M. Ángeles Sanromán

Recent developments and applications of immobilized laccase

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.

Screening of supports and inducers for laccase production by Trametes versicolor in semi-solid-state conditions

Abstract The production of laccase by Trametes versicolor under semi-solid-state conditions has been studied. Several supports (polyurethane foam, wheat straw, barley straw, wood shavings and barley bran) have been tested in order to determine the most suitable for laccase production by the above-mentioned microorganism. Barley bran led to the highest activity levels, reaching maximum values of about 1200 U/l. In this stage of research, several factors affecting laccase production (veratryl alcohol, xylidine, fresh support and the initial C/N ratio) were investigated. Xylidine was shown to be the best inducer of laccase activity, attaining values of about 1700 U/l. Moreover, the addition of fresh support not only prolonged culture lifetime but also enhanced activity levels, reaching in all the cases values higher than 2000 U/l. In addition, the decolourisation of three structurally different dyes by cultures grown on the best operating conditions determined in the present study, was monitored in order to assess the degrading capability of the ligninolytic complex secreted by such cultures. The decolourisation of all the dyes tested was almost total (85–96%) after 6 days of incubation.

PAHs soil decontamination in two steps: Desorption and electrochemical treatment

The presence of carcinogenic polycyclic aromatic hydrocarbons (PAHs) in soils poses a potential threat to human health if exposure levels are too high. Nevertheless, the removal of these contaminants presents a challenge to scientists and engineers. The high hydrophobic nature of PAHs enables their strong sorption onto soil or sediments. Thus, the use of surfactants could favour the release of sorbed hydrophobic organic compounds from contaminated soils. In this work, five surfactants, namely Brij 35, Tergitol NP10, Tween 20, Tween 80 and Tyloxapol, are evaluated on the desorption of PAHs [benzanthracene (BzA), fluoranthene (FLU), and pyrene (PYR), single and in mixture] from a model sample such as kaolin. In all cases, the best results were obtained when Tween 80 was employed. In order to obtain the global decontamination of PAHs, their electrochemical degradation is investigated. It is concluded that the order of increasing degradation for single compounds is BzA>FLU>PYR when they are subject to the same electrochemical treatment. In addition, there is a direct relationship between the ionization potential and the electrochemical degradation of PAH.

Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27

In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.

Impact of ionic liquids on extreme microbial biotypes from soil

This work aims at identifying, amongst extreme soil biotypes at locations of high salinity and high hydrocarbon load, microbial strains able to survive short or long-term exposure to the presence of selected ionic liquids. We have evaluated the impact of ionic liquids on the diversity of the soil microbiota to identify which microbial strains have higher survival rates towards ionic liquids, and consequently those which might possibly play a major role in their biotic fate. To the best of our knowledge, this is the first study of this kind. Soils, from a region in Portugal (Aveiro) were sampled and the bacterial and fungal strains able to survive after exposure to high concentrations of selected ionic liquids were isolated and further characterised. We have mainly focused on two types of cations: imidazolium – the most commonly used; and cholinium – generally perceived as benign. The surviving microbial strains were isolated and taxonomically identified, and the ionic liquid degradation was analysed during their cultivation. The continuing exposure of the microbial strains to petroleum hydrocarbons is likely to be the basis for their acquired resistance to some imidazolium salts; also, the higher capacity of fungi – compared to bacteria – to grow, even during their exposure to these liquid salts, became evident in this study.

Production of Thermostable Lipolytic Activity by Thermus Species

A quantitative screening for intra‐ and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.

Removal of PAHs and pesticides from polluted soils by enhanced electrokinetic-Fenton treatment.

In this study, electrokinetic-Fenton treatment was used to remediate a soil polluted with PAHs and the pesticide pyrimethanil. Recently, this treatment has emerged as an interesting alternative to conventional soil treatments due to its peculiar advantages, namely the capability of treating fine and low-permeability materials, as well as that of achieving a high yield in the removals of salt content and inorganic and organic pollutants. In a standard electrokinetic-Fenton treatment, the maximum degradation of the pollutant load achieved was 67%, due to the precipitation of the metals near the cathode chamber that reduces the electro-osmotic flow of the system and thus the efficiency of the treatment. To overcome this problem, different complexing agents and pH control in the cathode chamber were evaluated to increase the electro-osmotic flux as well as to render easier the solubilization of the metal species present in the soil. Four complexing agents (ascorbic acid, citric acid, oxalic acid and ethylenediaminetetraacetic acid) in the Fenton-like treatment were evaluated. Results revealed the citric acid as the most suitable complexing agent. Thereby its efficiency was tested as pH controller by flushing it in the cathode chamber (pH 2 and 5). For the latter treatments, near total degradation was achieved after 27 d. Finally, phytotoxicity tests for polluted and treated samples were carried out. The high germination levels of the soil treated under enhanced conditions concluded that nearly complete restoration was achieved.

On the hunt for truly biocompatible ionic liquids for lipase-catalyzed reactions

One of the challenges in the field of biocatalysis is the search for efficient reaction media avoiding enzyme deactivation. This work shows for the first time that water + hydrophilic ionic liquids mixtures enhance (up to 50%) the enzymatic activity of lipases, opening new opportunities for enzyme extraction and biocatalysis.

Effective heterogeneous electro-Fenton process of m-cresol with iron loaded actived carbon

The degradation of m-cresol (MC) has been investigated by a heterogeneous electro-Fenton process using iron loaded activated carbon (Fe-AC) as the heterogeneous electro-Fenton catalyst. Experimental results demonstrated that MC was effectively removed through an electro-Fenton process. Calculated TOC removal and overall energy consumption showed that the use of a low iron concentration (28 mg L−1) increases the efficiency of the process. The reactions followed a pseudo-first order kinetic equation and kinetic coefficients confirm that the MC reduction, when it is alone, is faster than in the presence of a similar compound, tert-butylhydroquinone (TBHQ) (from 0.0935 to 0.0692 min−1); therefore TBHQ exerts an antioxidative protection effect. In all cases, it is concluded that heterogeneous electro-Fenton treatment with Fe-AC follows a two-step process: adsorption and oxidation; allowing removal rates higher than in the literature. In addition, the reusability of this catalyst was demonstrated by operating it in continuous mode. Finally, LC-MS analysis allowed the development of a plausible degradation route.

Enhanced selective metal adsorption on optimised agroforestry waste mixtures

The aim of this work is to ascertain the potentials of different agroforestry wastes to be used as biosorbents in the removal of a mixture of heavy metals. Fern (FE), rice husk (RI) and oak leaves (OA) presented the best removal percentages for Cu(II) and Ni(II), Mn(II) and Zn(II) and Cr(VI), respectively. The performance of a mixture of these three biosorbents was evaluated, and an improvement of 10% in the overall removal was obtained (19.25mg/g). The optimum mixture proportions were determined using simplex-centroid mixture design method (FE:OA:RI=50:13.7:36.3). The adsorption kinetics and isotherms of the optimised mixture were fit by the pseudo-first order kinetic model and Langmuir isotherm. The adsorption mechanism was studied, and the effects of the carboxylic, hydroxyl and phenolic groups on metal-biomass binding were demonstrated. Finally, the recoveries of the metals using biomass were investigated, and cationic metal recoveries of 100% were achieved when acidic solutions were used.