René Carmona

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.

Simultaneous detection of the absorption spectrum and refractive index ratio with a spectrophotometer: monitoring contaminants in bioethanol

The optical properties of a biofuel resulting from the fungi-treated lignocellulosic biomass in an ethanol matrix were studied. The matrix simulates the case that the bioethanol is contaminated by sugars, water and colour pigments that reduce the quality of the biofuel and compromise the combustion process. It is suggested that by applying a spectrophotometer only, it is possible to obtain valid information, i.e. the spectral features of the contaminants as well as the refractive index ratio of bioethanol. This allows for simultaneous purity and density detection of biomass-derived liquids or liquid biofuels, in comparison to a reference representing an ideal bioethanol (pure ethyl alcohol, ethanol of 99.5% purity (v/v)).

Bioenergy II: Biological Pretreatment with Fungi as a Tool for Improvement of the Enzymatic Saccharification of Eucalyptus globulus Labill to Obtain Bioethanol

This study is focused on the effect of the application of biological pretreatment of Eucalyptus globulus Labill wood pieces on the cellulose digestibility by depolymerizing enzymes. Wood chips were incubated with five different white-rot fungi (WRF) for 30, 45 and 60 days at 25°C. The effect of the fungal action was determined as weight losses, changes in chemical composition and released sugars in the wood chips. Enzymatic hydrolysis was conducted in a discontinuous reactor at 37°C, using a commercial cellulase preparation from Trichoderma reesei. The enzyme/substrate ratio was 0.04 g/g. The highest weight losses were obtained using Stereum hirsutum and Lentinus edodes for all the incubation times, reaching 27.2% and 25.8% at 60 days, respectively. The lowest weight losses were produced by Coriolus versicolor and Pleurotus ostreatus, reaching 6.0 and 9.1%, respectively after 60 days. For all the tested fungi, the yield of reducing sugars and glucose increased significantly over the untreated controls, with Stereum hirsutum producing after 30 days pretreatment the best values (140 mM reducing sugars and 53 mM glucose); after this time no additional increments were observed. Pre-treated wood pieces showed changes in chemical composition in comparison to control samples. Total extractable substances contents are higher in wood pieces subjected to fungal action. However, lignin and alpha-cellulose values are lower in comparison to control samples. Some net holocellulose consumption could be observed, mainly during pretreatment with S. hirsutum by 45 and 60 days. Altogether these results support the potential of Eucalyptus globulus pre-treatment with S. hirsutum and L. edodes by periods not longer than 30 days, as a tool to increase the wood accessibility to depolymerizing action of hydrolytic enzymes. This preliminary study contributes to the identification of fungal pretreatment conditions for more effective cellulose degradation, a vital step in the utilization of monomer sugars process from cellulose to produce ethanol. Additional analysis of the solid and liquid fractions after saccharification is necessary to complement this information.

Evaluation of the Lignolytic Effect of the White‐Rot Fungi Ceriporiopsis Sp, Pleurotus Sp, and Phlebia Sp on Industrial Pinus Radiata Logs

Abstract Pretreatment of pinus Radiata logs with the white‐rot fungi Ceriporiopsis sp (9C), Pleurotus sp (9P), and Phlebia sp (24P), under field conditions was studied. The lignin content was evaluated by gravimetric techniques (Klason lignin), FTIR spectroscopy, and gas chromatography. A control sample and samples treated for 90 days with each of the fungi were analyzed. The ability of the fungi to degrade the lignin was in the order Phlebia sp (24P) > Ceriporiopsis sp (9C) > Pleurotus sp (9P). The authors thank FONDEF for financial support through research grant project No. D97I‐2032.