Using low-shear aerated and agitated bioreactor for producing two specific laccases by trametes versicolor cultures induced by 2,5-xylidine: Process development and economic analysis.

Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes.

Performance, body development and ingestive behavior of Nelore and crossbred heifers finished in a grazing system.

The aim of this experiment was to evaluate growth, body development and ingestive behavior of Nelore and crossbred heifers. Twenty-two contemporary heifers (eight Nelore, seven Nelore × Angus (½ Angus) and seven Nelore × Pantaneiro (½ Pantaneiro) crosses) were evaluated. The variables evaluated were weight, subcutaneous fat thickness [assessed by ultrasound in the longissimus dorsi (SFT) and biceps femoris (SFTP8)] morphometric measures and ingestive behavior (determined between 7 am and 5 pm). The daily gain differed between the breeds (P < 0.05), being greater for the ½ Pantaneiro than Nelore and equal to ½ Angus at the end of the study. The ½ Angus heifers differed (P < 0.01) from the other breeds for SFT (4.36 mm), with no difference between Nelore (2.77 mm) and ½ Pantaneiro (3.38 mm). The SFTP8 was greater (P < 0.01) (5.36 mm) in ½ Angus heifers than t others crossbreed, and greater in ½ Pantaneiro than in Nelore (4.28 vs 3.29 mm). The heart girth and rump width between the ilia were larger (P < 0.01) for the ½ Angus than ½ Pantaneiro and Nelore (169 cm; 41.6 cm, respectively). Substernal height was greatest (P < 0.01) for the Nelore (69.1 cm), reflecting the greater anterior and posterior height. The ½ Angus spent the longest time (P < 0.05) walking and less time grazing. Idleness also differed (P < 0.05) throughout the day. Crossbred heifers exhibited better performance and development than Nelore. The grazing pattern of ½ Pantaneiro heifers was identical to Nelore, but walked less, gained more weight, and exhibited better carcass finishing.

Xylan, Xylooligosaccharides, and Aromatic Structures With Antioxidant Activity Released by Xylanase Treatment of Alkaline-Sulfite-Pretreated Sugarcane Bagasse.

Xylanase enzymes are useful to fractionate plant biomass, producing xylan, xylooligosaccharides (XOS), and antioxidant-derived XOS. In a biorefinery, pretreated biomass can be digested with xylanase prior to cellulose saccharification, enhancing the product portfolio in the process. With this vision, this study highlighted a wide range of new products attainable from alkaline-sulfite-pretreated sugarcane bagasse by treatments with endo-xylanase under controlled conditions. The developed process provided a crude extract corresponding to 29.7% (w/w) of pretreated sugarcane bagasse. The crude extract included a relatively polymeric glucuronoarabinoxylan fraction, DP2-DP6 xylooligosaccharides, and aromatic compounds. The enzymatically produced extract was fractionated with increasing ethanol concentrations [up to 90% (v/v)], providing precipitation of varied polymeric xylan fractions (48% (w/w) of the crude extract) with average molar masses ranging from 28 kDa to 3.6 kDa. The fraction soluble in 90% ethanol was subjected to adsorption on 4% (w/v) activated charcoal and eluted with an ethanol gradient from 10% to 70% (v/v), thus providing xylooligosaccharides and aromatic fractions. Most of the xylooligosaccharides (74% of the eluted sugars) were washed out in 10%-30% ethanol. DP2 and DP3 structures predominated in the 10% ethanol fraction, while DP5 structures were significantly enriched in the 30% ethanol fraction. Higher ethanol concentrations desorbed xylooligosaccharides associated with higher amounts of aromatic compounds. Total aromatics, phenolic structures, and p-hydroxycinnamates predominated in the fractions desorbed with 60% and 70% ethanol. The antioxidant activity of produced fractions correlated with their phenolic contents. Compiled results indicate that a wide variety of products can be prepared from pretreated biomass using xylanase-aided extraction procedures. Recovered fractions presented different features and specific application prospects. Beyond polymeric xylan with low lignin contamination, xylooligosaccharides or even lignin-carbohydrate complexes with antioxidant activity can be included in the biorefinery portfolio based on the currently developed fractionation studies.

High-solid enzymatic hydrolysis of sugarcane bagasse and ethanol production in repeated batch process using column reactors.

Alkaline sulfite pretreated sugarcane bagasse was enzymatically hydrolyzed in a packed-bed column reactor and a bubble column reactor was evaluated to produce ethanol from the hydrolysate. Initial solid loadings of 9-16% were used in column reactor in the hydrolysis step, and the use of lower value (9%) resulted in 41 g L-1 of glucose in the hydrolysate, corresponding to 87% of cellulose hydrolysis yield. This yield was reduced to 65% for a solid loading of 16%, corresponding to a glucose concentration of 54 g L-1. Subsequently, Saccharomyces cerevisiae and Scheffersomyces stipitis were used for ethanol production in medium based on hydrolysate previously obtained, using different aeration flowrates (0.3, 0.5 and 0.7 vvm). In simple batch fermentation using S. cerevisiae, higher ethanol yield (0.40 g.g-1) and productivity (1.58 g.L-1.h-1) were achieved using 0.5 vvm. When S. stipitis was used in simple batch co-fermentations, the maximum ethanol productivities were obtained using 0.5 and 0.7 vvm (0.64 and 0.63 g.L-1.h-1, respectively). Successive repeated batches resulted in average ethanol concentration of 38 g.L-1 and fermentation efficiency of 82%, when using S. cerevisiae. For S. stipitis, those values were, respectively, 36 g.L-1 and 50%, with volumetric productivity increased along the cycles. Thus, the potential of the bioreactors as simple systems for use in the biological steps of biorefineries was demonstrated. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02932-3.

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall.

The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total pCA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that pCA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the pCA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in pCA content on hemicelluloses and generated a C4 plant that is promising for valorizing pCA production in biorefineries.

The spawning method affects the reproductive efficiency of piracanjuba fish (Brycon orbignyanus) in restocking programs / El método de desove afecta la eficiencia reproductiva de piracanjuba (Brycon orbignyanus) en programas de repoblación / O método de desova afeta a eficiência reprodutiva de piracanjuba (Brycon orbignyanus) em programas de repovoamento

Abstract Background: Preserving the genetic diversity of wild fish is an important consideration for restocking programs, as inbreeding can compromise progeny survival as well as impact the resilience of natural populations. Objective: To evaluate the influence of spawning method: semi-natural (SN) or strip-spawning (ST), and the number of breeders (1♀:3♂ and 2♀:6♂) on the reproductive efficiency and genetic diversity of B. orbignyanus progeny destined for restoration of wild stocks. Methods: Rates of fertilization, hatching and broodfish mortality were recorded. For genetic evaluations (allele frequency, observed and expected heterozygosity, Shannon index, inbreeding coefficient, molecular variance analysis, and genetic differentiation), breeders (n=24), and their progenies (90 larvae/treatment) were sampled and analyzed using eight microsatellite markers. Results: Higher fertilization and hatching rates, and lower broodfish mortality were observed for the SN method (p<0.05), whereas the number of breeders did not affect these parameters (p>0.05). Interaction between spawning method and number of breeders was not significant (p>0.05). The amplified microsatellite loci produced a total of 30 alleles, with sizes between 80 and 225 bp and their frequencies indicated an increase (p<0.05) of genetic diversity in the progenies, but low genetic differentiation between treatments (p>0.05). Conclusion: The spawning methods and number of breeders tested increased equally the genetic diversity of the progeny, with low genetic differentiation between treatments. In contrast, rates of fertilization, hatching and brood fish mortality revealed that the SN method resulted in the best reproductive efficiency due to the handling stress and injuries caused by ST. Thus, SN proves to be the most suitable spawning-method for B. orbignyanus in restocking programs.

Resumen Antecedentes: Mantener la diversidad genética de los peces salvajes es una consideración importante para los programas de repoblación, ya que la endogamia puede comprometer la supervivencia de la progenie y afectar la supervivencia de las poblaciones naturales. Objetivo: Evaluar la influencia del método de desove (seminatural - SN o en franjas - ST) y el número de reproductores (1♀:3♂ y 2♀:6♂) sobre la eficiencia reproductiva y la diversidad genética de progenies de B. orbignyanus destinados a la restauración de poblaciones silvestres. Métodos: Se monitorearon las tasas de fertilización, eclosión y mortalidad de reproductores. Para las evaluaciones genéticas (frecuencias alélicas, heterocigosidad observada y esperada, índice de Shannon, coeficiente de endogamia, análisis de varianza molecular y diferenciación genética) los reproductores (n=24) y su progenie (90 larvas/tratamiento) se muestrearon y analizaron utilizando ocho marcadores microsatélites. Resultados: La interacción entre el método de desove y el número de reproductores no fue significativa (p>0,05). Se obtuvieron mejores tasas de fecundación y eclosión (p<0,05), y una menor mortalidad de reproductores (p<0,05) con el método SN, mientras que el número de reproductores no tuvo efecto (p>0,05). Los loci de microsatélites amplificados produjeron un total de 30 alelos con tamaños entre 80 y 225 pb, y sus frecuencias indicaron un aumento (p<0,05) en la diversidad genética de las progenies, pero una baja diferenciación genética entre tratamientos (p>0,05). Conclusión: Los métodos de desove y el número de reproductores evaluados aumentaron de la misma manera la diversidad genética de las progenies, con baja diferenciación genética entre tratamientos. En contraste, las tasas de fecundación, eclosión y mortalidad de peces reproductores revelaron que el SN tuvo la mejor eficiencia reproductiva, un hecho relacionado con el estrés del manejo y las lesiones causadas por el ST. Por lo tanto, el SN demuestra ser el método de desove más adecuado para B. orbignyanus en los programas de repoblación.

Resumo Antecedentes: A manutenção da diversidade genética dos peixes selvagens é uma importante consideração para programas de repovoamento, já que a endogamia pode comprometer a sobrevivência da progênie, além de impactar na resiliência das populações naturais. Objetivo: Avaliar a influência do método de desova (semi-natural - SN ou por extrusão - ST) e número de reprodutores (1♀:3♂ e 2♀:6♂) na eficiência reprodutiva e na diversidade genética de progênie de B. orbignyanus destinados à recuperação de estoques selvagens. Métodos: Taxas de fertilização, eclosão e mortalidade de reprodutores foram monitorados. Para avaliações genéticas (frequências alélicas, heterozigosidade observada e esperada, índice de Shannon, coeficiente de endogamia, análise de variância molecular e diferenciação genética), reprodutores (n=24) e sua progênie (90 larvas/tratamento) foram amostrados e analisados utilizando oito marcadores microssatélites. Resultados: Interação entre método de desova e número de reprodutores não foi significante (p>0,05). Melhores taxas de fertilização e eclosão (p<0,05), e menor (p<0,05) mortalidade de reprodutores foram observados para o método SN, enquanto que o número de reprodutores não afetou esses parâmetros (p>0,05). Os loci microssatélites amplificados produziram um total de 30 alelos, com tamanhos entre 80 e 225 pb e suas frequências indicaram aumento (p<0,05) da diversidade genética nas progênies, mas baixa diferenciação genética entre os tratamentos (p>0,05). Conclusão: Os métodos de desova e números de reprodutores avaliados aumentaram igualmente a diversidade genética das progênies, com baixa diferenciação genética entre tratamentos. Em contraste, as taxas de fecundação, eclosão e mortalidade de reprodutores revelaram que SN obteve a melhor eficiência reprodutiva, fato relacionado com o estresse de manipulação e injurias causadas por ST. Por isso, SN se mostrou como o método de desova mais adequado para B. orbignyanus em programas de repovoamento.

A city cluster risk-based approach for Sars-CoV-2 and isolation barriers based on anonymized mobile phone users' location data.

Given the recent outbreak of Sars-CoV-2, several countries started to seek different strategies to control contamination and minimize fatalities, which are usually the primary objectives for all strategies. Secondary objectives are related to economic factors, therefore ensuring that society would be able is to keep its essential activities and avoid supply disruptions. This paper presents an application of anonymized mobile phone users' location data to estimate population flow amongst cities with an origin-destination matrix. The work includes a clustering analysis of cities, which may enable policymakers (and epidemiologists) to develop public policies giving the appropriate consideration for each set of cities within a Province or State. Risk measures are included to analyze the severity of the spread among the clusters, which can be ranked. Then, intelligence can be obtained from the analysis, and some clusters could be isolated to avoid contagion while keeping their economic activities. Therefore, this analysis is reproducible for other states of Brazil and other countries and can be adapted for districts within a city, especially considering the possibility of a second wave COVID-19 pandemic.

Determinants of farmers' loyalty to dairy processors in Minas Gerais, Brazil / Determinantes da fidelidade dos produtores de leite aos laticínios em Minas Gerais, Brasil

ABSTRACT: The current study explores variables associated with the loyalty of dairy farmers to dairy processors in the Brazilian context. A multivariate discrete choice (Logit) model and alternative formulations assess the associations between loyalty metrics and farm and processor characteristics for a sample of 32 dairy farmers in 16 municipalities at the Zona da Mata in Minas Gerais. Twenty-two dairy processors were identified as milk buyers in the area studied, but each farmer indicated that they could sell to an average of five alternative buyers of milk. Farmers' attributes such as production scale or the technological level are not statistically significantly associated with loyalty in this sample. The current milk price paid to farmers in our sample is not associated with increased loyalty (sales to a single processor for 6 or more years) in all estimated models; although, further research on this impact is merited to inform buyer-pricing policy. Variables associated with increased loyalty include payment of premiums for quality, farmer years of experience and cooperation among farmers in the purchase of inputs. Delayed payment is associated with reduced loyalty. We could not determine the effect of participation in technical assistance programs offered by processors on loyalty, because in our sample all farmers received free university-provided technical assistance. The payment of a premium based on milk volume was also unassociated with loyalty determination. The small size of our sample limits the ability to generalize our results but provides exploratory results that facilitate future investigation.

RESUMO: O objetivo deste estudo é explorar as variáveis importantes associadas à fidelidade dos produtores de leite aos laticínios no contexto brasileiro. Um modelo de escolha discreta multivariada (Logit) e formulações alternativas foram usadas para avaliar as associações entre métricas de fidelidade e características de fazendas e processadores em uma amostra de 32 produtores de leite de 16 municípios da Zona da Mata, em Minas Gerais. Vinte e dois laticínios foram identificados como compradores de leite na região estudada, entretanto, cada produtor indicou que tem, em média, a possibilidade de vender o leite para cinco empresas diferentes. Os atributos dos agricultores, como escala de produção ou nível tecnológico, não foram estatisticamente associados significativamente à fidelidade. O preço atual do leite pago aos agricultores nessa amostra não está associado a probabilidade de aumento da fidelidade (vendas para um único laticínio por seis anos ou mais) em todos os modelos estimados. No entanto, pesquisas adicionais sobre esse impacto são necessárias para subsidiar políticas de preços aos produtores. As variáveis associadas ao aumento da fidelidade incluem pagamentos de prêmios pela qualidade, anos de experiência dos agricultores e a cooperação entre os agricultores na compra de insumos. O atraso no pagamento está associado à redução de fidelidade. Não foi possível determinar o efeito da participação em programas de assistência técnica oferecidos pelos processadores na fidelidade, provavelmente porque todos os produtores estudados recebem assistência técnica de graça de uma Universidade. O pagamento de prêmios com base no volume de leite também não foi associado à determinação da fidelidade. O pequeno tamanho de nossa amostra limita a capacidade de generalizar nossos resultados, mas fornece resultados exploratórios que facilitam investigações futuras.

EgPHI-1, a PHOSPHATE-INDUCED-1 gene from Eucalyptus globulus, is involved in shoot growth, xylem fiber length and secondary cell wall properties.

MAIN CONCLUSION: EgPHI-1 is a member of PHI-1/EXO/EXL protein family. Its overexpression in tobacco resulted in changes in biomass partitioning, xylem fiber length, secondary cell wall thickening and composition, and lignification. Here, we report the functional characterization of a PHOSPHATE-INDUCED PROTEIN 1 homologue showing differential expression in xylem cells from Eucalyptus species of contrasting phenotypes for wood quality and growth traits. Our results indicated that this gene is a member of the PHI-1/EXO/EXL family. Analysis of the promoter cis-acting regulatory elements and expression responses to different treatments revealed that the Eucalyptus globulus PHI-1 (EgPHI-1) is transcriptionally regulated by auxin, cytokinin, wounding and drought. EgPHI-1 overexpression in transgenic tobacco changed the partitioning of biomass, favoring its allocation to shoots in detriment of roots. The stem of the transgenic plants showed longer xylem fibers and reduced cellulose content, while the leaf xylem had enhanced secondary cell wall thickness. UV microspectrophotometry of individual cell wall layers of fibers and vessels has shown that the transgenic plants exhibit differences in the lignification of S2 layer in both cell types. Taken together, the results suggest that EgPHI-1 mediates the elongation of secondary xylem fibers, secondary cell wall thickening and composition, and lignification, making it an attractive target for biotechnological applications in forestry and biofuel crops.

The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials.

The ability of white-rot fungi to degrade polysaccharides in lignified plant cell walls makes them a suitable reservoir for CAZyme prospects. However, to date, CAZymes from these species are barely studied, which limits their use in the set of choices for biomass conversion in modern biorefineries. The current work joined secretome studies of two representative white-rot fungi, Phanerochaete chrysosporium and Trametes versicolor, with expression analysis of cellobiohydrolase (CBH) genes, and use of the secretomes to evaluate enzymatic conversion of simple and complex sugarcane-derived substrates. Avicel was used to induce secretion of high levels of CBHs in the extracellular medium. A total of 56 and 58 proteins were identified in cultures of P. chrysosporium and T. versicolor, respectively, with 78-86% of these proteins corresponding to plant cell wall degrading enzymes (cellulolytic, hemicellulolytic, pectinolytic, esterase, and auxiliary activity). CBHI predominated among the plant cell wall degrading enzymes, corresponding to 47 and 34% of the detected proteins in P. chrysosporium and T. versicolor, respectively, which confirms that Avicel is an efficient CBH inducer in white-rot fungi. The induction by Avicel of genes encoding CBHs (cel) was supported by high expression levels of cel7D and cel7C in P. chrysosporium and T. versicolor, respectively. Both white-rot fungi secretomes enabled hydrolysis experiments at 10 FPU/g substrate, despite the varied proportions of CBHs and other enzymes present in each case. When low recalcitrance sugarcane pith was used as a substrate, P. chrysosporium and T. versicolor secretomes performed similarly to Cellic® CTec2. However, the white-rot fungi secretomes were less efficient than Cellic® CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic® CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes highlighted T. versicolor enzymes for providing high glucan conversions, even at lower proportion of CBHs, probably because the other enzymes present in this secretome and CBHs lacking carbohydrate-binding modules compensate for problems associated with unproductive binding to lignin.

Resting time before slaughter restores homeostasis, increases rigor mortis time and fillet quality of surubim Pseudoplatystoma spp.

This study assesses the respiratory dynamics related to stress parameters and resting time before slaughter, in the quality of surubim (Pseudopatystoma spp.) fillets. A completely randomized design was conducted using five treatments: resting time before slaughter of 0, 2, 4, 8 and 24 hours, with 15 fish sampled per treatment. Time 0 corresponded to the treatment without resting time, where the fish were slaughtered immediately after arriving at the processing plant. The resting time did not affect the electrolyte balance, hemoglobin, plasma, hepatic glycogen, myofibrillar fragmentation index (MFI) and water holding capacity (WHC) of surubins. However, with increased resting time, there was a significant decrease in muscle glycogen and an increase in blood pH and blood bicarbonate levels. Additionally, respiratory parameters showed an increase in pO2 and, consequently, in O2 saturation and a decrease in pCO2.The hematocrit and MCV values of the surubins after 24 hours of resting decreased significantly. In the first hours of resting, the highest values of erythrocytes and CHCM were observed. The lowest level of stress was observed for fish having 24 hours of resting. Fish having longer resting periods (8 and 24 hours) presented fillets with a higher pH (P <0.05) and the rigor mortis establishment time was shorter for the first 2 hours and 24 hours of resting time. There was a linear decrease in fillet lightness and an increase in the intensity of red (CIE a*) color up to 24 hours when resting was increased. In CIE b*, a linear decrease (P <0.05) of the yellow intensity of the fillets was observed as the surubim resting time increased. A resting time of 4 to 8 hours before slaughter is effective in reestablishing homeostasis after transporting surubim, providing fillets with higher quality and a greater length of the pre-rigor mortis period.

Enzyme-aided xylan extraction from alkaline-sulfite pretreated sugarcane bagasse and its incorporation onto eucalyptus kraft pulps.

Hemicellulose-rich substrates produced in the lignocellulose biorefinery context can yield macromolecular xylan structures with assorted application in the chemical industry. Xylan presents natural affinity to cellulose and its incorporation onto fibers increases the physical processability of pulp; however, current studies diverge on how molar mass affects xylan interaction with cellulose. In the current work, xylans with varied structural characteristics were prepared from alkaline-sulfite pretreated sugarcane bagasse with aid of an alkaline-active xylanase and selective precipitations using different ethanol concentrations. Prepared xylan fractions, containing low levels of lignin contamination (4-9%) and molar masses ranging from 2.3 kDa to 34 kDa, were incorporated onto eucalyptus pulp fibers up to 4.7 g xylan/100 g pulp. The efficiency of xylan incorporation onto cellulosic fibers was dependent on the xylan structures, where low molar mass and low substitution degree favored high incorporation levels.

Differentiation of Tracheary Elements in Sugarcane Suspension Cells Involves Changes in Secondary Wall Deposition and Extensive Transcriptional Reprogramming.

Plant lignocellulosic biomass, mostly composed of polysaccharide-rich secondary cell walls (SCWs), provides fermentable sugars that may be used to produce biofuels and biomaterials. However, the complex chemical composition and physical structure of SCWs hinder efficient processing of plant biomass. Understanding the molecular mechanisms underlying SCW deposition is, thus, essential to optimize bioenergy feedstocks. Here, we establish a xylogenic culture as a model system to study SCW deposition in sugarcane; the first of its kind in a C4 grass species. We used auxin and brassinolide to differentiate sugarcane suspension cells into tracheary elements, which showed metaxylem-like reticulate or pitted SCW patterning. The differentiation led to increased lignin levels, mainly caused by S-lignin units, and a rise in p-coumarate, leading to increased p-coumarate:ferulate ratios. RNAseq analysis revealed massive transcriptional reprogramming during differentiation, with upregulation of genes associated with cell wall biogenesis and phenylpropanoid metabolism and downregulation of genes related to cell division and primary metabolism. To better understand the differentiation process, we constructed regulatory networks of transcription factors and SCW-related genes based on co-expression analyses. Accordingly, we found multiple regulatory modules that may underpin SCW deposition in sugarcane. Our results provide important insights and resources to identify biotechnological strategies for sugarcane biomass optimization.

The secretome of two representative lignocellulose-decay basidiomycetes growing on sugarcane bagasse solid-state cultures.

Secretome evaluations of lignocellulose-decay basidiomycetes can reveal new enzymes in selected fungal species that degrade specific substrates. Proteins discovered in such studies can support biorefinery development. Brown-rot (Gloeophyllum trabeum) and white-rot (Pleurotus ostreatus) fungi growing in sugarcane bagasse solid-state cultures produced 119 and 63 different extracellular proteins, respectively. Several of the identified enzymes are suitable for in vitro biomass conversion, including a range of cellulases (endoglucanases, cellobiohydrolases and ß-glucosidases), hemicellulases (endoxylanases, α-arabinofuranosidases, α-glucuronidases and acetylxylan esterases) and carbohydrate-active auxiliary proteins, such as AA9 lytic polysaccharide monooxygenase, AA1 laccase and AA2 versatile peroxidase. Extracellular oxalate decarboxylase was also detected in both fungal species, exclusively in media containing sugarcane bagasse. Interestingly, intracellular AA6 quinone oxidoreductases were also exclusively produced under sugarcane bagasse induction in both fungi. These enzymes promote quinone redox cycling, which is used to produce Fenton's reagents by lignocellulose-decay fungi. Hitherto undiscovered hypothetical proteins that are predicted in lignocellulose-decay fungi genomes appeared in high relative abundance in the cultures containing sugarcane bagasse, which suggests undisclosed, new biochemical mechanisms that are used by lignocellulose-decay fungi to degrade sugarcane biomass. In general, lignocellulose-decay fungi produce a number of canonical hydrolases, as well as some newly observed enzymes, that are suitable for in vitro biomass digestion in a biorefinery context.

Functional characterization and comparative analysis of two heterologous endoglucanases from diverging subfamilies of glycosyl hydrolase family 45.

Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0 °C and 80.9 °C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70 °C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis.

Descending stairs: Good or bad task to discriminate women with patellofemoral pain?

BACKGROUND: There is no consensus on kinematics alterations during descending stairs in females with patellofemoral pain (PFP). In addition, there are no studies that have evaluated the three dimensional kinematics of the trunk, pelvis, hip, knee, and ankle using a multi-segmental model of the foot simultaneously during this task in patients with PFP and evaluated the subphases of stair descent. The objectives of this study were to compare the three dimensional kinematics of the trunk, pelvis, and lower limbs during different subphases of stair descent and identify the discriminatory capacity of the kinematic variables among women with PFP and healthy women. METHODS: In this cross-sectional study, thirty-four women with PFP and thirty-four pain free women between 18 and 35 years-old were submitted to three-dimensional kinematic evaluation during stair descent. RESULTS: It was observed that kinematic differences between the groups occurred in the first double support phase of the stair descent, with the variables of internal rotation of the hindfoot in relation to the tibia in the initial contact (2.1°; sensitivity = 68.6%, specificity = 61.8%) and contralateral pelvic drop in load response (1.3°, sensitivity = 65.7%, specificity = 63.7%) presenting the best ability to discriminate women with and without PFP. CONCLUSION: Our results suggest that kinematic changes during stair descent should be used with caution during the evaluation and decision-making process in women with PFP.

Fate of p-hydroxycinnamates and structural characteristics of residual hemicelluloses and lignin during alkaline-sulfite chemithermomechanical pretreatment of sugarcane bagasse.

BACKGROUND: Preparing multiple products from lignocellulosic biomass feedstock enhances the profit and sustainability of future biorefineries. Grasses are suitable feedstocks for biorefineries as they permit a variety of possible by-products due to their particular chemical characteristics and morphology. Elucidating the fate of p-hydroxycinnamates (ferulates-FAs and p-coumarates-pCAs) and major structural components during bioprocessing helps to discriminate the sources of recalcitrance in grasses and paves the way for the recovery of p-hydroxycinnamates, which have multiple applications. To address these subjects, we assessed sugarcane bagasse biorefining under alkaline-sulfite chemithermomechanical (AS-CTM) pretreatment and enzymatic saccharification. RESULTS: The mass balances of the major bagasse components were combined with 2D-NMR structural evaluation of process solids to advance our understanding of sugarcane bagasse changes during biorefining. AS-CTM pretreatment provided a high yield and thoroughly digestible substrates. The pretreated material was depleted in acetyl groups, but retained 62 and 79% of the original lignin and xylan, respectively. Forty percent of the total FAs and pCAs were also retained in pretreated material. After pretreatment and enzymatic hydrolysis, the residual solids contained mostly lignin and ester-linked pCAs, with minor amounts of FAs and non-digested polysaccharides. Saponification of the residual solids, at a higher alkali load, cleaved all the ester linkages in the pCAs; nevertheless, a significant fraction of the pCAs remained attached to the saponified solids, probably to lignin, through 4-O ether-linkages. CONCLUSION: AS-CTM pretreatment provided soundly digestible substrates, which retain substantial amounts of xylans and lignin. Acetyl groups were depleted, but 40% of the total FAs and pCAs remained in pretreated material. Ester-linked pCAs detected in pretreated material also resisted to the enzymatic hydrolysis step. Only a more severe saponification reaction cleaved ester linkages of pCAs from residual solids; nevertheless, pCAs remained attached to the core lignin through 4-O ether-linkages, suggesting the occurrence of an alkali-stable fraction of pCAs in sugarcane bagasse.

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids.

Sugarcane bagasses from three experimental sugarcane hybrids and a mill-reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and ß-glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70-100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944-951, 2018.

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

BACKGROUND: New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. RESULTS: Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O-methyl-glucuronic acid and p-coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. CONCLUSIONS: Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Limitation of cellulose accessibility and unproductive binding of cellulases by pretreated sugarcane bagasse lignin.

BACKGROUND: The effectiveness of the enzymatic hydrolysis of cellulose in plant cell wall is strongly influenced by the access of enzymes to cellulose, which is at least in part limited by the presence of lignin. Although physicochemical treatments preceding the enzymatic catalysis significantly overcome this recalcitrance, the residual lignin can still play a role in the process. Lignin is suggested to act as a barrier, hindering cellulose and limiting the access of the enzymes. It can also unspecifically bind cellulases, reducing the amount of enzymes available to act on cellulose. However, the limiting role of the lignin present in pretreated sugarcane bagasses has not been fully understood yet. RESULTS: A set of sugarcane bagasses pretreated by five leading pretreatment technologies was created and used to assess their accessibility and the unproductive binding capacity of the resulting lignins. Steam explosion and alkaline sulfite pretreatments resulted in more accessible substrates, with approximately 90% of the cellulose hydrolyzed using high enzyme loadings. Enzymatic hydrolysis of alkaline-treated (NaOH) and steam-exploded sugarcane bagasses were strongly affected by unproductive binding at the lowest enzyme loading tested. Analysis of the extracted lignins confirmed the superior binding capacity of these lignins. Sulfite-based pretreatments (alkaline sulfite and acid sulfite) resulted in lignins with lower binding capacities compared to the analogue pretreatments without sulfite (alkaline and acidic). Strong acid groups present in sulfite-based pretreated substrates, attributed to sulfonated lignins, corroborated the lower binding capacities of the lignin present in these substrates. A more advanced enzyme preparation (Cellic CTec3) was shown to be less affected by unproductive binding at low enzyme loading. CONCLUSIONS: Pretreatments that increase the accessibility and modify the lignin are necessary in order to decrease the protein binding capacity. The search for the called weak lignin-binding enzymes is of major importance if hydrolysis with low enzyme loadings is the goal for economically viable processes.