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HOW TO DETERMINE PHYTOPLANKTON?. Si lvana V. Rodrigues. Determination of phytoplynkton composition and biovolume Utermöhl method: : Advantage: asy sampling, long storage times Disadvantage: requires a lot of time, and specialists

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slide1
HOW TO DETERMINE

PHYTOPLANKTON?

Silvana V. Rodrigues

Determination of phytoplynkton composition and biovolume

Utermöhl method::

  • Advantage: asy sampling, long storage times
  • Disadvantage: requires a lot of time, and specialists
  • Results: relative contribution of algas classes x biovolume
slide2
HOW TO DETERMINE

PHYTOPLANKTON ?

peridinina

Dinoflagelados

Clorophyta

Cryptophyta

Cyanobacterias

aloxanthin

slide4
Importance of chlorophyll a
  • 1.000 milhão tons produzidas por ano na terra e no mar
  • indicator único da biomassa aquática
  • parâmetro bioquímico mais freqüentemente medido

em oceanografia

Cloroplasto

fig.cox.miami.edu/.../phts/c8.10x21.overview.jpg

struggle.net/history/images/

molecule.jpgwww.molecularexpressions.com

slide5
Function of pigments in photosynthetic organisms

chlorophyll a:

light absorption (“Light harvesting complexes”)

electron donor and acceptor in reative centers

Carotenoids:

Light absorption

Protection of chlorophyll (“quenching “ of Chl photoinduced triplet

state ) and quenching of O2 singlet state .

slide7
Characteristics which make it possible to use algal pigments

(chlorophylls, carotenoids and phycobiliproteins) as chemotaxonomic

markers

  • They are present in all photosynthetic algae, but absent in most bacteria,

protozoa and detritus

  • Many occur only in specific classes or even genera, allowing the

determination of phytoplankton taxonomic composition at least at class level,

or better

  • They are strongly coloured, and in the case of chlorophylls and phycobiliproteins

are fluorescent, what allows their detection with high sensitivity,

  • Most of them are labile and esily dgraded after cell death, allowing to

distinguish living from dead cells

slide8
Hystorical overview
  • 1952: chlorophyll was recognized as a selective phytoplankton marker, in the presence of other biological components (zooplankton, bacteria, detritus)
  • 1984-1987: HPLC methods for the determination of chls, carotenoids and phytoplankton degradation products
  • Use of pigment chemotaxonomy for recognition, in field samples, of phytoplanktonic classes not detected since then, because of preservation problems or filtration losses.
    • alloxanthin (Cryptophyta)
    • chlor b (Chlorophyta and Prasinophyta)
    • zeaxanthin (Cyanobacteria)
    • 19’-hexanoiloxifucoxanthin (Prymnesiophyta)
    • divynil-chlorophyill a (Proclorophyta)
slide9
Chlorophylls:
      • 132 -Metilcarboxilates of -
  • Mg-phytoporphyrin (double bond in D ring): Cl c, Mg-phytoclhorin: Cl a, Cl b

Phytil at C-173 (Cl a and b)

Propionic acid at C17: Cl a and b

Acrílic acid at C17: Cl c

Mg coordination complexes with cyclic tetra-pyrrols

Macrocicles with five member rings

slide10
Chlorophylls:
      • 132 -Metilcarboxilates of -
  • Mg-phytoporphyrin (double bond in D ring): Cl c, Mg-phytoclhorin: Cl a, Cl b

Phytil at C-173 (Cl a and b)

Propionic acid at C17: Cl a and b

Acrílic acid at C17: Cl c

Oxo substituent at C-131

methyl-carboxilate groups at C-132 -

slide11
chlorophyll b

chlorophyll a

DV-chlorophyll a

DV-chlorophyll b

Molecule drawings:N. Montoya

slide12
chlorophyll c1

chlorophyll c2

chlorophyll c3

Molecule drawings:N. Montoya

slide13
Degradation by chemical processes:
  • Molecules become chemically and fotochemically
  • more labile in organic solvents
  • than in the cells
  • Loss of metal
  • Chla  Phaeophitin
  • in organic solvents
  • In dilute acids
  • under high intensity of light
slide14
Degradation by chemical processes:
  • Epimerization

(HPLC: in SiO2):

  • Allomerization

(oxidation by O2):

Cl  enolate  Cla’, b’

  • Chl a  132 Hydroxiclhorophyll a
  • Chl a Cl a - Hyidroxilactone.

Both processes

can be minimized

by decreasing the temperature

In alcoholic or hydro-alcoholic solutions

Specially in pH >7

slide15
Degradation by chemical processes:

Loss of phytil group

Cl chlorophyillide

In methanol or ethanol in basic medium

slide16
Biodegradation:

Loss of metal:

Mg-dequelatase

Formation of phaeophytins

  • To cyclic tetra-pirrols

perifercally modified

(enzymatically,

Specially in the absence of

light and O2):

Decarboximetilation

Formation of

pirophaeophytins e

pirophaeophorbides

Hydrolisis of the phytil

ester

(chlorophyllase)

chlorophillide formation

Allomerization

Epimerization (Chl-oxidase)

slide17
Biodegradation:
  • To linear tetra
  • pirrols

5

4

Normally by oxidative opening

of the macrocycle ring, between

C-4 and C-5,

C-5 stays as an aldehyde

slide18
Carotenoids

Derive from carotene:

C40H56

β- β- carotene

Isoprenoid

units

Polyen:

Absorbtion

of light.

COLOUR

-carotene: ,-carotene

-carotene: ,-carotene

-carotene: ,-carotene

-carotene: ,-carotene

lycopene: ,-carotene

slide19
Properties

More stable in phytoplankton and in plants than chlorophylls: they don‘t

have N, so can‘t be used in enzymatic amino-acid building.

Example:

Leaves lose the green colour in autumn (chlorophyll),

But don‘t lose colours due to carotenoids

slide20
Polyene chain is responsible for instability:
  • Oxidation by air or peroxides
  • Electrophyle addition ( H+ and Lewis acids)
  • Isomerization E/Z caused by heat, light or chemicals,
  • Undergo reactions at the ends of the molecules
  • Production of artefacts
slide21
Acetil-CoA

Geranylgeranyldiphosphate

Geranylgeranyldiphosphate

Biosynthesis:

occurs in

thylakoid

membranes

Phytoene

Dessaturation

Lycopene

Ciclization

,  -carotene

,  -carotene

Hydroxilation

Hydroxilation

lutein

Zeaxanthin

Deepoxidation

Dark

Epoxidation

Light

Anteraxanthin

Can occur in the dark

Depends a lot on light

Dark

Epoxidation

Light

Deepoxidation

Violaxanthin

VIOLAXANTHIN

CICLE

Rearrangement

Neoxanthin

slide22
DIADINOXANTHIN CICLE

Diadinoxantin

epoxidation

+ 2H + O2 - H2O

DARK

LIGHT

+ 2H - H2O

Diatoxanthin

slide23
Carotenoids

C40H56

β- β- carotene

Aldehydes,

ketones

Enzimatic

hydroxilation

Acetates

(OCOMe)

e lactones

Carboxi

(CO2H),

carbometoxi

(CO2Me)

ou metoxi

(OMe)

Hydroxi-

carotenoids

as fatty acid esters,

or as

Glycosides or

glycosylesters,

others as

sulphates

Epoxidation

slide24
Xantophylls

Isoprenoids

Zeaxanthin

isomers

Lutein

slide25
Acetilenic

Diatoxanthin

Alenic

fucoxanthin

Norcarotenoids

( skeleton C37)

Peridinin

C39H50O7

slide26
In acid medium

Epoxides rearrange (5,6 to 5,8 form)

7

6

8

5

violaxanthin

7

6

8

5

neoxanthin

slide27
In basic medium:
  • In general stable
  • exception:

esters are hydrolysed

some compounds suffer structural change (fucoxanthin, peridinin)

fucoxanthin

slide32
Amphidinium carterae (Dinophyta)

Rzi =[lpigmi]/[chlorophyll a]

Rz =[peridinin]/[chlorophyll a]

chlorophyll c2

chlorophyll a

dinoxanthin

peridinin

diadinoxanthin

slide33
Dunaliella tertiolecta (Chlorophyta)

Rzi =[lpigmi]/[chlorophyll a]

Rz =[lutein]/[chlorophyll a]

chlorophyll b

chlorophyll a

neoxanthin

violaxanthin

lutein

anteraxanthin

slide35
Retention times and mean absorption properties (inHPLC eluant) of the major pigments detected in Erythrobacter longus (ATCC 33941) and isolates NAP1, MG3, and NJ3Y. Peak numbers correspond to those indicated in Fig. 5. Solvents and caroteneid band ratios from the literature data: 1 solvent=methanol+ water (4:1) containing 40mM NH4OH, %(III/II)=0; 2 solvent= methanol, %(III/II)=0; 3 solvent=acetone, %(III/II)=33; 4, 5 solvent=diethyl ether; 6 solvent=acetone, %(III/II)=21

Michal Kobližek Arch Microbiol (2003) 180 : 327–338

slide36
Reverse-phase HPLC

chromatograms (360 nm) for

acetone extracts prepared from

whole cell pellets of a Erythrobacter

longus ATCC 33941,

b NAP1, c MG3, and d NJ3Y.

Peak identities: 1 erythroxanthin

sulfate, 2 bacteriorubixanthinal,

3 zeaxanthin, 4 bacteriochlorophyll

a, 5 bacteriophaeophytin

a, and 6 β,β-carotene

Michal Kobližek Arch Microbiol (2003) 180 : 327–338

slide37
HPLC chromatogram of fuorescent pigments from a surface sample

(2 m depth) collected at station C354-004. Excitation was at 365 nm,

emission at 780 nm, with 20-nm slits. These wavelengths were chosen to

maximize the signal from BChla, while minimizing the signal from the more

abundant pigments, Chla and Chlb. (Inset) Fluorescence emission spectrum of

the peak eluting at 16.7 min in (A). Excitation was at 365 nm and slits were

20 nm.

Zbigniew S. Kolber et al, Science 292, 2492-2495; 2001.

slide39
Pigmentos

Em geral são moléculas lábeis, atingem o sedimento em vários estágios de degradação.

Degradação dos pigmentos originais

principalmente na água e na superfície do sedimento, durante a deposição

(Hodgson et al., 1997)

  • Na água:
  • rápida e extensa
  • (≤95 % dos compostos em poucos dias)
  • digestão por herbívoros,
  • enzimática, na senescência celular
  • oxidação química, microbiológica e pela luz.

Fatores que afetam

a taxa

de degradação:

  • Tempo para chegar
  • ao fundo
  • Tipo de pigmento
  • Grau de ataque
  • químico e biológico
  • Nos sedimentos:
  • taxa de degradação menor, especialmente em condições anóxicas. Depende de:
  • intensidade de luz e da
  • bioturvação invertebrada
slide40
DEGRADATIN PRODUCTS:
  • degradation to uncoloured compounds
  • conversion to cis-carotenoids and phaeopigments more difficult to identify (Steenbergen et al., 1994 apud Hodgson et al., 1997).

Separation and quantification of pigments in sediments

More complex than in phytoplankton samples, due to the variety of degradation or transformation products (Mendes et al. 2007) .

slide41
Chlorophyll b: occurs mainly ingreen algae and vascular plants,

Chlorophylls c: in diatoms, dinophlagellates and some brown algae

Kowalewska et al., 2004.

Chl a‘ and phaeophytin:

degradação products due to

Environmental stress

Pirophaeophitins and steril

Chlorins: degradation

products due to zooplankton

Phaeophorbides:

Degradation products due

to zooplankton

Jeffrey, 1997 apud Kowalewska et al., 2004).

slide42
Fossile Pigments:

Used in paleoclimatic and paleoenvironmental issues

Chlorophylls :

More labile than carotenoids , but phaephitins are persistent in sedimentary records

Carotenoids:

Stability depends on structure (decreases with the increase of the number of functional gruoups).

slide43
Carotenoids:

Estáveis, abundantes

(adaptado de Buchaca & Catalan 2008)

slide44
Chlorophylls :

(adaptado de Buchaca & Catalan 2008)

slide46
Chlorophylls

Phaeophytin a

Chlorophyll a

- Mg

- Mg

- Phytil

- Mg, -COOMe

Phaephorbide a

Pirophaephytin a

Jeffrey et al.;1997

slide47
Polyene chain: chromophore

UV7VIS: Electronic transitions

Main

transition

Vibrational

fine

structure

slide48
Calculation of % III/II for a caroteneid

II

III

0

0

Vibrational

fine

structure

slide49
Molecular structure x spectroscopic properties

Lenght   

Chromophore (polyene chain):

slide50
Molecular structure x spectroscopic properties

Geometrical cis-trans isomers:

small hypsochromic effect

Significant hypochromic effect

Reduction of vibrational fine structure

Appearance of a cis-peak (≈ 142 nm below the longest maximum

of the all-rans,measurd in hexane

Beta-Rings: fine structure much reduced, max shorter than in the

acyclic

Acetylenic groups: replacement of d.bond to triple bond - 15-20 nm

shorter wavelength

Allenic groups

Carbonyl groups

Britton, 1995, Carotenoids,

3 vol, Birkhäuser

slide51
Molecular environment x spectroscopic properties

1: displacement of max to longer wavelength

slide53
HPLC method with improved resolution, LC–MS analysis and the automated acquisition of MS/MS data for pigments
  • extracts from a sediment (Priest Pot, Cumbria, UK),
  • a microbial mat (les Salines de la Trinital, South Catalonia, Spain)
  • a culture (C. phaeobacteroides):

SEPARATION OF A GREAT NUMBER OF PIGMENTS, INCLUDING NOVEL BACTERIOCHLOROPHYLL DERIVATIVES.

Airs, 2001

slide56
HPLC coupled both to UV photodiode array detection and to atmospheric pressure mass spectrometric techniques (HPLC–DAD-APIMS)

Pigments ( chlorophylls, carotenoid), galactolipids, alkaloids, sterols and mycosporine-like amino acids,

Frassanito 2005

slide59
Chemotaxonomic estimation of phytoplankton communities in aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.
  • Extraction must be quantitative for all pigments
  • HPLC separation must be able to:
  • separate simultaneously groups of molecules of very different polarities
  • Resolve very similar compounds, for instance isomers
slide60
Extraction of phytoplankton pigments

Acetone 90 %

Acetone 100 %

Methanol

Acetone :Methanol ( 1:1)

N,N-dimetilformamide (DMF)

Buffered Methanol ( 2% NH4Ac 0,5 M)

Solvents:

Procedure:

Sonication or criogenic homogenization

„overnight“ or immediate extraction

slide61
Separation (HPLC)

Filtration

GF/F 47mm

Extration:

Methanol: NH4Ac 0,5M (98:2) +

Sonification, ice-bath (30 s) +

Centrifugation (5 min, 4800 rpm)

slide62
Chromatographic separation of

Phytoplankton pigments

slide63
Separation with C30 columns:Development of a

computer-assisted method (Software Dry Lab)

Fase estacionária:

C30 (YMC, C30, 5µm,

polimérica

250x4,6 mm ID

Fase móvel:

A:CH3OH:TBA (28 mM)

70:30 (v/v)

B: CH3CH2OH

pH 6,5

Gradiente:

chlorophylls:

Fig A:30-100 % B, 50 min

Vazão: 1,2 ml/min

T: 47 oC

Carotenóides:

Fig B:25-63 % B, 35 min,

63-100%B/13 min

Vazão: 1,4 ml/min

T: oC

c3

DV, MV cl b

alo-, diato-xanthins

e luteína

c2

DV, MV cl a

c1

luteína

aloxanthin

diatoxanthin

Resolution:

otimization

for chlorophylls

And for carotenoids in

Sparate runs

Mistura-teste

Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49

Resolution: separation mono/divynil clh a, b

They don‘t separate in C18 !! (depends on aliphatic chain?)

slide64
Separation with C8 columns:

1) Development of a computer-assisted method (Software Dry Lab)

DV, MV cl b

Zeaxanthin, luteína,

DV, MV cl a

c3

Fase estacionária:

C8 (Eclipse XDB, 3,5 µm

150x4,6 mm ID

Fase móvel:

A:CH3OH:TBAA (28 mM)

70:30 (v/v), pH 6,5

B: CH3OH

c1 +clorofilídeo a

C2 +MgDVP

Mistura-teste.Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49

2) Zapata et al., 2000Mar. Ecol Progr. Ser. 195: 29-45, 2000

Fase móvel:

A:CH3OH : CH3CN : pirid.acet. (50:25:25); B: CH3OH : CH3CN : acetona (20:60:20)

slide65
C8, Zapata

R=0,8

cl b/DVcl b

R< 0,5

Zeaxanthin,

dihidroluteína

Clor c2

R>1

4k Hex

/9‘cis Neo

R> 1,25

MgDVP

R< 0,5

cl b/DVcl b

R= 0,8

R=1

C8, Van Heukelem

4k Hex

/9‘cis Neo

Não resolve

Pigment mixture, S. Wright, Course Notes

C8: better for chlorophyll c family

slide66
Comparison of method sensitivity with C18 and C8 columns

Fases estacionárias:

C8 (Symmetry C8, 3,5 µm

150 x 4,6 mm)

C18 (Supelcosil L-C18, 5 µM

250 x 4,6 mm)

Fase móvel:

Coluna C18:

adap. Kraay, 1992

A:CH3OH:H2O (85:15)

B: CH3CN.H2O (90:10)

C: Acet. Etila

(vazão 0,6 ml/min)

Coluna C8:

Zapata, 2000

Mendes et al., Limnol. Oceanogr. Methods 5, 2007, 363-370

C18: More sensitivity

Lower limit of detection

Better for low concentration pigments

slide67
Separation of complex samples, method

compatible with LC/MS

Método SCOR 1997

Fase estacionária:

2 colunas „in line“

Waters Spherisorb ODS2

3 µM

150 x 4,6 mm)

Fase móvel:

A: NH4Ac 0,01M

B: CH3OH

C: CH3CN

D: Acet. Etila

Gradiente:5%A, 85% B,

15 % C isocr.5 min,

0%A, 20% B,15%C,65% D,

95 min, 0%A, 1%B, 1%C,

98%D, 5 min,isocr. 5 min

Adequado para

LC/MS

Método Airs et Al.

Extrato de amostra de sedimento (Priest Pot)

Airs et al.;Journal of Chromatography a 917 (2001) 167-177

slide68
zeaxanthin

19,-butanoilfuco

luteina

Cl a + DV cla

Cl b + DV clb

peridinina

diatoxanthin

diadinoxanthin

Cl c2

aloxanthin

dinoxanthin

neoxanthin

violaxanthin

prasinoxanthin

fucoxanthin

Cl c3

Fase estacionária:

Spherisorb ODS1/ C18

250 x 4,6 mm – 5 m

Fase móvel:

A: CH3OH 0,3 M em

NH4Ac : ACN : H20

(51:36:13)

B: AcetEtila: ACN (70:30)

Vazão: 1,2 ml/min

Gradiente:0 a 25 % B em

5 min, isocr.5 min,

25% a 100% B

em 20 min.

Labor. UFF, Cromatógrafo Bischoffanalysentechn., Mistura-teste (DHI), 100µL injetados na fase A,

Separates:,-carotene, ,-carotene, Aloxanthin,Lutein, Neoxanthin,

Violaxanthin, Fucoxanthin, Diatoxanthin,Diadinoxanthin, Peridinina,

Dinoxanthin, Zeaxanthin, Mixoxantophyll, Equinenone, Cantaxanthin,

Astaxanthin, Okenone, Scytonemin-1, -2, Bacteriophaeophytin-a,

Bacteriochlorophyll-e, chlorophyll-a, Chlorophilide-a, Chl-a Allomer and Epimer,

phaeophytin- a1, a2, phaeophorbide -a1, -a2, -a3, -a3’, -a4, chlorophyll b,

phaeophytin -b1, -b2, chlorophyll –c1, -c2, -c3

Buchaca e Catalan (2008)

slide69
HOW TO DETERMINE PHYTOPLANKTON ?

ESTIMATION OF THE ABUNDANCE OF PHYTOPLANKTONIC COMMUNITY BY PIGMENT MARKERS

Based on the contribution, in terms of chlorophyll a,

of each group of taxonomical class (Chl a)c

to total chlorophyll a in the sample (Chl a)t :

(Chl a)t = (Chl a)c1 + (Chl a)c2 + (Chl a)c3 + ...... + (Chl a)cn

Calculation of(Chl a)cn ?

Easy !

slide70
METHOD 1:

Calculation of(Chl a)c by the choice of one marker pigment

for each class

Problem:

Fixed R

not necessarily

Corresponds

To the ratios

In the samples

(Chl a)t = Rzea/cla x (Zea) + Rlut/cla x (Lut) + .........+ Rfuco x (Fuco)

{

sample

(Chl a)c and

% of each class

Fixed

slide71
METHOD 2:

Multilinear regression

Sample 1: (Chl a)t1 = Rzea/cla x (Zea)1 + Rlut/cla x (Lut)1 + .........+ Rfuco x (Fuco)1

Sample 2: (Chl a)t2 = Rzea/cla x (Zea)2 + Rlut/cla x (Lut)2 + .........+ Rfuco x (Fuco)2

....................................................................................................................................

Sample n: (Chl a)tn = Rzea/cla x (Zea)n + Rlut/cla x (Lut)n + .........+ Rfuco x (Fuco)n

Unknown Rs, determined by pela resolution of a system

of n equations and n unknowns

Rs are determined, but many classes don‘t have a specific pigment

(Chl a)cn

% of ech class

slide72
MÉTODO 3:

Determinação da composição fitoplanctônica por análise fatorial

(MACKEY et al., 1996)

  • „Software „CHEMTAX: problema de análise fatorial:
  • matriz de dados S: concentrações encontradas para os pigmentos
  • no ambiente num conjunto de amostras
  • fatorizada em matrizes

F : matriz das razões dos pigmentos para as diferentes classes

de algas puras e

C : abundâncias de cada classe de alga em cada amostra

slide73
MATRIZ F:RazõesRi =[lpigmi]/[chlorophyll a para cada classe

C: contribuição de cada

classe (a ser determinada)

MATRIZ S:experimental

Amostra 1: (Chl a)t1 (Zea)1 (Lut)1 ....... (Fuco)1

Amostra 2: (Chl a)t2(Zea)2 (Lut)2 ....... (Fuco)2

.................. ............ ......... ........ .......

Amostra n: (Chl a)tn (Zea)n (Lut) ....... (Fuco)n

Clpras

ClDin

ClCryp

ClHapt3

ClHapt4

ClChlor

ClSyn

ClDiatom

F x C = S

slide74
Para uma fatorização de S que tenha um significado físico:

F : variável, Fo: dados da literatura (normalizados/Cl a)

Estimativa inicial da matriz de abundâncias das classes (Co):

calculada resolvendo-se a equação de mínimos quadrados:

minimizar:  S – Co Fo,

sob as condições: [Co]ij 0  i, j

 [Co]ij = 1  j

O resíduo é expresso por:

o = S – Co Fo

Um algoritmo de „decréscimo máximo“ do resíduo foi usado

(variação dos elementos de F, 10% a cada iteração)

slide75
Juturnaíba reservoir as a study model

Marcelo Marinho e Silvana V. Rodrigues

slide76
OBJETIVOS
  • Avaliar a aplicabilidade do método de análise de pigmentos por HPLC

para detecção das variações na biomassa e composição do fitoplâncton, comparando com os dados obtidos por microscopia

slide77
METODOLOGIA

Fitoplâncton

  • Coletas quinzenais - jun/96 - mai/97 (estação central)
  • Biovolume
    • método de sedimentação (Utermöhl, 1958)

Pigmentos

Amostra

(0,25 - 1,8 L)

Injeção e análise

HPLC

CONDIÇÕES CROMATOGRÁFICAS

  • Filtração (GF/C)
  • Congelamento
  • (CO2 sólido)
  • Coluna C18 - fase reversa
  • Gradiente alta pressão
  • (modificado de Garrido & Zapata, 1993)
  • Detecção - 440nm

Extração

Metanol 100%

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-1

-1

Biomass (chlorophyll a)

Contribution calculated by marker pigments

Razão Xan/Chl-a

CHEMTAX

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Biovolume
  • 0,2 L +Lugol’s solution
  • sedimentation method (Utermöhl, 1958)
      • biomass: product of population and mean unit volume of each species
      • (specific density of cells = 1 g/cm3,
      • cell size = mean of at least 30 measurements)
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Microcystis aeruginosa

20 mg/L

Anabaena spiroides

Cylindrospermopsis raciborskii

Biomass (Biovolume)

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Biomass (CHEMTAX) x Biomass (biovolume)

2 periods in both methods

CHEMTAX:

Period 1 (June - November 96):

3.7 - 36.4 mg/L chl a

Chlorophyceae, Cyanobacteria, Cryptophyceae

Period 2 (December 96- May 97):

46.9 - 254.4 mg/L chl a

81% to 99 % Cyanobacteria.

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CONCLUSIONS
  • High correlation between biovolume and Chl-a. Chl-a can be used as a parameter to estimate biovolume.
  • Interpretation of pigment data with CHEMTAX: better correlation with biovolume than that based on Xan/Chl-a ratios from unialgal cultures.
  • Only Chlorophyceae and Dinophyceae did not present significant correlation with cell count.
  • Similar general pattern of the phytoplankton community dynamics by cell count and pigment analysis: two periods and the Cyanobacteria bloom recorded.
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GUANABARA

BAY

RJ/BRAZIL

12SAMPLING SITES:

SAMPLING FREQUENCE:

- 12 CAMPAIGNS

- JANUARY TO AUGUST (SUMMER/AUTUMN) 2006

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HOMOGENEITY OF SAMPLES

WITHIN EACH DATA MATRIX

Data processing:

CHEMTAX:

Samples divided in 5

environmentally

different groups

5

2

3

4

1

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