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Light-Controlled Fermentations for Microbial Chemical and Protein Production

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Optimizing dark fermentation for hydrogen production: lessons from Thermoactinomyces mirandus.

Selina V Haller^1,2, Luise Ebert², Mira Mutschlechner²

¹Department Microbiology, Universität Innsbruck, Innsbruck, Austria.

Frontiers in Microbiology

|July 11, 2026

Summary

Optimizing dark fermentation (DF) with Thermoactinomyces mirandus significantly boosts biological hydrogen (H2) production. pH control is crucial for maximizing H2 yields from biomass, offering a renewable energy pathway.

Keywords:

biosynthesis carbon-to-nitrogen ratio competing pathways inhibitory factors lactose metabolism pH control process optimization substrate utilization

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Area of Science:

Biotechnology and Bioengineering
Renewable Energy
Microbial Physiology

Background:

Biological hydrogen (H2) production via dark fermentation (DF) is a promising renewable energy strategy.
Process efficiency in DF is often limited by strain-specific characteristics and suboptimal operating conditions.

Purpose of the Study:

To characterize the fermentation physiology of the thermophilic anaerobe Thermoactinomyces mirandus.
To optimize small-scale H2 production by T. mirandus through screening various conditions.

Main Methods:

Batch cultivations were performed at 52 °C to screen carbon sources, nitrogen sources, C/N ratios, and inhibitory factors.
Small-scale, closed-flask pH control was implemented and compared to uncontrolled conditions.
Fermentation product profiles and H2 production rates were analyzed under different conditions.

Main Results:

Yeast extract as a nitrogen source and fructose, xylose, or lactose as carbon sources supported the highest H2 yields.
Implementing pH control at pH 7.2 increased H2 yield 4.2-fold and improved lactose consumption to 99.4%.
An optimal pH range of 7.5-8.0 was identified for maximum H2 production rate (VHPR), and higher lactose loads increased VHPR but decreased yield.

Conclusions:

pH control is a critical factor for significantly enhancing H2 yield in T. mirandus fermentation.
Quantitative operating windows for T. mirandus were defined, including optimal pH and substrate loading.
The study provides a transferable workflow for optimizing DF processes and mitigating inhibitory factors.