Impacts of Kafirin Allelic Diversity, Starch Content, and Protein Digestibility on Ethanol Conversion Efficiency in Grain Sorghum

Seed protein and starch composition determine the efficiency of the fermentation process in the production of grain-based ethanol. Sorghum, a highly water- and nutrient-efficient plant, provides an alternative to fuel crops with greater irrigation and fertilizer requirements, such as maize. However, sorghum grain is generally less digestible because of extensive disulfide cross-linking among sulfur-rich storage proteins in the protein starch matrix. Thus, the fine structure and composition of the seed endosperm directly impact grain end use, including fermentation performance. To test the hypothesis that kafirin (prolamin) seed storage proteins specifically influence the efficiency of ethanol production from sorghum, 10 diverse genetic lines with allelic variation in the beta-, gamma-, and delta-kafirins, including three P-kafirin null mutants, were tested for ethanol yield and fermentation efficiency. Our selected lines showed wide variation in grain biochemical features, including total protein (9.96-16.47%), starch (65.52-74.29%), and free amino nitrogen (FAN) (32.84-73.51 mg/L). Total ethanol yield (ranging from 384 to 426 L/metric ton), was positively correlated to starch content (R-2 = 0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (R-2 = 0.52). Increases in FAN content enhanced fermentation efficiency (R-2 = 0.65). The highest ethanol producer was elite stay green breeding line B923296, and the line with the highest fermentation efficiency at the 72 h time point was inbred BTx623. A large-seeded genotype, KS115, carrying a novel gamma-kafirin allele, was rich in FAN and exhibited excellent short-term fermentation efficiency at 85.68% at the 20 h time point. However, the overall ethanol yield from this line was comparatively low at 384 L/metric ton, because of insufficient starch, low digestibility, and high crude protein. Multivariate analysis indicated an association between the beta-kafirin allele and variation in grain digestibility (P = 0.042) and FAN (P = 0.036), with subsequent effects on ethanol yield. Reversed-phase HPLC profiling of the alcohol-soluble kafirin protein fraction revealed diversity in protein content and composition across the lines, with similarities in peak distribution profiles among beta-kafirin null mutants compared with normal lines.