Orange G and Remazol Brilliant Blue R decolorization by white rot fungi Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus

Thirty different white rot strains were screened for Orange G and Remazol Brilliant Blue R (RBBR) decolorization on agar plates. Three promising strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus, selected on the basis of this screening, were used for decolorization study in liquid media. All three strains efficiently decolorized both Orange G and RBBR, but they differed in decolorization capacity depending on cultivation conditions and ligninolytic enzyme production. Two different decolorization patterns were found in these strains: Orange G decolorization in I. resinosum and P. calyptratus was caused mainly by laccase, while RBBR decolorization was effected by manganese peroxidase (MnP); in D. squalens laccase and MnP cooperated in the decolorization processes.     

Lignin modifying enzymes of Coriolopsis polyzona and their role in olive oil mill wastewaters decolourisation

In order to decolourise olive oil mill wastewaters (OOMW) efficiently, production and differential induction of ligninolytic enzymes by the white rot Coriolopsis polyzona, were studied by varying growth media composition and/or inducer addition. Among various possible inducers, veratryl alcohol appeared to be the most efficient to enhance specific productions of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase by a factor of 18.5, 20.8 and 55, respectively. Ligninolytic enzymes were better produced in glucose based medium with a low nitrogen level (2.2 mM) under O2 atmosphere. The addition of 5 mM veratryl alcohol resulted in a maximal production of LiP, whereas maximal MnP and laccase were obtained at 10 mM. LiP production was totally repressed in presence of 100 microM Mn2+. The extrapolation of these conditions on OOMW based media was carried out at different effluent dilutions and the possible role of the different ligninolytic enzymes in OOMW decolourisation was studied. A better effluent decolourisation was obtained under LiP induction condition (5 mM veratryl alcohol) than when LiP was repressed (100 microM Mn2+). Furthermore, high levels of laccase had a detrimental effect on OOMW decolourisation concomitant to the formation of soluble polymeric aromatic compounds.     

Phanerochaete chrysosporium吸附载体的选择及染料降解研究

研究了游离细胞与载体吸附培养、不同载体材料对Phanerochaete chrysosporium进行连续染料脱色及产酶能力的影响。结果表明,P. chrysosporium可在载体上良好生长,甚至生长到载体内部。木屑、玉米芯、花生壳3种载体材料中,以木屑载体吸附培养物的持续脱色和产酶效果最佳,该培养物经三轮连续脱色后对染料RB5仍能达到最高95%的脱色率,并产生596 U/L锰依赖过氧化物酶（MnP）和1 326 U/L木质素过氧化物酶（LiP）,对染料的持续脱色和产酶能力明显优于游离细胞培养物。     

Phanerochaete chrysosporium吸附载体的选择及染料降解研究

研究了游离细胞与载体吸附培养、不同载体材料对Phanerochaete chrysosporium进行连续染料脱色及产酶能力的影响。结果表明,P.chrysosporium可在载体上良好生长,甚至生长到载体内部。木屑、玉米芯、花生壳3种载体材料中,以木屑载体吸附培养物的持续脱色和产酶效果最佳,该培养物经三轮连续脱色后对染料RB5仍能达到最高95%的脱色率,并产生596 U/L锰依赖过氧化物酶（MnP）和1 326 U/L木质素过氧化物酶（LiP）,对染料的持续脱色和产酶能力明显优于游离细胞培养物。     

Study of the degradation of dyes by MnP of Phanerochaete chrysosporium produced in a fixed-bed bioreactor

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium in a fixed-bed tubular bioreactor, filled with cubes of nylon sponge, operating in semi-solid-state conditions, was studied. Maximum individual manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1293 and 225 U/l were detected.The in vitro decolourisation of two structurally different dyes (Poly R-478, crystal violet) by the extracellular liquid obtained in the above-mentioned bioreactor was monitored in order to determine its degrading capability. The concentration of some compounds (sodium malonate, manganese sulphate) from the reaction mixture was optimised in order to maximise the decolourisation levels. A percentage of Poly R-478 decolourisation of 24% after 15 min of dye incubation was achieved.On the other hand, a methodology for a long treatment of these dyes based on the continuous addition of MnP enzyme and H(2)O(2) was developed. Moreover, this enzymatic treatment was compared with a photochemical decolourisation process. The former allowed to maintain the degradation rate almost constant for a long time, resulting in a decolourisation percentage of 70% and 30% for crystal violet and Poly R-478, respectively, after 2 h of treatment. As for the latter, it was not able to degrade Poly R-478, whereas crystal violet reached a degradation of 40% in 2 h.     

Oxidative dechlorination of methoxychlor by ligninolytic enzymes from white-rot fungi

Ligninolytic enzymes, manganese peroxidase (MnP), laccase, and lignin peroxidase (LiP), from white-rot fungi were used in an attempt to treat methoxychlor (MC), a chemical widely used as a pesticide. MnP and laccase in the presence of Tween 80 and 1-hydroxybenzotriazole (HBT), respectively, and LiP were found to degrade MC, and MnP-Tween 80 decreased MC levels by about 65% after a 24-h treatment. MC was converted into methoxychlor olefin (MCO) and 4,4'-dimethoxybenzophenone by MnP-Tween 80 or laccase-HBT treatment. These results indicate that ligninolytic enzymes from white-rot fungi can catalyze the oxidative dechlorination of MC. Moreover, a metabolite MCO was also degraded by MnP-Tween 80 or laccase-HBT treatment.     

Hydrolytic and ligninolytic enzyme activities in the Pb contaminated soil inoculated with litter-decomposing fungi

The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at H?lv?l? (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.     

Removal of estrogenic activity of natural steroidal hormone estrone by ligninolytic enzymes from white rot fungi

Natural steroidal hormone estrone (E1) was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. E1 decreased by 98% after 5 d of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, which suggested that the disappearance of E1 is related to ligninolytic enzymes produced extracellularly by white rot fungus. Therefore, E1 was treated with MnP and laccase prepared from the culture of white rot fungi. HPLC analysis demonstrated that E1 disappeared completely in the reaction mixture after 1 h of treatment with either MnP or laccase. Using the yeast two-hybrid assay system, it was also confirmed that both enzymatic treatments completely removed the estrogenic activity of E1 after 2 h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of E1.     

Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes

Bisphenol A (BPA) and nonylphenol (NP) were treated with manganese peroxidase (MnP) and laccase prepared from the culture of lignin-degrading fungi. Laccase in the presence of 1-hydroxybenzotriazole (HBT), the so-called laccase-mediator system, was also applied to remove the estrogenic activity. Both chemicals disappeared in the reaction mixture within a 1-h treatment with MnP but the estrogenic activities of BPA and NP still remained 40% and 60% in the reaction mixtures after a 1-h and a 3-h treatment, respectively. Extension of the treatment time to 12 h completed the removal of estrogenic activities of BPA and NP. The laccase has less ability to remove these activities than MnP, but the laccase-HBT system was able to remove the activities in 6 h. A gel permeation chromatography (GPC) analysis revealed that main reaction products of BPA and NP may be oligomers formed by the action of enzymes. Enzymatic treatments extended to 48 h did not regenerate the estrogenic activities, suggesting that the ligninolytic enzymes are effective for the removal of the estrogenic activities of BPA and NP.     

二鼠李糖脂对白腐菌降解稻草中木质纤维素的影响

通过固态发酵方式采用白腐菌对稻草中木质纤维素进行降解,并研究了生物表面活性剂二鼠李糖脂对该降解过程的影响。结果表明,不同浓度的二鼠李糖脂能在不同程度上提高降解过程水溶性有机碳的含量,添加0.007%和0.021%二鼠李糖脂的实验组最高TOC(total organic carbon)浓度较对照组分别提高了83.6%和54.5%,这有利于黄孢原毛平革菌的生长,且延缓了菌体的衰退。添加临界胶束浓度0.007%和0.021%浓度的二鼠李糖脂可使LiP(lignin peroxidase)酶活分别提高85.7%和41.2%,二鼠李糖脂对MnP(manganese peroxidase)酶活没有显著影响。生物表面活性剂的介入促进了白腐菌对稻草中木质素的降解,添加0.007%二鼠李糖脂可使木质素降解率提高54%。     

Antioxidant metabolism of coffee cell suspension cultures in response to cadmium

The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) were investigated. Cd accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied CdCl(2) concentration in the external medium. At 0.05mM CdCl(2), growth was stimulated, but at 0.5mM CdCl(2), the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at the higher CdCl(2) concentration. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased, particularly at the higher concentration of CdCl(2). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was increased at the lower CdCl(2) concentration used, but could not be detected in cells growing in the higher CdCl(2) concentration after 24h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl(2) treatment and one GR isoenzyme was shown to specifically respond to CdCl(2). The results suggest that the higher concentrations of CdCl(2) may lead to oxidative stress. The main response appears to be via the induction of SOD and CAT activities for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione for the synthesis of Cd-binding peptides, which may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion.     

PCDD/Fs-induced oxidative damage and antioxidant system responses in tobacco cell suspension cultures

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) are ubiquitous contaminants and can be considerably accumulated by natural plants. In order to elucidate the biochemical and physiological responses of plant to PCDD/Fs, tobacco Bright Yellow-2 (BY-2) cells were selected as model plant and treated with time- and concentration-dependent PCDD/Fs. The toxic effect and oxidative stress caused by PCDD/Fs were evident, which could be indicted by the reduction in fresh mass, the increase in malondialdehyde (MDA) content, and the damage of tobacco cell ultrastructure. PCDD/Fs tolerance was correlated with changes in antioxidant system and hormones of tobacco cells. Superoxide dismutase (SOD) and peroxidase (POD) exhibited peak enzyme activities at the PCDD/Fs concentration of 1000 ng WHO₉₈-TEQ g⁻¹ fresh weight. Glutathione reductase (GR) enzyme activity increased monotonically at high level PCDD/Fs, but the activity of catalase (CAT) was only slightly affected at all treatment. Meanwhile, the exposure to PCDD/Fs resulted in the changes of hormones content. With the increase of exposure concentration of PCDD/Fs, the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) increased, whereas the concentration of jasmonates (JAs) decreased. The above results suggest that tobacco cells had the ability to cope with the oxidative stress induced by low concentration of PCDD/Fs through increasing the activities of antioxidant enzymes and alternating plant hormones levels. However, oxidative stress and toxicity would burst out when plant cells were exposed to the high levels of PCDD/Fs.     

Aerobic degradation of di- and trichlorobenzenes by two bacteria isolated from polluted tropical soils

Two polychlorinated biphenyl (PCBs)-degrading bacteria were isolated by traditional enrichment technique from electrical transformer fluid (Askarel)-contaminated soils in Lagos, Nigeria. They were classified and identified as Enterobacter sp. SA-2 and Pseudomonas sp. SA-6 on the basis of 16S rRNA gene analysis, in addition to standard cultural and biochemical techniques. The strains were able to grow extensively on dichloro- and trichlorobenzenes. Although they failed to grow on tetrachlorobenzenes, monochloro- and dichlorobenzoic acids, they were able to utilize all monochlorobiphenyls, and some dichlorobiphenyls as sole sources of carbon and energy. The effect of incubation with axenic cultures on the degradation of 0.9 mM 1,4-dichlorobenzene, 0.44 mM 1,2,3- and 0.43 mM 1,3,5-trichlorobenzene in mineral salts medium was studied. Approximately, 80-90% of these xenobiotics were degraded in 200 h, concomitant with cell increase of up to three orders of magnitude, while generation times ranged significantly (P<0.05) from 17-32 h. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were detected in crude cell-free extracts of cultures pre-grown with benzoate, with the latter enzyme exhibiting a slightly higher activity (0.15-0.17 micromolmin(-1) mg of protein(-1)) with catechol, suggesting that the meta-cleavage pathway is the most readily available catabolic route in the SA strains. The wider substrate specificity of these tropical isolates may help in assessing natural detoxification processes and in designing bioremediation and bioaugmentation methods.     

Remazol Brilliant Blue R decolourization by the laccase from Trametes trogii

The decolourization of the recalcitrant dye RBBR by the culture filtrate of Trametes trogii and its isolated laccase was investigated. Both filtrates from Cu-induced cultures as well as purified laccase decolourized the dye RBBR. The purified laccase decolourized the dye down to 97% of 100 mg l(-1) initial concentration of RBBR when only 0.2U ml(-1) of laccase was used in the reaction mixture. The effects of different physicochemical parameters were tested and optimal decolourization rates occurred at pH 5 and at a temperature of 50 degrees C. Decolourization of RBBR occurred in the presence of metal ions which could be found in textile industry effluents. Of all the metal ions tested, FeCl2 was the most inhibiting for the decolourization. HBT was shown to have no effect on the decolourization of RBBR at low concentration, while at a concentration of 5 mM it slightly inhibited decolourization. The presence of aromatic compounds was found to be inhibiting for the decolourization at a concentration of 10 mM, but not at 0.1 mM, while at 1 mM only ortho-diphenols were inhibiting. Probing the effect of methanol it was found that higher concentrations caused a decrease in the decolourization rate of RBBR. The effect of laccase inhibitors on the decolourization of RBBR was tested with L-cysteine, SDS and EDTA. It was demonstrated that L-cysteine was the most inhibiting substrate for the decolourization while SDS was only inhibiting at 10 mM concentration and ETDA was not inhibiting at all tested concentrations.     

Ligninase-mediated transformation of 4,4′-dibromodiphenyl ether (BDE 15)

The structurally related hydroxylated polybrominated diphenyl ether (PBDE) like hydroxylated 4,4′-dibromodiphenyl ether widely occur in precipitation, surface water, and biotic media. The origins of hydroxylated PBDEs (OH-PBDEs) are of particular interest due to their greater toxic potencies than the corresponding PBDEs. We studied the transformation behavior and products of 4,4′-dibromodiphenyl ether (BDE 15) mediated by lignin peroxidase (LiP), an extracellular enzyme that is produced by certain white rot fungus and is widely present in the natural environment. We found that BDE 15 can be effectively transformed through the reaction mediated by LiP, and two different mono-OH-dibromodiphenyl ethers were identified by using gas chromatography–mass spectrometry (GC-MS) and GC-MS/MS. In particular, we compared the reaction behavior for systems variously containing natural organic matter (NOM) and/or veratryl alcohol (VA), a metabolite that certain fungus produces along with LiP in nature. It was found that the VA’s enhancement effect on LiP performance was impaired by the presence of NOM. The findings in this study provide useful information for better understanding the origins of OH-PBDEs found in the environment.     

Physiological and molecular responses of the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis> to polychlorinated biphenyl contamination in soil

Polychlorinated biphenyls (PCBs) are a class of man-made organic compounds ubiquitously present in the biosphere. In this study, we evaluated the toxic effects of different concentrations of PCBs in two natural soils (i.e. red soil and fluvo-aquic soil) on the earthworm Eisenia fetida. The parameters investigated included anti-oxidative response, genotoxic potential, weight variation and biochemical responses of the earthworm exposed to two different types of soils spiked with PCBs after 7 or 14 days of exposure. Earthworms had significantly lower weights in both soils after PCB exposure. PCBs significantly increased catalase (CAT), superoxide dismutase (SOD), and guaiacol peroxidase (POD) activity in earthworms exposed to either soil type for 7 or 14 days and decreased the malondialdehyde (MDA) content in earthworms exposed to red soil for 14 days. Of the enzymes examined, SOD activity was the most sensitive to PCB stress. In addition, PCB exposure triggered dose-dependent coelomocyte DNA damage, even at the lowest concentration tested. This response was relatively stable between different soils. Three-way analysis of variance (ANOVA) showed that the weight variation, anti-oxidant enzyme activities, and MDA contents were significantly correlated with exposure concentration or exposure duration (P < 0.01). Furthermore, weight variation, CAT activity, and SOD activity were significantly affected by soil type (P < 0.01). Therefore, the soil type and exposure time influence the toxic effects of PCBs, and these factors should be considered when selecting responsive biomarkers.     

Treatment of phenolic wastewater by horseradish peroxidase immobilized by bioaffinity layering

Horseradish peroxidase was immobilized by bioaffinity layering and used for the treatment of wastewater containing p-chlorophenol. For this purpose, lectin Concanavalin A was bound to Sephadex beads. The glycoenzyme peroxidase was layered upon this Con A layer. Subsequently, alternate layers of the enzyme and Con A were applied. The most efficient design consisted of three layers of Con A and peroxidase each. This immobilized enzyme preparation retained 80% of the activity of the free peroxidase used for immobilization. PEG at the concentration of 0.1 mg ml(-1) was found to prevent enzyme inactivation by the products, although it increased the process time. Thus 60 U ml(-1) of enzyme completely converted the p-chlorophenol (into products) in 4 min in the absence of PEG. On the other hand, only 0.05 U ml(-1) of enzyme was required for this purpose in the presence of PEG but the process required 60 min. Peroxidase converts phenol molecules into free radicals. These free radicals then polymerize and get precipitated. As a further means of minimizing exposure of the enzyme to free radicals and enhancing the reusability, it was decided to remove the enzyme from reaction medium after 10 min. With this strategy, the bioaffinity layered peroxidase preparation could be reused five times without any loss of activity.     

Operation of a two-phase partitioning bioreactor for the oxidation of anthracene by the enzyme manganese peroxidase

A study was conducted to determine the potential of a two-phase partitioning bioreactor (TPPB) for the treatment of a poorly soluble compound, anthracene, by the enzyme manganese peroxidase (MnP) from the fungus Bjerkandera sp. BOS55. Silicone oil was used as the immiscible solvent, which contained anthracene at high concentrations. The optimization of the oxidation process was conducted taking into account the factors which may directly affect the MnP catalytic cycle (the concentration of H(2)O(2) and malonic acid) and those that affect the mass transfer of anthracene between the organic and the aqueous phase (solvent and agitation speed). The main objective was carried out in terms of improved efficiency, i.e., maximizing the anthracene oxidized per unit of enzyme used. The TPPB reached nearly complete oxidation of anthracene at a conversion rate of 1.8mgl(-1)h(-1) in 56h, which suggests the application of enzymatic TPPBs for the removal of poorly soluble compounds.     

Correlation of gas chromatographic properties of chlorobenzenes and polychlorinated biphenyls with the occurrence of reductive dechlorination by untamed microorganisms

To understand the dechlorination ability of chlorobenzenes (CBs) and polychlorinated biphenyls (PCBs) by untamed microorganisms under anaerobic condition and to correlate gas chromatographic properties with the occurrence of reductive dechlorination, introduction of CBs and PCBs in the culture medium inoculated with microorganisms from sludge and sediment, respectively, were performed. Three kinds of culture media preparing from sludge, river water and a synthetic medium were used in the experiments. HCB was degraded to 1,3,5-trichlorobenzene (1,3,5-TCB) and 1,3-dichlorobenzene (1,3-DCB) in both sludge medium and synthetic medium with inoculated microorganisms. Three PCB congeners including 2,3,4-, 3,4,5- and 2,3,4,5-CBp (chlorinated biphenyl) were not found to be dechlorinated in the river water medium with inoculation culture but to be dechlorinated in the synthetic medium. MNDO methodology was used to compute theoretical dechlorination reaction heats and GC-ECD techniques were used to estimate chromatographic data of CB and PCB congeners. Both CB and PCB congeners showed that dechlorination by untamed microorganisms under anaerobic mixed cultures were more likely to occur when larger amounts of energy were released and greater deltaln RRT value between the parent congener and the daughter product was observed. Deltaln RRT provided a more precise information on the singularity of PCBs ortho-dechlorination in an aspect of thermodynamic favorable rule.     

Comparison of lignin peroxidase and horseradish peroxidase for catalyzing the removal of nonylphenol from water

Concentrations of aqueous-phase nonylphenol (NP), a well-known endocrine-disrupting chemical, are shown to be reduced effectively via reaction with lignin peroxidase (LiP) or horseradish peroxidase (HRP) and hydrogen peroxide. We systematically assessed their reaction efficiencies at varying conditions, and the results have confirmed that the catalytic performance of LiP toward NP was more efficient than that of HRP under experimental conditions. Mass spectrum analysis demonstrated that polymerization through radical–radical coupling mechanism was the pathway leading to NP transformation. Our molecular modeling with the assistance of ab initio suggested the coupling of NP likely proceeded via covalent bonding between two NP radicals at their unsubstituted carbons in phenolic rings. Data from acute immobilization tests with Daphnia confirm that NP toxicity is effectively eliminated by LiP/HRP-catalyzed NP removal. The findings in this study provide useful information for understanding LiP/HRP-mediated NP reactions, and comparison of enzymatic performance can present their advantages for up-scale applications in water/wastewater treatment.