Ankom 220

Lignocellulosic material preparation and microbial consortium cultivation were performed as described previously [4 (link)]. Briefly, corn stalks obtained locally from Wuhan, China were air-dried and alkali-treated before use. The relative contents of cellulose, hemicellulose, and lignin were respectively 37.1%, 24.1%, and 12.1% in air-dried corn stalk and 62.4%, 17.6%, and 5.7% in alkali-treated corn stalk. The microbial consortium TMC7 was enriched from thermophilic compost using a reduplicative subcultivation process [4 (link)] and preserved in 20%glycerol (v/v) at -80°C. TMC7 was activated and cultured in 350 ml of sterilized PCS medium (1 g/l peptone, 2 g/l yeast extract, 2 g/l CaCO₃, 5 g/l NaCl, 0.35 g/l MgSO₄•7H₂O, and 1 g/l K₂HPO₄) supplemented with 1% (w/v) alkali-treated corn stalk. The cultures were incubated in a 500 mL flask with a loose aluminum cap under static conditions in the dark at 65°C for 7 days. The weight loss of corn stalk and lignocellulosic components was determined according to a gravimetrical method with uninoculated medium serving as a control [14 (link)]. The components of the cellulosic substrates were analyzed on a Ankom220 fiber analysator (Ankom, USA) by Van Soest [15 ] detergent fiber analysis.

Another five plants were randomly selected from each maize inbred line for determination of hemicellulose, cellulose, and lignin contents. The entire third basal internode was cut off, heated in an air oven at 105 °C for 30 min, and then dried to a constant weight at 80 °C. The DWUL of the third internode was calculated by dividing the dry weight of the internode by its length. The dried sample was then crushed and passed through a 40-mesh screen. Approximately 1 g of the sifted sample (M1) was placed in a filter bag (with a weight of M0) for measurement of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) contents using an ANKOM 220 fiber analyzer (ANKOM Technology Corp., Fairport, NY, USA) according to a previous study [55 (link)]. Hemicellulose, cellulose, and lignin percentages were calculated as follows: hemicellulose (%) = (NDF – ADF)/M1; cellulose (%) = (ADF – ADL)/M1; and lignin (%) = (ADL – M0)/M1. The percentage of each compound was multiplied by DWUL to obtain hemicellulose (HWUL), cellulose (CWUL), and lignin (LWUL) weights per unit length of the third basal internode (mg cm^− 1). Each component was measured three times per sample.

Samples of BSG untreated and treated with the appropriate EFE and in various doses were oven‐dried to constant weight aimed to determine dry matter (DM) content and then were ground to 1 mm (Cyclotec 1093 Sample Mill; Tecator, Höganäs, Sweden). Before that, the sample was examined to analyze crude protein (CP), ether extracts (EE), and ash conferring to the procedures described by the Association of Official chemists Analytical Chemists (1995 ). Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were tested by using a fiber analyzer (ANKOM 220, ANKOM Technology, Macedon, NY) according to the technique defined by Van Soest et al. (1991 (link)). The soluble dry matter (DMS) was estimated following the protocol of Elwakeel et al. (2007 (link)). Nonfiber carbohydrates (NFC) are estimated with the formula of the National Research Council (NRC) (2001 (link)) (Equation 1): $$\text{NFC}(\%)=100‐\left(\text{EE}(\%)+\text{CP}(\%)+\text{NDF}(\%)+\text{Ash}(\%)\right)$$