Influence of initial RSC and glucose concentrations on the production of iturin A
In our previous study [4], the optimal initial RSM and glucose concentrations for iturin A production by B. subtilis 3–10 in submerged fermentation were found to be 90 and 20 g/L, respectively. Therefore, 20 g/L initial glucose was used to test the influence of initial RSC concentration on iturin A production by B. amyloliquefaciens CX-20. As shown in Fig. 1a, the maximum iturin A final concentration 0.39 g/L was obtained when the initial RSC concentration was 90 g/L. With the increase of RSC concentration from 30 to 90 g/L, the production of iturin A increased. However, the iturin A production started to decrease upon further increase of RSC concentration from 90 to 150 g/L. An interesting phenomenon was that the final reducing sugar concentrations increased almost linearly with the initial RSC concentrations. This might be positively related to the reducing sugar released from RSC during fermentation process. The initial reducing sugar concentration slightly decreased, which was speculated to be related to the Maillard reaction caused by the high-temperature sterilization due to the covalent bonds formed between a free reactive -NH2 group of an amino acid and the carbonyl group of a reducing sugar [27]. The concentration of final free ammonium nitrogen (FFAN) also increased linearly from 162.50 to 1266.95 mg/L with the RSC concentration increasing from 30 to 150 g/L. Compared with the FFAN concentration, the initial free ammonium nitrogen (IFAN) concentration increased slightly (Fig. 1b). This indicated that B. amyloliquefaciens CX-20 had a strong ability to hydrolyze the insoluble nitrogen source in RSC to produce soluble form, which was not surprisingly considering the strong intrinsic protease activity of many Bacillus [4]. Genome and transcriptome analysis of B. amyloliquefaciens CX-20 demonstrated that it could not only produce proteases that hydrolyze proteins into peptides and amino acids, but also phytase, xylanase, cellulase and lipase enzyme, which was similar to Aspergillus oryzae and could result in the release of phosphate and the production of simple sugars to be used as carbon source for the growth of the microorganisms [28]. Therefore, Bacillus has intrinsic advantages for direct bio-utilization of RSC for the production of microbial metabolites [4, 5].
According to above results, the initial RSC concentration of 90 g/L was fixed to determine the optimum concentration of glucose and further improve the production of iturin A. Subsequently, the effects of different glucose concentrations ranging from 0 to 100 g/L on iturin A production were explored. It was clearly demonstrated that the optimal initial glucose concentration for iturin A production was 60 g/L, and the corresponding maximum iturin A concentration reached 0.82 g/L. Nevertheless, 0.10 g/L of iturin A was still produced even without adding glucose (Fig. 1c). As a complex mixture, RSM can not only be used as a nitrogen source [4, 5, 15, 17, 29], but can also provide carbon [14, 16] for the growth and metabolism of microorganisms. According to our results, RSC could also be used as both a carbon source and a nitrogen source. As a carbon source, RSC seemed to be efficient, since the final concentration of reducing sugars was 2.05 g/L from initial 4.22 g/L after 72 h fermentation. This further proved that B. amyloliquefaciens CX-20 could produce many enzymes to hydrolyze the carbohydrates of RSC into reducing sugars. It has been reported that rapeseed oil could be used as a source of carbon to ferment microbial products such as lipase [23], erythromycin [30] and isocitric acid [31]. However, whether the residual oil in RSC could be used as a carbon source for iturin A production by B. amyloliquefaciens CX-20 was still unclear. But RSC was insufficient to support the fermentation and synthesis of iturin A by B. amyloliquefaciens CX-20 as the sole carbon source. Accordingly, with the increase of initial glucose concentration from 0 to 60 g/L, the production of iturin A continued rising, but decreased when the initial glucose concentration was raised above 60 g/L. Although the final concentration of reducing sugars increased with the increase of initial glucose concentration, the change trend of the FFAN concentration (decreased from 771 to 522 mg/L) was opposite (Fig. 1d). This indicated that higher glucose or RSC concentrations could mutually promote the corresponding substrate consumption, but might not be necessary for improving iturin A production.
Influence of lipase loading on the production of iturin A from RSC
Different concentrations of lipase (ranging from 0 to 10 U/mL) were added into the medium containing the optimal concentrations of 90 g/L RSC and 60 g/L glucose at the beginning of the process to enable simultaneous hydrolysis and fermentation. As shown in Fig. 2, with the increase of lipase concentration from 0 to 0.5 U/mL, the production of iturin A gradually increased. However, with the further increase of lipase concentration (from 0.5–10 U/mL), the production of iturin A began to decrease. When the concentration of lipase was 0.5 U/mL, the iturin A production reached a maximum of 1.14 g/L, which represented a 38.15% increase over the fermentation without any lipase addition. By contrast, when the concentration of lipase reached 10 U/mL, the final concentration of iturin A decreased to 0.59 g/L, which was even 27.94% lower than that without any lipase addition. The change trend of the final reducing sugar concentration was similar to that of iturin A production. Lipases are a group of enzymes that hydrolyze the ester bonds in triacylglycerides to form fatty acids and glycerol, or catalyze the synthesis of esters under certain conditions [32]. The lipase used in this study was a mixture used as a feed additive to aid animal digestion. Therefore, we speculated that the appropriate addition of lipase might help hydrolyze the residual oil in RSC into fatty acids and glycerol. Notably, glycerol has been proved to be a more suitable carbon source for iturin A production than glucose (data not shown). This helps explain why the FFAN concentrations with added lipase was lower than that without lipase, since the released glycerol could also promote the consumption of the nitrogen source (Fig. 2b). However, excess lipase had a negative effect on the production of iturin A.
Influence of lipids and lipase on the fermentation of iturin A
As shown in Table 1, the content of protein in RSC was 33.5% while the protein content in RSM was 39.4%. Therefore, the protein content in 90 g/L RSC was equal to that of 76.52 g/L RSM, and the latter was added to substitute 90 g/L RSC to simulate the protein content in the original medium. Because the crude fat content of RSM was 1.6% while that of RSC was 14.4%, the crude fat content of the medium containing 90 g/L RSC was 12.96 g/L, while that of the medium containing 76.52 g/L RSM was 1.22 g/L. The difference of crude fat between the two media was 11.74 g/L. In order to explore the effect of crude fat on iturin A production, 0, 6, 12 or 24 g/L of natural rapeseed oil was added into the medium containing 76.52 g/L RSM (Fig. 3a). After 72 h of fermentation, the final concentration of iturin A without added oil was 0.95 g/L, which was 15.56% higher than that of the 90 g/L RSC medium. With the increase of oil concentration, the trend of iturin A production was gradually decreasing. When 12 g/L rapeseed oil was added into the medium, the iturin A production decreased to 0.79 g/L. Although the production of iturin A decreased 17.46% compared to that produced without any oil addition, its value was very close to that produced with 90 g/L RSC (0.82 g/L). When 6 g/L rapeseed oil was added to the medium, the iturin A production decreased to 0.93 g/L, which was very close to the value obtained without adding oil. Because the lipopeptide products possess surfactin activity and cause foam formation, it is difficult to control the fermentation, which also restricts the industrialization of lipopeptide production [2]. Rapeseed oil has been found to be an efficient antifoam compound [33]. Our results also demonstrated that when the concentration of added rapeseed oil was lower than 0.6%, there was almost no negative effect on iturin A production. Therefore, rapeseed oil seemed to also be a suitable antifoam compound for iturin A production. However, when 24 g/L rapeseed oil was added into the medium, the iturin A production decreased to 0.66 g/L.
From the results shown in Fig. 2, we found that an appropriate concentration of lipase could improve the production of iturin A. Conversely, its production would be reduced when the concentration of lipase was too high. The oil content of RSM was lower than that of RSC (Table 1). Therefore, in theory, the optimal concentration of lipase for RSM should decrease accordingly. As expected, when the concentration of lipase was 0.1 U/mL, the iturin A production had a slight increase, from 0.95 g/L to 1.01 g/L, but when the lipase concentration was increased to 1 U/mL, the final concentration of iturin A was slight lower than that without any lipase added (0.95 g/L vs. 0.93 g/L). When the lipase concentration was increased to 5 U/mL, the final concentration of iturin A drastically decreased to 0.53 g/L, which was only 55.14% of that produced without any added lipase (Fig. 3b). At the same time, the final reducing sugar concentration gradually increased with the increase of lipase concentration when the lipase concentration was more than 0.1 U/mL.
Although the production of iturin A decreased slightly when RSC was used as nitrogen source if the lipase concentration was increased to 5 U/mL, the production of iturin A apparently decreased when RSM containing an equal protein content was used as the nitrogen source. Therefore, proper proportions of rapeseed oil and lipase appeared to be crucial for optimal iturin A production. As shown in Fig. 3b, a lipase concentration of 5 U/mL had a significant influence on the production of iturin A from RSM, and was chosen to explore the appropriate ratio of rapeseed oil to lipase. As shown in Fig. 3c, with the increase of rapeseed oil concentration from 0 to 12 g/L, iturin A production continued rising and reached a maximum of 1.02 g/L, which was almost equal to the iturin A production (1.03 g/L) produced from 90 g/L RSC with 5 U/mL lipase, when the addition of rapeseed oil was 12 g/L. However, when the concentration of rapeseed oil was increased to 24 g/L, the final concentration of iturin A decreased to 0.61 g/L, which was only 59.42% of that obtained with 12 g/L rapeseed oil.
There are many possible explanations why lipase and rapeseed oil could reduce each other’s negative effects on iturin A production. The most likely one is related to microbial growth. Therefore, we examined the growth curves of Bacillus under several representative conditions. As shown in Fig. 4, when 12 g/L rapeseed oil or 5 U/mL lipase was added separately into the medium containing 76.52 g/L RSM, both the specific growth rates (0.56 and 0.52 h− 1 from 1.03 h− 1) and the ultimate maximum viable cell count (about 8 × 108 and 3 × 108 mL− 1 from about 8 × 109 mL− 1) were significantly reduced compared with the medium without either rapeseed oil or lipase. However, when 12 g/L rapeseed oil and 5 U/mL lipase were added at the same time, both the specific growth rate (0.80 h− 1) and the ultimate maximum viable cell count (about 2 × 109 mL− 1) showed an obvious recovery.