Recently, considering the tight supply and demand of essential commodities such as alternative proteins and biopolysaccharide raw materials, Professor Fei Qiang and his team at Xi'an Jiaotong University proposed a new paradigm for the synthesis of biological macromolecules by using methanophiles to convert methane gas.
图 | 费强（来源：费强）
When developing this new technology, they found that there were two key scientific and technical constraints on the development of this technology: first, the mechanism of cellular metabolism regulation of the strain was not clear, and second, the targeted synthesis strategy of the target product was not sound.
In view of the above difficulties, they developed a high-density fermentation technology that can efficiently synthesize cell proteins and polysaccharide polymers for methanophile cell factories, focusing on the two technical routes of breeding natural strains and modified model strains.
Among them, the dry weight of the cell is more than 14 g/L, the spatiotemporal production efficiency of protein is close to 20 g/L/day, the protein content is more than 70%, and the content of biological polysaccharides is more than 30%, and the above-mentioned indicators such as biological carbon sequestration capacity and product conversion efficiency are at the international advanced level.
With the optimization and improvement of this technology, the above production figures are expected to increase by 25% in a short period of time.
While solving the production bottleneck, they also deeply analyzed the synergistic adaptation mechanism of carbon and nitrogen metabolic flows in cell factories.
By introducing a controllable induction strategy based on nitrogen-oxygen nutrition, the artificial directional synthesis of target products was strengthened, the production efficiency of proteins and biopolysaccharides was significantly improved, and the one-step transformation from one-carbon molecules to biomacromolecules was realized.
（来源：Green Chemistry）
In general, the methane protein preparation technology developed this time provides a practical solution for the food security and the "cultivated land red line" in mainland China.
Fei Qiang said that the crude protein content of methane protein produced this time is more than 70%, rich in essential amino acids such as leucine, threonine, lysine and phenylalanine, and all 18 amino acids account for more than 85% of the protein, which is a simple protein type.
The structure ratio of methane protein is close to that of fishmeal, far better than that of soybean meal, and can be used to replace existing protein feeds.
Based on the industrial production of 10 million tons of methane protein (70% protein), it is equivalent to 23 million tons of imported soybeans (30% protein).
Compared to growing soybeans, the production of methane protein can save more than 500 times more arable land and 3,000 times more freshwater resources, without the need for large amounts of fertilizers and pesticides, and is not affected by factors such as seasons and climate.
In addition, the newly developed high-density fermentation technology enables the targeted co-production of biomacromolecular components such as intracellular polysaccharides and exopolysaccharides.
After testing, the structure of the intracellular polysaccharide is highly similar to that of amylopectin, and it has good biocompatibility and degradability, and can be used as a pharmaceutical film and hydrogel. Exopolysaccharides can be used as stabilizers, emulsifiers and wound dressings for medical aesthetic products.
It can be seen that the use of shale gas, coalbed methane or biogas to prepare biomacromolecules can not only increase the carbon added value of methane, but also achieve efficient bioenergy storage and carbon sink, which provides a new strategy for the development of new quality productivity.
"Asking for protein from microorganisms"
According to reports, methane, the second largest greenhouse gas, has a 20-year cycle greenhouse effect more than 80 times that of carbon dioxide. Methane gas comes from a wide range of sources, with coalbed methane and biogas being the main sources in addition to the well-known natural gas and shale gas resources.
In 2015, the United States transitioned from a natural gas importer to a pure natural gas exporter with advanced shale gas extraction technology.
As the world's largest country in terms of proven shale gas reserves, the mainland ranked second in the world in terms of shale gas extraction in 2020, and with the National Development and Reform Commission (NDRC) and the National Energy Administration (NEA) jointly releasing the 14th Five-Year Plan for Modern Energy System, the mainland is expected to achieve new breakthroughs in shale gas production and development.
The methane content of shale gas is more than 95%, and as a low-density, high-heat gas, the high-value development and efficient storage of shale gas have also become the focus of the development of shale gas utilization technology in the mainland.
In addition, due to the poor profit model of restricted biogas, it is difficult to promote biogas projects, resulting in insufficient utilization of agricultural and forestry organic waste carbon resources.
At present, methane is mainly used for combustion to produce heat and electricity, and the utilization method is relatively simple and the added value is low. Moreover, all the carbon in methane is converted into carbon dioxide during the combustion process, resulting in carbon emissions and waste of carbon resources, and the economy of carbon atoms is very low.
With the proposal of the "dual carbon" goal, the mainland attaches great importance to the development of methane emission reduction and utilization technology. One-carbon biomanufacturing technology can prepare a variety of functional bio-based products while achieving efficient methane fixation.
Methanophiles are a special class of environmental microorganisms that are able to grow using methane as the only carbon source. Therefore, methane bioconversion technology can be carried out spontaneously at room temperature and pressure, with less toxic by-products and carbon dioxide emissions.
It is also reported that soybean meal is the most important source of feed protein for aquaculture, and the mainland's soybean raw materials have long relied on imports, with a foreign dependence of more than 80%, which has become one of the biggest shortcomings of the mainland's agriculture.
Under the current increasingly severe and complex international situation, the soybean supply situation is not optimistic, which seriously endangers 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 the relevant departments of the mainland have repeatedly put forward the demand for "protein from microorganisms".
At present, the European Union has approved microbial protein for feed additives, and technology companies such as Calysta in the United States and Unibio in Denmark have also begun to develop scale-up production technologies, and considerable progress has been made.
However, the world's mainstream microbial protein core production technology and production strains are still mastered by European and American enterprises, which indirectly restricts the independent technology development of the mainland.
Cultures: "chips" in biomanufacturing
A few years ago, top universities in Europe and the United States had accumulated many years of research results and valuable experience in methane bioutilization, while there were very few domestic studies and needed to make breakthroughs.
As an academic leader in biochemical engineering at Xi'an Jiaotong University, Fei Qiang has long carried out cutting-edge scientific and technological research on the development and efficient utilization of biomanufacturing raw materials.
In order to solve the problem of single utilization of methane gas and low added value of products, Fei Qiang led the members of the research group in 2017 to develop a one-carbon bioconversion technology using methane as a raw material.
Considering the growth of the global population and the increasing living standards of people, and the production capacity of traditional animal and plant proteins has reached the upper limit, there will be a shortage of protein in the future, so they chose cellular protein as one of the main products to carry out the project design.
Once the topic was identified, they focused on the core elements of biomanufacturing: microbial strains and high-density fermentation technology.
Strain is the "chip" in the field of biomanufacturing, which largely determines the production capacity and product quality, while the mainland is weak in the research and development and preservation of industrial strains, and is still in the stage of catching up with the advanced level of foreign countries.
It is difficult to form the core competitiveness of the strain without independent intellectual property rights, and it is easy to be "stuck" at a critical moment in the future.
In 2017, with the support of the Shaanxi Provincial Key R&D Program, Fei Qiang led the research team members to collect samples from various wetland environments in Shaanxi Province for several times.
After hundreds of strain breeding and identification, more than 10 strains of methanophilic bacteria were finally obtained from the rice fields at the foot of the Qinling Mountains and the samples of Niubeiliang.
It has the advantages of fast growth rate and strong stress resistance, and can naturally synthesize high value-added products such as excellent proteins, active polysaccharides, and natural products.
In 2019, Fei Qiang, as the backbone of the project, received the key special support of the National Key R&D Program "Synthetic Biology", and with the help of cutting-edge technologies such as biosynthesis, he deeply excavated the key functional genes and metabolic regulatory mechanisms in microorganisms, and improved genetic modification tools and artificial cell construction methods.
Through a comprehensive exploration and analysis of key mechanisms, they have successfully established a set of methane biomanufacturing platform technologies centered on methanophile cell factories, and realized 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 the support of the key project of the National Key R&D Program "Green Biomanufacturing".
The team members completed thousands of shake flask experiments, built a variety of culture systems from scratch, verified the effects of gas source components, medium composition and concentration on strains, and realized the efficient biotransformation and utilization of methane and biogas.
In the past 3 years, the research group has gone through hundreds of batches of fermentation experiments and successfully developed a high-density fermentation process and strategy for methanophiles.
During this time, they completed bioreactor systems from 0.3L to 30L, achieved 100x scale-up at the laboratory level, and finally created a high-density fermentation process for methanophils for different target products.
It is worth mentioning that although the fermentation work mentioned in this paper occupies a small space, there are dozens of "unideal" fermentation experiments hidden behind it to guide them to summarize their experience.
Finally, they not only obtained the optimal process, but also systematically summarized the synthesis mechanism of methanophile proteins and polysaccharides, and proposed a new strategy to induce the synthesis of biological macromolecules based on nutrient regulation strategies for the first time.
In 2023, they collaborated with a large-scale biogas production platform to explore a continuous high-density fermentation process for a 100L bioreactor system, which preliminarily verified the reliability of the process scale-up and laid a solid foundation for the industrial production of methane protein.
Efforts should be made to ensure the independent property rights of the complete set of processes and equipment for methane biomanufacturing
In fact, during the strain selection and process optimization, they encountered many difficulties, the project was stalled for a while, the confidence and motivation of the students were greatly damaged many times, and the mentality of everyone was often up and down.
Due to the lack of a high-throughput breeding system adapted to the gas carbon source, the methanophile screening process is time-consuming and laborious, and a large number of consumables are often required.
Methanophiles often grow in synergy with other microorganisms in the natural environment, and there is a strong interaction dependence between them, and it is extremely difficult to isolate and obtain a single strain of methanophiles.
Fei Qiang was most impressed by the fact that a master's student spent a year screening methanophiles that may be rich in carotenoids, but because of the infection during preservation, after 20 rounds of screening, he still failed to obtain a single strain, and finally had to give up purification and turn to a mixed bacterial system for research.
Not only that, but the optimization of the fermentation process of methanophiles is also full of twists and turns. Methane is a gaseous carbon source with very low solubility, and the gas-liquid mass transfer rate is a major limiting factor for the growth of methanophiles.
The gas-liquid mass transfer rate of the fermenter system and the shake flask system was too different, which made it difficult and inefficient for them to explore the fermentation conditions in the shake flask system.
At the beginning, they continued to use model strains such as Escherichia coli to optimize fermentation optimization, mainly to optimize the concentration of media components, temperature, pH and other conditions for single factor and response surface, but with little success. A Ph.D. student had not made any substantial progress for nearly a year and a half after taking over this part of the work.
On the other hand, Fei Qiang believes that it is the unique challenges of methanophila research that drives his team to continue to innovate and insist on doing original research.
In this study, some of the problems they encountered were difficult to find reference solutions in other microbial studies. This forced them to go back to the source and dig deeper into the unique metabolic regulation mechanism of methanophiles.
In the most typical case, fed-batch fermentation, which is commonly used in model strains, not only failed to increase the cell density of methanogens, but also led to a decrease in protein yield.
At first, they were puzzled, but with the help of transcriptomics, they found that the cells were in a state of nutritional imbalance and their growth was stagnant later in the process, and the carbon flow was used to synthesize exopolysaccharides.
This discovery led them to the development of a continuous fermentation process to improve protein production efficiency and led them to wonder if there might be some unknown mechanism to control the synthesis of proteins and polysaccharides by methanophiles.
最终，相关论文以《嗜甲烷菌生物合成可食用产品的可控性诱导新策略》（A novel nutritional induction strategy flexibly switching the biosynthesis of food-like products from methane by a methanotrophic bacterium）为题发在 Green Chemistry[1]。
Ph.D. student Zixi Gao is the first author, and Professor Fei Qiang is the corresponding author of the first unit.
图 | 相关论文（来源：Green Chemistry）
As this pioneering work is of great significance for the development of alternative proteins and biopolysaccharide functions. In view of this, this paper was also invited by Green Chemistry to be published as a cover paper.
However, the research group hopes not only to develop a "laboratory technology", but also pays great attention to the industrialization of the technology, hoping that this technology can truly play its value.
At present, they have cooperated with companies with rich experience in fermentation engineering to promote the engineering scale-up of technology.
At the same time, it has also carried out fruitful work with local governments and upstream and downstream enterprises in the industry with rich shale gas resources, and has reserved rich resources for industrialization.
As the first scientific research team in mainland China to develop methane biomanufacturing technology using methane as raw material, they will continue to carry out precise coupling regulation around the material metabolism and energy metabolism of methanophils.
By strengthening the interaction between intracellular carbon-nitrogen metabolism, the targeted synthesis of specific amino acids can be realized, and the methane bioconversion efficiency and the production efficiency of the target product can be improved at the molecular level.
In view of the wide market space of biosynthetic polysaccharides in the fields of food, medical treatment, environment, materials and other fields, they will also further carry out physicochemical and biological activity efficacy experiments for the application of methane polysaccharides in medical aesthetic raw materials and agricultural fertilizers.
In order to adapt to industrial production, they will first focus on the development of a new biological reaction system with a high gas-liquid mass transfer rate, so as to adapt to the high-density fermentation of methanophiles and the production process of target products using different methane gas sources, and have independent intellectual property rights to ensure that the complete set of production processes and equipment for methane biomanufacturing have independent intellectual property rights.
In the near future, they will conduct continuous pilot production validation under industry-wide conditions based on different methane gas sources, including shale gas bases, livestock and poultry farms, and natural gas tailings, to demonstrate the large-scale production of methane proteins or polysaccharides and their carbon reduction potential.
Resources:
1.Gao, Z., Guo, S., Chen, Y., Chen, H., Fu, R., Song, Q., ... & Fei, Q. (2024). A novel nutritional induction strategy flexibly switching biosynthesis of food-like products from methane by a methanotrophic bacterium. Green Chemistry.
Operation/Typesetting: He Chenlong