Methylomonas methanica and Methylosinus sp. co-cultivated with methylotrophs under saline conditions for microbial protein production from biogas as alternatives to natural-gas- based production using Methylococcus capsulatus

Microbial protein (MP) is a promising alternative protein source that can be produced using methane as a substrate derived from undervalued resources. Methylococcus capsulatus is one of the most prominent microbial species employed for industrial production, while other potentially suitable methanotrophs remain underexplored. Here, we selected the alternative methanotrophs Methylosinus sp. and Methylomonas methanica with high growth rates and potential for halotolerance and compared them to M. capsulatus. Methylosinus sp. and Methylomonas methanica achieved up to 1.87-fold higher maximum specific growth rates and 2.52-fold higher biomass concentrations, compared to M. capsulatus. When the pH was allowed to acidify below optimum for growth, the protein content of M. methanica (45% ± 5% CDW) was 1.55 times compared to pH-controlled conditions. Cultivation at an increased salt content (20 g NaCl L −1) boosted the protein content of M. methanica to 81% ± 7% CDW, showing its applicability for MP production using saline water. Co-culturing M. methanica with the methylotrophic yeast Komagataella phaffii led to a 1.27-fold higher essential amino acid content, which indicates an improvement in protein quality, while the use of synthetic biogas increased the protein yields up to 1.5-fold, compared to purified methane. In summary, M. methanica co-cultured with selected methylotrophs has the potential to enable more sustainable MP production under saline conditions using biogas, as an alternative to natural gas-based production with M. capsulatus. These strategic upstream choices could bring us closer to a future-proof protein production system.

The global population is anticipated to reach 9.8 billion by 2050 (United Nations, 2024), which intensifies the demand for protein-rich foods (Tilman et al., 2011). Livestock production is responsible for more than 30% of the global anthropogenic greenhouse gas emissions (Crippa et al., 2021), while occupying 70% of the arable land (Steinfeld et al., 2006). Alternative plant-based proteins, such as soy, perform better than animal proteins in these aspects, but still contribute to detrimental effects, including tropical deforestation (Tilman et al., 2011), which further intensifies the need for more sustainable alternative proteins. Microbial protein (MP), or single-cell protein, the protein-rich biomass of microorganisms, such as bacteria, fungi, yeasts, and microalgae, has the potential to be a more sustainable alternative (Nasseri et al., 2011). It can be used for a variety of food (and feed) applications, including meat and dairy analogues, snacks or beverages (Pereira et al., 2024). The production of MP requires substantially fewer resources than conventional protein sources–ca. 40,000 times less land and 20 times less water compared to beef–while emitting only a fraction of the greenhouse gases (Smetana et al., 2023). MP could be a highly attractive feedstock for industrial applications independent of geographical or seasonal constraints (Ritala et al., 2017), as it can be produced with ca. 10 times higher protein yields per kilogram of feedstock and ca. 10 times higher nitrogen usage efficiency than conventional meat (Pikaar et al., 2017). Among the potential MP sources, bacteria are particularly promising due to their high protein content which ranges from 50% to 82% in cell dry weight (% CDW), depending on the microbial species and cultivation conditions (Ravindra, 2000; Sakarika et al., 2023). This approaches the protein content of traditional meat sources, such as raw chicken (90% in dry weight–DW) (MyFoodData, 2026), and even surpassing other animal-derived proteins, like whole egg (48%–50% DW) (American Egg Board, 2021) and plant-based sources, such as soybean (36%–45% DW)​ (Wang and Cavins, 1989). Bacterial MP often contain high levels of all essential amino acids (EAA) for human nutrition, making it a complete protein source, and also contain other components, like lipids, carbohydrates, vitamins, minerals and fibres (Ravindra, 2000; Nasseri et al., 2011). One of the greatest advantages of bacteria is their ability to use a wide range of substrates, including gaseous carbon sources, like methane (CH 4), which can be found in biogas–a more renewable methane source (European Biogas Association, 2023). The conversion of side streams to biogas that is subsequently used as carbon source can circumvent potential safety concerns associated with the liquid fraction of side streams that could include toxic components, such as pesticides and heavy metals or pathogenic microorganisms (Pieja et al., 2017). This makes bacterial MP based on biogas considerably promising for industrial applications (Verbeeck et al., 2020).

Converting methane to MP requires methanotrophic microorganisms–also known as methane-oxidising bacteria (MOB), which are able to utilise methane as the sole carbon and energy source (Hanson and Hanson, 1996). Among MOB, Methylococcus capsulatus has been extensively studied for MP production, and is already commercialised in feed products, such as FeedKind™ (Ritala et al., 2017). However, methane-based cultivation remains constrained by gas-liquid mass transfer and generally achieves lower biomass productivities than microorganisms utilizing soluble carbon substrates (Joergensen and Degn, 1987) which has long challenged large-scale implementation. Nevertheless, there are important benefits to gas fermentation of CH 4 to MP which we illustrated above. However, the low tolerance to impurities, such as H 2 S at 0.5% v/v in the gas phase (Pei et al., 2022), limits its potential future applications to the use of natural gas or upgraded biogas instead of using biogas directly. Other MOB, especially γ-proteobacteria, could be suitable candidates, as they use the ribulose monophosphate (RuMP) pathway, which enables more efficient carbon assimilation than the serine pathway (Hanson and Hanson, 1996). This pathway is associated with higher biomass yields, lower carbon and energy losses, and improved substrate and oxygen utilization efficiency, making methanotrophs employing RuMP attractive for MP production and other industrial methane bioconversion processes (Sakarika et al., 2022). Among these, Methylomonas methanica has been identified as a promising candidate due to its high methane oxidation rate and rapid growth (Hoefman et al., 2012; Kim et al., 2018), although quantitative cultivation data remain limited. This MOB species can perform well under higher salinity (Bowman et al., 1993; Lettau et al., 2022) with a potential for industrial applications such as Calysta (Silverman et al., 2019; 2021). The α-proteobacterium Methylosinus sp. Has also been identified as a fast-growing species (Hoefman et al., 2012; Kim et al., 2018). Despite the use of the less efficient serine pathway for methane assimilation by this MOB (Hanson and Hanson, 1996), reporting lower growth yields than γ-proteobacteria (Scheutz et al., 2009), the species Methylosinus sp. Has attracted industrial interest in MP production (Silverman et al., 2019; 2021). Production (Silverman et al., 2019; 2021).

Co-culturing MOB with methylotrophs–the wider microbial group that is able to assimilate one-carbon (C1) compounds (Anthony, 1982) – could enhance MP production (Asmamaw and Fassil, 2014). One suggested mechanism is that non-MOB methylotrophs could consume metabolic intermediates released during methane oxidation, such as methanol, formate, and acetate, which may otherwise accumulate and negatively affect MOB metabolism (Anthony, 1982), thereby improving growth efficiency and protein yields. Limited studies have explored the co-cultivation of MOB with non-MOB methylotrophs (Stock et al., 2013). The methylotrophic bacterium Methylorubrum extorquens (previously known as Methylobacterium extorquens) and yeast Komagataella phaffii (previously known as Pichia pastoris) were selected due to their established use in methylotrophic biotechnology (Ochsner et al., 2015; Zahrl et al., 2017). Furthermore, their metabolic flexibility, including the ability to utilize compounds such as acetate, formate and methanol, makes them promising candidates for co-cultivation with MOB. These metabolic interactions may not only affect the overall growth efficiency but could also influence the allocation of carbon and, potentially, the allocation between cellular components and composition of the resulting biomass. However, the mechanisms underlying such effects remain largely unexplored.

Despite the increasing interest in methane-based MP, quantitative comparisons between alternative methanotrophs remain scarce and are often limited to qualitative observations, individual strains, or different cultivation conditions, making direct comparisons difficult. Likewise, the potential benefits of co-cultivation with non-methanotrophic methylotrophs and the influence of cultivation conditions relevant for industrial implementation have received little attention. Consequently, it remains unclear which microbial candidates and cultivation strategies offer the greatest potential to improve methane-based MP production.

This study explored alternative candidates for methane-based MP production beyond the conventional M. capsulatus, which is currently the main methanotroph in industrial methane-based MP production. We hypothesized that alternative MOB and MOB–methylotroph co-cultures improve biomass production, carbon utilization efficiency, and protein quality. To test this hypothesis, M. methanica and Methylosinus sp. Were quantitatively compared with M. capsulatus in terms of maximum specific growth rate, biomass concentration, protein content, and EAA composition. In addition, co-cultivation with the non-MOB methylotrophs K. phaffii and M. extorquens was evaluated to determine its effect on biomass production and protein quality. Finally, the best-performing MOB was evaluated under cultivation strategies with direct industrial relevance, including pH control, increased salinity, and the use of biogas instead of purified methane. These conditions were selected because they represent realistic future operating scenarios that may improve process sustainability while introducing trade-offs between biomass productivity and protein quality. Specifically, pH acidification may reduce the need for base addition, the use of saline medium may enable the use of saline or alternative water sources, and biogas could provide a more sustainable methane source than purified methane.

Microorganisms

The MOB M. capsulatus LMG 26900 and Methylosinus sp. LMG 26262 were purchased from the Belgian Coordinated Collection of Microorganisms (BCCM/LMG, Ghent, Belgium) and M. methanica DSM 25384 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The methylotrophic yeast K. phaffii ATCC 76273 was purchased from the American Type Culture Collection (ATCC, Virginia, United States) and the methylotrophic bacterium Methylorubrum extorquens DSM 1338 from DSMZ.

Inoculum preparation

For the MOB, 120 mL gas-tight serum vials (Sigma-Aldrich, Germany) were filled with 20 mL of diluted nitrate mineral salt (dNMS) medium (Dedysh and Dunfield, 2014) and supplemented with 30% v/v methane (99.95% purity, B50 Air Liquide) to a CH 4:air ratio of 0.3:1 (v/v) corresponding to a CH 4:O 2 ratio comparable to that used successfully in our previous study (Kerckhof et al., 2021), using gas sterilised through 0.2 µm filters (Minisart®, Sartorius, Germany). The methylotrophs were grown in 100 mL Erlenmeyer flasks (ROTILABO®, Carl Roth, Germany), filled with 20 mL Luria-Bertani (LB) medium (Thermo Fisher Scientific, United States), supplemented with 0.30% v/v sterile methanol (99.8+ %, Chem-Lab, Belgium), and sealed with metal caps (Ø37/39 mm, Carl Roth, Germany).

All cultures were grown in axenic conditions, which were routinely verified by Sanger sequencing throughout the study. They were inoculated at 10% v/v and incubated at 28 °C under orbital shaking of 120 rpm (KS 4000 I control, IKA, Germany), except for M. capsulatus that was incubated at 37 °C, as suggested by the culture collection for optimal growth (Hanson and Hanson, 1996). Prior to the experiments, all cultures were washed three times using sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, United States), followed by centrifugation at 20,817 g for 5 min (Centrifuge 5,430, Eppendorf, Belgium), and resuspended in autoclaved PBS to correct their optical cell density at 600 nm (OD 600) to ca. 0.30 (Spectronic™ 200, Thermo Scientific, United States).

Experimental methodology

The study comprised three main experiments, with the goal to assess the performance of the selected methanotrophs in comparison to the conventional M. capsulatus and their co-culture with the selected methylotrophs and select the most optimal combination at the targeted operational conditions (Section 2.4). First, we compared different MOB in terms of maximum specific growth rate, biomass concentration and protein content, where the individual performance of M. methanica and Methylosinus sp. Was compared to M. capsulatus, which acted as the positive control (Figure 1). Next, the performance of the MOB co-cultures with the non-MOB methylotrophs K. phaffii or M. extorquens was compared to the corresponding pure cultures, with the latter acting as a positive control (Figure 1). For the third and last experiment, the effect of cultivation conditions that can act as stressors (i.e., pH acidifications during growth, increased salt content in the cultivation medium and use of biogas) was evaluated on the best-performing MOB based on the previous experiments, comparing the performance of the pure culture under these suboptimal growth conditions, to their performance under optimal conditions (Figure 2). These conditions were selected as they reflect potentially more sustainable future applications. Cultivation at lower pH could reduce the need for base addition, increased salinity reflects the use of saline water sources, such as water affected by saltwater intrusion or suitably treated saline process waters, thereby reducing the reliance on freshwater. The use of methane-rich biogas represents a more sustainable alternative to fossil natural gas or upgraded biomethane. The objective was to evaluate the trade-offs between productivity and product quality under these conditions. In this case, the pure cultures grown under optimal conditions acted as positive control.

Experimental design and procedures

All experiments were conducted in axenic conditions, using 1 L Scott bottles (GL45 Duran®, Carl Roth, Germany), filled with 200 mL ammonium mineral salts (AMS) medium (Supplementary Table S1), inoculated at 10% v/v and sealed with halogenated black butyl rubber stoppers (GL45, Carl Roth, Germany) and polypropylene screw caps with aperture (Duran®, GL45, VWR International, United States). The bottles were autoclaved with air in the headspace, after which the required gaseous substrates were added by injection using a sterile 50 mL syringe. Ammonium (NH 4 +) was selected as nitrogen source, primarily due to its potential to be recovered from side streams, thereby aligning with the core objectives of resource recovery (Pikaar et al., 2017). For the co-cultures, the pure cultures were diluted with PBS to achieve cell counts within the same order of magnitude (2–5 × 10 8 cells mL -1), measured by flow cytometry (Section 2.5), and mixed in equal ratios of 1:1 (v/v) with 10% v/v of total inoculum per co-culture, to ensure comparable initial contributions of both microorganisms. Pure cultures of the non-MOB methylotrophs were grown as a positive control, while bottles without inoculum served as a negative control. In all cases, the negative control consisted of bottles containing the mineral medium, carbon source and air (all sterile), but no inoculum. Bottles were randomly assigned to the different treatments and randomly positioned in the incubator to minimize potential positional effects. Microbial growth was monitored until the stationary phase was reached, considering the different growth duration in each experiment (6–12 days, Supplementary Tables S2, S3). The OD 600 was measured daily (Spectronic™200, Thermo Fisher Scientific, United States), while biomass concentration (g CDW L -1), headspace pressure (kPa), and gas composition (% v/v) were recorded at the start and end of growth. The pH was monitored at the start and end of growth, except in the experiment assessing the effect of pH adjustment, where it was measured daily (Section 2.3). Headspace pressure was recorded using a digital manometer (DPGA-06, Dwyer Instruments Inc., US0041, United States) and pH using a pH meter (Multi-parameter analyser C3020, Consort, Belgium). At the end of the experiment, 4 mL (2 × 2 mL) suspension samples were taken. One of the tubes was immediately stored at −20 °C as raw sample, while the other tube was centrifuged at 20,817 g for 5 min (Centrifuge 5,430, Eppendorf, Belgium), subsequently washed three times with PBS and then stored as two separate 2 mL Eppendorf tubes (supernatants and pellet) at −20 °C for later protein measurements. The remaining biomass was stored in 50 mL Falcon® tubes (Corning, United States) at −20 °C for later analysis of the biomass concentration.

In the control experiment, the bottles were supplemented with 30% v/v methane (99.95% purity, B50 Air Liquide) that has been filter-sterilized (0.2 µm) to a CH 4:air ratio of 0.3:1 (v/v), except for the experiment using different methane sources and for the positive control (non-MOB methylotrophs), which received 0.30% v/v (about 2.40 g/L) sterile methanol (99.8+ %, Chem-Lab, Belgium) instead of methane. The cultures were incubated under orbital shaking of 120 rpm (KS 4000 I control, IKA, Germany) at temperatures selected according to the reported optimal growth conditions of each organism. Specifically, cultures were incubated at 28 °C, except for M. capsulatus, which was incubated at 37 °C for optimal growth (Hanson and Hanson, 1996). All experiments were performed in biological triplicates.

To assess the effect of pH, a subset of pure cultures of M. methanica were grown in media with an increased buffer concentration (1.0 M phosphate buffer) compared to the control (0.2 M phosphate buffer), and the pH was adjusted on daily basis by using 2.0 M of NaOH, to achieve a stable pH of 6.84 ± 0.06 during growth. For the control condition, no pH adjustments were made, with an initial pH of the medium of 6.93 ± 0.04.

To assess the effect of higher salt levels, NaCl was added to the cultivation medium prior to growth at 10, 20, and 35 g NaCl L -1, while no NaCl was added in the control (0 g NaCl L -1). This resulted in a conductivity of 2, 8, 11, and 18 m cm -1 for 0, 10, 20, and 35 g NaCl L -1), respectively. The selected salinity levels represent different potential operational scenarios: 10 and 20 g NaCl L -1 represent intermediate salinities relevant to brackish or concentrated saline process waters as well as the partial use of seawater, whereas 35 g NaCl L -1 approximates seawater salinity and represents the potential substitution of freshwater with seawater.

The impact of different methane sources was assessed using purified methane as a control, with a composition of 99.995% v/v CH 4 (B50, Air Liquide, France), without the presence of other substrates. This was compared with two types of biogas, containing varying levels of CO 2. Real biogas was collected in 10 L airtight sampling gas bags (Tedlar® PVF, HedeTech Co. Ltd., China) from a single batch of a local maize farm and had an initial composition of 78% ± 3% v/v CH 4 and of 22% ± 1% v/v CO 2. Synthetic biogas was also used. It was manually prepared by mixing CH 4 and CO 2 (both >99.995% v/v, B50, Air Liquide, France) in 10 L airtight sampling gas bags to achieve an initial composition of 65% ± 1% v/v CH 4 and 35% ± 2% v/v CO 2. The biogas compositions were measured by gas chromatography (Section 2.6). Neither of the biogas types contained any detectable amount of H 2 S or other gases than CH 4 or CO 2. The gases were injected to the bottles at 30% v/v at ambient conditions, resulting in a CH 4:air ratio of 0.3:1 for purified methane, and a CH 4:CO 2:air ratio of 0.23:0.07:1 for real biogas and 0.20:0.11:1 for synthetic biogas (in all cases v/v). The gas compositions (CH 4, O 2, CO 2 and N 2) were measured at the start and end of the growth (stationary phase) (Section 2.5).

Analytical techniques

The biomass concentration (g CDW L -1) was quantified as total suspended solids (TSS), using a standard-level analytical balance (HR-250AZ, A&D Company Limited, Japan) and oven at 105 °C (UN450pLus, Memmert GmbH, Germany), according to Standard Methods 2540D and E (APHA, 1997). Growth curves were obtained for each culture by creating calibration curves for each methanotrophic species, relating OD 600 to microbial biomass concentration. Biomass concentration was then plotted in function of time. Maximum specific growth rates (µ max, in h -1) were determined, based on data points obtained from the exponential growth phase, as described by Richards (1959) (Supplementary Table S2).

To compare the protein content between the different cultivation conditions evaluated in this study the total (crude) protein concentration (g protein L -1) was quantified through the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, United States), without prior pre-treatment (Sakarika et al., 2020; 2023). The assay was applied consistently throughout the experiments employing different microbial strains. The corresponding media were included as controls to correct for potential background interference. Bovine serum albumin (BSA) was used for the calibration curve. The absorbance was measured at 562 nm, using a multimode plate reader (Infinite® 200 PRO, TECAN, Switzerland). The protein content (% in cell dry weight, or % CDW) was then determined by dividing the total (crude) protein (g protein L -1) by the biomass concentration (g CDW L -1) and multiplying by 100.

Protein quality was assessed through measuring the EAA content (in g kg −1 CDW) by an external accredited lab (NutriControl, Netherlands), applying ion exchange chromatography and photometric detection at 570 nm, as previously described (European Commission (EC) No 152/2009, Annex III F). The EAA histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine were analysed. Prior to analysis, the samples were stored at −20 °C, and freeze-dried under nitrogen atmosphere to a temperature of −55 °C (ALPHA 1–2 LDplus, Fisher Scientific, United States). To obtain enough biomass for EAA analysis, the biomass of the biological triplicates was consolidated as one sample and was presented as one data point. Consequently, differences in amino acid profiles should be interpreted as indicative trends rather than statistically confirmed differences. The EAA content of microbial biomass was calculated considering 5% of moisture in the final product, as previously described by (EC No 152/2009). The daily consumption needed to cover EAA requirements was determined for the microbial biomass, and compared with three types of common food products (soybean, whole egg and raw chicken) (WHO/FAO/UNU, 2007; U.S. Department of Agriculture, 2024). The calculations were based on the limiting EAA (WHO/FAO/UNU, 2007) and an average body weight (62 kg) (Walpole et al., 2012), assuming each food source was the sole dietary intake on a certain day. Given that the EAA samples have been pooled, these comparisons are intended as benchmarks and not as statistical comparisons.

The gas composition (% v/v) was measured through gas chromatography using thermal conductivity detection (Compact GC 4.0, Global Analyser Solutions, the Netherlands). CH 4, O 2, and N 2 were quantified using a Porabond Q column (15 m × 0.32 mm × 10 μm) maintained at 50 °C, and helium as the carrier gas supplied at 3 bar at a flow rate of 0.1 mL/min. H 2 was quantified using a separate channel with a Molsieve 5 A column (30 m × 0.32 mm × 30 μm) maintained at 60 °C and nitrogen as the carrier gas supplied at 3 bar at a flow rate of 0.1 mL/min. Both channels used a 25 μL sample loop. The injector temperature was 70 °C, and the detector temperature was 110 °C. The detection limit of the thermal conductivity detector was 100 ppmv for each quantified gas. Gas consumption of CH 4 (g), protein yield (g protein g -1 CH4) and biomass yield (g CDW g -1 CH4) were calculated as previously described (Khoshnevisan et al., 2019).

Data analysis

Statistical analysis was conducted using the software package JASP (Version 0.19.3, Team 2024). Descriptive statistics were reported as mean values with standard deviations (SD), with maximum specific growth rates presented as mean ± SD of biological triplicates (n = 3). Protein content, protein yields, and pH were calculated from three biological replicates, with technical triplicates averaged prior to statistical analysis. Statistical comparisons were therefore performed using biological replicates only (n = 3). Mann-Whitney U Tests were applied to examine significant differences in rank-distributions. Statistical significance was set at p ≤ 0.05.

Comparison of different MOB in terms of maximum specific growth rate, biomass concentration and protein content

To identify alternative methanotrophs with improved potential for MP production, the growth performance of M. methanica and Methylosinus sp. Was compared with the conventional industrial species M. capsulatus. These species were evaluated based on their maximum specific growth rate, biomass concentration, and protein content under the tested cultivation conditions.

Methylosinus sp. And M. methanica both showed significantly (p < 0.05) higher maximum specific growth rates than M. capsulatus under the conditions applied here (Figure 3a). Methylosinus sp. Was the fastest grower (μ max = 0.75 ± 0.11 h -1, p = 0.005), corresponding to a 1.9-fold higher growth rate than M. capsulatus, followed by M. methanica (0.65 ± 0.14 h -1, p = 0.012, 1.6-fold higher), compared to M. capsulatus (0.40 ± 0.01 h -1). Methylomonas methanica and Methylosinus sp. Surpassed M. capsulatus in terms of biomass concentration in the stationary phase. Methylomonas methanica yielded a biomass concentration of 0.53 ± 0.08 g CDW L -1, which was 2.5 times higher than M. capsulatus (0.21 ± 0.04 g CDW L -1, p < 0.001). Methylosinus sp. Achieved a biomass concentration of 0.39 ± 0.06 g CDW L -1 (p < 0.001). Methylococcus capsulatus grew slower and achieved lower biomass concentration compared to M. methanica and Methylosinus sp., but it had a superior protein content (58% ± 4% CDW), significantly (both p < 0.001) surpassing M. methanica (45% ± 5% CDW; 1.3-fold lower) and Methylosinus sp (18% ± 4% CDW; 3.2-fold lower) (Figure 4a).

Overall, these results indicate that the alternative methanotrophs achieved higher growth rates and biomass production, but M. capsulatus still produced biomass with the highest protein content.

Comparison of pure MOB with co-cultures in terms of maximum specific growth rate, biomass concentration and protein content

To evaluate whether co-cultivation with non-methanotrophic methylotrophs could improve methane-based MP production, the three selected methanotrophs, M. capsulatus, M. methanica, and Methylosinus sp., were cultivated in combination with K. phaffii or M. extorquens. The co-cultures were compared with the corresponding pure cultures based on maximum specific growth rate, biomass concentration, protein content, and, for the most promising methanotroph of this work, EAA composition.

The maximum specific growth rate of the co-culture of M. capsulatus with K. phaffii was significantly higher (0.48 ± 0.03 h -1, p = 0.009), compared to M. capsulatus in pure culture (μ max 0.40 ± 0.01 h -1) (Figure 3a; Supplementary Table S3). The biomass concentration of the co-culture with K. phaffii in the stationary phase was not significantly different (p = 0.057) from the pure culture of M. capsulatus (0.21 ± 0.04 g CDW L -1). When M. capsulatus was co-cultured with M. extorquens, the growth was slower (0.35 ± 0.01 h 1, p = 0.006; 1.1-fold lower), compared to the pure culture of M. capsulatus. The biomass concentration in the stationary phase was not significantly different (p = 0.655) compared to the pure culture of M. capsulatus. The co-cultures had similar protein content to the pure culture of M. capsulatus (58% ± 4% CDW) (p = 0.352 with K. phaffii; p = 0.965 with M. extorquens) (Figure 4a).

The co-culture of Methylosinus sp. With K. phaffii (0.66 ± 0.02 g CDW L -1, p = 0.092) or M. extorquens (0.60 ± 0.07 g CDW L -1, p = 0.131) achieved similar biomass concentrations to the pure culture of Methylosinus sp (0.75 ± 0.11 g CDW L -1) (Figure 3a). The protein content was 28% ± 9% CDW (p = 0.101) when Methylosinus sp. Was grown with K. phaffii and 19% ± 8% CDW (p = 0.329) with M. extorquens, which was comparable to the pure culture of Methylosinus sp (18% ± 4% CDW) (Figure 4a).

The maximum specific growth rates (p = 0.700 and 0.775) and biomass concentrations in the stationary phase (p = 0.057 and 0.184) of the co-cultures of M. methanica with K. phaffii (0.60 ± 0.08 h -1) or M. extorquens (0.63 ± 0.05 h -1) did not substantially differ from the pure culture of M. methanica (0.65 ± 0.14 h -1, Figure 3a). The protein content of the co-culture with K. phaffii was significantly higher (64% ± 6% CDW, p < 0.001) than the pure culture of M. methanica (45% ± 5% CDW) (Figure 4a). The co-culture with M. extorquens had similar protein content as the pure culture of M. methanica (42% ± 7% CDW, p = 0.329). This trend was also reflected in the higher EAA content for the co-culture with K. phaffii (126 g kg -1 CDW), in contrast to the lower EAA content in the co-culture with M. extorquens (87 g kg -1 CDW), compared to M. methanica in pure culture (99 g kg -1 CDW) (Figure 5; Supplementary Table S4). If we presume that microbial biomass from these (co-)cultures would be the only dietary human protein intake on a certain day, 111 g of dry product of the co-culture with K. phaffii (limited by lysine) would be required to cover daily EAA requirements, which is lower than the amounts that would need to be consumed of M. methanica in pure culture (148 g, limited by histidine) (Figure 6; Supplementary Table S5). In contrast, 258 g of dry product of the co-culture with M. extorquens would be required to cover their daily EAA requirements (limited by histidine), nearly double that of the co-culture with K. phaffii.

In summary, co-cultivation had species-dependent effects on methane-based MP production. It did not substantially affect biomass concentration or protein content of the total biomass for the co-cultures with M. capsulatus or Methylosinus sp., although K. phaffii slightly increased the growth rate of M. capsulatus. In contrast, co-cultivation of M. methanica with K. phaffii significantly improved the protein content of the biomass and EAA composition without compromising the growth performance, resulting in the highest nutritional quality among the tested co-cultures. Based on these findings we identify the M. methanica-K. phaffii co-culture as a promising co-culture for further development of methane-based MP production.

The effect of stressors on M. methanica in terms of maximum specific growth rate, biomass concentration and protein content

Following the selection of M. methanica as a promising alternative methanotroph amongst the ones tested in this work, the effects of cultivation conditions that are relevant for future industrial implementation were evaluated. Specifically, the influence of pH acidification during growth, increased salinity of the cultivation medium, and the use of methane-rich biogas instead of purified methane were assessed in terms of growth performance, biomass production, and protein quality. These conditions were selected because they represent potentially more sustainable production scenarios, allowing the trade-offs between production capacity and quality to be evaluated.

Acidification during growth leads to increased protein content and lower biomass concentration

In the condition without pH adjustment, the pH of the medium dropped from 6.84 ± 0.12 to 4.60 ± 0.32 (p < 0.001) between start and stationary phase of growth of M. methanica (Supplementary Figure S1). When the pH was adjusted on a daily basis, the pH levels remained stable (p = 0.077). Maintaining a stable pH during cultivation significantly affected the growth of M. methanica, resulting in a 1.3-fold higher μ max (0.82 ± 0.03 h- 1, p = 0.047), compared to no pH adjustment (0.65 ± 0.14 h- 1). The biomass concentration was 3.8 times higher with a stable pH during growth (1.98 ± 0.05 g CDW L- 1, p < 0.001), than without a stable pH (0.53 ± 0.08 g CDW L- 1) (Figure 3b). In contrast, the protein content decreased to 29% ± 12% CDW (p = 0.008) when pH was adjusted, which is 0.64 times the value achieved with no adjustment (45% ± 5% CDW) (Figure 4b; Supplementary Table S6).

Increased salt concentration increases the protein content

The NaCl addition to the cultivation medium significantly (p < 0.05) increased the protein content of M. methanica, compared to the control without NaCl (Figure 4d). At 10 and 35 g NaCl L -1, the protein content reached 60% ± 8% CDW and 72% ± 5% CDW (both p = 0.001), which are 1.3-fold and 1.6-fold higher respectively, compared to 45% ± 5% CDW for the control without NaCl. The highest protein content was 1.8 times higher compared to the control condition at 20 g NaCl L -1, reaching 81% ± 7% CDW (p < 0.001). The higher salt concentration in the cultivation medium had a limited effect on the growth of M. methanica in terms of μ max which ranged from 0.59 ± 0.05 to 0.65 ± 0.14 h -1, and biomass concentration, which ranged from 0.46 ± 0.03 to 0.61 ± 0.07 g CDW L -1. No significant differences were observed at 10 g NaCl L -1 (p = 0.700 and 0.057), 20 g NaCl L -1 (p = 0.400 and 0.051) and 35 g NaCl L -1 (p = 0.781 and 0.421, respectively) (Figure 3c; Supplementary Table S6).

Biogas enhances the protein content and protein yields, compared to purified methane

When M. methanica was cultivated using biogas as methane source, the pH levels remained stable throughout the growth (p = 0.077 for synthetic biogas; p = 0.100 for real biogas). The use of synthetic and real biogas, both led to increased protein content, compared to purified methane (Figure 4c; Supplementary Table S6). This effect was stronger for synthetic biogas, which had 1.5 times the protein content (67% ± 12% CDW, p = 0.008) of the M. methanica biomass produced using purified methane (45% ± 5% CDW). The use of real biogas resulted in 1.3 times the protein content (58% ± 9% CDW, p = 0.002) achieved with purified methane. The protein yields were also significantly higher with biogas as methane source. Specifically, protein yields per unit of consumed CH 4 increased to 0.56 ± 0.076 g protein g -1 CH4 when using synthetic biogas (p = 0.005), and to 0.40 ± 0.14 g protein g -1 CH4 using real biogas (p = 0.049), compared to purified methane (0.37 ± 0.12 g protein g -1 CH4) (Supplementary Table S7). The use of synthetic and real biogas did not affect the growth in terms of μ max (p = 0.400 and 0.700) and biomass concentrations (p = 0.063 and 0.156, respectively), compared to purified methane (Figure 3d). Biomass yields per unit of consumed CH 4 (p = 0.051 and 0.057) also showed no significant differences (Supplementary Table S7).

In summary, the tested cultivation conditions demonstrated that process modifications can substantially impact the balance between biomass productivity and protein quality. Preventing pH acidification favored biomass production but reduced protein content, whereas increased salinity and cultivation on biogas enhanced the protein content without significantly affecting growth performance. These findings indicate that cultivation conditions can be tailored according to the desired balance between productivity and biomass quality for methane-based MP production.

Methylosinus sp. and M. methanica as faster-growing MOB compared to M. capsulatus

Methylosinus sp. And M. methanica performed better than M. capsulatus when grown as pure cultures, achieving up to 1.87-fold higher maximum specific growth rate (μ max 0.75 ± 0.11 h −1). Despite this, M. capsulatus had a higher protein content (58% ± 4% CDW) compared to Methylosinus sp (18% ± 4% CDW) and M. methanica (45% ± 5% CDW). It should be noted that the different methanotrophs were cultivated at their reported optimal temperatures (M. capsulatus: 37 °C; Methylosinus sp. And M. methanica: 28 °C), as recommended for optimal growth of each species. Methane was supplied in excess throughout cultivation and remained detectable in the headspace at the end of the experiments, indicating that methane availability was not limiting under the applied conditions. Under the conditions applied in this work, the achieved μ max of M. capsulatus (0.40 ± 0.01 h −1), biomass concentration (0.21 ± 0.04 g CDW L -1) and protein content (40%–53% CDW) were in line with reported values (Whittenbury et al., 1970; Joergensen and Degn, 1987; Steinberg et al., 2017; Rasouli et al., 2018). In contrast, data for M. methanica remain limited (Kulkarni et al., 2021; Silverman et al., 2021), and, to our knowledge, no such data are available for Methylosinus sp., making this study one of the first to provide quantitative analysis and comparison with M. methanica.

Although Methylosinus sp. And M. methanica have been identified as fast-growing MOB (Hoefman et al., 2012; Kim et al., 2018), previous studies only rely on qualitative visual assessments of growth during selective plating for fast-growing Methylosinus sp. The limited quantitative studies on M. methanica report comparable biomass concentrations (0.25–0.26 g CDW L -1), but slightly lower maximum specific growth rates (μ max 0.16–0.40 h -1) than the 0.65 ± 0.14 h -1 observed in this study (Kulkarni et al., 2021; Silverman et al., 2021). The protein content of M. capsulatus achieved in this work falls within the commonly reported range for bacteria (i.e., 50%–82% CDW) (Ravindra, 2000; Nasseri et al., 2011). However, lower values for other bacteria (∼20% CDW) have been reported in MP studies, including mixed MOB cultures (i.e., 15%–51% CDW) (Khoshnevisan et al., 2019; Valverde-Pérez et al., 2020; Zha et al., 2021), and HOB (i.e., 18%–82% CDW) (Sakarika et al., 2020), supporting the present findings for Methylosinus sp. And M. methanica while demonstrating the variability across species and cultivation conditions. It has previously been demonstrated that growth rate is directly related to macromolecular composition and cell size (Sakarika et al., 2023), which illustrates that growth rates under the current experimental conditions will affect the outcome–nonetheless, these were largely uniform across the MOB.

Protein quality in M. methanica could be improved by co-culturing with Komagataella phaffii

Co-culturing M. methanica with K. phaffii did not have any impact on growth performance, however, the protein content of the co-culture was 1.42 times (64% ± 6% CDW) that of the pure culture of M. methanica (45% ± 5% CDW). Moreover, the EAA content was 28% higher for the co-culture (126 g kg -1 CDW), compared to the pure culture. Therefore, the co-culture biomass had higher protein content and improved protein quality than the pure culture of M. methanica.

When benchmarking against common food products, the microbial biomass of the co-culture had a lower protein content than animal products, like raw chicken (86% DW) and whole egg (48%–50% DW), or plant proteins such as soybean meal (36%–45% DW) (U.S. Department of Agriculture, 2024). However, the content of sulfur-containing EAA, like methionine, was higher in the co-culture at 6% of EAA, surpassing that of soybean meal which amounts to 4% of EAA (WHO/FAO/UNU, 2007). However, protein content and amino acid composition alone do not fully determine the suitability of microbial biomass as a food ingredient. Additional factors, including protein digestibility, nucleic acid content, and regulatory requirements, must also be evaluated before food applications can be considered. Given that the plant-based meat alternatives–dominated by soybean–currently account for the majority (i.e., 67%) of the global meat alternative market (Statista, 2025), these findings support the potential of MP as a future food ingredient.

The underlying mechanism behind the incremental protein content and protein quality in the co-culture of M. methanica and K. phaffii is not yet well understood. The MOB are hypothesized to benefit from co-cultivation with non-MOB methylotrophs that consume the liquid byproducts of methanotrophy that could be toxic to MOB (Oshkin et al., 2014), leading to improved growth for the overall co-culture. While this effect was observed for both co-cultures of M. capsulatus (i.e., with K. phaffii or with M. extorquens), such an interaction does not appear to explain our observations, as neither the co-culture with K. phaffii nor with M. extorquens resulted in higher growth, but with a higher protein content, relative to M. methanica in pure culture. This implies the involvement of other, potentially more complex physiological and metabolic mechanisms (Oshkin et al., 2014), which could not be fully explored in the present study.

In the case of M. methanica co-cultivated with M. extorquens, both growth performance and protein content were marginally lower than in the pure culture of M. methanica. During pre-cultivation (i.e., the preparation of cultures for the main experiment), M. extorquens consistently had slower growth compared to K. phaffii under the same methanol concentrations, indicating that its limited growth may be attributed to the different substrate conditions rather than interactions between the species that compose the co-culture. The co-culture of M. methanica with K. phaffii likely involves different interactions, given the marginally impaired growth, but significantly higher protein content and better EAA profile. Because protein content is expressed as a percentage of CDW rather than as total protein production, the observed increase cannot be explained solely by the presence of additional K. phaffii biomass in the co-culture. However, the respective contributions of each microorganism to the final protein content and composition cannot be distinguished with the experimental approach used here. Therefore, although the co-culture biomass exhibited improved protein content and amino acid quality, the underlying mechanisms remain to be elucidated.

Boosting the protein content in M. methanica

Increased salinity enhanced the protein content in M. methanica, with the highest value recorded at 81% ± 7% CDW when the medium was supplemented with 20 g NaCl L -1. The genus Methylomonas is known for its halotolerance, with recorded MMO activity up to 120 g NaCl L -1 (Kalyuzhnaya, 2016; Bussmann et al., 2021; Lettau et al., 2022), however, there is no indication that members of this genus are inherently halophilic. While some marine Methylomonas strains requiring NaCl for growth, for instance, 1 g NaCl L -1 for strains DR1 and IR1 (Lees et al., 1991), others tend to be salt-sensitive, like those from freshwater or terrestrial environments (Abin et al., 2024). The strain explored in this study, M. methanica DSM 25384, was obtained from an accredited laboratory (DSMZ, Germany) and was originally isolated through methane enrichment cultures inoculated with seawater, suggesting a certain degree of halotolerance. A previous study employing the same strain, tested its salt tolerance up to 20 g NaCl L -1 (Bussmann et al., 2021), reporting the highest growth under control conditions without salt addition (0% NaCl). Nevertheless, in our work, the impact of salt addition on growth performance was limited. The study did not report any protein quantification, hence no direct comparison of protein content could be made. The observed increase in the crude protein content at higher salt concentrations during growth indicates a change in the relative protein content of the biomass, however, the underlying mechanisms cannot be resolved from the present data. Osmotic adaptation may involve changes in cellular composition and biomass allocation, and the observed differences could therefore reflect multiple mechanisms. Further targeted analyses would be required to determine the contribution of changes in the protein content and other stress-induced changes in biomass composition.

The use of biogas resulted in higher protein yields compared to purified methane, which is consistent with previous studies where biogas also enhanced protein yields in mixed methanotrophic cultures (Khoshnevisan et al., 2019; Zha et al., 2021). While this increase in protein yield was observed for biomass grown in both synthetic and real biogas, a corresponding increase in biomass yield was not consistently observed, which suggests differences in biomass composition or carbon allocation. Further understanding of the underlying carbon and electron partitioning would require dedicated mass balance and compositional analyses. The different gas composition of biogas compared to purified methane may contribute to this response by altering the methane availability and dissolved CO 2 concentrations. The lower CH 4 fraction in biogas may therefore affect the biomass composition, although the mechanisms underlying the increased protein yield were not investigated in the present study. However, as CH 4 remained available in excess throughout the cultivation, as indicated by its presence in the headspace at the end of the experiment, the increased protein yield cannot be directly attributed to methane limitation. Similarly, although the presence of CO 2 in biogas may influence the cultivation environment through dissolved CO 2 and associated changes in medium composition and conductivity, the stable pH observed throughout the cultivation suggests that CO 2 -induced acidification was not a major factor. Therefore, the enhanced protein yield observed with biogas may result from responses to the altered gas composition, but the underlying mechanisms require further investigation.

Cultivation conditions shape microbial protein production: Trade-off between biomass productivity and protein content

This study demonstrated a clear trade-off between biomass productivity and protein content in M. methanica under varying cultivation conditions. When the pH was controlled during growth, the maximum specific growth rate of M. methanica was 1.26 times higher (μ max 0.82 ± 0.0.3 h -1), and the biomass concentration 3.8 times (1.98 ± 0.05 g CDW L -1), compared to cultivation without pH control where the pH was lower. The decrease in pH below the optimal pH range of growth for M. methanica, i.e., pH 5.50–8.50 (Semrau et al., 2010), explains the reduced growth. In contrast, the protein content was 1.6 times lower (29% CDW) in M. methanica when pH was controlled, than without pH control (45% CDW). A similar effect was observed when increasing salinity, resulting in a higher protein content but lower growth in M. methanica (Section 4.3), further demonstrating the inverse relationship between growth performance and protein content (Sakarika et al., 2023).

These findings reveal strong parallels between acidified and saline cultivation conditions: both resulted in lower growth performance, but higher protein content. Whereas pH control enhanced biomass productivity, it reduced protein content. Conversely, increased salinity raised protein content but limited growth. This highlights the critical role of cultivation conditions for MP production (Sakarika et al., 2020), impacting the trade-off between production rate and product quality (Wortel et al., 2018; Sakarika et al., 2020; 2023; Banerjee and Mukhopadhyay, 2023). However, the overall protein productivity (Supplementary Tables S2, S5, S6) represents a balance between biomass formation and protein content. For example, despite the lower protein content, pH-controlled cultivation resulted in a higher average protein productivity (2.7 mg protein L -1 h -1) compared to cultivation without pH adjustment (1.1 mg protein L -1 h -1), due to the substantially higher biomass concentration. Similarly, increased salinity enhanced protein productivity by increasing the protein content of the biomass, despite lower growth performance. Therefore, while MP production may prioritize maximizing protein content, it may benefit from maximizing overall biomass (and thus protein) productivity. For example, a process that achieves superior biomass productivity, but lower protein content, might ultimately yield more protein over time, compared to a process with lower biomass productivity but higher protein content (Van Peteghem et al., 2025).

Limitations and future outlook

The conversion of CH 4 into MP represents a promising strategy to valorize this energy-rich gas (Pieja et al., 2017). However, both technical and microbial metabolism-related challenges could currently hinder its industrial adoption. This study identified Methylosinus sp. And M. methanica as a promising alternatives to the conventionally used M. capsulatus, with respect to biomass production under the cultivation conditions evaluated. However, these findings were obtained in lab-scale experiments and therefore require validation in controlled bioreactors, where gas-liquid mass transfer, mixing and oxygen availability, among others, differ substantially. Consequently, the performance improvements observed here should not be directly extrapolated to industrial production. Nevertheless, the results demonstrate that exploring these alternative methanotrophs may represent a promising strategy for improving MP production.

Additionally, M. capsulatus benefits from years of industrial application (Ritala et al., 2017) and regulatory approval (European Commission, 2010; United States Food and Drug Administration, 2022). In contrast, alternative methanotrophs would require a complete safety evaluation and regulatory approval before commercialization for food (or feed) applications, which represents an additional barrier beyond process optimization. Therefore, the implementation of alternative strains depends not only on their cultivation performance but also on their technical scalability and regulatory acceptance.

Using biogas as a more sustainable methane source, compared to natural gas, makes MP production more sustainable and supports circular economy through the recovery of carbon and reactive nitrogen (Matassa et al., 2016; Angelidaki et al., 2018). Even though this study demonstrated the potential of biogas for MP production, the mechanisms in M. methanica remain poorly understood, and this requires further research. Moreover, the impact of biogas impurities such as H 2 S, NH 3, VOCs (European Biogas Association, 2023) was not fully assessed here. These could either inhibit MOB growth (Cáceres et al., 2013) or improve protein quality by, e.g., increasing the content of sulfur-rich amino acids (Areniello et al., 2024).

Using saline water in MP production could enhance the final protein content, as demonstrated in M. methanica (§ 4.3), while reducing competition with other microorganisms (Imhoff, 1986). This study demonstrated halotolerance in M. methanica up to 35 g NaCl L -1, the equivalent of ocean water, suggesting great promise given the rise in anticipated saltwater intrusion into freshwater reservoirs (Walter et al., 2013), and offering a potentially more sustainable alternative to freshwater use. However, these approaches require further assessments to evaluate their robust use in industrial applications.

Methylosinus sp. And M. methanica were the fastest-growing MOB under the cultivation conditions evaluated in this study, achieving 1.88-fold and 1.63-fold higher maximum specific growth rates, respectively, than the conventionally used M. capsulatus. However, M. capsulatus presented the highest protein content, highlighting that microbial species and strain selection depends on the production targets. Maintaining a stable pH further increased the biomass concentration of M. methanica, while increased salinity boosted the protein content 1.8-fold. Co-culturing M. methanica with K. phaffii improved the protein quality yielding a 1.27-fold higher EAA content, while both synthetic and real biogas increased the protein yield compared to purified methane, with the largest improvement (about 1.5-fold) observed for synthetic biogas. These findings provide new insights into MP production from biogas and identify the use of saline water and co-cultures as well as the use of biogas as promising strategies for future optimization and upscaling of methane-based MP production. Future studies should, among others, target deciphering the dynamics and interactions of the individual co-culture partners, including how these interactions may influence carbon allocation and biomass composition, complement bulk protein measurements with more targeted biomass composition analyses, and define the carbon partitioning to better understand the mechanisms underlying changes in protein production and yield. Finally, strain selection and process optimization should be tailored to the desired balance between biomass productivity, protein content, and protein quality for efficient MP production.