Characteristics of lignocellulolytic enzymes
The composition of lignocellulolytic enzyme cocktail for high-viscosity corn mash would be screened in the lignocellulolytic enzyme library. The lignocellulolytic enzyme library contained the main enzyme system from T.reesei and the coenzyme system from A.niger. Although the commercial cellulase was produced from T.reesei, the enzyme system from T.reesei has some drawbacks . The main enzyme system from T.reesei was incompleted and unbalanced, such as no genes encoding cellobiose dehydrogenases and feruloyl esterase, few monooxygenases and β-glucosidase expressed in the T.reesei extracellular protein [11, 12]. These key coenzymes may need to be introduced from other species to improve the enzymatic efficiency . For high-viscosity, high-sugar corn mash, the coenzymes such as FAE, GH61 and β-glucosidase were overexpressed up to 15% of total extracellular enzymes for optimization of enzyme system composition. It is reported that this FAE, GH61 and β-glucosidase from Penicillum picerum had unique enzymology characteristics in our previous paper [13, 14]. The FAE and GH61 reduced biomass recalcitrance by increasing xylose/arabinose ratio and decreasing HBI of crude biomass, which could reduce lignocellulose degradation recalcitrance, providing favorable conditions for enzymatic hydrolysis [13,14]. The novel lignocellulolytic cocktail was balanced enzymatic ratio with filter paper activity of 40.8 FPU/mL, xylanase of 1320.6 IU/mL, and feruloyl esterase of 22.5 IU/mL and additional side activities, including amylase of 4.5 IU/mL.
Considering the cost of using enzymes, lignocellulolytic enzyme was selected for further experiments to test at a lower enzyme dosage. Corn mash fermentation was performed at different enzyme loadings (2–200FPU/L) to facilitate the screening of optimum enzyme dosage. The ethanol concentration, sugar reduction, and viscosity of corn mash corresponding to different enzyme dosages are presented in Table 1. The ethanol yield increased with increased cellulase dosage until 50FPU/L. Further increased enzyme dosage (100–200FPU/L) did not increase the ethanol yield. The enzyme dosage ranges of 20–40FPU/L did not result in maximal promotion in alcohol concentration and yield. Therefore, 50FPU/L was selected as the optimal cellulase dosage in the SSF. At the optimum enzyme dosage of 50FPU/L mash, the ethanol yield increased by 13.3% compared with the reference mash.
The application of lignocellulolytic enzymes preparation decreased the mash viscosity and increased the yeast-fermentation efficiency. This result was consistent with a previously published report. The results of Czarnecki and Nowak indicate the beneficial effects of lignocellulolytic enzymes (e.g., xylanase, cellulase, and glucanase) on rye mashes, such as a decreased viscosity, enhanced starch saccharification, and increased productivity of ethanol [15, 16].
The novel lignocellulolytic enzymes were applied at different fermentation scales from 0.3–70 L. At the 0.3, 1, 5, and 70 L batch-fermentation scales, the reductions in corn mash viscosity were 46.3%, 31.6%, 35.5%, and 40.9% compared with the reference mash (33.5 ± 1.5 Pa·s), respectively. The treatment of corn mashes with the novel lignocellulolytic enzymes resulted in increasing concentrations of ethanol by 12.4%, 12.0%, 11.8%, and 12.9% compared with the reference mash. The highest yield of ethanol in the corn mash digested with the novel lignocellulolytic enzyme reached 117.0 ± 0.1 g/L at the 70 L batch fermentation, whereas that in the control reached only 103.6 ± 1.0 g/L. After the lignocellulolytic enzyme addition, the residual starch content decreased to 5.34± 0.26%, whereas that without lignocellulolytic enzyme was 6.58 ± 0.86% (Table 2). The residual cellulose content of corn mash decreased to 7.48 ± 0.10%, whereas that without lignocellulolytic enzyme was 12.64 ± 0.52%. Approximately 1.24% of starch and 5.16% of cellulose in the corn mash were further hydrolyzed. This result indicates that a part of the residual cellulose in corn mash was mostly degradable by the lignocellulolytic enzyme. The starch conversion with the cellulase cocktail improved because cellulase disrupted the cell wall structure of the grain and promoted starch release. Furthermore, the cellulase cocktail possibly contained amylases.
Further degradation of residual SSF broth after ethanol evaporated by lignocellulolytic enzymes cocktail
To elucidate the promotion effect of lignocellulolytic enzymes on alcoholic fermentation clearly, the lignocellulolytic enzymes were added into the residual broth in SSF. The 36 h SSF broth (containing unconverted solids) with the ethanol evaporated was used for another round of separate hydrolysis and fermentation (SHF), which reduced the ethanol inhibition on the cellulase activity. As shown in Fig. 1, approximately 7.0 g/L of glucose was released from the saccharification of the residual broth with the lignocellulolytic enzymes, whereas no glucose was released from that without cellulase addition. Then, the activated yeast was added into the saccharification broth for another 36 h ethanol fermentation. Further ethanol production can yield 6.44 g/L after lignocellulolytic enzymes addition. The residual starch contents with and without lignocellulolytic enzymes addition were 5.06 ± 0.34% and 6.02± 0.86%, respectively (Table 3). The residual cellulose contents of corn mash with and without lignocellulolytic enzymes addition were 10.07 ± 0.10% and 13.20 ± 0.52%, respectively. Approximately 0.96% of starch and 3.13% of cellulose in the corn mash were further hydrolyzed. Adding lignocellulolytic enzyme resulted in 13.58 g of sugar production, including the release of 1.43 g of soluble residual sugar and 12.15 g of glucose from starch and cellulose. These results indicate that the lignocellulolytic enzymes did promote the release of more glucose from residual starch and cellulose for ethanol increase but not yeast-fermentation efficiency. Certainly, the hydrolysis efficiency of starch and cellulose in SHF was apparently lower than that in SSF.
The main co-product of corn mash fermentation was DDGS, which can increase the economics of this process. A summary of DDGS composition is provided in Table 4. The residual cellulose content of DDGS decreased from 33.12% to 21.20%, while the starch content of DDGS decreased from 14.07% to 11.26% (Table 4). These results showed that the lignocellulolytic enzymes addition did promote the further hydrolysis of residual starch and cellulose in corn mash. The crude protein in DDGS was determined to be 29.63% with the addition of the novel lignocellulolytic enzymes, and 24.12% without the novel lignocellulolytic enzymes. No difference in DDGS color was observed (Supplemental Fig.1). The value of DDGS, which is sold as animal feed, can be increased by increasing the protein content.
The addition of novel lignocellulolytic enzymes for ethanol yield improvement does not require any additional equipment or control system. The only additional cost would be that of the lignocellulolytic enzymes. Ten tons of corn mash can produce one ton of ethanol. The ten tons of corn mash need add lignocellulolytic enzyme input of RMB 75 yuan (FPAase of lignocellulolytic enzyme calculated as 40 FPU/mL; RMB 6000 yuan/ton lignocellulolytic enzymes). The estimated ethanol increase can improve by 10% (conservative computation) in alcoholic industrialization, which is equivalent to increasing the income by RMB 600 yuan (6000 yuan/ton ethanol) from producing 1 ton of ethanol. The net revenue can increase to RMB 525 Yuan (600-75) from producing 1 ton of ethanol. The fuel ethanol production in Jilin Province is 0.6 million tons per year, while the fuel ethanol production in China will be approximately 10.0 million tons per year in near future. This technology can increase the net revenue of fuel ethanol in Jilin Province by RMB 3.2billion yuan, while the net revenue of fuel ethanol in China by RMB 52.5 billion yuan per year. Moreover, the co-product will add a certain revenue due to the protein content increase of DDGS.