Gemma Eibes

Degradation of estrogens by laccase from Myceliophthora thermophila in fed-batch and enzymatic membrane reactors

Several studies reported that natural and synthetic estrogens are the major contributors to the estrogenic activity associated with the effluents of wastewater treatment plants. The ability of the enzyme laccase to degrade these compounds in batch experiments has been demonstrated in previous studies. Nevertheless, information is scarce regarding in vitro degradation of estrogens in continuous enzymatic bioreactors. The present work constitutes an important step forward for the implementation of an enzymatic reactor for the continuous removal of estrone (E1) and estradiol (E2) by free laccase from Myceliophthora thermophila. In a first step, the effect of the main process parameters (pH, enzyme level, gas composition (air or oxygen) and estrogen feeding rate) were evaluated in fed-batch bioreactors. E1 and E2 were oxidized by 94.1 and 95.5%, respectively, under the best conditions evaluated. Thereafter, an enzymatic membrane reactor (EMR) was developed to perform the continuous degradation of the estrogens. The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane, which allowed the recovery of enzyme while both estrogens and degradation products could pass through it. The highest removal rates at steady state conditions were up to 95% for E1 and nearly complete degradation for E2. Furthermore, the residual estrogenic activity of the effluent was largely reduced up to 97%.

Immobilization of Laccase by Encapsulation in A Sol-Gel Matrix and Its Characterization and Use for the Removal of Estrogens

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol–gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2–22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5–17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10–30% and tolerance to different inactivating agents (NaN3, ZnCl2, CoCl2, CaCl2, methanol, and acetone) was 20–40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed‐bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10–15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17β‐estradiol (E2), and 17α‐ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady‐state conditions. In addition, a 63% decrease in estrogenic activity was detected.

Continuous operation of a fluidized bed reactor for the removal of estrogens by immobilized laccase on Eupergit supports.

The feasibility of the operation of a fluidized bed reactor for the removal of estrogens by immobilized laccase was investigated in order to improve the degradation yields and enzyme stability previously obtained with packed bed reactors. High removal levels (between 76 and 90%) and significantly prolonged stability of the biocatalyst over 16 days were attained. In parallel, a decrease up to 90% in the estrogenic activity of the effluent was measured. Thus, the technology presented seems a promising tool to increase the applicability of laccases in bioremediation processes.

Strategies for the design and operation of enzymatic reactors for the degradation of highly and poorly soluble recalcitrant compounds

The presence of recalcitrant compounds in both wastewaters and soils is an important environmental problem. Oxidative enzymes from white-rot fungi have been successfully utilised for the in vitro degradation of xenobiotics, such as the azo dye Orange II and the polycyclic aromatic hydrocarbon anthracene (compounds with high and low solubilities, respectively). Two different reactor configurations are proposed: (i) an enzymatic membrane reactor for the treatment of soluble compounds, consisting of a continuous stirred tank reactor coupled to an ultrafiltration membrane to facilitate the retention and recycling of enzyme; and (ii) a two-phase enzymatic reactor for the degradation of poorly soluble compounds, consisting of an immiscible solvent, which contains the contaminant at high concentrations, and the aqueous phase containing the enzyme and cofactors involved in the catalytic cycle. In this paper, factors affecting the design and operation of both systems are discussed, and experimental results concerning the efficiency and stability of the processes are presented.

Enzymatic technologies for remediation of hydrophobic organic pollutants in soil.

Worldwide there are numerous contaminated sites as a result of the widespread production and use of chemicals in industrial and military activities as well as poor schemes of waste disposal and accidental spillages. The implementation of strategies for decontamination and restoration of polluted sites has become a priority, being bioremediation with biological agents a promising alternative. Enzyme-based technologies offer several advantages over the use of microbial cells, provided that the biocatalyst meets specific requirements: efficiency to remove the target pollutant/s, non-dependency on expensive coenzymes or cofactors, enzyme stability, and an affordable production system. In this mini-review, the direct application of enzymes for in situ soil bioremediation is explored, and also novel ex situ enzymatic technologies are presented. This new perspective provides a valuable insight into the different enzymatic alternatives for decontamination of soils. Examples of recent applications are reported, including pilot-scale treatments and patented technologies, and the principles of operation and the main requirements associated are described. Furthermore, the main challenges regarding the applicability of enzymatic technologies for remediation of hydrophobic organic pollutants from soil are discussed.

Application of response surface methodology to study the removal of estrogens in a laccase-mediated continuous membrane reactor

Abstract A three-level Box–Behnken factorial design combined with response surface methodology (RSM) was applied as a tool to study the laccase-catalyzed removal of three estrogenic compounds: estrone (E1), estradiol (E2), and ethinylestradiol (EE2), in a continuous enzymatic membrane reactor (EMR). Three main factors affecting the treatment efficiency were considered: enzyme activity, hydraulic residence time (HRT) and oxygenation rate. As expected, laccase activity and HRT showed large effects and, interestingly, the relevance of oxygen in improving the oxidation kinetics through raising the dissolved oxygen above saturation levels was demonstrated. When considering elimination rates as the response, optimal conditions were: 1,000 U/L of laccase, 1 h HRT and 60 mgO2/(L·h) of oxygenation rate, predicting 2.82–3.24 mg eliminated/(L·h), (71–81% of oxidation). These optimum conditions were successfully validated, and 75% of estrogenicity reduction was achieved. On the other hand, only 100 U/L were found as optimal to maximize the efficacy of the enzyme: E1 was oxidized by 0.06 mg/(L·h·U), although the removal of estrogenicity decreased to 60%. The methodology was also applied to maximize the reduction of estrogenic activity: the highest values assayed [1,000 U/L, HRT 4 h and 60 mgO2/(L·h)] provided 99% detoxification.

Biocatalytic Generation of Mn(III)-Chelate as a Chemical Oxidant of Different Environmental Contaminants

The objective of this study was to investigate the enzymatic generation of the Mn3+‐malonate complex and its application to the process of oxidizing several organic compounds. The experimental set‐up consisted of an enzymatic reactor coupled to an ultrafiltration membrane, providing continuous generation of Mn3+‐malonate from a reaction medium containing versatile peroxidase (an enzyme produced by Bjerkandera adusta strain BOS55), H2O2, MnSO4, and malonate. The effluent of the enzymatic reactor was introduced into a batch‐stirred reactor to oxidize three different classes of compounds: an azo dye (Orange II), three natural and synthetic estrogens, and a polycyclic aromatic hydrocarbon (anthracene). The enzymatic reactor provided the Mn3+ complex under steady‐state conditions, and this oxidative species was able to transform the three classes of xenobiotics considerably (90–99%) with negligible loss of activity.

Bifidobacterial growth stimulation by oligosaccharides generated from olive tree pruning biomass

This work aims to evaluate the prebiotic potential of oligosaccharides (OS) obtained from autohydrolysis of olive tree pruning biomass (OTPB). Two selected fractions (F1 and F2) were characterized and used in in vitro fermentations by two Bifidobacterium spp. (B. adolescentis and B. longum) and one fecal inoculum. The fraction F1 presented a lower average degree of polymerization (DP) mainly with OS ranging from 3 to 6 DP, whereas the fraction F2 corresponded to a pool of unsubstituted and acetylated oligomers with DP between 4 and 19. In the fermentation by Bifidobacterium, F1 supported a higher biomass formation, OS consumption and organic acids production than F2. With the fecal inoculum, the accumulation of organic acids, as the sum of acetate, propionate and butyrate, was similar for F1 and F2 (107 and 101mM, respectively). The bifidobacteria counts also increased during the incubation time for both OS fractions.

Continuous removal of endocrine disruptors by versatile peroxidase using a two‐stage system

The oxidant Mn3+‐malonate, generated by the ligninolytic enzyme versatile peroxidase in a two‐stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol‐A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17β‐estradiol, and 17α‐ethinylestradiol) were removed from wastewater at degradation rates in the range of 28–58 µg/L·min, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn3+‐malonate (hydraulic retention time as well as Na‐malonate and H2O2 feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3–8.8 mg/L) and environmental (1.2–6.1 µg/L) concentrations. Finally, when the two‐stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14‐fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (∼8 h). This study demonstrates the feasibility of the two‐stage enzymatic system for removing EDCs both at high and environmental concentrations.

Fostering the action of versatile peroxidase as a highly efficient biocatalyst for the removal of endocrine disrupting compounds.

Response surface methodology (RSM) was used to optimize the removal of five endocrine disrupting compounds (EDCs) by the enzyme versatile peroxidase (VP): bisphenol A (BPA), triclosan (TCS), estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). The optimal variables of enzyme activity (90-100 U L(-1)), sodium malonate (29-43 mM) and MnSO4 (0.8-1 mM) led to very high removal rates of the five pollutants (2.5-5.0 mg L(-1) min(-1)). The structural elucidation of transformation products arising from the enzymatic catalysis of the EDCs was investigated by Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Liquid Chromatography Electrospray Time-of-Flight Mass Spectrometry (LC-ESI-TOF-MS). The presence of dimers and trimers, indicative of oxidative coupling, was demonstrated.