Table Of ContentALGAE AND THEIR BIOTECHNOLOGICAL POTENTIAL
ALGAE AND THEIR
BIOTECHNOLOGICAL POTENTIAL
Proceedings of the 4th Asia-Pacific
Conference on Algal Biotechnology,
3-6 July 2000 in Hong Kong
Editedby
Feng Chen
DepartmentofBotany.
TheUniversity ofHong Kong
and
YueJiang
DepartmentofBotany,
The University ofHong Kong
....
"
Springer-Science+Business Media, B.Y.
AC.I.P.Cataloguerecordforthisbook isavailablefrom theLibrary ofCongress.
ISBN978-90-481-5886-7 ISBN978-94-015-9835-4(eBook)
DOI10.1007/978-94-015-9835-4
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©2001SpringerScience+BusinessMediaDordrecht
OriginallypublishedbyKluwerAcademicPublishersin2001.
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Contents
Preface ix
1 Polyunsaturated FattyAcids: Biological Significance, Biosynthesis,and 1
Production by Microalgaeand Microalgae-likeOrganisms
c:s.
Yap andF.Chen
2 Application ofStatistically-Based ExperimentalDesigns for Optimizing 33
EicosapentaenoicAcid Production byNitzschia /aevis
Z.Y. Wenand F.Chen
3 Optimization ofNitrogen Sources for theProduction ofEicosapentaenoic 55
Acid by the Diatom Nitzschia /aevisinHeterotrophicCultures
Z.Y. WenandF.Chen
4 Effects of Nitrogen Sourceand Vitamin B on DocosahexaenoicAcid 69
12
Production byCrypthecodiniumcohnii
Y.Jiang,F.Chen andH.S. Li
5 Neural Networks for Modellingand Predictingthe Ch/orellaprotothecoides 79
Cultivation Processes
G.Y.Zhang,S.Y.Guo, L. Li,W.S. Zhou andM.Y.Cai
6 Modellingofa Continuous Algal Production System Using Intelligent 93
Methods
N.Clarkson,K.O.Jones andAJ.Young
7 High Yield Production ofLutein by Heterotrophic Chlorellaprotothecoides 107
in Fed-Batch Systems
X.M.Shiand F.Chen
8 Induction ofAstaxanthin Formation inthe Green Microalga Chlorococcum 121
sp. by ReactiveOxygen Species (ROS) under MixotrophicConditionsof
Growth
R.Y.N.Maand F.Chen
v
vi
9 PreparativeIsolation and Purification ofAstaxanthin from the Green 127
Microalga Chlorococcumsp.byHigh-SpeedCounter-Current
Chromatography
RB.Li andF.Chen
10 Changes inContent,Constituentsand DistributionofConstitutiveand 135
ExcretedSugarsofSpirulina(Arthrospira)maxima inNutrient-Limited
Batch Cultures
J.L.Xia,Z.Y.NieandJ.M.Levert
11 Growth,NutrientAssimilationand CadmiumRemoval bySuspended and 147
Immobilized ScenedesmusacutusCultures: Influence ofImmobilization
Matrix
R.O.Cafiizares-Villanueva,S.Gonzalez-MorenoandA.R.Dominguez
Bocanegra
12 Metal Sorption byMicroalgae for Employment inBiotreatmentof 163
EnvironmentalHeavyMetal Contamination
P.Mathad,S.B.AngadiandR.D.Mathad
13 ToxicEffectofTributyltin (TBT)onDifferent Green Microalgal Species 181
N.F.Y.Tam,Y.S.WongandA.M.Y.Chong
14 CatalyticDegradation ofthe Herbicide Glyphosate bythe Paddy Field 195
Isolates ofCyanobacteria
T.BalakumarandV.Ravi
15 Effect ofPost-CollectionStorageTimeand Seasononthe Antibacterial 207
ActivityofSelectedSouthern African MarineMacroalgae
V.Vlachos,A.T.CritchleyandA.VonHoly
16 Hormesis inBioassayofMacroalgalFungal Propagules 215
M.Barreto,C.J.StrakerandA.T.Critchley
17 BiologicalActivitiesofExtracts from SeveralSpeciesofRohdomelaceae 227
from Fujian Coasts ofChina
Y.Zheng
18 Studies ofthe Pharmacologyand ToxicologyofSpirullnamaxima 233
(SMNJU.02)
Z.L.LiuandD.H.Cao
vii
19 Characterization ofthe icfGGene ClusterImplicated inthe Regulation of 251
Carbon Metabolism inthe CyanobacteriumSynechocystissp. PCC6803
L.Gonzalez,O.Basso,S.BeduandC.C.Zhang
20 PreliminaryStudieson the Genetic Transformation ofSpirulinaplatensis 263
X.c. Zhang,Y.X.Mao,G.G.Wang,RH.Zhang,G.P.YangandZ.H.Sui
21 Effect of Temperature on the Desaturase Gene Translation in Spirulina 271
platensisStrain Cl.
A.Hongsthong,P.Deshnium,K.Paithoonrangsarid,P.Phapugrangkul,
M.Tanticharoen andS.Cheevadhanarak
22 Application ofaTelemetrySystem to StudyingMicroalgalDynamics and 279
Red Tides inHong Kong
I.H.Y.Lamand1.1.Hodgkiss
23 The PitfallsofUsingDifferentClassificationSystems to Quantify 293
BiodiversityofCyanobacteria: ACase Studyfrom Hong Kong Rocky
Shores
S.Nagarkar
Index 303
Preface
The term 'algae' is a very difficult one to define. It may appear in textbooks ofBotany,
Zoology and Microbiology. Ingeneral, algae are organisms that include seaweeds and a
numberofsingle-celledand multicellular microscopicforms, Algae are ubiquitous;they
inhabitoceans, freshwater bodies, rocks,soils and trees. There may be over 50,000 algal
speciesonthe earth.
Man's uses ofalgae have a long history. In China, marine algae were used as
food asfarback as600-800BC. Inrecent decades, there has been renewedinterest inthe
utilization of algae as sources of health food and high-value chemicals and
pharmaceuticals,and foraquaculture, agricultureand wastewatertreatment. Even so, the
biotechnological potential of algae is still far from fully exploited. With the aim of
promoting algal biotechnology particularly in the Asia-Pacific region, the Fourth Asia
Pacific Conference on Algal Biotechnology was planned and successfully held in Hong
Kong during 3-6 July 2000. The Conference attracted more than 250 participants from
30 countries and regions. Some 230 papers were presented at the Conference. Among
them,40 papers were selected after peer review. Apart ofthe selected papers have been
published in Journal of Applied Phycology (Vol. 13 No.4). This book consists of
another part of the selected papers, which deal with the various aspects of algal
biotechnologywith emphasis onthebiotechnologicalpotential ofalgae.
This book cannot bemade possible withoutthe help and effort ofmany. First of
allwe are indebtedtothe authors ofthe various chapters fortheir excellentcontributions.
Second, we would like to thank the reviewers whose critical comments and constructive
suggestions have helped to improve the quality ofthis book greatly. Third, we would
liketo thank the generous support ofthe Innovation and Technology Commission ofthe
Governmentofthe Hong Kong Special Administrative Region. Finally, wewould liketo
acknowledge the assistance ofMartine van Bezooijen and the other staffat Kluwer in
producingthe book.
Feng (Steven)Chen
Vue Jiang
Hong Kong,September2001
ix
POLYUNSATURATED FATTY ACIDS: BIOLOGICALSIGNIFICANCE,
BIOSYNTHESIS, AND PRODUCTION BYMICROALGAE AND
MICROALGAE-LIKEORGANISMS
C.Y. YAPandF.CHEN
Department ofBotany, The University of Hong Kong. Pokfulam Road,
Hong Kong, P R.China
1.Abstract
Growing interest in the nutritional and pharmaceutical importance of polyunsaturated
fatty acids (PUFAs) has created an increasing demand for purified PUFAs. As the
traditional sources are insufficient for satisfying this demand, alternative sources are
being sought. Microalgaeare agreat sourceof manyhighlyvaluableproductsand they
are considered a potential alternative for the large-scale production of PUFAs.
Investigations havebeenactivelycarriedoutforscreeningofpotentialmicroalgalstrains
and developmentof feasibleculturetechniquesfor the commercialproductionof these
vitalcompounds.
2.Introduction
Polyunsaturated fatty acids (PUFAs) (Fig. I), especiallythose long-chain fatty acids in
the n-3 (originally named 00-3) family, have entered the biomedical and nutraceutical
arenasastheyperformmanyvitalfunctions inbiologicalmembranesandasprecursorsof
avarietyof lipidregulatorsofcellularmetabolism(Berdanier,2000;Hwang,2000).The
beneficialeffectofn-3PUFAswasfirstnoticedbyDyerbergandBangintheearly 1970s.
They reported that the Inuit (Greenland Eskimos)population had a lower incidence of
heart disease thanthat of an equivalentpopulationof Danes.They also foundthatthese
Greenland Eskimos had a favourable plasma lipid profile: with low levels of
triglycerides, plasma cholesterol and very low-density lipoproteins (VLDL) and high
levels of high-density lipoproteins (HDL) (Dyerberg et aI., 1975). As the Eskimos
consumealargeamountofmarinemammalsandarcticfishintheirdietthatarerichinn
3 fatty acids, n-3 PUFAs are presumedto be a major factor responsiblefor the healthy
effectof fishoil.ThereportbyDyerberget al. (1975) hadspurredmanyinvestigatorsto
performepidemiologicalstudiesinothercountriesto investigatethe beneficialeffectsof
n-3 PUFAs on humans. It has become clear that dysfunctions of PUFA-derived
eicosanoids may lead to illnesses and disorders including cardiovascular, respiratory,
gastrointestinal, renal, dermal and immune diseases, cerebral and ocular
underdevelopment, as well as carcinogenesis(Horrocks and Yeo, 1999). Other studies
relatingtoPUFAssuchasthebiosynthesismechanismsofPUFAs,exploitationofsources
of PUFAs, and productionof PUFAs havealsobeencarriedoutextensively.This review
F. ChenandY.Jiang(eds.),AlgaeandtheirBiotechnologicalPotential, 1-32.
©2(0) KluwerAcademicPublishers.
2 C.Y.YAPAND F. CHEN
first introduces the biological significance and beneficial effects ofpolyunsaturated fatty
acids, then briefly describes the biosynthesis ofPUFAs and finally discusses the various
sources of PUFAs and the techniques involved in the microbial production ofPUFAs,
with emphasison the biotechnologicalpotentialofmicroalgae.
COOH
all-cis-5,8,II,14-arachidonicacid(AA,20:4n-6)
COOH
all-cis-4,7,10,13,16,I9-docosahexaenoicacid(DHA,22:6n-3)
Figure I. Chemicalstructure oftwo biologically important PUFAs:AA andDHA. Trivial nomenclatureof
PUFA:thelirstnumberdenotesthenumberofcarbonatoms. Thenumberafterthecolondenotesthenumberof
doublebonds,andthenumberaftern-denotesthepositionofthelastdoublebondfromthemethylendoffatty
acids.
3. Significanceofpolyunsaturated fatty acids
3.1. BIOLOGICALFUNCTIONS
Polyunsaturatedfatty acids (PUFAs)are classifiedmainly into four families designated n
3, n-6, n-7 and n-9. The n-3 and n-6 families offatty acids predominate in plants and
animals. The parent fatty acid ofthe n-3 family isa-linolenic acid and ofthe n-6 family,
linoleic acid. These precursor acids are elaborated into longer chains and more highly
unsaturated fatty acids, for examples, arachidonic acid (20:4n-6) and docosahexaenoic
acid (22:6n-3) byaseries ofdesaturationsand elongations.
PUFAs are structural components ofcell and organelle membranes (mainly as sn-2
phospholipids). They are crucial for regulating the membrane structure, fluidity, phase
transitions and permeability as well as for the control ofmembrane-associated processes
(Berdanier, 2000; Gill and Valivety, 1997). PUFAs also act as the precursors of many
metabolites that regulate vital biological functions. In plants, PUFAs are converted by a
variety ofenzymes to various oxygenated compounds, acting as anti-infectives, wound
response mediators, chemotactic agents, aroma and flavour compounds (Gill and
Valivety, 1997). In lower animals, such as insects and marine invertebrates, PUFA
derived metabolites mediate cellular processes and ecological responses including
metamorphosis, reproduction,chemotaxis and immune function (Gill and Valivety, 1997).
PUFAPRODUCTION BY MICROALGAE 3
In higher animals, long-chain PUFAs are precursors of a diverse series of oxygenated
fatty acids termed 'eicosanoids' that are crucial to the development and the proper
maintenance of homeostasis (Fig. 2) (Hwang, 2000). PUFA-derived eicosanoids in
humans including prostaglandins, prostacyclins, thromboxanes and leukotrienes are
produced through two main pathways: the cyclo-oxygenase and lipoxygenase pathways,
each iscatalyzed by a distinct group ofenzymes (Fig.3).These compounds have a short
lifespan.They exert potent biological activities even at very low concentrations and they
are linked to many physiological and pathophysiological syndromes (Gurr, 1999; Gurr
and Harwood, 1991).
3.2.ESSENTIAL FATTYACIDS:LINOLEIC ACID AND a-LINOLENIC ACID
Higher animals includinghumans are unable toproduce fatty acids over CI8 as they only
possess M, .15,.16and .19desaturases (lacking .112and .115);they cannot form linoleic
acid (LA, l8:2n-6) and a-linolenic (ALA, 18:3n-3) from oleic acid de novo (Fig. 4).
However,they can further elaborate LAand ALA to longer PUFAs(Gurr, 1999). LA and
ALA are hence considered to be essential fatty acids (EFA) and must be obtained from
the diet, asthey are the parent acids ofthe n-3and n-6 families ofPUFAs.
Linoleic acid is abundant in several seed oils and its major sources come from the
seeds of sunflower, com and soybean (White, 2000). Diets deficient in LA or having
unusual ratios ofLA to ALA induce changes in the PUFA composition ofneuronal and
glial membranes. Such changes have been linked to alterations in retina and brain
functions (Fernstrom, 1999).Linoleic acid deficiency may also lead to skin lesions (Gurr,
1999).
A group of isomers of linoleic acid collectively termed conjugated linoleic acid
(CLA) has received considerable attention in recent years.They are oxidatively unstable
compounds that may form in small amounts during partial hydrogenation, during
oxidation, and during normal or abusive heating. Food lipids originating from ruminant
animals (beef, dairy and lamb) also contain CLA. CLA appears to exhibit
anticarcinogenic and antiatherogenic properties (Ha et aI., 1987, 1989; Hunter, 2000).
Effort to confirm and extend the potential benefits ofCLA is expected to be an area of
continuingresearch interest.
The main dietary sources ofa-linolenic acid are canola oil, rapeseed and soybean
oil (White, 2000). A study ofa rural population in France and the UK confirmed that
ALA could lower the clotting activity of platelets and the response of platelets to
aggregation by thrombin (Renaud, 1995; Renaud et al., 1986). There are researches
indicatingthat ALA rich diets may improve some aspects ofcardiovascularfunctions and
protect against heart attacks but further confirmation is needed (Gurr, 1999).
Nevertheless, ALA is essential as it is the parent acid ofthe physiologically important
long-chainPUFAsofthe n-3 family.ALA isconvertedto longer-chainn-3 PUFAs by the
same desaturases used for the n-6 or n-9 families, but the extent to which ALA is
convertedto long-chain PUFAsinhumans isnotknown (Hwang,2000).
