Multi-step approach to add value to corncob: production of biomass-degrading enzymes, lignin and fermentable sugars

This work presents an integrated and multi-step approach for the recovery and/or application of the lignocellulosic fractions from corncob in the production of high value added compounds as xylo-oligosaccharides, enzymes, fermentable sugars, and lignin in terms of biorefinery concept. For that, liquid hot water followed by enzymatic hydrolysis were used. Liquid hot water was performed using different residence times (1050 minutes) and holding temperature (180200 °C), corresponding to severities (log(R0)) of 3.364.64. The most severe conditions showed higher xylo-oligosaccharides extraction (maximum of 93%) into the hydrolysates and higher recovery of cellulose on pretreated solids (maximum of 65%). Subsequently, hydrolysates and solids were used in the production of xylanases and cellulases, respectively, as well as, pretreated solids were also subjected to enzymatic hydrolysis for the recovery of lignin and fermentable sugars from cellulose. Maximum glucose yield (100%) was achieved for solids pretreated at log(R0) of 4.42 and 5% solid loading.Michele Michelin is a recipient of a FCT fellowship (SFRH/BPD/ 100786/2014). ThisstudywassupportedbythePortugueseFoundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-010145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. Héctor Ruiz would like to thank the financial support to the Mexican Science and Technology Council (CONACYT, Mexico) for the Basic Science Project-2015-01 (Ref. 254808) and the Energy Sustainability Fund 2014-05 (CONACYT-SENER), Mexican Centre for Innovation in Bioenergy (Cemie-Bio), and Cluster of Bioalcohols (Ref. 249564). We thank Dr. Nelson Lima from MUM (Micoteca da Universidade do Minho, PT) that gently provided the Trichoderma reesei fungi.info:eu-repo/semantics/publishedVersio

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

Identification of Brazilian wild-type isolates of Aspergillus fumigatus based on polyphasic approach including MALDI-TOF MS technique

Currently is accpeted that the majority of the unknown species in the world are in the tropics and Brazil possibly holds more than 25% of this biodiversity. In the filamentous fungi case the number of new species described for science in Brazil is very small when compared with the potentiality of the region. In addition, there is expected to find a large biological variability inside of described and new fungal species. Species belonging to the Aspergillus genus are responsible for invasive aspergillosis. This is gaining prominent position because of their capability to affect immunocompromised and immunosuppressed patients and aspergillosis is most commun mould infection more recognised worldwide. A better understanding about the regional fungal traits can help to a fast and effective treatment of patients with aspergillosis. In order to set up a collection containing regional fungal strains with clinical relevance to attend the great variability (morphology, biochemical, genomics and proteomics patterns) of the Brazilian fungal population 13 wild-type Aspergillus isolates from section Fumigati were studied. They were isolated from the five official Brazilian regions (North, Northeast, Centre-east, Southeast and South) and identified based on a polyphasic approach which is. based on morphological, biochemical, molecular biology and spectral analyses by MALDI-TOF MS. After collected, purified and identified all 13 Aspergilli isolates were preserved at Filamentous Fungi Collection of Ribeirão Preto (CFF-RP), São Paulo, Brazil. Data obtained from the polyphasic approach led to a final fungal identification at species level. Overall, MALDI-TOF MS was the fast technique for identify the species. However, molecular biology was the gold standard for the species identification once it was used to validate data obtained from the other techniques mentioned above. Moreover, either MALDI-TOF MS or molecular biology should not be used as a single step for fungal identification.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil)

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