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1.  Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6. 
The anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 was analyzed. Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold. In contrast, in crude extracts, the azo reductase activity was not stimulated. A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells. The same increase could be observed with different bacterial strains. This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6. The addition of 1,2-dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS-) increased the activity of amaranth reduction. The presence of bacterial cells was needed to maintain the reduction process. Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.
PMCID: PMC168674  PMID: 9293019
2.  The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria 
A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.
PMCID: PMC92004  PMID: 10742223
3.  Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium. 
Applied and Environmental Microbiology  1991;57(11):3144-3149.
Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.
PMCID: PMC183939  PMID: 1781678
4.  Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains 
Journal of Bacteriology  2004;186(12):3862-3872.
A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.
doi:10.1128/JB.186.12.3862-3872.2004
PMCID: PMC419928  PMID: 15175300
5.  Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases 
Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.
doi:10.1128/AEM.69.6.3448-3455.2003
PMCID: PMC161523  PMID: 12788749
6.  Characterization of Azo Reduction Activity in a Novel Ascomycete Yeast Strain 
Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.
doi:10.1128/AEM.70.4.2279-2288.2004
PMCID: PMC383148  PMID: 15066823
7.  Localization of the Enzyme System Involved in Anaerobic Reduction of Azo Dyes by Sphingomonas sp. Strain BN6 and Effect of Artificial Redox Mediators on the Rate of Azo Dye Reduction 
The effect of different artificial redox mediators on the anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 or activated sludge was investigated. Reduction rates were greatly enhanced in the presence of sulfonated anthraquinones. For strain BN6, the presence of both cytoplasmic and membrane-bound azo reductase activities was shown.
PMCID: PMC1389254  PMID: 16535698
8.  Soybean peroxidase-mediated degradation of an azo dye– a detailed mechanistic study 
BMC Biochemistry  2013;14:35.
Background
Peroxidases are emerging as an important class of enzymes that can be used for the efficient degradation of organic pollutants. However, detailed studies identifying the various intermediates produced and the mechanisms involved in the enzyme-mediated pollutant degradation are not widely published.
Results
In the present study, the enzymatic degradation of an azo dye (Crystal Ponceau 6R, CP6R) was studied using commercially available soybean peroxidase (SBP) enzyme. Several operational parameters affecting the enzymatic degradation of dye were evaluated and optimized, such as initial dye concentration, H2O2 dosage, mediator amount and pH of the solution. Under optimized conditions, 40 ppm dye solution could be completely degraded in under one minute by SBP in the presence of H2O2 and a redox mediator. Dye degradation was also confirmed using HPLC and TOC analyses, which showed that most of the dye was being mineralized to CO2 in the process.
Conclusions
Detailed analysis of metabolites, based on LC/MS results, showed that the enzyme-based degradation of the CP6R dye proceeded in two different reaction pathways- via symmetric azo bond cleavage as well as asymmetric azo bond breakage in the dye molecule. In addition, various critical transformative and oxidative steps such as deamination, desulfonation, keto-oxidation are explained on an electronic level. Furthermore, LC/MS/MS analyses confirmed that the end products in both pathways were small chain aliphatic carboxylic acids.
doi:10.1186/1471-2091-14-35
PMCID: PMC4028748  PMID: 24308857
Azo dye; Degradation; Enzyme; Mediator; LC/MS; Metabolites
9.  Decolorization and biodegradation of reactive sulfonated azo dyes by a newly isolated Brevibacterium sp. strain VN-15 
SpringerPlus  2012;1(1):37.
Azo dyes constitute the largest and most versatile class of synthetic dyes used in the textile, pharmaceutical, food and cosmetics industries and represent major components in wastewater from these industrial dying processes. Biological decolorization of azo dyes occurs efficiently under low oxygen to anaerobic conditions. However, this process results in the formation of toxic and carcinogenic amines that are resistant to further detoxification under low oxygen conditions. Moreover, the ability to detoxify these amines under aerobic conditions is not a wide spread metabolic activity. In this study we describe the use of Brevibacterium sp. strain VN-15, isolated from an activated sludge process of a textile company, for the sequential decolorization and detoxification of the azo dyes Reactive Yellow 107 (RY107), Reactive Black 5 (RB5), Reactive Red 198 (RR198) and Direct Blue 71 (DB71). Tyrosinase activity was observed during the biotreatment process suggesting the role of this enzyme in the decolorization and degradation process, but no-activity was observed for laccase and peroxidase. Toxicity, measured using Daphnia magna, was completely eliminated.
doi:10.1186/2193-1801-1-37
PMCID: PMC3566399  PMID: 23396675
Azo dyes; Textile wastewater; Decolorization; Biodegradation; Detoxification; Brevibacterium; Tyrosinase; Carcinogenic aromatic amine
10.  Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress 
BMC Plant Biology  2012;12:140.
Background
Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Results
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
Conclusions
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
doi:10.1186/1471-2229-12-140
PMCID: PMC3460772  PMID: 22882870
Grapevine; Dehydrin; Stress-induced expression; Powdery mildew; Promoter
11.  In situ analysis of denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column. 
A diesel fuel-contaminated aquifer was bioremediated in situ by the injection of oxidants (O2 and NO3-) and nutrients in order to stimulate microbial activity. After 3.5 years of remediation, an aquifer sample was excavated and the material was used (i) to isolate bacterial strains able to grow on selected hydrocarbons under denitrifying conditions and (ii) to construct a laboratory aquifer column in order to simulate the aerobic and denitrifying remediation processes. Five bacterial strains isolated from the aquifer sample were able to grow on toluene (strains T2 to T4, T6, and T10), and nine bacterial strains grew on toluene and m-xylene (strains M3 to M7 and M9 to M12). Strains T2 to T4, T6, and T10 were cocci, and strains M3 to M7 and M9 to M12 were rods. The morphological and physiological differences were also reflected in small sequence variabilities in domain III of the 23S rRNA and in the 16S rRNA. Comparative sequence analyses of the 16S rRNA of one isolate (T3 and M3) of each group revealed a close phylogenetic relationship for both groups of isolates to organisms of the genus Azoarcus. Two 16S rRNA-targeted oligonucleotide probes (Azo644 and Azo1251) targeting the experimental isolates, bacteria of the Azoarcus tolulyticus group, and Azoarcus evansii were used to investigate the significance of hydrocarbon-degrading Azoarcus spp. in the laboratory aquifer column. The number of bacteria in the column determined after DAPI (4',6-diamidino-2-phenylindole) staining was 5.8 x 10(8) to 1.1 x 10(9) cells g of aquifer material-1. About 1% (in the anaerobic zone of the column) to 2% (in the aerobic zone of the column) of these bacteria were detectable by using a combination of probes Azo644 and Azo1251, demonstrating that hydrocarbon-degrading Azoarcus spp. are significant members of the indigenous microbiota. More than 90% of the total number of bacteria were detectable by using probes targeting higher phylogenetic groups. Approximately 80% of these bacteria belonged to the beta subdivision of the class Proteobacteria (beta-Proteobacteria), and 10 to 16% belonged to the gamma-Proteobacteria. Bacteria of the alpha-Proteobacteria were present in high numbers (10%) only in the aerobic zone of the column.
PMCID: PMC168503  PMID: 9172330
12.  Azo Reductase Activity of Intact Saccharomyces cerevisiae Cells Is Dependent on the Fre1p Component of Plasma Membrane Ferric Reductase 
Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae Δfre1 and Δfre1 Δfre2 mutant strains, but not the Δfre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae Δfre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.
doi:10.1128/AEM.71.7.3882-3888.2005
PMCID: PMC1168983  PMID: 16000801
13.  Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus. 
Applied and Environmental Microbiology  1992;58(11):3598-3604.
Five 14C-radiolabeled azo dyes and sulfanilic acid were synthesized and used to examine the relationship between dye substitution patterns and biodegradability (mineralization to CO2) by a white-rot fungus and an actinomycete. 4-Amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid were used as representative compounds having sulfo groups or both sulfo and azo groups. Such compounds are not known to be present in the biosphere as natural products. The introduction of lignin-like fragments into the molecules of 4-amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid by coupling reactions with guaiacol (2-methoxyphenol) resulted in the formation of the dyes 4-(3-methoxy-4-hydroxyphenylazo)-[U-14C]benzenesulfonic acid and 4-(2-sulfo-3'-methoxy-4'-hydroxy-azobenzene-4-azo)-[U-14C]benzenesulf oni c acid, respectively. The synthesis of acid azo dyes 4-(2-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid and 4-(4-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid also allowed the abilities of these microorganisms to mineralize these commercially important compounds to be evaluated. Phanerochaete chrysosporium mineralized all of the sulfonated azo dyes, and the substitution pattern did not significantly influence the susceptibility of the dyes to degradation. In contrast, Streptomyces chromofuscus was unable to mineralize aromatics with sulfo groups and both sulfo and azo groups. However, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed that lignocellulolytic fungi and bacteria can be used for the biodegradation of anionic azo dyes, which thus far have been considered among the xenobiotic compounds most resistant to biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC183150  PMID: 1482182
14.  Isolation and Characterization of Paracoccus sp. GSM2 Capable of Degrading Textile Azo Dye Reactive Violet 5 
The Scientific World Journal  2014;2014:410704.
A potential bacterial strain GSM2, capable of degrading an azo dye Reactive Violet 5 as a sole source of carbon, was isolated from textile mill effluent from Solapur, India. The 16S rDNA sequence and phenotypic characteristics indicated an isolated organism as Paracoccus sp. GSM2. This strain exhibited complete decolorization of Reactive Violet 5 (100 mg/L) within 16 h, while maximally it could decolorize 800 mg/L of dye within 38 h with 73% decolorization under static condition. For color removal, the most suitable pH and temperature were pH 6.0–9.0 and 25–40°C, respectively. The isolate was able to decolorize more than 70% of five structurally different azo dyes within 38 h. The isolate is salt tolerant as it can bring out more than 90% decolorization up to a salt concentration of 2% (w/v). UV-Visible absorption spectra before and after decolorization suggested that decolorization was due to biodegradation and was further confirmed by FT-IR spectroscopy. Overall results indicate the effectiveness of the strain GSM2 explored for the treatment of textile industry effluents containing various azo dyes. To our knowledge, this could be the first report on biodegradation of Reactive Violet 5 by Paracoccus sp. GSM2.
doi:10.1155/2014/410704
PMCID: PMC4030516  PMID: 24883397
15.  Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4▿  
Journal of Bacteriology  2007;189(7):2660-2666.
Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and Vmax for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
doi:10.1128/JB.01418-06
PMCID: PMC1855793  PMID: 17237179
16.  Genome Sequence of Sphingomonas xenophaga QYY, an Anthraquinone-Degrading Strain 
Genome Announcements  2013;1(1):e00031-12.
Sphingomonas xenophaga QYY is an efficient anthraquinone-degrading strain. Here, we present a 4.2-Mb assembly of the first genome sequence of S. xenophaga. We have annotated 36 coding sequences (CDSs) encoding aromatic catabolism and 216 CDSs responsible for toxic resistance and stress response, which may provide insights into the degradation of complex aromatics.
doi:10.1128/genomeA.00031-12
PMCID: PMC3569308  PMID: 23405319
17.  Microbial Populations Associated with Treatment of an Industrial Dye Effluent in an Anaerobic Baffled Reactor 
Fluorescent in situ hybridization (FISH) using 16S and 23S rRNA-targeted probes together with construction of an archaeal 16S ribosomal DNA (rDNA) clone library was used to characterize the microbial populations of an anaerobic baffled reactor successfully treating industrial dye waste. Wastewater produced during the manufacture of food dyes containing several different azo and other dye compounds was decolorized and degraded under sulfidogenic and methanogenic conditions. Use of molecular methods to describe microbial populations showed that a diverse group of Bacteria and Archaea was involved in this treatment process. FISH enumeration showed that members of the gamma subclass of the class Proteobacteria and bacteria in the Cytophaga-Flexibacter-Bacteroides phylum, together with sulfate-reducing bacteria, were prominent members of a mixed bacterial population. A combination of FISH probing and analysis of 98 archaeal 16S rDNA clone inserts revealed that together with the bacterial population, a methanogenic population dominated by Methanosaeta species and containing species of Methanobacterium and Methanospirillum and a relatively unstudied methanogen, Methanomethylovorans hollandica, contributed to successful anaerobic treatment of the industrial waste. We suggest that sulfate reducers, or more accurately sulfidogenic bacteria, together with M. hollandica contribute considerably to the treatment process through metabolism of dye-associated sulfonate groups and subsequent conversion of sulfur compounds to carbon dioxide and methane.
doi:10.1128/AEM.67.7.3226-3235.2001
PMCID: PMC93005  PMID: 11425746
18.  Decolorization and partial mineralization of a polyazo dye by Bacillus firmus immobilized within tubular polymeric gel 
3 Biotech  2011;2(1):67-78.
The degradation of C.I. Direct red 80, a polyazo dye, was investigated using Bacillus firmus immobilized by entrapment in tubular polymeric gel. This bacterial strain was able to completely decolorize 50 mg/L of C.I. Direct red 80 under anoxic conditions within 12 h and also degrade the reaction intermediates (aromatic amines) during the subsequent 12 h under aerobic conditions. The tubular gel harboring the immobilized cells consisted of anoxic and aerobic regions integrated in a single unit which was ideal for azo dye degradation studies. Results obtained show that effective dye decolorization (97.8%), chemical oxygen demand (COD) reduction (91.7%) and total aromatic amines removal were obtained in 15 h with the immobilized bacterial cell system whereas for the free cells, a hydraulic residence time of 24 h was required for an equivalent performance in a sequential anoxic and aerobic process. Repeated-batch experiments indicate the immobilized cells could decolorize C.I. Direct red 80 and reduce medium COD in five successive batch runs with enhanced activity obtained after each consecutive run, thus suggesting its stability and potential for repeated use in wastewater treatment. UV–visible spectrophotometry and HPLC analysis were used to confirm the partial mineralization of the dye. Data from this study could be used as a reference for the development of effective industrial scale biotechnological process for the removal of dyes and their metabolites in textile wastewater.
doi:10.1007/s13205-011-0035-3
PMCID: PMC3339580  PMID: 22582158
Decolorization; Mineralization; Azo dye; Tubular gel; Bacillus firmus; Chemistry; Bioinformatics; Agriculture; Stem Cells; Biomaterials; Biotechnology; Cancer Research
19.  Decolorization and partial mineralization of a polyazo dye by Bacillus firmus immobilized within tubular polymeric gel 
3 Biotech  2011;2(1):67-78.
The degradation of C.I. Direct red 80, a polyazo dye, was investigated using Bacillus firmus immobilized by entrapment in tubular polymeric gel. This bacterial strain was able to completely decolorize 50 mg/L of C.I. Direct red 80 under anoxic conditions within 12 h and also degrade the reaction intermediates (aromatic amines) during the subsequent 12 h under aerobic conditions. The tubular gel harboring the immobilized cells consisted of anoxic and aerobic regions integrated in a single unit which was ideal for azo dye degradation studies. Results obtained show that effective dye decolorization (97.8%), chemical oxygen demand (COD) reduction (91.7%) and total aromatic amines removal were obtained in 15 h with the immobilized bacterial cell system whereas for the free cells, a hydraulic residence time of 24 h was required for an equivalent performance in a sequential anoxic and aerobic process. Repeated-batch experiments indicate the immobilized cells could decolorize C.I. Direct red 80 and reduce medium COD in five successive batch runs with enhanced activity obtained after each consecutive run, thus suggesting its stability and potential for repeated use in wastewater treatment. UV–visible spectrophotometry and HPLC analysis were used to confirm the partial mineralization of the dye. Data from this study could be used as a reference for the development of effective industrial scale biotechnological process for the removal of dyes and their metabolites in textile wastewater.
doi:10.1007/s13205-011-0035-3
PMCID: PMC3339580  PMID: 22582158
Decolorization; Mineralization; Azo dye; Tubular gel; Bacillus firmus
20.  Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase from Sphingomonas xenophaga BN6 by Random Mutagenesis 
Journal of Bacteriology  2001;183(7):2322-2330.
The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.
doi:10.1128/JB.183.7.2322-2330.2001
PMCID: PMC95140  PMID: 11244073
21.  Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. 
Journal of Bacteriology  1993;175(21):6890-6901.
From a soil isolate, Pseudomonas strain C18, we cloned and sequenced a 9.8-kb DNA fragment that encodes dibenzothiophene-degrading enzymes. Nine open reading frames were identified and designated doxABDEFGHIJ. Collectively, we refer to these genes as the DOX pathway. At the nucleotide level, doxABD are identical to the ndoABC genes that encode naphthalene dioxygenase of Pseudomonas putida. The DoxG protein is 97% identical to NahC (1,2-dihydroxynaphthalene dioxygenase) of P. putida. DoxE has 37% identity with cis-toluene dihydrodiol dehydrogenase. DoxF is similar to the aldehyde dehydrogenases of many organisms. The predicted DoxHIJ proteins have no obvious sequence similarities to known proteins. Gas chromatography with a flame ionization detector and mass spectroscopy confirmed that the DOX proteins convert naphthalene to salicylate and converting phenanthrene to 1-hydroxy-2-naphthoic acid. doxI mutants convert naphthalene to trans-o-hydroxybenzylidenepyruvate, indicating that the DoxI protein is similar to NahE (trans-o-hydroxybenzylidenepyruvate hydratase-aldolase). Comparison of the DOX sequence with restriction maps of cloned naphthalene catabolic pathway (NAH) genes revealed many conserved restriction sites. The DOX gene arrangement is identical to that proposed for NAH, except that the NAH equivalent of doxH has not been recognized. DoxH may be involved in the conversion of 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoat e to cis-o-hydroxybenzylidenepyruvate. doxJ encodes an enzyme similar to NahD (isomerase). Our findings indicate that a single genetic pathway controls the metabolism of dibenzothiophene, naphthalene, and phenanthrene in strain C18 and that the DOX sequence encodes a complete upper naphthalene catabolic pathway similar to NAH.
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PMCID: PMC206814  PMID: 8226631
22.  Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038 
The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.
PMCID: PMC1389094  PMID: 16535479
23.  Metabolism of naphthalene, 2-methylnaphthalene, salicylate, and benzoate by Pseudomonas PG: regulation of tangential pathways. 
Journal of Bacteriology  1975;124(2):679-685.
Naphthalene is metabolized by Pseudomonas PG through 1,2-dihydroxynaphthalene and salicylate to catechol, which is then degraded by the meta pathway. 2-Methylnaphthalene, but not 1-methylnaphthalene, also serves as a growth substrate and is metabolized by the same route, through 4-methylcatechol. The same nonspecific meta pathway enzymes appear to be induced by growth on either naphthalene or 2-methylnaphthalene. The level to which 2-hydroxymuconic semialdehyde hydrolase is induced is low and probably of no metabolic significance. Growth on salicylate or catechol, both intermediates of naphthalene degradation, or benzoate results in induction of the ortho pathway, the alternative route for catechol dissimilation. No induction of 1,2-dihydroxynaphthalene oxygenase was found in salicylate-grown cells. Anaerobic growth on a succinate-nitrate medium in the presence of various inducers indicates that cis, cis-muconate, or one of its metabolites is the inducer of the ortho pathway enzymes. The inducer or inducers of the early enzymes of naphthalene degradation and of the meta pathway enzymes must be an early intermediate of the naphthalene pathway above salicylate.
PMCID: PMC235954  PMID: 1184575
24.  Aerobic and Anaerobic Toluene Degradation by a Newly Isolated Denitrifying Bacterium, Thauera sp. Strain DNT-1 
A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.
doi:10.1128/AEM.70.3.1385-1392.2004
PMCID: PMC368410  PMID: 15006757
25.  Application of response surface methodology for optimization of decolorization and mineralization of triazo dye Direct Blue 71 by Pseudomonas aeruginosa 
3 Biotech  2013;4(6):605-619.
The decolorization and degradation of Direct Blue 71 were investigated using a mono culture of Pseudomonas aeruginosa. The bacterium was able to decolorize the dye medium to 70.43 % within 48 h under microaerophilic conditions. The medium was then aerated for 24 h to promote the biodegradation of the aromatic amines generated from azo bond cleavage. Reduction in total organic carbon in dye medium was 42.58 % in the microaerophilic stage and 78.39 % in the aerobic stage. The degradation metabolites formed were studied using UV–vis techniques, high performance liquid chromatography, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy analysis. Data obtained provide evidence for the formation of aromatic amines and their subsequent oxidative biodegradation by a single strain of P. aeruginosa during successive microaerophilic/aerobic stages in the same flask. The influence of incubation temperature (20–45 °C), medium pH (5–10) and initial dye concentration (25–150 mg/L) on decolorization was evaluated to greatly influence decolorization extent. The optimal decolorization conditions were determined by response surface methodology based on three-variable central composite design to obtain maximum decolorization and to determine the significance and interaction effect of the variables on decolorization. The optimal conditions of response were found to be 35.15 °C, pH 8.01 and 49.95 mg/L dye concentration giving an experimental decolorization value of 84.80 %. Very high regression coefficient between the variables and the response (R2 = 0.9624) indicated a good evaluation of experimental data by polynomial regression model.
doi:10.1007/s13205-013-0192-7
PMCID: PMC4235883
Response surface methodology; Azo dye; Pseudomonas aeruginosa; Decolorization; Degradation

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