For decades, a large number of studies have proved that livestock emit large amounts of greenhouse gases during the breeding process. With animal husbandry as the representative, related food and its industrial chain have become a nightmare that affects the global environment, and the impact on the ecosystem is quite extensive.
Previously, according to the statistics of the Food and Agriculture Organization of the United Nations and the World Resources Institute, the animal husbandry, including cattle breeding, accounted for nearly 15% of the total global emissions; while the carbon dioxide generated by all vehicles accounted for only 24%. . In 2018, “Science” magazine published a report stating that the food and related industry chain “contributed” 26% of global greenhouse gas emissions.
In order to effectively reduce the food carbon footprint and achieve sustainable food manufacturing technology. Scientists from the Wys s Institute of Harvard University have started a new attempt. They use microbial fermentation to build a more environmentally friendly food manufacturing process. The technology will use carbon dioxide as a fermentation raw material to produce various biological products, and its first product goal will be to use carbon dioxide to synthesize food-grade fat. Edible fats are fats that people can eat or cook directly, such as soybean oil, peanut oil, olive oil, mutton oil, butter, and so on.
Precise fermentation technology adds “flavor” to food
Today, with the rapid development of “carbon cycle” and “carbon fixation” technologies, companies such as LanzaTech, Cemvita, Carbon Recycling International and others have already put the “gas fermentation” label on the hot search.
For a long time, inspired by traditional fermentation processes such as bread and beer making, the use of various microorganisms to quickly and effectively carry out large-scale biomass synthesis has been a common topic.
In recent years, the precision fermentation industry that uses various modified microorganisms as “cell factories” is gradually emerging on synthetic biology flights that fly higher and higher. More and more biotechnology companies have poured into this field, with the primary goal of solving the carbon emission problem of animal husbandry and aquaculture. The products manufactured by this type of company are mainly protein and fat, and artificial meat is involved on the application side. Category, dairy industry and related condiments, etc.
If protein is the basic raw material for food, then fat adds “flavor” to the food. Specifically in the field of synthetic fats, in addition to the hard work of microbial fermentation technology, synthetic biology companies such as Hoxton Farms, which manufacture fat and meat based on cell synthesis methods, are also sharing the cake in the field of synthetic fats.
At present, these start-up companies have gradually begun to gain the attention of the investment market. Among them, cheese merchant Change foods’ total financing reached 3.1 million US dollars, while Nourish Ingredients, which focuses on vegetable fats, received a total of 14.4 million US dollars in financing.
In the current situation, whether fermenting microorganisms or culturing cells are used, the manufacturing process of various commercial engineered microorganisms generally requires the addition of starch, sugar or cellulose as food sources to produce ethanol or lactic acid, and then the subsequent synthesis reaction.
Although a step in the right direction, sugar-based fermentation technology will put pressure on the planting industry and is not universal in the world. In general, these companies have not opened up the upper reaches of the reaction chain.
Symbiotic flora with personalized production function
In May 2021, Circe Bioscience Inc. (hereinafter referred to as Circe), a spin-off from the Wyss Institute, was incorporated in Delaware. The company’s first goal will be to use engineered microorganisms to convert carbon dioxide into food-grade fat.
This start-up is derived from the Circe project of the Wyss Institute, which means “the recycling industry of the cell factory”. Two scientists Shannon Nangle and Marika Ziesack from the Wyss Institute at Harvard University became the co-founders.
In their project, the hookworm Cupriavidus necator (hereinafter referred to as C.necator, formerly known as Ralstonia eutropha) has become a “synthetic code” to open up the production chain.
About 60 years ago, C.necator H16, a typical strain of this category, was isolated from the soil. Since then, it has become the most studied chemoautotrophic bacteria with the best genome characteristics. In the past few decades, C.necator has become a hot research object with the synthesis of polyhydroxyalkanoate projects.
Similar to plants, this type of bacteria fixes carbon based on the Calvin cycle pathway, which can use hydrogen as an electron donor to reduce carbon dioxide to organic matter. In recent years, this bacterium has been favored by researchers as a “potential stock” for a carbon fixation platform.
In 2020, the Circe project team designed a “symbiotic flora” culture model based on modified C.necator and E. coli, and produced three main products by fixing carbon dioxide: sucrose, PHA and lipochitooligosaccharides.
According to the product function division between steps, the technology is mainly divided into two test parts: the
first part uses the “symbiotic flora” to fix carbon dioxide to increase the yield while the product sucrose can be used as feed for heterotrophic bacteria; the
second part , Based on the fermentation process of modified strains to manufacture the required products.
First, by expressing sucrose synthase related to cyanobacteria to construct metabolic pathways, the research team designed a symbiosis system based on E. coli and C.necator to increase sucrose production.
Metabolic pathway based on engineered C.necator
Subsequently, the research team designed the PHA production process and selectively changed the product composition by combining different thioesterases and PHA synthetase to directly produce copolymers from carbon dioxide. In addition, the research team also designed a route for C.necator to use carbon dioxide to produce LCOs, which is a plant growth promoter.
By comparing the data, it is found that the sucrose yield obtained by the symbiotic colony system is 2 to 3 times the yield of the C.necator cultivation system alone; compared with the yield of the wild-type strain, it is increased to 30 times. Researchers speculate that this result may be due to the thermodynamics of heterotrophic bacteria being effectively utilized by autotrophic bacteria.
At the end of the first part of the experiment, the research team used Escherichia coli in the symbiotic colony to successfully produce violet and carotene. This will lay the foundation for achieving longer-term production goals.
In addition, the production of PHA has been further improved after pathway modification, and the cell dry weight ratio of the product has increased to 30% to 60% from the previous single digit depending on the composition of the product. The produced LCOs has a titer of 1.4 mg/L, which is equivalent to the yield of its natural source of Brachyrhizobium. The research team applied the obtained LCOs to germinated seeds and corn plants and observed increases in various growth parameters.
During the research process, free fatty acids produced based on the PHA synthesis pathway attracted the attention of the research team. The value is that the use of modified strains that can express different enzymes will be able to synthesize specific fatty acid molecules of various chain lengths.
By using different fatty acid molecules such as short-chain, medium-chain, and long-chain, Circe will be able to imitate and produce different types and sources of fat molecules, such as plant-derived cocoa butter, linseed oil, or animal-derived milk fat, etc. , And then used to prepare delicious and climate-friendly food.
This product molecule that can be precisely designed and manufactured gives Circe the confidence to enter the food industry. The research team said that after proper design and modification, this type of bacteria can produce any number of various products under suitable growth conditions. Its potential products include, but are not limited to: alcohols, fatty acids, alkanes, polymers and amino acids of various chain lengths.
”Our vision for the company is to produce different fats according to the needs of the market. Tell us which fat you want, and we will design microorganisms to make that molecule.” said Shannon Nangle, the company’s CEO. Currently, the company has applied for patents on metabolic pathways and processes and other technologies.
On the whole, this research not only proves the versatility of its modified strains for production, but also explores the linear production from carbon dioxide to the final product.
At this stage, how to achieve high-efficiency, low-cost, and sustainable supply of sugar raw materials has become a major bottleneck problem in the development of bioenergy and biorefinery.
Through gene editing and metabolic engineering, Circe has constructed a more concise metabolic production pathway. According to reports, the company will first introduce triglycerides (TAG) that can replace milk fat. Another short-term production target is PHA, which is used to make biodegradable plastic packaging, textiles and personal care ingredients.
In the future, its symbiotic colony production system may be able to produce more biological products in a purely “self-sufficient” manner.
Researches in domestic universities have made progress repeatedly, and large-scale production is to be tested
In my country, the autotrophic fermentation technology based on C.necator strain has also received continuous attention from researchers.
In 2018, Song Hao and others from the School of Chemical Engineering and Technology of Tianjin University constructed a microbial electrosynthesis (MES) reaction system based on C.necator H16 to increase the production of poly-3-hydroxybutyrate.
In 2019, the Bi Changhao team of the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, constructed a biological pathway for the synthesis of fatty acids from C.necator bacteria. Its research was supported by the Chinese Academy of Sciences’ key deployment of the “Carbon dioxide artificial biotransformation” project. In 2020, the team has improved the strain to increase the production of polyhydroxybutyrate.
There is no doubt that the promotion and use of biological products based on fermentation technology will help reduce the consumption of resources, land and energy in animal husbandry, breeding, and agricultural production. In addition, the “carbon fixation” technology is gaining momentum. Regardless of whether it is due to increasingly severe environmental issues or from the perspective of relevant favorable policies launched by countries around the world, this field will become more popular.
For consumers, the risks of biosynthetic foods are still unclear at this stage. However, based on the theory of fermentation synthesis, this type of food can guarantee sufficient protein, nutrients and wonderful flavor, and it will also have obvious advantages in the control of cholesterol, hormones and antibiotics. In the future, the elderly, allergic people and vegetarians may be able to choose this kind of personalized products according to their needs.
Prior to this, Circe will first focus on solving the problem of expanding production scale.
Like most studies, the fermentation process of Circe was born in a laboratory flask. So far, the production scale of the process has been upgraded to a fermentation tank with a capacity of 10 liters.
In this regard, Dr. Luo Yu, the founder of IKELAI Biotechnology, who focuses on the research of biocatalysis and synthetic biology methods, said that Circe will respond to the safety of the system, rationally cultivate and control a large number of microorganisms and Its metabolites will become an important issue.
Since the production environment of C.necator requires a stable and strict ratio of carbon dioxide, hydrogen and air, after the expansion of the device, the risk of explosion caused by hydrogen will become the first limiting factor.
In addition, the permissible survival density and growth cycle of the fermentation bacteria are also difficult. Although the symbiotic colony system shows the advantages of simultaneously benefiting two kinds of bacteria, it is used in industrial production, the growth density and survival period of microorganisms will involve cost issues. However, the growth cycle of wild-type C.necator itself is longer.
Finally, although the coordinated production of multiple strains can be achieved technically, the ensuing problems also include more complex intermediate products and whether continuous production can be achieved under the condition of continuous accumulation of metabolites. Including the subsequent more complicated separation and purification steps, which will be closely related to cost issues, and are also the main reason why this type of technology is currently limited in industrial applications.
However, Luo Yu also said that based on microbial fermentation technology, it can directly synthesize the same molecules as natural animal and plant products. This technology has repeatedly achieved breakthroughs in recent years. There are gratifying progress in both synthesis efficiency and synthesis range. In addition, the national policy has long been “preferring” sustainable development technology companies, and synthetic foods will have great market demand and development space under the requirements of relevant regulations and standards.
”If it can’t be scaled up, it has no value.” Circe’s Chief Scientific Officer Marika Ziesack said that the next step for the research team is to continue to expand the response, and eventually form the ability to cooperate with carbon dioxide supply companies (such as various high-emission factories). And directly raise the scale of microorganisms
