Response of bioluminescent bacteria to sixteen azo dyes

Recombinant bioluminescent bacteria were used to monitor and classify the toxicity of azo dyes. Two constitutive bioluminescent bacteria,Photobacterium phosphoreum andEscherichia coli, E. coli GC2 (lac::luxCDABE), were used to detect the cellular toxicity of the azo dyes. In addition, four stress-inducible bioluminescentE. coli, DPD2794 (recA::luxCDABE), a DNA damage sensitive strain; DPD2540 (fabA::luxCDABE), a membrane damage sensitive strain; DPD2511 (katG::luxCDABE), an oxidative damage sensitive strain; and TV1061 (grpE::luxCDABE), a protein damage sensitive strain, were used to provide information about the type of toxicity caused by crystal violet, the most toxic dye of the 16 azo dyes tested. These results suggest that azo dyes result in serious cellular toxicity in bacteria, and that toxicity monitoring and classification of some azo dyes, in the field, may be possible using these recombinant bioluminescent bacteria.     

Biodegradation of bioaccessible textile azo dyes by Phanerochaete chrysosporium

Azo dyes are important chemical pollutants of industrial origin. Textile azo dyes with bioaccessible groups for lignin degrading fungi, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), were synthesised using different aminobenzoic and aminosulphonic acids as diazo components. The inocula of the best biodegradation assays were obtained from a pre-growth medium (PAM), containing one of the synthesised dyes. The results of the dye biodegradation assays were evaluated every 7 days, by the decrease of the absorbance at the maximum wavelength of the dye, by the decrease of the sucrose concentration in the culture medium and by the increase of the biomass during the 28 days of assay. It was observed that the extent of dye biodegradation depended on the sucrose concentration, on the degraded dye structure and, on the dye present in the PAM medium.     

Biodegradation of bioaccessible textile azo dyes by Phanerochaete chrysosporium

Azo dyes are important chemical pollutants of industrial origin. Textile azo dyes with bioaccessible groups for lignin degrading fungi, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), were synthesised using different aminobenzoic and aminosulphonic acids as diazo components. The inocula of the best biodegradation assays were obtained from a pre-growth medium (PAM), containing one of the synthesised dyes. The results of the dye biodegradation assays were evaluated every 7 days, by the decrease of the absorbance at the maximum wavelength of the dye, by the decrease of the sucrose concentration in the culture medium and by the increase of the biomass during the 28 days of assay. It was observed that the extent of dye biodegradation depended on the sucrose concentration, on the degraded dye structure and, on the dye present in the PAM medium.     

Correlation of anaerobic biodegradability and the electrochemical characteristic of azo dyes

Some experiments were conducted to study some electrochemical factors affecting the bacterial reduction (cleavage) of azo dyes, knowledge of which will be useful in the wastewater treatments of azo dyes. A common mixed culture was used as a test organism and the reductions of Acid Yellow 4, 11, 17 and Acid Yellow BIS were studied. It was found that the azo dyes were reduced at different rates, which could be correlated with the reduction potential of the azo compounds in cyclic voltammetric experiments. Acid Yellow BIS (E _r − 616.75 mV) was reduced at the highest rate of 0.0284 mol g dry cell weight⁻¹ h⁻¹, Acid Yellow 11 (E _r − 593.25 mV) at 0.0245 mol g dry cell weight⁻¹ h⁻¹ and Acid Yellow 4 (E _r − 513 mV) at 0.0178 mol g dry cell weight⁻¹ h⁻¹. At the same time, the decolourization rate of Acid Yellow 17 (E _r − 627.5 mV) was 0.0238 mol g dry cell weight⁻¹ h⁻¹, which was affected by the nature of chlorine substituent. Reduction of these azo dyes did not occur under aeration conditions. These studies with a common mixed culture indicate that the reduction of azo dyes may be influenced by the chemical nature of the azo compound. The reduction potential is a preliminary tool to predict the decolourization capacity of oxidative and reductive biocatalysts.     

Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes

Selection for utilization of carboxy-Orange I [1-(4-carboxyphenylazo)-4-naphthol] in the chemostat yielded Pseudomonas strain K24 which was unable to grow on carboxy-Orange II [1-(4-carboxyphenylazo)-2naphthol] while selection for growth on carboxy-Orange II had previously led to strain KF 46 which did not utilize carboxy-Orange I. Orange I azoreductase of strain K24, the key enzyme of dye degradation, was purified 80-fold with 17% yield to electrophoretic homogeneity and compared to the previously purified Orange II azoreductase of strain KF46. Common properties of the two enzymes were their monomeric structure, their specificity for NADPH and NADH as cosubstrates, the range of their K _m values for substrates and cosubstrates as well as their reactivity towards a series of substrate analogs. They differed from each other with respect to molecular weight (21,000 and 30,000) and in the absolute requirement of Orange I azoreductase for a hydroxy group in the 4 position of the naphthol ring of the substrate molecule as compared to the requirement for substrates with a 2-naphthol moiety by Orange II azoreductase. The pure enzymes did not exhibit immunological cross-reaction with each other. Crude extracts of strains K24 and KF46 and of azoreductase-negative strains isolated at different stages of the adaptation experiments, however, contained material which cross-reacted (CRM) with both anti Orange I azoreductase serum and anti Orange II azoreductase serum. The CRM may represent a common precursor protein of the azoreductases in strains K24 and KF46.Abbreviations Orange I 1-(4-sulfophenylazo)-4-naphthol - carboxy-Orange I 1-(4-carboxy phenylazo)-4-naphthol - Orange II 1-(4-sulfophenylazo)-2-naphthol - carboxy-Orange II 1-(4-carboxyphenylazo)-2-naphthol - SDS sodium dodecyl sulfate - DCAB 4,4-dicarboxyazobenzene - CRM cross reacting material - anti OrIar serum antiserum against Orange I azoreductase - anti OrIIar serum antiserum against Orange II azoreductase Enzymes Orange I azoreductase or NAD(P)H 1-(4-sulfophenylazo)-4-naphthol oxidoreductase (EC 1.6.6-) - Orange II azoreductase or NAD(P)H 1-(4-sulfophenylazo)-2-naphthol oxidoreductase (EC 1.6.6-)     

Exploring threshold operation criteria of biostimulation for azo dye decolorization using immobilized cell systems

This follow-up study provided an evaluation on threshold operation criteria of biostimulation in immobilized cell systems (ICSs) with Aeromonas hydrophila onto packing materials Porites corals. Essential nutrients in appropriate flow rate for biostimulation were inevitably required to maintain maximum attached cell population for cost-effective biodecolorization. With the method of “graphical reconstruction”, the most economically feasible strategy of medium stimulation for color removal was quantitatively revealed. Our findings pointed out no matter what operation mode of reactor was (e.g., suspended batch cultures or ICS) color removal efficiency for A. hydrophila still strongly depended upon intrinsic kinetics and chemical reactivities of azo dyes. Mass transport effects in ICS might not play most significant roles to limit dye biodecolorization of A. hydrophila (except Reactive red 198, Reactive green 19), as relative rankings of color removal rates of various dyes were almost in parallel with those in suspended batch cultures.     

Batch tests for assessing decolourisation of azo dyes by methanogenic and mixed cultures

Most of the published studies on azo dye colour removal involve anaerobic mixed cultures and there is some interest in the knowledge of how dye reduction occurs, if by facultative, strictly anaerobic or both bacterial trophic groups present in classic anaerobic digestors. This paper describes the behaviour of methanogenic and mixed bacteria cultures on the colour removal in batch systems, of a commercial azo dye, C.I. Acid Orange 7, used in paper and textile industries. The aim of this study is to demonstrate, by analysing dye decolourisation, that it occurs with mixed cultures as well as with strictly anaerobic (methanogenic) cultures. Tests were performed with a range of dye concentrations between 60 and 300 mg l⁻¹. The influence of dye concentration on the carbon source removal and decolourisation processes was studied. The effect of carbon source concentration on colour removal was also analysed for both cultures. The degradation rates in mixed and methanogenic cultures were compared. The consumption of carbon source was monitored by COD analysis and dye degradation by ultraviolet-visible spectrophotometry and thin layer chromatography.     

Surfactant modified barley straw for removal of acid and reactive dyes from aqueous solution

A barley straw was modified by a surfactant, cetylpyridinium chloride, and used as an adsorbent for acid (acid blue 40) and reactive dye (reactive black 5) adsorption in aqueous solution. Characterization of the modified barley straw was performed using N₂ adsorption, titration, and FT-IR analysis. It was found that the surfactant modified barley straw exhibits higher adsorption to acid blue 40 than reactive black 5 and adsorption of the dyes is influenced by several parameters such as dye initial concentration, adsorbent dosage, solution pH, and adsorption temperature. Adsorption isotherms show that maximum adsorption of acid blue 40 and reactive black 5 is 1.02 × 10⁻⁴ and 2.54 × 10⁻⁵ mol/g, respectively. Desorption studies show that both dyes are strongly bounded with the adsorbent and exhibit low desorption.     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

Decolourization of reactive azo dyes with microorganisms growing on soft wood chips

The decolourization of a mixture of 200 mg L⁻¹ each of Reactive Black 5 and Reactive Red 2 dye was studied in batch experiments using microorganisms growing on forest residue wood chips in combination with or without added white-rot fungus, Bjerkandera sp. BOL 13. The study was performed as a first stage in the development of a relatively simple treatment process for textile wastewater, designed to work in developing countries. Forest residue wood chips contain a mixture of fungi and bacteria which is an advantage when complex molecules should be degraded. The wood chips furthermore provide the microorganisms with carbon source which make the addition of e.g. glucose unnecessary. The results showed that the microorganisms growing on the forest residue wood chips decolourized the mixture of the two dyes; adding extra nutrients approximately doubled the decolourization rate. The time needed for decolourization was approximately 18 days when nutrients were added. Lignocellulosic material is complex and so were the analysis, microorganisms were therefore transferred to ordinary soft wood chips from forest residue wood chips. Decolourization was measured with spectrophotometer and in order to determine intermediates HPLC was used.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Microbial decolorization of azo dyes and dye industry effluent by Fomes lividus

The white rot fungus, Fomes lividus, was isolated from the logs of Shorea robusta in the Western Ghats region of Tamil Nadu, India. The fungus was tested for decolorization of azo dyes such as orange G (50 M) congo red (50 M) amido black 10B (25 M) and also for colour removal from dye industry effluents. The results revealed that the fungus could remove only 30.8% of orange G in the synthetic solution, whereas congo red and amido black 10B were removed by 74.0 and 98.9% respectively. A dye industry effluent was treated by the fungus in batch and continuous mode. In batch mode treatment, a maximum decolorization of 84.4% was achieved on day 4, and in continuous mode a maximum decolorization of 37.5% was obtained on day 5. The colour removal by the basidiomycete fungus might be due to adsorption of the dyes to the mycelial surface and metabolic breakdown. These results suggested that the batch mode treatment of Fomes lividus is one of the most efficient ways for colour removal in dye industry effluents.     

In vivo and laccase-catalysed decolourization of xenobiotic azo dyes by a basidiomycetous fungus: characterization of its ligninolytic system

Bioremediation is considered a promising eco-efficient alternative for industrial wastewater treatment. Particular attention is currently being given to biological degradation of synthetic dyes and more specifically to colour removal by fungi. This work looks at the extracellular enzymatic system of strain Euc-1. Its ability to decolourize 14 xenobiotic azo dyes was evaluated and compared with the well-known species Phanerochaete chrysosporium. Strain Euc-1 is a mesophilic white-rot basidiomycete, the main secreted ligninolytic enzyme being laccase (0.38 U ml^–1). Although low manganese-dependent peroxidase activity (0.05 U ml^–1) was also detected, neither lignin peroxidase nor aryl alcohol oxidase could be found in batch culture. Optimum pH values of 4.0 and 5.0 were obtained in the laccase-catalysed oxidation of guaiacol and syringaldazine, respectively. Laccase activity increased with the temperature rise up to 50–60 °C and remarkable thermal stability was observed at 50 °C with a half-life of 12 h and no deactivation within the first 2 h. Solid-plate decolourization studies showed that basidiomycete Euc-1 decolourized 11 azo dyes whereas P. chrysosporium only two. Moreover, it is shown that purified laccase from basidiomycete Euc-1 efficiently decolourizes the azo dye acid red 88.     

Rapid decolourization of azo dyes by a new isolated higher manganese peroxidase producer: Phanerochaete sp. HSD

In the present paper, a strain of higher MnP producer, Phanerochaete sp. HSD, was screened and the important medium components influencing MnP production were optimized using fractional factorial design and central composite experimental design; statistical analysis suggested diammonium tartrate and Mn²⁺ were the important factors and under the optimum conditions, MnP activity reached 2613 ± 22 U/l, accorded with the predicted value from response surface analysis. The feasibility of using this fungus to decolourize azo dyes was examined too. Results indicated that crude enzyme solution of it could decolourize three azo dyes efficiently and speedily: for 120 and 350 mg/l of Congo red, 95% decolourization rate was observed at the 5th and 8th hour; for 200, 350 and 600 mg/l methyl orange, 95% decolourization rate was obtained at the 5th, 6th and 9th hour; furthermore, the decolourization rates of 150 and 300 mg/l of Eriochrome black T were up to 97.1% and 91.4% at the 7th and 13th hour, respectively. In addition, MnP played a crucial role in the decolourization process.     

Evaluation of colour removal in synthetic saline wastewater containing azo dyes using an immobilized halotolerant cell system

Studies were carried out to evaluate the colour removal capacity of a moderately halotolerant bacterium, Bacillus firmus, in synthetic saline wastewater medium (SSWM) under static condition. The bacterial strain effectively decolourized Polar red B (an azo dye) in a wide range of sodium chloride (1-6%, w/v), dye (5-100 mg/L) and SDS (0.1-5.0 mg/L) concentrations and at pH range of 6-10 after 24 h of incubation. Cell immobilization studies indicated that colour removal was significantly higher (p < 0.05) in immobilized halotolerant cell systems than with free cells of B. firmus especially at salt concentrations higher than 4%. Results suggest the potential of using the immobilized halotolerant cell system for effective treatment of dye-contaminated saline wastewaters.     

Influence of selected physical parameters on the biodegradation of acrylamide by immobilized cells of Rhodococcus sp.

The influences of concentration of acrylamide, pH, temperature, duration of storage of encapsulated cells and presence of different metals and chelators on the ability of immobilized cells of a Rhodococcus sp. to degrade acrylamide were evaluated. Immobilized cells (3 g) rapidly degraded 64 and 128 mM acrylamide in 3 and 5 h, espectively, whereas free cells took more than 24 h to degrade 64 mM acrylamide. An acrylamide concentration of 128 mM inhibited the growth of the free cells. Immobilized bacteria were slow to degrade acrylamide at 10 °C. Less than 60% of acrylamide was degraded in 4 h. However, 100% of the compound was degraded in less than 3 h at 28 °C and 45 °C. The optimum pH for the degradation of acrylamide by encapsulated cells was pH 7.0. Less than 10% of acrylamide was degraded at pH 6.0, while ca. 60% of acrylamide was degraded at pH 8.0 and 8.5. Copper and nickel inhibited the degradation, suggesting the presence of sulfhydryl (-SH) groups in the active sites of the acrylamide degrading amidase. Iron enhanced the rates of degradation and chelators (EDTA and 1,10 phenanthroline) reduced the rates of degradation suggesting the involvement of iron in its active site(s) of the acrylamide-degrading-amidase. Immobilized cells could be stored up to 10 days without any detectable loss of acrylamide-degrading activity.     

Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors.

Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%). Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity. Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity. Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS). Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye. Hydrogen, added in bulk, was clearly the preferred electron donor. Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge. However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction. The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye. The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria.     

Proposed pathways for the reduction of a reactive azo dye in an anaerobic fixed bed reactor

Some process has been proposed for azo dye degradation and anaerobic bioreactors are one of them, since for their reduction, the dye has to be the electron acceptor. An anaerobic fixed bed bioreactor packed with activated carbon (AC) is proposed to degradate the Reactive Red 272 azo dye. In the present paper a dye degradation mechanism in an anaerobic environment is explained. It is very important to consider the interaction dye-microorganism-AC, because the groups in the AC surface take part in the reaction besides being an excellent carrier for microorganism and an adsorbent for the dye. The aromatic compounds produced in the dye reduction are partially degraded as a function of inlet dye concentration and reactor residence time. In anaerobic environment the aromatic compounds are decomposed through hydroxylation, carboxylation and redox reactions, due to enzymatic reactions.