Fed‑Batch PHB Production from Glycerol using the LAMBDA Minifor Fermenter-Bioreactor

Polyhydroxybutyrate (PHB) is a biodegradable bioplastic produced intracellularly by many bacterial species when an excess of carbon is present under nitrogen-limited conditions. Due to its biodegradability and material properties comparable to conventional plastics, PHB is widely studied as a sustainable alternative to petroleum-based polymers.

In a recent study conducted at Universidad Autónoma Metropolitana (Mexico), researchers investigated PHB production from glycerol, a major by-product of biodiesel production, using controlled fed-batch fermentation.

The goal was to evaluate PHB production performance under well-defined and reproducible process conditions.

To ensure stable cultivation and precise environmental control, the experiments were performed using the LAMBDA Minifor laboratory fermenter.

Reference: León Santiesteban, H. H., Aguirre Aguilar, J., Beltrán, D. Á., Contreras Larios, J. L., Reyes Chilpa, R., García Martínez, J. C., & González Brambila, M. M. (2026). Bioplastic Production in Circular Economy Paths with Glycerol and Whey. Catalysts, 16(2), 178. doi.org/10.3390/catal16020178

Use of the LAMBDA Minifor Fermenter

The LAMBDA Minifor fermenter was used as the primary bioreactor for the cultivation of Bacillus megaterium and Bacillus subtilis, two known PHB-producing microorganisms. 

Each strain was cultivated separately under identical fed-batch fermentation conditions, enabling direct comparison of PHB production performance.

The Minifor enabled stable and reproducible control of key fermentation parameters, including:

• Temperature control at 30 °C

• Continuous aerobic cultivation

• Gentle mixing  to maintain homogeneous conditions

• Long-duration fed batch fermentation runs up to 180 hours

• Operation under a high carbon-to-nitrogen (C:N) ratio, promoting intracellular PHB accumulation

These controlled conditions are critical for studying bioplastic production, as PHB synthesis occurs primarily when microbial growth slows and excess carbon is redirected toward polymer storage.

Fermentation Method

Fed-batch fermentation was performed using the LAMBDA Minifor laboratory fermenter with a working volume of approximately 1.7 L.

The culture medium contained:

• Glycerol and glucose as carbon sources

• Ammonium sulfate as a limiting nitrogen source

• A high C:N ratio (~210–220:1) to induce PHB synthesis

Cultivation was carried out under controlled aerobic conditions at 30 °C. Separate fermentations were performed using Bacillus megaterium and Bacillus subtilis

Additional experiments were conducted with and without trace elements in the medium. 

Limiting trace elements increased metabolic stress, promoting intracellular PHB production rather than continued biomass growth.

Samples were collected during fermentation to monitor:

• Biomass concentration
• Substrate consumption
• PHB production

Bacillus megaterium showed the highest productivity, reaching PHB concentrations of approximately 3.1 g L⁻¹. PHB accumulation occurred mainly during the stationary phase, when microbial growth slowed and carbon was redirected toward polymer synthesis.

Bacillus subtilis also produced PHB under the same fed-batch fermentation conditions, although at lower concentrations.

In both cases, PHB synthesis increased once active biomass growth slowed.

Limiting trace elements increased PHB production in both strains by promoting intracellular polymer accumulation rather than biomass formation.

This highlights the importance of controlled nutrient limitation and stable process conditions for efficient intracellular biopolymer production.

Conclusion

The LAMBDA Minifor laboratory fermenter provides a reliable platform for:

• Fed-batch PHB production
• Bioplastic research
• Microbial fermentation studies

Its ability to maintain stable temperature, aeration, and mixing over extended cultivation periods enables reproducible and comparable results across experiments.

The system is particularly suited for process development where precise control of nutrient limitation and reproducibility are critical.

Applications include:

• PHB and bioplastics research
• Fed-batch fermentation process development
• Microbial strain evaluation
• Laboratory-scale bioprocess optimization