Identification of Exiguobacterium artemiae Strains Tolerant to Xenobiotics in Wastewater from the Bogota River, Colombia

Abstract

The Bogotá River receives a large amount of domestic and industrial wastewater, which has significant ecological impacts. Although these effluents charge several bacteria that represent a risk to human, animal, and vegetable health, some species can also resist high concentrations of contaminants. This study aimed to investigate the microbial diversity present in the wastewater of the Bogotá River through metataxonomic analysis. Additionally, the tolerance of a strain of Exiguobacterium artemiae isolated from these waters to xenobiotics was evaluated. The results showed a clear predominance of the phylum Proteobacteria. At the family level, the most prominent were Moraxellaceae, Comamonadaceae and Rhodobacteraceae, while at the genus level, Acinetobacter predominated over the other genera. Three bacterial isolates numbered 10, 13, and 18 were evaluated for their ability to grow in media supplemented with heavy metals such as chromo (Cr), cobalt (Co), cadmium (Cd), nickel (Ni), and several textile dyes. All three isolates grew in the presence of heavy metals and textile dyes, highlighting isolate number 13 for higher resistance to Co and Cr and isolate 18 for Co and Ni. Isolate 18 was identified via WGS as a strain related to Exiguobacterium artemiae 255-15, with 94.4% similarity. Genes and pathways associated with xenobiotic degradation routes, mainly aromatic compounds, EthD family reductases, FMN-dependent NADH-azoreductases, and chromate efflux transporters, were identified, suggesting their potential in the bioremediation of water contaminated with xenobiotics.

Keywords:
Pollution; whole-genome sequencing; Exiguobacterium artemiae; xenobiotics; efflux pumps.

HIGHLIGHTS

Severe pollution from wastewater harms the Bogotá River

Proteobacteria was the most abundant phyla in the Bogotá River.

Proteobacteria Firmicutes and Bacteroidota were the most abundant phyla in the Bogotá River

Exiguobacterium artemiae has genetic mechanism to resist heavy metal toxicity and other xenobiotics.

Exiguobacterium artemiae is a potential bioremediation candidate due to its resilience and detoxofication.

GRAPHICAL ABSTRACT

INTRODUCTION

The Bogota River is considered one of the most contaminated effluents of Colombia because of the continued exposure to several contamination sources from the beginning of the river at Paramo until its outlet in the Magdalena River in Girardot town. Along its 313 Km extension, the Bogotá River receives a high charge of pollutants, including xenobiotics, such as pharmaceuticals, pesticides, surfactants, hydrocarbons, dyes, and endocrine-disrupting compounds. Additionally, heavy metals like lead (Pb), cadmium (Cd), chromium (Cr), mercury (Hg), arsenic (As), and nickel (Ni) are also significant contaminants. These pollutants mainly originate from domestic sewage, industrial discharges from tanneries and metalworking operations, mining activities, and agricultural runoff [¹]. Xenobiotic pollution is concerning because it remains in the environment due to its biological activity and ability to persist in river sediments over time. The xenobiotic persistence in water bodies represents a significant risk to the health of aquatic organisms and humans, as they can induce cytotoxic, genotoxic, and mutagenic effects, and have been associated with cancer development [²,³]. Likewise, these pollutants can exert direct effects on the microbial communities of the river system, altering their structure and composition, and affecting their ecological functions [⁴].

On the other hand, heavy metals are particularly problematic because they are non-degradable and tend to bioaccumulate in living organisms, since their concentrations exceed environmental safety guidelines, posing ongoing risks to aquatic ecosystems and human health [⁵]. It has also been reported that metals such as Cd, zinc (Zn) and copper (Cu) can alter microbial diversity and favor the selection of antibiotic-resistant communities in urban river sediments [⁴], even though many bacteria possess beneficial properties, such as the ability to transform or immobilize metals through various metabolic pathways [⁶]. Similarly, these pollutants are being released into the Magdalena River, which serves as a primary source of water and fishing for a significant portion of the Colombian population [⁷]. This situation underscores how human activities disrupt ecosystems by introducing contaminants, leading to indirect adverse effects on the environment and human health [⁸].

One of the main activities in southern Bogotá and the surrounding towns is the leather industry, followed by extractive mining. The leather manufacturing uses chemicals, such as lime, tannins, dyes, chromium sulfates, acids, and solvents, some of which are retained in low proportions (<20%) in the final product, resulting in a high load of pollution in wastewater, directly affecting water bodies and soil [⁹]. On the other hand, mining results in high concentrations of heavy metals such as mercury (Hg) and Pb, which have been found above the maximum levels allowed in water bodies [⁷]. In addition, other compounds, such as polycyclic aromatic hydrocarbons (PAHs), trace elements, such as arsenic (As), strontium (Sr), silver (Ag) [¹⁰], chlorides, sulfates, phenols, and nitrates [¹¹]; and persistent organic pollutants (POPs), which are used in the plastics, textiles, and cosmetics industries, among others. They have also been found in sediments near the Magdalena River in Colombia and in the Biobío region in Chile, mainly in industrialized areas [¹²]. The presence of organic compounds and metals at concentrations above the maximum values allowed in bodies of water highlights the noncompliance with the regulations established by government institutions, which threatens the health of the communities and the deterioration of the rivers, as happens with the Bogotá River [¹³]. In addition to organic and inorganic pollutants, rivers are a transmission route for diseases caused by viruses, parasites, and bacteria [¹⁴,¹⁵]. In addition, Giardia spp. and Cryptosporidium spp. have been identified in the upper basin of the Bogotá River, as well as in water purification systems, suggesting parasitic contamination and considerable sanitary risk [¹⁴]. Similarly, bacteria constitute the majority group of isolates from different water sources, with members of the Enterobacteriaceae family being the most representative indicators of fecal contamination, especially Escherichia coli. Its presence indicates poor water quality and insufficient or nonexistent treatment [¹⁶-¹⁸]. In addition to Enterobacteriaceae, some investigations have detected virulent genotypes of Helicobacter pylori in samples from the Bogotá River [⁵]. Various genomic studies have identified bacterial communities belonging mainly to the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria, and Fusobacteria in different water sources, such as rivers, and wastewater from industries such as paper, leather and mining [¹⁹-²¹]. These phyla include a great diversity of bacterial genera that contribute to the cycle of elements and the degradation of pollutants, using them as sources of carbon and energy. In bioremediation processes, bacteria can transform pollutants into CO₂ and H₂O or convert them into less toxic intermediates for other species, which can then incorporate them into their metabolism [²²].

Metagenomic analyses and laboratory tests have revealed species that can transform or degrade xenobiotics. Some genera, such as the Christensenellaceae-R-7-group, Anaerolinea, Desulfovibrio, Candidatus Solibacter, and Leptolinea, have been detected in wastewater from the paper industry, where they could play a key role in the oxidation and detoxification of complex pollutants [²³]. The textile industry is one of the main contributors to wastewater worldwide. It contains a wide variety of dyes, the most common being azo derivatives [²⁴]. Genera such as Exiguobacterium, Kurthia, Kluyvera, Cedecea, Enterobacter, Aeromonas, Lysinibacillus, Acinetobacter, and Comamonas have been isolated from the effluents of this industry and have optimized the degradation of direct red dye (DR-28), which is known to be carcinogenic and mutagenic [²⁵]. On the other hand, studies by Dellamatrice and coauthors (2017) revealed that species of the genera Anabaena, Phormidium, and Synechococcus are capable of degrading textile dyes such as Indigo, RBBR, and Sulfur Black [²⁶].

The spread of residual pollutants, including heavy metals, in soils and water bodies threatens the biodiversity of ecosystems. Recent studies have identified heavy metals in wastewater, such as Zn, aluminum (Al), iron (Fe), and Cr [⁹,²⁷], and high concentrations of Cu, Pb, and Zn in surface soils contaminated with oil [²⁸]. Bacteria have developed resistance to heavy metals, which allows them to survive in highly polluted environments. An example of this is Pseudomonas aeruginosa CH07, which is resistant to Cd and Hg [²⁹]. Other genera with high levels of Hg resistance (> 50 ppm) include Proteus, Xanthomonas, Alteromonas, Aeromonas, and some members of the Enterobacteriaceae family [³⁰], and some bacteria have managed to eliminate cadmium and arsenic with efficiencies of 93% and 94%, respectively [³¹,³²]. Species of the genera Kosakonia, Klebsiella, Acinetobacter, and Serratia, which were isolated from tanneries' effluents, have demonstrated their ability to reduce Cr(VI) and tolerate the presence of Ni, Zn, Cd, and Hg [⁹,²⁷]. Biological processes show great promise for treating industrial wastewater, as chemical methods, including precipitation, sedimentation, chemical extraction, and oxidation, often involve chemical agents that can produce toxic byproducts harmful to the environment [⁹,²⁷,³³]. We analyzed bacterial communities via metataxonomic analysis in the Bogotá River and evaluated bacterial species with extended tolerance to heavy metals such as cobalt (Co), Cr, Cd, and Ni, as well as their resistance to different textile dyes. The bacterium Exiguobacterium artemiae was isolated from the Bogotá River and characterized by whole-genome sequencing (WGS), and the presence of genes encoding enzymes associated with xenobiotic degradation was analyzed, highlighting its ecological role in polluted environments.

MATERIAL AND METHODS

Sampling and physicochemical analysis of waters from the Bogota River

To conduct this study, three distinct water samples were collected from the Bogotá River at its confluence point with the Magdalena River at coordinates of 4°17′20.6″ N, 74°47′45.3″ W (Figure 1). This site, situated at the terminal stretch of the Bogotá River, is characterized by a high level of pollution that is discharged into the Magdalena River, a major source of water for the country. Each sample consisted of three liters of water, which were divided into three one-liter sterile amber bottles. The samples were stored in polystyrene refrigerators at a temperature of 4°C and transported to the laboratory for analysis. The physicochemical parameters measured in situ included pH, dissolved oxygen (%), conductivity (mS), and turbidity (NTU). The chemical oxygen demand (COD), nitrate and phosphate contents (mg/L) were determined via a portable multiparameter photometer (SCHOTT) and a Water quality test kit (Merck) as previously described [³⁴]. For microbiological analysis, the water samples were centrifuged at 8500 × g for 20 minutes to obtain the sediment, which was stored at -20 °C until further processing.

Figure 1
A. Geographical location of the Bogotá River in the sampling point. B. The confluence point between the Bogota and Magdalena rivers converges in Girardot, Cundinamarca (Colombia).

Bacterial screening and chromium tolerance

For bacterial isolation, an aliquot of sediment from the Bogotá River, previously obtained by centrifugation, was transferred to 50 mL Falcon tubes containing 10 mL of nutrient broth. The tubes were incubated at 32 °C for 24 hours. From this inoculum, serial dilutions of 1 × 10⁻¹ to 1 × 10⁻⁷ were prepared in sterile 0.9% saline solution, and 100 µL of the 1 × 10⁻³ to 1 × 10⁻⁶ dilutions were streaked onto nutrient agar plates using a glass rake to obtain individual colonies. The plates were incubated at 32 °C for 24 hours and twenty strains identified sequentially were selected. To assess chromium tolerance, a qualitative test was performed on plates with minimal salt medium (MSM): (6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 1 g/L NH₄Cl, 0.5 g/L NaCl, 0.246 g/L MgSO₄ · 7H₂O, 0.01 g/L CaCl₂, and 0.5% (w/v) glucose), according to [²³], supplemented with 5 and 10 mM potassium dichromate (K₂Cr₂O₇) as a source of Cr(VI) (pH 6.9). The isolates were incubated at 32 °C for 48 hours. At the end of the incubation, three isolates (10, 13, and 18) that exhibited good growth in the presence of Cr were selected for subsequent tests. All the assays were performed in triplicate.

Growth in the presence of Co, Ni, Cd, and Cr

Resistance to heavy metals was evaluated in isolates 10, 13, and 18 via a quantitative test in MSM supplemented with 5 mg/L of each metal (Co, Ni, Cd, or Cr) and 2 mg/L additional potassium dichromate (K₂Cr₂O₇) at pH 6.9. Briefly, 50 mL Falcon tubes were prepared with 5 mL of MSM supplemented with the corresponding metal. Each tube was inoculated with the strain adjusted to an optical density (OD) of 0.7 at 546 nm. The cultures were incubated at 32 °C with shaking at 180 rpm for 72 hours. The absorbance of the samples in triplicate was measured at 546 nm, and a blank composed of the culture medium and the corresponding metal was used as a reference.

Growth in the presence of azo dyes

The growth capacity of strains 10, 13, and 18 was evaluated in the presence of azo dyes used in the textile industry (black avitera, black terasil, and black lanaset). The qualitative test was carried out by inoculating each strain in Petri dishes with MSM agar (6 g/l Na₂HPO₄, 3 g/L KH₂PO₄, 1 g/L NH₄Cl, 0.5 g/L NaCl, 0.246 g/L MgSO₄ · 7H₂O, 0.01 g/L CaCl₂), supplemented with 2 mg/L of each dye as a source of carbon and energy. The plates were incubated at 32 °C for 24-48 hours. All tests were carried out in triplicate.

Bacterial strain 18 was subjected to decolourization of the three azo dyes under shaking conditions. Freeze strain was growth in the nutritive agar plate at 35°C O.N for adaptation. Then, for the preinoculum preparation, between 3 and 5 UFC were incubated in 7 mL of nutritive broth and incubated at 32°C,180 rpm, O.N. After that, bacterial culture was adjusted to an optical density (OD) of 0.7 at 546 nm and inoculated in 30 mL of MSM broth, supplemented with 0.5% (w/v) glucose and 2mg/L of each dye and incubated for 48 horas at 32°C,180 rpm by duplicate. After incubation, 1 mL of culture was centrifuged at 12,000 rpm for 5 minutes. The optical density of the clear supernatant was measured at 490 nm at final point. The decolourization activity in terms of percentage was calculated according to the formula below.

$$\%ofdecolourization=\left(initialabsorbance-finalabsorbance/initialabsorbance\right)x100$$

Metataxonomic analysis of water samples from the Bogotá River

To determine the microbial diversity in the waters of the Bogotá River, the samples were concentrated via a vacuum pump and 0.45 µm cellulose filters (Sartorius Biolab Products). Genomic DNA extraction was carried out via the DNeasy PowerLyzer Microbial Kit. The meta-taxonomic analysis was subsequently carried out via the rDNA 16S molecular marker, specifically in the variable regions V3--V4, via the oligonucleotides Bakt_341F (5'-CCTACGGGNGGCWGCAG-3') and Bakt_805R (5'-GACTACHVGGGTATCTAATCC-3') [³⁵]. Sequencing was performed on the Illumina MiSeq platform, generating paired-end reads (2 x 300 bp). An initial quality assessment of the raw reads was conducted using FastQC to evaluate sequencing quality metrics. Reads were subsequently filtered based on their Phred quality scores, retaining only high-quality sequences (Q ≥ 20). Raw data were initially processed with Kraken2 [³⁶] for taxonomic assignment via the SILVA v. 138 bacterial classifier. The results were uploaded to R software (v. 4.2.2; University of Auckland, Auckland, New Zealand) and subsequently normalized to 115,932 reads. Assignments containing fewer than 50 reads or fewer in every taxonomic category were removed. Taxonomic composition graphs were generated via the ggplot2 package.

Genomic and functional analysis of Exiguobacterium artemiae

Among the isolates showing enhanced growth in the presence of cobalt and nickel, strain 18 was selected for whole-genome sequencing and taxonomic identification. Genomic DNA extracted from bacterial strain was sequenced using an Illumina NovaSeq 6000 platform in paired-end mode (2 × 150 bp) at the Sequencing Unit of the University of Antioquia (Colombia). Sequencing libraries were prepared using the TruSeq Nano DNA Library Preparation Kit, generating paired-end reads of 150 bp suitable for downstream genome assembly and analysis.

Genome assembly was performed using SPAdes, followed by post-assembly filtering to remove contigs shorter than 1,000 bp using seqmagick. Assembly quality was first evaluated using QUAST by comparison with the reference genome of Exiguobacterium artemiae strain 255-15 (NCBI accession GCA_000019905.1), allowing the assessment of structural assembly metrics. Assembly completeness was subsequently assessed through the identification of conserved single-copy orthologs using BUSCO, applying the bacilli_odb10 lineage dataset selected for its taxonomic proximity to Exiguobacterium. Genomic relatedness was then evaluated by calculating average nucleotide identity (ANI) with fastANI, using the same reference genome.

Genome annotation was carried out via BlastKOALA [³⁷] and the BV-BRC platform [³⁸]. Subsystem prediction and metabolic network reconstruction were performed in the SEED database and the RAST server [³⁹].

A list of the identified KEGG Orthologies (KOs) was generated, and the presence of enzymes involved in the degradation pathways of xenobiotics was examined. In addition, the NCBI database was consulted to identify genes that encode proteins previously reported for their role in xenobiotic catabolic process. The sequence is found in NCBI with the following codes: Assembly: ASM3334281v1, GenBank GCA_033342815.1, and RefSeq: GCF_033342815.1. Protein network analysis and functional enrichment were performed via the STRING server to infer associations with specific biological processes related to microbial metabolism in polluted environments [⁴⁰]. Enzyme structures (obtained from the AlphaFold database, https://alphafold.ebi.ac.uk/) with functional annotations such as azo-reductases (azoR) were structurally docked with azo dyes (obtained from Drugbank https://go.drugbank.com/) via Autodock Vina implemented on the Swissdock server to evaluate their substrate affinity [⁴¹].

Data analysis

Bacterial isolation and growth in the presence of heavy metals were performed in at least three different cultures. The determinations were taken in triplicate, and the means and standard errors were calculated to determine significant differences (p <0.01).

RESULTS

Analysis of the physicochemical parameters of the water sample

The physicochemical parameters of the water from the Bogotá River allow us to evaluate its quality, which can have an important impact on the water bodies that receive it. The physicochemical parameters were 67.30% for DO, 0.33 mS/cm for EC, and 334 mg/L for COD (Table 1). The physicochemical profile corresponds to a freshwater system with moderate mineralization but severe organic pollution, consistent with wastewater-impacted or highly contaminated surface water under aerobic stress conditions. These values reflect poor water quality, according to the 2023 assessment of the lower basin of the Bogotá River conducted by local environmental authorities. Moreover, the high concentration of nitrates (102 mg/L) exceeds the permissible limit (11 mg/L) in wastewater intended for agricultural use, according to Law 1256 of 2021 of the Colombian Ministry of the Environment and Sustainable Development. The turbidity values (655 NTU) exceeded the allowed values according to national environmental regulations.

Table 1
Physicochemical analysis of the water from the Bogotá River.

Parameter	Value	Value according to the ICA
Dissolved Oxygen (DO)	67.30%	0.673
Electrical Conductivity (EC)	0.33 mS/cm	0
pH	7.7	1
Turbidity	655 NTU	-
COD (Chemical Oxygen Demand)	334 mg/L	0.13
Nitrates	102 mg/L	-
Phosphates	144 mg/L	-

Taxonomic diversity in the waters of the Bogotá River

Following normalization and removal of low-representative taxonomic groups (< 50 reads), the final dataset consisted of 116,391 reads, averaging 701 reads per sample. In total, 166 taxonomic identifiers (Taxon IDs) were detected, distributed across 7 phyla, 9 classes, 30 orders, 52 families, and 122 genera (Supplementary Table S1), which accounted for 99.6%, 99%, 97%, 95%, and 82% of the assigned reads, respectively.

At the phylum level, Proteobacteria dominated the bacterial community, accounting for over 95% of the total relative abundance across all samples (Figure 2A). At the family level, community structure was primarily shaped by three predominant families, Moraxellaceae (48%), Comamonadaceae (13%), and Rhodobacteraceae (12%), which collectively represented approximately 73% of the total abundance (Figure 2B). At the genus level, Acinetobacter emerged as the most abundant taxon, comprising 54% of the total reads, followed by sequences assigned to uncultured bacteria (5%) (Figure 2C). No substantial differences in taxonomic composition were observed among the three replicates, as reflected by the highly similar relative abundance patterns across all taxonomic levels analyzed. The dominance of the same major phyla, families, and genera in each replicate, together with the comparable proportional contributions of the most abundant taxa, indicates a stable and reproducible microbial community structure across samples.

Figure 2
Taxonomic diversity of bacteria in the Bogotá River. Taxonomic diversity at the phylum (A), family (B), and genus (C) levels. Each column shows the replicates for each category. The Y-axis shows the reported abundance value.

Isolation of bacterial strains and resistance to heavy metals

Twenty bacterial isolates were recovered from the analyzed sediments and tested for growth on MSM supplemented with chromate (5 and 10 mM Cr6+). Among these isolates, those identified as 10, 13, and 18 showed the best growth and were selected for metal tolerance testing.

The three selected isolates were cultured in 5 mg/mL of heavy metals such as cobalt (Co), nickel (Ni), cadmium (Cd), and chromium (Cr) for 72 hours in MSM. The results shown in Figure 3 indicate that all three isolates grew (tolerant) in the presence of the metals, with isolate 18 showing the best growth for all metals, especially nickel (p < 0.01). This allows us to infer that this strain could be resistant or tolerant to the toxicity that these metals generally cause to most organisms. The growth of isolate 13 was similar to that of isolate 18, with greater resistance to Co and Cr. Finally, isolate 10 presented lower growth values but similar tolerances to all the metals tested.

Figure 3
Bacterial growth in the presence of heavy metals. The graph shows the growth measured via absorbance (546 nm) for isolates 10, 13, and 18 in the presence of 5 mg/mL cobalt (Co), nickel (Ni), cadmium (Cd), or chromium (Cr). A significant difference (p <0.01) is represented with the symbol *. The data represents the average of three replicates for each trial.

Growth in the presence of azo dyes

Isolates 10, 13, and 18, resistant to heavy metals, were evaluated for their growth on media containing azo dyes. The strains were cultured on MSM supplemented with 2 mg/ml of Avitera Black, Terasil Black, and Lanaset Black. The results revealed that all bacterial isolates showed abundant growth (+++) in the dyes tested, although strain 18 showed less growth (+) in Lanaset Black (Table 2). These findings suggest the ability of these isolates to utilize azo dyes as a carbon and energy source without altering their metabolism, perhaps by destabilizing the azo functional group (-N=N-), which is generally toxic to many organisms.

Table 2
Bacterial growth in the presence of azo dyes (Terasil Black, Avitera Black, and Lanaset Black).

Isolate	Black Terasil	Black Avitera	Black Lanaset
10	+++	+++	+++
13	+++	+++	+++
18	+++	+++	+

Considering the strains' ability to grow in the presence of azo dyes, we performed a decolorization assay using E. artemiae. The results show that this strain maintains its growth capacity in the presence of these dyes, achieving a decolorization percentage of 15.8% for lanaset black and 17.5% for avitera black (Table 3). However, E. artemiae did not show a significant effect on the terasil black dye.

Table 3
Decolorization test of terasil, avitera, and lanaset black dyes by E. artemiae.

Dye	D.O (24h)	% decolorization
NT	0,218	-
NL	0,174	15,85
NA	0,192	17,5

Genomic and functional analysis of Exigoubacterium artemiae

The draft genome assembly of strain 18 consisted of 40 contigs, with a total length of 3,229,202 bp was obtained, with a GC content of 47.12%. Assembly continuity was reflected by an N50 value of 146,209 bp. The completeness of the gene space was assessed using BUSCO with the bacilli_odb10 dataset, which detected 99.3% complete BUSCO orthologs, all as single copies. A mere 0.7% of expected genes were categorized as missing, with no fragmented or duplicated BUSCOs identified, indicating a highly complete gene repertoire. Average nucleotide identity analysis using fastANI yielded 94.51% identity between the genome of strain 18 and the reference genome of Exiguobacterium sibiricum strain 255-15 (GCA_000019905.1). This assignment was robustly confirmed and complemented by a phylogenomic analysis (Maximum likelihood) based on 111 concatenated genes, which reconstructed the evolutionary history of the group with high support and allowed the confident statistical assignment (100) of bacterial isolate 18 to the genus Exiguobacterium. Furthermore, this analysis demonstrated the formation of a monophyletic group between isolate 18 and the reference genome of E. sibiricum, thereby consolidating its identification at the species level. Recently, a taxonomic reclassification inside of the genus Exiguobacterium was proposed, considering E. sibiricum as a later heterotypic synonym of E. artemiae; thus, sequence comparison of isolate 18 in the Nucleotide database at NCBI resulted in E. artemiae [⁴²]. This ANI value exceeds commonly accepted thresholds for genus-level relatedness and approaches species delineation cutoffs (approximately 95 - 96%) [⁴³], supporting a close phylogenetic relationship between strain 18 and E. artemiae. Further taxonomic resolution of the E. artemiae isolate revealed a 99% identity with the E. artemiae strain 9AN (seq. accession GCA_025234645.1) according to 16S rRNA sequence comparison in the BacDive database (https://bacdive.dsmz.de/strain/18118).

Functional annotation with the KEGG database identified approximately 3300 genes, 1703 of which were assigned to one of the functional categories of KEGG (Supplementary Table S2). Among these genes, 12 genes are involved in xenobiotic degradation and genes that confer resistance to heavy metals. Table 4 shows the 7 genes associated with xenobiotic degradation, which encode the following enzymes: ring-cleaving dioxygenase, EthD family reductase, nitronate monooxygenase, FMN-dependent NADH-azoreductase (azoR), Multicopper oxidase, aromatic amino acid hidroxylase and catechol 2,3 dioxygenase. These proteins are associated with the catabolic process of aromatic compounds and xenobiotic degradation according to the biological process annotation. Table 5 shows the five genes identified that encode efflux pumps and transporters with functional annotations related to resistance to heavy metals.

Table 4
Genes involved in xenobiotic metabolism in E. artemiae.

Enzyme	Degradation Pathway	Locus Tag	Access Code NCBI	UniProt
Ring-cleaving dioxygenase	Degradation of aromatic compounds	R7V76_RS02430	MDW2884425.1	B1YLN1
EthD family reductase	Ethyl tert-butyl ether degradation	R7V76_RS03990	MDW2884729.1	B1YIJ4
Nitronate monooxygenase	Degradation of nitroaromatic compounds	R7V76_RS04030	MDW2884737.1	B1YIK2
FMN-dependent NADH-azoreductase azoR	Degradation of azo dyes	R7V76_RS10910	MDW2886100.1	B1YHK7
Multicopper oxidase domain-containing protein	Degradation of aromatic compounds	R7V76_RS09790	MDW2885879.1	U1LJE8
Aromatic am ino acid hydroxylase	Degradation of aromatic compounds	R7V76_RS11305	WP_318092558.1	B1YMF3
Catechol 2,3-dioxygenase	Degradation of aromatic compounds	R7V76_RS03330	WP_318091250.1	B1YHY5

Table 5
Genes involved in heavy metal resistance in E. artemiae.

Protein name	Locus Tag	Access Code NCBI	UniProt
Chromate efflux transporter	R7V76_RS02365	WP_318091137.1	B1YLL8
Formate/nitrite transporter family protein	R7V76_RS08455	WP_012369453.1	B1YJJ9
Arsenite efflux transporter metallochaperone ArsD	R7V76_RS12930	WP_318092970.1	U1N1P1
Metalloregulator ArsR/SmtB family transcription factor	R7V76_RS08435	WP_214767312.1	B1YJJ5
Heavy metal translocating P-type ATPase	R7V76_RS03215	WP_318091232.1	B1YHW0

The enzymes involved in xenobiotic degradation identified in the genome of strain 18 E. artemiae are listed. The locus tag column references the annotated gene and the NCBI Access Code number of the genome deposited in GenBank: GCA_033342815.1. Protein network analysis (16 nodes, edge 27) and functional enrichment (PPI enrichment p-value: 8.35e-06) revealed associations with monocarboxylic acid catabolic process (1.31e-07), microbial metabolism in diverse environments (1.31e-07), short-chain dehydrogenase/reductase, and xenobiotic catabolic process (1.10e-05) (Figure 4).

Figure 4
Local network cluster (STRING) enrichment of genes related to xenobiotic metabolism of E. artemiae.

In the catabolic process of xenobiotic compounds, azoreductases (azoRs) play a key role in mitigating dye contamination. For their study, the three-dimensional structures of the azoR from E. artemiae (identification code AF-B1YHK7-F1-v4) and Bacillus anthracis (3P0R PDB accession) were retrieved from the AlphaFold and PDB databases, respectively. Structural comparison revealed a sequence alignment score = 593.1 and the RMSD between 175 pruned atom pairs was 0.948 angstroms; (across all 208 pairs: 1.682). On the other hand, available azobased colorants including aniline, reactive red 1, acid yellow 54 free acid, and methyl red were retrieved from the DrugBank database. Docking analysis showed potential interactions between azoR and azo-based dyes with higher affinity for acidic ligands carrying sulfonyl groups such as the acid yellow 54 free acid (Supplementary Table S3) [⁴⁴,⁴⁵].

DISCUSSION

Among the various water sources in Colombia, the Bogota River faces significant pollution issues [⁴⁶]. Although these rivers are used for agricultural production and for supplying drinking water, their water quality is contaminated due to excessive accumulation of nutrients from agricultural and industrial sources and urban discharges [⁴⁷,⁴⁸]. This pollutant discharge reduces the water oxygen concentration and alters biological diversity owing to the presence of xenobiotics such as heavy metals, which represent a risk to human and animal health and other ecosystems [¹¹]. This study provides fundamental data for understanding the impact of pollution on biological diversity and microbial adaptation to extreme environments to enable their use in bioremediation or as biological bioindicators of pollution.

The physicochemical parameters of the lower basin of the Bogota River reflect its high degree of contamination, which begins in the upper basin with the impact of tannery industries. Despite being freshwater (based on EC), the very high COD combined with reduced DO indicates intense organic contamination and active oxygen consumption. Consistently, the values obtained in our study for dissolved oxygen (0.673), chemical oxygen demand (0.13), and electrical conductivity (0.00) indicate that the water of the Bogotá River has poor quality since these values are below the range of acceptable or good values (0.71-1.0), depending on the water quality index [¹¹]. Although the Bogotá River in its lower basin has received water from another river cleaner that can gradually dilute the level of contamination, this study sampled at the connection with the Magdalena River continues to show a high load of organic and inorganic matter that can be oxidized, causing a high chemical oxygen demand and a decrease in dissolved oxygen in water [⁴⁹]. On the other hand, the high content of phosphates may be related to household discharges with high concentrations of detergents or industrial effluents that use phosphate salts in their processes. Likewise, the nitrate concentration usually indicates contamination by human or animal waste or runoff from agricultural fertilizers, leading to large eutrophication processes and reducing the survival of various aquatic species [⁵⁰]. These parameters are key indicators of water quality and reflect the impact of wastewater and urban discharges on the Bogotá River. Although this study did not consider the typical seasonal variations in Colombia (dry and rainy seasons), it is important to note that these variations significantly influence the physicochemical and microbiological parameters of the water. This has been documented in previous research, such as that of Páez and coauthors (2025) [⁵¹], who analyzed rivers in Urabá, Colombia, and Debassi and coauthors (2025) [⁵²], who studied surface and groundwater, as well as treated and untreated wastewater in Algeria.

Despite the high levels of contamination of the Bogota River, several bacterial communities may accomplish important ecological roles. Notably, the analysis of taxonomic diversity revealed a high prevalence of the phylum Proteobacteria in the analyzed sample with high wastewater discharge [¹⁶-¹⁸] (Figure 2), which has been identified in previous studies performed in the Seine River in France and the Scheldt in Belgium [¹⁹-²¹]. In particular, these phyla play a fundamental role in biological and chemical processes that occur in contaminated environments, such as maintaining the active nitrogen, sulfur, and carbon cycles that are fundamental in the decomposition of organic matter [⁵³]. These phyla not only suggest that the microbiological ecosystem is in the process of adapting to altered conditions of eutrophication and contamination but also that they may also be involved in pollutant degradation and may become an alternative for bioremediation [⁵⁴-⁵⁶]. Additionally, the genera Acinetobacter and the bacteria of the family Rhodobacteraceae were predominant in the analysis of taxonomic diversity in this study (Figure 2), and these microorganisms are closely related to water contaminated specifically by their metabolic adaptations and ability to degrade organic compounds such as hydrocarbons [⁵⁷,⁵⁸], hydrogen sulfide and tolerance to other toxic compounds, which makes these microorganisms potential agents for bioremediation [⁵⁹].

Three bacterial strains (10, 13, and 18) were isolated and grown in the presence of heavy metals. Strain 13 exhibited higher resistance to cobalt (Co) and chromium (Cr) (Figure 3), while strain 18 demonstrated superior resistance to all tested metals. This enhanced tolerance could be attributed to the strain's ability to cope with the toxicity of these metals, potentially through biochemical or physiological mechanisms such as the production of metal-binding proteins, active expulsion via efflux pumps, and bioaccumulation, among others. Consequently, strain 18 presents significant potential for applications in biotechnology and bioremediation. Genome sequencing revealed that strain 18, with the greatest growth in all metals (nickel and cobalt), corresponded to E. artemiae. Taxonomic resolution was confirmed genotypically at the BacDive database [⁶⁰], assessing the species identity without any ambiguity or further taxonomic determination. The genus Exiguobacterium is widely distributed because of its adaptability and ability to reduce the effects of numerous pollutants, including heavy metals and organic pollutants, in soil and water matrices [¹,⁵⁶,⁶¹]. Exiguobacterium has also been identified in various places impacted by human activity, such as oilfields [⁶²,⁶³], contaminated soils [⁶³], wastewater from industrial processes [⁶⁴], treatment plants [⁶⁵], and has also been found in the skin microbiome of horses suggesting its source from tannery industrial residues [⁶⁶]. In particular, E. artemiae has been described in the Siberian permafrost [⁶⁷]. However, it has also been reported in natural wetlands [⁶⁸] and built [⁶⁹], as well as in polluted rivers [⁷⁰]. Different studies have reported that E. artemiae can survive under adverse temperature conditions [⁷¹], salinity, or even oxygen levels [⁷²]. Exiguobacterium species have been classified as bioemulsifiers through lipopeptide production which has led to high interest in its industrial, biotechnological, and bioremediation applications [⁷³].

In accordance with previous studies, growth tests of E. artemiae were carried out on azo dyes, such as Avitera black, Terasil and Lanaset, to evaluate their potential in environmental bioremediation [⁷⁴]. These colorants are frequently used in the textile, leather, cosmetics, food, plastics, and paper industries [⁷⁵, ⁷⁶]. As in many countries, in Colombia, stricter regulations have been established for its use and disposal because of its high toxicity and the risk of releasing dangerous compounds into the environment. The isolated strains were subjected to heavy metal stress and grown with these azo dyes. In general, the three strains (10-13-18) had abundant growth in the three tested dyes, although strain 18 showed less growth in the Lanaset black dye (Table 2). These findings correlate with previous studies that reported energy generation in addition to bleaching and detoxification processes [⁷⁷,⁷⁸]. These findings indicate that these microorganisms use azo dyes as a source of carbon and energy. The results of the decolorization assay showed that E. artemiae was able to degrade these dyes, although with low percentages of decolorization (Table 3). One of the possible mechanisms involved in this degradation could be related to the production of enzymes such as FMN- and NADH-dependent azoreductase (azoR), whose gene was identified in its genome. Azoreductases have been identified in the degradation of dyes such as Reactive Black 5 by Staphylococcus sp. strain MEH038S [⁷⁹].

Although this process is not very common, azo dyes are complex organic compounds that contain an azo group (-N = N-) in their structure, which confers resistance to degradation. The low yields observed in this study may be related to a low production of oxidoreductase and oxidase enzymes which produce the mineralization of the dyes or in turn with the production of intermediate metabolites such as amines that can be toxic to the cell [⁸⁰], decreasing its viability. Currently, certain microorganisms have developed mechanisms to decompose it and produce smaller metabolites, facilitating its survival [⁷⁷,⁷⁸,⁸¹].

The genomic and functional analysis of the E. artemiae genome revealed the presence of 1703 genes in the functional category of the KEGG database. Five genes encode proteins of interest related to resistance to heavy metals, including chromate efflux transporters, formate/nitrite transporter family proteins, arsenite efflux transporter metallochaperone ArsD, the metalloregulator ArsR/SmtB family transcription factors, and heavy metal-translocating P-type ATPases (Table 5). These enzymes have been previously reported in other heavy metal resistance studies. For example, one of the most studied mechanisms of chromate resistance is encoded by the chrA gene, which has been studied mainly in Bacillus, Geobacter, Flavobacterium, Methanosarcina, and Serratia [⁸²-⁸⁴]. In addition, it is a chromate efflux transporter protein, a heavy metal translocator P-type ATPase [⁸⁴], and ArsD, a metal chaperone for the efflux transporter of arsenite, has been reported as a transporter of metal cations such as chromate [⁸⁵], Cu, Zn, Cd [⁸⁶,⁸⁷] and arsenite [⁸⁸,⁸⁹] outside the cell. These findings suggest that, by expressing the genes that encode these molecules, E. artemiae has mechanisms to overcome the toxicity of these compounds and prevent oxidative damage. The genomic analysis of E. artemiae revealed the presence of proteins that favor other mechanisms of cell survival, as well as transcription factors that control the expression of genes involved in resistance to heavy metals. Our study revealed that E. artemiae expresses a gene involved in the expression of the formate/nitrite transporter (TNF) family of proteins. This protein is not directly related to resistance to heavy metals, but it is reported to facilitate the transport of anions such as formate and nitrite through the cell membrane [⁹⁰,⁹¹]. These proteins play indirect roles in cellular homeostasis and the response to stress conditions. On the other hand, E. artemiae expresses a transcription factor of the ArsR/SmtB family, a transcriptional regulator that acts as a heavy metal sensor in the cellular environment by detecting toxic concentrations, modulating the expression of resistance genes, and promoting the activation of the detoxification and efflux system [⁹²]. A particular example of this is the case of Thermus thermophilus, a transcription factor member of the ArsR/SmtB family that regulates arsenic resistance genes in this species and is recognized for inhabiting extremely high-temperature environments [⁹³].

With respect to xenobiotic degradation, E. artemiae may transform recalcitrant compounds into less toxic metabolites through specific metabolic pathways. One of these routes is related to catechol 2,3-dioxygenase, which is found in this species and participates in the degradation of chlorocyclohexane and chlorobenzene, as well as that of xylene and styrene, converting them into metabolizable molecules and, for this reason, is related to the degradation of polycyclic aromatic hydrocarbons (HPAs) such as aromatic amines and benzene derivatives [⁸⁰]. This is supported by studies carried out on the Rhodococcus jostii RHA1 strain isolated from soils with polychlorinated biphenyl compounds (PCBs), which is a species that is characterized by the presence of this enzyme [⁹⁴]. Other enzymes reported in the genomic analysis of E. artemiae include the EthD family reductase, FMN-dependent NADH-azoR, catechol 2,3-dioxygenase, and nitronate monooxygenase, which are enzymes related to the degradation of organic and organo-halogen pollutants, among other compounds [⁹⁵]. Some of these enzymes have been reported to be responsible for the reduction of azo compounds [⁷⁹], aromatic compounds [⁹⁶] and nitro-chemicals [⁹⁷] including fertilizers, pesticides or industrial dyes [⁹⁸], such as those evaluated in this study, and other enzymes have been reported to be responsible for the elimination of nitro groups and halogen groups from organic compounds [⁹⁹]. E. artemiae not only expresses genes that encode enzymes related to the elimination of groups but also expresses genes encoding enzymes responsible for the modification of groups, as in the case of the hydroxylation of aromatic groups, which is catalyzed by the enzyme aromatic amino acid hydroxylase [¹⁰⁰], facilitating the elimination of this compound through catabolic routes. The docking analysis between azoR and azo dyes showed a likely affinity suggesting a possible specific substrate for those ligands. Thus, the azoR enzyme from E. artemiae might be important in a bioremediation strategy because it enables the environmentally friendly degradation of azo dyes, which are prevalent pollutants in industrial effluents [¹⁰¹]. AzoR enzymes are used to detoxify and mineralize dye-contaminated wastewater, reducing toxicity and environmental impact [¹⁰²]. Their biotechnological application involves the use of microbial strains that produce these enzymes to biologically decolorize and degrade azo dyes [¹⁰³].

The presence of organofluorine compounds in various industrial applications has led to increases in the concentration of F- in natural environments [¹⁰⁴]. Owing to their abundant presence in the environment and their toxic effects, some bacteria have evolved mechanisms of resistance to fluoride. However, little is known about these strategies and pathways [¹⁰⁵]. E. artemiae contains the crcB gene, which encodes a fluoride efflux transporter that plays a role in resistance to high concentrations of halogens.

The heavy metal translocator P-type ATPases constitute a large family of ion-transport proteins, such as H+, Na+, K+, Mg2+, Ca2+, Ag+/Ag2+, Zn2+, Co2+, Pb2+, Ni 2+, Cd 2+, and Cu+/Cu2+ [¹⁰⁶]. The P-type ATPase of E. sibiricum shares 99% homology with the zinc/cadmium/lead-transporting P-type ATPase of Escherichia coli (strain K12), an enzyme that combines the hydrolysis of ATP with the export of Zn, Cd, Pb, Co, Ni and Hg with higher activity when metals are present as metal-thiolate complexes [⁸⁶,⁸⁷]. Additionally, 97% similarity was shown with the Cd, Zn and Co-transporting ATPase from Bacillus subtilis (strain 168), an enzyme capable of combining the hydrolysis of ATP with the transport of Cd, Zn and Co out of bacterial cells. However, it does not transport copper [⁸⁶,⁸⁷].

In conclusion, a set of oxidative enzymes, transcriptional regulators, and efflux transporters encoded in the E. artemiae genome might reflect its ability to survive harsh conditions with heavy metals and xenobiotic compounds and make it a potential candidate for bioremediation of contaminated water or, in general, in water and soil bioremediation processes. However, it is still necessary to expand studies with a specific characterization at the metabolic level to determine the ability of this bacterium to degrade xenobiotics and, specifically, what type of molecules it degrade and even identify the metabolites that it releases as products of waste. Identifying species with enhanced azoR activity is vital for bioremediation and biotechnological applications because it would allow for the development of effective and sustainable strategies to degrade azo dyes in water bodies and the optimization of microbial consortia for specific remediation needs.

• Funding:
  This research was funded by The Research Council from Universidad Antonio Nariño; internal grant number 2022207 and The APC was funded by Universidad Antonio Nariño.
• Institutional Review Board Statement:
  Not applicable.
• Informed Consent Statement:
  Not applicable.

Acknowledgments:

We thank Antonio Nariño University for financing and supporting this work. We thank the National Center for Genomic Sequencing of the University of Antioquia and Colquímicos for donating the dyes.

Supplementary Material

Supplementary material was deposited in figshare under accession DOI 10.6084/m9.figshare.31329796

Supplementary PDF

Supplementary PDF

Supplementary PDF

Supplementary PDF

Use of Generative Artificial Intelligence

The authors declare that RUBRIQ (https://rubriq.com/), an AI-powered editing tool, was used under full human supervision for text proofreading. No confidential or sensitive data were uploaded to such tool, and all AI-assisted content was checked, corrected, and approved by the authors, who take full responsibility for the integrity and originality of the manuscript.

Data Availability Statement:

Research data are available in the NCBI databases. Assembly: ASM3334281v1, GenBank GCA_033342815.1, and RefSeq: GCF_033342815.1

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• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Marcos Pileggi

Publication Dates

• Publication in this collection
  17 Apr 2026
• Date of issue
  2026

History

• Received
  26 Mar 2025
• Accepted
  03 Feb 2026