Hydrogen Production Mechanism

Low sunlight utilization efficiency due to a large chlorophyll antenna size

Fully pigmented cells at the surface of the culture over-absorb incoming sunlight (i.e., they absorb more than can be utilized by photosynthesis), and ‘heat dissipate’ most of it. This is alleviated by the truncated, or smaller Chlantenna size of the
photosystems. The research seeks to develop green algae with a “truncated light-harvesting chlorophyll antenna”, which produce more H2 per bioreactor surface area.

Slow rate of H2 production due to non-dissipation of proton gradient across thylakoid membranes

The rate of photobiological H2production from water is limited by proton accumulation inside the algal thylakoids. This barrier is being eliminated upon a genetic insertion of proton channels into the algal thylakoid membranes. The proper application of such proton channels across thylakoid membranes could substantially enhance photobiological H2 production.  Moreover, it would also alleviate other competitive processes, such as inhibition of H2 production by electron flow to CO .

Discontinuity of H2photo-production due to co-generation of O2, an inhibitor of the [Fe]-hydrogenase

model of the Chlamydomonas reinhardtii [Fe]-hydrogenase. The O2-sensitive catalytic site cluster is identified by yellow, red and purple space-filled atoms. The alpha helices, shown in green, line one of two hydrophobic gas pathways, which allows gas diffusion between the active site and the surface of the protein. The viewer is looking straight down one such pathway. The research seeks to engineer the gas pathways toprevent O2 from reaching the catalytic site, but not H2 from diffusing out of the protein.

Discontinuity of H2 photo-production due to co-generation of O2, an inhibitor of the [Fe]-hydrogenase

An approach to bypass the O2-sensitivity is to temporally separate normal photosynthesis from Hproduction.  This was successfully implemented upon sulfur nutrient deprivation of the algae, which acted as a metabolic switch, causing anaerobiosis and inducing H2 photo-production by the cells in a process that could be sustained for 3-4 days (see figure below). This breakthrough lead to the design of molecular biological approaches to limit sulfur nutrient availability to the chloroplast and to extend anaerobiosis of the culture, thus, genetically bypassing Barrier Z (see next slide).

Discontinuity of H2photo-production due to co-generation of O2, an inhibitor of the [Fe]-hydrogenase

Sulfate uptake, assimilation, and cysteine biosynthesis by the chloroplast in the green alga, Chlamydomonas reinhardtii, is required for oxygenic photosynthesis. Sulfate anions are transported from the environment into the chloroplast through the “plasma membrane”and a “chloroplast sulfate transport system.” The research seeks to genetically interfere with
the function of the chloroplast sulfate transporter in order to impede oxygen evolution and to generate green algae in which photosynthesis is less active than cellular respiration. Such strains would be sulfur-limited, perform oxygenic photosynthesis under anaerobic conditions and constitutively produce H2.

Discontinuity of H2 photo-production due to co-generation of O2, an inhibitor of the [Fe]-hydrogenase

An O2-tolerant [NiFe]-hydrogenase has been identified from the photosynthetic bacteria, Rubrivivaxgelatinosus andThiocapsa roseopersicina. This O2 -tolerant [NiFe]-hydrogenase will be genetically expressed in a cyanobacterium for continuous photo-production of H2 and O2 from water.

Integrated Biological H2 Production

Illustrative Scenario: Green algae, cyanobacteria, and photosynthetic bacteria are co-cultured anaerobically in a photoreactor, and dark anaerobic bacteria in a fermentor. Feedstock for the dark anaerobic bacteria is derived from the cell biomass/sugars of the algae, cyanobacteria and photosynthetic bacteria. Additional feedstock for the dark anaerobic bacteria is derived from lignocellulosic products. The small organic molecule by-products of the dark, anaerobic, bacterial fermentation are subsequently utilized as feedstock for the algae, cyanobacteria and photosynthetic bacteria. The research seeks to implement specific aspects of this Integrated Biological H2 Production System.

Limitation due to the high nitrogen/carbon (N/C) ratio in photosynthetic bacteria

To extend the absorption spectrum of H2-photoproduction to the infrared region (700-900 nm), anoxygenic photosynthetic bacteria would be included to work in tandem with green algae and cyanobacteria. Hydrogen in photosynthetic bacteria, e.g. Rhodospirillum rubrum, is generated by the nitrogenase enzyme. This enzyme is expressed only under conditions of inorganic nitrogen limitation (low N/C ratio). To maximize H2 -production activity in photosynthetic bacteria, it is important to alleviate the positive suppression of gene expression by inorganic nitrogen in the medium. The research seeks to apply molecular engineering techniques to achieve constitutive expression of the nitrogenase enzyme under high N/C ratios in the medium.

The fermentation hydrogen molar yield (mol H2 /mol substrate) is too low due to various biological limitations

There is enough energy in glucose to produce 12 mol of H2, yet biologically the maximal molar yield is 4. Most laboratories, however, reported an even lower H2 molar yield around 2. The simultaneous production of waste organic acids andsolvents lower the H2 molar yield. One effective strategy is to perform metabolic engineering to re-direct microbial pathways preferentially toward Hproduction. New pathways must also be discovered to harness all of the energy stored in sugar substrates.

Glucose feedstock is a major  cost driver for economic Hproduction via fermentation

Challenge: glucose is too expensive to support economic H2 production.

Solution: identify microbes that can produce H2  from glucose-rich cellulose and hemicellulose,
both of which are major constituents of abundant lignocellulosic biomass.

Barrier X: Truncated chlorophyll antenna size strains in green algae weredeveloped.
Barrier Y: A thylakoid-spanning artificial proton channel was designed. Barrier Z-I: [Fe]-hydrogenase O2 -diffusion barriers were identified.
Barrier Z-II: Parameters affecting continuity of H2 production were identified.
Barrier Z-III: Chloroplast sulfate permease genes were repressed to lower sulfate uptake and the Photosynthesis/Respiration ratio.
Barrier Z-IV: Strains with O2 -tolerant [NiFe]-hydrogenase were identified.
Barrier AA: A tubular photo-bioreactor was tested.
Barrier AF: A high N/C ratio nitrogenase de-repressed photosynthetic bacterium strain was obtained.
Barrier AI: A model microbe was selected for pathway engineering.Barrier AK: Microbes producing H2 from cellulose and hemicellulose were screened and identified.

Problems and Improvement Strategies in no particular order

State Transistions

In response to light conditions, PSI and PSII rearrange their light harvesting attenae complexes. There are two states, 1 and 2. State 2 is induced by anaerobiosis and is the state occupied by C. reinhardtii during H2 production.

Electron Cycling

I don’t really know much about this yet.

Co-productions of gases

If an oxygen resistane hydrogenase is produced and H2 and O2 evolution are contemporaneous, there may be difficulties in separating the two gases. Additionally, it could be dangerous to store the two together. This necessitates either temporal separation, or a method for removing the O2.

Starch accumulation

The algae lives off accumulated starch while it is producing H2. One method of improvement is to modify how much starch is accumulated before H2 production. I am not sure if carbon is being fixed during H2 production.


Algae produces protective pigments that are supposed to absorb damaging light, but mught also reduce the efficiency of the system by absorbing otherwise useful photons.

Excess excitation of the P680 reaction center results in the production of raeactive oxygen species (ROS). In order to prevent this, the chloroplast engages in non-photochemical quenching (NPQ).
Protective pigments

From wikipedia:

hotoinhibition is a reduction in a plant’s (or other photosynthetic organism’s) capacity for photosynthesis caused by exposure to strong light (above the saturation point). Photoinhibition is not caused by high light per se, but rather absorption of too much light energy compared with the photosynthetic capacity, i.e. any excess energy that the photosystem cannot handle is damaging. Too much light energy affects (photosystem II (P680)) more than photosystem I (PSI), and it has been hypothesized that the excess energy damages either the oxidizing (donor) or reducing (acceptor) side of PSII, damaging the water oxidizing complex on the donor side or blocking the flow of electrons to plastoquinone on the acceptor side (Hall & Rao 1999).

Photoinhibition is often reversible, i.e. dynamic photoinhibition, and does in that case not inflict permanent damage to the photosystem. However, severe photoinhibition over a long time may cause highly reactive free oxygen radicals to form, which degrade photosynthetic components, i.e. chronic photoinhibition or photodamage. Particularly vulnerable is one of the main core proteins of photosystem II, protein D1, encoded by the gene psbA.

Plants and algae have several mechanisms to protect against photoinhibition, e.g. through the xanthophyll cycle.

The xanthophyll cycle involves conversions of pigments from a non-energy-quenching form to energy-quenching forms. This is a way to reduce the absorption cross-section of the light harvesting antenna, and thus to reduce the amount of energy that reaches the photosynthetic reaction centers. Reducing the light harvesting antenna is one of the main ways of protecting against photoinhibition and changes in the xanthophyll cycling takes place on a time scale of minutes to hours [1]. In higher plants there are three carotenoid pigments that are active in the xanthophyll cycle: violaxanthin, antheraxanthin and zeaxanthin. During light stress violoxanthin is converted to antheraxanthin and zeaxanthin, which functions as photoprotective pigments. This conversion is done by the enzyme violaxanthin de-epoxidase. [2]

In diatoms and dinoflagellates the xanthophyll cycle consists of the pigment diadinoxanthin, which is transformed into diatoxanthin (diatoms) or dinoxanthin (dinoflagellates), at high light.

High H2 production mutants

One mode of improvement is simply screening mutants for high H2 production. I am not sure if the genetic bases for the improvements have been unraveled yet.

I have read that most of the mutants appear to be respiratory mutants, meaning mutants that consume more oxygen during repsiration than wild type algae. From the perspective of improving efficiency of the hydorgen production, this type of mutant is relatively uninteresting.

Quantum efficiency

I understand this to mean that some parts of the electron transport chain may not have enough capacitance to deal with a backlog of electrons. Tuning the ratios to store electrons that would otherwise floresce might be useful