Primera evidencia de actividad enzimática nitrilasa en Xylaria sp., y su relación con la biosíntesis de ácido indol-3-acético / First evidence of nitrilase enzymatic activity of Xylaria sp. and its relationship with the biosynthesis of indole-3-acetic acid

Resumen Muchos de los hongos degradadores de madera están implicados en la síntesis de metabolitos bioactivos de naturaleza antimicrobiana y terapéutica, así como de compuestos de importancia biotecnológica, incluyendo derivados indólicos, entre otros. Estos hongos brindan ciertos beneficios ecológicos a las plantas, entre los que se destacan la protección contra fitopatógenos y la promoción del crecimiento radicular. Xylaria sp. es un hongo degradador de celulosa (lignocelulolítico) con potencial biotecnológico. El ácido indol-3-acético (AIA) desempeña un papel sumamente importante en las interacciones planta-microorganismo, ya que es esencial para la fisiología y el correcto desarrollo morfológico vegetal. Se sabe que las enzimas nitrilo-hidrolíticas (nitrilasas) están involucradas en la síntesis de compuestos indólicos en las plantas, no obstante, se dispone de poca información acerca de la naturaleza de estas enzimas en el reino de los hongos. A través de una aproximación bioquímica y de genética molecular, se demuestra por primera vez que Xylaria sp. posee actividad enzimática nitrilasa utilizando compuestos ricos en nitrógeno y carbono como sustrato. La cepa estudiada aumentó sus niveles de expresión génica relativa y mostró crecimiento micelial, ambos en presencia de compuestos químicos como cianobenceno y KCN. Los resultados de este trabajo sugieren que el microorganismo es capaz de degradar moléculas nitrogenadas complejas. Por otra parte, mediante biofertilización con extractos fúngicos, se observó que Xylaria sp. promueve el desarrollo del sistema radicular de plántulas de Arabidopsis thaliana, además de sintetizar AIA.

Abstract Endophytic fungi inhabit plant tissues internally and asymptomatically, and many of them are involved in the synthesis of bioactive metabolites of antifungal and therapeutic nature, as well as other compounds of biotechnological importance including indole derivatives, among many others. Ecologically, they provide some benefits to plants including protection against phy-topathogens and promotion of root growth. In this sense, Xylaria sp. is a cellulose-decomposing fungus with biotechnological potential. It is worth mentioning that indole-3-acetic acid (IAA) also plays an extremely important role in plant-micro-organism interactions, as it is essential for physiology and proper plant morphological development. It is known that nitrile-hydrolytic enzymes (nitrilases) are involved in the synthesis of plant indole compounds; however, relatively little information is available concerning the nature of these enzymes in the fungal kingdom. In view of the above, through a biochemical and molecular-genetic approach, it has been demon-strated for the first time that Xylaria sp. carries out nitrile-hydrolytic enzyme activity using nitrogen and carbonrich compounds as substrate. The studied strain increased its relative gene expression levels and showed mycelial growth, both in the presence of chemical compounds such as cyanobenzene and KCN. Thus, the results of this work suggest that the micro-organism is capable of degrading complex nitrogenous molecules. On the other hand, through fungal biofertilization, it was observed that Xylaria sp. promotes the development of the root system of Arabidopsis thaliana seedlings, in addition to synthesizing IAA.

[First evidence of nitrilase enzymatic activity of Xylaria sp. and its relationship with the biosynthesis of indole-3-acetic acid]. / Primera evidencia de actividad enzimática nitrilasa en Xylaria sp., y su relación con la biosíntesis de ácido indol-3-acético.

Endophytic fungi inhabit plant tissues internally and asymptomatically, and many of them are involved in the synthesis of bioactive metabolites of antifungal and therapeutic nature, as well as other compounds of biotechnological importance including indole derivatives, among many others. Ecologically, they provide some benefits to plants including protection against phytopathogens and promotion of root growth. In this sense, Xylaria sp. is a cellulose-decomposing fungus with biotechnological potential. It is worth mentioning that indole-3-acetic acid (IAA) also plays an extremely important role in plant-micro-organism interactions, as it is essential for physiology and proper plant morphological development. It is known that nitrile-hydrolytic enzymes (nitrilases) are involved in the synthesis of plant indole compounds; however, relatively little information is available concerning the nature of these enzymes in the fungal kingdom. In view of the above, through a biochemical and molecular-genetic approach, it has been demonstrated for the first time that Xylaria sp. carries out nitrile-hydrolytic enzyme activity using nitrogen and carbon-rich compounds as substrate. The studied strain increased its relative gene expression levels and showed mycelial growth, both in the presence of chemical compounds such as cyanobenzene and KCN. Thus, the results of this work suggest that the micro-organism is capable of degrading complex nitrogenous molecules. On the other hand, through fungal biofertilization, it was observed that Xylaria sp. promotes the development of the root system of Arabidopsis thaliana seedlings, in addition to synthesizing IAA.

Lignocellulolytic Biocatalysts: The Main Players Involved in Multiple Biotechnological Processes for Biomass Valorization.

Human population growth, industrialization, and globalization have caused several pressures on the planet's natural resources, culminating in the severe climate and environmental crisis which we are facing. Aiming to remedy and mitigate the impact of human activities on the environment, the use of lignocellulolytic enzymes for biofuel production, food, bioremediation, and other various industries, is presented as a more sustainable alternative. These enzymes are characterized as a group of enzymes capable of breaking down lignocellulosic biomass into its different monomer units, making it accessible for bioconversion into various products and applications in the most diverse industries. Among all the organisms that produce lignocellulolytic enzymes, microorganisms are seen as the primary sources for obtaining them. Therefore, this review proposes to discuss the fundamental aspects of the enzymes forming lignocellulolytic systems and the main microorganisms used to obtain them. In addition, different possible industrial applications for these enzymes will be discussed, as well as information about their production modes and considerations about recent advances and future perspectives in research in pursuit of expanding lignocellulolytic enzyme uses at an industrial scale.

Solid-State Fermentation of Steam-Exploded Opuntia ficus-indica Cladodes Using Trichoderma reesei CCT-2768 for the Production of Cellulolytic Enzymes.

The sustainable development of the drylands, i.e., regions with limited availability of water, depends on the exploitation of the few biomass types that can thrive in such conditions, such as the Opuntia ficus-indica, a plant of the Cactaceae family. In the present study, the cladodes of O. ficus-indica were used as a substrate by the fungus Trichoderma reesei CCT-2768 for the production of cellulolytic enzymes through solid-state fermentation. Firstly, the extraction of the mucilage, soluble components of industrial interest, was evaluated. Temperature, water-to-biomass ratio, and time of mixture were varied using an experimental design and impacted, especially, the pectin removal. Then, the lignocellulosic residue was used for the production of enzymes; the effect of the water activity, biomass pretreatment, mineral supplementation, temperature, and inoculum size on the enzymatic production were investigated using two sets of experimental designs. The steam explosion pretreatment exposed the fiber to the microbial action and boosted the enzyme production, provided that the medium was supplemented with salts. This combination has improved the production of xylanase, CMCase, FPase, and polygalacturonase by 27, 62, 98, and 185%, respectively. The temperature of 35 °C was determined as the optimal for the production of FPase, xylanase, and polygalacturonase, while no effect was observed on the production of CMCase and ß-glucosidase. The optimization of the enzymatic production performed in this study can potentially provide a new application for the Opuntia biomass and improve the sustainable development of the drylands.

Analysis of carbohydrate-active enzymes and sugar transporters in Penicillium echinulatum: A genome-wide comparative study of the fungal lignocellulolytic system.

Penicillium echinulatum 2HH is an ascomycete well known for its production of cellulolytic enzymes. Understanding lignocellulolytic and sugar uptake systems is essential to obtain efficient fungi strains for the production of bioethanol. In this study we performed a genome-wide functional annotation of carbohydrate-active enzymes and sugar transporters involved in the lignocellulolytic system of P. echinulatum 2HH and S1M29 strains (wildtype and mutant, respectively) and eleven related fungi. Additionally, signal peptide and orthology prediction were carried out. We encountered a diverse assortment of cellulolytic enzymes in P. echinulatum, especially in terms of ß-glucosidases and endoglucanases. Other enzymes required for the breakdown of cellulosic biomass were also found, including cellobiohydrolases, lytic cellulose monooxygenases and cellobiose dehydrogenases. The S1M29 mutant, which is known to produce an increased cellulase activity, and the 2HH wild type strain of P. echinulatum did not show significant differences between their enzymatic repertoire. Nevertheless, we unveiled an amino acid substitution for a predicted intracellular ß-glucosidase of the mutant, which might contribute to hyperexpression of cellulases through a cellodextrin induction pathway. Most of the P. echinulatum enzymes presented orthologs in P. oxalicum 114-2, supporting the presence of highly similar cellulolytic mechanisms and a close phylogenetic relationship between these fungi. A phylogenetic analysis of intracellular ß-glucosidases and sugar transporters allowed us to identify several proteins potentially involved in the accumulation of intracellular cellodextrins. These may prove valuable targets in the genetic engineering of P. echinulatum focused on industrial cellulases production. Our study marks an important step in characterizing and understanding the molecular mechanisms employed by P. echinulatum in the enzymatic hydrolysis of lignocellulosic biomass.

Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared from Secretomes of Filamentous Fungi Isolated from Amazonian Biodiversity.

The use of lignocellulosic biomass (LCB) has emerged as one of the main strategies for generating renewable biofuels. For the efficient use of such feedstock, pre-treatments are essential. The hydrolysis of cellulose - major component of LCB - demands enzymatic cocktails with improved efficiency to generate fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzyme platforms. In this study, two enzymatic cocktails were developed for hydrolysis of two lignocellulosic biomasses: industrial cellulose pulp and cassava peel. The solid biomass ratio in relation to the protein content of the enzyme cocktail was performed by experimental design. The optimized cocktail for the hydrolysis of cellulose pulp (AMZ 1) was composed, in protein base, by 43% of Aspergillus sp. LMI03 enzyme extract and 57% of T. reesei QM9414, while the optimal enzyme cocktail for cassava peel hydrolysis (AMZ 2) was composed by 50% of Aspergillus sp. LMI03 enzyme extract, 25% of the extract of P. citrinum LMI01 and 25% of T. reesei. The ratio between solids and protein loading for AMZ 1 cocktail performance was 52 g/L solids and 30 mg protein/g solids, resulting in a hydrolytic efficiency of 93%. For the AMZ 2 cocktail, the hydrolytic efficiency was 78% for an optimized ratio of 78 g/L solids and 19 mg protein/g solids. These results indicate that cocktails formulated with enzymatic extracts of P. citrinum LMI01, Aspergillus sp. LMI03, and T. reesei QM9414 are excellent alternatives for efficient hydrolysis of plant biomass and for other processes that depend on biocatalysis.

An overview of Trichoderma reesei co-cultures for the production of lignocellulolytic enzymes.

Biorefineries are core facilities for implementing a sustainable circular bioeconomy. These facilities rely on microbial enzymes to hydrolyze lignocellulosic substrates into fermentable sugars. Fungal co-cultures mimic the process of natural biodegradation and have been shown to increase certain enzyme activities. Trichoderma reesei and its many mutant strains are major cellulase producers and are heavily utilized as a source of carbohydrate-active enzymes. Several reports have demonstrated that T. reesei co-cultures present higher enzyme activities compared with its monocultures, especially in the context of ß-glucosidase activity. The performance of T. reesei during co-culturing has been assessed with several fungal partners, including Aspergillus niger, one of the most recurrent partners. Various aspects of co-cultivation still need further investigation, especially regarding the molecular interactions between fungi in controlled environments and the optimization of the resulting enzyme cocktails. Since plenty of genetic and physiological data on T. reesei is available, the species is an outstanding candidate for future co-culture investigations. Co-cultures are still a developing field for industrial enzyme production, and many aspects of the technique need further improvement before real applications. KEY POINTS: • T. reesei co-cultures are an alternative for producing lignocellulolytic enzymes. • Several reports suggest an increase in certain enzyme activities in co-cultures. • More in-depth investigations of co-cultures are necessary for advancing this field.

Growth of Leucoagaricus gongylophorus Möller (Singer) and production of key enzymes in submerged and solid-state cultures with lignocellulosic substrates.

The aim of this study was to characterize the growth of the fungus Leucoagaricus gongylophorus LEU18496, isolated from the fungus garden of the nest of leaf cutter ants Atta mexicana. The fungus garden was cultivated in an artificial laboratory nest and the fungus further grown in submerged (SmC) and solid state (SSC) cultures with sugarcane bagasse, grass or model substrates containing CM-cellulose, xylan or lignin. The CO2 production rate with grass in SmC (Vmax 34.76 mg CO2 Lgas-1 day- 1) was almost four times than SSC (Vmax 9.49 mg CO2 Lgas-1 day- 1), while the production rate obtained in sugarcane bagasse in SmC (Vmax 16.02 mg CO2 Lgas-1 day- 1) was almost three times than that for SSC (Vmax 5.42 mg CO2 Lgas-1 day- 1). In addition, the fungus grew with defined carbon substrates mixtures in SmC, but at different rates, first xylan, followed by CM-cellulose and lignin. Endoglucanase and xylanase activities (U mgprotein-1) were detected in all cultures, the specific activity was higher in the fungus-garden, 5.2 and 1.8; followed by SSC-grass, 1.5 and 0.8, and SSC-bagasse, 0.9 and 0.8, respectively. Laccase activity in the fungus-garden was 44.8 U L- 1 and 10.9 U L- 1 in the SSC-grass. The gongylidia structures observed by environmental scanning electron microscopy were ca. 40 µm and the hyphae width ca. 5 µm. The results show that L. gongylophorus from A. mexicana have promising applications for the treatment of plant residues to release fermentable sugars and the production of high value lignocellulolytic enzymes such as endoglucanase, xylanase or laccases.

Evaluating Eucalyptus leaf colonization by Brasilonema octagenarum (Cyanobacteria, Scytonemataceae) using in planta experiments and genomics.

BACKGROUND: Brasilonema is a cyanobacterial genus found on the surface of mineral substrates and plants such as bromeliads, orchids and eucalyptus. B. octagenarum stands out among cyanobacteria due to causing damage to the leaves of its host in an interaction not yet observed in other cyanobacteria. Previous studies revealed that B. octagenaum UFV-E1 is capable of leading eucalyptus leaves to suffer internal tissue damage and necrosis by unknown mechanisms. This work aimed to investigate the effects of B. octagenarum UFV-E1 inoculation on Eucalyptus urograndis and to uncover molecular mechanisms potentially involved in leaf damage by these cyanobacteria using a comparative genomics approach. RESULTS: Leaves from E. urograndis saplings were exposed for 30 days to B. octagenarum UFV-E1, which was followed by the characterization of its genome and its comparison with the genomes of four other Brasilonema strains isolated from phyllosphere and the surface of mineral substrates. While UFV-E1 inoculation caused an increase in root and stem dry mass of the host plants, the sites colonized by cyanobacteria on leaves presented a significant decrease in pigmentation, showing that the cyanobacterial mats have an effect on leaf cell structure. Genomic analyses revealed that all evaluated Brasilonema genomes harbored genes encoding molecules possibly involved in plant-pathogen interactions, such as hydrolases targeting plant cell walls and proteins similar to known virulence factors from plant pathogens. However, sequences related to the type III secretory system and effectors were not detected, suggesting that, even if any virulence factors could be expressed in contact with their hosts, they would not have the structural means to actively reach plant cytoplasm. CONCLUSIONS: Leaf damage by this species is likely related to the blockage of access to sunlight by the efficient growth of cyanobacterial mats on the phyllosphere, which may hinder the photosynthetic machinery and prevent access to some essential molecules. These results reveal that the presence of cyanobacteria on leaf surfaces is not as universally beneficial as previously thought, since they may not merely provide the products of nitrogen fixation to their hosts in exchange for physical support, but in some cases also hinder regular leaf physiology leading to tissue damage.

Biotransformation of lignocellulosic biomass into industrially relevant products with the aid of fungi-derived lignocellulolytic enzymes.

Lignocellulosic material has drawn significant attention among the scientific community due to its year-round availability as a renewable resource for industrial consumption. Being an economic substrate alternative, various industries are reevaluating processes to incorporate derived compounds from these materials. Varieties of fungi and bacteria have the ability to depolymerize lignocellulosic biomass by synthesizing degrading enzymes. Owing to catalytic activity stability and high yields of conversion, lignocellulolytic enzymes derived from fungi currently have a high spectrum of industrial applications. Moreover, these materials are cost effective, eco-friendly and nontoxic while having a low energy input. Techno-economic analysis for current enzyme production technologies indicates that synthetic production is not commercially viable. Instead, the economic projection of the use of naturally-produced ligninolytic enzymes is promising. This approach may improve the economic feasibility of the process by lowering substrate expenses and increasing lignocellulosic by-product's added value. The present review will discuss the classification and enzymatic degradation pathways of lignocellulolytic biomass as well as the potential and current industrial applications of the involved fungal enzymes.

A simple and accurate method for specific quantification of biomass in mixed cultures of filamentous fungi by quantitative PCR / Un método simple y preciso para la cuantificación específica de biomasa en cultivos mixtos de hongos filamentosos por PCR cuantitativa

Abstract Production of lignocellulolytic enzymes by filamentous fungi have a great potential at industrial level due to their widespread applications. Mixed fungal cultures and particularly mixed fungal biofilms constitute a promising fermentation system for an enhanced enzyme production. However, it has not been addressed how much of this enhancement depends on the mixed biomass proportion. In this sense, the aim of this study was to develop a method to specifically and accurately quantify mixed fungal biomass. For this purpose, mixed biofilm cultures composed of Aspergillus niger and Trichoderma reesei, two filamentous fungi used industrially for cellulase production, were collected from 48 to 120 h of growth; mycelia were pulverized, and DNA was extracted for qPCR assays with specific primers for each fungus. Primers were designed from non-conserved regions of sequences of actin and β-tubulin genes of both A. niger and T. reesei. Specificity of these primers was tested in silico and experimentally. A statistically significant correlation was obtained between qPCR-calculated biomass and dry weight biomass data. By this method, it was possible to detect changes on mycelia proportions in biofilms over time, suggesting a competitive interaction between these two fungi. In conclusion, this method allows a specific and accurate quantification of mixed fungal biomass and could be also applied to different mixed culture systems for studying microbial interactions.

Resumen La producción de enzimas lignocelulolíticas por hongos filamentosos tiene un gran potencial a nivel industrial debido a sus diversas aplicaciones. Los cultivos fúngicos mixtos y particularmente las biopelículas fúngicas mixtas constituyen un sistema de fermentación prometedor para una mayor producción enzimática. Sin embargo, no se ha abordado cuánto de esta mejora depende de la proporción de biomasa mixta. En este sentido, el objetivo de este estudio fue desarrollar un método para cuantificar de forma específica y precisa la biomasa fúngica mixta. Para este propósito, se recolectaron cultivos mixtos de biopelículas de 48 a 120 h de crecimiento compuestos por Aspergillus niger y Trichoderma reesei, dos hongos filamentosos utilizados industrialmente para la producción de celulasas; el micelio se pulverizó y el ADN se extrajo para ensayos de qPCR con cebadores específicos para cada hongo. Los cebadores se diseñaron a partir de regiones no conservadas de las secuencias de los genes de actina y β-tubulina de A. niger y T. reesei. La especificidad de estos cebadores se probó in silico y experimentalmente. Se obtuvo una correlación estadísticamente significativa entre la biomasa calculada mediante qPCR y los datos de biomasa en peso seco. Mediante este método, fue posible detectar cambios en las proporciones de los micelios en las biopelículas a lo largo del tiempo, lo que sugiere una interacción competitiva entre estos dos hongos. En conclusión, este método permite una cuantificación específica y precisa de la biomasa fúngica mixta y también podría aplicarse a diferentes sistemas de cultivo mixto para estudiar interacciones microbianas.

High-quality draft genome sequence of a biofilm forming lignocellulolytic Aspergillus niger strain ATCC 10864.

Filamentous fungus Aspergillus niger has high industrial value due to their lignocellulolytic enzyme activities and ATCC 10864 is one of the few type strains of A. niger which has a unique biofilm forming capability. Here we report the first draft genome sequence of A. niger ATCC 10864 strain. The genome of A. niger ATCC 10864 is 36,172,237 bp long and comprise of 310 scaffolds with 49.5% average GC content. A total of 10,804 protein-coding genes were predicted among which 10,761 genes were with putative functions. A. niger ATCC 10864 genome coded for 709 putative carbohydrate active enzyme families distributed in six functional categories and among them glycoside hydrolases (GHs) represent the most number of families (279). Genes that include pepA, brlA, exgA, LaeA, rodA, GCN have also been identified in this study, which may play a role in biofilm formation. This high-quality draft genome sequence will facilitate our understanding of the mechanisms behind fungal biofilm formation and higher lignocellulolytic enzyme production.

Fungal decomposers of leaf litter from an invaded and native mountain forest of NW Argentina.

The impact of plant species invasions on the abundance, composition and activity of fungal decomposers of leaf litter is poorly understood. In this study, we isolated and compared the relative abundance of ligninocellulolytic fungi of leaf litter mixtures from a native forest and a forest invaded by Ligustrum lucidum in a lower mountain forest of Tucuman, Argentina. In addition, we evaluated the relationship between the relative abundance of ligninocellulolytic fungi and properties of the soil of both forest types. Finally, we identified lignin degrading fungi and characterized their polyphenol oxidase activities. The relative abundance of ligninocellulolytic fungi was higher in leaf litter mixtures from the native forest. The abundance of cellulolytic fungi was negatively related with soil pH while the abundance of ligninolytic fungi was positively related with soil humidity. We identified fifteen genera of ligninolytic fungi; four strains were isolated from both forest types, six strains only from the invaded forest and five strains were isolated only from the native forest. The results found in this study suggest that L. Lucidum invasion could alter the abundance and composition of fungal decomposers. Long-term studies that include an analysis of the nutritional quality of litter are needed, for a more complete overview of the influence of L. Lucidum invasion on fungal decomposers and on leaf litter decomposition.

Insights from the genome of a high alkaline cellulase producing Aspergillus fumigatus strain obtained from Peruvian Amazon rainforest.

Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil.

Flammulina velutipes: An option for "alperujo" use.

BACKGROUND: Two-phase olive-mill wastes (or "alperujo") exhibit highly phytotoxic properties, mainly due to phenols. A valuable option for alperujo is its agricultural use, provided that no phytotoxic effects occur. AIMS: The present investigation was aimed at evaluating the efficacy of two strains of the lignin-degrading fungus Flammulina velutipes to colonize alperujo in order to produce edible mushrooms and to achieve its detoxification. METHODS: Some important cultural characters related to mushroom production (earliness, biological efficiency and quality of basidiomes) were estimated. The production of lignocellulolytic enzymes, phenol removal and detoxification of the substrate was evaluated. RESULTS: High biological efficiencies (70.8%) were obtained at 12°C with F. velutipes strain BAFC 670/06 in a substrate containing poplar wood shavings and 90% of alperujo. The nature of the substrate did not seem to exert an important influence on pileus and stem morphology; nevertheless shortest stems were observed at higher temperatures. Endo-ß-1,4-glucanase, endo-ß-1,4-xylanase, laccase and Mn-peroxidase activities were detected in the extracts recovered from the solid-state cultures. Both F. velutipes strains were effective in removing the phenolic compounds. The initial concentration in the substrate with 90% alperujo was reduced in the case of F. velutipes BAFC 1763 by 84.31%, and 40.15% by F. velutipes BAFC 670/06. Germinability experiments on Raphanus sativus, showed that alperujo phytotoxicity was significantly reduced by F. velutipes cultures. CONCLUSIONS: The experimented changes by the spent mushroom substrate resulting from F. velutipes cultivation with high amount of alperujo would allow its reuse for agricultural purposes.

Production of lignocellulolytic enzymes from floriculture residues using Pleurotos ostreatus / Producción de enzimas lignocelulolíticas a partir de residuos de floricultura empleando Pleurotus ostreatus / Produçâo de enzimas lignoceluliticas a partir de residuos de floricultura utilizando Pleurotus ostreatus

Floriculture is a vital agro-industrial sector in the Colombian economy; the export of flowers positively impacts employment and the balance of trade. However, this industry could negatively impact the environment if its waste products are not handled properly. These flower residues, rich in lignin, hemicellulose and cellulose, could be a cost-effective raw material to produce enzymes. Here, we evaluate the production of lignocellulolytic enzymes by degradation of Chrysanthemum and Rosa residues using Pleurotus ostreatus, and manganese sulfate and copper sulfate as inductors. From the two residues, we obtained laccase, manganese peroxidase, endoglucanase, exoglucanase, and (3-glucosidase. The use of inductors, favored all enzyme activities except for (3-glucosidase. The enzymes that displayed the highest activity were laccase (4,693.4 U/L and 2,640 U/L from the residues of Chrysanthemum and Rosa, respectively) and (3-glucosidase (9,513 U/L and 6,811.9 U/L). The enzyme that showed the lowest activity was endoglucanase (11.5 U/L and 15.4 U/L). Under the conditions evaluated, the best substrate for enzyme production is Chrysanthemum wastes; the extracts obtained had higher enzymatic activity than the extracts from Rosa residues.

En Colombia la floricultura es un sector agro-industrial importante, con impactos positivos en el empleo y la balanza comercial. Sin embargo, tiene impacto negativo en el medio ambiente porque genera alto volumen de residuos. Estos residuos, ricos en lignina, hemicelulosa y celulosa, podrían ser una materia prima de bajo costo para la producción de enzimas. En este trabajo se estudió en la producción de enzimas lignocelulíticas por la degradación con Pleurotus ostreatus de residuos de Chrysanthemum y Rosa, usando como inductores sulfato de manganeso y de cobre. A partir de ambos residuos se obtuvieron lacasa, manganeso peroxidasa, endoglucanasa, exoglucanasa y p-glucosidasa. Los inductores favorecieron todas las actividades enzimáticas, excepto para p-glucosidasa. Las enzimas que tuvieron mayores actividades fueron lacasa (4,693.4 U/L y 2,640 U/L a partir del residuo de Chrysanthemum y Rosa, respectivamente) y p-glucosidasa (9,513 U/L y 6,811.9 U/L). La enzima que tuvo menor actividad fue endoglucanasa (11.5 U/L y 15.4 U/L). Bajo las condiciones evaluadas, el mejor residuo para producción de enzimas fue Chrysanthemum, porque los extractos tuvieron mayor actividad enzimática que los producidos a partir de Rosa.

Na Colombia, a floricultura è um importante setor da indùstria agrícola, com impactos positivos sobre o sector laboral na balança comercial. Além disto, tem impacto negativo sobre o meio ambiente, pois gera grandes volumes de resíduos. Estes resíduos, com altos conteùdos em lignina, hemicelulose e celulose, podem ser uma matèria-prima de baixo custo para a produçâo de enzimas. Neste traballio foi estudada a produçâo da enzimas lignoceluliticas pela degradaçâo com Pleurotus ostreatus de residuos de Chrysanthemum e Rosa, utilizando como indutores sulfatos de cobre e manganês. A partir destes resíduos foram obtidos lacase, manganês peroxidase, endoglucanase, exoglucanase e β-glicosidase. O uso de indutores favoreceu as atividades enzimáticas, exceto β-glicosidase. As enzimas que apresentaram atividades mais elevadas foram lacase (4,693.4 U/L e 2,640 U/L a partir do Chrysanthemum e Rosa, respetivamente) e β-glicosidase (9,513 U/L e 6,81.9 U/L). A enzima que apresentou menor atividade foi a endoglucanase (11.5 U/L e 15.4 U/L). Nas condiçôes testadas, o melhor resíduo para a produçâo da enzima foi Chrysanthemum, porque os extratos tinham uma atividade enzimática mais elevada que aqueles produzidos a partir de Rosa.

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils.

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, ß-d-glucosidase, cellobiohydrolase, ß-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated ß-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal-Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.(AU)

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Grape stalks as substrate for white rot fungi, lignocellulolytic enzyme production and dye decolorization / Uso del escobajo como sustrato para el crecimiento de hongos de la pudrición blanca, la producción de enzimas ligninolíticas y la decoloración de tinturas

The aim of this work was to evaluate the potential of grape stalks, an agroindustrial waste, for growth and lignocellulolytic enzyme production via solid-state fermentation, using the following three white rot fungi: Trametes trogii, Stereum hirsutum and Coriolus antarcticus. The decolorization of several dyes by the above mentioned cultures was also investigated. Similar values of dry weight loss of the substrate were measured after 60 days (33-43 %). C. antarcticus produced the highest laccase and Mn-peroxldase activities (33.0 and 1.6 U/g dry solid). The maximum endoglucanase production was measured in S. hirsutum cultures (10.4 U/g), while the endoxylanase peak corresponded to T. trogii (14.6 U/g). The C. antarcticus/grape stalk system seems potentially competitive in bioremediation of textile processing effluents, attaining percentages of decolorization of 93, 86, 82, 82, 77, and 58 % for indigo carmine, malachite green, azure B, remazol brilliant blue R, crystal violet and xylidine, respectively, in 5 h.(AU)

El objetivo de este trabajo fue evaluar el potencial del escobajo, un residuo agroindustrial, como sustrato para el crecimiento y la producción de enzimas lignocelulósicas de tres hongos causantes de pudrición blanca en la madera: Trametes trogii, Stereum hirsutum y Coriolus antarcticus. Para ello se utilizaron técnicas de fermentación en estado sólido. También se ensayó la decoloración de colorantes industriales sobre estos cultivos. La pérdida de peso seco del sustrato fue similar después del día 60 (33-43 %). C. antarcticus produjo las mayores actividades de lacasa y Mn-peroxidasa (33,0 y 1,6 U/g peso seco). La mayor actividad endoglucanasa fue medida en cultivos de S. hirsutum (10,4 U/g), y la mayor actividad endoxilanasa en T. trogii (14,6 U/g). El sistema C. antarcticus/escobap mostró un importante potencial para su aplicación en la biorremediación de efluentes textiles, con porcentajes de decoloración de 93, 86, 82, 82, 77 y 58 % para índigo carmín, verde de malaquita, azure B, azul R brillante de remazol, cristal violeta y xilidina, respectivamente, en 5 h.(AU)