Characterization of Bacillus nealsonii strain KBH10 capable of reducing aqueous mercury in lab-scale reactor

The environmental release of Mercury is continuously increasing with high degree of mobility, transformation and amplified toxicity. Improving remediation strategies is becoming increasingly important to achieve more stringent environmental safety standards. This study develops a lab-scale reactor for bioremediation of aqueous mercury using a biofilm producing bacterial strain, KBH10 isolated from mercury polluted soil. The strain was found resistant to 80 mg/L of HgCl2 and identified as Bacillus nealsonii via 16S rRNA gene sequence analysis. The strain KBH10 was characterized for optimum growth parameters and its mercury biotransformation potential was validated through mercuric reductase assay. A packed-bed column bioreactor was designed for biofilm-mediated mercury removal from artificially contaminated water and residual mercury was estimated. Strain KBH10 could grow at a range of temperature (20–50 °C) and pH (6.0–9.0) with optimum temperature established at 30 °C and pH 7.0. The optimum mercuric reductase activity (77.8 ± 1.7 U/mg) was reported at 30 °C and was stable at a temperature range of 20–50 °C. The residual mercury analysis of artificially contaminated water indicated 60.6 ± 1.5% reduction in mercury content within 5 h of exposure. This regenerative process of biofilm-mediated mercury removal in a packed-bed column bioreactor can provide new insight of its potential use in mercury bioremediation.

Indigeneous Streptomyces spp. isolated from Cyperus rotundus rhizosphere indicate high mercuric reductase activity as a pontential bioremediation agent

Rahayu HM, Putri WA, Khasanah AU, Sembiring L, Purwestri YA. 2021. Indigenous Streptomyces spp. isolated from Cyperus rotundus rhizosphere indicate high mercuric reductase activity as a potential bioremediation agent. Biodiversitas 22: 1519-1526. The purification and characterization of mercuric reductase of four indigenous Streptomyces spp. from Cyperus rotundus L. rhizosphere in mercury-contaminated area have been investigated. Cell-free extract was obtained by disrupting cells using sea sand at 4 °C followed by centrifugation. Mercuric reductase was purified by ammonium sulfate precipitation, dialysis, and chromatography column (DEAE Sepharose anion column chromatography). The determination of optimum pH and temperature of mercuric reductase activity was measured based on the number of NADPH2 oxidized to NADP per mg protein per minute using a spectrophotometer. The molecular weight of mercuric reductase was determined using SDS-PAGE. Result showed that the highest specific activity of mercuric reductase was recorded from Streptomyces spp. BR28. The optimum pH and temperature of cell-free extract enzyme mercuric reductase were 7.5 and 80 °C, respectively. The enzyme was purified to 431.87-fold with specific activity 21918.95 U/mg protein. SDS PAGE showed that the molecular weight of mercuric reductase in Streptomyces spp. BR 28 ranged from 50 kDa to 75 kDa. It can be concluded that Streptomyces isolates contain mercuric reductase and have potential as mercury bioremediation agent to overcome mercury contamination in the environment.

Mercury-Tolerant Ensifer medicae Strains Display High Mercuric Reductase Activity and a Protective Effect on Nitrogen Fixation in Medicago truncatula Nodules Under Mercury Stress

Mercury (Hg) is extremely toxic for all living organisms. Hg-tolerant symbiotic rhizobia have the potential to increase legume tolerance, and to our knowledge, the mechanisms underlying Hg tolerance in rhizobia have not been investigated to date. Rhizobial strains of Ensifer medicae, Rhizobium leguminosarum bv. trifolii and Bradyrhizobium canariense previously isolated from severely Hg-contaminated soils showed different levels of Hg tolerance. The ability of the strains to reduce mercury Hg2+ to Hg0, a volatile and less toxic form of mercury, was assessed using a Hg volatilization assay. In general, tolerant strains displayed high mercuric reductase activity, which appeared to be inducible in some strains when grown at a sub-lethal HgCl2 concentration. A strong correlation between Hg tolerance and mercuric reductase activity was observed for E. medicae strains, whereas this was not the case for the B. canariense strains, suggesting that additional Hg tolerance mechanisms could be playing a role in B. canariense. Transcript abundance from merA, the gene that encodes mercuric reductase, was quantified in tolerant and sensitive E. medicae and R. leguminosarum strains. Tolerant strains presented higher merA expression than sensitive ones, and an increase in transcript abundance was observed for some strains when bacteria were grown in the presence of a sub-lethal HgCl2 concentration. These results suggest a regulation of mercuric reductase in rhizobia. Expression of merA genes and mercuric reductase activity were confirmed in Medicago truncatula nodules formed by a sensitive or a tolerant E. medicae strain. Transcript accumulation in nodules formed by the tolerant strain increased when Hg stress was applied, while a significant decrease in expression occurred upon stress application in nodules formed by the Hg-sensitive strain. The effect of Hg stress on nitrogen fixation was evaluated, and in our experimental conditions, nitrogenase activity was not affected in nodules formed by the tolerant strain, while a significant decrease in activity was observed in nodules elicited by the Hg-sensitive bacteria. Our results suggest that the combination of tolerant legumes with tolerant rhizobia constitutes a potentially powerful tool in the bioremediation of Hg-contaminated soils.

Thermal Stability of a Mercuric Reductase from the Red Sea Atlantis II Hot Brine Environment as Analyzed by Site-Directed Mutagenesis

ABSTRACTThe lower convective layer (LCL) of the Atlantis II brine pool of the Red Sea is a unique environment in terms of high salinity, temperature, and high concentrations of heavy metals. Mercuric reductase enzymes functional in such extreme conditions could be considered a potential tool in the environmental detoxification of mercurial poisoning and might alleviate ecological hazards in the mining industry. Here, we constructed a mercuric reductase library from Atlantis II, from which we identified genes encoding two thermostable mercuric reductase (MerA) isoforms: one is halophilic (designated ATII-LCL) while the other is not (designated ATII-LCL-NH). The ATII-LCL MerA has a short motif composed of four aspartic acids (4D414–417) and two characteristic signature boxes that played a crucial role in its thermal stability. To further understand the mechanism behind the thermostability of the two studied enzymes, we mutated the isoform ATII-LCL-NH and found that the substitution of 2 aspartic acids (2D) at positions 415 and 416 enhanced the thermal stability, while other mutations had the opposite effect. The 2D mutant showed superior thermal tolerance, as it retained 81% of its activity after 10 min of incubation at 70°C. A three-dimensional structure prediction revealed newly formed salt bridges and H bonds in the 2D mutant compared to the parent molecule. To the best of our knowledge, this study is the first to rationally design a mercuric reductase with enhanced thermal stability, which we propose to have a strong potential in the bioremediation of mercurial poisoning.IMPORTANCEThe Red Sea is an attractive environment for bioprospecting. There are 25 brine-filled deeps in the Red Sea. The Atlantis II brine pool is the biggest and hottest of such hydrothermal ecosystems. We generated an environmental mercuric reductase library from the lowermost layer of the Atlantis II brine pool, in which we identified two variants of the mercuric reductase enzyme (MerA). One is the previously described halophilic and thermostable ATII-LCL MerA and the other is a nonhalophilic relatively less thermostable enzyme, designated ATII-LCL-NH MerA. We used the ATII-LCL-NH enzyme as a parent molecule to locate the amino acid residues involved in the noticeably higher thermotolerance of the homolog ATII-LCL MerA. Moreover, we designed a novel enzyme with superior thermal stability. This enzyme might have strong potential in the bioremediation of mercuric toxicity.

Biochemical Properties of Mercuric Reductase from Local Isolate of Bacillus sp for Bioremediation Agent

Mercuric reductase is the important enzyme which catalyzes a reduction of a toxic Hg2+ to non-toxic Hg0. The enzyme which has been potentially used as mercury bioremediation agent is produced by mercury resistant bacteria. These research aims are to determinate the resistance level of a local Bacillus sp to HgCl2 in media, to determine the mercuric reductase activity from the bacteria, and to determine the biochemical properties of the mercuric reductase. The Bacillus sp was grown in the Nutrient Broth media with various of 0; 20; 40; 60; 120; and 160 µM HgCl2 to know the response of the bacteria against mercury, The cell growth of Bacillus sp was measured by optical density (OD) method of at λ 600 nm. The mercuric reductase activity was assayed in the solution of MRA (Mercury Reductase Assay), then the oxidized NADPH was observed by the spectrophotometry method at λ340 nm. The result showed that the Bacillus sp has been resistant to media containing mercury at 120 µM, but the microbial growth was decreased by 50% in media containing mercury 80 µM. The Bacillus sp could produce highly the mercuric reductase enzyme at 16 hours of growth time with enzyme activity as 0.574 Unit/µg. The mercuric reductase from the bacteria has an optimum activity at pH 6 and temperature 37 °C