Exploring Co-cultivation Strategies of Nitrogen-Fixing Bacteria and Recombinant Yeast for Sustainable Biomanufacturing

Abstract

To reduce reliance on energy-intensive, chemically synthesised nitrogen sources in microbial cultivation, we investigated the integration of the nitrogen-fixing bacterium Azotobacter vinelandii into a bioconversion process with the recombinant yeast Saccharomyces cerevisiae. Our objective was to develop a more sustainable approach by utilising biological nitrogen fixation to supply ammonium directly within co-cultures, thereby reducing external nitrogen inputs and associated energy costs.
We began by assessing five A. vinelandii strains performance in co-culture with S. cerevisiae. Co-cultivation experiments were conducted in defined Burk’s media modified to accommodate both microbes, with and without supplementary ammonium, allowing us to evaluate A. vinelandii’s ability to sustain yeast growth under nitrogen-limiting conditions. Flow cytometry enabled us to track population dynamics and physiological changes over time, while complementary plate-count assays provided quantitative estimates of viable cell numbers.
Our results revealed that one engineered strain of A. vinelandii exhibited significantly higher ammonium output and co-culture growth. Under ammonium-free conditions, S. cerevisiae growth reached levels comparable to ammonium-supplemented controls when paired with A. vinelandii, indicating effective nitrogen providing. Moreover, the ammonium supplied by A. vinelandii was sufficient to support vanillylamine production. The resulting titer and conversion were comparable to those of control culture supplemented with high concentrations of ammonium.
Finally, we compared three flask-scale cultivation strategies to assess their effectiveness. We analysed metabolite profiles and ammonium uptake rates to elaborate on possible metabolic interactions of each mode. Simultaneous cultivation offered operational simplicity but suffered from competitive resource depletion and required a robust detection method to monitor subpopulation. Sequential cultivation supported yeast growth, although further optimisation is required. Sequential supernatant allowed greater control over bioconversion but required downstream processing, suggesting a promising route for scale-up.
In summary, this study demonstrates the feasibility of integrating engineered A. vinelandii with S. cerevisiae to create a self-sufficient nitrogen supply within the bioprocess, and identifies sequential supernatant as a more feasible strategy for upscale industrial application.

Popular Abstract

A New Era in Biomanufacturing: Microbes Helping Microbes
Producing ammonia for growing microbes in labs and biotechnology factories usually requires a lot of energy and chemicals. In this project, we explored a more natural and sustainable way to provide ammonia by using a helpful soil bacterium called Azotobacter vinelandii. This bacterium can capture nitrogen from the air and turn it into a form that other organisms can use—like a natural provider.
We worked with both wild-type and genetically modified versions of A. vinelandii and tested how well they could grow alongside baker’s yeast (Saccharomyces cerevisiae), a well-known production strain used in food, medicine, and biofuel industries. In nature, these two organisms do not live together and have their own niches. Our goal was to investigate if the bacteria could feed nitrogen, in the form of ammonium, to the yeast without needing extra N-containing chemical inputs.
After evaluating five different bacterial strains, we found one that stood out—it grew well with yeast and still produced more ammonium. When we removed chemical nitrogen from the system, the yeast still managed to grow and produce the desired target products—thanks to the bacteria, showing that this synthetic co-culture really works.
We also evaluated three different ways of growing the two microbes together to find the best strategy. Co-cultivation, where the microbes grow together, was very interesting and allowed for their growth. However, it requires a robust monitoring method to accurately quantify their proportions, as well as live and dead cells in the co-culture population. Unfortunately, this may require special optimisation. The method where we first grew the bacteria, then transferred their ammonium-rich spent media to the yeast, worked especially well. Although it takes a bit more time, it offers better control and could be useful for large-scale production in the future.
Overall, this study shows that combining natural nitrogen-fixers like A. vinelandii with yeast could help make industrial bioprocesses more sustainable and less dependent on fossil-derived chemicals.

• author: Haniyya, Haniyya
• supervisor: Magnus Carlquist
• organization: Applied Microbiology; Biotechnology; Biotechnology (MSc)
• course: KMBM01 20251
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: A. vinelandii, nitrogen-fixing, S. cerevisiae, synthetic co-cultures, vanillylamine, applied microbiology
• language: English
• id: 9202004
• date added to LUP: 2025-06-19 11:20:03
• date last changed: 2025-06-19 11:20:03