Evaluation of The Stability of Nitrogen-Fixing Bacterial Co-cultures under Different Cultivation Conditions

Abstract

The Haber-Bosch process is a widely used method for industrial nitrogen fixation, converting nitrogen gas (N₂) into ammonia (NH₃). However, it is energy-intensive, requiring high temperatures and pressures. Biological nitrogen fixation (BNF) may offer a sustainable alternative, utilizing bacteria like Azotobacter vinelandii to convert nitrogen gas into ammonia. Co-culture systems combining A. vinelandii with microorganisms like Pseudomonas putida could support nitrogen-dependent bioproduction. A. vinelandii showed that it can grow and produce ammonium in both Burk’s medium and M9 medium without ammonium supplementation, although the microorganism requires more time adaptation in M9 medium without ammonium. Furthermore, the bacteria can produce more ammonium in stationary phase without carbon sources. Below 1.25 mM of ammonium, P. putida has a slow growth rate, while in supplementation of 5 mM of ammonium P. putida has the highest growth rate. Two cultivation strategies were evaluated for the ability of P. putida to proliferate using ammonium produced by A. vinelandii. First, a sequential growth strategy was evaluated, where P. putida sustain growth in spent medium from A. vinelandii supplemented with glucose and trace elements. Secondly a simultaneous growth strategy was evaluated, in which a mix of A. vinelandii and P. putida formed a synthetic co-culture. Using flow cytometry and plate count methods, it was found that both strategies are possible, and that P. putida indeed could proliferate using ammonium supplied by A. vinelandii.

Popular Abstract

Who are superheroes?

Could you imagine two superheroes can tackle bigger problems if they work together?
Many industries currently use fossil fuels to produce chemicals like ammonia, which requires a lot of energy. However, there's a growing demand for renewable alternatives. Industrial biotechnology offers a solution by using microorganisms to produce chemicals sustainably. The process will be more sustainable following the principles of green chemistry. Industrial biotechnology utilizes microorganisms in the process to produce chemicals, or uses products from microorganisms, for example, enzymes. Another alternative could be to use nitrogen-fixing bacteria. In the process, bacteria will be able to convert air into ammonium through their metabolism.
Two microorganisms are trying to solve a big problem. Not only do they try to reduce the production of synthetic ammonia, but they are also trying to produce valuable chemicals, which can contribute to reducing dependence on fossil fuels. One bacterium used is called Pseudomonas putida, which can produce valuable chemicals, for example, muconic acid, which can be used as a raw material for biopolymers. Unfortunately, P. putida needs ammonia to produce the valuable chemicals, but it is better if it could be generated using non-fossil resources. Another bacterium that can produce ammonium is called Azotobacter vinelandii. Perhaps P. putida can get help from this bacterium to supply ammonium. In this project, we have evaluated how they work together.
A. vinelandii requires good food (optimal medium) and needs time to produce a lot of ammonium. On the other hand, P. putida will grow and probably produce the valuable chemicals when there is enough ammonium in their food. In my study, the growth and ammonium production in different types of media were evaluated. We found that specific mineral media with the right combination of salts, pH, and sugars allowed both microorganisms to grow. Furthermore, there are two ways the two bacteria can complement each other.
In the sequential growth operation, A. vinelandii produced ammonium and P. putida used it as a medium in a subsequent cultivation step. For this option, another carbon source and trace elements needed to be added. The results showed P. putida could grow, although the growth was slow. A low concentration of ammonium was probably a big challenge in this operation.
In the simultaneous growth operation, by using advanced instruments, the amount of those bacteria could be counted. A. vinelandii and P. putida were distinguished by using advanced technology called flow cytometry. My study revealed that P. putida indeed could grow in the presence of A. vinelandii that supplied the ammonium.
In conclusion, the results of the project point to the ammonium-producing A. vinelandii being a possible alternative to synthetic ammonium as a nitrogen source to support the growth of P. putida. However, further optimization is needed before it is a viable option for industrial biomanufacturing.
Will the superheroes achieve their goal together?

• author: Syaadah, Wida
• supervisor: Magnus Carlquist; Khaled Youssef
• 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, P. putida, ammonium, co-culture, flow cytometry, applied microbiology
• language: English
• id: 9198829
• date added to LUP: 2025-06-30 13:59:49
• date last changed: 2025-06-30 13:59:49