• Energy Research

Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, β-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, β-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and β-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.

AbstractMexico is one of the largest agricultural producers in Latin America and generates a large amount of agricultural residue. The aim of this study was to establish the usefulness of four of the main Mexican crops (corn, wheat, sugarcane, and Agave) as feedstock for lignocellulosic bioethanol production. The lignocellulosic residue ratio (RR), defined as weight of residue (in tons) per ton of product, was measured by sampling crop fields in 11 geographic regions of Mexico. The chemical composition, assessed by Fourier‐transform infrared spectroscopy (FTIR) and carbon‐13 nuclear magnetic resonance (13C NMR), and structural composition (extractives, cellulose, hemicellulose, and lignin contents), heating value, and metal content of these lignocellulosic residues were measured. Biorefinery locations, and their theoretical bioethanol production, were suggested using the gravity center method and techno‐economic criteria. The highest RR (1.1 ton of straw per ton of grain) was obtained for wheat straw followed by corn (0.8 ton of stover per ton of grain), sugar cane (0.15 ton of bagasse per ton of cane), and Agave (0.2 ton of bagasse per ton of stem). The composition of these biomasses varied significantly depending on the parental material in extractive compounds, lignocellulosic matter, ashes, hemicellulose, lignin, O‐alkyl C, aromatic C, and carboxyl C. The cellulose crystallinity index and the heating value exhibited small variations among biomasses. Copper, Zn, Cd, and Ni content in the biomasses generally exceeded the European Norm (EN‐Plus FprEN 1496 1‐2 B) for solid biofuels. In total, these agricultural biomasses could be used as feedstock for 34 biorefineries in Mexico with a total bioethanol production potential of approximately 1246 million L year–1. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd

Abstract Second-generation ethanol process uses lignocellulose, but a pre-treatment is required to degrade lignin before saccharification. Biological pre-treatment, using phenoloxidases, represents a cheap and eco-friendly option. Mould contamination can be overcome by heat pre-conditioning of substrates. Pasteurisation can be a mild and sustainable option. A 5-level response-surface experimental design was performed to test the effect of different time and temperatures on lignocellulolytic activities and substrate dephenolisation. Substrate microbial communities were characterised via catabolic profiles to determine functional diversity changes after pasteurisation. Temperatures ranging from 70 to 75 °C and time from 5 to 10 h led to the highest laccase activities and 30% of substrate dephenolisation was achieved while avoiding mould contamination. The per cent of O-Alkyl (polysaccharides) did not vary significantly, meaning that polysaccharides were not extensively consumed. Microbial communities were less catabolically diversified (H’ = 1.2) but more active (AWCD = 0.9) and could degrade polysaccharides.

L'éthanol a été désigné comme un inducteur de laccase. Cependant, il existe des rapports controversés sur son efficacité avec certains champignons. Dans cette étude, nous avons émis l'hypothèse que l'éthanol laccase induite chez Pycnoporus sanguineus dépend des conditions nutritionnelles en azote. Pour le prouver, nous avons évalué la production de laccase dans des cultures submergées de P. sanguineus, avec différentes concentrations d'azote et avec ou sans ajout d'éthanol dans une expérience factorielle. Afin d'analyser les effets des facteurs sur les variables de réponse, une ANOVA factorielle et des modèles de surface de réponse ont été réalisés. Il a été constaté que la source d'azote était le principal facteur qui affectait la production de laccase chez P. sanguineus. Les traitements à l'extrait de levure (2 g/L) et à l'éthanol (3 g/L) ont induit l'activité laccase la plus élevée (31,01 ± 4,9 U/L), tandis que les traitements à l'urée ont atteint l'activité la plus faible (moins de 1,6 U/L). L'éthanol a eu des effets positifs et synergiques sur la production de laccase, conformément au modèle de réponse de surface, tant que de simples sources d'azote (urée) n'étaient pas disponibles. Nous suggérons que la laccase chez P. sanguineus est régulée par un mécanisme de répression de l'azote catabolique ; l'activité de la laccase est fortement inhibée par l'urée utilisée comme source d'azote et elle diminue lorsque la quantité d'urée augmente ; au contraire, un effet synergique positif a été observé entre l'extrait de levure et l'éthanol sur la production de laccase. Se ha señalado el etanol como inductor de lacasa. Sin embargo, hay informes controvertidos sobre su eficiencia con algunos hongos. En este estudio, planteamos la hipótesis de que la lacasa de etanol inducida en Pycnoporus sanguineus depende de las condiciones de nutrientes de nitrógeno. Para demostrarlo, evaluamos la producción de lacasa en cultivos sumergidos de P. sanguineus, con diferentes concentraciones de nitrógeno y con o sin etanol añadido en un experimento de diseño factorial. Con el fin de analizar los efectos de los factores sobre las variables de respuesta, se realizó un ANOVA factorial y modelos de superficie de respuesta. Se encontró que la fuente de nitrógeno fue el principal factor que afectó la producción de lacasa en P. sanguineus. Los tratamientos con extracto de levadura (2 g/L) y etanol (3 g/L) indujeron la actividad de lacasa más alta (31.01 ± 4.9 U/L), mientras que los tratamientos con urea alcanzaron la actividad más baja (menos de 1.6 U/L). El etanol tuvo efectos positivos y sinérgicos en la producción de lacasa, de acuerdo con el modelo de respuesta de superficie, siempre que no se dispusiera de fuentes simples de nitrógeno (urea). Sugerimos que la lacasa en P. sanguineus está regulada por un mecanismo catabólico de represión de nitrógeno; la actividad de la lacasa está fuertemente inhibida por la urea utilizada como fuente de nitrógeno y disminuye cuando aumenta la cantidad de urea; por el contrario, se observó un efecto positivo sinérgico entre el extracto de levadura y el etanol en la producción de lacasa. Ethanol has been pointed out as a laccase inducer. However, there are controversial reports about its efficiency with some fungi. In this study, we hypothesized that ethanol laccase induced in Pycnoporus sanguineus depends on nitrogen nutriment conditions. To prove this, we assessed laccase production in submerged cultures of P. sanguineus, with different nitrogen concentrations and with, or without ethanol added in a factorial designed experiment. In order to analyze the effects of factors on the response variables, a factorial ANOVA, and response-surface models were performed. It was found that the nitrogen source was the main factor that affected laccase production in P. sanguineus. The treatments with yeast extract (2 g/L) and ethanol (3 g/L) induced the highest laccase activity (31.01 ± 4.9 U/L), while the treatments with urea reached the lowest activity (less than 1.6 U/L). Ethanol had positive and synergic effects on laccase production, in accordance with the surface response model, as long as simple nitrogen sources (urea) were not available. We suggest that laccase in P. sanguineus is regulated by a catabolic nitrogen repression mechanism; laccase activity is strongly inhibited by urea used as nitrogen source and it decreases when the amount of urea increases; contrarily, a synergic positive effect was observed between yeast extract and ethanol on laccase production. تمت الإشارة إلى الإيثانول كمحفز للكاز. ومع ذلك، هناك تقارير مثيرة للجدل حول كفاءتها مع بعض الفطريات. في هذه الدراسة، افترضنا أن الإيثانول لاكاز المستحث في Pycnoporus sanguineus يعتمد على ظروف التغذية النيتروجينية. لإثبات ذلك، قمنا بتقييم إنتاج laccase في الثقافات المغمورة من P. sanguineus، مع تركيزات مختلفة من النيتروجين ومع أو بدون إضافة الإيثانول في تجربة مصممة للعوامل. من أجل تحليل تأثيرات العوامل على متغيرات الاستجابة، تم إجراء ANOVA عاملي ونماذج سطح الاستجابة. وجد أن مصدر النيتروجين كان العامل الرئيسي الذي أثر على إنتاج اللوكاز في P. sanguineus. أدت العلاجات باستخدام مستخلص الخميرة (2 جم/لتر) والإيثانول (3 جم/لتر) إلى أعلى نشاط للاكاز (31.01 ± 4.9 وحدة/لتر)، في حين وصلت العلاجات باستخدام اليوريا إلى أدنى نشاط (أقل من 1.6 وحدة/لتر). كان للإيثانول آثار إيجابية وتآزرية على إنتاج اللاكاز، وفقًا لنموذج الاستجابة السطحية، طالما لم تكن مصادر النيتروجين البسيطة (اليوريا) متوفرة. نقترح أن اللاكاز في P. sanguineus يتم تنظيمه بواسطة آلية قمع النيتروجين التقويضي ؛ يتم تثبيط نشاط اللاكاز بقوة بواسطة اليوريا المستخدمة كمصدر للنيتروجين وينخفض عندما تزداد كمية اليوريا ؛ على النقيض من ذلك، لوحظ تأثير إيجابي تآزري بين مستخلص الخميرة والإيثانول على إنتاج اللاكاز.

The use of synthetic dyes for laccase induction in vivo has been scarcely explored. We characterized the effect of adding different synthetic dyes to liquid cultures of Pycnoporus sanguineus on laccase production. We found that carminic acid (CA) can induce 722 % and alizarin yellow 317 % more laccase than control does, and they promoted better fungal biomass development in liquid cultures. Aniline blue and crystal violet did not show such positive effect. CA and alizarin yellow were degraded up to 95 % during P. sanguineus culturing (12 days). With this basis, CA was selected as the best inducer and used to evaluate the induction of laccase on solid-state fermentation (SSF), using sugarcane bagasse (SCB) as substrate, in an attempt to reach selective delignification. We found that laccase induction occurred in SSF, and a slight inhibition of cellulase production was observed when CA was added to the substrate; also, a transformation of SCB under SSF was followed by the 13C cross polarization magic angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). Results showed that P. sanguineus can selectively delignify SCB, decreasing aromatic C compounds by 32.67 % in 16 days; O-alkyl C region (polysaccharides) was degraded less than 2 %; delignification values were not correlated with laccase activities. Cellulose-crystallinity index was increased by 27.24 % in absence of CA and 15.94 % when 0.01 mM of CA was added to SCB; this dye also inhibits the production of fungal biomass in SSF (measured as alkyl C gain). We conclude that CA is a good inducer of laccase in liquid media, and that P. sanguineus is a fungus with high potential for biomass delignification.