Diverse hydrocarbon degradation genes, heavy metal resistome, and microbiome of a fluorene-enriched animal-charcoal polluted soil.

Environmental compartments polluted with animal charcoal from the skin and hide cottage industries are rich in toxic heavy metals and diverse hydrocarbon classes, some of which are carcinogenic, mutagenic, and genotoxic, and thus require a bio-based eco-benign decommission strategies. A shotgun metagenomic approach was used to decipher the microbiome, hydrocarbon degradation genes, and heavy metal resistome of a microbial consortium (FN8) from an animal-charcoal polluted site enriched with fluorene. Structurally, the FN8 microbial consortium consists of 26 phyla, 53 classes, 119 orders, 245 families, 620 genera, and 1021 species. The dominant phylum, class, order, family, genus, and species in the consortium are Proteobacteria (51.37%), Gammaproteobacteria (39.01%), Bacillales (18.09%), Microbulbiferaceae (11.65%), Microbulbifer (12.21%), and Microbulbifer sp. A4B17 (19.65%), respectively. The microbial consortium degraded 57.56% (28.78 mg/L) and 87.14% (43.57 mg/L) of the initial fluorene concentration in 14 and 21 days. Functional annotation of the protein sequences (ORFs) of the FN8 metagenome using the KEGG GhostKOALA, KofamKOALA, NCBI's conserved domain database, and BacMet revealed the detection of hydrocarbon degradation genes for benzoate, aminobenzoate, polycyclic aromatic hydrocarbons (PAHs), chlorocyclohexane/chlorobenzene, chloroalkane/chloroalkene, toluene, xylene, styrene, naphthalene, nitrotoluene, and several others. The annotation also revealed putative genes for the transport, uptake, efflux, and regulation of heavy metals such as arsenic, cadmium, chromium, mercury, nickel, copper, zinc, and several others. Findings from this study have established that members of the FN8 consortium are well-adapted and imbued with requisite gene sets and could be a potential bioresource for on-site depuration of animal charcoal polluted sites.

Depletion of hydrocarbons and concomitant shift in bacterial community structure of a diesel-spiked tropical agricultural soil.

Bacterial community of a diesel-spiked agricultural soil was monitored over a 42-day period using the metagenomic approach in order to gain insight into key phylotypes impacted by diesel contamination and be able to predict end point of bioattenuation. Soil physico-chemical parameters showed significant differences (P < 0.05) between the Polluted Soil (PS) and the Unpolluted control (US)across time points. After 21 days, the diesel content decreased by 27.39%, and at the end of 42 days, by 57.11%. Aromatics such as benzene, anthanthrene, propylbenzene, phenanthrenequinone, anthraquinone, and phenanthridine were degraded to non-detected levels within 42 days, while some medium range alkanes and polyaromatics such as acenaphthylene, naphthalene, and anthracene showed significant levels of degradation. After 21 days (LASTD21), there was a massive enrichment of the phylum Proteobacteria (72.94%), a slight decrease in the abundance of phylum Actinobacteriota (12.74%), and > 500% decrease in the abundance of the phylum Acidobacteriodota (5.26%). Day 42 (LASTD42) saw establishment of the dominance of the Proteobacteria (34.95%), Actinobacteriota, (21.71%), and Firmicutes (32.14%), and decimation of phyla such as Gemmatimonadota, Planctomycetota, and Verrucromicrobiota which play important roles in the cycling of elements and soil health. Principal component analysis showed that in PS moisture contents, phosphorus, nitrogen, organic carbon, had greater impacts on the community structure in LASTD21, while acidity, potassium, sodium, calcium and magnesium impacted the control sample. Recovery time of the soil based on the residual hydrocarbons at Day 42 was estimated to be 229.112 d. Thus, additional biostimulation may be required to achieve cleanup within one growing season.

Chromium contamination accentuates changes in the microbiome and heavy metal resistome of a tropical agricultural soil.

The impacts of hexavalent chromium (Cr) contamination on the microbiome, soil physicochemistry, and heavy metal resistome of a tropical agricultural soil were evaluated for 6 weeks in field-moist microcosms consisting of a Cr-inundated agricultural soil (SL9) and an untreated control (SL7). The physicochemistry of the two microcosms revealed a diminution in the total organic matter content and a significant dip in macronutrients phosphorus, potassium, and nitrogen concentration in the SL9 microcosm. Heavy metals analysis revealed the detection of seven heavy metals (Zn, Cu, Fe, Cd, Se, Pb, Cr) in the agricultural soil (SL7), whose concentrations drastically reduced in the SL9 microcosm. Illumina shotgun sequencing of the DNA extracted from the two microcosms showed the preponderance of the phyla, classes, genera, and species of Actinobacteria (33.11%), Actinobacteria_class (38.20%), Candidatus Saccharimonas (11.67%), and Candidatus Saccharimonas aalborgensis (19.70%) in SL7, and Proteobacteria (47.52%), Betaproteobacteria (22.88%), Staphylococcus (16.18%), Staphylococcus aureus (9.76%) in SL9, respectively. Functional annotation of the two metagenomes for heavy metal resistance genes revealed diverse heavy metal resistomes involved in the uptake, transport, efflux, and detoxification of various heavy metals. It also revealed the exclusive detection in SL9 metagenome of resistance genes for chromium (chrB, chrF, chrR, nfsA, yieF), cadmium (czcB/czrB, czcD), and iron (fbpB, yqjH, rcnA, fetB, bfrA, fecE) not annotated in SL7 metagenome. The findings from this study revealed that Cr contamination induces significant shifts in the soil microbiome and heavy metal resistome, alters the soil physicochemistry, and facilitates the loss of prominent members of the microbiome not adapted to Cr stress.

Functional characterization of the ABC transporters and transposable elements of an uncultured Paracoccus sp. recovered from a hydrocarbon-polluted soil metagenome.

Environmental microorganisms usually exhibit a high level of genomic plasticity and metabolic versatility that allow them to be well-adapted to diverse environmental challenges. This study used shotgun metagenomics to decipher the functional and metabolic attributes of an uncultured Paracoccus recovered from a polluted soil metagenome and determine whether the detected attributes are influenced by the nature of the polluted soil. Functional and metabolic attributes of the uncultured Paracoccus were elucidated via functional annotation of the open reading frames (ORFs) of its contig. Functional tools deployed for the analysis include KEGG, KEGG KofamKOALA, Clusters of Orthologous Groups of proteins (COG), Comprehensive Antibiotic Resistance Database (CARD), and the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT V6) for antibiotic resistance genes, TnCentral for transposable element, Transporter Classification Database (TCDB) for transporter genes, and FunRich for gene enrichment analysis. Analyses revealed the preponderance of ABC transporter genes responsible for the transport of oligosaccharides (malK, msmX, msmK, lacK, smoK, aglK, togA, thuK, treV, msiK), monosaccharides (glcV, malK, rbsC, rbsA, araG, ytfR, mglA), amino acids (thiQ, ynjD, thiZ, glnQ, gluA, gltL, peb1C, artP, aotP, bgtA, artQ, artR), and several others. Also detected are transporter genes for inorganic/organic nutrients like phosphate/phosphonate, nitrate/nitrite/cyanate, sulfate/sulfonate, bicarbonate, and heavy metals such as nickel/cobalt, molybdate/tungstate, and iron, among others. Antibiotic resistance genes that mediate efflux, inactivation, and target protection were detected, while transposable elements carrying resistance phenotypes for antibiotics and heavy metals were also annotated. The findings from this study have established the resilience, adaptability, and survivability of the uncultured Paracoccus in the hydrocarbon-polluted soil.

Metagenomic insights into the microbial community structure and resistomes of a tropical agricultural soil persistently inundated with pesticide and animal manure use.

Persistent use of pesticides and animal manure in agricultural soils inadvertently introduced heavy metals and antibiotic/antibiotic resistance genes (ARGs) into the soil with deleterious consequences. The microbiome and heavy metal and antibiotic resistome of a pesticide and animal manure inundated agricultural soil (SL6) obtained from a vegetable farm at Otte, Eiyenkorin, Kwara State, Nigeria, was deciphered via shotgun metagenomics and functional annotation of putative ORFs (open reading frames). Structural metagenomics of SL6 microbiome revealed 29 phyla, 49 classes, 94 orders, 183 families, 366 genera, 424 species, and 260 strains with the preponderance of the phyla Proteobacteria (40%) and Actinobacteria (36%), classes Actinobacteria (36%), Alphaproteobacteria (18%), and Gammaproteobacteria (17%), and genera Kocuria (16%), Sphingobacterium (11%), and Brevundimonas (10%), respectively. Heavy metal resistance genes annotation conducted using Biocide and Metal Resistance Gene Database (BacMet) revealed the detection of genes responsible for the uptake, transport, detoxification, efflux, and regulation of copper, cadmium, zinc, nickel, chromium, cobalt, selenium, tungsten, mercury, and several others. ARG annotation using the Antibiotic Resistance Gene-annotation (ARG-ANNOT) revealed ARGs for 11 antibiotic classes with the preponderance of ß-lactamases, mobilized colistin resistance determinant (mcr-1), macrolide-lincosamide-streptogramin (MLS), glycopeptide, and aminoglycoside resistance genes, among others. The persistent use of pesticide and animal manure is strongly believed to play a major role in the proliferation of heavy metal and antibiotic resistance genes in the soil. This study revealed that agricultural soils inundated with pesticide and animal manure use are potential hotspots for ARG spread and may accentuate the spread of multidrug resistant clinical pathogens.

Impact of spent engine oil contamination on the antibiotic resistome of a tropical agricultural soil.

Profiling of hydrocarbon-contaminated soils for antibiotic resistance genes (ARGs) is becoming increasingly important due to emerging realities of their preponderance in hydrocarbon-inundated matrices. In this study, the antibiotic resistome of an agricultural soil (1S) and agricultural soil contaminated with spent engine oil (AB1) were evaluated via functional annotation of the open reading frames (ORFs) of their metagenomes using the comprehensive antibiotic database (CARD) and KEGG KofamKOALA. CARD analysis of AB1 metagenome revealed the detection of 24 AMR (antimicrobial resistance) gene families, 66 ARGs, and the preponderance (69.7%) of ARGs responsible for antibiotic efflux in AB1 metagenome. CARD analysis of 1S metagenome revealed four AMR gene families and five ARGs. Functional annotation of the two metagenomes using KofamKOALA showed 171 ARGs in AB1 and 29 ARGs in 1S, respectively. Majority of the detected ARGs in AB1 (121; 70.8%) and 1S (16; 55.2%) using KofamKOALA are responsible for antibiotic efflux while ARGs for other resistance mechanisms were also detected. All the five major antibiotic efflux pump systems were detected in AB1 metagenome, though majority of the ARGs for antibiotic efflux belong to the RND (resistance-nodulation-cell division) and MFS (major facilitator superfamily) efflux systems. Significant differences observed in the ARGs recovered from 1S and AB1 metagenomes were statistically validated (P < 0.05). SEO contamination is believed to be responsible for ARGs increase in AB1 metagenome via mechanisms of cross-resistance especially with efflux pumps. The detection of these ARGs is of great public health concern in this era of multidrug resistant isolates resurgence.

Remarkable shift in structural and functional properties of an animal charcoal-polluted soil accentuated by inorganic nutrient amendment.

BACKGROUND: Soils polluted with animal charcoal from skin and hide cottage industries harbour extremely toxic and carcinogenic hydrocarbon pollutants and thus require a bio-based eco-friendly strategy for their depuration. The effects of carbon-free mineral medium (CFMM) amendment on hydrocarbon degradation and microbial community structure and function in an animal charcoal-polluted soil was monitored for 6 weeks in field moist microcosms consisting of CFMM-treated soil (FN4) and an untreated control (FN1). Hydrocarbon degradation was monitored using gas chromatography-flame ionization detector (GC-FID), and changes in microbial community structure were monitored using Kraken, while functional annotation of putative open reading frames (ORFs) was done using KEGG KofamKOALA and NCBI's conserved domain database (CDD). RESULTS: Gas chromatographic analysis of hydrocarbon fractions revealed the removal of 84.02% and 82.38% aliphatic and 70.09% and 70.14% aromatic fractions in FN4 and FN1 microcosms in 42 days. Shotgun metagenomic analysis of the two metagenomes revealed a remarkable shift in the microbial community structure. In the FN4 metagenome, 92.97% of the population belong to the phylum Firmicutes and its dominant representative genera Anoxybacillus (64.58%), Bacillus (21.47%) and Solibacillus (2.39%). In untreated FN1 metagenome, the phyla Proteobacteria (56.12%), Actinobacteria (23.79%) and Firmicutes (11.20%), and the genera Xanthobacter (9.73%), Rhizobium (7.49%) and Corynebacterium (7.35%), were preponderant. Functional annotation of putative ORFs from the two metagenomes revealed the detection of degradation genes for aromatic hydrocarbons, benzoate, xylene, chlorocyclohexane/chlorobenzene, toluene and several others in FN1 metagenome. In the FN4 metagenome, only seven hydrocarbon degradation genes were detected. CONCLUSION: This study revealed that though CFMM amendment slightly increases the rate of hydrocarbon degradation, it negatively impacts the structural and functional properties of the animal charcoal-polluted soil. It also revealed that intrinsic bioremediation of the polluted soil could be enhanced via addition of water and aeration.

Unravelling the antibiotic and heavy metal resistome of a chronically polluted soil.

The antibiotic and heavy metal resistome of a chronically polluted soil (3S) obtained from an automobile workshop in Ilorin, Kwara State, Nigeria was deciphered via functional annotation of putative ORFs (open reading frames). Functional annotation of antibiotic and heavy metal resistance genes in 3S metagenome was conducted using the Comprehensive Antibiotic Resistance Database (CARD), Antibiotic Resistance Gene-annotation (ARG-ANNOT) and Antibacterial Biocide and Metal Resistance Gene Database (BacMet). Annotation revealed detection of resistance genes for 15 antibiotic classes with the preponderance of beta lactamases, mobilized colistin resistance determinant (mcr), glycopepetide and tetracycline resistance genes, the OqxBgb and OqxA RND-type multidrug efflux pumps, among others. The dominance of resistance genes for antibiotics effective against members of the Enterobacteriaceae indicate possible contamination with faecal materials. Annotation of heavy metal resistance genes revealed diverse resistance genes responsible for the uptake, transport, detoxification, efflux and regulation of copper, zinc, cadmium, nickel, chromium, cobalt, mercury, arsenic, iron, molybdenum and several others. Majority of the antibiotic and heavy metal resistance genes detected in this study are borne on mobile genetic elements, which facilitate their spread and dissemination in the polluted soil. The presence of the heavy metal resistance genes is strongly believed to play a major role in the proliferation of antibiotic resistance genes. This study has established that soil is a huge repertoire of antibiotic and heavy metal resistome and due to the intricate link between human, animals and the soil environment, it may be a major contributor to the proliferation of multidrug-resistant clinical pathogens.