**1. Introduction**

Agro-industrial residues are important sources of lignocellulosic biomass, the main feedstock to be used as a substitute for petrol in a circular bioeconomy. These organic materials are generated in large volumes as a result of agricultural and agro-industrial activities and are in great part subtilized, especially in developing countries that are the main suppliers of commodities. According to Magalhães Jr. et al. [1], around 900 million tons of biomass from agro-industrial residues such as sugarcane bagasse, cereal straws and oil palm solid wastes will be generated in South America in 2025, and their most probable destination, if not left in the field, is burning for energy generation and composting, the most traditional processing methods. However, considering that more than 60% of their mass can be converted to fermentable sugars, these residues could be valorized through microbial processes

to produce second generation (2G) ethanol and other biofuels, organic acids, biomaterials and many other commercial bioproducts.

Nowadays, 2G ethanol is the product with the most mature stage of technological development obtained in lignocellulose biorefineries. However, the cost of 2G ethanol is still not competitive with that of petroleum-based liquid fuels [2], in part because of the cost of enzymes that are necessary to convert fibrous carbohydrates into fermentable sugars. Another step that impacts the overall cost and lacks technological maturity is the pretreatment, necessary to prepare the recalcitrant structure of lignocellulosic biomass to enzymatic hydrolysis or saccharification. In general, the aim of the pretreatment is to enhance the recovery of glucose and other reducing sugars from lignocellulosic biomass in the saccharification step, and this is usually accomplished through the decrease of the crystallinity of cellulose, degree of polymerization, lignin content and moisture content, associated to an increase of available surface area [3].

Several methods have been reported to pretreat lignocellulosic biomass, most of them thermochemical processes. These include steam explosion with or without alkali washing, dilute acid hydrolysis, alkaline pretreatment, organosolv, ammonia fiber expansion, liquid hot water, wet oxidation and others [3]. Alkaline pretreatment is especially efficient in solubilizing lignin, while mild acid pretreatment is more directed to the hemicellulose fraction.

In 2018, the global sugarcane production was of 1.9 billion tons, and Brazil was the main producer accounting for 39% of the total production, followed by India with 20% [4]. In the harvest of 2018/2019, India has surpassed Brazil producing more than 700 million tons, but for 2019/2020 the projections are very similar for both countries [5]. The Brazilian production estimative for the harvest of 2019/2020 is of around 643 million tons [6]. Sugarcane bagasse is a porous residue of cane stalks generated after the crushing and extraction of sugarcane juice. It is composed mainly by cellulose (32–44%), hemicellulose (27–32%), lignin (19–24%) and ashes (4.5–9%). Sugar mills generate approximately 270–280 kg of bagasse (with 50% moisture) per metric ton of sugarcane [7,8].

Soybean is one of the most important sources of vegetable protein for human and animal nutrition and one of the most economical sources of oil for food and biofuel production. According to the United States Department of Agriculture [9], the world production of soybean for 2019/2020 is estimated at 338 million metric tons. The world's leading producer is Brazil and the second is the United States, responsible for 37% and 27% of the global production, respectively. From one ton of soybean with around 13% moisture, 50 kg of soybean husks are obtained [10]. The composition of soybean husks depends on the dehulling process, so they may contain varying amounts of cellulose (29–51%), hemicelluloses (10–25%), lignin (1–4%), pectins (4–8%), proteins (11–15%) and minor extractives [11].

The global production of oil palm is estimated at 73 million metric tons for the period of 2019/2020, Indonesia and Malaysia being the leading producers with 58% and 26% of the total, respectively [9]. The milling of oil palm to extract the oil generates a solid fibrous residue, named oil palm empty fruit bunches (EFB). It is estimated that for each ton of fresh fruit bunches processed, 220 kg of EFB are generated. This residue is composed of around 30, 25 and 25% (mass percentages) of cellulose, hemicellulose and lignin, respectively [12].

The present work aimed at evaluating the effect of different enzyme preparations and combinations in the saccharification of these three important agro-industrial residues, the sugarcane bagasse, soybean husks and oil palm empty fruit bunches, pretreated by mild acid and alkali. The enzyme pools were represented by cellulase and xylanase complexes of *Penicillium verruculosum* (B1 host preparation), by the B1-XylA preparation obtained through the recombinant expression of *Penicillium canescens* xylanase A in the *P. verruculosum* B1 host strain, and by the F10 preparation obtained by recombinant expression of *Aspergillus niger* β-glucosidase in the B1 host strain. All preparations were crude, which could significantly reduce the cost associated to the enzymatic saccharification step.
