Bioleaching of multiple metals from contaminated sediment by moderate thermophiles

Marine Pollution Bulletin xxx (2015) xxx–xxx

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Bioleaching of multiple metals from contaminated sediment by moderate thermophiles Min Gan, Shiqi Jie, Mingming Li, Jianyu Zhu ⇑, Xinxing Liu ⇑ School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China Key Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha 410083, China

a r t i c l e

i n f o

Article history: Received 22 January 2015 Revised 12 June 2015 Accepted 23 June 2015 Available online xxxx Keywords: Bioleaching Multiple metals Moderate thermophiles Leaching model Community dynamic

a b s t r a c t A moderately thermophilic consortium was applied in bioleaching multiple metals from contaminated sediment. The consortium got higher acidification and metals soubilization efficiency than that of the pure strains. The synergistic effect of the thermophilic consortium accelerated substrates utilization. The utilization of substrate started with sulfur in the early stage, and then the pH declined, giving rise to making use of the pyrite. Community dynamic showed that A. caldus was the predominant bacteria during the whole bioleaching process while the abundance of S. thermotolerans increased together with pyrite utilization. Solubilization efficiency of Zn, Cu, Mn and Cd reached 98%, 94%, 95%, and 89% respectively, while As, Hg, Pb was only 45%, 34%, 22%. Logistic model was used to simulate the bioleaching process, whose fitting degree was higher than 90%. Correlation analysis revealed that metal leaching was mainly an acid solubilization process. Fraction analysis revealed that metals decreased in mobility and bioavailability. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Due to the drastic industrialization and urbanization process, heavy metal contamination has become a worldwide environmental concern. Metal contamination is considered to be the most serious pollution source in aquatic environments (Akinci and Guven, 2011; Jiang et al., 2014). Metal undergoes a series of transition in water body like sorption and precipitation (Du Laing et al., 2009). Meanwhile, metals combined in sediment would be released to waters with physical or chemical variation and then assimilated by aquatic organisms, which may pose a potential hazard to the environment and human health (Chen and Lin, 2001; Mil-Homens et al., 2014). It is urgent to find a sound and effective method to solve this problem. The treatment of contaminated sediments can be achieved by either physical or chemical methods, while these techniques still show some limitations in practice such as high-energy requirements, high cost, low efficiencies, and operational difficulties (Chen and Lin, 2001). Bioleaching has already been successfully applied in biometallurgy for extracting metals from low-grade ores as a cost-effective and environmental friendly technique ⇑ Corresponding authors at: School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China. E-mail addresses: [email protected] (J. Zhu), [email protected] (X. Liu).

(Rohwerder et al., 2003; Zhang et al., 2008). The metals can be extracted from ores based on the acidification effect of the bacteria activities (indirect mechanism), or solubilized from sulfide by enzymatic oxidation directly (direct mechanism) (Akinci and Guven, 2011; Liu et al., 2008). Bioleaching technique offered a feasible alternative in bioremediation area, and it has attracted growing attentions in recent years (Wen et al., 2013, 2012). The primary microorganisms have been reported for bioleaching of heavy metals in soils, including heterotrophic microbe (e.g. Aspergillus niger, Penicillium simplicissimum, Penicillium purpurogenum, Rhodotorula rubra) and heterotrophic microbe (Acidithiobacillus spp.). The heterotrophic microorganisms produce low molecular weight organic acids during bioleaching, which can form water-soluble complexes with metals, while it also brings organic pollution. The mesophilic chemolithotrophic Acidithiobacillus spp. like Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are actively applied in heavy metal bioremediation areas (Park et al., 2014; Shahrabi-Farahani et al., 2014). However, the mesophilic bacteria are easily influenced by the dissolved organic matter, high content heavy metals and low pH (Fang and Zhou, 2006; Wang et al., 2014; Zheng et al., 2009). There are still no reports existed on the use of moderate thermophilic bacteria in the case of heavy metals bioremediation area, although the rates of bioleaching of metals from ores by moderate thermophiles have been demonstrated to be higher than those of mesophiles, and in another case, even higher

http://dx.doi.org/10.1016/j.marpolbul.2015.06.040 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Gan, M., et al. Bioleaching of multiple metals from contaminated sediment by moderate thermophiles. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.06.040

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M. Gan et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

than those of extreme thermophiles for its resistant to higher pulp densities and heavy metal concentrations (Ilyas et al., 2014, 2010). In addition, in summer or other high temperature conditions, the work environment exceeds optimal growth temperature of mesophilic microbes (>30 °C), which will limit the activity of the microorganism, while thermophilic bacteria have well adaptability, which can greatly enhance the bioleaching efficiency and decrease the operating cost. Based on this consideration, moderately thermophilic bacteria Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus are first applied in the sediment bioleaching in this research. Few literatures exist on any interactive effect of the process variables during the bio-removal of heavy metals from solid wastes. In this study, the dynamic of these essential elements during bioleaching have been systematic investigated. Single and mixed cultures of S. thermotolerans and A. caldus were used separately to compare efficiency of metal solubilization in flasks experiment. Based on the conclusion above, a more detailed tank bioleaching experiment was conducted to investigate the substrates utilization, leaching mode, components correlation and community dynamic. The geochemical characterization is of primary significance to assess the toxic effect of sediment (Beolchini et al., 2009), so the fraction dynamic of metals in different bioleaching stage were analyzed. In addition, Logistic empirical model was used to fit the heavy metals solubilization process. Knowing microbial ecology is important for optimizing the leaching parameters and improving the heavy metal solubilization efficiency (Zeng et al., 2010). Community dynamic and its corresponding Canonical Correspondence Analysis (CCA) were investigated. This study investigated the feasibility of bioleaching of multiple metals from contaminated sediment by moderate thermophiles and the dynamic change in bioleaching process, which provided useful information for controlling and optimizing the bioleaching process.

2.2. Shake flask experiment with pure and mixed cultures Shake flask experiments were conducted to investigate the effect of pure S. thermotolerans, A. caldus and the mixed culture on bioleaching. The experiment was done in 500 mL shake flask with a 200 mL bioleaching volume and 5% solid content (m/v) at 45 °C, 180 rpm. And 2.0 g sulfur and 2.0 g pyrite (FeS2) were added to the bioleaching system as joint substrates. These two bacterial strains were preserved by the Key Laboratory of Biometallurgy, Ministry of Education, and cultivated in 9 K medium (3 g/L (NH4)2SO4, 0.1 g/L KCl, 0.5 g/L K2HPO43H2O, 0.5 g/L MgSO47H2O, 0.01 g/L Ca(NO3)2) with FeSO47H2O and elemental sulfur respectively. The initial concentration of each strain was 0.75  107 cell/mL which was determined by microscopic counting. Based on the results of flask experiment, a mixed culture was adopted in subsequent stirred tank reactor experiment. The flask experiments were performed in duplicate to guarantee dependability. The error bar represented the standard deviation of the parallel experiment data, which was calculated using the STDEV function. 2.3. Stirred tank reactor bioleaching experiment The subsequent bioleaching experiment was conducted in 2 L stirred tank reactor with 1.5 L bioleaching volume at 180 rpm, which was set in a 45 °C constant temperature water bath. 75 g dry sediment was added into 1.5 L deionized water (solid content 5 % (m/v)), at the same time, 15 g sulfur and 15 g pyrite were added as the corporate substrate. 1 mL leachate was taken out from the conical flask and replenished by corresponding pH 9 K medium. pH, ORP and SO2 were monitored every two days. Cell density 4 was measured by microscope count method. Fraction analysis were conducted according to the selective sequential extraction method following Tessier et al. (1979). 2.4. Community dynamics analysis

2. Materials and method 2.1. Sediment Sediment was sampled from Xiawan Port of Xiangjiang River (Zhuzhou city, Hunan province, China; N27.8554401652, E113.0786195397). Long term affected by smelter and chemical industry, Xiawan Port of Xiangjiang River is the most seriously heavy metals polluted area in China (Chen et al., 2004; Zhu et al., 2013). pH of the sediment was measured after it brought back to laboratory. The samples were naturally dried and sieved through one hundred meshes. Metals content was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (Baird, PS-6, USA). Table 1 showed that the content of most metals was tremendously exceeded the secondary standard environmental quality standard of China, as shown above, leading to that sediments become toxic for most biological components.

Table 1 Metal content in sediment. Sl. No.

Metal

Total content (mg/kg)

Standard value (mg/kg)

1 2 3 4 5 6 7 8

Al Cd Zn Pb Hg As Mn Cu

79358.3 83.8 4294.3 581.5 81.2 83.6 2417.8 394.1

/ 1 300 350 1 20 / 100

DNA of the bacteria in stirred tank reactor was extracted every three days. The leaching solution was allowed to settle down for 10 min and then 10 mL solution filtered through 0.45 lm filter paper. Then the filtrate was centrifuged at 12,000 rpm with sigma centrifuge for 5 min to get the cells. Considering the multiple metals complex on the surface of bacteria, the harvested bacteria were pretreated with 180uL lysozyme buffer at 37 °C for 30 min. 2422 g Tris was dissolved in 1L distilled water and adjusted pH to 8.0 then added 0.744 g EDTA-Na2. Then, 0.4 g lysozyme and 240 lL Triton was added into 20 mL solution above, which comprised the lysozyme buffer. The subsequent extraction procedure operated according Tiangen bacteria extraction kit. The integrity of extracted genomic DNA was validated by electrophoresis with 1% (w/v) agarose gel. The target gene arsB of S. thermotolerans were amplified with primers St-F (50 -CCGTGTTATTAGCAGGAGCAATTAGAACAGTCGGCA TA-30 ) and St-R (50 -TGCGGCTCGACTTCTCACGGGCATAGCGATCAAA CG-30 ); Target 16S rRNA fragments of A. caldus were amplified with primers Ac-F (50 -TGCGGCTCGACTTCTCAC-30 ) and Ac-R (50 -GGGCA TAGCGATCAAACG-30 ) (Wang et al., 2012). Conventional PCR was performed with a Bio-Rad. The PCR program, the method of purifying PCR products and the calculation of DNA copy numbers referenced from previous research. The PCR products were diluted serially from 106 to 1010 copies/mL to construct standard curves. The real-time PCR was carried out with iCycler iQ Real-time PCR detection system (Bio-Rad Laboratories Inc., Hercules, USA). The detailed steps for quantitative analysis of community were described (Li et al., 2011; Wang et al., 2012; Zeng et al., 2010; Zhang et al., 2009). CCA was used to explore the potential correlations between microbial community structure and environmental

Please cite this article in press as: Gan, M., et al. Bioleaching of multiple metals from contaminated sediment by moderate thermophiles. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.06.040

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variables, and the key leaching parameters which shape the structure of microbial community during bioleaching (Yu et al., 2014). The CCA analysis was conducted with the Canoco for Windows software version 4.5. 3. Results and discussion 3.1. Dynamic changes of pH, ORP, iron and cell density in flask experiment Fig. 1 shows the dynamic variation of pH, ORP, iron and cell density during bioleaching with S. thermotolerans, A. caldus and the mixed culture. As reflected in pH and ORP (Fig. 1a and b), the activation period lasted about 13 days, which illustrates the initial environment is not conducive to moderate thermophiles. There was a sudden decrease in pH approximately from the 10th day. Depending on the rapid sulfur oxidation and pyrite utilization, within 6 days after bioleaching activation, pH in the system with mixed culture dropped roughly from 7.4 to 2.5. Since A. caldus possesses excellent oxidation ability on both sulfur and pyrite, its acidification efficiency rank second to the mixed culture. With the decreasing of pH, the oxidation–reduction potential (ORP) significant increased. The ORP of the system adopted with the mixed culture reached 550 mV for the effective sulfur, pyrite utilization and ferrous oxidation. The change trend of iron concentration showed that pyrite utilization started from the 14th day while mass utilization began approximately from the 22nd day. High pH of the system may inhibit pyrite utilization in early stage. The difference of iron concentration between the pure strain and mixed culture is not obvious, but as a whole, the mixed culture got the highest concentration. The ferrous concentration in system with pure A. caldus is higher than that of other two groups, which means that S. thermotolerans could accelerate the ferrous iron oxidation (Fig. 1c). That the bioleaching system adopted with mixed culture has the highest acidification efficiency, reduction oxidation potential and iron concentration among these groups (Fig. 1a–c) proved sulfur and pyrite can be synergistically utilized by the consortium. Sulfur and pyrite can be both utilized by S. thermotolerans and A. caldus. Meanwhile, the produced ferrous iron and released dissolved organic matter from sediments or dead cells in leaching process can subsequently oxidized by S. thermotolerans (Halinen et al., 2009), which can reduce the toxic effect to autotrophic bacteria and facilitate the bioleaching process (Zeng et al., 2010). The proliferation of A. caldus, S. thermotolerans and the mixed culture on the 16th day reached highest (Fig. 1d), and cell density was 5.12  109/mL, 3.52  109/mL, and 3.96  109/mL respectively. The bioleaching system with A. caldus gets the highest cell density but not the best acidification effect, which reconfirmed the synergistic effect in substrates utilization by the thermophilic consortium. 3.2. Metals leaching efficiency on the 17th and 31st day in flask experiment Fig. 2 shows the metals soubilization efficiency on the 17th and 31st day. The results indicate that difference of leaching efficiency existed in the middle stage of the bioleaching between the pure and mixed culture system. As a whole, the mixed culture system gets the highest leaching efficiency on the 17th day. Zn soubilization efficiency reached 56.4% with the mixed culture, while it reached 41.3% and 51.5% in system with S. thermotolerans and A. caldus respectively. The soubilization of Cu, Mn in different groups are similar with that of Zn. Bioleaching efficiency of Pb, Hg and As in mixed culture was 5.63%, 4.38%, 16.37% respectively. The corresponding leaching efficiency of these metals were 4.23%, 4.12%,

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13.37% and 4.93%, 5.38%, 14.78% in system with S. thermotolerans and A. caldus respectively. Results showed that soublization efficiency of these refractory elementals in mixed culture is superior to that of pure strains. Proton and ferric iron in the bioleaching system are direct responsible for the soubilization of metals (Velgosova et al., 2014), based on this mechanism, so the synergistic effect in substrates utilization can promote the metal release. Results demonstrated a similar soubilization of metals at the end point between different combinations, which can be contributed to the same leaching environment. 3.3. pH, ORP and iron variation in tank bioleaching The activation stage lasted for 5 days in tank reactor (Fig. 3a), which is much more efficient than that in flask experiment. The discrepancies of oxygen content and mass transfer effect between reactors may directly generate the difference in activation period. pH dropped rapidly from the 7th to 17th day. High acidification rate can be attributed to the efficient substrate utilization by the consortium. The pH decrease trend is not evident after eighteen days, while the acidification efficiency cannot be neglected for pH, which was presented as a log scale (Fig. 3a). Redox potential varied from 100 mV to 500 mV during the bioleaching process and a remarkable downtrend was observed during 17th day to 42rd day. ORP is another crucial factor influencing the bioleaching process. Metal sulfides oxidization rate and organic compounds degradation rate would increase correspondingly with ORP increase (Calmano et al., 1993; Peng et al., 2009). According to Eq. (1), it can be found that redox potential was consistent with the dynamic of Fe3+/Fe2+ ratio. In this period, incomplete ferrous oxidation (Fig. 3c) leaded to the high Fe2+/Fe3+ ratio and the low redox potential. This phenomenon implied that the abundance of ferrous oxidation bacteria was insufficient, or the oxidation ability was inhibited during this period.

E ¼ E0 þ

nF ln aFe3þ =aFe2þ RT

ð1Þ

3.4. Bacteria growth, sulfate and iron production As shown in Fig. 3 b, the cell density of the moderately thermophile consortium reached 6.96  109/mL in the 16 days, which illustrated the community well adapted to the initial bioleaching system. Then, it reduced gradually from the 17th day, meanwhile, environment factors changed dramatically including pH declining, ORP rising and multiple metals radical accumulation (Figs. 3a and 5). These factors bring the damage to bacteria and community structure transition (Govarthanan et al., 2014). Cell density entered into a rapid descending stage when intimidated by more severe environmental factors after 32 days. Sulfate in system was derived from sulfur and pyrite utilization by the bacteria. Sulfate concentration preserved a stable increase trend, but its production rate dropped when bacteria declined in later stage (Fig. 3 b). As shown in Eqs. (2) and (3) (Chandra and Gerson, 2010; Zhang et al., 2008), significant amounts of proton is produced in pyrite utilization process, and the acidification efficiency is much higher than that of sulfur. Fig. 3c showed that ferric and ferrous concentration maintained in a pretty low level in early bioleaching stage and then increased by a wide margin with pH and community change (Figs. 3a and 4a). Variation of iron concentration showed that mass utilization of pyrite began on the twelfth day. Through comparing with the results in flask experiment, the fact can be deduced that high pH may inhibit pyrite utilization, bacteria proliferation and ferrous oxidation. A suitable pH environment is indispensable for pyrite decomposition. Ferrous oxidation percentage maintained in an extraordinary low level from the 10th to 30th

Please cite this article in press as: Gan, M., et al. Bioleaching of multiple metals from contaminated sediment by moderate thermophiles. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.06.040

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Fig. 1. Variation of pH, ORP, iron concentration and cell density (mean ± SD) with pure and mixed strains in flask experiments during bioleaching. (a) pH, (b) redox potential, (c) ferrous and ferric iron concentration and (d) cell density.

Fig. 2. Metal Cu, Zn, Mn, Cd, Pb, Hg, As soubilization efficiency on the 17th (a) and 31st (b) day in flask bioleaching.

day. However, it was almost completely oxidized on the 37th day. Higher ferric percentage is beneficial to pyrite utilization (Eq. (2)). The abundance of ferrous oxidizing bacteria may be augmented in this process. Based on the analysis above, we can find that joint substrates provide multiple choices for bacteria and facilitate its adaption in unfavorable condition. bacteria

þ FeS2 þ 14Fe3þ þ 8H2 O ƒƒƒ! 15Fe2þ þ 2SO2 4 þ 16H bacteria

2S0 þ 3O2 þ 2H2 O ƒƒƒ! 2H2 SO4

ð2Þ ð3Þ

3.5. Community dynamics analysis As shown in Fig. 4a, S. thermotolerans and A. caldus was co-inoculated into the system with 0.5  108/mL each species at the beginning of the bioleaching. In the first eight days, the density of A. caldus increased to 2.38  109/mL, while the proliferation of S.

thermotolerans was unremarkable only reached 2.6  109/mL. Hereafter, S. thermotolerans entered into a rapid proliferation period until the 17 day. The increasing of S. thermotolerans was tightly related to lower pH, the rising ferrous concentration and the adaption to substrates. The ratio of A. caldus far exceed that of S. thermotolerans, making A. caldus could be recognized as the predominant strain in the system. Similar shift trend of A. caldus was also observed in chalcopyrite moderately thermophilic bioleaching process (Johnson et al., 2008; Zeng et al., 2010). A. caldus, a pure sulfur-oxidizing bacterium, can utilize both elemental sulfur and pyrite. As for S. thermotolerans, its sulfur oxidation ability was inhibited because it was initially cultured by ferrous sulfate. Since the oxidation of sulfur can produce more growth energy than the oxidation of ferrous iron (Sand and Gehrke, 2006). A. caldus can thus get more energy than the iron-oxidizing S. thermotolerans for their growth and propagation. Sulfate production and ferric/ferrous ratio (Fig. 3b and c) confirmed that the acidification in this period mainly relied on mass utilization of sulfur, which is

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Fig. 3. Variation of pH, redox potential, iron and sulfate concentration in stirred tank reactor during bioleaching. (a) dynamic change of pH and redox potential, (b) cell density and sulfate concentration change, (c) Fe(II), Fe(III) and total iron concentration change.

Fig. 4. Community change and CCA tri-plot. (a) Community change during bioleaching, (b) CCA tri-plot shows the correlations between microbial community and environmental parameters. Arrows on the graph represent environmental parameters. Triangles represent species. Circles represent samples from different stages of bioleaching. Circles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 represent samples on day 5, 8, 12, 14, 17, 20, 23, 26, 29, 35, 38, 41 respectively.

Fig. 5. Solubilization of the metals during bioleaching. The dots represent practical soubilization efficiency, and lines represent the model fitting to the practical soubilization efficiency.

consistent with the efficient sulfur oxidizing ability of A. caldus. The abundance of S. thermotolerans was sharply decreased from the 17th day. This may be caused by the soubilization of heavy metals. A. caldus maintained in a relatively high density from the 12th to 35th day, during which was also the most important acidification period. A. caldus basically exceeded S. thermotolerans in the whole bioleaching process, which was determined by its broad pH adaption ability, excellent heavy metal resistance and efficient utilization to substrate (Dopson et al., 2003; Olson et al., 2003; Watkin et al., 2009). Canonical Correspondence Analysis (CCA) in Fig. 4 b clearly presents the potential correlations between microbial community and environmental parameters (pH, ORP, Fe3+, Fe2+ and metals) during bioleaching. The environmental parameters exhibited a basically linear relationship due to the simple binary bacteria composition. On the CCA analysis, spots clustering together indicate similar community structures. It revealed that A. caldus was tightly related to the initial acidification in early stage. While S. thermotolerans plays a more important role in later ferrous iron

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oxidation process. The interaction between microorganisms plays a pivotal role in improving the heavy metals bioleaching efficiency (Zeng et al., 2010). Understanding the community structure can provide valuable information for optimizing process parameters and improving bioleaching efficiency. 3.6. Heavy metals bioleaching model and correlation analysis

Logistic Equation y ¼ A2 þ ðA1  A2Þ

  x p  1þ x0

ð4Þ

The influences of pH on solubilization efficiency of different metals are not consistent. As shown in Fig. 5, the solubilization of Zn, Cu, Mn and Cd began in a relatively high pH around 6. The fastest solubilization period concentrated on pH 5–2. However, the bioleaching effect was not notable after 20 days even with slowly decreasing pH. This is because the fraction is easily affected by acid like exchangeable fraction, carbonate fraction and Fe–Mn oxide fraction released. The solubilization efficiency of Cu, Cd is the highest, and then followed with Mn and Zn, reached nearly 90%. The bioleaching efficiency of some other elements like As, Hg, Pb was only 44.70%, 33.85%, 22.40% respectively, which is consistent with previous researches (Chen and Lin, 2004; Fonti et al., 2013). Bioleaching is a complex process which could be affected by multifactor including pH, ORP, oxygen, substrate, bacteria, temperature, Fe3+/Fe2+ ratio. It is difficult to establish a model which covering all influence factors. As metals solubilization efficiency is tightly correlated with pH decline in early stage, and it decreased with easily combined fraction released in subsequent bioleaching

process, solubilization efficiency presented as ‘S’ form. Logistic model is first chosen to simulate the bioleaching process. It can be found from Fig. 5 and Table 2 (supplementary materials) that the Logistic model curves and parameters (R-Square > 90%) are well fitted with this process. According to the definition of Logistic model, metals soubilization can be correspondingly divided into five stages including the beginning stage, acceleration, turning, deceleration and saturation stage. Parameter A2 was basically consistent with the metal solubilization efficiency and can be recognized as an important parameter to evaluate the leaching difficulty. Establishing metals bioleaching kinetic model can deepen theoretical basis and be beneficial to improve the bioleaching efficiency. Table 3 (supplementary materials) presents the potential correlations of environmental factors. Spearman correlation coefficient between pH and soubilization efficiency Al, Cu, Zn, Hg, Mn, Pb, Cd, As are 0.97, 0.93, 0.94, 0.86, 0.94, 0.97, 0.83, 0.97 respectively (Table 3). Coefficient of Pearson and Kendall were also higher than 0.71758. Pearson, Spearman and Kendall correlation index revealed that metals solubilization is highly negative correlated with pH decline, which illustrates metals leaching are mainly an acid solubilization process. Previous studies (Chen and Lin, 2001; Liu et al., 2008; Pathak et al., 2009; Rulkens et al., 1995) regarded direct or indirect mechanism (Eqs. (5) and (6)) are both involved in metals solubilization, while this study indicated mainly the indirect mechanism was involved in sediment bioleaching, because only little sulfide existed in sediment. Generally, it is deemed that the direct mechanism only apply to sulfide bioleaching (Rodrıguez et al., 2003; Tributsch, 2001). As for the function of iron, correlation coefficient between total iron

Table 2 Logistic fitting curve parameter. A1

Al Cu Zn Hg Mn Pb Cd As

A2

x0

p

Statistics

Value

Error

Value

Error

Value

Error

Value

Error

Reduced Chi-Sqr

Adj. R-Square

3.14356 9.44425 2.26261 0.6418 1.77408 1.38854 2.43124 4.12728

0.72182 4.8486 7.0498 2.92049 8.01973 0.84443 2.53307 1.17855

53.35724 98.14382 85.67337 43.12968 80.98157 181558.3 91.41869 43.17831

23.20104 6.08685 7.32601 10.18533 6.21977 7.35E+08 1.7491 2.50104

48.13627 9.01743 10.16769 15.46961 8.29056 98905.47 6.01105 24.3516

19.19929 1.22717 1.5742 6.15519 1.47791 3.39E+08 0.34795 1.15047

2.14484 1.66958 2.07403 1.34934 2.14641 1.18282 1.9352 4.79109

0.50025 0.37335 0.66245 0.51263 0.76022 0.39269 0.2022 0.92407

1.00928 24.1657 54.78935 8.88609 67.80145 0.8408 6.46584 4.64191

0.98699 0.96899 0.94045 0.94006 0.91715 0.98363 0.99176 0.98209

Table 3 Pearson, Spearman and Kendall correlation analysis parameters.

Pearson Corr. (pH) Sig. (pH) Spearman Corr. (pH) Sig. (pH) Kendall Corr. (pH) Sig.(pH) Pearson Corr. (ORP) Sig. (ORP) Spearman Corr. (ORP) Sig. (ORP) Kendall Corr. (ORP) Sig. (ORP) Pearson Corr. (Fe3+) Sig. (Fe3+) Spearman Corr. (Fe3+) Sig. (Fe3+) Kendall Corr. (Fe3+) Sig. (Fe3+)

pH

ORP

Al

Cu

Zn

Hg

Mn

Pb

Cd

As

1 / 1 / 1 / 0.93797 0.0000 0.77332 0.0000 0.58203 0.0000 / / / / / /

0.93797 0.0000 0.77332 0.0000 0.58203 0.0000 1 / 1 / 1 / / / / / / /

0.9816 0.0000 0.96503 0.0000 0.90909 0.0000 0.90105 0.0001 0.74825 0.0051 0.57576 0.0092 0.87828 1.71E04 0.95622 1.18E06 0.87025 8.93E05

0.72832 0.0072 0.93007 0.0000 0.78788 0.0004 0.52132 0.0822 0.71329 0.0092 0.51515 0.0197 0.55873 0.05898 0.94221 4.61E06 0.80918 2.70E04

0.76773 0.0036 0.94406 0.0000 0.81818 0.0002 0.59907 0.0396 0.74126 0.0058 0.54545 0.0136 0.64626 0.02317 0.9352 8.06E06 0.77865 4.56E04

0.84519 0.0005 0.85814 0.0004 0.71758 0.0013 0.6707 0.0170 0.64098 0.0247 0.38169 0.0857 0.6326 0.02728 0.84912 4.75E04 0.67692 0.00243

0.73302 0.0067 0.94406 0.0000 0.81818 0.0002 0.5795 0.0483 0.74126 0.0058 0.54545 0.0136 0.61706 0.03256 0.9352 8.06E06 0.77865 4.56E04

0.95333 0.0000 0.96503 0.0000 0.90909 0.0000 0.86445 0.0003 0.74825 0.0051 0.57576 0.0092 0.87999 1.60E04 0.95622 1.18E06 0.87025 8.93E05

0.62327 0.0304 0.82517 0.0010 0.66667 0.0027 0.41573 0.1789 0.56643 0.0548 0.33333 0.1314 0.44906 0.14307 0.80911 0.00143 0.62597 0.00483

0.97834 0.0000 0.96503 0.0000 0.87879 0.0001 0.85892 0.0003 0.73427 0.0065 0.54545 0.0136 0.7871 0.00238 0.95972 7.80E07 0.87025 8.93E05

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Fig. 6. Fraction behavior of metals in different stage. (0): Sediment before bioleaching, (20): the sediment in the 20th day, (47): the sediment in the 47th day; F1: exchangeable form, F2: carbonate associated form, F3: Fe–Mn oxide-associated form, F4: organic associated form, F5: residual form.

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M. Gan et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

and solubilization efficiency of Pb, Hg, As are higher than that of other easy leached metals on average. This result illustrates ferric ion play a unique role in metals leaching with low solubility product. bacteria

MS þ 2O2 ƒƒƒ! MSO4 Indirect mechanism

ð5Þ

H2 SO4 þ sediment  M ! sediment þ M xþ þ SO2 4 Direct mechanism

ð6Þ

Cd, and mainly existed in organic associated and residual forms after bioleaching (Hg 51.64% and As 74.1%) (Fig. 6e and b). Metals in exchangeable, carbonate and Fe/Mn oxide-bonded fraction are considered to be more mobile and bioavailable while organic fraction and residual fraction are more stable and non-bioavailable. Metals are mainly bounded to the stable fraction after bioleaching, resulting in the minimizing of metals mobility and bioavailability. It implies that metals are not only reduced in total content but also decreased in mobility and bioavailability after bioleaching.

3.7. Fraction behavior of metals during bioleaching

4. Conclusions

The geochemical characterization of the sediments is of primary importance in bioremediation (Dell’Anno et al., 2009). Moreover, a geochemical characterization with a detailed analysis of heavy metal speciation can provide important insights on the potential fate of each heavy metal (Beolchini et al., 2009). Percentage of metals fraction in different bioleaching stages are shown in Fig. 6. The results indicate that metals fractions are radically changed after bioleaching not just in total quantity. In the initial sediment, Zn (Fig. 5h) mainly existed in residual form up to 2335 mg/kg, and followed by in carbonate associated and Fe-Mn oxide-associated form (782 and 692 mg/kg). The content of carbonate associated and Fe– Mn oxide-associated form Zn, decreased up to 78 and 124 mg/kg after 20 days. While the content of exchangeable form increased from 43 to 191 mg/kg. It can speculate that metal fraction transformed from relatively stable state to exchangeable form, and this transformation is facilitated to metals soubilization. As for the transformation of residual form, it decreased from the initial content to 590 and 39 mg/kg on the 20 and 47 day. The variation in chemical forms of Cu is showed in Fig. 5d. Cu mainly existed in organic associated (129 mg/kg) and residual forms (90 mg/kg) before the bioleaching. Almost all the exchangeable, carbonate associated and Fe–Mn oxide-associated forms Cu were solubilized after 47 days, above which only account for 6.4% in total content after bioleaching. All five component speciation of Mn, Zn, Cu, Cd are significantly reduced especially the exchangeable and carbonate associated fraction. As for Pb, Hg and As, the bioleaching efficiency was not so high as the former metals. According to previous researches, the partitioning of chemical forms was affected by physicochemical property of the bioleaching system, and the chemistry of metals was also an important parameter in determining the forms (Chen et al., 2005). The exchangeable, carbonate associated, Fe–Mn oxide-associated and organic associated form Pb was 75.15, 79.53, 105.17 and 103.65 mg/kg before bioleaching, while the content of these forms decreased to 70.65, 63.52, 67.43, 70.65 mg/kg and 17.75, 9.50, 30.14, 12.80 mg/kg on the 20th and 47th day. It illustrated that the above form of heavy metals was sensitive to the variation in pH, ORP and ferric ions and relatively easy to be released in bioleaching. As for the residual form Pb, its content showed a contrary tendency with the above mentioned fraction, which initial content was 168.57 mg/kg and increased to 208.14 and 342.65 mg/kg on the 20th and 47th day. It indicated that Pb gradually adsorbed or converted to residual form after mobilization. As bioleaching processed, sulfate produced along with substrates utilization, and PbSO4 was formed after sulfate combined with the mobilized Pb. It was comparably stable even acidic condition for the low solubility constant (1.06  108). In later stage, lots of ferric ion was produced with pyrite utilization (Fig. 3c), which further stabilized the conversion of Pb for the facilitation on formation of stable lead jarosite like substance. Although the content of residual form Pb increased, it was a stable state with low bioavailability (Beolchini et al., 2009). The fraction transformation of Hg, As was similar to Pb, which content decreased while not such remarkable like Cu, Zn,

In this study, a moderately thermophilic consortium S. thermotolerans and A. caldus were used for bioleaching of multiple metals from contaminated sediment. The mixed consortium has higher bioleaching efficiency than that of pure strains due to the synergistic effect. The utilization of joint substrate would be altered with environment change. Logistic empirical model was used to simulate the metals solubilization, which was highly fitted with this process. Metal leaching was mainly an acid solubilization process. Bacteria community structure would shift with environmental factors change. Microbial ecology dynamic can provide valuable information for us to optimize the leaching parameters. Metals reduced in total content and decreased in bioavailability. Acknowledgements This research was supported by the National Natural Science Foundation of China (51174239), the Fundamental Research Funds for the Central Universities of Central South University (2015zzts089) and the Hunan Provincial Co-Innovation Center for Clean and Efficient Utilization of Strategic Metal Mineral Resources. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.marpolbul.2015. 06.040. References Akinci, G., Guven, D.E., 2011. Bioleaching of heavy metals contaminated sediment by pure and mixed cultures of Acidithiobacillus spp. Desalination 268, 221–226. Beolchini, F., Dell’Anno, A., De Propris, L., 2009. Auto- and heterotrophic acidophilic bacteria enhance the bioremediation efficiency of sediments contaminated by heavy metals. Chemosphere 74, 1321–1326. Calmano, W., Hong, J., Foerstner, U., 1993. Binding and mobilization of heavy metals in contaminated sediments affected by pH and redox potential. Water Sci. Technol. 28, 223-223. Chandra, A., Gerson, A., 2010. The mechanisms of pyrite oxidation and leaching: a fundamental perspective. Surf. Sci. Rep. 65, 293–315. Chen, S.-Y., Lin, J.-G., 2001. Bioleaching of heavy metals from sediment: significance of pH. Chemosphere 44, 1093–1102. Chen, S.-Y., Lin, J.-G., 2004. Bioleaching of heavy metals from livestock sludge by indigenous sulfur-oxidizing bacteria: effects of sludge solids concentration. Chemosphere 54, 283–289. Chen, Y., Wu, F., Lu, H., Yao, C., 2004. Analysis on the water quality changes in the Xiangjiang River from 1981 to 2000. Resourc. Environ. Yangtze Basin 13, 508– 512. Chen, Y.X., Hua, Y.M., Zhang, S.H., Tian, G.M., 2005. Transformation of heavy metal forms during sewage sludge bioleaching. J. Hazard. Mater. 123, 196–202. Dell’Anno, A., Beolchini, F., Gabellini, M., 2009. Bioremediation of petroleum hydrocarbons in anoxic marine sediments: consequences on the speciation of heavy metals. Mar. Pollut. Bull. 58, 1808–1814. Dopson, M., Baker-Austin, C., Koppineedi, P.R., Bond, P.L., 2003. Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic microorganisms. Microbiology 149, 1959–1970. Du Laing, G., Rinklebe, J., Vandecasteele, B., 2009. Trace metal behaviour in estuarine and riverine floodplain soils and sediments: a review. Sci. Total Environ. 407, 3972–3985.

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Please cite this article in press as: Gan, M., et al. Bioleaching of multiple metals from contaminated sediment by moderate thermophiles. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.06.040