Nitrate-Medium (NMS): the NMS medium consists of Solution A and Solution B: Solution A (per 1 L): 5.24 g K₂HPO₄·3H₂O, 2.62 g KH₂PO₄, 0.04 g FeSO₄·7H₂O, 3.0 g MgSO₄·7H₂O, 3.0 g NaCl, 10.0 g KNO₃ (nitrogen source), 0.0167 g FeCl₃·6H₂O, 0.151 g CaCl₂. Solution B (per 1 L): 0.3 g MnSO₄·H₂O, 0.24 g NaMoO₄·H₂O, 0.34 g ZnSO₄·7H₂O. Mix 10 ml of Solution A and 0.1 ml of Solution B, then adjust to 100 ml with deionized water (Park et al., 2024).

Ammonium-Medium (AMS): the AMS medium similarly comprises Solution A and Solution B: Solution A (per 1 L): 5.24 g K₂HPO₄·3H₂O, 2.62 g KH₂PO₄, 0.04 g FeSO₄·7H₂O, 3.0 g MgSO₄·7H₂O, 3.0 g NaCl, 5 g NH₄Cl (nitrogen source), 0.0167 g FeCl₃·6H₂O, 0.151 g CaCl₂. Solution B: Identical to NMS. Mix 10 ml of Solution A and 0.1 ml of Solution B, then adjust to 100 ml with deionized water.

Nitrogen-Free Medium (NFMS): the NFMS medium also consists of Solution A and Solution B. Solution A (per 1 L): 5.24 g K₂HPO₄·3H₂O, 2.62 g KH₂PO₄, 0.04 g FeSO₄·7H₂O, 3.0 g MgSO₄·7H₂O, 3.0 g NaCl, 0.0167 g FeCl₃·6H₂O, 0.151 g CaCl₂. Solution B: identical to NMS and AMS. Mix 10 ml of Solution A and 0.1 ml of Solution B, then adjust to 100 ml with deionized water.

Copper Supplementation for MMO Regulation (El Ghazouani et al., 2012). For pMMO expression: add 0.1 ml of filter-sterilized CuSO₄ stock solution to achieve a final Cu²⁺ concentration of ~5 μmol/L. For sMMO expression: omit CuSO₄ supplementation to maintain a Cu²⁺-free condition (0 μmol/L).

The Methylosinus trichosporum OB3b was used in the experiment, from Professor Xing Xinhui's research group at Tsinghua University, following the aforementioned protocols, 100 ml of NMS, AMS, and NFMS media were prepared in 250 ml culture flasks. The media were sterilized at 121 °C for 20 min using an autoclave and allowed to cool to room temperature before inoculation.

MOB seed culture was aseptically inoculated at 10% (v/v) of the medium volume, and the target OD₆₀₀ for harvesting MOB seed culture is 0.8 ± 0.06. Subsequently, the headspace of each flask was purged and replaced with one of the following gas mixtures (balanced with air): CH₄ : Air = 1:0 (100% methane); CH₄ : Air = 1:1 (50% methane); CH₄ : Air = 1:2 (33% methane); CH₄ : Air = 1:3 (25% methane); CH₄ : Air = 2:1 (67% methane); CH₄ : Air = 3:1 (75% methane). Following inoculation and gas exchange, the flasks were incubated in a rotary shaker maintained at 30 °C and 180 rpm.

Methane consumption was directly determined and calculated using headspace gas chromatography under the following analytical conditions: a GC7900 gas chromatograph equipped with a combined column system (OV-101 capillary column and TDX-01 molecular sieve column) and a thermal conductivity detector was employed for effective separation of methane, carbon dioxide, oxygen, and nitrogen. The operating parameters included a column temperature of 50 °C, an injection port temperature of 100 °C, a detector temperature of 120 °C, hydrogen as carrier gas, and a operating current of 120 mA. Manual injections were performed using a 1 ml gas-tight syringe to sample the headspace of culture bottles. Standard curven was obtained by external standard calibration using a series of pure methane standards (0.1–1.0 ml). Cultivation followed a 24-h ventilation protocol over a 7-day period. Daily methane consumption was determined by measuring headspace methane via GC immediately after ventilation (pre-Tn) and 24 h later before the next ventilation (post-Tn). The daily consumption was calculated as the difference between the pre- and post-Tn methane volumes derived from the standard curve, and the total consumption was obtained as the cumulative sum over the 7-day cultivation period.

Every 24 h, 3 ml of bacterial culture was sampled, with distilled water serving as the blank control, with three sets of parallel repetitions for each condition and all data were analyzed using analysis of variance. The OD₆₀₀ was measured using a UV-Vis spectrophotometer to assess microbial growth dynamics at different time points. By using the logistic function to fit the cell growth curve, the lag phase (λ) and maximum specific growth rate (μ_max) of the bacterial cells under this condition can be obtained, and a dynamic mathematical model of the methane oxidizing bacterium Methylosinus trichosporum OB3b can be established. The formula equation is:

y represents OD₆₀₀, A₁ represents the initial concentration of the fermentation broth, A₂ represents the maximum concentration of the fermentation broth, X represents the cell growth time, X₀ represents the proportional constant of the maximum growth rate, and P represents the bacterial growth index.

After the completion of cell culture, collect 10 ml of bacterial solution and centrifuge at 4 °C and 8,000 r/min for 15 min to obtain bacterial cell precipitate. Rinse the cell precipitate twice with 0.05 mol/L phosphate buffer, and then add 2 ml of buffer to resuspend the precipitate. MMO activity was determined via the propylene epoxidation assay (Smith et al., 2011). Reaction setup: a 10 ml reaction vial was loaded with 2 ml of cell suspension and sealed. Using a disposable syringe, 4 ml of headspace air was removed and immediately replaced with 4 ml of premixed gas (propylene:air = 1:1 v/v). The vial was resealed and inverted for incubation in a rotary shaker (30 °C, 200 rpm) to facilitate epoxidation over 30 min. Reaction Termination: post-reaction, the vial was inverted and stored at 4 °C overnight to allow phase separation. The sample was then transferred to a 5 ml centrifuge tube and clarified by centrifugation (10,000 rpm, 15 min). The supernatant was collected for MMO activity analysis. Epoxide Quantification: propylene oxide (PO) concentration was measured via gas chromatography under the following conditions: column: BD-5HT capillary column (30 m × 0.25 mm × 0.25 μm). Temperatures: column: 170 °C (isothermal), Injector: 250 °C, flame ionization detector (FID) detector: 250 °C, Injection: manual, 0.5 μl splitless, retention time of PO: ~2.86 min. The calculation formula for MMO activity is as follows:

C_epoxypropane: concentration calculated from the gas chromatographic peak area via the epoxypropane standard curve (nmol/ml); V: total volume of the reaction system (ml); t: reaction time (min); W_DCW: dry cell weight (mg). Unit: nmol/(min·mg dcw), where “mg” refers to milligram of dry cell weight.

Nitrogenase Activity Assay via Acetylene Reduction Method (Montes-Luz et al., 2023). The nitrogenase activity was determined using the acetylene reduction assay (ARA). Briefly, 15 ml of bacterial culture was transferred into a 150 ml serum bottle, supplemented with 0.1% (v/v) methanol as an electron donor. The bottle was sealed with a butyl rubber stopper and aluminum crimp cap. For the reaction initiation: 10% of the headspace volume was removed using a disposable syringe. An equivalent volume of purified acetylene gas was immediately injected. The bottle was resealed to maintain gas-tight conditions. The reaction was carried out in a rotary shaker at 30 °C and 150 rpm for 60 min. Post-incubation, 1 μl of headspace gas was sampled using a gas-tight syringe and analyzed for ethylene production using a SCION456 gas chromatograph equipped with a flame ionization detector (FID). Chromatographic conditions were as follows: column: BD-5HT capillary column (30 m × 0.25 mm × 0.25 μm). Temperature program: column: 50 °C (isothermal), Injector: 200 °C, FID: 250 °C, Injection: manual split mode, 1 μl injection volume, Ethylene retention time: ~2.93 min. The calculation formula for Nitrogenase activity is as follows:

C_ethylene: concentration derived from the gas chromatographic peak area via the ethylene standard calibration curve (nmol/ml); V: headspace volume of the reaction vial (ml); t: reaction time (min); W_DCW: dry cell weight (mg). Unit: nmol/(min·mg dcw), where “mg” refers to milligram of dry cell weight.

Determination of Cell Dry Weight (CDW) by Constant Weight Method. The cellular dry weight was measured using the constant weight method. Initially, cell pellets were collected via low-temperature centrifugation at 4 °C and 8,000 rpm for 15 min. The harvested biomass was subsequently washed twice with 0.05 M phosphate buffer (pH 7.0) to remove residual medium components. The processed cell pellets were then transferred to a constant-temperature drying oven and dehydrated at 70 °C until reaching constant weight. The dried samples were precisely weighed using an analytical balance with 0.1 mg resolution. For protein content analysis: (1) the lyophilized samples were reconstituted in 50 ml of distilled water. (2) Cell disruption was performed using an ultrasonic homogenizer under the following parameters: power output: 300 W, Pulsing cycle: 2 s ON, 4 s OFF, Total duration: 10 min. (3) The resulting cell lysate was collected for subsequent protein quantification. SCP Quantification via Bradford Assay: the SCP content was determined using the Coomassie Brilliant Blue G-250 dye-binding method (Yang et al., 2014). The assay principle relies on: formation of protein-dye complexes exhibiting maximum absorbance at 595 nm. Linear correlation between absorbance and protein concentration (0–1,000 μg/ml range). Colorimetric detection using a UV-Vis spectrophotometer. The SCP yield formula is as follows:

Y_SCP denotes the yield of SCP, ΔW_CDW represents the cell dry weight (g/L), and △W_CH4 signifies the total amount of methane consumed during the cultivation process. ∑_△6WCH4represents the cumulative methane consumption across six ventilation intervals.

Based on experimental data from Figure 1 and Table 1, the physiological metabolism of MOB was significantly modulated by gas-phase composition. Under CH₄:air = 3:1 (v/v), the system exhibited suboptimal performance: maximum OD₆₀₀ (0.63), cell dry weight (0.28 ± 0.004 g/L), and MMO-specific activity (118.72 nmol/(min·mg_dcw)) were all below optimal levels. This phenomenon arose from three metabolic constraints: (1) CO₂ accumulation: from high methane metabolism induced proton motive force imbalance; (2) insufficient oxygen partial pressure (DO < 15% saturation) hindered copper cofactor binding in pMMO, reducing electron transport chain activity; (3) hypoxic microenvironments activated Fnr-regulated anaerobic metabolic compensation pathways, triggering energy diversion.

Comparative experiments revealed that in pure methane systems (CH₄:air = 1:0), nitrate-dependent pMMO-expressing strains exhibited metabolic suppression (OD₆₀₀ < 0.5). Incremental oxygen supplementation to CH₄:air = 1:3 induced metabolic state transitions: (1) elevated DO enhanced cytochrome c oxidase-mediated respiratory phosphorylation efficiency; (2) Cu²⁺-assisted stabilization of pMMO tetrameric conformations; (3) biphasic gas-liquid mass transfer optimization yielded peak kinetic performance: maximum specific growth rate (μ_max = 0.022 h⁻¹); CDW (0.44 ± 0.02 g/L) and soluble protein content (20.73 ± 0.18%) increased by 56.4% and 32.5%, respectively, compared to the 3:1 group.

Notably, the lag phase was reduced to 15.31 ± 0.3 h (representing a 26% reduction compared with the 3:1 group). Based on this observation, it is speculated that elevated oxygen levels promoted expression of the nitrogen assimilation gene cluster (nasAB-nrtP) through activation of the OmpR/PhoB two-component system, thereby facilitating rapid metabolic network reprogramming. These findings furnish key metabolic node parameters for implementing gas-phase regulation strategies in industrial-scale SCP production.

Comparative analysis of Figure 2 and Table 2 revealed that under strict anaerobic conditions (CH₄:air = 1:0), the ammonium nitrogen-sMMO expressing MOB cultivation system exhibited significant metabolic inhibition. Specific manifestations included: biomass index OD₆₀₀ reaching only 0.73, cell dry weight 0.29 ± 0.016 g/L, key enzymatic parameter MMO-specific activity declining to 46.99 nmol/(min·mg·dwc), and soluble protein content (17.78 ± 0.88%) being significantly lower than other treatment groups. This metabolic inhibition originated from impaired electron transport chain function in microaerophilic strains under hypoxic conditions, leading to reduced oxidative phosphorylation efficiency in sMMO-catalyzed methane hydroxylation and consequent impact on carbon assimilation flux. With gradual air supply introduction, elevated gas-phase oxygen partial pressure markedly activated the methane oxidation metabolic network. Under optimal CH₄:air = 1:3 conditions, experimental groups demonstrated typical exponential growth phase characteristics: OD₆₀₀ increased by 62.98% to 1.19, cell dry weight reached 0.56 ± 0.014 g/L, maximum specific growth rate (μ_max) 0.025 h⁻¹. Concurrently detected soluble protein content rose to 21.38 ± 0.46%, with lag phase shortened to 12.44 ± 0.31 h, indicating that oxygen's synergistic effects not only enhanced sMMO-mediated methane oxidation-assimilation flux but also optimized metabolic coupling efficiency between TCA cycle and amino acid synthesis pathways, increasing per-biomass SCP yield by 156%. This gas ratio-dependent metabolic regulation mechanism provides crucial theoretical foundation for industrial SCP production based on dynamic gas supply strategies.

Systematic analysis of Figure 3 and Table 3 reveals the underlying mechanisms of gas composition effects on methanotrophic bacterial metabolism regulation. In pure methane systems (CH₄:Air=1:0), nitrogen-dependent sMMO-expressing strains exhibited the lowest specific growth rate (μ = 0.013 h⁻¹) due to dual constraints: firstly, MMO enzymatic kinetics became DO-limited under strictly anaerobic conditions, significantly reducing methane oxidation metabolic flux; secondly, undetectable nitrogenase activity indicated nitrogen assimilation pathway blockage forming a growth-limiting bottleneck.

With air introduction (CH₄:Air=1:3), while elevated dissolved oxygen enhanced methane oxidation rate, excessive oxygen partial pressure caused irreversible oxidative damage to nitrogenase complexes, reducing nitrogen fixation efficiency. This directly manifested in physiological parameters: OD₆₀₀ = 0.52, cell dry weight 0.21 ± 0.018 g/L, and soluble protein content 6.7 ± 0.28%, significantly lower than other groups.

Optimal performance occurred at CH₄:Air = 1:2, where precise DO control enabled metabolic flux redistribution: maintaining sMMO's optimal redox potential while creating microaerobic conditions to protect nitrogenase active sites. Experimental data showed: maximum specific growth rate reached 0.016 h⁻¹, lag phase shortened to 26.49 ± 0.3 h, OD₆₀₀ = 0.62 and cell dry weight 0.28 ± 0.008 g/L, and soluble protein content increased to 13.4 ± 0.28%. This discovery provides critical parameters for industrial SCP production optimization, suggesting dynamic gas regulation to balance MMO catalytic efficiency and nitrogenase stability can overcome substrate competition inhibition in conventional cultivation systems.

Although the closed batch culture system used in this study did not monitor gas consumption or supplement reaction gases in real time, the core objective of the experimental design is to establish a benchmark correlation between initial gas ratios and metabolic responses under different nitrogen source modes (such as growth kinetics parameters, enzyme activity thresholds, etc.). Although the gas phase ratio undergoes dynamic changes during the reaction process, all comparative experiments maintain consistent closed conditions, thus obtaining the optimal initial ratio threshold with clear comparative value.