research on micro algae
TRANSCRIPT
Research on Microalgae 2010
Wageningen UR
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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
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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
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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.
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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.
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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.
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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 )
Chlorophyceae (Green algae)
Rhodophyceans (Red algae)
Haptophyceae
Prasinophyceae
Cryptophyceae
Xanthophyceae
Eustigmatophyceae
Dinophyceans
Euglenopyhceans
Cyanophyceae (blue-green
algae)
Skeletonema, Thalassiosira, Phaeodactylum,
Chaetoceros
Chlorella, Dunaliella, Scenedesmus, Haematococcus,
Nannochloris
Porphyridium cruentum, Galdieria
Isochrysis, Pavlova
Tetraselmis (Fig.1), Pyramimonas
Chlamydomonas, Rhodomonas, Chroomonas
Olistodiscus
Nannochloropsis (Fig. 2)
Crypthecodinium, Alexandrium, Gymnodinium,
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.
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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!
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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 m
2 (perpendicular to the sun‟s rays, earth surface) – or, expressed as energy, 1100 J per m
2 per second (or,
1100 W m-2
).
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.
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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 %.
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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.
Thermophilic (warm water adapted) microalgae on the other hand, grow at temperatures up to 75 oC
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.
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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.
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 MgSO4 - 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 (> 5 g L
-1), 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
Element
gram
pro
gram Mw
Ratio
Molar
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
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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:
A bubble column is a transparent
cylinder. The culture is mixed
with air bubbles.
Figure 2:
A standard 2-liter desktop fermentor for
laboratory use.
Figure 3:
A 200 L stainless steel bubble
column fermentor with
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”
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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 effective with specific volumetric productivities up to 3 g DHA L
-1 day
-1 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.
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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 enthalpy of combustion of 547.8 kJ×Cmol
-1.
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 tonnes
.ha
-1.year
-1.
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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.
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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.
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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 can be produced is 115,000 L ha
-1 year
-1. 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 year
-1 of oil – this is still significantly more than can be obtained from energy crops (the areal productivity of
palm oil is 6,000 L ha-1
year-1
).
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
18
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.
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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
biological systems as integrated
wholes, using tools of modeling,
simulation, and comparison to
experiment. Metabolic flux
analysis,genomics, transcriptomic
s andproteomics are used to get
insight how microalgae function
and how metabolic routes can be
optimized to obtain high product
yields.
Focusing on the cultivation
of microalgae from lab-
scale to commercial
systems. In this
section, shake flask
experimentsare discussed
up to commercial systems.
Further, also heterotrophic
algae and their cultivation
are discussed.
Focusing on
the isolation of products
from the microalgae
and biorefiningthem into
products with
applications in different
industry sectors.
Attention is paid to
the isolation, biorefining,
conversion and purificatio
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
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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
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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
22
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)
23
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 very low (0.005 g L
-1) 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 bottleneck; however, at high biomass concentrations (>10 g L
-1) 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
24
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.
25
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 set temperature (20-37°C) and with a low light intensity (50-100 umol m
-2s
-1). 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.
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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!
27
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 rate of autotrophic growth. Growth rates are typically 0.3-1 d
-1. 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, range from 80-200 m
3 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
The highest published biomass densities in heterotrophic productions, are about 120 g dw L-1
. Normally, heterotrophic microalgal cultures are taken to 70 – 100 g dw L
-1. 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 With the green alga, Chlorella sorokiniana (Fig. 1), a heterotrophic growth of about 1 g dw L
-1 h
-1 has been
obtained. With the heterotrophic diatom Nitzschia alba, 0.8 g dw L-1
h-1
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 illuminated photobioreactors; 1.2 g dw L
-1 h
-1 has been recorded with Arhtrospira. However, the reactor
geometry and cost implications are not favorable for the photobioreactor production. For comparison, the most productive industrial yeast fermentations can reach 25 g dw L
-1 h
-1 .
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
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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 LEDs (see figure). The light intensity at the reactor surface can be varied between 0 and 2500 μmol m
-2 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