Dual augmentation for aerobic bioremediation of MTBE and TCE pollution in heavy metal-contaminated soil

In this work we isolated from soil and characterized several bacterial strains capable of either resisting high concentrations of heavy metals (Cd²⁺ or Hg²⁺ or Pb²⁺) or degrading the common soil and groundwater pollutants MTBE (methyl-tert-butyl ether) or TCE (trichloroethylene). We then used soil microcosms exposed to MTBE (50 mg/l) or TCE (50 mg/l) in the presence of one heavy metal (Cd 10 ppm or Hg 5 ppm or Pb 50 or 100 ppm) and two bacterial isolates at a time, a degrader plus a metal-resistant strain. Some of these two-membered consortia showed degradation efficiencies well higher (49–182% higher) than those expected under the conditions employed, demonstrating the occurrence of a synergetic relationship between the strains used. Our results show the efficacy of the dual augmentation strategy for MTBE and TCE bioremediation in the presence of heavy metals.     

Heavy-metal stress induced accumulation of chitinase isoforms in plants

Plant chitinases belong to so-called pathogenesis related proteins and have mostly been detected in plants exposed to phytopathogenic viruses, bacteria or fungi. A few studies revealed that they might also be involved in plant defence against heavy metals. This work was undertaken to monitor the accumulation of chitinases in a set of heavy-metal stressed plants and bring evidence on their involvement during this kind of stress. Roots of different plant species including Vicia faba cvs. Aštar and Piešťansky, Pisum sativum, Hordeum vulgare, Zea mays and Glycine max were exposed to different concentrations of lead (300 and 500 mg l⁻¹ Pb²⁺), cadmium (100 and 300 mg l⁻¹ Cd²⁺) and arsenic (50 and 100 mg l⁻¹ As³⁺). In each case, the toxicity effects were reflected in root growth retardation to 80–10% of control values. The most tolerant were beans, most sensitive was barley. Extracts from the most stressed roots were further assayed for chitinase activity upon separation on polyacrylamide gels. Our data showed that in each combination of genotype and metal ion there were 2–5 different chitinase isoforms significantly responsive to toxic environment when compared with water-treated controls. This confirms that chitinases are components of plant defence against higher concentrations of heavy metals. In addition, accumulation of some isoforms in response to one but not to other metal ions suggests that these enzymes might also be involved in a more (metal) specific mechanism in affected plants and their biological role is more complex than expected.     

Employment of Rhizobacteria for the Inoculation of Barley Plants Cultivated in Soil Contaminated with Lead and Cadmium

In laboratory experiments, the rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens7, Agrobacterium radiobacter 10, and Flavobacterium sp. L30 were found to have a relatively high resistance to the toxic heavy metals lead and cadmium (except that strain L30 was found to be sensitive to Cd). When introduced by means of seed bacterization, the heavy metal–resistant strains actively colonized the rhizosphere of barley plants cultivated in uncontaminated and contaminated soils. In both pot and field experiments, seed bacterization improved the growth of barley plants and the uptake of nutrient elements from soil contaminated with Pb and Cd. The bacterization also prevented the accumulation of Pb and Cd in barley plants, thereby mitigating the toxic effect of these heavy metals on the plants.     

Chromate reduction by PVA-alginate immobilized <Emphasis Type="Italic">Streptomyces griseus</Emphasis> in a bioreactor

Microbial reduction of toxic Cr⁶⁺ to the less toxic Cr³⁺ is potentially a useful bioremediation process. Among the matrices tested for whole cell immobilization of an efficient chromate-reducing Streptomyces griseus strain, PVA-alginate was the most effective and was used for reduction of Cr(VI) in a bioreactor. Cr⁶⁺ reduction efficiency decreased as Cr⁶⁺ was increased from 2 to 12 mg l⁻¹ but increased with an increase in biomass concentration. However, increasing the flow rate from 2 to 8 ml h⁻¹ did not significantly affect Cr⁶⁺ reduction. The reduction was faster in simulated effluent than in synthetic medium and complete removal of 8 mg Cr⁶⁺ l⁻¹ from effluent and synthetic medium occurred in 2 and 12 h, respectively. Our results indicate that immobilized S. griseus cells could be applied for the large-scale bioremediation of chromate-containing effluents and wastewaters.     

Isolation and Molecular Characterization of Heavy Metal-Resistant Alcaligenes faecalis from Sewage Wastewater and Synthesis of Silver Nanoparticles

Environmental pollution with toxic heavy metals is increasing throughout the world alongside industrial development. Microorganisms and microbial products can be highly efficient bioaccumulators of soluble and particulate forms of metals, especially dilute external solutions. Microbe related technologies (Biotechnology) may provide an alternative or additive conventional method for metal removal or metal recovery. This study dealt with isolation, identification and characterization of heavy metal-resistant (Pb²⁺, Cd²⁺, Al³⁺, Cu²⁺, Ag²⁺ and Sn²⁺) bacteria from sewage wastewater at Taif Province, Saudi Arabia. Nine bacterial isolates were selected by using an enrichment isolation procedure based on high level of heavy metal resistance. All the isolates showed high resistance to heavy metals with Minimum Inhibitor Concentration (MIC) ranging from 800 μg/ml to 1400 μg/ml. All nine resistant isolates showed multiple tolerances to heavy metals. On the basis of morphological, biochemical and 16S rRNA characterization, the most potent isolates (Cd1-1, Ag1-1, Ag1-3 and Sn1-1) were identified as Alcaligenes faecalis. Scanning electron microscope analysis showed that the morphology of Alcaligenes faecalis Ag1-1 was unchanged after growth in medium without and with addition of Ag²⁺ indicative Ag²⁺ is not toxic to the isolate under the conditions tested. The ability of Alcaligenes faecalis Ag1-1 to synthesize sliver nanoparticles was examined. The heavy metal-resistant bacteria obtained could be useful for the bioremediation of heavy metal-contaminated environment.     

Hyperaccumulation of nickel by two Alyssum species from the serpentine soils of Iran

Serpentine soils, which contain relatively high concentrations of nickel and some other metals, are the preferred substrate for some plants, especially those that accumulate Ni in their tissues. In temperate regions more Ni-hyperaccumulator plants are found in Alyssum than in any other genus. In this study, serpentine soils of two areas (Marivan and Dizaj) in the west/northwest of Iran and also perennial Alyssum plants growing on these soils were analyzed for Ni and some other metals. The highest concentrations of total metals in the soils of these areas for Ni, Cr, Co and Mn were 1,350, 265, 94 and 1,150 μg g⁻¹, respectively, while concentrations of Fe, Mg and Ca reached 3.55%, 16.8% and 0.585% respectively. The concentration of exchangeable Ni in these soils is up to 4.5 μg g⁻¹. In this study two Alyssum species, A. inflatum and A. longistylum, have been collected from Marivan and Dizaj, respectively. Analysis of leaf dry matter shows that they can contain up to 3,700 and 8,100 μg Ni g⁻¹, respectively. This is the first time that such high Ni concentrations have been found in these species. The concentrations of other metals determined in these species were in the normal range for serpentine plants, except for Ca, which was higher, up to 5.3% and 3.5%, respectively     

Removing heavy metals from synthetic effluents using “kamikaze” Saccharomyces cerevisiae cells

One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated waters. In this study, the heavy metal hypersensitive yeast strain pmr1∆ was investigated for the ability to remove Mn²⁺, Cu²⁺, Co²⁺, or Cd²⁺ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in removing Mn²⁺, Cu²⁺, or Co²⁺ from synthetic effluents containing 1–2 mM cations, with a selectivity $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} and also in removing Mn²⁺ and Cd²⁺ from synthetic effluents containing 20–50 μM cations, with a selectivity Mn²⁺ > Cd²⁺.     

Adsorption of Heavy Metals by Paenibacillus validus Strain MP5 Isolated from Industrial Effluent–Polluted Soil

The present investigation was carried out to isolate bacterial strains from soil/mud samples of metal-polluted environment to check whether the natural adaptation of microbes has equipped them for bioremediation of toxic heavy metals. The primary and secondary screening resulted in 50 mesophilic autotrophic isolates of microbial consortium adapted for metal tolerance and bioadsorption potentiality. The multimetal tolerance in bacterial strain was developed by sequential transfer to higher concentrations of Cd, Cr, Cu, Pb, Ni, and Zn. The isolates were checked for their biosolubilization potential with copper-containing metal sulfide ores, viz. chalcopyrite exhibited 64% and covellite 54% solubilization in the presence of 10^?3 M multiple heavy metals on the fifth day at 35°C and pH 6.0. Metal adsorption of highly potential isolate, i.e., Paenibacillus validus MP5, studied by inductively coupled plasma optical emission spectroscopy (ICP-OES), showed maximum adsorption of Zn 27%, followed by Ni and Cd 16%, Cr 15%, Co 9%, and Pb 7.5% in chalcopyrite, which suggested its possible role in decontamination of metal-polluted sites.     

Isolation and characterization of endophytic bacterium LRE07 from cadmium hyperaccumulator Solanum nigrum L. and its potential for remediation

Valuable endophytic strains facilitating plants growth and detoxification of heavy metals are required because the application of plant–endophyte symbiotic system is a promising potential technique to improve efficiency of phytoremediation. In this study, endophytic bacterium LRE07 was isolated from cadmium hyperaccumulator Solanum nigrum L. It was identified as Serratia sp. by 16S rRNA sequence analysis. The endophytic bacterium LRE07 was resistant to the toxic effects of heavy metals, solubilized mineral phosphate, and produced indoleacetic acid and siderophore. The heavy metal detoxification was studied in growing LRE07 cells. The strain bound over 65% of cadmium and 35% of zinc in its growing cells from single metal solutions 72 h after inoculation. Besides the high removal efficiencies in single-ion system, an analogous removal phenomenon was also observed in multi-ions system, indicating that the endophyte possesses specific and remarkable heavy metal remediation abilities.     

Degradation of Microcystin-RR by Sphingomonas sp. CBA4 isolated from San Roque reservoir (Córdoba – Argentina)

We report the aerobic biodegradation of Microcystin-RR (MC-RR) by a bacterial strain isolated from San Roque reservoir (Córdoba – Argentina). This bacterium was identified as Sphingomonas sp. (CBA4) on the basis of 16S rDNA sequencing. The isolated strain was capable of degrading completely MC-RR (200 μg l⁻¹) within 36 h. We have found evidence that MC-RR biodegradation pathway by this Sphingomonas sp. strain would start by demethylating MC-RR, affording an intermediate product, which is finally biodegraded by this strain within 72 h. Our results confirm that certain environmental bacteria, living in the same habitat as toxic cyanobacteria, have the capability to perform complete biodegradation of MC, leading to natural bioremediation of waterbodies. The bacterium reported here presents genetic homologies with other strains that degrade MC-LR. However, initial demethylation of MC-RR has been not described previously, raising questions on the probable presence of different biodegradation pathways for different MC variants.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Use of the microalga Scenedesmus obliquus to remove cadmium cations from aqueous solutions

The ability of a wild strain of Scenedesmus obliquus, isolated from a heavy metal-contaminated environment, to remove Cd²⁺ from aqueous solutions was studied at several initial concentrations. Viable biomass removed metal to a maximum extent of 11.4 mg_Cd/g at 1 mg_Cd/l, with most Cd²⁺ being adsorbed onto the cell surface. A commercially available strain (ACOI 598) of the same microalga species was also exposed to the same Cd concentrations, and similar results were obtained for the maximum extent of metal removal. Heat-inactivated cells removed a maximum of 6.04 mg_Cd/g at 0.5 mg_Cd/l. The highest extent of metal removal, analyzed at various pH values, was 0.09 mg_Cd/g at pH 7.0. Both strains of the microalga tested have proven effective in removing a toxic heavy metal from aqueous solutions, hence supporting their choice for bioremediation strategies of industrial effluents.     

Nickel Tolerance and Accumulation by Filamentous Fungi From Sludge of Metal Finishing Industry

Industrialization, urbanization and increased vehicular traffic have resulted in increased contamination of our environment by heavy metals. The long persistence of heavy metals in nature has in turn resulted in development of metal resistant microbial strains. These strains are minimizing heavy metals toxicity, either by metal complexation or precipitation and other mechanisms. Characterization of fungal diversity was done in contaminated soil of the Wazirpur industrial area throughout the year. In this area highly acidic hazardous solid waste produced high concentration of heavy metals (Ni, Cu, Cr, Fe, Mn). Nickel toxicity is a major environmental concern. Due to long persistence of this waste in the environment without any treatment, many fungal isolates from the surrounding environment settle on the upper surface of waste. Few of them are capable of growing in the toxic conditions. More than 20 strains were isolated, most of them belonging to species of Aspergillus, Penicillium, Fusarium and Mucor genera. Seasonal variation in fungal diversity was significant. Four filamentous fungal isolates were found to be resistant for nickel (II) and a strain of Papulaspora sepedonoides reported first time for bioremediation of Ni (II) in this investigation, which is absorbing 62.33 μmol Ni gr^?1. These fungal isolates showed a high level (100–10000 mg kg^?1) of resistance for Ni (II) salt and removing Ni (II) from solution. Metal uptake varied with fungi. The toxicity also was influenced by different factors like pH and composition of growth medium.     

Overexpression of a Single Membrane Component from the Bacillus mer Operon Enhanced Mercury Resistance in an Escherichia coli Host

Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 μM Hg²⁺, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.     

Simultaneous phenanthrene and cadmium removal from contaminated soil by a ligand/biosurfactant solution

Surfactants and inorganic ligands are pointed as efficient to simultaneous removal of heavy metals and hydrophobic organic pollutants from soil. However, the biosurfactants are potentially less toxic to soil organisms than other chemical agents. Thus, in this study the efficiency of combinations of iodide (I⁻) ligand and surfactants produced by different bacterial species in the simultaneous removal of cadmium (Cd²⁺) and phenanthrene in a Haplustox soil sample was investigated. Four microbial surfactants and the synthetic surfactant Triton X-100 were tested with different concentrations of ligand. Soil samples contaminated with Cd²⁺ and phenanthrene underwent consecutive washings with a surfactant/ligand solution. The removal of Cd²⁺ increased with increased ligand concentration, particularly in solutions containing biosurfactants produced by the bacterial strains Bacillus subtilis LBBMA155 (lipopeptide) and Flavobacterium sp. LBBMA168 (mixture of flavolipids) and Triton X-100. Maximum Cd²⁺ removal efficiency was 99.2% for biosurfactant produced by Arthrobacter oxydans LBBMA 201 (lipopeptide) and 99.2% for biosurfactant produced by Bacillus sp. LBBMA111A (mixed lipopeptide) in the presence of 0.336 mol iodide l⁻¹, while the maximum efficiency of Triton X-100 removal was 65.0%. The biosurfactant solutions removed from 80 to 88.0% of phenanthrene in soil, and the removal was not influenced by the presence of the ligand. Triton X-100 removed from 73 to 88% of the phenanthrene and, differently from the biosurfactants, iodide influenced the removal efficiency. The results indicate that the use of a single washing agent, called surfactant-ligand, affords simultaneous removal of organic contaminants and heavy metals.     

Enhancement of emulsifier production by <Emphasis Type="Italic">Curvularia lunata</Emphasis> in cadmium,zinc and lead presence

The influence of cadmium, zinc and lead on fungal emulsifier synthesis and on the growth of filamentous fungus Curvularia lunata has been studied. Tolerance to heavy metals established for C. lunata was additionally compared with the sensitivity exhibited by strains of Curvularia tuberculata and Paecilomyces marquandii—fungi which do not secrete compounds of emulsifying activity. Although C. lunata, as the only one out of all studied fungi, exhibited the lowest tolerance to heavy metals when grown on a solid medium (in conditions preventing emulsifier synthesis), it manifested the highest tolerance in liquid culture - in conditions allowing exopolymer production. Cadmium, zinc and lead presented in liquid medium up to a concentration of 15 mM had no negative effect on C. lunata growth and stimulated emulsifier synthesis. In the presence of 15 mM of heavy metals, both the emulsifier and 24-h-old growing mycelium exhibited maximum sorption capacities, which were determined as 18.2 ± 2.67, 156.1 ± 10.32 mg g⁻¹ for Cd²⁺, 22.2 ± 3.40, 95.2 ± 14.21 mg g⁻¹ for Zn²⁺ and 51.1 ± 1.85, 230.0 ± 28.47 mg g⁻¹ for Pb²⁺ respectively. The results obtained by us in this work indicate that the emulsifier acts as a protective compound increasing the ability of C. lunata to survive in heavy metal polluted environment. Enhancement of exopolymer synthesis in the presence of Cd²⁺, Zn²⁺ and Pb²⁺ may also suggest, at least to some extent, a metal-specific nature of emulsifier production in C. lunata. Due to accumulation capability and tolerance to heavy metals, C. lunata mycelium surrounded by the emulsifier could be applied for toxic metal removal.     

Cu,Zn-superoxide dismutase is differently regulated by cadmium and lead in roots of soybean seedlings

The cadmium (Cd²⁺) and lead (Pb²⁺)-induced changes in Cu,Zn-SOD gene expression on the level of mRNA accumulation and enzyme activity were analyzed in roots of soybean (Glycine max) seedlings. The Cd²⁺ caused the induction of copper–zinc superoxide dismutase (Cu,Zn-SOD) mRNA accumulation, at each analyzed metal concentration (5–25 mg/l), whereas in Pb²⁺-treated roots this effect was observed only at the medium metal concentrations (50–100 mg/l of Pb²⁺). The analysis of Cu,Zn-SOD activity proved an increase in enzyme activity during Cd²⁺/Pb²⁺ stresses, however in Pb²⁺-treated plants the activity of enzyme was not correlated with respective mRNAs level. Presented data suggest that different metals may act on various level of Cu,Zn-SOD expression in plants exposed to heavy metals stress.     

Sensitivity of bacterial vs. acute Daphnia magna toxicity tests to metals

The objectives of this study were to evaluate the sensitivity of two bacterial tests commonly used in metal toxicity screening — the Vibrio fischeri bioluminescence inhibition test and the Pseudomonas putida growth inhibition test — in comparison to the standard acute Daphnia magna test, and to estimate applicability of the selected methods to the toxicity testing of environmental samples. The D. magna acute test proved to be more sensitive to cadmium (Cd), zinc (Zn) and manganese (Mn) than the two bacterial assays, whereas P. putida seems to be the most sensitive species to lead (Pb). Manganese appears to be slightly toxic to D. magna and non-toxic to the two selected bacteria. This leads to the conclusion that even in regions with high background concentrations, manganese would not act as a confounding factor. Low sensitivity of V. fischeri to heavy metals questions its applicability as the first screening method in assessing various environmental samples. Therefore, it is not advisable to replace D. magna with bacterial species for metal screening tests. P. putida, V. fischeri and/or other bacterial tests should rather be applied in a complex battery of ecotoxicological tests, as their tolerance to heavy metals can unravel other potentially present toxic substances and mixtures, undetectable by metal-sensitive species.     

Adsorption of Copper and Cadmium by Cu- and Cd-Resistant Bacteria and Their Composites with Soil Colloids and Kaolinite

Abstract

Remediation of toxic metals by bacteria offers a relatively inexpensive and efficient way for the decontamination of soil and associated environments. The present study was carried out to investigate the surface characteristics, adsorption, and remobilization of Cd and Cu on bacteria and their composites with soil colloidal components, which are the most active constituents in soils. The bacterial strain NTG-01 (Enterobacter aerogenes), which was both Cd- and Cu-resistant, was isolated from a heavily Cu-contaminated soil of the mining area in Daye suburb of Hubei Province, China. Batch laboratory experiments with NTG-01 and soil colloids were performed to quantify adsorption of Cu and Cd. The surface area of kaolinite and the soil colloids from an Alfisol and Ultisol increased by 3.0–8.8% after the introduction of the bacteria. In the presence of bacterial cells, the negative charges of soil colloid systems increased and the positive charges decreased, shifting pH from 4.0 to 6.5. Our results demonstrate that bacteria promote the adsorption of Cd and Cu by kaolinite and soil colloid systems. However, the heavy metals bound by the bacterial composites could also be easily released by NH₄NO₃ and EDTA. Caution should be taken when using such bacterial strains in bioremediation of heavy metal-contaminated soils.     

Phytoremediation—A Novel and Promising Approach for Environmental Clean-up

ABSTRACT

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering of phytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.