Anaerobic batch reactor treating acid mine drainage: Kinetic stability on sulfate and COD removal

Anaerobic batch reactor treating acid mine drainage: Kinetic stability on sulfate and COD removal

Journal of Water Process Engineering 31 (2019) 100825 Contents lists available at ScienceDirect Journal of Water Process Engineering journal homepag...

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Journal of Water Process Engineering 31 (2019) 100825

Contents lists available at ScienceDirect

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Anaerobic batch reactor treating acid mine drainage: Kinetic stability on sulfate and COD removal


Josiel Martins Costa , Renata Piacentini Rodriguez, Giselle Patrícia Sancinetti ⁎

Laboratory of Anaerobic Biotechnology - Science and Technology Institute, Federal University of Alfenas (UNIFAL-MG), Rodovia José Aurélio Vilela, 11999, BR 267, Km 533, 37715-400, Poços de Caldas, MG, Brazil



Keywords: Bioreactors Electron flow Heavy metals Kinetic parameters Sulfate-reducing bacteria

The leaching of an acid solution containing metals and dissolved sulfate constitutes an environmental problem known as acid mine drainage (AMD). An anaerobic sequencing batch reactor designed for AMD treatment was evaluated regarding its capacity to accommodate an increase in metals concentrations. Time profiles were performed to verify chemical oxygen demand (COD) and sulfate removal kinetics. The resulting apparent kinetic constant values showed a decrease in the reaction rate at the same time that sulfate removal increased. An increase in Fe2+ concentration from 100 to 400 mg L−1 decreased the sulfate removal rate (K ap S ) from ap (0.20 ± 0.03) to (0.072 ± 0.004) h−1, and the COD removal rate (K COD ) from (0.057 ± 0.003) to (0.027 ± 0.002) h−1. Even with the reduction in the COD and sulfate removal rates, the system was stable for the cycle time of 48 h in all operation phases.

1. Introduction Sulfide minerals present in mining waste are oxidized in the presence of water which implies an environmental problem that affects the soil, aquatic organisms, humans, and animals [1,2]. Studies of remediation techniques applied at actual mining sites, such as lime neutralization, wetlands, and permeable reactive barriers, provide insight into the long-term implications of AMD remediation. Therefore, despite available remediation approaches, AMD treatment remains a challenge [3]. Chemical precipitation, desalination, reverse osmosis, and ion exchange are other examples of processes used for removing sulfate from sulfate-rich wastewater, however, all of them have a significant cost. Alternatively, high-efficiency anaerobic biotechnology has encouraged researchers to develop the application of this technology for complex wastewaters [4]. Anaerobic wastewater treatment has been performed using several distinct reactor configurations [5–8]. These include, for example, by anaerobic sequencing batch reactor (ASBR), which offers such advantages as good solids retention time, the elimination of the secondary sedimentation step, efficient operating controls, and simple operation [9]. Several studies on metal and sulfate removal from wastewater indicate the ASBR is a good low-cost alternative [10–14]. The kinetics of the system can be affected by several factors, such as microorganism immobilization properties, substrate diffusion in the biofilm, stirring speed, long cellular retention times, a high biomass ⁎

concentration, bioreactor configuration, and environmental conditions (e.g., sulfide concentration, temperature, and pH). A deep understanding of the phenomena that occur in the system, such as the mass transfer between granule and substrate, is important for process improvement [9,15]. Considering the need to carry out studies to elucidate the fundamental phenomena that occur in the microenvironment of a particle and in its neighborhood, the main goal of this study was to verify the influence of increasing metals concentrations on the kinetics of COD and sulfate removal in an ASBR designed to treat AMD. Changes in the electron flow to the sulfidogenic process related to high metals concentration were also evaluated. 2. Methodology 2.1. Reactor setup and materials A bench-scale reactor, equipped with a paddle stirrer and a thermal bath (kept at 50 rpm and 30 °C, respectively) was used to treat synthetic acid mine drainage. The total volume reactor (7 L) was filled with 5.5 L of synthetic AMD every 48 h cycle. The biomass was contained during the entire reactor operation in a perforated stainless steel plate basket. The synthetic wastewater included sulfate and ethanol as a sole carbon source, both at 1500 mg L−1, from phase I to IV. In the adaptation phase, the COD and sulfate concentration was 1000 mg L−1. The

Corresponding author. E-mail address: [email protected] (J. Martins Costa). Received 11 February 2019; Received in revised form 26 March 2019; Accepted 8 April 2019 2214-7144/ © 2019 Elsevier Ltd. All rights reserved.

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Considering the maintenance of residual SO42−, the integral method was used, along with a modified first order kinetic model, with residual concentration, adjusted by non-linear regression (Levenberg-Marquardt method using OriginPro 8.0 software), as demonstrated by other authors for sulfate [9,12], and for COD kinetic parameters [20,21], whose expression is presented in Eq. (3), where S0 is the initial concentration (mg L-1) and t is time (h). For COD analysis, a first-order model was used, regardless the residual.

Table 1 ASBR phases with the addition of metals and time operation. Phase

Fe2+ (mg L−1)

Zn2+ (mg L−1)

Cu2+ (mg L−1)

Time operation (day)

Adaptation I II III IV

3.4 100 200 300 400

7.2 20 20 20 20

– 5 5 5 5

65 58 42 40 40

S(t) = SSR + (So

sludge, used as inoculum, was obtained from an up-flow anaerobic sludge bed reactor (UASB) used to treat poultry slaughterhouse effluent. Metals concentrations were defined according to the toxicity of sulfate-reducing bacteria (SRB) for these metals [16,17]. The composition of synthetic AMD was: Na2SO4, MgSO4.7H2O, FeSO4.7H2O, ZnCl2, and CuSO4.7H2O. The concentration of Mg2+ was 8.7 mg L−1 in all phases only as a nutrient, and the concentrations of Fe2+ and Zn2+ were 3.4 mg L−1 and 7.2 mg L−1, respectively, in the adaptation phase. The affluent pH was corrected closed to value 4 before cycle. The metals composition of wastewater varied among the five phases of this study, according to Table 1, which also shows the operating time in each phase.

SSR )e

Kap S t


3. Results and discussion 3.1. Removal data Table 2 presents the average results for pH, sulfide, sulfate, COD, and metals removals after 245 days. The sulfate removal rate increased as metals were added [13]. In general, the reactor operation was stable in all operation phases, demonstrating effluent pH values in the range of neutrality, which are adequate for microbiological development and allows adequate disposal in the water bodies. High removals of SO42−, COD, and metals (Fe2+, Cu2+, and Zn2+) were obtained.

2.2. Experimental procedure

3.2. Sulfidogenic activity and electron flow changes

The system was evaluated as a function of organic matter removal in the form of COD, sulfate, and metals removal. pH analyses of the affluent, effluent, and sulfide production were also performed. The time cycle was set to 48 h [10,12,14]. All analyses followed the procedures given by the Standard Methods for the Examination of Water and Wastewater [18].

To evaluate the competition between SRB and the other microbial groups (methanogenesis/fermentative), the electron flow to sulfate reduction was calculated based on the fraction of COD removed by sulfate reduction, taking into account that 0.67 g of COD are oxidized per gram of sulfate reduced [22] (Eq. (4)). Fig. 1 shows the proportion of electrons utilized by SRB and MPA/FB.

2.3. Kinetic adjustment

CODSO4 (%) = 0.67 × SO24

Temporal profiles were performed at the end of each phase, allowing the apparent kinetic parameters to be identified. The microbial growth was assumed to be constant, due to the slow growth of the biomass and the short duration of the temporal profile. Considering that mass transfer occurs between the granule and the synthetic AMD, and that it is the limiting step of the removal process using the first order model, it is possible to obtain Eq. (1), where dS/dt is the sulfate consumption rate (mg L−1 h−1), S is the substrate concentration (mg L−1) −1 and K ap ) [12,19]. S is the apparent kinetic constant (h

The increase in metals concentration throughout the phases increased the electron flow to sulfate-reducing process. The increase of Fe2+ concentration to 100, 200, 300, and 400 mg L−1 increased the electron flow used by SRB to (34 ± 7), (46 ± 7), (62 ± 10), and (63 ± 12)%, respectively. Even with decreasing COD removal, sulfidogenesis was favored by increased metals concentrations. Metals remove sulfide from the bulk liquid by precipitation, which reduces the inhibition of sulfide in the microbial community, due to the fact that sulfide combines with the iron in cytochromes or any other metalcontaining compounds that affect cellular functionality [23]. In addition, an overbalance in the chemical equilibrium of the sulfate reduction to sulfide can change the thermodynamics of the reaction, as the presence of metals consumes the sulfide in the solution, favoring the formation of the product [12]. The microbial interactions between SRB and MPA/fermentative are complex and variable depending on the operating conditions, reactor design, and environmental factors. In a down-flow fixed structured bed

dS = dt

K ap S S


From Eq. (1) for batch reactor design, the residual parameter (SSR) was added, as expressed in Eq. (2).

dS = dt

K ap S (S SSR )



removed (%)

Table 2 Operating results of the ASBR in phases I, II, III, and IV. Phase






pH effluent Sulfate affluent (M) Sulfate effluent (M) COD affluent (M) COD effluent (M) COD/Sulfate Sulfide effluent (M) Fe2+ removal (%) Zn2+ removal (%) Cu2+ removal (%)

6.8 ± 0.2 0.011 ± 0.003 0.007 ± 0.003 0.007 ± 0.003 0.002 ± 0.002 0.7 ± 0.3 0.001 ± 0.001 – – –

7.3 ± 0.2 0.017 ± 0.002 0.009 ± 0.002 0.017 ± 0.001 0.001 ± 0.001 1.0 ± 0.1 0.002 ± 0.001 99 ± 1 100.0 ± 0.1 97 ± 1

7.4 ± 0.1 0.016 ± 0.001 0.006 ± 0.001 0.016 ± 0.002 0.002 ± 0.001 1.0 ± 0.1 0.003 ± 0.001 99.5 ± 0.3 100.0 ± 0.1 97 ± 2

7.5 ± 0.2 0.016 ± 0.001 0.004 ± 0.002 0.015 ± 0.001 0.0025 ± 0.0004 1.0 ± 0.1 0.005 ± 0.001 99.8 ± 0.1 100.0 ± 0.1 95 ± 4

7.3 ± 0.2 0.016 ± 0.002 0.004 ± 0.001 0.015 ± 0.001 0.003 ± 0.001 1.0 ± 0.1 0.004 ± 0.001 99.8 ± 0.1 100.0 ± 0.1 93 ± 3


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metals concentrations. With lower concentrations of metals, it was observed that the sulfate removal rate was higher. Note that for each profile, there was a characteristic time for the stabilization of sulfate removal. As an example, the profile of phases III and IV shows that the stabilization of the removal occurs near the end of the cycle time (approximately 45 h), which may be related to the high metals concentrations present in these phases and the lower K ap S values, caused by a longer time required for sulfate removal. For the adaptation phase, I, and II, stabilization was observed at approximately 20, 30, and 33 h, respectively, characterizing higher K ap S values as compared to phases III and IV. The comparison of the kinetic constant with other research is complex due to variations in experimental conditions, inoculum characteristics, and bioreactor configuration. Nonetheless, some previous works have shown similarity to some of our operating conditions, inoculum, COD/SO4−2 ratio, time cycle, and electron donors [12,26,27]. ap However, even so, the K ap S values were not similar. The K S values obtained here were 0.44 ± 0.04, 0.4 ± 0.1, and 0.000235 h-1, respectively, highlighting an obstacle to true comparisons of sulfate kinetic behavior. Our results suggest a connection between the stoichiometry and the kinetics of the SRB process [28]. The COD kinetics analysis employed a first-order model, since data showed no evidence of residues, in contrast with models that describe a non-zero plateau. The use of a simpler model releases an extra degree of freedom for the fit, which is harmless here since no conclusion is drawn ap from the likelihood of this model. The K COD values showed a decrease in the COD removal rate with an increase in the metals concentration, which did not compromise the total removal since there was stabilization of the removal after 48 h. The COD kinetics were modified by the presence of metals, which did not present a perfect fit, since the previous studies that considered the presence of residual did not use wastewater containing sulfate and metals [20,21].

Fig. 1. Electron flow to sulfate reduction (SRB) and the methanogenesis/fermentative (MPA/FB) process.

reactor (DFSBR) for AMD treatment, the electron flow to SRB was stable around 38% when the Fe2+ concentration was increased from 100 to 400 mg L−1 [8]. Factors, such as the origin of the inoculum, the transport of substrate within the granule, the site of sulfate reduction, and its proximity to the site of methanogenesis/sulfidogenesis, determine the toxicity of the sulfide and the competitiveness interactions, thus potentially causing a unique scenario [24]. Sulfate removals from 72.6–92.5% were obtained using an up-flow anaerobic packed-bed reactor containing wastewater with the metals Cd2+, Ni2+, Fe2+, Cu2+, Pb2+, and Zn2+. The biomass characterization revealed that the precipitation of metals was associated with the outer and inner cell surfaces of SRB as a result of the sulfide generated by them [25]. 3.3. Kinetic analysis The temporal profiles constructed to evaluate the kinetic behavior of COD and SO42− removal are shown in Fig. 2. Table 3 shows the parameters obtained, the kinetic constants, and the adjustment coefficients. The theoretical residual sulfate concentration (SSR) decreased, with the exception of the adaptation phase, as the sulfate removal increased. Comparing the model with the real values of sulfate effluent, a similarity is observed mainly when considering the standard deviation, indicating that the model well-represented the data. Considering the parameter K ap S as an indicator of the reaction rate, it can be observed that from the addition of the metals Fe2+, Zn2+, and Cu2+ (phase I to phase IV), there was a decrease in the reaction rate, which may have been due to the high concentration of metals added into the system. This decrease in the reaction rate did not impair the sulfate removal for the 48 h cycle, as seen in Fig. 2, since the reaction rate became stable after a certain time within the expected cycle time. Further, according to Table 2, sulfate removal increased with increasing

4. Conclusions The results obtained in this study are crucial to understand the biological kinetics of AMD treatment and to optimize bioreactor operation procedures. Biological sludge was a suitable option for the AMD treatment with high metals concentration. The kinetic behavior of COD and sulfate removal through kinetic adjustment of the first order was representative apart from the increase in metals concentration. The ap COD (K COD ) and sulfate (K ap S ) apparent kinetic constant showed that there was a decrease in the reaction rate at the same time that COD and sulfate removal increased. This may have been due to the high metals concentration throughout the phases, resulting in a longer time required for COD and SO42− removal. It was verified that the increase of

Fig. 2. COD (a) and sulfate (b) temporal profiles at the end of each operational phase. Notes: (●) experimental point. (―) model used. 3

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Table 3 Kinetic parameters for COD and sulfate removal. Parameters −1

S0 (mg L ) SSR ± σ (mg L−1) Sulfate effluent ± σ (mg L−1)

−1 Kap S ± σ (h )

R2S COD0 (mg L−1) CODE ± σ (mg L−1) ap K COD ± σ (h−1)







789 ± 55 445 ± 3 698 ± 316 0.16 ± 0.01

1531 ± 107 797 ± 16 857 ± 144 0.20 ± 0.03

1532 ± 107 499 ± 26 622 ± 108 0.14 ± 0.02

1407 ± 98 308 ± 20 425 ± 176 0.08 ± 0.01

1387 ± 97 307 ± 16 413 ± 101 0.072 ± 0.004






1471 ± 103 208 ± 148 0.08 ± 0.01

1744 ± 122 95 ± 44 0.057 ± 0.003

1547 ± 108 168 ± 80 0.066 ± 0.002

1754 ± 123 237 ± 42 0.041 ± 0.002

1435 ± 100 327 ± 54 0.027 ± 0.002






Fe2+ concentration from 100 to 400 mg L-1 favored sulfidogenesis, with a flow of electrons used by SRB from (34 ± 7) to (63 ± 12)%. For the 48 h cycle time, the sulfate and COD removal rates were stable in all phases, which shows that the metals concentration applied was not toxic to the microbial consortium.

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