Citric Acid by-Product Fermentation by Bacillus subtilis I9: A Promising Path to Sustainable Animal Feed

Citric acid by-products in animal feed pose a sustainability challenge. <i>Bacillus</i> species are commonly used for fermenting and improving the nutritional quality of feedstuffs or by-products. An experiment was conducted to enhance the nutritional value of citric acid by-products through fermentation with <i>Bacillus subtilis</i> I9 for animal feed. The experiment was carried out in 500 mL Erlenmeyer flasks with 50 g of substrate and 200 mL of sterile water. Groups were either uninoculated or inoculated with <i>B. subtilis</i> I9 at 10⁷ CFU/mL. Incubation occurred at 37 °C with automatic shaking at 150 rpm under aerobic conditions for 0, 24, 48, 72, and 96 h. Inoculation with <i>B. subtilis</i> I9 significantly increased <i>Bacillus</i> density to 9.3 log CFU/mL at 24 h (<i>p</i> < 0.05). CMCase activity gradually increased, reaching a maximum of 9.77 U/mL at 72 h. After 96 h of fermentation with inoculated <i>B. subtilis</i> I9, the citric acid by-product exhibited a significant decrease (<i>p</i> < 0.05) in crude fiber by 10.86%, hemicellulose by 20.23%, and cellulose by 5.98%, but an increase in crude protein by 21.89%. Gross energy decreased by 4% after inoculation with <i>B. subtilis</i> in comparison to the uninoculated control (<i>p</i> < 0.05). Additionally, the non-starch polysaccharide (NSP) degradation due to inoculation with <i>B. subtilis</i> I9 significantly reduced (<i>p</i> < 0.05) NSP by 24.37%, while galactose, glucose, and uronic acid decreased by 22.53%, 32.21%, and 18.11%, respectively. Amino acid profile content increased significantly by more than 12% (<i>p</i> < 0.05), including indispensable amino acids such as histidine, isoleucine, lysine, methionine, phenylalanine, tryptophan, and valine and dispensable amino acids like alanine, aspartic acid, glutamic acid, glutamine, glycine, proline, and tyrosine. Furthermore, citric acid by-products inoculated with <i>B. subtilis</i> I9 exhibited changes in the cell wall structure under scanning electron microscopy, including fragmentation and cracking. These results suggest that fermenting citric acid by-products with <i>B. subtilis</i> I9 effectively reduces dietary fiber content and improves the nutritional characteristics of citric acid by-products for use in animal feed.