CHLAMYDOMONAS REINHARDTII - A FUTURE HYDROGEN SOURCE?
by Ellenor Olssen, Uppsala/Sweden, Sept. 2001
Eukaryotic green algae were discovered to produce hydrogen under anaerobic conditions already during the 1940's. This was the beginning of a great journey that still has not been finished to search for algae that can produce enough hydrogen to run for example a fuel cell. Since the algae use only sunlight and water to make hydrogen, the gas that is produced in this way can be regarded as a renewable fuel. One of the algae that has been getting lots of attention and been the focus of many resource projects is Chlamydomonas reinhardtii, a unicellular, biflagellated green algae belong to the order Volvocales. This essay tries to give the answer to in which respect Chlamydomonas reinhardtii can be used for this purpose and what has already been done. I will write about both its prospects as a model organism, the mechanism behind hydrogen production in algae and the likelihood for a future large-scale factory of hydrogen based on Chlamydomonas reinhardtii.
Chlamydomonas as a model organism
Like all the other members of the family Chlamydomonadaceae, Chlamydomonas reinhardtii is a unicellular, uninucleate organism with a cup-shaped chloroplast (fig. 1). It originally lives in freshwater but can easily be grown on agar plates or in liquid in laboratory and has no requirement for vitamins. Its biggest advantage as a model organism has for a long time been its ability to be used for tetrad analysis. The foundation of this method is that all meiotic products form a single zygospore can be collected and investigated. This means that Chlamydomonas reinhardtii is one of few algae were the life cycle can be fully investigated in a laboratory.
Figure 1: Chlamydomonas reinhardtii.
Chlamydomonas also grows quickly and has a doubling time of less then 10h hours (under optimal
conditions: around 5h), equivalent to many cyanobacteria for example Synechocystis PCC 6803. It also
has a short life cycle (<2 weeks), which also makes experiments on the organism easier. For studies of
photosynthesis another advantages is that the algae can grow without sunlight because of its ability to
use acetate as a carbon source. Thus mutants incapable of performing photosynthesis can be cultivated
heterotrophically.
These algae have 17 chromosomes and the organism is usually haploid. One problem when to clone genes
of this organism is that the more or less 100 Mbp long genome unfortunately is extremely GC-rich (62%).
The high GC content results in unfavourable codon preference for heterologous expression, and can
present diffuculties for polymerase-based manipulations due to secondary-structure effects. Since these
characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in
principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such
genes would help to avoid the need for special additives of bases during in vitro polymerase-based
manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. One way
to improve amplification in PCR is therefore to select primers with 45-50% GC-content. The mitochondrial
genome is only 15,7-kb and has already been sequenced in 1994. The chloroplast genome on the other
hand is 195-kb and DNA sequencing has been completed. It has so far been shown that the gene
sequence largely resembles that of land plants chloroplasts but that the order of the genes clearly
differs. More than 50 mutations on nuclear gene loci that affect the chloroplast biogenesis and
photosynthetic functions have already been described. This is very important since the alga also has
been getting attention because of its advantages in studies of photosynthetic mechanisms. Compared
with Arabidopsis. Chlamydomonas is also the only known eukaryote where nucleus, chloroplast, and
mitochondria can all be transformed!
The production of hydrogen in algae
Research on hydrogen production is today done mainly on two organisms, cyanobacteria (for example
Nostoc sp.) and, as already has been mentioned Chlamydomonas reinhardtii. The bacteria use
nitrogenase while the crucial enzyme in Chlamydomonas is a hydrogenase. These are completely different
enzymes and while the nitrogenase ordinary is involved in nitrogen fixation the hydrogenase is believed to
be involved in photosynthesis. If the two enzymes are to be compered it can be concluded that even
though hydrogenase have a efficiency that is much higher than for nitrogenase it is also unfortunately
more sensitive towards oxygen, than the nitrogenase, which makes hydrogen production much more
difficult in Chlamydomonas.
Briefly, the mechanism behind hydrogen production is that the hydrogenase combines protons and
electrons from reduced ferredoxins to create hydrogen. One possible reason why Chlamydomonas
harbours a hydrogenase is to be able to lower the redox state in the cell. This makes sense considering
that ferredoxins (small iron-sulphur proteins) are involved in the electron transport in photosynthesis are
the natural redox partners of algal hydrogenase (fig 2 and 3).
Figure 2: A picture showing the basic reactions that take place during photosynthesis. Take special notice about the oxygen that is created of water in PS-II.
If the photosynthetic dark reaction is slowed down by some reason it is important for Chlamydomonas to
get rid of all excess electrons from photosynthesis and avoid an "electron cue" in the photosystems since
they easily can create damage in the cell. For example, ferredoxin can reduce oxygen to form superoxide
(O2-) (Mehler reaction), which is extremely dangerous for biological membranes. This is one reason why
the algae need to be in an anaerobic environment to be able to produce hydrogen. When no oxygen is
present ferredoxin "gives" the electrons to hydrogenase instead of oxygen and hydrogen is produced
(anaerobic Mehler reaction). It is also known that oxygen lowers or inhibits the activity of the
hydrogenase and that the expression of the gene that encodes for hydrogenase is induced by anaerobic
conditions.
There might also be another reason why Chlamydomonas posses, the hydrogenase that was discovered
together with the two-step-process (see below). Without sulphur is it impossible for any
photosynthesising organism to live since it is a vital part of the photosystem proteins. A sulphur
deficiency therefore makes the photosynthesis (esp. photosystem II) to "turn off" and since that also
leads to an oxygen deficiency the algae can not store or burn energy the ordinary way by producing
carbohydrates, proteins or fat and by respiration, respectively. It therefore uses an alternative, more
ineffective, way whereby hydrogen is released.
Research has also shown that Chlamydomonas, under anaerobic conditions, can use an alternative
energy source to get the electrons needed for hydrogen production. It is at this moment not clear what
this energy source is exactly or how it is done but it seem to be an endogenous substrate with carbon
dioxide being one by-product.
Figure 3; After energy has been captured by chlorophylls in the form of photons, several reactions take place to transport the energy and turn it into sugar. Some of the components in these reactions are Fe-S proteins, which can be found in photosystem I.
Methods of enhanced hydrogen production
Early experiments have shown that the easiest way to produce hydrogen has been to decrease the light
intensity. In dim light the photosynthesis releases less oxygen and the cells become anaerobe (remember
the compensation point) and the hydrogenase is able to operate. But only a small amount of hydrogen
can be produced, not enough for commercial production. The light can be increased to a level where very
little oxygen is produced together with hydrogen. The biggest problem then is to separate hydrogenase
from oxygen. There are at present two different approaches to this problem in Chlamydomonas. They can
in short be called the "one-stage-process" and the "two-stage-process".
Single-stage-process: In the single stage process photosynthetic oxygen and hydrogen is evolved at
the same time. Unfortunately, this method has not really been very successful. The biggest problem is
the inhibition of the hydrogenase by oxygen, which is hard to avoid since oxygen is created during the
first step in the photosynthesis all the time (fig 4). One solution to this problem might be to use classic
genetics. Mutant prokaryotic organisms with a hydrogenase that can tolerate higher concentrations of
oxygen do for example already exist. The idea is therefore to do the same to Chlamydomonas and create
mutants that contain a more oxygen tolerating hydrogenase. If this were possible the
single-stage-process would be much more advanced compared to the two-stage-process, in terms of
yield, cost and effectiveness. The reason for that is the ability for Chlamydomonas to produce hydrogen
continuously. To be able to detect these mutants a chemochromic sensor is used. Chlamydomonas
colonies a re grown on an agar plate and light is shone from beneath. Over the agar plate is a thin film
sensor placed that contain tungsten oxide and palladium which change colour when exposed to
nanomolar quantities of hydrogen. It is therefore easy to detect and isolate those colonies that contain
the right mutant.
Two-stage-process: In this method hydrogen- and oxygen gas production is separated either in time or
in space. By incubating the algae in nutrients that lack sulphur it is possible to inhibit the oxygenic
photosynthesis. The reason for this is that sulphur is a very important component in the photosystem II
repair cycle. Without sulphur it is impossible to produce either cysteine or methionine and the protein
biosynthesis is therefore heavily impaired. That leads to a collapse of the D1 protein (32-kDa reaction
centre protein), which is essential for photosystem II and needs to be constantly replaced. During
sulphur deprivation photosynthesis and respiration starts to go down, even thought the light is on. Since
photosynthesis decline much quicker then respiration an equilibrium point is reached after a while, usually
after 22 hours (fig 4). After that time the amount of oxygen that is used in respiration is greater then the
oxygen released by photosynthesis and the algae can be defined as anaerobic. It is at this point that
hydrogen is produced in larger amounts.
Figure 4; This diagram shows the relationship between oxygen consumption by respiration and oxygen production by photosynthesis when Chlamydomonas reinhardtii is grown in sulphur deficiency.
When this is done in real life the algae are allowed to grow and feed in a normal way for some time to be able to store energy. Thereafter they are moved to a new bottle with no sulphur present and when all the oxygen is consumed (after 22 hours) hydrogen starts to bubble up from the top of the bottles into a hydrogen collection tubes. The algae can produce hydrogen for up to four days before it needs to refill its energy storage.
What the future has in hold
The future looks very bright right now for Chlamydomonas as a hydrogen producer. It is true that in the two-stage-process of producing hydrogen there is a need to increase the yield by a factor of hundred or more. But lots of research is done and new physiological methods are on its way. The combination of classical genetics and gene technology might also solve many of the problems that the oxygen-intolerance of the hydrogenase creates. A complete genome sequencing have not yet been done but is on its way and might also come with new ideas and solutions. It should also be mentioned that a company has already been established in California and is named "Melis Energy" after the founder Anastasios Melis, professor at University of California, Berkeley. That is the same researcher who (with some help from colleges of course) discovered the two-stage-process. The company's objective is to create the world's first non-polluting, biotechnical energy source on the base of green algae.
Reference
- R.M. Dent, M Han and K.K. Niyogi, 2000. Functional Genomics of Plant Photosynthesis in the Fast Lane Using Chlamydomonas reinhardtii. Trends in Plant Science Vol.6. No.8:364-371.
- M.L. Ghirardi, L Zhing, J.W. Lee, T Flynn, M Seibert, E Greenbaum and A Melis, 2000. Microalgae: A Green Source of Renewable H2. Tibtech vol18:506-511.
- E.H.Harris, 2001. Chlamydomonas as a Model Organism. Annu.Rev.Plant Physiol.Plant.Mol.Biol 52:363-406.
- A. Melis, L Zhang, M Forestier, M.L. Ghirardi and M Seibert, 2000. Sustained Photobiological Hydrogen Gas Production upon Reversible Inactivation of Oxygen Evolution in the Green Alga Chlamydomonas reinhardtii. Plant Physiology, 122:127-135.
- L. Taiz and E Zeiger, 1998. Plant Physiology, 2nd ed. Sinauer Associates Inc, USA.
- R. Wünschiers, R. Schulz and H. Senger, 2001. Electron pathways involved in H2-metabolism in the green alga Scenedesmus obliquus. Biochimica Biophysica Acta, 1503:271-278.
- Webpage: http://www.melisenergy.com