By

TRANSGENIC ALGAE AS A GREEN “CELL-

FACTORY”

SpeakerChudasama Amar A.

M.Sc. (PMBB)Reg. No.-J4-01355-2014

Department of Biotechnology

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Content

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Introduction Emerging economies and the growing world

population rely heavily on the natural resources.

3Outlook for future energy source (IEA, 2010)

Continue… Sustainable production methods for food and energy are

necessary, if we do not want to convert all available nature into agricultural land. So, there is a need for alternatives.

The society focuses increasingly on sustainability and therefore on the recycling of waste streams, sustainable production and efficient use of energy.

The cultivation of microalgae can make an important contribution to the transition to a more sustainable society or bio based economy.

Barbosa (2003) 4

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• Microalgae, also called phytoplankton by biologists, they are very small plant-like organisms between 1-50 micrometres in diameter without roots or leaves.

• Together with the sea weeds (large aquatic plants), microalgae are so called aquatic biomass.

• Only a few tens of thousands out of a total have been described in literature and more to be known.

• With so many unknown algae species an almost inexhaustible source of possibilities exists.

What is Microalgae ?

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• Size and Shape :Algae are range in size, from the invisible (microscopic) example volvox to the visible (macroscopic) example kelp

• Solitary unicellular algaeTheir shape are round, oval, or pear-shaped algae of this group (i.e. Chlorella)

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BODY CHARACTERISTICS

• Multicellular algae• These are forms of thread, filament and

sheet-like multicellular algae.• The example is Oedogonium which have

filament shape body.

• Unicellular algae in colony• Cells are dependent on one another for their

survival.• The protoplast of each cell is connected to

another by pores on the cell wall.• The colony shape is like a disc, a ball, or

net.• The example is Hydrodictyon which have

shape like a net

Kinross, (2001) 10

Microalgae are ubiquitous Algae are not only suitable for environmentally friendly production of many

commodities, but also for the treatment of waste streams.

They grow excellent on e.g. carbon dioxide from flue gases, residual water of agro-industrial companies and even diluted digestive from manure.

In return they produce valuable raw materials. Algae recycle nutrients that thus remain available in the nutrient cycle, instead of being wasted and pollute the water.

11Tokarski, (2010)

POOL

SNOW

SOIL

TREES

ROCKS

HOT WATER SPRINGS

WALLS

ROOFS

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• Microalgae are very common (hundreds of thousands species exist) and occur both in freshwater and seawater where they form the basis for most food chains.

• The only difference between the algae grown in low molarity and high molarity salt solutions is the appearance of one extra polypeptide at high salt concentration.

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Normally microalgae are not visible by the naked eye, but if the water is eutrophic, massive algal blooms occur, changing the water in a green, brown, blue or orange liquid mass.

Reijnders, (2007) 14

Picture of Algal Blooms

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Major group of ALGAE The genetic analysis and ranking of all types of microalgae is

still in progress and there is not yet a complete and consistent classification. At the moment taxonomists have distinguished the following main groups:

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Why Micro Algae As A Cell Factory ?• Algae are a diverse group of autotrophic organisms that have the ability

to grow rapidly, efficiently use light energy, fix atmospheric CO2, and produce more biomass per acre than vascular plants

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How can we utilize?

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• On a global scale microalgae produce more than 75% of the oxygen required for animals and humans.

• Microalgae are sunlight-driven cell factories that convert carbon dioxide to many added-value compounds can be produced for applications in food, feed, cosmetics and feedstock for the chemical industry and they also have potential as sustainable energy carriers.

• Economically feasible production of value added compounds with microalgae is possible because microalgae produce biomass and specific biomass ingredients directly from solar irradiation at high photosynthetic efficiencies and high volumetric and areal productivities.

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PER HA MICRO ALGAE AND PLANTS TREES TO REDUCE THE CARBON COMPARISON

Micro algae reduce the

carbon 58-90 ton /Ha / YEAR

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Plants trees reduce the carbon 25

ton /Ha / YEAR>

RECOMBINANT DNA TECHNOLOGY(BASIC)

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WHAT IS RECOMBINANT TECHNOLOGY ?

It is a process of joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.

Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize and manipulate genes. 

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SIX STEPS OF RECOMBINANT DNA

1. Isolating (vector and target gene)2. Cutting (Cleavage)3. Joining (Ligation)4. Transforming 5. Cloning6. Selecting (Screening)

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GENETICALLY MODIFIED ALGAL STRAINS

• As a result of increased research on eukaryotic algae and cyanobacteria a large amount of data, protocols and publications on the molecular biology of algae has become available.

• Due to the rapid evolving DNA-sequencing methods and DNA-data analysis software, sequencing a genome is now within the reach of every medium-sized research program.

Jinkerson et al., (2010) 24

In the late 1980s, several eukaryotic marine microalgae and seaweeds were successfully transformed by different Transformation Methods, e.g.

Microinjection in the marine macro-green alga Acetabalaria sp.,

Plasmid Vectors in the marine diatom Cyclotella cryptica

Gene Gun or the Biolistic Method in the macro-red alga Eucheuma sp. and brown alga respectively .

 

 

Neuhaus et al., (1986)

Dunahay et al., (1995)

Qin et al., (1998)

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• Entering the new century, the trend of the genetic engineering of marine algae has been to apply The Transgenic Marine Algae As Cell Factories And Marine Bioreactors.

• In the last decades research on algae for the production of Bio-fuel, Food, Feed or Chemicals has expanded rapidly.

• Since then successful genetic transformation of approximately 30 algal species has been demonstrated.

León-Bañares et al., 2004; 26

Example Of Transgenic Algae

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WHAT IS TRANSFORMATION?• Transformation may be referred to as a stable genetic

change brought about by the uptake of naked DNA (DNA without associated cells or proteins) from its surroundings through the cell membranes.

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TRANSFORMATION METHODS USED IN MICROALGAE GENETIC ENGINEERING.

S. Qin et al. / Biotechnology Advances 30 (2012) 29

CONJUGATIVE DNA TRANSFER• Trans-conjugation is the transfer of DNA by direct cell-to-cell contact

or by a bridge-like connection between two cells.

• The versatility of gene transfer by transconjugation in marine cyanobacteria was first demonstrated in one strain of marine Synechocystis and one strain of marine Pseudanabaena.

Sode et al., (1992) 30

ELECTROPORATION• Electroporation can transfer extrinsic genes independent of the cell's

ability and universally to different genera.

• The first usage in marine cyanobacterium was performed in marine unicellular Synechococcus sp. NKBG042902-YG 1116.

• Efficient electroporation-mediated transformation was achieved in both wild-type and cell wall-deficient eukaryotic Chlamydomonas reinhardtti strains.

• The efficiency of electroporation was better than that obtained with the glass beads method to introduce exogenous DNA to algal cells.

Shimogawara et al., (1998)31

THE PHENOMENA

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• Direct gene transfer by the biolistic method (micro-particle bombardment) has been proven to be the most efficient method and is highly reproducible in introducing exogenous DNA into algal cells.

• This method has been successfully employed for the transformation of many microalgal nuclear and chloroplast expression systems, and it is not surprising that biolistic transformation remains the most useful tool for transgenic studies of marine macroalgae regardless of their cell walls and life cycle.

(Matsunaga et al., 1991)

BIOLISTIC TRANSFORMATION

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• Agitation with glass beads has been used to efficiently introduce foreign DNA into microalgae and was first reported in the freshwater alga C. reinhardtii (Kindle, 1990).

• The genetic transformation system for Dunaliella salina, which lacks a rigid cell wall, by glass beads has been successfully established and the glass beads method is more efficient and repeatable, more easily controlled and less physically destructive to cells than electroporation and particle bombardment for the transformation of D. salina when these three methods were conducted and compared at the same time.

• The main drawback of this method is its inability to transfer DNA into cells with thick cell walls.

(Coll., 2006)

GLASS BEADS

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• As a direct physical method that is able to penetrate intact cell walls, the microinjection method does not necessarily require a protoplast regeneration system.

• Additionally, microinjection allows the introduction of DNA (or other substances) under microscopical control into specific targets.

• A high yield and stable nuclear transformation was achieved in the marine unicellular green alga Acetabularia mediterranea by microinjecting SV40 DNA.

• This method was successfully used to tackle the problem of the nuclear transport of algal proteins.

• Regardless of its complicated and delicate procedure, microinjection, could be considered to be a highly efficient and low cost transformation method for marine algae.

(Neuhaus et al., 1986)

MICROINJECTION

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Indoor production systems For small scale algae production mostly for research purposes a wide

range of cultivation systems is used. Algae can be cultivated in simple photobioreactors.

Closed systems are typically referred to as photo bioreactors. They can be placed outdoors in some cases they are placed inside greenhouses to allow more controlled conditions.

The major advantage of using these systems is the increased surface area for a certain culture volume.

Other advantages of PBR’s are lower contamination risk, easier mixing which improves mass transfer, and easier control of temperature, pH and nutrient supply. However, the cost of installation and operation is much higher than those of open pond systems.

ALGAE CULTIVATION

Roesler (1998) 37

Method Of Algae Production: State of Art

Wijffels, (2008)

FLAT PANEL REACTORS

FERMENTERS

ERLENMEYERS

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OPEN PONDS Algae is grown in a pond in the open air Simple design and low capital costs Less efficient than other systems Other organisms can contaminate the pond and potentially damage or

kill the algae CLOSED LOOP PONDS Raceways are shallow, annular channels where mixing takers place

using paddle wheels.Similar to open ponds but not exposed to the atmosphere and use of a

sterile source of carbon dioxide Could potentially be directly connected to carbon dioxide sources

(such as smokestacks) and thus use the gas before it is every released into the atmosphere.

Wijffels, (2010)39

Algae ponds are not the most effective cultivation system. Theoretically up to 10% of the energy from solar light can be converted in chemical energy in biomass via photosynthesis, while the remaining part is lost as heat.

In practice however, this photosynthetic efficiency is much lower. This is caused by the limited penetration off sunlight into the turbid algae pond.

As a result only the algae cells near the surface receive a lot of light which they cannot efficiently convert into biomass.

 

Continue…

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Different Methods

Source Proviron

Flat Glass plate reactors

Bioreactive façade

Raceway ponds

Algae Ponds

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Comparison of Open and Closed Systems for Microalgae

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Alagal Products

• The algal cell contains many useful substances and microalgae are cultivated increasingly for the production of valuable raw materials. For example, it is possible to produce oil, proteins, starch and pigments.

• Applications of these materials are numerous, ranging from biodiesel and bio-plastics to colorants and hamburgers.

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Major products derived out from Microalgae

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•Algae have been used as a food source and for treatment of various ailments from many decades.

•Algae can form numerous compounds that are currently present in nutraceuticals and have the potential to become more intensively exploited.

•Different types of algae, specifically microalgae, that could become more prevalent in food supplements and nutraceuticals are Nostoc, Botryococcus, Anabaena, Chlamydomonas, Scenedesmus, Synechococcus, Parietochloris, and Porphyridium etc.

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New Products That Have Been Made By Algae Through Genetic Modification

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Biofuel production

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The Economist 2009

Comparing Potential Biofuel Crops

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Nutraceutical Oil from Microalgae The oils from some algae have high levels of unsaturated fatty

acids. For example, Parietochloris incisa is very high in arachidonic acid, where it reaches up to 47% of the triglyceride pool.

Some varieties of algae contain the long-chain, essential omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Fish oil contains the omega-3 fatty acids, but the original source is algae (microalgae in particular), which are eaten by marine life such as copepods and are passed up the food chain.

Algae have emerged in recent years as a popular source of omega-3 fatty acids for vegetarians who cannot get long-chain EPA and DHA from other vegetarian sources such as flaxseed oil, which only contains the short-chain alpha-linolenic acid (ALA).

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Pigments The natural pigments (carotenoids and chlorophylls) produced by

algae can be used as an alternative to chemical dyes and coloring agents.

The presence of some individual alga pigments, together with specific pigment concentrations ratios, are taxon-specific: analysis of their concentrations with various analytical methods, particularly high-performance liquid chromatography (HPLC), can therefore offer deep insight into the taxonomic composition and relative abundance of natural alga populations in sea water samples.

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Pollution control Sewage can be treated with algae, reducing the usage of large

amounts of toxic chemicals that would otherwise be needed. Algae can be used to capture fertilizers in runoff from farms. When

subsequently harvested, the enriched algae itself can be used as fertilizer.

Aquariums and ponds can be filtered using algae, which absorb nutrients from the water in a device called an algae scrubber, also known as an algae turf scrubber (ATS) .

Agricultural Research Service scientists found that 60–90% of nitrogen runoff and 70–100% of phosphorus runoff can be captured from manure effluents using a horizontal algae scrubber, also called an algal turf scrubber (ATS).

Researchers collected and dried the nutrient-rich algae from the ATS and studied its potential as an organic fertilizer. They found that cucumber and corn seedlings grew just as well using ATS organic fertilizer as they did with commercial fertilizers

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Private company active in microalgae production for commercialization of goods

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CASE STUDY

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Material and MethodC. reinhardtii Strains,

Transformation, and Growth Conditions

Plasmid construction

Southern and Northern Blots

Protein Expression, Western Blotting, and ELISA

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Results

1.• De Novo Synthesis of a lsc Antibody Gene in C. reinhardtii

Chloroplast Codon Bias

2.• Construction of a Chimeric C. reinhardtii Chloroplast lsc

Antibody Gene.

3.• Southern Blot Analysis of HSV8-lsc Transgenic Chloroplast

4.• Accumulation of HSV8-lsc mRNA in Transgenic Strain

5.• Analysis of HSV8-lsc Protein Accumulation in Transgenic

C. reinhardtii Chloroplasts.

6.• Characterization of HSV8-lsc Antibodies Expressed in E.

coli and Chloroplast.

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Fig. 1. Restriction maps of HSV8-lsc genes for expression in C. reinhardtii chloroplasts

(A)Relevant restriction sites delineating the rbcL 5 UTR (BamHI NdeI), the HSV8 coding region and flag tag (NdeIXbaI), and the rbcL 3 UTR (XbaIBamHI), as well as relevant restriction sites of the atpA 5 UTR (BamHI NdeI), the HSV8 coding region and flag tag (NdeIXbaI), and the rbcL 3 UTR (XbaIBamHI) are shown.

(B) Restriction map showing the site of integration of the HSV8-lsc genes into plasmid p322 for integration into the C. reinhardtii chloroplast genome. p322 DNA includes the 5.7-kb region from EcoRI to XhoI in the C. reinhardtii chloroplast genome corresponding to position 44,877– 50,577 .Double headed arrows indicate regions corresponding to the probes used in the Southern blot analysis. Black boxes indicate, from left to right, psbA exon 5 and the 5S and a small portion of the 23S ribosomal RNA genes, respectively. 58

Fig. 2. Southern and Northern blot analysis of HSV8-lsc in C. reinhardtii chloroplast transformants.

(A) C. reinhardtii DNAs were prepared as described in experimental procedures, digested with EcoRI and XhoI, and subjected to Southern blot analysis. Filters were hybridized with the radioactive probes indicated by the double-headed arrows in Fig. 2B.

(B) Detection of chloroplast-expressed HSV8-lsc mRNA in transgenic C. reinhardtii strains. Total RNA isolated from untransformed (WT), atpA HSV8-lsc transformed (10-6-3), and rbcL transformed (20-4-4) strains was separated on denaturing agarose gels and blotted to nylon membrane. The membranes were either stained with methylene blue (Bottom) or hybridized with a psbA cDNA probe (Middle) or a hsv8-specific probe (Top). 59

Fig. 3. Expression of HSV8-lsc proteins in bacteria and chloroplast.

(A) Twenty micrograms of crude protein from E. coli, WT C. reinhardtii, and transgenic lines 10-6-3 and 20-4-4 were separated by SDSPAGE and either stained with Coomassie blue (Left) or blotted to nitrocellulose membrane and decorated with an anti-flag antibody (Right). (B) Proteins from E. coli and C. reinhardtii expressing the Hsv8-lsc antibody were separated into soluble and insoluble pellets by centrifugation. Twenty micrograms of protein were either stained with Coomassie blue (Left) or blotted to nitrocellulose membrane and decorated with an anti-flag antibody (Right). (C) Soluble proteins from C. reinhardtii transgenic line 10-6-3 were either treated with (Bme) or without (no Bme) reducing agent before separation on SDSPAGE. Proteins were blotted to nitrocellulose membrane and decorated with anti-flag antibody.

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Fig. 5. Effect of growth condition on accumulation of HSV8-lsc in C. reinhardtii.

Before harvest, C. reinhardtii transgenic lines 10-6-3 and 20-4-4 were maintained at either 1 106 cells per ml or 1 107 cells per ml. Cultures were grown either under continuous illumination or under a 12-h light12-h dark cycle. Total soluble protein (20 g) was separated by SDSPAGE, blotted to nitrocellulose membrane, and decorated with anti-Flag antibody.

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Summary Here they demonstrate the efficient expression of a unique large single-chain (lsc)

antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii.

they achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5 and 3 RNA elements.

This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo.

Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification.

they further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5 and 3 elements used to drive expression of the antibody gene.

These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain. 62

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Objective

Validate a set of genetic tools that enable protein targeting to distinct subcellular locations.

Develop two complementary methods for multigene engineering in the eukaryotic green microalga Chlamydomonas reinhardtii.

Develop The tools enable advanced metabolic and genetic engineering to promote microalgae biotechnology and product commercialization.

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Material and Method

Algal strains, transformations and growth conditions Plasmid construction Fluorescence microscopy Fluorescence activated cell sorting GUS activity assay

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Continue…

Chlamydomonas matings

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Figure 1. Chlamydomonas transformation vectors for protein targeting to specific

subcellular locations.

(A–D) - Schematic representation of Chlamydomonas targeting vectors.

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Continue…

E–P. Microscopy images of cells transformed with pBR28 (E–G), pBR29 (H–J), pBR30 (K–M), and pBR31 (N–P). Top row are live cell images of the fluorescent proteins targeted to the nucleus (E), mitochondria (H), chloroplast (K) or ER (N, O). (I) The cell is costained with the mitochondrial dye Mitotracker. (N) Cross section through the top of a cell expressing mCherry in the ER allows for the visualization of the cortical ER network. (O) Cross section through the middle of the same cell as in (N). The chloroplast membranes are visualized in (F), (L) and (P). Merged images are shown in the bottom row. 68

Figure 2. Gene stacking using a multicistronictransformation vector.

(A). A schematic representation of the Chlamydomonas multicistronic expression vector. The expression of the cassette is under the control of the hsp70/rbcs2 promoter (P).

(B). PCR analysis of the multicistron cassette genome integration.

(C). Live cell fluorescence microscopy of a clone expressing the multicistronic vector.

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Summary

Here they validate a set of genetic tools that enable protein targeting to distinct subcellular locations, and present two complementary methods for multigene engineering in the eukaryotic green microalga Chlamydomonas reinhardtii.

The tools described here have enable advanced metabolic and genetic engineering to promote microalgae biotechnology and product commercialization.

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Concluding remarks Algal technology is a sustainable technology that may

contribute to the solution of societal problems like climate change and fossil fuel depletion and genetically modified algae will be part of that technology.

In view of their potential as rapid-growing photosynthetic cell factories, algae are attracting increasing attention for sustainable production of biodiesel, but also many other products such as proteins, colorants, vitamins, CO2 capture, etc. is achievable.

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Thank You

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