In Situ Corn Fiber Conversion for Ethanol Improvement by The Addition of Novel Lignocellulolytic Enzyme Cocktail

Background: The technology of converting corn mashes to ethanol has been mature, but corn mashes has high-viscosity and high-sugar characteristics which hindered cellulose utilization and yeast-fermentation efficiency. The excessive viscosity of corn mash is caused by the presence of non-starch polysaccharides, such as cellulose in cereal grains. Corn kernel fiber (mostly cellulose) is typically unconverted in the process. Results: A novel lignocellulolytic enzymes cocktail with strong substrate specificity was prepared for high-viscosity, high-sugar corn mash. The in situ conversion of corn mashes with novel lignocellulolytic enzymes at the optimum cellulase dosage of 50 FPU/L resulted in 12.4%, 12.0%, 11.8%, and 12.9% increased ethanol concentration compared with the reference mash at 0.3, 1, 5, and 70 L batch-fermentation scales, respectively. The highest yield of ethanol from corn mash digested with the prepared novel lignocellulolytic enzyme reached 117.0 ± 0.1g/L at the 70 L batch fermentation, which was a 12.9% increase in ethanol yield. Adding the lignocellulolytic enzymes caused the greatest decrease in viscosity of corn mash by 40.9% compared with the reference mash (33.5 ± 1.5 Pa·s), whereas the residual sugars decreased by 56.3%. Simultaneously, the application of novel lignocellulolytic enzymes increased the value of dried distiller’s grain with solubles by increasing the protein content and decreasing the residual cellulose and starch content.Conclusion: The application of novel lignocellulolytic enzymes significantly improved the alcohol concentration, productivity, and yield. With the same amount of material, the application of the novel enzymes cocktail can enhance the ethanol yield by more than 10%. The in situ conversion of cellulose promoted the release of contents, including starch and protein, which can decrease the fermentation broth viscosity and improve the rheological property, thereby improving the ethanol yield. Thus, this technology can increase the net revenue of fuel-ethanol industrialization and promote the technological progress of renewable energy.

Recombinant bacteriophage LysKB317 endolysin mitigates Lactobacillus infection of corn mash fermentations

Background Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. Results In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing 1 species of Acetobacter, 8 species of Lactobacillus, 1 species of Pediococcus, 3 species of Streptococcus, and 1 species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5–7.5 up to at least 48 h. Maximum activity was observed at 50 °C up to at least 72 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 3-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 was similar to uncontaminated controls. Conclusion Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.

Recombinant Bacteriophage LysKB317 Endolysin Mitigates Lactobacillus Infection of Corn Mash Fermentations

Background Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. Results In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing one species of Acetobacter, eight species of Lactobacillus, one species of Pediococcus, three species of Streptococcus, and one species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5 – 7.5 up to at least 48 h. Maximum activity was observed at 50°C up to at least 72 hrs. In addition, LysKB317 was stable in 30% ethanol up to at least 72 hrs. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 3-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 were similar to uncontaminated controls. Conclusion Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.

Recombinant Bacteriophage LysKB317 Endolysin Mitigates Lactobacillus Infection of Corn Mash Fermentations

Background Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. Results In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing one species of Acetobacter, eight species of Lactobacillus, one species of Pediococcus, three species of Streptococcus, and one species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5 – 7.5 up to at least 48 h. Maximum activity was observed at 50°C up to at least 72 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 4-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 were similar to uncontaminated controls. Conclusion Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.