Microflora involved in textile dye waste removal

Textile dyes are heavily used in factories for coloring different cloth materials. This work was designed to identify microorganisms capable of removing textile dyes, either by biodegradation or by biosorption. We expected to isolate microorganisms adapted to high dye concentrations from sites near textile industry complex. An experiment was conducted to study the efficiency of the isolates in removing textile dyes. The tested dyes were used as carbon and nitrogen sources for isolation of soil and/or water microorganisms capable of removing textile dyes wastes from factories effluent. The results indicated the low efficiency of both bacteria and actinomycetes in clean-up the effluent from the waste dyes in 10-21 days. On the other hand six fungal isolates were obtained by plating factory effluent on Martin's medium and media containing dyes as the sole source of carbon and nitrogen for growth. These isolates fell in two genera, Aspergillus and Trichoderma. Results of these studies revealed the potential capacity of these fungi to decolorize the tested dyes in comparatively short time (2-24 hours) indicating strong efficiency of dye bioremediation by the fungal isolates. Since the process involved is mostly fast interaction between the fungal mycelium and the dye in the media, the possible mechanism could be based on a biosorption of such chemicals on the intact fungal biomass, rather than direct biodegradation of the compounds.     

Assessment of textile dye remediation using biotic and abiotic agents

The aim of the current work was to assess the removal of direct and reactive dyes using biotic and abiotic agents. Removal of dyes and their derivatives from aqueous solutions was investigated using sugarcane bagasse, sawdust, rice straw, charcoal and fungal biomass as dye removing agents. Seven fungal strains known to have high capacity in removing textile dyes were used. Results of this study indicated that Penicillium commune, P. freii, and P. allii removed 96, 64 and 65%, respectively, of direct violet dye after two hours of incubation. In addition, the use of rice straw was shown to be more efficient in dye removal, than was bagasse or sawdust. Rice straw was effective in removing 72% of direct violet dye within 24 hours. However, with reactive dyes, removal activity was reduced to 27%. Similar trends were recorded with the other tested biotic agents, fast removal of reactive dye was not found after 48 hours of contact time. Results of this study indicate that low-cost, renewable, bioadsorption agents are relatively effective in removing textile dyes from solution.     

Evaluation of biotoxicity of textile dyes using two bioassays

The toxicity of eight textile dyes was evaluated using two bioassays namely: Ames test and seed germination test. The Ames test is widely used for the evaluation of hazardous mutagenic effect of different chemicals, as a short-term screening test for environmental impact assessment. The eight-textile dyes and Eithidium bromide dye (as positive control) were tested with five "his" Salmonella typhimurium strains: TA 100; TA 98; TA 1535; TA 1537; TA 1538. Using six concentrations of each dye (2.5 microg/ml, 4.5 microg/ml, 9 microg/ml, 13.5 microg/ml, 18 microg/ml, and 22.5 microg/ml) revealed that, most of the dyes were mutagenic for the test strains used in this study. The high concentrations of dye eliminated microbial colonies due to the high frequency of mutation causing lethal effect on the cells.In this work the phytotoxicity of different soluble textile dyes was estimated by measuring the relative changes in seed germination of four plants: clover, wheat, tomato and lettuce. The changes in shooting percentages and root length as affected by dye were also measured. Seed germination percent and shoot growth as well as root length were recorded after 6 days of exposure to different concentrations of textile dyes in irrigation water. The results show that high concentrations of dyes were more toxic to seed germination as compared with the lower concentrations. However, the low concentrations of the tested dyes adversely affected the shooting percent significantly.     

Adsorptive removal of textile dyes from aqueous solutions by dead fungal biomass

Dead fungal biomass prepared from Phanerochaete chrysosporium and Funalia trogii was tested for their efficiency in removal of textile dyes. The effects of contact time, initial dye concentration, amount of dead biomass and agitation rate on dye removal have been determined. Removal of all dyes required a very short time (60 min). Experimental results show that, P. chrysosporium was more effective than F. trogii . An increase in the amount of dead biomass positively affected of the dye removal. The removal efficiency of different amount of biomass was in order 1 g > 0.5 g > 0.2 g > 0.1 g. The highest removal was obtained at 150-200 rpm. Slightly lower removing activities were found at lower agitation rates. This study showed that it was possible to remove textile dyes by dead biomass of P. chrysosporium .     

Evaluation of laser dye mutagenicity using the ames/salmonella microsome test

Twenty-five laser dyes and four analogs were tested for mutagenicity in the Ames/Salmonella test. Seven dyes and two analogs gave positive mutagenic responses with bacterial strains TA1538 and TA98. Of two widely used families of laser dyes (coumarins and rhodamines), four coumarin samples, but none of the rhodamine samples, were mutagenic. All mutagenic compounds require enzyme activation for positive response except two terphenyl analogs, which are mutagenic with or without activation. Using high-performance liquid chromatography (HPLC), it was determined that five mutagenic dye samples had multiple components. The dyes themselves may not be the mutagenic agents in all cases (as with Nile Blue) but may contain impurities that are mutagenic. One dye, adicyanome-thylene (DCM) (≥95% pure), was mutagenic at doses below 0.5 μg/plate on strains TA1538 and TA98. DCM also induced reversions in strains TA96, TA97, TA100, TA102, and TA104, although less efficiently. This study indicates the need for further toxicological testing of these types of compounds. The mutagenic components of these dye mixtures, whether it is the dye or a contaminant, presents a possible hazard to those handling them. Therefore, practices and procedures for the safe handling of specific dyes should be reviewed in light of these findings.     

Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.     

A promising Ames battery for mutagenicity characterization of new dyes

When testing new products, potential new products, or their impurities for genotoxicity in the Ames test, the quantity available for testing can be a limiting factor. This is the case for a dye repository of around 98,000 substances the Max Weaver Dye Library (MWDL). Mutagenicity data on dyes in the literature, although vast, in several cases is not reliable, compromising the performance of the in silico models. In this report, we propose a strategy for the generation of high‐quality mutagenicity data for dyes using a minimum amount of sample. We evaluated 15 dyes from different chemical classes selected from 150 representative dyes of the MWDL. The purity and molecular confirmation of each dye were determined, and the microplate agar protocol (MPA) was used. Dyes were tested at the limit of solubility in single and concentration‐response experiments using seven strains without and with metabolic activation except for anthraquinone dyes which were tested with eight strains. Six dyes were mutagenic. The most sensitive was YG1041, followed by TA97a > TA98 > TA100 = TA1538 > TA102. YG7108 as well as TA1537 did not detect any mutagenic response. We concluded that the MPA was successful in identifying the mutagenicity of dyes using less than 12.5 mg of sample. We propose that dyes should be tested in a tiered approach using YG1041 followed by TA97a, TA98, and TA100 in concentration‐response experiments. This work provides additional information on the dye mutagenicity database available in the literature.     

Astrazon Red dye decolorization by growing cells and pellets of Funalia trogii

The dye decolorization activity of fungal pellets has been compared with another method based on the decolorization of dye by growing cells. The pellet method was more advantageous than the growing cell method. The growing cells of F. trogii decolorized 21% of the dye in distilled water medium and 16% in stock basal medium in 24 h. On the other hand, Funalia trogii pellets rapidly decolorized the Astrazon Red dye, mono-azo textile dye, in 24 h, without any visual sorption of any dye to the pellets. The effect of various supplements on longevity of decolorization by free pellets was also tested. Glucose and cheese whey supplementation improved dye decolorization performance of the pellets and remained high and stable for 10 days. We also tested the dye decolorization ability of pellets immobilized on activated carbon. These pellets showed the stable dye decolorization activity during the repeated batch experiments. The study revealed that dye decolorization by pellets is more effective method than the growing cell method.     

Decolorization of diazo-dye Reactive Blue 172 by Pseudomonas aeruginosa NBAR12

A novel bacterial strain capable of decolorizing textile dyes was isolated from dye contaminated soil obtained from industrial estate of Ahmedabad, Gujarat, India. The bacterial isolate Pseudomonas aeruginosa NBAR12 was capable of decolorizing 12 different dyes tested with decolorization efficiency varying in the range of 80 to 95%. Maximum extent as well as rate of Reactive Blue 172 (RB 172) decolorization was observed when glucose (2 g x l(-1)) and yeast extract (2.5 g x l(-1)) were supplemented in the medium. The optimum dye pH and temperature for dye decolorization was found to be 7 and 40 degrees C, respectively. The decolorizing activity was found to increase with increasing the dye concentration from 50 to 400 mg x l(-1). The dye decolorization was strongly inhibited at 500 mg dye l(-1) in the medium. High performance thin layer chromatography analysis indicated that dye decolorization occurred due to the breakdown of dye molecules into colorless end products.     

Screening of filamentous fungi for the decolorization of a commercial reactive dye

The aim of this work is to verify the ability of 19 isolates of 13 different fungal species to decolorize the reactive dye blue‐BF‐R. The isolates of Pleurotus pulmonarius, P. ostreatus, P. ëous, P. citrinopileatus, Lentinus edodes, Phanerochaete chrysosporium, Schizophyllum commune, Agaricus blazei, Ganoderma sp. and four isolates obtained from textile effluent were evaluated in minimum liquid medium. In addition, seven of them were also evaluated on solid medium, and both media were both added 0.5 g dye/l. All isolates evaluated on solid medium decolorized the dye. The isolates Phanerochaete chrysosporium CCB478 and Lentinus edodes CCB047 were the ones that presented the fastest and slowest growth, respectively. Despite the isolate of the textile effluent had grown on solid medium, it did not decolorize the dye. All the isolates of the genus Pleurotus, except the isolate Pleurotus ëous CCB440, decolorized the dye in liquid medium. They presented decolorization percentage ranging from 39% to 51%. The absorbance ratio (Abs₅₉₀/Abs₄₅₅) of the culture medium inoculated with these isolates decreased throughout the experiment indicating the fungal dye degradation. The others presented decolorization percent below 8%. The isolates of Pleurotus, except the isolate Pleurotus ëous CCB440, were able to decolorize and to degrade the commercial reactive dye blue‐BF‐R. The results indicate their potential to be used in the treatment of effluents containing this dye. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In vitro genotoxicity of dyes present in colored smoke munitions

Genetic toxicology studies were conducted on organic dyes and mixtures used in colored smoke munitions. The dyes studied included Solvent Red 1; two different batches (Lot 1 and Lot 2) of Disperse Red 11; terephthalic acid; and a mixture of 25 parts Solvent Red 1, 5 parts Disperse Red 11, and 16 parts terephthalic acid. The dyes were evaluated for their ability to produce mutations in Salmonella bacterial strains and in Chinese hamster ovary (CHO) cells. The dyes were also tested in CHO cells to determine cytotoxicity and the induction of sister chromatid exchanges and chromosome aberration. None of the dyes were genotoxic in the standard Ames assay using bacterial strain TA1535 or TA100 with or without the addition of S-9 or in TA98 and TA1538 without S-9. With S-9, Disperse Red 11 (Lot 2) showed significant mutagenic activity in TA98 and TA1538 which increased as a function of S-9 concentration. However, the maximum level of mutagenic activity detected was low (3.8 revertants/micrograms). The azo dye Solvent Red 1 was also negative in a pre-incubation assay designed to reduce azo compounds to free amines. Solvent Red 1 was cytotoxic to mammalian cells, caused a significant increase in SCE, but was not mutagenic or clastogenic. Disperse Red 11 (Lot 1 and Lot 2) were not cytotoxic or clastogenic but produced an increase in cell cycle time and SCE frequency. Only Disperse Red 11 (Lot 2) increased mutations in the CHO/hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay. The mutagenic activity of the dye mixture was not significant, suggesting no synergistic interaction between the dyes. These studies demonstrated that none of the dyes was clastogenic and that a contaminant in Disperse Red 11 (Lot 2) may be responsible for the weak mutagenic activity in both mammalian and bacterial cell systems.     

Mutagenicity tests of fabric-finishing agents in salmonella typhimurium: Fiber-reactive wool dyes and cotton flame retardants

Thirty-nine fabric-finishing agents were tested for mutagenic activity in Salmonella typhimurium. Twenty-four fiber-reactive wool dyes and three acid dyes (not fiber-reactive) were screened by spot tests in strains TA100, TA98, TA1535, and TA1537. Among these dyes, seven bromoacrylamide dyes and one vinyl sulfone dye were mutagenic. Additionally, one of the three acid dyes was mutagenic in spot tests. The mutagenicity of the acid dye was due to an impurity or breakdown product rather than to the dye itself; the origin of the activities of the other dyes is unknown. No mutagenicity was observed among five chlorotriazine or four sulfonyl-ethane sulfonic acid dyes. Eight phosphorus-containing flame retardants (phosphonium, phosphine, phosphine oxide, and phosphonic acid derivatives) and methyl-N-methylolcarbamate, which is employed to obtain a flame-retardant finish on cotton, were tested for mutagenicity in strains TA100, TA98, TA1535, and TA1537, using quantitative incorporation assays. All were nonmutagenic. Two of three bromoalkyl-substituted triazine flame retardants were mutagenic in strains TA100 and TA1535. It is unknown whether this activity is due to impurities or to the parent compound. The flame retardants tested were either in actual commercial use or in experimental development for potential commercial processes. These results indicate the need for early testing of potential fabric-finishing agents and processes.     

Combining different assays and chemical analysis to characterize the genotoxicity of waters impacted by textile discharges

Waters receiving textile discharges can exhibit genotoxic and mutagenic activity, which has been related to the presence of dyes and aromatic amines as synthesis precursors or byproducts. The aim of this study was to identify dyes and aromatic amines in water samples impacted by textile discharges, and to evaluate the genotoxic responses of these samples using the Salmonella/microsome assay in strains TA98 and YG1041, and the Fpg‐modified comet assay in the RTL‐W1 fish cell line. The genotoxicity of river samples downstream of the discharge was greater than the upstream samples in both of the Ames tests. The Fpg‐modified comet assay detected similar levels of DNA damage in the upstream and downstream samples. Mutagenicity was not detected with TA98, except for the Quilombo River samples, but when YG1041 was used as the tester strain mutagenicity was detected for all sites with a very different profile in upstream sites relative to the other sites. The mutagenic response strongly indicated that aromatic amines or dyes were contributing to the mutagenic activity downstream. The impact of textile discharges was also confirmed by chemical analysis, because the highest concentrations of azo dyes and aromatic amines were detected in the river downstream. This study shows the value of combining assays measuring complementary endpoints to better characterize the mutagenicity of environmental samples, with the advantage that this approach provides an indication of what classes of compounds are responsible for the effect. Environ. Mol. Mutagen. 57:559–571, 2016. © 2016 Wiley Periodicals, Inc.     

The influence of extracellular H2O2 production on decolorization ability in fungi

A set of 50 randomly chosen fungal strains belonging to different basidiomycete species was tested for H2O2 and ligninolytic enzyme production and for decolorization of synthetic dyes Orange G and Remazol Brilliant Blue R. The decolorization capacity of individual strains was influenced by the level of H2O2 and laccase activity. The strains producing H2O2 at a concentration of 1.0-1.5 microM exhibited the most efficient decolorization; higher or lower H2O2 concentration reduced this ability. None of the strains without a detectable laccase activity was able to decolorize the tested dyes.     

Toxicity and mutagenicity of anthraquinones from Aspergillus chevalieri.

More than 100 strains of the Aspergillus glaucus group were cultivated on synthetic media for 11 days at 28 degrees C. Organic extracts of fungal material were screened by thin-layer chromatography (TLC) for the mycotoxins aflatoxins B1,2 and G1,2, sterigmatocystin, ochratoxin A, gliotoxin, patulin, and xanthocillin X. None of these toxins were produced in detectable amounts under experimental conditions. Nevertheless, organic extracts exhibited high toxicity after intraperitoneal (i.p.) administration in mice. Aspergillus chevalieri strain ZT 8268 was selected for further investigation of its toxic metabolites. The main toxic action was attributed to the four anthraquinone derivatives, physicion, physcionanthrone B, physciondianthrone, and erythroglaucin, which were isolated and identified. No toxic effects were found after oral administration. Using the Salmonella/mammalian microsome test, mutagenic activity (frame-shift) was detected in strain TA 1537 in the presence of S-9 liver microsome preparation.     

Decolourisation of diverse industrial dyes by some Phlebia spp. and their comparison with Phanerochaete chrysosporium

Three species of Phlebia, viz. P. brevispora, P. fascicularia and P. floridensis have been evaluated for their potential to decolourise eight industrial dyes including; reactive yellow, reactive orange, reactive red, rathidol scarlet, coracryl black, coracryl pink, coracryl violet and coracryl red. The cultures used for the present study were pre adapted by growing these on yeast glucose agar medium supplemented with Poly-R 478, a reference dye. The fungal cultures were grown in mineral salts broth and harvested after different incubation periods to obtain their cell free enzyme extracts which were then used to assess their ability to decolourise the above mentioned dyes. The extracts obtained from the cultures grown for six days significantly decolourised the tested dyes. The study revealed Phlebia spp. to be better dye decolourisers than Phanerochaete chrysosporium.     

Hydrogen peroxide mutagenicity towards Salmonella typhimurium

In bioassays conducted under controlled, comparable conditions, weak direct mutagenicity responses were observed for hydrogen peroxide in the standard (Ames test) agar plate incorporation bioassay with Salmonella typhimurium strains TA97, TA98, TA102, and TA1537, in a 20 min preincubation test with strains TA97, TA98, TA100, TA102, TA1537, and TA1538, and in a liquid incubation modification using strain TA1537. These results conclusively demonstrate that hydrogen peroxide is a weak mutagen, especially in strains that are sensitive to oxidative damage under suitable bioassay conditions.     

Colorimetric assay for antifungal susceptibility testing of Aspergillus species

A colorimetric assay for antifungal susceptibility testing of Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, and Aspergillus ustus) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 microg of XTT/ml with 25 microM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10(2) CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.     

Mutagenic screening of marker grenade dyes by the Salmonella reversion assay, L5178Y/TK+/- mouse lymphoma assay, and in vivo sister chromatid exchange analysis in mice

Two dyes (C.I. Solvent Yellow No. 33 and a mixture of C.I. Solvent Yellow No. 33 and C.I. Solvent Green No. 3) were tested for mutagenicity in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay, and also for sister chromatid exchange (SCE) induction in vivo in C57B1/6J mice. In addition, a greater than 99.9% pure sample of the yellow dye [2-(2'-quinolyl)-1,3-indandione] was tested with and without exogenous activation in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay. Neither C.I. Solvent Yellow No. 33 nor the C.I. Solvent Yellow No. 33 and Solvent Green No. 3 mixture was positive for inducing SCEs in vivo. All three dyes were tested in the standard plate incorporation test in seven Salmonella strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. The dyes were negative with and without exogenous activation in TA98, TA1535, and TA1538. One test with TA1537 was positive with the greater than 99.9% purified yellow dye. All three dyes gave weakly positive results (less than a twofold increase) with S-9 in TA100 and were clearly positive in TA102 and TA104 both with and without S-9. They also induced mutation at the thymidine kinase locus in mouse lymphoma cells, produced both large- and small-colony trifluorothymidine-resistant mutants, and were clastogenic. The purified yellow dye was further tested for SCE induction in mouse lymphoma cells and was determined to give a slightly positive response in the presence of S-9.     

Structure--activity relationship studies on the mutagenicity of some azo dyes in the Salmonella/microsome assay

Analogs of Direct Black 19 and Direct Black 38 were synthesizedand tested in the Salmonella/microsome assay. Those dyes whichgave positive responses in strains TA98 and TA1538 would beexpected to be metabolized to p-phenyl-enediamine by the livermicrosomal enzymes (S9). Pure p-phenylenediamine is non-mutagenicin this assay but becomes mutagenic after it is oxidized. Thusthe positive response of our synthetic azo compounds are mostlikely due to the formation of oxidized p-phenylenediamine.Modification of the moieties that can be metabolized to p-phenylenediamineby sulfonation, carboxylation or copper complexation eliminatedthe mutagenic responses.