Low-Abundance Members of the Firmicutes Facilitate Bioremediation of Soil Impacted by Highly Acidic Mine Drainage From the Malanjkhand Copper Project, India

Abstract

Sulfate- and iron-reducing heterotrophic bacteria represented minor proportion of the indigenous microbial community of highly acidic, oligotrophic acid mine drainage (AMD), but they can be successfully stimulated for in situ bioremediation of an AMD impacted soil (AIS). These anaerobic microorganisms although played central role in sulfate- and metal-removal, they remained inactive in the AIS due to the paucity of organic carbon and extreme acidity of the local environment. The present study investigated the scope for increasing the abundance and activity of inhabitant sulfate- and iron-reducing bacterial populations of an AIS from Malanjkhand Copper Project. An AIS of pH 3.5, high soluble SO₄ ^2- (7838 mg/l) and Fe (179 mg/l) content was amended with nutrients (cysteine and lactate). Thorough geochemical analysis, 16S rRNA gene amplicon sequencing and qPCR highlighted the intrinsic metabolic abilities of native bacteria in AMD bioremediation. Following 180 days incubation, the nutrient amended AIS showed marked increase in pH (to 6.6) and reduction in soluble -SO₄ ^2- (95%), -Fe (50%) and other heavy metals. Concomitant to physicochemical changes a vivid shift in microbial community composition was observed. Members of the Firmicutes present as a minor group (1.5% of total community) in AIS emerged as the single most abundant taxon (∼56%) following nutrient amendments. Organisms affiliated to Clostridiaceae, Peptococcaceae, Veillonellaceae, Christensenellaceae, Lachnospiraceae, Bacillaceae, etc. known for their fermentative, iron and sulfate reducing abilities were prevailed in the amended samples. qPCR data corroborated with this change and further revealed an increase in abundance of dissimilatory sulfite reductase gene (dsrB) and specific bacterial taxa. Involvement of these enhanced populations in reductive processes was validated by further enrichments and growth in sulfate- and iron-reducing media. Amplicon sequencing of these enrichments confirmed growth of Firmicutes members and proved their sulfate- and iron-reduction abilities. This study provided a better insight on ecological perspective of Firmicutes members within the AMD impacted sites, particularly their involvement in sulfate- and iron-reduction processes, in situ pH management and bioremediation.

Keywords: Firmicutes; acid mine drainage; bioremediation; biostimulation; dissimilatory sulfate reduction; metagenomics; quantitative PCR.

FIGURE 1

Distribution of relative abundance of 16S rRNA gene sequences detected in microcosm setup (amended and unamended) at phylum-level (cumulative abundance > 0.1%) and class-level (top 18) taxonomic resolution. WPGA based agglomerative hierarchical clustering of treatments on the basis of abundance of phylum using Bray–Curtis dissimilatory matrix is represented. U denotes uncultured member.

FIGURE 2

Heat map based relative abundance of distribution of top 50 Clostridiales members in all the microcosm setup. U and O denote uncultured member and others, respectively.

FIGURE 3

Distribution of taxonomic groups at genera level in 0_Day, C_6M and C+L_6M. The 50 most abundant genera (cumulative abundance > 0.2%) associated with each sample is visualized in ternary plots. The position in the triangle indicates the relative abundance of each taxon among the three samples. U and O denote uncultured member and others, respectively.

FIGURE 4

Rank abundance based analysis of top 100 OTUs in the microcosm setup. Rank abundance profile of top 100 OTUs from each sample is depicted (A) C_6M, (B) H_6M, (C) C+L_6M, and (D) 0_Day. (E–H) represent the percent distribution of top 100 OTUs across the samples. (I) Venn diagram shows the shared and unique OTUs across the samples (considering top 100 OTUs from each sample). Detail taxonomic affiliations of 100 OTUs are presented in Supplementary Table S2

FIGURE 5

Taxonomic distribution pattern (at class-level) of top 100 OTUs in each setup.

FIGURE 6

Distribution of shared OTUs (from top 100 OTUs) between the samples (A) 0_Day and H_6M and (B) C+L_6M and C_6M. U denotes uncultured member.

FIGURE 7

Quantitative PCR based analysis of gene copy number of bacteria/specific taxa and dsrB gene among the microcosm setup.

FIGURE 8

Taxonomic distribution of enrichment setups from C_6M and C+L_6M. (A) Phylum-level distribution of enriched populations in Clostridium specific (Clos) and Facultative anaerobic (FA) enrichment. (B) Distribution of genera in Clostridium specific enrichment and Facultative anaerobic enrichment. (C) Shift in microbial diversity (represented at genera-level) when enriched population of Clos_C_6M and FA_C_6M were subjected to iron and sulfate reducing medium. Clos_IRM: distribution pattern of Clostridium specific enrichment in iron reducing medium; Clos_SRM: distribution pattern of Clostridium specific enrichment in sulfate reducing medium; FA_IRM: distribution pattern of Facultative anaerobic enrichment in iron reducing medium and FA_SRM: distribution pattern of Facultative anaerobic enrichment in iron reducing medium. U and O denote uncultured member and others, respectively.

FIGURE 9

Denaturing gradient gel electrophoresis (DGGE) profile of treatments (H_5M, C_5M, and C+L_5M) at 150 days of incubation. Red arrow indicates distinct band while blue represents common bands. ^∗Represents the organisms detected in both treatment and enrichment of C_6M and C+L_6M.