Engineering Cupriavidus necator for medium-chain-length polyhydroxyalkanoate production under two-stage fed-batch fermentation

Plastics that break down naturally in the environment have long been a goal for scientists seeking to replace the persistent materials that clog landfills and oceans. Among the most promising candidates are a family of biodegradable plastics called polyhydroxyalkanoates, or PHAs. These are not synthetic chemicals manufactured in a lab, but rather natural storage materials that certain bacteria produce when they have more food than they need. Think of these bacteria as tiny factories that stockpile energy in the form of plastic granules inside their cells. The properties of the resulting plastic depend heavily on the building blocks, or monomers, that make up the chain. Shorter building blocks create a rigid, brittle material that snaps easily, while longer blocks introduce flexibility and stretch, making the plastic more like rubber. For decades, researchers have struggled to get the most efficient bacteria to produce these longer, more flexible blocks, limiting the range of applications for these eco-friendly materials.

A team of researchers at the National Science and Technology Development Agency in Thailand has taken a significant step forward in solving this problem. They worked with a well-known bacterium called Cupriavidus necator, which is famous for its ability to turn various waste products into plastic. However, this native bacterium naturally produces only the short, brittle building blocks. To change this, the scientists engineered the bacterium by adding a specific gene from a different type of bacteria, Pseudomonas putida, which is known for its ability to assemble the longer, flexible blocks. This genetic modification created a new strain, which the researchers named mcl-C19. When this new strain was fed a mixture of molasses and used cooking oil, it began to produce a hybrid plastic containing both the short, rigid blocks and the longer, flexible ones. This combination resulted in a material with a wider range of properties, potentially making it useful for many more applications than the brittle versions produced by the original bacteria.

The researchers did not stop at simply creating the new strain; they also developed a specific feeding strategy to maximize production. Bacteria need a balanced diet to grow their cells, but they only start stockpiling plastic when they are starved of certain nutrients while still having plenty of carbon. To exploit this, the team used a two-stage process. In the first stage, they fed the bacteria a nutrient-rich mixture to help them multiply rapidly and build a large population. Once the population was high, they switched to the second stage, where they added pulses of carbon-rich food—specifically molasses and used palm oil—without adding extra nitrogen. This created an unbalanced environment that forced the bacteria to divert their energy into making plastic instead of growing new cells. By carefully controlling this process in a large tank, the engineered bacteria accumulated a massive amount of plastic, reaching a concentration of 13 grams per liter, which represented nearly half of the total weight of the cells.

What makes this achievement particularly notable is the diversity of the plastic produced. The engineered bacteria did not just make one type of long block; they created a complex mixture containing six different types of building blocks, ranging from short chains of four carbon atoms to longer chains of ten. The specific mix of these blocks changed depending on how the bacteria were fed. When the researchers used a simple batch process where everything was added at once, the plastic had one composition. When they used the two-stage pulse-feeding method, the proportions of the different blocks shifted. This suggests that scientists can fine-tune the physical properties of the plastic—making it stiffer or more stretchy—simply by adjusting the feeding schedule and the type of waste food used. The study confirmed that using a mixture of molasses and used palm oil was the most effective way to generate the desired long-chain blocks, with the new strain producing four times more of these flexible components than the unmodified parent strain.

The work also demonstrated that this process can be scaled up from small flasks to large industrial bioreactors without losing efficiency. The results in a two-liter tank were nearly identical to those in smaller containers, indicating that the method is robust enough for larger production. While the amount of plastic produced was high, the researchers noted that the percentage of the cell weight made up of plastic could still be improved. They acknowledged that further genetic tweaks might be needed to push the bacteria to convert even more of their resources into plastic rather than other cellular components. However, the current results show that by combining a simple genetic change with a smart feeding strategy, it is possible to turn common waste materials into a versatile, biodegradable plastic with properties that can be tailored for specific needs. This approach offers a practical pathway toward a bio-based economy where plastic production relies on renewable resources and generates materials that can safely return to the environment.