Addition Of Redox Mediators Research Articles

Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators

Trametes trogii BAFC 463 culture fluids (containing 110 U ml −1 laccase; 0.94 U ml −1 manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 °C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu +2, Pb +2 or Cd +2) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.

Decolorization of synthetic dyes by crude laccase from a newly isolated Trametes trogii strain cultivated on solid agro-industrial residue

A new dye-decolorizing white-rot fungus was isolated and identified as Trametes trogii based on its ITS-5.8S rRNA gene sequence analysis and morphological characteristics. Laccase was the only lignolytic enzyme produced by this strain during solid substrate fermentation (SSF) in soybean cake, a solid agro-industrial residue used for the first time in enzyme production. The extracellular crude enzyme from T. trogii in solid substrate fermentation showed good activity in synthetic dye color removal, decolorizing 85.2% Remazol Brilliant Blue R (50mgl−1), 69.6% Reactive Blue 4 (35mgl−1), and 45.6% Acid Blue 129 (83.3mgl−1) without the addition of redox mediators, 90.2% Acid Red 1 (10mgl−1), and 65.4% Reactive Black 5 (18.3mgl−1) with the addition of 1mM 1-hydroxybenzotriazole in 30min. Native polyacrylamide gel electrophoresis (Native-PAGE) of the crude enzyme and effects of laccase inhibitors on decolorization corroborated the laccase as the major enzyme involved in the decolorization of dyes. The comparison of color removal by the crude culture filtrates and by the whole fungal culture on the solid substrate revealed the former was more advantageous.

In-situ Cr(VI) reduction with electrogenerated hydrogen peroxide driven by iron-reducing bacteria

Cr(VI) was reduced in-situ at a carbon felt cathode in an air-cathode dual-chamber microbial fuel cell (MFC). The reduction of Cr(VI) was proven to be strongly associated with the electrogenerated H(2)O(2) at the cathode driven by iron-reducing bacteria. At pH 2.0, only 42.5% of Cr(VI) was reduced after 12h in the nitrogen-bubbling-cathode MFC, while complete reduction of Cr(VI) was achieved in 4h in the air-bubbling-cathode MFC in which the reduction of oxygen to H(2)O(2) was confirmed. Conditions that affected the efficiency of the reduction of Cr(VI) were evaluated experimentally, including the cathodic electrolyte pH, the type of iron-reducing species, and the addition of redox mediators. The results showed that the efficient reduction of Cr(VI) could be achieved with an air-bubbling-cathode MFC.

Biodegradation of a simulated textile effluent by immobilised-coated laccase in laboratory-scale reactors

Laccase from Trametes pubescens was immobilised on alumina pellets and coated with polyelectrolytes. It was shown that this approach enhanced both laccase stability and reusability. Further, the immobilised-coated laccase was applied to the decolouration of a simulated textile effluent in laboratory-scale reactors. The simulated textile effluent was based on the recalcitrant diazo dye Reactive Black 5 (0.5 g/L). It was found that the decolouration was due to two processes: dye adsorption on the immobilisation support and coating and dye degradation by the laccase enzyme. The adsorption process represented less than 10% of colour removal for all cases, so decolouration was mainly due to laccase action. The decolouration was performed in both batch and continuous modes. A complete decolouration of the effluent was obtained in 30–36 h for the former and 48 h for the latter without the addition of redox mediators. In addition, the decolourised effluent showed lower phytotoxicity than the original one. These encouraging results make the process suitable for its potential implementation at industrial scale.

Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis

Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around − 0.4 V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results.

Production and synthetic dyes decolourization capacity of a recombinant laccase from Pichia pastoris

To produce and purify a recombinant laccase from Pichia pastoris and to test its ability in decolourization of synthetic dyes. A cDNA encoding for a laccase was isolated from Pycnoporus sanguineus and was expressed in P. pastoris strain SMD1168H under the control of the alcohol oxidase (AOX1) promoter. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as cultivation temperature, pH, copper concentration and methanol concentration, were investigated. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a molecular mass of about 62.8 kDa. The purified enzyme showed a similar behaviour to the native laccase produced by P. sanguineus. Four different synthetic dyes including azo, anthraquinone, triphenylmethane and indigo dyes could be efficiently decolourized by the purified recombinant laccase without the addition of redox mediators. Heterologous production of P. sanguineus laccase in P. pastoris was successfully achieved. The purified recombinant laccase could efficiently decolourize synthetic dyes in the absence of mediators. This study is the first report on the synthetic dye decolourization by the recombinant P. sanguineus laccase. The decolourization capacity of this recombinant enzyme suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents.

Microbial reductive dechlorination of trichloroethene to ethene with electrodes serving as electron donors without the external addition of redox mediators

In situ bioremediation of industrial chlorinated solvents, such as trichloroethene (TCE), is typically accomplished by providing an organic electron donor to naturally occurring dechlorinating populations. In the present study, we show that TCE dechlorinating bacteria can access the electrons required for TCE dechlorination directly from a negatively polarized (-450 mV vs. SHE) carbon paper electrode. In replicated batch experiments, a mixed dechlorinating culture, also containing Dehalococcoides spp., dechlorinated TCE to cis-dichloroethene (cis-DCE) and lower amounts of vinyl chloride (VC) and ethene using the polarized electrode as the sole electron donor. Conversely, neither VC nor ethene formation occurred when a pure culture of the electro-active microorganism Geobacter lovleyi was used, under identical experimental conditions. Cyclic voltammetry tests, carried out on the filter-sterilized supernatant of the mixed culture revealed the presence of a self-produced redox mediator, exhibiting a midpoint potential of around -400 mV (vs. SHE). This yet unidentified redox-active molecule appeared to be involved in the extracellular electron transfer from the electrode to the dechlorinating bacteria. The ability of dechlorinating bacteria to use electrodes as electron donors opens new perspectives for the development of clean, versatile, and efficient bioremediation systems based on a controlled subsurface delivery of electrons in support of biodegradative metabolisms and provides further evidence on the possibility of using conductive materials to manipulate and control a range of microbial bioprocesses.

Simultaneous production of laccase and decolouration of the diazo dye Reactive Black 5 in a fixed-bed bioreactor

In this paper the production of laccase and the decolouration of the recalcitrant diazo dye Reactive Black 5 (RB5) by the white-rot fungus Trametes pubescens immobilised on stainless steel sponges in a fixed-bed reactor were studied. Laccase production was increased by 10-fold in the presence of RB5 and reached a maximum value of 1025 U/l. Enhanced laccase production in the presence of RB5 in this fungus is an added advantage during biodegradation of RB5-containing effluents. The decolouration of RB5 was due to two processes: dye adsorption onto the fungal mycelium and dye degradation by the laccase enzymes produced by the fungus. RB5 decolouration was performed during four successive batches obtaining high decolouration percentages (74%, 43% and 52% in 24 h for the first, third and four batch, respectively) without addition of redox mediators. Also, the in vitro decolouration of RB5 by the concentrated culture extract, containing mainly laccase, produced in the above bioreactor was studied. The decolouration percentages obtained were considerably lower (around 20% in 24 h) than that attained with the whole culture.

Degradation of Polycyclic Aromatic Hydrocarbons by Rigidoporus lignosus and its Laccase in the Presence of Redox Mediators

The metabolism of polycyclic aromatic hydrocarbons (PAHs) was studied in vivo and in vitro in systems consisting of Rigidoporus lignosus and its laccase, in the presence of so-called "mediator" compounds. The static culture of the native fungal strain was able to metabolize anthracene and 2-methylanthracene, but not 9-nitroanthracene. The addition of redox mediators 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) or violuric acid (VA) led to a significant increase in the degradation of substrates. The oxidation of PAHs was not significant when purified laccase was used without the addition of mediators. The addition of these compounds increased the oxidation of all substrates by approximately 70-80% after 72 h of incubation. The degradation rate was highest for 2-methylanthracene in the presence of VA.

Cathodic Decolourisation of Dyes in Concentrates from Nanofiltration and Printing Pastes

Direct cathodic reduction of dyes which contain an azo-goup in the chromophore was successfully used for decolourisation of intensively coloured concentrates from Nanofiltration treatment of textile effluents. Based on laboratory scale experiments, a technical multi-cathode electrolyser was applied for full scale decolourisation experiments at cell currents from 40 to 80 A. The absorbance of the treated wastes decreased from 60 to 80% of the initial value at an energy consumption of 2–8 kWh m−3. Experiments with addition of redox mediator indicate a significant increase in decolourisation rate; however chemical consumption is increased for 0.5–1.5 kg m−3 of waste. The decolourisation of reactive dye containing printing pastes was also achieved at the laboratory scale, where decolourisation of 60–80% was achieved.

Effect of redox mediator, AQDS, on the decolourisation of a reactive azo dye containing triazine group in a thermophilic anaerobic EGSB reactor

The feasibility of thermophilic (55 °C) anaerobic treatment applied to colour removal of a triazine contained reactive azo dye was investigated in two 0.53 l expanded granular sludge blanket (EGSB) reactors in parallel at a hydraulic retention time (HRT) of 10 h. Generally, this group of azo dyes shows the lowest decolourisation rates during mesophilic anaerobic treatment. The impact of the redox mediator addition on colour removal rates was also evaluated. Reactive Red 2 (RR2) and anthraquinone-2,6-disulfonate (AQDS) were selected as model compounds for azo dye and redox mediator, respectively. The reactors achieved excellent colour removal efficiencies with a high stability, even when high loading rates of RR2 were applied (2.7 g RR2 l −1 per day). Although AQDS addition at catalytic concentrations improved the decolourisation rates, the impact of AQDS on colour removal was less apparent than expected. Results show that the AQDS-free reactor R2 achieved excellent colour removal rates with efficiencies around 91%, compared with the efficiencies around 95% for the AQDS-supplied reactor R1. Batch experiments confirmed that the decolourisation rates were co-substrate dependent, in which the volatile fatty acids (VFA) mixture was the least efficient co-substrate. The highest decolourisation rate was achieved in the presence of either hydrogen or formate, although the presence of glucose had a significant impact on the colour removal rates.

An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization

The plant pathogen basidiomycete S. rolfsii secretes two laccases (SRL1 and SRL2) with molecular weights of 55 and 86 kDa, respectively. Laccase production was shown to be inducible by the addition of 2,5-xylidine to the cultural media. After treatment with a combination of chitinase and β-1,3-glucanase, two different laccases were isolated from the sclerotia depending on the stage of sclerotia development. The more prominent laccase, SRL1, was purified and found to decolourize the industrially important wool azo dye Diamond Black PV 200 without the addition of redox mediators. The enzyme (pI 5.2) was active in the acidic pH range, showing an optimal activity at pH 2.4, with ABTS as substrate. The optimum temperature for activity was determined to be 62 °C. Enzyme stability studies revealed that SRL1 was notably stable at 18 °C and pH 4.5, retaining almost full activity after a week. Oxidation of tyrosine was not detectable under the reaction conditions but the enzyme did oxidize a variety of the usual laccase substrates. SRL1 was strongly inhibited by sodium azide and fluoride. Dye solutions decolourized with the immobilized laccase were successfully used for redyeing.

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q ⨪ B) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential ( E h) to 200–300 mV is the result of increasing the lifetime of Q ⨪ B, as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q ⨪ B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q ⨪ B, the flash-induced redox changes of Q ⨪ B were observed at low E h values unless E h reached a value at which Q B underwent reduction at equilibrium to form Q BH 2. The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q BH 2) of the secondary acceptor.

On the lack of ATP-induced midpoint potential shift for cytochrome b-566 in plant mitochondria.

Recently proposed mechanisms of site II energy transduction that assign a key role to cytochrome b-566 are based on the finding that the apparent midpoint potential of b-566 in animal mitochondria increases by more than 250 mV upon addition of ATP [Chance et al. (1970) Proc. Nat. Acad. Sci. USA 66, 1175-1182]. However, since it has never been shown that the redox mediators used in the midpoint potential measurements equilibrate directly with b-566, the observed midpoint potential shift could merely reflect reversed electron transport. In mung bean mitochondria, the apparent midpoint potential of b-566 is known to be unaffected by addition of ATP [Dutton and Storey (1971) Plant Physiol. 47, 282-288]. In the present work, mung bean b-566 is shown to undergo an ATP-induced reduction similar to that observed for b-566 in animal mitochondria. However, in mung bean mitochondria the reduction is found to be rapidly relaxed by addition of redox mediator (phenazine methosulfate, PMS) and concomitantly PMS causes a marked, antimycinsensitive stimulation of ATPase activity. These results suggest that the ATP-induced reduction in mung bean mitochondria is due to reversed electron transport and that PMS can effectively short-circuit reversed electron transport in this system, bringing it close to equilibrium. Moreover, since mung bean and animal b-566 are identical in all other respects tested, the results support the idea that the apparent midpoint potential shift in animal mitochondria is also merely due to reversed electron transport, and that the mediators are now not effective enough to bring the system to equilibrium.