Luciana Pereira

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

Dyes—Environmental Impact and Remediation

Dyes are an important class of synthetic organic compounds used in many industries, especially textiles. Consequently, they have become common industrial environmental pollutants during their synthesis and later during fibre dyeing. Textile industries are facing a challenge in the field of quality and productivity due to the globalization of the world market. As the highly competitive atmosphere and the ecological parameters become more stringent, the prime concern of the textile processors is to be aware of the quality of their products and also the environmental friendliness of the manufacturing processes. This in turn makes it essential for innovations and changes in these processes, and investigations of appropriate and environmentally friendly treatment technologies or their residues. The large-scale production and extensive application of synthetic dyes can cause considerable environmental pollution, making it a serious public concern. Legislation on the limits of colour discharge has become increasingly rigid. There is a considerable urgent need to develop treatment methods that are effective in eliminating dyes from their waste. Physicochemical and biological methods have been studied and applied, although each has its advantages and disadvantages, with the choice being based on the wastewater characteristics, available technology and economic factors. Some industrial-scale wastewater treatment systems are now available; however, these are neither fully effective for complete colour removal nor do they address water recycling.

Thermal modification of activated carbon surface chemistry improves its capacity as redox mediator for azo dye reduction.

The surface chemistry of a commercial AC (AC(0)) was selectively modified, without changing significantly its textural properties, by chemical oxidation with HNO(3) (AC(HNO3)) and O(2) (AC(O2)), and thermal treatments under H(2) (AC(H2)) or N(2) (AC(N2)) flow. The effect of modified AC on anaerobic chemical dye reduction was assayed with sulphide at different pH values 5, 7 and 9. Four dyes were tested: Acid Orange 7, Reactive Red 2, Mordant Yellow 10 and Direct Blue 71. Batch experiments with low amounts of AC (0.1 g L(-1)) demonstrated an increase of the first-order reduction rate constants, up to 9-fold, as compared with assays without AC. Optimum rates were obtained at pH 5 except for MY10, higher at pH 7. In general, rates increased with increasing the pH of point zero charge (pH(pzc)), following the trend AC(HNO3) < AC(O2) < AC(0) < AC(N2) < AC(H2). The highest reduction rate was obtained for MY10 with AC(H2) at pH 7, which corresponded to the double, as compared with non-modified AC. In a biological system using granular biomass, AC(H2) also duplicated and increase 4.5-fold the decolourisation rates of MY10 and RR2, respectively. In this last experiment, reaction rate was independent of AC concentration in the tested range 0.1-0.6 g L(-1).

Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes

Laccases are useful biocatalysts for many diverse biotechnological applications. In this study we have established efficient and reliable expression systems and high-throughput screenings for the recombinant CotA-laccase from Bacillus subtilis. The expression levels of cotA-laccase were compared in five different Escherichia coli host strains growing in 96-well microtiter plates under different culture conditions. Lower coefficients of variance (around 15%) were achieved using crude cell lysates of BL21 and KRX host strains growing under microaerobic conditions. Reproducible high-throughput screenings for the decolorization of high redox potential azo and anthraquinonic dyes were developed and optimized for identification of variants with increased redox potential. The enzymatic assays developed were tested for the screening of one mutant library from CotA-laccase created by error-prone PCR.

Metallic nanoparticles: microbial synthesis and unique properties for biotechnological applications, bioavailability and biotransformation

Abstract The impact of nanotechnology in all areas of science and technology is evident. The expanding availability of a variety of nanostructures with properties in the nanometer size range has sparked widespread interest in their use in biotechnological systems, including the field of environmental remediation. Nanomaterials can be used as catalysts, adsorbents, membranes, water disinfectants and additives to increase catalytic activity and capability due to their high specific surface areas and nanosize effects. Thus, nanomaterials appear promising for new effective environmental technologies. Definitely, nanotechnology applications for site remediation and wastewater treatment are currently in research and development stages, and new innovations are underway. The synthesis of metallic nanoparticles has been intensively developed not only due to its fundamental scientific interest but also for many technological applications. The use of microorganisms in the synthesis of nanoparticles is a relatively new eco-friendly and promising area of research with considerable potential for expansion. On the other hand, chemical synthesis occurs generally under extreme conditions (e.g. pH, temperature) and also chemicals used may have associated environmental and human health impacts. This review is an overview of current research worldwide on the use of microorganisms during the biosynthesis of metallic nanoparticles and their unique properties that make them good candidates for many applications, including in biotechnology.

Fate of aniline and sulfanilic acid in UASB bioreactors under denitrifying conditions.

Two upflow anaerobic sludge blanket (UASB) reactors were operated to investigate the fate of aromatic amines under denitrifying conditions. The feed consisted of synthetic wastewater containing aniline and/or sulfanilic acid and a mixture of volatile fatty acids (VFA) as the primary electron donors. Reactor 1 (R1) contained a stoichiometric concentration of nitrate and Reactor 2 (R2) a stoichiometric nitrate and nitrite mixture as terminal electron acceptors. The R1 results demonstrated that aniline could be degraded under denitrifying conditions while sulfanilic acid remains. The presence of nitrite in the influent of R2, caused a chemical reaction that led to immediate disappearance of both aromatic amines and the formation of an intense yellow coloured solution. HPLC analysis of the influent solution, revealed the emergence of three product peaks: the major one at retention time (R(t)) 14.3 min and two minor at R(t) 17.2 and 21.5 min. In the effluent, the intensity of the peaks at R(t) 14.3 and 17.2 min was very low and of that at R(t) 21.5 min increased (∼3-fold). Based on the mass spectrometry analysis, we propose the structures of some possible products, mainly azo compounds. Denitrification activity tests suggest that biomass needed to adapt to the new coloured compounds, but after a 3 days lag phase, activity is recovered and the final (N(2) + N(2)O) is even higher than that of the control.

UV/TiO2 Photocatalytic Degradation of Xanthene Dyes

UV/titanium dioxide (TiO2) degradation of two xanthene dyes, erythrosine B (Ery) and eosin Y (Eos), was studied in a photocatalytic reactor. Photocatalysis was able to degrade 98% of Ery and 73% of Eos and led to 65% of chemical oxygen demand removal. Experiments in buffered solutions at different initial pH values reveal the pH dependence of the process, with better results obtained under acidic conditions due to the electrostatic attraction caused by the opposite charges of TiO2 (positive) and of anionic dyes (negative). Batch activity tests under methanogenic conditions showed the high toxicity exerted by the dyes even at low concentrations (~85% with initial concentration of 0.3 mmol L−1), but the end products of photocatalytic treatment were much less toxic toward methanogenic bacteria, as detoxification of 85 ± 5% for Eos and 64 ± 7% for Ery were obtained. In contrast, the dyes had no inhibitory effect on the biogenic‐carbon biodegradation activity of aerobic biomass, obtained by respirometry. The results demonstrate that photocatalysis combining UV/TiO2 as a pretreatment followed by an anaerobic biological process may be promising for the treatment of wastewaters produced by many industries.

Effect of different carbon materials as electron shuttles in the anaerobic biotransformation of nitroanilines

Aromatic amines resulted from azo dyes biotransformation under anaerobic conditions are generally recalcitrant to further anaerobic degradation. The catalytic effect of carbon materials (CM) on the reduction of azo dyes is known and has been confirmed in this work by increasing threefold the biological reduction rate of Mordant Yellow 1 (MY1). The resulting m‐nitroaniline (m‐NoA) was further degraded to m‐phenylenediamine (m‐Phe) only in the presence of CM. The use of CM to degraded anaerobically aromatic amines resulted from azo dye reduction was never reported before. In the sequence, we studied the effect of different CM on the bioreduction of o‐, m‐, and p‐NoA. Three microporous activated carbons with different surface chemistry, original (AC0), chemical oxidized with HNO3 (ACHNO3), and thermal treated (ACH2), and three mesoporous carbons, xerogels (CXA and CXB) and nanotubes (CNT) were assessed. In the absence of CM, NoA were only partially reduced to the corresponding Phe, whereas in the presence of CM, more than 90% was converted to the corresponding Phe. ACH2 and AC0 were the best electron shuttles, increasing the rates up to eightfold. In 24 h, the biological treatment of NoA and MY1 with AC0, decreased up to 88% the toxicity towards a methanogenic consortium, as compared to the non‐treated solutions. Biotechnol. Bioeng. 2016;113: 1194–1202.

Ciprofloxacin wastewater treated by UVA photocatalysis: contribution of irradiated TiO2 and ZnO nanoparticles on the final toxicity as assessed by Vibrio fischeri

Photocatalysis has become an attractive process to treat wastewater since it allows a rapid and efficient degradation of micropollutants in water. A solution of ciprofloxacin (CIP) was photocatalytically treated by ultraviolet A light (UVA) and titanium dioxide (TiO2) or zinc oxide (ZnO) nanoparticles. Toxicity of CIP and of the treated CIP solutions, as well as the toxicity of TiO2 and ZnO irradiated nanoparticles, was evaluated towards Vibrio fischeri. The lowest concentration of CIP tested, 10 μg L−1, leads to 50% of luminescence inhibition. Regarding irradiated nanoparticles, ZnO presented higher bacteria luminescence inhibition than TiO2, 97 and 38%, respectively. Due to high toxicity of ZnO, it was only possible to evaluate the CIP solution treated by UVA/TiO2. Initially, the toxicity decreased with the time of the process, but after 15 min the toxicity increased significantly (55%) and after 45 min of treatment, was 70%. High-performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR) analysis proved that the initial decrease of toxicity was caused by CIP adsorption on catalyst surface, which latter increased due to the generation of by-products and toxicity contribution of soluble nanoparticles. Ten by-products were identified by liquid chromatography-mass spectrometry (LC-MS) and the mechanism of CIP photocatalytic degradation was proposed.

UV/Tio2 photocatalytic reactor for real textile wastewaters treatment

Textile dye wastewaters are characterized by strong colour, salts and other additives, high pH, temperature, chemical oxygen demand (COD) and biodegradable materials. Being aesthetically and environmentally unacceptable, these wastewaters need to be treated before their discharge. Anaerobic bioprocesses have been proposed as being environmentally friendly and relatively cheap; however, when applied to real effluent with a complex composition, they can fail. In this study, a photoreactor combining UV light and TiO2, immobilized in cellulosic fabric, was applied for the treatment of two industrial textile wastewaters. High colour and COD removal, and detoxification, were achieved for both wastewaters, at controlled pH of 5.5. Effluents showed very poor biodegradability due to their complex composition; thus, the proposed process is an efficient alternative.