Physiology and Cultivation of
Algae and Cyanobacteria
1.
Definition
• Algae formal tax. standing, polyphyletic origin, artificial assemblage of O2
evolving photosynthetic organisms
• Algae vs. Plants
the same storage compounds, similar defence strategies against predators & parasites
Plants
•hi degree of differentiation
•repr. organs surrounded by jacket of
sterile cells
•multicell. embryo remains
developmentaly & nutrit. independent
on parents
•meristems on root/shoot apices
•digenetic life cycles with alterations
betw. hapl. gametophyte & dipl.
sporophyte
Algae
•don’t have roots, stems, leaves, not well defined
vasc. tissue
•don’t form embryo
•mono- & digenetic life cycles
•occur in dissimilar forms (micro algae, macro a.
multicellular, colonies, branched,…)
•less complexity of the thalli
•hi diversity 0.2 – 60m
ecology & habitats
reserve & structural
polysacharides
evolutionary origin
•1 – 10 mil. species
•½ primary production in biosphaera
Klasification
• under constant revision
(Van Den Hoek et al. 1995)
Occurrence & distribution
Aquatic
• almost everywhere (from freshwater spring to salt lakes)
• tolerance of wide range of pH, temp., turbidity, O2 & CO2 conc.
• planctonic
» unicellular, suspended throughout lighted regions of all water (inc. polar ice)
• benthic
» within sediments
» limited to shallow areas (because of rapid attenuation of light with depth)
» attached to stones – epilithic, on mud/sand – epipelic
» on oter algae/plants – epiphytic, on animals – epizoic
• marine benthic – after habitat
– supralitoral – above high-tide level within reach wave spray
– intertidal – exposed to tidal cycles
– sublitoral – from extreme low-water to cca 200m deep
• ocean – 71% of earth surface, more than 5000 spec. of planctonic algae
– phytoplancton
» base of marine food chain
» produce 50% of O2 we inhale - life
» death – blooms – too large populations (decrease water transparency, prod. toxins & poisons)
– kelps
» giant algae – temperate pelagic marine environment, till 60m submerged forests
» also beneath polar ice sheet
» can survive at very low depth
– record of 268m u.s.l. – dark blue red algae (blue-green ligh, 0.0005% of surface intensity)
» have accesory pigments, chanel the energy to chl a
• accesory & protective pigments – give algae wide variety of colors <> names
Occurrence & distribution
Freshwater fytoplancton & benthic algae
» base of aquatic food chain
» not exhibit size range of marine relatives
Subaerial
» life on land
» tree trunks, animal fur, snow, hot springs, desert rocks
» activity – convert rock > soil
− to minimize soil erosion & increase
water retention & nutrient availability for plants
Symbiosis
• lichens, corals
» to survive in environments that they could not alone
Structure o thallus
Unicells & unicell colonial algae
• solitary cells, unicells with/w-out flagela, motile
(Ochromonas)/non-motile (Nannochloris)
• colony
– aggregates of several single cells held together ±organized
– grow – cell division
– each cell can survive solely
• coenobium
– colony with number of cells & arrangement determined at the
time of origin (e.g. Volvox – motile, Pediastrum – non-motile)
Structure o thallus
Filamentous algae
– result from cell division in plane perpendicular to axis of
filament – cell chain
─simple
└branched – true/false
─uniseriate – 1 layer of cells
└multiseriate – up to multiple layer
Syphonous algae
– siphonous/coenocytic construction of tubular filaments
lacking transverse cell walls
– unicellular but multinucleate (coenocytic)
Structure o thallus
Parenchymatous & pseudoparenchymatous algae
– mostly macroscopic
• parenchymatous
» originated from division of primary filament (all
directions)
» lost filamentous structure
• pseudoparenchymatous
» originated from close aggregation of branched
filaments, forming thallus held together with
mucilages (red algae)
Nutrition
• algae = phototrophs
• most algal divisions – contain colorless heterotrophic spec.
– osmotrophy, phagotrophy
– auxotrophy – cannot synthesize essential components (vitamin B12, fatty acids,..) and have to
import them
• algae can use wide spectrum of nutritional strategies
combining:
– phototrophy
– heterotrophy
– mixotrophy (relative contribution of photo.&hetero. can vary)
» often in extreme environment (limiting light,…)
– after nutritional strategies:
– obligate heterotrophic algae – primarily heterotrophs, but capable phototrophy in limiting
prey concentration (Gymnodium gracilentum - Dinophyta)
– obligate phototrophic algae – primarily phototrophs, but capable phagotrophy/osmotrophy
when light is limiting (Dinobryon divergens - Heterocontophyta)
– facultative mixotrophic algae – can equally well grow as photo-/heterotrophs (Fragilidinium
subglobosum - Dinophyta)
– obligate mixotrophic algae – primary mode is phototrophy & phago-&/osmotrophy provides
essential substances (e.g.photoauxotrphs, Euglena gracilis - Euglenophyta)
Reproduction
• vegetative by division of single cell or
fragmentation of colony
• asexual by production of motile spores
• sexual by union of gametes
– vegetative & asexual
» allow stability of addapted genotypes from generation to the
next
» fast & economical increase of number of individual
» lack genetic variability
– sexual
» involves - plasmogamy (union of cells)
- karyogamy (union of nuclei) - chromosome/gene
association & meiosis >> genetic recombination
» allow variation, but is more costly
Vegetative & Asexual reproduction
• Binary fission & Cellular bisection
– simplest form
– parent org. divides into two equal parts of the same hereditary info as
parent
– unicellular a. - longitudinal
- transverse
– growth of population - lag > exponential > log > stationary (plateau) phase
– in multicellular a. & colonies - leads to the growth of individual
• Zoospore, Aplanospore & Autospore
– zoospores - flagelate motile spores that may be produced within parental
vegetative cell (Clamydomonas - Chlorophyta)
– aplanospores - aflagelate spores that begin their development within
parent cell wall before being released
- can dvelop into zoospores
– autospores - aflagelate daughter cells released from ruptured cell wall of
parental cell, - replicas of vegetative cells that produce them & lack the
capacity to develop into zoospore (Nannochloropsis - Heterocontophyta,
Chlorella - Chlorophyta)
spores - may be produced within - ordinary cells
- specialized sporangia
Vegetative & Asexual reproduction
• Autocolony formation
– coenobium/colony - each cell can produce new colony similar to
parent.
– cell division produce multicellular group (not the unicellular
individuals) > differs from the parent in cell size not in number
e.g. Volvox (Chlorophyta)
– gonidia - series of cells which produce
a hollow sphaere within the hollow
of parental colony (released after its rupture)
• Fragmentation
– ± random process whereby non-coenobic colonies/filaments break
into two/several fragments having capacity to develop into new
individual
Vegetative & Asexual reproduction
• Resting stages
– under unfavourable conditions (desiccation)
– thick-walled cells
• hypnospores & hypnozygotes
– thick-walled, produced ex novo from cells previosly separated from parent cells
» hypnospores - Ulothrix spp., Chlorococcum (Chlorophyceae)
» hypnozygotes - Spyrogyra spp. (Chlorophyceae), Dinophyta
– enables algae to survive temporary drying out of small water bodies & allow
transport to another (e.g. via birds)
• statospores
– endogenous cysts formed within vegetative cells by members of Chrysophyceae
e.g. Ochromonas spp.
» cyst walls consist of silica >> preserved as fossils
– spherical, ellipsoidal, often ornamented with spines or other projections
– wall - with pores sealed by unsilicified bung
– within cysts lie nucleus, chloroplasts, reserve material
– after dormancy - germination - form one/several flagelate cells
• akinetes occurrence in blue-green algae
– enlarged vegetative cells that develop thickened wall in response to limiting env.
nutrients or light (e.g. Anabaena cylindrica - Cyanophyta)
– extremely resistant to drying & freezing
– long-term anaerobic storage of genetic material, remain viable in sediments for
many years in hard conditions
– in suitable conditions > germination into new vegetative cells
Sexual reproduction
Gametes
– morphologicaly identical/different with/from vegetative cells (a. group speciphic
sign)
– haploid DNA content
– possible different gamete types
• isogamy - both gametes types motile & indistinguishable
• heterogamy - gametes differ in size
» anisogamy - both gametes are motile, 1. small - sperm
2. large - egg
» oogamy - 1. motile, small - sperm
2. non-motile, very large - egg
Algae exhibit 3 different life cycles with variation within differrent groups
• main difference - where meiosis occure & type of cells it produces & whether
there is more than one free-living stages
Sexual reproduction
Haplontic or zygotic life cycle
– single predominant haploide vegetative phase, with meiosis after
germination of zygote
» Chlamydomonas (Chlorophyta)
Diplontic or gametic life cycle
– single predominant diploid vegetative phase
– meiosis gives rise to haploid gametes
» Fucus (Heterocontophyta), Diatoms
Haplontic or zygotic life cycle
Diplontic or gametic life cycle
Sexual reproduction
Diplohaplontic or sporic life cycle
– present alternations of generation between two different phases
consisting of haploide gametophyte & diploid sporophyte
• gametophyte - produce gamete by mitosis
• sporophyte - produce spore by meiosis
– alternation of generations can be
• isomorphic - both phases morphologicaly identical
» Ulva (Chlorophyta)
• heteromorphic - with predominance of
– sporophyte - Laminaria (Heterocontophyta)
– gametophyte - Porhyra (Rodophyta)
Diplohaplontic or sporic life cycle
-isomorphic alternation of generations
Diplohaplontic or sporic life cycle
-heteromorphic alternation of generations
Physiology and Cultivation of
Algae and Cyanobacteria
2.
Summaries of the ten algal divisions
• Historically
– classified on basis of: pigmentation, chem. structure of
storage products, thylakoid membrane organization,
chemistry, structure of cell wall, number, arrangement,
and ultrastructure of flagella, occurrence of special
features, sex.cycles
• Recently
– comparison of sequence of 5S; 18S; 28S rRNA, internal
genetic coherence
Cyanophyta
– together with prochlorophyta – non-motile G- eubacteria
• 1-cell. to filament (un-/branched), & colonial aggr.
• widely distrib.; planctonic, blooms, picoplancton, benthic, soil, mud,
hot springs; symbiotic
• Pigm.: chl a, blue & red phycobilins, carotenes
• phycobilisomes in rows on outher surface of thylakoids
• Thylacoids – free in cytoplasm, non-stacked, singled&equidistant
• Res.polysach.: cyanophycean starch (granules betw. thylakoids),
cyanophycin
• some marine contain gas vesicles
• some filamentous form heterocysts & akinetes
• some produce hepato-, neurotoxins
Prochlorophyta
• 1-cell. / filamentous (un-/branched)
• free-living component of pelagic nanoplancton, obligate symbionts within
didemnid ascidians & holoturians
• mainly limited to tropical&subtropical env.
• Pigm.: chl a, chl b, β-carotene, xanthophyls lack phycobilins,
• Thylakoids – free in cytoplasm, stacked
• Res.polysach.: cyanophycean starch (starch-like)
Cyan.&Prochlor.
• able to fix nitrogen
• contain polyhedral bodies (carboxysomes) with RuBisCO
• in cell wall peptidoglycane layer
• obligate photoautotrophs
• asexual reproduction (cell division / fragmentation of colonies)
Glaucophyta
• 1-cell., flagellate, dorsiventral construction, 2 unequal flagella inserted
in shallow depression below apex
• marine, rare freshw., solme soil
• Pigm.: chl a, acces.pigm.: phycoerythrocyanin, phycocyanin,
allophycocyanin in phycobilisomes, carotenoids
• Chlpl. lie ni spec. vacuole, present thin peptidoglycan wall betw. 2
outher plastid membranes
• Thylacoids non-stacked
• ctDNA in center of chlpl. near carboxysomes (RuBisCO)
• Res.polysach.: starch (granules in cytoplasm outside of chlpl.)
• photoautotrophs with blue-green plastids – cyanelles
– presumed to be phylogenetically derived from endosymbiotic cyanobacterium
• unknown sex. reproduction
Rhodophyta
• red algae, mostly seaweeds, can free-living 1-cell.
• mostly marine
• lack flagellate stages
• Pigm.: chl a, phycobiliproteins in phycobilisomes
• Chlpl. 2-membrane enclosure
• thylakoids – non-stacked, single&equidistant within chlpl.
• ctDNA scattered throughout chlpl.
• Res.polysach.: floridean starch & α-1.4-glucan polysach. – grains in
cytoplasm
• mostly photoautotrophs
• Repr.
– in majority – cytokinesis incomplete >> pit connection >> proteinacous
plug >> plug
– sexual.: isomorphic / heteromorphic diplohaplontic life cycle
Heterokontophyta
• when 2 flagella they are different
• flagelate cells – heterokont – long mastigonemate flagellum directed forward during
swimming & short smooth fl., points backwards along the cell
• 1-cell-to-colonial, filamentous, siphonous, multicellular complex kelp; various type of
flagellum
• mostly marine (can freshw,&terestrial)
• Pigm.: prepoderance of carotenoids over chlorophylls
– chl a, c1, c2, c3, access.: b-carotene, fucoxanthin, vaucheriaxanthin
• thylakoids stacked in three – lamellae
• one lamellae usually runs along whole chlpl. = girdle lamellae
• chlpl. 2-membrane & fold of ER
• ctDNA – ring-shaped
• Res.polysach.: chrysolaminarin in cytoplasm in spec. vacuole
• eyespot – layer of globules, enclosed within chlpl. together with photoreceptor loc. in
smooth flagellum, forms photoreceptive apparatus
• photoautotrophy can be combine with heterotrophy
• sex. reproduc.: life cycle – haplontic (Chrysophyceae), diplontic (Bacillariophyceae),
diplohaplontic (Phaeophyceae)
Haptophyta
• mostly 1-cell., motile, palmelloid / coccoid (rare colonial/filament.)
• generaly marine
• flagellate cells – 2 naked flagella inserted laterally or apically (may have diff.
lenght)
• haptonema – typically long organelle flagellar structure (diff ultrastruct.)
• Pigm.: chl a, c1, c2, access.: fucoxanthin, β-carotene, xanthins
• Chlpl. enclosed within fold of ER
• Thylakoids – stacked in three, no girdle lamellae
• ctDNA – nucleoid scattered in chlpl.
• can be eyespot – row of globules inside chlpl., no associated flagellar struct.
• Res.polysach.: chrysolaminarin
• Cell surface – tiny celulosic scales or calcified scales bearing spoke-like fibrils
• Phototrophs / heterotrophs, phagotrophy in forms that lack cell covering
• Sex. reprod.: heteromorphic diplohaplontic life cycle
Cryptophyta
• 1-cell. flagellate
• assymetric cells, dorsiventrally constructed
• 2 unequal hary flagella, subapically inserted, emerging from above gullet loc.
on ventral side of cell – lined by ejectosomes
• free-swimming in freshw. & marine
• Pigm.: chl a, c2, phycobilins in thylakoid lumen > in phycobilisomes
• Chlpl. – 1 - 2 per cell, surrounded by fold of ER - in these intermembrane
space – peculiar organemme – nucleomorph - ? nucleus of red algal symbiont
• Thylakoids in pairs, no girdle lamellae
• Pyrenoid projects out from the inner side of chlpl.
• ctDNA condensed in small nucleoid inside chlpl.
• Res.polysach.: starch ganules in periplastidial space
• eyespot sometimes inside plastid, no association with flagella
• Cell enclosed in stiff, proteinaceous periplast – polygonal plates
• mostly photosynthetic nutrition, can be heterotrophs
• Repr.: primary longitudinal cell division, can be sexual
Dinophyta
• 1-cell., flagelates (can be ameboid, coccoid, palmelloid or filament.)
• 2 flagella with independent beating pattern (1st - training, 2nd - girdling) >> rotatory whirling motion
• characteristic cell covering components beneath cell membrane (layer of flat, polygon. vesicles –
empty or celulose filled)
• dinokont type – theca – bi-partite armor (upper, anterior [epiconus]; lower, posterior [hypoconus])
separated by groove (cingulum) loc. transversal flagellum; smaller groove (sulcus) – extended
posteriorly – host longitudinal flagellum
• important freshw. & marine microplancton
• consumed by filterfeeders; parasites; endosymbionts of tropical corals
• Pigm.: chl a, b, c1, c2, fucoxanthin, carotenoids, xanthophylls
• Chlpl. (if present) surrounded by 3 membranes
• Thylakoids stacked in 3; ctDNA in small nodules scattered in whole chlpl.
• complex photoreceptive system “compound eye” (Warnowiaceae) – lens & retinoid
• Dinocaryon – eukaryotic nucleus, chromosomes condensed during mitosis, karyotheca unbroken –
endomitosis
• Res.polysach.: starch - grains in cytoplasm; oil droplets in some genera
• At cell suraface – trichocysts – discharge explosively when stimulated
• Photoautotrophy & hi. diversity of nutrit. types
• can form blooms, possib. bioluminiscence
• Sex. reproduct. – haplontic life cycle; can form hypnospores
Euglenophyta
• mostly unicell.flagellates, can be colonial
• widely distributed, freshwater, brakish & marine, abundant in hi. heterotrophic
env.
• flagella arise from bottom of cavity – reservoir (loc. in anterior end of cell);
can live in mud; presence of pellicle – proteinaceous wall inside cytoplasm –
spiral construction
• Pigm.: chl a & b, β & γ-carotenes, xanthins, some spec. can absent plastids
• Chlpl.- 3 membrane envelope
• Thylakoids – group of three without girdle lamella
• photoreceptive system – orange eyespot loc. free in cytoplasm; true
photoreceptor loc. at base of flagellum
• Res.polysach.: paramylon (β-1,3-glucan); granules inside cytoplasm(not in
chlpl.)
• obligate mixotrophs, require vitamins of B group; colorless can be
phagotrophs, osmotrophs
• Reproduction – only asexual
Chlorarachniophyta
• naked, unicell., form net-like plasmodium via filopodia
• life cycle forms – amoeboid, coccoid, flagellate stages
• ovoid zoospores bear single flagellum
• marine
• Pgm.: chl a & b
• Chlpl. – 4-membrane envelope, each has prominent projecting
pyrenoid
• Thylakoids – stacked in one to three
• nucleomorph present btw. 2nd and 3th membrane originated from green
algal endosymbiont
• Res.polysach.: paramylon (β-1,3-glucan)
• phototrophs & phagotrophs engulfs bacteria, flagelates & eukaryotic
algae
• Repr. – mostly asexual – mitosis or zoospore formation
– heterogamy rarely
Chlorophyta
• great range of somatic differentiation (flagellates to differentiated multicell. thalli)
• thallus organization <--> basis of classification
• flagella -wide diversity in number & arrangement (1-8 in apical / subapical region)
• zoids are isokont (similar struct. but can differ in lenght)
• freshwater, marine & terestrial
• Pigm.: chl. a & b, β & γ-carotenes, xanthophylls
• Chlpl. – 2-membrane envelope
• Thylakoids – stacked, form grana
• pyrenoid (if present) in chlpl.
• ctDNA – circular
• Res.polysach.: starch – most important – grain inside chlpl.
• glucan & β-1,4-mannan can present in cell wall
• eyespot (if present) loc. inside chlpl.
• photoautotrophs, can be heterotrophs
• Repr. – sex. – variety of life cycle – group specific
– similarity to higher plants - Trentepohliaceae – cell division using phrogmoplast disc where
the cells will divide
• probably land plants derived directly from these freshwater algae
Endosymbiosis & origin of
eukaryotic algae
• procaryotic ancestor → endosymbiotic events → primary plastid
• secondary / tertiary endosymbiosis → plastid (sec. eukaryotic cell)
• arrangement of cellular compartments inside the the other – info about evolut.
history
• Cyanobacteria
– evolved >2.8 bill. years ago
– fundamental roles in ocean carbon, oxygen, nitrogen fluxes
– turning point in biogeochemistry of Earth
– photosynthesis – energy of VIS, oxidation of H2O, reduction of CO2
– CO2 + H2O + light chl a> (CH2O)n + O2
Three major algal lineages of primary plastids
• Glaucophyta lineage
– plastids – thin peptidoglycan cell wall & cyanobacter.-like pigments
» cyanobacterial ancestry
– neither green nor red plastid present
– any secondary plastid derived from Glacophyta is known
• Chlorophyta lineage “green algae”
– green algal plastid
– 2-membrane system surround.
• chl b – sec. pigment, phycobiliproteins lost
– other – prochlorophyte based hypothesis
– major role in oceanic food webs & carbon cycle from -2.2 bill. years until Permian extinction (-250mil y.)
– origin of land plants
– >Secondary endosymbiosis → Euglenophyta (3-membrane plastid) & Chlorarachniophyta (4-membrane plastid
• Rodophyta leneage “ red algae”
– red algal plastid
– 2-membrane system
• chl a & phycobiliproteins organized into phycobilisomes attached on thylakoid membrane
• Thylakoids with phycobilisomes don’t form stacks – similar to cyanobacteria
– >Secondary endosysmbiosis → Cryptomonades (Cryptophyta)
• (4-membrane plastid) + chl c (also Haptophyta, Heterocontophyta & Dinophyta)
– stacked thylakoids found in lineages lacking phycobilisomes
– >a few groups of Dinoflagelates Tertiary endosymbiosis – uptake of secondary plastid containing
endosymbiont
– All these groups are relatively modern org.
– dinoflagelates & coccolithophorids rise paralel with dinosaurs
– diatoms rise with mammals
members of red lineages in shallow seas in Jurassic period provide petroleum
Endosymbiosis & origin of
eukaryotic algae
Physiology and Cultivation of
Algae and Cyanobacteria
3.
Photosynthesis
• light
• structure & function
– membrane structure
– pigments
– photosystems
• photosynthesis – reactions
– light dependent
– light independent
• energy transfers
Light
• sunlight vs. PAR
• units; W m-2 s-1; µmol m-2 s-1
• spectrum
• absorption, transmittation, reflection,
scattering, interference
• environmental accessibility (spectrum, int..)
Structure & function
• thylakoid membrane structure
• pigments
• photosystems
Photosynthetic reactions
• general equation:
» nCO2 + nH2O + light (CH2O)n + nO2
• light dependent reactions
• light interception, l. energy transfer
• excitation, charge separation
• ETR
» linear
» cyclic
• O2 evolution
• light independent reactions
• CBB – Calvin Benson Bassham Cycle
• RuBisCO
• CA
Chl a
Role of Carbon Concentrating
Mechanism
• Carbonic Anhydrase
• periplasmic space, carboxysomes (b-g algae),
pyrenoid (algae)
• CA vs. RuBisCO
• mechanisms of HCO3
- uptake
– active transp. via CMP (enough ATP)
– symport Na+ HCO3
- - via icpB complex
– Na+/H+ antiport help
– difusion
• CO2 uptake - difusion
Energy transfers & regulations
• excitation energy (excess) dissipation pathways
– photosynthesis
– state stransitions
– heat production (Xanthophyll cycle)
– fluorescence
• photoaclimation
– light dependent motions; state transitions; non-photochemical processes
• photoinhibition
– photomodification
– photodamage
• photorespiration & chlororespiration
• RuBisCo – generation of glycolate
• enigma; PQ pool reducion
Photosynthesis–Irradianceresponsecurve
(Pvs.Ecurve)
Measurement of photosynthetic
production
Methods:
– gravimetric
– turbidimetric
– fluorometric
– gasometric
– IRGA; O2 measurement
Physiology and Cultivation of
Algae and Cyanobacteria
4.
Biogechemical role of algae
• Energy flow in the environment
• Limiting nutrients
– Liebig’s Law of the Minimum
µ = µmax [LM] / [LM] + Km
• Cycles of nutrients
• Phosphorus
• Nitrogen
• Sulphur
• Carbon & Oxygen
• environmental factors
Phosphorus
• PO4
3•
membranes, coenzymes, DNA, RNA, ATP
• orthophosphate; metaphosphate
(polyphosphate); “organic” phosphate
Nitrogen
• diazotrophs
• N-fixation (N2 >> NH3)
• assimilation (NO3
-&NH4
+ >> organic N)
• mineralization (organic N >> NH4
+)
• nitrification (NH4
+&NO2
- >> NO3
-)
• denitrification (NO3
- >> NO, N2O, N2)
• nitrogenase; MoFe(V)-protein; PS I;
• Nostoc, Anabaena, Trichodesmium,
Katagnymene
Silicon
• [Si(OH)4]; (SiO2 . nH2O)
• silica-forming organism
• Chrysophyceae
• Bacillariophyceae
• Dictyochlorophyceae
• cell wall; sediments
Sulphur
• aminoacids (Cys, Pro, cystine)
• SO2, SO4
2-, H2S,
• APS (adenosine-5’-phosphosulphate)
• PAPS (3’-phosphoadenosine-5’-
phosphosulphate)
• DMS (dimethyl sulphide)
• DMSP (dimethylsulphonium propionate)
Carbon & Oxygen
• CO2, H2O, CO, O2, HCN, CH4, CaCO3,
CFC, HCFC
• photosynthesis
• oxidizing atmosphere
• coccoliths
Algae & Men
• macroalgae (commerce - 42 countries)
• food
– Laminaria (China, N.,S.Korea, Japan, Philipines, Chile,
Norway, Indonesia, U.S., India)
– Porphyra, Kappaphycus, Undaria, Euchema, Gracilaria
– Nori (Porphyra yezzoensis) – 13mil. t/y
• microalgae
• carotenoids, pigmenst, proteins, vitamins, …
– nutraceuticals, pharmaceuticals, animal feed additives,
cosmetics, fertilizers
– Dunaliella, Haematococcus, Arthrospira, Chlorella
Physiology and Cultivation of
Algae and Cyanobacteria
5.
Overview
• Collection of the samples
• Isolation & purification of algal culture
• Culturing
– Methods
– Equipments & material
– Conditions
– Culturing media
Collection of the samples
• purpose specific
• sample specifics (nature & environment)
• time
• concentration
• type of vessel/container
• removal of the unwanted organisms (filter)
• transfer conditions & storing
Isolation & purification of algal
culture
• Equipments & suplies
– Microscopes
– Filters & sieves
– Glasseare, Plasticware, Utensils
• Methods
– Sterile manipulation
– Isolation techniques
Equipments & suplies
• Microscopes
– dissecting (80x,..)
– inverted
– lighting
• dark-field
• fiber optic light source
• fluorescent lamp
• epifluorescence
• Filters & sieves
– woven screens
– nylon netting
– membrane filters (material)
– differential filtration
• Flow-box
Glasseare, Plasticware, Utensils
• borosicate glassware
• plasticware (ready-to-use, culture-grade)
• sterilization technique & sterility
• dust-proof cabinet / clean containers
• caps & splips
• sterile filtration apparatus
• sterile spatula
• pens, labels
• parafilm
• growth chambers
– test tubes, flask, culture flask, Erlenmeyer flask, Petri dish, plugs, two-steps screw
– cultivator, tank, bioreactor
Sterilization & sterile manipulation
Methods of isolation & purification
• Enrichment culture
• Single-cell isolation
• Size separation >> filtering
• Density separation >> centrifugation
• Dilution
• Isolation with use of agar
– streaking
– spray
• Isolation with use of phototaxis
• Automatization (flowcytometer)
Culturing techniques
• chemicals
• equipment – balance, ph-meter, autoclave, filtration,
ultrasonic washer, refrigerator, cultivator
• conditions – ph, temp, irradiation, photoperiode,..
• glasware – E. flask, reagent bottles, pipetes, flask, tubes, P.
dishes, spatulas, funels, filter holder, syringe, ..
• water
• agar
• soil
Media
• stock solutions
– macronutrients
– trace elements
– vitamins
– chelators
– soil extracts
• preparation of media
• synthetic media
• enriched media
• soil water media
• solidified media – agar
• Freshwater media
• Seawater media
• natural & artificial
Physiology and Cultivation of
Algae and Cyanobacteria
6.
Culture methods
• Bath cultures
– common, simple, low cost, closed system, volume-limited
– any flow of nutritions & products
– Erlenmeyer flasks, tubes, Petri dishes
– growth curve phases – lag, acceleration, exponential, retardation,
stationary, decline
Culture methods
• Continuous cultures
– resources are potentially infinite
– cultures are maintained at chosen point on the growth
curve by regulated addition of fresh medium
– air pump – CO2 source, mixing
– categories of contin. cult.:
• turbidostat cultures
• chemostat cultures
• Semi-continuous cultures
– periodic fresh medium addition & harvesting
Culture methods
• Commercial-scale cultures
– volume of cca. 102 – 109 l
– large open ponds, circular ponds with rotating
arm, raceway ponds, large bags, tube system
– factors to be considered:
• biology of alga; the cost of land; labor; energy; water; nutrients;
climate (if outdoors); type of product
• light utilization efficiency; ability to control temp.; hydrodynamic
stress; ability to maintain culture unialgal or axenic; scale up ability
– Chlorella, Spirulina, Dunaliella
Methods used for
algal culture growth evaluation
• direct
– fresh/dry mass determination
– counting – number of cells (colonies)
– cell volume
– protein content
– calorific value
– flow-cytometry & epifluorescence microscopy
• indirect
– turbidity; optical density
– chlorophyll content
Endogenous rhythms
• circadian rhythms
• molecular feedback loop ~ 24h~ environmental light-dark
cycles ~ positive/negative phase shifts according to phaseresponse
curve of particular response
• necessity to transfer sunlight-sensitive processes into the night
(cell division in night)
• lost under certain conditions (e.g. constatnt light, bright light,
growth of Euglena on organic medium)
– phototaxis
– timing of cell division
– photosynthetic capacity
– bioluminiscence
– gene expression
– sensitivity to UV
Endogenous rhythms
• biweekly (circa-semilunar) rhythms
• wave action (sea may reduce gamete concentration of marine org.
with external fertilization)
• e.g.:brown alga Dictyota dichotoma releases eggs twice a month in
the field; synchronization signal every second full-moon light
• day-length effect (photoperiodism)
• circadian timer measures length of night and triggers photoperiodic
response
• LD (12:12~light:dark) – induce upright thali formation
• SD (8:16)– induce reproductive organs formation
• circannual rhythmicity
• sequence of short and long days over the years results cyclic
reproductive stages formation (e.g. Ascophyllum, Laminaria)
• signal probably temporal sequence of different physiological stages
Documetation
• Microcopy (light & fluorescence)
– drawings
– photography – classic & digital
• algal culture collections
• Web pages of culture collections in the world:
http://wdcm.nig.ac.jp/hpcc.html
• macroalgae ~ herbarium