GJ11 producing γ-PGA by a glutamate-independent manner
Without sodium glutamate added in the medium, GJ11 produced γ-PGA at about 25.0 g/L. With the increase of sodium glutamate added in the medium, the γ-PGA production was also increased in the broth. However, the γ-PGA production was not increased continuously when the sodium glutamate concentration was more than 40.0 g/L (Fig. 1a). This was consistent with the result of SDS-PAGE (Fig. 1b). Thereby, GJ11 is a glutamate-independent γ-PGA producer, and an addition of glutamate can further improve its γ-PGA production. We further used gel permeation chromatography to determine the molecular weight of γ-PGA produced by GJ11. The retention time of standard γ-PGA was about 8.39 min, while the retention time of γ-PGA produced by GJ11 was about 6.92 min (Fig. 1c). Thereby, the molecular weight of γ-PGA produced by GJ11 is higher than that of the γ-PGA standard with a molecular weight of 580 kD.
Compatibility of GJ11 to FA powder
FA powder was dissolved in water as the medium for culturing GJ11. The results showed that the biomass of GJ11 was gradually increased with the increase of FA powder (< 40 g/L), indicating that FA powder could provide nutrients to support the bacterial growth. After FA powder was increased to more than 40 g/L, the biomass of GJ11 was decreased (Fig. S1a). We further found that, with an increase of FA powder used for culturing GJ11, the pH value of the medium was decreased due to the presence of organic acids (Fig. S1b). The low pH value suppressed the bacterial growth. Thereby, we adjusted the pH value of the medium to 7.0, and fount it was later decreased to ~ 6.5 after sterilization (Fig. S1c).
The original fermentation medium with FA powder was adjust to pH 7.0 for culturing GJ11. As shown in Fig. S1d, the biomass of the culture reached its maximum when FA powder was used at 40 g/L. When the concentration of FA powder was more than 40 g/L, the biomass decreased dramatically when FA powder added in the fermentation medium increased. The γ-PGA production reached the highest value, ~ 42 g/L, when no FA powder was added into the fermentation medium. Then, the production decreased as FA powder added in the fermentation medium increased. Thereby, the excessive FA powder is unfavorable for the bacterial growth and γ-PGA production.
Effects of carbon and nitrogen sources on γ-PGA production
We found that FA powder was unfavorable for GJ11 to produce γ-PGA in the original fermentation medium. Thereby, we further detected whether FA powder (40 g/L) could substitute some nutrients in order to reduce the cost of γ-PGA production. Glucose is known as the most efficient carbon source for producing γ-PGA currently. Thus, we studied whether FA powder could substitute glucose in the fermentation medium. As shown in Fig. 2a, the biomass and γ-PGA production in the broth were both decreased without an addition of glucose. Under the glucose concentration of 70 g/L, the biomass and γ-PGA production were both increased when the concentration of glucose added in the medium increased. When the glucose concentration was more than 70 g/L, the biomass and γ-PGA production was no longer increased with the increase of glucose. Besides glucose, citrate acid is regarded as a common carbon source for γ-PGA production. As shown in Fig. 2b, the increase of citrate sodium could improve γ-PGA production rather than biomass. This was probably due to the fact that citrate acid was mainly contributed to biosynthesizing glutamate, a monomer for biosynthesizing γ-PGA. When the concentration of citrate sodium was more than 10 g/L, the production of γ-PGA was no longer increased with the increase of citrate sodium added in the medium.
Although GJ11 is a glutamate-independent γ-PGA producer, addition with glutamate could improve its γ-PGA production. As shown in Fig. 2c, the biomass was not significantly influenced by addition with sodium glutamate, but the γ-PGA production was increased with the increase of sodium glutamate added in the medium. The highest γ-PGA production was obtained when sodium glutamate was added at a final concentration of 10 g/L, and higher concentrations of sodium glutamate could not improve but reduce γ-PGA production in the broth.
The glutamate-independent producer can use inorganic nitrogen sources for biosynthesizing γ-PGA [21]. As shown in Fig. 2d, an addition of NaNO3 could improve both biomass and γ-PGA production in the broth. When the concentration of NaNO3 was more than 2 g/L, the biomass was no longer increased with the increase of NaNO3, but the γ-PGA production was still increased with the increase of NaNO3 (< 12 g/L). However, another inorganic nitrogen source, NH4Cl, could neither improve γ-PGA production nor increase biomass in the broth (Fig. 2e). Moreover, the bacterial growth was inhibited by NH4Cl when it was added into the medium at a final concentration more than 2 g/L. Additionally, the biosynthesis of γ-PGA was reduced when NH4Cl was added into the medium at a final concentration more than 4 g/L.
Effects of inorganic salts on γ-PGA production
Inorganic salts have been reported to be important for γ-PGA production [26, 27]. As shown in Fig. 3a, KH2PO4 could improve both biomass and γ-PGA production. However, when the concentration of KH2PO4 was more than 0.3 g/L, the biomass was no longer increased with the increase of KH2PO4 added in the medium. Moreover, the biomass was reduced when the concentration of KH2PO4 was more than 0.7 g/L. For γ-PGA production, the excessive KH2PO4 (> 0.9 g/L) was also unfavorable for GJ11 to produce γ-PGA.
It has been reported that Mn2+ can improve cell growth, prolong cell viability, and assist the utilization of different carbon sources and increase γ-PGA production [26]. As shown in Fig. 3b, an addition of MnSO4 could not significantly improve γ-PGA production, indicating that Mn2+ in the FA powder was enough for GJ11 to produce γ-PGA. Moreover, with the increase of MnSO4 added in the medium, γ-PGA production in the broth was gradually decreased.
Mg2+ has been reported to be necessary for the activity of PgsBCA in biosynthesizing γ-PGA [27]. Our results showed that an addition of MgSO4 could not improve biomass and γ-PGA production. When the concentration of MgSO4 increased, γ-PGA production in the broth was gradually reduced (Fig. 3c). Thereby, an addition of Mg2+ is not necessary for γ-PGA production in the medium with FA powder.
We further investigated whether an addition of Ca2+ could improve γ-PGA production. We found that an addition of CaCl2 could not significantly improve biomass and γ-PGA production in the medium with FA powder. Moreover, the excessive Ca2+ inhibited γ-PGA production (Fig. 3d). The results indicated that Ca2+ was already enough for producing γ-PGA in the medium with FA powder.
As shown in Fig. 3E, an addition of FeCl3 in the medium with FA powder was favorable for improving biomass rather than γ-PGA production. However, excessive Fe3+ (> 0.02 g/L) could not further improve biomass in the broth.
Orthogonal test for optimizing fermentation medium
On the basis of our results, we further optimized the fermentation medium with orthogonal test. Glucose, citrate sodium, sodium glutamate, NaNO3, KH2PO4, and FA powder were selected for further optimization according to the orthogonal experiments (L18(37)). As shown in Table 2, glucose, NaNO3, sodium glutamate, and citrate sodium improved γ-PGA production, while FA powder was negative for γ-PGA production. According to the R-value obtained from the orthogonal tests, we found that γ-PGA production was successively affected by sodium glutamate, NaNO3, citrate sodium, FA powder, glucose, and KH2PO4. The optimal combination of the medium was A3B3C2D1E3F3, corresponding to 80 g/L glucose, 20 g/L NaNO3, 0.7 g/L KH2PO4, 20 g/L FA powder, 20 g/L sodium glutamate, and 20 g/L citrate sodium, which resulted in the highest γ-PGA production of 35.54 g/L. On the other hand, glucose and FA powder improved the bacterial biomass, while NaNO3, sodium glutamate, citrate sodium, and KH2PO4 were unfavorable for the bacterial growth. On the basis of R-value, we found that the biomass was successively affected by glucose, NaNO3, citrate sodium, FA powder, sodium glutamate, and KH2PO4. The bacterial growth was negatively related with γ-PGA production. In this study, we mainly focused on γ-PGA production. Thus, our optimized medium contained 80 g/L glucose, 20 g/L NaNO3, 0.5 g/L KH2PO4, 20 g/L FA powder, 20 g/L sodium glutamate, and 20 g/L citrate sodium. Compared to original fermentation medium, the cost of sodium glutamate and citrate sodium were both decreased around one third due to an addition of FA powder in the medium. Moreover, FA powder could be a substitute for NH4Cl, MgSO4, MnSO4, CaCl2, and FeCl3 in the original fermentation medium.
Table 2
Results of Orthogonal Experiment
Treatment | A | B | C | D | E | F | γ-PGA (g/L) | OD600 |
1 | 1 | 1 | 1 | 1 | 1 | 1 | 15.39 | 4.91 |
2 | 1 | 2 | 2 | 2 | 2 | 2 | 22.08 | 3.04 |
3 | 1 | 3 | 3 | 3 | 3 | 3 | 27.14 | 2.15 |
4 | 2 | 1 | 1 | 2 | 2 | 3 | 22.51 | 5.27 |
5 | 2 | 2 | 2 | 3 | 3 | 1 | 26.83 | 5.52 |
6 | 2 | 3 | 3 | 1 | 1 | 2 | 25.53 | 3.61 |
7 | 3 | 1 | 2 | 1 | 3 | 2 | 29.86 | 6.88 |
8 | 3 | 2 | 3 | 2 | 1 | 3 | 26.69 | 7.03 |
9 | 3 | 3 | 1 | 3 | 2 | 1 | 22.78 | 7.38 |
10 | 1 | 1 | 3 | 3 | 2 | 2 | 17.69 | 4.98 |
11 | 1 | 2 | 1 | 1 | 3 | 3 | 33.06 | 3.00 |
12 | 1 | 3 | 2 | 2 | 1 | 1 | 21.68 | 4.42 |
13 | 2 | 1 | 2 | 3 | 1 | 3 | 19.09 | 5.20 |
14 | 2 | 2 | 3 | 1 | 2 | 1 | 20.87 | 4.12 |
15 | 2 | 3 | 1 | 2 | 3 | 2 | 29.21 | 4.45 |
16 | 3 | 1 | 3 | 2 | 3 | 1 | 24.31 | 7.01 |
17 | 3 | 2 | 1 | 3 | 1 | 2 | 22.23 | 7.20 |
18 | 3 | 3 | 2 | 1 | 2 | 3 | 35.54 | 4.43 |
K1 | 138.44 | 128.86 | 145.19 | 160.25 | 130.62 | 131.87 | | |
K2 | 144.04 | 151.76 | 155.08 | 146.48 | 141.48 | 146.60 | | |
K3 | 161.42 | 161.88 | 142.24 | 135.77 | 170.41 | 164.04 | | |
k1 | 23.07 | 21.48 | 24.20 | 26.71 | 21.77 | 21.98 | | |
k2 | 24.01 | 25.29 | 25.85 | 24.41 | 23.58 | 24.43 | | |
k3 | 26.90 | 26.98 | 23.71 | 22.63 | 28.40 | 27.34 | | |
R | 3.83 | 5.50 | 2.14 | 4.08 | 6.63 | 5.36 | | |
Optimization of fermentation conditions for γ-PGA production
On the basis of optimized fermentation medium, we detected the effect of medium pH on γ-PGA production. As shown in Fig. 4a, with the increase of original pH value of medium, the γ-PGA production was gradually decreased. At pH 7.0, the biomass achieved its highest value. This result was consistent with a previous literature [21]. We also investigated the effect of liquid volume on γ-PGA production, and found the production was decreased when the liquid volume increased (Fig. 4b). The biomass reached its highest amount when the liquid volume was 50 mL which was loaded in a 250 mL flask. However, excessive liquid volume was unfavorable for the bacterial growth. As shown in Fig. 4c, the γ-PGA production and biomass were both influenced by the inoculation amount. When the inoculation amount was more than 3%, the γ-PGA production was gradually decreased with the increase of inoculation amount. Similarly, the biomass was decreased with the increase of inoculation amount (> 5%).
Verification of optimized fermentation medium and conditions for γ-PGA production
We cultured GJ11 in the optimized fermentation medium and conditions, and found glucose in the medium was not significantly consumed by GJ11 during the first 12 h, corresponding to our result that no γ-PGA was accumulated in the broth. During 12–36 h, the biomass was dramatically increased, corresponding to a rapid decrease of residual glucose in the broth. Consistently, γ-PGA was rapidly biosynthesized in this period, with a maximum production of 41.47 g/L and a high productivity of 1.15 g/(L·h). During 36–48 h, the biomass, residual glucose, and γ-PGA production had no significant change. During 48–96 h, the biomass was increased again, accompanied by a decrease of residual glucose in the broth. In this period, γ-PGA production was gradually decreased (Fig. 5a), suggesting that some γ-PGA was consumed as carbon and nitrogen source for the bacterial growth. FA in the medium could not be used by GJ11, which was consistent with the previous report [18]. However, other organic acids could be gradually consumed by GJ11, accompanied by a gradually increased pH value of broth (Fig. 5b).
We further verified the influence of FA powder on biomass and γ-PGA production in the optimized formula. We found that the optimized fermentation medium with 20 g/L FA powder could produce γ-PGA at 42.55 g/L, while the medium without FA powder only produced 2.87 g/L γ-PGA (Fig. 5c). These results indicated that FA powder was important for γ-PGA production in our optimized formula because it probably contained some nutrients for the bacterial cells to produce γ-PGA.
HR induced by γ-PGA and its hydrolyzates
Lei et al. have reported that γ-PGA could protect plants against abiotic stress, such as high and low temperature [28]. In our study, we used γ-PGA purified from the fermentation broth of GJ11 to treat plants, then invested whether HR could be triggered in the leaves. The results showed that γ-PGA could not induce neither HR nor ISR to protect plants form the pathogen (Pst DC3000) infection (Fig. 6a and b). We deduced that this might be due to the high molecular weight of γ-PGA. Thus, we digested γ-PGA into γ-PGA hydrolysates with smaller molecular weights. We found that, as hydrolysis time increased, more and more γ-PGA was digested into the hydrolysates with smaller molecular weights (Fig. 6c).
After hydrolysis, the solution was adjusted to pH 7.0, then used for injecting the tobacco leaves. As shown in Fig. 6d, the 5 h hydrolysates could trigger HR in the leaves significantly. Consistently, the plants with ISR triggered by irrigating roots with 5 h hydrolysates showed a significant resistance against the pathogen (Pst DC3000) infection (Fig. 6e). We further found that 5 g/L γ-PGA hydrolysates was more effective for inducing the resistance of plants against Pst DC3000 infection (Fig. 6f).
H2O2 accumulation and callose deposition induced by γ-PGA hydrolyzates
Many activators produced by beneficial microorganisms can trigger ISR to protect plants from pathogens infection by eliciting defence-related responses, such as ROS (e.g. H2O2) accumulation and callose deposition [24]. In order to know whether γ-PGA hydrolyzates could induce the defence-related responses, the roots of A. thaliana were treated with γ-PGA hydrolyzates, then infected by Pst DC3000. The results showed that treatment with γ-PGA hydrolyzates alone could not induce significant H2O2 accumulation (Fig. 7a) and callose deposition (Fig. 7b) in the leaves. However, inoculation with pathogen alone could elicit H2O2 accumulation and callose deposition. Our further investigation showed that pre-treatment with γ-PGA hydrolyzates could elicit mild but effective plant immunity to rapidly response to Pst DC3000 infection, accompanied by significant H2O2 accumulation (Fig. 7a) and callose deposition (Fig. 7b) in the leaves.
We used different defense-compromised lines of Arabidopsis, including NahG, jar1-1, ein2, and npr1, to detect the possible signals induced by γ-PGA hydrolyzates. Compared to the control (water), after inoculation with Pst DC3000 for 12 h, the lines, including Col-0, NahG, and jar1-1, were all observed to have H2O2 accumulation (Fig. 7c) and callose deposition (Fig. 7d), while the lines, such as ein2 and npr1, were not. After 24 h, pre-treatment with γ-PGA hydrolyzates significantly enhance H2O2 accumulation (Fig. 7c) and callose deposition (Fig. 7d) in the lines Col-0, NahG, and jar1-1, rather than in the lines ein2 and npr1. These results suggested that the ISR induced by γ-PGA hydrolyzates is dependent on NPR1, and the ET signal, rather than the SA and JA signals in plants.
We further recovered the pathogen in different lines, and found that pre-treatment with γ-PGA hydrolyzates could significantly reduce the amount of Pst DC3000 in Col-0, NahG, and jar1-1 when compared to that in CK (Fig. 7e). However, the pathogen amounts recovered in ein2 and npr1 was similar between the group pre-treated with γ-PGA hydrolyzates and the control (CK). The results further verified that the ISR induced by γ-PGA hydrolyzates is dependent on the ET signaling and NPR1 in plants.