J. Peter Jones

Utilization of cross‐linked laccase aggregates in a perfusion basket reactor for the continuous elimination of endocrine‐disrupting chemicals

A perfusion basket reactor (BR) was developed for the continuous utilization of insolubilized laccase as cross‐linked enzyme aggregates (CLEAs). The BR consisted of an unbaffled basket made of a metallic filtration module filled with CLEAs and continuously agitated by a 3‐blade marine propeller. The agitation conditions influenced both the apparent laccase activity in the reactor and the stability of the biocatalyst. Optimal laccase activity was obtained at a rotational speed of 12.5 rps and the highest stability was reached at speeds of 1.7 rps or lower. The activity and stability of the biocatalyst were affected drastically upon the appearance of vortices in the reaction medium. This reactor was used for the continuous elimination of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA), and triclosan (TCS). Optimization of EDC elimination by laccase CLEAs as a function of temperature and pH was achieved by response surface methodology using a central composite factorial design. The optimal conditions of pH and temperature were, respectively, 4.8 and 40.3°C for the elimination of p353NP (a branched isomer of NP), 4.7 and 48.0°C for BPA, and 4.9 and 41.2°C for TCS. Finally, the BR was used for the continuous elimination of these EDCs from a 5 mg L−1 aqueous solution using 1 mg of CLEAs at pH 5 and room temperature. Our results showed that at least 85% of these EDCs could be eliminated with a hydraulic retention time of 325 min. The performances of the BR were quite stable over a 7‐day period of continuous treatment. Furthermore, this system could eliminate the same EDCs from a 100 mg L−1 solution. Finally, a mathematical model combining the Michaelis–Menten kinetics of the laccase CLEAs and the continuous stirred tank reactor behavior of the BR was developed to predict the elimination of these xenobiotics. Biotechnol. Bioeng. 2009;102: 1582–1592.

Laccase immobilization and insolubilization: from fundamentals to applications for the elimination of emerging contaminants in wastewater treatment

Over the last few decades many attempts have been made to use biocatalysts for the biotransformation of emerging contaminants in environmental matrices. Laccase, a multicopper oxidoreductase enzyme, has shown great potential in oxidizing a large number of phenolic and non-phenolic emerging contaminants. However, laccases and more broadly enzymes in their free form are biocatalysts whose applications in solution have many drawbacks rendering them currently unsuitable for large scale use. To circumvent these limitations, the enzyme can be immobilized onto carriers or entrapped within capsules; these two immobilization techniques have the disadvantage of generating a large mass of non-catalytic product. Insolubilization of the free enzymes as cross-linked enzymes (CLEAs) is found to yield a greater volume ratio of biocatalyst while improving the characteristics of the biocatalyst. Ultimately, novel techniques of enzymes insolubilization and stabilization are feasible with the combination of cross-linked enzyme aggregates (combi-CLEAs) and enzyme polymer engineered structures (EPESs) for the elimination of emerging micropollutants in wastewater. In this review, fundamental features of laccases are provided in order to elucidate their catalytic mechanism, followed by different chemical aspects of the immobilization and insolubilization techniques applicable to laccases. Finally, kinetic and reactor design effects for enzymes in relation with the potential applications of laccases as combi-CLEAs and EPESs for the biotransformation of micropollutants in wastewater treatment are discussed.

Decolorization Of Textile Dye Solutions

Results are presented on the use of ozone to decolorize textile dye solutions. The results describe the rates of reaction and the stoichiometry for the use of ozone to decolorize a simulated wastewater containing a bisazo acid dye (Acid Red 158). These rates of reaction are not sensitive to pH and are only mildly affected by temperature. The effects of a combination of an uncolored organic compound on the rates of reaction were investigated because if ozone reacts preferentially with uncolored compounds, its use might not be economical. Guar gum used in the textile industry as an aid in dyeing carpets increases the consumption of ozone by 20–60% for the conditions studied and has a small effect on the reaction rate. The competition found is not severe enough to make ozone treatment uneconomical.

Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters.

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3 U/g for laccase and 167.4 U/g for tyrosinase, exhibited high enzymatic activity at pH5-8 and temperature at 5-30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis-Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in order to evaluate the potential efficiency of the biocatalyst. In batch mode the combi-CLEA transformed more than 80% to nearly 100% of acetaminophen from the municipal wastewater and more than 90% from the hospital wastewater. UPLC-MS analysis of acetaminophen metabolites showed the formation of its oligomers as dimers, trimers and tetramers due to the laccase and 3-hydroxyacetaminophen due to the tyrosinase.

Laccase-Based CLEAs: Chitosan as a Novel Cross-Linking Agent

Laccase from Coriolopsis Polyzona was insolubilized as cross-linked enzyme aggregates (CLEAs) for the first time with chitosan as the cross-linking agent. Concentrations between 0.01 and 1.867 g/L of chitosan were used and between 0.05 and 600 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The laccase was precipitated using ammonium sulphate and cross-linked simultaneously. Specific activity and thermal stability of these biocatalysts were measured. Activities of up to 737 U/g were obtained when 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used as a substrate. Moreover, the stability of these biocatalysts was improved with regards to thermal degradation compared to free laccase when exposed to denaturing conditions of high temperature and low pH. The CLEAs stability against chemical denaturants was also tested but no significant improvement was detected. The total amount of ABTS to be oxidized during thermal degradation by CLEAs and free laccase was calculated and the insolubilized enzymes were reported to oxidize more substrate than free laccase. The formation conditions were analyzed by response surface methodology in order to determine an optimal environment for the production of efficient laccase-based CLEAs using chitosan as the cross-linking agent. After 24 hours of formation at pH 3 and at 4°C without agitation, the CLEAs exhibit the best specific activity.

Hybrid bioreactor (HBR) of hollow fiber microfilter membrane and cross-linked laccase aggregates eliminate aromatic pharmaceuticals in wastewaters

Widespread detection of numerous micropollutants including aromatic pharmaceuticals in effluents of wastewater treatment plants has prompted much research aimed at efficiently eliminating these contaminants of environmental concerns. In the present work, a novel hybrid bioreactor (HBR) of cross-linked enzymes aggregates of laccase (CLEA-Lac) and polysulfone hollow fiber MF membrane was developed for the elimination of acetaminophen (ACT), mefenamic acid (MFA) and carbamazepine (CBZ) as model aromatic pharmaceuticals. The MF alone showed removals of the three drugs varying approximately from 50 to 90% over the course of 8h in the filtrate of aqueous solution. Synergistic action of the MF and CLEA-Lac during operation achieved eliminations from aqueous solution of around 99%, nearly 100% and up to 85% for ACT, MFA and CBZ, respectively. Under continuous operation, the HBR demonstrated elimination rates of the drugs from filtered wastewater up to 93% after 72h for CBZ and near complete elimination of ACT and MFA was achieved within 24h of treatment. Concomitantly to the drugs eliminations in the wastewater, the CLEA-Lac exhibited 25% residual activity while being continuously recycled with no activity in the filtrate. Meanwhile, the filtrate flowrate showed only minor decline indicating limited fouling of the membrane.

Elimination of Bisphenol A and Triclosan Using the Enzymatic System of Autochthonous Colombian Forest Fungi

Bisphenol A (BPA) and triclosan (TCS) are known or suspected potential endocrine disrupting chemicals (EDCs) which may pose a risk to human health and have an environmental impact. Enzyme preparations containing mainly laccases, obtained from Ganoderma stipitatum and Lentinus swartzii, two autochthonous Colombian forest white rot fungi (WRF), previously identified as high enzyme producers, were used to remove BPA and TCS from aqueous solutions. A Box-Behnken factorial design showed that pH, temperature, and duration of treatment were significant model terms for the elimination of BPA and TCS. Our results demonstrated that these EDCs were extensively removed from 5 mg L−1 solutions after a contact time of 6 hours. Ninety-four percent of TCS and 97.8% of BPA were removed with the enzyme solution from G. stipitatum; 83.2% of TCS and 88.2% of BPA were removed with the L. swartzii enzyme solution. After a 6-hour treatment with enzymes from G. stipitatum and L. swartzii, up to 90% of the estrogenic activity of BPA was lost, as shown by the yeast estrogen screen assay. 2,2-Azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) was used as a mediator (laccase/mediator system) and significantly improved the laccase catalyzed elimination of BPA and TCS. The elimination of BPA in the absence of a mediator resulted in production of oligomers of molecular weights of 454, 680, and 906 amu as determined by mass spectra analysis. The elimination of TCS in the same conditions produced dimers, trimers, and tetramers of molecular weights of 574, 859, and 1146 amu. Ecotoxicological studies using Daphnia pulex to determine lethal concentration (LC50) showed an important reduction of the toxicity of BPA and TCS solutions after enzymatic treatments. Use of laccases emerges thus as a key alternative in the development of innovative wastewater treatment technologies. Moreover, the exploitation of local biodiversity appears as a potentially promising approach for identifying new efficient strains for biotechnological applications.

Control of methanol vapours in a biotrickling filter: performance analysis and experimental determination of partition coefficient.

Methanol vapours were treated in a biotrickling filter (BTF) packed with inert polypropylene spheres. The effects of the nitrogen concentration in the nutrient solution, the empty bed residence time (EBRT) and the methanol inlet concentration, on the BTF performance, were all examined. The elimination capacity (EC), the biomass and the carbon dioxide production rates were all increased with the rising of the nitrogen concentration and the EBRT. The EC also rose with increasing methanol inlet load (IL) when the methanol inlet concentration and the EBRT were varied, from 0.3 to 37.0 g m(-3), and from 20 to 65 s, respectively. The BTF reached its maximum EC level of 2160 g m(-3) h(-1) when it was operated at an IL level of 3700 g m(-3) h(-1). The input methanol was removed through two mechanisms: biodegradation and absorption in the liquid phase. The partition coefficient for the methanol in the BTF was determined at five EBRTs and along the packed bed. It generally followed the Henry model, having an average value of 2.64 x 10(-4)[mol L(-1)](gas)/[mol L(-1)](liquid).

Analysis and comparison of biotreatment of air polluted with ethanol using biofiltration and biotrickling filtration.

This study analyses the performance of ethanol biofiltration with percolation (biotrickling filtration, BTF) comparing to a conventional biofilter (biofiltration, BF). Two biofilters packed with clay balls were operated in a range of inlet concentrations of ethanol in the air varying from 0.47 to 2.36 g m−3. For both the BF and BTF, the specific growth rate (μ) and the elimination capacity (EC) decreased with the ethanol inlet concentration, presenting a kinetic of substrate inhibition. A Haldane-type model was adjusted for both biofilters in order to model both EC and μ as a function of the ethanol inlet concentration in the gas. The maximum EC was similar for both biofilters, at around 46 g m−3 h−1, whereas the maximum μ was 0.0057 h−1 for the BF and 0.0103 h−1 for the BTF. The maximum of ethanol removed, occurned at the lowest inlet concentration of (0.47 g m−3), and reached 86% for the BF and 74% for the BTF.

Solubility of methane in pure non-ionic surfactants and pure and mixtures of linear alcohols at 298 K and 101.3 kPa

The emissions of methane (CH4), a powerful greenhouse gas (GES), contribute to the increase in GES concentration level in the atmosphere. For this reason, the importance of controlling CH4 emissions of anthropogenic origin has increased over the last decades. Physicochemical and biological processes are available for treating CH4. For this reason, such properties as the solubility of CH4 in aqueous solutions and organic solvents are of great relevance in different applications in environmental engineering and biotechnology. In this study, the solubility of CH4 was determined at 298 K and 101.3 kPa in organic solvents, such as polyoxyethylenesorbates (Tween 20, Tween 40, and Tween 60), and linear alcohols (methanol, ethanol, and butan-1-ol) alone and in their admixtures. Admixtures of methanol with butan-1-ol exhibited the highest solubility of CH4, of around 0.49 g m−3 of solvent, whereas the solubility of CH4 in linear alcohols varied from 0.167 g m−3 to 0.41 g m−3 of solvent. In the case of Tweens, CH4 solubility decreased with the hydrophilic-lipophilic balance (HLB) number.