Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse.

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Data are available via ProteomeXchange with identifier PXD046075. Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L-1), while WBE promoted the higher release of xylose (5.71 g L-1). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

First report on the enzymatic profile of Kretzschmaria zonata.

Enzymes from phytopathogenic fungi are desirable for biotechnological applications and a highly virulent phytopathogen shows great appeal for enzymes production. To assess the biotechnological potential of Kretzschmaria zonata, a plant pathogenic fungus, we analyzed its enzymatic profile after growth on six different types of lignocellulosic biomasses. The fungus was able to produce a wide variety of enzymes with superior xylanase activity. The corn cob induced the highest specific activity of xylanase, 56.30 U/mg of protein, as well as other important enzymatic activities such as endoglucanase, 11.20 U/mg of protein; pectinase, 4.52 U/mg of protein; and ß-glucosidase, 2.77 U/mg of protein. The highest release of xylose, 0.88 g/L, was observed after saccharification of 10% of alkaline pretreated sugarcane bagasse by a commercial cocktail supplemented with the crude extract from K. zonata after growth on corn cob. The fungus extract is rich in hemicellulases and accessory enzymes and the result showed synergism between the enzymes present in the commercial mixture and in the K. zonata extract. This is the first report concerning the biotechnological potential of the fungus K. zonata, especially regarding to its ability to produce plant biomass degrading enzymes related to second generation ethanol production.

Brachiaria brizantha Grass as a Feedstock for Ethanol Production

HIGHLIGHTS Brachiaria brizantha proved to be a promising biomass for ethanol production. Fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural.

Abstract Different lignocellulosic biomasses are found worldwide and each country has its own important industrial crop that can be converted into high-value products, such as ethanol. Therefore, evaluation of new biomasses to be used in biorefineries is important to decrease the dependence on non-renewable resources and to guarantee sustainable development. This work evaluated Brachiaria brizantha, a grass commonly used as animal forage, and the standard biomass for 2G-ethanol, sugarcane bagasse. The chemical compositions of both biomasses were determined and different times and temperature of acid pretreatment were tested. Morphological analysis via scanning electron microscopy showed more deconstructed fibers after harsher biomass pretreatments. Simultaneous saccharification and fermentation of pretreated Brachiaria brizantha presented higher efficiency than when using sugarcane bagasse as the carbon source. A biomass conversion of 46 % was achieved when Brachiaria brizantha grass was pretreated with 2% sulfuric acid for 60 minutes. Moreover, fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural. It was concluded that Brachiaria brizantha is a promising biomass for ethanol production.

Root collar rot, a new lethal disease on Tectona grandis caused by Kretzschmaria zonata in Brazil.

Tectona grandis L.f., known as teak, is one of the most valuable tropical hardwood species that has been extensively planted in tropical zones, covering about 6,8 million hectares (Kollert and Kleine 2017). Recent advances in silvicultural management and use of improved clones have enhanced productivity and wood quality of teak plantations in Brazil. However, the incidence of diseases has increased over time being a threat to sustainability of commercial teak plantations. Therefore, forest pathology studies have been conducted in Brazil to minimize the risks of losses caused by the diseases on teak, ensuring the expected economic profitability. In a recent disease survey conducted in Midwest of Brazil, almost one thousand teak trees showing typical die-back symptoms with root collar rot were found. The diseased trees showed undersize leaves displaying yellowish to pale brown color, followed by wilt, defoliation and death. At the base of the trunk, root collar rot was observed, with sloughing and deterioration of the bark exhibiting flattened and encrusted fungi fruiting bodies of gray to bright white color. Over the time, the wood of infected trees develops black zone lines and soft tissue due to both lignin and cellulose decay. The disease begins in the root and spreads to the collar of the tree, causing a collapse in sap flow leading to mortality. To discover the disease cause, samples of infected trees were collected to perform an accurate pathogen identification by polyphasic approach, as well as pathogenicity test. From isolation in Malt Extract Agar (MEA), one fungus showing white progressing to gray mycelial growth was consistently isolated. Two isolates named as GFP131 and GFP132 were characterized. Microscopic examination showed conidia aseptate, hyaline, ovoid to fusiform-ellipsoid shaped, measuring 6-8 x 2-4 µm; stromata with surface brown to dark brown; perithecia with variable shapes and ostioles papillate; and ascospores aseptate, dark brown, fusiform to ellipsoid, measuring 20-37 × 8-15 µm, displaying a straight germinal line slightly less than ascospore length. These morphological characteristics were similar to descriptions for genus Kretzschmaria (Rogers and Ju 1998; Stadler et al. 2013). Genomic DNA was extracted from mycelium, and the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4 was sequenced and then deposited under Genbank codes MH481853 and MH481854. A BLASTn search showed >99% identity with K. zonata sequence (KY660541). Phylogenetic inference by Maximum Likelihood method using Kimura 2-parameter model confirms that the isolates belong to Kretzschmaria zonata (Lév.) P.M.D. Martin. A pathogenicity test was established in a greenhouse with controlled conditions of temperature (28-30 °C) and humidity (80-90 %). Five plants were inoculated with GFP132 at the collar region with one mycelium disk of approximately 5 mm in diameter each, and the inoculated area was wrapped in plastic film. Disks of MEA culture media were placed on five additional plants as controls. Forty days after inoculation, all of the previously mentioned symptoms were observed for all inoculated plants, while control plants showed only scars at the inoculation point. The pathogen was reisolated from all five of the inoculated plants. Kretschmaria zonata has been reported on teak in Nigeria (West 1938) and in Mexico (Cibrian Tovar et al. 2014). However, this is the first report of K. zonata on T. grandis for Brazil and the first report anywhere to include Koch's postulates, proving the etiology of the disease.

Engineered GH11 xylanases from Orpinomyces sp. PC-2 improve techno-functional properties of bread dough.

BACKGROUND: Endo-1,4-ß-xylanases have marked hydrolytic activity towards arabinoxylans. Xylanases (xynA) produced by the anaerobic fungus Orpinomyces sp. strain PC-2 have been shown to be superior in specific activity, which strongly suggests their applicability in the bakery industry for the processing of whole-wheat flour containing xylans. In the present study, two xylanases from this source, the small wild-type xylanase SWT and the small mutant xylanase SM2 (V108A, A199T), were expressed in Escherichia coli, purified, characterized, tested for their ability to hydrolyze whole-wheat flour and applied in dough processing. RESULTS: Both purified SM2 and SWT showed high specific activity against oat spelt xylan and wheat arabinoxylan, exhibiting maximum activity at pH 3-7 and 60 °C. SM2 was more thermostable than SWT, which suggests that the mutations enhanced its stability. Both SWT and SM2 were able to hydrolyze whole-wheat flour, and evaluation of their applicability in dough processing by the sponge method indicated that use of these enzymes increased dough volume by 60% and reduced texture hardness by more than 50%, while gumminess and chewiness were reduced by 40%. CONCLUSION: The recombinant xylanases showed potential for application in bakery processing and can improve techno-functional properties in sponges. © 2018 Society of Chemical Industry.

Inhibitors Compounds on Sugarcane Bagasse Saccharification: Effects of Pretreatment Methods and Alternatives to Decrease Inhibition.

Considering bioethanol production, extensive research has been performed to decrease inhibitors produced during pretreatments, to diminish energy input, and to decrease costs. In this study, sugarcane bagasse was pretreated with NaOH, H2SO4, and water. The higher concentration of phenols, 3.3 g/L, was observed in biomass liquid fraction after alkaline pretreatment. Acid pretreatment was responsible to release considerable acetic acid concentration, 2.3 g/L, while water-based pretreatment was the only to release formic acid, 0.02 g/L. Furans derivatives were not detected in liquid fractions regardless of pretreatment. Furthermore, washing step removed most of the phenols from pretreated sugarcane bagasse. Saccharification of alkali-pretreated biomass plus polyethylene glycol (PEG) at 0.4% (w/v) enhanced 8 and 26% the glucose and the xylose release, respectively, while polyvinylpyrrolidone (PVP) also at 0.4% (w/v) increased the release by 10 and 31% of these sugars, respectively, even without washing and filtration steps. Moreover, these polymers cause above 50% activation of endoglucanase and xylanase activities which are crucial for biomass hydrolysis.

Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification.

Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily-accessible hosts, such as Pichia pastoris, can help in revealing these effects.

The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: A comparative study.

Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32 g/L) and xylose (9.00 g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84 U/mg of protein), ß-glucosidase (29.48 U/mg of protein), ß-xylosidase (2.95 U/mg of protein), pectinase (127.46 U/mg of protein) and laccase (2.49 U/mg of protein).

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse.

Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two "second generation" substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.

Production and application of an enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum with potential for hydrolysis of sugarcane bagasse.

Blending of the enzyme extracts produced by different fungi can result in favorable synergetic enhancement of the enzyme blend with regards to the main cellulase activities, as well as the inclusion of accessory enzymes that may not be as abundant in enzyme extracts produced by predominantly cellulase producing fungi. The Chrysoporthe cubensis:Penicillium pinophilum 50:50 (v/v) blend produced herein presented good synergy, especially for FPase and endoglucanase activities which were 76% and 48% greater than theoretical, respectively. This enzyme blend was applied to sugarcane bagasse previously submitted to a simple alkali pretreatment. Glucan hydrolysis efficiency reached an excess of 60% and xylan conversion exceeded 90%. Increasing the hydrolysis temperature from 45 to 50°C also resulted in a 16-20% increase in conversion of both glucan and xylan fractions. The blended enzyme extract obtained therefore showed great potential for application in the lignocellulose hydrolysis process.

The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in D-xylose, L-arabinose and steam-exploded sugarcane bagasse.

The interest in the conversion of plant biomass to renewable fuels such as bioethanol has led to an increased investigation into the processes regulating biomass saccharification. The filamentous fungus Aspergillus niger is an important microorganism capable of producing a wide variety of plant biomass degrading enzymes. In A. niger the transcriptional activator XlnR and its close homolog, AraR, controls the main (hemi-)cellulolytic system responsible for plant polysaccharide degradation. Sugarcane is used worldwide as a feedstock for sugar and ethanol production, while the lignocellulosic residual bagasse can be used in different industrial applications, including ethanol production. The use of pentose sugars from hemicelluloses represents an opportunity to further increase production efficiencies. In the present study, we describe a global gene expression analysis of A. niger XlnR- and AraR-deficient mutant strains, grown on a D-xylose/L-arabinose monosaccharide mixture and steam-exploded sugarcane bagasse. Different gene sets of CAZy enzymes and sugar transporters were shown to be individually or dually regulated by XlnR and AraR, with XlnR appearing to be the major regulator on complex polysaccharides. Our study contributes to understanding of the complex regulatory mechanisms responsible for plant polysaccharide-degrading gene expression, and opens new possibilities for the engineering of fungi able to produce more efficient enzymatic cocktails to be used in biofuel production.