Heavy Metal Resistant Bacterial Strains Research Articles

Isolation of heavy metal-resistant Staphylococcus epidermidis strain TWSL_22 and evaluation of heavy metal bioremediation potential of recombinant E. coli cloned with isolated cadD.

A heavy metal-resistant bacterial strain, TWSL_22 was isolated from an industrial effluent sample and tested for heavy metal tolerance and resistance. The strain was molecularly characterized as Staphylococcus epidermidis based on 16S rDNA gene analysis and the sequence was deposited in the NCBI repository (accession number KT184893.1). Metal removal activity (P<.001) of TWSL_22 was 99.99±0.001%, 74.43±2.51%, and 51.16±4.17% for Cd, Pb, and Cu, respectively. Highest MIC was observed for Cd. Antibiotic susceptibility assays revealed the strain TWSL_22 to be resistant to several antibiotics. The strain was screened for possible heavy metal-resistant genes and presence of cadA, copA, and cadD was confirmed by PCR. A DNA fragment containing complete sequence of cadD (618bp) was isolated and cloned into pET 21a(+), transformed into E. coli BL21 and designated as E. coli/cadDET. E. coli/cadDET showed high metal tolerance capacity and could remove over 82% of heavy metals (Zn2+, Cd2+, Cu2+, and Cr3+) in the industrial effluent.

Heavy metals-resistant bacteria (HM-RB): Potential bioremediators of heavy metals-stressed Spinacia oleracea plant

Microorganism technologies can provide a potential alternative to traditional methods of removing heavy metals to conserve agricultural soils. This study aimed to identify and characterize heavy metals-resistant bacteria (HM-RB) isolated from industry-affected soil and their desired impact as bioremediators of heavy metals-stressed spinach plants. Three of 135 isolates were selected based on a high level of resistance to heavy metals. Based on morphological and biochemical characteristics, the selected isolates were identified as Bacillus subtilis subsp. spizizenii DSM 15029T DSM (MA3), Paenibacillus jamilae DSM 13815T DSM (LA22), or Pseudomonas aeruginosa DSM 1117 DSM (SN36). Experiments were implemented to investigate the three isolated HM-RB ability on improving attributes of growth, physio-biochemistry, and components of the antioxidant defense system of spinach plant exposed to the stress of cadmium (Cd2+; 2mM), lead (Pb2+; 2mM) or 2mM Cd2++2mMPb2+. Compared to control, Cd2+ or Pb2+ stress markedly lowered plant fresh and dry weights, leaf contents of chlorophylls and carotenoids, rates of transpiration (Tr), net photosynthesis (Pn) and stomatal conductance (gs), relative water content (RWC), and membrane stability index (MSI). In contrast, contents of α.tochopherol (α.TOC), ascorbic acid (AsA), glutathione (GSH), proline, soluble sugars, Cd2+, and Pb2+, as well as activities of enzymatic and non-enzymatic antioxidants were markedly elevated. The application of HM-RB promoted the tolerance to heavy metal stress in spinach plants by improving Tr, Pn, gs, RWC, and MSI, while activities of enzymatic and non-enzymatic antioxidants were suppressed. These results reflected positively in promoting plant growth under heavy metal stress. Therefore, the application of HM-RB as potential bioremediators may be a promising strategy for promoting plant growth and productivity under heavy metal stress.

Interaction of Sewage Bacteria with Heavy Metals, Antibiotics and Plants

Environment pollution of toxic heavy metals and antibiotics is widespread due of massive industrialization and overuse of antibiotics. Moreover, metal contamination can function as a selective agent in the spread of antibiotic resistance, affecting both animals and plant species. Isolation, identification and characterization of three heavy metal resistant bacterial strains from sewage pipes of Lahore city was carried out in this study. On the basis of morphology and 16S ribosomal RNA (rRNA) gene sequencing, the isolates were identified as Escherichia coli strain MHR-1, Bacillus subtilis strain MHR-2, Exiguobacterium aurantiacum strain MHR-3. These isolates were resistant to Cadmium (Cd) at (&lt; 100 µg/mL), Chromium (Cr) (&lt; 500 µg/mL), Lead (Pb) (&lt; 400 µg/mL) and Arsenic (As) (&lt; 200 µg/mL). Moreover, the isolates tolerated a combined stress of 50 µg/mL for all the heavy metals tested. Antibiotic susceptibility of the bacteria towards the 9 antibiotics revealed: MHR-1 was resistant to 66%, intermediate to 12% and susceptible to 22%; MHR-2 was resistant 66%, intermediate to 22% and susceptible to 12%; MHR-3 was resistant to 22%, intermediate to 33% and susceptible to 45%. For antibiotic-heavy metal co-stress, about 78% of antibiotics showed synergistic effects while 11% showed antagonistic effects along with a stress of 50 µg/mL of all heavy metals tested. Test of isolates for their ability to affect seed growth with and without heavy metal stress, showed no significant interaction with plants to tolerate the heavy metal stress. This study proposes an effective approach to target antibiotic resistant bacteria with combine stress of heavy metals, and further investigation will lead in to better insight in devising an effective treatment for targeting resistant pathogens. Key Words: , , , , .

Microbial characterization of heavy metal resistant bacterial strains isolated from an electroplating wastewater treatment plant

Heavy metal pollution is one of the most widespread and complex environmental issues globally, posing a great threat to the ecosystem as well as human health. Bioremediation through heavy metal-resistant bacteria (HMRB) is currently the most promising technology to address this issue. To obtain HMRB to remediate heavy metal pollution potentially, 15 culturable HMRB strains were isolated from the sludge samples of an electroplating wastewater treatment plant (EWWTP), which belonged to the Bacillus, Shewanella, Lysinibacillus, and Acinetobacter genera. Their maximum tolerance concentrations to Cu2+, Ni2+, Mn2+, Co2+, and Cr2O72− were 40 mM, 10 mM, 200 mM, 40 mM, and 10 mM, respectively, and strain Mn1-4 showed much higher Mn2+ tolerance and removal effectiveness (3.355 g/L) than previously published reports. Moreover, multiple heavy metal-resistant genotypes and phenotypes were identified among these strains, of which strain Co1-1 carried the most of resistant gene sequences (10) and exhibited resistance to 7 categories of heavy metals, and the co-occurrence of heavy metal and antibiotic resistance were clearly observed in strain Ni1-3. In addition, flanked insert sequence (IS) elements on the heavy metal resistant genes (HMRGs) suggested that horizontal gene transfer (HGT) events may have resulted in multiple heavy metal resistance phenotypes and genotypes in these strains, and IS982 family transposase was presumed to result in the high Ni2+ tolerance in strain Ni1-3. This study expands our understanding of bacterial heavy metal resistance and provides promising candidates for heavy metal bioremediation.

Inoculation of soil with cadmium-resistant bacterium Delftia sp. B9 reduces cadmium accumulation in rice (Oryza sativa L.) grains

Bioremediation of heavy metal polluted soil using metal-resistant bacteria has received increasing attentions. In the present study, we isolated a heavy metal-resistant bacterial strain from a Cd-contaminated soil, and conducted pot experiments to evaluate the effect of bacterial inoculation in soil on soil Cd speciation, rice grain biomass and Cd accumulation. We find that the isolated bacterial strain is a Gram-negative bacterium, and named as Delftia sp. B9 based on the 16S rDNA gene sequence analysis. TEM–EDS manifests that Cd can be bioaccumulated inside cell, resulting in intracellular dissolution. The Cd contents of rice grain in the two rice cultivars (early and late rice) are all below the standard limit for Food Safety of People's Republic of China (0.2 mg/kg) after the treatment of both living and non-living cells. Non-living cells are more applicable than the use of living cells for the short time bioremediation. The average content of soil exchangeable fraction of Cd decreases whereas the residual fraction increases with bacterial inoculation. All our results suggest Delftia sp. B9 is able to the stabilization of Cd in soil and reduce Cd accumulation in rice grain, therefore, this strain is potentially suitable for the bioremediation of Cd-contaminated paddy soils.

Quick discrimination of heavy metal resistant bacterial populations using infrared spectroscopy coupled with chemometrics.

Lead and cadmium are frequently encountered heavy metals in industrially polluted areas. Many heavy metal resistant bacterial strains have a high biosorption capacity and thus are good candidates for the removal of toxic metals from the environment. However, as of yet there is no accurate method for discrimination of highly adaptive bacterial strains among the populations present in a given habitat. In this study, we aimed to find distinguishing molecular features of lead and cadmium resistant bacteria using Attenuated Total Reflectance-Fourier Transformed Infrared (ATR-FT-IR) spectroscopy and chemometric approaches. Our results demonstrated that both control and metal exposed E. coli and S. aureus strains could be successfully discriminated from each other using hierarchical cluster and principal component analysis methods. Moreover, we found that lead exposed bacterial strains could be successfully discriminated from cadmium exposed ones with a high heterogeneity value. These clear discriminations can be described by the ability of a bacterium to change its metabolism in terms of the content and structure of cellular macromolecules under heavy metal stress. In our case, cadmium and lead-induced genetic response systems in bacteria caused remarkable alterations in overall cellular metabolism. Bacteria deal with a heavy metal stress by altering nucleic acid methylations and lipid and protein synthesis. Heavy metal burden led to the development of relevant metabolic changes in proteins, lipids, and nucleic acids of the resistant bacteria described in this study. Our approach showed that infrared spectra obtained via ATR-FT-IR spectroscopy coupled with chemometric analysis can be utilized for rapid, low-cost, informative, reliable, and operator-independent discrimination of resistant bacterial populations.

Potential of Rhizobacteria for Improving Lead Phytoextraction in Ricinus communis

Heavy metal–resistant bacterial strains were isolated from heavy metal–contaminated soils and identified as Bacillus sp. and Leclercia adecarboxylata on the basis of their morphology and biochemical characters using the VITEK 2 Systems Version 05.02. The heavy metal and antibiotic resistance of the isolates were studied. A green house pot experiment was conducted to examine the bacterial ability to extract the lead in soils and for their effect on lead uptake by Ricinus communis in an artificially contaminated soil. Bacterial inoculated pots increased the biomass of the R. communis compared to the uninoculated control, and the root growth of the plant was also increased in the inoculated pot. The experimental data confirmed that lead‐resistant bacteria have a pronounced effect on heavy metal uptake in plants, which may provide a new bacterially assisted phytoremediation of metal‐contaminated soils. © 2013 Wiley Periodicals, Inc.

Isolation and phylogenetic analysis of zinc resistant Acinetobacter sp. and its potential for bioremediation

In this study, a gram negative, non- motile short rod-shaped and heavy metal resistant bacterial strain was isolated from effluents. The optimum temperature and pH for the growth of the strain were 33°C and 7, respectively. The minimum inhibitory concentrations (MICs) of zinc (Zn), copper (Cu), chromium (Cr) and mercury (Hg) against the isolate were determined. The isolate showed MICs of 5, 4, 3 and 2 mM when grown on Zn, Cu, Cr and Hg, respectively. The isolate was assessed for its ability to remove zinc from medium amended with different concentrations of zinc. Up to 65% of zinc was removed at the zinc concentration of 1 mM. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Acinetobacter with 98% similarity to Acinetobacter junni . It was then designated as Acinetobacter sp. HM_AF14. Based on the data obtained in this study, it can be concluded that Acinetobacter sp. HM_AF14 can be used for bioremediation of zinc from effluents. Keywords : Heavy metals, Acinetobacter sp., bioremediation, 16S rRNA, zinc removal African Journal of Biotechnology Vol. 12(26), pp. 4123-4128

Heavy metal resistant anaerobic bacterial strains from brewery digester sludge

This work focused on the study of the types of heavy-metal-resistant anaerobic bacteria from the sludge of a brewery wastewater treatment plant exposed to high concentrations of dissolved Cd (II), Cu (II) and Zn (II). Characterisations were carried out by polymerase chain reaction of the 16S rRNA gene of bacterial strain. Using special culture media, two types of strong heavy metal-resistant bacterial strains were isolated. The first was a sulphate-reducing bacterium identified as Clostridium ganghwense strain HY-42-06. This strain of sulphate-reducing bacteria tolerated Cd (II), Cu (II) and Zn (II) at the tested concentrations. The other type was identified as consisting of a mixture of Micrococcus luteus, Wolinella succinogenes, Sporosarscina sp. PIC-C28 and Alicycliphilus sp. R-24604. The results found that these four dominant strains tolerated Cd (II) at 20 mg/l, only Wolinella succinogenes cannot tolerate Cu (II) at 2 mg/l while none of them tolerated Zn (II) at 30 mg/l.

Tolerance and Biosorption of Heavy Metals by Cupriavidus metallidurans strain XXKD-1 Isolated from a Subsurface Laneway in the Qixiashan Pb-Zn Sulfide Minery in Eastern China

The heavy metal-resistant bacterial strain Cupriavidus metallidurans XXKD-1, was isolated from a 475 m subsurface laneway in the Qixiashan Pb-Zn mine, near Nanjing City in eastern China. The phylogenetic analysis based on the 16S rRNA gene sequence shows that the microorgamism has 99% similarity to C. metallidurans CH34. Minimal inhibitory concentrations (MICs) of metals for the bacterium were determined. The bacterial strain XXKD-1 exhibited high MIC values for various heavy metals and a large spectrum of antibiotic resistance. The order of toxicity of the metals to the bacterium was Cd2+ (1 mM) > Co2+ (2 mM) > Cr3+ (2.5 mM) > Ni2+ (3 mM) > Cu2+ (3.5 mM) > Mn2+ (6 mM) > Zn2+ (10 mM) in Tris minimal broth media. Metal bioaccumulation was determined during the course of growth. C. metallidurans strain XXKD-1 is a very efficient biosorbent. It can remove 74% Mn, 53% Cd, 52% Co, 48% Zn and 4% Cr in the cell walls and 4% Mn, 12% Cd, 11% Co, 9% Zn and 50% Cr in intracellular spaces during the active growth cycle (using initial heavy metal concentrations of 0.5 mM). However, it did not efficiently accumulate Cu and Ni, removing only 12% and 10% with total metal biosorption capacities of 3.8 mg/L (0.06 mM) and 3.1 mg/L (0.053 mM), respectively. Because C. metallidurans cells could grow in the presence of elevated concentrations of metals and present relatively high heavy metal biosorption capacities in aerobic conditions, this bacterium may have potential applications for the in-situ bioremediation of heavy metal-contaminated aqueous (or soil) systems in mine ecosystems.

Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil

A heavy metal-resistant bacterial strain was isolated from heavy metal-contaminated soils and identified as Burkholderia sp. J62 based on the 16S rDNA gene sequence analysis. The heavy metal- and antibiotic resistance, heavy metal solubilization of the isolate were investigated. The isolate was also evaluated for promoting plant growth and Pb and Cd uptakes of the plants from heavy metal-contaminated soils in pot experiments. The isolate was found to exhibit different multiple heavy metal and antibiotic resistance characteristics. Atomic absorption spectrometer analysis showed increased bacterial solubilization of lead and cadmium in solution culture and in soils. The isolate produced indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. The isolate also solubilized inorganic phosphate. Inoculation with the isolate was found to significantly ( p < 0.05) increase the biomass of maize and tomato plants. Increase in tissue Pb and Cd contents varied from 38% to 192% and from 5% to 191% in inoculated plants growing in heavy metal-contaminated soils compared to the uninoculated control, respectively. These results show that heavy metal-solubilizing and plant growth promoting bacteria are important for plant growth and heavy metal uptake which may provide a new microbial enhanced-phytoremediation of metal-polluted soils.

Characterization and Nickel Sorption Kinetics of a New Metal Hyper-accumulator Bacillus sp

The heavy metal-resistant bacterial strain SJ-101 has been isolated from fly ash contaminated soil. Based on the morphological and biochemical characteristics, the isolate SJ-101 was presumptively identified as Bacillus sp. The adsorption isotherms revealed the absolute adsorption capacity (Q°) of 244 mg Ni g−1 dry cell mass vis-à-vis 161 mg Ni g−1 synthetic resin (Amberlite IR-120). The higher relative adsorption capacity (K F ) of 7.37, and the intensity of adsorption (1/n) of 0.58 with dry cell biomass suggested higher affinity of Bacillus cells towards nickel ions. The data conform to the Langmuir adsorption model relatively better than the Freundlich model. The thermodynamic parameters indicated the feasibility, endothermic, and interactive nature of nickel adsorption process on the cell surface. Higher Ni tolerance and sorption capacity of Bacillus sp. SJ-101, explicitly signifies its implications in Ni bioremediation process.

Aspects of Plant-Microbe Interactions in Heavy Metal Polluted Soil

The role of soil microbiota, specifically rhizospheric microorganisms, in the development of phytoremediation techniques has to be elucidated in order to speed up the process and to optimize the rate of mobilization/absorption of pollutants. To meet such a purpose, several heavy metal resistant bacterial strains were isolated from a contaminated soil and from the rhizosphere of some spontaneous plants grown therein, such as Brassica sp., Trifolium repens, Trifolium pratense and Chenopodium album. Colonization of the rhizosphere is known to be helpful for bacteria, but their presence is also supposed to be functional to the plants, especially if in connection with their ability to produce Plant Growth Promoting (PGP) compounds (e.g. indole-acetic acid) or to protect the plant from pathogens. Therefore, in such an adverse environment, plants and bacteria surely take advantage by cooperating. Several bacterial isolates were tested and found to be capable of producing PGP compounds. Among them, a highly heavy metal resistant strain (Br-10) was isolated from the roots of a spontaneous plant belonging to the Brassicaceae family, whose members are known to accumulate heavy metals, and identified by phenotypic tests and 16S rDNA analysis as Serratia plymuthica. To evaluate its putative biocontrol activity, a screening of its antagonistic properties against various soil phytopathogens was also undertaken. Among the pathogenic fungi tested, a strain of Phytophtora megasperma f. sp. glycinea was found to be sensitive. Preliminary investigations of Br-10 resistance mechanisms, performed through hybridization studies on genomic DNA, suggested for S. plymuthica strain Br-10 the existence of a resistance mechanism different from that codified by the well-known czc gene cluster of Alcaligenes eutrophus that encodes for cadmium, zinc and cobalt resistance through an efflux system. By the use of Inductively Coupled Plasma analysis Cd was found to be preferentially adsorbed or accumulated rather than being forced out of the cell or not imported at all. Growth kinetics studies revealed that increasing Cd levels do not seem to affect cell growth up to the stationary phase, while having a clear impact on cell viability during the stationary phase.