Thelmo A. Lú-Chau

Fungal pretreatment: An alternative in second-generation ethanol from wheat straw

The potential of a fungal pretreatment combined with a mild alkali treatment to replace or complement current physico-chemical methods for ethanol production from wheat straw has been investigated. Changes in substrate composition, secretion of ligninolytic enzymes, enzymatic hydrolysis efficiency and ethanol yield after 7, 14 and 21 days of solid-state fermentation were evaluated. Most fungi degraded lignin with variable selectivity degrees, although only eight of them improved sugar recovery compared to untreated samples. Glucose yield after 21 days of pretreatment with Poria subvermispora and Irpex lacteus reached 69% and 66% of cellulose available in the wheat straw, respectively, with an ethanol yield of 62% in both cases. Conversions from glucose to ethanol reached around 90%, showing that no inhibitors were generated during this pretreatment. No close correlations were found between ligninolytic enzymes production and sugar yields.

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.

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.

Review of solid state fermentation for lignocellulolytic enzyme production: challenges for environmental applications

Within the context of increasing environmental concern, energy production from lignocellulosic substrates is gaining great interest. Enzymes have proven their efficiency in the degradation of the lignocellulosic complex but their use remains limited in environmental applications such as anaerobic digestion mainly due to their prohibitive cost. Therefore, solid state fermentation (SSF) emerges as an interesting alternative for the in situ production of lignocellulolytic enzymes. Various research efforts on the lab scale optimization of SSF are discussed. They are presented according to the type of inoculum used in the process: bacterial species and fungal species under both mesophilic and thermophilic conditions. In general, parameters that impact the SSF process include: substrate type and particle size, substrate pretreatment, inoculum, nutrient supplementation, moisture content, pH, aeration, temperature and mixing. Using different substrates, authors aim at maximizing enzyme production taking into account one to several of the indicated operational parameters. The reviewed research puts forward the adaptation of the operational parameters, enzyme production cost and loading, enzyme mixture quality and efficiency and finally reactor design as the main challenges for environmental large-scale application.

Alkali treatment of fungal pretreated wheat straw for bioethanol production

Abstract Bioconversion of lignocellulosic materials into ethanol requires an intermediate pretreatment step for conditioning biomass. Sugar yields from wheat straw were previously improved by the addition of a mild alkali pretreatment step before bioconversion by the white-rot fungus Irpex lacteus. In this work, an alternative alkaline treatment, which significantly reduces water consumption, was implemented and optimized. Sugar recovery increased 117% with respect to the previously developed alkaline wash process at optimal process conditions (30°C, 30 minutes and 35.7% (w/w) of NaOH). In order to further reduce operational costs, a system for alkali recycling was implemented. This resulted in the treatment of 150% more wheat straw using the same amount of NaOH. Finally, enzymatic hydrolysis was optimized and resulted in a reduction of enzyme dose of 33%.

Comprehensive investigation of the enzymatic oligomerization of esculin by laccase in ethanol : water mixtures

The enzymatic polymerization of phenolic compounds arouses increasing interest due to the production of derivatives with improved biological activity. The reaction yield, the molecular mass, the structure and the properties of synthesized polymers can be controlled by the reaction conditions such as solvent and type of enzyme and substrate. In this study, the oxidative oligomerization of esculin by laccase from Trametes versicolor was performed in the presence of ethanol, a biocompatible co-solvent for food and nutraceutical applications. The formation of a precipitate was associated with the oligomerization reaction except for the medium with 50% (v/v) ethanol, due to the low reaction yield. The evaluation of antioxidant activity of the monomer and products showed that the pellet fraction from the reaction with esculin at 2 g L−1 in acetate buffer led to the highest activities. The presence of esculin oligomers was confirmed by MALDI-TOF analysis, which identified a repetition unit of 338 Da with a degree of polymerization up to 9 as well as other oligomers, mainly in the pellet fraction, with a repetition unit of 176 Da which are attributed to be esculetin oligomers. Additionally, size exclusion chromatography (SEC) and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the products.

Review of lignocellulolytic enzyme activity analyses and scale-down to microplate-based assays

With the increasing use of enzymes in environmental applications, there is a need for analytical methods adapted to large factorial experiments. Existing reference methods are chemical and labor intensive and unsuitable to analyze in parallel a large number of samples. Based on an extensive literature review and on experimental results, this work compares reference and microplate adapted methods to define the most adequate filter paper, carboxymethylcellulase, β-glucosidase and xylanase activity tests. In the adapted methods, the total reaction volume was reduced from 2.2-24.5 mL to 0.21-0.24 mL. Statistical analysis of the activities measured on enzyme mixtures by applying the 96-well plate reduced methods showed that they were not significantly different to the activities obtained with reference tests.

Application of flow cytometry for monitoring the production of poly(3‐hydroxybutyrate) by Halomonas boliviensis

In this study, a flow cytometry (FC) protocol was implemented to measure poly(3‐hydroxybutyrate) (PHB) content in a halophilic bacterium, to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB. A clear correlation between the PHB concentration determined by GC and the fluorescence signal obtained from stained bacteria by using FC was observed. Additionally, the heterogeneity of bacterial population as a function of PHB content was measured.

Application of Fungal Pretreatment in the Production of Ethanol From Crop Residues

Abstract The fungal pretreatment of lignocellulosic biomass for the production of ethanol is a low-cost, safe, and environmentally friendly process that can be used as an alternative to the more energy-demanding physicochemical pretreatments. It is based on the special features of white-rot fungi (WRF), which have the ability to degrade lignin by using extracellular ligninolytic enzymes. Fungal pretreatment has the advantage of selectively removing lignin and increase the digestibility of cellulose and hemicellulose at mild operational conditions, without producing inhibitors. Fungal pretreatment has been applied to different crop residues for producing ethanol, demonstrating that it is capable of handling feedstocks of variable origin. However, the integration of the fungal pretreatment in a biorefinery needs to be adapted to the special characteristics of WRF, to take full advantage of its great potential and to overcome the inherent drawbacks