Research on Microalgae

2010
Wageningen UR
 Index

1. Algae .........................................................................................................................................3
2. Facts on Algae .........................................................................................................................4
2.1 Difference between micro- and macroalgae .........................................................................4
2.2 Photosyntesis……….……………………………………………………………….…………5

2.3 Species .......................................................................................................................................6

2.4 Growing algae ...........................................................................................................................7
2.4.1 Light ..................................................................................................................................8
2.4.2 Temperature ...................................................................................................................10
2.4.3 Nutrients ..........................................................................................................................11
2.4.4 Reactor ............................................................................................................................12
2.5 Interesting products ................................................................................................................13
2.6 Production potentials ..............................................................................................................14
3. Applications ............................................................................................................................15
3.1 Bulk Chemicals........................................................................................................................15
3.2 Food & Feed ............................................................................................................................16
3.3 Fine Chemicals........................................................................................................................17
3.4 Energy ......................................................................................................................................17
3.4.1 Biofuels .................................................................................................................................17
3.4.2 Feasibility for Energy ...........................................................................................................18
4. Technologies ..........................................................................................................................19
4.1 Systems Biology......................................................................................................................19
4.1.1 Metabolic flux analysis ........................................................................................................19
4.1.2 Metabolomics techniques ..................................................................................................20
4.1.3 Genomics ..............................................................................................................................21
4.1.4 Transcriptomics....................................................................................................................22
4.1.5 Proteomics ............................................................................................................................22
4.1.6 Bioinformatics .......................................................................................................................23
4.2 Production ................................................................................................................................23
4.2.1 Open systems ......................................................................................................................24

1
4.2.2 Shake flask cultivation ........................................................................................................25
4.2.3 Lab-scale photobioreactors ................................................................................................25
4.2.4 Pilot-scale photobioreactors ……………………………………….………….................25

4.2.5 Commercial scale photobioreactors ..................................................................................26

4.2.6 Heterotrophic organisms.....................................................................................................26
4.2.6.a Growth ...............................................................................................................................27
4.2.6.b Production potential .........................................................................................................27
4.3 Biorefinery ................................................................................................................................28
4.3.1 Conversion ...........................................................................................................................28
4.3.2 Purification ............................................................................................................................29
5. Projects ...................................................................................................................................29
5.1 High density cultures of microalgae .....................................................................................29
5.2 Harvesting of microalgae .......................................................................................................29
5.3 Harnessing the sun for microalgae cultures ........................................................................30
5.4 Biofuels from microalgae: Scenario studies for algae plants ............................................31
5.5 Optimization of lipid production in microalgae ....................................................................32
5.6 Physico-chemical properties of proteins isolated from microalgae ..................................33

2
 1. Algae

Microalgae represent a promising feedstock for the production of biofuels as well as the production of other
bulk chemicals, food and feed. They are attractive alternatives compared to terrestrial oleaginous species
because their productivity can be boosted to higher levels and they do not have to compete for land suitable for
agriculture. These advantages are based on the simple fact that microalgae can be contained in a watery
environment in which all growth factors can be optimized as for example carbon dioxide and nutrient levels,
temperature and light distribution.
Growing microalgae in a contained environment, being either raceway ponds or closed low-cost outdoor
photobioreactors, is technically challenging and the associated costs and energy requirement need to de
drastically reduced before this process can be successfully commercialized.

Within Wetsus, the Dutch centre of excellence of sustainable water technology, a research theme „biofuels
from microalgae‟ was started in 2008 (www.wetsus.nl). The objective of this research program is to realize
breakthroughs leading to the successful commercialization of a microalgae production process for biofuels
feedstock. This research theme is supported by 13 companies listed below and carried out by 7 PhD
researchers focusing on different issues related to this process:

 Enhancement of lipid productivity. Current oil-accumulating microalgae species accumulate lipids in a non-
growing phase. The objective is to steer metabolism in the direction of concurrent microalgae growth and
lipid accumulation.
 Enhancement of photosynthetic efficiency. In well-designed photobioreactors light is the growth-limiting
factor. The objective of this study is to enhance the conversion efficiency of (sun)light into lipid-rich
biomass by modifying light distribution on the reactor surface.
 Carbon dioxide supply and oxygen removal. Depletion of carbon dioxide (CO2) or the accumulation of
oxygen (O2) directly limit productivity, and the associated gas transfer reflects a major energy input for
microalgae production processes. The objective of this study is therefore to enhance the rates of transfer of
CO2 and O2.
 Microalgae biorefinery. The objective of this study is the development of a simple process to extract
functional proteins for food and feed in order to be able to use a larger fraction of the microalgae biomass
and make value out of it.
 Microalgae harvesting. Microalgae are unicellular and difficult to concentrate from the dilute product
streams. The objective is to develop a simple pre-concentration step based on flocculation.
 Scenario analysis. The whole microalgae production process chain is complex and depends on several
external factors. As an example microalgae production can be combined with CO2 removal from
combustion gasses and nutrient removal from wastewater. The objective of this scenario analysis is to
specify the critical factors in the process design and to search towards solutions that anticipate to the
developments in the environment of algae production plants.

3
 2. Facts on Algae

The ultimate and ideal energy carrier for durable technologies is solar irradiation. The most efficient
method to benefit from solar irradiation to produce biomass is growing microalgae. Microalgal
biotechnology is a relatively young field and presently the market is mainly determined by a
few species (Spirulina, Chlorella and Dunaliella). It is expected that the commercial market will expand
with other promising species for valuable and more diverse products. The biodiversity of microalgae is
enormous and each species produces its own unique product(s). Because only 10% of the species are
identified microalgae represent an almost untapped resource. It has been estimated that there
are between 200,000 and several million species, compared with about 250,000 species of higher
plants.

Microalgae have an enormous potential. This is supported by the comparison, in terms of

development, with both microbial fermentations and agriculture. Due to the development in
both technology and strains (in case of fermentation) and crops (in agriculture), the productivity of
present systems is about 5000 times higher than the original natural production systems. Production
of microalgae is still based on traditional technologies with wild type strains. It is a great challenge to
realize breakthroughs in both photobioreactor technology and strain development.

Especially marine microalgae are rich in high-value bioactive components like vitamins, ω-3 fatty
acids, pigments, antioxidants and sterols. Only a small number of these compounds have been
commercialized at large scale. Development of new products from microalgae has always been limited
by the technology, as described above. Especially for products for which algae need to be grown as
monocultures, the available technology is seen as a bottleneck. Most of the commercial systems
applied are open ponds for the production of Spirulina, Chlorella and Dunaliella. Apart from that,
microalgae are produced at aquaculture sites in which they serve as feed.

2.1 Difference between micro- and macroalgae

Two kinds of algae exist: macroalgae, also known as seaweed and microalgae. Microalgae are very small
plant-like organisms (+/- 1 to 50 μm), which can be seen with the aid of a microscope. Unlike higher plants,
microalgae do not have roots, stems and leaves. Microalgae, capable to perform photosynthesis, are important
for life on earth; they produce approximately half of the atmospheric oxygen and use simultaneously the
greenhouse gas carbon dioxide to grow photoautotrophically.
In addition, life in oceans, seas and lakes is dependent on microalgae, because these are at the bottom end of
the food chain.

Macroalgae are seaweed or kelp - aquatic “plants” that are cultivated either directly in the sea, attached to solid
structures like poles and rafts, or, in some cases, as small individual plants, kept in suspension in agitated
ponds. Macroalgae are produced for their content of gelling substances: agar, alginates and carrageenans –
and for food: the annual global production of seaweed is several million tons. Compared to other types of
aquaculture, the production of seaweed is only surpassed by freshwater fishes. Presently there is also interest
in seaweeds as a feedstock for production of biofuels.
In general, microalgae are cultured in photobioreactors while macroalgae are cultured in natural environments.
When we refer to algae on this website, we refer to microalgae.

The biodiversity of microalgae is enormous and they represent an almost untapped resource. It has been
estimated that about 200,000-800,000 species exist of which about 35,000 species are described. Over 15,000
novel compounds originating from algal biomass have been chemically determined (Cardozo et al. 2007). Most
of these microalgae species produce unique products like carotenoids, antioxidants, fatty acids, enzymes,
polymers, peptides, toxins and sterols.
The chemical composition of microalgae is not an intrinsic constant factor but varies over a wide range, both
depending on species and oncultivation conditions. It is possible to accumulate the desired products in
microalgae to a large extend by changing environmental factors liketemperature, illumination, pH, CO2 supply,
salt and nutrients.

4
2.2 Photosynthesis
The key process for microalgae to obtain energy is photosynthesis. Photosynthesis is the process of using light
energy (hν) to fix carbon dioxide into hydrocarbons and discharge oxygen as waste product (Eq.1).

CO2 +H2O ----> Cn(H2O)n + O2 Equation 1

Photosynthesis consists of light and dark reactions (see diagram). In the light reaction, pigments capture light
to generate ATP and NADPH2. ATP serves as chemical energy and NADPH2 serves as reducing power. In the
dark reaction, these energy-rich components are used in the Calvin cycle to convert carbon dioxide into
organic molecules catalyzed by enzymes. Light reactions take place in the thylakoid membranes of
chloroplasts. These thylakoid membranes contain the photosynthetic apparatuses consisting of light absorbing
pigments and an electron transport chain. Firstly, the photosystem antenna complex, composing out of
chlorophyll supported by accessory pigments, absorbs photons with wavelengths between 400 and 700 nm
(Photosynthetic Active Radiation).

In the reaction center of the photosystems, chlorophyll absorbs one photon and releases one electron. These
electrons are transported via the electron transport chain to photosystem II where reduction takes place and
NADPH2 is generated. Via photolysis of water into oxygen and protons, the electron is regenerated at the
chlorophyll. As a result, a proton gradient across the thylakoid membrane is created and this gradient is used
by ATP synthase to generate ATP.

In the Calvin-Benson cycle, enzymes starting with ribulose-biphosphate carboxylase (Rubisco) use ATP and
NADPH to synthesize three-carbon-sugars (C3-sugars) from carbon dioxide. Then, C3-sugars are combined to
form molecules of glucose. Glucose can be converted to polysaccharides which serve as building materials or
to fatty acids which serve as building blocks for membrane lipids or as a source of energy storage. The
enzymes in the dark reaction are temperature dependent and therefore predominantly define the
optimal temperature in which the species can grow.

5
2.3 Species
Microalgae comprise a large number of very different, generally photoysynthetic and generally unicellular
organisms. Genetically, the variability between the different groups of microalgae is much larger that for
example among the terrestrial plants.

The number of registered species varies between 25-40 thousands. The species are normally grouped
in classes, (up to 24 ) that share many biochemical and physical characteristics.

The major classes and a few examples of cultured species or genus examples are given below:

Bacillariophyceae (Diatoms ) Skeletonema, Thalassiosira, Phaeodactylum,

Chlorophyceae (Green algae) Chaetoceros
Rhodophyceans (Red algae) Chlorella, Dunaliella, Scenedesmus, Haematococcus,
Haptophyceae Nannochloris
Prasinophyceae Porphyridium cruentum, Galdieria
Cryptophyceae Isochrysis, Pavlova
Xanthophyceae Tetraselmis (Fig.1), Pyramimonas
Eustigmatophyceae Chlamydomonas, Rhodomonas, Chroomonas
Dinophyceans Olistodiscus
Euglenopyhceans Nannochloropsis (Fig. 2)
Cyanophyceae (blue-green Crypthecodinium, Alexandrium, Gymnodinium,
algae) Chattonella, Karenia
Euglena
Spirulina, Synechococcus, Synechocystis, Cyanidium

The Cyanophyceae are prokaryotes, that is, their DNA is not organized in a nucleus and their DNA replication
and protein synthesis mechanisms are very different from that of the other groups, which are all eukaryotes.
The Cyanophyceae were the first photosynthetic organisms that appeared in the evolution and are in terms
of productivity and ability to grow in extreme environments, still highly competitive.

But, because of the use of new, more modern molecular technologies, these classes are frequently re-
arranged. A number of the species are maintained in culture collections, either cryopreserved or in an actively
metabolizing state.

6
2.4 Growing algae
Taking an interest in the growth of algae, it is important to realize that there is a large difference between
cultivating macroalgae and microalgae. When we refer to algae on this website, we refer to microalgae.
Microalgae are microscopic, generally unicellular organisms that grow in freshwater and seawater. The water,
in which they are cultivated, is referred to as medium or substrate.

The cultures can reach concentrations of up to 0.2 – 10 g of dry matter (dw) per liter – about 1000 times more
concentrated than the densest natural algal blooms. Microalgae are produced for their content of oils, pigments
or special carbohydrates or to use directly as food additives. New applications of microalgal culture include
products of pharmaceutical interest with genetically modified algae – also referred to as molecular factories.
The omega-3 fatty acids which are among the most popular health food components, are originally produced
by microalgae. They are transported through the food chains and recently, microalgal production of fatty acids
as food ingredients has been commercialized.

There is an increased interest in microalgae as feedstock of oil for the chemical industry and biodiesel. For
that, the scale of production needs to be increased and the cost price of production needs to be decreased
drastically. Recently, Many companies and research groups developed activities in this area.

Algae normally grow by photosynthesis (called autotrophic growth). Some of them, however, can also utilize
various organic carbon sources like glucose or acetate, either as a supplement to photosynthesis or replacing
it completely (heterotrophic growth). The heterotrophs are the most amenable to large volume - highly dense
cultures, as light is more difficult to ”feed “ to a culture than for example, a concentrated sugar solution!

7
2.4.1Light
Photosynthetically active radiation (PAR) is the part of the solar radiation spectrum that ranges from 400 to
700 nm in wavelength (Fig. 1). The PAR range of the spectrum contains 43 % of the energy of the total solar
light spectrum. This is at the same time also the part of the spectrum that is visible to the human eye. The light
may be measured either as a stream or flux of photons, or as radiation energy. A photon is the smallest
possible unit of light, emitted when an electron in an atom changes orbit from an outer, high energy position, to
a position closer to the nucleus. The photons in blue light (wavelength around 400 nm) are more energy-rich
than photons in the red end (700 nm). Full sunlight may reach 2100 µmoles of photons (PAR) per second per
2 2
m (perpendicular to the sun‟s rays, earth surface) – or, expressed as energy, 1100 J per m per second (or,
-2
1100 W m ).

Figure 1: Relative intensity on energy basis of sunlight at ground level

Ultraviolet light is the radiation in the range 10-400 nm. At the lower wavelength end, the UV spectrum is
adjacent to X-rays.

Infrared light covers a wide range of wavelengths, from 750 nm to 1 mm and borders to the microwaves. Also,
the infrared radiation cannot be utilized by microalgae. Unlike the UV-radiation, it is not directly detrimental, but
may create a thermostating problem as it contains almost half the energy of the sunlight and is readily
absorbed in water.

8
Microalgal efficiency on light

Algae cannot use ultraviolet nor infrared light - neither do they make more benefit from energy-rich “blue”
photons than from the “red” photons. Figure 2 shows the microalgal sunlight energy utilization budget – only 9
% of the total sunlight energy may end up bound in biomass.

Figure 2: Sunlight energy budget in

microalgal photosynthesis.
The maximum potential energy
conservation in biomass is 9 %.

9
 2.4.2 Temperature
Microalgae have a wide range of temperature optima:

 The psychrophilic (cold water adapted) microalgae grow at temperatures below 0 °C and typically
have temperature growth maxima just a few degrees above zero. They are found in the polar regions.
Diatoms like Nitzschia and Amphiphrora and the cryptomonad genus, Chlamydomonas and the green
alga, Chlorella are frequently found psychrofiles.

o
Thermophilic (warm water adapted) microalgae on the other hand, grow at temperatures up to 75 C
and are found in and around hot springs. Only cyanobacteria
like Synechococcus and Synechocystis are found among the true thermophilic photosynthetic
organisms.
 Psychrophilic and thermophilic algae are intensively studied in the search for novel enzymes.
 Mesophilic algae cover the span between the psychrophilic and thermophilic. The ability of growing
at different temperatures in this range, however, also varies considerably. Chlorella sorokiniana for
example, is unusually thermotolerant (see fig. 1) and grows from 5 to 45 degrees, with an optimum
around 35 °C, while the temperature span of other microalgal species may be much more
narrow. Many of the industrially most interesting algae have temperature optima of about 25 degrees.

Temperature effect on the conversion of light

Light and temperature affects growth simultaneously in most microalgae. Light optimum of growth varies with
temperature – so to obtain a good annual productivity it is essential that these relationships are accurately
known for the cultured species. This knowledge is usually described in 2-factor growth models.

Temperature effects on metabolites

Many microalgae, particularly cold water adapted, increase the proportion of polyunsaturated fatty acids when
cultivated at lower temperatures.
Also, the proportion of different lipid classes may change with the cultivation temperature.

Industrial implications
For an algal species to have and industrial production potential, it is important that the optimum growth
temperature is sufficiently high, as cooling usually is considerably more expensive than heating! Even under
Nordic temperate conditions, on a sunny day, temperature can rise rapidly in photobioreactors because of their
large surface:volume ratios.

For Nordic temperate locations, the use of waste heat in the late autumn months and early spring months
usually is economically attractive.

10
 2.4.3 Nutrients
Algae need a rather limited number of elements for growth, that are supplied as minerals (salts),
called nutrients. Carbon dioxide is normallynot referred to as a nutrient.

A couple of elements – nitrogen (N) and phosphorous (P), sodium (Na), sulphur (S), potassium (K) and
magnesium (Mg) are required in rather large amounts and hence are referred to
as macronutrients. Micronutrients provide a number of elements that are required in very small quantities, like
manganese (Mn), zinc (Zn), copper (Cu), molybdenum (Mo) and cobalt (Co). Iron (Fe), chloride (Cl), calcium
(Ca) and borium (Bo) are required in intermediate quantities. Soil extract is sometimes included if an algae has
an unknown requirement – in many cases, however, the unknown requirement is a vitamin.

gram Ratio
pro Molar
Element gram Mw pro 1 C
C 0.541 12 1
H 0.074 1 1.64
O 0.296 16 0.41
N 0.0822 14 0.13
S 0.005 32.1 0.0035
P 0.00182 31 0.0013
K 0.0064 39.1 0.0036
Mg 0.00139 24.3 0.0013
Ca 7.25E-07 40.1 4.01E-07
Na 0.00098 23 0.00095
Si 0.000125 28.1 9.87E-05

Diatoms furthermore need silica (Si) in large quantities for producing their cell walls.

Some microalgae have vitamin requirements – vit. B1 (Thiamin), B12 (cyanocobalamin) and vit. H (biotin) are
often included in media recipes.

On top of this, the cultivation medium should have:

- a certain osmolarity, which may be regulated with salts as NaCl and MgSO 4
- a certain pH, which may be regulated with buffers or controlled by a pH controller.

A number of standard media recipes exist, such as F/2, Walne medium etc. and frequently, researchers modify
them so the composition reflects the elemental composition of the cultured species. For high density cultures
-1
(> 5 g L ), higher concentrations of nutrients are needed and new media have to be developed. However, it is
important that all the minerals stay in solution – i.e., that no precipitates are formed. Otherwise, one cannot be
sure that the algae have access to sufficient quantities of all the required elements!
Culture collections (see links page) provide small cultures at a certain fee (usually, € 50-100 per
delivery). Usually, they also provide media recipes that have been found suitable for the species and,
sometimes also ready made media.

Nutrients for heterotrophically growing algae

A number of algal species may grow in the dark on organic substances, such as glucose, acetate, glycerol or
aminoacids. In addition, they require the same nutrients as the photosynthetically growing algae

11
 2.4.4 Reactor
A reactor is an enclosed environment. In principle, a pond might be called a reactor – but it is customary to
distinguish between “open pond cultivation” and “photobioreactor cultivation” of microalgae.
Also, a small shake-flask or a test-tube culture is not called a reactor.

For microalgal cultivation, two main categories of reactors exist, photobioreactors and fermentors:

 Photobioreactors typically are tanks with transparent walls through which illumination of the culture
takes place – but illumination may also be immersed – fluorescent tubes or LEDs can be immersed
directly in the culture. Photobioreactors can be chemically sanitized – but cannot(1) operate
axenically(2). A frequently used photobioreactor is the bubble column (Fig. 1).
 Fermentors are normally stainless steel tanks and can be used for heterotrophic cultivation of
microalgae, but small desktop fermentors are often made with a glass vessel (Fig. 2). Fermentors
can be illuminated from inside, but the light transfer is very limited. Therefore internal illumination is
sedlom applicated (Fig. 3).

Figure 1: Figure 2: Figure 3:

A bubble column is a transparent A standard 2-liter desktop fermentor for A 200 L stainless steel bubble
cylinder. The culture is mixed laboratory use. column fermentor with
with air bubbles. internal illumination.
(1)
At present, the only way to sterilize large constructions is by using steam at 121 °C at 1.3 bar. Over 200 L, only steel constructions
can be built to withstand these conditions. Inserted glass tubes or glass windows can accommodate illumination.

(2)
Axenically means “without other organisms present”

12
2.5 Interesting products
Commercial productions
The history of microalgal cultivation extends about 125 years back, the first industrial productions being the
Japanese Chlorella productions for food supplements, shortly after the Second World War. The production
took place outdoor in circular or centrally agitated ponds. The traditional ponds are still being used, although
being gradually replaced by heterotrophic production systems. About 2000 tons of dryChlorella is being
produced annually, mostly in Japan. About half is produced by heterotrophic methods. The algal biomass is
either used directly or for a glucan extract (Chlorella growth factor).

The largest production is that of Arthrospira (former known as Spirulina), with about 3000 tons produced per
year (exclusively autotrophic). This production mostly occurred in open raceway ponds and used directly
as food supplements. Development of Arthrospira cultivation started at the Berkely University in California with
the establishment of the companies Earthrise and later Cyanotech. Arthrospira cultivation is growing rapidly in
India and China. Arthrospira can grow at a high alkalinity, which eliminates many competing algae, and is
therefore well suited for open ponds. Astaxanthin is the pink pigment found in salmon and shrimp. Natural
astaxanthin is made from the green alga Haematococcus pluvialis that may contain up to 6 %. About 500 tons
ofHaematococcus biomass is produced annually, autotrophically or mixotrophically, inphotobioreactors or
raceway ponds.

ß-carotene is produced with the green alga Dunaliella, of which 1200 tons biomass is produced annually, partly
in lagoons, partly in raceway ponds. Dunaliella can grow at high salinity, which provides the competitive
advantage that makes it suitable for open pond cultivation.

Polyunsaturated fatty acids DHA

The dinophycean Crypthecodinium cohnii and the thraustochytrids, Schizochytrium andUlkenia, yield high
volumetric productivities of the polyunsaturated fatty acids, EPA and DHA. The DHA production is the most
-1 -1
effective with specific volumetric productivities up to 3 g DHA L day with Schizochytrium, about half that
for Crypthecodinium. Productions are entirely heterotrophic.

Experimental productions
EPA is produced with the diatom Phaeodactylum (autotrophic), Nitzschia alba or N.
protothecoides (heterotrophic). The carotenoids lutein can be produced with Nitzschia
protothecoides (heterotrophic), Muriellopsis and a range of Chlorella species (autotrophic). Oil production for
biodiesel can be produced by a wide range of algal species, mostly autotrophic. Ethanol or hydrogen
fermentation can be done withChlamydomonas.

13
2.6 Production potentials

The photosynthetic efficiency (PE) is defined as

the fraction of light energy fixed as chemical
energy during photoautotrophic growth. Minimally
10 light photons (quanta) are required to produce
one mol of O2. Taking a representative biomass
composition (CH1.78O0.36N0.12) this corresponds to
14 quanta needed to fix one mol of CO2 into
biomass, based on ammonium as a nitrogen
source. Finally, one mol of CO2 fixed, results in
one Cmol of biomass (= 21.25 g dry weight) with an
-1
enthalpy of combustion of 547.8 kJ×Cmol .

In photosynthesis only light of wavelengths

between 400 and 700 nm is used. This represents
42.3% of the energy of the total spectrum of
sunlight and it is called photosynthetic active
radiation (PAR). The average energy content of
these quanta is 218 kJ/mol quanta. Combining all
these data it is calculated that maximally 9% of
sunlight energy (considering all wavelengths) can
be converted into chemical energy as new
biomass. Only considering the PAR range the
efficiency is 21.4 %. Based on solar irradiation
data as can be found e.g.
on http://re.jrc.ec.europa.eu/solarec/index.htm it
can be calculated that the maximal theoretical
biomass productivity in the south of Spain is 280
. -1. -1
tonnes ha year .

14
 3. Applications
Currently, algae production primarily targets food and feed markets. These activities will remain
important and will surely grow. Yet, we expect that algae production for non-food purposes will prove
even more important within a decade or so, both in tonnage and economic value. Given their
unequalled growing rate among photosynthetic organisms, algae hold great promise for the cost
efficient large scale production of biofuels, bulk & commodity chemicals and fine chemicals.

3.1 Bulk Chemicals

The chemical industry is a highly energy intensiveone. In the Netherlands, it represents some 25% of
total energy consumption in the form of fossil input and the energy that is needed to convert it into bulk,
commodity and fine chemicals. Much of this required energy is needed for the incorporation of oxygen and
nitrogen atoms into fossil fuel-typical „(hydro)carbon-only‟ molecules that lack this heteroatom functionality.
Without it, bulk products like polyesters would not exist. By nature, biomass is rich in oxygen and nitrogen
atoms. New biomass (unlike the old biomass in oil, coal and gas) is rich in oxygen and nitrogen atoms. This
provides a chemical and thermodynamical shortcut to many functionalized compounds. This translates into
lower energy input, fewer conversions and a greatly reduced dependence on efficient heat exchange, i.e., the
possibility for cost effective production at small scale. This shortcut has always been there, but is now heavily
(re)investigated from the realization that the era of cheap and plentiful fossil resources will soon come to an
end. The biobased chemical industry is still in its infancy, compared to the century-old petrochemical industry if
one compares the width of the bulk & commodity chemicals portfolio. But the potential is huge and progress is
fast. Products like (the biofuel) ethanol and (the biofuel by-product) glycerol are increasingly used as platform
chemicals for others bulk products. E.g., glycerol can be used to produce propanediol, propylene glycol,
branched polyesters, nylons and glyceraldehyde. A new challenge is the production of N-containing
compounds that we now look upon as typical petrochemicals. E.g., butane-1,4-diamine can also be produced
from the (biobased) amino acid lysine. AFSG Biobased Products has determined that the majority of the bulk &
commodity chemicals produced by the petrochemical industry surrounding Rotterdam harbor can be derived
from biomass within 10-20 years.
As a feedstock for bulk & commodity chemicals, biomass produced by fast growing algae is „as good as any
biomass‟, as long as it provides the required fatty acids, carbohydrates and/or bulk proteins (i.e. amino acids)
at the right price. Given the latter requirement, carbohydrate-derived chemicals would obviously benefit most
from algae that mainly produce carbohydrates, particularly those that can be efficiently converted. Likewise,
the production of N-containing chemicals would benefit most from protein-rich algae sources, particularly those
containing N-rich amino acids like lysine and arginine. Given that there are tens of thousands of algae species,
careful selection is the first step to take. This may be further aided by biotechnological tools to actively
steer algae metabolism in desired directions.

15
3.2 Food & Feed
The composition of a number of microalgae is compared with food sources in Table 1. It is shown that
microalgae are rich in proteins, carbohydrates and fatty acids. Carbohydrates and lipids in microalgae are
especially interesting because of the functionality of these ingredients, such as the high concentration of ω-3
fatty acids.

Table 1: Composition of several food sources and microalgae (in % of dry weight) From: Becker E.W. (1994)
Microalgae Biotechnology and Microbiology. Cambridge University Press, Cambridge

Product Proteins Carbohydrates Lipids

Bakers yeast 39 38 1
Meat 43 1 34
Milk 26 38 28
Rice 8 77 2
Soy beans 37 30 20
Anabaena cylindrica 43-56 25-30 4-7
Chlamydomonas reinhardtii 48 17 21
Chlorella vulgaris 51-58 12-17 14-22
Porphyridium cruentum 28-39 40-57 9-14
Scenedesmus obliquus 50-56 10-17 12-14
Spirulina maxima 60-71 13-16 6-7
Synechococcus spec. 63 15 11

Despite the promises of the new non-feed application markets, nowadays, thefeed & food markets are still
the major worldwide outlets of commercial algae production. In relation to food applications microalgae are
mainly used for theirhigh value ingredients (Fig. 1) such as vitamins (e.g. C and D2), ω-3 fatty acids, pigments
and antioxidants (β-carotene, astaxanthin and lutein). In the future we expect microalgae will also be used as a
source of proteins and polysaccharides as co-products in biofuel and chemical production after biorefinery.
Food applications are mainly based on the use of the complete biomass such asChlorella and Arthrospira. In
addition, there is a large market of natural β-carotene from Dunaliella.

Our core expertise, however, lies on microalgae productionand valorization. In food & feed, the major
applications are dried algae like Chlorella andArthrospira and isolated, high value compounds like carotenoids
(e.g. beta-carotene) from Dunaliella. These algae and algae compounds find their way as functional food
and feed ingredients (Fig. 2).
Within WUR, we have a specific interest in the metabolomics of carotenoids biosynthesisin the alga Dunaliella
salina. We employ metabolic profiling strategies to create a better understanding of the effects of physiological
conditions on carotenoids metabolism to generate leads for the maximization of carotenoids production. For
the same goal, the „milking‟ of carotenoids from this algal species is studied in detail. Microalgae are also
studied as feed for shellfish and fish production, in particular that of the blue musselMytilus edulis and sole.

16
3.3 Fine Chemicals
For fine chemicals, the same advantages in using biomass versus fossil feedstock apply as those outlined
under Bulk & Commodity Chemicals. A cheap yet complex O-rich compound like glucose would be an
extremely expensive fine chemical, if it were to be produced starting from fossil feedstock. Given the higher
price level, lower scale and broader palette of fine chemicals versus bulk chemicals, secondary metabolites are
also relevant sources here.
Fine chemicals are an extremely diverse group. So far, the most important algal based fine chemicals are food
grade products like beta-carotene, pigments, vitamins, polysaccharides and essential fatty acids. New non-
food fine chemicals that are currently under investigation include bioactive substances like pharmaceutically
active polyketides and antifouling agents, but also polyunsaturated fatty acids that act as reactive diluents in
paints. The potential for algal based fine chemicals is however much larger than the above mentioned
examples. Given the enormous variety of algal species and the compounds they produce, the critical success
factor is often the efficient purification of a desired product or precursor thereof from the complex total biomass.

3.4 Energy
Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels
(photograph). Microalgae, like higher plants, produce storage lipids in the form of triacyglycerols (TAGs) which
can be used to synthesize fatty acid methyl esters (a substitute for fossil-derived diesel fuel). Microalgae
represent a very attractive alternative compared to terrestrial oleaginous species, because their productivity is
much higher and it does not compete for land suitable for agricultural irrigation or consumption by humans or
animals, providing therefore food security.

To date, commercial application of microalgae has concentrated on compounds that have a very high
value per kilo (e.g. carotenoids). To be a feasible source for biodiesel, the current price for microalgae
production needs to be reduced by two orders of magnitude. In addition, the scale of production of lipids from
microalgae would need to be three orders of magnitude greater than the scale currently possible for high-value
compounds. These ambitious goals are feasible because the potential productivity of microalgae is tenfold
greater than that of agricultural crops.

However, the promises of several companies in the field combined with expectations from the market have led
to unrealistic predictions for the potential of microalgae. There are companies that promise to produce an
amount of biodiesel from microalgae that is either near or in some cases higher than the maximum amount
achievable. In areas with high irradiation a theoretical maximum productivity of 280 tonnes of dry biomass per
year can be produced. If we then assume a lipid content of 40% in the microalgae, the total amount of oil that
-1 -1
can be produced is 115,000 L ha year . However, these productivities are unrealistic at this point in time.
-
With state-of-the-art technology, it might be possible to produce in the order of magnitude of 20,000 L ha
1 -1
year of oil – this is still significantly more than can be obtained from energy crops (the areal productivity of
-1 -1
palm oil is 6,000 L ha year ).

3.4.1 Biofuels
The global fuel retailing market is immense: in 2007, the total annual turnover was 996 billion dollar for almost
the same volume in liters. Biofuels are increasingly entering this fossil fuel dominated arena, often aided by
policy, incentives and directives for political and environmental reasons. In Europe, the Fuel Directive of the EC
aims at the replacement of 5.75% and 10% of fossil-based fuel by biofuels in 2010 and 2020, respectively. To
be competitive and independent from fluctuating support from (local) policy on the long run, biofuels should
equal or beat the cost level of fossil fuels. Here, algae based fuels hold great promise, directly related to the
potential to produce morebiomass/ha-year than any other form of biomass. We feel that the break-even point
for algae-based biofuels is within reach in about 10 years.

To realize this huge potential, the knowledge and technology levels will need to be pushed, which is
why algae-based biofuel is often referred to as ‘third generation biofuel’. We aim to strengthen our

17
position at the forefront of these developments, and invite interested commercial parties to join us in
the process.

The estimated ten years to reach the break-even point also takes into account that the competing prices of oil,
gas and coal will increase beyond the peak levels of around 140$/barrel that were reached shortly before the
credit crunch that started in 2008. Our current aim is to lower the production costs of algae biomass to around
0.40 Euro/kg dry matter (2008 price levels). Both the oil and carbohydrate fractions in the algal biomass are
important, with routes to algal biodiesel and bioethanol, respectively. A non-technological advantage lies in the
societal need to both feed the world and provide energy in the form of biofuels. When produced in the right
way, the large scale production of algae biomass does not compete for food production, unlike said traditional
food crops and crops that use the same arable land.

3.4.2 Feasibility for Energy

For Delta, an energy companies in the Netherlands, we performed a feasibility study on the production of
microalgae, in which 3 production technologies were compared: open pond , horizontal tubular
photobioreactor and a flat panel photobioreactor.
The analysis was based on state-of-the-art technology for the solar conditions in the Netherlands. Estimations
were conservative, which means that for reaching estimated productivities there is no need to develop systems
or processes further than what is now possible. In this analysis we also assumed that nutrients for the growth
medium and CO2had to be bought. The end product of the process we designed is an algal paste with a dry
matter content of 20%. Extraction of oil and esterification was not considered.

Two different plant sizes were evaluated (1 and 100 ha). We report here the values estimated for a scale of
100 ha. Microalgae biomass can be produced cheaper in photobioreactors than in raceway ponds, but this is
achieved at the expense of higher energy consumption. When comparing the two photobioreactors, the
horizontal tubular reactor and flat panel show a similar biomass production cost. Regarding energy balance,
flat panels perform a bit better, even though both systems have a negative balance.

There is no practical experience with cultivation of microalgae for energypurposes. Photobioreactors have only
been applied for the production of biomass of high value, i.e. more than 100 €/kg DW. As a consequence,
processes have never been optimized for applications where the value of biomass is less than 1 €/kg DW.
Process development for the production of microalgae for energy purposes still needs to be done. In order to
analyze the effect of some parameters on biomass / energy costs a sensitivity analysis was made. In this way
we could determine whether and how costs could be realistically reduced. With the present status of the
technology, production costs were calculated at 4.02 €/kg biomass (153.5 € / GJ) but could become as low as
0.42 €/kg biomass (16.0 € / GJ).
Development of the technology combined with the usage of the remaining biomass components, which are not
required for biofuelproduction, in other applications (a biorefinery approach in which 100% biomass is
valorized) the commercial production of microalgae could become a realistic option for the biofuel market.

18
 4. Technologies
Wageningen UR focuses on many different aspects of microalgae. They can mainly be categorized in three
areas of focus:

Systems biology studies complex Focusing on the cultivation Focusing on

biological systems as integrated of microalgae from lab- the isolation of products
wholes, using tools of modeling, scale to commercial from the microalgae
simulation, and comparison to systems. In this and biorefiningthem into
experiment. Metabolic flux section, shake flask products with
analysis,genomics, transcriptomic experimentsare discussed applications in different
s andproteomics are used to get up to commercial systems. industry sectors.
insight how microalgae function Further, also heterotrophic Attention is paid to
and how metabolic routes can be algae and their cultivation the isolation, biorefining,
optimized to obtain high product are discussed. conversion and purificatio
yields. n of products from
microalgae.

4.1 Systems Biology

4.1.1 Metabolic flux analysis

In algae carbon dioxide and inorganic compounds like nitrate and phosphate are converted into biomass and
different types of products like, for example, lipids. Light is used as energy source. This is done through a
complex set of biochemical reactions that take place in different compartments with the most relevant
being the chloroplast, mitochondria and the cytosol. To obtain more insight in metabolism metabolic flux
balancing is a useful tool.
Using the known stoichiometry of the biochemical reactions a metabolic network model can be constructed
(Figure 1). Next, by assuming steady state and constructing mass balances over the intracellular metabolites
the rates with which these biochemical reactions take place (the fluxes) can be connected to the consumption
of substrates and production of biomass and other compounds like lipids. These network models can be used
essentially in two ways:

1. One is that by measuring the rates with which substrates are consumed and biomass and products
are formed, the intracellular flux distribution can be calculated. Next metabolic flux distributions under
different conditions can be compared. For example, the flux distribution under conditions where no
lipids are produced can be compared with the flux distribution under conditions where more or

19
 less lipid formation occurs. This can give insight in possible metabolic bottlenecks. Especially, in
combination with measurement of gene expression this technique can give insight into regulation
mechanisms.
2. The second option is to use these models to calculate optimal solutions using linear optimization
techniques. For example, one can calculate the flux distribution resulting in optimal biomass growth or
product formation. This gives insight in why algae have a certain metabolic flux distribution. In
addition, metabolic reactions can be added to the network or deleted from the network and the effect
on biomass and product formation can be studied in silico. In this way these models can help
improving, for example, product formation by either engineering the conditions in the environment of
the algae or by genetic engineering.

Example of a simplified metabolic network model for Algae:

4.1.2 Metabolomics techniques

The metabolome represents the collection of all metabolites in a plant or algal cell. Metabolites are the final of
gene and proteins expression.Transcriptomics and proteomics reveal part of the genomics story of a cell,
metabolomics can give an instantaneous snapshot of the metabolite profile of a cell. The WUR metabolomics
facility provide a multi-technology platform comprising integrated dedicated and non-targeted (MS)
technologies to generate the broadest overview of the cell metabolome composition:

 HPLC and GC separation technologies

 3 GC mass spectrometers and one GC-TOF mass spectrometer
 One Ultima LC(TOF) mass spectrometer + one UPLC LTQ-Orbitrap mass spectrometer
 Flow Injection Analyses
 MS and MS/MS detection analysis
 A range of detection techniques (e.g PDA, EC, Fluorescence)
 Automated robotic extraction facilities

20
  Full bioinformatics support
 MetAlignTM software for automated sample comparison
 Targeted and non-targeted analyses of complex extracts
 Total protocol design
 On line antioxidant analysis
 Head space trapping and analysis
 Pre- and post-column derivatisation

Comparison of the metabolite profile (flavonoids) versus antioxidant capacity

of different metabolites in two different onion cultivars

4.1.3 Genomics
The genome is the complete set of inherited genetic information encoded in the DNA. It is the DNA information
that makes up an organism. Genomics is the study of an organism's entire genome, the sum total of all an
individual organism's genes.

Genes, fragment of DNA, also called "coding" DNA, contain the chemical recipe that determines particular
traits, for example the ability of specific algae to produce EPA or DHA. The genome of complex organisms, like
human, animals, plants and algae comprise about 20,000-40,000 genes.
Genes generally comprise only a relative small part of the total genome, a substantial part of the genome is
"noncoding" DNA. Within these noncoding regions of the genome is the information that determines in which
cell types and at what stages in the life of an organism the genes are active.

Genomics is the study of the entire set of DNA sequences, both coding and noncoding DNA. It is the study of
all the genes of a cell or tissue at the DNA (genotype), mRNA (transcriptome) and protein (proteome) levels.

Specific genomics includes activities which are intended at determining the entire DNA sequence of
organisms, genome wide genetic mapping of markers linked to specific traits (e.g SNP detection), the analyses

21
of intragenomic phenomena such as heterosis, epistasis or other interactions between loci within the genome,
and the analyses of gene expression (see transcriptomics). In a somewhat wider definition genomics also
includes proteomics (unraveling the total proteome of a cell or organism) and metabolomics (analyzing the full
spectrum of metabolites in a cell or organism).

Over the past decade, genomics has sparked an extraordinary biological revolution. The information and
technology of genomics has considerably improved our understanding of plant evolution, the mechanisms of
disease resistance or adaptation to the environment, the control of metabolic pathways and the role of genes in
shape, color and taste of plants and fruits.

Genomics of algae is still in its infancy.

4.1.4 Transcriptomics
The transcriptome of an organism, for example an algal cell, is the set of messenger RNA (mRNA) molecules
or "transcripts," produced in one cell or a population of cells. The transcriptome can cover the transcripts of an
entire organism, or a specific subset of transcripts present in only one particular cell type (this is relevant for
multicellular algae).

Transcription of DNA into mRNA is one of the first steps in the regulation of cellular processes by the genes of
that cell. The transcriptome can vary with growth conditions or with developmental stage of the cell. Information
about the transcript levels is needed for understanding how genes control the cellular processes in response to
changes inside or outside the cell. It may also reveal how genes interact to each other in so-called gene
regulatory networks.

Transcriptomics, also called genome-wide expression profiling, is one of the tools which is used to understand
how a cell controls biological processes: for example metabolic pathways leading to economically valuable
compounds, or cellular processes which control growth rate of cell division. Transcriptomics help to resolve
questions such as: what are the functional roles of different genes and in what cellular processes do they
participate? How are genes regulated and how do genes and gene products interact? How do gene expression
levels differ in various cell types and states and how is gene expression changed by various treatments or
environmental stimuli?

Common technologies for genome-wide or high-throughput analysis of gene expression are cDNA
microarrays, oligo-microarrays, cDNA-AFLP, SAGE and cDNA sequencing. Microarrays are particularly
valuable when the genome of the organism of interest is already known and many thousands of individual
cDNA‟s or oligo‟s are available. For a few organisms prefabricated expression microarrays are available. cDNA
sequencing is particularly suitable when the genome sequence is unknown and when cDNA are hardly
available.

4.1.5 Proteomics

The active components in every cell are proteins. Enzymes, which are proteins as well, are continually
controlling pools of molecules which play a key role in cellular and metabolic processes. Structural proteins are
important in e.g. membrane permeability, intercellular communication or cell wall architecture, etc. As such,
proteomics research is relevant for analyzing those biological processes closely linked to the phenotype.

Proteomics technologies and instrumentation

Several tools for the profiling and quantification of highly complex protein mixtures available at WUR. We use
two-dimensional gelelectrophoresis and/or highly sensitive mass spectrometry to separate and
identify thousands of proteins or peptides. Technologies and tools available are:

 Qualitative and quantitative analyses of protein profiles

 1D and 2D protein gel electrophoresis, including fluorescent staining methods (eg. DIGE)

22
  Gel image analysis software
 Multidimensional LC-MS/MS analysis (Qtof Synapt and Orbitrap FTMS)
 Peptide sequencing for protein identification
 Analyses of post-translational modifications e.g.Phosphorylation / Glycosylation / N-terminal
acetylation
 Bioinformatics
 Protein-protein interaction studies (transcription factors)
 Automated robotic sample preparation facilities

4.1.6 Bioinformatics

The applied bioinformatics research at WUR facilitates high throughput „omics‟ research, by providing the
computational infrastructure for data acquisition,
management and analysis. Applied Bioinformatics develops and implements algorithms, software and
databases. We provide bioinformatics services in fields ranging from functional genomics to diagnostics. Our
research program focuses on comparative genomics, gene function
prediction, proteome and metabolome analysis.

Bioinformatics technologies

 Data analyses in genomics, proteomics and metabolomics

 Grid and cluster computing
 Automated genome annotation and visualization
 Text and data mining
 Development of molecular markers
 Sequence analysis for diagnostics
 Laboratory information management systems (LIMS)
 Analyses of heterogeneous „omics‟ data
 Custom software and database development
 Consultancy

4.2 Production
At first glance, cultivation of phototrophs such as microalgae seems easy. They seem to grow easily in
ponds in the garden. However, you should realize that microalgal concentrations in these ponds are
-1
very low (0.005 g L ) andproductivity is almost zero, because nutrients and light are limiting it.
Microalgae grown in open or closed systems, regardless of configuration, need light, nutrients and
mixing; however, shear forces and high oxygen levels should be prevented (Fig. 1).
In these systems, light is the most difficult parameter to provide, because it cannot be stored and
ideally should be distributed in such a way that low light intensities are provided everywhere in
the photobioreactor. However, in most systems, ideal light intensities occur only in a very small part.
Photosynthetic yields drop dramatically at the walls (or top) where photoinhibition and heat dissipation
occur due to too high light intensities. Further inside the culture, light cannot penetrate because of cell
shading and microalgae are subjected to darkness.
Another bottleneck in growing microalgae are nutrients like nitrogen and phosphorus, but also
dissolved carbon dioxide as the inorganic carbon source for photosynthetic biomass production. At
low biomass concentrations, carbon dioxide supply and removal of excess oxygen is mostly not a
-1
bottleneck; however, at high biomass concentrations (>10 g L ) they can become rate limiting.

Mixing of liquid culture is required to suspend biomass, to promote contact between liquid nutrient
medium and cells and to prevent gradients of nutrients, pH and temperature inside the reactor. On
small scale, mixing is rather easy; however, at large scale, mixing becomes more difficult and can
become rate limiting. Mostly, aeration is used to mix microalgae cultures and in that case

23
detrimental shear stress should be prevented. In small closed systems, maintaining pH
and temperature at optimal range is quite easy. But, at commercial scale, temperature control becomes
expensive and serious attention should be given if temperature control is worthwhile.

4.2.1 Open systems

All kinds of very interesting cultivation systems for microalgae have been developed. Here, open
systems are addressed. Open systems are mostly natural lakes or open ponds. Two types of open
ponds exist: circular ponds with a rotating arm or long channel ponds, single or connected to each
other, that are mixed with a paddle wheel (raceway pond) (Figure 1). The type frequently used on
a commercial basis are raceway ponds. These ponds are usually no more than thirty centimeters deep
and are mixed via a paddle wheel that circulates water withnutrients and microalgae. Sunlight falls on
the reactor surface and is absorbed by the culture, implicating that photon flux decreases with
increasing depth (Figure 2). As can be seen, most of the volume is exposed to very low light
intensities. In this zone, light energy will be converted efficiently, but the productivity is low, because
little light is available. Close to the reactor surface, light intensities are very high and because of
photoinhibition and/or photosaturation, the light efficiency and thus productivity will be very low.

In these systems, due to a long light path and bad mixing, photosynthetic efficiency is low and
consequently, biomass concentration and volumetric productivity are very low. Another disadvantage
is that water temperature and light conditions in open systems cannot be controlled. For that, the
growing season is largely dependent on location and, aside from tropical areas, is limited to warmer
months where more light is available. Nevertheless, this is a very successful concept because of
simplicity and low costs. Scaling up is very easy and areas filled with ponds up to 150 ha
are commercially used. However, because the system is open, this system is vulnerable for
contaminations. Therefore, only a few species that can grow in selective environments, can be grown
in these open systems; high salinity for Dunaliella salinaand high alkalinity with Spirulina platensis.
Another approach is to use large inoculum amounts produced in closed photobioreactors and take
advantage of fast-growing species such as Chlorella.

24
 Light intensity (solid line) and productivity
(dotted line) in an open pond at high light
intensities

4.2.2 Shake flask cultivation

When a culture from a culture collection arrives or is collected from outside waters/soils, it should be cultivated
in small volumes before it can be used as inoculum for photobioreactors. This can be done in a light climate
cabinet under continuous light or light with a day/night rhythm of 16h/8h. Cultures are grown at a
-2 -1
set temperature (20-37°C) and with a low light intensity (50-100 umol m s ). They are orbital shaken (100
r.p.m.) to keep the microalgae into suspension. Air is enriched with 2% carbon dioxide and is blown into the
headspace to keep the pH constant and to ensure that there is sufficient carbon available for the microalgae.
After sufficient growth (depending on species: growth 1-6 weeks), the culture inside the flasks can be used as
inoculum for photobioreactors.

4.2.3 Lab-scale photobioreactors

Many microalgae species are promising to produce a wide range of compounds with interest for different
industry sectors. To be able to produce these products, monocultures have to be produced and for that,
photobioreactors have to be used. A photobioreactor can be described as an enclosed, illuminated culture
vessel designed for controlled biomass production of phototrophic liquid cell suspension cultures. On lab-scale
two different photobioreactors are available, bubble columns and flat panel photobioreactors. Bubble columns
have the advantage that they are easy to construct; flat panel photobioreactors have the advantage that they
have shorter optical paths and due to the flat design the illuminated area is exactly known. Short optical paths
lead to higher biomass concentrations and increased volumetric productivities. These lab-scale
photobioreactors have a volume of about 0.5-50 L and are mixed by air and eventually enriched by CO2.
Control of pH can be done by adding carbon dioxide to the air or by pumping acid if pH is too high. Light is
mostly provided by artificial light that can be thoroughly controlled, allowing accurate calculations
of photosynthetic efficiencies. Fluorescent tubes were mostly used in the past, but nowadays most researchers
switch to LED lights, which give a more homogenous light distribution and less heat. Temperature can be
controlled by pumping water through a water jacket.

4.2.4 Pilot-scale photobioreactors

Pilot plant scale photobioreactors can be used to produce inoculum for commercial production systems, to
produce a small quantity of biomass or to produce specialty productsfor a small market. An example is the
bubble column, a vertical tubular reactor. Scalability of this system is limited since, when putting several
systems close to each other, they will shadow each other. Changing to a horizontal tubular
photobioreactor consisting of long horizontal tubes eliminates this problem. However, this has its own scaling
problem: algae will consume nutrients and CO2 while producing O2 (which inhibits algal growth at elevated
concentrations), so growth conditions deteriorate further along the tube and therefore tube length is limited.
Up-scaling can be achieved by installing individual modules with optimized size vs. tube length ratios. To make
optimal use of surface area receiving solar irradiation, flat photobioreactors can be used. Also for these
systems, with the current technology available, scaling up consists in installing multiple optimal modules.

25
These systems have the highest photosynthetic efficiency due to its largest surface to volume ratio, which
promotes dilution of the light intensity over a bigger surface area.
Presently, photosynthetic efficiencies on solar basis, found in different systems outdoors, are 1 -2 % for
raceway ponds, 2-4 % for horizontal tubular photobioreactors and 3-5 % for flat panels. Photosynthetic
efficiency will depend on reactor configuration, process conditions and microalgae strain.

4.2.5 Commercial scale photobioreactors

Most of the commercial available products from algae are produced in open ponds (Figure 1). The main reason
for using these systems are that they are very cheap to build, operation is simple and scaling up is very easy.
Disadvantages are low volumetric productivities, vulnerability to contamination and high evaporative losses.
The most commonly used open system is a raceway pond, single or several of these ponds connected in
series. These ponds are usually no more than thirty centimeters deep and are mixed via a paddle wheel that
circulates water with nutrients and microalgae. Ponds can produce about 20 ton biomass/ha/year.

However, many species cannot be grown in these open ponds because they are easily overgrown by other
microorganisms and because some products (used in cosmetics and for human consumption) require a
constant quality and safety is a major parameter. For these products, closed systems that can be fully
controlled have to be used. As closed photobioreactors, only the tubular photobioreactor (Figure 2) is used
commercially because it can be easily constructed and it can have a ten times higher volumetric productivitiy
than open ponds. This type of reactor consists of many tubes of about 3-10 cm in optical path (path that light
has to travel, i.e. tube diameter) and 25-100 meter long and is operated at biomass concentrations of 1 to 5
grams per liter dry weight. The system has as main drawbacks the high investment and operation costs and a
poor mass transfer which can lead to oxygen build up in the reactor to inhibiting levels and consequently
growth inhibition. Biomass production is about 20-60 ton/ha/yr depending on optical density and the capacity of
the degassers.

Flat panel photobioreactors have been considered too expensive to be viable. However, innovative ways to
construct flat panels cheaper are being developed and for example plastic bags in metal racks can favor this
type of reactor over tubular reactors. In the future, innovative cheap designs of this type of reactor will probably
be used instead of tubular systems.

4.2.6 Heterotrophic organisms

In the last decade, a couple of successful industrial heterotrophic microalgal productions have been
established. Many microalgae can assimilate organic substances to cover variable part of their carbon and
energy requirements. To cover their entire energy requirements and be able to grow in complete darkness, the
organic substances are respired in mitochondria with oxygen as electron acceptor, a process similar to the
respiration in animal cells. Some algae, such as Chlamydomonas (Fig 1), may also use a slightly modified
process to respire acetate, the so-called glyoxylate pathway. In Chlamydomonas the process is regulated so
that it takes place only in the dark.

Chlamydomonas is also able to ferment starch, which was produced during the day, into ethanol under
anaerobic conditions. So far, fermentation has been demonstrated only in a few microalgae species (Fig. 2).
Algae capable of growing in the dark, are called true heterotrophs, while algae that require light but are able to
supplement the metabolism with organic substances, are called mixotrophs. Very few species, however, can
grow in darkness in addition being able to grow also as true autotrophs – i.e., in light without organic
supplements.

Organic substances that may be respired, include glucose, acetate, glycerol, TCA cycle intermediates (for
example citric acid) and a number of amino acids. Only glucose, acetate and glycerol may play a role as
substrates in industrial productions. These substances are small molecules and algae are not normally able to
metabolize large molecules such as proteins or even complex particles – but some algae, most notably in the
classes Dinophyceans and Prymnesiophyceans are able to engulf large molecules and particles in a process
called pinocytosis or fagocytosis, depending on the size of particles.
Some Dinophyceans have lost their ability to form chloroplasts, but are able to retain functional chlorplast from
ingested algae!

26
A good place to look for heterotrophic species is among decaying seaweed where the decomposition
processes result in a rich variety of dissolved organic substances.

4.2.6.a Growth
Heterotrophic growth of microalgae is usually slower than autotrophic growth, generally about 2/3 of the growth
-1
rate of autotrophic growth. Growth rates are typically 0.3-1 d . But due to practical limitations on the transfer
of light into photobioreactors, heterotrophic cultures can grow in relatively higher densities in large-volume
reactors. A reactor for heterotrophic production is referred to as a fermentor even though the growth process is
respiration – and not fermentation. This means that the algae require oxygen.

The volume of the fermentors that currently are being used for industrial heterotrophic microalgal cultivation,
3
range from 80-200 m and the fermentors provide temperature control, pH – and oxygen control and agitation –
usually by impellers. Furthermore, the fermentor is kept bacteria free. This is a requirement when organic
substances like glucose, acetate or yeast extract are being used. The bacteria-free operation is established by
initial steam-sterilizing the fermentor – usually with the nutrient solution in it and ensuring, that all streams that
subsequently are added to the fermentor, are sterile. A large-volume bacteria-free starter culture is
required, typically several hundred liters. In the case of small desktop fermentors, the entire fermentor can be
steam sterilized in an autoclave – for larger fermentors, steam is added under pressure so that the temperature
in all compartments is kept at 121 ˚C for at least 20 minutes.

The two main types of cultivation processes are:

• Batch – the reactor is filled with medium and inoculated and the whole culture is harvested after the end of
the cultivation period – typically, a few days only.
• Continuous – the reactor is constantly diluted with new growth medium and, to keep the volume constant, a
similar volume of culture is harvested. This operation can be carried on until the reactor gets contaminated with
bacteria or needs cleaning.
The resulting productivity per unit reactor volume is very high – but so are investment and operating costs.

4.2.6.b Production potential

-1
The highest published biomass densities in heterotrophic productions, are about 120 g dw L . Normally,
-1
heterotrophic microalgal cultures are taken to 70 – 100 g dw L . With heterotrophic production, the productivity
(rate of production) is indicated as a volumetric productivity: the amount of algae or product that is
produced per unit reactor volume per time. With autotrophic production, the productivity can also be indicated
as areal productivity – the amount of algae or product that can be produced per unit time per unit area,
occupied by thephotobioreactor (reactor footprint) or, as a specific area productivity – for instance, related to
the plane projection of the bioreactor surface. Aslight is inevitably the limiting factor with industrial
photobioreactors, areal productivity is most frequently used to indicate productivity. These differences make
the two systems difficult to compare in terms of productivity.

Biomass productivity
-1 -1
With the green alga, Chlorella sorokiniana (Fig. 1), a heterotrophic growth of about 1 g dw L h has been
-1 -1
obtained. With the heterotrophic diatom Nitzschia alba, 0.8 g dw L h can be obtained. The volumetric
biomass productivity of thraustochytrids (heterotrophic primitive algae used for fatty acid production) is
reported to be in the same range. But, similar volumetric productivities can be obtained with very thin, strongly
-1 -1
illuminated photobioreactors; 1.2 g dw L h has been recorded with Arhtrospira. However, the reactor
geometry and cost implications are not favorable for the photobioreactor production. For comparison, the most
-1 -1
productive industrial yeast fermentations can reach 25 g dw L h .

Product productivity
With some microalgal productions – like for health food, the biomass productivity is important, while for others,
the volumetric productivities of a specific compound is of greater interest.

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4.3 Biorefinery
Biorefinery is defined as the co-production of a spectrum of bio-based products (food, feed,
materials, chemicals) and energy (fuels, power, heat) from biomass. The separately discussed product
markets under Applications might suggest that each algal product market is on its own. This is however not the
case, particularly if the aim is to enter high volume, low price markets like biofuels. The algae
production, isolation and processing costs are more easily covered by a value chain that also includes high
value streams. These may represent only a minor fraction of the total biomass, but may contribute to the
economics in a major way. For example, in the EOS project AlgiCoat, in which we collaborate with Akzo Nobel,
Essent and Ingrepro, the first generation value chain includes (in order of decreasing value) coating
components, biofuels, and waste stream biomass, which is used as a simple source ofenergy in power plants.
Of course, this cascading concept only works if one can isolate individual streams from the algae biomass for
individual applications. This is exactly what biorefinery should bring about and identical to the refinery of crude
mineral oil (fossil biomass!) into multiple streams via cracking and subsequent processing.

There are many individual „enabling technologies‟ behind a biorefinery process. These include mechanical
pretreatment, heat treatment, chemical/enzymatic cell wall degradation, fermentation, isolation/purification,
conversion etc.. At WUR, we have ample expertise with these techniques from the biorefinery of all major
forms of biomass. The total chain of processing events is always tailor-made and optimized for a given
biomass source and the applications aimed at. For unicellular organisms like algae, the first steps in the total
biorefinery process differ strongly from that for plant-based material. Instead of mechanical harvesting and
pretreatment of the crude plant material, efficient isolation of dispersed cells from the production medium is
required. No lignin or hemicellulose is present, but many algae have cell walls that need to be broken down to
allow efficient isolation of compounds of interest. In the later stages where individual compounds are isolated
and converted, processes are more similar.

. Unless direct milking of a desired algal product is possible (see Purification for an important example), the
first step in microalgae value creation is the isolation (harvesting) thereof from the production medium. There
are several techniques available for this, including:
- evaporation (usable in areas where both sunlight and water are abundant)
- filtering over micro screens
- centrifugation (often in combination with micro screens)
- flocculation, using agents like alum, ferric chloride or organic polyelectrolytes
- froth flotation: the water and algae are aerated into a froth, which can be removed easily from the water.

High volume, low value applications like biofuels pose a great challenge since cost- and energy effective
isolation procedures are essential. From an energy perspective,flocculation and froth flotation techniques are
particularly interesting. Centrifugation is an energy intensive processing step that, if unavoidable, should be
optimized for energy efficiency to lower the cost price of the end product. Which technique is best is highly
dependent on the algal species involved. In general, research on large scale algae isolation is still in its
infancy.

The isolation of individual components from algal biomass is discussed under Purification.

4.3.1 Conversion

Many isolated algal components will need to be (bio) chemically converted to match the
exact application needs. A simple example is the conversion of isolated fatty acid
triglyceride oils into methylesters for the production of biodiesel. Here, the required
(transesterification) conversion technology is readily available at Wageningen UR. Some
optimization is needed to determine optimal conversion and application conditions, given
the particular triglyceride ‘blend’ produced by the algae. For the unique aliphatic, long
chain oils produced by species like Botryococcus braunii, conversion to biofuels requires a
dedicated approach. For all major biomass streams including algae, the conversion
to energy, energy carriers, chemicals and materials is a core expertise of AFSG-Biobased
Products. Mastered technologies include chemical, biochemical (enzymatic) and
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fermentative processes. For thermochemical conversions and appropriate matching of
biomass-based combustion engine fuels and energy plant feedstock, we collaborate with
our strategic partner ECN.

4.3.2 Purification
Purification strategies for individual components from algal biomass are highly diverse, as a direct
consequence of the complexity anddiversity of the algae biomass „matrices‟ and the physico-chemical
properties of the compounds of interest therein. Beta-carotenoids can be isolated from the production medium
by milking of the living cells, using an apolar solvent like dodecane to extract the carotenoids. The milking also
shifts the equilibrium, causing the algae to produce more desired product. Most relevant components however
can only be purified by harvesting and processing total algae biomass. Processing may involve the
degradation of the algal cell wall first, so that the cell contents become accessible to subsequent purification
techniques.

5. Projects

5.1 High density cultures of microalgae

Algae are a diverse group of plant like organisms. Like plants, most algae use photosynthesis to convert
carbon dioxide, water and some trace elements into biomass, secondary metabolites and oxygen. Algae differ
from plants because they don't have true roots, leaves and other typical plant-like structures. In this project, we
selected the alga Monodus subterraneus that produces an unsaturated 3-fatty acid (eicosapentaenoic acid).
Nowadays, algae are commercially produced in ponds. Contamination and low biomass concentrations are
disadvantages of these „open‟ systems. In this project, we focus on „closed‟ photobioreactors and we try to
solve different engineering aspects of photobioreactors.
So, why are algae still produced in open ponds commercially? High biomass concentrations introduced a new
bottleneck in algal production systems: growth inhibition. Growth inhibition occurs at high cell concentrations
when the growth inhibitor is not removed. At this moment, it is prevented by daily replenishment of the medium.
In literature, two possible origins of growth inhibition are described: autoinhibition (growth inhibition due to an
inhibitor produced by the alga itself) or medium depletion (nutrient limitation or nutrient imbalance).

Aim
Our goal is to determine what causes the growth inhibition in high-density cultures of Monodus subterraneus.

Method
We will grow Monodus in flat panel photo bioreactors (Fig.1). When algal growth stops, and can only be
prevented by replenishment of the medium, the nature of the growth inhibition will be investigated in bioassays
(24-well plates).

5.2 Harvesting of microalgae

Introduction
In commercial algae production harvesting is generally done by centrifugation. However, the costs and energy
demands for harvesting the algal biomass by these methods are high. In the technical and economical analysis
on microalgae for biofuels it was shown that the investment costs for the centrifuges contributed up to 34% of
the total investment on equipment. The study also showed that the centrifuge used 48.8% of the total energy
consumption.

In a feasibility study, it was found that the total costs for concentrating the microalgae from 0.3 g/L to 100 g/L
(10% dry matter) can be reduced from 2.72 Euro/kg (for centrifugation) to about 0.7 Euro/kg when the algae
are pre-concentrated to 5% dry matter. This can be achieved by flocculation combined with flotation or
sedimentation prior to further concentration by centrifugation or filtration. In addition the energy demand

29
decreased from 4.76 kWh/kg to 0.4-0.6 kWh/kg.

Flocculation can be achieved in different ways (induced flocculation, auto- and bioflocculation or
electroflocculation), but in general flocculation of the algal biomass is still poorly understood. The optimal
conditions of the algae and the culture medium needed for effective flocculation are often unpredictable, which
makes it difficult to find ways to control the harvesting process. In addition, after harvesting oil needs to be
extracted from the biomass and often the cell wall is a big barrier to facilitate extraction and the thickness of the
cell wall is affected by the conditions of the cells at the time of harvesting.

Aim
The development of pre-concentration processes for different oleaginous algae based on induced flocculation,
electroflocculation, bioflocculation and autoflocculation. The algae can be different in cell size, cell shape,cell
wall thickness and show different algal surface properties, such as the zeta potential and the surface tension of
the algal suspension. These factors are expected to be crucial parameters for the flocculation processes. The
algal aggregate size and size distribution as well as the aggregate density are important parameters for further
concentration via flotation or sedimentation.

We want to derive mechanistic as well as kinetic models for the flocculation of algae based on the
experimental results from our tests and validate the models. The derived mechanistic models should predict
the effectiveness of the flocculation method for a given algae and the size and density of the algal flocks
obtained. The kinetic model should predict the speed at which algal cells will flocculate at given medium and
cell conditions. These models will help us to develop effective integrated harvesting processes for the different
algae studied.

5.3 Harnessing the sun for microalgae cultures

Introduction
The importance of photoautotrophic microorganisms is based on their potential use for high value compounds,
as heavy metals storage cells, for biofuels or as potential source of biomass in animal feed.
Efficient utilization of light inside the photobioreactors is one of the major problems in bioreactor design for
microalgae biomass production. To overcome this problem reactors have been developed in which optical
techniques can be used to dilute sunlight as well as photobioreactors with a short light-path.

Aim
The aim of this project is to maximize the photosynthetic efficiency of the growing microalgae in a new
photobioreactor prototype in which outdoor sunlight conditions will be simulated.

Research
The prototype used is a flat panel photobioreactor with a light-path of 14 mm, which is illuminated with red
-2 -
LEDs (see figure). The light intensity at the reactor surface can be varied between 0 and 2500 μmol m s
1
(PAR, 400-700 nm) to reach exactly the same intensity as measured with a light sensor placed outdoors. In
that way the photobioreactor can be operated at outdoor light conditions with optimal control of all other
parameters. The reactor will be operated in Huelva (Andalucía, Spain) to profit from the high-irradiance clear-
sky conditions.
The use of species differing in photosynthetic capacity and/or optimum temperature could show the
applicability of the production process for different microalgae in this photobioreactor. As such, we
use Chlorella sorokiniana as a reference strain because of its high growth rate and its high tolerance to high
light and temperature. Besides, Nannochloropsis oculata will enable us to judge photobioreactor performance
for microalgae with a lower growth rate and higher sensitive to high light and temperature.

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5.4 Biofuels from microalgae: Scenario studies for algae plants
Introduction
Biodiesel production with algae is most efficient in large scale production plants (Barbosa, Reith et al. 2007),
nevertheless the production of biodiesel with algae is not yet competitve with current diesel. Besides the
development of new technology the success for algae-based biodiesel production is also related with the
interaction of the production systems and their environment. One route to develop competitive production
processes is to integrate component and energy flows in production plants with the production of valuable
byproducts and to make efficient use of the opportunities of the environment of algae production plants.
To evaluate the interaction of different production systems and cultivation technology and to find bottlenecks in
the applications this project on scenario studies is performed.

Figure 1. Interaction of algae plants with their environment

Aim
The scenario study project aims the development of an approach to evaluate the use of micro-algae in
biodiesel production systems. The approach starts with current technology and from the inputs of the system
the outputs are predicted. Then the system is assessed with respect to economic and sustainability criteria.
The scenario study approach concerns also features that allow:
- to find most suitable cultivation and processing technology
- to specify development directions for the system and applied technology
- to find critical points in the system and applied technology
- to manage limitations in available knowledge
- to develop robust and flexible systems

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5.5 Optimization of lipid production in microalgae
Motivation
Currently mankind is looking for alternative energy sources because of the depletion of the Earth‟s fossil fuel
stock. An important focal point in the development of renewable resources is biodiesel production by
microorganisms. Microalgae, which largely contributed to fossil fuel formation millions of years ago, are
considered to be good candidates. Some species are known to accumulate lipids up to 45% of their dry weight.
A major advantage of the use of microalgae for biodiesel production is that they can grow on non-arable land
with high aerial productivities. Therefore, the production of algal biodiesel will not compete with the production
of agricultural crops. Another advantage is that the lipids produced by microalgae are very similar to those
obtained from oil producing plants such as soy or maize. Therefore, the current existing biodiesel production
methods can be applied to microalgal oil (see Figure 1).

Figure 1: Microalgal biodiesel is a renewable energy source because CO2 from the atmosphere is fixated and
sunlight is used as an energy source. Another advantage is that existing technologies can be used for the
production of biodiesel from the extracted algal oils.

Technological challenge
Some microalgal species are able to accumulate lipids under certain environmental conditions, for example
during depletion of important nutrients such as nitrogen or phosphate. However, little is known about the
mechanism of lipid accumulation. Also, the accumulation of lipids is not high enough in many species and
furthermore, nutrient-depletion-induced lipid accumulation is characterized by a decrease in growth rate
rendering the production process of microalgal biodiesel suboptimal (see Figure 2).

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Figure 2: Some microalgal species are able to accumulate lipids under nutrient depleted circumstances. This
type of lipid accumulation is always coupled to growth reduction. Understanding the mechanisms of lipid
accumulation can help to direct the algal metabolism towards simultaneous growth and lipid formation.

The aim of this study is to get a better understanding of the mechanism of lipid production in microalgae and
through this improve lipid productivity. A metabolic model will be designed based on the genome of
Chlamydomonas reinhardtii, a model-organism for green microalgae, with special attention to the lipid
metabolism. Such a model can be used to describe the internal fluxes of the cell and in combination with
experimental data it can be used to get insight into the mechanism of lipid accumulation. With this insight the
possibilities of uncoupling growth-rate reduction and lipid accumulation will be explored, which should lead to
an improved production process for microalgal biodiesel.

5.6 Physico-chemical properties of proteins isolated from microalgae

Background
Microalgae contain large amounts of proteins (up to 47% [w/w]). This abundance and their possible application
as ingredients for food products make algae proteins an interesting target for study. The proteins are one
amongst many interesting compounds that could be obtained microalgae (biorefinery). However, microalgae
proteins can only be applied as food ingredient if their techno-functional properties are satisfactory after
isolation. Within this project the possibilities to gain high quality proteins for food and non-food applications will
be explored.

Problem definition
Microalgae have already been considered as a source of food proteins for a long time. However, no isolation
method has been published for the mild, preparative isolation of proteins from microalgae cells. All published
analytical methods require the application of harsh conditions and thereby destroying the functional properties
of the protein present. In this project, an isolation procedure will be developed to obtain functional protein
fractions from algae as part of the biorefinery concept. To allow efficient use of the proteins, a thorough
characterization of their chemical structure and properties is needed. Therefore the obtained protein fractions
and purified proteins from algae will be characterized with respect to protein composition, structure, thermal
stability and other physico-chemical properties.

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Experimental set-up

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