Decolorization of water and oil-soluble azo dyes by <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 μg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 μg/ml Sudan I and II; or 1.5 μg/ml Sudan III and IV in deMann–Rogosa–Sharpe broth at 37°C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.     

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.     

Mutagenicity testing of certified food colors and related azo, xanthene and triphenylmethane dyes with the Salmonella/microsome system.

Thirty-seven azo, xanthene and triphenylmethane dyes including FD and C colors currently approved for use in the U.S.A. and a number of delisted food colors, were tested in the Salmonella/microsome system. In addition to direct plate tests with five tester strains (TA1535, TA100, TA1537, TA1538, TA98), the azo dyes were also assayed after chemical reduction to their component amines. Also, a selected group of azo dyes was subjected to liquid tests (both aerobic with microsomes and anaerobic) and to plate tests involving initial 16 h anaerobic incubations to facilitate microbial reduction of the azo bond. None of the presently listed FD and C colors was mutagenic in any of the test modifications. Among formerly listed colors only Butter Yellow (p-dimethylaminoazobenzene), a recognized animal carcinogen, was mutagenic in the aerobic liquid test. Several other azo dyes were either directly mutagenic, viz. Acid Alizarin Yellow R and Alizarin Yellow GG; required microsomal activation, viz. Acid Alizarin Red B and Methyl Red; or required chemical reduction and microsomal activation, viz. Acid Alizarin Violet N and Sudan IV. Of the non-azo dyes tested only two xanthene dyes appeared to be mutagenic, viz. 9-(2-sulfophenyl)-6-hydroxy-3-isoxanthenone and its 2,4,5,7-tetrabromo derivative.     

Reduction of azo dyes by intestinal anaerobes.

Reduction of seven azo dyes (amaranth, Ponceau SX, Allura Red, Sunset Yellow, tartrazine, Orange II, and methyl orange) was carried out by cell suspensions of predominant intestinal anaerobes. It was optimal at pH 7.4 in 0.4 M phosphate buffer and inhibited by glucose. Flavin mononucleotide caused a marked enhancement of azo reduction by Bacteroides thetaiotaomicron. Other electron carriers, e.g., methyl viologen, benzyl viologen, phenosafranin, neutral red, crystal violet, flavin adenine dinucleotide, menadione, and Janus Green B can replace flavin mononucleotide. These data suggest that an extracellular shuttle is required for azo reduction.     

Reduction of azo dyes by intestinal anaerobes.

Reduction of seven azo dyes (amaranth, Ponceau SX, Allura Red, Sunset Yellow, tartrazine, Orange II, and methyl orange) was carried out by cell suspensions of predominant intestinal anaerobes. It was optimal at pH 7.4 in 0.4 M phosphate buffer and inhibited by glucose. Flavin mononucleotide caused a marked enhancement of azo reduction by Bacteroides thetaiotaomicron. Other electron carriers, e.g., methyl viologen, benzyl viologen, phenosafranin, neutral red, crystal violet, flavin adenine dinucleotide, menadione, and Janus Green B can replace flavin mononucleotide. These data suggest that an extracellular shuttle is required for azo reduction.     

Degradation of azo dyes by oxidative processes--laccase and ultrasound treatment

Azo dyes are of synthetic origin and their environmental fate is not well understood. They are resistant to direct aerobic bacterial degradation and form potentially carcinogenic aromatic amines by reduction of the azo group. This study shows that applying the oxidative processes of enzymatic treatment with laccase and ultrasound treatment, both alone and in combination, leads to dye degradation. Laccase treatment degraded both Acid Orange and Direct Blue dyes within 1-5 h but failed in the case of Reactive dyes, whereas ultrasound degraded all the dyes investigated (3-15 h). When applied as multi-stage combinations the treatments showed synergistic effects for dye degradation compared with individual treatments. Bulk light absorption (UV-Vis) and ion pairing HPLC were used for process monitoring. Additionally, mass spectrometry was used to elucidate the structures of intermediates arising from ultrasound treatment.     

Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis

Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E. faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent Km values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent Vmax was 86.2 microM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

Oil-soluble dyes for marking Spodoptera frugiperda (Lepidoptera: Noctuidae)

Although various biological aspects of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) have been examined, adult movement and dispersal of this insect pest is not well understood. Release-recapture techniques by using marked insects is a useful approach for dispersal studies; however, the marking technique should not significantly affect insect biology or behavior. Therefore, the effect of different concentrations of oil-soluble dyes (Solvent Blue 35 [C.I. 61554], Sudan Red 7B [C.I. 26050], Sudan Black B [26150], Sudan Orange G [C.I. 11920], and Sudan I 103624 [C.I. 12055]) on development, mortality, and fecundity of S. frugiperda was evaluated. Dyes were added to artificial diet used to feed larvae. Larval and pupal development and mortality, adult longevity, and female fecundity were evaluated. High concentrations (400 and 600 ppm) of all dyes led to longer larval and pupal stages. Adult life span and number of eggs were not affected by the dyes. Sudan Red 7B marked both adults and eggs very well. Solvent Blue 35 marked both adults and eggs, but the blue-marked eggs could not be distinguished from some bluish eggs laid by nonlabeled females. Adults and eggs were not adequately marked by the Sudan Black B, Sudan Orange G, and Sudan I 103624 (a yellow dye).     

Bacterial decolorization and degradation of azo dyes

Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such as textile, food, cosmetics and paper printing. They are generally recalcitrant to biodegradation due to their xenobiotic nature. However microorganisms, being highly versatile, have developed enzyme systems for the decolorization and mineralization of azo dyes under certain environmental conditions. Several genera of Basidomycetes have been shown to mineralize azo dyes. Reductive cleavage of azo bond, leading to the formation of aromatic amines, is the initial reaction during the bacterial metabolism of azo dyes. Anaerobic/anoxic azo dye decolorization by several mixed and pure bacterial cultures have been reported. Under these conditions, this reaction is non-specific with respect to organisms as well as dyes. Various mechanisms, which include enzymatic as well as low molecular weight redox mediators, have been proposed for this non-specific reductive cleavage. Only few aerobic bacterial strains that can utilize azo dyes as growth substrates have been isolated. These organisms generally have a narrow substrate range. Degradation of aromatic amines depends on their chemical structure and the conditions. It is now known that simple aromatic amines can be mineralized under methanogenic conditions. Sulfonated aromatic amines, on the other hand, are resistant and require specialized aerobic microbial consortia for their mineralization. This review is focused on the bacterial decolorization of azo dyes and mineralization of aromatic amines, as well as the application of these processes for the treatment of azo-dye-containing wastewaters.     

Anaerobic Metabolism of 1-Amino-2-Naphthol-Based Azo Dyes (Sudan Dyes) by Human Intestinal Microflora

The rates of metabolism of Sudan I and II and Para Red by human intestinal microflora were high compared to those of Sudan III and IV under anaerobic conditions. Metabolites of the dyes were identified as aniline, 2,4-dimethylaniline, o-toluidine, and 4-nitroaniline through high-performance liquid chromatography and liquid chromatography electrospray ionization tandem mass spectrometry analyses. These data indicate that human intestinal bacteria are able to reduce Sudan dyes to form potentially carcinogenic aromatic amines.     

Special traits of decomposition of azo dyes by anaerobic microbial communities

We present the results of an investigation into the special traits of conversion of azo dyes Acid Orange 6, Acid Orange 7, Methyl Orange, and Methyl Red under anaerobic conditions in comparison to aerobic conditions. In the presence of oxygen, only Methyl Red underwent decomposition, while under oxygen-free conditions, all remaining substances were fully decolourised under the action of a methanogenous consortium of microorganisms. The products of reduction of the azo bond are determined in the case of each dye. Introduction of additional acceptors of electrons (sulfate and nitrate) had a negative influence on the discoloration of azo dyes. Addition of ethanol as an available organic cosubstrate accelerated decomposition of azo dyes both under methanogenous and sulfate- and nitrate-reducing conditions. There is no direct correlation between the rates of conversion of azo dyes under anaerobic conditions or their toxicity to acetoclastic methanogens. Changes in the morphological composition of the community decolouring an azo dye depended on the duration of its impact on microorganisms. The mechanism of the reduction of the azo bond under the action of substances acting as mediators is explained. These substances are products of the metabolism of the microbial community in anaerobic conditions. It is shown that the supposed mediators NADH and sulfide efficiently decolourise azo dyes in a cell-free system, while riboflavin significantly increased the rate of conversion of substrates in recurrent cycles of discoloration only in the presence of an anaerobic microbial consortium.     

Mutagenicity of azo dyes: structure-activity relationships.

Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.     

微生物对偶氮染料的脱色及其基因工程研究进展

偶氮染料广泛应用在纺织印染、造纸印刷等行业中。染料废水的排放将会导致严重的环境污染,使用微生物处理染料废水是解决此问题的有效方法。该文概述了微生物对偶氮染料的脱色的研究,包括细菌对偶氮染料的脱色,真菌对偶氮染料的脱色,脱色产生的芳香胺并进一步被降解,以及基因工程技术在微生物对偶氮染料脱色的研究进展。     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

Evaluation of metabolism of azo dyes and their effects on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> metabolome

Dyes containing one or more azo linkages are widely applied in cosmetics, tattooing, food and drinks, pharmaceuticals, printing inks, plastics, leather, as well as paper industries. Previously we reported that bacteria living on human skin have the ability to reduce some azo dyes to aromatic amines, which raises potential safety concerns regarding human dermal exposure to azo dyes such as those in tattoo ink and cosmetic colorant formulations. To comprehensively investigate azo dye-induced toxicity by skin bacteria activation, it is very critical to understand the mechanism of metabolism of the azo dyes at the systems biology level. In this study, an LC/MS-based metabolomics approach was employed to globally investigate metabolism of azo dyes by Staphylococcus aureus as well as their effects on the metabolome of the bacterium. Growth of S. aureus in the presence of Sudan III or Orange II was not affected during the incubation period. Metabolomics results showed that Sudan III was metabolized to 4-(phenyldiazenyl) aniline (48%), 1-[(4-aminophenyl) diazenyl]-2-naphthol (4%) and eicosenoic acid Sudan III (0.9%). These findings indicated that the azo bond close to naphthalene group of Sudan III was preferentially cleaved compared with the other azo bond. The metabolite from Orange II was identified as 4-aminobenzene sulfonic acid (35%). A much higher amount of Orange II (~90×) was detected in the cell pellets from the active viable cells compared with those from boiled cells incubated with the same concentration of Orange II. This finding suggests that Orange II was primarily transported into the S. aureus cells for metabolism, instead of the theory that the azo dye metabolism occurs extracellularly. In addition, the metabolomics results showed that Sudan III affected energy pathways of the S. aureus cells, while Orange II had less noticeable effects on the cells. In summary, this study provided novel information regarding azo dye metabolism by the skin bacterium, the effects of azo dyes on the bacterial cells and the important role on the toxicity and/or inactivation of these compounds due to microbial metabolism.     

Degradation analysis of Reactive Red 198 by hairy roots of <Emphasis Type="Italic">Tagetes patula</Emphasis> L. (Marigold)

Tagetes patula L. (Marigold) hairy roots were selected among few hairy root cultures from other plants tested for the decolorization of Reactive Red 198. Hairy roots of Tagetes were able to remove dye concentrations up to 110 mg L^−l and could be successively used at least for five consecutive decolorization cycles. The hairy roots of Tagetes decolorized six different dyes, viz. Golden Yellow HER, Methyl Orange, Orange M2RL, Navy Blue HE2R, Reactive Red M5B and Reactive Red 198. Significant induction of the activity of biotransformation enzymes indicated their crucial role in the dye metabolism. UV–vis spectroscopy, HPLC and FTIR spectroscopy analyses confirmed the degradation of Reactive Red 198. A possible pathway for the biodegradation of Reactive Red 198 has been proposed with the help of GC–MS and metabolites identified as 2-aminonaphthol, p-aminovinylsulfone ethyl disulfate and 1-aminotriazine, 3-pyridine sulfonic acid. The phytotoxicity study demonstrated the non-toxic nature of the extracted metabolites. The use of such hairy root cultures with a high ability for bioremediation of dyes is discussed.     

Evaluation of impact of exposure of Sudan azo dyes and their metabolites on human intestinal bacteria

Sudan azo dyes are banned for food usage in most countries, but they are illegally used to maintain or enhance the color of food products due to low cost, bright staining, and wide availability of the dyes. In this report, we examined the toxic effects of these azo dyes and their potential reduction metabolites on 11 prevalent human intestinal bacterial strains. Among the tested bacteria, cell growth of 2, 3, 5, 5, and 1 strains was inhibited by Sudan I, II, III, IV, and Para Red, respectively. At the tested concentration of 100 μM, Sudan I and II inhibited growth of Clostridium perfringens and Lactobacillus rhamnosus with decrease of growth rates from 14 to 47%. Sudan II also affected growth of Enterococcus faecalis. Growth of Bifidobacterium catenulatum, C. perfringens, E. faecalis, Escherichia coli, and Peptostreptococcus magnus was affected by Sudan III and IV with decrease in growth rates from 11 to 67%. C. perfringens was the only strain in which growth was affected by Para Red with 47 and 26% growth decreases at 6 and 10 h, respectively. 1-Amino-2-naphthol, a common metabolite of the dyes, was capable of inhibiting growth of most of the tested bacteria with inhibition rates from 8 to 46%. However, the other metabolites of the dyes had no effect on growth of the bacterial strains. The dyes and their metabolites had less effect on cell viability than on cell growth of the tested bacterial strains. Clostridium indolis and Clostridium ramosum were the only two strains with about a 10 % decrease in cell viability in the presence of Sudan azo dyes. The present results suggested that Sudan azo dyes and their metabolites potentially affect the human intestinal bacterial ecology by selectively inhibiting some bacterial species, which may have an adverse effect on human health.     

Decolorization and degradation of textile dyes with biosulfidogenic hydrogenases

Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480-620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds.     

Sudan azo dyes and Para Red degradation by prevalent bacteria of the human gastrointestinal tract

Sudan azo dyes have genotoxic effects and ingestion of food products contaminated with Sudan I, II, III, IV, and Para Red could lead to exposure in the human gastrointestinal tract. In this study, we examined thirty-five prevalent species of human intestinal bacteria to evaluate their capacity to degrade Sudan dyes and Para Red. Among these tested bacterial strains, 23, 13, 33, 30, and 29 out of 35 species tested were able to reduce Sudan I, II, III, IV, and Para Red, respectively, to some extent. Bifidobacterium infantis, Clostridium indolis, Enterococcus faecalis, Lactobacillus rhamnosus, and Ruminococcus obeum were able to reduce completely all four tested Sudan dyes and Para Red. Escherichia coli and Peptostreptococcus magnus were the only two strains that were not able to reduce any of the tested Sudan dyes and Para Red to any significant extent. Metabolites of the reduction of the tested Sudan dyes and Para Red by E. faecalis were isolated and identified by HPLC and LC/ESI-MS analyses and compared with authentic standards. Thus it appears that the ability to reduce Sudan dyes and Para Red except Sudan II is common among bacteria in the human colon.