Enhancement of nitroaromatic compounds anaerobic biotransformation using a novel immobilized redox mediator prepared by electropolymerization

Functionalized polypyrrole (PPy) composites were prepared by incorporation of a model redox mediator, anthraquinonedisulphonate (AQDS), as doping anion during the electropolymerization of pyrrole (Py) monomer on active carbon felt (ACF) electrode. Then, the resulting composite, ACF/PPy/AQDS as a novel immobilized redox mediator for catalyzing anaerobic biotransformation of the model nitroaromatic compounds (NACs), such as nitrobenzene (NB), 2,4- and 2,6-dinitrotoluene (DNT), were investigated in detail. The results showed that ACF/PPy/AQDS exhibited good catalytic activity and stability, and its addition effectively accelerated the NACs anaerobic reduction to the corresponding amino compounds. In order to estimate the relationship between community dynamics and the function of immobilized redox mediator, a combined method based on fingerprints (ribosomal intergenic spacer analysis, RISA) and 16S rRNA gene sequencing was used. The results indicated that the existence of ACF/PPy/AQDS made the potent AQDS-reducing bacteria keeping predominant in the catalytic systems. Based on the results above, it can be concluded that this novel immobilized redox mediator is feasible and potentially useful to enhance NACs anaerobic reduction.     

Enhanced bio-decolorization of azo dyes by quinone-functionalized ceramsites under saline conditions

Anthraquinone-2-sulfonate was immobilized on ceramsites (AQS-ceramsites) using a novel adsorption/covalence coupling method and their effects on the anaerobic bio-decolorization rates of azo dyes by salt-tolerant AQS-reducing (STAR) community were investigated. The results showed that AQS-ceramsites mediated specific bio-decolorization rates of four azo dyes Acid Yellow 36, Reactive Red 2, Acid Red 27 and Acid Orange 7 increase 2.3–6.4 fold than those lacking ceramsites in the presence of 50 g/L NaCl. Moreover, repeated experiments with AQS-ceramsites showed that the decolorization efficiencies of azo dyes could remain over 98% of their original value. These results indicated that AQS-ceramsites functioning as redox mediators exhibited good catalytic activity and stability under saline conditions. The dynamics of the STAR community structure revealed by PCR-DGGE also showed that the presence of AQS-ceramsites made STAR bacteria keeping predominant in the catalytic system. Therefore, it can be concluded that this novel solid redox mediator is potentially useful for the treatment of saline dye wastewater.     

Autocatalysis in Reactive Black 5 biodecolorization by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> W1

Autocatalysis in biological decolorization of Reactive Black 5 (RB5) by Rhodopseudomonas palustris W1 was investigated in batch assays. An improvement of 1.5-fold in decolorization rate of RB5 was obtained by supplementing decolorization metabolites from 200 mg l(-1) RB5. Liquid chromatography-mass spectrometry and cyclic voltammetric analysis revealed that the constituent of dye precursors, from azo bonds breakage, with quinone-like structure and reversible oxidation-reduction activity can be used as redox mediators and was responsible for the catalytic reduction of RB5. The required amount of metabolites for catalytic decolorization was quite small, indicating its possible application in real textile wastewater treatment. Furthermore, decolorization metabolites of RB5 were shown as effective in catalyzing anaerobic decolorization of Direct Yellow 11, an azo dye without autocatalyic activity.     

Immobilized redox mediators for electrochemical NAD(P)<Superscript>+</Superscript> regeneration

The applicability of dissolved redox mediators for NAD(P)⁺ regeneration has been demonstrated several times. Nevertheless, the use of mediators in solutions for sensor applications is not a very convenient strategy since the analysis is not reagentless and long stabilization times occur. The most important drawbacks of dissolved mediators in biocatalytic applications are interferences during product purification, limited reusability of the mediators, and their cost-intensive elimination from wastewater. Therefore, the use of immobilized mediators has both economic and ecological advantages. This work critically reviews the current state-of-art of immobilized redox mediators for electrochemical NAD(P)⁺ regeneration. Various surface modification techniques, such as adsorption polymerization and covalent linkage, as well as the corresponding NAD(P)⁺ regeneration rates and the operational stability of the immobilized mediator films, will be discussed. By comparison with other existing regeneration systems, the technical potential and future perspectives of biocatalytic redox reactions based on electrochemically fed immobilized mediators will be assessed.     

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.     

Identification of quinoide redox mediators that are formed during the degradation of naphthalene-2-sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Kinetics during the redox biotransformation of pollutants mediated by immobilized and soluble humic acids

The aim of this study was to elucidate the kinetic constraints during the redox biotransformation of the azo dye, Reactive Red 2 (RR2), and carbon tetrachloride (CT) mediated by soluble humic acids (HA_s) and immobilized humic acids (HA_i), as well as by the quinoid model compounds, anthraquinone-2,6-disulfonate (AQDS) and 1,2-naphthoquinone-4-sulfonate (NQS). The microbial reduction of both HA_s and HA_i by anaerobic granular sludge (AGS) was the rate-limiting step during decolorization of RR2 since the reduction of RR2 by reduced HA_i proceeded at more than three orders of magnitute faster than the electron-transferring rate observed during the microbial reduction of HA_i by AGS. Similarly, the reduction of RR2 by reduced AQDS proceeded 1.6- and 1.9-fold faster than the microbial reduction of AQDS by AGS when this redox mediator (RM) was supplied in soluble and immobilized form, respectively. In contrast, the reduction of NQS by AGS occurred 1.6- and 19.2-fold faster than the chemical reduction of RR2 by reduced NQS when this RM was supplied in soluble and immobilized form, respectively. The microbial reduction of HA_s and HA_i by a humus-reducing consortium proceeded 1,400- and 790-fold faster than the transfer of electrons from reduced HA_s and HA_i, respectively, to achieve the reductive dechlorination of CT to chloroform. Overall, the present study provides elucidation on the rate-limiting steps involved in the redox biotransformation of priority pollutants mediated by both HA_s and HA_i and offers technical suggestions to overcome the kinetic restrictions identified in the redox reactions evaluated.     

Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Anaerobic degradation of azo dye Drimaren blue HFRL in UASB reactor in the presence of yeast extract a source of carbon and redox mediator

This paper presents results on anaerobic degradation of the azo dye blue HFRL in a bench scale Upflow anaerobic sludge blanket (UASB) reactor operated at ambient temperature. The results show that the addition of yeast extract (500 mg/L) increased color removal (P < 0.05) from 62 to 93% despite the low chemical oxygen demand (COD) removal (~35%) which happened due to volatile fatty acids (VFA) accumulation. There were no differences in color removal (~91%) when yeast extract (500 mg/L) was used in the presence or absence of glucose, suggesting that yeast extract acted as source of redox mediator (riboflavin) and carbon. The specific rate of dye removal increased along the operational phases and depended on the presence of yeast extract, suggesting progressive biomass acclimatization. Analysis of bacterial diversity by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR–DGGE) method showed there was biomass selection along the bioreactor operation and no evidence of azo dye degrading bacteria predominance. This strengthens the hypothesis that color removal happens extracellularly by the reduction of azo bond by reduced redox mediators, such as riboflavin, which is present in high amount in the yeast extract.     

Use and improvement of microbial redox enzymes for environmental purposes

Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed.     

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.     

Redox-mediated decolorization of Direct Red 23 and Direct Blue 80 catalyzed by bioaffinity-based immobilized tomato (Lycopersicon esculentum) peroxidase

The aim of this study was to investigate the role of concanavalin A (Con A)-cellulose-bound tomato peroxidase for the decolorization of direct dyes. Cellulose was used as an inexpensive material for the preparation of bioaffinity support. Con A-cellulose-bound tomato peroxidase exhibited higher efficiency in terms of dye decolorization as compared to soluble enzyme under various experimental conditions. Both Direct Red 23 and Direct Blue 80 dyes were recalcitrant to the action of enzyme without a redox mediator. Six compounds were investigated for redox-mediating property. Immobilized peroxidase decolorized both dyes to different extent in the presence of all the used redox mediators. However, 1-hydroxybenzotriazole emerged as a potential redox mediator for tomato peroxidase catalyzed decolorization of direct dyes. These dyes were maximally decolorized at pH 6.0 and 40 degrees C by soluble and immobilized peroxidase. The absorption spectra of the untreated and treated dyes exhibited a marked difference in the absorption at various wavelengths. Immobilized tomato peroxidase showed a lower Michaelis constant than the free enzyme for both dyes. Soluble and immobilized tomato peroxidase exhibited significantly higher affinity for Direct Red 23 compared to Direct Blue 80.     

Potential applications of the oxidoreductive enzymes in the decolorization and detoxification of textile and other synthetic dyes from polluted water: a review

Recently, the enzymatic approach has attracted much interest in the decolorization/degradation of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical and biological treatments, which pose serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. The potential of the enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases, and azoreductases) has been exploited in the decolorization and degradation of dyes. Some of the recalcitrant dyes were not degraded/decolorized in the presence of such enzymes. The addition of certain redox mediators enhanced the range of substrates and efficiency of degradation of the recalcitrant compounds. Several redox mediators have been reported in the literature, but very few of them are frequently used (e.g., 1-hydroxybenzotriazole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone). Soluble enzymes cannot be exploited at the large scale due to limitations such as stability and reusability. Therefore, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.     

Potential Applications of the Oxidoreductive Enzymes in the Decolorization and Detoxification of Textile and Other Synthetic Dyes from Polluted Water: A Review

ABSTRACT

Recently, the enzymatic approach has attracted much interest in the decolorization/degradation of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical and biological treatments, which pose serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. The potential of the enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases, and azoreductases) has been exploited in the decolorization and degradation of dyes. Some of the recalcitrant dyes were not degraded/decolorized in the presence of such enzymes. The addition of certain redox mediators enhanced the range of substrates and efficiency of degradation of the recalcitrant compounds. Several redox mediators have been reported in the literature, but very few of them are frequently used (e.g., 1-hydroxybenzotriazole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone). Soluble enzymes cannot be exploited at the large scale due to limitations such as stability and reusability. Therefore, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.     

Biological sulphate reduction and redox mediator effects on azo dye decolourisation in anaerobic–aerobic sequencing batch reactors

In this work, the anaerobic period of an anaerobic–aerobic sequencing batch reactor was found to allow the reductive decolourisation of azo dyes. 1-l reactors were operated in 24-h cycles comprising anaerobic and aerobic reaction phases, fed with a simulated textile effluent including a reactive type (Remazol Brilliant Violet 5R) or an acid type (Acid Orange 7) azo dye. The aim was to assess the role of different redox phenomena in the anaerobic decolourisation process. Selective inhibition of sulphate reducing bacteria was carried out in the sulphate-containing, reactive dye fed reactor, resulting in nearly complete, though reversible and inhibition of decolourisation. The acid dye fed reactor's supplementation with sulphate, though resulting in sulphate reduction, did not improve decolourisation. Other redox mediators, namely quinones, were more effective in promoting electron transfer to the azo bond. Bio-augmentation of the acid dye fed reactor with a pure sulphate reducer strain known to decolourise azo dyes, Desulfovibrio alaskensis, was also carried out. Decolourisation was improved, but apparently as a result of the carbon source change required to support D. alaskensis growth. A chemically mediated reduction of the azo bond coupled to biological sulphate reduction, thus seemed to account for the high decolourisation yields of both dyes.     

Microbial hydrogen production with immobilized sewage sludge

Municipal sewage sludge was immobilized to produce hydrogen gas under anaerobic conditions. Cell immobilization was essentially achieved by gel entrapment approaches, which were physically or chemically modified by addition of activated carbon (AC), polyurethane (PU), and acrylic latex plus silicone (ALSC). The performance of hydrogen fermentation with a variety of immobilized-cell systems was assessed to identify the optimal type of immobilized cells for practical uses. With sucrose as the limiting carbon source, hydrogen production was more efficient with the immobilized-cell system than with the suspended-cell system, and in both cases the predominant soluble metabolites were butyric acid and acetic acid. Addition of activated carbon into alginate gel (denoted as CA/AC cells) enhanced the hydrogen production rate (v(H2)) and substrate-based yield (Y((H2)/sucrose)) by 70% and 52%, respectively, over the conventional alginate-immobilized cells. Further supplementation of polyurethane or acrylic latex/silicone increased the mechanical strength and operation stability of the immobilized cells but caused a decrease in the hydrogen production rate. Kinetic studies show that the dependence of specific hydrogen production rates on the concentration of limiting substrate (sucrose) can be described by Michaelis-Menten model with good agreement. The kinetic analysis suggests that CA/AC cells may contain higher concentration of active biocatalysts for hydrogen production, while PU and ALSC cells had better affinity to the substrate. Acclimation of the immobilized cells led to a remarkable enhancement in v(H2) with a 25-fold increase for CA/AC and ca. 10- to 15-fold increases for PU and ALSC cells. However, the ALSC cells were found to have better durability than PU and CA/AC cells as they allowed stable hydrogen production for over 24 repeated runs.     

Impact and application of electron shuttles on the redox (bio)transformation of contaminants: A review

During the last two decades, extensive research has explored the catalytic effects of different organic molecules with redox mediating properties on the anaerobic (bio)transformation of a wide variety of organic and inorganic compounds. The accumulated evidence points at a major role of electron shuttles in the redox conversion of several distinct contaminants, both by chemical and biological mechanisms. Many microorganisms are capable of reducing redox mediators linked to the anaerobic oxidation of organic and inorganic substrates. Electron shuttles can also be chemically reduced by electron donors commonly found in anaerobic environments (e.g. sulfide and ferrous iron). Reduced electron shuttles can transfer electrons to several distinct electron-withdrawing compounds, such as azo dyes, polyhalogenated compounds, nitroaromatics and oxidized metalloids, among others. Moreover, reduced molecules with redox properties can support the microbial reduction of electron acceptors, such as nitrate, arsenate and perchlorate. The aim of this review paper is to summarize the results of reductive (bio)transformation processes catalyzed by electron shuttles and to indicate which aspects should be further investigated to enhance the applicability of redox mediators on the (bio)transformation of contaminants.     

Basic and applied aspects in the microbial degradation of azo dyes

Azo dyes are the most important group of synthetic colorants. They are generally considered as xenobiotic compounds that are very recalcitrant against biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several microorganisms are able, under certain environmental conditions, to transform azo dyes to non-colored products or even to completely mineralize them. Thus, various lignolytic fungi were shown to decolorize azo dyes using ligninases, manganese peroxidases or laccases. For some model dyes, the degradative pathways have been investigated and a true mineralization to carbon dioxide has been shown. The bacterial metabolism of azo dyes is initiated in most cases by a reductive cleavage of the azo bond, which results in the formation of (usually colorless) amines. These reductive processes have been described for some aerobic bacteria, which can grow with (rather simple) azo compounds. These specifically adapted microorganisms synthesize true azoreductases, which reductively cleave the azo group in the presence of molecular oxygen. Much more common is the reductive cleavage of azo dyes under anaerobic conditions. These reactions usually occur with rather low specific activities but are extremely unspecific with regard to the organisms involved and the dyes converted. In these unspecific anaerobic processes, low-molecular weight redox mediators (e.g. flavins or quinones) which are enzymatically reduced by the cells (or chemically by bulk reductants in the environment) are very often involved. These reduced mediator compounds reduce the azo group in a purely chemical reaction. The (sulfonated) amines that are formed in the course of these reactions may be degraded aerobically. Therefore, several (laboratory-scale) continuous anaerobic/aerobic processes for the treatment of wastewaters containing azo dyes have recently been described.     

Decolorization of anthraquinone dye intermediate and its accelerating effect on reduction of azo acid dyes by Sphingomonas xenophaga in anaerobic–aerobic process

Decolorization of 1-aminoanthraquinone-2-sulfonic acid (ASA-2) and its accelerating effect on the reduction of azo acid dyes by Sphingomonas xenophaga QYY were investigated. The study showed that ASA-2 could be efficiently decolorized by strain QYY under aerobic conditions according to the analysis of total organic carbon removal and UV-VIS spectra changes. Moreover, strain QYY was able to reduce azo acid dyes under anaerobic conditions. The effects of various operating conditions such as carbon sources, temperature, and pH on the reduction rate were studied. It was demonstrated that ASA-2 used as a redox mediator could accelerate the reduction process. Consequently the reduction of azo acid dyes mediated by ASA-2 and the decolorization of ASA-2 with strain QYY could be achieved in an anaerobic-aerobic process.     

Arthrobacter sp. lipase immobilization for preparation of enantiopure masked β-amino alcohols

Recent reports on immobilization of lipase from Arthrobacter sp. (ABL, MTCC 5125; IIIM isolate) on insoluble polymers have shown altered properties including stability and enantioselectivity. Present work demonstrates a facile method for the preparation of enantiopure β-amino alcohols by modulation of ABL enzyme properties via immobilization on insoluble as well as soluble supports using entrapment/covalent binding techniques. Efficacies of immobilized ABL on insoluble supports prepared from tetraethylorthosilicate/aminopropyltriethoxy silane and soluble supports derived from copolymerization of N-vinyl pyrrolidone-allylglycidyl ether (ANP type)/N-vinyl pyrrolidone-glycidyl methacrylate (GNP type) for kinetic resolution of masked β-amino alcohols have been studied vis-à-vis free ABL enzyme/wet cell biomass. The immobilized lipase on different insoluble/soluble supports has shown 21–110 mg/g protein binding and 30–700 U/g activity for hydrolyzing tributyrin substrate. The findings have shown a significant enhancement in enantioselectivity (ee 99%) vis-à-vis wet cell biomass providing ee 70–90% for resolution of β-amino alcohols.