A cow’s digestive system may seem like an unlikely model for producing sustainable chemicals. But inside the rumen, the large fermentation chamber in a cow’s stomach, microbes routinely do something that remains difficult to reproduce in a laboratory: work together to break down tough plant material and extract useful chemicals from it.

A research team led by UC Santa Barbara chemical engineering and bioengineering professor Michelle O’Malley has received an $800,000 grant from the National Science Foundation to understand how those microbial partnerships work, then recreate and engineer them in the laboratory.

The three-year project will recreate part of that microbial teamwork in the laboratory, pairing anaerobic fungi like those found in a cow’s digestive system with bacteria that consume the fungi’s byproducts. The fungi thrive without oxygen and are exceptionally good at breaking down tough plant material. The bacteria then convert compounds released by the fungi into energy-rich molecules known as volatile fatty acids, including medium-chain fatty acids (MCFAs) and other useful products that can serve as building blocks for fuels, polymers, coatings, and other products.

“What we’re really trying to do with this grant is to develop a basic-science understanding of how cross-kingdom microbial partnership actually happens,” said O’Malley. “We’re building protocols and experimental tools so that researchers can know whether they have formed a microbiome successfully, rapidly screen it for activity, and understand why they see certain outcomes.” 

Letting Microbes Specialize
Much of modern biomanufacturing relies on engineering one microorganism to perform several jobs. Nature, on the other hand, often divides the work among specialists.

In the rumen, anaerobic fungi attack lignocellulose, the fibrous material that makes plant stems and leaves tough, and release lactate, acetate, ethanol, and other fermentation products as they break the fiber down. Nearby bacteria then consume those compounds and convert them into fatty acids with potential uses in fuels and materials. 

“Fungi and bacteria are working together to spread the load of creating these products,” O’Malley said. “We want to peer under the hood of the microbes and understand the metabolic foundations that lead to particular outcomes.” 

The team will build simplified versions of those communities in the laboratory, changing the organisms and growth conditions to see what makes the partnerships stable and productive.

David Valentine, a distinguished professor of earth science and co-principal investigator, said that studying microbes individually can leave out a fundamental part of their biology.

“If we don’t know how a microbe interacts with a community, do we really understand the individual microbe?” Valentine said. “Microbes have been evolving for billions of years in communities, and then we come along and study them in isolation. Of course, we are going to miss all the back and forth between the community members this way.” 

Valentine’s group will help track what passes between the fungi and bacteria, examining what each organism consumes, what it produces, and how those exchanges affect the community.

“I expect the research will take us in exciting analytical directions beyond the routine analysis of reactants and products,” he said.

The goal is not simply to find a combination that works, but to understand why it works well enough that researchers can reproduce the result elsewhere.

A Biofoundry Built for Difficult Microbes
Much of that work will take place in UCSB’s Biofoundry for Extreme and Exceptional Fungi, Archaea, and Bacteria, known as ExFAB.

The NSF-supported biofoundry was established to make unusual and difficult-to-study microorganisms more accessible. In particular, ExFAB allows researchers to investigate microbes that cannot tolerate oxygen or are hard to grow and genetically manipulate using conventional methods.

“We have some great tools available on campus to track consumption, production and exchange of molecules, including the mass spectrometer suite at the ExFAB Biofoundry and the isotope facilities at the Marine Science Institute,” said Valentine, co-principal investigator of ExFAB.  

Directed by O’Malley, ExFAB combines liquid-handling robots, incubators, analytical instruments, and a flow cytometer inside a specially designed oxygen-free chamber. The setup allows researchers to run many carefully controlled anaerobic experiments at once rather than working through cultures one by one. 

For this project, the team plans to test more than 500 combinations of nutrients and growth conditions and develop fluorescent tools to track how fungal and bacterial populations change over time. 

“This is much less about reaching a particular rate or yield with a microbiome, and much more about developing tools for the microbiome community, which will be able to apply to this system and many others,” said O’Malley.  “I’m most excited that we’re making a platform that nobody has made before, so that we can eventually leverage AI on all of this data to get better, faster outcomes. But this effort is really about the measurement and creating the tools.”  

The team will also develop genetic tools to improve both sides of the partnership. Researchers will modify anaerobic fungi to change what they provide to their bacterial partners and identify bacterial genes that help stabilize the community and influence the fatty acids produced. 

The collaboration also includes UC Berkeley researchers Benjamin Rubin and Brady Cress and UCSB project scientist Elaine Kirschke. 

Taking Teamwork to the Next Level
The immediate project centers on turning plant and agricultural waste into useful medium-chain fatty acids. Its broader value may lie in uncovering principles that can be applied to other microbial communities.

“I think it will be a win if we can decipher interactions between microbes that boost the reliability and efficiency of product production,” Valentine said. “Ideally our research will hit upon general rules that can be applied to improve anaerobic processes more generally.” 

Those rules could extend beyond biomanufacturing. NSF notes that the methods and models developed through the project could also contribute to understanding other microbial communities, including the human gut microbiome.