The effect of inhibitor compounds in the production of inulinase enzyme by fermentation

The usage of lignocellulosic sources as an alternative to the pure carbon sources in the production of value-added products such as inulinase by the biotechnological process has been recently increased. However, some pretreatment methods for the use of these sources in the medium lead to the formation of inhibitors that will affect the fermentation medium. In this situation, it is very important to know the tolerance of the microorganism to inhibitory compounds to be used in the production of the value-added product. In this study, inulinase fermentations were performed by Aspergillus nigerA42 (ATCC® 204407) using different concentrations of HMF (1.0-6.0 g/L), furfural (0.1-2.0 g/L), phenol (0.1-5.0 g/L), acetic acid (2.5-12.5 g/L) and formic acid (1.5-7.0 g/L) as an inhibitor. The ten different mathematical models (MGM, MLM, MRM, Stannard, Weibull, MMF, Asymmetric, Baranyi, Huang, Fitzhugh) were applied to fermentations in which the best Iase activity was obtained for each inhibitor and control fermentation. In addition, the Placket-Burman design was used to investigate the synergistic effect of mix fermentation, which contains all possible inhibitors from lignocellulosic material after data analysis. Finally, the lignocellulosic compounds such as barley husk, oat husk, wheat bran, and rye bran were hydrolyzed with dilute acid and inulinase fermentations were performed. The highest and lowest Iase activity was obtained for HMF 268.74 U/mL (1 g/L HMF), 62.76 U/mL (6 g/L HMF); for furfural 197.35 U/mL (0.1 g/L furfural), 5.14 U/mL (1.0 g/L furfural), for phenol 117.39 U/mL (0.1 g/L phenol), 12.57 U/mL (5 g/L phenol), for acetic acid 340.32 U/mL (2.5 g/L), 3.24 U/mL (12.5 g/L) and for formic acid 136.83 U/ml (1.5 g/L formic acid), 312.69 U/mL (7 g/L formic acid). 10 different models were applied to fermentations with highest Iase activity. The models were compared with some model parameters such as RMSE, MAE, AF, BF, R2. The most compatible models for the production of inulinase was determined to Stannard for HMF, Weibull for furfural, MGM for phenol, Huang for acetic acid, and Weibull for formic acid. Plackett-Burman Design results showed that, the Iase activity was determined as 215.77 U/mL in fermentations which all inhibitors were at minimum concentration (HMF=3.0 g/L, furfural= 0.1 g/L, phenol= 0.1 g/L, acetic acid= 2.5 g/L, formic acid= 3.,0 g/L). The enzyme activity was not statistically different from control fermentation (P>0.05). Finally, inulinase fermentations were carried out by hydrolysis of barley, oat, wheat bran and rye bran with dilute acid and Iase activity was found to be 202.29, 42.03, 244.13 and 254.60 U/mL, respectively. In this study, the tolerance ranges of different inhibitor (HMF, furfural, phenol, acetic acid, and formic acid) concentrations of Aspergillus niger A42 (ATCC® 20447) were determined. Thus, it will be predetermined whether the microorganism will work at inhibitor concentrations likely to be released by lignocellulosic materials. This is considered to be important in deciding the necessity of processes such as detoxification. In addition, the mixture fermentations were optimized for the inhibitor mixture to be tolerated by the microorganism (HMF=3.0 g/L, furfural= 0.1 g/L, phenol= 0.1 g/L, acetic acid= 2.5 g/L, formic acid= 3.,0 g/L). It was demonstrated that the inhibitor compound concentrations in barley, rye bran and wheat bran hydrolysates are tolerated by the microorganism and no detoxification process is required. It was thought that all fermentations performed would be important in deciding the necessity of processes such as detoxification in fermentations with lignocellulosic materials. KEYWORDS: Inulinase, inhibitor, Aspergillus niger, acetic acid, phenol, fermentation, formic acid, furfural, HMF.