Growing global concerns about climate change, CO2 reduction, and fossil fuel depletion have stimulated interest in a circular bioeconomy. This awareness has prompted investigation into the biosynthesis of various value-added chemicals from renewable biomass [1], [2], [3], [4]. The carbohydrate content of the cellulose and hemicellulose in biomass is considered the most promising feedstock in biochemical production because it is an abundant, inexpensive, and non-edible feed source [1]. Cellulose is a linear homopolymer of linked β-1,6 glucan that has been extensively utilized in a saccharification process to release D-glucose, which then could be utilized either as a feedstock for bioethanol or converted into a chemical product [2], [3], [4], [5]. On the other hand, hemicellulose, which is a complex polysaccharide composed of various sugars, has received less attention [1]. Hemicellulose is often hydrolysed to simple sugars for fermentation, but it has a unique sugar structure that makes it a rare sugar compound with prebiotic qualities that could realize a market value higher than that of the monosaccharide D-glucose [6].

Xyloglucan, a major hemicellulose found in plant cell walls and seeds, is composed of a β-1,4-linked D-glucose backbone decorated with α-1,6-linked xylose side chains in a repeated pattern with every four glucose molecules left undecorated. In some plant species, these xylose side chains are substituted by β-1,2-linked galactose or fucose residues [7]. Isoprimeverose (α-D-xylopyranosyl-(1→6)-β-D-glucopyranose), a disaccharide consisting of one unit of glucose and xylose, is a unique sugar motif found in the complex structure of tamarind xyloglucan [8]. Due to its specific structure, oligoxyloglucan derivatives, including isoprimeverose, have been reported to exhibit prebiotic properties in the gut microbiome population [9], [10]. The valorization of tamarind xyloglucan into isoprimeverose also holds significant economic potential because commercially available isoprimeverose is relatively expensive (>500 USD/10 mg) by comparison with the cost of tamarind xyloglucan [11], which constrains the study and application of isoprimeverose. Enzymatic hydrolysis is a promising green process that is used to produce valuable rare sugars from hemicellulose. However, the construction of highly productive recombinant microorganisms is essential in order to achieve a cost-effective enzyme for bioprocesses.

Aspergillus oryzae is a filamentous fungus renowned for its secretion of robust enzymes and is generally recognized as safe (GRAS) [12], [13]. A. oryzae is historically used in Asian food fermentation and as a source of industrial enzymes such as amylase, lipase, and protease that are used in food and brewing, for biomass degradation, and in biodiesel conversion [12], [14]. The availability of complete genome sequences and advances in genetic engineering tools have expanded the versatility of A. oryzae for bioproduction [15], [16], [17], [18]. Previous studies have used complex genetic engineering of A. oryzae to produce a cellulase enzyme cocktail of lactic acid, kojic acid, and 2,3-butanediol [19], [20], [21], [22]. These findings collectively highlight the usefulness of A. oryzae for bioproduction from renewable biomass.

Our research group has developed a process for the production of isoprimeverose from tamarind xyloglucan using an enzyme cocktail from genetically engineered A. oryzae. Using this process, two self-cloning A. oryzae strains expressing extracellular endoglucanase (EG) and specific isoprimeverose oligoxyloglucanase (IpeA) were successfully constructed. EG hydrolyzes unbranched β-1,4-glycan from tamarind xyloglucan polysaccharide, and IpeA releases isoprimeverose from the non-reducing end by hydrolyzing the β-1,4-glycan backbone from xylo-oligosaccharides [23], [24]. The combination of these enzymes is essential for liberating isoprimeverose from tamarind xyloglucan, and a more than 90 % conversion ratio from 20 g/L of tamarind xyloglucan was achieved in our previous study [25]. As far as could be ascertained, commercial IpeA has only been reported as a fraction in the enzyme mixture of Driselase (Sigma-Aldrich) [26], which is not ideal for large-scale isoprimeverose production. The current study presented an opportunity to develop a process for the mass production of IpeA. However, in order to cultivate filamentous fungi in submerged fermentation, certain practicalities in liquid transfer handling had to be overcome due to mycelia aggregate formation. Controlling filamentous growth would potentially have a positive effect on oxygen and nutrient transfer rates, which could enhance productivity under large-scale production.

In the present study, therefore, we investigated the effects that fermentation parameters could exert on large-scale fermentation. We tested different types and concentrations of the source of carbon. We tried different levels of pH. Also, we tried two different impeller shapes: a Disk Turbine (DT) and a MAXBLEND® (MB). As a result, we verified the scalability of A. oryzae fermentation from flask level to a 5 L bioreactor.