Method for automated high performance closed batch cultivation of gas-utilizing methanogens

Area of Science:

• Microbial biotechnology
• Bioprocess engineering
• Synthetic biology

Background:

• Advancing microbial cell factories for gas fermentation requires understanding their physiological and biotechnological limits.
• Current methods often face limitations in controlling headspace gas pressure, hindering optimal microbial growth and product formation.
• Automated control systems are crucial for precise regulation in closed cultivation systems.

Purpose of the Study:

• To construct and operate a novel Gas and Pressure Controller (GPC) for automated control of headspace gas pressure in closed cultivation bottles.
• To investigate the physiological and biotechnological characteristics of autotrophic, hydrogenotrophic methanogenic archaea under precisely controlled gas conditions.
• To quantify novel physiological limits by eliminating gas limitations during growth and methane formation.

Main Methods:

• Development and implementation of the Gas and Pressure Controller (GPC) for automated gassing, sparging, monitoring, and regulation of headspace volume in real-time.
• Cultivation of four autotrophic, hydrogenotrophic methanogenic archaea (Methanothermobacter marburgensis, Methanotorris igneus, Methanocaldococcus jannaschii, Methanocaldococcus villosus) using the GPC.
• Testing the GPC's functionality with Methanococcus maripaludis for long-term, autonomous cultivation.

Main Results:

• Unprecedented high maximum specific methane productivity (qCH4) values were determined for the tested archaea, including M. marburgensis (169.59 ± 12.52 mmol g−1 h−1) and M. igneus (420.21 ± 89.46 mmol g−1 h−1).
• The qCH4 for M. marburgensis was over 10-fold higher than in conventional closed batch cultures and comparable to fed-batch systems.
• The GPC demonstrated reliable, safe, and autonomous long-term functionality with M. maripaludis.

Conclusions:

• The novel GPC device enables optimal headspace pressure control, offering flexibility for various gas-fermenting biotechnological processes.
• The device facilitates near-optimal cultivation conditions in semi-continuous closed batch mode, aiding in the analysis of limiting factors.
• The GPC supports automated biomass production of autotrophic, hydrogenotrophic methanogens.