Biomanufacturing holds the promise of using the power of biology to produce sustainable replacements for numerous petroleum-derived items we use in our daily lives. The biomanufacturing industry often relies on batch/fed-batch fermentation, a process in which microbes are cultivated in a bioreactor to yield a specific product in a single production cycle (i.e. grow, rinse, repeat). However, batch fermentation can be inefficient and expensive, which keeps bio-based products from being cost-competitive. Addressing these challenges in fermentation is crucial for unlocking the full potential of biomanufacturing.
Scientists at the Advanced Biofuels and Bioproducts Process Development Unit (ABPDU) and Pow.Bio have now successfully demonstrated a fermentation platform that can drastically reduce CapEx and OpEx, paving the way for “high-yield, low-cost” 2nd generation biomanufacturing.
Pow.Bio has developed a platform for continuous fermentation, in which a fermentation process is run over longer periods of time and product is continuously extracted.
“Using continuous fermentation, you can make more product in the same amount of time as traditional batch fermentation,” said Maggie Stoeva, senior scientist at Pow.Bio. “You don’t need to invest in bigger reactors. This helps bring down production costs.”
Continuous biomanufacturing is challenging, hence why it has yet to be widely adopted. Bioreactors running continuous fermentations often become contaminated with unwanted microbes, and the microbial production hosts can experience genetic instability, both of which decrease productivity.
Pow.Bio’s two-chamber continuous fermentation platform separates growth and production to alleviate these issues. In this system, one bioreactor is exclusively used for the growth of microbes. Then, these microbes are fed into a separate bioreactor dedicated to production of the target molecule. Careful media formulation represses growth in the second reactor, limiting the outgrowth of non-productive or contaminant cells. Product is regularly extracted and microbes are recycled back into the second bioreactor to maintain productivity.
The company partnered with ABPDU to test and scale up this process, as part of a collaboration funded by the Department of Energy’s Bioenergy Technologies Office. Mevalonate, a precursor to several commodity chemicals, was used as an example product, using a genetically engineered E. coli strain provided by Visolis. Transferring the process to ABPDU required implementation of customized bioreactor controls necessary to maintain a constant density of cells in the growth chamber, ensuring the cells were always maintained at their maximum growth rate. A membrane system was then deployed to retain cells in the production chamber, increasing cell density and overall productivity of the bioreactor.
“The ABPDU was our partner throughout this work and answered our questions about what this process would look like at a different facility,” Stoeva said. “Having their expertise to work through that was very helpful.”
The collaborative effort successfully scaled up the process to 30 L and maintained high productivity over 500 hours. The proof-of-concept process demonstrated contamination control upon repeated intentional contamination, and generated four times more product compared with the optimized fed-batch fermentation.
“Working on continuous fermentation has been a learning experience for all of us. Our collaboration enabled us in not only getting the complex engineering system up and running, but also advancing cutting edge science in industrial biomanufacturing,” said David Chang, process engineer at ABPDU. “It is really great that I was able to develop a technology that can be applied globally.”
In addition, the process ran just as smoothly with sugars produced from corn stover, a more sustainable alternative to traditional corn or sugarcane-derived feedstocks.
“This project came with a lot of unique challenges, but the results were really encouraging and ultimately worth the effort,” said Laura Fernandez, a senior process engineer at ABPDU who led the fermentation effort in the lab.
“In an industry with thin margins, being able to operate continuously at maximum productivity is potentially transformative,” said Eric Sundstrom, staff scientist at ABPDU. “Continuous fermentation has long been thought of as theoretically ideal but too challenging to implement in practice. This project helps demonstrate that those challenges are not insurmountable.”
Building off this progress, Pow.Bio has scaled the process up further to a 200 L production capacity at their facility.
“That initial scale-up we did at ABPDU was on the road to this larger scale that we’ve now done in-house,” Stoeva said. “That was very important for proving our technology.”
The company’s next step is to test their continuous fermentation process in even larger reactors and in other molecules to demonstrate that it can work across a broad range of hosts and products. They are currently partnering with the Agile BioFoundry to adapt their process to a Rhodosporidium toruloides host producing fatty alcohols, which are useful in a wide range of products. The ABPDU will be involved in this project to screen R. toruloides strains and select candidates to transition to Pow.Bio’s system.
Pow.Bio’s long-term goal is to help enable continuous fermentation for the biomanufacturing industry.
“We aim to share our technology through partnerships and in-house capabilities to run continuous fermentations at a meaningful scale for biomanufacturing companies,” Stoeva said.