Dynamics of Microbial Communities during the Removal of Copper and Zinc in a Sulfate-Reducing Bioreactor with a Limestone Pre-Column System

Abstract

Biological treatment using sulfate-reducing bacteria (SRB) is a promising approach to remediate acid rock drainage (ARD). Our purpose was to assess the performance of a sequential system consisting of a limestone bed filter followed by a sulfate-reducing bioreactor treating synthetic ARD for 375 days and to evaluate changes in microbial composition. The treatment system was effective in increasing the pH of the ARD from 2.7 to 7.5 and removed total Cu(II) and Zn(II) concentrations by up to 99.8% and 99.9%, respectively. The presence of sulfate in ARD promoted sulfidogenesis and changed the diversity and structure of the microbial communities. Methansarcina spp. was the most abundant amplicon sequence variant (ASV); however, methane production was not detected. Biodiversity indexes decreased over time with the bioreactor operation, whereas SRB abundance remained stable. Desulfobacteraceae, Desulfocurvus, Desulfobulbaceae and Desulfovibrio became more abundant, while Desulfuromonadales, Desulfotomaculum and Desulfobacca decreased. Geobacter and Syntrophobacter were enriched with bioreactor operation time. At the beginning, ASVs with relative abundance <2% represented 65% of the microbial community and 21% at the end of the study period. Thus, the results show that the microbial community gradually lost diversity while the treatment system was highly efficient in remediating ARD.

Keywords: acid rock drainage; anaerobic; bioreactor; community; diversity; dynamics; metagenomics; microbial; sulfate-reducing.

Figure 1

Scheme of the treatment system. (1) Peristaltic pump, (2) limestone pre-column (height: 25 cm, internal diameter (i.d.): 5.5 cm), (3) limestone pre-column effluent and/or bioreactor influent, (4) biological reactor (height: 43.2 cm, i.d.: 5.5 cm), (5) biogas, (6) sampling port of sludge.

Figure 2

(A) pH variation in the operation time of the treatment system fed with a pH-2.7 synthetic ARD containing sulfate (2000 mg L⁻¹), acetate as electron donor (2.5 g COD L⁻¹), Cu(II) (15 mg L⁻¹ during periods II and III) and Zn(II) (15 mg L⁻¹ during period III): limestone pre-column influent (●), limestone pre-column effluent/bioreactor influent (), and bioreactor effluent (). (B) Sulfate reduction (primary axis-left) and sulfide production (secondary axis-right) in the treatment system fed with a synthetic ARD containing sulfate (2000 mg L⁻¹), acetate as electron donor (2.5 g COD L⁻¹), Cu(II) (15 mg L⁻¹ during periods II and III) and Zn(II) (15 mg L⁻¹ during period III): sulfate (●) and sulfide (▲) in the influent and sulfate () and sulfide () in the effluent. (C) Concentration of soluble Cu(II) and soluble Zn(II) during the operation of the treatment system fed with a synthetic ARD containing sulfate (2000 mg L⁻¹), acetate as electron donor (2.5 g COD L⁻¹), Cu (II) (15 mg L⁻¹ during periods II and III) and Zn(II) (15 mg L⁻¹ during period III): limestone pre-column influent (●), limestone pre-column effluent/bioreactor influent (), and bioreactor effluent ().

Figure 2

(A) pH variation in the operation time of the treatment system fed with a pH-2.7 synthetic ARD containing sulfate (2000 mg L⁻¹), acetate as electron donor (2.5 g COD L⁻¹), Cu(II) (15 mg L⁻¹ during periods II and III) and Zn(II) (15 mg L⁻¹ during period III): limestone pre-column influent (●), limestone pre-column effluent/bioreactor influent (), and bioreactor effluent (). (B) Sulfate reduction (primary axis-left) and sulfide production (secondary axis-right) in the treatment system fed with a synthetic ARD containing sulfate (2000 mg L⁻¹), acetate as electron donor (2.5 g COD L⁻¹), Cu(II) (15 mg L⁻¹ during periods II and III) and Zn(II) (15 mg L⁻¹ during period III): sulfate (●) and sulfide (▲) in the influent and sulfate () and sulfide () in the effluent. (C) Concentration of soluble Cu(II) and soluble Zn(II) during the operation of the treatment system fed with a synthetic ARD containing sulfate (2000 mg L⁻¹), acetate as electron donor (2.5 g COD L⁻¹), Cu (II) (15 mg L⁻¹ during periods II and III) and Zn(II) (15 mg L⁻¹ during period III): limestone pre-column influent (●), limestone pre-column effluent/bioreactor influent (), and bioreactor effluent ().

Figure 3

(a) Analysis performed on Bray–Curtis distances or dissimilarities for six sludge samples during the three different operation periods of the sulfate-reducing bioreactor with the limestone pre-column system. Two samples or biological replicates were collected in each period. Period I (adaptation phase): day 0 (●) and day 24 (○). Period II (15 mg Cu(II) L⁻¹): day 181 (■) and day 217 (□). Period II (15 mg Cu(II) L⁻¹ and 15 mg Zn(II) L⁻¹: day 356 (▲) and day 372 (). (b) Relative abundances of most abundant amplicon sequence variants (ASVs) at genus level in six sludge samples during the three different operation periods of the sulfate-reducing bioreactor with the limestone pre-column system. Two samples or biological replicates were collected in each period.

Figure 4

Clusters and heat maps plotting log₁₀ of the counts for methanogenic archaea, SRB and syntrophic bacteria amplicon sequence variants (ASVs).

Figure 5

Phylogenetic tree based on V3 and V4 regions of 16SrRNA sequences datasets, showing communities of methanogenic archaea (red), SRB (blue) and syntrophic bacteria (yellow). The branches correspond to amplicon sequence variants (ASVs) reported in this study. Bootstrap values are shown as circles.