840.119:Hydrogen Production by Chlamydomonas reinhardtii cells in Sulfur-deprived Environments

Abstract
Many recent developments have been made in the research of Chlamydomonas reinhardtii for hydrogen production. The purpose of this webpage is to assist in understanding the recent developments being written about in academic journals regarding hydrogen production, and to help clarify the idea of using a biological organism to produce hydrogen for energy use.

State of hydrogen production and utilization
The main reason many are looking at hydrogen fuel for energy is to curb the worldwide use of crude oil to produce fuel for vehicles. The molecular hydrogen would combine with molecular oxygen and produce water as a byproduct. The use of hydrogen in vehicles is currently being researched by major car companies and many are confident that hydrogen fuel will be utilized in vehicles.[1]

The problems involved with crude oil supplies, cost, and availablity will get worse as other countries around the world become more industrialized and need oil-consuming vehicles like the United States. Currently, as of 2006, 97% of fuel used for transportation comes from crude oil.[1]

The vast majority of molecular hydrogen is currently made by a process that heats up petroleum and natural gas to high temperatures. Producing hydrogen using crude oil defeats the purpose of using hydrogen as a clean alternative fuel to gasoline although on a small scale, using only natural gas to produce hydrogen would result in half as much carbon emissions as using current gasoline for cars. The other ways of producing hydrogen gas is the electrolysis of water (running electricity through water to split the water into hydrogen and oxygen) using electricity produced from wind, solar, or hydroelectric generators. The main problem with electrolysis is that humans need electricity, and some electricity is still made from fossil fuels. Therefore, more electricity would need to be produced from fossil fuels if electricity from renewable energy is used for hydrogen production.[1]

The main objective of the current research is lowering the cost of the hydrogen produced by the Chlamydomonas reinhardtii. These objectives have been outlined below:
 * 1) Increasing the hydrogen production yield per gram of organism.
 * 2) The long recovery time between switching the Chlamydomonas reinhardtii from sulfur deprived to sulfur replete conditions.
 * 3) Current expense of the reactor material.[2]

State of Chlamydomonas reinhardtii research
Many articles have been written about the ability to produce hydrogen from green algae, and this has been known for over 60 years [3]. The state of Chlamydomonas reinhardtii research is making hydrogen production more efficient and less expensive.

Objectives
The main objective of this website is to review current trends in microbial hydrogen production research by specifically focusing on recent research in molecular hydrogen production by Chlamydomonas reinhardtii in sulfur deprived media. We will accomplish this by providing background information about molecular hydrogen production. We will identify the mechanisms that produce hydrogen in the green algea, Chlamydomonas reinhardtii, and describe some techniques in which hydrogen is extracted.

Background
Some forms of green algae can produce molecular hydrogen gas utilizing the near infrared region of sunlight using the [FeFe]-hydrogenase. The [FeFe]-hydrogenase enzymes in Chlamydomonas reinhardtii absorb light from 400-700nm with a light conversion to hydrogen of 13% to 15%. Relative to other photosynthetic organisms a 13% to 15% light conversion is very efficient. The formation of hydrogen can be simplified as 2H+ + 2FD- -> H2 + 2FD, where the FD is the main [FeFe]-hydrogenase enzyme that produces the molecular hydrogen [6]. Molecular hydrogen formation could also be simplified to a substitution of electron carriers by the photosynthetic cells. By exposing the cells to specific conditions we are able to modify photosynthesis so that oxygen will not act as the final electron carrier of the electron transport chain, rather hydrogen will allow the cells to release molecular hydrogen as opposed to molecular oxygen. It has been found that Chlamydomonas reinhardtii also is a low maintenance organism so because of its high efficiency, useful production mechanism, and low maintenance requirements Chlamydomonas reinhardtii appears to be a useful organism for the industrial scale production of molecular hydrogen [4].

Chlamydomonas reinhardtii alone
Chlamydomonas reinhardtii is put in an oxygen deprived environment. If molecular oxygen is present, the pathway for hydrogen production is irreversibly inhibited [3]. The deprivation of sulfur acts as a metabolic switch between oxygen production (sulfur present), which require oxygen scavengers that are compounds or organisms that adsorb all molecular oxygen that is present in order to prevent the irreversibly inhibited hydrogen production (no sulfur present)[5]. The absence of sulfur then expresses the hydrogenase enzyme, which will use both water oxidation, and catabolism of starch to produce molecular hydrogen in an acidic environment. Without an acidic environment, which is done by adding acetic acid, the molecular hydrogen will not be produced.

It is often convenient to consider the process of molecular hydrogen production to be one that contains three phases:

Dark Phase--This phase involves the culture and growth of Chlamydomonas reinhardtii cells without the presence of light. By growing the cells in dark conditions we allow the organism to allot resources to the expression and synthesis of the [FeFe] hydrogenase enzyme, an enzyme crucial to the process of hydrogen formation.

Light Phase--This phase involves the exposure of the cells to light. This phase follows the dark phase and is responsible for hydrogen production. The enzymes that were previously synthesized in the Dark Phase are now put to work and through a modified form of photosynthesis hydrogen is produced. It should be considered that the utilized cells have a limited capacity to produce hydrogen. This means that our cells have a very limited life or a method must be used in order to rejuvenate the hydrogen production capabilities by the cells.

Regenerative Phase--This phase involves the introduction of sulfur to the media in which the cells were grown. During this phase no hydrogen is produced because the exposure of the cells to sulfur-rich conditions allows normal photosynthesis to occur leading to the production of molecular oxygen. In both the Dark Phase and Light Phase the cells are grown in media that has been depleted of hydrogen.

Sulfur-deprived Conditions
Sulfur-deprived media are required for cellular proliferation during the Light Phase and the Dark Phase. This is because in the absence of sulfur the mechanism of electron transport within the chloroplasts is modified via partial inactivation of photosystem II by the organisms. Photosystem II is the unit of the chloroplast responsible for the transfer of electrons to final electron carrier oxygen allowing the cell to produce and excrete molecular oxygen, the common byproduct of photosynthesis. During its partial inactivation photosystem II is no longer able to use important enzymes in the transport of electrons to hydrogen and a buildup of electrons occurs in the chloroplasts. Still, water must be split (source of electrons) in order to allow energy rich compounds to be produced as a result of photosynthesis. Under these conditions the cell must find a way to allow the electrons to drain out (usually through an electron acceptor) as the cell cannot simply turn off its ability to produce the electrons. Protons become the cell’s choice for an electron acceptor, which will alleviate the burden of a clogged electron transport chain [4].

Another effect of sulfur deprived conditions includes the development of anaerobic conditions within the cell. This is important because any oxygen present will bind to the [FeFe] hydrogenase enzymes, which will prevent the cell from producing molecular hydrogen. Though the effects of sulfur-deprived media may optimize the yield of molecular hydrogen, the cell is still restricted to a certain capacity of hydrogen that can be produced. This limitation gives a finite lifetime on the cell’s ability to produce molecular hydrogen. To restore the cell’s ability to produce molecular hydrogen, sulfur must be introduced into the media. The reason for the limitation is that the element, sulfur, is present in two critical enzymes methionine and cysteine. By removing free inorganic sulfur from the media the cell is not able to synthesize anymore of these amino acids necessary for overall protein synthesis or repair. This leads to protein degradation, which means the cell is slowly decaying as it produces molecular hydrogen. By feeding the cell inorganic sulfur atoms, the molecular hydrogen production capacity is restored and the cells are once again able to produce molecular hydrogen [4].

Immobilization
One of the largest challenges of optimizing molecular hydrogen production by Chlamydomonas reinhardtii cells is the transfer of the cells from sulfur deficient conditions to sulfur rich conditions (for regenerative purposes) and then back to sulfur deficient conditions (for further hydrogen production). Recent research in immobilization has provided a new technique to eliminate this challenge. Prior to the development of immobilizations, cells were suspended in aqueous media with either sulfur rich or deficient conditions present. This posed a problem for scientists because the cells had to be filtered out of the media to be transferred to the next media in the cycle of molecular hydrogen production. The filtration process was very time consuming and so was not feasible on an industrial scale. Another dilemma that plagued the free suspension in liquid media technique was the inability to make the media with cells very concentrated. This restricted the amount of light that could interact with the cells decreasing the overall yield of molecular hydrogen. To avoid difficulties with media transition or cellular concentration immobilization techniques were developed [6].

Techniques
Basic techniques in immobilization include the attachment of Chlamydomonas reinhardtii cells to a surface and then running media over the cellular coated surface at a certain flow velocity. Such an apparatus would then be exposed to light when necessary and the flowing media could easily alternate between sulfur depleted and sulfur excess conditions [6].

To optimize such a technique the correct surface had to be developed for attaching the Chlamydomonas reinhardtii cells. Laurinavichene et al has developed a surface that has proven to provide the greatest hydrogen production. The surface utilizes the high cellular binding potential the cells have with porous glass surfaces. First, long thin strips of porous glass are placed in a large cylinder with the thin strips of glass placed along the walls of the container. After sterilizing the container and thin strips of glass, media containing free Chlamydomonas reinhardtii cells was added to the container. The cells were then allowed to bind to the surfaces of all of the thin strips of porous glass. After rinsing off the glass strips to remove any unbound cells, the primary media, tris-acetate-phosphate (TAP), was added to the thin strips of porous glass. The porous glass strips were then arranged in a matrix, which is depicted in the schematic of the photobioreactor as the glass textile. Such an arrangement of glass sheets provides a high surface area to volume ratio allowing the cells to bind a greater area of surface. This is important for optimizing the concentration of cells that are attached to the glass matrix [6].

The glass matrix is then placed in the photobioreactor as shown in the schematic below. This apparatus is used to expose the cultures to light and consisted of two glass plates bound together by clamps and grease. Each glass plate also included two inlet tubes for argon and media exposure. Argon flow was necessary to increase the apparatus’ ability to mix media that were being presented. Silicon partitions were present on the inside of the glass walls in order to direct the flow of media and argon. The media of choice would then be inserted into the inlet tube and pushed through by argon to the outlet tube. The exhaust coming out of the outlet tube would then contain media, unbound cells, argon, and molecular hydrogen or oxygen depending on the conditions. The matrix of the glass strips, previously described, was placed between the two plates of the apparatus. Since the surrounding plates were made of glas,s light could shine on the photobioreactor in the form of cool flourescent light allowing the photoreaction to occur [6].

The concentration of algal chlorophyll (a and b) was measured using extraction with an ethanol solvent. This provided a technique that could indicate the ability for the culture to make successive amounts of molecular hydrogen. The level of chlorophyll indicated the current ability for the cell to continue making molecular hydrogen during the Light Phase as low amounts indicated that the capacity to form molecular hydrogen was dwindling. This method provides a precise technique for determining when the media needed to be exchanged from sulfur deficient to sulfur rich and vice versa. By running ethanol extractions on the media output, the amount of culture that was coming out with the output media could be analyzed providing an indicator of the cells’ ability to bind to the glass matrix and how this ability changed over time or media exposure [6].

Gas chromatography was performed on the gaseous output in order to measure the concentrations of molecular hydrogen and oxygen produced in the photobioreactor [6].

A schematic of the photobioreactor is shown below [6].



Potential Impact
The potential impact of producing hydrogen by organisms that utilize the sunlight to produce hydrogen is unmeasurable. If hydrogen is able to be made via the Chlamydomonas reinhardtii organism (or others) on an economically competitive level as fossil fuel or electrical hydrogen production, then the world would use far less carbon based fuel for energy needs.

Associated Risks
The risks involved with the project involve taking adequate precautions, since molecular hydrogen is prone to explosions. In order for the hydrogen production to be useful to humankind a large amount of hydrogen would need to be produced at one place for shipment. Therefore, proper precautions would be mandatory to ensure no hydrogen explosions at the hydrogen production plant. With mass production of  Chlamydomonas reinhardtii, it would be necessary to take precautions to guard against the dumping of unused algae into local lakes or ponds so that the aquatic life would survive near a plant making hydrogen from algea.

Ethical Issues
Currently there are no ethical issues with the use of algae for hydrogen production making this one of the main benefits of utilzing  Chlamydomonas reinhardtii's ability to produce molecular hydrogen.