Biohydrogen is now being commercially produced as a “Biofuel”. It is the advanced biofuel, which makes the use of living biomass or organisms for its production. Biohydrogen is now considered best among all the biofuels as it accounts to fulfil all the energy demands as it can obtain from the sustainable resources like:

  • Biological splitting of the water molecule
  • By the conversion of biomass
  • By the solar thermal splitting of water

Biohydrogen is a type of biofuel like others i.e. bioethanol, biodiesel, bio-oil etc. Hydrogen can produce by both chemical and biological method. The method, by which the hydrogen is produced biologically by the use of microorganisms, in a bioreactor will refer as Biohydrogen. In simple words, we can say the biological conversion of hydrogen into biohydrogen via microorganisms is known as biohydrogen.

Content: Biohydrogen

  1. Definition of Biohydrogen
  2. Milestones in the discovery of Biohydrogen
  3. Generations of biofuels
  4. Ideal properties of hydrogen as a Biofuel
  5. Limitations of hydrogen as a Biofuel
  6. Production of Biohydrogen
  7. Factors affecting Biohydrogen production
  8. Future prospects of Biohydrogen

Definition of Biohydrogen

Biohydrogen is a biofuel which is the source of energy that uses living microorganisms for the conversion of hydrogen into biohydrogen via the fermentation and photolysis process in a specialized container or a bioreactor.

Milestones in the discovery of Biohydrogen

1939Hans GaffronDiscovered the switching of algae between the production of H2 and O2
1997Ananstasios MalisDiscovered the cause of algae to switch from producing H2 by the depletion of sulphur
2006Researchers from the University of BielefeldDiscovered large amount of hydrogen by genetically modifying the single cell Chlamydomonas reinhardtiin
2007Ananstasios MalisDiscovered the conversion of solar energy to chemical energy in tax X mutants of Chlamydomonas reinhardtiin

Generations of biofuels

There are three generations of biofuel which includes:

  1. Biofuel made from food crop and their residues termed as “First-generation biofuel”.
  2. Biofuel made from non-food crops or wastes termed as “Second-generation biofuel”.
  3. And the biofuel made by the use of microorganisms is termed as “Third-generation biofuel” or “Advanced biofuel”.

Among these three generations of biofuel, Biohydrogen comes in the category of third-generation biofuel or advanced biofuel. Third-generation biofuel has certain advantages over the first and second-generation biofuel.

The production of the first-generation biofuel has increased the price rates of the food items, whereas the third generation biofuels would not.

Second-generation biofuel required more surface area or land for the production of the biofuel, whereas advanced biofuel requires smaller areas for the production and are having the efficiency to capture solar energy 10 times to that of the second generation.

Ideal properties of hydrogen as a Biofuel

In everyone’s mind, there will be a question, that why hydrogen? So, we will discuss some ideal properties of hydrogen, that why it is used as a “Biofuel”.

There are some unique properties of hydrogen which make it ideal for the production as a biofuel:

  • Hydrogen has three times the high energy density than petrol and diesel.
  • Hydrogen is highly combustible which can use as a fuel.
  • Combustion of hydrogen only yields water which does not contribute to the gaseous pollutants.
  • The efficiency of providing energy is more, with its little use.
  • Acts as an energy carrier as hydrogen traps sunlight, wind, water as a renewable source.
  • Hydrogen can be used as a transport fuel, by storing it as a “Metal hydride”.
  • It can be easily produced by the renewable source of energy like solar, wind, water etc.

Limitations of hydrogen as a Biofuel

  • Storage of hydrogen is difficult.
  • There is a limitation of the cost of competitive technology for the production of biofuel.
  • The utilization of hydrogen is quite difficult.
  • Hydrogen can easily escape from the atmosphere to space.
  • The presence of oxygen poisons the microbes, which produce hydrogen.

Production of Biohydrogen

The production of biohydrogen is through two biological methods:

methods of biohydrogen production

  • Through the fermentation process: It involves two methods namely photo fermentation and dark fermentation.
  • Through the photolysis: It also involves two methods namely direct photolysis and indirect photolysis.


Biohydrogen can produce by the process of fermentation. The fermentation of Biohydrogen is carried out by the use of microorganisms like bacteria. The process is either carried out in the presence of light i.e. photo fermentation or in the absence of light i.e. dark fermentation.


This process makes the use of photosynthetic organisms and the additional light source. The organisms used in the photo fermentation carry out the process of photosynthesis by the use of photosystem-I only for the production of hydrogen. For the splitting of water, these organisms not only requires an additional light source but also utilize organic acids like acetic acid to generate hydrogen, by donating its electron.


In photofermentation, we can see in the equation, there is no evolution of oxygen, therefore it is a type of anoxygenic photosynthesis.

Dark fermentation

It is a process which makes the use of carbohydrate as an energy or carbon source. Dark fermentation does not require a source of light energy. Other than carbohydrate like glucose, it can also use other substrates like organic compounds, polymers (starch, cellulose etc.), algae biomass etc.

Dark fermentation is a very complex process to carry out, which requires a series of biochemical reactions.

Breakdown of glucose into pyruvate and NADH: During this step, glucose converts into pyruvate by the phosphorylation of NAD into NADH.

dark fermentation step 1

Conversion of pyruvate to acetyl CoA: The conversion of pyruvate into acetyl CoA can be catalysed by the use of two enzymes.

Catalysis by ferredoxin oxidoreductase

dark fermentation step 2

Catalysis by formate lyase

dark fermentation step 3

Reoxidation of ferredoxin: This step involves the reoxidation of ferredoxin by the Fe-fe hydrogenase enzyme.

dark fermentation step 4

Production of hydrogen

dark fermentation step 5


It makes the use of photoautotrophic organisms such as microalgae and cyanobacteria. These organisms use light as an energy source and carbon dioxide as a carbon source for the splitting of hydrogen.


Therefore in biophotolysis, the production of hydrogen is under anaerobic conditions.

Direct photolysis

It makes the use of solar light as a light source and photosynthetic algae to convert water into chemical energy or to produce hydrogen.

Direct photolysis involves two steps for the generation of Biohydrogen:

Absorption of sunlight by the photosystem-II of algae leads to the oxidation of water into electron, proton and oxygen molecules.

direct photolysis step 1

Absorption of sunlight by the photosystem-I of algae leads to the transfer of an electron to the ferredoxin and to the hydrogenase enzyme through the electron transport chain. Recombination of proton and electron for the production of hydrogen gas.

direct photolysis step 2

This process also refers as One stage photolysis, where hydrogen can be produced directly by the use of water, light energy and algae photosystem.

Indirect photolysis

This process makes the use of photosynthetic microorganisms like microalgae, cyanobacteria etc. Indirect photolysis also converts solar energy into the chemical energy by the series of two steps:

In the first step, there is the production of biomass by the photosynthetic system. The second step involves the utilization of the biomass rich in carbohydrates to produce Biohydrogen.

indirect photolysis

Indirect photolysis involves the removal of both hydrogen and oxygen at different steps during the process just to avoid the sensitivity of the hydrogenase enzyme. It involves two stages to complete a reaction, so it refers as Two-stage photolysis.

Cyanobacteria used in the process of indirect photolysis are Gloebacter sp., Synechocystic sp. Etc.

Table showing different biological processes for the production of Biohydrogen:

Biological processes for the production of hydrogenOrganisms usedYield of hydrogenEnd product
Photo fermentationPhototrophic bacteria like purple sulfur and non-sulfur bacteria0.16H2 and CO2
Dark fermentationFermentative bacteria like Clostridium, Citrobacter and Enterobacter sp.65-75H2 , CO2 and VFA
Direct photolysisGreen algae0.07H2 and O2
Indirect photolysisCyanobacteria0.36H2 and O2

Factors affecting Biohydrogen production

There are several factors affect Biohydrogen production:

Temperature: Thermophilic bacteria gives the maximum yield of hydrogen than the mesophilic bacteria. In simple words, the temperature is a factor which depends upon the type of microorganisms and substrate used for hydrogen production.

pH: Hydrogen-producing microorganisms work well below the pH of 5.5-6.0.

Hydraulic retention time: For, satisfactory hydrogen yields, the Hydraulic retention time should be in between 8-14 hours. Hydraulic retention time also influenced by several factors, like the composition of the substrate, type of substrate, type of microorganisms etc.

The partial pressure of hydrogen: The partial pressure of the hydrogen should be low which can increase the yield of hydrogen production by 68%.

Future prospects of Biohydrogen

Biohydrogen is considered to be a “Future fuel” because of its high energy density, zero-emission of carbon dioxide and other factors which we have discussed.

In addition to this, several studies are also going on to improve the technology and the production of biohydrogen. There is a scope of development in each area, and our technology is improving day by day.

Therefore, the use of oxygen-tolerant hydrogenase, increased production of hydrogen with the use of a minimal substrate, development of a cost-effective method for the commercial production of hydrogen etc. are the areas of improvement.

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