Influence of fermentation time on surfactin production by B. velezensis YA215
To enhance surfactin yield, we optimized the fermentation duration in SS medium supplemented with branched-chain amino acids. Specifically, 1 g/L of L-Leu, L-Ile, and L-Val were added, and the effect of different fermentation times (12, 24, 36, 48, and 60 h) on surfactin content was analyzed using high-performance liquid chromatography (HPLC). As illustrated in Fig. 1, surfactin content peaked at 36 h without any branched-chain amino acid supplementation. When L-Val and L-Ile were added, the surfactin levels also reached their maximum at 36 h, mirroring the trend seen in the control group. However, with L-Leu addition, the peak surfactin content was achieved earlier, at 24 h. Among the four groups, the highest surfactin yield was observed in the L-Val group, significantly surpassing the other groups, while the yields in the L-Leu and L-Ile groups were lower than the control SS group. These findings indicate that branched-chain amino acids not only alter the timing of maximum surfactin production by B. velezensis YA215 but also affect the overall surfactin yield, providing essential theoretical insights for optimizing fermentation conditions.
Impact of branched-chain amino acids concentration on surfactin production by B. velezensis YA215
Following the optimization of fermentation time with branched-chain amino acid supplementation in SS medium, the concentration of these amino acids was further optimized. Different concentrations of L-Leu, L-Ile, and L-Val were added to the fermentation medium, and the surfactin content in the fermentation supernatant was measured using HPLC at the previously optimized fermentation times. As shown in Fig. 2, surfactin content increased with the concentration of L-Leu and L-Ile, peaking at 1 g/L. For L-Val, surfactin content also increased with concentration but reached its maximum at 0.5 g/L. Among the three amino acids, L-Val addition resulted in the highest surfactin production, while L-Ile addition resulted in the lowest.
These experimental results provide critical data for optimizing surfactin production, particularly regarding the timing and concentration of branched-chain amino acid supplementation. The data offer a solid experimental foundation for increasing surfactin yield and have significant implications for future research and applications in this field(Zhou et al. 2019). Previous studies have demonstrated that the addition of branched-chain amino acids can significantly impact surfactin synthesis during microbial fermentation(Balakrishnan et al. 1996). Researchers have observed that adding these amino acids at specific fermentation time points enhances the yield of the target product, aligning with our finding that surfactin content maximized at 36 h with L-Val addition. Furthermore, our results highlight potential optimization opportunities concerning branched-chain amino acid concentrations. Specifically, surfactin production peaked at 1 g/L for L-Leu and L-Ile, corroborating findings by Yao et al., which indicated that moderate amino acid concentrations promote surfactin production during biological fermentation(Yao et al. 2014).
Impact of branched-chain amino acids supplementation on biomass and lipopeptide production by B. velezensis YA215
Under the optimized fermentation conditions, the influence of branched-chain amino acids on the growth and lipopeptide production of B. velezensis YA215 was thoroughly examined. Supplementing the SS medium with branched-chain amino acids affected both the biomass and lipopeptide yield of the YA215 strain. As depicted in Fig. 3A, a slight decrease in biomass was observed at 36 h of fermentation with the addition of branched-chain amino acids, although the reduction was not pronounced. Figure 3B shows that the pH of the fermentation supernatant decreased after 36 h, especially with the addition of Ile and Val, potentially contributing to the biomass reduction. However, this decline in biomass did not exhibit a clear trend. In contrast, lipopeptide yield varied with branched-chain amino acid supplementation, with a significant increase in lipopeptide production observed in the Val-supplemented group compared to the control, whereas Leu and Ile supplementation resulted in decreased lipopeptide yields (Fig. 3C). This variation in lipopeptide yield differs from the previously discussed surfactin production trends, as lipopeptides comprise surfactin, iturin, and fengycin families(Ongena et al. 2008), and changes in surfactin alone do not fully represent lipopeptide yield trends. These results underscore the multifaceted roles of branched-chain amino acids in microbial fermentation systems, affecting not only specific metabolite yields but also biomass and environmental parameters such as pH. This understanding provides valuable insights into the regulatory mechanisms of microbial fermentation processes. Moreover, in microbial metabolic engineering applications, it is crucial to consider the comprehensive effects of branched-chain amino acid supplementation on the overall production process to design and optimize fermentation conditions more accurately. This study not only offers a solid experimental foundation for surfactin production but also provides valuable references for engineering improvements in related microbial fermentation systems. Additionally, the observed increase in lipopeptide yield with branched-chain amino acid supplementation, particularly with Val, aligns with findings by Zhou et al., who reported that appropriate supplementation of branched-chain amino acids stimulates target product production in similar microbial fermentation experiments, likely due to their role in metabolic pathways(Zhou et al. 2019).
Effect of branched-chain amino acids on the antibacterial activity of lipopeptides from B. velezensis YA215
Following the optimization of fermentation conditions, the antibacterial activity of lipopeptides extracted from fermentation supernatants with branched-chain amino acid supplementation was evaluated using the acid precipitation and ethanol extraction method. The antibacterial effect against E. coli and S. aureus was assessed using an inhibition zone assay with equal lipopeptide concentrations across four sample groups. As illustrated in Fig. 4 and detailed in Table 1, lipopeptides from fermentation media supplemented with the three amino acids exhibited superior antibacterial effects against S. aureus and E. coli compared to the control group. Notably, the antibacterial effect was significantly higher in the Val-supplemented samples than in those supplemented with Leu and Ile. Furthermore, the lipopeptides demonstrated a stronger inhibitory effect against E. coli than S. aureus. As bioactive molecules, lipopeptides are categorized into surfactin, iturin, and fengycin families(Ongena et al. 2008), each exhibiting different antibacterial properties. Surfactin, for instance, shows notable antibacterial activity but limited antifungal activity(Buchoux et al. 2008), whereas iturin possesses strong antifungal activity(Hua et al. 2023, Yaraguppi et al. 2023), and fengycin, with lower hemolytic activity, displays potent antifungal properties(Fu et al. 2022, He et al. 2023). Previous studies have also indicated that surfactin is the primary compound responsible for antibacterial activity. Our prior research corroborated these findings, identifying surfactin as the principal antibacterial component produced by the YA215 strain(Yu et al. 2022). The consistent trend in surfactin yield changes with amino acid supplementation further supports the correlation between surfactin content and lipopeptide antibacterial efficacy. These findings provide valuable clues for understanding the bioactivity of lipopeptides produced by the YA215 strain and their mechanisms of antibacterial action, offering useful references for microbial metabolic engineering in the production of bioactive substances.
Table 1
Lipopeptide inhibition results of B. velezensis YA215 strain.
Sample | S.aureus | E.coli |
Diameter of inhibition zone(mm) |
SS | 10.03 ± 0.21a | 15.17 ± 0.15a |
SS + Leu | 11.73 ± 0.21b | 17.3 ± 0.29b |
SS + Ile | 12.83 ± 0.35c | 18.7 ± 0.32c |
SS + Val | 15 ± 0.56d | 24 ± 0.26d |
Note: different letters indicate significant differences between groups (p < 0.05). |
Effect of branched-chain amino acids on the hydrolyzed fatty acids of lipopeptides from B. velezensis YA215
The increased antibacterial activity of surfactin is not solely linked to its production quantity but also closely associated with structural and conformational changes. In this section, the relationship between the enhanced antibacterial activity of surfactin and its structural modifications is explored. By hydrolyzing lipopeptides extracted from four sample groups, changes in fatty acids were analyzed to infer structural information about surfactin. Initially, the extracted lipopeptides were subjected to high-temperature hydrolysis with hydrochloric acid under identical concentration conditions, resulting in free β-hydroxy fatty acids. These acids were then reacted with methanol under acidic conditions to form β-hydroxy fatty acid methyl esters, which were subsequently detected using GC-MS. Comparative analysis of the total ion chromatograms (Fig. 5) and the extracted ion chromatograms with an m/z of 103 (Fig. 6) revealed that the total types of β-hydroxy fatty acids did not change with branched-chain amino acid supplementation; all four sample groups exhibited 14 chromatographic peaks. In the mass spectrometry analysis, β-hydroxy fatty acid methyl esters generated M-1, M-18, and M-50 fragment ion peaks due to the loss of H atom, H2O, and (CH3OH + H2O) respectively. Through comparison with the NIST database, it was determined that these 14 fatty acid methyl esters had molecular weights ranging from 230 to 300, differing by 14 units, corresponding to β-hydroxy fatty acid methyl esters with varying carbon chain lengths. β-Hydroxy fatty acid methyl esters with the same carbon chain length exhibited n, iso, or anteiso structures due to branching differences(Cheng et al. 2013). It was found that the retention time of β-hydroxy fatty acid methyl esters had a linear relationship with their carbon number, and the retention time followed the order iso < anteiso < n for esters with the same carbon number(Hosono et al. 1983). Based on this analysis, the lipopeptide samples contained β-hydroxy fatty acids with various chain lengths and branching structures, such as iso C12, iso C13, anteiso C13, n C14, iso C14, iso C15, anteiso C15, n C15, iso C16, n C16, iso C17, anteiso C17, and n C18, highlighting the diversity and structural complexity of the fatty acid chains.
Further analysis of the β-hydroxy fatty acid content variation with different carbon chain lengths (Fig. 7) showed that, compared to the control group, the proportions of C12 and C13 β-hydroxy fatty acids decreased with branched-chain amino acid addition. With Val supplementation, the content of C14 β-hydroxy fatty acids increased compared to the control, while other chain lengths decreased. With Leu supplementation, the contents of C15 and C17 β-hydroxy fatty acids increased, whereas other chain lengths decreased. With Ile supplementation, the contents of C16, C17, and C18 β-hydroxy fatty acids increased, while other chain lengths decreased.
Further exploration of the conformational changes in β-hydroxy fatty acids with different chain lengths (Fig. 8) indicated that, compared to the control group, the contents of n C12 β-hydroxy fatty acids, iso C13 β-hydroxy fatty acids, and anteiso C13 β-hydroxy fatty acids decreased with branched-chain amino acid addition. With Val supplementation, the proportion of iso C14 β-hydroxy fatty acids increased from 13.30–23.80%, and iso C16 from 4.22–8.31%, while other conformations decreased. With Leu supplementation, the proportions of iso C15, anteiso C15, iso C17, and anteiso C17 β-hydroxy fatty acids increased, especially iso C15, which rose from 11.42–26.81%, while other conformations decreased. With Ile supplementation, the proportions of n C14, anteiso C15, n C16, anteiso C17, n C17, and n C18 β-hydroxy fatty acids increased, particularly n C16, which significantly rose from 10.37–20.54%, while other carbon chain lengths decreased.
The analysis of the experimental data revealed that the addition of branched-chain amino acids altered the content and configuration of β-hydroxy fatty acids of various chain lengths. This phenomenon might be due to the specific structural effects of these branched chains on the formation and antibacterial activity of surfactin. Liu et al. discovered that adding Arg and Gln to the culture medium of B. subtilis T8942 increased the even-numbered β-hydroxy fatty acids (C14, C16), while adding Cys, His, Met, Ser, or Thr increased the odd-numbered β-hydroxy fatty acids (C13, C15) (Liu et al. 2012). Furthermore, they noted that the addition of branched-chain amino acids (L-Val, L-Leu, and L-Ile) led to significant changes in hydroxy fatty acid residues: L-Leu markedly increased the proportion of iso odd-numbered hydroxy fatty acid residues; L-Ile enhanced the proportion of anteiso odd-numbered hydroxy fatty acid residues; and L-Val elevated the proportion of iso even-numbered hydroxy fatty acid residues. Our experimental results also indicated that Val addition significantly increased the content of C14 β-hydroxy fatty acids, while Leu and Ile additions notably increased the contents of C15, C16, C17, and C18 β-hydroxy fatty acids, highlighting the impact of branched-chain amino acids on surfactin's structure and configuration.
Impact of branched-chain amino acids on the peptide chains of surfactin in B. velezensis YA215
The changes in the peptide chain composition of surfactin under the same concentration conditions in four sample groups were analyzed using LC-MS/MS, providing critical insights into its molecular structure. Table 2 shows that the SS fermentation produced surfactin with peptide chains Glu-Leu-Leu-Val-Asp-Leu-Leu and Glu-Leu-Leu-Val-Asp-Leu-Val, connected to C13-C16 fatty acid chains, known as [Leu 7]surfactin and [Val 7]surfactin, respectively, as well as [Leu 7]surfactin linked to C12 fatty acid chains. Upon Leu addition, [Val 7]surfactin was converted to [Leu 7]surfactin, resulting in a single peptide chain structure of [Leu 7]surfactin. With Ile addition, the Val at the 7th position in the peptide chains of C12, C14, and C16 surfactin was replaced by Leu, unifying the peptide chain structure as [Leu 7]surfactin. The C13 surfactin peptide chain structure remained consistent with the control SS. However, the C15 surfactin peptide chains exhibited two structures: [Leu 7]surfactin and [Leu/Ile 4]surfactin, where Val at the 4th position was replaced by Leu or Ile. Upon Val addition, the C13 surfactin peptide chain displayed two structures: one with Leu at the 7th position replaced by Val, forming [Val 7]surfactin, and another consistent with the control SS, [Leu 7]surfactin. The peptide chain structures of C12, C14, C15, and C16 surfactin were consistent with the control SS, each containing two structures, but the proportion of [Val 7]surfactin notably increased (Fig. 9). Quantitative analysis indicated that the proportion of C14 [Val 7]surfactin increased by 3.29 times compared to the control SS, aligning with the previously observed fatty acid changes. This could be attributed to the addition of Val in the SS medium, enhancing the antibacterial activity of lipopeptides.
Additionally, it was observed that adding L-Val and L-Ile to the medium selectively increased the yield of [Val 7]surfactin, whereas the addition of L-Leu resulted in a lower yield of [Val 7]surfactin. This selective effect may be associated with the cross-relationships between amino acid biosynthesis and metabolic pathways. This finding is consistent with previous studies on the regulatory role of amino acids in lipopeptide biosynthesis. Peypoux et al. found that supplementing the medium with L-Val or L-Ile as nitrogen sources selectively enhanced the production of Val7-surfactin (Peypoux et al. 1992).
Table 2
Results of changes in the structure of the peptide chain of surfactin.
Retention Time (min) | Main LC-MS Peak(m/z) | β-OH fatty acids | Peptide sequence |
SS |
12.99–14.10 | 994.57 | C12 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
13.57 | 1008 | C13 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.29 | 1008 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
14.09 | 1022 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.64 | 1022 | C15 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
14.37 | 1036 | C15 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
15.27 | 1036 | C16 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
15.01 | 1050 | C16 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
SS + Leu |
12.71–14.10 | 994 | C12 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
13.54 | 1008 | C13 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.26 | 1008 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
14.06,14.60 | 1022 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.51,15.37 | 1036 | C15 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.92 | 1050 | C16 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
SS + Ile |
12.71–14.10 | 994 | C12 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
13.53 | 1008 | C13 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
| 1008 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
14.07,14.60 | 1022 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.40,14.94,15.30 | 1036 | C15 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
15.15,16.18 | 1050 | C16 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
15.66 | 1050 | C15 | N-Glu-Leu-Leu-Leu/Ile-Asp-Leu-Leu-C |
SS + Val |
12.71–13.20 | 994 | C12 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
13.20–14.10 | 994 | C13 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
13.57 | 1008 | C13 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.29 | 1008 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
14.04 | 1022 | C14 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
14.59 | 1022 | C15 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
14.49 | 1036 | C15 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |
15.20 | 1036 | C16 | N-Glu-Leu-Leu-Val-Asp-Leu-Val-C |
15.01 | 1050 | C16 | N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C |