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Tolerance and metabolic response of Pseudomonas taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors
© The Author(s) 2018
- Received: 22 November 2017
- Accepted: 6 July 2018
- Published: 19 July 2018
Bio-conversion of lignocellulosic biomass to high-value products offers numerous benefits; however, its development is hampered by chemical inhibitors generated during the pretreatment process. A better understanding of how microbes naturally respond to those inhibitors is valuable in the process of designing microorganisms with improved tolerance. Pseudomonas taiwanensis VLB120 is a natively tolerant strain that utilizes a wide range of carbon sources including pentose and hexose sugars. To this end, we investigated the tolerance and metabolic response of P. taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors including organic acids (acetic acid, formic acid, and levulinic acid), furans (furfural, 5-hydroxymethylfurfural), and phenols (vanillin).
The inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However, P. taiwanensis was able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when P. taiwanensis VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors.
In this study, the tolerance and metabolic response of P. taiwanensis VLB120 to biomass hydrolysate-derived inhibitors was investigated. P. taiwanensis VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors compared to most wild-type microbes reported in the literature. It adopts different resistance mechanisms, including detoxification, efflux, and repair, which require additional energy and resources. Thus, targeting redox and energy metabolism in strain engineering may be a successful strategy to overcome inhibition during biomass hydrolysate conversion and lead to development of more robust strains.
- Biomass hydrolysate inhibitors
- Inhibitor tolerance
- Pseudomonas taiwanensis
Pseudomonas taiwanensis is an obligate aerobe, biofilm-forming organism that was isolated from soil at the Institute of Microbiology, University of Stuttgart, Germany [1–4]. It can thrive in diverse habitats, and is known for its ability to colonize soil and participate in soil biochemical processes [5, 6]. The potential of P. taiwanensis for the degradation and bioremediation of a wide variety of chemicals, including natural and synthetic compounds, such as caprolactam , naphthalene , and toluene, has attracted a great research interest . Furthermore, the strain utilizes a wide range of organic molecules as carbon sources including pentose/hexose sugars and aromatic hydrocarbons .
Unlike other industrially relevant Pseudomonas putida strains, such as P. putida KT2440, P. putida DOT-T1E, and P. putida S12, P. taiwanensis VLB120 is the only known Pseudomonas strain that is able to utilize xylose as the sole carbon and energy source without any genetic modifications . These remarkable features of P. taiwanensis emphasize its potential for the production of high-value products, such as n-butanol from low-cost renewable feedstocks through rational metabolic engineering as shown in a variety of heterologous microorganisms, including those cultivated aerobically such as P. putida .
While the physiology of P. taiwanensis VLB120 matches the basic requirements for growth on biomass hydrolysate, its exposure to biomass hydrolysate-derived inhibitors including acetic acid, formic acid, levulinic acid, furfural, 5-HMF, and vanillin has not yet been characterized. These compounds influence the growth of microorganisms in various ways, including DNA mutation, membrane disruption, intracellular pH drop, and other cellular targets [10, 11]. Therefore, understanding how P. taiwanensis metabolically respond to inhibitors and identifying which metabolic pathways and metabolites are involved can hasten the development of the strain to a production strain. These information can also be used to design other robust strains that are not able to grow on biomass hydrolysate naturally. Hence, the main aim of this work was to determine the tolerance and metabolic response of P. taiwanensis VLB120 toward the main inhibitory compounds present in lignocellulosic biomass hydrolysates.
Strain and culture mediums
Pseudomonas taiwanensis VLB120 was obtained from the Institute of Applied Microbiology, RWTH Aachen, Germany. The cell culture medium used on this study consisted of (L−1): 2.12-g NaH2PO4∙2H2O, 2-g (NH4)2SO4, 10-mg EDTA, 0.1-g MgCl2∙6H2O, 2-mg ZnSO4∙7H2O, 1-mg CaCl2∙2H2O, 5-mg FeSO4∙7H2O, 0.2 mg Na2MoO4∙2H2O, 0.2-mg CuSO4∙5H2O, 0.4-mg CoCl2∙6H2O, 1-mg MnCl2∙2H2O, and 4.5-g glucose as a carbon source . Unless stated otherwise, all chemicals and reagents used in this study were purchased from Sigma-Aldrich (Chemical Co, USA).
Inhibitors threshold concentration test
The inhibitor threshold concentration affecting growth was evaluated using the Growth Profiler 960 (EnzyScreen, Heemstede, The Netherlands). The inhibitory compounds were added into minimal medium supplemented with 4.5 g L−1 of glucose in different concentration levels. The media pH was adjusted to 7.0 ± 0.03 with 5 M of sodium hydroxide before inoculation. The same medium without inhibitory compounds was used as control.
Bioreactor-batch growth experiment
Bioreactor-batch cultivations were performed to characterize the metabolic response of P. taiwanensis VLB120 under stress conditions. The experiments were performed in 1.3-L bioreactors (SARTORIOUS ®) with 0.5-L working volume. Cultures were inoculated at OD of approx. 0.05 and fermentation temperature, stirrer speed, and pH were set at 30 °C, 800 rpm, and 7.0, respectively. Cultures were supplied with air at a flow rate of 1 slpm, and minimum dissolved oxygen saturation level was 40%. The whole fermentation process was monitored by continuously measuring the CO2 percentage in the off-gas. All cultures were performed in triplicates and batch cultures were run for 24 h.
Sample preparation for metabolome analysis
During bioreactor-batch growth experiments, supernatants were collected along the cultivation to quantify optical density at 600 nm (Spectrophotometer VWR UV-1600PC, USA) as well as extracellular metabolites. Samples for extracellular metabolite analysis were spun down at 10,000g for 5 min and stored at − 20 °C for further use. Samples for intracellular metabolite measurement were rapidly harvested (3 mL) with an electronic pipette at optical density of 0.4–0.6 (OD600 nm), and filtered with fast filtration system as described previously . Immediately after the filtration process, quenching and extraction of metabolites were performed by adding 2 mL of 75% (v/v) boiling ethanol (70 °C) and 25 µL of fully labeled 13C cell extracts as an internal standard (IS) to the filtered cells and heated for 1 min. The cells were re-extracted by adding additional 1.5 mL of boiling ethanol at 70 °C. The samples were concentrated by evaporating the organic solvent for 5 h at 25 °C using a vacuum concentrator (SAVANT, SpeedVac, Thermo Fisher Scientific, San Diego, CA, USA) followed by lyophilization (LABCONCO, FreeZone, Kansas City, MO, USA) overnight at − 40 °C. All dried extracts were re-suspended in 250 μL of LC–MS grade water, which is compatible with the initial mobile phase of the LC–MS method and stored at − 80 °C until analysis.
Measurement of inhibitors and extracellular metabolites
The concentration of inhibitors and extracellular metabolites was measured by high-performance liquid chromatography (HPLC). More specifically, quantification of furfural, 5-HMF, vanillin, and their corresponding acid in media was performed on a Dionex Ultimate 3000 HPLC equipped with a Supelco Discovery HS F5-3 HPLC column (150 × 2.1 mm × 3 µm) and a UV detector (260, 277, 304, and 210 nm). Samples (1 µL) were analyzed using a gradient method with mobile phase A: 10-mM ammonium formate, pH 3, and B: acetonitrile. A flow rate of 0.7 mL min−1 was used and the column was held at 30 °C. The program started with 5% of solvent B for 0.5 min and increased linearly to 60% over 5 min. The gradient was thereafter increased to 90% B over 0.5 min and kept at this condition for 2 min. Finally, returned to 5% B and equilibrated until 10 min.
Concentrations of glucose, gluconate, acetic acid, formic acid, and levulinic acid were determined using a Dionex Ultimate 3000 HPLC with an Aminex® HPX-87X Ion Exclusion (300 × 7.8 mm) column (Bio-Rad, Hercules, CA) and RI-150 refractive index detector. Gluconate was measured by UV monitoring at 210 nm. The mobile phase consisted of 5-mM H2SO4, the flow rate was 0.6 mL min−1 and the column was kept at 60 °C. Samples were held at 5 °C during the analysis and 20-µL sample volume injected.
Measurement of intracellular metabolites
Metabolite measurement was performed on AB SCIEX Qtrap1 5500 mass spectrometer (AB SCIEX, Framingham, MA, USA) ion-pairing techniques operated in negative mode as previously described . A sample of 20 uL was injected on to an XSELECT HSS XP (150 × 2.1 mm × 2.5 μm) (Waters, Milford, MA, USA) column, which was equilibrated for 10 min before injecting with 100% eluent A (10 mM tributylamine, 10 mM acetic acid (pH 6.86), 5% methanol, and 2% 2-propanol). Gradient elution was set to 0% of eluent B (2-propanol) for the first 5 min, and increased to: 2% (5–9 min), 6% (9–12 min), 11% (12–13.5 min), 28% (13.5–15.5 min), and 53% (15.5–22.5 min), and returned back to 0% (22.5–23 min) and equilibrated for 10 min (23–33 min) with 100% eluent A. The flow rate was 0.4 mL min−1 (0–15.5 min), 0.15 mL min−1 (16.5–23 min), and 0.4 mL min−1 (27–33 min); oven temperature was set to 40 °C. The mass spectrometer was operated in multiple-reaction-monitoring (MRM) mode. The optimized parameters for 0.4-mL min−1 flow rate were as follows: ion-spray voltage, − 4.5 kV; curtain gas and CAD gas, 40 and 12, respectively. The capillary temperature was 500 °C.
HPLC and LC–MS data were processed using Chromeleon™ 7.1.3 (Thermo Scientific™) and Multi-Quant™ 3.0.2 (AB SCIEX™), respectively. For absolute quantification of intracellular metabolites, isotope ratio-based approach was used as previously described [15, 16]. This technique was performed using cell extracts grown in fully U-13C-labeled glucose as an internal standard for quantifying the intracellular metabolites of P. taiwanensis VLB120 grown on naturally labeled glucose. All statistical analyses were done using R (R Development Core Team ) and SIMCA (Umetrics, Umea, Sweden).
Utilization of biomass hydrolysate sugars by P. taiwanensis VLB120
Hydrolysis of lignocellulosic biomass results in a mixture of sugars including the hexoses glucose, galactose, and mannose, and the pentoses xylose and arabinose . In most cases, these mixtures can only be metabolized partly or sequentially, with glucose being the preferred carbon source [19–23].
Growth of P. taiwanensis VLB120 was also assessed on other carbon sources including sodium acetic acid, sodium benzoate, glycerol, and mixture of different carbon sources. The results (Additional file 1 Fig. S1) indicate that P. taiwanensis VLB120 was able to grow on these compounds as sole source of carbon and energy.
Effect of biomass hydrolysate inhibitors on P. taiwanensis VLB120 growth
Effects of hydrolysis-derived inhibitors on the lag phase of P. taiwanensis VLB120
Concentration [g L−1]
Lag phase (h)a
The effect of vanillin was comparable to that of furfural and 5-HMF. The lag phase was prolonged by 15 h, while the specific growth rate was reduced by 38% and the final biomass titer was decreased by 18% at a concentration level of 3 g L−1 of the corresponding inhibitory compounds. By increasing the concentration of vanillin to 4 g L−1, the final biomass titer was reduced by 90% and the lag phase was prolonged to 33 h (Table 1). A complete inhibition of growth of P. taiwanensis was observed when the concentration of 5-HMF, furfural, and vanillin exceeded 3, 4, and 4 g L−1, respectively (Additional file 1: Fig. S2). This might be caused by the pH drop due to the formation of the corresponding acid form of the added inhibitors.
Acetic acid and formic acid showed a similar inhibitory effect on cell growth. Both compounds slightly increased the final biomass of P. taiwanensis VLB120, as shown in Additional file 1: Fig. S2, but reduced the growth rate as their concentration increased (Fig. 3). The main difference of these two inhibitors was observed as the concentration of acetic acid exceeded 6 g L−1 where after the lag phase was clearly elongated compared to formic acid (Table 1), similar as described previously for yeast .
Determination of inhibitory threshold concentrations affecting P. taiwanensis VLB120 growth
The concentrations resulting in a 50% reduction of P. taiwanensis VLB120 growth (IC50) with 5-HMF and furfural are highly comparable with the reported values for Thermoanaerobacter pseudethanolicus 39E , Bacillus coagulans MXL-9 , S. cerevisiae CBS1200 , and Zymomonas mobilis ATCC 10988 . Based on IC50 values, 5-HMF provided the strongest inhibition followed by furfural and vanillin, respectively. These results are in line with the previous reports which confirmed that furfural and 5-HMF were identified as main inhibitors in biomass hydrolysates [26, 31–33]. In contrast, IC50 values for acetic acid and formic acid were above the highest tested concentration (10 g L−1), which means that this concentration was not high enough to reduce the growth of P. taiwanensis VLB120 by 50%. These values are higher compared to well-known production strains such as E. coli (IC50 2.5-g L−1 formic acid and 9.0-g L−1 acetic acid) . This indicates that P. taiwanensis VLB120 is highly tolerant to acetic acid and formic acid when glucose is used as a sole carbon source.
Degradation capacity of lignocellulosic biomass-derived inhibitors by P. taiwanensis VLB 120
A number of microorganisms have evolved different strategies including reduction and oxidation processes to detoxify inhibitory compounds [35–38]. For instance, Gluconacetobacter xylinus oxidizes furfural and 5-HMF directly to furoic acid and 5-hydroxymethyl-2-furoic acid, respectively . Microorganisms such as E. coli and S. cerevisiae not possessing oxidative degradation pathways for furan aldehydes  use their native oxidoreductases to reduce furan aldehydes to furan alcohols under anaerobic conditions [34, 40].
In this study, the metabolic response and degradation potential of lignocellulosic biomass-derived inhibitory compounds by P. taiwanensis VLB 120 was investigated using a targeted metabolomics approach. Since some of these inhibitors have structural similarity and share the same degradation pathway, only acetic acid, levulinic acid, furfural, and vanillin were considered for the metabolomics study. For a reliable quantitative metabolomics analysis, 2 g L−1 of each inhibitory compound was chosen based on half maximal inhibitor concentration (IC50) value assuming that this concentration level is sufficiently high to affect cell behavior and metabolism without being lethal.
Based on the supernatant analysis, P. taiwanensis VLB120 is able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively (Fig. 5c, d). Vanillic acid was further metabolized to protocatechuic acid and eventually entered the central carbon pathway via the β-ketoadipate route [41, 42], whereas 2-furoic acid was secreted outside the cells as the conversion of furfural to 2-furoic acid carried out on the outer surface of the cells  and remained in the fermentation broth without further conversion.
There was no significant growth of cells observed until the majority of furfural and vanillin in the medium were converted to their corresponding acid, which would also explain the long lag phase. This indicates that the presence of these inhibitors in the media obstructed the growth of P. taiwanensis VLB120. However, their corresponding acids had a less toxic effect and, therefore, allowed growth of P. taiwanensis VLB120. These findings are in agreement with the previous studies that proved the aldehyde form as the most toxic one of several aromatic inhibitory compounds, whereas the corresponding acids were less toxic, while the alcohol form was the least toxic one [25, 44–46].
Acetic acid was completely consumed from the fermentation broth after 8 h of cultivation (Fig. 5a). This indicates that acetate was activated to acetyl-CoA and completely metabolized from the fermentation broth via the TCA cycle to carbon dioxide, which agrees with findings of Matano et al.  and Gebhardt et al. .
Physiological parameters of P. taiwanensis VLB120 during growth on glucose in the presence of inhibitory compounds
Specific growth rate
0.58 ± 0.02
0.45 ± 0.06
0.19 ± 0.01
0.33 ± 0.04
0.69 ± 0.03
Specific glucose uptake
g g−1CDW h−1
10.45 ± 0.58
8.13 ± 1.69
1.72 ± 0.12
4.25 ± 1.70
7.44 ± 0.42
Specific gluconate production rate
g g−1CDW h−1
10.70 ± 0.39
7.72 ± 1.72
1.49 ± 0.10
3.74 ± 1.52
6.04 ± 0.88
Biomass yield on glucose
0.05 ± 0.00
0.06 ± 0.00
0.10 ± 0.00
0.12 ± 0.02
0.09 ± 0.01
Pseudomonas taiwanensis VLB120 completely metabolized glucose to gluconate in the pretense of acetic acid. This could be related to the direct utilization of acetic acid as an additional carbon and energy source.
Effect of inhibitory compounds on the P. taiwanensis VLB120 metabolome composition
Comparative analyses of the primary and key intermediate metabolites were considered to investigate the metabolic response of P. taiwanensis VLB120 to lignocellulose-derived inhibitors. For each tested inhibitor, intracellular metabolites were extracted with boiling ethanol from exponentially growing P. taiwanensis VLB120 cultures at an optical density (OD600 nm) of 0.4–0.6.
In total, 80 metabolites from different classes, including sugars phosphates, amino acids, organic acids, redox cofactors, nucleosides/bases, and nucleotides, were quantified across all conditions. These metabolites do not cover the entire metabolome of P. taiwanensis VLB120; however, they possess an essential role in central metabolism. To provide comparative information regarding the metabolic differences among each group, a principal component analysis (PCA) was performed.
Nucleotide monophosphates (e.g., AMP, CMP, IMP, GMP, and UMP) seemed to have high influence in separating the samples treated with acetic acid from the rest of the groups. This could be related to the requirement of ATP to convert acetate to acetyl-CoA which results in the production of AMP. The observed low intracellular concentration of acetyl-CoA was mainly related to its utilization for re-generation of ATP via the TCA cycle [51, 52].
The mechanisms that lead to the observed change of intracellular concentrations of other classes of metabolite (e.g., sugar phosphates, organic acids, and amino acids) could be the consequence of changes in cellular energetics and redox state of the cell. Metabolites including ATP and NADPH are generally reported to have key functions in the survival of any organism in a stressful environment [53, 54]. This is due to the fact that microorganisms require both NADPH-dependent detoxification and ATP-dependent efflux to cope with inhibitors . Since those metabolites are a fundamental requirement for the maintenance of metabolism, energy generation, and growth, their perturbations may induce widespread changes in metabolism [53, 54, 56–62].
To investigate the role of cellular energetics and redox carrier metabolites in P. taiwanensis VLB120 during growth with inhibitory compounds, the level of ATP and NADPH/NADP ratio was determined in both control sample and samples treated with inhibitors. Since several enzymes are regulated by the ratio between reduced and oxidized cofactors , NADPH/NADP ratio was considered in steady of absolute concentration of NADPH.
At the time of sampling, the oxidation of vanillin and furfural to their corresponding acids were ongoing, while acetic acid was metabolizing. The concentration of levulinic acid remained constant throughout the fermentation process.
Similarly, the increased level of NADPH/NADP ratio provided reducing power to deal with the oxidative stress that caused by the inhibitors . This observation is in agreement with a previous study, showing that Pseudomonas fluorescens produced high NADPH to cope with oxidative stress . NADPH diminishes oxidative stress and provides the reductive environment necessary for cellular activities . For instance, the production of ATP via oxidative phosphorylation cannot be effective for aerobic organism growing under stress conditions unless it is equipped with enough supply of NADPH that provides a reductive environment [57, 58, 62]. In the presence of acetic acid, NADPH/NADP ratio was slightly impaired, while the concentration of ATP was unchanged, which was also in reasonable agreement with a previous study . The observed minimal effect of acetic acid on that metabolite could be related to the direct utilization of acetate by P. taiwanensis as an additional carbon and energy source.
Overall, there appeared to be a metabolic shift in P. taiwanensis to enhance the levels of ATP and NADPH/NADP ratio to cope with the stress imposed by inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors.
In this study, the tolerance and metabolic responses of P. taiwanensis VLB120 to biomass hydrolysate-derived inhibitors were investigated. The overall results suggest that the tested inhibitors affect P. taiwanensis VLB120 physiology in various ways with respect to lag phase, specific growth rate, and biomass yield. To overcome these effects, P. taiwanensis VLB120 adopt different resistance mechanisms, including detoxification, efflux, and repair, which require additional cellular energy and resources. P. taiwanensis VLB120 went through metabolic rearrangement to generate more ATP and NADPH to mitigate the stress imposed by inhibitors.
In general, efficiently use of biomass hydrolysate as fermentation media requires microorganism that can utilize both C6 and C5 sugars and able to tolerate the inhibitory compounds formed during biomass pretreatment process. P. taiwanensis VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors and efficiently utilize glucose, xylose, and galactose as a carbon and energy source. This indicates that the physiology of P. taiwanensis VLB120 matches the aforementioned basic requirements for growth on biomass hydrolysate.
MK supervised the study and involved in scientific discussion. GW designed and performed experiments, analyzed and interpreted the data, and wrote the manuscript. Both authors read and approved the final manuscript.
We thank Dr. Konstantin Schneider for the valuable discussion and contribution to the paper. We also thank Prof. Dr. Andreas Schmid (Department of Solar Materials, The Helmholtz Center for Environmental Research, UFZ, Germany) for supplying P. taiwanensis VLB120 strain. We also thank Yasin Yildiz Dastan for his help during fermentation setup.
The authors declare that they have no competing interests.
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