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Article

Screening of Methanotrophic Strain for Scale Applications: Methane Emission Reduction and Resource Utilization

by
Chen Di
1,†,
Weijia Yu
1,† and
Yongze Lu
1,2,3,*
1
School of Energy and Environment, Southeast University, Nanjing 210096, China
2
Key Laboratory of Water Safety and Aquatic Ecosystem Health of Xizang, Xizang Minzu University, Xianyang 712082, China
3
Key Laboratory of Water Pollution Control and Ecological Restoration of Xizang, National Ethnic Affairs Commission, Xizang Minzu University, Xianyang 712082, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sustainability 2025, 17(8), 3687; https://doi.org/10.3390/su17083687
Submission received: 3 March 2025 / Revised: 14 April 2025 / Accepted: 16 April 2025 / Published: 18 April 2025
(This article belongs to the Section Environmental Sustainability and Applications)
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Abstract

Methanotrophs hold significant potential in global methane mitigation and resource recovery. However, the limited rate of cell proliferation remains a significant constraint for large-scale applications. Therefore, screening efficient methanotrophic strains that are suitable for industrial applications to mitigate methane and exploring potential methane resource utilization pathways are of great importance for sustainable development. Gradient dilution and the streak plate method were employed to isolate methanotrophic strains from a previously domesticated methane-oxidizing microbial consortium. We isolated a highly efficient strain, M6, which exhibited a 230% increase in growth rate compared to the laboratory model strain Methylocystis bryophila (M. bryophila). Taxonomic analysis revealed that strain M6 is classified as Methylocystis parvus. Genomic data indicated a diverse range of metabolic functions. In addition to utilizing methane, strain M6 can also utilize citrate to generate energy and intermediate products, addressing issues related to insufficient methane supply or low methane mass transfer efficiency. Metabolic adaptability ensures the stability of its application. The optimal cultivation conditions for strain M6 were determined, characterized by mild and easily implementable parameters. Based on the analysis of the genome and metabolic pathways, strain M6 exhibits potential for the synthesis of bioproducts, such as proteins, lipids, and polyhydroxyalkanoates (PHAs), with the fermentation process not requiring cost-intensive carbon sources, making it both economical and sustainable.

1. Introduction

Methane, as the second significant greenhouse gas after carbon dioxide, contributes approximately 20% to global warming [1]. Its global warming potential over a 20-year period is approximately 84 times greater than that of carbon dioxide [2]. Methane is significantly more efficient at trapping heat in the atmosphere compared to carbon dioxide, meaning that even small amounts of methane emissions can have a substantial impact on the greenhouse effect [3]. The concentration of methane has been steadily rising in recent years, rapidly increasing from around 750 ppb during the early industrial period to 1942.94 ppb in October 2024 (https://gml.noaa.gov/ccgg/trends_ch4/, accessed on 20 February 2025) [4], becoming an accelerator of short-term climate change. Controlling methane emissions is crucial for mitigating climate change [5]. The Global Methane Pledge commits to reducing methane emissions by 30% relative to 2020 levels by 2030 [6].
Methanotrophs, which are widely distributed in nature [7,8,9,10], play a crucial role in global atmospheric methane balance due to their unique ability to utilize methane as both a carbon source and an energy source [11]. Model analyses suggested that with the help of methanotrophs, the rise in average atmospheric temperature over the next 100 years could be reduced by 0.22 °C compared to scenarios without these bacteria [12]. In addition to mitigating methane emissions, methanotrophs can also serve as potential cellular factories for various bioproducts, such as methanol, single-cell proteins, and bioplastics [13,14,15], thereby facilitating the resource utilization of methane. Although methanotrophs hold significant potential in methane reduction and resource utilization, their efficient growth and scaling-up of production remain major limiting factors for large-scale applications [13]. Therefore, selecting highly active methanotrophic strains with rapid proliferation characteristics is of great importance for sustainable development.
In 1906, the first methanotrophic strain was successfully isolated [16]. Subsequently, an increasing number of methanotrophs were isolated and characterized. In 1970, Whittenbury et al. isolated over 100 species of Gram-negative methanotrophs and introduced the classification system comprising Type I, Type II, and Type X [17]. Type I and Type II bacteria utilize formaldehyde through the ribulose monophosphate (RuMP) cycle and serine cycle, respectively. Type X bacteria also utilize formaldehyde via the RuMP cycle, but differ from Type I species in that they express low levels of serine cycle enzymes, specifically ribulose-1,5-bisphosphate carboxylase, which is involved in the Calvin–Benson–Bassham (CBB) cycle [16]. Furthermore, Type II and Type X bacteria possess nitrogen-fixing capabilities, whereas most Type I bacteria lack this ability [18].
Given the significant role of methanotrophs in mitigating climate change and their high industrial value, along with the challenges associated with their large-scale application for sustainable development, this study seeks to address these issues. We isolated different methanotrophic strains from a methane-oxidizing microbial consortium and selected strains with efficient growth for further investigation. Taxonomic classification was performed through multi-phase analysis to determine their taxonomic position, while whole-genome sequencing was utilized to predict their metabolic functions. Additionally, cultivation conditions were optimized. This research enriches the existing microbial resource bank of methanotrophs and is of great significance for reducing methane emissions, mitigating the greenhouse effect, and promoting the resource utilization of methane.

2. Materials and Methods

2.1. Sample Source and Model Strain

Methane-oxidizing microbial consortia, previously domesticated in the laboratory, were selected for the isolation of pure methanotrophic strains [19]. The microbial consortia were cultivated for over 400 days in a reactor, with methane serving as the sole carbon source. The model methanotrophic strain Methylocystis bryophila (DSM-21852) was selected for comparison, and it was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). The model methanotrophic strain M. bryophila is a typical Type II methanotroph that assimilates formaldehyde via the serine pathway.

2.2. Medium

This study used DSMZ medium 1409, which contains the following components (per liter): 0.4 g of KNO3, 50 mg of MgSO4·7H2O, 10 mg of CaCl2·2H2O, 0.1 g of K2HPO4, 10 mg of NaCl, and 1 mL of trace element solution. The trace element solution contains (per liter): 5.0 g of Ethylene Diamine Tetraacetic Acid (EDTA), 2.0 g of FeSO4·7H2O, 0.1 g of ZnSO4·7H2O, 0.2 g of CoCl2·6H2O, 0.1 g of CuCl2·2H2O, 20 mg of NiCl2·6H2O, and 30 mg of Na2MoO4. The pH of the medium was adjusted to 6.20 ± 0.05 using 1 M of NaOH or 1 M of HCl [20]. For solid media, 20 g·L−1 of agar powder was added.

2.3. Isolation and Storage of Strains

2.3.1. Preliminary Enrichment of Strains

A total of 200 mL of DSMZ medium 1409 was added to a serum bottle and sealed with an isobutyl rubber stopper. The headspace was replaced with 50% CH4 and 50% O2 premixed gas using a syringe, and the bottle was then sterilized in an autoclave (Boxun, Shanghai, China). A 1 g sample of the methane-oxidizing microbial consortium was obtained from the laboratory domesticated reactor, dissolved in 50 mL of sterile water. Filtration is used to remove fine particles from the sample, and the filtered bacterial suspension is transferred to a serum bottle for further enrichment and cultivation. The serum bottle was subsequently incubated at 25 °C and 180 rpm in a shaker incubator. The growth of the microbial population was monitored by measuring the turbidity of the culture, which typically became cloudy after 5–7 days, indicating a high bacterial concentration. At this stage, 3 mL of the culture supernatant was pipetted into a newly sterilized culture bottle. The cultivation steps were repeated to remove contaminating microorganisms, thereby obtaining a more stable methanotrophic culture.

2.3.2. Strains Screening

Methanotrophs were isolated using the continuous dilution and streak plate method. Briefly, 1 mL of the bacterial suspension was serially diluted in 10-fold increments to concentrations of 10−3, 10−4, and 10−5. A total of 0.2 mL of each diluted suspension was plated onto agar plates containing the solid culture medium described in Section 2.1. The plates were placed in anaerobic culture bags, where the gas was replaced with 50% CH4 and 50% O2, and incubated at 25 °C. The growth of the methanotrophs was monitored regularly. Streaking was repeated on fresh culture plates, and colonies exhibiting distinctive morphological features of methanotrophs were isolated under the same conditions. After purification, some colonies were transferred to liquid culture medium for further cultivation. The biomass of the methanotrophs was evaluated by measuring the optical density at 600 nm using a UV-visible spectrophotometer (UV9100, LabTech, Beijing, China) [21]. Nitrate determination was conducted using standard methods to assess the nitrogen assimilation ability of the methanotrophs [19]. Additionally, individual colonies were transferred to slant solid culture medium tubes, with a small amount of methane added, sealed with paraffin, and stored at 4 °C for future use.

2.4. Strain Identification

2.4.1. Morphological Observation

The pure cultured strains were plated onto DSMZ medium 1409 solid plates and incubated in a 50% CH4 + 50% O2 premixed gas atmosphere. The colony characteristics on the plates were observed. Single colonies were then selected and inoculated into DSMZ medium 1409, and the growth pattern in liquid culture medium was examined.

2.4.2. Physiological and Biochemical Characterization

Gram Staining: A drop of bacterial suspension was taken from the liquid culture medium and placed on a glass slide, which was then dried and fixed over an alcohol lamp. Crystal violet dye (ammonium oxalate solution) was added for 1 min, followed by rinsing with distilled water. Iodine solution was applied for 1 min and rinsed with distilled water. 95% ethanol was added dropwise until no purple color remained (approximately 20–30 s), after which the slide was rinsed with distilled water and dried using blotting paper. Finally, the slide was stained with safranin for 1–2 min, rinsed, dried, and examined under a microscope (Olympus, Japan).
Starch Hydrolysis Test: A pipette was used to transfer the bacterial suspension onto a starch hydrolysis medium plate, which was then incubated at 30 °C. After incubation, iodine solution was applied to the plate. If a clear, colorless zone appeared around the bacterial growth, indicating starch hydrolysis, the result was considered positive. Otherwise, the result was negative.
Methyl Red Test: This test was used to assess the ability of bacteria to ferment glucose and produce acid. The bacterial suspension was inoculated into peptone water medium and incubated for 2 to 4 days. Methyl red reagent was then added to the culture medium. If a significant amount of acid was produced from glucose fermentation, the medium turned red, indicating a positive result in the methyl red test. If a small amount of acid was produced, or if the acid was further converted into other substances (e.g., alcohols, ketones, aldehydes, gases, or water), the medium turned yellow, indicating a negative result in the methyl red test.
Catalase Test: A loop was used to pick a colony from the solid plate and transfer it to a test tube. Then, 2 mL of 3% hydrogen peroxide solution was added to the tube. After 30 s, the presence of bubbles indicated a positive result, whereas the absence of bubbles indicated a negative result.
Citrate Utilization Test: Sodium citrate was used as the sole carbon source in the medium, which also contained bromothymol blue as a pH indicator. If the bacteria were able to utilize citrate, it would be broken down into carbonates, making the medium alkaline. Consequently, the indicator would change from green to deep blue. If the bacteria were unable to utilize citrate, no color change would occur in the medium.

2.4.3. 16S rDNA Sequencing and Phylogenetic Tree Construction

Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (NEB, USA). The 16S rDNA was amplified from the purified DNA using the universal bacterial primers 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). The PCR products were sequenced, and the preparatory steps for sequencing included PCR amplification, TA cloning, transformation, and plasmid extraction procedures. The obtained 16S rDNA sequence was subjected to a BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 24 November 2023) search against GenBank to identify homologous sequences. The sequencing results were then compared with publicly available 16S rDNA sequences in GenBank to perform nucleotide homology analysis. Phylogenetic analysis was performed using the Neighbor-Joining method in MEGA 11.0 software to determine the taxonomic status of methanotrophic strain.

2.5. DNA Extraction and Whole-Genome Analysis

The strain was cultured in DSMZ medium 1409 to an OD600 of approximately 1.0. Samples were quickly collected by centrifugation (4000 rpm, 4 °C, 10 min), and the biomass was frozen immediately in liquid nitrogen and stored at −80 °C for subsequent whole-genome sequencing.
The genomic DNA of the samples was extracted, and its purity and integrity were assessed using agarose gel electrophoresis. Libraries were constructed using both the Nanopore and Illumina platforms, followed by quality control of the libraries. Once the libraries passed quality control, they were sequenced on the Nanopore PromethION (Oxford nanopore, Oxford, UK) and Illumina NovaSeq PE150 (Illumina, San Diego, CA, USA) platforms, with sequencing depth and library concentration adjusted to meet the target data output.
To ensure the accuracy and reliability of subsequent data analysis, the raw sequencing data were filtered to remove low-quality reads. The quality-controlled valid data were then assembled using SMRT Link v5.1.0 software, resulting in an initial genome assembly that represented the basic genomic features of the sample. The assembly was optimized and corrected using Arrow v2.3.2 software, followed by alignment analysis and further correction. Ultimately, a complete chromosome sequence was obtained. Finally, bioinformatics methods were employed for genome composition and gene function analysis. Functional annotation of coding gene sequences is performed using databases to predict gene functions. The basic steps of functional annotation are as follows: (1) Perform Diamond alignment (evalue ≤ 105) of the predicted gene protein sequences against multiple functional databases. (2) Filter alignment results: For each sequence, select the alignment with the highest score for annotation (identity ≥ 40% and coverage ≥ 40%).

2.6. Optimization of Culture Conditions

The culture conditions of the methanotrophic strain were optimized to improve its metabolic activity. The liquid culture medium DSMZ medium 1409, as described in Section 2.1, was used for cultivation. The initial pH of the medium was optimized by incubating cultures within a pH range of 5.0 to 9.0 with increments of 1.0. The incubation temperature was varied between 15 °C and 40 °C (in 5 °C intervals). Different methane-to-oxygen ratios in the headspace were tested (3:1, 2:1, 3:2, 1:1, and 2:3). Additionally, Cu2+ concentrations in the medium were adjusted to 0.00, 5.00, 10.00, 20.00, and 40.00 μmol/L to identify the optimal Cu2+ concentration. Cultures were incubated in a shaking incubator at 200 rpm, with three replicates for each treatment. After 96 h of incubation, the biomass of the methanotrophs was measured.

2.7. Data Analysis

The experimental samples were set up in three parallel groups, and the data are presented as the mean ± standard deviation (SD). The experimental data were organized and analyzed using Microsoft Excel 2021 software. Analysis of variance (ANOVA) was used to compare significant differences between different treatments, with p < 0.05 considered statistically significant. Tables were created using Microsoft Excel 2021, and figures were generated using Origin 2021 software.

3. Results

3.1. Strain Screening and Metabolic Characterization

Six methanotrophic strains were successfully isolated using the streak plate method. These strains were then cultured in methane-enriched media, and two strains with significantly faster growth rates (named M4 and M6) were selected for further comparison with the laboratory-preserved M. bryophila strain regarding cell proliferation and nitrogen assimilation performance (Figure 1). The results showed that, compared to M. bryophila, both M4 and M6 strains exhibited faster growth rates, reaching the stationary phase on day 3, with strain M6 showing a slightly faster growth rate. In contrast, the model strain M. bryophila only reached a similar biomass level by day 10. Moreover, both M4 and M6 strains demonstrated stronger nitrogen assimilation capabilities, achieving nitrate removal rates of 47.48% and 55.41% on day 3, while M. bryophila reached a stable nitrogen removal rate of 47.02% on day 8. These results indicated that strain M6 met the expected selection criteria and was suitable for further research.
Figure 1c illustrates the change in gases (CH4, O2, and CO2) during the cultivation of M. bryophila and strain M6. The gas consumption and production trends closely mirrored the patterns of cell proliferation. Strain M6 exhibited a significantly higher gas metabolism rate compared to M. bryophila, reaching the stationary phase by day 3, whereas M. bryophila displayed a more gradual metabolic profile. The stoichiometric analysis and growth kinetics calculations were performed (Table 1) following the method described by Schill et al. [22]. M. bryophila consumed 1 mL of CH4 while consuming 1.11 mL of O2, producing 0.33 mL of CO2, and increasing cell numbers by 1.28 × 108, with a maximum specific growth rate of 0.28. In contrast, strain M6 consumed 1 mL of CH4 while consuming 1.35 mL of O2, producing 0.38 mL of CO2, and increasing cell numbers by 1.71 × 108, with a maximum specific growth rate of 0.78.

3.2. Traditional Taxonomic Characteristics of Strain M6

The morphological characteristics of the methanotrophic strain M6 are shown in Figure S1. On agar plates with methane as the carbon source, strain M6 formed light brown, smooth-surfaced colonies with well-defined edges, measuring approximately 0.8–1.2 mm in diameter. The surface of the colonies was slightly raised, and the colonies were easy to pick up (Figure S1a). In liquid medium, the strain appeared as a white, turbid suspension with a white bacterial film adhering to the walls of the culture bottle (Figure S1b). Under an optical microscope (×10, Figure S1c), strain M6 appeared as spherical or short rod-shaped cells.
The physiological and biochemical characteristics of the methanotrophic strain M6 are presented in Figure 2. The results of the Gram-stain experiment indicate that strain M6 is Gram-negative. Both the starch hydrolysis and methyl red tests were negative, whereas the catalase test and citrate utilization test yielded positive results.

3.3. Phylogenetic Analysis of Strain M6

To further classify strain M6, its 16S rDNA gene was sequenced (NCBI: SAMN38194746), and a phylogenetic tree was constructed to determine its taxonomic position. The results indicated that strain M6 is most closely related to Methylocystis parvus strain 54 (Figure 3). Based on the species-level classification threshold, it was preliminarily determined that strain M6 belongs to Methylocystis parvus. Therefore, strain M6 is classified as a Type II aerobic methanotroph.

3.4. Metabolic Network Analysis of Strain M6

3.4.1. Genome Sequencing and Assembly

In this study, the whole-genome of strain M6 (NCBI: SAMN47852635) was sequenced using the Nanopore PromethION and Illumina NovaSeq PE150 platforms. As shown in Table 2 and Figure S2, the sequence assembly resulted in a circular chromosome, with no plasmids detected. The genome size of strain M6 is 4,148,822 bp with a GC content of 64.15%. A total of 4702 coding genes were identified, accounting for 87.32% of the genome. A total of 51 tRNA genes and 9 rRNA genes were identified. The whole-genome sequencing results of strain M6 were annotated by aligning with the Gene Ontology (GO) (Figure S3), Kyoto Encyclopedia of Genes and Genomes (KEGG) (Figure S4), and Clusters of Orthologous Groups (COG) (Figure S5) databases to obtain functional information for the bacterium.

3.4.2. KEGG Database Annotation

A total of 2087 genes in the methanotrophic strain M6 were functionally annotated in the KEGG database, accounting for 44.4% of the total genome. In the KEGG database annotation, the metabolic functional genes identified in strain M6 are categorized into major pathways (Figure 4), including Amino Acid Metabolism (150 genes), Biosynthesis of Other Secondary Metabolites (40 genes), Carbohydrate Metabolism (161 genes), Energy Metabolism (177 genes), Glycan Biosynthesis and Metabolism (25 genes), Lipid Metabolism (60 genes), Metabolism of Other Amino Acids (48 genes), and Nucleotide Metabolism (68 genes). Additionally, genes associated with methane oxidation (40 genes) and nitrogen metabolism (27 genes) were also detected in strain M6. The key genes related to methane oxidation, the serine cycle, nitrogen metabolism, and PHA synthesis in strain M6 are presented in Table S1.

3.5. Optimization of Culture Conditions for Strain M6

The culture conditions for strain M6, were optimized to assess the impact of external environmental factors on its growth. Biomass measurements were recorded after 96 h of cultivation to evaluate the influence of various parameters. The results indicated that pH, temperature, methane-to-oxygen volume ratio, and copper ion concentration significantly influenced the growth of strain M6. As shown in Figure 5a, strain M6 exhibited strong growth within a pH range of 6 to 8 with optimal growth observed at pH 7. In contrast, growth was significantly inhibited in acidic (pH < 6) and alkaline (pH > 8) environments, with minimal or no growth observed under these extreme conditions. As shown in Figure 5b, strain M6 demonstrated strong adaptability and optimal growth within a temperature range of 25–30 °C. However, growth rates were markedly reduced at temperatures below 20 °C and above 35 °C, indicating the strain’s sensitivity to temperature fluctuations. As depicted in Figure 5c, the biomass of strain M6 after 96 h of cultivation initially increased and then declined with an increasing methane-to-oxygen volume ratio. The highest biomass was observed at the methane-to-oxygen volume ratio of 1:1, suggesting that this ratio is optimal for maximizing growth. Figure 5d demonstrates that in the absence of copper ions (Cu2+), strain M6 displayed extremely low growth activity. As the Cu2+ concentration increased, the biomass initially increased and then slightly declined. The highest biomass was observed at a copper ion concentration of 15 μmol/L, indicating this concentration to be optimal for supporting the strain’s growth.

4. Discussion

Methanotrophs are a group of microorganisms that are capable of utilizing methane as both the carbon and energy source via assimilatory and dissimilatory metabolic pathways. They play a critical role in mitigating global warming. Screening high-efficiency strains that are suitable for large-scale applications, elucidating their metabolic functions, and analyzing potential methane utilization pathways are crucial for sustainable development.

4.1. Rapid Proliferation and Nitrogen Assimilation Capability

The methane metabolism of methanotrophs begins with the catalytic action of methane monooxygenase (MMO), which activates the strong C-H bond in methane and oxidizes it to methanol using oxygen at ambient temperature and pressure [23]. Methanol is subsequently oxidized by methanol dehydrogenase to formaldehyde, which is further converted into formate or biomass [13]. Ultimately, methane is oxidized into carbon dioxide, providing energy for cellular metabolism. Strain M6 and M. bryophila exhibited different growth characteristics. In terms of gas changes, M6 required more O2 as an electron acceptor while consuming the same volume of CH4, resulting in more CO2 production. From a cell growth perspective, M6 exhibited a higher maximum specific growth rate, with a μmax of approximately 2.79 times that of M. bryophila, demonstrating superior rapid proliferation characteristics.
Strain M6 exhibited a rapid cell proliferation rate, indicating its strong methane assimilation and dissimilation capabilities, as well as its high overall metabolic activity, highlighting its potential for large-scale applications. Nitrogen is an essential element for the synthesis of amino acids, proteins, and other cellular components, with proteins acting as the primary carriers of life activities. Additionally, nitrogen is required for nucleic acid synthesis during cell proliferation. Strain M6 demonstrated a high nitrate assimilation rate, facilitating biomass synthesis and the realization of biological functions. This capability forms the foundation for methane emission reduction and resource utilization.

4.2. Phylogenetic Affiliation and Metabolic Characteristic

Methanotrophs are classified into Type I, Type II, and Type X based on their formaldehyde assimilation pathways [17]. Subsequently, through genomic sequencing, aerobic methanotrophs have been broadly categorized into two major proteobacterial groups: γ-proteobacteria (Group I) and α-proteobacteria (Group II) [24]. The γ-proteobacteria (Group I) encompass the previously classified Type I and Type X methanotrophs, while the α-proteobacteria (Group II) include the formerly recognized Type II methanotrophs. The α-proteobacteria consist of four genera: Methylocella, Methylocapsa, Methylocystis, and Methylosinus. The γ-proteobacteria are further subdivided into 12 genera: Methylothermus, Methylosoma, Methylosphaera, Methylosarcina, Methylomonas, Methylohalobius, Methylomicrobium, Methylococcus, Methylocaldum, Methylobacter, Clonothrix, and Crenothrix. The 16S rDNA sequencing results indicate that strain M6 belongs to Methylocystis parvus, a Type II methanotroph that assimilates formaldehyde via the serine pathway.
Physiological and biochemical characterization revealed the fundamental metabolic properties of strain M6. Gram-staining confirmed it as a Gram-negative bacterium. The negative result in the starch hydrolysis test indicated the strain’s inability to utilize starch. The methyl red test, which assesses the production of acidic end products during glucose metabolism, yielded a negative result for strain M6, suggesting minimal acid production, likely due to its inability to metabolize glucose. The catalase test was positive, demonstrating the strain’s ability to decompose hydrogen peroxide via catalase, reflecting its metabolic adaptability, which indicates potential stability in industrial-scale applications. The positive citrate utilization test confirmed the strain’s ability to generate energy and intermediate metabolites through citrate metabolism, a trait that may address challenges related to insufficient methane supply or low methane mass transfer efficiency in practical applications.

4.3. Metabolic Functional Diversity

Based on whole-genome sequencing results (Table S1), the methane metabolic pathway of strain M6 was further analyzed, and the corresponding genes were identified. Strain M6 oxidizes methane to methanol via methane monooxygenase (pmoA), which is subsequently oxidized to formaldehyde by methanol dehydrogenase (mxaF). Genes related to the serine pathway (glyA, agxt, hprA, gck, eno, ppc, mdh, mtkA, mcl) were identified for formaldehyde assimilation, representing a typical metabolic pathway of Type II methanotrophs. The growth of methanotrophic bacteria requires a substantial amount of nitrogen (0.25 mol N per 1 mol C assimilation) [25] and exhibits the capability to assimilate nitrate [26]. Whole-genome sequencing of strain M6 identified 27 nitrogen metabolism-related genes, including key genes (Table S1) such as the nitrate transport gene (nrtA), nitrate reductase gene (nasC), and nitrite reductase gene (nirB).
In the KEGG annotation, most of annotated functional genes were categorized under metabolism, demonstrating a remarkable diversity in metabolic functions. Specifically, functional proteins involved in nucleotide metabolism, lipid metabolism, energy metabolism, carbohydrate metabolism, and amino acid metabolism were abundantly represented. This metabolic versatility underscores the potential of methanotrophs as cellular factories for the production of various high-value products, a subject that has been extensively studied. The diverse metabolic capabilities of strain M6 provide a promising avenue for methane resource utilization, highlighting its potential in biotechnological applications aimed at methane valorization.

4.4. Mild Cultivation Conditions

The cultivation of Methanotrophs is a complex, dynamic biological process. The growth and metabolic activity of the strain are influenced not only by its intrinsic properties but also by external factors such as nutrient availability and environmental conditions [27], including temperature, pH, methane-to-oxygen ratio, and copper ion concentration. Temperature and pH directly affect the overall enzymatic activity of the cells. Methane serves as both the carbon and energy source for methanotrophs, and oxygen acts as a critical electron acceptor. Maintaining a balanced ratio of carbon and oxygen is essential for their optimal growth [28,29]. Methane monooxygenase (MMO), existing in both soluble (sMMO) and particulate (pMMO) forms, is regulated by copper ions (Cu2+). Cu2+ not only functions as the active center for the expression of sMMO and pMMO but also plays a key role in the synthesis of pMMO [30]. However, highCu2+ concentrations can induce heavy metal toxicity in cells [31]. Single-factor experiments were conducted to determine the optimal cultivation conditions for the rapid proliferation of strain M6. The results indicated that the optimal pH was around 7, the optimal temperature was 30 °C, the optimal methane-to-oxygen volume ratio was 1:1, and the optimal Cu2+ concentration was 15 μmol/L. These cultivation conditions are mild, easily controllable, and highly feasible for industrial applications. The ability to maintain these conditions with minimal complexity and cost highlights the potential of strain M6 for scalable and sustainable biotechnological processes. Considering that the 1:1 methane and oxygen ratio poses a potential explosion risk from an industrial safety perspective, it is essential to employ technical measures to reduce the risk of explosion. In the study by Li et al. [32], a membrane-aerated biofilm reactor was used to supply methane and oxygen, with gases diffusing directly from the membrane surface into the water without bubble formation. This reduced the violent mixing of gases with water, decreasing the escape of flammable gases from the liquid phase to the gas phase, thus mitigating the formation of explosive mixtures and achieving the long-term stable operation of the reactor.

4.5. Methane Emission Reduction and Resource Utilization

Methanotrophs can produce a variety of valuable products from methane. By utilizing methane as the sole carbon source, methanotrophs can synthesize bioplastics, biofuels, feed additives, ectoine, and various other high-value compounds. Given the genome and metabolic pathways of strain M6, analyzing its potential pathways for resource utilization is of significant importance. Strain M6 exhibits a high nitrate assimilation rate and contains a large number of genes related to amino acid metabolism (150 genes), suggesting its potential for microbial protein synthesis, which could contribute to alleviating the global food crisis [19,33]. Additionally, the strain possesses a substantial number of functional proteins related to lipid metabolism (60 genes), highlighting its potential for synthesizing microbial lipids. These lipids can serve as raw materials for biodiesel, surfactants, cosmetics, and other industrial applications, playing a critical role in the chemical and energy sectors [24,34]. Furthermore, strain M6 also exhibits potential for polyhydroxyalkanoates (PHAs) production, a biodegradable polymer. This process begins with the oxidation of methane to methanol by methane monooxygenase (MMO), followed by the conversion of methanol to formaldehyde via methanol dehydrogenase (MDH). Formaldehyde is then assimilated into central metabolism through the serine cycle, generating acetyl-CoA. Under nutrient-limiting conditions (e.g., nitrogen or phosphorus deprivation), acetyl-CoA is polymerized into PHA granules by PHA synthase and stored intracellularly. PHA synthase plays a crucial role in the synthesis of PHAs via the serine pathway in Type II methanotrophs [35]. Whole-genome sequencing of strain M6 revealed the presence of the gene phaC (Table S1), which encodes PHA synthase, suggesting that strain M6 has the potential for PHAs biosynthesis. PHAs can be used to produce biodegradable plastics, drug carriers, and other materials, providing environmental and sustainability advantages [36]. Other potential applications of strain M6 remain to be explored.
Methanotrophs employ methane, a greenhouse gas, as a substrate for bioconversion, distinguishing them from microorganisms that rely on cost-intensive carbon sources such as glucose, fructose, or xylose. This unique characteristic makes gas fermentation processes based on methanotrophs economically viable and sustainable.

5. Conclusions

In this study, a methanotrophic strain, M6, belonging to Methylocystis parvus, was successfully screened for its high growth efficiency and suitability for large-scale applications. In addition to its ability to utilize methane, strain M6 can also metabolize citrate to generate energy and intermediate metabolites, which addresses challenges related to insufficient methane supply or low methane mass transfer efficiency during practical applications. Its antioxidant mechanisms and metabolic adaptability ensure its stability under operational conditions. The optimal cultivation conditions for strain M6 were determined to be mild and easily achievable, further enhancing its industrial feasibility. Beyond methane emission reduction, strain M6 demonstrates significant potential for synthesizing high-value products, including microbial protein, microbial lipids, and polyhydroxyalkanoates (PHAs). Notably, the fermentation process does not rely on cost-intensive carbon sources such as glucose, fructose, or xylose, making it both economically viable and environmentally sustainable. This study characterized the basic metabolic traits of strain M6 under atmospheric pressure conditions but lacks an assessment of the strain’s pressure and osmotic stress responses, which warrants further research to provide a more solid theoretical foundation for the industrial application of the strain. It should also be noted that this study focused on the basic metabolic traits of strain M6, but lacked an assessment of the strain’s pressure tolerance and osmotic stress responses. This aspect is crucial for providing a more solid theoretical foundation for the industrial application of the strain and warrants further investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su17083687/s1, Figure S1: Morphological characteristics of methanotrophic strain M6; Figure S2: Whole-genome map of methanotrophic strain M6; Figure S3: GO annotation classification of the whole-genome sequence of strain M6; Figure S4: KEGG annotation classification of the whole-genome sequence of strain M6; Figure S5: COG annotation of the whole-genome sequence of strain M6; Table S1: The key genes related to methane oxidation, the serine cycle, Nitrogen metabolism and PHA synthesis in strain M6.

Author Contributions

Conceptualization, C.D.; methodology, W.Y.; software, C.D.; validation, W.Y.; formal analysis, C.D. and W.Y.; investigation, W.Y.; resources, Y.L.; data curation, W.Y.; writing—original draft preparation, C.D.; writing—review and editing, Y.L.; visualization, W.Y.; supervision, Y.L.; project administration, Y.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Innovation Project on Emission Peak and Carbon Neutrality of Jiangsu Province (BK20220038).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
RuMPRibulose monophosphate
CBBCalvin–Benson–Bassham
GOGene Ontology
KEGGKyoto Encyclopedia of Genes and Genomes
COGClusters of Orthologous Groups
MMOMethane monooxygenase
PHAPolyhydroxyalkanoate
MDHMethanol dehydrogenase

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Figure 1. Metabolic characteristics of methanotrophic strains: (a) growth curve; (b) nitrate concentration variation curve; (c) gas content variation curve.
Figure 1. Metabolic characteristics of methanotrophic strains: (a) growth curve; (b) nitrate concentration variation curve; (c) gas content variation curve.
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Figure 2. Physiological and biochemical characteristics of the methanotrophic strain M6: (a) Gram-stain test, (b) starch hydrolysis test, (c) methyl red test, (d) catalase test (negative: no bubble formation; M6: bubble formation occurs), (e) citrate utilization test.
Figure 2. Physiological and biochemical characteristics of the methanotrophic strain M6: (a) Gram-stain test, (b) starch hydrolysis test, (c) methyl red test, (d) catalase test (negative: no bubble formation; M6: bubble formation occurs), (e) citrate utilization test.
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Figure 3. Phylogenetic tree based on 16S rDNA sequences.
Figure 3. Phylogenetic tree based on 16S rDNA sequences.
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Figure 4. Number of genes annotated to metabolism in KEGG.
Figure 4. Number of genes annotated to metabolism in KEGG.
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Figure 5. Effect of different cultivation conditions on the growth of strain M6: (a) pH, (b) temperature, (c) methane/oxygen volume ratio, (d) Cu2+ concentration.
Figure 5. Effect of different cultivation conditions on the growth of strain M6: (a) pH, (b) temperature, (c) methane/oxygen volume ratio, (d) Cu2+ concentration.
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Table 1. Stoichiometric analysis and growth kinetics calculation of strains M. bryophila and M6.
Table 1. Stoichiometric analysis and growth kinetics calculation of strains M. bryophila and M6.
StrainV(O2)/V(CH4)Y(CO2/CH4)Y(Cells/CH4) *μmax
M. bryophila1.110.331.28 × 1080.28
M61.350.381.71 × 1080.78
* Calculated by logarithmic growth period data.
Table 2. Genomic features of strain M6.
Table 2. Genomic features of strain M6.
IndicatorValue
Number of scaffolds1
Genome size (bp)4,148,822
G + C content (mol%)64.15
Number of genes4702
Gene length/Genome length87.32
tRNA51
sRNA0
rRNA9
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Di, C.; Yu, W.; Lu, Y. Screening of Methanotrophic Strain for Scale Applications: Methane Emission Reduction and Resource Utilization. Sustainability 2025, 17, 3687. https://doi.org/10.3390/su17083687

AMA Style

Di C, Yu W, Lu Y. Screening of Methanotrophic Strain for Scale Applications: Methane Emission Reduction and Resource Utilization. Sustainability. 2025; 17(8):3687. https://doi.org/10.3390/su17083687

Chicago/Turabian Style

Di, Chen, Weijia Yu, and Yongze Lu. 2025. "Screening of Methanotrophic Strain for Scale Applications: Methane Emission Reduction and Resource Utilization" Sustainability 17, no. 8: 3687. https://doi.org/10.3390/su17083687

APA Style

Di, C., Yu, W., & Lu, Y. (2025). Screening of Methanotrophic Strain for Scale Applications: Methane Emission Reduction and Resource Utilization. Sustainability, 17(8), 3687. https://doi.org/10.3390/su17083687

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