Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe–metal interactions studies

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H₂S and by a direct enzymatic process involving hydrogenases and c₃ cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria–metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.     

Magnesium-induced biofilm development in Arthrobacter sp. SUK 1201 and removal of hexavalent chromium

This study reports the influence of Mg ions on the development and architecture of biofilms by a chromium resistant and reducing bacterium Arthrobacter sp. SUK 1201 and their utilization in the removal of toxic hexavalent chromium. Among the different metal ions tested, Mg(II) greatly influenced the biofilm growth in peptone yeast extract glucose medium. Both Scanning and Confocal Laser Scanning Microscopy revealed that biofilms formed under the induction of Mg(II) had characteristic higher cell densities. The cells remain embedded in thick porous layers of extracellular polymeric substances as evident from the fluorescein isothiocyanate labeled lectin concanavalin A and 4, 6- diamino-2-phenylindole staining. COMSTAT analysis also indicated maximum thickness and roughness coefficient of the biofilm grown in presence of Mg(II). Biofilms of Arthrobacter sp. SUK 1201 developed under such Mg (II) influenced condition showed complete removal of 0.5 mM Cr(VI) in mineral salts medium. The biofilm of this isolate grown in presence of Mg(II) was also able to remove 60µM Cr(VI) from mine seepage water suggesting its possible implication in effective bioremediation of chromium polluted environments.     

Chromate removal by yeasts isolated from sediments of a tanning factory and a mine site in Argentina

Twenty-one yeast-like microorganisms were isolated from tannery effluents and from a nickel–copper mine in Argentina. They were tested for their Cu(II), Ni(II), Cd(II) and Cr(VI) tolerance in qualitative assays on solid medium. Three isolates were selected for their multiple tolerance to the different heavy metals and highest tolerance to Cr(VI). According to morphological and physiological analysis and 26S rDNA D1/D2 domain sequences the isolates were characterized as: Lecythophora sp. NGV-1, Candida sp. NGV-9 and Aureobasidium pullulans VR-8. Resistance of the three strains to high Cr(VI) concentrations and their ability to remove Cr(VI) were assessed using YNB-glucose medium supplemented with 0.5 and 1 mM Cr(VI). Chromate removal activity was estimated by measuring remaining Cr(VI) concentration in the supernatant using the colorimetric 1,5-diphenylcarbazide method and total chromium was determined by flame atomic absorption spectroscopy. The results indicate that the initial Cr(VI) concentration negatively influenced growth and the specific growth rate but stimulated the metabolic activity of the three strains; resistance to Cr(VI) by these strains was mainly due to reduction of Cr(VI) rather than chromium bioaccumulation. This study showed the potential ability of these strains as tools for bioremediation of Cr(VI) from contaminated sites.     

Reduction and precipitation of chromate by mixed culture sulphate-reducing bacterial biofilms

The ability of sulphate-reducing bacterial biofilms to reduce hexavalent chromium (Cr(VI)) to insoluble Cr(III), a process of environmental and biotechnological significance, was investigated. The reduction of chromate to insoluble form has been quantified and the effects of chromate on the carbon source utilization and sulphate-reducing activity of the bacterial biofilms evaluated. Using lactate as the carbon/energy source and in the presence of sulphate, reduction of 500 micromol l-1 Cr(VI) was monitored over a 48-h period where 88% of the total chromium was removed from solution. Mass balance calculations showed that ca 80% of the total chromium was precipitated out of solution with the bacterial biofilm retaining less than 10% of the chromium. Only ca 12% of the chromate added was not reduced to insoluble form. Although Cr(VI) did not have a significant effect on C source utilization, sulphate reduction was severely inhibited by 500 micromol-1 Cr(VI) and only ca 10% of the sulphate reducing activity detected in control biofilms occurred in the presence of Cr(VI). Low levels of sulphide were also produced in the presence of chromate, with control biofilms producing over 10-times more sulphide than Cr(VI)-exposed biofilms. Sulphide- or other chemically-mediated Cr(VI) reduction was not detected. The biological mechanism of Cr(VI) reduction is likely to be similar to that found in other sulphate-reducing bacteria.     

Effect of Rhamnolipids on Chromium-Contaminated Kaolinite

Hexavalent chromium Cr(VI) is a common environmental pollutant that is treated by its reduction to the trivalent form Cr(III). The latter can be re-oxidized to the toxic form, Cr(VI), under specific conditions. A study was conducted on the removal of Cr(III) to eliminate the hazard imposed by its presence in soil as there has been some evidence that organic compounds can decrease its sorption. The effect of addition of negatively-charged biosurfactants (rhamnolipids) on chromium contaminated kaolinite was studied. Results showed that the rhamnolipids have the capability of extracting 25% portion of the stable form of chromium, Cr(III), from the kaolinite, under optimal conditions. The removal of hexavalent chromium was also enhanced compared to water by a factor of 2 using a solution of rhamnolipids. Results from the sequential extraction procedure showed that rhamnolipids remove Cr(III) mainly from the carbonate and oxide/hydroxide portions of the kaolinite. The rhamnolipids had also the capability of reducing close to 100% of the extracted Cr(VI) to Cr(III) over a period of 24 days. This study indicated that rhamnolipids could be beneficial for the removal or long–term conversion of chromium Cr(VI) to Cr(III).     

Soil chromium bioremediation: Synergic activity of actinobacteria and plants

The aim of this work was to evaluate a strategy to reduce the bioavailable chromium fraction in soil, using a Cr(VI) resistant microorganism, Streptomyces sp. MC1, under non sterile conditions, with maize plants as bioindicator and/or bioremediator.Soil samples were contaminated with 100, 200 and 400 mg kg⁻¹ of Cr(VI) or Cr(III). Bioavailable chromium (35%) was only detected in samples with Cr(VI). Soil samples with Cr(VI) 200 mg kg⁻¹ were inoculated with Streptomyces sp. MC1, and bioavailable chromium decreased up to 73%.Zea mays seedlings were planted in soil samples contaminated with chromium. Plantlets accumulated chromium mainly as Cr(III), and biomass decreased up to 88%. Streptomyces sp. MC1 was inoculated in soil samples contaminated with 200 mg kg⁻¹ of Cr(VI) and Z.mays seedlings were planted.Streptomyces sp. MC1 caused Z.mays biomass increase (57%), chromium accumulation and bioavailable chromium decreased up to 46% and 96%, respectively.This work constitutes the first contribution of cooperative action between actinobacteria and Z.mays in the bioremediation of Cr(VI) contaminated soil. The large removal capacity of bioavailable chromium by Streptomyces sp. MC1 and Z.mays infers that they could be successfully applied together in bioremediation of soils contaminated with Cr(VI).     

Biosorption of Chromium(VI) and Lead(II): Role of Spirulina platensis in the Treatment of Industrial Effluent

Biosorption is the process of removal of any chemical molecules by the treatment of biological material. Industrialization resulted in the discharge of various toxic heavy metals into water bodies, which poses serious health hazards to humans and animals. In the present study, live Spirulina platensis was used as a biosorbent for the removal of the heavy metals chromium (Cr(VI)) and lead (Pb(II)) from the aqueous samples. S. platensis were cultured in the presence of different concentrations of heavy metals. The growth of the algal cells was found to be decreased by 59% and 36% in media containing 50 ppm Cr(VI) and Pb(II), respectively. To assess the biosorption of heavy metals, at different time intervals, the spent culture media were used to detect Cr(VI) by atomic absorption spectroscopy method and Pb(II) by 4-(2-pyridylazo)resorcinol indicator method. Results suggested that there was a significant uptake of Cr(VI) and Pb(II) from the medium by S. platensis, with corresponding decrease of metals in the medium. When metal salt solutions or industrial effluent samples were passed through the column containing immobilized live S. platensis in calcium alginate beads, the concentration of Cr(VI) was found to be reduced drastically. The present study indicates the application of S. platensis for the bioremediation of heavy metals from the samples obtained from industrial effluents.     

Biosorption of chromium (Cr(III)/Cr(VI)) on the residual microalga Nannochloris oculata after lipid extraction for biodiesel production

In order to increase the economic feasibility of biodiesel production from microalgae, the residual biomass after biodiesel production can be utilized as biosorbent for heavy metal removal. In this study, biosorption of chromium by residual Nannochloris oculata after lipid extraction was investigated. Increased surface area of N. oculata was observed after lipid extraction. Cr(III) removal increased as the pH increased from 2 to 6, while Cr(VI) removal was highest at pH 2 and it decreased with the increase in pH. Cr(VI) was reduced to Cr(III) in the presence of biomass under acidic conditions; X-ray photoelectron spectroscopy revealed that the converted Cr(III) was bound to the biomass. Chromium removal was significantly enhanced at high chromium concentrations, which indicates that surface reactions may occur at high chromium/biomass ratios. FTIR study indicated that phosphate and carboxyl functional groups of the biomass were mainly responsible for chromium binding.     

The effects of hydraulic retention time (HRT) on chromium(VI) reduction using autotrophic cultivation of Chlorella vulgaris

Bioprocess and Biosystems Engineering - Chromium is an acutely toxic heavy metal that is known to be a carcinogen. Of the two predominant forms of chromium, Cr(III) and Cr(VI), Cr(III) has only...     

Characterization of PGP Traits of a Hexavalent Chromium Resistant Raoultella sp. Isolated from the Rice Field near Industrial Sewage of Burdwan District,WB, India

Chromium (Cr) is the most toxic at its hexavalent state. Widespread use of chromium for various anthropogenic activities causing rapid decline of the agricultural productivity is now a major global concern. The purpose of this study was to isolate the plant growth promoting (PGP) chromium-resistant bacteria and characterize it before being applied for bioremediation. A potent Cr-resistant rhizobacterium (CrS2) was isolated from the rice field near an industrial sewage and identified as Raoultella sp. based on 16S rDNA sequence homology with some phenotypic characteristics. The strain exhibited Cr(VI) resistance up to 25 mM and also possesses some important PGP traits. The selected CrS2 strain has varied degrees of resistance to other toxic heavy metals/metalloids like arsenic, cadmium, and lead. The removal capacity of chromium was studied in broth cultures. The appropriate growth media for the strain is peptone yeast glucose media with glucose (0.5%) and peptone (1%) as carbon and nitrogen sources, respectively. The strain removed substantial amount of chromium after media optimization. The chromate reductase (EC.1.6.5.2) activity was constitutive in nature of this strain. Thus, the strain CrS2 may be exploited for bioremediation of Cr(VI) in Cr-contaminated agricultural soil, where it might also enhance plant growth promotion.     

Influence of transition metals on Streptomyces coelicolor and S. sioyaensis and generation of chromate-reducing mutants

Bacteria-assisted bioremediation is widely recognized as a low-cost method to minimize the consequences of soil pollution with toxic metals originating from industrial sites. Strains used in bioremediation have to deal with high metal load via biosorption, reduction, bioprecipitation, metal sequestration, and/or chelation. Actinobacteria, and streptomycetes in particular, are considered a perspective group for bioremediation as natural soil inhabitants with extensive secondary metabolism. Nevertheless, there is no reference information on survival of the model streptomycetes in the presence of the most abundant metal pollutants. Also, there are no reports describing the selection approaches towards improvement of bioremediation properties. In this work, the resistance of Streptomyces coelicolor M145 and Streptomyces sioyaensis Lv81 to certain transition metals and their growth under different pH values are described for the first time. Spontaneous chromate-resistant S. sioyaensis Lv81-138 strain was selected in the course of this work. Strain Lv81-138 is the most efficient actinobacterial Cr(VI) reducer reported so far, capable of converting 12 mmol/L of Cr(VI) into Cr(III) in a medium supplemented with 50 mmol/L K₂CrO₄.     

Modulation of chromium(VI) toxicity by organic and inorganic sulfur species in yeasts from industrial wastes

Two chromium(VI) resistant yeast strains (Candida sp. and Rhodosporidium sp.) were isolated from industrial wastes. Four different yeasts, three from the Industrial Yeast Collection and one of pharmaceutical origin, were also studied in relation to chromate toxicity and its alleviation by sulfur species. The growth of yeasts from industrial wastes was inhibited by 50% by high concentrations of Cr(VI): Candida sp. by 4 mM Cr(VI) and Rhodosporidium sp. by 10 mM Cr(VI) in Sabouraud Broth medium. The other Cr(VI)-sensitive yeasts were inhibited by 0.1 mM Cr(VI). The general mechanism of chromium resistance in Candida sp. and Rhodosporidium sp. was due to reduced uptake of chromium, but not to biological reduction from Cr(VI) to Cr(III). In Cr(VI)-sensitive yeasts, chromium was accumulated as much as 10-fold, as in Saccharomyces cerevisiae. Cr(VI) toxicity in Candida sp. was modulated from Cr(VI)-resistance to Cr(VI)-hypersensitivity depending on the addition of methionine, cysteine, sulfate and djenkolic acid. If Candida sp. was grown in the presence of S-amino acids, especially methionine, it was more resistant than if the sulfur source was sulfate. When sulfate transport was enhanced by addition of djenkolic acid, Candida sp. became hypersensitive. Rhosporidium sp. was always resistant to Cr(VI) because sulfate transport was inefficient and it assimilated sulfur as S-amino acids. Cr(VI)-sensitive yeasts required larger amounts of S-amino acids, especially methionine, to tolerate Cr(VI) toxicity. Cysteine was toxic for C.famata 6016 above 50 microM.     

Bioaccumulation and biosorption of chromium by Aspergillus niger MTCC 2594

Chromium toxicity is of prime concern due to chrome tanning processes in the leather sector. Chrome tanning results in the discharge of toxic levels of chromium causing pollution hazards. Chromium levels of Cr(III) and Cr(VI) were high above permissible limits in chrome samples after chrome tanning. The potential of Aspergillus niger MTCC 2594 to accumulate chromium as well as its biosorption capacity is investigated in this study. Bioaccumulation of Cr(III) and Cr(VI) in the spent chrome liquor has resulted in a 75-78% reduction of the initial Cr content in 24-36 h. A. niger biomass is found to be very effective in the biosorption of Cr(III) and Cr(VI) in spent chrome liquor. Maximum adsorption of 83% for biosorption of Cr(III) at 48 h and 79% of Cr(VI) at 36 h in spent chrome liquor is observed. The biosorption characteristics fit well with Langmuir and Freundlich isotherms and the adsorption parameters are evaluated. The biosorption of Cr also follows Lagergren kinetics. A. niger biomass is effectively used for the biosorption of chromium with 79-83% Cr removal in 36-48 h.     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

Growth stimulatory effect of Ochrobactrum intermedium and Bacillus cereus on Vigna radiata plants

AIMS: This study assessed the plant growth-promoting ability of the bacterial strains Ochrobactrum intermedium (isolate CrT-1) and Bacillus cereus (isolate S-6). METHODS AND RESULTS: Two chromium resistant bacterial strains isolated from chromium-contaminated wastewater and soils were identified as O. intermedium CrT-1 and B. cereus S-6. These strains were inoculated on seeds of mungbean Vigna radiata var NM-92, which were germinated and grown under chromate salts (300 microg ml(-1) of CrCl(3)or K(2)CrO(4)). The data show that Cr(VI) was more toxic because of its better availability to plants roots when compared with Cr(III). The major part of Cr(VI) supplied to the seedlings was reduced to Cr(III) in the rhizosphere by the bacterial strains, thus lowering the toxicity of chromium to seedlings. CONCLUSIONS: Strains have significant Cr(VI) resistance and reduction potential and have ability to enhance mungbean plant growth under chromium stress. SIGNIFICANCE AND IMPACT OF THE STUDY: These strains could be utilized for the growth of economically important cash crops as well as for the bioremediation of chromium-polluted soils.     

Removal of chromium from industrial waste by using eucalyptus bark

Several low cost biomaterials such as baggase, charred rice husk, activated charcoal and eucalyptus bark (EB) were tested for removal of chromium. All the experiments were carried out in batch process with laboratory prepared samples and wastewater obtained from metal finishing section of auto ancillary unit. The adsorbent, which had highest chromium(VI) removal was EB. Influences of chromium concentration, pH, contact time on removal of chromium from effluent was investigated. The adsorption data were fitted well by Freundlich isotherm. The kinetic data were analyzed by using a first order Lagergren kinetic. The Gibbs free energy was obtained for each system and was found to be -1.879 kJ mol(-1) for Cr(VI) and -3.885 kJ mol(-1) for Cr(III) for removal from industrial effluent. The negative value of deltaG0 indicates the feasibility and spontaneous nature of adsorption. The maximum removal of Cr(VI) was observed at pH 2. Adsorption capacity was found to be 45 mg/g of adsorbent, at Cr(VI) concentration in the effluent being 250 mg/l. A waste water sample containing Cr(VI), Cr(III), Mg, and Ca obtained from industrial unit showed satisfactory removal of chromium. The results indicate that eucalyptus bark can be used for the removal of chromium.     

Simultaneous bioremediation of Cr(VI) and lindane in soil by actinobacteria

Environments co-contaminated with metals and organic compounds are difficult to remediate. Actinobacteria is an important group of microorganisms found in soils, with high metabolic versatility and potential for bioremediation. In this paper, actinobacteria were used to remediate soil co-contaminated with Cr(VI) and lindane. Five actinobacteria, tolerant to Cr(VI) and lindane mixture were selected: Streptomyces spp. A5, A11, M7, and MC1, and Amycolatopsis tucumanensis DSM 45259. Sterilized soil samples were inoculated with actinobacteria strains, either individually or as a consortium, and contaminated with Cr(VI) and lindane, either immediately or after 7 days of growth, and incubated at 30 °C during 14 days. All actinobacteria were able to grow and remove both contaminants, the consortium formed by Streptomyces spp. A5, M7, MC1, and A. tucumanensis showed the highest Cr(VI) removal, while Streptomyces sp. M7 produced the maximum lindane removal. In non-sterile soil samples, Streptomyces sp. M7 and the consortium removed more than 40% of the lindane, while Streptomyces sp. M7 demonstrated the greatest Cr(VI) removal. The most appropriate strategy for bioremediation of Cr(VI) and lindane co-contaminated soils would be the inoculation with Streptomyces sp. M7.     

Advanced kinetic model of the Cr(VI) removal by biomaterials at various pHs and temperatures

Recently, a new and simple kinetic model was derived from a basic concept of the redox reaction between Cr(VI) and biomaterials, and successfully described the removal behavior of Cr(VI) under various Cr(VI) and biomaterial concentrations. However, this model did not consider the effects of pH and temperature on the Cr(VI) removal by biomaterials. In this study, a new efficient biomaterial, pine needle, capable of removing Cr(VI) was used as a model one to study the Cr(VI) removal by biomaterials. Analysis of chromium species in aqueous and solid phases revealed that the removal mechanism of Cr(VI) by pine needle was its reduction into Cr(III). The removal rate of Cr(VI) increased with a decrease in pH or with an increase of temperature. Finally, an advanced kinetic model in the form of -d[Cr(VI)]/dt = Ae(Ea/RT)[H+]n[Cr(VI)][OCs] was derived, and successfully predicted the time-dependent Cr(VI) concentration at various pHs (2-4) and temperatures (10-55 degrees C).     

Use of Experimental Factorial Design for Optimization of Hexavalent Chromium Removal by a Bacterial Consortium: Soil Microcosm Bioremediation

Hexavalent chromium Cr(VI) is regularly introduced into the environment through diverse anthropogenic activities. It is highly toxic, mutagenic and carcinogenic, and because of its solubility in water, chromate contamination can be difficult to contain. Bacteria can reduce chromate to insoluble and less toxic trivalent chromium Cr(III), and thus increasing attention is paid to chromate bioremediation to reduce its ecotoxicological impacts. In this study, the factorial design 2³ was employed to optimize critical parameters responsible for higher Cr(VI) removal by a bacterial consortium. The factors considered were pH, temperature, and inoculum size at two markedly different levels. All three dependent variables have significant effect on Cr(VI) reduction. Optimal Cr(VI) removal by the bacterial consortium occurred at pH 9, temperature 37°C, and inoculum size OD = 3. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.984, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. In addition, the effect of bioaugmentation of Cr(VI)-polluted soil microcosms with the bacterial consortium was investigated using the best factor levels. Contaminated soil by 20 and 60 mg/Kg of Cr(VI) showed reductions of 83% and 65% of initial Cr(VI) by the bacterial consortium, suggesting that this bacterial consortium might diminish phytoavailable Cr(VI) in soil and be useful for cleaning up chromium-contaminated sites.     

Enhanced exopolymer production and chromium stabilization in Pseudomonas putida unsaturated biofilms

Chromium-contaminated soils threaten surface and groundwater quality at many industrial sites. In vadose zones, indigenous bacteria can reduce Cr(VI) to Cr(III), but the subsequent fate of Cr(III) and the roles of bacterial biofilms are relatively unknown. To investigate, we cultured Pseudomonas putida, a model organism for vadose zone bioremediation, as unsaturated biofilms on membranes overlaying iron-deficient solid media either containing molecular dichromate from potassium dichromate (Cr-only treatment) or with deposits of solid, dichromate-coated hematite (Fe+Cr treatment) to simulate vadose zone conditions. Controls included iron-deficient solid medium and an Fe-only treatment using solid hematite deposits. Under iron-deficient conditions, chromium exposure resulted in lower cell yield and lower amounts of cellular protein and carbohydrate, but providing iron in the form of hematite overcame these toxic effects of Cr. For the Cr and Fe+Cr treatments, Cr(VI) was completely reduced to Cr(III) that accumulated on biofilm cells and extracellular polymeric substances (EPSs). Chromium exposure resulted in elevated extracellular carbohydrates, protein, DNA, and EPS sugars that were relatively enriched in N-acetyl-glucosamine, rhamnose, glucose, and mannose. The proportions of EPS protein and carbohydrate relative to intracellular pools suggested Cr toxicity-mediated cell lysis as the origin. However, DNA accumulated extracellularly in amounts far greater than expected from cell lysis, and Cr was liberated when extracted EPS was treated with DNase. These results demonstrate that Cr accumulation in unsaturated biofilms occurs with enzymatic reduction of Cr(VI), cellular lysis, cellular association, and extracellular DNA binding of Cr(III), which altogether can facilitate localized biotic stabilization of Cr in contaminated vadose zones.