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Biological processes of hydrogen production have significant advantages compared to conventional thermal processes. Firstly, they are partly more sustainable, as they use renewable energy sources for production of hydrogen. Secondly, they are less energy-intensive, because most processes are conducted at moderate temperatures and pressure. Additionally, biological processes are environmentally more acceptable and CO2-neutral, they can use a wide range of substrates, as well waste materials, as feedstocks (Das et al., 2001).

The biological processes for hydrogen production can be divided into 4 systems (Levin et al., 2004):

  1. Biophotolysis
  2. Photo-Fermentation
  3. Biological CO-conversion
  4. Dark Fermentation


Biophotolysis of water can occur directly by use of green algae or indirectly by use of cyanobacteria.

During photosynthesis in green algae, water is oxygenated and oxygen is produced. In this process, electrons are generated, which are transmitted to ferredoxine. Through a reversible hydrogenase enzyme, protons are reduced to hydrogen, with aid of the electrons, adsorbed on ferredoxin. Advantageous is, that only water and light energy is necessary for this process (compare reaction equation of direct biophotolysis).

Disadvantageous is, that the hydrogenase activity is highly inhibited, because of produced oxygen. Therefore, photosynthetic production of oxygen and hydrogen have to be separated temporally and/or spatially. Using this process, hydrogen production rates of 0.07 mmol/(h.l) can be produced.

During indirect biophotolysis, cyanobacteria can produce hydrogen (compare reaction equation of indirect biophotolysis).

Cyanobacteria possess a few enzymes, involved in hydrogen production. On the one hand, nitrogenases catalyze the production of hydrogen as by-product of the nitrogen reduction to ammonium. On the other hand, hydrogenases act in two directions – hydrogen can be can synthesized and oxygenated. Maximal production rates are 0.355 mmol/(h.l).


Photo-Fermentation is another photo-synthetic process, where not-sulfuric purpurbacteria produce hydrogen. Purpurbacteria can produce hydrogen of organic acids as well as directly of biomass (compare reaction equation of photo-fermentation).

The enzyme nitrogenase catalyze the reaction. Advantageous is, that in this process, no oxygen is produced and therefore enzyme activity is not inhibited. Disadvantageous is the high nitrogenase energy consumption as well as the low conversion efficiency of light energy. Hydrogen synthesis rates of at most 0.161 mmol/(h.l) could be reached.

Biological CO-conversion

Several photo-heterotrophic bacteria can grow in the dark and use CO as sole carbon source. During production of ATP, carbon dioxide and hydrogen are released (compare reaction equation of biological CO-conversion).

Disadvantageous is the mass transfer of gaseous CO into the bacteria suspension. Maximal hydrogen production rates of 96 mmol/(h.l) are reached.


Hydrogen can be produced by anaerobic microorganisms under light exclusion by using high-carbonic substrates. This fermentations can be conducted at different temperatures: mesophilic at 25-40 °C, thermophilic at 40-65 °C, extremely thermophilic at 65-80 °C or hyperthermophilic at temperatures above 80 °C.

Compared to previous processes, not only hydrogen and carbon dioxide is produced during dark-fermentation, also traces of methane, carbon monoxide or hydrogen sulfide may be contained in biogas. Therefore it is important to control growth of methane producing bacteria. This can be achieved on the one hand by heat inactivation of the inoculum, setting low pH-values, on the other hand by high dilution rates and therefore elution of the slowly growing methanogenic bacteria (Angenent et al., 2004).

Due to different metabolic pathways with various end products, diverse theoretical hydrogen yields are possible during dark-fermentation. If acetic acid is produced as end product, a theoretical maximum of 4 mol hydrogen can be achieved from one mol glucose (compare reaction equation of dark-fermentation to acetate). This value is called Thauer-Limit (Thauer et al., 1977).

If butyric acid is the end product, the theoretical maximum halves to 2 mol hydrogen per mol glucose (compare reaction equation of dark-fermentation to butyrate). Also propionate or other reduced end products, e.g. ethanol or lactic acid can be produced and the hydrogen yield is further reduced.

Fermentations for biological hydrogen production offer several advantages for industrial application, as high productivities and growth rates can be achieved.

Dark-Fermentation depends on experimental environmental conditions, such as pH value, temperature, cell density or substrate. Therefore, an improvement of those parameters is essential for optimal production of hydrogen. Maximal hydrogen production rates of 8-121 mmol/(h.l) could be achieved, depending on different temperatures.

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