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.

Comparative Methods for Quantification of Sulfate-Reducing Bacteria in Environmental and Engineered Sludge Samples

“…DNA was extracted by standard protocols, including several purification steps for eliminating humid acids that interfere with subsequent molecular techniques [30,36,46]. A volume of 12 mL of sludge or bacterial suspension was centrifuged for 30 min at 4000× g. The supernatant was discarded, and the pellet was used for DNA extraction using DNeasy ® PowerSoil ® Kit (QIAGEN GmbH., Hilden, Germany) according to the protocol provided by the supplier and a previous study [27].…”

“…This technique provides relative cell counts and requires specialized sequencing equipment and bioinformatic analysis. Therefore, its application is justified in studies seeking to understand the composition and structure of complex microbial communities [16,30]. In studies where precise absolute cell counts are necessary, such as those involving cell concentration assessments or process performance evaluations, alternative, more direct, and specific methods, such as cell counting, should be employed [3,31,32].…”

“…A sludge sample was obtained from an SR lab-scale bioreactor, initially inoculated with anaerobic sludge from a lagoon. This bioreactor was used for the bioprecipitation of copper and zinc (Figure 1a, II), whose sulfate-reducing activity had been monitored for two years [30,46]. The Shannon index (alpha-diversity) was 2.72 [30].…”

“…This bioreactor was used for the bioprecipitation of copper and zinc (Figure 1a, II), whose sulfate-reducing activity had been monitored for two years [30,46]. The Shannon index (alpha-diversity) was 2.72 [30]. The sample was collected in sterile plastic recipients, stored at 4 • C, and used for later comparison of enumeration methods.…”

“…The bottle headspace was flushed with nitrogen gas, and all bottles were incubated for 45 days in darkness in a climate-controlled chamber at 30 ± 2 • C. The enriched SR sludge was analyzed immediately by conventional methods. Likewise, genomic DNA extracts from serial dilutions of enriched SR sludge were obtained and stored at −80 • C. The Shannon index was 3.64 [30]. Following the previous procedure, two independent SR sludge samples (standard and control) were prepared to compare enumeration methods later.…”