Production of Catalases by Aspergillus niger Isolates as a Response to Pollutant Stress by Heavy Metals

Isolates of Aspergillus niger, selected from the coal dust of a mine containing arsenic (As; 400 mg/kg) and from the river sediment of mine surroundings (As, 1651 mg/kg, Sb, 362 mg/kg), growing in minimal nitrate medium in the phase of hyphal development and spore formation, exhibited much higher levels of total catalase activity than the same species from the culture collection or a culture adapted to soil contaminated with As (5 mg/L). Electrophoretic resolution of catalases in cell-free extracts revealed three isozymes of catalases and production of individual isozymes was not significantly affected by stress environments. Exogenously added stressors (As⁵⁺, Cd²⁺, Cu²⁺) at final concentrations of 25 and 50 mg/L and H₂O₂ (20 or 40 mM) mostly stimulated production of catalases only in isolates from mines surroundings, and H₂O₂ and Hg²⁺ caused the disappearance of the smallest catalase I. Isolates exhibited a higher tolerance of the toxic effects of heavy metals and H₂O₂, as monitored by growth, than did the strain from the culture collection.     

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.     

一株十溴联苯醚高效好氧降解菌的筛选、鉴定及降解特性

【目的】从广东贵屿镇电子垃圾拆解地采集的沉积物样品中分离十溴联苯醚(BDE-209)高效好氧降解菌,并考察其对BDE-209的降解特性。【方法】通过生理生化实验和16S rRNA测序鉴定菌种,正交实验优化降解条件,并分析不同降解体系及影响因素对菌降解BDE-209的影响。【结果】鉴定结果显示,该BDE-209好氧降解菌为短短芽孢杆菌(Brevibacillus brevis)。B.brevis对1 mg/L BDE-209 5 d的降解率可达54.38%。正交实验结果表明,B.brevis降解BDE-209的最优条件为:pH 7,投菌量3 g/L,温度30°C。降解特性研究结果显示B.brevis对BDE-209降解的最佳菌龄为36 h,最佳氮源为(NH4)2SO4,B.brevis对Cu2+、Cd2+有较好的耐受性,但Cu2+和Cd2+的存在会影响其对BDE-209的降解。当Cu2+浓度在1 5 mg/L,Cd2+浓度在0.3 0.5 mg/L范围内时,B.brevis对BDE-209降解均可达50%以上。【结论】B.brevis对BDE-209有很好的降解效率,研究结果对BDE-209的好氧微生物降解及环境中BDE-209的生物修复具有较好的科学意义和应用价值。     

Production of catalases byComamonas spp. and resistance to oxidative stress

Bacterial isolates Comamonas terrigena N3H (from soil contaminated with crude oil) and C. testosteroni (isolated from the sludge of a wastewater treatment plant), exhibit much higher total catalase activity than the same species from laboratory collection cultures. Electrophoretic resolution of catalases revealed only one corresponding band in cell-free extracts of both C. testosteroni cultures. Isolates of C. terrigena N3H exhibited catalase-1 and catalase-2 activity, whereas in the collection culture C. terrigena ATCC 8461 only catalase-1 was detected. The environmental isolates exhibited much higher resistance to exogenous H2O2 (20, 40 mmol/L) than collection cultures, mainly in the middle and late exponential growth phases. The stepwise H2O2-adapted culture of C. terrigena N3H, which was more resistant to oxidative stress than the original isolate, exhibited an increase of catalase and peroxidase activity represented by catalase-1. Pretreatment of cells with 0.5 mmol/L H2O2 followed by an application of the oxidative agent in toxic concentrations (up to 40 mmol/L) increased the rate of cell survival in the original isolate, but not in the H2O2-adapted variant. The protection of bacteria caused by such pretreatment corresponded with stimulation of catalase activity in pretreated culture.     

Diversity and heavy metal tolerance of endophytic fungi from Dysphania ambrosioides,a hyperaccumulator from Pb–Zn contaminated soils

The diversity and heavy metal (HM) tolerance of endophytic fungi (EF) associated with Dysphania ambrosioides, a hyperaccumulator from two Pb–Zn contaminated sites were investigated. A total of 237 culturable EF were isolated and identified to 43 taxa based on morphological characteristics and rDNA internal transcribed spacer analysis, of which 13 occurred as endophytes of both sites, while other taxa were only found in either site. The colonization rate, dominant genera, community structure of EF as well as the HM content in the plant from two sites were significantly different. We suggest that these differences may result from the difference in the soil HM content: lower HM content in the soil, more EF in the plant, which may enhance the plant HM accumulation and thus result higher HM in it. HM tolerance tests indicated that 50% of the isolates exhibited HM tolerance. Among them, two isolates exhibited better HM tolerance, of which FT2G59 could tolerate Pb, Zn, and Cd, and the minimum inhibitory concentration (MIC) of them were 30–50, >?680, 20–30?mmol/l, respectively. While, the isolate FT2G7 could tolerate Cd, and the MIC was 30–50?mmol/l. These isolates may have potential application in phytoremediation.     

Metal Contamination in Sediment of One of the Upper Reaches of the Yangtze River: Mianyuan River in Longmenshan Region,Southwest of China

Metal contamination in sediment of the Mianyuan River (one of the major upper reaches of the Yangtze River) in Longmenshan Region (China) was investigated in 2012. Means of metal concentrations in sediment (<74μm) were Cr: 59.93 ± 19.8% mg/kg; As: 7.21 ± 50.2% mg/kg; Se: 0.45 ± 66.3% mg/kg; Pb: 19.89 ± 29.3% mg/kg; Zn: 78.98 ± 31.9% mg/kg; Cd: 0.69 ± 28.3% mg/kg; Ba: 0.71 ± 34.0% g/kg; Mn: 0.55 ± 62.2% g/kg. This study suggested: (1) concentrations of Cd, As, Cr, and Pb in Mianyuan River sediment were lower than those of the middle and lower reaches of the Yangtze River; (2) the increase of metals during the period from 2006 to 2009 was probably related to the destruction of tailings piles by the Wen Chun earthquake in 2008; (3) organic materials decided the distribution of Cd, Se, As, Ba, and Mn in the upstream sediment, while the iron and manganese minerals controlled the distribution of Ba, Cr, and Zn in the downstream sediment; (4) sources of Cd, Se, and As were geogenic, while sources of Cr, Zn, Ba, and Mn were anthropogenic; (5) the source of Pb in the upstream sediment was probably automobile exhaust, but that of Pb in the downstream sediment was geogenic.     

Anti-Helicobacter pylori effects of IgY from egg york of immunized hens

Effects of egg york containing IgY specific for Helicobacter pylori on the bacterial growth and intragastric infection were investigated in comparison with a proton-pump inhibitor pantoprazole. For in vitro anti-bacterial activity test, H. pylori (1×10(8) CFU/mL) was incubated with a serially diluted IgY for 3 days. As a result, IgY fully inhibited the bacterial growth at 16 mg/mL, which was determined to a minimal inhibitory concentration. In vivo elimination study, male C57BL/6 mice were infected with the bacteria by intragastric inoculation (1×10(8) CFU/mouse) 3 times at 2-day intervals, and 2 weeks later, orally treated twice a day with 50, 100, 200 or 500 mg/kg IgY for 18 days. After the final administration, biopsy sample of the gastric mucosa was assayed for the bacterial identification via urease, oxidase, catalase, nitrate reduction and H(2)S tests in addition to microscopic examination for mucosal inflammation. In CLO kit test, 75, 50, 12.5 and 12.5% of the animals revealed positive reaction following treatment with 50, 100, 200 and 500 mg/kg IgY, respectively, resulting in a superior efficacy at 200 mg/kg than 30 mg/kg pantoprazole that displayed 75% elimination. The CLO test results were confirmed by bacterial identification. Microscopic examination revealed that H. pylori infection caused severe gastric mucosal inflammation, which were not observed in the CLO-negative mice following treatment with IgY or pantoprazole. Taken together, IgY inhibited the growth of H. pylori, and improved gastritis and villi injuries by eliminating the bacteria from the stomach. The results indicate that IgY could be a good candidate overcoming tolerance of antibiotics for the treatment of H. pylori-mediated gastric ulcers.     

Effect of Cd-containing wood ash on the microflora of coniferous forest humus

The use of wood ash in forestry has been questioned because the cadmium (Cd) concentration of ash, which varies between 1 and 20 mg kg(-1) ash, exceeds the level allowed for fertilizers (3 mg kg(-1)) used in agriculture. To investigate the combined and separated effects of Cd and ash on the forest humus microflora, pumice or wood ash, spiked with a water-soluble (CdCl(2)) or -insoluble (CdO) form of Cd at three levels (0, 400 and 1000 mg kg(-1)), were applied at a fertilization level of 5000 kg ha(-1) in a laboratory microcosm study. The trial consisted of 60 microcosms (five replications per treatment), which were incubated in darkness at +20 degrees C and a constant relative air humidity of 60%. After two months the humus in the microcosms was sampled. Analyses of CO(2) evolution to measure the overall microbial activity and of phospholipid fatty acid (PLFA) pattern to measure microbial community structure were performed. The substrate-use patterns of Biolog EcoPlates were analyzed as a measure of bacterial functionality. Finally the bacterial (3)H-thymidine incorporation in the presence of different concentrations of Cd and the number of colony forming units (cfu) of bacteria on nutrient agar in the presence of 0, 5 and 20 mg Cd l(-1) agar were applied to measure Cd tolerance. The use of pumice (pH of humus under the pumice 4.0) did not induce any changes in the above variables compared to two untreated microcosms (humus pH 3.9). Pumice was therefore used to distribute the Cd evenly over the humus surface in order to estimate the possible effect of Cd without ash (pH of humus under the ash 7.0). The application of ash increased the microbial activity, changed the PLFA and substrate-use patterns and increased cfu compared to the humus under pumice. The form and level of Cd in the ash had no further effect on this result. In the humus under pumice the level, but not the form of Cd decreased the microbial activity and changed the PLFA pattern compared to the unspiked pumice. None of the treatments induced bacterial tolerance to Cd. Ash thus protected the humus microflora from the harmful effects of Cd.     

Iodine sensitivity of bacteria isolated from iodinated water systems

Fourteen bacterial isolates, predominantly Pseudomonas sp., from two water systems disinfected by iodinated anion-exchange resins were studied and compared with an isolate of Pseudomonas aeruginosa from a povidone-iodine solution and four other isolates. Pseudomonas cepacia and P. aeruginosa grown in brain heart infusion were 3 to 5 logs less sensitive to 1 mg/L I2 (pH 7.2, 1 min) when compared with cultures grown in phosphate buffer. Another P. cepacia isolate was the least sensitive culture when grown in brain heart infusion (1 log decrease) but was more sensitive after cultivation in phosphate buffer (5 logs). Isolates from an iodinated potable water system, including P. cepacia, Staphyloccus warneri, and a Bacillus sp., were all less sensitive to iodine than a "resistant" P. aeruginosa and three other isolates when grown in brain heart infusion. A clinical isolate of P. aeruginosa exhibited intermediate sensitivity. The sensitivity of bacteria to iodine is thus highly variable, depending on the organism as well as the growth conditions.     

Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane

Contamination by heavy metals is one of the major environmental problems in many countries and these contaminants reach from various sources such as traffic cars and other activities. Soil and phylloplane samples were collected from eight traffic and two non-traffic sites in Sohag city, Egypt. Heavy metal contents of Cd2?, Zn2? and Pb2? of soil and phylloplane samples were determined and revealed high levels of Zn2? and Pb2? in traffic samples. A total of 112 bacterial and 62 fungal isolates were obtained from soil and phylloplane. Bacterial isolates were characterized on the basis of morphological, physicochemical and biochemical characteristics; and 16S rRNA gene sequences. Fungal isolates were identified according to morphological characterization. Minimal inhibitory concentrations (MICs) of Cd2?, Zn2? and Pb2? for each isolate were detected. All bacterial and fungal isolates demonstrated resistance to lead with MICs >0.528 mM and >0.211, respectively. Moreover, the maximum MICs of cadmium and zinc for bacteria were 0.821 mM and 1.471 mM, respectively, where as, MICs for fungi were 0.328 mM and 0.588 mM, respectively. The most resistant bacterial and fungal isolates were Pseudomonas aeruginosa RA65 and Penicillium corylophyllum, respectively. Therefore, P. aeruginosa RA65 was selected for further investigations. Growth curve study showed that 0.264 mM lead had no efficiently effect on the growth of P. aeruginosa RA65. Plasmid isolation evidenced by transformation studies indicated that P. aeruginosa RA65 harbored a single plasmid (~9.5 kb) which mediated heavy meal resistance. Consequently, these microbial isolates could be potentially used in bioremediation of heavy metal-contaminated environment.     

R-factor in Proteus vulgaris from ulcerative disease of fish,Channa punctatus

A Proteus vulgaris isolated from external ulcers of the fresh water fish Channa punctatus showed multidrug resistance and heavy metal tolerance. The isolate from the ulcer showed resistance to chloramphenicol (Ch), nalidixic acid (Nx), streptomycin (Str) and tetracycline (Tet) with minimum inhibitory concentration (MIC) values of 750, 150, 75 and 125 microg/ml, respectively. The isolate showed growth in medium containing cadmium (Cd2+), up to a concentration of 2.5 mM indicating its heavy metal tolerance. Resistance to Ch, Str, Tet and Cd2+ of the isolate was lost after plasmid curing. Presence of plasmid DNA in the wild type and its absence in the cured P. vulgaris suggested that the resistance were plasmid mediated.     

Identification and characterization of heavy metal-resistant unicellular alga isolated from soil and its potential for phytoremediation

A unicellular alga displaying a high growth rate under heterotrophic growth conditions was isolated from soil and identified as Chlorella sorokiniana. The optimal temperature for growth was 35 degrees C and the optimal pH was 6.0-7.0. Glucose, sucrose, galactose, maltose, and soluble starch served as carbon sources supporting growth under dark conditions. The cell yield was 50 g/l (wet weight) in a heterotrophic medium containing 3% glucose. Isolated unicellular algae were highly resistant to heavy metals such as Cd(2+), of which the minimal inhibitory concentration was 4 mM. Algae were capable of taking up the heavy metal ions Cd(2+), Zn(2+) and Cu(2+) at 43.0, 42.0 and 46.4 microg/mg dry weight, respectively. Growth inhibition of Oryza sative shoots by 5 ppm Cd(2+) in hydroponic medium was completely prevented by the addition of 0.25 mg of wet Chlorella cells. These results indicated that this isolate was potentially useful for phytoremediation by preventing environmental dispersion of heavy metals.     

镉和铅对鲫鱼肝胰脏过氧化氢酶活性的影响

采用急性毒性实验方法,研究了不同ρCd2+和ρPb2+及二者混合溶液,染毒24、48、72、96 h后,对鲫鱼肝胰脏过氧化氢酶(CAT)活性的影响.结果表明:在实验剂量范围(ρCd2+为0.5～6.0 mg/L,ρPb2+为10.0～40.0 mg/L),0.5、2.0 mg/L的Cd2+对CAT活性有诱导作用,6.0 mg/L的Cd2+对CAT活性有抑制作用;Pb2+胁迫对CAT活性随着染毒时间的延长而逐渐降低;2.0 mg/L Cd2+与20.0 mg/L Pb2+混合胁迫对CAT活性有明显的诱导作用;0.5mg/L Cd2++10.0 mg/L Pb2+,6.0 mg/L Cd2++40.0 mg/L Pb2+混合胁迫对CAT活性有明显抑制作用.Cd2+和Pb2+胁迫对CAT活性的影响呈剂量效应,二者关系曲线为抛物线,抛物线顶点是鲫鱼对Cd2+和Pb2+污染从适应到中毒反应的阈值,可间接作为检测环境污染情况的指标.     

Bioremediation of toxic heavy metals using acidothermophilic autotrophes

Investigations were carried out to isolate microbial strains from soil, mud and water samples from metallurgically polluted environment for bioremediation of toxic heavy metals. As a result of primary and secondary screening various 72 acidothermophilic autotrophic microbes were isolated and adapted for metal tolerance and biosorption potentiality. The multi-metal tolerance was developed with higher gradient of concentrations of Ag, As, Bi, Cd, Cr, Co, Cu, Hg, Li, Mo, Pb, Sn and Zn. The isolates were checked for their biosolubilization ability with copper containing metal sulfide ores. In case of chalcopyrite 85.82% and in covellite as high as 97.5% copper solubilization occurred in presence of 10(-3) M multi-heavy metals on fifth day at 55 degrees C and pH 2.5. Chemical analyses were carried out by inductively coupled plasma spectroscopy (ICP) for metal absorption. The selected highly potential isolate (ATh-14) showed maximum adsorption of Ag 73%, followed by Pb 35%, Zn 34%, As 19%, Ni 15% and Cr 9% in chalcopyrite.     

Biosorptive removal of arsenic from drinking water

A biomass derived from the plant Momordica charantia has been found to be very efficient in arsenic(III) adsorption. An attempt was made to use this biomass for arsenic(III) removal under different conditions. The parameters optimized were contact time (5-150 min), pH (2-11), concentration of adsorbent (1-50 g/l), concentration of adsorbate (0.1-100mg/l), etc. It was observed that the pH had a strong effect on biosorption capacity. The optimum pH obtained for arsenic adsorption was 9. The influence of common ions such as Ca(2+), Mg(2+), Cd(2+), Se(4+), Cl(-), SO(4)(2-), and HCO(3)(-), at concentrations varying from 5 to 1000 mg/l was investigated. To establish the most appropriate correlation for the equilibrium curves, isotherm studies were performed for As(III) ion using Freundlich and Langmuir adsorption isotherms. The pattern of adsorption fitted well with both models. The biomass of M. charantia was found to be effective for the removal of As(III) with 88% sorption efficiency at a concentration of 0.5mg/l of As(III) solution, and thus uptake capacity is 0.88 mg As(III)/gm of biomass. It appears that this biomass should be used as a palliative food item. Further it also appears that the dietary habits may play a role in the toxic effects of ingested arsenic.     

Physiological effects of dietary cadmium acclimation and waterborne cadmium challenge in rainbow trout: respiratory, ionoregulatory, and stress parameters

A suite of respiratory, acid-base, ionoregulatory, hematological, and stress parameters were examined in adult rainbow trout (Oncorhynchus mykiss) after chronic exposure to a sublethal level of dietary Cd (500 mg/kg diet) for 45 days and during a subsequent challenge to waterborne Cd (10 microg/L) for 72 h. Blood sampling via an indwelling arterial catheter revealed that dietary Cd had no major effects on blood gases, acid-base balance, and plasma ions (Ca(2+), Mg(2+), K(+), Na(+), and Cl(-)) in trout. The most notable effects were an increase in hematocrit (49%) and hemoglobin (74%), and a decrease in the plasma total ammonia (43%) and glucose (49%) of the dietary Cd-exposed fish relative to the nonexposed controls. Dietary Cd resulted in a 26-fold increase of plasma Cd level over 45 days (approximately 24 ng/mL). The fish exposed to dietary Cd showed acclimation with increased protection against the effects of waterborne Cd on arterial blood P(aCO2) and pH, plasma ions, and stress indices. After waterborne Cd challenge, nonacclimated fish, but not Cd-acclimated fish, exhibited respiratory acidosis. Plasma Ca(2+) levels declined from the prechallenge level, but the effect was more pronounced in nonacclimated fish (44%) than in Cd-acclimated fish (14%) by 72 h. Plasma K(+) was elevated only in the nonacclimated fish. Similarly, waterborne Cd caused an elevation of all four traditional stress parameters (plasma total ammonia, cortisol, glucose, and lactate) only in the nonacclimated fish. Thus, chronic exposure to dietary Cd protects rainbow trout against physiological stress caused by waterborne Cd and both dietary and waterborne Cd should be considered in determining the extent of Cd toxicity to fish.     

UV-resistant Acinetobacter sp. isolates from Andean wetlands display high catalase activity

Andean wetlands are characterized by their extreme environmental conditions such as high UV radiation, elevated heavy metal content and salinity. We present here the first study on UV tolerance and antioxidant defense of four Acinetobacter strains: Ver3, Ver5 and Ver7, isolated from Lake Verde, and N40 from Lake Negra, both lakes located 4400 m above sea level. All four isolates displayed higher UV resistance compared with collection strains, with Ver3 and Ver7 being the most tolerant strains not only to UV radiation but also to hydrogen peroxide (H(2)O(2)) and methyl viologen (MV) challenges. A single superoxide dismutase band with similar activity was detected in all studied strains, whereas different electrophoretic pattern and activity levels were observed for catalase. Ver3 and Ver7 displayed 5-15 times higher catalase activity levels than the control strains. Analysis of the response of antioxidant enzymes to UV and oxidative challenges revealed a significant increase in Ver7 catalase activity after H(2)O(2) and MV exposure. Incubation of Ver7 cultures with a catalase inhibitor resulted in a significant decrease of tolerance against UV radiation. We conclude that the high catalase activity displayed by Ver7 isolate could play an important role in UV tolerance.     

Pyrene and Phenanthrene Influence on Soil Microbial Populations

Two studies were conducted to evaluate microbial populations in polycyclic aromatic hydrocarbon-contaminated soil. Captina silt loam was freshly exposed to (1) 0 or 2000?mg pyrene/kg and sampled after 10- and 61-wk incubation and (2) 0 or 505?mg pyrene + 445?mg phenanthrene/kg and sampled after a 21-wk incubation. Microbial numbers were determined by plate-count techniques. Isolated bacteria, selected degraders, and wholesoil extracts were analyzed by fatty acid methyl ester analysis (FAME). In the pyrene experiment, pyrene did not affect total bacterial or fungal numbers, but pyrene degraders increased from undetectable levels to 7.09 log₁₀ degraders/g in the contaminated soil. The FAME analysis of bacterial isolates detected no pyrene effect, but wholesoil FAME indicated an increase in the contaminated soil of a fatty acid characteristic of protozoa and a major fatty acid detected in isolated degraders. In the pyrene + phenanthrene experiment, the contaminants had no impact on bacterial, fungal, or actinomycete numbers but increased degrader numbers. No effect of pyrene + phenanthrene was detected by isolate FAME, but whole-soil FAME indicated an effect similar to that in the pyrene experiment. The results indicate that pyrene, although not impacting microbial numbers, may have altered the soil microbial composition and that Captina silt loam can develop an effective degrader population under tested conditions.     

Cadmium biosorption by yeast, Candida tropicalis CBL-1, isolated from industrial wastewater

The present study is aimed at assessing the ability of metal-resistant yeast, Candida tropicalis CBL-1, to uptake metal from liquid medium. The minimum inhibitory concentration of Cd(II) against Candida tropicalis CBL-1 was 2,800 mg/L. The yeast could also tolerate Zn(II) (3,100 mg/L), Hg(II) (2,400 mg/L), Ni(II) (2,200 mg/L), Cr(VI) (2,000 mg/L), Pb(II) (1,100 mg/L), and Cu(II) (2,200 mg/L). The yeast isolate showed typical growth curves but lag and log phases extended in the presence of cadmium. The yeast isolate showed optimum growth at 30oC and pH 7. The metal processing ability of the isolate was determined in a medium containing 100 mg/L of Cd(II). Candida tropicalis CBL-1, could reduce Cd(II) 59%, 64% and 70% from the medium after 48, 96 and 144 h, respectively. C. tropicalis CBL-1 was also able to remove Cd(II) 46% and 60% from the wastewater after 6 and 12 days, respectively. Cd produced an increase in glutathione and non-protein thiols level by 37% (17.50±0.8-24.0±1.2) and 18% (3.30±0.7- 3.90±0.8) at 100 mg/L concentration, respectively. Metal tolerance and accumulation together with changes in the GSH status and non-protein thiols under Cd exposure were studied in C. tropicalis.