The yield differences of Xcn1 between strains of X. neamtophlia
The difference of Xcn1 production of different strains in TSB medium was shown in Figure 1 (a). The results showed that Xcn1 yield of X. nematophila All was the highest with 94.15 μg/mL. The Xcn1 yield of X. nematophila YL001 was 57.80 μg/mL in the middle level. X. nematophila AN6 and X. nematophila Mex showed low yield of Xcn1 with 25.60 μg/mL and 30.63 μg/mL, respectively. In this study, the Xcn1 production of X. nematophila All was significantly higher than that of X. nematophila Mex, X. nematophila AN6 and X. nematophila YL001 strains, which indicated that strain was an important factor of determining Xcn1 production.
Effect of different media on Xcn1 production
The results of cell growth and Xcn1 production of X. nematophila YL001 in different media were shown in Figure 1 (b-c). The results indicated that PP3 medium showed a maximum Xcn1 production (87.21 μg/mL) followed by BR medium (69.71 μg/mL) and KB medium (67.61 μg/mL). Lower Xcn1 production was observed in YS medium (11.12 μg/mL). The maximum cell growth in PP3 medium was found to be high when compared with other media. PP3 medium was selected as the original medium for the carbon and nitrogen sources selection studies because it was most suitable for cell growth and Xcn1 production of X. nematophila YL001. Medium played a decisive role in the growth and whole fermentation process of microorganisms [36, 37]. Previous studies have shown that PP3+ medium is the most suitable for the growth of X. nematophila YL001 [36]. However, in our study, PP3 medium is more suitable for the growth of X. nematophila YL001 than PP3+ medium. To some extent, the reason for this phenomenon is that the culture conditions are different. In addition, the yield of Xcn1 in PP3+ medium was significantly lower than that in PP3 medium. And our purpose was to improve the yield of Xcn1, so PP3 medium was selected as the original medium.
Effect of various nutrient sources on cell growth and Xcn1 production
Based on the original medium, single factor evaluation of inorganic salts (10 g/L), carbon source (5 g/L), and nitrogen source (20 g/L), were respectively conducted to explore the effect on cell growth and the production of Xcn1. Figure 2 (a) indicated that among the various nitrogen sources studied, the X. nematophila YL001 produced the maximum Xcn1 in proteose peptone (81.82 μg/mL), followed by tryptone (69.19 μg/mL) and yeast extract (63.34 μg/mL). The DCW in yeast extract (25.07 g/L) was found to be high when compared with other nitrogen sources. Among carbon source, maltose showed a maximum Xcn1 production (106.31 μg/mL) followed by fructose (101.67 μg/mL) and starch (98.57 μg/mL). Lower Xcn1 production was observed with the sucrose (37.06 μg/mL). The maximum DCW was found in starch (27.01 g/L) when compared with other carbon source (Figure 2 b). The effects of supplementation with various minerals were evaluated and were found to have potential impact on Xcn1 production. Among the various inorganic salt studied, the X. nematophila YL001 produced the maximum Xcn1 in K2HPO4 (112.30 μg/mL) and the minimum Xcn1 in NaCl (71.30 μg/mL). The DCW in Na2SO4 (25.47 g/L) was found to be high when compared with other inorganic salt (Figure 2 c).
The optimal concentrations of nitrogen, carbon and inorganic salts were determined by controlling their doses. Thus, proteose peptone (20 g/L), maltose (10 g/L) and K2HPO4 (5 g/L) were chosen as the source of nitrogen, carbon and inorganic salt for further experiments, respectively (Figure 2 d). By optimizing the nutrition of medium, the yield of Xcn1 was enhanced from 50.73 μg/mL to 117.99 μg/mL. Among the nitrogen sources, although yeast extract was the most suitable for the growth of X. nematophila YL001, proteose peptone was the most suitable for Xcn1 production. Fructose and starch were suitable for X. nematophila YL001 growth as carbon sources, but maltose was most suitable for Xcn1 production. In several inorganic salts, Na2SO4 and MgSO4 were suitable for the growth of X. nematophila YL001, but K2HPO4 was the most suitable for the production of Xcn1. These results indicate that the growth of X. nematophila YL001 is different from biosynthetic process of Xcn1. Previous studies showed that X. nematophila YL001 produced the most antibiotics when proteose peptone was used as nitrogen source [36]. When glucose and maltose were used as carbon source, X. nematophila YL001 produced a lot of antibiotics with concentration of 243.3 U/mL and 233.3 U/mL [36]. Going further, it was consistent with previous studies and also indicated that Xcn1 was the main active component of the antibiotic produced by X. nematophila YL001 [23, 41].
Optimization of medium constituents by response surface method
After determination of nitrogen sources, carbon sources and inorganic salts in the medium, the combined effects of concentration of medium constituents on Xcn1 production were further investigated. Table 1 showed experimental range and levels of the independent variables. The experimental results listed in Table 2 were analyzed using multiple regressions. The production of Xcn1 was found to be maximum at run 11 (113.65 μg/mL). The second-order polynomial model was proposed to evaluate the optimum levels of these selected variables and was shown in the following equation:
![](data:image/png;base64,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)
The optimum levels which were suitable for the maximum yield of Xcn1 were 1.274% maltose, 2.083% proteose peptone and 0.377% K2HPO4 in the media. In this study, the determination coefficient (R2) confirmed the importance of statistical design, exhibiting minor experimental error and a fit regression equation. The R2 of this designed CCD model was 0.9443. The F value of the model is 18.38, while the P value is less than 0.0001, indicating that the model has significance (Table 3). The saddle or elliptical nature of contour plot showed the significance of the good interactions between the respective variables. Figure 3 showed the contour plot and 3D response surface plot for the Xcn1 yield generated by the predicted CCD model. According to the model equation, we predicted that a maximum Xcn1 yield of 113.65 μg/mL could be achieved at 12.74 g/L maltose, 20.83 g/L proteose peptone and 3.77g/L K2HPO4. By HPLC, the content of Xcn1 was 117.99 μg/mL with the optimized medium, which was close to the predicted response. Compared with the output of Xcn1 of TSB medium (50.67 μg/mL), the output of Xcn1 of the optimized PP3 medium (113.65 μg/mL) increased by 112.65%.
Optimization of cultural conditions by a-factor-at-a-time method
Based on the above efforts, the fermentation conditions of X. nematophila YL001 was optimized further to improve the production of Xcn1. In order to get the most suitable conditions for the production of Xcn1, 6 kinds of fermentation conditions, such as the temperature, initial pH, inoculum size, rotation speed, liquid loading and fermentation time, were optimized for the fermentation. The production of Xcn1 was assessed at different temperatures (15-35°C) and reached the maximum value of 119.87 μg/mL at 25°C (Figure 4 a). It showed that 25°C was the optimal fermentation temperature. Significantly, further increase in the temperature reduced the yield of Xcn1. Over different pH ranges (5-9) were assessed for Xcn1 production (Figure 4 b). Fermentation medium favoured pH 7 for maximum production of Xcn1 (125.72 μg/mL). Furthermore, an increase or decrease in the pH of the medium decreased the production of Xcn1. The yield of Xcn1 increased with the increased inoculum size but it decreased when inoculum size was over 10% (Figure 4 c). Thus, 10% was selected as the optimal inoculum size. The production of Xcn1 was assessed at different rotating speeds (50-200rpm) and reached the maximum value of 125 μg/mL at 100 rpm (Figure 4 d). There was no significant change in the yield of Xcn1 when the speed continued to increase. So 100 rpm was selected as the optimal rotating speed. The effect of medium loading on the production of Xcn1 was investigated by changing the volume of the medium. It showed that the optimal yield of Xcn1 was 153.56 μg/mL with 75mL medium in 250mL flasks (Figure 4 e). Length time of fermentation was also influenced toward the yield of Xcn1. The production of Xcn1 was assessed at different fermentation time (24-120h). There was no significant increase in the production of Xcn1 when the fermentation time increased (Figure 4 f). Consequently, 48h was selected as the optimal fermentation time.
Through the optimization of fermentation conditions, the yield of Xcn1 reached 153.56 μg/mL. Temperature and pH had significant influence on Xcn1 production of X. nematophila YL001, and high or low temperature and pH both led to a sharp decrease in Xcn1 production (Figure 5). Several other environmental conditions, such as inoculum size, rotation speed, liquid loading and fermentation time, could not make such a huge change in Xcn1 production. In this study, 7.0 was the most suitable pH for Xcn1 production. However, this was not consistent with the results obtained in previous studies. The weak alkaline pH environment was found to be beneficial for the production of Xcn1. And the production of Xcn1 were 2.49-fold higher at pH 8.5 relative to that at pH 7.0 [31]. To some extent, it may be due to the combined effects of various environmental conditions on Xcn1 production. Accordingly, more precise control of pH and temperature would help to increase the yield of Xcn1. In addition, pH7.0 was the most suitable for the growth of X. nematophila YL001 [38]. Thus, it needs to be further explored the relationship between the metabolism of growth of X. nematophila YL001 and the biosynthesis pathway of Xcn1.
Effect of precursor substances on Xcn1 production
Biosynthetic precursors, such as arginine, leucine, urea and acetic acid, affected biosynthesis of Xcn1 that natural product produced by X. nematophila YL001. Markedly, arginine influenced the production of Xcn1 and its optimal concentration was 3 mmol/L with the productivity of Xcn1 was 132.78 μg/mL (Figure 5). In order to determine the best occasion of adding, arginine (3 mmol/L) was added to the fermentation medium at the 0th, 6th, 12th, 18th, 24th, 30th h, during fermentation. The production of Xcn1 was assessed at 48h and reached the maximum value of 173.99 μg/mL with added arginine at the 12th h (Figure 5 e). Therefore, the 12th hour was selected as the optimal time to add arginine of 3 mmol/L to the fermentation medium. The yield of Xcn1 was increased to 173.99 μg/mL by adding precursors properly. Four precursors were involved in the biosynthesis of Xcn1 directly or indirectly. The synthesis of arginine by microorganism needed urea to provide nitrogen. In the form of arginyl thioester, arginine was condensed with N-acyl-D-asparaginyl thioetser into the long chain of Xcn1 [23, 42]. This process occurred in the initial stage of Xcn1 long chain biosynthesis, hence arginine was the most helpful precursor for Xcn1 biosynthesis. This is consistent with the results of our study. Moreover, leucine and acetic acid were involved in the long chain synthesis of Xcn1, respectively, to form peptide bonds and extend the carbon chain [23, 42]. If precursors, such as arginine, leucine and acetic acid, were added in proper proportion at the right time, the production of Xcn1 would be further improved.
Previous studies evaluated the content of antibacterial active substances in fermentation broth of X. nematophila YL001 according to the size of bacteriostatic circle [36]. The above methods are not accurate enough, so we have adopted high performance liquid chromatography in this study. High performance liquid chromatography method was provided a more precise and accurate method for the Xcn1 detection and was proved to be a powerful tool for the optimization Xcn1 production by X. nematophila YL001. This method has broad prospects in follow-up study.
Microbial natural products are important sources for antibiotic discovery whether medicine or pesticides [43, 44]. However, the low yield limits the development and utilization of microbial natural products. In order to improve the output of antibiotic by microbes, biosynthesis metabolic regulation and fermentation process optimization had been researched [45, 46]. In an industrial point of view, to improve the production, various methods and techniques should be screened for Xcn1 production. We added some precursors of Xcn1 biosynthesis to the medium and optimized the fermentation process to increase the production of Xcn1 in this study. Furthermore, the fermentation of X. nematophila YL001 should be studied in pilot scale to further to optimize the fermentation conditions in the fermentation tank, so as to improve the output of Xcn1 and lay a foundation for its industrial production. With the use of macroporous resin X-5 in a fermentation setting, X. nematophila CB6 improved its ability to produce Xcn1 [40]. Perhaps, this method could be used to increase the production of Xcn1 in the fermentation broth of X. nematophila YL001.