are the quadratic coefficients. Analysis of variance (ANOVA) in a statistical software package was used to perform statistical analysis of the model (Zhang, Li, Zhang, Wang, & Xing, 2012). Subsequently, the optimized conditions of microbial co-cultivation obtained from RSM were applied for succinic acid production from xylan and untreated corn cob through CBP.
2.4  Determination of xylanase and β-xylosidase activities
Xylanase activity was determined using dinitrosalicylic acid (DNS) method (Cui et al., 2009). The fermentation liquid (1 mL) was added in 1 mL PBS buffer (50 mM pH 6.5) containing 1% (w/v) xylan and incubated for 10 min at 55 °C. The reaction was stopped by the addition of 2 mL of DNS. Finally, the absorbance of the mixture was measured at 540 nm according to the xylose standard curve. One unit of xylanase activity was defined as the amount of enzyme that released 1 μmol of xylose equivalent per minute under the assay conditions.
β-xylosidase activity was determined by the amount of p-nitrophenyl (pNP) released from p-nitrophenyl-β-d-xylopyranoside (pNPX) (Jiang et al., 2018). 1mL of 8 mM pNPX dissolved in 50 Mm, pH 6.5 phosphate buffer solution was preheated at 55 ° C for 3 minutes, then 1 mL of enzyme solution was added and the reaction was performed for 10 minutes. The reaction was stopped by the addition of 1 mL of 1M sodium carbonate solution and the increasing absorbance at 405 nm measured after cooling. The enzyme activity was calculated using pNP as the standard. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of pNP per minute from pNPX under the above conditions.
2.5 Analytical methods
Concentrations of xylose, glucose and organic acids were quantified by high performance liquid chromatography (HPLC) (UitiMate 3000 HPLC system, Dionex, USA) using a UVD 170U ultraviolet detector at a wavelength of 215 nm, and an ion exchange chromatographic column (Bio Rad Aminex HPX-87H column, USA). The products were eluted at 55 °C with 5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min (Dai et al., 2017).
3      RESULTS AND DISCUSSION
3.1 Efficient degradation of xylan by T. thermosaccharolyticum M5
Hemicellulose is one of main constituents in lignocellulose, while xylan is the major portion of hemicellulose in plant cell wall (Borchardt, 2013). Previous studies have shown that the newly isolated T. thermosaccharolyticum M5 exhibited relatively high capacity of xylan degradation owing to its efficient expression xylan degrading enzymes, including xylanase and β-xylosidase, indicating that it would be a good partner with succinic acid producer to achieve succinic acid production from lignocellulose rich in hemicellulose (Jiang et al., 2018). Hence, the degradation capability of strain M5 for different concentrations of xylan was first investigated.
As seen in Fig. 2A, strain M5 was capable of degrading up to 100 g/L of xylan. With the increase of xylan concentration, higher xylose concentration was accumulated, which reached the highest level of 11.23 g/L when 80 g/L of xylan was used as the substrate. However, when xylan concentration was further increased above 100 g/L, xylose accumulation was decreased. This phenonium may be attributed to the product feedback inhibition to xylanase and β-xylosidase caused by the high concentration of xylose. Indeed, when additional 5 and 10 g/L of xylose were initially added to xylanase reaction systems, xylanase activity was decreased by 18% and 53%, respectively, while β-xylosidase activity was decreased by 22% and 61%, respectively when compared to the control with no addition of xylose (data not shown). These results suggested that the simultaneous removal or utilization of reducing sugars may help maintain high hemicellulose degrading enzymes activities.
In addition, pH also affects the activities of xylanase and β-xylosidase (Fig 2B). With initial xylan concentration of 80 g/L, different pH values ranging from 5.5 to 8.0 was maintained. Under pH of 6.5, strain M5 showed the most efficient xylan degradation. Although the optimum pH for the growth of strain M5 was alkaline, however, it has been proved that xylan degradation ability was better under acidic conditions (Xiaojing et al., 2014). What’s more, strain M5 could hardly utilize xylose when growing at 37℃ (Fig 2C), which meant that strain M5 would not compete with succinic acid producer for xylose within co-cultivation system.