating process of seawater before bioleaching experiment reduced the D.O. in seawater, Samples TA-1 to TA-4 in the presence of seawater showed a lower D.O. at the beginning. The D.O. in each sample after a half-month bioleaching was 5.46 mg/L, 5.02 mg/L, 3.84 mg/L, 3.29 mg/L and 2.71 mg/L, separately. The D.O. declined obviously because of reactions showed in Eqs. (2)–(5). Therefore, an extreme low D.O. of 3.29 mg/L and 2.71 mg/L showed in Samples TA-3 and TA-4, which was in response to the relatively low bacteria concentration and copper recovery. CuFeS2 + 4Fe3+ + 2H2O + 3O2 → Cu2+ + 5Fe2+ + 4H+ + 2SO42− (2) CuS + 4Fe3+ + 2H2O + O2 → Cu2+ + 4Fe2+ + 4H+ + SO42− (3) Cu2S + 6Fe3+ + 2H2O + O2 → 2Cu2+ + 6Fe2+ + 4H+ + SO42−(4)
3.4.2. XRD analysis The XRD analysis of residue ores in Samples TA-0 to TA-4 were conducted respectively after bioleaching experiments, which showed clearly that silica (SiO2) was the major mineral phase in those samples. XRD analysis results of residue ores in Samples TA-0 to TA-4 after bioleaching showed great variations in mineral phases whether in the presence of or in the absence of seawater. Four main phases were detected by XRD analysis in Sample TA-0 in the absence of seawater: SiO2, (NH)4Fe3(SO4)2(OH)6, 4Cu2S·CuS and Cu2S. In bioleaching system in the presence of seawater, there were four phases (SiO2, (NH)4Fe3(SO4)2(OH)6, peptidoglycan and 4Cu2S·CuS) in Sample TA-1 mainly. And SiO2, (NH)4Fe3(SO4)2(OH)6, 4Cu2S·CuS and Cu2S owned the most proportion in Sample TA-2. However, the same phases including SiO2, KFe3(SO4)2(OH)6, (NH)4Fe3(SO4)2(OH)6, 4Cu2S·CuS and Cu2S were found significantly in Samples TA-3 and TA4. It could be identified clearly that the same phases including SiO2, (NH)4Fe3(SO4)2(OH)6 and 4Cu2S·CuS were obtained in all experimental samples. There was no KFe3(SO4)2(OH)6 was found in Samples TA-0 and TA-2, where no or low proportion of seawater were added. While in Samples TA-3 and TA-4 treated with high proportion of seawater, a relatively high proportion of KFe3(SO4)2(OH)6 were noticed, which may cause a great obstacle to bioleaching according to previous studies (Bevilaqua et al., 2013). The results in Fig. 1(a) showed that Cu2+ concentration of Samples TA-3 and TA-4 in the presence of high proportion of seawater was less than that of Samples TA-0 to TA-2. It was therefore clear that significant effect of seawater on bioleaching might be related to precipitation like KFe3(SO4)2(OH)6. And the possible reasons of the effects of seawater on bioleaching were as follows. 4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O
(5)
H2SO3 + 2Fe3+ + H2O → 2Fe2+ + 4H+ + SO42−
(6)
[Fe(H2O)6]3+ + H2O → [FeOH(H2O)5]2+ + H3O+ 2+
[FeOH(H2O)5] 3+
+ H2O → [Fe(OH)2(H2O)4]
+
+
+ 2[Fe(OH)2(H2O)4] [Fe(H2O)6] PFe3(SO4)2(OH)6↓ + 2H3O+ + 10H2O
+
4. Conclusions The data in this study represented the firstly detailed investigation of the response of bacterial community to seawater in low-grade copper sulfide ore bioleaching. High copper recovery (84.70%) was obtained after adding 20.00% seawater. Seawater had significant effects on bacterial community structure and little difference showed between blank sample (no seawater) and sample adding 20.00% seawater. Bacterial richness decreased with increasing proportion of seawater. Low proportion of seawater was beneficial to copper extraction and dominant bioleaching bacteria (Acidithiobacillus ferrooxidans). Such study should provide a good reference for low-grade copper sulfide ore bioleaching and better application of seawater.
(7) +
+ H3O
2SO42−
(8) +
+
P
→ (9)
CRediT authorship contribution statement Wei Chen: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing review & editing, Supervision, Project administration, Funding acquisition. Shenghua Yin: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Aixiang Wu: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Leiming Wang: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original
A large amount of Fe2+ inside leaching solution of each sample was oxidized to Fe3+ under acid condition. High Fe3+ concentrations promoted the hydrolysis reactions between H2O and Fe3+, and the reaction processes were shown in Eqs. (5)–(9). The following ions including Ca2+, Mg2+, SO42−, CO32–, Na+, Cl− and K+ were contained in seawater mainly (Suyantara et al., 2018). However, K+ and SO42− were both significant components of precipitation indicated in Eq. (9). Based on the results showed in previous Section 3.3.1, adding high proportion of seawater accelerated the formation of precipitation like KFe3(SO4)2(OH)6, which thus decreased bacteria activity. 8
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draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Xun Chen: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Science Foundation for Excellent Young Scholars of China (51722401), the Key Project of National Natural Science Foundation of China (51734001) and the Fundamental Research Funds for the Central Universities (FRF-TP-18003C1). References Atzori, G., Mancuso, S., Masi, E., 2019. Seawater potential use in soilless culture: a review. Sci. Hortic. 249, 199–207. Bevilaqua, D., Lahti, H., Suegama, Patrícia H., Garcia, O., Benedetti, A.V., Puhakka, J.A., Tuovinen, O.H., 2013. Effect of na-chloride on the bioleaching of a chalcopyrite concentrate in shake flasks and stirred tank bioreactors. Hydrometallurgy 138 (113), 1–13. Brierley, C., Brierley, J., 2013. Progress in bioleaching-part B: applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 97, 7543–7552. Chen, L., Tsui, M.M.P., Lam, J.C.W., Hu, C., Wang, Q., Zhou, B., Lam, P.K.S., 2019. Variation in microbial community structure in surface seawater from pearl river delta: discerning the influencing factors. Sci. Total Environ. 660, 136–144. Davis-Belmar, C.S., Cautivo, D., Demergasso, C., Rautenbach, G., 2014. Bioleaching of copper secondary sulfide ore in the presence of chloride by means of inoculation with chloride-tolerant microbial culture. Hydrometallurgy 150, 308–312. Fagan, M.A., Ngoma, I.E., Chiume, R.A., Minnaar, S., Sederman, A.J., Johns, M.L., Harrison, S.T.L., 2014. MRI and gravimetric studies of hydrology in drip irrigated heaps and its effect on the propagation of bioleaching micro-organisms. Hydrometallurgy 150, 210–221. Feng, S., Yang, H., Zhan, X., Wang, W., 2014. Novel integration strategy for enhancing chalcopyrite bioleaching by acidithiobacillus sp. in a 7-L fermenter. Bioresour. Technol. 161, 371–378. Feng, S., Yang, H., Wang, W., 2016. Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages. Bioresour. Technol. 200, 186–193. Giuseppe, G., Kazumasa, T., Tatsuya, K., Chiharu, T., 2019. Mechanochemical activation of chalcopyrite: relationship between activation mechanism and leaching enhancement. Miner. Eng. 131, 280–285. Gong, Y., Ma, F., Xue, Y., Jiao, C., Yang, Z., 2019. Failure analysis on leaked titanium tubes of seawater heat exchangers in recirculating cooling water system of coastal nuclear power plant. Eng. Fail. Anal. 101, 172–179. Hao, X.D., Liang, Y.L., Yin, H.Q., Liu, H.W., Zeng, W.M., Liu, X.D., 2017. Thin-layer heap bioleaching of copper flotation tailings containing high levels of fine grains and microbial community succession analysis. Int. J. Miner. Metall. Mater. 24 (4), 360–368. https://doi.org/10.1007/s12613-017-1415-4. Hiroyoshi, N., Miki, H., Hirajima, T., Tsunekawa, M., 2001. Enhancement of chalcopyrite leaching by ferrous ions in acidic ferric sulfate solutions. Hydrometallurgy 60 (3),
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