Second-generation ethanol production from wheat straw: fungi, fungal enzymes, and applications

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 19/07/2013La paja de trigo es una materia prima barata que no compite con los recursos alimentarios y una alternativa para producir combustibles de segunda generación (2G) como el bioetanol. Para el aprovechamiento de los azúcares de este residuo son imprescindibles tres etapas: pretratamiento, hidrólisis enzimática y fermentación de los azucares a etanol. En este contexto, el objetivo de este trabajo fue buscar alternativas para mejorar diferentes etapas del proceso de producción de etanol 2G a partir de paja de trigo. En primer lugar se muestrearon 21 hongos basidiomicetos en condiciones de fermentación en estado sólido (SSF), durante 3 semanas de incubación, para evaluar si el biopretratamiento puede ser una alternativa a los tratamientos físico-químicos. Sólo 6 hongos mejoraron la recuperación de azúcares fermentables respecto de la paja no biopretratada. La especie más eficiente fue Irpex lacteus, que rindió valores de recuperación de xilosa del 62% y de glucosa del 66% con una conversión a etanol del 90%. Para optimizar estos rendimientos, I. lacteus se sometió a diferentes condiciones de SSF. Sólo la adición de sales de Mn2+ mejoró la recuperación de glucosa (un 6%). Los estudios de secretómica revelaron por qué I. lacteus es tan eficiente durante el biopretratamiento, permitieron describir las enzimas implicadas en el proceso y identificar una nueva peroxidasa decoloradora de tintes (DyP). La enzima pura fue muy estable a pH ácido, altas temperaturas y elevadas concentraciones de H2O2, y además mejoró significativamente la digestibilidad de la celulosa de paja de trigo al suplementarla al cóctel de hidrólisis enzimática. Con otro fin biotecnológico, las enzimas del crudo de I. lacteus y la peroxidasa versátil de Pleurotus eryngii también fueron capaces de polimerizar moléculas como lignanos, péptidos, proteínas y arabinoxilanos feruloilados, generando productos con propiedades nuevas o diferentes.Wheat straw is a cheap raw material which does not compete with food resources, constituting an alternative to produce second-generation (2G) biofuels such as bioethanol. To obtain the fermentable sugars from this residue, three steps are required: pretreatment, enzymatic hydrolysis, and sugars fermentation to ethanol. In this context, the aim of this work was to search alternatives to improve different process steps in 2G ethanol production from wheat straw. First of all, 21 basidiomycetes fungi were screened in solid state fermentation conditions (SSF), during 3 weeks of incubation, to evaluate if biopretreatment could be an alternative to physico-chemical pretreatments. Only 6 fungi increased the sugar yields compared to the non-biopretreated wheat straw. The most efficient species was Irpex lacteus, rendering a xylose yield of 62% and glucose yield of 66% with a 90% conversion to ethanol. With the objective of enhancing these yields, I. lacteus was subjected to different SSF conditions. Only Mn2+ addition increased glucose recoveries (6%). Secretomic studies revealed why I. lacteus was the most efficient fungus during the biopretreatment, allowed describing the enzymes implicated in that process, and identifying a new dye-decolorizing peroxidase (DyP). The purified enzyme was very stable to acid pH, high temperatures, and H2O2 concentrations. In addition, DyP increased significantly the cellulose digestibility of wheat straw when it was supplemented to the enzyme hydrolysis cocktail. Concerning other biotechnological applications, the crude´s enzymes of I. lacteus and the versatile peroxidase of Pleurotus eryngii were also capable of cross-linking diverse molecules such as lignans, peptides, proteins, and feruloylated arabinoxylans, generating products with new or different properties.Fac. de Ciencias BiológicasTRUEunpu

Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment

BACKGROUND: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases. RESULTS: In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H(2)O(2) producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases. CONCLUSION: The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis

Promoting microbial utilization of phenolic substrates from bio-oil

The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70 wt% Tween® 20 and 30 wt% Span® 80. Pseudomonas putida KT2440 grew to an optical density (OD600) of 1.0 ± 0.2 when supplied with 1.6 wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002 g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization

Un enfoque multiómico permite entender cómo Pleurotus eryngii transforma el material lignocelulósico no leñoso

1 p.Pleurotus eryngii es un hongo de prados y pastizales de interés biotecnológico por su capacidad para transformar el material lignocelulósico no leñoso. En este estudio combinamos análisis transcriptómicos, exoproteómicos y metabolómicos con objeto de ofrecer una explicación sobre los aspectos enzimáticos relacionados con la degradación de la paja de trigo. Durante la fase temprana de crecimiento encontramos un conjunto de enzimas extracelulares inducidas y constitutivas formado por glicosil hidrolasas, polisacárido liasas y carbohidrato esterasas activas sobre polisacáridos,lacasas activas sobre lignina, y una cantidad sorprendente de aril-alcohol oxidasas (AAOs). A tiempos largos identificamos una mayor diversidad y abundancia de enzimas, representada por oxidorreductasas implicadas en la despolimerización de celulosa y lignina, muchas de ellas inducidas desde la fase temprana de crecimiento. Estas enzimas oxidativas incluyeron monooxigenasas líticas de polisacáridos (LPMOs), celobiosa deshidrogenasa implicada en la activación de las LPMOs, y peroxidasas ligninolíticas (principalmente manganeso peroxidasas), junto a una gran abundancia de AAOs productoras de H2O2. Algunas de las enzimas más relevantes activas sobre polisacáridos aparecieron unidas a módulos de unión a celulosa. Esto se relacionó con el hábitat de P. eryngii.También elucidamos aspectos del catabolismo intracelular de compuestos aromáticos, un tema poco investigado en los basidiomicetos degradadores de lignina. Este enfoque multiómico revela que, aunque la descomposición de la paja de trigo no se traduce en grandes cambios (de acuerdo con análisis de 2D-NMR, entre otros), se produce la activación de enzimas hidrolíticas y oxidativas de gran interés biotecnológico en procesos dirigidos al aprovechamiento de la biomasa vegetal.Proyectos GENOBIOREF (BIO2017-86559-R), MICINN (cofinanciado con fondos FEDER); PIE-202120E019 y PIE-201620E081, CSIC; y contratos DE-AC02-05CH11231 y DE-AC36-08GO28308, U.S. DOEPeer reviewe

Producción de etanol de segunda generación a partir de paja de trigo: hongos, enzimas fúngicas y aplicaciones

La paja de trigo es una materia prima barata que no compite con los recursos alimentarios y una alternativa para producir combustibles de segunda generación (2G) como el bioetanol. Para el aprovechamiento de los azúcares de este residuo son imprescindibles tres etapas: pretratamiento, hidrólisis enzimática y fermentación de los azucares a etanol. En este contexto, el objetivo de este trabajo fue buscar alternativas para mejorar diferentes etapas del proceso de producción de etanol 2G a partir de paja de trigo. En primer lugar se muestrearon 21 hongos basidiomicetos en condiciones de fermentación en estado sólido (SSF), durante 3 semanas de incubación, para evaluar si el biopretratamiento puede ser una alternativa a los tratamientos físico-químicos. Sólo 6 hongos mejoraron la recuperación de azúcares fermentables respecto de la paja no biopretratada. La especie más eficiente fue Irpex lacteus, que rindió valores de recuperación de xilosa del 62% y de glucosa del 66% con una conversión a etanol del 90%. Para optimizar estos rendimientos, I. lacteus se sometió a diferentes condiciones de SSF. Sólo la adición de sales de Mn2+ mejoró la recuperación de glucosa (un 6%). Los estudios de secretómica revelaron por qué I. lacteus es tan eficiente durante el biopretratamiento, permitieron describir las enzimas implicadas en el proceso y identificar una nueva peroxidasa decoloradora de tintes (DyP). La enzima pura fue muy estable a pH ácido, altas temperaturas y elevadas concentraciones de H2O2, y además mejoró significativamente la digestibilidad de la celulosa de paja de trigo al suplementarla al cóctel de hidrólisis enzimática. Con otro fin biotecnológico, las enzimas del crudo de I. lacteus y la peroxidasa versátil de Pleurotus eryngii también fueron capaces de polimerizar moléculas como lignanos, péptidos, proteínas y arabinoxilanos feruloilados, generando productos con propiedades nuevas o diferentes

Microbial biotechnology in the effort to end hunger

Comisión Interministerial de Ciencia y Tecnología PID20211234690BI0

Microbial biotechnology in the effort to end hunger

Comisión Interministerial de Ciencia y Tecnología, (Grant/Award Number: 'PID20211234690BI00'

Characterization of a Novel Dye-Decolorizing Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat Straw

9 páginas, 7 figuras, 4 tablas -- PAGS nros. 4316-4324(2013)Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H2O2 concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (kcat/Km of 1.6 × 106 s-1 M-1) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzymeThis work has been carried out with funding from the Spanish project PRI-PIBAR-2011-1402 and EU FP7 project Peroxicats (KBBE-2010-4-265397). D.S. thanks the Spanish Ministry of Economy for a FPU fellowshipPeer reviewe

Mixed Carboxylic Acid Production by Megasphaera elsdenii from Glucose and Lignocellulosic Hydrolysate

Volatile fatty acids (VFAs) can be readily produced from many anaerobic microbes and subsequently utilized as precursors to renewable biofuels and biochemicals. Megasphaera elsdenii represents a promising host for production of VFAs, butyric acid (BA) and hexanoic acid (HA). However, due to the toxicity of these acids, product removal via an extractive fermentation system is required to achieve high titers and productivities. Here, we examine multiple aspects of extractive separations to produce BA and HA from glucose and lignocellulosic hydrolysate with M. elsdenii. A mixture of oleyl alcohol and 10% (v/v) trioctylamine was selected as an extraction solvent due to its insignificant inhibitory effect on the bacteria. Batch extractive fermentations were conducted in the pH range of 5.0 to 6.5 to select the best cell growth rate and extraction efficiency combination. Subsequently, fed-batch pertractive fermentations were run over 230 h, demonstrating high BA and HA concentrations in the extracted fraction (57.2 g/L from ~190 g/L glucose) and productivity (0.26 g/L/h). To our knowledge, these are the highest combined acid titers and productivity values reported for M. elsdenii and bacterial mono-cultures from sugars. Lastly, the production of BA and HA (up to 17 g/L) from lignocellulosic sugars was demonstrated

Structures of wall heterogalactomannans isolated from three genera of entomopathogenic fungi

9 páginas, 5 figuras, 4 tablas -- PAGS nros. 862-870O-linked heterogalactomannans with similar structural features have been purified from the fungal walls of the entomopathogenic fungi Lecanicillium muscarium, Beauveria bassiana, Beauveria brongniartii, and Cordyceps sphingum. Their composition and structure have been determined using acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear magnetic resonance spectroscopy (NMR). All structures have an α-(1 → 6)-mannose backbone, but one of the two strains of L. muscarium included in this study contained an acidic heterogalactomannan instead of the neutral polysaccharide isolated in the rest of the species analyzed. Sequence analysis of the internal transcribed spacer (ITS) region of this strain indicated that it belongs to the related genus Simplicillium, displaying low identity (83 %) with the closest Lecanicillium species. This is a new demonstration of the structural diversity of fungal wall heteromannans and validates their interest as chemotaxonomic markers. The production of a pullulan-like extracellular polysaccharide in strain CBS 413.70C of L. muscarium is also reportedThis work was supported by Grants DGI-CTQ2006-10874-C02-01 and 02 from Ministerio de Educación y Ciencia of Spain and CTQ-2009-10547 from Spanish Ministerio de Ciencia e Innovación (MICINN)Peer reviewe