Susana Rodríguez-Couto

Uses of Laccases in the Food Industry

Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in the last decades due to their ability to oxidise both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes, including the food industry. Thus, laccases hold great potential as food additives in food and beverage processing. Being energy-saving and biodegradable, laccase-based biocatalysts fit well with the development of highly efficient, sustainable, and eco-friendly industries.

Transformation pathway of Remazol Brilliant Blue R by immobilised laccase

This study deals with the biotransformation products obtained from the transformation of the anthraquinonic dye Remazol Brilliant Blue R (RBBR) by immobilised laccase from the white-rot fungus Trametes pubescens. A decolouration percentage of 44% was obtained in 42h. RBBR transformation products were investigated using ultraviolet-visible (UV-vis) spectrum scan and High Performance Liquid Chromatography/Mass Spectrometry (LC-MS) analysis. Two compounds were identified as the transformation intermediates (m/z 304.29 and m/z 342.24) and other two as the final transformation products (m/z 343.29 and m/z 207.16). As a result a metabolic pathway for RBBR transformation by laccase was proposed. No backward polymerisation of the transformation products resulting in recurrent colouration was observed after laccase treatment of RBBR. It was also found that the biotransformation products of RBBR showed less phytotoxicity than the dye itself.

Cost analysis in laccase production

In this paper the cost of producing the enzyme laccase by the white-rot fungus Trametes pubescens under both submerged (SmF) and solid-state fermentation (SSF) conditions was studied. The fungus was cultured using more than 45 culture medium compositions. The cost of production was estimated by analyzing the cost of the culture medium, the cost of equipment and the operating costs. The cost of the culture medium represented, in all cases, the highest contribution to the total cost, while, the cost of equipment was significantly low, representing less than 2% of the total costs. The cultivation under SSF conditions presented a final cost 50-fold lower than the one obtained when culturing under SmF conditions at flask scale. In addition, the laccase production under SSF conditions in tray bioreactors reduced the final cost 4-fold compared to the one obtained under SSF conditions at flask scale, obtaining a final price of 0.04 cent €/U.

Removal of synthetic dyes by an eco‐friendly strategy

In this paper an eco‐friendly strategy for the removal of synthetic dyes from aqueous solutions was proposed. Thus, in a first step the dyes were adsorbed onto sunflower seed shells (SS) by using a batch technique. Subsequently, in a second step, these dyed SS were used as support‐substrates to produce laccase by the white‐rot fungus Trametes pubescens under semi‐solid‐state conditions. The effect of inducer addition on laccase production was studied. The optimum conditions (addition of both 0.5 mM Cu+2 and 50 μM tannic acid on the 3rd day of cultivation) led to a maximum laccase activity of 30272 U/L. Further, the system was efficiently scaled‐up to laboratory bioreactors producing a maximum activity of 40172 U/L together with a total decolouration of the adsorbed dye. In addition, SDS‐PAGE showed that laccases were the only enzymes present in the extracellular fluid. Therefore, apart from dye removal this approach allowed the production of high titres of laccase, which was obtained almost pure (only needed to be ultra‐filtrated).

Biodegradation pathway and detoxification of the diazo dye Reactive Black 5 by Phanerochaete chrysosporium

The in vivo biodegradation of the diazo dye Reactive Black 5 (RB5) by Phanerochaete chrysosporium immobilised on cubes of nylon sponge and on sunflower-seed shells (SS) in laboratory-scale bioreactors was investigated. The SS cultivation led to the best results with a decolouration percentage of 90.3% in 72 h for an initial RB5 concentration of 100 mg/L. It was found that the addition of 0.4 mM veratryl alcohol (VA) into the medium considerably increased the decolouration rate in SS cultivation. However, the addition of VA had no effect in the nylon cultivation. Thin layer chromatography (TLC) revealed that RB5 was transformed into one metabolite after 24 h. UV-vis spectroscopy and Fourier Transform Infrared (FT-IR) also confirmed the biodegradation of RB5. Toxicity of RB5 solutions before and after fungal treatment was assayed using Sinorhizobium meliloti as a sensitive soil microorganism. P. chrysosporium transformed the toxic dye RB5 into a non-toxic product.

A promising inert support for laccase production and decolouration of textile wastewater by the white-rot fungus Trametes pubescesns

Cubes of nylon sponge, cubes of polyurethane foam (PUF), cuttings of stainless steel sponges and the commercial carriers Kaldnes™ K1 were tested as inert supports for laccase production by the white-rot fungus Trametes pubescens under semi-solid-state fermentation conditions. The cultures operating with Kaldnes™ K1 led to the highest laccase activity (3667 U/l). In addition this support could be re-utilised, making the whole process more economical. Subsequently, the decolouration of simulated textile wastewater (STW) by T. pubescens grown on the different tested supports under semi-solid-state fermentation conditions was studied. Decolouration percentages around 66-80% were obtained in 96 h. It was found that STW decolouration was due to two mechanisms: laccase action (biodegradation) and adsorption onto fungal mycelium, save for the PUF cultures in which decolouration was mainly due to adsorption onto the support. Further, the decolouration of STW by Kaldnes™ K1 cultures in three successive batches of 96 h each was studied. Decolouration percentages of 51.3, 70.0 and 69.8%, were attained for each batch, respectively.

Design of Stable and Powerful Nanobiocatalysts, Based on Enzyme Laccase Immobilized on Self-Assembled 3D Graphene/Polymer Composite Hydrogels

Graphene-based materials appear as a suitable answer to the demand for novel nanostructured materials for effective nanobiocatalytic systems design. In this work, a design of stable and efficient nanobiocatalysts made of enzyme laccase immobilized on composite hydrogels [reduced graphene oxide (rGO)/polymer] is presented. The composite hydrogel supports were synthesized by self-assembly of graphene oxide nanoplatelets in the frame of a polymer latex matrix, where the polymer nanoparticles were adsorbed onto the GO surface, creating hybrid nanoplatelets. These hybrids self-assembled when ascorbic acid was added as a GO reducing agent and formed three-dimensional porous structures, greatly swollen with water, e.g., the composite hydrogels. The hydrogels were used as a support for covalent immobilization of the laccase. The performance of the nanobiocatalysts was tested in the oxidative degradation of the recalcitrant synthetic dye Remazol Brilliant Blue R in aqueous solutions. The biocatalysts showed strong dye discoloration ability and high stability as they preserved their catalytic action in four successive batches of dye degradation. The presented biocatalysts offer possibilities for overcoming the main disadvantages of the enzyme catalysts (fragile nature, high cost, and high loading of the enzyme), which would lead to a step forward toward their industrial application.

Production of laccase and decolouration of the textile dye Remazol Brilliant Blue R in temporary immersion bioreactors

Laccase production by Trametes pubescens grown on sunflower-seed shells (SS) under solid-state fermentation (SFF) conditions in temporary immersion bioreactors was studied. Three immersion cycles were considered: 1 min immersed and 9 min non-immersed, 1 min immersed and 30 min non-immersed and 1 min immersed and 60 min non-immersed. The latter led to the highest laccase activities (4000-6000 Ul(-1)). Also, the in vitro and in vivo decolouration of the recalcitrant textile dye Remazol Brilliant Blue R (RBBR) was assessed. It was found that RBBR (133.33 mg l(-1)) was efficiently decolourised by T. pubencens grown on SS under SSF conditions in temporary immersion bioreactors in five successive batches. The percentage of RBBR decolouration was higher than 55% in 4h and around 70% in 24h in all the batches. However, it was found that RBBR decolouration by the crude culture filtrates was more advantageous. Thus, an RBBR decolouration percentage of nearly 80% in 2h was obtained.

Morphology and laccase production of white‐rot fungi grown on wheat bran flakes under semi‐solid‐state fermentation conditions

In this paper, we studied the laccase production and the growth morphology of different white-rot fungi, i.e. Pleurotus ostreatus, Trametes pubescens, Cerrena unicolor and Trametes versicolor, cultured under semi-solid-state fermentation conditions using wheat bran flakes as a natural low-cost support substrate. Trametes versicolor exhibited the highest laccase activity per gram of total dry matter, followed by P. ostreatus (63.5 and 58.2Ug(-1) , respectively). In addition, they showed a time profile of laccase production that was quite similar. Growth morphology was studied using environmental microscopic images and analyzed by discrete Fourier transformation-based software to determine the mean diameter of the hyphae, the number of hypha layers and the global micromorphology. The four strains exhibited different micromorphologies of growth. Pleurotus ostreatus presented narrow hyphae, which formed many thick clumps, T. pubescens and T. versicolor showed clumps of different sizes and C. unicolor showed thick hyphae that formed larger clumps, but in less amounts.

Biodegradation of bisphenol A by the newly-isolated Enterobacter gergoviae strain BYK-7 enhanced using genetic manipulation

Endogenous bacterial strains possessing a high bisphenol A (BPA)-tolerance/degradation activity were isolated from different outlets of petrochemical wastewater in Iran using the enrichment cultivation approach. Two bacterial isolates with high efficiency for BPA degradation in basal medium and petrochemical wastewater were identified as Enterobacter gergoviae strain BYK-7 and Klebsiella pneumoniae strain BYK-9 using morphology, 16s rDNA analysis and MALDI-TOF mass spectrometry systems. Due to the pathogenicity of K. pneumoniae, the E. gergoviae strain was selected for further studies. This strain with very high BPA tolerance (up to 2000 mg L−1) degraded 23.10 ± 0.126 mg L−1 BPA in basal medium, 31.35 ± 4.05 mg L−1 BPA in petrochemical wastewater and 53.50 ± 0.153 mg L−1 BPA in nutritious medium within 8, 72 and 48 h, respectively. Biostimulation by mineral salts and ethanol was effective in the BPA-degradation activity of the E. gergoviae. In addition, recombinant E. gergoviae [pBRbisd] was able to degrade 45.02 ± 0.334 mg L−1 BPA in basal medium within 48 h. These results point out this strain as a very promising organism for BPA removal in industrial wastewater.