Chromium(VI) bioaccumulation capacities of adapted mixed cultures isolated from industrial saline wastewaters

Enrichment mixed cultures tolerating relatively high concentrations of chromium and salt ions were isolated and their bioaccumulation properties improved by adaptation. Mixed cultures were enriched in Nutrient Broth media containing 25-300 mg l(-1) Cr(VI) and 0%, 2%, 4%, 6% (w/v) NaCl. Bioaccumulation of Cr(VI) was studied in a batch system as a function of initial pH (7, 8 and 9), Cr(VI) and NaCl concentrations. Increasing NaCl and Cr(VI) concentrations led to significant decreases in percentage uptake and dried weight of mixed cultures but increased maximum specific chromium uptake. The maximum specific chromium uptake value at pH 8 was 58.9 mg g(-1) for 316.1 mg l(-1) Cr(VI) in the absence of NaCl, while at pH 9 it was 130.1 mg g(-1) in media including 194.5 mg l(-1) Cr(VI) and 2% NaCl concentrations. At 4% NaCl, the maximum Cr(VI) uptake of 127.0 mg g(-1) for 221.1 mg l(-1) Cr(VI) occurred at pH 9, while at 6% NaCl the maximum Cr(VI) uptake of 114.9 mg g(-1) for 278.1 mg l(-1) Cr(VI) was found at pH 7.     

Inhibitory and stimulating effect of single and multi-metal ions on hexavalent chromium reduction by Acinetobacter sp. Cr-B2

Potential application of chromium reducing bacteria for industrial scale wastewater treatment demands that effect of presence of other metal ions on rate of Cr(VI) reduction be investigated, as industrial wastewaters contain many toxic metal ions. In the current study, the effect of different heavy metal ions (nickel, zinc, cadmium, copper, lead, iron) on chromium reduction by a novel strain of Acinetobacter sp. Cr-B2 that shows high tolerance up to 1,100 mg/L and high Cr(VI) reducing capacity was investigated. The alteration in Cr(VI) reduction capacity of Cr-B2 was studied both in presence of individual metal ions and in the presence of multi-metal ions at different concentrations. The study showed that the Cr(VI) reduction rates decreased in presence of Ni²⁺, Zn²⁺ and Cd²⁺ when present individually. Pb²⁺ at lower concentration did not show significant effect while Cu²⁺ and Fe³⁺ stimulated the rate of Cr(VI) reduction. In the studies on multi-metal ions, it was observed that in presence of Cu²⁺ and Fe³⁺, the inhibiting effect of Ni²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ on Cr(VI) reduction was reduced. Each of these metals affect the overall rate of Cr(VI) reduction by Cr-B2. This work highlights the need to consider the presence of other heavy metal ions in wastewater when assessing the bioreduction of Cr(VI) and while designing the bioreactors for the purpose, as rate of reduction is altered by their presence.     

Isolation and Characterization of Chromium(VI)-Reducing Bacteria from Tannery Effluents

Two chromium-resistant bacteria (IFR-2 and IFR-3) capable of reducing/transforming Cr(VI) to Cr(III) were isolated from tannery effluents. Isolates IFR-2 and IFR-3 were identified as Staphylococcus aureus and Pediococcus pentosaceus respectively by 16S rRNA gene sequence analyses. Both isolates can grow well on 2,000 mg/l Cr(VI) (as K₂Cr₂O₇) in Luria-Bertani (LB) medium. Reduction of Cr(VI) was found to be growth-associated in both isolates and IFR-2 and IFR-3 reduced 20 mg/l Cr(VI) completely in 6 and 24 h respectively. The Cr(VI) reduction due to chromate reductase activity was detected in the culture supernatant and cell lysate but not at all in the cell extract supernatant of both isolates. Whole cells of IFR-2 and IFR-3 converted 24 and 30% of the initial Cr(VI) concentration (1 mg/l) in 45 min respectively at 37°C. NiCl₂ stimulated the growth of IFR-2 whereas HgCl₂ and CdCl₂ significantly inhibited the growth of both isolates. Optimum temperature and pH for growth of and Cr(VI) reduction by both isolates were found to be between 35 and 40°C and pH 7.0 to 8.0. The two bacterial isolates can be good candidates for detoxification of Cr(VI) in industrial effluents.     

Selection and properties of mutant yeast Pichia guilliermondii strains resistant to chromium (VI)

Yeast Pichia guilliermondii strains L3 and L2, exposed to UV mutagenesis, produced over 80 mutants capable of growing on media containing 1.5 mM bichromate (Cr(VI)). The mutations making the strains resistant to Cr(VI) were dominant or semidominant. The mutants varied in Cr(VI) resistance, the degree of chromium accumulation in the cells (from 0.1 to 11.6 mg/g dry cells), and the degree of Cr(VI) reduction (from 50% to complete disappearance of bichromate from the culture liquid). Chromium accumulation in mutant cells depended on medium composition, Cr(VI) concentration, and the time of exposure to Cr(VI). The resistance to bichromate can be caused by various reasons: decrease in chromium absorption, altered ability to reduce Nr(VI), or damage of sulfate transport mechanisms.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Selection and properties of mutant yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> strains resistant to chromium (VI)

Yeast Pichia guilliermondii strains L3 and L2, exposed to UV mutagenesis, produced over 80 mutants capable of growing on media containing 1.5 mM bichromate (Cr(VI)). The mutations making the strains resistant to Cr(VI) were dominant or semidominant. The mutants varied in Cr(VI) resistance, the degree of chromium accumulation in the cells (from 0.1 to 11.6 mg/g dry cells), and the degree of Cr(VI) reduction (from 50% to complete disappearance of bichromate from the culture liquid). Chromium accumulation in mutant cells depended on medium composition, Cr(VI) concentration, and the time of exposure to Cr(VI). The resistance to bichromate can be caused by various reasons: decrease in chromium absorption, altered ability to reduce Cr(VI), or damage of sulfate transport mechanisms.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 204–209.Original Russian Text Copyright © 2005 by Babyak, Ksheminskaya, Gonchar, Yanovich, Fedorovich.     

Distinct and effective biotransformation of hexavalent chromium by a novel isolate under aerobic growth followed by facultative anaerobic incubation

A bacterial isolate (G161) with high Cr(VI)-reducing capacity was isolated from Cr(VI)-contaminated soil and identified as Leucobacter sp. on the basis of 16S rRNA gene sequence analysis. The isolate was a Gram-positive, aerobic rod. The hexavalent chromate-reducing capability of the isolate was investigated under three conditions of oxygen stress. The isolate was found to reduce Cr(VI) under all conditions but performed most effectively during aerobic growth followed by facultative anaerobic incubation. Under these conditions, the isolate tolerated K₂Cr₂O₇ concentrations up to 1,000 mg/l and completely reduced 400 mg/l K₂Cr₂O₇ within 96 h. The strain reduced Cr(VI) over a wide range of pH (6.0–11.0) and temperatures (15–45 °C) with optimum performance at pH?8.0 and 35 °C. The presence of other metals, such as Ca²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺, induced no effect or else played a stimulatory role on Cr(VI)-reduction activity of the strain. The strain was tested for Cr(VI) removal in wastewaters and proved capable of completely reducing the contained Cr(VI). This is the novel report of a bacterial growth and Cr(VI)-reduction process under sequential aerobic growth and facultative anaerobic conditions. The study suggested that the isolate possesses a distinct capability for Cr(VI) reduction which could be harnessed for the detoxification of chromate-contaminated wastewaters.     

Hexavalent Chromium Reduction and Accumulation by <Emphasis Type="Italic">Acinetobacter</Emphasis> AB1 Isolated from Fez Tanneries in Morocco

Hexavalent chromium reduction and accumulation by Acinetobacter AB1 isolated from Fez tanneries effluents were tested. The effects of some environmental factors such as pH, temperature, and exposure time on Cr(VI) reduction and resistance were investigated. We found that this strain was able to resist to concentrations as high as 400 mg/l of Cr(VI). Moreover, pH 10 and the temperature 30°C constitute favourable conditions to the growth and reduction of Acinetobacter AB1. Complete reduction of Cr(VI) was observed at low initial Cr(VI) concentrations of 50 mg/l after 72 h of incubation. Furthermore, Transmission electron microscope (TEM) analysis showed morphological changes in AB1 strain due 48H exposure to 100 mg/l chromate concentration and revealed circular electron dense (dark black point) inclusion within the cell cytoplasm suggesting chromium deposition within the cells.     

Preliminary studies on continuous chromium(VI) biological removal from wastewater by anaerobic-aerobic activated sludge process

The long-term continuous chromium(VI) removal from synthetic wastewater affected by influent hexavalent chromium (Cr(VI)) and glucose concentrations were studied with an anaerobic-aerobic activated sludge process. It was observed that before activated sludge was acclimated, the chromium in the effluent increased immediately as the influent chromium increased. However, both Cr(VI) and total chromium (TCr) in the effluent significantly decreased after acclimation. In the acclimated activated sludge, the chromium removal efficiency was 100% Cr(VI) and 98.56% TCr at influent Cr(VI) levels of 20 mg/day, 100% Cr(VI) and 98.92% TCr at influent Cr(VI) levels of 40 mg/day, and 98.64% Cr(VI) and 97.16% TCr at influent Cr(VI) levels of 60 mg/day. The corresponding effluent Cr(VI) and TCr concentrations were 0 and 0.012 mg/l, 0 and 0.018 mg/l, and 0.034 mg/l and 0.071 mg/l, respectively. When the influent glucose increased from 1125 to 1500 mg/l at influent Cr(VI) dosage of 60 mg/day, the Cr(VI) and TCr removal efficiency with the acclimated activated sludge improved from 98.64% and 97.16% to 100% and 98.48%, respectively, and the chromium concentration in the effluent decreased from 0.034 mg/l of Cr(VI) and 0.071 mg/l of TCr to 0 (Cr(VI)) and 0.038 mg/l (TCr). The effluent COD and turbidity was around 40 mg/l and 0, respectively, after the activated sludge was acclimated. Further studies showed that after the activated sludge was acclimated, its specific dehydrogenases activity (SDA) and protein contents increased. The SDA and protein increased respectively 15% and 10% when influent Cr(VI) increased from 20 to 60 mg/day.     

Bioremediation of toxic chromium from electroplating effluent by chromate-reducing Pseudomonas aeruginosa A2Chr in two bioreactors

The chromate-reducing ability of Pseudomonas aeruginosa A2Chr was compared in batch culture, with cells entrapped in a dialysis sac, and with cells immobilized in an agarose-alginate film in conjunction with a rotating biological contactor. In all three systems, the maximum Cr(VI) reduction occurred at 10 mg Cr(VI)/l. Whereas at 50 mg Cr(VI)/l concentration, only 16% of the total Cr(VI) was reduced, five spikings with 10 mg chromate/l at 2-h intervals led to 96% reduction of the total input of 50 mg Cr(VI)/l. Thus maximum Cr(VI) reduction was achieved by avoiding Cr(VI) toxicity to the cells by respiking with lower Cr(VI) concentrations. At 10 mg Cr(VI)/l, the pattern of chromate reduction in dialysis-entrapped cells was almost similar to that of batch culture and 86% of the bacterially reduced chromium was retained inside the dialysis sac. In electroplating effluent containing 100 mg Cr(VI)/l, however, the amount of Cr(VI) reduced by the cells immobilized in agarose-alginate biofilm was twice and thrice the amount reduced by batch culture and cells entrapped in a dialysis sac, respectively.     

The reduction of Cr(VI) by bacteria of the genus Pseudomonas

Non-nitrate-reducing collection bacteria from the genus Pseudomonas were found to be able to use hexavalent chromium as a terminal electron acceptor. The reduction of Cr(VI) was accompanied by an increase in the cell biomass. At the Cr(VI) concentrations in the medium lower than 15 mg/l, the non-nitrate-reducing pseudomonads reduced Cr(VI) less efficiently than did denitrifying pseudomonads. In contrast, at the Cr(VI) concentrations higher than 30 mg/l, Cr(VI) was reduced more efficiently by the non-nitrate-reducing pseudomonads than by the denitrifying pseudomonads.     

The Reduction of Cr(VI) by Bacteria of the Genus Pseudomonas

Non-nitrate-reducing collection bacteria from the genus Pseudomonas were found to be able to use hexavalent chromium as a terminal electron acceptor. The reduction of Cr(VI) was accompanied by an increase in the cell biomass. At Cr(VI) concentrations in the medium lower than 15 mg/l, the non-nitrate-reducing pseudomonads reduced Cr(VI) less efficiently than did denitrifying pseudomonads. In contrast, at Cr(VI) concentrations higher than 30 mg/l, Cr(VI) was reduced more efficiently by the non-nitrate-reducing pseudomonads than by the denitrifying pseudomonads.     

Anaerobic digestion of cattail with rumen culture in the presence of heavy metals

The anaerobic digestion of cattail by rumen cultures in the presence of Cu(II), Cd(II) or Cr(VI) was investigated in this study. Three cases were respectively observed for the different metal dosages: promoted cattail degradation and methane production at a low heavy metal concentration, e.g., Cu(II) 2.4 mg/l, Cd(II) 1.6 mg/l, Cr(VI) 4.0 mg/l; reduced cattail degradation efficiency and methane production at a middle metal level; a severe inhibition to the cattail degradation at a high heavy metal dosage. The inhibition kinetics of Cu(II) on the digestion of cattail by rumen cultures was also analyzed and a simplified Andrews equation was used to describe such an inhibition. The inhibition constants for Cu(II) on the degradation of cattail, production of volatile fatty acids and formation of methane were estimated as 7.4, 9.5 and 6.4 mg/l, respectively. Comparative experimental results suggest that the order of toxicity degree of the tested metals on the rumen cultures was: Cd(II) > Cu(II) > Cr(VI).     

利用细菌还原有毒Cr(Ⅵ)为Cr(Ⅲ)

Overthelastfewdecadesenvironmentalcontaminationwithheavymetalshasincreaseddrastically .Heavymetalsfoundinwastewatersareharmfultotheenvironmentandtheireffectsonbiolo gicalsystemareverysevere.Anefficientandcheaptreatmentfortheirremovalandreuseofspentmetalsfromwastewaterneedstobedeve loped .Theremovaloftoxicmetalsfromtheenvironmentbymi croorganismshaspotentialasaneffectivemeansofremediatingheavymetalswastes.Microbe basedtechnologiescanprovideanalternativetoconventionalmethodsformetalremoval[1 ] .…     

Microsomal metabolism of hexavalent chromium. Inhibitory effect of oxygen and involvement of cytochrome P-450

The reduction of hexavalent chromium (Cr(VI] by rat liver microsomes was studied. With 15-120 microM Na2CrO4 microsomes (0.5 mg protein/ml) effectively reduced Cr(VI) in the presence of NADPH provided anaerobic conditions. Phenobarbital (PB) and Aroclor 1254 (PCB) pretreatment increased microsomal Cr(VI) reduction while CoCl2 reduced the rate. The rates with 30 microM Na2CrO4 were: 6.4 +/- 0.1, 7.8 +/- 0.7, 13.4 +/- 0.5, 2.95 +/- 0.09 nmol Cr.mg prot.-1 min-1 for control, PB, PCB and cobalt pretreated microsomes respectively. Kinetic studies gave a Michaeli-Menten like first-order kinetics with increases both in Km and Vmax values after pretreatment with PB or PCB. CO partly inhibited the microsomal Cr(VI) reduction. The CO-sensitive reduction rate was directly correlated to the cyt. P-450 content of the different microsomal preparations. Substituting NADH for NADPH gave approximately 27% lower activity with 30 microM Na2CrO4. This activity was neither inducible by cyt. P-450 inducers nor influenced by CO. Oxygen 1.0% and 0.10% gave approximately 100% and 30% inhibition of Cr(VI) reduction (30 microM Na2CrO4) respectively, and an uncompetitive like inhibitory pattern was found. No redox cycling of Cr(VI) was seen. 51Cr binding to the microsomes was approximately 10% after complete reduction of 30 microM Na2CrO4. Externally added FMN, Fe3+-ADP and nitrobenzen stimulated microsomal Cr(VI) reduction. A 60% higher reduction rate of Cr(VI) by isolated hepatocytes was found during anaerobic in comparison with aerobic conditions.     

Moderate selenium dosing inhibited chromium (VI) toxicity in chicken liver

This study aimed to clarify the effect of selenium (Se) on chromium (VI) [Cr(VI)]‐induced damage in chicken liver. A total of 105 chickens were randomly divided into seven groups of 15. Group I received deionized water; group II received Cr(VI) (7.83 mg/kg/d) alone; and other groups orally received both Cr(VI) (7.83 mg/kg/d) and Se of different doses (0.14, 0.29, 0.57, 1.14, and 2.28 mg/kg/d). The levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), Ca²⁺‐ATPase, and mitochondrial membrane potential (MMP) were measured. Results showed that Cr(VI) increased MDA content and decreased GSH content, T‐SOD activity, Ca²⁺‐ATPase activity, and MMP level. Meanwhile, Se co‐treatment (0.14, 0.29, and 0.57 mg/kg/d) increased the viability of the above indicators compared with Cr(VI)‐treatment alone. In addition, histopathologic examination revealed that Cr(VI) can cause liver damage, whereas Se supplementation of moderate dose inhibited this damage. This study confirmed that Se exerted protective effect against Cr(VI)‐induced liver damage.     

Diversity of chromium-resistant and -reducing bacteria in a chromium-contaminated activated sludge

AIMS: This study attempts to establish a relationship between the Cr(VI) resistance of the culturable microbial community and the Cr(VI) resistance and Cr(VI)-reducing ability of representative strains of each population, in order to assess whether these are exclusive characteristics of one microbial group or abilities shared among many groups. METHODS AND RESULTS: A group of 48 Cr(VI)-resistant isolates, with different colony types, was isolated from chromium-contaminated activated sludge. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein patterns and fatty acid methyl ester analysis identified six populations, representing 54% of the isolated bacteria, as belonging to the genera Acinetobacter and Ochrobactrum. The remaining populations included strains identified as species of the beta-Proteobacteria and high G + C Gram-positive bacteria. The Cr(VI) resistance and reduction ability of the strains were tested. All but two isolates grew in the presence of 1 mmol l(-1) Cr(VI). During enrichment, all isolates were able to survive to 2 mmol l(-1) Cr(VI) and complete Cr(VI) reduction was achieved. Representative strains of each population were able to partially reduce (5.4-39.1%) the Cr(VI) present in the growth medium. CONCLUSIONS: Most of the identified isolates have never been reported to be Cr(VI)-resistant and/or Cr(VI)-reducing strains. The mechanisms of Cr(VI) resistance and reduction may differ from group to group; therefore, it is evident that both Cr(VI) resistance and reduction are shared abilities and not an exclusive characteristic of a single group, possibly reflecting horizontal genetic transfer resulting from selective pressure in this contaminated environment. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study of a microbial community under chronic chromate stress and, as the success of microbial-based metal remediation technologies requires a better understanding of the microbial community and the population response to metal stress, it may contribute to the implementation of a strategy of bioremediation of chromate-contaminated environments.     

Characterization of microbial fuel cells enriched using Cr(VI)-containing sludge

Microbial fuel cells (MFCs) were successfully enriched using sludge contaminated with Cr(VI) and their characteristics were investigated. After enrichment, the charge of the final 10 peaks was 0.51 C +/- 1.16%, and the anodic electrode was found to be covered with a biofilm. The enriched MFCs removed 93% of 5 mg/l Cr(VI) and 61% of 25 mg/l Cr(VI). 16S rDNA DGGE profiles from the anodic electrode indicated that beta-Proteobacteria, Actinobacteria, and Acinetobacter sp. dominated. This study is the first to report that electrochemically active and Cr(VI)-reducing bacteria could be enriched in the anode compartment of MFCs using Cr(VI)-containing sludge and demonstrates the Cr(VI) removal capability of such MFCs.     

Immune response and disease resistance of Oreochromis mossambicus to Aeromonas hydrophila after exposure to hexavalent chromium

The objective of this study was to investigate the effect of chronic exposure to sublethal concentrations of hexavalent chromium (K2Cr2O7) on the immune response and disease resistance of Oreochromis mossambicus (Peters) to bacterial Aeromonas hydrophila infection. Fish (45 to 50 g) were exposed to 0.005, 0.05, 0.5, and 5 mg l(-1) [0.01, 0.1, 1, and 10% LC50, respectively] of hexavalent chromium Cr (VI) for 28 d. The specific immune response was assessed by antibody response to A. hydrophila by bacterial agglutination assay, and to sheep red blood cells (SRBC) by plaque forming cell (PFC) assay. In addition, nonspecific immune mechanisms were assessed by serum lysozyme activity and reactive nitrogen intermediates, the latter in terms of nitric oxide (NO) production by peripheral blood leucocytes. Overall immunity was assessed by disease resistance against live virulent A. hydrophila. The study clearly indicated that chronic exposure of fish to 0.5 and 5 mg l(-1) of chromium (VI) decreased both nonspecific and specific parameters of the immune system, which resulted in a lower disease resistance to A. hydrophila. Interestingly, 0.05 mg l(-1) of Cr (VI) enhanced disease resistance and both nonspecific and specific immune responses to A. hydrophila. Our study revealed a concentration-dependent modulation of the immune system by chromium (VI), as demonstrated by suppressive or stimulatory effects on lymphocytes, lysozyme, phagocytic killing mechanisms, and disease resistance in O. mossambicus.     

Oxidative stress response and fatty acid changes associated with bioaccumulation of chromium [Cr(VI)] by a fresh water cyanobacterium Chroococcus sp.

Cr(VI) at 2.5, 5, 7.5 and 10 mg/l was removed over 1–5 days by a freshwater cyanobacterium, Chroococcus sp. 2.5 mg Cr(VI)/l gave the optimum rate. With 5 mg Cr(VI)/l, activities of superoxide dismutase and catalase were increased. Amounts of palmitic (16:0), stearic (18:0) and oleic acid (18:1) in the cell also increased after exposure to Cr(VI).