Co-cultivation of methane oxidizing bacteria and photosynthetic bacteria for single cell protein production

Co-cultivation of methane oxidizing bacteria and photosynthetic bacteria for single cell protein production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-cultivation of methane oxidizing bacteria and photosynthetic bacteria for single cell protein production Jianxiong Zhang, Zengwu Song, Jiaying Xin, Lirui Sun, Tianyu Cui, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7286353/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Single cell protein (SCP) is widely used in food and feed due to its high protein content, rich essential amino acids, low fat content, and presence of various trace elements. It can serve as a dietary supplement in animal diets or as a substitute for certain proteins. Methane-oxidizing bacteria (MOB), which utilize methane as their sole carbon and energy source and are not limited by factors such as land or light, are a significant asset for SCP production. Their co-culture system with photosynthetic bacteria (PSB) can further enhance SCP production efficiency. By comparing monoculture and co-culture data, it was confirmed that a synergistic interaction based on intracellular substance exchange exists between two mixed microorganisms: adding intracellular substances from MOB increased the maximum OD 600 of PSB by 19% and cell dry weight by 10%, while adding intracellular substances from PSB increased the OD 600 of MOB by 32% and cell dry weight by 2.06 times. Under nitrogen-limited conditions, the co-culture system achieved 2.26 times higher OD 600 and 2.6 times greater cell dry weight compared to monoculture, demonstrating that the nitrogen-fixing activity of PSB effectively supplemented nitrogen sources. In normal nitrogen-supplied medium, the co-culture increased methane consumption by 13% (0.1252 g vs. 0.1108 g), SCP yield by 8% (0.349 vs. 0.323 g DCW/g CH 4 ), and cell dry weight by 21%. 16S rRNA analysis revealed that MOB dominated the co-culture system (46.25%), with photosynthetic cyanobacteria as a secondary component (10.9%), validating the ecological structure of this synergistic system. The research outcomes provide a novel and efficient co-culture model for optimizing industrial SCP production. Methane-oxidizing bacteria photosynthetic bacteria Methane Single cell protein co-culture system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Methane oxidizing bacteria and photosynthetic bacteria synergistically promote growth Enhancement of single-cell protein production under nitrogen-limited conditions through biological nitrogen fixation by photosynthetic bacteria Co-culture system-driven dual improvement of methane assimilation efficiency and single-cell protein yield 1. Introduction As the second most significant greenhouse gas globally, methane exhibits a global warming potential over 30 times greater than carbon dioxide (Tays et al., 2018 ). Methane-oxidizing bacteria (MOB), serving as pivotal drivers of natural methane cycles, mediate sequential methane→methanol→formaldehyde conversions through methane monooxygenase (MMO) and methanol dehydrogenase (MDH) (Zhang et al., 2008 ) -catalyzed cascade reactions, ultimately converting methane to CO 2 via the ribulose monophosphate (RuMP) or serine pathways, playing crucial roles in greenhouse gas mitigation (Shindell et al., 2012 ; (Strong et al., 2015 ). Recent advancements in synthetic biology and metabolic engineering have transformed these gram-negative bacteria from environmental remediators into versatile biomanufacturing platforms, owing to their unique C1 metabolic network (Khmelenina et al., 2019 ; (Panagiotis et al., 2019 ). Approximately 85–95% of their biomass derives from methane conversion, achieving single-cell protein (SCP) yields exceeding 60% while co-synthesizing high-value products like polyhydroxybutyrate (PHB) and exopolysaccharides (Dong et al., 2017 ; (Teixeira et al., 2018 ). MOB derived SCP has demonstrated potential to partially replace fishmeal protein, alleviating feed protein shortages. Multiple studies have validated the safety and superiority of MOB produced SCP in aquaculture applications, Jintasataporn et al. conducted comparative feeding trials with whiteleg shrimp ( Penaeus vannamei ), demonstrating that the SCP-supplemented group exhibited significantly higher feed conversion ratios than those fed conventional fishmeal-based diets (Jintasataporn et al., 2021 ). In parallel, Romarheim et al. documented the intestinal protective effects of Methylococcus capsulatus -derived SCP in Atlantic salmon (Salmo salar), where dietary supplementation effectively mitigated soybean meal-induced enteritis, confirming SCP's role in maintaining gastrointestinal health (Romarheim et al., 2013 ). However, monoculture systems face challenges including feedback inhibition by metabolic byproducts (e.g., organic acids, methanol) and oxygen transfer limitations. The integration of photosynthetic bacteria (PSB) presents an innovative solution: Cyanobacteria and other oxygenic photosynthetic species assimilate CO 2 through light-driven carbon fixation while releasing O 2 , simultaneously alleviating CO 2 -mediated inhibition of methane oxidation and providing essential electron acceptors for MOB metabolism (Sánchez-Baracaldo and Cardona, 2020 ). Concurrently, PSB generate SCP suitable as nutritional additives for baked goods. For instance, Hafsa et al. demonstrated that incorporating 1–3% spirulina-derived SCP increased bread protein content from 8.18–9.90%, with sensory evaluation confirming superior color attributes at 1% supplementation (Hafsa et al., 2014). Furthermore, PSB integration broadens substrate flexibility, enabling utilization of methane-rich waste streams (e.g., industrial effluents, biogas) to establish circular "waste-to-protein" economies (Liang et al., 2024 ; (Parsy et al., 2024 ). Cyanobacteria are also key participants in the atmospheric nitrogen cycle, and their nitrogen fixation ability is driven by oxygen sensitive nitrogenase (encoded by the nifHDK gene) (Berman-Frank et al., 2003 ). They coordinate the contradiction between oxygen producing photosynthesis and nitrogen fixation through spatiotemporal separation or spatial isolation strategies (Alleman and Peters, 2023 ; (Zeng and Zhang, 2022 ). When nitrogen deficiency occurs, the accumulation of 2-ketoglutarate in cells triggers the activation of the global nitrogen regulatory factor NtcA and the main regulatory factor HetR, which induces heteromorphic cell differentiation through phosphorylation and synergistic regulatory networks (negatively feedback regulated by genes such as ask1930 and alr3234), enabling cyanobacteria to efficiently fix nitrogen under long-term nitrogen deficiency conditions (Harish and Seth, 2020 ; (Roumezi et al., 2020 ; (Videau et al., 2016 ). Existing co-culture studies have revealed symbiotic synergies: Hill et al. conducted co-culture experiments with the alkaliphilic Methylomicrobium alcaliphilum 20Z and the cyanobacterium Synechococcus sp. PCC 7002, demonstrating that the microbial consortium exhibited prolonged growth duration and enhanced biomass accumulation compared to monoculture groups (Hill et al., 2017 ). This interspecies synergy provides novel insights into optimizing single-cell protein (SCP) production through synthetic microbial consortia engineering. Rasouli et al. successfully converted wastewater contaminants into fishmeal-comparable SCP using microalgae-MOB consortia, validating the technology's environmental and economic benefits (Rasouli et al., 2018 ). These findings establish that MOB-PSB co-culture systems, through coupled carbon-oxygen cycling and metabolic interactions, provide an innovative paradigm for green SCP production. This study employed selective media for MOB and PSB isolation/purification, with colony morphology characterized via plate culture techniques. Transmembrane substance exchange experiments analyzed interspecies metabolic interactions, systematically evaluating synergistic effects. Nitrogen metabolism regulation was investigated through comparative cultivation under nitrogen-limited and nitrogen-sufficient conditions to quantify PSB-mediated modulation of methane assimilation efficiency. Carbon source manipulation experiments elucidated PSB-enhanced biomass accumulation mechanisms in MOB. Dynamic relative abundance changes in co-culture systems were resolved using 16S rRNA gene sequencing. The findings provide theoretical and technical foundations for constructing high-efficiency SCP biosynthesis systems through microbial consortia engineering, offering innovative solutions for methane bio-mitigation and advancing the "Dual Carbon" strategic goals. 2. Materials and Methods 2.1 Microbial activation and enrichment The methane-oxidizing mixed microbial consortium, which enrichment culture with methane as a sole source of carbon and energy under light, were cultivated using medium formulations detailed in Table S1. For MOB cultivation, a copper-free mineral salts medium was prepared by combining 10 mL of Solution A and 0.1 mL of Solution B in 250 mL side-arm Erlenmeyer flasks, adjusted to 100 mL final volume. Flasks were sealed with sealing film, with rubber stoppers, tubing, and glass side-arms autoclaved in high-temperature-resistant sterilization pouches. Sterilization was performed at 121°C (0.1 MPa) for 20 minutes. Post-sterilization, components were UV-treated for 30 minutes in a laminar flow hood after cooling to ambient temperature. Inoculation procedures involved ethanol-sterilized handling, with 10% (v/v) inoculum volume. Flasks were charged with methane : oxygen : carbon dioxide : nitrogen (2:1:1:1, v/v) using a vacuum pump and incubated at 30°C with 180 rpm orbital shaking. Gas mixtures were refreshed daily under 24 W LED illumination (12 h light/dark cycle) for 6 days. Cultures were subsequently stored at 4°C. PSB enrichment protocol: Under photoautotrophic conditions with CO 2 supplementation (no methane), MOB growth was suppressed while PSB proliferated. Cultures were charged with pure CO 2 , maintained under continuous 24 W LED illumination, and incubated at 30°C/180 rpm for 15 days. Two sequential cultivation cycles yielded axenic PSB cultures. MOB enrichment protocol: Under strict dark conditions with CH 4 :O 2 (2:1, v/v) and CO 2 exclusion, PSB growth ceased while MOBs thrived. Light-blocking membranes were applied during incubation at 30°C/180 rpm (6-day cycle). Two iterative cultivation rounds under these chemoautotrophic conditions produced monobacterial MOB cultures. Under nitrogen-limited conditions, the medium was prepared without NH 4 Cl or KNO 3 supplementation. When methane, methanol, or formate served as carbon sources, a gas mixture of methane: oxygen: carbon dioxide : nitrogen (2:1:1:1, v/v) was directly injected into the culture flasks. Methanol (0.1% v/v) and formate (0.1% v/v) were replenished daily. 2.2 Extraction of substances inside and outside cells. A 50 mL aliquot of cultured microbial suspension was subjected to centrifugation at 8000 × g for 15 min (4°C) to separate extracellular components (supernatant) from cellular biomass (pellet). The pelleted cells were resuspended in 50 mL deionized water and transferred to an ultrasonic processing vessel. Cellular lysis was performed using an ultrasonic disruptor (300 W output) with cyclic operation parameters: 2-second pulses followed by 4-second intervals, sustained for 10 minutes to achieve complete membrane disintegration, yielding intracellular constituents. 2.3 Analysis method Every 24 hours, 3 mL of bacterial culture was sampled, with distilled water serving as the blank control. The OD 600 was measured using a UV-Vis spectrophotometer to assess microbial growth dynamics at different time points. By using the logistic function to fit the cell growth curve, the lag phase ( λ ) and maximum specific growth rate ( μ max ) of the bacterial cells under this condition can be obtained, and a dynamic mathematical model of the methane oxidizing bacterium Methylosinus trichosporum OB3b can be established. The formula equation is: y represents OD 600 , A 1 represents the initial concentration of the fermentation broth, A 2 represents the maximum concentration of the fermentation broth, X represents the cell growth time, X 0 represents the proportional constant of the maximum growth rate, and P represents the bacterial growth index. After cultivation, cells were harvested via low-temperature centrifugation (4°C) and wash with physiological saline three times and then freeze dry to constant weight using a freeze dryer to determine the dry cell weight (DCW). Methane concentration was quantified using a gas chromatograph (GC7900, Tianmei, China) equipped with a thermal conductivity detector (TCD) (Xie et al., 2023). The carrier gas was hydrogen, with the column oven, injection port, and detector temperatures set at 50°C, 100°C, and 120°C, respectively. Manual injection was performed using a 1 mL disposable syringe with a 1 mL sample volume. Protein concentration was measured using the Coomassie Brilliant Blue assay (Yang et al., 2014). The SCP yield was defined as the mass of SCP produced per unit mass of methane consumed, expressed as g DCW/g CH 4 . Y SCP represents the SCP yield (g DCW/g CH 4 ), △W DCW denotes the dry cell weight, △W CH4 represents the total methane consumption during cultivation, △ 6 W CH4 indicates the methane consumption across six gas-exchange intervals. The density of CH 4 was calculated using the ideal gas law: PV=nRT Based on the ideal gas law, the density of methane was calculated to be 0.6667 g/L under standard conditions (20°C, 1 atm). 2.4 Microbial Analysis This study employed 16S rRNA gene sequencing to characterize methane-oxidizing mixed microbial consortium Nine biological replicates were centrifuged and aliquoted into 5 mL sterile tubes, followed by cryopreservation at -40°C in medical-grade freezers. The preserved samples were subsequently submitted to Magichand Technology Co., Ltd. (Guangdong, China) for high-throughput sequencing analysis. 3. Results and discussion 3.1 Microscopic and macroscopic morphology of MOB and PSB Microscopic examination of the cultured bacteria revealed distinct morphological characteristics: PSB predominantly exhibited an elliptical shape with chlorophyll pigmentation, while MOB appeared as short rods (Fig. 1 A-B). In the MOB- photosynthetic bacteria co-culture system, both elliptical photosynthetic cells and numerous small rod-shaped MOB cells were observed (Fig. 1 C-D). Notably, the cell diameter of PSB measured approximately 2–5 times larger than that of MOB, consistent with established bacterial size ranges (Fig. 1 E-F). Colony morphology on solid media demonstrated systematic differences: PSB formed dark green, confluent colonies, whereas MOB colonies displayed light pink pigmentation (Fig. 1 G-H). Liquid co-culture experiments showed progressive darkening of the culture broth over time, indicating robust growth of both MOB and PSB (Figure S1). 3.2 Mutual growth effects of intracellular and extracellular substances between MOB and PSB 3.2.1 Effects of MOB intracellular and extracellular substances on PSB growth Since there was no carbon source in the culture medium and growth relied solely on photosynthesis by the PSB, a 26-day cultivation was conducted. As shown in Fig. 2 , it was found that the PSB supplemented with intracellular substances of MOBs achieved a maximum OD 600 of 1.18 ± 0.05, a dry cell weight of 0.367 ± 0.036 g/L, and a true protein content of 11.2 ± 0.5%. In contrast, the PSB supplemented with extracellular substances of MOBs showed a maximum OD 600 of 1.09 ± 0.05, a dry cell weight of 0.36 ± 0.026 g/L, and a true protein content of 9.8 ± 0.3%. The PSB without any MOB-derived intracellular or extracellular substances exhibited a maximum OD 600 of 0.99 ± 0.03, a dry cell weight of 0.333 ± 0.032 g/L, and a true protein content of 9.32 ± 0.45%. In summary, both intracellular and extracellular substances of MOBs promoted the growth of PSB to some extent. The intracellular substances of MOBs had the most significant impact on the growth of PSB, while the extracellular substances also exhibited a certain growth-promoting effect. 3.2.2 The influence of intracellular and extracellular substances of PSB on the growth of MOB Photosynthetic cyanobacteria contain vitamins B1, B2 (riboflavin), B12, folic acid, etc., and vitamin B12 is excreted into the medium as the PSB grow (Bonnet et al., 2010 ). Vitamin B1 can act as a cofactor in the enzyme complex of transketolase in the pentose phosphate pathway (Palacios et al., 2014 ). MOB can convert riboflavin into the coenzyme FAD (Okamoto et al., 2014 ; (Pradhan et al., 2019 ), which serves as a coenzyme for the reductase component of sMMO and participates in the metabolism of MOB. The addition of trace amounts of vitamin B12 during cultivation can enhance sMMO activity and promote the growth of MOB. Hiroyuki et al. also confirmed that vitamin B12 can promote the growth of MOB and increase MMO activity (Iguchi et al., 2011 ). Folic acid is a precursor of tetrahydrofolate, an essential growth factor for bacteria, and is closely related to the methylation of deoxyribonucleotides into thymidine, which enhances the metabolic vitality of the bacterial cells. From the growth curve (Fig. 3 A and Table 1 ), it can be observed that when intracellular substances of PSB were added to the MOB culture medium, with 5 mL of water added as the control group, the MOB with added intracellular substances of PSB reached a maximum OD 600 of 1.175 ± 0.02, a lag phase of 15.3713 h, and a maximum specific growth rate of 0.0178 h − 1 . MOBs with added extracellular substances of PSB reached a maximum OD 600 of 0.93 ± 0.03, a lag phase of 21.2173 h, and a maximum specific growth rate of 0.0158 h − 1 . The control group without any added intracellular or extracellular substances had a maximum OD 600 of only 0.885 ± 0.03, a lag phase of 22.6685 h, and a maximum specific growth rate of 0.0151 h − 1 . The dry cell weight of MOBs with added intracellular substances of PSB was 0.485 ± 0.024 g/L, with a true protein content of 19.85 ± 0.36%, while the dry cell weight of MOB with added extracellular substances was 0.27 ± 0.01 g/L, with a true protein content of 10.3 ± 0.57%. The control group without any added intracellular or extracellular substances had a dry cell weight of 0.236 ± 0.012 g/L and a true protein content of 9.97 ± 0.46%. The results indicate that both intracellular and extracellular substances of PSB have a certain promoting effect on the growth of MOB. However, the MOB with added intracellular substances of PSB exhibited significantly higher maximum OD 600 and dry cell weight compared to the control group, along with the shortest lag phase. This may be due to the presence of growth factors such as vitamins B1, B2 (riboflavin), B12, and folic acid in the intracellular substances of PSB, which can significantly enhance MMO activity, stimulate metabolic activity, and promote the growth of MOBs. This suggests that the intracellular substances of PSB have a more pronounced promoting effect on MOB. The MOB with added extracellular substances of PSB showed higher maximum OD 600 , dry cell weight, SCP yield, and maximum specific growth rate compared to the control group, as well as a shorter lag phase. This may be because the extracellular substances added were the supernatant obtained after centrifuging PSB, which contains growth factors such as vitamin B12 produced during the metabolic process of PSB. These factors promote the growth of MOB and increase SCP yield, indicating that the extracellular substances of PSB also enhance the growth of MOB. Table 1 The lag phase and maximum growth rate obtained by fitting the growth curve. OD max λ (h) µ max (h − 1 ) Intracellular substances 1.175 ± 0.02 15.3713 0.0178 Extracellular substances 0.93 ± 0.03 21.2173 0.0158 Water 0.885 ± 0.03 22.6685 0.0151 3.3 The impact of PSB on MOB In nitrogen-limited medium (Fig. 4 A and Table 2 ), the maximum OD 600 of MOB was only 0.227 ± 0.023, with a lag phase of 30.2869 h, a maximum specific growth rate of 0.0064 h − 1 , and a cell dry weight of 0.05 ± 0.013 g/L. In contrast, the co-culture of MOB and PSB achieved a maximum OD 600 of 0.511 ± 0.052, a lag phase of 25.6448 h, a maximum specific growth rate of 0.0178 h − 1 , and a cell dry weight of 0.13 ± 0.018 g/L. Under nitrogen-limited conditions, the pure culture of MOB exhibited severely restricted growth, with all parameters at markedly low levels. This unequivocally reflects the potent suppression of MOB metabolic activity by nitrogen source deficiency, resulting in significantly impeded cellular proliferation and severely constrained biomass accumulation. In stark contrast, the MOB-PSB co-culture system demonstrated substantial growth advantages: 1) Elevated biomass as evidenced by maximum OD 600 and cell dry weight values substantially exceeding those of pure MOB culture, indicating significantly enhanced system-wide biomass yield; 2) Enhanced metabolic activity, confirming that the presence of PSB markedly accelerated the growth rate of MOB; 3) Optimized environmental adaptability, with the shortened lag phase demonstrating the co-culture system's accelerated acclimation to nitrogen limitation stress. The symbiotic relationship between MOB and PSB effectively alleviated nitrogen limitation stress. Through synergistic resource exchange and metabolic cross-feeding, it achieved concomitant enhancement of growth kinetic parameters and biomass production. This synergy establishes a theoretical framework for applying microbial co-culture technologies in low-nitrogen environments. The study also explored the impact of PSB on methane assimilation by MOB. The maximum OD 600 for MOB growth was 1.027 ± 0.04 (Fig. 4 B), with a lag phase of 20.0871 h, a maximum specific growth rate of 0.0180 h − 1 (Table 2 ), and a cell dry weight of 0.358 ± 0.015g/L (Fig. 4 D). In contrast, the co-culture had a lag phase of 18.5359 h, a maximum specific growth rate of 0.0213 h − 1 , and a cell dry weight of 0.435 ± 0.032 g/L. The total methane consumption for MOB alone was 0.1108 g, while the co-culture consumed 0.1252 g (Fig. 4 C). The SCP yield for MOB was 0.323 g DCW/g CH 4 , whereas the co-culture achieved 0.349 g DCW/g CH 4 . The methane consumption and SCP yield of the co-culture were significantly higher than those of MOB alone. This may be due to the presence of PSB, which produce growth factors such as vitamins B1, B2, and B12, enhancing the activity of methane monooxygenase and thereby promoting methane assimilation. Additionally, Methylosinus trichosporium OB3b can oxidize ammonia to hydroxylamine, which is then converted to nitrite. Nitrite inhibits the activity of formate dehydrogenase (FDH) in Methylosinus trichosporium OB3b, suppressing methane uptake. Photosynthetic cyanobacteria possess nitrite reductase, which converts nitrite back to ammonia, reducing its inhibitory effect on MOB and promoting methane absorption. Besides nitrite, high concentrations of carbon dioxide produced by MOB can also inhibit their growth. PSB absorb carbon dioxide under light conditions, producing oxygen required by MOB, thereby enhancing methane assimilation. This demonstrates that the presence of PSB can promote the growth of MOB. Table 2 The lag phase and maximum growth rate obtained by fitting the growth curve. OD max λ (h) µ max (h − 1 ) under restricted nitrogen source conditions MOB 0.227 ± 0.023 30.2869 0.0064 Co-culture 0.511 ± 0.052 25.6448 0.0178 Under normal medium conditions MOB 1.027 ± 0.04 20.0871 0.0180 Co-culture 1.03 ± 0.05 18.5359 0.0213 Furthermore, the study investigated the effects of methane, methanol, and formate as carbon sources on the co-culture system. The growth curves revealed that when methane was used as the carbon source (Fig. 5 and Table 3 ), the co-culture achieved a maximum OD 600 of 1.09 ± 0.025, with a lag phase of 18.7128 h and a maximum specific growth rate of 0.0214 h − 1 . When methanol was the carbon source, the co-culture reached a maximum OD 600 of 0.72 ± 0.013 on the fifth day, with a lag phase of 22.6036 h and a maximum specific growth rate of 0.0464 h − 1 . For MOB alone with methanol as the carbon source, the maximum OD 600 was 0.62 ± 0.037 on the fourth day, with a lag phase of 23.4109 h and a maximum specific growth rate of 0.0666 h − 1 . This suggests that using methanol, an intermediate metabolite of methane, as the carbon source can increase the maximum specific growth rate. However, the decline in OD 600 by the fourth or fifth day indicates that methanol accelerates bacterial growth but also prematurely induces the decline phase. The initial OD 600 values of the co-culture and MOB alone were similar, but the co-culture started with a higher OD 600 , suggesting that methanol addition caused cell lysis in PSB, releasing intracellular materials into the medium and delaying the decline phase compared to MOB alone. When formate was used as the carbon source, a continuous decline in OD 600 was observed from the beginning to the sixth day for both strains, likely due to excessive formate altering the medium's pH, making it unsuitable for cell survival and leading to the death of MOB and PSB. Table 3 The lag phase and maximum growth rate obtained by fitting the growth curve. carbon source OD max λ (h) µ max (h − 1 ) Co-culture methane 1.09 ± 0.025 18.7128 0.0214 methanol 0.72 ± 0.013 22.6036 0.0464 MOB methane 0.62 ± 0.037 23.4109 0.0666 3.4 Microbial composition analysis The full sequence of 16S rRNA of methane-oxidizing mixed microbial consortium was amplified, and after amplification, cloning and sequencing were performed. Homology analysis was conducted by comparing the sequences using the BLAST tool on the National Center for Biotechnology Information (NCBI) website. The comparative analysis results showed that the PSB present in methane-oxidizing mixed microbial consortium were cyanobacteria . In the culture with only MOB (Fig. 6 ), the relative abundance of Methylosinus was 32.04%, that of Methylophilus was 42.29%, and that of cyanobacteria was 4.7%. In the culture with only PSB, the relative abundance of Methylosinus was 7.9%, that of cyanobacteria was 48.58%, and that of Methylophilus was 0.96%. In the co-culture of MOB and PSB, the relative abundance of Methylosinus was 46.25%, that of cyanobacteria was 10.9%, and that of Methylophilus was 19.3%. Methylophilus utilizes intermediate metabolites from MOB growth, such as methanol, formate, and formaldehyde, for its own growth. Comparing the cultures of MOB alone and the co-culture, it was found that the relative abundance of Methylophilus was higher in the MOB-only culture than in the co-culture. This suggests that the presence of PSB promotes the uptake of intermediate metabolites like methanol and formate by MOB, thereby limiting the growth of Methylophilus . In the PSB-only culture, the relative abundance of Methylophilus was very low because no methane was supplied during cultivation, preventing MOB from growing and thus eliminating the production of intermediate metabolites like methanol and formate. In the MOB-PSB co-culture system, the relative abundance of PSB was relatively low, but throughout the cultivation process, they played a supportive role in the growth of MOB. 4. Conclusion This chapter reveals the synergistic mechanisms and application potential of a co-culture system involving MOB and PSB. Selective cultivation experiments demonstrated that the two types of strains significantly promote each other's growth through the secretion of metabolites, with intracellular substances playing a particularly prominent role. Under nitrogen-limited conditions, the OD and dry cell weight of the co-culture system increased by 2.26 times and 2.6 times, respectively, compared to monocultures, confirming that PSB can supplement nitrogen sources for MOB and enhance SCP production. Experiments with nitrogen-containing media showed that PSB eliminate growth-inhibiting factors produced by MOB, thereby increasing methane assimilation. Carbon source screening indicated that methane is the most suitable for the co-culture system, while methanol triggers the lysis of PSB, and excessive formic acid leads to cell death. 16S rRNA sequencing confirmed that MOB dominate the co-culture system, while PSB primarily play a supporting role: not only by providing growth factors such as vitamins B1, B2, and B12 but also by clearing metabolic inhibitors through material cycling. This technology breaks through the traditional single-strain cultivation model, establishing a "carbon-negative biomanufacturing" system based on natural material cycling—utilizing the greenhouse gas methane to produce high-value protein while achieving carbon sequestration. The study suggests that the integration of synthetic ecology and process engineering will advance this technology as a core solution for sustainable protein production, offering innovative approaches to addressing global food security and climate change. Declarations Acknowledgments This research was financially supported by central government support for local university reform and development fund - talent cultivation support program project (high-level talents) (304017). Thanks to the research group of Professor Xing Xinhui from Tsinghua University for providing the strains. References Alleman, A. B. and Peters, J. W. (2023) Mechanisms for Generating Low Potential Electrons across the Metabolic Diversity of Nitrogen-Fixing Bacteria. Appl Environ Microb, 89(5). Berman-Frank, I., Lundgren, P. and Falkowski, P. (2003) Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res Microbiol, 154(3): 157-164. Bonnet, S., Webb, E. A., Panzeca, C., Karl, D. M., Capone, D. G. and Sañudo-Wilhelmy, S. A. (2010) Vitamin B12 excretion by cultures of the marine cyanobacteria Crocosphaera and Synechococcus. Limnol Oceanogr, 55(5): 1959-1964. Dong, T., Fei, Q., Genelot, M., Smith, H., Laurens, L. M. L., Watson, M. J. and Pienkos, P. T. (2017) A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense. Energ Convers Manage, 140: 62-70. Hafsa, Yaiche Achour, Amel, Doumandji, Samia, Sadi and Sidahmed, Saadi (2014) Evaluation of nutritional and sensory properties of bread enriched with spirulina. Harish and Seth, K. (2020) Molecular circuit of heterocyst differentiation in cyanobacteria. J Basic Microb, 60(9): 738-745. Hill, E. A., Chrisler, W. B., Beliaev, A. S. and Bernstein, H. C. (2017) A flexible microbial co-culture platform for simultaneous utilization of methane and carbon dioxide from gas feedstocks. Bioresource Technol, 228: 250-256. Iguchi, H., Yurimoto, H. and Sakai, Y. (2011) Stimulation of Methanotrophic Growth in Cocultures by Cobalamin Excreted by Rhizobia . Appl Environ Microb, 77(24): 8509-8515. Jintasataporn, O., Chumkam, S., Triwutanon, S., LeBlanc, A. and Sawanboonchun, J. (2021) Effects of a Single Cell Protein ( Methylococcus capsulatus , Bath) in Pacific White Shrimp ( Penaeus vannamei ) Diet on Growth Performance, Survival Rate and Resistance to Vibrio parahaemolyticus , the Causative Agent of Acute Hepatopancreatic Necrosis Disease. Frontiers in Marine Science, 8: 764042. Khmelenina, V. N., But, S. Y., Rozova, O. N. and Trotsenko, Y. A. (2019) Metabolic Features of Aerobic Methanotrophs: News and Views. Curr Issues Mol Biol, 33: 85-99. Liang, J. S., Zhang, P. Y., Zhang, R., Chang, J. N., Chen, L., Zhang, G. M. and Wang, A. J. (2024) Bioconversion of volatile fatty acids from organic wastes to produce high-value products by photosynthetic bacteria: A review. Environ Res, 242: 117796. Okamoto, A., Saito, K., Inoue, K., Nealson, K. H., Hashimoto, K. and Nakamura, R. (2014) Uptake of self-secreted flavins as bound cofactors for extracellular electron transfer in Geobacter species. Energ Environ Sci, 7(4): 1357-1361. Palacios, O. A., Bashan, Y. and de-Bashan, L. E. (2014) Proven and potential involvement of vitamins in interactions of plants with plant growth-promoting bacteria-an overview. Biol Fert Soils, 50(3): 415-432. Panagiotis, T., Benyamin, K., Zhu, X. Y., Zha, X. and Irini, A. (2019) Methane oxidising bacteria to upcycle effluent streams from anaerobic digestion of municipal biowaste. Journal of Environmental Management, 251: 109590. Parsy, A., Monlau, F., Guyoneaud, R. and Sambusiti, C. (2024) Nutrient recovery in effluents from the energy sectors for microalgae and cyanobacteria biomass production: A review. Renewable & Sustainable Energy Reviews, 191: 114207. Pradhan, S. K., Singh, N. R., Dehury, B., Panda, D., Modi, M. K. and Thatoi, H. (2019) Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study. J Cell Biochem, 120(10): 16990-17005. Rasouli, Z., Valverde-Pérez, B., D'Este, M., De Francisci, D. and Angelidaki, I. (2018) Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs. Biochem Eng J, 134: 129-135. Romarheim, O. H., Landsverk, T., Mydland, L. T., Skrede, A. and Overland, M. (2013) Cell wall fractions from prevent soybean meal-induced enteritis in Atlantic salmon (Salmo salar). Aquaculture, 402: 13-18. Roumezi, B., Xu, X. M., Risoul, V., Fan, Y. P., Lebrun, R. and Latifi, A. (2020) The Pkn22 kinase of Nostoc PCC 7120 is required for cell differentiation via the phosphorylation of HetR on a residue highly conserved in genomes of heterocyst-forming cyanobacteria. Front Microbiol, 10: 3140. Sánchez-Baracaldo, P. and Cardona, T. (2020) On the origin of oxygenic photosynthesis and Cyanobacteria. New Phytol, 225(4): 1440-1446. Shindell, D., Kuylenstierna, J. C. I., Vignati, E., van Dingenen, R., Amann, M., Klimont, Z., Anenberg, S. C., Muller, N., Janssens-Maenhout, G., Raes, F., Schwartz, J., Faluvegi, G., Pozzoli, L., Kupiainen, K., Höglund-Isaksson, L., Emberson, L., Streets, D., Ramanathan, V., Hicks, K., Oanh, N. T. K., Milly, G., Williams, M., Demkine, V. and Fowler, D. (2012) Simultaneously Mitigating Near-Term Climate Change and Improving Human Health and Food Security. Science, 335(6065): 183-189. Strong, P. J., Xie, S. and Clarke, W. P. (2015) Methane as a Resource: Can the Methanotrophs Add Value? Environ Sci Technol, 49(7): 4001-4018. Tays, C., Guarnieri, M. T., Sauvageau, D. and Stein, L. Y. (2018) Combined Effects of Carbon and Nitrogen Source to Optimize Growth of Proteobacterial Methanotrophs. Front Microbiol, 9: 2239. Teixeira, L. V., Moutinho, L. F. and Romao-Dumaresq, A. S. (2018) Gas fermentation of C1 feedstocks: commercialization status and future prospects. Biofuel Bioprod Bior, 12(6): 1103-1117. Videau, P., Rivers, O. S., Hurd, K., Ushijima, B., Oshiro, R. T., Ende, R. J., O'Hanlon, S. M. and Cozy, L. M. (2016) The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in Anabaena sp. strain PCC 7120. P Natl Acad Sci USA, 113(45): E6984-E6992. Xie, J., Sun, X. K., Du, H. G., Chen, D. W. and Wang, Y. (2023) Exploring the Effects of Different Methane and Oxygen Concentrations on the Methane-Oxidizing Bacteria Mixed Community. J Environ Eng, 149(12): 04023081. Yang, Y. G., Xiang, Y. B., Xia, C. Y., Wu, W. M., Sun, G. P. and Xu, M. Y. (2014) Physiological and electrochemical effects of different electron acceptors on bacterial anode respiration in bioelectrochemical systems. Bioresource Technol, 164: 270-275. Zeng, X. L. and Zhang, C. C. (2022) The Making of a Heterocyst in Cyanobacteria Annual Review of Microbiology. Annual Review of Microbiology, 76: 597-618. Zhang, Y. X., Xin, J. Y., Chen, L. L., Song, H. and Xia, C. U. (2008) Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol. J Nat Gas Chem, 17(1): 103-109. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7286353","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512545882,"identity":"f84b0c8d-941c-4f3c-acd0-e4ab3f4758c4","order_by":0,"name":"Jianxiong Zhang","email":"","orcid":"","institution":"Harbin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Jianxiong","middleName":"","lastName":"Zhang","suffix":""},{"id":512545883,"identity":"6cd60417-928e-4e9c-ae4b-39c0e936a0de","order_by":1,"name":"Zengwu Song","email":"","orcid":"","institution":"Harbin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Zengwu","middleName":"","lastName":"Song","suffix":""},{"id":512545884,"identity":"2a3d9c3b-cb9b-434e-9fe1-7597c2170230","order_by":2,"name":"Jiaying Xin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACZgYGgwQGBh4Q+wBDhYScPIlazlgYGzaQZCVjW0UiUCN+YHCc90DBg5rDMub8awwPF86TSGBsYH746AYeLZLNfAkGCccO81jOeGNweOY2iTx2BjZj4xw8WviZeQwMEtgO8xjcOJZwmHebRDFjAw+bND4tbGAt/2Ba5kgkNhwgoAVsS2IbUMv55gOHeRuI0CLZDNLSlw60hfnA4RnHJIwNmwn4xeD8GTPDH9+s7Q3OH2z+XFBTJyfP3vzwMT4tIO8YMDA0MzBIJICilYEBSuIFzA8YGOqAvjpAjOJRMApGwSgYiQAAgTBKnEn8K+EAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4010-7171","institution":"Harbin University of Commerce","correspondingAuthor":true,"prefix":"","firstName":"Jiaying","middleName":"","lastName":"Xin","suffix":""},{"id":512545885,"identity":"33e01cd4-bce1-4d52-b3d8-39242648b975","order_by":3,"name":"Lirui Sun","email":"","orcid":"","institution":"Harbin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Lirui","middleName":"","lastName":"Sun","suffix":""},{"id":512545886,"identity":"de328076-d08c-447f-bb70-8854ef3c7c54","order_by":4,"name":"Tianyu Cui","email":"","orcid":"","institution":"Harbin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Cui","suffix":""},{"id":512545887,"identity":"1696f31a-fe68-4900-b965-d4f82c84d765","order_by":5,"name":"Jinhui Xie","email":"","orcid":"","institution":"Harbin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Jinhui","middleName":"","lastName":"Xie","suffix":""},{"id":512545888,"identity":"87d958d7-f00f-4b56-8747-60165c9e1537","order_by":6,"name":"Haixin Bi","email":"","orcid":"","institution":"Harbin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Haixin","middleName":"","lastName":"Bi","suffix":""},{"id":512545889,"identity":"23aa0d0d-3db2-46e4-84f3-49ffd17b04f0","order_by":7,"name":"Chungu Xia","email":"","orcid":"","institution":"Lanzhou Institute of Chemical Physics","correspondingAuthor":false,"prefix":"","firstName":"Chungu","middleName":"","lastName":"Xia","suffix":""}],"badges":[],"createdAt":"2025-08-04 03:08:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7286353/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7286353/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91419154,"identity":"5238ae71-1e82-4b38-ab81-fc124c9b1ce3","added_by":"auto","created_at":"2025-09-16 09:54:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":463444,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic cell morphology of PSB (A-B), co-culture (C-D), and MOB (E-F); Pictures of plates coated with PSB (G) and plate delineation pictures of species of MOB-growing bacteria (H).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/77025228a89044d4534a50e4.png"},{"id":91419173,"identity":"6088a08e-8505-4e67-b832-5693f62e4292","added_by":"auto","created_at":"2025-09-16 09:54:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150238,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of intracellular and extracellular substances of MOB on the growth of PSB (A), protein content and cell dry weight (B).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/39904c92cc00f3b697c672e8.png"},{"id":91419149,"identity":"c8e7583a-639f-4715-9403-75c001634cd0","added_by":"auto","created_at":"2025-09-16 09:53:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57043,"visible":true,"origin":"","legend":"\u003cp\u003eFitted growth curves of MOB during the addition of intracellular and extracellular substances to PSB, single-cell protein production of MOB during the addition of intracellular and extracellular substances in PSB.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/30f33b4a822c12365d7fa1fa.png"},{"id":91419182,"identity":"d1909b5c-5ae5-4139-8da2-593b5dbfe946","added_by":"auto","created_at":"2025-09-16 09:54:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":111284,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of microorganisms in MOB and co-culture systems under restricted nitrogen source conditions (A) and normal medium conditions (B), methane uptake (C) and single-cell protein production (D) by MOB and co-culture.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/0913620535db551ed0a76b3a.png"},{"id":91419233,"identity":"3079d269-18d7-46d3-9884-598567f233e2","added_by":"auto","created_at":"2025-09-16 09:54:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38050,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of microorganisms in MOB and co culture systems under different carbon source conditions.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/374afa916daf533dce517cc8.png"},{"id":91419187,"identity":"433c5c58-c65c-45f4-a144-3e7ce045e3c3","added_by":"auto","created_at":"2025-09-16 09:54:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41058,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance plotted from 16S rRNA data.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/b7048a4780210f59d2d4c048.png"},{"id":91449427,"identity":"3995502f-c067-4f65-a024-59455edc62d7","added_by":"auto","created_at":"2025-09-16 15:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1504443,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7286353/v1/8be0f95f-9612-49de-b3da-4d94923cde12.pdf"}],"financialInterests":"","formattedTitle":"Co-cultivation of methane oxidizing bacteria and photosynthetic bacteria for single cell protein production","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eMethane oxidizing bacteria and\u0026nbsp;photosynthetic bacteria\u0026nbsp;synergistically promote growth\u003c/li\u003e\n \u003cli\u003eEnhancement of single-cell protein production under nitrogen-limited conditions through biological nitrogen fixation by photosynthetic bacteria Co-culture system-driven dual improvement of methane assimilation efficiency and single-cell protein yield\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eAs the second most significant greenhouse gas globally, methane exhibits a global warming potential over 30 times greater than carbon dioxide (Tays et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Methane-oxidizing bacteria (MOB), serving as pivotal drivers of natural methane cycles, mediate sequential methane\u0026rarr;methanol\u0026rarr;formaldehyde conversions through methane monooxygenase (MMO) and methanol dehydrogenase (MDH) (Zhang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) -catalyzed cascade reactions, ultimately converting methane to CO\u003csub\u003e2\u003c/sub\u003e via the ribulose monophosphate (RuMP) or serine pathways, playing crucial roles in greenhouse gas mitigation (Shindell et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; (Strong et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Recent advancements in synthetic biology and metabolic engineering have transformed these gram-negative bacteria from environmental remediators into versatile biomanufacturing platforms, owing to their unique C1 metabolic network (Khmelenina et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; (Panagiotis et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Approximately 85\u0026ndash;95% of their biomass derives from methane conversion, achieving single-cell protein (SCP) yields exceeding 60% while co-synthesizing high-value products like polyhydroxybutyrate (PHB) and exopolysaccharides (Dong et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; (Teixeira et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). MOB derived SCP has demonstrated potential to partially replace fishmeal protein, alleviating feed protein shortages. Multiple studies have validated the safety and superiority of MOB produced SCP in aquaculture applications, Jintasataporn et al. conducted comparative feeding trials with whiteleg shrimp (\u003cem\u003ePenaeus vannamei\u003c/em\u003e), demonstrating that the SCP-supplemented group exhibited significantly higher feed conversion ratios than those fed conventional fishmeal-based diets (Jintasataporn et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In parallel, Romarheim et al. documented the intestinal protective effects of \u003cem\u003eMethylococcus capsulatus\u003c/em\u003e-derived SCP in Atlantic salmon (Salmo salar), where dietary supplementation effectively mitigated soybean meal-induced enteritis, confirming SCP's role in maintaining gastrointestinal health (Romarheim et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, monoculture systems face challenges including feedback inhibition by metabolic byproducts (e.g., organic acids, methanol) and oxygen transfer limitations.\u003c/p\u003e\u003cp\u003eThe integration of photosynthetic bacteria (PSB) presents an innovative solution: Cyanobacteria and other oxygenic photosynthetic species assimilate CO\u003csub\u003e2\u003c/sub\u003e through light-driven carbon fixation while releasing O\u003csub\u003e2\u003c/sub\u003e, simultaneously alleviating CO\u003csub\u003e2\u003c/sub\u003e-mediated inhibition of methane oxidation and providing essential electron acceptors for MOB metabolism (S\u0026aacute;nchez-Baracaldo and Cardona, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Concurrently, PSB generate SCP suitable as nutritional additives for baked goods. For instance, Hafsa et al. demonstrated that incorporating 1\u0026ndash;3% spirulina-derived SCP increased bread protein content from 8.18\u0026ndash;9.90%, with sensory evaluation confirming superior color attributes at 1% supplementation (Hafsa et al., 2014). Furthermore, PSB integration broadens substrate flexibility, enabling utilization of methane-rich waste streams (e.g., industrial effluents, biogas) to establish circular \"waste-to-protein\" economies (Liang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; (Parsy et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cyanobacteria are also key participants in the atmospheric nitrogen cycle, and their nitrogen fixation ability is driven by oxygen sensitive nitrogenase (encoded by the nifHDK gene) (Berman-Frank et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). They coordinate the contradiction between oxygen producing photosynthesis and nitrogen fixation through spatiotemporal separation or spatial isolation strategies (Alleman and Peters, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; (Zeng and Zhang, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When nitrogen deficiency occurs, the accumulation of 2-ketoglutarate in cells triggers the activation of the global nitrogen regulatory factor NtcA and the main regulatory factor HetR, which induces heteromorphic cell differentiation through phosphorylation and synergistic regulatory networks (negatively feedback regulated by genes such as ask1930 and alr3234), enabling cyanobacteria to efficiently fix nitrogen under long-term nitrogen deficiency conditions (Harish and Seth, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; (Roumezi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; (Videau et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExisting co-culture studies have revealed symbiotic synergies: Hill et al. conducted co-culture experiments with the alkaliphilic \u003cem\u003eMethylomicrobium alcaliphilum\u003c/em\u003e 20Z and the cyanobacterium \u003cem\u003eSynechococcus\u003c/em\u003e sp. PCC 7002, demonstrating that the microbial consortium exhibited prolonged growth duration and enhanced biomass accumulation compared to monoculture groups (Hill et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This interspecies synergy provides novel insights into optimizing single-cell protein (SCP) production through synthetic microbial consortia engineering. Rasouli et al. successfully converted wastewater contaminants into fishmeal-comparable SCP using microalgae-MOB consortia, validating the technology's environmental and economic benefits (Rasouli et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These findings establish that MOB-PSB co-culture systems, through coupled carbon-oxygen cycling and metabolic interactions, provide an innovative paradigm for green SCP production.\u003c/p\u003e\u003cp\u003eThis study employed selective media for MOB and PSB isolation/purification, with colony morphology characterized via plate culture techniques. Transmembrane substance exchange experiments analyzed interspecies metabolic interactions, systematically evaluating synergistic effects. Nitrogen metabolism regulation was investigated through comparative cultivation under nitrogen-limited and nitrogen-sufficient conditions to quantify PSB-mediated modulation of methane assimilation efficiency. Carbon source manipulation experiments elucidated PSB-enhanced biomass accumulation mechanisms in MOB. Dynamic relative abundance changes in co-culture systems were resolved using 16S rRNA gene sequencing. The findings provide theoretical and technical foundations for constructing high-efficiency SCP biosynthesis systems through microbial consortia engineering, offering innovative solutions for methane bio-mitigation and advancing the \"Dual Carbon\" strategic goals.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1 Microbial activation and enrichment\u003c/p\u003e\n\u003cp\u003eThe methane-oxidizing mixed microbial consortium, which enrichment culture with methane as a sole source of carbon and energy under light, were cultivated using medium formulations detailed in Table S1. For MOB cultivation, a copper-free mineral salts medium was prepared by combining 10 mL of Solution A and 0.1 mL of Solution B in 250 mL side-arm Erlenmeyer flasks, adjusted to 100 mL final volume. Flasks were sealed with sealing film, with rubber stoppers, tubing, and glass side-arms autoclaved in high-temperature-resistant sterilization pouches. Sterilization was performed at 121\u0026deg;C (0.1 MPa) for 20 minutes. Post-sterilization, components were UV-treated for 30 minutes in a laminar flow hood after cooling to ambient temperature. Inoculation procedures involved ethanol-sterilized handling, with 10% (v/v) inoculum volume. Flasks were charged with methane : oxygen : carbon dioxide : nitrogen (2:1:1:1, v/v) using a vacuum pump and incubated at 30\u0026deg;C with 180 rpm orbital shaking. Gas mixtures were refreshed daily under 24 W LED illumination (12 h light/dark cycle) for 6 days. Cultures were subsequently stored at 4\u0026deg;C.\u003c/p\u003e\n\u003cp\u003ePSB enrichment protocol: Under photoautotrophic conditions with CO\u003csub\u003e2\u003c/sub\u003e supplementation (no methane), MOB growth was suppressed while PSB proliferated. Cultures were charged with pure CO\u003csub\u003e2\u003c/sub\u003e, maintained under continuous 24 W LED illumination, and incubated at 30\u0026deg;C/180 rpm for 15 days. Two sequential cultivation cycles yielded axenic PSB cultures.\u003c/p\u003e\n\u003cp\u003eMOB enrichment protocol: Under strict dark conditions with CH\u003csub\u003e4\u003c/sub\u003e:O\u003csub\u003e2\u003c/sub\u003e (2:1, v/v) and CO\u003csub\u003e2\u003c/sub\u003e exclusion, PSB growth ceased while MOBs thrived. Light-blocking membranes were applied during incubation at 30\u0026deg;C/180 rpm (6-day cycle). Two iterative cultivation rounds under these chemoautotrophic conditions produced monobacterial MOB cultures.\u003c/p\u003e\n\u003cp\u003eUnder nitrogen-limited conditions, the medium was prepared without NH\u003csub\u003e4\u003c/sub\u003eCl or KNO\u003csub\u003e3\u003c/sub\u003e supplementation. When methane, methanol, or formate served as carbon sources, a gas mixture of methane: oxygen: carbon dioxide : nitrogen (2:1:1:1, v/v) was directly injected into the culture flasks. Methanol (0.1% v/v) and formate (0.1% v/v) were replenished daily.\u003c/p\u003e\n\u003cp\u003e2.2 Extraction of substances inside and outside cells.\u003c/p\u003e\n\u003cp\u003eA 50 mL aliquot of cultured microbial suspension was subjected to centrifugation at 8000 \u0026times; g for 15 min (4\u0026deg;C) to separate extracellular components (supernatant) from cellular biomass (pellet). The pelleted cells were resuspended in 50 mL deionized water and transferred to an ultrasonic processing vessel. Cellular lysis was performed using an ultrasonic disruptor (300 W output) with cyclic operation parameters: 2-second pulses followed by 4-second intervals, sustained for 10 minutes to achieve complete membrane disintegration, yielding intracellular constituents.\u003c/p\u003e\n\u003cp\u003e2.3 Analysis method\u003c/p\u003e\n\u003cp\u003eEvery 24 hours, 3 mL of bacterial culture was sampled, with distilled water serving as the blank control. The OD\u003csub\u003e600\u003c/sub\u003e was measured using a UV-Vis spectrophotometer to assess microbial growth dynamics at different time points. By using the logistic function to fit the cell growth curve, the lag phase (\u003cem\u003e\u0026lambda;\u003c/em\u003e) and maximum specific growth rate (\u003cem\u003e\u0026mu;\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) of the bacterial cells under this condition can be obtained, and a dynamic mathematical model of the methane oxidizing bacterium Methylosinus trichosporum OB3b can be established. The formula equation is:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"122\" height=\"40\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003ey represents OD\u003csub\u003e600\u003c/sub\u003e, A\u003csub\u003e1\u003c/sub\u003e represents the initial concentration of the fermentation broth, A\u003csub\u003e2\u003c/sub\u003e represents the maximum concentration of the fermentation broth, X represents the cell growth time, X\u003csub\u003e0\u003c/sub\u003e represents the proportional constant of the maximum growth rate, and P represents the bacterial growth index.\u003c/p\u003e\n\u003cp\u003eAfter cultivation, cells were harvested via low-temperature centrifugation (4\u0026deg;C) and wash with physiological saline three times and then freeze dry to constant weight using a freeze dryer to determine the dry cell weight (DCW). Methane concentration was quantified using a gas chromatograph (GC7900, Tianmei, China) equipped with a thermal conductivity detector (TCD) \u0026nbsp;(Xie et al., 2023). The carrier gas was hydrogen, with the column oven, injection port, and detector temperatures set at 50\u0026deg;C, 100\u0026deg;C, and 120\u0026deg;C, respectively. Manual injection was performed using a 1 mL disposable syringe with a 1 mL sample volume. Protein concentration was measured using the Coomassie Brilliant Blue assay \u0026nbsp;(Yang et al., 2014). The SCP yield was defined as the mass of SCP produced per unit mass of methane consumed, expressed as g DCW/g CH\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"97\" height=\"49\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"125\" height=\"26\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003eSCP\u0026nbsp;\u003c/sub\u003erepresents the SCP yield (g DCW/g CH\u003csub\u003e4\u003c/sub\u003e),\u0026nbsp;△W\u003csub\u003eDCW\u0026nbsp;\u003c/sub\u003edenotes the dry cell weight,\u0026nbsp;△W\u003csub\u003eCH4\u0026nbsp;\u003c/sub\u003erepresents the total methane consumption during cultivation,\u0026nbsp;△\u003csub\u003e6\u003c/sub\u003eW\u003csub\u003eCH4\u003c/sub\u003e indicates the methane consumption across six gas-exchange intervals.\u003c/p\u003e\n\u003cp\u003eThe density of CH\u003csub\u003e4\u003c/sub\u003e was calculated using the ideal gas law:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePV=nRT\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on the ideal gas law, the density of methane was calculated to be 0.6667 g/L under standard conditions (20\u0026deg;C, 1 atm).\u003c/p\u003e\n\u003cp\u003e2.4 Microbial Analysis\u003c/p\u003e\n\u003cp\u003eThis study employed 16S rRNA gene sequencing to characterize methane-oxidizing mixed microbial consortium Nine biological replicates were centrifuged and aliquoted into 5 mL sterile tubes, followed by cryopreservation at -40\u0026deg;C in medical-grade freezers. The preserved samples were subsequently submitted to Magichand Technology Co., Ltd. (Guangdong, China) for high-throughput sequencing analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Microscopic and macroscopic morphology of MOB and PSB\u003c/h2\u003e\u003cp\u003eMicroscopic examination of the cultured bacteria revealed distinct morphological characteristics: PSB predominantly exhibited an elliptical shape with chlorophyll pigmentation, while MOB appeared as short rods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). In the MOB- photosynthetic bacteria co-culture system, both elliptical photosynthetic cells and numerous small rod-shaped MOB cells were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Notably, the cell diameter of PSB measured approximately 2\u0026ndash;5 times larger than that of MOB, consistent with established bacterial size ranges (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). Colony morphology on solid media demonstrated systematic differences: PSB formed dark green, confluent colonies, whereas MOB colonies displayed light pink pigmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). Liquid co-culture experiments showed progressive darkening of the culture broth over time, indicating robust growth of both MOB and PSB (Figure S1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Mutual growth effects of intracellular and extracellular substances between MOB and PSB\u003c/h2\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Effects of MOB intracellular and extracellular substances on PSB growth\u003c/h2\u003e\u003cp\u003eSince there was no carbon source in the culture medium and growth relied solely on photosynthesis by the PSB, a 26-day cultivation was conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, it was found that the PSB supplemented with intracellular substances of MOBs achieved a maximum OD\u003csub\u003e600\u003c/sub\u003e of 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, a dry cell weight of 0.367\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036 g/L, and a true protein content of 11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%. In contrast, the PSB supplemented with extracellular substances of MOBs showed a maximum OD\u003csub\u003e600\u003c/sub\u003e of 1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, a dry cell weight of 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026 g/L, and a true protein content of 9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%. The PSB without any MOB-derived intracellular or extracellular substances exhibited a maximum OD\u003csub\u003e600\u003c/sub\u003e of 0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, a dry cell weight of 0.333\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032 g/L, and a true protein content of 9.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45%. In summary, both intracellular and extracellular substances of MOBs promoted the growth of PSB to some extent. The intracellular substances of MOBs had the most significant impact on the growth of PSB, while the extracellular substances also exhibited a certain growth-promoting effect.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 The influence of intracellular and extracellular substances of PSB on the growth of MOB\u003c/h2\u003e\u003cp\u003ePhotosynthetic cyanobacteria contain vitamins B1, B2 (riboflavin), B12, folic acid, etc., and vitamin B12 is excreted into the medium as the PSB grow (Bonnet et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Vitamin B1 can act as a cofactor in the enzyme complex of transketolase in the pentose phosphate pathway (Palacios et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). MOB can convert riboflavin into the coenzyme FAD (Okamoto et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; (Pradhan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which serves as a coenzyme for the reductase component of sMMO and participates in the metabolism of MOB. The addition of trace amounts of vitamin B12 during cultivation can enhance sMMO activity and promote the growth of MOB. Hiroyuki et al. also confirmed that vitamin B12 can promote the growth of MOB and increase MMO activity (Iguchi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Folic acid is a precursor of tetrahydrofolate, an essential growth factor for bacteria, and is closely related to the methylation of deoxyribonucleotides into thymidine, which enhances the metabolic vitality of the bacterial cells. From the growth curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), it can be observed that when intracellular substances of PSB were added to the MOB culture medium, with 5 mL of water added as the control group, the MOB with added intracellular substances of PSB reached a maximum OD\u003csub\u003e600\u003c/sub\u003e of 1.175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, a lag phase of 15.3713 h, and a maximum specific growth rate of 0.0178 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. MOBs with added extracellular substances of PSB reached a maximum OD\u003csub\u003e600\u003c/sub\u003e of 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, a lag phase of 21.2173 h, and a maximum specific growth rate of 0.0158 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The control group without any added intracellular or extracellular substances had a maximum OD\u003csub\u003e600\u003c/sub\u003e of only 0.885\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, a lag phase of 22.6685 h, and a maximum specific growth rate of 0.0151 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The dry cell weight of MOBs with added intracellular substances of PSB was 0.485\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024 g/L, with a true protein content of 19.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36%, while the dry cell weight of MOB with added extracellular substances was 0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g/L, with a true protein content of 10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57%. The control group without any added intracellular or extracellular substances had a dry cell weight of 0.236\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 g/L and a true protein content of 9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46%. The results indicate that both intracellular and extracellular substances of PSB have a certain promoting effect on the growth of MOB. However, the MOB with added intracellular substances of PSB exhibited significantly higher maximum OD\u003csub\u003e600\u003c/sub\u003e and dry cell weight compared to the control group, along with the shortest lag phase. This may be due to the presence of growth factors such as vitamins B1, B2 (riboflavin), B12, and folic acid in the intracellular substances of PSB, which can significantly enhance MMO activity, stimulate metabolic activity, and promote the growth of MOBs. This suggests that the intracellular substances of PSB have a more pronounced promoting effect on MOB. The MOB with added extracellular substances of PSB showed higher maximum OD\u003csub\u003e600\u003c/sub\u003e, dry cell weight, SCP yield, and maximum specific growth rate compared to the control group, as well as a shorter lag phase. This may be because the extracellular substances added were the supernatant obtained after centrifuging PSB, which contains growth factors such as vitamin B12 produced during the metabolic process of PSB. These factors promote the growth of MOB and increase SCP yield, indicating that the extracellular substances of PSB also enhance the growth of MOB.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe lag phase and maximum growth rate obtained by fitting the growth curve.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOD\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eλ\u003c/em\u003e (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003e\u0026micro;\u003c/em\u003e \u003csub\u003emax\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntracellular substances\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.3713\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0178\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtracellular substances\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.2173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0158\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWater\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.885\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22.6685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0151\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 The impact of PSB on MOB\u003c/h2\u003e\u003cp\u003eIn nitrogen-limited medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the maximum OD\u003csub\u003e600\u003c/sub\u003e of MOB was only 0.227\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023, with a lag phase of 30.2869 h, a maximum specific growth rate of 0.0064 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and a cell dry weight of 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013 g/L. In contrast, the co-culture of MOB and PSB achieved a maximum OD\u003csub\u003e600\u003c/sub\u003e of 0.511\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052, a lag phase of 25.6448 h, a maximum specific growth rate of 0.0178 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and a cell dry weight of 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 g/L. Under nitrogen-limited conditions, the pure culture of MOB exhibited severely restricted growth, with all parameters at markedly low levels. This unequivocally reflects the potent suppression of MOB metabolic activity by nitrogen source deficiency, resulting in significantly impeded cellular proliferation and severely constrained biomass accumulation. In stark contrast, the MOB-PSB co-culture system demonstrated substantial growth advantages: 1) Elevated biomass as evidenced by maximum OD\u003csub\u003e600\u003c/sub\u003e and cell dry weight values substantially exceeding those of pure MOB culture, indicating significantly enhanced system-wide biomass yield; 2) Enhanced metabolic activity, confirming that the presence of PSB markedly accelerated the growth rate of MOB; 3) Optimized environmental adaptability, with the shortened lag phase demonstrating the co-culture system's accelerated acclimation to nitrogen limitation stress. The symbiotic relationship between MOB and PSB effectively alleviated nitrogen limitation stress. Through synergistic resource exchange and metabolic cross-feeding, it achieved concomitant enhancement of growth kinetic parameters and biomass production. This synergy establishes a theoretical framework for applying microbial co-culture technologies in low-nitrogen environments.\u003c/p\u003e\u003cp\u003eThe study also explored the impact of PSB on methane assimilation by MOB. The maximum OD\u003csub\u003e600\u003c/sub\u003e for MOB growth was 1.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), with a lag phase of 20.0871 h, a maximum specific growth rate of 0.0180 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and a cell dry weight of 0.358\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, the co-culture had a lag phase of 18.5359 h, a maximum specific growth rate of 0.0213 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and a cell dry weight of 0.435\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032 g/L. The total methane consumption for MOB alone was 0.1108 g, while the co-culture consumed 0.1252 g (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The SCP yield for MOB was 0.323 g DCW/g CH\u003csub\u003e4\u003c/sub\u003e, whereas the co-culture achieved 0.349 g DCW/g CH\u003csub\u003e4\u003c/sub\u003e. The methane consumption and SCP yield of the co-culture were significantly higher than those of MOB alone. This may be due to the presence of PSB, which produce growth factors such as vitamins B1, B2, and B12, enhancing the activity of methane monooxygenase and thereby promoting methane assimilation. Additionally, \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e OB3b can oxidize ammonia to hydroxylamine, which is then converted to nitrite. Nitrite inhibits the activity of formate dehydrogenase (FDH) in \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e OB3b, suppressing methane uptake. Photosynthetic cyanobacteria possess nitrite reductase, which converts nitrite back to ammonia, reducing its inhibitory effect on MOB and promoting methane absorption. Besides nitrite, high concentrations of carbon dioxide produced by MOB can also inhibit their growth. PSB absorb carbon dioxide under light conditions, producing oxygen required by MOB, thereby enhancing methane assimilation. This demonstrates that the presence of PSB can promote the growth of MOB.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe lag phase and maximum growth rate obtained by fitting the growth curve.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOD\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eλ\u003c/em\u003e (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003e\u0026micro;\u003c/em\u003e \u003csub\u003emax\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eunder restricted nitrogen source conditions\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMOB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.227\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.2869\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0064\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCo-culture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.511\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25.6448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0178\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eUnder normal medium conditions\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMOB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20.0871\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0180\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCo-culture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18.5359\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0213\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFurthermore, the study investigated the effects of methane, methanol, and formate as carbon sources on the co-culture system. The growth curves revealed that when methane was used as the carbon source (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the co-culture achieved a maximum OD\u003csub\u003e600\u003c/sub\u003e of 1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025, with a lag phase of 18.7128 h and a maximum specific growth rate of 0.0214 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. When methanol was the carbon source, the co-culture reached a maximum OD\u003csub\u003e600\u003c/sub\u003e of 0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013 on the fifth day, with a lag phase of 22.6036 h and a maximum specific growth rate of 0.0464 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. For MOB alone with methanol as the carbon source, the maximum OD\u003csub\u003e600\u003c/sub\u003e was 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037 on the fourth day, with a lag phase of 23.4109 h and a maximum specific growth rate of 0.0666 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This suggests that using methanol, an intermediate metabolite of methane, as the carbon source can increase the maximum specific growth rate. However, the decline in OD\u003csub\u003e600\u003c/sub\u003e by the fourth or fifth day indicates that methanol accelerates bacterial growth but also prematurely induces the decline phase. The initial OD\u003csub\u003e600\u003c/sub\u003e values of the co-culture and MOB alone were similar, but the co-culture started with a higher OD\u003csub\u003e600\u003c/sub\u003e, suggesting that methanol addition caused cell lysis in PSB, releasing intracellular materials into the medium and delaying the decline phase compared to MOB alone. When formate was used as the carbon source, a continuous decline in OD\u003csub\u003e600\u003c/sub\u003e was observed from the beginning to the sixth day for both strains, likely due to excessive formate altering the medium's pH, making it unsuitable for cell survival and leading to the death of MOB and PSB.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe lag phase and maximum growth rate obtained by fitting the growth curve.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecarbon source\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOD\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eλ\u003c/em\u003e (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003e\u0026micro;\u003c/em\u003e \u003csub\u003emax\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCo-culture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emethane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18.7128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0214\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22.6036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0464\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMOB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emethane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.4109\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0666\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Microbial composition analysis\u003c/h2\u003e\u003cp\u003eThe full sequence of 16S rRNA of methane-oxidizing mixed microbial consortium was amplified, and after amplification, cloning and sequencing were performed. Homology analysis was conducted by comparing the sequences using the BLAST tool on the National Center for Biotechnology Information (NCBI) website. The comparative analysis results showed that the PSB present in methane-oxidizing mixed microbial consortium were \u003cem\u003ecyanobacteria\u003c/em\u003e. In the culture with only MOB (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the relative abundance of \u003cem\u003eMethylosinus\u003c/em\u003e was 32.04%, that of \u003cem\u003eMethylophilus\u003c/em\u003e was 42.29%, and that of cyanobacteria was 4.7%. In the culture with only PSB, the relative abundance of \u003cem\u003eMethylosinus\u003c/em\u003e was 7.9%, that of cyanobacteria was 48.58%, and that of \u003cem\u003eMethylophilus\u003c/em\u003e was 0.96%. In the co-culture of MOB and PSB, the relative abundance of \u003cem\u003eMethylosinus\u003c/em\u003e was 46.25%, that of \u003cem\u003ecyanobacteria\u003c/em\u003e was 10.9%, and that of \u003cem\u003eMethylophilus\u003c/em\u003e was 19.3%. \u003cem\u003eMethylophilus\u003c/em\u003e utilizes intermediate metabolites from MOB growth, such as methanol, formate, and formaldehyde, for its own growth. Comparing the cultures of MOB alone and the co-culture, it was found that the relative abundance of \u003cem\u003eMethylophilus\u003c/em\u003e was higher in the MOB-only culture than in the co-culture. This suggests that the presence of PSB promotes the uptake of intermediate metabolites like methanol and formate by MOB, thereby limiting the growth of \u003cem\u003eMethylophilus\u003c/em\u003e. In the PSB-only culture, the relative abundance of \u003cem\u003eMethylophilus\u003c/em\u003e was very low because no methane was supplied during cultivation, preventing MOB from growing and thus eliminating the production of intermediate metabolites like methanol and formate. In the MOB-PSB co-culture system, the relative abundance of PSB was relatively low, but throughout the cultivation process, they played a supportive role in the growth of MOB.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis chapter reveals the synergistic mechanisms and application potential of a co-culture system involving MOB and PSB. Selective cultivation experiments demonstrated that the two types of strains significantly promote each other's growth through the secretion of metabolites, with intracellular substances playing a particularly prominent role. Under nitrogen-limited conditions, the OD and dry cell weight of the co-culture system increased by 2.26 times and 2.6 times, respectively, compared to monocultures, confirming that PSB can supplement nitrogen sources for MOB and enhance SCP production. Experiments with nitrogen-containing media showed that PSB eliminate growth-inhibiting factors produced by MOB, thereby increasing methane assimilation. Carbon source screening indicated that methane is the most suitable for the co-culture system, while methanol triggers the lysis of PSB, and excessive formic acid leads to cell death. 16S rRNA sequencing confirmed that MOB dominate the co-culture system, while PSB primarily play a supporting role: not only by providing growth factors such as vitamins B1, B2, and B12 but also by clearing metabolic inhibitors through material cycling. This technology breaks through the traditional single-strain cultivation model, establishing a \"carbon-negative biomanufacturing\" system based on natural material cycling\u0026mdash;utilizing the greenhouse gas methane to produce high-value protein while achieving carbon sequestration. The study suggests that the integration of synthetic ecology and process engineering will advance this technology as a core solution for sustainable protein production, offering innovative approaches to addressing global food security and climate change.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis research was financially supported by central government support for local university reform and development fund - talent cultivation support program project (high-level talents) (304017). Thanks to the research group of Professor Xing Xinhui from Tsinghua University for providing the strains.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlleman, A. B. and Peters, J. W. (2023) Mechanisms for Generating Low Potential Electrons across the Metabolic Diversity of Nitrogen-Fixing Bacteria. Appl Environ Microb, 89(5).\u003c/li\u003e\n\u003cli\u003eBerman-Frank, I., Lundgren, P. and Falkowski, P. (2003) Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res Microbiol, 154(3): 157-164.\u003c/li\u003e\n\u003cli\u003eBonnet, S., Webb, E. A., Panzeca, C., Karl, D. M., Capone, D. G. and Sa\u0026ntilde;udo-Wilhelmy, S. A. (2010) Vitamin B12 excretion by cultures of the marine cyanobacteria \u003cem\u003eCrocosphaera\u003c/em\u003e and \u003cem\u003eSynechococcus.\u003c/em\u003e Limnol Oceanogr, 55(5): 1959-1964.\u003c/li\u003e\n\u003cli\u003eDong, T., Fei, Q., Genelot, M., Smith, H., Laurens, L. M. L., Watson, M. J. and Pienkos, P. T. (2017) A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, \u003cem\u003eMethylomicrobium buryatense.\u003c/em\u003e Energ Convers Manage, 140: 62-70.\u003c/li\u003e\n\u003cli\u003eHafsa, Yaiche Achour, Amel, Doumandji, Samia, Sadi and Sidahmed, Saadi (2014) Evaluation of nutritional and sensory properties of bread enriched with spirulina.\u003c/li\u003e\n\u003cli\u003eHarish and Seth, K. (2020) Molecular circuit of heterocyst differentiation in cyanobacteria. J Basic Microb, 60(9): 738-745.\u003c/li\u003e\n\u003cli\u003eHill, E. A., Chrisler, W. B., Beliaev, A. S. and Bernstein, H. C. (2017) A flexible microbial co-culture platform for simultaneous utilization of methane and carbon dioxide from gas feedstocks. Bioresource Technol, 228: 250-256.\u003c/li\u003e\n\u003cli\u003eIguchi, H., Yurimoto, H. and Sakai, Y. (2011) Stimulation of Methanotrophic Growth in Cocultures by Cobalamin Excreted by \u003cem\u003eRhizobia\u003c/em\u003e. Appl Environ Microb, 77(24): 8509-8515.\u003c/li\u003e\n\u003cli\u003eJintasataporn, O., Chumkam, S., Triwutanon, S., LeBlanc, A. and Sawanboonchun, J. (2021) Effects of a Single Cell Protein (\u003cem\u003eMethylococcus capsulatus\u003c/em\u003e, Bath) in Pacific White Shrimp (\u003cem\u003ePenaeus vannamei\u003c/em\u003e) Diet on Growth Performance, Survival Rate and Resistance to \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, the Causative Agent of Acute Hepatopancreatic Necrosis Disease. Frontiers in Marine Science, 8: 764042.\u003c/li\u003e\n\u003cli\u003eKhmelenina, V. N., But, S. Y., Rozova, O. N. and Trotsenko, Y. A. (2019) Metabolic Features of Aerobic Methanotrophs: News and Views. Curr Issues Mol Biol, 33: 85-99.\u003c/li\u003e\n\u003cli\u003eLiang, J. S., Zhang, P. Y., Zhang, R., Chang, J. N., Chen, L., Zhang, G. M. and Wang, A. J. (2024) Bioconversion of volatile fatty acids from organic wastes to produce high-value products by photosynthetic bacteria: A review. Environ Res, 242: 117796.\u003c/li\u003e\n\u003cli\u003eOkamoto, A., Saito, K., Inoue, K., Nealson, K. H., Hashimoto, K. and Nakamura, R. (2014) Uptake of self-secreted flavins as bound cofactors for extracellular electron transfer in \u003cem\u003eGeobacter\u003c/em\u003e species. Energ Environ Sci, 7(4): 1357-1361.\u003c/li\u003e\n\u003cli\u003ePalacios, O. A., Bashan, Y. and de-Bashan, L. E. (2014) Proven and potential involvement of vitamins in interactions of plants with plant growth-promoting bacteria-an overview. Biol Fert Soils, 50(3): 415-432.\u003c/li\u003e\n\u003cli\u003ePanagiotis, T., Benyamin, K., Zhu, X. Y., Zha, X. and Irini, A. (2019) Methane oxidising bacteria to upcycle effluent streams from anaerobic digestion of municipal biowaste. Journal of Environmental Management, 251: 109590.\u003c/li\u003e\n\u003cli\u003eParsy, A., Monlau, F., Guyoneaud, R. and Sambusiti, C. (2024) Nutrient recovery in effluents from the energy sectors for microalgae and cyanobacteria biomass production: A review. Renewable \u0026amp; Sustainable Energy Reviews, 191: 114207.\u003c/li\u003e\n\u003cli\u003ePradhan, S. K., Singh, N. R., Dehury, B., Panda, D., Modi, M. K. and Thatoi, H. (2019) Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study. J Cell Biochem, 120(10): 16990-17005.\u003c/li\u003e\n\u003cli\u003eRasouli, Z., Valverde-P\u0026eacute;rez, B., D\u0026apos;Este, M., De Francisci, D. and Angelidaki, I. (2018) Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs. Biochem Eng J, 134: 129-135.\u003c/li\u003e\n\u003cli\u003eRomarheim, O. H., Landsverk, T., Mydland, L. T., Skrede, A. and Overland, M. (2013) Cell wall fractions from prevent soybean meal-induced enteritis in Atlantic salmon (Salmo salar). Aquaculture, 402: 13-18.\u003c/li\u003e\n\u003cli\u003eRoumezi, B., Xu, X. M., Risoul, V., Fan, Y. P., Lebrun, R. and Latifi, A. (2020) The Pkn22 kinase of \u003cem\u003eNostoc \u003c/em\u003ePCC 7120 is required for cell differentiation via the phosphorylation of HetR on a residue highly conserved in genomes of heterocyst-forming cyanobacteria. Front Microbiol, 10: 3140.\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Baracaldo, P. and Cardona, T. (2020) On the origin of oxygenic photosynthesis and Cyanobacteria. New Phytol, 225(4): 1440-1446.\u003c/li\u003e\n\u003cli\u003eShindell, D., Kuylenstierna, J. C. I., Vignati, E., van Dingenen, R., Amann, M., Klimont, Z., Anenberg, S. C., Muller, N., Janssens-Maenhout, G., Raes, F., Schwartz, J., Faluvegi, G., Pozzoli, L., Kupiainen, K., H\u0026ouml;glund-Isaksson, L., Emberson, L., Streets, D., Ramanathan, V., Hicks, K., Oanh, N. T. K., Milly, G., Williams, M., Demkine, V. and Fowler, D. (2012) Simultaneously Mitigating Near-Term Climate Change and Improving Human Health and Food Security. Science, 335(6065): 183-189.\u003c/li\u003e\n\u003cli\u003eStrong, P. J., Xie, S. and Clarke, W. P. (2015) Methane as a Resource: Can the Methanotrophs Add Value? Environ Sci Technol, 49(7): 4001-4018.\u003c/li\u003e\n\u003cli\u003eTays, C., Guarnieri, M. T., Sauvageau, D. and Stein, L. Y. (2018) Combined Effects of Carbon and Nitrogen Source to Optimize Growth of Proteobacterial Methanotrophs. Front Microbiol, 9: 2239.\u003c/li\u003e\n\u003cli\u003eTeixeira, L. V., Moutinho, L. F. and Romao-Dumaresq, A. S. (2018) Gas fermentation of C1 feedstocks: commercialization status and future prospects. Biofuel Bioprod Bior, 12(6): 1103-1117.\u003c/li\u003e\n\u003cli\u003eVideau, P., Rivers, O. S., Hurd, K., Ushijima, B., Oshiro, R. T., Ende, R. J., O\u0026apos;Hanlon, S. M. and Cozy, L. M. (2016) The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in \u003cem\u003eAnabaena\u003c/em\u003e sp. strain PCC 7120. P Natl Acad Sci USA, 113(45): E6984-E6992.\u003c/li\u003e\n\u003cli\u003eXie, J., Sun, X. K., Du, H. G., Chen, D. W. and Wang, Y. (2023) Exploring the Effects of Different Methane and Oxygen Concentrations on the Methane-Oxidizing Bacteria Mixed Community. J Environ Eng, 149(12): 04023081.\u003c/li\u003e\n\u003cli\u003eYang, Y. G., Xiang, Y. B., Xia, C. Y., Wu, W. M., Sun, G. P. and Xu, M. Y. (2014) Physiological and electrochemical effects of different electron acceptors on bacterial anode respiration in bioelectrochemical systems. Bioresource Technol, 164: 270-275.\u003c/li\u003e\n\u003cli\u003eZeng, X. L. and Zhang, C. C. (2022) The Making of a Heterocyst in Cyanobacteria Annual Review of Microbiology. Annual Review of Microbiology, 76: 597-618.\u003c/li\u003e\n\u003cli\u003eZhang, Y. X., Xin, J. Y., Chen, L. L., Song, H. and Xia, C. U. (2008) Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol. J Nat Gas Chem, 17(1): 103-109.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Methane-oxidizing bacteria, photosynthetic bacteria, Methane, Single cell protein, co-culture system","lastPublishedDoi":"10.21203/rs.3.rs-7286353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7286353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSingle cell protein (SCP) is widely used in food and feed due to its high protein content, rich essential amino acids, low fat content, and presence of various trace elements. It can serve as a dietary supplement in animal diets or as a substitute for certain proteins. Methane-oxidizing bacteria (MOB), which utilize methane as their sole carbon and energy source and are not limited by factors such as land or light, are a significant asset for SCP production. Their co-culture system with photosynthetic bacteria (PSB) can further enhance SCP production efficiency. By comparing monoculture and co-culture data, it was confirmed that a synergistic interaction based on intracellular substance exchange exists between two mixed microorganisms: adding intracellular substances from MOB increased the maximum OD\u003csub\u003e600\u003c/sub\u003e of PSB by 19% and cell dry weight by 10%, while adding intracellular substances from PSB increased the OD\u003csub\u003e600\u003c/sub\u003e of MOB by 32% and cell dry weight by 2.06 times. Under nitrogen-limited conditions, the co-culture system achieved 2.26 times higher OD\u003csub\u003e600\u003c/sub\u003e and 2.6 times greater cell dry weight compared to monoculture, demonstrating that the nitrogen-fixing activity of PSB effectively supplemented nitrogen sources. In normal nitrogen-supplied medium, the co-culture increased methane consumption by 13% (0.1252 g vs. 0.1108 g), SCP yield by 8% (0.349 vs. 0.323 g DCW/g CH\u003csub\u003e4\u003c/sub\u003e), and cell dry weight by 21%. 16S rRNA analysis revealed that MOB dominated the co-culture system (46.25%), with photosynthetic cyanobacteria as a secondary component (10.9%), validating the ecological structure of this synergistic system. The research outcomes provide a novel and efficient co-culture model for optimizing industrial SCP production.\u003c/p\u003e","manuscriptTitle":"Co-cultivation of methane oxidizing bacteria and photosynthetic bacteria for single cell protein production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 09:53:26","doi":"10.21203/rs.3.rs-7286353/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aec84d53-f90d-4d89-9a48-839735509890","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-16T15:00:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-16 09:53:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7286353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7286353","identity":"rs-7286353","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}