Assessing potential effects of a laccase extract over the enzymatichydrolysis of Eucalyptus bark residues

Lignocellulosic materials are rising as an alternative to petroleum, from which biofuels and numerous compounds may be produced. Eucalyptus barks, abundantly generated by pulp & paper mills, are a good example of such materials, being typically used for energy production. Holocellulose conversion of these materials is usually made by enzyme preparations, mainly acting on the hydrolysis of complex cellulose into monomer sugars. These materials, however, can still present a substantial amount of lignin, a well-documented enzymes barrier. This work aimed to assess how a laccases extract can influence the hydrolysis of eucalyptus bark and the best conditions for their action. Eucalyptus bark residues (EBR) were initially subjected to autohydrolysis with a severity (S0) of 3.84 [1]. The pre-treated solid was then hydrolyzed using Cellic CTec2, combined with a laccases-mediated treatment employing an extract prepared by the group of Maria de Lourdes Polizelli [2]. Potential effects of laccases were estimated through the quantification of the glucose produced over time and differences in the profile of enzymes adsorption onto the solid. The effects of laccases over the hydrolysis of EBR seemed to be dependent of numerous factors. For a solids load of 2 %, laccases addition simultaneously with cellulases had no positive effects but when added 24 h before cellulases, glucose production increased 11 %, possibly from an inferior electron donors competition with LPMOs on Cellic Ctec. Increasing laccases dosage from 2 to 10 IU/g solid led to a visible reduction of hydrolysis efficiency, suggesting possible toxicity/inhibition effects above a given level. Applying a washing step showed to be efficient in removing some of the formed phenolics, while its overall benefit seemed to depend on the extension of laccases action before being washed. When an efficient laccases treatment was conducted before the washing step, involving reduced mass transfer limitations and an adequate period of time, subsequent enzymatic hydrolysis produced nearly 30 % more glucose for a 8 % solids load. In accordance, there was also a significant increase on the levels of free Cel7A after hydrolysis of this new solid, suggesting important modifications on the levels and structure of its lignin. The utilization of laccases on the hydrolysis of lignocellulosic biomass may represent an interesting element for more efficient and economic processes.This work had the financial support from the Portuguese Foundation for Science and Technology under the scope of Project EcoTech (POCI-01-0145-FEDER-032206). The authors also acknowledge RAIZ for kindly providing the residues of eucalyptus bark.info:eu-repo/semantics/publishedVersio

Biogas production through co-digestion of enzymatically pretreated corn bran and cow manure

Biogas production from wastes is an alternative that contributes positively to the environment and minimize the dependence on fossil energy sources. Additionally, the reuse of biomasses helps to reduce the waste production, but a pretreatment is required to use it in the anaerobic digestion. Here biogas was produced through co-digestion of enzymatically pretreated corn bran and cow manure. Firstly, it was selected the most hydrolysable waste (barley bagasse, sugar cane bagasse, elephant grass, thick orange pie, average orange pie, wheat bran, coffee grounds, orange peel, white sludge, vinasse, corn bran, soy bran, soy peel, cotton bran, cassava husk, cassava flour, banana peel, corn bran, sorghum stem, sorghum seed, total sorghum and wet distiller grain) by the crude extracts containing amylase (secreted by Aspergillus brasiliensis), xylanase (Aspergillus tamarii Kita) and cellulase (Trichoderma reesei, Novozymes®). Later on, different mixtures of these enzymes were studied using simplex-centroid designs. The most hydrolyzed waste by each enzyme individually (measured by reducing sugar using dinitrosalicylic acid, DNS) at 50°C, 120 rpm and 24 h were corn bran, banana peel and sorghum seed. Then, the simplex-centroid designs resulted in model equations and respective response surface contours. Amylase extract had a significant positive influence on corn bran hydrolysis by maximizing the reducing sugar yield when it was used individually (35g/L of reducing sugar). After it, the pretreated corn bran and a cow manure (1:2 g of volatile solids) were employed for biogas production in batch assays. It was found a biogas accumulation of 326 mL in the 12nd day of anaerobic codigestion, which were similar to the control (containing 35 g/L of glucose alone) and 53% higher than that found with corn bran without enzymatic pretreatment. In conclusion, it was observed that the crude extract optimized for amylase production affected significantly the corn bran hydrolyses and consequently the biogas production in a co-digestion with cow manure.CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico process 142139/2017-3)FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo process 2018/07522-6)FCT (Fundação para a Ciência e Tecnologia)info:eu-repo/semantics/publishedVersio

Nanocellulose production: exploring the enzymatic route and residues of pulp and paper industry

Increasing environmental and sustainability concerns, caused by current population growth, has promoted a raising utilization of renewable bio-resources for the production of materials and energy. Recently, nanocellulose (NC) has been receiving great attention due to its many attractive features such as non-toxic nature, biocompatibility, and biodegradability, associated with its mechanical properties and those related to its nanoscale, emerging as a promising material in many sectors, namely packaging, regenerative medicine, and electronics, among others. Nanofibers and nanocrystals, derived from cellulose sources, have been mainly produced by mechanical and chemical treatments; however, the use of cellulases to obtain NC attracted much attention due to their environmentally friendly character. This review presents an overview of general concepts in NC production. Especial emphasis is given to enzymatic hydrolysis processes using cellulases and the utilization of pulp and paper industry residues. Integrated process for the production of NC and other high-value products through enzymatic hydrolysis is also approached. Major challenges found in this context are discussed along with its properties, potential application, and future perspectives of the use of enzymatic hydrolysis as a pretreatment in the scale-up of NC production.This work was carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)- LISBOA-01-0145-FEDER-022059, supported by Operational Programme for Competitiveness andInternationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Programme (Lisboa 2020)and by North Portugal Regional Operational Program (Norte 2020) under the Portugal 2020 PartnershipAgreement, through the European Regional Development Fund (ERDF) and was supported by the PortugueseFoundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 and through Project EcoTech (POCI-01-0145-FEDER-032206/FAPESP 2018/07522-6) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope ofNorte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Using a polyphasic approach to identify wild Brazilian fungal strains

[Aim] The species must be delineated based on a polyphasic approach, including morphology, physiology, profile of secondary metabolites and molecular biology [1]. According to Santos et al. [2;3] it is clearer that spectral analyses add value to the polyphasic approach. This work aimed to perform a polyphasic approach based on morphological, biochemical and spectral analysis by MALDI-TOF ICMS for identify Aspergilli isolates from different environments of Brazil

Identification of brazilian aspergilli based on a polyphasic approach including MALDI-TOF ICMS

Publicado em "Biological resource centres : closing the gap between science and society : abstracts book...". ISBN 978-972-97916-5-9Microbial culture collections were established to preserve cultures of fungi and bacteria for taxonomical studies. However, nowadays they are also important for the ex situ conservation of microbial biodiversity. They are responsible for collecting, cataloguing, identifying and preserving strains for biomedical research, teaching, industry, agriculture, etc. Hence, this current work performed a polyphasic study based on morphology, biochemistry and MALDI-TOF ICMS to identify aspergilli from different environments in Brazil. Thirteen isolates of Aspergillus spp. deposited at the Filamentous Fungi Collection of Ribeirão Preto (CFF-RP) were analysed. Strains were grown on Czapek-Dox Agar (CZA) and Malt Extract Agar (MEA) at 30°C for morphology. Biochemical characterisation (production of ochratoxin A and fumonisin B2) was performed by HPLC. The MALDI-TOF ICMS analysis were performed on an Axima LNR system (Kratos Analytical, Shimadzu, Manchester, UK) equipped with a nitrogen laser (337 nm), using a mass range from m/z=2000 to 20000 Da and Escherichia coli DH5α strain for external calibration. The fungal identifications were performed using SARAMIS software (AnagnosTech mbH, Postdam-Golm, Germany). One A. thermomutatus was an OTA producer. In contrast, fumonisin B2 was no detected for all strains studied. The MALDI-TOF ICMS results corroborated the morphological identifications. Of the 13 isolates, 38, 31, 15.5 and 15.5% were A. fumigates, A. niveus, A. thermomutatus and A. ochraceus, respectively. These results contribute to knowledge about microbial biodiversity from the Brazilian environment

Effect of oxygen transfer rate on cellulases production in stirred tank and internal-loop airlift bioreactors

In an aerobic process, such as enzymes production by fungi, the oxygen supply into fermentation medium is an important factor in order to achieve good productivities. Oxygen has an important role in metabolism and microorganism growth, being of extreme importance the control of both the dissolved oxygen transfer rate into the bioreactor and the oxygen consumption by the microorganism [1,2]. Dissolved oxygen transfer rate can be analyzed and described by means of the mass transfer coefficient, KLa, being one of the most important parameters for the design and operation of mixing/sparging of aerobic bioreactors. (…

Cloning, purification, and biochemical characterization of an esterase from Aspergillus nidulans

A large accumulation of agro-industrial waste from different segments is generated daily and is often not properly managed. There are now other fronts in research to give a destination to these residues; these studies are generally aimed at obtaining new and better enzymes and the formulation of enzymatic cocktails that contain (for example, cellulases and hemicellulases) responsible for the degradation of lignocellulosic material. The plant cell wall is mainly composed of cellulose, hemicellulose, and lignin, forming a complex structure. Xylan is one of the main constituents of hemicellulose. To degrade this structure, enzymatic hydrolysis must occur synergistically with xylanolytic enzymes, such as endo-beta-1,4-xylanases, -xylosidases, and acetyl xylan esterase (AXE). In the current work, we reported the purification and biochemical characterization of an acetyl xylan esterase (AxeCE3) from Aspergillus nidulans. The axeCE3 gene was cloned into the pEXPYR vector and transformed into A. nidulans A773 for protein expression. The enzyme AxeCE3 was purified and characterized for its biochemical properties. AxeCE3 showed activity over a wide range of pH (3.0-9.0) and temperature (30-70 °C), with maximum activity at 55 °C, pH 7.0. Regarding the stability at temperature, AxeCE3 showed values above 90% of residual activity after 24 h of incubation at 45 and 50 °C. In relation to stability at pH, AxeCE3 maintained more than 90% of its residual activity after being incubated at 25 °C for 24 h between the pH range 3.0 to 9.0. It was also verified the effect of possible inhibitors (ethylenediamine tetraacetic acid (EDTA), Furfural, and 5- Hydroxymethylfurfural (5-HMF)) on the enzyme activity. AxeCE3 maintained 88% of relative activity at 5 mM EDTA, 43% and 82% at 50 mM furfural and 5-HMF, respectively. The results showed that AxeCE3 has interesting properties to use in the development in the formulation of enzymatic cocktails for the hydrolysis of lignocellulosic residues.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and process 301963/2017-7. Research scholarships were granted to RCA and DA by FAPESP (Grant No: 2020/00081-4 and No: 2020/15510-8), to GSA by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Finance Code 001).info:eu-repo/semantics/publishedVersio

Pretreatment of brewers' spent grains for cellulases production by Aspergillus niger van Tieghem

Successful utilization of cellulosic materials as a renewable carbon source is dependent on the development of economically feasible process technologies both for the production of biomass-degrading enzymes, and for the enzymatic hydrolysis of cellulosic materials to low molecular weight products. Significant cost reduction is required in order to enhance the commercial viability of cellulase production technology and biomass pretreatment can be an essential processing step for this purpose. Thus, the aim of this work was to evaluate the performance of pretreated brewers´ spent grains on the improvement of cellulases production by A. niger van Tieghem. For this, brewers´ spent grains was submitted to autohydrolysis treatment. Initially, the material was dried, milled and sieved (1.0 mm screen). Water was added to the sample in a closed and pressurized vessel (solid/liquid ratio 1:10 w/v), and the system heated to 180, 190 or 200ºC for 10, 35 or 50 min. The liquor obtained (hemicelluloses fraction) was separated from the solids (cellulose/lignin) by filtration and both fractions were used together or not as carbon source on fermentation: 1% (w/v) treated solid fraction; 1% (w/v) solids plus 10% (v/v) liquor, or only liquor. Carboxymethylcellulose, avicel and untreated brewers´ spent grains were used as control. The inocullum was done in Mandels medium and the cultivation conditions were 30ºC/100 rpm for 6 days. Carboxymethylcellulase (CMCase) and avicelase were assayed by DNS using 1% (w/v) carboxymethylcellulose in sodium acetate buffer, pH 4.0 and 1% (w/v) avicel in the same buffer, pH 5.0, respectivelly, while β-glucosidase was detected by p-nitrophenolate released using 5 mM pnp-β-D-glucoside in sodium citrate buffer, pH 4.5. One unit of enzymatic activity was defined as the amount that liberated 1 μmol of product per minute on assay conditions. The results showed that the liquor obtained at 190ºC/50 min autohydrolysis was quite favorable to CMCase and avicellase production, since the enzyme production was significantly higher than with other sources. However, the effect of the treatment on β-glucosidase production was not as significant as the control. These results show that by using autohydrolysis liquor as an alternative substrate, the performance of the bioprocess for cellulase production can be improved

Evaluation of autohydrolysis process for cellulases production by Aspergillus niger van Tieghem using corncob biomass

Lignocellulosic residues, such as corncob, are a complex matrix composed by cellulose, hemicellulose and lignin that can be used for different biotechnological applications (e.g. enzymes production). However, the applications of these crude residues as substrate for enzymes production are often inefficient. An efficient hydrolysis of these residues requires a pretreatment (e.g. autohydrolysis) that lead to a more accessible structure for microorganisms attack. Recently, fungi have received significant attention as a source of new thermostable enzymes for use in many biotechnological applications, including biomass degradation (cellulases are key enzymes for efficient biomass degradation) [1]. The enzymatic degradation of cellulose to glucose is achieved by the cooperative action of endoglucanases (EC 3.1.1.4, hydrolyze randomly the internal glycosidic linkages), exoglucanases (cellobiohydrolases, CBH, EC 3.2.1.91, hydrolyze cellulose chains by removing cellobiose mainly from the non-reducing ends) and _-glucosidases (EC 3.2.1.21, cleave cellooligosaccharides and cellobiose to glucose) [2]. In this context, this work evaluates the inclusion of pretreated corncob in the nutrient media for cellulose production by Aspergillus niger van Tieghem in comparison with non-treated corncob. Autohydrolysis pretreament conditions used were 180, 190 and 200 ºC for 10, 30 and 50 min, and two fractions were obtained: solid and liquid fractions enriched by cellulose and hemicellulose, respectively. Three different mixtures (for each condition) were used as carbon source in Mandels medium [3] during the cellulases production by the A. niger van Tieghem: a solid fraction (1% w/v in medium), a liquid fraction (100 % v/v in medium), and a mixture of the solid and liquid fractions (1% w/v + 10% v/v in medium). Fermentation conditions were at 30ºC, 100 rpm, and the cellulases and _-xylosidase were quantified by Miller [4] and Kersters-Hilderson [5] methods, respectively, after 6 days of fermentation. Interestingly, the results showed that the highest cellulases production was obtained when the microorganism grows in medium containing the hemicellulose fraction (or liquid fraction) as carbon source. The exoglucanase and endoglucanase production using the liquid fraction obtained at 200ºC for 30 min, were three and twenty times higher, respectively, than the production obtained using corncob untreated, as carbon source. In relation to _-g production, the best autohydrolysis condition was 180ºC for 30 minutes; this production was fifteen percent higher than the production detected with crude corncob. This work shows the potential of autohydrolysis retreatment of lignocellulosic residues as a strategy to increase and add-value the cellulase production by filamentous fungi

Production of an enzymatic cocktail by Aspergillus awamori grown on corn straw with stirred tank bioreactor

Increased agroindustrial activity has led to the accumulation of large amounts of lignocellulosic material (LCM). LCM is nature's most abundant source of renewable carbon, representing a valuable industrial substrate with potential for many applications. Thus, the objective of this work was to screen for different holocellulases and to analyze the production of an Aspergillus awamori enzyme cocktail grown in corn straw (CS) using a stirred tank bioreactor. Screening was performed with A. clavatus, A. flavus, A. terreus, A. niveus, A. awamori and A. brasiliensis cultivated in minimal medium (MM), with 1% CS at 30 ºC or 37 ºC (for A. niveus), 120 rpm, for 5 days. Xylanase (XYN) and endoglucanase (EG) activities were evaluated by formation of reducing sugars using dinitrosalicylic acid (DNS). Cellobiohydrolase (CBH), -glucosidase (BGL) and -xylosidase (BXL) were determined by cleavage of PNP--D-cellobioside, PNP--D-glucopyranoside and PNP--D- xylopyranoside, respectively. After screening, a pre-inoculum was prepared with the best enzyme producer using a 500 mL MM flask and incubated at 30 °C, 120 rpm for 48 hours. The increase of enzyme production was performed in a Benchtop BioFlo 310 bioreactor, with 4.5 L of MM and 1% of CS, and was then inoculated the best enzyme producer. Cultivation was performed at 30 °C, pH 6.5, 275 rpm, air flow 2 v.v.m., for 5 days. During the screening, all fungi presented EG, CBH, BGL, XYN and BXL activities. However, A. awamori was chosen to continue the experiments because of its BXL activity which was 12.6 times higher than that produced by A. niveus. At the scale-up stage, XYN production (47.80 U/ mL) increased 4.1-fold compared to flask activity (11.52 U/mL). BXL also showed 1.6-times higher activity, as well as EG, CBH and BGL, which improved 2.3, 3.3 and 1.2 times their activities, respectively. It was concluded that the staggering of cocktail production improved the enzymatic activities and that corn straw is an excellent source of induction. Furthermore, this cocktail has the potential to be applied in the hydrolysis of different LCM due to the range of holocellulases present.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Institutos Nacionais de Ciência e Tecnologia (INCT)Fundação para a Ciência e a Tecnologia (FCT)info:eu-repo/semantics/publishedVersio