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Good afternoon, I am Victor Chepurnov, production and R”D manager of Belgian company Tomalgae. I would like to thank everyone for attending this presentation. Today I shall talk on microalgae industry, very briefly about myths (or initial expectations), more about realities, with a special attention to microalgae for aquaculture. Hopefully original illustrations of various microalgae I scattered over the presentation will help to diversify the lecture.

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As, for instance, freshwater benthic diatom Cymatopleura; cells of two different sizes are illustrated

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An introduction to algae, what would I recommend to read?

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Mitigation and Adaptation Strategies for Global Change

January 2013, Volume 18, Issue 1, pp 5-12

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A quote from the article

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blue-green algae (or cyanobacteria) were mentioned – here is an illustration of one of these – Merismopedia – cell are arranged in rows, forming colonies held together by a mucilaginous matrix

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Filamentous blue-greem alga Oscellatoria serves as substratum for attachment of stalk-formimg cells of diatom Achnanthes

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INTRODUCTION TO ALGAE: WHAT TO WATCH?

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https://www.youtube.com/watch?v=CB2XlpD

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Again, Russell Chapman, Algae: the most important plants, in YouTube, “well worth the view, whether an algae veteran or an newbie to this industry”

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“Here are two benthic marine diatoms that live in sand; Pleusigma (sigmoid cell) and Toxonidea

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“Here are two benthic marine diatoms that live in sand; Pleusigma (sigmoid cell) and Toxonidea

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Water70.8%

Land29.2%

Basic fact about the Earth: more than 70 percent of its surface is covered with water

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Land94%

Water6%

The origin of our food (protein equivalent) – currently only 6% comes from water, the rest from land … TAKING INTO ACCOUNT THAT, ON LAND, WE MAINLY ‘PRODUCE’ FOOD WHILE IN THE WATER WE STILL MAINLY ‘COLLECT’ FOOD.

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Water Land

Both in the water and on land the primary biomass is produced by photosynthetic (autotrophic) organisms

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In the Water —

Algae (lower plants)

On Land — Higher (vascular)

plants

By Algae (so-called lower plants) in the water and by higher (vascular) plants on land

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Closterium, belongs to desmids, a a distinctive group within the green algae (Chlorophycaea)

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Algae-46.2%

(48.5 Gt C yr–1)

Terrestrial Plants -53.8%

(56.4 GT C yr–1)

Global net primary biomass production (via photosynthesis), algae vs terrestrial plants, approximately half and half

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MicroalgaeMacroalgae(seaweeds)

Algae

Algae are (often, traditionally or artificially) subdivided into two groups, microscopic algae and seaweeds (macroalgae)

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Microalgae97%

Macroalgae(seaweeds)

3%

Algae

Remarkably, these are microalgae that are the major aquatic primary producers. Phytoplankton serves as the base of the aquatic food web, providing food for larger animals and indirectly for humans, whose fisheries completely depend upon the plankton. So Microalgae are the foundation of life in the water

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Some typical marine coastal planktonic algae from North Sea, almost all are diatoms

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Proteins 40 - 60 10 - 20

Fats 5 - 40 1 - 5

PUFAs +++ +

Vitamins +++ +

Other BAC +++ +

Proteins, fats, unsaturated fatty acids, vitamins, other biologically active compounds … obviously, Microalgae are more nutritious and richer biochemically, if compared with seaweeds and terrestrial crops

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Algae Industry; world annual production: Our dominant, important , nutritious microalgae – only 15 000 t , seeweeds – much more, 17 million

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Typically microalgal food supplements are advertised something like this… superfood

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A culture of green microalga Chlorococcum induced to accumulate visibly large amount of neutral lipids. This illustration shows a culture of green microalga Chlorococcum induced to accumulate visibly large amount of neutral lipids.

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Further on, to give a brief general overview of microalgae industry, I shall refer several sources of knowledge and expertise I do trust, since these are largely compatible with my own experience that counts 25 years in science and about 10 years in mass microalgae cultivation.

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As a preamble to my following considerations. Despite their apparent simplicity, microalgae mass cultures are a difficult undertaking and most current R&D efforts appear to lack a full understanding of the complexities involved, or knowledge of what has already been tried, and all too often failed, before. John Benemann

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“As pioneers go, in the modern business of algae biofuels and co-products, possibly no one has built a longer record of accomplishment than Dr. John Benemann” Careful reading of his interview to algae industry magazine in 2010 would be a good start, in my opinion.

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JOHN R. BENEMANN, Ph.D.

CEO, MicroBio Engineering, Inc., PO Box 15821, San Luis Obispo, CA 93406 Cell: 925 352 3352 www.microbioengineering.com jbenemann@microbioengineering.com

Short Bio: B.S. Chemistry and Ph.D. Biochemistry,

University of California Berkeley; postdoctoral

fellow, U.C. San Diego. Associate Researcher in

Depts. of Civil Engineering and Plant and Microbial

Biology, U.C. Berkeley. With Dr. Joseph Weissman,

founded two algal biotechnology and aquaculture

companies, EnBio, Inc., and, later, SeaAg, Inc.

Associate Professor, Dept. Applied Biology, Georgia

Institute of Technology and Adjunct Professor,

University Hawaii. Since 2007, CEO of MicroBio

Engineering, Inc., San Luis Obispo, California, a

consulting engineering and R&D company in algal

technologies and wastewater treatment founded with

Prof. Tryg Lundquist, California State Polytechnic

University, Cal Poly. Founding director, Algae

Biomass Organization (ABO). Consultant and advisor

to U.S. and international agencies and companies.

Extensive background in algal biotechnology,

from photosynthesis and genetics to process

design and engineering of large-scale cultivation

systems.

Then, I also recommend his open access article Microalgae for Biofuels and Animal Feeds published in 2013.

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Next, a famous handbook of Microalgae Culture edited by Amos Richmond. Personally, I find extremely useful and informative a preface to this book ‘An introduction into the state of the art’ written by the Editor, just five pages. If you don’t have the book, the preface can easily be found in internet.

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Two rather straightforward statements from the preface: - The original concept…. - -similarly, …

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Next, some principal facts on current microalgae industry, from Benemann’s publication of 2013

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Need microalgae production ponds of various scales for your projects or production companies? Can be professionally designed and constructed by MicroBio Engineering firm headed by John Benemann.

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Back to the statement on that … as an illustrative example, I would recommend to look at the history of growth and achievements of Cyanotech Company. They also have very informative web-site.

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Cyanotech – large-scale production of blue-green alga Spirulina and astaxanthin-containing green alga (red colour) Haematococcus in open raceway ponds.

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Ponds of red colour contain Haematococcus cultures cultivated for astaxantin

Ponds of red colour contain Haematococcus cultures cultivated for astaxantin

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Astaxantin-contaiing cells of Haematoccocus under microscope

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Further principal facts on commercial microalgal production

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And what would be the future

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And what would be the future

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The most dangerous and destructive are primitive, unicellular organisms which, in the presence of their natural favourite food, microalgae, exhibit simply enormous potential to multiply, typically exceeding the growth potential of the microalgae. In this context …

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Very important statement, in my opinion, from a publication released by Cyanotech. ….Indeed, persistent contamination of closed cultures systems has led to serious problems for microalgae companies in California, Hawaii (not Cyanotech), and Europe—in all three cases it led to bankruptcy for these companies.”

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Back to the preface of the handbook of microalgal culture; now more optimistic

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Back to the preface of the handbook of microalgal culture; now more optimistic

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Three chapter in this book are dedicated to aquaculture

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Further on

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Also: Benemann 2015 , Overview of Aquaculture feeds from Microalgae, Aquaculture America 2015

New Orleans, Louisiana, February 20, 2015

Brief Conclusions Regarding Microalgae Aquafeeds , from Benemanns presentation at Aquaculture forum, 2015, in Dubai Current: Live feeds, on-site production, costs are extremely variable but in average ~$100-200/kg dw (dry basis) Mid-term: Astaxanthin for salmon feed from Haematococcus if the price of algae has been reduced to at least ~$20/kg dw algae; integrated aquaculture with “green water” algal blooms food chains and waste treatment Longer-term: Fish meal/oil replacement if the price of algae dropped to (~$2/kg dw) Future: Replace commodity feeds, algae price should be less than ($<1/kg dw)

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Pretty wide choice especially among planktonic diatoms and green algae diatom All the four industrially produced cultivars are also listed often, but these are not among the most popular.

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Alternative to on-site microalgae production – concentrated and preserved biomass

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Further, I have tried to sort and generalize available information and my own practical experience on microalgae cultivars using their various characteristics including harvesting and processing the biomass. If green – positive (or relevant, acceptable) If orange – negative (problematic or even hopeless) That is what eventually came out… a few illustrative examples

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Isochrysis –problematic moments – unstable cultures, because of grazing, basically impossible to dry, without damage to cell integrity. Key reason of difficulties – absence of tough cell wall.

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Nannochloropsis: One serious problem – digestibility (except for rotifers).

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— Fast rate of growth

— Nutritional value (low omega3 FA)

— Digestibility (Chlorella)

— Buoyancy (Tetraselmis)

— ‘Resistance’ to grazing

— Concentrate

— Drying

— Rehydration and (or) resuspension

Two most commonly produced microalgae, Chlorella and Spirulina, plus green alga Tetraselmis. Not positive for all the three – low contents of omega PUFAs. Than, digestibility of Chlorella and buoyancy of Tetraselmis.

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60 — Small cell size

— Fast rate of growth

— Nutritional value

— Digestibility

— Buoyancy

— ‘Resistance’ to grazing

— Concentrate

— Drying

— Rehydration and (or) resuspension

Rhodomonas – popular to rear copepods. Could grow faster. Delicate cell wall does not allow obtaining cell concentrates of sufficient quality

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61 — Small cell size (fusiform)

— Fast rate of growth

— Nutritional value

— Digestibility

— Buoyancy

— ‘Resistance’ to grazing

— Concentrate

— Drying

— Rehydration and (or) resuspension

BACILLARIOPHYTA (D IATOMS) :

PHAEODACTYLUM

Phaeodactylum: For many applications, the cells are too large. Often the cultures are very unstable, because of grazing problem by heterotrophic flagellates.

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Companies producing concentrated and processed algae. Reed mariculture is world’s largest producer of marine microalgae concentrates, cultivate various species. Concentrates are liquds or frozen

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Tomalgae, our company, focuses on production and development of its own microalgal cultivar and development of various products based on it.

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• Fast rate of growth (specific control)

• Nutritional value (specific control)

• Digestibility

• Buoyancy

• ‘Resistance’ to grazing (rigorous control)

• Concentrate (specific)

• Drying

• Rehydration and (or) resuspension (specific)

The cultivar of Tomalgae is a diatom belonging to taxonomic order Thalassiosirales. According to the list of criteria suitable for applications in aqualture, all are green (positive). To achieve this, special developments and adjustments were required, for six of the nine attributes considered.

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Cells intact!

Illustrated that biomass concentration, friz-drying and rehydration do not bring a damage to cell integrity.

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Apparently the most innovative part of Tomalgae technology is a breeding program (controlled sexual crosses) for their cultivar that substantially contributes to sustainability of the production and gives a real chance for demanded domestication (genetic manipulations using classical tools – mixing genomes and creating new combinations of genes by means of sex).

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Why is conventional breeding approach so crutial for technological developments of many diatom species? Fundamentally Because, as leading diatomist Professor David Mann specified… after…. Diatom Gomphonema acuminatum is illustrated; it has both sexy shape and sexual life cycle.

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Gomphonema cells are also able to move, sometimes in search of its sexual partner and as illustrated can form very nice branched colonies

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And, as a final remark, again a quote from John Benemann

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