There are several photosynthetic and non-photosynthetic microorganisms that can produce hydrogen gas namely, green algae, cyanobacteria, photosynthetic bacteria and dark fermentative bacteria Sporanaerobium hydrogeniformans which produces hydrogen as a byproduct of its metabolism. Firmicutes and Bacteroides are the most prevalent hydrogen-producing bacteria and account for 92% of bacterial species in the human large intestine, where 51% are Firmicutes and the other 41% are Bacteroides. Microbes, such as green algae, consume water in the presence of sunlight and produce hydrogen as a byproduct. a much sought-after green energy source. Its production takes place in the unique catalytic center of the unicellular algae and is only possible if certain cofactors of the relevant proteins are present. Photosynthesis in cyanobacteria and green algae splits water into hydrogen ions and electrons. A new genus of anaerobic lignocellulose-degrading bacteria found in the hot spring which has the ability to produce hydrogen gas as a byproduct of its metabolism could be a source of clean and renewable energy. Escherichia coli is capable of producing hydrogen under anaerobic growth conditions.

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Isolation Methodology

The isolation of H2PB typically involves the following steps:

1. Sample Collection

• Environment selection: Hydrogen-producing bacteria can be found in various environments, including anaerobic digesters, soil, sewage sludge, and aquatic sediments.
• Sample collection: Collect samples aseptically to prevent contamination.

2. Enrichment Culture

• Media preparation: Prepare a suitable enrichment medium, such as Reinforced Clostridial Medium (RCM), containing a carbon source (e.g., glucose, acetate) and nitrogen source.
• Inoculation: Inoculate the enrichment medium with the collected sample.
• Incubation: Incubate the culture under anaerobic conditions at an appropriate temperature (e.g., 37°C) for a specific period.

3. Isolation of Pure Cultures

• Serial dilution: Perform serial dilutions of the enriched culture to reduce the microbial load.
• Plate inoculation: Spread the diluted cultures on selective agar plates (e.g., Reinforced Clostridium Agar) to isolate individual colonies.
• Colony purification: Pick individual colonies and re-streak on new agar plates to obtain pure cultures.

4. Hydrogen Production Screening

• Hydrogen production assay: Screen the isolated colonies for hydrogen production using gas chromatography (GC) or other suitable techniques.
• Positive selection: Select colonies exhibiting high hydrogen production rates for further characterization.