Cell growth, PHA content, and substrate consumption during co-feeding of aromatic compounds and glycerol to P. putida KT2440.
Pseudomonas putida are natural producers of mcl-PHAs due to their unique metabolic versatility to synthesize polymers when grown on various carbon sources, such as glucose, glycerol, fatty acids, and lignin derivatives [29, 41]. P. putida KT2440 exhibits a strong ability to convert lignin derivatives into PHA as a sole carbon and energy source through the β-ketoadipate pathway [13, 25, 39]. Herein, the physiological characteristics and mcl-PHA synthesis capabilities of P. putida KT2440 were investigated using 10 g·L-1 glycerol as the main carbon source with the addition of 0.5 g·L-1 benzoate, vanillin, or vanillic acid as the co-metabolic substrate, with a fixed NH4Cl concentration of 0.8 g·L-1. Figure 1 depicts the cell dry weight (CDW), PHA content/yield, main carbon source consumption, and nitrogen consumption compared to the co-metabolism of glycerol/aromatic compounds over time.
As shown in Fig. 1a, with 10 g·L-1 glycerol alone, cells grew slowly and reached 0.4 g·L-1 cell dry weight (CDW) at 12 h, followed by rapid growth until 48 h, then gradually reached 2.6 g·L-1 CDW at 72 h. With the addition of 0.5 g·L-1 benzoate, vanillin, or vanillic acid with 9.5 g·L-1 glycerol, the cell growth rapidly increased to 3.0 g·L-1, 2.8 g·L-1 and 3.0 g·L-1 CDW, respectively, at 72 h. Results indicated that the addition of 0.5 g·L-1 aromatic monomer (i.e. benzoate, vanillin, and vanillic acid) stimulated cell growth as the cell dry weight at 72 h increased by 16.1%, 9.4% and 15.0%, respectively, compared to when feeding glycerol alone.
The same trend was also revealed in PHA content/yield. As shown in Fig. 1b, with 10 g·L-1 glycerol alone, the PHA content decreased from 6.8% (0.009 g·L-1) to 2.9% (0.01 g·L-1) of CDW at 12 h and to 1.1% (0.01 g·L-1) of CDW at 24 h, then significantly increased to 20.7% of CDW (0.5 g·L-1) at 72 h. With the addition of 0.5 g·L-1 of benzoate, vanillin, and vanillic acid, PHA content/yield followed the same declining trend during the first 24 h, and continuously grew rapidly to 30.9% (0.9 g·L-1), 26.7% (0.7 g·L-1) and 28.0% (0.8 g·L-1) of CDW, respectively, at 72 h. Compared to 10 g·L-1 glycerol alone, the addition of 0.5 g·L-1 benzoate, vanillin, and vanillic acid with 9.5 g·L-1 glycerol increased the PHA content by 49.3%, 29.0% and 35.3% of the CDW at 72 h, respectively. Among the three aromatic monomers, benzoate showed the strongest effects in terms of enhancing cell growth and PHA content.
In addition, concomitant assimilation of glycerol, aromatic compounds, and the nitrogen source was observed. Glycerol was rapidly consumed within 72 h, both with and without the addition of aromatic compounds (Fig. 1c), although the addition of aromatic compounds slightly slowed down the glycerol consumption, but did not reduce the extent of its consumption at 72 h. All the aromatic compounds in the co-feeding treatment were nearly completely consumed within the first 12 h (Fig. 1d). With all four different combinations of carbon sources, the nitrogen concentration dramatically declined during the first 24 h and was gradually exhausted by 48 h, while the addition of aromatic compounds appeared to accelerate nitrogen consumption (Fig. 1e). These results suggested that PHA started to accumulate after 24 h, when the nitrogen supply entered a limited level.
Compared to genetic modification [23, 24], the co-feeding of multiple carbon substrates is a relatively simple and straightforward approach to reduce costs and improve PHA yield. Effects of the co-feeding strategy on PHA synthesis vary depending on the different combinations of carbon sources. A previous study reported the successful enhancement of PHA content to 32.4% of CDW by co-feeding glycerol with octanoate compared to 1.3% of CDW with glycerol alone by P. putida mt-2 [29]. On the contrary, some previous studies reported that some aromatic compounds have negative effects on cell growth and PHA synthesis. Kenny et al. [31] reported that although co-feeding glycerol with terephthalic acid (TA) to P. putida Go16 enhanced cell growth and PHA yield compared to feeding terephthalic acid alone, co-feeding reduced cell growth and PHA yield when compared to feeding glycerol alone, resulting in a total CDW from 19.1 g·L-1 (glycerol alone) to 11.7–14.7 g·L-1 and total PHA yield from 6.3 g·L-1 (glycerol alone) down to 4.4–5.2 g·L-1. Moreover, Curley et al. [32] also reported that co-feeding 5 mM 5-(4-tolyl)-valeric acid (an aromatic compound, TVA) with 15 mM nonanoic acid enhanced the cell growth and PHA content to 1.4 g·L-1 and 15.0% of the CDW, respectively, compared to 0.6 g·L-1 and 0.03% of the CDW with 10 mM TVA control, respectively, but still produced a lower CDW and PHA content than when nonanoic acid was fed alone. In another studies, the PHA content reached around 70.0% of CDW with 1 g·L-1 (6 mM) nonanoic acid in P. putida, which is significantly higher than that when co-feeding TVA with nonanoic acid [42]. Appropriate aromatic compounds that can be fed together with glycerol and enhance the cell growth and PHA content in P. putida when compared to feeding glycerol alone have been rarely reported.
Glycerol is the main waste product of biodiesel production through the transesterification of animal fats and vegetable oils [23]. Glycerol has become a promising substrate for PHA production. In addition, lignin is considered as a low-value waste product from biorefinery processes [16]. In this study, the results of co-feeding of glycerol and lignin-derived aromatic compounds, including benzoate, vanillin, and vanillic acid, showed substantial enhancement on cell growth and PHA content compared to feeding glycerol alone. This provides a unique strategy to lower production costs and improve PHA content/yield. The PHA compositions and their underlying mechanisms were further investigated in the following experiments.
Effects of lignin derivatives on PHA compositions.
The GC-MS analysis of the PHA samples from the co-feeding of different aromatic monomers with glycerol confirmed the presence of five monomers, including 3-hydroxyhexanoate (3HHX, C6:0), 3-hydroxyoctanoate (3HO, C8:0), 3-hydroxydecanoate (3HD, C10:0), 3-hydroxydodecoate (3HDD, C12:0), and 3-hydroxytetradecanoate (3HTD, C14:1/C14:0) (Figs. S1 and S2, Table 1). 3HD (C10:0) was the predominant monomer under all fermentation conditions and accounted for up to 63.3% of the total PHA monomer composition, which was consistent with previous reports [24, 43]. Compared to the PHA grown on glycerol only, with the addition of aromatic compounds, the 3HD (C10:0) and 3HDD (C12:0) content decreased to around 58.9% and 7.6%, respectively, while the content of shorter chain length monomers increased from 3.5% to 4.5% for 3HHX (C6:0) and from 20.4% to 23.9% for 3HO (C8:0). Unsaturated and saturated 3HTD (C14:1, C14:0) remained at around 4% and less than 1% under all the tested fermentation conditions. Our results showed that the addition of aromatic compounds decreased the proportion of long chain monomer (C10:0 and C12:0) of PHA polymer and increased the short-chain monomer (C6:0 and C8:0) proportion. Previous reports indicated that compared to glycerol alone, co-feeding terephthalate (TA) with glycerol also decreased the amount of 3-hydroxydecanoic acid (3HD, C10:0) and 3-hydroxydodecanoic acid (3HDD, C12:0) and increased the amount of 3-hydroxyoctanoic acid (3HO, C10:0) and 3-hydroxyhexanoic acid (3HX, C6:0) [31]. Besides, previous proteomics studies revealed that the β-oxidation pathway was upregulated when P. putida was fed solely with vanillic acid or lignin [13]. Therefore, the addition of lignin derivatives might elevate the fatty-acid degradation pathway and decrease the number of long-chain monomers (C10 and C12) in favour of producing more reducing equivalents and short-chain monomers (C8 and C6). Thus, it indicates that the manipulation of PHA polymer composition is possible through the substrate co-feeding strategy.
Table 1
Monomer compositions of PHA accumulated from different carbon sources a.
| 3HHX | 3HO | 3HD | 3HDD | 3HTD |
| C6:0 | C8:0 | C10:0 | C12:0 | C14:1 | C14:0 |
| mol % | mol % | mol % | mol % | mol % | mol % |
Glycerol 10 g·L− 1 | 3.5 ± 0.3 | 20.4 ± 1.2 | 63.3 ± 3.4 | 8.3 ± 0.7 | 4.4 ± 0.4 | 0.1 ± 0.01 |
Glycerol 9.5 g·L− 1 + Benzoate 0.5 g·L− 1 | 4.3 ± 0.3 | 23.5 ± 1.3 | 58.9 ± 3.9 | 7.8 ± 0.6 | 4.8 ± 0.3 | 0.7 ± 0.08 |
Glycerol 9.5 g·L− 1 + Vanillin 0.5 g·L− 1 | 4.5 ± 0.3 | 23.3 ± 1.4 | 59.5 ± 3.5 | 7.9 ± 0.6 | 4.3 ± 0.3 | 0.5 ± 0.04 |
Glycerol 9.5 g·L− 1 + Vanillic acid 0.5 g·L− 1 | 4.5 ± 0.3 | 23.9 ± 1.6 | 59.4 ± 3.2 | 7.6 ± 0.6 | 4.2 ± 0.4 | 0.4 ± 0.03 |
a 3-hydroxyhexanoate (C6:0, 3HHX), 3-hydroxyoctanoate (C8:0, 3HO), 3-hydroxydecanoate (C10:0, 3HD), 3-hydroxydodecanoate (C12:0, 3HDD), 3-hydroxytetradecanoate (C14:1, C14:0, 3HTD) were determined by GC/MS. |
HMBC NMR analysis was used to characterize the monomer composition of PHA produced by P. putida KT2440 with glycerol alone (Fig. S3a, table S1) or co-feeding with benzoate (Fig. S3b, table S1). The HMBC spectra usually contains multiple bond correlations, including two or three bond couplings between 1H and 13C. Several residual one-bond correlation (HSQC-like) peaks were also observed doublets (two peaks in the 1H dimension), an artefact of incomplete filtering of one-bond correlations [44]. As shown in figures S3a and S3b, 1H resonances at 2.00 and 2.34 ppm are coupled to alkene carbons at 122.9 and 133.9 ppm indicating they are allylic (adjacent to a double bond) CH2 groups [45], distinct from the more common 1H resonances from CH2 groups in the alkyl chains (around 1.26 ppm). The 13C signals of 122.9 ppm and 133.9 ppm correspond to the double bond between Δ5 and Δ6 of C14 [41, 45], whose residual one bond correlations are visible at the left side of Fig. S3b. The proton signal at 5.16 ppm is assigned to the H-3 CH group, along the backbone of the PHA polymer and connecting with the adjacent monomer residue through the ester bond (-C(O)-O-CH-); the carbon of carbonyl group is C-1 of the preceding residue and has a multiple bond correlation H-3. The shoulder peak at 2.46 ppm and 2.55 ppm are the diastereotopic C-2 CH2 group and are clearly coupled with the adjacent C-1 CH in the COSY spectrum (Fig. S4a). Consistently, all of the multiple bond couplings of H-2 demonstrated two peaks closely together. The multiple bond couplings of saturated groups such as CH2 or CH3 are crowded in the upper right corner of the HMBC spectra. The detailed assignments of each monomer were carefully noted in the spectra, cross validated by COSY NMR (Fig. S4a) and HSQC NMR spectra (Fig. S4b) and listed in Table S2. The overlapping peaks were assigned to the same value and also corrected as based on previous publications [45–47]. Notably, the assignments of the longer alkyl chains are approximate, and the accurate values may vary due to the overlap in both the proton and carbon dimensions. Moreover, several unassigned peaks remain in the HMBC (notably at δ1H 2.28 ppm, with cross peaks to δ13C 24.9, 29.6, and 173.2 ppm), HSQC (δ1H/13C 2.28/31.1 ppm), and COSY spectra; these may correspond to undiscovered components in the PHA polymer. Overall, the HMBC spectra of PHA from cells grown on glycerol alone or glycerol with benzoate take a similar pattern, indicating that the PHA monomer composition is from C6 to C14 and confirming the monomer characterization by GC-MS.
Effects of the ratio of glycerol to benzoate on cell growth and PHA synthesis.
To gain more detailed insights into regulation patterns of PHA synthesis, the effects of the ratio of glycerol to benzoate (10 g·L-1 total carbon source and 0.8 g·L-1 NH4Cl) on CDW (Fig. 2a), PHA content/yield (Fig. 2b), glycerol utilization (Fig. 2c), and aromatic utilization (Fig. 2d) were investigated. As shown in Fig. 2a, for all six combinations of glycerol and benzoate, the cell dry weight generally increased until 72 h and maintained nearly constant until after 120 h total hours of fermentation. Results showed that the addition of 1 g·L-1 benzoate as a co-substrate with 9 g·L-1 glycerol led to significant improvement in both cell growth and PHA accumulation compared to feeding glycerol alone. After 72 h of fermentation, compared with 10 g·L-1 glycerol alone, the CDW and PHA content of P. putida KT2440 with 9 g·L-1 glycerol and 1 g·L-1 benzoate increased from 2.6 g·L-1 to 2.9 g·L-1 (increased by 11.8%) and from 22.8% (0.6 g·L-1) to 37.2% of CDW (1.1 g·L-1, increased by 63.2%), respectively. Although the PHA content with 10 g·L-1 glycerol continuously improved to 28.3% of CDW (0.7 g·L-1) till 96 h, the PHA content was still lower than that with glycerol 9 g·L-1 plus benzoate 1 g·L-1. When the benzoate concentration was higher than 1 g·L-1, the cell growth and PHA synthesis were repressed. In particular, PHA synthesis nearly ceased and CDW was reduced by more than one-half with feedstocks such as glycerol 6 g·L-1 + benzoate 4 g·L-1 or glycerol 5 g·L-1 + benzoate 5 g·L-1, although up to 97% of benzoate and glycerol in the medium were consumed. These results indicated that as the benzoate concentration increased to 1 g·L-1, the PHA content and cell dry weight (CDW) reached the highest value of 37.2% (1.1 g·L-1) and 2.9 g·L-1, respectively. Further increased benzoate loading decreased both cell growth and PHA content.
Similar to previous studies [5, 10], the simultaneous consumption of a dual carbon source, including glycerol and benzoate by P. putida under co-feeding conditions, was observed. The glycerol concentrations decreased for all six scenarios and reached nearly zero within 120 h. In particular, during the 48 h to 72 h period, with glycerol 9 g·L-1 plus 1 g·L-1 benzoate, the consumption of glycerol was significantly accelerated to reach near-complete consumption at 72 h, faster than all other scenarios, including through feeding glycerol alone. In contrast, the benzoate concentration decreased at a nearly linear consumption rate of 1 g·L-1 per 24 h for all five scenarios. These results showed that when the benzoate concentration was lower than 1 g·L-1, PHA biosynthesis started to rise. Also, 1 g·L-1 benzoate co-feeding with 9 g·L-1 glycerol improved cell growth, PHA content, and glycerol consumption when compared to feeding glycerol alone, although the acceleration of glycerol consumption occurred in a delayed manner. On the other hand, when the initial benzoate was higher than 1 g·L-1, the resulting cell growth and PHA content were lower than those with feeding of glycerol only, which might be due to the degradation of PHA and other biomass-derived precursors in order to defend against oxidative stress generated by benzoate [14]. Addition of benzoate and its corresponding oxidative stress generation have been extensively studied in P. putida. The addition of benzoate and its corresponding oxidative stress generation have been extensively studied in P. putida. In specific, when the benzoate concentration was higher than 1 g·L-1, P. putida cells faced elevated oxidative stress, which in turn exaggerated the detoxification process [48]. Previous proteomics studies indicated that four categories of enzymes were up-regulated during the benzoate utilization process, including enzymes related to the benzoate degradation pathway, enzymes related to general metabolism (e.g. TCA cycle), enzymes related to detoxification (e.g. ABC transporter), and oxidative stress-related proteins such as catalase/peroxidase [48, 49]. The detoxification process raises energy demand, which means that cells would divert the carbon fluxes to central metabolism and other energy-generating pathways to meet the demand, which in turn decreases the cell growth and PHA synthesis fluxes. Thus, whenever the benzoate was nearly consumed and the cell was relieved from the oxidative stress, the PHA synthesis process would then activate as long as the glycerol was still abundant in the medium.
Effects of nitrogen availability on cell growth and PHA accumulation using glycerol with or without benzoate.
Results indicated that the addition of a small amount of benzoate (1 g·L-1) increased cell growth and PHA accumulation, which suggests that mild oxidative stress might be beneficial. However, the specific mechanisms remain unclear. One possible explanation is the addition of benzoate increased the total equivalent carbon amount, since one molar benzoate is equivalent to seven molars of available carbon and thus increases the carbon and nitrogen ratio. The presence of an appropriate amount of nitrogen source is critical to both cell growth and PHA accumulation. In our previous publication, PHA accumulation rate was reportedly higher under nitrogen deficient conditions than that under nitrogen sufficient conditions [25]. Therefore, further investigation is needed to confirm the regulation dependence of PHA synthesis for co-feeding glycerol with benzoate among varied nitrogen source concentrations. Since the amount of benzoate addition did affect PHA synthesis, investigating the relationship between benzoate addition and the nitrogen source would guide future PHA optimization process design. Herein, the effects of benzoate, the nitrogen source and the glycerol ratio on cell growth and PHA accumulation were investigated in this study. The total carbon source remained the same at 10 g·L-1 in various combinations, including glycerol 10 g·L-1 (108.6 mM, total C 325.8 mM), glycerol 9 g·L-1 (97.7 mM) plus benzoate 1 g·L-1 (8.2 mM, total C 350.5 mM), glycerol 5 g·L-1 (54.3 mM) plus benzoate 5 g·L-1 (40.9 mM, total C 449.2 mM), and benzoate 10 g·L-1 (81.9 mM, total C 573.3 mM). The nitrogen source, NH4Cl, also varied in different concentrations, including 0.2 g·L-1 (3.7 mM), 0.5 g·L-1 (9.3 mM), 0.8 g·L-1 (15 mM), and 1.1 g·L-1 (20.6 mM). The cell growth and PHA content were investigated under different glycerol/benzoate/nitrogen scenarios.
Figure 3 shows the effects of NH4Cl concentration on the CDW (Fig. 3a) and PHA content (Fig. 3b) using 10 g·L-1 carbon source. As shown in Fig. 3a, the CDW with 1 g·L-1 benzoate (8.2 mM) plus 9 g·L-1 glycerol (97.7 mM, the white column) was higher than that with 10 g·L-1 glycerol alone under all tested nitrogen concentrations. The CDW with 1 g·L-1 benzoate plus 9 g·L-1 glycerol reached the highest amount, at 3.1 g·L-1 with 1.1 g·L-1 NH4Cl, whereas the CDW only reached 2.4 g·L-1 with 10 g·L-1 glycerol alone. Higher benzoate concentrations inhibited cell growth and the CDW was maintained at 0.8 g·L-1 with 0.2–0.8 g·L-1 NH4Cl and decreased to 0.5 g·L-1 CDW with 1.1 g·L-1 NH4Cl. When the ratio of benzoate/glycerol was raised to 5/5 and 10/0, cell growth was suppressed and dropped below 0.9 g·L-1 CDW. As shown in Fig. 3b, when 1 g·L-1 of benzoate was co-metabolized with 9 g·L-1 glycerol, the PHA content with various levels of nitrogen supply were all higher than those from 10 g·L-1 of glycerol. In particular, the maximum PHA content was 45.7% CDW (with 0.2 g·L-1 NH4Cl) and the PHA content decreased as the NH4Cl concentration increased. If the benzoate/glycerol ratio changed to 5/5 and 10/0, the PHA content reached its maximum at 39.6% and 5.6% of CDW at 0.2 g·L-1 NH4Cl, respectively, and further decreased significantly as the nitrogen concentration increased. In addition, different timings of nitrogen depletion occurred under different initial nitrogen source conditions, which can affect the cell growth and PHA accumulation. The PHA content using glycerol 9 g·L-1 plus benzoate 1 g·L-1 was 1.2 g·L-1, which was 1.7 times the value achieved from glycerol 10 g·L-1 with NH4Cl at 0.8 g·L-1. Although the maximum yield of 3.1 g·L-1 CDW was achieved under the nitrogen sufficient condition of NH4Cl at 1.1 g·L-1, PHA synthesis was seriously inhibited. P. putida KT2440 nearly stopped accumulating PHA when the NH4Cl and benzoate concentration exceeded 0.2 g·L-1 and 5 g·L-1, respectively. Results showed that even though the addition of benzoate increased the total equivalent carbon amount, the addition of only 1 g·L-1 benzoate resulted in the highest PHA accumulation content. Theoretically, the PHA content should be further enhanced as the amount of benzoate amount addition increased. However, the results showed that the addition of 9 g·L-1 glycerol and 1 g·L-1 benzoate led to the highest cell growth and PHA content at all tested initial nitrogen concentrations. Besides, the cell growth and PHA content both decreased as the benzoate amount was higher than 1 g·L-1 in spite of different initial glycerol/nitrogen ratios. This result indicated that the effects of benzoate addition on CDW and PHA content was independent from the nitrogen availability, which ruled out the possible correlation of increased carbon to nitrogen ratio with PHA synthesis.
Metabolite profiles and regulatory patterns of glycerol and benzoate co-metabolism in P. putida KT2440.
PHA is accumulated as carbon and as a reductive-power storage polymer in P. putida KT2440 under demanding physiological conditions. Maintaining the intracellular redox balance of cells plays a pivotal role in PHA biosynthesis. The pyridine nucleotides NAD(P)+ and NAD(P)H are involved in both catabolism and anabolism as the most important redox carriers. Consequently, changes of their intracellular concentrations lead to altered metabolic network patterns [50]. Additional, accurate identification and quantitation of metabolite responses to substrate alteration can provide a detailed physiological characterization of the microorganism and valuable guidance for strategies to enhance PHA synthesis. In this study, NAD(P)+/NAD(P)H concentrations, ratios and metabolite profile analysis using NMR were investigated to gain insights on the regulatory patterns of glycerol and benzoate co-metabolism in P. putida KT2440.
NMR spectroscopy is a commonly used analytical tool in metabolomics due to its nondestructive, nonbiased, automatable and reproducible features. In addition, by combining one dimensional 1H, 13C, and 31P NMR techniques (Figs. S5-S8, Table S3), NMR metabolomics analysis is particularly effective at detecting and characterizing compounds that are less tractable polar compounds such as sugars, organic acids, phospholipids, and nucleoside compounds (NADP(H)), which mainly compose intracellular metabolite components [51]. In order to investigate the regulatory patterns of the co-feeding strategy of glycerol and benzoate to P. putida KT2440, 1H NMR spectroscopy was first used to determine extracellular metabolite profiles. Samples were taken at 72 h of fermentation, using glycerol 10 g·L-1, glycerol 9 g·L-1 plus benzoate 1 g·L-1, glycerol 5 g·L-1 plus benzoate 5 g·L-1, and benzoate 10 g·L-1 as carbon sources, respectively (Fig. S5). When feeding with 10 g·L-1 glycerol alone (Fig. S5a), the remaining glycerol was still abundant. In contrast, the glycerol peaks were in lower abundance and no benzoate peaks were detected after co-feeding 9 g·L-1 glycerol and 1 g·L-1 benzoate (Fig. S5b), indicating that the addition of 1 g·L-1 benzoate enhanced the consumption of glycerol. However, as the benzoate concentration increased to 5 g·L-1 (Fig S5c), compared to the other feeding conditions, more glycerol remained in the medium, suggesting that further elevated benzoate concentration suppressed glycerol utilization, which were consistent with the HPLC results before (Figs. 2c and 2d). Higher peak intensity of benzoate was exhibited in Figure S5d, reflecting the fact that more benzoate remained unutilized in the medium.
The intracellular metabolites exhibited a distinct profile pattern when compared with the supernatant. Both 1H (Fig. S6) and 13C NMR spectroscopy (Fig. S7) were used to determine the intracellular metabolite profiles using the same sampling time as the supernatant. Trehalose was clearly identified among all of the feeding conditions and exhibited the highest peak intensity with 10 g·L-1 glycerol alone (Figs. S6a and S7a). A rapid declining trend of the trehalose signal was observed both in the 1H NMR (Figs. S6b-S6d) and 13C NMR spectra (Figs. S7b-S7d) as the benzoate concentration increased from 1 g·L-1 to 5 g·L-1 and maintained a low level when progressing towards 10 g·L-1. The succinate and acetate compounds stayed at a low level for 10 g·L-1 glycerol and 9 g·L-1 glycerol + 1 g·L-1 benzoate, then spiked to a higher level when the benzoate concentration increased to 5 g·L-1 and went back to lower levels again for 10 g·L-1 benzoate (Figs. S6a-S6d). Acetate acts here as an important intracellular scavenging carbon source for maintaining the Acetyl-CoA balance [52, 53]. Our results showed that a 5 g·L-1 benzoate concentration might inhibit the acetate re-cycling process, which correlates with acetate accumulation. Also, as an indicator, the accumulation of succinate might suggest that the reduction route of the TCA cycle was activated with the addition of benzoate, which generated more reducing currency (NADPH) to cope with the excess oxidative stress [43]. On the other hand, succinate is the junction between the benzoate degradation pathway and the TCA cycle [54]. An enhanced benzoate concentration might therefore contribute to the observed succinate accumulation. However, too high of a benzoate concentration, such as 10 g·L-1, might slow down the benzoate utilization due to inhibited cell growth, which was correlated with a lower abundance of succinate and acetate (Figs. S6d and S7d).
In addition, 31P NMR was used to characterize the intracellular phosphate-containing extracts (Fig. S8). 31P NMR results showed different phosphate profiles when co-feeding glycerol with benzoate at various levels compared to when feeding glycerol or benzoate as the sole carbon source. The signals of NADP+ and NADPH were identified among all of the treatments, and it was found that the NADP+/NADPH abundance increased in the intracellular extracts as the benzoate concentration increased with the co-feeding of glycerol (Figs. S8a-S8c). Metabolite profiling using NMR techniques could provide solid results in metabolite characterization. Results suggested that a higher level of PHA synthesis was realized by increasing NADH, NADPH, and glycerol utilization, likely through improved reduction power generation pathways such as the ED pathway and the reduction route of the TCA cycle [9]. It indicated that oxidative stress could manipulate the PHA synthesizing process by altering the intracellular NADPH content, since NADPH is crucial for maintaining antioxidant defences [55]. The increased NADPH can also be diverted to the PHA biosynthesis pathways and the building up of biomass in order to maintain the redox balance of the cell. Reducing equivalent was upregulated with the addition of benzoate, which might be the directly correlated reason for PHA enhancement. Therefore, cross-evaluation for the intracellular redox state by measuring the NAD+/NADH and NADP+/NADPH ratios was needed to further confirm the findings.
In Fig. 4, the intracellular NAD+/NADH (Fig. 4a) and NADP+/NADPH ratios (Fig. 4b) over time using different combinations of glycerol and benzoate were elucidated in order to understand the relationship between the redox balance and PHA production. The total pool of NADH was larger than that of NADPH. As shown in Fig. 4a, the ratio of NAD+/NADH at 12 h increased dramatically within the test range, especially from 10.2 with glycerol 10 g·L-1 to 18.4 with glycerol 9.5 g·L-1 and benzoate 0.5 g·L-1. As the cultivation time progressed, the ratio of NAD+/NADH declined during both the exponential and the late-log phase. For all glycerol/benzoate treatments, the NADH concentration increased during fermentation and reached its highest level at 48 h, followed by a significant decline at 72 h (Table S4). The NAD+/NADH ratio is an important component of the redox state of a cell, and NADH links the TCA cycle to cellular energy generation. The central TCA pathway is regulated by cellular energy level and redox balances, which precisely matches NADH production to respiratory demand. The downward trend in the NAD+/NADH ratio and the upward trend in NADH concentration revealed that oxidative stress was strengthened and that the carbon fluxes in the TCA cycle switched to the PHA cycle to some extent by adding a small amount of benzoate (1 g·L-1 or 0.5 g·L-1) into the glycerol fermentation system (Figs. 1 and 2). In addition, when adding 2 g·L-1 benzoate as the co-feeding substrate, the NADH concentration was slightly higher than that with 10 g·L-1 glycerol as fermentation persisted until 72 h (Table S4). It is plausible that more benzoate entered the β-ketoadipate pathway and is converted into succinate and acetyl-CoA, which in turn entered the TCA cycle and produced more NADH in P. putida KT2440.
When P. putida grew on benzoate and glycerol, the ratio of NADP+/NADPH dramatically declined during the whole fermentation period (72 h) (Fig. 4b), but all NADP+/NADPH ratios from the three different combinations of glycerol and benzoate loading were lower than those with glycerol alone. In addition, with 0.5 g·L-1 benzoate + 9.5 g·L-1 glycerol and 1 g·L-1 benzoate + 9 g·L-1 glycerol, the NADPH concentration increased as the fermentation was prolonged, while the NADPH concentration with 10 g·L-1 glycerol alone decreased during the first 24 h, and then increased until 72 h. Notably, the intracellular NADPH concentration increased significantly during the late logarithmic phase in which large amounts of PHA and biomass accumulated. The yields of PHA and CDW using glycerol 9.5 g·L-1 plus benzoate 0.5 g·L-1 or glycerol 9 g·L-1 plus benzoate 1g·L-1 were much higher than the values from glycerol 10 g·L-1 (Figs. 1 and 2). While NADH participates in catabolic reactions, NADPH predominantly acts as a reducing agent in anabolic reactions, such as the biosynthesis of PHA biopolymers and active biomass [56]. In a previous publication, when P. putida grew on glucose as the sole carbon source, the NADP+ concentration was higher than NADPH [57]. The NADP+/NADPH ratio was around 1.21 and the NAD+/NADH ratio was 11.3, whereas the NADP+/NADPH ratio was even higher in the mutant strain. For example, in the glucose dehydrogenase inactive strain (P. putida KT2440Δgcd), the NADP+/NADPH ratio was around 3.4, which is higher than the wild type strain. In the pyruvate dehydrogenase overexpression strain (P. putida KT2440 AcoA), the NADP+/NADPH ratio (7.44) was even higher than with the wild type. These results suggested that a mutation of enzymes in carbohydrate utilization pathways hampered the reduction power generation while increasing the NADP+/NADPH ratio at the same time. However, the downward trend of NADP+/NADPH in co-feeding conditions suggested the activation of enzymes involved in reducing power generation pathways, which still needs further confirmation through proteomics analysis.
Cellular proteome profiles of co-feeding glycerol with benzoate in P. putida.
In general, bacteria have evolved several strategies to be able to adapt to fluctuating environments to ensure their survival. One of them is up-regulating or down-regulating the synthesis of certain proteins while metabolizing available nutrients [58]. Therefore, to identify potential enzymes and pathways involved in the co-feeding of glycerol and benzoate in P. putida, the global proteome profiles of intracellular extracts (n = 3 for each condition) were analyzed. Strains were fed on 10 g·L-1 glycerol or 9 g·L-1 glycerol + 1 g·L-1 benzoate as their carbon sources for 3 days. A total of 1926 proteins were quantified, including 249 up-regulated proteins and 214 down-regulated proteins in the glycerol and benzoate co-feeding condition, compared to the glycerol control (q value < 0.05 and fold changes > 1.3). The list of up-regulated and down-regulated proteins (Table S5-S6) was first merged and then subjected to gene ontology (GO) enrichment analysis. Biological process (BP) and KEGG pathway enrichment analyses were performed to compare the bacterial physiological and metabolic responses to glycerol and co-feeding glycerol + benzoate conditions.
The top 15 enriched biological processes with a p value of less than 0.01 are presented in descending form in Fig. 5a. Notably, the addition of benzoate activates a series of up-regulations of chemotaxis proteins (e.g. chemotaxis histidine kinase CheA, Table S5) in the signal transduction process of P. putida. Moreover, the locomotion process was exclusively upregulated, demonstrating that P. putida activated the biosynthesis of flagellum to facilitate chemotaxis for tumbling and swimming away from the toxic benzoate compound [58, 59]. In addition, eleven out of 15 biological processes contained both up- or down-regulated proteins, indicating that the metabolic pathways were dynamically altered with the addition of benzoate. Therefore, pathway enrichment analysis was further performed to examine the metabolic response that occurs when co-feeding glycerol with benzoate.
The top 10 enriched KEGG pathways with a p value less than 0.01 are also presented in descending form in Fig. 5b. Moreover, the overview of the significantly expressed proteins among the enriched KEGG pathways were highlighted in the heatmap afterwards for both up- and down-regulated proteins (Figs. 6a and 6b) and finally mapped into the metabolic pathway map (Fig. 7). The introduction of benzoate into the carbon source receipt of solo glycerol could act as both a toxic and a nutritional signal, triggering a series of both detoxification and degradation systems of the aromatic compound in P. putida KT2440. Our results demonstrated that after exposure to the co-feeding conditions of glycerol and benzoate, a precise metabolic response at the level of aromatic degradation pathways was accompanied with a general stress response in different levels, indicated by the induction of enzymes known to respond to oxidative stress and energy generation. Interestingly, the metabolic response appears to reflect a shift of intracellularly available carbon sources towards energy and precursor generation, for instance, which is usually exhibited as a survival strategy to overcome environmental stress in P. putida [60–62].
The enriched KEGG pathways revealed that the addition of benzoate significantly impacted the aromatic degradation pathways (Fig. 5b). In Figs. 6a and 7, enzymes involved in the catechol branch of the β-ketoadipate pathway [54] showed significant up-regulation. In particular, 1,2-dioxygenase (catA1) and muconate cycloisomerase 1 (catB) demonstrated a huge, 21.4-fold and 104.3-fold up-regulation, respectively. Besides, the protocatechuate branch [49] was also up-regulated and exhibited lower fold-change compared to the catechol branch, where 4-carboxymuconolactone decarboxylase (pcaC) and 3-oxoadipate enol-lactonase (pacD) demonstrated a 1.4-fold and 1.5-fold up-regulation, respectively. In addition, shikimate degradation, which is connected with the protocatechuate branch, was also up-regulated. For instance, quinate dehydrogenase (quiA) and 3-dehydroshikimate dehydratase (quiC) displayed 1.3-fold, 1.5-fold, and 2.0-fold up-regulation respectively, suggesting that there were multiple pathways involved in aromatic degradation [63].
Glycerol utilization is negatively regulated by GlpR repressor, which binds to the up-stream binding sites of glp clusters such as the glpF and glpD genes, repressing the glycerol utilization and generating a prolonged lag phase [52]. Glycerol 3-phosphate (G3P) in turn binds to glpR repressor and relieves the glpR-associated repression in a positive feedback loop [64]. Proteomics results indicated that the glpR repressor did not show noteworthy differences in performance under any conditions (additional file 2, Table S7), suggesting the regulation of glp clusters by glpR is not affected by the co-feeding of carbon sources. However, a down-regulation happened to glycerol-3-phosphate dehydrogenase (glpD) under co-feeding conditions (Fig. 6b), resulting in the accumulation of glycerol 3-phosphate (G3P) and thus relieving the glpR repression. Therefore, glycerol utilization was enhanced under co-feeding conditions, which was also consistent with previous HPLC (Figs. 1c and 2c) and metabolite profile (Figs. S5a-S5c) results.
Notably, consistent with the metabolic profiles results before (Figs. S6a-S6c), proteomics analysis demonstrated that the addition of benzoate activates the trehalose degradation enzymes, suggesting the participation of other inner carbon sources in the energy generation process (Fig. 6a). Trehalose plays a dual role both in osmoregulation and in the metabolism of linear or branched glycogen in P. putida KT2440 [65]. Due to lack of the ostAB genes that directly connect trehalose with UDP-D-glucose in P. putida KT2440, trehalose degradation is by-passed with maltose (bifunctional trehalose synthase B/maltokinase, treSB) and glycogen (maltooligosyltrehalose synthase/trehalohydrolase, treY/treZ) and finally leads to the ED pathway through glucose-1-P (glycogen phosphorylase, glgP). On the other hand, glycogen is also degraded to dextrin through the up-regulated 4-alpha-glucanotransferase (malQ, 1.7-folds), which relates to glycan degradation for generating oligomers or monomers of glucose [66]. Here we also observed an up-regulated polysaccharides transporter (PP_3126, 1.48-folds) which could export the polysaccharides such as dextrin to periplasm space [67], following the hydrolases to produce glucose for further utilization. Consistently, enzymes involved in glucose degradation in peripheral pathways (i.e. direct phosphorylation to glucose 6-phosphate or conversion to gluconate [68, 69]) were up-regulated, including glucose dehydrogenase (gcd), D-gluconate kinase (gnuK), and glucokinase (glk), with 1.5-fold, 1.4-fold and 1.7-fold up-regulation, respectively.
Enzymes involved in the Entner-Doudoroff (ED) pathway were differentially expressed under co-feeding conditions, for instance, Glucose 6-phosphate-1-dehydrogenase (zwf-1), 6-phosphogluconolactonase (pgl), and KHG/KDPG aldolase (eda) were 2.8-fold, 1.8-fold, and 1.7-fold up-regulated, respectively (Fig. 6a, Table S5). In contrast, several enzymes within the EMP pathway were down-regulated, such as enolase (eno), phosphoglycerate kinase (pgk), and pyruvate kinase II (pykA), indicating that the suppressed EMP pathway and activated ED pathway is more favorable for producing NADPH. During heterotrophic growth on glycerol, the Entner-Doudoroff (ED) pathway and pyruvate metabolism play a key role in PHA biosynthesis according to previous transcriptome and fluxomic analysis of P. putida KT2440 [43]. Bhaganna et al. [33] investigated the cellular response and performed the proteomics study with the presence of benzene (5.2 mM) and glycerol (0.52 M) in P. putida KT2440. Benzene stress inhibited cell growth, while glycerol protected cell systems by upregulating glucose-6-phosphate dehydrogenase, isocitrate lyase, and enoyl-CoA hydratase, which are involved in the ED-pathway, TCA cycle, and fatty-acid beta-oxidation pathway, respectively. Moreover, Chavarria et. al. [70] reported that under oxidative stress, the ED pathway was activated for generating the reducing equivalent (NADPH) in P. putida. These results are consistent with our findings, which reinforce the hypothesis that the addition of benzoate with glycerol triggers oxidative stress and activates the ED pathway for generating excess reductive equivalent.
Amino acids are the main component of the precursor of biomass synthesis. The catabolism of amino acid is also a necessary process to produce energy under unfavorable growth conditions [71, 72]. However, the enriched biological process and KEGG pathway analysis showed the suppression in both the amino acid biosynthesis and degradation pathways (Figs. 5a and 5b), indicating that the addition of benzoate not only inhibited the biosynthesis of amino acids, but also refrained from obtaining energy at the expense of the amino acid (L-arginine) degradation process. Alternatively, the reductive tricarboxylic acid cycle, ED pathway, and carbohydrate degradation processes were upregulated for the generation of energy, in contrast to the downregulation of the oxidative glyoxylate cycle. Isocitrate dehydrogenase (icd, idh) is a critical branching enzyme between the TCA cycle and glyoxylate shunt, and defends against oxidative damage to the process of generating reducing power (NADPH) [9]. The switch between the TCA cycle and glyoxylate shunt is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK), which activates isocitrate dehydrogenase (icd) by dephosphorylation following the activation of enzymes in the TCA cycle and the inhibition of the glyoxylate shunt, respectively [73]. Isocitrate dehydrogenase kinase/phosphatase (aceK) was up-regulated 1.3-fold, which performing kinase/phosphatase on isocitrate dehydrogenase (icd) and switch off of glyoxylate shunt which was in consistent with the down regulated proteins such as isocitrate lyase (aceA) and malate synthase G (glcB). Moreover, other enzymes involved in the TCA cycle were also up-regulated, including aconitate hydratase (acnA1, 1.5-folds), succinate dehydrogenase (sdhA, 1.4-folds; sdhB, 1.5-folds; sdhC, 1.7-folds), class 2 fumarate hydratase (fumC2, 1.4-folds), and malate:quinone oxidoreductase (mqo3, 1.3-folds) in P. putida KT2440, suggesting the activation of the reductive branch of TCA cycle to produce reductive power such as ATP and NAD(P)H [62]. Previous publication has reported that [73] the abundance of oxaloacetate and pyruvate in Pseudomonas aeruginosa are key activators of isocitrate dehydrogenase (idh) and isocitrate dehydrogenase kinase/phosphatase (aceK), which follows the dephosphorylation of isocitrate dehydrogenase (icd), activating the TCA cycle and inhibiting the glyoxylate shunt. Moreover, in the case of co-feeding glucose with benzoate in P.putida KT2440, isocitrate lyase (aceA) and isocitrate dehydrogenase (icd) were both down-regulated [5]. Our results were partially consistent with previous reports with regards to the up-regulation of aceK and insignificant changes in icd, whereas other isocitrate dehydrogenase (idh) was down-regulated, indicating that there might be additional mechanisms of isocitrate dehydrogenase (idh) under co-feeding conditions in P. putida KT2440.
Some of the enzymes involved in cell wall biosynthesis were down-regulated with the glycerol and benzoate co-feeding conditions (Fig. 6b). For example, N-acetyl-β-muramate 6-phosphate phosphatase (mupP), beta-N-acetylglucosaminidase (nagZ), glucans biosynthesis protein G (opgG), and dTDP-glucose pyrophosphorylase (rfbA), involved in peptidoglycan biosynthesis, were down-regulated. Besides, UDP-3-O-acylglucosamine (lpxD), which participates in Lipid A biosynthesis and is located on the surface of the membrane, was also down-regulated. The bacterial cell wall provides structural integrity and plays an important role in regulating cellular envelope balance under stress conditions [74]. The suppression of cell wall synthesis proteins might lead to cell wall rearrangement or deficiency, redirecting intracellular carbon sources into reducing equivalent generation processes that could help the cell overcome the stress [75].
Bacteria express several enzymes that play roles in the detoxification of reactive oxygen species (ROS) [15]. The proteomics results (Figs. 6a and 6b) confirmed that P. putida KT2440 responds to oxidative stress by up-regulating hydroperoxidase (katE), catalase-peroxidase (katG), and Cytochrome c551 peroxidase (ccpA) with hydrogen peroxidase alleviating capacity, accompanied by the aromatic degradation process. This data is in agreement with several previous reports that observed a significant abundance of the above-mentioned protein induced when Pseudomonas was grown on stress conditions [48, 76, 77]. Besides, the glutathione reduction system was also activated in responding to oxidative stress, for instance, glutathione peroxidase (PP_1686) and glutathionyl-hydroquinone reductase (gpr) were up-regulated 1.6- and 2.5-fold, respectively. These enhanced enzymes indicated that the aromatic degradation involved oxidative conditions by generating hydrogen-peroxidase, corresponding to the activation of antioxidant systems in P. putida, which was widely reported as being in lignin-derived compound degradation in P. putida [15, 48, 49]. Conversely, other oxidative stress response enzyme systems, such as such as thioredoxin (trx, trxA, and trxB), peroxiredoxin (ahpC), and glutathione system (gor, gshB), were down-regulated. Due to the reducing energy requirement (e.g. NADH or NADPH) in order to recycle the small antioxidant molecules, the expression of these enzymes might be dynamically altered [48]. The same trend was observed for NADH dehydrogenase (ndh) and NAD(P)H dehydrogenase (PP_1644), showing the interconversion of NAD+/NADH and NADP+/NADPH was also suppressed.
The fatty-acid metabolism was directly correlated with PHA synthesis and being found more active during the co-feeding of glycerol with benzoate (Fig. 6a). The fatty-acid β-oxidation was up-regulated, which breaks down the fatty acids and produces NADH and acetyl-CoA for the cell to maintain redox homeostasis and more importantly, as the precursor for PHA synthesis. For instance, long-chain-fatty acid-CoA ligase (fadD2) and medium-long chain acyl-CoA dehydrogenase (fadE) were up-regulated 1.7-fold and 1.4-fold, respectively. The oxidative stress generated by the aromatic compound degradation might be the reason for inducing the up-regulation of enzymes in fatty-acid oxidation, which was consistent with previous publications [13, 37]. In contrast, several enzymes related to fatty-acid biosynthesis were down-regulated (Figs. 6a and 6b). In particular, Acetyl-coenzyme A synthetase (acsA1) and the biotin carboxyl carrier protein of acetyl-CoA carboxylase (accB) were down-regulated, which is related to the initial step of acetyl-CoA conversion. However, malonyl-CoA-ACP transacylase (fabD), the rate-limiting step in fatty-acid biosynthesis, was up-regulated. Previous studies reported that the up-regulation of fabD enhanced growth by increasing the unsaturated longer chain length of fatty acids, which might be an alternative bacterial strategy to alleviate the n-butanol stress in E. coli [78, 79]. Furthermore, enzymes directly involved in PHA synthesis, such as poly(R)-3-hydroxyalkanoate polymerase 2 (phaC2), were greatly up-regulated, suggesting the enhancement of PHA synthesis under co-feeding conditions. Besides, enzymes linked between PHA biosynthesis and fatty-acid synthesis, such as (R)-3-hydroxydecanoyl-ACP: CoA transacylase (phaG), were down-regulated, limiting the fatty-acids from biosynthesis process from entering the PHA synthesis pathway. Therefore, our results demonstrated that the monomer composition of PHA is dynamically balanced between fatty-acid biosynthesis and β-oxidation. The proteomics results reinforced the GC-MS results, where the decreasing of long-chain monomers and increasing of short-chain monomers of the PHA polymer maintained the unsaturated fatty-acid content in co-feeding conditions. In addition, the PHA depolymerizing enzyme, poly(3-hydroxyalkanoate) depolymerase (phaB), was also up-regulated, indicating that PHA is also dynamically regulated in order to maintain cellular redox balance.
Overall, the mechanism of the co-feeding of benzoate and glycerol in P. putida KT2440 could possibly involve the following steps (Figs. 6a, 6b, and 7). First, the addition of benzoate stimulates glycerol consumption by relieving glpR repression, generating oxidative stress and thus influencing intracellular redox balance. Then, the redox balance could be potentially influenced by the increased NAD+/NADH ratio and decreased NADP+/NADPH ratio, corresponding to enhanced oxidative-stress response reactions (e.g., katE/G and gqr) and energy generation pathways, which includes the ED pathway (e.g., zwf-1, pgl, eda), the reduction TCA route (e.g., sdhA/B/C, fumC2, mqo3), the trehalose degradation pathway (e.g., treSB, treZ), and the fatty-acid β-oxidation pathway (e.g., fadD2, fadE). Lastly, the excess amount of reducing equivalents (NAD(P)H) are stored as PHA polymers through the up-regulation of PHA polymerase (phaC2). Therefore, the enhancement of PHA content is not due to the increased equivalent carbon and nitrogen ratio, but due to the increased inner reducing power pools (NAD(P)H). Besides, the addition of lignin breakdown products may trigger the redox regulation, manipulate the intracellular reducing power elevation, and influence the carbon efficiency under co-feeding conditions, which sheds light on a promising direction towards efficient lignin valorization. Further research efforts can be carried out to understand the balance of energy, reductant, and carbon source related to efficient electron transfer in order to maximize the PHA yield.