Enhanced biotreatment of acid mine drainage in the presence of zero-valent iron and zero-valent copper

Batch laboratorial experiments were conducted to evaluate the potential of Fe 0 , Cu 0 and sulfate-reducing bacteria (SRB) for reduction and removal of sulfate and heavy metals from synthetic acid mine drainage. The variation in solution pH indicates that the Fe 0 /Cu 0 bimetallic system provided favorable conditions for SRB growth and sulfate reduction. When the SO 42 (cid:1) concentration of wastewater was 3,000 mg/L, the SO 42 (cid:1) removal ef ﬁ ciency was 51.6% for the SRB system, 76.3% for the Fe þ SRB system, and 92.0% for the Fe/Cu þ SRB system. Moreover, Pb 2 þ , Cu 2 þ and Zn 2 þ ions were completely removed. The results demonstrate that the Fe/Cu þ SRB system had apparent advantages over the SRB system, especially at low pH. This study demonstrates that an Fe/Cu þ SRB system could be a promising technology for treating wastewater containing high concentrations of sulfate and heavy metals. laboratorial experiments were conducted to evaluate the of and

etc. The physical processes have disadvantages such as high infrastructure costs and high operating costs. Therefore, biological processes have been employed as an alternative to treat sulfate-containing wastewater (Singh et al. ; Rodriguez et al. ). Sulfates are non-toxic to organisms, but their reduction products (namely sulfides) have a highly poisonous effect on microbes including SRB (Singh et al. ). Thus, treating AMD by biological processes alone may be hampered by the presence of high concentrations of sulfides.
Zero-valent iron (ZVI, Fe 0 ), a strong reducing reagent (E 0 ¼ À0.447 V), in aqueous solution, acts as an electron donor under certain conditions and can carry out oxidation-reduction reactions without any external energy input. ZVI is expected to lower the oxidation-reduction potential (ORP), which is beneficial for the formation of an enhanced anaerobic environment for SRB (Kumar et al. ). Additionally, Fe 0 can release from Fe 0 to Fe 2þ (Fe 0 þ 2H þ ¼ Fe 2þ þ H 2 ) and thus buffer the solution pH (Liu et al. ) and reduce the toxicity of H 2 S by the formation of FeS precipitation (Zhang et al. ). Fe 0 can also lower mobility and toxicity of some heavy metals (Cu 2þ and Cr 6þ ) through reduction behavior. Therefore, However, the reactivity of Fe 0 tends to decline with time because of the accumulation of hydroxide precipitates and other corrosion products on the metal surface. It has been demonstrated that Cu 0 addition could provide high reaction potential and improve reduction capacity of Fe 0 , resulting in a higher pollutant removal efficiency even in a neutral pH range (Lai et al. ). Thus, this study proposed an Fe/Cu bimetallic packed anaerobic system to improve sulfate removal by SRB from AMD. The validity and applicability of the system is tested and compared under different conditions.

Materials
Iron shavings (Fe 0 ) and copper shavings (Cu 0 ) were collected from a local machining factory. The shavings used had a purity of greater than 99% and a grain size range of 14-18 mesh. The shavings (1 g) were mixed with 10 mL of 10% (v/v) HCl solution for 2 min and were then rinsed with nitrogen-sparged deionized water, and then rinsed in acetone. The shavings were dried with N 2 inside the anaerobic glove box before use.
The mixed SRB population was enriched from activated sludge obtained from the anaerobic digester at a local sewage treatment plant. For this, 5 g anhydrous Na 2 SO 4 was added into a 1-L bottle containing 1 L of anaerobic sludge. After 7 days of incubation at 37 C, 50 mL of sludge was transferred to another 1-L bottle containing 950 mL of autoclaved Postgate's B medium (Postgate ), and then incubated at 37 C for another 7 days. This procedure was repeated 7 times to enrich SRB. The resultant culture was centrifuged (8,000 g, 10 min), washed twice with physiological saline, and centrifuged again as indicated above to collect the pellet (namely the final consortium).
Light microscopy and scanning electron microscopy
Chemical oxygen demand (COD) was adjusted as required with glucose.
Batch tests were conducted in 1-L glass bottles, to test the effect of various parameters on the removal of sulfates.
The bottles had one inlet for probe insertion and two outlets for sampling and gas discharge. The bottles were connected with 500 mL aspirated bottles (gas holder) filled with 2.0 M NaOH solution to trap the H 2 S generated during incubation.
SRB consortium (about 5 g) was transferred to bottles with 900 mL artificial AMD. The headspace was flushed with N 2 /CO 2 gas (80:20, v/v) to exclude oxygen and bottles were sealed with butyl rubber septa to exclude oxygen permeation. Fe 0 and Cu 0 were added to the mixture at a final concentration of 2 and 0.2 g/L, respectively. The bottles were then incubated at 100 rpm and 37 C. There were four treatments with three replications as follows: (1) Fe/Cu; (2) SRB; (3) Fe þ SRB; (4) Fe/Cu þ SRB. In each treatment, the volume of AMD was identical (900 mL).

Analysis methods
Liquid samples were obtained from each bottle using a plastic syringe at a pre-determined time. The supernatant was collected for analysis after centrifugation at 8,000 rpm for

RESULTS AND DISCUSSION
Effect of Fe 0 and Cu 0 on sulfate reduction The performance of the systems was analyzed by taking into account the efficiency of sulfate reduction when the COD concentration of AMD was 3,600 mg/L, and the initial pH was 7.0. The effect of Fe 0 and Cu 0 on sulfate reduction is shown in Figure 1. As shown, no sulfate reduction occurred in the case of Fe/Cu. This indicates that sulfate could not be reduced by Fe 0 , and the adsorption of sulfate by Fe 0 or Cu 0 was negligible. The standard Gibbs free energy change (ΔG 0 ) is about 1,430 KJ/mol > 0. Thus, this reaction cannot take place spontaneously.

):
H 2 can then serve as an electron donor for stimulating the anaerobic biotransformation of reducible contaminants.  Effect of COD/SO 4 2À ratio on sulfate reduction In this work, glucose was provided as the sole organic substrate for microbial growth and sulfate reduction, and the relevant reaction is as follows: The COD mass used for the desulfurization was calcu-    (1)).
Several studies have been conducted to find the optimum COD/SO 4 2À ratios for SRB to reduce sulfate but the results were diverse. The best COD/SO 4 2À ratio was found to be 5.0 with sludge as the organic substrate (Al-Ani ), whereas SRB was predominant for a ratio below 1.7 using natural or synthetic substrates (Prasad et al. ).
When complex organics were used as substrate, higher optimum ratios were likely obtained because not all the carbon present was utilized by SRB. Glucose is an excellent carbon source for microbes, thus a low COD/SO 4 2À ratio was demonstrated in this study.

Effect of initial pH on sulfate reduction
The effect of initial pH on sulfate reduction was tested with pH decreasing from 7.0 to 3.0, when the concentrations of SO 4 2À and COD were respectively 3,000 and 3,600 mg/L, and the temperature of the system was 37 C. Figure 3 shows the changes of sulfate removal and effluent pH in the bottles after 60 days of incubation across the three systems. As shown, initial pH exerted an apparent effect on sulfate removal across various systems. In the SRB system, sulfate removal gradually declined with the initial pH decreasing from 7.0 to 4.0, and an apparent decrease of sulfate removal was observed with the decrease of pH from 4.0 to 3.0 (Figure 3(a)). In contrast, the influence of initial pH on sulfate removal in the Fe þ SRB and Fe/Cu þ SRB systems was slighter compared with that in the SRB system ( Figure 3). The sulfate removal of the SRB, Fe þ SRB and Fe/Cu þ SRB systems were 32.5%, 63.5% and 78.4% respectively with the initial pH at 4.0. Sulfate removal was only 23.6% in the SRB system with pH 3.0 (Figure 3(a)). Sulfate removal reached 58.2% and 74.3% in the Fe þ SRB and Fe/Cu þ SRB systems respectively under the same pH condition (Figure 3(b) and 3(c)). The optimal pH for SRB growth was around 6-7, whereas the performance of the Fe 0 -amended systems was good for sulfate removal when the initial pH was above 4.0.
A similar behavior of the effluent pH influenced by the changed initial pH was observed in the three systems ( Figure 3). The effluent pH in the SRB system was above 6.2 with the initial pH above 4.0. When the initial pH decreased to 3.0, the effluent pH was about 5.5 ( Figure   3(a)). In the Fe þ SRB and Fe/Cu þ SRB systems, effluent pH was above 6.5 with the initial pH from 3.0 to 7.0. The result of initial pH effect on sulfate removal and effluent pH suggests that Fe 0 could increase the pH of the systems to improve the SRB activity. In this study, the increase of effluent pH after incubation was primarily due to the release of bicarbonate alkalinity during sulfate reduction (Equation (3)), which is beneficial for acid neutralization and pH buffering (Singh et al. ). Additionally, in Fe 0 -amended systems, alkalinity can be generated by anoxic corrosion of Fe 0 (Equation (1)) and sulfate reduction (Equation (2)), as both processes involve the consumption of hydrogen ions.
Therefore, the pH values of the effluent from the Fe þ SRB and Fe/Cu þ SRB systems were higher than that from the SRB system.

Metal removal
To confirm the positive effect of Fe 0 , heavy metals removal was monitored during the first 96 h of reaction in the Fe, SRB, Fe þ SRB and Fe/Cu þ SRB systems with Pb 2þ , Cu 2þ and Zn 2þ concentrations of 50, 50 and 50 mg/L, respectively (Table 1). In the Fe system, the concentration of Zn 2þ was almost unchanged, since Zn 2þ could not be reduced by Fe 0 ; however, the Pb 2þ and Cu 2þ removal was almost 100% after 72 h, indicating the reduction and precipitation of Pb 2þ and Cu 2þ . In the SRB, Fe þ SRB and Fe/Cu þ SRB systems, the removal ratios of Pb 2þ , Cu 2þ and Zn 2þ were 100% after 48 h. At the end of experiments, the residual ratios of Fe 0 were 34.3% and 28.5% in the Fe þ SRB and Fe/Cu þ SRB systems, while the residual ratio of Cu 0 was 92.6% (data not shown). The SRB populations are consistent with calculated sulfate reduction efficiency (Figure 1). Although H 2 (g) concentrations were not measured, increments in pH were indicative of anaerobic Fe 0 corrosion which produced H 2 (g) (Equation (1)). SRB growth could be enhanced with the addition of H 2 as an electron donor (Ayala-Parra et al.

)
. Improved sulfate reduction efficiency in the presence of Fe 0 may therefore correspond to higher relative populations of H 2 oxidizing SRB.

Sulfur element balance
Sulfate conversion was analyzed with the contents of different forms of sulfur. Figure 5 shows the S balance in the three systems.   dissipated through other processes such as microbial assimilation, oxidation of sulfide into elemental sulfur, etc.
Apparently, the addition of Fe 0 greatly reduced the content of aqueous sulfide, which may lessen the inhibitory effect of sulfide on SRB.

Economic analysis
A rough economic analysis was performed on the operating costs, such as the costs of reagents and the costs of energy. It should be noted that this analysis is just an approximate tool to differentiate the trends in the operating costs associated with the use of combined treatment. A rigorous economic analysis should consider initial investment, prices at plant scale, maintenance and labor costs and so forth. Table 2 shows that the total reagent and power costs were $0.95/m 3 .
The cost of an advanced and complete treatment is always up to $5-7/m 3 for industrial wastewater (Oller et al. ), which is higher than that in the present study. This may be because the investments in equipment and construction, maintenance and labor costs were not included in the operating cost calculations in the present study.

CONCLUSIONS
The results of operating conditions on sulfate reduction in four systems indicate an enhanced activity of SRB by adding Fe 0 /Cu 0 . The highest sulfate reduction efficiency of 92.0% was obtained with the feed sulfate loading rate of 3,000 mg/L in the Fe/Cu þ SRB system, which was significantly higher than that of SRB and Fe þ SRB systems. The presence of Fe 0 could increase solution pH and decrease solution toxicity through the formation of metal sulfide precipitate. The sulfate reduction of the Fe þ SRB system could be further enhanced by adding Cu 0 . This study demonstrates that the Fe/Cu þ SRB system could be a promising technology for treating wastewater containing heavy metals and sulfate.