Screening method for Enzyme-based liquefaction of corn stover pellets at high solids.

Liquefaction of high solid loadings of unpretreated corn stover pellets has been demonstrated with rheology of the resulting slurries enabling mixing and movement within biorefinery bioreactors. However, some forms of pelleted stover do not readily liquefy, so it is important to screen out lots of unsuitable pellets before processing is initiated. This work reports a laboratory assay that rapidly assesses whether pellets have the potential for enzyme-based liquefaction at high solids loadings. Twenty-eight pelleted corn stover (harvested at the same time and location) were analyzed using 20 mL enzyme solutions (3 FPU cellulase/ g biomass) at 30 % w/v solids loading. Imaging together with measurement of reducing sugars were performed over 24-hours. Some samples formed concentrated slurries of 300 mg/mL (dry basis) in the small-scale assay, which was later confirmed in an agitated bioreactor. Also, the laboratory assay showed potential for optimizing enzyme formulations that could be employed for slurry formation.

Cellulolytic enzymes production guided by morphology engineering.

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. ß-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or ß-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Protective effects of non-catalytic proteins on endoglucanase activity at air and lignin interfaces.

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air-liquid interface provide rationale. This work investigated root causes including effects of the air-liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air-liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%-50% of its activity due to an air-liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.

Adaptive laboratory evolution of nanocellulose-producing bacterium.

Adaptive laboratory evolution through 12 rounds of culturing experiments of the nanocellulose-producing bacterium Komagataeibacter hansenii ATCC 23769 in a liquid fraction from hydrothermal pretreatment of corn stover resulted in a strain that resists inhibition by phenolics. The original strain generated nanocellulose from glucose in standard Hestrin and Schramm (HS) medium, but not from the glucose in pretreatment liquid. K. hansenii cultured in pretreatment liquid treated with activated charcoal to remove inhibitors also converted glucose to bacterial nanocellulose and used xylose as carbon source for growth. The properties of this cellulose were the same as nanocellulose generated from media specifically formulated for bacterial cellulose formation. However, attempts to directly utilize glucose proved unsuccessful due to the toxic character of the lignin-derived phenolics, and in particular, vanillan and ferulic acid. Adaptive laboratory evolution at increasing concentrations of pretreatment liquid from corn stover in HS medium resulted in a strain of K. hansenii that generated bacterial nanocellulose directly from pretreatment liquids of corn stover. The development of this adapted strain positions pretreatment liquid as a valuable resource since K. hansenii is able to convert and thereby concentrate a dilute form of glucose into an insoluble, readily recovered and value-added product-bacterial nanocellulose.

Deactivation and activation of lignocellulose degrading enzymes in the presence of laccase.

Cellulase and hemicellulase activities in a 1:1 ratio of enzymes extracted from Chrysoporthe cubensis and Penicillium pinophilum were evaluated in the presence of known monocomponent phenolic inhibitors and also with phenol mixtures derived from alkali pretreated sugarcane bagasse. The cellulolytic activities from C. cubensis:P. pinophilum displayed a much higher tolerance to phenolic inhibitors than equivalent enzyme activities obtained from Trichoderma reesei and Aspergillus niger. Enzymes from T. reesei and A. niger were deactivated at 0.3 and 1.5mg phenols/mg protein, respectively, as reported previously, while enzymes from C. cubensis:P. pinophilum resisted deactivation at 35mg phenols/mg protein. However, tolerance of xylanase with respect to phenols required the presence of laccase. Removal of laccase (enzyme) activity using sodium azide resulted in a 2x higher xylanase deactivation (from 40% to 80%). This paper identifies enzymes that are phenol tolerant, and whose adoption for lignocellulose hydrolysis could contribute to reductions in enzyme loading needed to hydrolyze alkali pretreated lignocellulosic substrates in the presence of lignin derived phenols.

Cisto broncogênico em hemicúpula diafragmática esquerda / Bronchogenic cyst in left hemidiaphragm

É descrito o caso raro de mulher com 46 anos de idade portadora de hemangioma de fígado e cisto broncogênico localizado na hemicúpula diafragmática esquerda, tratada com ressecção por cirurgia torácica videoassistida. A paciente teve boa evolução após a remoção de ambas as lesões e encontrase assintomática 21 meses após os procedimentos.

We described a rare case of a 46 year old female patient that had a liver hemangioma and a bronchogenic cyst located in the left hemidiaphragmatic dome treated through a video assisted thoracic surgery procedure. The patient had an uneventful recovery from both lesions and is asymptomatic 21 months following both procedures.

Quilotórax seguindo simpatectomia torácica / Chylothorax following thoracic sympathectomy

Descreve-se o caso de uma paciente submetida a simpatectomia torácica bilateral para tratamento de hiperidrose palmar por via posterior, que teve derrame pleural do tipo quiloso. O tratamento consistiu na ligadura doconduto torácico por intermédio de cirurgia torácica videoassistida, além de dieta pobre em gordura e triglicerídeo de cadeia média. A paciente obteve recuperação total.

The authors describe the case of a female patient who underwent bilateral thoracic sympathectomy for palmar hyperhidrosis by the posterior approach and developed pleural effusion diagnosed as a chylothorax. The treatment consisted in video assisted thoracic duct ligation, drainage of the pleural cavity and institution of total parental nutrition, along with a low fat and medium chain triglyceride diet. The patient had complete recovery.

Identification of two novel xylanase-encoding genes (xyn5 and xyn6) from Acrophialophora nainiana and heterologous expression of xyn6 in Trichoderma reesei.

Two novel genes, xyn5 and xyn6, coding for family 11 xylanases, were isolated from the thermotolerant filamentous fungus, Acrophialophora nainiana, by PCR using degenerate primers. The xyn6 gene was further expressed in Trichoderma reesei. DNA sequence analysis of xyn6 revealed an open reading frame (ORF) of 708 bp, interrupted by an intron of 58 bp. The xyn6 ORF encodes a predicted protein of 236 amino acid residues. The mature recombinant XynVI protein had a molecular mass of about 19 kDa, as estimated by gel electrophoresis. Analysis of the predicted amino acid sequence of XynVI paves the way for rational protein engineering by site-directed mutagenesis aiming to increase the thermostability of the heterologously-expressed xylanase.

A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules.

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.

Fungos anaeróbios / Anaerobic fungi

A descoberta de fungos anaeróbios obrigatórios no rume de ovelha por Orpin, em 1975, impulsionou estudos sobre tais microrganismos em laboratórios de diferentes partes do mundo. Estes estudos têm mostrado que os fungos anaeróbios não contêm mitocôndrias; em vez disso, apresentam organelas conhecidas como hidrogenossomas, que estão envolvidas na geração de energia. Estes fungos ainda produzem enzimas que são encontradas sob a forma de um complexo multiprotéico de alta massa molecular, similar ao celulossoma produzido por bactérias anaeróbias, ou individualmente. Diferentes enzimas hidrolíticas, produzidas por variadas espécies destes fungos, principalmente celulolíticas e hemicelulolíticas, têm sido isoladas e caracterizadas. Estas enzimas, em geral, têm atividades específicas similares ou maiores do que outras produzidas por fontes aeróbias correspondentes, tendo, portanto, grande potencial para aplicação industrial

Amylolytic activity of humicola sp

The thermophylic and cellulolytic fungus Humicola sp. secretes amylases in the liquid culture medium. This activity is induced by starch, maltose and cellobiose. Glucose impairs accumlation of amylolitic activity in the culture medium. The enzyme hydrolyzes starch, maltose and pullulan to glucose as the endproduct