Handbook of microalgal culture applied phycology and biotechnology edited by amos richmond, qiang hu

List of Contributors viiDepartment of Solar Energy and Environmental Physics Jacob Blaustein Institutes for Desert Research Ben-Gurion University of the Negev Midreshet Ben-Gurion 84990,

Handbook of Microalgal Culture Applied Phycology and Biotechnology

Second Edition

Edited by Amos Richmond, Ph.D., Prof Emeritus

Ben Gurion University of the Negev at Sede-Boker, IsraelThe Blaustien Institutes for Desert Research

This edition first published 2013 C 2004, 2013 by John Wiley & Sons, Ltd

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Library of Congress Cataloging-in-Publication Data

Handbook of microalgal culture : applied phycology and biotechnology / edited by Amos Richmond and Qiang Hu –Second edition

pages cm

Includes bibliographical references and index

ISBN 978-0-470-67389-8 (hardback) – ISBN 978-1-118-56716-6 – ISBN 978-1-118-56717-3 (emobi) –

ISBN 978-1-118-56718-0 (ePdf) – ISBN 978-1-118-56719-7 (ePub) 1 Algae culture–Handbooks, manuals, etc

2 Microalgae–Biotechnology–Handbooks, manuals, etc 3 Algology–Handbooks, manuals, etc I Richmond, Amos,editor of compilation II Hu, Qiang, 1960- editor of compilation

SH389.H37 2013

579.8–dc23

2013006646

A catalogue record for this book is available from the British Library

Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available inelectronic books

Cover images: C Amos Richmond and Qiang Hu

Cover design by Steve Thompson

Set in 9.5/12 pt Times by Aptara R Inc., New Delhi, India

1 2013

Robert A Andersen

Jiˇr´ı Masoj´ıdek, Giuseppe Torzillo, and Michal Kobl´ıˇzek

Yuan-Kun Lee, Wei Chen, Hui Shen, Danxiang Han, Yantao Li, Howland D T Jones, Jerilyn A Timlin,

and Qiang Hu

4 Strategies for Bioprospecting Microalgae for Potential Commercial Applications 69

William Barclay and Kirk Apt

Jerry J Brand, Robert A Andersen, and David R Nobles Jr.

Giuseppe Torzillo and Avigad Vonshak

Qiang Hu

Johan U Grobbelaar

William Barclay, Kirk Apt, and X Daniel Dong

10 Molecular Genetic Manipulation of Microalgae: Principles and Applications 146

Roshan Prakash Shrestha, Farzad Haerizadeh, and Mark Hildebrand

11 Biological Principles of Mass Cultivation of Photoautotrophic Microalgae 171

Amos Richmond

12 Theoretical Analysis of Culture Growth in Flat-Plate Bioreactors: The Essential Role of Timescales 205

Y Zarmi, G Bel, and C Aflalo

iii

iv Contents

Graziella C Zittelli, Natascia Biondi, Liliana Rodolfi, and Mario R Tredici

Emilio Molina Grima, Francisco Gabriel Aci´en Fern´andez, and Alfonso Robles Medina

C Meghan Downes and Qiang Hu

Jin Liu and Qiang Hu

Amha Belay

Michael A Borowitzka

Makoto M Watanabe and Yuuhiko Tanabe

Danxiang Han, Yantao Li, and Qiang Hu

21 Novel Sulfated Polysaccharides of Red Microalgae: Basics and Applications 406

Shoshana (Malis) Arad and Dorit van Moppes

Giuseppe Torzillo and Michael Seibert

Danxiang Han, Zhongyang Deng, Fan Lu, and Zhengyu Hu

24 IGV GmbH Experience Report, Industrial Production of Microalgae Under Controlled Conditions:

O Pulz, J Broneske, and P Waldeck

E Wolfgang Becker

R Cameron Coates, Emily Trentacoste, and William H Gerwick

Daniel J Barrera and Stephen P Mayfield

28 Molecular and Cellular Mechanisms for Lipid Synthesis and Accumulation in Microalgae:

Yantao Li, Danxiang Han, Kangsup Yoon, Shunni Zhu, Milton Sommerfeld, and Qiang Hu

Maria J Barbosa and Ren´e H Wijffels

Susan Blackburn

Contents v

Asher Brenner and Aharon Abeliovich

Drora Kaplan

33 Microalgae for Aquaculture: The Current Global Situation and Future Trends 615

Arnaud Muller-Feuga

Oded Zmora, Dan J Grosse, Ning Zou, and Tzachi M Samocha

35 Transgenic Marine Microalgae: A Value-Enhanced Fishmeal and Fish Oil Replacement 653

Jonathan Gressel

E Wolfgang Becker

37 The Enhancement of Marine Productivity for Climate Stabilization and Food Security 692

Ian S.F Jones and Daniel P Harrison

List of Contributors

Aharon Abeliovich†

Department of Biotechnology Engineering

Ben-Gurion University of the Negev

Beer-Sheva 84105, Israel

†Deceased

C Aflalo

Jacob Blaustein Institutes for Desert Research

Ben-Gurion University of the Negev

Midreshet Ben-Gurion 84990, Israel

Email: aflaloc@bgu.ac.il

Phone: 972-86596817

Fax: 972-86596802

Robert A Andersen

Senior Research Scientist

Friday Harbor Laboratories

Bigelow Laboratory for Ocean Sciences

P.O Box 380, East Boothbay, ME 04544, USA

Email: arad@bgu.ac.ilPhone: 972-8-6479069Fax: 972-8-9479067

Maria J Barbosa

Research Manager MicroalgaeFood and Biobased ResearchWageningen University and Research CenterP.O Box 17, 6700 AA Wageningen, The NetherlandsEmail: maria.barbosa@wur.nl

Phone: + 31 (0)317 480079 7Fax:+ 31 (0)317 483011

Daniel J Barrera

PhD StudentUniversity of California, San Diego

9500 Gilman Drive, La Jolla, CA 92093-0212, USAEmail: dbarrera@ucsd.edu

Phone: 858-869-3879

vi

List of Contributors vii

Department of Solar Energy and Environmental Physics

Jacob Blaustein Institutes for Desert Research

Ben-Gurion University of the Negev

Midreshet Ben-Gurion 84990, Israel

Universit`a degli Studi di Firenze

Piazzale delle Cascine 24, 50144 Firenze, Italy

Email: natascia.biondi@libero.it

Phone:+39-055-3288480

Fax: +39-055-3288272

Susan Blackburn

Head, Australian National Algae Culture Collection

CSIRO Marine and Atmospheric Research

G.P.O Box 1538, Hobart, Tasmania 7001, Australia

Phone: 512-4711589Fax: 512-2323402Director

Culture Collection of Algae (UTEX)University of Texas at Austin

205 W 24th Street, Austin, TX 78712, USAEmail: jbrand@austin.utexas.edu

Phone: (512) 4711589Fax: (512) 2323402

Asher Brenner

ProfessorUnit of Environmental EngineeringBen-Gurion University of the NegevBeer-Sheva 84105, Israel

Email: brenner@bgu.ac.ilPhone: 972-8-6479029

Wei Chen

Associate Research ProfessorLaboratory for Algae Research and BiotechnologyCollege of Technology and Innovation

Arizona State University

7001 E Williams Field RoadMesa, AZ 85212, USAEmail: wei.chen@asu.eduPhone: +1-480-727-5663Fax: +1-480-727-1475

viii List of Contributors

Zhongyang Deng

Associate Professor

School of Biological Engineering

Hubei University of Technology

Wuhan, Hubei 430068, China

New Mexico State University

Las Cruces, NM 88003-8001, USA

Email: cdownes@nmsu.edu

Phone: 575-202-5181

Francisco Gabriel Aci´en Fern´andez

Associate Professor of Chemical Engineering

Chemical Engineering Department

Center for Marine Biotechnology and Biomedicine

Scripps Institution of Oceanography

Skaggs School of Pharmacy and Pharmaceutical Sciences

University of California, San Diego

9500 Gilman Drive, La Jolla, CA 92093-0212, USA

Department of Plant Sciences

Weizmann Institute of Science

3754 Jenifer Street, NWWashington, DC 20015Email: dgrosse@terraqua.orgPhone: 202-258-9700Fax: 202-244-4667and

Adjunct Associate ProfessorUniversity of Maryland University CollegeGraduate Program in Environmental ManagementAdelphi, MD 20873, USA

Email: Dan.Grosse@faculty.umuc.eduPhone: 202-258-9700

Fax:202-244-4667

Emilio Molina Grima

Professor of Chemical EngineeringChemical Engineering DepartmentUniversity of Almer´ıa

04120 Almeria, SpainEmail: emolina@ual.esPhone:+34-950015032Fax:+34-950015484

Johan U Grobbelaar

Professor EmeritusDepartment of Plant SciencesUniversity of the Free StateBloemfontein 9300, South AfricaEmail: grobbeju@ufs.ac.zaPhone:+27-51-4012263

Farzad Haerizadeh

Metabolic Systems LeadSynthetic Biology R&DLife Technologies Corporation

5791 Van Allen WayCarlsbad, CA 92008, USAEmail: farzad.haerizadeh@lifetech.comPhone: 760-476-6156

Danxiang Han

Assistant Research ProfessorLaboratory for Algae Research and BiotechnologyCollege of Technology and Innovation

Arizona State University

7001 E Williams Field RoadMesa, AZ 85212, USAEmail: Danxiang.han@asu.eduPhone: +1-480-727-5661Fax: +1-480-727-1475

List of Contributors ix

Daniel P Harrison

Research Engineer

Ocean Nourishment Foundation

P.O Box 363, Glebe 2037 NSW, Australia

Professor and Co-Director

Laboratory for Algae Research and Biotechnology

College of Technology and Innovation

Arizona State University

7001 E Williams Field Road

Chinese Academy of Sciences

7 South Donghu Road, Wuhan, Hubei 430072, China

Email: huzy@ihb.ac.cn

Phone: +86-138-0864-8218

Fax: +86-27-6878-0016

Howland D.T Jones

Senior Scientist Biosciences

Sandia National Laboratories

Sede Boqer Campus, Midreshet Ben-Gurion 84990, IsraelEmail: droraka@bgu.ac.il

Phone: 972-8-6596835Fax: 972-8-6596909

Michal Kobl´ıˇzek

Senior ScientistDepartment of Phototrophic MicroorganismsInstitute of Microbiology

Academy of SciencesOpatovick´y ml´yn, CZ-37981 Tˇreboˇn, Czech RepublicEmail: koblizek@alga.cz

Phone: +420-384-340432;

Fax: +420-384-340415

Yuan-Kun Lee

Associate ProfessorDepartment of MicrobiologyYong Loo Lin School of MedicineNational University of SingaporeBlock MD4, 5 Science Drive 2, Singapore 117597Email: micleeyk@nus.edu.sg

Phone: +65-65163284Fax: +65-67766872

Yantao Li

Assistant Research ProfessorLaboratory for Algae Research and BiotechnologyCollege of Technology and Innovation

Arizona State University

7001 E Williams Field RoadMesa, AZ 85212, USAEmail: yantao.li@asu.eduPhone: +1-480-727-5662Fax: +1-480-727-1475

Jin Liu

Faculty Research AssociateLaboratory for Algae Research and BiotechnologyCollege of Technology and Innovation

Arizona State University

7001 E Williams Field RoadMesa, AZ 85212, USAEmail: gjinliu@gmail.comPhone: +1-480-727-1410Fax: +1-480-727-1475

x List of Contributors

Fan Lu

Professor

School of Biological Engineering

Hubei University of Technology

Wuhan, Hubei 430068, China

Email: lf1230nc@yahoo.com

Phone: +86-189-6173-3533

Jiˇr´ı Masoj´ıdek

Senior Scientist, Associate Professor

Department of Phototrophic Microorganisms

Institute of Microbiology

Academy of Sciences

Opatovick´y ml´yn, CZ-37981 Tˇreboˇn, Czech Republic

University of South Bohemia

Professor and Director

San Diego Center for Algae Biotechnology

John Dove Isaacs Chair of Natural Philosophy

Division of Biological Sciences

University of California, San Diego

2150C Bonner Hall, MC: 0368

9500 Gilman Dr., La Jolla, CA 92093-0368, USA

Email: smayfield@UCSD.edu

Phone: 858 822-7743

Alfonso Robles Medina

Professor of Chemical Engineering

Chemical Engineering Department

Department of Biotechnology Engineering

Ben-Gurion University of the Negev

713, Route de Mudaison

34670 Biallargues – FranceEmail: arnaud.muller-feuga@microphyt.euPhone: +33 6 14 79 68 92

David R Nobles Jr.

CuratorCulture Collection of Algae (UTEX)University of Texas

205 W 24th Street, Austin, TX 78712, USAEmail: dnobles@austin.utexas.edu

Phone: 512-4714019Fax: 512 4710354

Amos Richmond

Professor EmeritusBlaustien Institutes for Desert ResearchBen-Gurion University of the NegevSede-Boker, Israel

Email: amosri31@yahoo.comPhone: 052 6379666

Fax: 08 6596742

Liliana Rodolfi

ResearcherDipartimento di Scienze delle Produzioni Agroalimentari

e dell’AmbienteUniversit`a degli Studi di FirenzePiazzale delle Cascine 24, 50144 Firenze, ItalyEmail: liliana.rodolfi@unifi.it

Phone: +39-055-3288304Fax: +39-055-3288272

Fax: 361-937-6470

Michael Seibert

Professor EmeritusDepartment of Chemistry and GeochemistryColorado School of Mines

Golden, CO 80401, USAEmail: mike.seibert@nrel.govPhone: 1-303-384-6279Fax: 1-303-384-7836

College of Technology and Innovation

Arizona State University

7001 E Williams Field Road

Senior Scientist Biosciences

Sandia National Laboratories

Via Madonna del Piano, 10-I-50019 Sesto Fiorentino,Italy

Email: torzillo@ise.cnr.itPhone:+39-055-5225992Fax:+39-055-5225920

Mario R Tredici

ProfessorDipartimento di Scienze delle Produzioni Agroalimentari

e dell’AmbienteUniversit`a degli Studi di FirenzePiazzale delle Cascine 24, 50144 Firenze, ItalyEmail: mario.tredici@unifi.it

Phone: +39-055-3288306Fax: +39-055-3288272

Emily Trentacoste

PhD CandidateCenter for Marine Biotechnology and BiomedicineScripps Institution of Oceanography

University of California, San Diego

9500 Gilman Drive, La Jolla, CA 92093-0212, USAEmail: etrentacoste@ucsd.edu

Phone: 01-703-501-8682Fax: 01-858-534-0576

Hui Shen

Senior Research FellowDepartment of MicrobiologyYong Loo Lin School of MedicineNational University of SingaporeBlock MD4, 5 Science Drive 2, Singapore 117597Email: micshenh@nus.edu.sg

Phone: +65-65163284Fax: +65-67766872

Avigad Vonshak

ProfessorJacob Blaustein Institutes for Desert ResearchBen-Gurion University

Sede Boqer Campus 84990, IsraelEmail: avigad@bgu.ac.il

Phone: 972-8-6596799

xii List of Contributors

Assistant Research Professor

Laboratory for Algae Research and Biotechnology

College of Technology and Innovation

Arizona State University

7001 E Williams Field Road

Ben-Gurion University of the Negev

Midreshet Ben-Gurion, 84990 Israel

Arizona State University

7001 E Williams Field RoadMesa, AZ 85212, USAEmail: Shunni.Zhu@asu.eduPhone: +1-480-727-1410Fax: +1-480-727-1475

Graziella C Zittelli

ResearcherIstituto per lo Studio degli Ecosistemi, CNRVia Madonna del Piano 10, 50019 Sesto Fiorentino (FI),Italy

Email: graziella.chinizittelli@ise.cnr.itPhone: +39-055-5225950

Fax: +39-055-5225920

Oded Zmora

Larval and Live Feed ResearchInstitute of Marine and Environmental TechnologyDepartment of Marine Biotechnology

University of Maryland, Baltimore CountyColumbus Center, 701 East Pratt Street, Baltimore, MD

21202, USAEmail: zmorao@umbc.eduPhone: 410-234-8890Fax: 410-234-8896

Ning Zou

ProfessorPhycology InstituteLudong University186# Hongqi Middle Road, Yantai city 264025Shandong province, China

Email: ningzou76@hotmail.comPhone: 13325159079

Thirty-seven chapters that comprise this volume are

authored by some 70 scientists encompassing in their

respective works the major aspects of microalgal

biotech-nology Whatever merit this Handbook deserves is all due

to the high level of professional competence, experience,

and vision of its many contributors, who presented their art

in a well-supported, comprehensive manner, and to whom

the Editors extend their gratitude

The sharing of expertise, ideas, and know-how with all

interested in the field of microalgal biotechnology should

contribute to advance this novel science, unfolding its great

potential

Amos Richmond wishes to thank Yair Zarmi for his

con-tribution to Section 11.8 in Chapter 11 A pleasant duty is to

acknowledge the very useful assistance of Ori Even-Zahav

in the various technical aspects of the editorial work Also,the fine assistance of Ilana Saller in many chores associatedwith this publication is gratefully acknowledged Finally,

he is grateful to his wife Dalia for her generous support,encouragement, and patience along the many long hoursthe editorial work consumed

Qiang Hu wishes to thank his wife, Zhen Li, not onlyfor her understanding and support during the long daysand nights of his working on this book but also for hergreat patience and continuous love all over the years while

he has been chasing Microalgal Biotechnology the worldover, from China to Israel to Japan to the United States ofAmerica, and then to

xiii

The most prominent issue which has been dominating the

applied microalgal world for the past few years is seen in the

great surge of interest in the mass cultivation of microalgae

for promising products, first and foremost among which has

been microalgal fuel The self-propelled interest in

algae-based fuel is attracting several newcomers to this novel

biotechnology, who affect the current course of research

and promote ambitious entrepreneurship A concise

his-torical overview of microalgal biotechnology seems

there-fore essential, for providing a background and a

perspec-tive for understanding the current state of the art as well

as an evaluation of the present and the future research

trends

The formal beginning of this field of research and

appli-cations may be seen in the founding of the “Algae Mass

Culture Symposium” held in 1952 at Stanford University,

USA This was followed by the pioneering “Alga Culture –

From Laboratory to Pilot Plant,” a brilliant report and

con-clusions of this initial phase of studying the feasibility of

microalgal mass cultivation outdoors, which was edited by

J.S Burlew

A certain lull in research followed thereafter, essentially

since the initial summation of these early experiments

(con-ducted in the forties and early fifties) did not carry any great

promise for commercial microalgal endeavors

A new impetus to the idea of microalgae as a food source

for a growing world population was provided, in fact, by

the report of the “United Nations Advisory Committee to

Avert an Impending Protein Crisis,” predicting extreme

pro-tein deficiency for the growing world population by the year

2000 Research activity in mass cultivation of microalgae

was thus encouraged to develop microalgae as a source

of food and feed to avert hunger The major thrust in this

research took place initially in Italy, France, the Czech

Republic, Japan, USA, and Germany, providing the basis

for the first international meeting devoted solely to applied

phycology which was held in 1978, in Acre, Israel,

orga-nized as a joint venture of the Israeli and German

govern-ments That conference was attended by only a few scores

of researchers, joining from the world over, representing ineffect a significant segment of the total community of par-ties interested at the time in microalgal mass production.The interest in this field however grew steadily, and in theearly nineties, the International Society of Applied Phycol-ogy was established, first meeting in Florence The societywas serving as the scientific focal point for the efforts todevelop a cost-effective methodology for commercial massproduction of some dozen microalgal species The societymembership, at that time, did not exceed 150 members

It is to be noted that as experience in mass cultivation ofmicroalgae was augmenting along the past 40 years, a dis-appointing truth has been unfolding carrying a stern conclu-sion: mass production of microalgae is no means by which

to produce inexpensive products or chemicals, particularlyfood or feed commodities anticipated to replace conven-tional agricultural or marine sources All along this period,therefore, some leading researchers turned their attentionaway from major industrial commodities to smaller butlucrative markets, developing technologies by which toproduce unique, costly supplements from photoautotrophic

microalgae, for example, health-food pills based on ulina or Chlorella cell mass, astaxanthin, beta-carotene,

Spir-polysaccharides of red microalgae, polyunsaturated fattyacids (PUFA), and other products The rather high price tagthese microalgal products carry compensate for the highproduction costs of the algal cell mass and for the relativelysmall market volumes

The unique commercial successes however could notcover the fact that the original vision which promptedalgal biotechnology, that is, producing microalgal chem-icals, food, and feed to avert deficiency of basic suppliesneeded by the upward growing world populations, has notbeen successful to date

It may thus perhaps seem odd that despite this ground the idea to produce microalgae for fuel, which cir-culated in rather low tones following the upheaval (in themid seventies) in oil prices, started to amass popularitysometime in the beginning of this century It has presentlyxiv

back-Introduction xv

become a major issue in several scientific as well as

com-mercial meetings devoted to microalgae in general and to

algal fuel in particular Indeed, algal conferences presently

take place several times each year all over the world,

orga-nized by different groups and attended by several

thou-sands of interested parties Research money to investigate

microalgal fuel comes in rather generous volumes from

governments as well as private funds and commercial

orga-nizations

One naturally wonders about the factual basis for this

fer-vor, seemingly portraying sheer optimism based on

asser-tions that have been weakened with experience The fact

that certain microalgal species produce good quality

pro-tein at an output rate some 10 times higher than soybean

harvested from an equivalent area, or producing several

times more oil than the same area of palm oil trees led

to the assertion that microalgae could readily replace

con-ventional sources of food and feed, once suitable protocols

for mass production of the relevant algal species would be

developed

In reality, however, two basic points have become clear

First, microalgal overall productivity of cell mass (but not

secondary metabolites) is approximately equivalent to that

of irrigated and fertilized agricultural plants Second, the

production cost of a given quantity of algal cell mass or

product is, as a rule, very significantly higher than that of

the pertinent cell mass of conventional agricultural

com-modities

It has become generally accepted that it is the

forbid-dingly high cost of production of the cell mass of all

microalgal species grown presently, which has been

plac-ing to date an insurmountable barrier to cost-effective

large-scale algal fuel or, to a lesser extent, algal feed

commodities

The fact that essentially the sole limitation to industrial

microalgal oil is none but the cost of producing cell mass

and extracting oil is elucidated in great detail by Molina

Grima et al (Chapter 14) Using a small-scale

experimen-tal framework and calculating the exact expenses involved

in producing microalgal oil, they concluded that the

pro-duction cost of one metric ton of microalgal oil was around

30 000 euro (however, using free CO2 and wastewater as

fertilizer, the authors claim a further reduction of some

50% in cost would be possible) This cost figure obtained

by carefully adding all expenses, including algal oil

extrac-tion of the saponifiable microalgal mass clearly signifies

that although algal diesel is certainly technically feasible, it

is very far from becoming cost-effective Furthermore, their

energy balance calculations indicated that the open raceway

was the only mass production device used presently which

yielded a positive energy balance (i.e., ratio of total energyharvested to total energy consumed in the entire process inwhich microalgal cell mass turned to biodiesel) This find-ing adds an additional burden on the microalgal-diesel idea,since the open raceway is the least efficient cell mass pro-ducer relative to its footprint, yielding at best less than 45tons of dry cell mass per hectare per year (i.e., around 1%photosynthetic efficiency) David Walker, who thoroughlyrefuted the algal fuel concept (Chapter 11), suggested thatone acre of land or algal pond could supply the fuel to runone car per year Accordingly, hundreds of million hectares

of algal raceways would be required to supply only a part

of the global energy demand

Chini Zittelli et al (Chapter 13) also concluded that thecost of production of microalgal cell mass must be reduced

by significantly more than an order of magnitude to ply cost-effective raw material for oil extraction Valid evi-dence thus indicates the chances of achieving cost-effectivemicroalgae mass from which to extract oil economicallylook very distant

sup-In conclusion, the present state of knowledge in gal biotechnology is devoid of know-how by which to pro-duce much needed basic commodities in a large-scale andcost-effectively Clearly, the high order of a cost-effectivemicroalgal commodity requires much greater and moreintensive efforts in basic research It addresses, for example,genomic modification (M Hildebrand laboratory, Chapter10) for obtaining much improved strains for greater produc-tivity and physiological robustness, and conferring resis-tance to pests and contaminants and yielding high-valuecell content All of these go hand in hand with sophisti-cated engineering solutions for inexpensive, mass manu-facture of long-enduring photobioreactors, in which stronglight dilution is facilitated and which offer efficient, mass-produced mixing modes, harvesting, dewatering, and pro-cessing devices

microal-It should however be stressed, at this point, that the modity production target addressed and highlighted so far,although of high priority, represents only one developmenttrack out of great many possibilities open in microalgaeculture Indeed, an exciting aspect of microalgal biotech-nology, clearly reflected in this Handbook, is the myriadopportunities for producing materials and products offered

com-by the richness in the diversified microalgal world In fact,interesting research directions, many of which presented

in this Handbook, are being successfully developed withgood prospects for commercial recognition Some promis-ing examples in this direction follow: identifying microal-gae as a useful, inexpensive platform (compared with ani-mal cell culture), for addressing the growing interest in

xvi Introduction

recombinant proteins such as vaccines, therapeutic

anti-bodies, or industrial enzymes (S.P Mayfield laboratory,

Chapter 27)

The extraordinary variety of bioactive compounds of

commercial interest discovered in cyanobacteria and

microalgae calling for genetic manipulations and promising

metabolic pathway discoveries open valuable opportunities

in several branches of biotechnology (W.H Garwick

lab-oratory, Chapter 26) Another interesting theme concerns

sulfated polysaccharides produced by red microalgae This

product is being tested and used for applications in the

cos-metic industry as well as promising pharmaceutical

appli-cations (S Arad laboratory, Chapter 21) Production of

high-value products in heterotrophic microalgae represents

still another unique path for commercial utilizations (see

W Barclay, K Apt & D Dong, Chapter 9) Due to the

capacity to reach exceedingly high cell concentrations, the

heterotrophic mode of cultivation for suitable algal species

may yet prove very lucrative A branch of algae culture

with an enormous potential which so far has been rather

neglected concerns the preparation of new edible foods, an

enticing example for which is the cyanobacteria Nostoc, the

biology and biotechnology of which are described by

Danx-iang Han et al (Chapter 23) Certainly, the rich microalgal

world offers a plethora of novel genes and many potentially

useful products

Finally, in terms of a global approach to aid humanity

I suggest, joining some other algae researchers, that the

most promising as well as the most important target of

microalgal biotechnology at present goes back to the

orig-inal vision of supplementing food and feed to the growing

world demand This issue is presently assuming an

ever-greater importance, as global food prices are rising, steadily

becoming harder to reach by a growing section of

human-ity As world population and the standard of living keep

rising, a deficit of commodities such as fish meal is

evolv-ing rapidly W Becker (Chapter 36) notes, for example, that

for the expected total demand of 100 million tons of

prod-ucts from aquaculture in the coming decade, a deficiency of

10 million tons of PUFA is envisioned An increase in foodprices is thus to be expected without adequately satisfyingthe growing demand

Therein lies the arena in which microalgal biotechnologyhas a major and most important role to play in terms of theglobal situation, that is, providing PUFA and protein-richmicroalgal cell mass for partial replacement of fish meal(see J Gressel, Chapter 35) Supplies of fish and marineanimals could thereby rise to meet demand, potentially alle-viating protein shortages from growing world populations(see A Muller-Feuga, Chapter 33)

The price of fish meal has been presently stabilizedaround $1500/ton, having doubled in recent years due tooverfishing, dwindling wild fish resources A large dis-crepancy has long been existing between costs of fishmeal versus suitable microalgae used for larvae feeding

in hatcheries, for example, Isochrysis, Nannochloropsis, or Pavlova sp Despite richness in high-quality protein and

suitable PUFA, the high production cost of these and ilar nutritional microalgae did not permit their large-scale,commercial use as partial fish-feed replacement However,with expected improvements in algal genetics, produc-tion devices as well as production protocols for growingmicroalgal cell mass (an expected by-product of the well-financed research for commercial algal oil), the cost gapbetween fish meal and that of algae replacing fish mealshould become ever narrower with time as well as withpersistent, focused research efforts

sim-Chances are that long before microalgal oil will becomecost-effective, if ever, microalgae as a fish-meal replace-ment should reach the economic end zone, providing sev-eral million tons of protein and PUFA-rich marine and ani-mal feed, to the great benefit of humanity

Amos Richmond, PhDProf Emeritus, Ben-Gurion University

Sede-Boker, Israel

Part 1 The Microalgal Cell with Reference

to Mass Cultures

Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition Edited by Amos Richmond and Qiang Hu.

1 The Microalgal Cell

asexual reproduction (e.g., Chlorella, Nannochloropsis) Algal ultrastructure is also diverse, paralleling their

biochemical and physiological diversity Many genomes of microalgae have been sequenced, and these areproviding new insights into algal diversity Genomic research has corroborated known endosymbiotic eventsand has revealed unknown, or cryptic, such events Endosymbiosis has been a major factor in the production ofalgal diversity, and once it is better understood, this may be a practical means for producing new combinations

of traits that have commercial application The current state of algal taxonomy is summarized

Keywords algae; carbohydrate; chloroplast; endosymbiosis; genome; lipid; morphology; physiology;

phy-toplankton; protein

1.1 INTRODUCTION

Algae are primarily oxygen-releasing photosynthetic

organisms with simple body plans – no roots, stems, or

leaves Algae are usually aquatic organisms They do not

form a single monophyletic group and consequently

can-not be easily defined Although algae as a group are

ubiq-uitous, individual species occupy specific habitats Some

algae are attached to a substrate like plants, some are motile

like animals, some are simply suspended in water, some

grow loosely on soil, trees, and animals, and some form

symbiotic relationships with other organisms (e.g., corals,

lichens) The internal cell structure of algae varies greatly

Microalgae lack complex multicellular structures that are

found in seaweeds The cyanobacteria or blue-green algae

have a prokaryotic cell structure and closely resemble teria Eukaryotic algal cells have a nucleus and usually one

bac-or mbac-ore chlbac-oroplasts; they also have mitochondria, Golgibodies, endoplasmic reticulum, and other typical eukary-otic organelles Despite the difficulty in presenting a cleardefinition for algae, thousands of books, scores of scien-tific journals, and numerous internet websites are dedicatedsolely to compiling our knowledge of algae (Lee, 2008;Graham et al., 2009)

Microalgae appear in a wide variety of shapes and forms.This morphological variation occurs not only amongspecies but also among different life stages of the same

Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition Edited by Amos Richmond and Qiang Hu.

C

3

4 Robert A Andersen

Figure 1.1 Flagellate algal diversity (a)Pedinomonas, with one visible flagellum Scale bar= 5 μm (fromSkuja, 1956) (b)Dunaliella, with two equal flagella Scale bar= 10 μm (from Bold & Wynne, 1985) (c)Chlamydomonas, showing the biflagellate cell and four nonflagellate cells Scale bar= 10 μm (flagellatefrom Ettl, 1976; colony from Skuja, 1956) (d)Haematococcus, showing the flagellate cell and three

nonflagellate cells Scale bar= 10 μm (from Skuja, 1948) (e) Tetraselmis, a quadraflagellate marine alga.Scale bar= 5 μm (from Throndsen, 1993) (f) Pavlova, with two unequal flagella and a very short

haptonema Scale bar= 5 μm (after Throndsen, 1993) (g) Isochrysis, with two nearly equal flagella and twochloroplasts Scale bar= 5 μm (after Throndsen, 1993) (h) Synura, a colony with cells attached in the center.Scale bar= 10 μm (from Skuja, 1956) (i) Gymnodinium, a dinoflagellate with a circling transverse flagellumand a trailing longitudinal flagellum Scale bar= 25 μm (from Skuja, 1956) (j) Ochromonas, with two veryunequal flagella Scale bar= 10 μm (from Skuja, 1964) (k) Chrysochromulina, with a long haptonema arisingbetween the two flagella Scale bar= 5 μm (after Throndsen, 1993) (l) Euglena terricola, a large cell with oneflagellum emerging from a gullet Scale bar= 10 μm (from Skuja, 1956) (m) Dinobryon, an arbuscular colonyformed from loricas that surround each cell Scale bar= 10 μm (from Skuja, 1964) (n) Stephanosphaera, acolony where cells are attached laterally Scale bar= 10 μm (from Skuja, 1956) (o) Rhodomonas, a commonmarine biflagellate Scale bar=10 μm (from Skuja, 1948) (p) Volvox, a large colonial flagellate with

reproductive cells inside the otherwise hollow colony Scale bar= 35 μm (from West, 1904)

species The common forms are defined with adjectives

such as amoeboid, palmelloid (= capsoid), coccoid,

fil-amentous, flagellate, and sarcinoid (Figs 1.1 and 1.2)

Scientists use morphological life forms when generally

discussing algae and their stages; there are, however,

hundreds of thousands of algal species, and they do notalways fit neatly into a few convenient categories The firstalgae were morphologically simple organisms; today’s sim-plest morphologies, however, are frequently the result ofevolutionary reduction through which the algae are better

The Microalgal Cell 5

Figure 1.2 (Opposite )

6 Robert A Andersen

able to survive because of their simplicity In the following

text, algal forms are treated from simple to complex, and

this approach is strictly arbitrary (i.e., it does not reflect

“primitive” vs “advanced”)

Flagellates may be single cells where each cell is an

independent organism propelled through water with one

or more flagella (e.g., Pedinomonas, Chlamydomonas,

Gymnodinium, Ochromonas, Tetraselmis) (Fig 1.1)

Sev-eral to many flagellate cells may be joined together to

produce a motile colony (e.g., Dinobryon, Synura) Large

colonies, such as Volvox (Fig 1.1p), have hundreds of cells.

Most flagellate cells have two flagella, but marine

picoflag-ellates may have only one flagellum (e.g., Micromonas and

Pelagomonas) while Pyramimonas may have up to 16

flag-ella per cell Haptophyte algae usually have a haptonema

positioned between the two flagella (Fig 1.1k), and the

haptonema can be used for attaching to surfaces or

collect-ing particles of food Many common flagellate algae also

produce nonmotile stages, as shown for Chlamydomonas

and Haematococcus (Figs 1.1c and 1.1d) Changing the

environmental conditions can induce these alternate stages,

and the manipulation of stages can be used to advantage in

commercial facilities

Many microalgae have a nonmotile stage as the dominant

life form, and in some cases, no motile cells are ever found

in the life cycle (Fig 1.2) Amoeboid algae (e.g.,

Chlo-rarachnion, Chrysamoeba, Rhizochromulina) slowly creep

across substrates, including the marine snow particles in

oceans (Fig 1.2a) Amoeboid cells may capture bacteriausing pseudopods Coccoid algae reproduce by autospores

or zoospores, that is, mother cells undergo synchronizedmitotic divisions and the number of daughter cells is fixed

(e.g., 2, 4, 8, 16, 32) Single cells, such as sis are free, but commonly coccoid algae produce colonies (e.g., Chlorella, Oocystis, Scenedesmus) (Figs 1.2d–g and 1.2i) Some, such as Synechococcus (Fig 1.2c), exist today

Nannochlorop-as single cells or weakly connected cells, but their tors were filamentous algae Palmelloid algae have cellsembedded within a gelatinous matrix; usually the cells arenot physically connected to each other and only the gelholds them together The gelatinous mass may be plank-tonic or attached to a substrate (Fig 1.2j) The common

ances-flagellate Pavlova (Fig 1.1f), for example, produces large

palmelloid sheets when grown under certain culture ditions Sarcinoid colonies result from equal cell division

con-in three planes so that a cube is produced (Chlorosarccon-ina; Fig 1.2l) The oil-producing Botryococcus makes a crudely

parenchymatous colony (Fig 1.2k) Filaments are duced when cells attach end to end and form ribbon-like or chain-like assemblages In their simplest form,filaments are unbranched and consist of a single row of

pro-cells (uniseriate) such as Arthrospira/Spirulina (Fig 1.2n).

Complexity develops with side branches (Fig 1.2p) andmultiple rows of cells (multiseriate) The cyanobacterium

Nostoc forms large colonies that consist of uniseriate

tri-chomes embedded in a soft, inner colonial gel matrix;

Figure 1.2 Diversity of nonflagellate algae (a)Chrysamoeba mikrokonta, an amoeba with branching

pseudopods Scale bar= 5 μm (from Skuja, 1956) (b) Porphyridium purpureum, a single-celled red alga with

a stellate chloroplast Scale bar= 5 μm (from Hori, 1993b) (c) Synechococcus aeruginosus, the large

freshwater type species of this cyanobacterium Scale bar= 10 μm (from Geitler, 1932) (d) Nannochloropsissalina showing three elongate coccoid cells Scale bar= 2 μm (from Andersen et al., 1998) (e)

Nannochloropsis oculata showing four spherical coccoid cells Scale bar= 2 μm (from Andersen et al., 1998).(f)Chlorella vulgaris showing a large single cell (top), four autospores (bottom), release of autospores (right).Scale bar= 10 μm (from Fott, 1959) (g) Scenedesmus maximus, showing four laterally connected coccoidcells Scale bar= 10 μm (from Skuja, 1949) (h) Cosmarium ornatum, showing the typical semi-cell

construction of desmids Scale bar= 10 μm (from Skuja, 1956) (i) Oocystis gigas var incrassata showingeight cells within the old mother cell wall Scale bar= 20 μm (from Skuja, 1964) (j) Phacomyxa sphagnicola,

a palmelloid alga showing vegetative cells within a colonial gelatinous matrix Scale bar= 40 μm (fromSkuja, 1956) (k)Botryococcus braunii showing cells in packets and numerous oil droplets in each cell Scalebar= 10 μm (original) (l) Chlorosarcina superba showing a cuboidal colony Scale bar = 10 μm (from Skuja,1956) (m)Nostoc planctonicum showing an enlarged trichome (left), a long trichome, and the colony oftrichomes Scale bar= 5 μm (left, cells), = 25 μm (center, trichome), = 33 μm (colony) (from Geitler, 1932) (n)Spirulina/Arthrospira, showing different morphological forms of the spiraling trichome Scale bar= 10 μm(left),= 18 μm (center), = 5 μm (right) (from Geitler, 1932) (o) Ulothrix moniliformis, an unbranched filamentwith a well-defined gelatinous sheath Scale bar= 10 μm (from Skuja, 1956) (p) Cladophora sterrocladia,showing a typical branched filament shape Scale bar= 250 μm (from Skuja, 1949)

The Microalgal Cell 7

Figure 1.3 Diatom diversity (a)Thalassiosira decipiens, showing three cells connected by a chitinous strand.Scale bar= 10 μm (from Hendy, 1964) (b) Thalassiosira hyalina showing a filament of cells, each cell withnumerous chloroplasts Scale bar= 20 μm (from Hendy, 1964) (c) Chaetoceros pseudocrinitum showingcells connected by intertwined setae Scale bar= 20 μm (from Hendy, 1964) (d) Chaetoceros gracile, acommon single-cell species Scale bar= 5 μm (from Hendy, 1964) (e) Achnanthes lanceolata, a monoraphidspecies with two central raphes on the left valve and no raphes on the right valve Scale bar= 5 μm (fromPatrick & Reimer, 1966) (f)Navicula rhynchocephala showing the central raphes and numerous straiae Scalebar= 5 μm (from Patrick & Reimer, 1966) (g) Nitzschia linearis showing the marginal raphe and numerousstraiae Scale bar= 10 μm (Kalbe, 1980) (h) Phaeodactylum tricornutum showing the common

morphological shapes of the species Scale bar= 10 μm (original)

the outer colony surface has a tough, leathery consistency

(Fig 1.2m)

Diatoms are essentially silica-walled coccoid cells that

sometimes remain attached to form simple chains or

fil-aments (Figs 1.3a–1.3c) Diatoms are the most specious

group of algae, with estimates of up to one million or

more species (Round et al., 1990) Diatoms have cell walls

made of opaline silica, like that of window glass, and the

glass surfaces have numerous simple or complex “pores”that allow molecular exchanges between the cytoplasm andthe environment (Figs 1.4a and 1.4b) The cell has twovalves that are held together by girdle bands In a gen-eral sense, diatoms often categorized as centric or pen-nate; centric diatoms have valves that radiate from a centralregion (Figs 1.3a– 1.3d), whereas pennate diatoms havevalves that are bilaterally symmetrical (Figs 1.3e–1.3g)

8 Robert A Andersen

Figure 1.4 (a)Odontella sp showing the variety of pores and structures on the valve (V) and girdle band(G) (original) (b)Odontella aurita showing strutted processes connecting two valves (V) (original) Scalebar= 10 μm

The valves and the connecting girdle bands have

morphol-ogy mostly consistent within each species, thereby making

it possible to identify diatoms based upon the markings of

the silica walls

1.3 SEXUAL REPRODUCTION

Sexual reproduction may increase cell numbers or

pro-duce resistant stages (e.g., zygospores) that greatly

facil-itate the geographic distribution for the species The basic

elements of algal sexual reproduction are similar to those

of other eukaryotes; sperm and egg cells are formed, they

fuse (syngamy) and zygotes are formed However, many

variations occur among algae (Hori, 1993a, 1993b, 1993c)

For example, in the green algae Pandorina, there are not

just male/female gametes – there are at least 30 mating

types or syngens (Coleman, 2001) The sperm of pennate

diatoms are amoeboid while the spermatids of red seaweeds

are simple cells that drift in the oceans and reach a female

oogonium by chance The fusion of gametes is a

com-plex process that involves signal transduction at receptor

sites on flagella (Pan & Snell, 2000) or even the role of an

actin cytoskeleton in nonflagellate spermatia (Wilson et al.,

2003) A number of algal genera have no reports of sexual

reproduction (e.g., Chlorella, Euglena, Nannochloropsis,

Porphyridium), and there is debate whether these

observa-tions reflect the true absence of sex or the lack of thorough

attempts to find sex The ability to sexually reproduce has

important biotechnological implications because breeding

and selection can advance aquaculture just as breeding has

advanced the improvement of plants and animals for

agri-culture Conversely, if sex is not possible then a favorable

asexual strain may be maintained indefinitely That is, care

should be taken to preserve the strain so that mutations do

not alter the traits; cryopreservation is a good technique formaintaining strains without change

The ultrastructure of algae is more diverse than that foundamong animal and plant cells This reflects the broad phy-logenetic diversity of algae, their adaptation to many envi-ronments, and 3.5 billion years of evolutionary change.The cyanobacteria have relatively simple cells (Fig 1.5)

A cyanobacterial cell contains many sheet-like thylakoids,and these thylakoids appear as parallel lines in thin sectionsviewed in the transmission electron microscope (TEM).The cells divide by fission, or pinching, that converts onelarger cell into two smaller cells (Fig 1.5a) The ultrastruc-ture of eukaryotic cells is much more complex, their evo-lutionary history spans about 1.5–2.0 billion years, and thestructures vary significantly within and among algal classes.Eukaryotes possess a number of organelles and these areimportant metabolic compartments that allow specializa-tion (Martin, 2010) The general features of the eukaryoticalgae will be described (Fig 1.6)

1.4.1 Chloroplast

The chloroplast is the dominant organelle of eukaryoticalgae, and the sheet-like thylakoids contain the membrane-bound pigments that capture light for photosynthesis Thy-lakoid arrangement is consistent within algal groups butvaries among groups For example, charophycean plastidsresemble plant chloroplasts and have distinct grana (stacks

of thylakoids); haptophyte plastids have lamellae formedfrom three sheet-like thylakoids (Fig 1.7); heterokont plas-tids are similar to haptophytes but have an outer sac-likegirdle lamella surrounding the sheet-like lamellae Many

The Microalgal Cell 9

Figure 1.5. Synechococcus strain PCC7117 (a) Longitudinal section showing early cell division (arrowheads).(b) Transverse section, showing thylakoids Scale bar= 500 nm (unpublished, courtesy of Daiski Honda)

plastids have a pyrenoid (Fig 1.6) The pyrenoid is an

accumulation of RuBisCO (ribulose-1,5-bisphosphate

car-boxylase/oxygenase), the dominant protein involved in the

Calvin cycle of photosynthesis Interestingly, only green

algae store their photosynthates within the chloroplast

Thus, green algae have starch grains inside the plastid, but

for all other algae, carbohydrate or lipid storage is outside

the plastid (e.g., between the plastid and the chloroplast

ER in cryptophytes) (Ball et al., 2011) Lipid bodies are

Figure 1.6. Chroomonas mesostigmatica

Transmission electron micrograph (TEM) C,

chloroplast; E, eyespot; G, Golgi body; L, lipid body;

M, mitochondrion; Py, pyrenoid; S, starch granule

Scale bar= 600 nm (unpublished, courtesy of

Robert E Lee)

Figure 1.7. Phaeocystis TEM showing a secondaryplastid The lamellae (l) are composed of threethylakoids (arrows); the chloroplast is surrounded

by four membranes (see bracket region with fourmembranes plus a lamella) The outer chloroplastmembrane is continuous with the outer membrane(o) of the nuclear envelope, which is easily

distinguished from the inner membrane (i) of thenucleus (n) The plasma membrane (pm) is visible,the peripheral endoplasmic reticulum is visible (∗)and scales (s) surround the cell Scale bar= 250 nm(original)

10 Robert A Andersen

also common, and their appearance is usually related to the

physiological or environmental conditions of the algae, for

example, the number and size of lipid bodies increase when

the cells are under high light stress or nutrient starvation

The chloroplast contains its own DNA, typically circular

chromosomes; however, the DNA encodes for just a small

number of genes because most genes have been transferred

to the nucleus The dinoflagellate plastid genome often has

many small rings of DNA Many chloroplast genomes have

been sequenced (for review, see Green, 2011)

1.4.2 Mitochondrion

Mitochondria diversity is also greater among algae than for

animals and plants Green and red algae have

mitochon-drial cristae that are flattened like those of plants or

ani-mals However, the euglenoids have disc-like cristae, and

the haptophyte, heterokont, and dinoflagellate algae have

cristae that are tubular in shape For recent comments on

protistan mitochondria, see Shiflett & Johnson (2010)

1.4.3 Nucleus and mitosis

The nucleus of many algae is similar to that found in plants

or animals; however, significant differences occur The

typi-cal euglenoid nucleus always has condensed chromosomes,

and the dinoflagellate nucleus has unique chromosomes that

visually resemble a stack of coins Great diversity exists

for nuclear division or mitosis (see Lee, 2008; Graham

et al., 2009) The mitotic spindle may be formed inside a

persistent nuclear envelope, the spindle microtubules may

penetrate through a persistent but perforate envelope or

the nuclear envelope may break down like typical plants

and animals A highly reduced nucleus, named the

nucle-omorph, remains as evidence of secondary endosymbiosis

in the cryptophytes and chlorarachniophytes; for a review,

see Moore & Archibald (2009)

1.4.4 Golgi body and endoplasmic reticulum

Organelles such as the Golgi body and endoplasmic

retic-ulum are generally similar in structure to those of other

eukaryotes Algae use these organelles to produce organic,

silicate, or calcium carbonate scales as well as flagellar

hairs and other structures

1.4.5 Vacuoles

Eukaryotic cells may possess one or more types of vacuoles

Cells with rigid and complete cell walls often have a vacuole

that is at least analogous to the typical plant vacuole and the

vacuole functions to maintain a positive osmotic pressure

that in turn maintains cell and organismal rigidity Some

organisms, especially heterokont algae, use vacuoles for

storage products or byproducts derived from degradation

or remodeling of subcellular compartments, particularlyunder stress These algae produce low-molecular-weightcarbohydrates (laminarin, chrysolaminarin) in the cytosol,and because the molecules are small (e.g., 20–40 glucoseresidues) they affect the osmolarity of the cell To avoid

a surge in osmotic pressure, these small carbohydrates arekept within specialized vacuoles The heterokont and hapto-phyte algae commonly store oils in their cells The contrac-tile vacuole is an osmoregulatory organelle in freshwateralgae that removes osmotic water from cells Cells withtrue walls develop a positive osmotic pressure such thatthe wall keeps the protoplasm from bursting However, theprotoplasm of freshwater naked, thecate, or loricate cellswill expand as water enters the cell by osmosis If no water

is removed, these cells will burst Contractile vacuoles arerare in marine organisms because the saltwater is more orless isotonic with the protoplasm

1.4.6 Flagella and eyespots

Swimming algae are propelled by eukaryotic flagella Theflagellar axoneme, like that of animals and many non-flowering plants, has the nine pairs plus two microtubulesarrangement The diatom axoneme, however, has a 9+ 0arrangement (Manton & von Stosch, 1966) and the samearrangement occurs in algae whose flagella are not used

for swimming (e.g., Tetraspora, Lembi & Herndon, 1966).

Furthermore, the basic flagellum in many algae is enhanced

or modified The cryptophytes, typically, have bipartiteflagellar hairs on both flagella; heterokont algae have tri-partite flagellar hairs on the immature flagellum (bipar-

tite in Pelagomonas, absent in some Pinguiophyceae), and

some euglenoids produce hair-like scales (Bouck et al.,1978; Kugrens et al., 1987; Andersen et al., 1993; Kawachi

et al., 2002) Flagellar hairs change the swimming tion, that is, if the hairs are present, the cell swims for-ward, but if the hairs are removed, the cell swims back-wards (Sleigh, 1989) Flagella in certain groups also possess

direc-organic scales (e.g., Synura) Paraxonemal rods are found

in the flagella of certain algae (e.g., euglenoids, lates, dictyochophytes) The paraxonemal rod is contractile

dinoflagel-in ddinoflagel-inoflagellates but is noncontractile dinoflagel-in Euglena.

The flagellum of a swimming cell exerts considerableforce on the cell body and, therefore, the flagellum isanchored securely in the cell The flagellum undergoes atransition to form the basal body inside the cell and thebasal bodies are anchored with microtubular and fibrousroots These structures, in turn, either constitute or attach

to the cytoskeleton (see Andersen et al., 1991; Moestrup,2000) Flagella also undergo a maturation process When

The Microalgal Cell 11

a flagellum first forms from a nascent basal body, it is

termed the immature flagellum When the cell divides, the

immature flagellum is retracted, and it grows out again as a

mature flagellum; this process is termed flagellar

transfor-mation (Melkonian et al., 1987; Wetherbee et al., 1988)

Eyespots, or stigmata, are found on many swimming

cells The eyespot is red and represents a specialized lipid

accumulation that is associated with a flagellum The

eye-spot, often in concert with a paraflagellar body in the

flag-ellum, functions by providing phototaxis for the swimming

cell (Kreimer, 1994, 1999; J´ekely, 2009) That is, the cell

detects the direction of incident light and swims either

toward or away from the light source

1.4.7 Cell walls and coverings

The cell wall is a robust structure that completely encloses

the cytoplasm and allows the cell to increase its turgor

pressure without bursting Cell coverings, such as thecae,

loricas, scales, and coccospheres surround the cell body;

they provided protection but the cell must remain

osmot-ically balanced with the surrounding environment

There-fore, flagellate or amoeboid cells cannot have a true cell

wall Algae with true cell walls undergo cell division in

two ways If the mother cell wall is largely retained and a

new wall partitions the mother cell into two daughter cells,

this is termed desmoschisis These organisms can develop

a tough, rigid thallus because the cells are strongly bound

by their walls The brown, green, and red seaweeds – the

macroscopic algae – form walls by desmoschisis

Alter-natively, the mother cell wall can be completely dissolved

or discarded and the daughter cells must each produce an

entirely new cell wall; this is termed eleuteroschisis Some

organisms, like Chlorella, maintain a somewhat digested

and expanded mother cell wall, and this old wall holds the

daughter cells together as a colony

The biochemical composition of cell walls varies

amongst algal groups The cyanobacteria have a

peptido-glycan wall, often with associated layers or fibrils This

wall is a rich source of protein, and Spirulina is sold as a

health food rich in protein The cell walls of macroscopic

red algae consist of a cellulose microfibrillar scaffold that

is impregnated with polymers of sulfated galactans and

var-ious mucilages Agars and carrageenans (highly sulfated)

are commercially extracted from red algal walls and they

are used as thickeners and emulsifiers in a variety of

applica-tions Green algal cell walls may be composed of cellulose,

hemicellulose, pectic compounds, and glycoproteins

Thecae are thin organic coverings surrounding most, but

not all, of the cell Chlamydomonas and thecate (armored)

dinoflagellates are organisms with thecae The composition

of the theca varies, but often it has a cellulose microfibrillarinfrastructure Loricas are similar to thecae but, typically,there is a greater space between the cell and the lorica.Loricas are also frequently mineralized and may appearyellow or red in color Scale composition may be organic(cellulosic) materials, silicate glass, or calcium carbonatecrystals There are other cell coverings such as the pellicle

of euglenoids, the periplast of cryptophytes, and a widevariety of mucilaginous excretions

Strong cell walls may be advantageous if the cells passthrough pumps or strong mixing devices; however, thesewalls are often difficult to crack open when trying toextract cellular contents Walls also impede genetic trans-formation Conversely, the sheer forces of pumps easilydamage naked, scaled, or thecate cells, but it is easier toextract their contents or to employ genetic transformationtechniques Organisms with mineralized walls may requirespecial growth conditions, for example, diatoms require sil-ica The mineralization process can also be used to advan-tage, for example, coccolithophorid algae make scales withCaCO3, and they can be used as a CO2 bioscrubber forcarbon sequestration

1.5 BIOCHEMICAL ASPECTS

Large biomolecules are classified into broad major groups(e.g., carbohydrates, lipids, nucleic acids, and proteins).These groups are similar to those found in other livingorganisms, but again the algae provide an exceptional diver-sity of biomolecules This diversity has interested commer-cial companies in recent years, and as a consequence algaeare grown for the express purpose of harvesting these com-pounds – many examples are found in this book Thereare extensive publications on algal biochemistry, and theprecise biochemical product can be manipulated to somedegree by altering the growth conditions (Chapter 7; Hu,2004; Beer et al., 2009) For the purpose of this chapter, afew examples will be provided

1.5.1 Carbohydrates

The carbohydrate storage product in many algae is starch

or a starch-like product (e.g., green and red algae, tophytes, dinoflagellates) These starches have a primary

cryp-α-1,4-linked glucan molecular backbone, and typically the

backbone chain hasα-1,6-linked side chains (Viola et al.,

2001; Ball et al., 2011) The starches are large molecules(i.e., colloidal particles or larger particles) and starch grainsare easily visible in a light microscope Another group

of algae utilize a β-1,3-linked glucan backbone (e.g.,

heterokont algae, haptophytes, euglenoids) The degree

of polymerization varies significantly for these laminarin

12 Robert A Andersen

and paramylon products The smaller molecules, such as

chrysolaminarin, consist of fewer than 30 glucose residues,

and therefore to avoid osmotic problems, the molecules are

maintained in vacuoles At the other end of the spectrum,

large paramylon grains, such as those found in euglenoids,

are easily visible in a light microscope

1.5.2 Lipids

The diversity of algal lipids is also extensive (Wood,

1984) and the cellular lipid composition can be

manipu-lated (Hu et al., 2008; Wang et al., 2009), that is, under

low nitrogen conditions (e.g., in the stationary phase),

cells carry out photosynthesis and produce lipids from

photosynthetically fixed carbon (e.g., 3-phosphoglycerate)

Fatty acid and sterol diversity are found in cellular

mem-branes among algal groups; these lipids are more

diffi-cult to extract than the lipids accumulated as lipid

bod-ies or oil droplets Of the membrane lipids, galactolipids

(e.g., monogalactosyldiacylglycerol and

digalactosyldia-cylglycerol) are the major constituents of thylakoid

mem-branes on which photosynthetic machinery reside (Hu et al.,

2008) Nannochloropsis accumulates significant amounts

of membrane-bound eicosapentaenoic acid (EPA)

(Khozin-Goldberg & Iskandarov, 2011) A wide range of algae

pro-duce lipids as storage products (i.e., oleaginous algae), and

frequently the lipids can be observed as oil droplets in cells

(Fig 1.2k) These lipids are largely polyunsaturated fatty

acids (PUFAs), including the omega-3 PUFAs arachidonic

acid (AA), docosahexaenoic acid (DHA), and EPA The

heterokont algae (e.g., Chaetoceros, Nannochloropsis,

Pin-guiococcus) and the haptophytes (e.g., Pavlova, Isochrysis)

typically use oil droplets as a storage product, especially

when their carbohydrate storage is chrysolaminarin like

The Pinguiophyceae store EPA in large quantities (Kawachi

et al., 2002), and DHA is stored in many haptophyte algae

(Guschina & Harwood, 2006; Khozin-Goldberg &

Iskan-darov, 2011) General textbooks often report that green

algae store starch, but a considerable number of

chloro-phyte green algae (e.g., Scenedesmus, Chlorella) store oils

under stress (Guschina & Harwood, 2006) Other chapters

in this book address algal lipids in detail

1.5.3 Proteins

Algal proteins are also exceptionally diverse, and research

in specific areas, such as photosynthesis, has shown that

proteins can be manipulated by environmental changes

(Grossman et al., 1995) Cyanobacteria have

peptidogly-can cell walls and, therefore, are an excellent source

of proteins, that is, 40–60% of the dry weight is

pro-tein (e.g., Arthrospira/Spirulina, Synechococcus) (Becker,

2007) Green algae are also good sources (e.g., Chlorella, Scenedesmus); Euglena gracilis as well as Porphyridium

produce up to 30–60% protein by dry weight Protein-richcells are often actively growing/dividing cells (log phase)and, therefore, differ from stationary phase lipid-rich cells.Furthermore, some organisms sequester nitrogen when it isavailable in the environment and they store the excess nitro-gen in proteins; when nitrogen becomes limited, they digestthese storage proteins to release the nitrogen Many algaeproduce pyrenoids (Fig 1.6), which are accumulations ofthe enzyme RuBisCO (Kuchitsu et al., 1988) The enzymeplays a crucial role in photosynthetic carbon fixation, butthe pyrenoid accumulations are also a rich source of nitro-gen that can be tapped when nitrogen become deleted in theenvironment In a similar way, cryptophytes store nitrogen

in phycobiliproteins

1.6 BIODIVERSITY

The diversity of algae is amazing at several levels Speciesdiversity is measured by the number of described speciesand there is general agreement that many species havenot yet been described (Andersen, 1992; Norton et al.,1996) Diatoms have been called the “insects” of the algalworld because there may be millions of diatom species andbecause they are ubiquitous in distribution Conversely, theglaucophytes and dictyochophytes are groups with veryfew species, and they may be relic groups left over fromtimes past Algal diversity may also be measured in terms

of biochemical pathways, ecological roles, endosymbioticgenomes, morphology, reproductive strategies, and so forth.For example, the nontraditional, unusual, and even uniquebiochemical pathways and products of algae are described

in other chapters of this book The recent discovery of tic endosymbiotic genomes is significant, for example, pre-dominately green algal genes in diatoms (Moustafa et al.,2009) Endosymbiotic events, and even horizontal genetransfers, have been major genetic mixing pots that haveshuffled genomes, created gene duplications, and allowedfor gene replacements These have contributed significantly

cryp-to algal diversity at all levels

1.7 EVOLUTION AND SYSTEMATIC BIOLOGY 1.7.1 Evolutionary origins

Fossil prokaryotic cyanobacteria have been found in ments approximately 3.8 billion years ago, and at least sincethis date oxygen-releasing photosynthesis has occurred onearth Not only were these early algae efficient autotrophs,they produced so much free oxygen that it fundamen-tally changed life on earth (Falkowski & Knoll, 2007)

sedi-The Microalgal Cell 13

Ancient Precambrian stromatoliths first appeared about 3.5

billion years ago, and they are fossil remnants of massive

cyanobacterial growths in ancient times Stromatoliths are

still formed, but they are quite rare (e.g., Shark Bay,

Aus-tralia)

The origin of eukaryotes is not precisely known, but may

have occurred about 2 billion years ago (Knoll et al., 2006)

Currently, there is much debate about the eukaryotic origin;

analyses of entire genomes are providing both questions and

answers (Foster et al., 2009; Gribaldo et al., 2010; Koonin,

2010) We do know that the eukaryotic algae are not a

single evolutionary lineage, and therefore algae per se are

not a monophyletic group However, the plastid profoundly

defines eukaryotic algae from nonphotosynthetic protists

(e.g., “protozoa” and “aquatic fungi”), and the original

plas-tid genome traces back to a single primary endosymbiotic

event (Keeling, 2010) That is, about 2 billion years ago,

a nonphotosynthetic eukaryote engulfed a cyanobacterium;

rather than digesting it as food, the eukaryote "enslaved" the

cyanobacterium cell Over time, the enslaved cell became

a chloroplast, and its existence became deeply entwined

within the host cell Chloroplast division synchronized with

host cell division; genes were transferred from symbiont to

host genomes and optimized biochemical reactions inside

the host cytosol; and the structure and pigmentation of the

plastid evolved For a recent review of chloroplast pigments

(chlorophylls, carotenoids), see Roy et al (2011)

1.7.2 Cyanobacteria

The cyanobacteria, or blue-green algae, are the oldest

group of algae (Fig 1.5) Originally, their classification

was based strictly on gross morphology (Geitler, 1932)

However, both electron microscopy and molecular

phylo-genetic analysis have shown that the traditional

morpho-logical groups are not monophyletic groups, for example,

filaments have arisen independently several times

Cyto-logically and biochemically, the cyanobacteria are similar

to bacteria Most cyanobacteria possess chlorophyll a,

phy-cocyanin and phycoerythrin as light-harvesting molecules,

but chlorophylls b and d, as well as divinyl derivatives of

chlorophylls a and b, are found in a few organisms (e.g.,

Acaryochloris, Prochloron, Prochlorococcus) The storage

product is typically cyanophycean starch, a predominantly

α-1,4-linked polyglucan Ecologically, cyanobacteria are

autotrophs that photosynthesize and release oxygen, thus

they share this ecophysiology with eukaryotic algae Some

species are commercially valuable (e.g., Arthrospira), some

produce toxins that can taint and poison drinking water

(e.g., Microcystis), but most are innocuous organisms that

are ecologically significant but rarely recognized

Remark-ably, Prochlorococcus, a tiny oceanic picoplankton (0.5–

0.8μm in diameter), is the most abundant living organism

on the planet (Chisholm et al., 1992)

1.7.3 Eukaryotic super groups

The phylogenetic relationships of the eukaryotic algae wererarely considered in the light microscopy era (Fritsch,1935) The electron microscopy era was dominated by thediscovery of new ultrastructural diversity and the descrip-tion of new classes; nevertheless, new evolutionary rela-tionships began to emerge Recently, molecular biologyand phylogenetic analysis have contributed significantlytoward our understanding of relationships Multigene anal-ysis and genomic/proteomic analyses have helped recoverdeep branch relationships (Baldauf, 2003) Consequently,

we have some emerging super groups of algae althoughthere remains considerable debate (Hackett et al., 2007;Bodył et al., 2009; Reeb et al., 2009; Baurain et al., 2010;Burki et al., 2010; Parfrey et al., 2010; Green, 2011)

1.7.3.1 Algae with primary plastids

It is generally accepted that the glaucophyte, green, andred algae form a monophyletic group sometimes called

the Archaeplastida; recent genomic data supports the

monophyly of these three lineages that contain membrane-bound plastids (Price et al., 2012) The glau-cophytes may be more ancient because they have cyanelle-type photosynthetic organelles with peptidoglycan cellwalls, and they maintain the enzyme fructose biphosphatealdolase The green algae are a deeply divided lineage,with one branch containing many common organisms (e.g.,

double-Chlamydomonas, Chlorella, Ulva, Volvox) and another branch (e.g., Klebsormidium, Spirogyra, Chara) giving rise

to plants The evolutionary history of the red algae is morecomplex than first imagined (see Section 1.7.6), and fur-thermore, red algae have been captured and converted toplastids by secondary endosymbiotic events

1.7.3.2 Algae with secondary plastids

Secondary endosymbiotic events were once viewed as quent occurrences (Leedale, 1974), but the recent litera-ture argues for very few events (Keeling, 2010) The pri-mary morphological change is the occurrence of a sec-ondary plastid that typically has one or two additionalmembranes just outside the two chloroplast envelope mem-branes (Fig 1.7) One secondary endosymbiosis involving

fre-a red fre-algfre-ae must hfre-ave occurred efre-arly in the evolution ofeukaryotic life, and the ancestors diversified to form what

is sometimes called the chromalveolates (Cavalier-Smith,

14 Robert A Andersen

1999) This group is composed of the stramenopiles,

alve-olates, and rhizaria (SAR) as well as the cryptophytes

and haptophytes There is growing consensus for SAR:

stramenopiles include the heterokont algae, ¨oomycetes,

and thraustochytrids; alveolates consist of the

apicomplex-ans, ciliates, and dinoflagellates; rhizarians are largely

het-erotrophic amoebae but the group includes photosynthetic

chlorarachniophytes and Paulinella There is less support

for adding the Cryptophyceae and Haptophyceae to the

SAR to form the chromalveolates (Bodył et al., 2009;

Par-frey et al., 2010) even though they all contain chlorophyll

c; cryptophytes produce flagellar hairs similar to those of

heterokont algae, and haptophytes have chloroplasts and

storage products nearly identical with heterokont algae

The chlorarachniophytes and euglenoids also became

algae by two independent secondary endosymbioses

How-ever, rather than a red algal symbiont, it was a green

alga (see Keeling, 2010) These two groups are small, but

they do have some unusual biochemical and physiological

attributes that are contributed by their host cell evolutionary

ancestors

1.7.3.3 Algae with tertiary plastids

Dinoflagellates have added another level of complexity

Dinoflagellates began with a plastid derived by red algal

secondary endosymbiosis, but many dinoflagellates

aban-doned photosynthesis in favor of a phagotrophic existence

(see Section 1.7.8) In several independent cases,

nonpho-tosynthetic dinoflagellates regained photosynthesis by

ter-tiary endosymbiosis; the symbionts in these varied cases

were a diatom, a haptophyte, a cryptomonad, or a green

alga (Hackett et al., 2004)

1.7.3.4 Cryptic endosymbioses

The role of endosymbiosis in the evolution of algae is

appar-ently even more complex because genomic analyses reveal

cryptic endosymbioses For example, Moustafa et al (2009)

found that most plastid genes in diatoms had a green algal

origin This was completely unexpected and suggests that

evolutionary biologists must exercise caution when trying

to unravel the early history of eukaryotes Thus, over a

bil-lion years of evolutionary time, the occurrence of

endosym-biotic events, horizontal gene transfers, extinction of

inter-mediary forms, and perhaps additional yet undiscovered

factors have contributed to the complex evolutionary

his-tory of eukaryotic algae

1.7.4 Glaucophyte algae

Glaucophytes are sometimes considered among the most

basal of eukaryotic algae because their photosynthetic

cyanelles are like a cyanobacterial cell (e.g., chlorophyll

a, phycocyanin, phycoerythrin) This “preplastid” is

sur-rounded by a peptidoglycan cell wall and has other chemical features characteristic of prokaryotes The algaeare rarely encountered, but the common species can befound in acid bogs

bio-1.7.5 Green algae

Green algae have chlorophylls a and b, and most do not have

accessory light-harvesting pigments (for exceptions, seePrasinophyceae) The storage product is typically starch.Green algae are deeply divided into two groups, the chloro-phytes and charophytes While this division was recog-nized about 40 years ago based upon ultrastructural fea-tures (Pickett-Heaps & Marchant, 1972; Mattox & Stewart,1984; Stewart & Mattox, 1984), gene sequence data aswell as chloroplast and nuclear genome data continue tosupport this deep divergence (Timme & Delwiche, 2010).However, the relationships within each of the two lin-eages remain somewhat uncertain While the scaly flag-

ellate Mesostigma is probably the closest known living

green alga to the divergence of the two groups Ezpeleta et al., 2007), the chlorophycean lineage has severalnamed classes that are paraphyletic in many phylogeneticanalyses (e.g., Chlorophyceae, Mamiellophyceae, Prasino-phyceae, Trebouxiophyceae, Ulvophyceae) The phylogenywithin the charophyte lineage also is debated, particularlyabout which group is most closely related to land plants

(Rodr´ıguez-1.7.6 Red algae

Red algae contain chlorophyll a and phycobilisomes

(pig-ment complexes with allophycocyanin, phycocyanin, andphycoerythrin) that are located on the surface of unstackedthylakoid membranes Red algae are unique among eukary-otes in lacking both flagella and centrioles during theirentire life cycle Even 10 years ago, red algal diversity wasconsidered more or less framed, if not finalized However,studies on unicellular red algae have shown that the base

of the red algal tree is very diverse (Yoon et al., 2006).Seven classes are currently recognized Unicellular Cyani-diophyceae, which thrive in acidic hot springs, are posi-tioned at the base of the red algae; the Porphyridiophyceaeand Rhodellophyceae are also unicellular Among the 6000red algal species, 5800 belong to the Florideophyceae,which includes the large and commercially valuable sea-

weeds (e.g., Eucheuma, Gelidium, Gracilaria).

1.7.7 Heterokont algae

Heterokonts are perhaps the most diverse major group ofalgae, and they currently consist of about 16 classes They

The Microalgal Cell 15

vary from the giant kelps to picoplanktonic open ocean

species and include the ubiquitous diatoms The group

con-sists of three subgroups: Clade S1, Clade S2, and Clade S3

(Yang et al., 2012)

Clade S1 is composed of the Aurearenophyceae,

Chrysomerophyceae, Phaeophyceae,

Phaeothamnio-phyceae, Raphidophyceae, Schizocladiophyceae, and

Xanthophyceae Morphologically, they range from tiny

coccoid single cells to giant kelps The carotenoid pigments

are also diverse; some groups have the antheraxanthin–

violaxanthin light-harvesting carotenoid cycle, others

have the diatoxanthin–diadinoxanthin cycle, and the

Raphidophyceae have both types (Bjørnland &

Liaaen-Jensen, 1989; Mostaert et al., 1998) This group shares

some morphological features with the nonphotosynthetic

stramenopiles, and may be the earliest diverging group of

the three clades

Clade S2 includes the golden algae (Chrysophyceae,

Synurophyceae), the Eustigmatophyceae, and the

oil-producing algae (Pinguiophyceae) These algae are

dis-tinguished in antheraxanthin–violaxanthin light-harvesting

carotenoid cycle, and most species have two flagella with

well-developed microtubular root systems

Eustigmato-phyceae: these algae lack a chloroplast girdle lamella and

include the economically important Nannochloropsis

Pin-guiophyceae: this class uses EPA as a storage product,

and some members have odd flagellar features (Kawachi

et al., 2002) Chrysophyceae and Synurophyceae: these two

classes were originally distinguished based upon a series

of features (Andersen, 1987), but molecular studies

sug-gest they may be recombined Synchromophyceae: this odd

group of marine amoebae has only two species, but there

are some interesting links with Chlamydomyxa,

Leukarach-nion, and Chrysophyceae (Grant et al., 2009).

Clade S3 includes the diatoms (Bacillariophyceae),

the bolidomonads and Parmales (Bolidophyceae), the

sil-icoflagellates, pedinellids, and Rhizochromulina

(Dicty-ochophyceae) and the Pelagophyceae (oceanic

picoplank-ters, brown tide organisms, coastal macroalgae) The group

is distinguished morphologically by a reduced

flagel-lar apparatus (Saunders et al., 1995), biochemically by

light-harvesting carotenoids belonging to the diatoxanthin–

diadinoxanthin cycle (Bjørnland & Liaaen-Jensen, 1989),

and molecularly with multiple genes (Yang et al., 2012)

The diatoms are essentially single cells, sometimes held

together in chains, and the unifying character is their

siliceous cell walls (Round et al., 1990) Diatoms are rapidly

growing organisms that often produce large amounts of

oil, but they require silica for growth Originally, the

bolidophytes were limited to two picoflagellates, but the

silica-scaled Parmales are an alternate stage according to arecent molecular study (Ichinomiya et al., 2011) Bolido-phytes are closely related to diatoms The dictyochophytesare a small but distinct group that is largely composed

of marine organisms The pelagophytes are marine algae,

including open ocean picoplankters (e.g., Pelagococcus, Pelagomonas), coastal brown tide organisms (Aureococcus, Aureoumbra), and several benthic macrophytes forming gelatinous colonies (e.g., Chrysocystis, Chrysoreinhardia).

1.7.8 Dinoflagellates

These algae are predominately swimming organisms, andthey occur in both freshwater and seawater Typically, oneflagellum circles the cell in a cingulum while the sec-ond flagellum extends along a groove to beyond the cellposterior and pushes the cell Some dinoflagellates have

an armored cell covering made of thecal plates Manydinoflagellates lack thecal plates, and they are termed nakeddinoflagellates Both thecate and naked cells exist as photo-synthetic, heterotrophic, and mixotrophic organisms Pho-

tosynthetic cells typically use chlorophyll c2 and c3 aswell as peridinin, but dinoflagellates with tertiary endosym-

bionts (e.g., Dinophysis, Karenia, Kryptoperidinium, idodinium) have other pigments Photosynthetic dinoflag-

Lep-ellates frequently grow very slowly, often with a synthetic rate that barely outpaces respiration; however,dinoflagellates, may grow rapidly forming blooms in somecases (Smayda & Reynolds, 2001; Heil et al., 2005)

photo-1.7.9 Haptophytes

The haptophytes are almost exclusively marine and ish water organisms Most species have an unusualappendage – the haptonema – that may be used for attaching

brack-to substrates or for capturing food Isochrysis and Pavlova

have been important for aquaculture hatcheries becausethey grow rapidly, provide rapid and healthy growth forshellfish, are easy to maintain in large volume cultures,and produce significant amounts of PUFAs (Patil et al.,2007) Some haptophytes produce calcium carbonate scalestermed coccoliths, and this group is therefore called coc-colithophores Huge oceanic coccolithophore blooms occurand when cells sink to the bottom, they deposit significantamounts of calcium carbonate into ocean sediments (Fran-cois et al., 2002; Balch et al., 2010)

1.7.10 Cryptophytes

These red, brown, and green (rarely blue) flagellates arevery common in freshwater and coastal seas They haveseveral unusual features For example, the nucleomorph is

16 Robert A Andersen

a reduced nucleus that remains from a secondary

endosym-biosis, one or both flagella have bipartite tubular hairs, and

the cell is covered with a periplast consisting of special

plates (Fig 1.2) They utilize chlorophylls a and c as well

as phycobilins for harvesting light used in photosynthesis;

the photosynthetic storage product is starch Recent

phylo-genetic studies show a molecular relationship between the

cryptophytes and haptophytes; however, morphologically

and biochemically, the two groups have little in common

1.7.11 Euglenoids

These green flagellates are largely freshwater species

that occur in puddles, bogs, ponds, lakes, and rivers;

they are often abundant in waters with high ammonia

or urea content Colacium is an attached euglenoid that

lives in the cloaca of frogs and there are a few parasitic

taxa (e.g., Euglenomorpha, Kawkinea) The photosynthetic

euglenoids are evolutionarily related to trypanosomes and

other parasitic organisms They have a secondary

endosym-biotic “green” plastid, and they utilize chlorophylls a and

b; however, their storage product is a β-1,3-linked glucan,

paramylon The cell is covered with pellicular strips, and

the common Euglena and Phacus frequently change cell

shape by a process termed metaboly

1.7.12 Chlorarachniophytes

These are a small group of marine algae that occur

primar-ily in coastal waters, but the flagellate Bigelowiella is

com-mon in the Sargasso Sea and other oligotrophic open ocean

waters Chlorarachnion and Gymnochloris are amoeboid

organisms, but others such as Lotharella and Partenskyella

have dominant coccoid forms for at least some species The

“green” plastid resulted from a secondary endosymbiosis,

the photosynthetic pigments are chlorophyll a and b, and

the storage product is aβ-1,3-linked glucan (McFadden

et al., 1997)

1.7.13 Other photosynthetic alga-like organisms

In addition to the well-known algae, there are organisms

that are alga like, if not algae Paulinella chromatophora

was described long ago (Lauterborn, 1895); the amoeboid

cell is surrounded by a lorica composed of silica scales,

and the typical vegetative cell has two cyanelle-like or

plastid-like photosynthetic organelles Paulinella belongs

to the largely amoeboid group, Rhizaria Paulinella is very

exciting because it arose via primary endosymbiosis that

occurred only 60 million years ago; the other primary

endosymbiosis that led to all other plastids having occurred

about 2 billion years ago The genomes of this organism

have been sequenced and considerable research is being

carried out to understand how a recent endosymbiosis ates (Marin et al., 2005; Yoon et al., 2009; Reyes-Prieto

oper-et al., 2010, Mackiewicz oper-et al., 2011)

Hatena arenicola is exciting because it is in the very

early stage of secondary plastid formation (Okamoto

& Inouye, 2005) Hatena contains a Pyramimonas-like cell and photosynthesis occurs via the Pyramimonas-like chloroplast However, when Hatena undergoes cell divi- sion, the Pyramimonas-like plastid does not divide – the

synchrony of host and endosymbiont divisions has not been

established Therefore, one of the Hatena daughter cells

contains the plastid-like algae and the other daughter cell

must find a Pyramimonas cell and engulf it (Okamoto &

Inouye, 2006)

The picobiliphytes were primarily described from ronmental gene sequences, although some epifluorescenceimages were provided (Not et al., 2007) Yoon et al (2011)isolated single cells and sequenced the genomes for threeindividual cells Their results show that the picobiliphytesare not algae; rather, picobiliphytes are phagotrophic flag-ellates that eat cryptophyte-like prey These, therefore, arenot an algal group; they are heterotrophic flagellates thateat algae

envi-Finally, we have the description of Roombia truncata,

another colorless flagellate Okamoto et al (2009) have

shown that Roombia, cryptophytes, haptophytes,

katable-pharids, telonemids, centrohelids, and possibly the called picobiliphytes form a clade, the Hacrobia This studyhelps us to understand how the algal groups have evolved,and from a more practical viewpoint, it should lead to exper-iments where various organisms can be recombined to pro-duce new, valuable organisms for commercial purposes

Algae commonly grow in water, but certain species grow

on rocks, soils, snow, plants, and even animals (e.g., sloths);they also grow inside plants, rocks, and ice Algae arecommon symbionts living in lichens, ciliates, corals, flat-worms, and other animals (Round, 1981; Reisser, 1992).Algae contribute approximately half of the photosyntheticproductivity on earth, most of the production occurring inthe oceans (Falkowski & Raven, 2007) This seems unbe-lievable because plants are obvious and abundant on land.However, there are two important factors The oceans cover71% of the earth’s surface and 66% is open oceans whereonly phytoplankton exists Secondly, there is a fundamentaldifference between land plants and oceanic phytoplankton(Andersen, 2008) Land plant cells divide and accumulateover months, years, centuries, and even millennia in thecase of giant sequoia trees; therefore, biomass is obvious

The Microalgal Cell 17

and visible Conversely, when phytoplankton cells divide,

on average one of the daughter cells is eaten or dies; cells

do not accumulate into visible biomass Other marine life

seems more abundant (e.g., jelly fishes, crustaceans, fishes,

whales, sea birds) because the phytoplanktonic biomass is

accumulating in these marine animals

While most algae are oxygen-releasing photosynthetic

organisms (Larkum et al., 2003), there are many

exam-ples of algae without chloroplasts For example,

approxi-mately half of the dinoflagellates are heterotrophic

organ-isms unable to carry out photosynthesis Algae, such as

Ochromonas, are mixotrophic organisms; that is, they have

chloroplasts and photosynthesize, but they also engulf

bac-teria and other particles that are digested in food vacuoles

Finally, we assume that all algae are capable of taking

up occasional sugar molecules, amino acids, vitamins, and

other organic molecules; this process of moving molecules

across the plasma membrane is termed osmotrophy In some

cases, for example, Schizochytrium, osmotrophy is utilized

for mass culture

Seasonal succession is an interesting ecological process

for microalgae in nature Some algae will suddenly increase

in numbers, but soon other algae rapidly increase and

replace them The succession of algal species is repeated at

approximately the same time each year and events such as

the spring diatom bloom are predictable For most species,

it is not known where organisms reside during the slack

times; some produce cysts or other resting stages, but many

seemingly disappear, perhaps their numbers so reduced that

they became nearly impossible to find them Succession

may eventually be an important factor for large outdoor

polyculture ponds where algae are continuously grown and

harvested

ACKNOWLEDGMENT

I thank Robert E Lee and Daiske Honda for providing

TEM images, and I thank Hwan Su Yoon for reviewing the

manuscript

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Jiˇr´ı Masoj´ıdek1,2, Giuseppe Torzillo3, and Michal Kobl´ıˇzek1,2

1Department of Phototrophic Microorganisms, Institute of Microbiology, Academy of Sciences of the Czech

Republic, Opatovick´y ml´yn, Tˇreboˇn, Czech Republic

2Faculty of Science, University of South Bohemia, Braniˇsovsk´a 31, ˇCesk´e Budˇejovice, Czech Republic

3Institute of Ecosystem Study, Section of Florence, CNR, Sesto Fiorentino, Italy

Abstract

An understanding of photosynthesis is fundamental for microalgal biotechnology The process of thesis can be expressed as a light-driven redox reaction in which carbon dioxide is converted to carbohydratesand oxygen is released as a side-product This chapter describes the processes in detail from light capture tocarbon fixation The main techniques for measuring and monitoring photosynthetic processes in microalgalcultures are evaluated Finally, based on detailed knowledge, an estimate of a realistic, practically obtainable,maximum efficiency for photosynthetic solar energy conversion in microalgal cultures might reach 4–5%

photosyn-Keywords biomass productivity; Calvin cycle; carbon fixation; chlorophyll fluorescence; microalga; oxygen

evolution; photosynthesis; photosystem; photosynthetic efficiency; pigment

It’s not love or money that makes the world go round, it’s

photosynthesis.

2.1 THE PROCESS OF PHOTOSYNTHESIS

Photosynthesis is a unique process of sunlight energy

con-version in which inorganic compounds and light energy are

converted to organic matter by photoautotrophs Virtually

all forms of life on Earth depend directly or indirectly on

photosynthesis as a source of organic matter and energy for

their metabolism and growth

The earliest photoautotrophic organisms, anoxygenic

photosynthetic bacteria, evolved about 3 billion years ago

These bacteria use light energy to extract protons and

cyanobacteria and various eukaryotic algae and diatoms.

electrons from a variety of donor molecules, such as Fe2+

or H2S, and to reduce CO2to form organic molecules Inthis chapter, we focus on oxygen-producing photosyntheticmicroorganisms – prokaryotic cyanobacteria and eukary-otic algae; these microorganisms emerged later, about

2 billion years ago, and created our oxygenous atmosphere

on Earth

Cyanobacteria (blue-green algae) are frequently lular, while some species form filaments or aggregates Theinternal organisation of a cyanobacterial cell is prokary-otic, with a central region (nucleoplasm) rich in DNA and aperipheral region (chromoplast) containing photosyntheticmembranes The sheets of these photosynthetic membranesare usually arranged in parallel, close to the cell surface.Eukaryotic autotrophic microorganisms are traditionallydivided according to their light-harvesting photosynthetic

unicel-Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition Edited by Amos Richmond and Qiang Hu.

C

21

22 Jiˇr´ı Masoj´ıdek, Giuseppe Torzillo, and Michal Kobl´ıˇzek

Figure 2.1 Major products of the light and dark reactions of photosynthesis The process of oxygenic photosynthesis is divided into two stages, the so-called light reactions and dark reactions The light

reactions include light absorption, transfer of excitons, and electron and proton translocation resulting in theproduction of NADPH2, ATP, and O2 The other phase, the dark reactions that occur in the stroma, representsthe reduction of carbon dioxide and the synthesis of carbohydrates using the NADPH2and ATP produced inthe light reactions

pigments: Rhodophyta (red algae), Chrysophyceae (golden

algae), Phaeophyceae (brown algae), and Chlorophyta

(green algae) Their photosynthetic apparatus is organised

in special organelles, the chloroplasts, which contain

alter-nating layers of lipoprotein membranes (thylakoids) and

aqueous phases, the stroma (Staehelin, 1986)

Oxygenic photosynthesis can be expressed as a redox

reaction driven by light energy (harvested by chlorophyll

molecules) in which carbon dioxide and water are converted

to carbohydrates and oxygen This process is traditionally

divided into two stages, the so-called light reactions and

dark reactions (Fig 2.1) In the light reactions, which are

bound on photosynthetic membranes, the light energy is

converted to chemical energy – providing a biochemical

reductant NADPH2and a high-energy compound ATP In

the dark reactions, which take place in the stroma, NADPH2

and ATP are utilised in the sequential biochemical reduction

of carbon dioxide to carbohydrates

The classical description of photosynthetic activity is

based on measurements of oxygen evolution in proportion

to light intensity, the so-called light–response (P/I) curve

(Fig 2.2) The initial slope α = Pmax/Ik, where Ik

repre-sents the saturation irradiance and Pmax is the maximum

rate of photosynthesis In the dark, there is a net

consump-tion of oxygen as a consequence of respiraconsump-tion (the negative

part of the curve in Fig 2.2) Thus, gross photosynthesis is

considered as the sum of net photosynthesis (O2evolution)

and respiration (O2uptake) At low irradiance (light-limited

region), the rate of photosynthesis depends linearly on light

intensity With increasing light intensity, photosynthesis

becomes less and less efficient as the dark enzymatic

reac-tions utilising fixed energy become rate limiting Finally,

it reaches a plateau – the maximum (light-saturated) rate

of photosynthesis Pmax Under prolonged supra-optimal

irradiance, photosynthetic rates usually decline from the

light-saturated value This phenomenon is commonly

photosynthetic light-response curves, that is, thedependency of photosynthesis on irradiance Theinitial slope of the curve (α) is the maximum light

utilisation efficiency The intersection between themaximum rate of photosynthesisPmaxandα is the

light saturation (optimum) irradiance Atsupra-optimum irradiance, photosynthesis declines,which is commonly called down-regulation orphotoinhibition

referred to as photoinhibition of photosynthesis (see also

Chapter 6)

2.2 THE NATURE OF LIGHT

The energy for photosynthesis is delivered in the form oflight Light is an electromagnetic radiation and travels at

the speed c ∼ 3 × 108 m s−1 Based on its wavelength,electromagnetic radiation can be divided into several com-ponents (Fig 2.3) Radiation such as light is usually denoted

as having wavelengths between 10−3and 10−8m; gammaand X-rays have shorter wavelengths, while radio wavesare above 10−3m The visible part of the spectrum ranges

Photosynthesis in Microalgae 23

Colour spectrum

of white light

400 450

w Red

Figure 2.3 The spectrum of electromagnetic radiation showing, in particular, the visible light spectrum.

Photosynthetically active radiation (PAR) ranges from 400 to 700 nm

from the violet of about 380 nm to the far red at 750 nm,

this range being usually expressed in nanometres (1 nm

= 10−9 m) The wavelengths of visible light also

corre-spond to photosynthetically active radiation (PAR), that is,

radiation utilisable in photosynthesis

According to quantum theory, light energy is delivered in

the form of separated packages called photons (or quanta).

The energy of a single light quantum, or photon, is the

prod-uct of its frequency and Planck’s constant, that is, E = hν

(h= 6.626 × 10−34J s) Since energy is inversely related to

wavelength, a photon of blue light (about 400 nm) is more

energetic than that of red light (around 700 nm)

Photosyn-thetic pigments absorb the energy of photons and transfer

it to the reaction centre where it is utilised for

photochem-istry The photon should possess a critical energy sufficient

to excite a single electron from one pigment molecule and

initiate charge separation According to Einstein’s law, one

mole of a compound must absorb the energy of N photons

(N= 6.023 × 1023, the Avogadro number) to start a

reac-tion, that is, Nhν This unit is called an Einstein (E = 6.023

× 1023quanta)

In every day application, luminous flux is measured in

lumens (lm), which is defined as the light flux of one

can-dela into one steradian The intensity of illumination is then

expressed in lux (lm m−2), or historically in foot candles

(1 lm ft−2, i.e., 1 ft candle equals 10.76 lux) The definition

of lux is subjective, dependent on human vision, and

can-not be easily converted into other units; however, in some

technical areas it is recommended by EC legislation to

express the light provided by artificial light sources

Photobiologists prefer to measure light energy incident

on a surface, that is, radiant flux energy or irradiance,

in units of power per area (W m−2 or J m−2s−1) Sincephotochemical reactions in photosynthesis depend on thenumber of photons incident on a surface, irradiance might

be expressed as the number of quanta (photons) reachingunit surface area in unit time, that is, as photosyntheticphoton flux density measured inμmol quanta m−2s−1or

Thus, the approximate conversion factor for sunlight is 1

W m−2equivalent to about 4.6μmol photons m−2s−1

Dif-ferent types of instruments are used to measure irradiance;most of them measure PAR inμmol photons m−2s−1or in

W m−2

2.3 PHOTOSYNTHETIC PIGMENTS

All photosynthetic organisms contain organic pigments forharvesting light energy There are three major classes ofpigments: chlorophylls, carotenoids, and phycobilins.Chlorophyll (Chl) molecules consist of a tetrapyrrole ring

(polar head, chromophore) containing a central magnesium

atom, and a long-chain terpenoid alcohol (except for Chl

c) (Fig 2.4a) These molecules are noncovalently bound

to apoproteins Structurally, the various types of

chloro-phyll molecules designated a, b, c, and d differ in their

24 Jiˇr´ı Masoj´ıdek, Giuseppe Torzillo, and Michal Kobl´ıˇzek

III IV

V R

Figure 2.4 The structures of the three principal groups of pigments in algae and cyanobacteria – chlorophylls

(a), phycocyanobilin (b), and carotenoids [β-carotene (c) and violaxanthin (d)] All chlorophylls are

tetrapyrroles, where nitrogen atoms are coordinated around a Mg atom Chla and b differ in the R groupwhile Chlc does not contain a side chain of phytol Phycobiliproteins are open tetrapyrroles, which arecovalently linked to a protein Carotenoids are conjugated isoprenes with cyclic 6-carbon side groups,whereas xanthophylls such as violaxanthin, compared to carotenes, are oxygenated