Professional Documents
Culture Documents
Research On Micro Algae
Research On Micro Algae
2010
Wageningen UR
Index
1. 2. Algae .........................................................................................................................................3 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 2.4.2 2.4.3 2.4.4 Light ..................................................................................................................................8 Temperature ...................................................................................................................10 Nutrients ..........................................................................................................................11 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
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 nongrowing 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.
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, -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.
2.2 Photosynthesis
The key process for microalgae to obtain energy is photosynthesis. Photosynthesis is the process of using light energy (h) 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.
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 rearranged. A number of the species are maintained in culture collections, either cryopreserved or in an actively metabolizing state.
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!
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 moles of photons (PAR) per second per 2 2 m (perpendicular to the suns 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.
Figure 2: Sunlight energy budget in microalgal photosynthesis. The maximum potential energy conservation in biomass is 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.
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 pro Element gram C H O N S P K Mg Ca Na Si 0.541 0.074 0.296 0.0822 0.005 0.00182 0.0064 0.00139
Ratio Molar pro 1 C 1 1.64 0.41 0.13 0.0035 0.0013 0.0036 0.0013 4.01E-07 0.00095 9.87E-05
0.000125 28.1
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: A bubble column is a transparent cylinder. The culture is mixed with air bubbles.
(1)
Figure 3: A 200 L stainless steel bubble column fermentor with internal illumination.
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)
12
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
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 kJCmol . 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.
15
Product Bakers yeast Meat Milk Rice Soy beans Anabaena cylindrica Chlamydomonas reinhardtii Chlorella vulgaris Porphyridium cruentum Scenedesmus obliquus Spirulina maxima Synechococcus spec.
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), -3 fatty acids, pigments and antioxidants (-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 as Chlorella and Arthrospira. In addition, there is a large market of natural -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 mussel Mytilus edulis and sole.
16
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.
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 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 labscale 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.
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
2.
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. 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.
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 cDNAs or oligos are available. For a few organisms prefabricated expression microarrays are a vailable. 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.
24
Light intensity (solid line) and productivity (dotted line) in an open pond at high light intensities
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.
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.
27
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 m echanical 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
28
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.
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.
30
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
31
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).
32
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.
33
Experimental set-up
34