Brewers' spent grain as substrates for production of cellulolytic and hemicellulolytic enzymes by different Aspergillus species

Brewers' spent grain (BSG) is the major by-product of the brewing industry, representing around 85% of the total by-products generated. It is a lignocellulosic material containing about 38% cellulose, 29% hemicellulose, chiefly arabinoxylans, and 13% lignin. The production of cellulolytic and hemicellulolytic enzymes using this material as substrate represent an eco-friendly strategy for the lignocellulosic biomass hydrolysis, generating fermentable sugars that can be converted into high- added value products, such as bioethanol, lactic acid, xylitol and others. Thus, this work aimed to evaluate the potential of cellulolytic and hemicellulolytic enzymes production by some Aspergillus species cultivated in BSG. Fungi were grown in minimum media, pH 6.5, with 1% BSG and inoculum was done with 107spores/mL, cultivated at 30°C, 120 rpm, for 5 days. Every 24 hours 2 mL of the samples were collected. The enzymatic activity was performed after the incubation of the crude extract with 1% Linear arabinan, Xylan from beechwood, Xyloglucan, Locust bean gum and CMC, at 50°C for 60 minutes and the reducing sugars were determined using dinitrosalicylic acid (DNS). Synthetic substrates (2 mM of PNP--L-arabinofuranoside, PNP--D-xylopyranoside, PNP--Dglucopyranoside and PNP--D-cellobioside) were also used at the same conditions. The extract from A. niveus showed the best arabinanase (0.284 U/mL) and -glucosidase (0.126 U/mL) activities after 48 and 96 hours of cultivation, respectively. On the other hand, the extract from A. brasiliensis presented the best activities of -L-arabinofuranosidase (0.129 U/mL), -xylosidase (0.265 U/mL) and xylanase (2.15 U/mL) when cultivated for 48 hours. After 72 hours, this fungus also showed the best activities for xyloglucanase (1.06 U/mL), mannanase (0.617 U/mL) and endoglucanase (0.254 U/mL). The extract produced by A. flavus presented the best cellobiohydrolase activity with 0.113 U/mL after 120 hours of cultivation. It is important to mention that A. awamori, A. clavatus and A. terreus also showed good levels of different enzymes produced but they were not the best producers. These data suggest the great potential of different cellulolytic and hemicellulolytic enzymes production using BSG as substrate, which represents an eco-friendly destination for the residues and can generate high-added value products with great biotechnological application.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

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