1.

Introduction
There is an urgent need for the development of sustainable energy sources as an alternative to fossil  fuels.  Our
high demand and consumption of fossil  fuels  causes two problems. Firstly, by burning fossil  fuels  the carbon
dioxide generated has been recognized as the major contributor of global warming causing severe environmental and
economic damage. In the recently published Stern review a limited time of 10–20 years to react on climate change
was given. With the CO2 levels passing the 400 ppm threshold we already experience the destruction of reefs,
landscapes and the frequency of storms and flooding is increasing causing severe damage (Stern, 2006). Secondly,
the resources of fossil  fuels  are limited and our current demand does not allow the use of fossil  fuels  at the
same level and for the same price in the future. From the fossil  fuels,  oil is the most important and the one with the
lowest reserves. Despite an ongoing debate and different opinions about the true reserves there cannot be any doubt
that we will run out of oil this century (Asif and Muneer, 2007). Consequently, alternative  fuels  are required that
fulfill the following criteria: (i) no carbon dioxide should be released (or zero net carbon), (ii) a sustainable resource,
(iii) suitable as  fuel  for the transportation sector, (iv) provides a major part of the global energy demand, (v) should
be in an affordable price range (comparable to the current oil price).
This is a difficult task to achieve for any of the established alternatives to fossil  fuels.  Most  systems  (e.g.
nuclear power, wind power and photo voltaics) mainly produce electricity. Suitable large-scale storage and
transportation devices for electricity do not exist (while it can be converted into H2following electrolysis of water, this
process has around 30% conversion losses). However, around 60% of our energy demand is required as petrol
(mainly for the transportation sector). Other  systems,  as they exist today, are far from covering this demand
because their production efficiency is too low (e.g. biomass to liquid (BtL), conventional biodiesel, bioethanol). Hence
we currently do not have an available alternative that covers the main part of our energy requirements.
Nature has developed photosynthesis, a very efficient light harvesting and conversion  system,  which uses sunlight
to synthesize chemical energy carriers such as carbohydrates, lipids and proteins. Over millions of years nature has
collected the sun's energy and stored it as fossil  fuels  such as oil, coal and natural gas.
Besides the properties of photosynthetically produced fossil  fuels,  the potential of current photosynthetic 
systems  as a provider of clean CO2-neutral  fuels,  so called biofuels, has been recognized ([Powledge,
2008], Boswall, 2006 P. Boswall, Biofuels—the potential of field crops, Can. J. Plant Sci. 86 (2006), pp. 759–1759.
[Boswall, 2006] and [Lofstrom, 2005]). Although photosynthesis is the underlying mechanism for both kinds of  fuels,
 fossil  fuels  are a concentrated storage form of former biomass. However, in order to produce biofuels one has to
deal with the low energy density of sunlight, the low energy conversion efficiency and lower energy content of
biomass (biomass:  15 GJ/t vs. oil:  45 GJ/t) to compete with fossil  fuels  as an energy source.
For biofuels to be widely accepted in the energy market, research must focus on adapting and improving
photosynthetic organisms for biofuel production to meet this target. In this regard it is important to obtain a
fundamental understanding of the physiological processes in photosynthetic organisms which are responsible for
growth and synthesis efficiencies—we need to understand the organism as a whole. A newly designed  systems
biology  approach helps to gain insights into the regulation and networking of an entire organism and not only
isolated pathways and networks. With this knowledge, researchers will have the ability to perform a new level of
biotechnology: devoting the entire organism to the synthesis of biotechnologically relevant products and thereby be
able to tailor an organism suitable to produce biofuels at efficiency levels to compete with fossil  fuels.
The aim of this review is to demonstrate the potential of the new discipline of  systems biology  for biotechnology
and its application to the promising ‘new’ organism for biofuel (biohydrogen) production, Chlamydomonas reinhardtii.
 Abstract

With economic wealth the need for energy is rising. Hence we are facing two problems: to satisfy the increasing
energy demand and concomitantly deliver emission-free energy to avoid global warming. The process of
photosynthesis offers a natural and highly efficient method to produce emission-neutral biofuels. However, using
higher plants for such purposes causes several problems which are difficult to overcome and includes competition
with food producing agriculture in terms of arable land, the need for fresh water, low process efficiency and the
application of energy-intensive fertilizer in order to enhance growth performance. Photosynthetic microorganisms
and, in particular, microalgae offer an alternative approach. In this case production sites in photo-bioreactors can be
located on cheap, rural land and the organisms can be cultured in sea water rather than fresh water. However
microorganisms are not naturally adapted as efficient producers of biofuels. Due to the complex regulatory network
and mutual interaction of physiological processes and organelles, identifying the optimal production strategy is
impossible without a greater understanding of the complex interplay of all cellular processes.Systems biology has
emerged recently as a discipline to gain an understanding of these networks and their translation into a
mathematical in silico model. An in silico model allows simulating optimization steps and, therefore, provides a useful
method to identify targets for directed genetic/physiological modification to optimize the system for a biotechnological
approach.

2.2. Model organism and  systems biology

The idea of a model organism is to draw conclusions from a simplified  system  which allows one to extrapolate the
results to other organisms which are more difficult to investigate or, due to ethical obstacles, prevent research to the
same extent (e.g. using the mouse model to study humans). Model organisms are well characterized and usually
fulfill the basic requirements for the use of an organism for  systems biology  concomitantly, which are: (i) a fully
sequenced genome, (ii) an established range of genetic tools, (iii) easy handling, and (iv) high reproduction rates.
The ‘simple’ photosynthetic alga C. reinhardtii is an excellent model organism for a  systems biology  approach
compared to a complex vascular plant. An algae culture has only one type of cell and, if synchronized, can be at the
same stage of differentiation. This is a critical step in a  systems biology approach as the uniformity and
reproducibility of a sample determines data quality. Due to the complexity of the obtained data it is of utmost
importance that the environmental conditions can be easily controlled. A photo-bioreactor represents a sophisticated
culturing device providing the researcher with the necessary instrument to control environmental conditions but also
acquire data important for analyses and interpretation. C. reinhardtii is a single cell microalga, and not only a model
organism for the essential processes of photosynthesis in plants (Harris, 2001), but has also evolved as a model for
photosynthetic hydrogen production over the last 10 years (Melis and Happe, 2004). These circumstances make this
microalga a perfect candidate for a  systems biology  study with the target to improve its biotechnological
suitability.
For more than 60 years C. reinhardtii has existed as an established laboratory strain ([Proschold et al.,
2005], [Gutman and Niyogi, 2004] and [Harris, 2001]). With the completion of the nuclear genome sequence in 2007
(Merchant et al., 2007) (release of the last assembly, version 4, in spring 2008) all three genomes have now been
sequenced ([Maul et al., 2002], [Vahrenholz et al., 1993] and [Boer et al., 1985]). In addition to the sequence
information, transformation of all three sub-genomes is possible ([Remacle et al., 2006], [Kindle and Lee,
1998], [Shimogawara et al., 1998], [Dunahay, 1993], [Kindle, 1990] and [Boynton et al., 1988]) and a large mutant
collection, mainly managed by the Chlamy Center (http://www.chlamy.org), is accessible. C. reinhardtiican easily be
cultivated either photoautotrophically, mixotrophically (with light and organic acid) or heterotrophically (relying on
organic acids only). Its asexual and sexual reproduction can be controlled by synchronization by nitrate deprivation
(Harris, 1989). The doubling rate of green algae can be 5–6 h under laboratory conditions (Smith et al., 1990) and
24 h under mass culture ambient conditions (Ben-Amotz and Avron, 1990). Photoautotrophic cultures can easily be
grown to densities of more than 10 million cells per ml.
3. Biofuels
A biofuel is a solid, liquid or gaseous  fuel  derived from any biological carbon source including treated municipal
and industrial wastes (for an informative review see (Yuan et al., 2008)). With the problems we are now facing in
terms of global warming due to burning of fossil  fuels  and depleting resources there is a serious renaissance of
interest in renewable energy from biological sources (Gavrilescu and Chisti, 2005). A strategy to develop high
performing organisms has to consider the product of desire. Traditionally, land-based plants offer many options for
biofuel production and manufacturing costs are defined. Recently photosynthetic organisms such as (micro)algae or
cyanobacteria have been suggested as promising alternatives to land-based plants to provide biomaterial.

3.1. Biofuels from land-based plants

3.1.1. Biomass
The proportion of the energy supply attributed to biomass is high. As an indigenous energy source it covers 10–14%
of the worlds energy demand with particularly high acceptance in third world countries (Mckendry, 2002). Energy from
biomass is accepted as a CO2-emission neutral source: Only the carbon which has been previously fixed is released
into the environment by combustion. However, the energy demand during processing questions this fact. So far
biomass energy is derived mainly from residue or waste. For an improvement in energy farming, selection of high-
performing plants and genetic engineering has to take place. Currently established sources of biomass are willow,
poplar, Miscanthus and switchgrass. In particular high-yield C4 plants (plants with a more efficient
CO2 accumulation  system)  such as the latter two are promising candidates for efficient and competitive biomass
production. A critical factor which determines the energy conversion route is the cellulose/hemicellulose/lignin
proportions. Lignin is a component which is still difficult to access and plants with high cellulose/lignin content are
favored for this reason. The choice of plant cannot be generalized as it depends on local conditions such as climate
and soil as well as political  fuel  strategies. An advantage of biomass is its diversity in conversion options:
electricity/heat, liquid/gaseous  fuel  and chemical feedstock are all potential products. However, while established
production-flows offer an advantage, because production costs and volumes are known, a suitable organism still
remains to be designed. In order to improve biomass production and reduce costs, plants with low demands to soil
and water are necessary: A high-yield achieving crop such as Miscanthus combined with a high calorific value plant
like willow would already halve the land requirements. However, activities to provide the genetic engineering tools of
these plants have been started already. Poplar is the first tree whose genome has been sequenced
(http://genome.jgi-psf.org/Poptr1/Poptr1.home.html) and further complementing research is ongoing like the
generation of cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform (Ralph et al., 2006).

3.1.2. Biodiesel
A further established technology is the extraction of oil from plants. Soybean in the U.S. and rapeseed in Europe are
the two species established for biodiesel production. However land-based plants with even higher oil content are
being used such as coconut, palm oil and sunflower. Extracted vegetable oils can be converted into biodiesel by
transesterification and used in conventional combustion engines (Demirbas, 2007b). This method uses the seeds
usually for the fabrication of the product. Remaining biomass cannot be used or has to be utilized by additional
processing, such as BtL or anaerobic digestion, to increase efficiency. Besides its character as a renewable energy
source, an advantage of biodiesel over conventional diesel is its property of being biodegradable within a short time
(Zhang et al., 1998). Although biodiesel is an established  fuel  (as an additive of conventional diesel or even pure
biodiesel) still around 80% of the total production costs of biodiesel are derived from the feedstock (Demirbas,
2007a). This clearly puts pressure on the production of cheap vegetable oil sources.

3.1.3. Bioethanol
Since the 1980s ethanol has been an established alternative to fossil  fuels  in Brazil. It is produced mainly from
sugar and starch (sugar cane, corn). With the increasing demand on fossil  fuel  replacements the USA, Europe and
 other states are using or considering the substitution of petrol with ethanol. Advantages of bioethanol are clean
combustion and the closed CO2 cycle, which allows released CO2 to be fed back into the production cycle. To
increase the production efficiency from the feedstock, cellulose needs to be used, in addition to sugars. However, a
further acidic or enzymatic cleavage step is necessary to make the product available for ethanol production, which is
conducted in 3 steps: (1) yeast fermentation, (2) distillation/rectification and (3) dehydration to achieve a purity of
99.5%. The two major cost factors of the process are the biomass feedstock which contributes 40% to the overall
costs and the enzymes needed for the breakdown of cellulose. Chandel et al. (2007) postulate that advances in
pretreatment of biomaterial or an integrated approach with application of tailor-made enzymes for efficient and cost-
effective cellulose breakdown in combination with genetically engineered plants, may be used to tackle these
problems associated with bioethanol production.
Overall, the general drawbacks of the use of land-based plants for biofuels are:
● competition for food, fresh water and valuable agricultural areas,

● low efficiency due to low sunlight to biofuel conversion factors (<1% (Mckendry, 2002),
● high harvesting costs (harvesting biomass is one of the most significant contributors to the overall production
costs),
● difficulty in the application of GMOs in some countries,
● variation in material quality,
● existing capacity cannot cover larger demands (due to low efficiency and competition (Chisti, 2007).

3.2. Biofuels from microalgae

The first reports on the potential of microalgae as a  fuel  source were published as early as the 1980s ([Sawayama
et al., 1995], [Nagle and Lemke, 1990] and [Chisti, 1980–1981]). Recently microalgae have been rediscovered as
promising candidates for biotechnological applications (León-Bañares et al., 2004) and efficient energy production 
systems  ([Chisti, 2007], [Dismukes et al., 2008] and [Schenk et al., 2008]) for several reasons. Due to their single
cell structure algae are more efficient energy collectors than land-based plants (Dismukes et al., 2008). The range of
biotechnologically interesting products is much larger ([Spolaore et al., 2006] and [Pulz and Gross, 2004]) and the
desired product to biomass ratio is higher as no energy has to be spent to build up plant organs like roots or callus.
Since algae grow in water there is no competition for arable land. And by using salt-water cultivation  systems  the
demand for fresh water can be drastically reduced as sea water could be used instead of freshwater. In terms of
product supply algae harvest is not restricted to a few harvesting periods or so strongly dependent on weather
conditions. By improving growth performance by means of genetic engineering or physiological regulation, harvest
periods could even be shortened further. Cultivation in closed photo-bioreactor  systems  can be coupled to CO2-
sequestration more easily: CO2 from fossil combustion is pumped into the vessel  system  and used to increase
CO2 availability. The CO2 is fixed as carbohydrates by the cells and the easily accessible carbon source accelerates
growth with the benefit of a higher yield. A concomitant advantage of such a  system  is the increased mixing of the
culture by the gas being pumped in. The tolerance of some alga species to toxic compounds also allows their use in
bioremediation (Muñoz and Guieysse, 2006) and remaining biomass following product separation can be used in the
generation of biofertilizer (Vaishampayan et al., 2001) which can be used to improve conventional agriculture. The
prospects for genetic improvement of algae species are bright. For some species genome sequences are available or
sequencing is already in progress (e.g. Phaeodactylum tricornutum, Thalassiosira pseudonana,Dunaliella
salina, Chlorella vulgaris, etc.) and due to smaller genome sizes and new advanced sequencing technologies the
completion of further genome sequencing projects can be expected soon. For example, Botryococcus braunii, which
has the highest oil content reported so far (Banerjee et al., 2002), is scheduled as a 2009 JGI (Joint Genome
Institute) genome sequencing project.

3.2.1. Biomass
Similar to land-based plants algae offer a variety of different biofuel options. In the simplest way algae are being used
for the production of biomass. While land-based plants are mostly of the dry biomass type, algae belong to the wet
 biomass group which imposes different conversion procedures. While dry biomass is processed by gasification,
pyrolysis or combustion, for wet biomass the favored strategy involves biologically mediated procedures, like
fermentation, due to energy consuming drying costs of the wet material (Mckendry, 2002). As a result of its high
water content, electricity production from algae culture would not be favored due to the negative impact of water on
the process of thermal combustion. However, with respect to overall biomass yield, microalgae have the advantage
over land-based plants. Biomass production can even be conducted in low-cost salt-water open pond  systems  as
has been proven for Dunaliella salina for the production of β-carotene in Hutt Lagoon, Western Australia (Borowitzka
et al., 1984).

3.2.2. Biodiesel
In terms of biodiesel  fuel,  microalgae offer very promising perspectives. It has been published that oil content in
some algae species is >50% of dry mass (Chisti, 2007) and that production rates are substantially higher than
bioethanol production from sugar cane (Chisti, 2008). The extraction of fatty acids derived from microalgae and its
conversion to biodiesel has already been proven (Belarbi et al., 2000) and while production efficiency is expected to
be much higher for the cultivation of algae in photo-bioreactors than for land-based crops, proven production costs do
not yet exist. Currently, crude palm oil is the cheapest available bio-oil available at 0.52 US$/L. In contrast, oil from
microalgae costs around 2.80 US$/L. To be competitive with crude petrol oil (without considering any costs for
environmental damage, etc.) the target price would have to be 0.48 US$/L as calculated by Chisti (2007). To achieve
this target Chisti (2007) suggests employing a three step approach: (i) improving the production strategy by a
biorefinery based approach, utilizing valuable side products (e.g. proteins) or producing methane from remaining
biomass; (ii) genetic engineering to optimize algae for biodiesel production and adaptation to artificial conditions in
photo-bioreactors; (iii) design of sophisticated photo-bioreactors.

3.2.3. Biohydrogen
Besides the culturing advantages that algae provide, some algae are able to produce a unique biofuel which cannot
be provided by any land-based plants: hydrogen (H2). Regardless of the technical obstacles of dealing with H2 (e.g.
storage, development of new engines, etc.), H2 is a clean  fuel,  only producing water when burned, and when used
in  fuel  cells, offers the highest conversion efficiency ( 60–70%). Admittedly, due to the high temperature the
combustion of H2 in air produces high levels of toxic mono-nitrogen oxides (NOx). However, this can be avoided using
special H2 combustion engines but results in decreased efficiency ([Jin and Ishida, 2000] and [Takahashi et al.,
2003]). For biohydrogen to compete with fossil  fuels  production efficiencies have to be improved and according to
a study by the US Department of Energy, hydrogen production in Chlamydomonas has to take place at an efficiency
of 7% under outdoor conditions to be commercially viable. While maximum efficiency for this process has been
calculated to be 10–16% (Rupprecht et al., 2006), light to H2 conversion efficiencies of only around 1.5% have been
published under laboratory conditions (100 μE/m2 s continuous white light at room temperature (Kruse et al., 2005a).
To increase the energy yield by a further 5%, the remaining biomass from a H2-producing power plant should be
converted to  fuel  via BtL or anaerobic digestion. In addition, the process could benefit from carbon sequestration
(Hankamer et al., 2007a).
Overall, the drawbacks of microalgae biofuel production include:
● Little experience with the development of closed large-scale photo-bioreactors.

● High material costs for closed, highly efficient bioreactor  systems.

● No existing infrastructure and production pipeline.
● High energy requirement for cultivation (e.g. mixing).
● Expensive harvesting (cells need to be separated from medium which is time and/or energy consuming
(Borowitzka, 1992).
● Some aspects of the technology required are not yet established which causes high prototype costs.
3.3. Energy availability
Clearly, product output depends on the energy input of a photosynthetic organism's leaves or cells. However, there is
also a minimal energy requirement for cellular maintenance, to ensure survival, while other processes consume
energy to protect the cell. These photo protective processes use up energy by emitting excess light or by regulation
of antenna sizes and localization ([Wollman, 2001] and [Rochaix, 2007]). In addition, proteins which can be found in
the mitochondrion, such as AOX (alternative oxidase), help to regulate the redox status of the cell (ref). Energy can
be stored as carbohydrates, lipids or proteins and in which form and what amount of energy is stored depends on the
environmental situation. Firstly, an idea of how much energy is finally available for the synthesis of a certain product
needs to be considered:

Energy available to the organism: Etotal=Eabs−En/abs

where Etotal is the entire amount of energy which hits the earth surface, Eabs is the absorbed part of the light, whereas
En/abs represents the photons which are not absorbed, because the cell or leaf was missed or the properties of the
photon do not allow it to be absorbed.

Energy which cannot be absorbed is lost. Photosynthetic organisms – depending on their light harvesting antenna 
system  – are only able to collect certain parts of the light spectrum. In general  50% of the sun's energy is lost as
the photon's energy is not compatible with the light harvesting  system (UV light, green, far red and infra red).

Energy available for energy demanding processes: Ephysiol=Eabs−En/conv

where Ephysiol is the energy which is available for the organism's physiological processes, En/conv is the energy which
has been absorbed but cannot be used.

Photons which have been absorbed have to be converted to ATP and reduction equivalents (NAD(P)H) which
subsequently can be used to produce carbohydrates, lipids and proteins. However, not all of the incoming photons
can be converted. Plant protection mechanisms emit absorbed photons as fluorescence light and heat depending on
the status of the cell and environmental factors such as light intensity and temperature. For the absorption of light the
light-harvesting (LHC) proteins play a major role. As well as their ability to collect photons they also have a regulatory
function and dissipate excess light energy as heat or fluorescence via energy-dependent non-photochemical
quenching (NPQ). The disadvantage of this energy release is the loss of already absorbed photons (up to 80–95%
([Polle et al., 2002] and [Polle et al., 2003]) and considerable reduction of photon conversion efficiency (Kruse et al.,
2005a). The percentage of photons that are quenched depends on the environmental situation and the organism.

Energy available for product synthesis: Eprod=Ephysiol−Ehouse

where Eprod is the energy available for the synthesis of the desired product, Ehouse is the energy the organism needs to
maintain its integrity.

Finally, an organism cannot funnel all energy into the synthesis of one product. The cell's minimal energy demand for
maintenance has to be secured; otherwise productivity would decrease due to slow growth rates. This reveals in
principle the main targets of an optimization process and splits the involved processes into the regulation of:
energy input,

energy conversion, and

energy distribution (Fig. 1).
 Full-size image (69K)

Fig. 1. Overview of the energy availability and flow within an organism like C. reinhardtii. A photosynthetic single cell
or a single cell organism absorbs light of a certain wavelength which hits the cells surface. Only absorbed light can be
used for photosynthetic processes to run cellular metabolism. However, mechanisms of photo protection, cell
maintenance and storage take away energy which could be used for the synthesis of the product of desire. Thus, to
optimize production the entire organism's energy distribution machinery has to be adjusted to funnel energy
resources to the desired process.

At all of these steps optimization can be achieved and targets have already been defined in several publications
including external energy supply, the size of the light harvesting antenna and culturing conditions ([Doebbe et al.,
2007], [Mussgnug et al., 2007], [Melis and Happe, 2004], [Kruse et al., 2005a],[Kosourov et al., 2002], [Mus et al.,
2005], [Hankamer et al., 2007a] and [Hankamer et al., 2007b]). Furthermore, although listed as distinct parts of a
process, it should also be considered that these steps are interconnected and depend on each other.
This is the point where  systems biology  demonstrates its strength. In order to truly optimize the  system  for a
certain production flow the interplay of the components of a biological  system  has to be analyzed.