Metagenomic and culture-dependent approaches unveil active microbial community and novel functional genes involved in arsenic mobilization and detoxification in groundwater

Abstract

Background: Arsenic (As) and its species are major pollutants in ecological bodied including groundwater in Bangladesh rendering serious public health concern. Bacteria with arsenotrophic genes have been found in the aquifer, converting toxic arsenite [As (III)] to less toxic arsenate [As (V)] that is easily removed using chemical and biological trappers. In this study, genomic and metagenomic approaches parallel to culture-based assay (Graphical abstract) have made it possible to decipher phylogenetic diversity of groundwater arsenotrophic microbiomes along with elucidation of their genetic determinants.

Results: Seventy-two isolates were retrieved from six As-contaminated (average As concentration of 0.23 mg/L) groundwater samples from Munshiganj and Chandpur districts of Bangladesh. Twenty-three isolates harbored arsenite efflux pump (arsB) gene with high abundance, and ten isolates possessing arsenite oxidase (aioA) gene, with a wide range of minimum inhibitory concentration, MIC_As (2 to 32 mM), confirming their role in arsenite metabolism. There was considerable heterogeneity in species richness and microbial community structure. Microbial taxa from Proteobacteria, Firmicutes and Acidobacteria dominated these diversities. Through these combinatorial approaches, we have identified potential candidates such as, Pseudomonas, Acinetobacter, Stenotrophomonas, Achromobacter, Paraburkholderia, Comamonas and Klebsiella and associated functional genes (arsB, acr3, arsD, arsH, arsR) that could significantly contribute to arsenite detoxification, accumulation, and immobilization.

Conclusions: Culture-dependent and -independent shotgun metagenomic investigation elucidated arsenotrophic microbiomes and their functions in As biogeochemical transformation. These findings laid a foundation for further large-scale researches on the arsenotrophic microbiomes and their concurrent functions in As biogeochemical transformation in As-contaminated areas of Bangladesh and beyond.

Keywords: AMRs; Arsenotrophic bacteria; Arsenotrophic genes; Bioremediation; VFGs; Whole-genome shotgun sequencing.

Fig. 1

Graphical abstract showing overview of the study. This study was carried out to assess the diversity and transformation potentials of the arsenic-affected groundwater microbiomes using both culture-dependent and independent (shotgun metagenomics) approaches

Fig. 2

Minimum inhibitory concentration (MIC) of the arsenite tolerant bacteria. The MIC values detected ranged from 4 32 mM. Red and green stars indicate the presence of arsenite efflux pump (arsB) and arsenite oxidase (aioA) genes in the isolated bacteria, respectively. Each bar plot (deep maroon color) indicated the MIC (mM) value of each genus

Fig. 3

Phylogenetic tree of 16S rRNA gene sequences of arsenite tolerant groundwater bacteria. The maximum-likelihood tree was generated by MEGA 7.0 software, and visualized by iTOL v5.6.1. Nodal confidence in the resulting phylogenetic relationships was assessed using the bootstrap test (1000 replicates). Methanosarcina spp. was used as an outgroup. Different color codes (red: Pseudomonas, blueberry: Kluyvera, copper: Acinetobacter, violet: Strenotrophomonas, leafy green: Achromobacter, purple: Paraburkholderia, cyan: Comamonas, dark green: Lysinibacillus, and yellow: Methanosarcina) indicated different genera. Each reference and isolated strain’s GenBank accession number is displayed after the strain name

Fig. 4

Diversity of microbiomes in arsenic-contaminated groundwater. A Box plots illustrating within-sample (alpha) microbial diversity. Alpha diversity measured on the Observed species, Chao1, ACE, Shannon, Simpson, and InvSimpson indices showed significant variations (p = 0.013, Kruskal–Wallis test). B Principal coordinate analysis (PCoA) (measured on the Bray–Curtis distance method) showing significant microbiome diversity in different samples i.e., Chandpur and Munshiganj (p = 0.001, Kruskal–Wallis test)

Fig. 5

Taxonomic composition of microbiomes. Venn diagrams illustrate the unique and shared bacterial genomes in the arsenic-contaminated ground water samples of Munshiganj and Chandpur. A Venn diagram comparison of bacteria at an order level, B Venn diagram showing unique and shared bacterial families, C Shared and unique bacterial genera distribution between Munshiganj and Chandpur, and D Venn diagrams representing unique and shared species of bacteria two study areas. The blue circle indicates the microbiota that was shared between the study locations

Fig. 6

The taxonomic profile of the top 35 bacterial genera found in arsenic-contaminated groundwater. The 35 most prevalent bacterial genera are listed in order of decreasing relative abundance in six samples, with the remaining genera classified as ‘Other genera.’ Each stacked bar plot indicates the abundance of bacteria in the relevant category of samples. In contrast, the last two bar graphs represent the total relative abundance of bacterial taxa in Munshiganj and Chandpur district GW samples

Fig. 7

The species-level taxonomic profile of bacteria. The heatmap illustrates the hierarchical grouping of sample groups according to the relative abundance of the top 70 bacterial species revealed in the Munshiganj (M1-M4) and Chandpur GW metagenomes (C1-C2). The heatmap’s relative values (after normalization), shown by colors, represent the degree of bacterial species aggregation or content among samples based on the study region (Munshiganj and Chandpur), and criteria (pathogenic, opportunistic and non-pathogenic). The color bar (red to blue) depicts the row Z-scores (2 to -1.5), with red indicating high abundance and blue indicating low abundance. On the left, the color of the squares shows the relative number of bacterial species within each category. Additionally contains the distribution and relative abundance of the bacterial species found in the research metagenomes. The distribution and relative abundance of the bacterial species in the study metagenomes are also available in Data S1

Fig. 8

The taxonomic composition of the top 30 archaeal genera in arsenic-contaminated groundwater. The 29 most prevalent archaeal genera are listed in order of decreasing relative abundance in six samples, with the remaining genera classified as ‘Other genera.’ Each stacked bar plot indicates the abundance of archaea in the respective samples category. In contrast, the last two bar graphs represent the total relative abundance of archaeal genera in Munshiganj (M1- M4) and Chandpur (C1- C2) district GW samples

Fig. 9

Antimicrobial resistance genes (AMRGs) detected in arsenic-contaminated GW microbiomes. Metagenome sequencing data was used to search for open reading frames (ORFs) compared against the ResFinder database to identify AMRGs with over 95% sequence identity. The relative values in the heatmap (after normalization), depicted by colors, indicate the aggregation degree or content of AMRGs in the samples according to study region (Munshiganj and Chandpur) and types (biocides, drugs, metals, and multi-compound). The color bar (red to blue) displays the row Z-scores (2 to -1): red color indicates high abundance; blue color represents low abundance. The color of the squares on the left shows the relative abundance of the respective AMRGs in each group