Chemicals and other materials

All chemicals were of analytical grade (gradient grade in the case of chromatography solvents), unless stated otherwise. 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, purity > 98%) was obtained from AppliChem (Darmstadt, Germany). All other chemicals were purchased from Merck, Sigma-Aldrich and Th. Geyer GmbH (Renningen, Germany). The enzyme mixtures Celluclast 1.5 L and Viscozyme L were obtained from Sigma-Aldrich (Merck Group, Darmstadt, Germany). Celluclast 1.5 L, a cellulase from T. reesei, had an enzymatic activity of 756 glucanase units (GU)/g and was delivered at a concentration of 1.22 g/mL. Viscozyme L, a commercial cellulolytic enzyme mixture from Aspergillus sp., is a blend of beta-glucanases, pectinases, hemicellulases, and xylanases. According to the manufacturer (Novozymes Corp., Bagsvӕrd, Denmark) the main enzymatic activity of Viscozyme L was represented by beta-glucanase at 108 GU/g, corresponding to a concentration of 1.21 g/mL.

Source and maintenance of fungal strains

The investigated fungi included the species S. chlorohalonata, S. rugosoannulata (Duong et al. 2022a), S. commune, G. trabeum, T. reesei, P. chrysogenum, and G. butleri (Duong et al. 2022b). S. chlorohalonata A-2008–2 (DSM 27588), S. rugosoannulata (DSM 11372), G. trabeum (DSM 1398), and P. chrysogenum (DSM 848) were obtained from the strain collection of the Department of Applied Microbial Ecology at the Helmholtz Centre for Environmental Research-UFZ (Leipzig, Germany). They are also available from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). G. butleri (DSM 2917), S. commune (DSM 11223), and T. reesei (DSM 769) were derived from the DSMZ. The fungal strains were maintained on 2% (w/v) malt extract agar plates (1.5% agar; pH 5.7) at 28 °C in the dark.

Fungal cultivations on wheat straw and sample preparation for non-calorimetric analytical procedures

Wheat straw pretreatment through axenic fungal cultivation and the subsequent extraction of water-soluble compounds are comprehensively described in Duong et al. (2022a,b). Briefly, fungi were grown on milled and prewetted sterile wheat straw (0.5 g dry mass, about 2 mm particle size) in previously sterilised calorimetric polypropylene vials (total volume 40 mL) equipped with venting PTFE membrane screw caps. For preparation of fungal inocula, agar plugs (derived from the edges of fungal colonies grown on malt extract agar plates as described above) were homogenised in 2% malt extract medium (one agar plug per 1 mL of malt extract medium) with the help of an ULTRA-TURRAX® (IKA-Werke GmbH & Co. KG, Staufen, Germany). Thereafter, 0.5 mL of the resulting fungal suspension was used to inoculate one calorimetric vial, respectively. After inoculation, vials were closed with the sterile venting membrane screw caps mentioned before. Further vials containing untreated (i.e. without autoclaving and fungal inoculation) and autoclaved wheat straw (without fungal inoculation), respectively, were also established for comparison. For fungal cultivation and recording of calorimetric data, the vials were placed in independent measurement channels in a MC CAL isothermal microcalorimeter (C3 Prozess- und Analysentechnik GmbH, Haar b. München, Germany). The working temperature was set to 28.00 ± 0.01 °C. All further details can be retrieved from Duong et al. (2022a,b). After total incubation periods of either 32 (in the case of S. rugosoannulata and S. chlorohalonata) or 20 days (all other fungi), the vials were harvested and stored at − 20 °C. Thereafter, 0.1 M McIlvaine buffer (McIlvaine 1921) (pH 7.0) was applied in order to extract water-soluble compounds from the solid materials in a step in the following referred to as “first extraction” (please also refer to Fig. 1). Aqueous supernatants resulting from the extractions were stored at − 20 °C until further analyses described below. Total dry masses of solid fractions remaining from the first extraction were determined as previously described (Singh et al. 2014; Duong et al. 2022a).

Fig. 1

Schematic overview of aqueous extractions and enzymatic digestion of lignocellulosic samples and the sugar fractions derived thereof

Enzymatic digestion of samples

Solids remaining from total dry mass determination were homogenised with a ball mill (Pulverisiette 23; Fritsch, Idar-Oberstein, Germany) at 50 oscillations per second for 5 min and stored at ambient temperature under dry conditions in the dark until enzymatic digestion (hydrolysis) was carried out. The commercial enzyme preparations Celluclast 1.5 L (a cellulase) and Viscozyme L (a blend of beta-glucanases, pectinases, hemicellulases and xylanases) were applied based on previously published data (López-Gutiérrez et al. 2021) in mixture at 121 and 15.8 GU/g dry lignocellulosic solid, respectively. Enzymatic hydrolysis was carried out in 0.1 M Na-citrate buffer (pH 4.8) containing 0.2 g/L tetracycline in addition in order to suppress bacterial growth in the reaction mixtures. Lignocellulosic solid samples were applied at 2.5% (w/v). Incubations were carried out at 150 rpm and 40 °C for 18 h, hereby concomitantly extracting water-soluble sugar fractions (further on referred to as “second extraction”; Fig. 1). After that, samples were immediately frozen at − 20 °C and stored at this temperature until analysis. Additional lignocellulosic solids were always incubated in parallel, employing Na-citrate buffer in the absence of hydrolysing enzymes. Samples of untreated (not autoclaved) and autoclaved wheat straw without fungal pretreatment were also incubated in the presence and absence of hydrolysing enzymes, respectively, and served as controls. All incubations described above were carried out in triplicate.

Overview of the sugar fractions obtained from the different aqueous extraction steps and enzymatic digestion

After thawing the samples obtained from different aqueous extraction steps and enzymatic digestion as described above, these were centrifuged at 4 °C and 20,817 × g for 10 min (Eppendorf centrifuge 5430-R, rotor type FA-45–30-11; Eppendorf, Hamburg, Germany). The resulting aqueous supernatants were kept at − 20 °C for further analyses, which included the determination of total reducing sugars based on the dinitrosalicylic acid (DNSA) method, the determination of total sugars after acidic hydrolysis using the phenol–sulphuric acid method, and the determination of glucose, xylose, and fructose with the help of ultra-performance liquid chromatography (UPLC) as described below, respectively. A schematic summary of the different extraction and enzymatic digestion steps and the sugar fractions derived thereof is illustrated in Fig. 1. Three important sugar fractions were defined. The “readily bioavailable sugar” fraction refers to the sum of sugars derived from the first and the second extraction without enzymatic digestion. In a technical process such as biogas or bioethanol production from lignocellulosic substrates, this water-soluble sugar fraction could be expected to be immediately available to fermenting microbes. Another sugar fraction would become available to fermenting microorganisms only after digestion with hydrolytic enzymes (Fig. 1), which might be produced by the microorganisms themselves or exogenously added. Finally, the sum of these two fractions is further on referred to as “total bioaccessible sugars” (Fig. 1), representing both sugars that would immediately be available and those becoming available to fermenting microbes over time.

Total reducing sugar determination based on the DNSA method

Amounts of total reducing sugars in aqueous extracts of solid substrates were determined using the dinitrosalicylic acid (DNSA) method (Bailey 1988; Gonçalves et al. 2010). Briefly, 25 µL DNSA reagent (1% (w/v) DNSA, 30% (w/v) potassium sodium tartrate, 1,6% (w/v) NaOH) was added to 25-µL sample in the wells of a 96-well microplate (flat bottom). Subsequently, the microplate (covered with a lid) was placed on the orbital shaker at 150 rpm for 30 s, followed by incubation in the oven at 85 °C for 10 min. After that, the microplate was cooled down on ice, and 250 µL of distilled water was immediately added to the wells. The absorbance was read at 530 nm, using a GENios Plus microplate reader (Tecan, Männedorf, Switzerland). Calibration of the method was carried out using a mixture of equal amounts of D-glucose and D-xylose, based on essentially comparable amounts of glucan and xylan in wheat straw as reported before (García-Torreiro et al. 2016).

Determination of total sugars after acidic hydrolysis based on the phenol–sulphuric acid method

Amounts of total sugars in aqueous extracts of solid substrates were photometrically determined after acidic hydrolysis as previously described (Singh et al. 2014; Duong et al. 2022a), based on the phenol–sulphuric acid method (Dubois et al. 1956).

Ultra-performance liquid chromatography (UPLC) analysis of individual sugars in lignocellulosic samples

Aqueous supernatants (0.5 mL) of samples arising from the enzymatic digestion and second extraction step (Fig. 1) were placed in 1.5-mL Eppendorf tubes, supplemented with 0.5 mL acetonitrile, thoroughly mixed, and stored at − 20 °C until further use. Before analysis, samples were centrifuged at 20,817 × g and 4 °C for 10 min (Eppendorf centrifuge 5430-R). Aliquots (3.3 μL) of the resulting supernatants were directly subjected to an Aquity™ UPLC system (Waters, Eschborn, Germany) comprising of a binary solvent manager (BSM), a sample manager (SM), and an evaporative light scattering detector (ELSD) and equipped with an Acquity UPLC BEH Amide column (1.7 μm particle size; 2.1 × 100 mm; Waters) operated at a column temperature of 35.0 °C. The ELSD conditions were gain, 500; data rate, 10 pps; gas pressure, 30.0 psi; mode, cooling; and drift tube temperature, 50.0 °C. The following solvents served as mobile phases: solvent A, 80/20 acetonitrile/water and 0.2% TEA (triethylamine), and solvent B, 50/50 acetonitrile/water and 0.2% TEA. The following elution profile was applied: isocratic elution at 1% B for 0.18 min, linear increase to 99% B until 10.00 min, isocratic elution at 99% B until 10.25 min, linear decrease to 1% B until 10.50 min, and isocratic elution at 1% B until 13.00 min (0.130 mL/min flow rate).

Calibration of the method was carried out using external standards. Glucose, xylose, and fructose showed the best matches with retention times of peaks in samples and were applied for calibration in a concentration range of 16 to 2000 mg/L. Other sugars such as arabinose, galactose, mannose, maltose, sorbitol, and mannitol were also tested but did not yield sufficient matches with peak retention times in samples. Representative UPLC-ELSD chromatograms of glucose, xylose, and fructose in both a standard mixture and a sample, related calibration curves derived from exponential regression, and an overview of the retention times of different sugars in UPLC analyses can be found in the Supplementary Information (Figs. S1, S2, S3, and Table S1). Calibrations based on external standards were included in each analysis run, also to ensure the identity of detected peaks as these displayed slight shifts in retention times in different analytical runs (Supplementary Fig. S1 and S2, and Table S1).

Isothermal microcalorimetry

The isothermal microcalorimetry approach underlying the present work was previously comprehensively described (Duong et al. 2022a,b). Briefly, heat production rates of fungal wheat straw cultures in calorimetric vials were determined against reference vials containing 3 mL of sterile tap water, using the MC CAL isothermal microcalorimeter mentioned before at 28.000 ± 0.001 °C. The aforementioned amount of water was chosen to ensure that the heat capacity of the sample and the reference were approximately equal. In order to ensure a sufficient oxygen concentration and to prevent the accumulation of produced CO2 in the calorimetric vials, the latter were aseptically opened from a quarter-minute to a half-minute on each day of the working week (i.e. from Monday to Friday). The software OriginPro 2020 (OriginLab Corp., Northampton, MA, USA) was used to evaluate the calorimetric signals (Duong et al. 2022a,b). Metabolic heat- (i.e. the integral of the heat production rate of fungal wheat straw cultures over time) related parameters such as metabolic heat yield coefficients (YQ/X) derived from Duong et al. (2022a,b) were used to investigate possible correlations with amounts of sugars in the investigated water-extractable sugar fractions.

Statistical analyses

Linear and non-linear correlation analyses were performed using OriginPro 2020 as outlined in the text. Unpaired two-sample (two-sided) Student’s t-tests were performed using Microsoft® Excel® 2013 (version 15.0.5327.1000; Microsoft Corporation, Redmond/WA, USA).