Recently, considering the global supply and demand tension of essential goods such as alternative proteins and biopolymer raw materials, Professor Fei Qiang from Xi'an Jiaotong University and his team proposed a new paradigm of converting methane gas into biological macromolecules using methanotrophic bacteria.

During the development of this new technology, they found that there are two key scientific and technological issues that have constrained the development of the technology: one is that the cellular metabolic regulation mechanism of the bacterial strains is not clear; the other is that the directed synthesis strategy for the target products is not perfect.

In response to the above difficulties, they focused on the two technical routes of selecting natural strains and modifying model strains, and developed high-density fermentation technology that can efficiently synthesize cellular proteins and polysaccharide polymers for methanotrophic bacterial cell factories.

Among them, the dry weight of the bacteria exceeds 14 grams per liter, the spatiotemporal production efficiency of protein is close to 20 grams per liter per day, the protein content exceeds 70%, and the content of biopolysaccharides exceeds 30%. The above indicators such as biological carbon fixation capacity and product conversion efficiency are at the international advanced level.

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With the optimization and improvement of this technology, the above production data is expected to be further improved by 25% in a short period of time.While addressing production bottlenecks, they deeply analyzed the synergistic adaptation mechanism of carbon-nitrogen metabolic flux in the cell factory.

By introducing a controllable induction strategy based on nitrogen-oxygen nutrition, the artificial directed synthesis of the target product was enhanced, significantly improving the production efficiency of proteins and polysaccharides, achieving a one-step transformation from one-carbon molecules to biological macromolecules.

Overall, the methane protein preparation technology developed this time provides a feasible plan for the food security and protection of the "arable land red line" of our country.

Fei Qiang said that the crude protein content of the methane protein produced this time exceeds 70%, rich in essential amino acids such as leucine, threonine, lysine, and phenylalanine, all 18 kinds of amino acids account for more than 85% of the protein, which is a pure protein type.

The structural ratio of methane protein is close to fish meal, far better than soybean meal, and can be used to replace existing protein feed.Based on the industrial production of 10 million tons of methane protein (with a protein content of 70%), it is equivalent to the equivalent of 23 million tons of imported soybeans (with a protein content of 30%).

Compared with the cultivation of soybeans, the production of methane protein can save more than 500 times the amount of arable land and 3000 times the amount of freshwater resources. It is not only free from the need to apply a large amount of chemical fertilizers and pesticides, but also not affected by factors such as seasons and climate.

In addition, through the high-density fermentation technology developed this time, it is possible to achieve the directional co-production of intracellular polysaccharides and extracellular polysaccharides and other biological macromolecular components.

The detection shows that the structure of this intracellular polysaccharide is highly similar to that of branched starch, which has good biocompatibility and degradability, and can be used as medical films and hydrogels. Extracellular polysaccharides can be used as stabilizers, emulsifiers, and wound dressings for medical beauty products.

It can be seen that the preparation of biological macromolecules using shale gas, coalbed methane, or biogas can not only enhance the carbon value-added of methane but also achieve efficient biological energy storage and carbon sequestration, providing a new strategy for the development of new quality productive forces."Demanding Protein from Microorganisms"

It is reported that as the second largest greenhouse gas, the greenhouse effect of methane over a 20-year period is more than 80 times that of carbon dioxide. Methane sources are widespread, in addition to the well-known natural gas and shale gas resources, coalbed methane and biogas are also major sources of methane.

In 2015, the United States, with its advanced shale gas extraction technology, achieved a transformation from a natural gas importer to a pure natural gas exporter.

As the country with the world's largest proven reserves of shale gas, China's shale gas extraction volume ranked second in the world in 2020. With the joint release of the "14th Five-Year Plan for Modern Energy System" by the National Development and Reform Commission and the National Energy Administration, it is expected that China's shale gas production and development efforts will achieve new breakthroughs.

The methane content in shale gas is more than 95%, and as a low-density, high-energy gas, the high-value development and efficient storage of shale gas have also become the focus of the development of China's shale gas utilization technology.In addition, due to the poor profitability of biogas, the promotion of biogas projects is difficult, leading to insufficient utilization of organic waste carbon resources in agriculture and forestry.

At present, methane is mainly used for combustion to generate heat and electricity, with a relatively single utilization method and low added value. Moreover, the carbon in methane is entirely converted into carbon dioxide during the combustion process, resulting in carbon emissions and waste of carbon resources, with low carbon atom economy.

With the proposal of the "dual carbon" targets, China attaches great importance to the development of methane emission reduction and utilization technologies. One-carbon biomanufacturing technology can achieve efficient fixation of methane while preparing a variety of functional bio-based products.

Methanotrophic bacteria are a special class of environmental microorganisms that can grow using methane as the sole carbon source. Therefore, methane bioconversion technology can spontaneously proceed under normal temperature and pressure, with fewer toxic by-products and carbon dioxide emissions.

It is also reported that soybean meal is the most important source of protein for the breeding industry, and China's soybean raw materials have long been heavily dependent on imports, with a foreign dependence exceeding 80%, which has become one of the biggest weaknesses of China's agriculture.In the current increasingly severe and complex international situation, the supply situation of soybeans is not optimistic, which seriously endangers the national development and economic security.

The biomanufacturing of microbial protein has become one of the main ways to solve the shortage of protein resources, and relevant departments in our country have also proposed the demand of "asking for protein from microorganisms" many times.

At present, the European Union has approved the use of microbial protein as a feed additive, and technology companies such as the United States Calysta and Denmark Unibio have also started to develop and expand production technology, and have made considerable progress one after another.

However, the core production technology and production strains of the mainstream microbial protein in the world are still controlled by European and American enterprises, which indirectly restricts the development of our country's independent technology.

(Note: The translation is given in English, as requested.)Microorganisms: The "Chip" of the Biomanufacturing Field

A few years ago, top universities in Europe and America had already accumulated years of research achievements and valuable experience in the utilization of methane, while domestic research was minimal and urgently needed a breakthrough.

As the academic leader in biochemical engineering at Xi'an Jiaotong University, Fei Qiang has long been engaged in cutting-edge scientific and technological research and development around the development and efficient utilization of biomanufacturing raw materials.

In response to the issue of the single utilization method of methane gas and the low added value of products, in 2017, Fei Qiang led the team members to develop a one-carbon biotransformation technology using methane as the raw material.

Considering the global population growth and the continuous improvement of people's living standards, while the production capacity of traditional animal and plant proteins has reached its limit, there will be a shortage of protein supply in the future. Therefore, they chose cellular protein as one of the main products to carry out the subject design.After finalizing the research topic, they focused on the core elements of biomanufacturing (microorganism strains and high-density fermentation technology) for development.

The strain is the "chip" of the biomanufacturing field, which largely determines production capacity and product quality. However, our country is relatively weak in the development and preservation of industrial strains and is still in the stage of catching up with the advanced level abroad.

Without strains with independent intellectual property rights, it is difficult to form core competitiveness, and it is easy to be "strangled" at critical moments in the future.

In 2017, with the support of the key research and development plan project in Shaanxi Province, Fei Qiang led the research team members to various wetland environments in Shaanxi to collect samples many times.

After hundreds of times of strain selection and identification, more than ten strains of methane-utilizing bacteria were finally obtained from samples in the paddy fields at the foot of the Qinling Mountains and Niubeiliang, etc.It possesses advantageous traits such as rapid growth rate and strong stress resistance, and is capable of naturally synthesizing high-value products such as high-quality proteins, active polysaccharides, and natural products. Some of the production methods of these products have obtained national patent inventions and have completed the transformation of achievements.

In 2019, as a key member of the project, Fei Qiang received support from the National Key R&D Program "Synthetic Biology" key special project. With the aid of cutting-edge technologies such as biosynthesis, he deeply explored the key functional genes and metabolic regulatory mechanisms within microorganisms, and improved genetic modification tools and artificial cell construction methods.

Through comprehensive exploration and analysis of the key mechanisms, they successfully established a complete set of methane biomanufacturing platform technology centered on methanogenic bacteria cell factories, achieving the biosynthesis of high-value products including fine chemicals, biomaterials, and pharmaceutical intermediates using genetically engineered bacteria.

After a long period of accumulation, in 2021, Fei Qiang, as the project leader and chief scientist, received support from the National Key R&D Program "Green Biomanufacturing" key special project.

Team members, by completing thousands of batches of shake flask experiments, built various cultivation systems from scratch, verified the impact of gas source components, culture medium components, and concentrations on strains, and achieved efficient biotransformation and utilization of methane and biogas.Over the past three years, the research group has gone through hundreds of batches of fermentation experiments, successfully developing a high-density fermentation process and strategy specifically for methanotrophic bacteria.

During this period, they completed the development from a 0.3L to a 30L bioreactor system, achieving a 100-fold scale-up at the laboratory level, and ultimately created a high-density fermentation process for methanotrophic bacteria tailored to different target products.

It is worth mentioning that although the fermentation work mentioned in their paper occupies a very small part, it is backed by dozens of "unsatisfactory" batches of fermentation experiments that have guided them to summarize their experiences.

In the end, they not only obtained the optimal process but also systematically summarized the synthesis mechanisms of methanotrophic bacterial proteins and polysaccharides, and for the first time proposed a new strategy for inducing the synthesis of biological macromolecules based on nutritional regulation strategies.

In 2023, they collaborated with a large-scale biogas production platform to explore the continuous high-density fermentation process of a 100L bioreactor system, preliminarily verifying the scalability of the process, laying a solid foundation for the industrial production of methane proteins.Strive to ensure the independent intellectual property rights for the entire process and equipment of methane biotechnology manufacturing.

In fact, during the strain selection and process optimization, they encountered many difficulties, and the project was once at a standstill, with the students' confidence and enthusiasm being greatly frustrated multiple times, and everyone's mentality also fluctuated frequently.

Due to the lack of a high-throughput screening system adapted to gaseous carbon sources, the screening process for methanotrophic bacteria is time-consuming and labor-intensive, often requiring a large amount of consumables.

In the natural environment, methanotrophic bacteria often grow in symbiosis with other microorganisms, and there is a strong interdependent relationship between them, making it extremely difficult to isolate a single strain of methanotrophic bacteria.

The most impressive thing for Fei Qiang was that a master's student, after a year of hard work, finally screened a methanotrophic bacterium that might be rich in carotenoids, but it might have been contaminated during preservation. After twenty rounds of screening, a single strain was still not obtained, and in the end, he had to give up purification and turn to research with a mixed microbial system.Not only that, but the optimization of the fermentation process for methanotrophs has also been full of twists and turns. Methane is a gaseous carbon source with an extremely low solubility, and the gas-liquid mass transfer rate is the main limiting factor for the growth of methanotrophs.

The gas-liquid mass transfer rate in the fermentation tank system is much different from that in the shake flask system, which leads to many difficulties and low efficiency when they explore fermentation conditions in the shake flask system.

At first, they followed the fermentation optimization research of model strains such as Escherichia coli, mainly focusing on single-factor and response surface optimization of culture medium component concentrations, temperature, and pH, but the results were minimal. A doctoral student who took over this part of the work had not made any substantial progress for nearly a year and a half.

On the other hand, Fei Qiang believes that it is the unique challenges of methanotroph research that drive their team to continuously innovate and persist in conducting original research.

In this study, some of the problems they encountered are difficult to find reference solutions in related studies of other microorganisms. This has forced them to trace back to the source and delve deeply into the unique metabolic regulatory mechanisms of methanotrophs.The most typical case is that the commonly used batch-fed fermentation in the model strain not only failed to increase the cell density of methanotrophic bacteria, but also led to a decrease in protein yield.

At first, they couldn't figure it out. With the help of transcriptomics, they found that the cells were in a state of nutritional imbalance and growth stagnation in the later stage of the process, and the carbon flow was used to synthesize extracellular polysaccharides.

This discovery led them to develop a continuous fermentation process to improve protein production efficiency, and guided them to start thinking about whether there might be some unknown mechanism that controls the synthesis of proteins and polysaccharides by methanotrophic bacteria.

Finally, the related paper was published in Green Chemistry with the title "A novel nutritional induction strategy flexibly switching the biosynthesis of food-like products from methane by a methanotrophic bacterium" [1].

Doctoral student Gao Zixi is the first author, and Professor Fei Qiang is the corresponding author of the first unit.Due to the significant implications of this pioneering work for the development of alternative proteins and the functions of bio-polymers, this thesis has been invited by Green Chemistry to be published as a cover paper.

However, the research team aspires not only to develop a "laboratory technique" but also pays great attention to the industrial application of the technology, hoping that it can truly realize its value.

At present, they have started cooperation with companies that have rich experience in fermentation engineering to promote the engineering scale-up of the technology.

In the meantime, they have also carried out fruitful work with local governments rich in shale gas resources and enterprises in the industry chain, preparing abundant resources for industrialization.

As the first research team in our country to develop methanotrophic bacteria bio-manufacturing technology based on methane as raw material, they will continue to focus on the precise coupling and control of the material metabolism and energy metabolism of methanotrophic bacteria in the follow-up.By enhancing the interaction between carbon-nitrogen metabolism within cells to achieve the directed synthesis of specific amino acids, the efficiency of methane biotransformation and the production efficiency of target products can be improved at the molecular level.

Considering the broad market space for biopolymers in fields such as food, medical, environmental, and materials, they will also conduct further physicochemical and bioactivity efficacy experiments on the application of methane polysaccharides in medical beauty raw materials and agricultural microbial fertilizers.

To adapt to industrial production, they will first focus on developing a new type of bioreactor system with a higher gas-liquid mass transfer rate, to adapt to the high-density fermentation of methanotrophic bacteria and the production process of target products using different methane gas sources, ensuring that the complete set of methane biomanufacturing production processes and equipment have independent intellectual property rights.

Soon after, they will carry out continuous pilot production verification under full industrial conditions according to different methane gas sources, including shale gas bases, livestock and poultry farms, and natural gas tailings, thereby demonstrating the large-scale production of methane protein or polysaccharides and their carbon emission reduction potential.