Phenotypic and genomic analysis of multiple heavy metal-resistant Micrococcus luteus strain AS2 isolated from industrial waste water and its potential use in arsenic bioremediation.

Multiple heavy metal-resistant bacterium, Micrococcus luteus strain AS2, was isolated from industrial waste water of District Sheikhupura, Pakistan. The isolated bacterium showed minimum inhibitory concentrations of 55 and 275 mM against arsenite and arsenate. The bacterial strain also showed resistance against other heavy metal ions, i.e., lead, cadmium, chromium, mercury, nickel, and zinc, apart from arsenic. The optimum temperature and pH were 37 °C and 7, respectively. The antioxidant enzymes such as catalase were significantly increased under arsenite stress. The increase in 43.9% of GSH/GSSG and 72.72% of non-protein thiol was determined under15 mM arsenite stress. Bacterial genome was sequenced through Illumina and Nanopore and genes related to arsenic and other heavy metals were identified and blast (tblastx) on NCBI. Through scanning electron microscopy, no morphological changes were observed in bacterial cells under arsenite stress. The peaks appeared in EDX showed that there is surface adsorption of arsenite in bacterial cell while it was confirmed from Fourier transformed infrared spectroscopy analysis that there is some interaction between arsenite and functional groups present on the surface of bacterial cell. The SDS-PAGE analysis of whole-cell proteins under 15 mM arsenite stress clearly revealed that there is upregulation of some proteins in ranged of 60 to 34 kDa. The bioremediation efficiency (E) of bacterial biomass was 72% after 2 h and 99% after 10 h. The bioremediation efficiency of bacterial biomass is an indicator for the isolated bacterium to employ as a potential candidate for the amelioration of sites contaminated with arsenic.

Use of heavy metals resistant bacteria-a strategy for arsenic bioremediation.

A large number of industries release their untreated wastes in the environment causing an increase in the concentration of toxic pollutants including heavy metal ions in ground and drinking water which is above the WHO limit. The presence of toxic pollutants in the industrial wastes pollutes our environment. Arsenic (As) is a ubiquitous toxic metalloid. Its amount varies in different parts on the earth, and its concentration is increasing in our environment day by day both by natural and anthropogenic activities. It is found in two forms; one is arsenate (As5+) and other is arsenite (As3+) and the latter is more toxic due to high mobility across the cell membrane. The long-term use of arsenic-containing water causes arsenicosis. High arsenic consumption, revealed by skin harms, color change, and spots on hands and feet, may cause skin cancer and affect lungs and kidneys. Hypertension, a state of high blood pressure, and lack of insulin which causes diabetes and many other disorders which relate to reproduction are the consequences of arsenic contamination. Several methods have been employed to decontaminate arsenic pollution, but the bioremediation by using biomass of bacteria, algae, fungi, and yeasts is the most compromising approach and has gained much attention from researchers in the last few decades. The microbial detoxification of arsenic can be achieved by reduction, oxidation, and methylation. High bioremediation potential and feasibility of the process make bacteria an impending foundation for green chemistry to exterminate arsenic in the environment.

Identification of arsenic oxidizing genes fragment in Microbacterium sp. strain 1S1 and its cloning in E. coli (DH5a).

Microbacterium sp. strain 1S1, an arsenic-resistant bacterial strain, was isolated with 75 mM MIC against arsenite. Brownish precipitation with silver nitrate appeared, which confirmed its oxidizing ability against arsenite. The bacterial genomic DNA underwent Illumina and Nanopore sequencing, revealing a distinctive cluster of genes spanning 9.6 kb associated with arsenite oxidation. These genes were identified within an isolated bacterial strain. Notably, the smaller subunit (aioB) of the arsenite oxidizing gene at the chromosomal DNA locus (Prokka_01508) was pinpointed. This gene, aioB, is pivotal in arsenite oxidation, a process crucial for energy metabolism. Upon thorough sequencing analysis, only a singular megaplasmid was detected within the isolated bacterial strain. Strikingly, this megaplasmid did not harbor any genes responsible for arsenic resistance or detoxification. This intriguingly indicates that the bacterial strain relies on the arsenic oxidizing genes present for its efficient arsenic oxidation capability. This is especially true for Microbacterium sp. strain 1S1. Subsequently, a segment of genes linked to arsenic resistance was successfully cloned into E. coli (DH5a). The fragment of arsenic-resistant genes was cloned in E. coli (DH5a), further confirmed by the AgNO3 method. This genetically engineered E. coli (DH5a) can decontaminate arsenic-contaminated sites.

Brevibacterium sp. strain CS2: A potential candidate for arsenic bioremediation from industrial wastewater.

A multiple metal-resistant Brevibacterium sp. strain CS2, isolated from an industrial wastewater, resisted arsenate and arsenate upto 280 and 40 mM. The order of resistance against multiple metals was Arsenate > Arsenite > Selenium = Cobalt > Lead = Nickel > Cadmium = Chromium = Mercury. The bacterium was characterized as per morphological and biochemical characteristics at optimum conditions (37 ℃ and 7 pH). The appearance of brownish color precipitation was due to the interaction of silver nitrate confirming its oxidizing ability against arsenic. The strain showed arsenic processing ability at different temperatures, pH, and initial arsenic concentration which was 37% after 72 h and 48% after 96 h of incubation at optimum conditions with arsenite 250 mM/L (initial arsenic concentration). The maximum arsenic removal ability of strain CS2 was determined for 8 days, which was 32 and 46% in wastewater and distilled water, respectively. The heat-inactivated cells of the isolated strain showed a bioremediation efficiency (E) of 96% after 10 h. Genes cluster (9.6 kb) related to arsenite oxidation was found in Brevibacterium sp. strain CS2 after the genome analysis of isolated bacteria through illumine and nanopore sequencing technology. The arsenite oxidizing gene smaller subunit (aioB) on chromosomal DNA locus (Prokka_01508) was identified which plays a role in arsenite oxidation for energy metabolism. The presence of arsenic oxidizing genes and an efficient arsenic oxidizing potential of Brevibacterium sp. strain CS2 make it a potential candidate for green chemistry to eradicate arsenic from arsenic-contaminated wastewater.

Bacillus sp. strain MRS-1: A potential candidate for uranyl biosorption from uranyl polluted sites.

The uranyl tolerance of a metal-resistant Bacillus sp. strain MRS-1, was determined in this current study. This was done due to a rise in anthropogenic activities, such as the production of uranium-based nuclear energy, which contributes to environmental degradation and poses risks to ecosystems and human health. The purpose of the research was to find effective strategies for uranium removal to minimize the contamination. In this paper, the biosorption of uranyl was investigated by batch tests. Bacteria could continue to multiply up to 350 ppm uranyl concentrations, however this growth was suppressed at 400 ppm, that generally accepted as the minimum concentration for bacterial growth inhibition. The optimal conditions for uranyl biosorption were pH 7, 20 °C, and a contact duration of 30 min with living bacteria. According to the findings of an investigation that used isotherm and kinetics models (Langmuir, Freundlich and pseudo second order), Bacillus sp. strain MRS-1 biosorption seemed to be dependent on monolayer adsorption as well as certain functional groups that had a strong affinity for uranyl confirmed by Fourier Transform Infrared Spectroscopy (FTIR) analysis. The shifts/sharping of peaks (1081-3304 cm-1) were prominent in treated samples compared to control one. These functional groups could be hydroxyl, amino, and carboxyl. Our findings showed that Bacillus sp. strain MRS-1 has an elevated uranyl biosorption ability, with 24.5 mg/g being achieved. This indicates its potential as a powerful biosorbent for dealing with uranium contamination in drinking water sources and represents a breakthrough in the cleanup of contaminated ecosystems.

Proteomics and transcriptomic analysis of Micrococcus luteus strain AS2 under arsenite stress and its potential role in arsenic removal.

The proteomics and transcriptomic analysis of Micrococcus luteus strain AS2 was carried out through 2D gel electrophoresis and RT-PCR. Seven protein spots were selected randomly from the gel and identified through mass spectrometry. Four proteins including putative metal-dependent hydrolase TatD, thioredoxin reductase, DNA-directed RNA polymerase subunit alpha and chaperone protein DnaK were upregulated while superoxide dismutase [Mn], 3-oxoacyl-[acyl-carrier-protein] reductase FabG, and putative alkyl/aryl-sulfatase YjcS were down-regulated under arsenite stress. No significant difference was observed in aioB gene expression analysis in the presence and absence of arsenite. The optimum arsenite processing ability was determined at 37°C (90%) and at pH 7 (92%). The maximum metal processing ability was determined at 250 mM arsenite/L (90%) while the minimum was estimated at 1250 mM arsenite/L (42%). The maximum arsenite removal ability of strain AS2 determined after 8 days was 68 and 82% from wastewater and distilled water, and the organism can be a good bioresource for green chemistry to eradicate environmental arsenite.

Complete Genome Sequences of Highly Arsenite-Resistant Bacteria Brevibacterium sp. Strain CS2 and Micrococcus luteus AS2.

The complete genome sequences of two highly arsenite-resistant Actinomycetales isolates are presented. Both genomes are G+C rich and consist of a single chromosome containing homologs of known arsenite resistance genes.