Optimization of Medium and Oxygen Supply Conditions for PQS and PYO Synthesis in Pseudomonas aeruginosa
1. Medium Screening: Evaluation of PB and LB Media on PYO Synthesis in Pseudomonas aeruginosa
Design: Pseudomonas aeruginosa PAO1 was cultured in LB medium and PB medium respectively. The bacterial growth, PYO production level, and β-galactosidase activity in the two media were compared.
Results: In PB medium, P. aeruginosa significantly synthesized PYO, and the culture broth exhibited a typical green color; in LB medium, the bacterial broth was mainly yellow, with significantly reduced or undetectable PYO production. At both 24 h and 48 h time points, the β-galactosidase activity of the strain in PB medium was significantly higher than that in LB medium.
Conclusion: LB medium may be unfavorable for PYO synthesis in P. aeruginosa. PB medium should be selected for subsequent experiments to ensure that the bacterium maintains its PYO-producing characteristics.
2. Composite Medium Construction: Integrating PB Medium and MMO-MUG to Support Dual-Strain Growth
Design: PB medium was mixed with commercial MMO-MUG medium to construct a composite medium system. P. aeruginosa and Escherichia coli were inoculated separately, cultured in test tubes with shaking, and bacterial growth and PYO production were evaluated.
Results: In the composite medium, when P. aeruginosa was cultured alone, PYO synthesis was significant, and the broth showed an obvious green color; when E. coli was cultured alone, growth was excellent. This indicates that the composite medium can support the growth and characteristic expression of both strains simultaneously. Based on the test tube culture results, further cultivation was carried out in 1.5 mL centrifuge tubes, achieving strain culture in smaller volumes and laying the foundation for subsequent 96-well plate high-throughput experiments.
Conclusion: The constructed composite medium can synergistically support E. coli growth and P. aeruginosa PYO synthesis, showing good compatibility and applicability.
3. Optimization of Small-Volume Culture Systems: Evaluation of Centrifuge Tubes and 96-Well Plates for High-Throughput Detection
Design: To meet the requirements of high-throughput detection, the effects of different small-volume containers (test tubes, 1.5 mL centrifuge tubes, 96-well plates) on bacterial growth and PYO synthesis were compared. The PB+MMO-MUG composite medium system was reduced to 1.2 mL, placed in the above containers, and both shaking and static culture conditions were set. P. aeruginosa or E. coli was inoculated, and changes in medium color were observed.
Results:
In the test tube system, under both shaking and static conditions, the P. aeruginosa culture broth showed an obvious green color, and E. coli showed yellow, indicating good bacterial growth and PYO synthesis.
In the centrifuge tube and 96-well plate systems, even under shaking conditions, neither strain caused obvious color changes in the medium.
Conclusion: Small-volume closed systems such as centrifuge tubes and 96-well plates may severely limit the growth and metabolism of aerobic bacteria due to insufficient gas exchange. Subsequent optimization of oxygen supply strategies under small-volume culture conditions is required, such as using gas-permeable sealing films, increasing shaking intensity, or intermittent aeration, to ensure normal microbial growth and signal molecule expression.
4. Exploration of Oxygen Supply Methods: Evaluation of Multiple Oxygenation Strategies for Promoting PYO Synthesis
4.1 Sodium Bicarbonate
Background: PYO synthesis in P. aeruginosa strictly depends on high-density quorum sensing and aerobic metabolism in the late growth stage. Under static culture conditions, conventional dissolved oxygen supply is often insufficient to support this process. Therefore, exploring new methods to effectively improve oxygen mass transfer efficiency in the medium is crucial.
Literature review: Studies have shown that bicarbonate in solution can form and stabilize nanobubbles under certain conditions. These nanobubbles can significantly increase the specific surface area of the gas-liquid interface, promote oxygen transfer to the liquid phase, and have been found to activate anaerobic growth and metabolic pathways in P. aeruginosa [1]. This finding suggests that sodium bicarbonate may indirectly affect bacterial physiological status and metabolic flux by altering the physicochemical microenvironment of the medium.
Hypothesis: Adding sodium bicarbonate to a static culture system may enhance oxygen dissolution and retention through the nanobubble effect, thereby maintaining an aerobic microenvironment in the late growth stage and ultimately promoting PYO synthesis.
Design: Different concentrations of sodium bicarbonate (0.01 M to 0.08 M) were added to 1.2 mL PB medium and PB+MMO-MUG composite medium. P. aeruginosa PAO1 was inoculated and cultured statically, with the non-addition group as a control. The bacterial broth color was observed and OD₆₀₀ was measured.
Results: The OD₆₀₀ of the 0.01 M sodium bicarbonate group was slightly higher than that of the control, showing a slight growth promotion, but the broth did not show an obvious green color; as the concentration increased, the OD₆₀₀ changed irregularly, and no significant PYO synthesis was observed in any centrifuge tube group.
Conclusion: Although sodium bicarbonate promoted bacterial growth to a certain extent, it did not effectively activate the PYO synthesis pathway, possibly due to low nanobubble generation efficiency. The addition form should be optimized or combined with other gas-producing substrates in the future.
4.2 Oxygen Pump Aeration
Design: In a 1.2 mL composite medium (centrifuge tube system) inoculated with P. aeruginosa, forced aeration was performed for 2, 5, 8, and 10 minutes, with the non-aerated group as a control. All groups were statically cultured for 24 h, and bacterial growth was observed.
Results: After aeration treatment, no obvious coloration was observed.
Conclusion: Aeration in small closed systems such as centrifuge tubes is still insufficient to effectively support PYO synthesis and significantly increases the risk of contamination. Sterile aeration methods or closed ventilation strategies should be explored to optimize oxygen supply while maintaining sterility.
4.3 Hydrogen Peroxide
Design: Different concentrations of H₂O₂ (0–1 mM) were added to 1.2 mL composite medium, P. aeruginosa was inoculated and cultured statically, and OD₆₀₀ was measured at 24 h and 48 h, with color changes observed.
Results: Compared with the control group, there was no significant difference in OD₆₀₀ in any H₂O₂ group, and bacterial growth was not promoted; no green color was observed in any group, and PYO production was not increased.
Conclusion: Under the experimental conditions, H₂O₂ did not effectively improve dissolved oxygen or activate PYO synthesis, possibly due to rapid decomposition or local toxicity. More stable, slow-release oxygenation strategies should be explored.
5. Under the premise that multiple oxygenation methods had shown no effect, we began to suspect that the issue might lie with the PB+MMO composite medium and proceeded to verify this hypothesis.
Design:
Results: Whether hydrogen peroxide was added or not, the performance of the composite medium was not significantly different from that of LB medium, indicating the need to change the culture medium.
5.1 Comparison of LB, TSB, and BHI Media
Design: Pseudomonas aeruginosa PAO1 was cultured in LB, TSB, and BHI media respectively, and bacterial growth in the three media was compared.
Results: BHI medium supported the best growth of P. aeruginosa PAO1, so it should be used in subsequent experiments.
5.2 Further Exploration of Oxygen Supply Methods
5.2.1 Hydrogen Peroxide
Design:
Results: Compared with the control group, there was no significant difference in OD₆₀₀ among the groups treated with different concentrations of H₂O₂, and bacterial growth was not promoted; none of the groups showed a green color, and PYO production was not increased.
Conclusion: Under the experimental conditions, H₂O₂ did not effectively improve dissolved oxygen levels or activate PYO synthesis, possibly due to its rapid decomposition rate or local toxic effects.
5.2.2 Sodium Percarbonate
Design: Different proportions of oxygen-releasing tablets (mainly composed of sodium percarbonate) were added to BHI medium. After inoculation with Pseudomonas aeruginosa, the cultures were incubated statically, with the non-addition group serving as the control. Color changes were observed and OD₆₀₀ values were measured at 24 h and 48 h.
Results: Compared with the control group, none of the experimental groups showed a green coloration of the medium. However, when 0.01 g of sodium percarbonate was added, the OD₆₀₀ value significantly increased.
Conclusion: The addition of 0.01 g sodium percarbonate oxygen-releasing tablets showed a certain effect in static culture, but the effect was not obvious and could not be observed visually. Further observation in 97-well quantitative plates is required.
6. Medium Modification: Replacement of Pancreatic Peptone with Bacterial Peptone to Form New PB Medium
6.1 Sodium Percarbonate
Background: To achieve PYO-based detection, the core challenge is to continuously supply sufficient oxygen to high-density P. aeruginosa. In a static, closed culture system, conventional physical aeration methods are difficult to apply, so exploring a chemical strategy capable of in-situ, slow-release oxygen supply is particularly important.
Sodium percarbonate, as a solid peroxide, can slowly release oxygen and hydrogen peroxide upon contact with water; the latter can be further decomposed in solution to produce additional oxygen [4]. This property has led to its wide application as a long-acting oxygenation agent in aquaculture and wastewater treatment. Based on this, we propose the hypothesis that applying sodium percarbonate in a static quantitative plate culture system might directly increase the dissolved oxygen concentration in the medium through its slow oxygen release, thereby providing the essential electron acceptor for PYO synthesis in Pseudomonas aeruginosa during the late growth stage and ultimately promoting PYO production.
To test this hypothesis, this study added commercial oxygen-releasing tablets containing sodium percarbonate to the culture medium, aiming to investigate whether this chemical oxygenation method could effectively overcome the oxygen limitation bottleneck in static cultures.
Design: Oxygen tablets (mainly sodium percarbonate) were added to PB medium in 97-well plates at concentrations of 0.002–0.01 g. P. aeruginosa was inoculated and cultured statically for 24 h, with the non-addition group as a control. Color changes were observed and β-galactosidase activity was measured.
Results: Compared with the control group, no medium showed green color in any experimental group, and PYO synthesis was not significant; β-galactosidase activity was not significantly increased.
Conclusion: Sodium percarbonate oxygen tablets did not effectively improve dissolved oxygen or promote PYO synthesis in static culture, possibly due to a mismatch between oxygen release rate and system closure.
6.2 Potassium Nitrate
Background:
The synthesis of PYO by Pseudomonas aeruginosa in the late growth phase is strictly oxygendependent. However, in static, sealed culture systems, the respiratory activity of high-density bacterial populations can easily lead to local microenvironmental hypoxia, thereby limiting PYO production. To overcome this oxygen supply bottleneck, we drew inspiration from the adaptive metabolic strategies employed by P. aeruginosa in hypoxic environments within the host.
Literature studies have shown that in the microoxic environment of the cystic fibrosis patient lung, P. aeruginosa can initiate denitrification through the Dnr regulator to maintain energy metabolism [5]. The core of this pathway lies in the bacterium's ability to use nitrate as a terminal electron acceptor, replacing oxygen for anaerobic respiration, thereby enabling growth and survival under hypoxic conditions.
Based on this theory, we hypothesized that exogenous nitrate addition under static, hypoxic culture conditions might similarly provide an alternative respiratory pathway for P. aeruginosa. If the bacteria can maintain basic energy supply through denitrification, they may allocate more metabolic resources to the synthesis of secondary metabolites, including PYO. Therefore, we selected potassium nitrate as an exogenous electron acceptor to verify whether it could alleviate oxygen limitation in static cultures and thereby promote PYO synthesis.
Design:
Potassium nitrate (KNO₃) at concentrations of 10–100 mmol/L was added to PB medium. After inoculation with P. aeruginosa, the cultures were incubated statically in 97-well quantitative plates, with the non-addition group serving as the control. Color changes were observed and β-galactosidase activity was measured.
Results:
None of the KNO₃-supplemented experimental groups exhibited green coloration, and no significant PYO production was observed; nitrate reduction resulted in gas production, causing the sealing film to bulge and even leak. β-galactosidase activity was not significantly increased.
Conclusion:
Potassium nitrate, as an exogenous electron acceptor, did not effectively promote PYO synthesis; instead, the gas production compromised the stability of the culture system.
KNO₃ CK
6.6.3 Membrane Material Screening
Background
The production of pyocyanin (PYO) and the quorum sensing signal molecule PQS by Pseudomonas aeruginosa is tightly regulated by the quorum sensing system and is highly induced during the late growth stage at high cell density [6]. Notably, PYO not only serves as the final product of this regulatory network but also functions as a terminal signaling molecule that activates the SoxR regulon to further regulate downstream gene expression, forming a complex positive feedback loop. From the perspective of synthetic mechanism, PYO biosynthesis strictly depends on oxygen; its key modification enzyme, PhzS, a flavin-containing monooxygenase, must use molecular oxygen (O₂) as a substrate to complete the catalytic reaction [7].
Therefore, effective PYO detection requires ensuring that bacteria reach the late growth stage, which not only satisfies the cell density requirement for quorum sensing activation but also provides the necessary oxygen environment for PYO synthesis. However, previous experiments have shown that under conventional sealed culture conditions, dissolved oxygen in the medium cannot be maintained until the late growth stage, resulting in severe limitation of PYO synthesis. This poses a challenge for establishing a stable PYO-dependent detection method.
To address this limitation, this study proposes to establish a culture system capable of maintaining the micro-aerobic environment required for the late growth stage of bacteria by systematically screening and optimizing the gas permeability of sealing membrane materials for quantitative plates. This approach ensures efficient PYO synthesis and provides technical support for developing a reliable PYO detection method.
Design
To establish a reliable system suitable for static culture in 97-well plates, five membrane materials with both gas permeability and water resistance (EPTFE, nano-breathable film for building doors and windows, PVDF, PTFE, and TPU) were screened for sealing plates inoculated with Pseudomonas aeruginosa. Their ability to maintain a sterile environment while promoting oxygen transfer to support PYO synthesis was evaluated.
Results
The EPTFE membrane performed best, with the culture system showing obvious green coloration, indicating effective oxygen transfer and significant promotion of PYO synthesis; the PVDF membrane showed some PYO production but with severe evaporation and system instability; the PTFE and TPU membrane groups showed no obvious green coloration due to insufficient gas permeability; the nano-breathable film for building doors and windows interfered with interpretation due to its pink color and showed no clear PYO production.
Conclusion
Gas exchange efficiency is a key limiting factor for PYO synthesis in static cultures. Using a highly gas-permeable, low water-permeable EPTFE membrane for sealing can effectively ensure oxygen supply while maintaining sterility, making it an ideal strategy for small-volume high-throughput culture.
Note: CK: Heat-sealed aluminum foil film provided with the 97-well quantitative plate was used as the control; a–e: Experimental groups using PVDF (polyvinylidene fluoride) membrane, PTFE (polytetrafluoroethylene) membrane, EPTFE (expanded polytetrafluoroethylene) membrane, nano-breathable film for building doors and windows, and TPU (thermoplastic polyurethane) film, respectively.
References
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