Biology of Hydrogen Production

We will use hydrogen production in microorganisms as a primary example for how a potential biological energy based technology might be developed. The approaches that can be taken to engineer microbes so that they produce useful quantities of hydrogen can in principle be used to produce other compounds and materials.

All life requires metabolism, a complex web of redox chemistry. The energetic output of metabolism is a collection of molecules that the cell can use to perform useful work. Metabolism requires an input of energy, either from the breaking of chemical bonds (e.g. the multi-step breakdown of glucose to generate ATP and CO2) or, in the case of photosynthetic organisms, light. Plants, algae, cyanobacteria and photosynthetic bacteria can use light energy to raise electrons into higher energy states. These electrons are fed into the electron transport chain and their reducing power is used by the organism to drive forward a range of chemical reactions that would otherwise be energetically unfavorable.

In the case of plants, algae and cyanobacteria, the source of excitable electrons is water. The excited electrons are stripped from water which then splits into oxygen and protons. Hence, this form of photosynthesis is called oxygenic photosynthesis. In anoxygenic photosynthesis carried out by the photosynthetic bacteria (i.e. the green and purple, non-sulfur bacteria), electrons come from broken down organic substrates such as acetate and butyrate, and light is used to excite these to higher energy, enabling them to carry out additional energy yielding reactions as these molecules are eventually oxidized to CO2. In forms of photosynthesis systems, protons are generated which are used to sustain a pH gradient where movement of protons is coupled to the production of ATP.

Hydrogen is produced in microorganisms by enzymes capable of reducing free protons (H+) to dimolecular hydrogen (H2). Examples of these enzymes include the uptake and reversible hydrogenases, and the nitrogenases. The production of hydrogen by these enzymes is usually coupled to some other biochemical process. The energy used by these enzymes is usually multiple steps from an organism's central energy inputs (photosynthesis or oxidative phosphorylation) and is provided in the form of electron carriers such as ferredoxin or NADPH and energy yielding molecules like ATP. There is tremendous diversity of sequences in these classes of enzymes, there are several kinds of cofactors they use, and there are many differences in how they are regulated by and integrated into the metabolism of the organisms in which they are found.

Obtaining useful amounts of hydrogen from microorganisms will require increasing the efficiency of hydrogenases and overcoming other obstacles. One problem is that some hydrogenases and nitrogenases are inhibited by oxygen. Oxygen is produced by photosystem II (PSII) during oxygenic photosynthesis. Organisms have evolved mechanisms to deal with this incompatibility, generally by temporal regulation of these processes (e.g. activate nitrogenase in the dark) or by spatial separation (e.g. the differentiation of subset of cells in a population into specialized oxygen impermeable cells where nitrogen fixation or hydrogen evolution can occur).

Technological approaches to circumvent these problems might be to combine enzymes or pathways with desirable properties from different organisms to generate a robust hybrid pathway. Other approaches will involve understanding and controlling the cellular regulation of the gene products required for hydrogen production.

Questions:

• What organisms have the greatest potential to be engineered to produce hydrogen?
• What enzyme complexes should we modify and how?
• Which enzymes and metabolic pathways could we combine to make a chimeric, hydrogen producing organism?