NEW RESULTS IN THE RESEARCH OF HARDLY KNOWN TRACE ELEMENTS AND THEIR ROLE IN THE FOOD CHAIN

PROCEEDINGS OF INTERNATIONAL SYMPOSIUMBudapest, HUNGARY, September 1988

EDITOR:Prof. Dr. István PAIS

UNIVERSITY OF HORTICULTURE AND FOOD INDUSTRY

BUDAPEST1988

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EFFECT OF TITANIUM ON GROWTH AND PHOTOSYNTHETIC PIGMENT COMPO-

SITION OF CHLORELLA PYRENOIDOSA. I. EFFECT OF TITANIUM ASCORBATE

ON GROWTH AND PROTEIN CONTENT OF CHLORELLA

L. SIMON , F. KISS , Á. BALOGH and I. PAISX

Biological and Chemical Department of Teacher’s Training Col­

lege, Nyíregyháza, Hungary

Chemical Department of University of Horticulture and Food

Industry, Budapest, Hungary

Introduction

All over the world is continuous search fór new sources of

food and feed to solve malnutritional problems of the world po-

pulation. Microalgae very efficiently bioconvert the solar ener-

gy and produce large amount of algal biomass every year. Micro­

algae are useful as food fór humán consumption, are good sour­

ces of vitamins and proteins, serve as feed fór animals, bio-

fertilizers in fields, give renewable fuel. Most of the micro­

algae currently used fór these purposes are from the prokaryotic

cyanobacteria( blue-green algae ) or from Chlorophyta

( greert algae ) including Chlorella and Scenedesmus gén éra

( 1 , 2 ).

The interest in Chlorella species as potential and actual

sources of single cell protein( SCP ) continues despite of prob­

lems with digestibility, toxicity, high cost of production and

- 77 -

incomplete amino-acid composition( 2 , 3 , 4 ) . One possible way

to enhance the algal yield is optimálisátion of the composition

of inorganic nutrition médium. Enhancement of growth and protein

content of Chlorella sp. was reached with optimalisation of prin-

cipial elements in culture media( 5 , 6 ) . Besides well-known

microelements , vanadium, nickel, tungsten, cobalt and gallium

had positive effect on growth and chemical composition of this

alga( 2 , 7 , 8 , 9 ). 0.2 ^uM titanium was added as Ti^SO^)^

in the generál nutrition médium of green algae( Chlorophyceae )

and blue-green alga SpirulinaC Cyanobacteria. ) ( 2 ).

The purpose of this study was to investigate the effect of

titanium ascorbate on growth, dry matter accumulation and prote­

in content of Chlorella pyrenoidosa green alga.

Materials and methods

Algal cultures

Axenic cultures of Chlorella pyrenoidosa green algae( strain

IAM-C 28. University of Tokyo ) were cultured in sterile pyrex3

glass vessels containing 500 cm of autoclaved inorganic liquid

médium. The médium consists of 2.02 g KNO^, 1.13 g KHpPO^, 0.87

g K2HP0v 0.24 g MgS04 , 0.034 g CaCl2 , 1.0 g NaHCO^ 0.56 mg Fe

added as ferric citrate, 1.43 mg H^BO^, 0.905 mg MnC^. 4 £^0 ,

0.111 mg ZnS04 . 7 ^ 0 , 0.0395 mg CuSO^. 5 H20, 0.0126 mg

Na2Mo04 . 2 H20 , 0.0115 mg NH4V03 and 0.0245 mg C o C N O ^ . 6 HgO

per litre.

The algae were kept at 25 °C and permanently illuminated2

with white fluorescent light( 18 W/m ). The cultures were supp-

- 78 -

lied with constant and definitive aeration( bubbling mixture of

sterile air and 5 % CC^ ) which served as carbon source and sta-

bilised the algal suspension homogenity and pH.

Treatment of algae with Ti-ascorbate and ascorbic acid

Growth of algal cultures was followed photometrically by

measurement of changes in optical density( OD ) at a wawelength

of 600 nm( 5 ). Untreated algal cultures, reached approximately

0.1 g dry weight/liter algal suspension( OD = 0.2 ), were homo-

genised well and divided intő pyrex glass vessels under axenic

conditions. The cultures were treated with 0.1 ^uM , 0.5 ^uM ,

1 yuM , 5 ^uM , 10 ^uM and 20 ^uM quantity of titanium as tita­

nium ascorbate. Control cultures remained untreated or were trea­

ted with 27.2 yuM , 136 ^uM , 272 ,uM and 544 ^uM amount of as­

corbic acid. Algal cultures were autotrophically cultivated fór

72 hours.

Determination of dry weight and relative protein content

of algae

3Dry weight of algae was determined by centrifuging 25 cm

of cell suspension( 500 g , 10 minutes ), washed twice with cold

bidistilled water and drying until constancy of weight at 90 °C

( 7 ).

3To measure proteins 400 cm of algal cell suspension was

harvested by centrifuging( 1500 g , 15 minutes ). The frosen

cells were ground in a mortar with quartz sand and 0.05 M po-

tassium-phosphate buffer( pH = 7.0 ) fór 30 minutes at 4 °C.

The ruptured cells were centrifugedC 15000 g , 30 minutes ) at

- 79 -

4 °C. Protein content of crude extract was measured by dye-bin-

ding protein assay method from triplicate readings( 10 ). Re-

sults were quantified from the standard equation fór bovine se-

rum albumin.

Each experiment was replicated three times and results were

evaluated statistically.

Results and discussion

Commercial plánt conditioning product called TITAVIT is a

water soluble organic complex of titanium ascorbate containing

1 g of Ti and 100 g of ascorbic acid/liter of titanium ascorba­

te. Fór this reason adding different micromolar concentrations

of titanium intő algal cell cultures we have added 27.2 times

higher micromolar amounts of ascorbic acid with titanium ascor­

bate solution at the same time.

100 yuM ascorbic acid causes 30 % inhibition of cell divi-

sion in Chlorella pyrenoidosa cultures( 11 ). Therefore investi-

gating the effect of titanium added as titanium ascorbate on

growth of Chlorella, algal cultures treated with proportional

ascorbic acid concentrations served as Controls besides untrea­

ted cultures. 27.2 ^uM, 136 ,uM, 272 ^uM, 544 ^uM quantity of

ascorbic acid corresponds to 1 ^uM, 5 ^uM, 10 ^uM, 20 ^uM amounts

of titanium present in titanium ascorbate.

Figure 1. and 2. show changes in optical density( 600 nm )

during 72 hours of propagation in titanium treated( added as ti­

tanium ascorbate in basal médium ) and ascorbic acid treated

cells of Chlorella pyrenoidosa green alga. 1 ^uM amount of tita­

nium significantly enhanced growth of algae. Lower concentrations

- 80 -

ura 009 •íiieuap x®3Tid 0

S9 IoO CNJ j

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,OJ O 2

^ - bű •»

o v£>M c nPm O rH

-s

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¥- °?l a f i<y -h £h O Eh

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- 81 -

( 0.5 , 0.1 ^uM Ti ) had no significant effect on algal growth,

while higher titanium concentrations( 5 , 10 , 20 ^uM Ti )

strongly inhibited the growth of Chlorella pyrenoidosa. Lower

ascorbic acid amounts( 27.2 ,uM AA ) had no effect on algal

growth, while ascorbic acid added at higher concentrations

( 136 , 272 , 544 ^uM AA ) inhibited the growth of algae.

Figure 3- and 4. show the effect of different titanium and

ascorbic acid concentrations on dry matter accumulation of Chlo­

rella cells after 72 hours of cultivation. Optical density mea-

sured at 600 nm shows close correlation with dry matter content

of algae. 1 ^uM titanium treatment enhanced dry matter producti-

on of algae significantly by 12.5 %( Table 1. ). Higher titanium

and ascorbic acid amounts decreased dry weight of algae.

Chlorella pyrenoidosa dry matter consists of 57 % of pro-

tein( 2 ), therefore enhancement of dry weight may alsó affect

protein amount in cells. Complete extraction of totál protein

content of algae is rather lenghty procedure or needs cell homo-

genizer to rupture all cells( 4 ). Data obtained by our protein

extraction method are suitable to compare relative changes in

protein content caused by titanium treatment after 72 hours of

cultivation( Figure 5. ). Algae treated with 0.5 /uM and 1 ^uM

titanium yield 5 % and 15 % higher relative protein content than

control cells, respectively.

Sunmarizing our results we can point out that 1 ^uM amount

of titanium added as titanium ascorbate in liquid inorganic mé­

dium stimulated growth of Chlorella pyrenoidosa, enhanced dry

matter accumulation and relative protein content of algae. Inhi-

bitory effect of higher titanium ascorbate concentrations are

- 82 -

Table 1.: Dry weigths of Chlorella pyrenoidosa after 72 hours

of propagation under authotrophic conditions

( 18 W/m2 , 5 % C02 in air , 25 °C )

TreatmentsDry weight

g/l

Relative dry weight

%

1. Control 1.200 100

2 . + 0.1

3. + 0.5

yUM Ti

it ii

1.220

1.260NS

101.7

105

4. + 1 II II 1.350x 112.5

5. + 5 II fi 1.110 92.5

6 . +10 II fi 0.990 82.5

7. +20 fi II 0.70 58.3

8 . + 27. 2 /UM AA 1.21 100.8

9. + 136 ti n 1.18 98.3

10. + 272 ii ii 0.95 79.2

11. + 544 ii ii 0.88 73.3

Ti = treated with different a/nounts of titanium as titanium

ascorbate

AA = treated with different amounts of ascorbic acid

Control = untreated

x = significant at the 5 % level

NS = nőt significant

- 83 -

1 i 1 i ' ¥ X / 3 B 2 t B j o m 8 i a M 2

toCo 01•HrH+* rHa3<DUO+*c a)a>©o oC "OO *H<«*XJ

oG<DM-HHZo ►» 0*? P, oT-4o aj 4•HrH ajH >a> *hOh -mo O rHroa}32o<M <MO <M Oo-i-> 03O 4-> ha>.n 3bű O-H.Cwa>£ (VJ t"-í- & h X} 0)o -*->MpHo aj

03 (DG O

+-> 03a) oh na+J T-«g o a> C o a>

r o co P« oS fl <0 í*3 r-Í öJVH rH >fi a> cd f-« +■»-*-> O rH •HH 3 +* <C ü

) +» h ) J2 3 < bű o1 -H

O M I ^ <

- 84 -

due to additive inhibitory effects of ascorbic acid and titanium

present in titanium ascorbate. Our new results show that 50 %

reduction of ascorbic acid content, chelating compound of tita­

nium ascorbate, did nőt influence stability of the complex. Ef­

fect of titanium ascorbate with reduced ascorbic acid content

on algal growth needs further investigations.

NC•HQ)

c o n t r o l 0,1 0^ 1 5 10 20J i . M Ti

PIG. 5.rRelative amount of protein in cella of titanium treated Chlorella pyrenoidosa

- 85 -

Investigating the effect of titanium ascorbate on uptake

and utilization of macro- and micronutrients in algal cells may

help us to understand the positive effect of this compound on

Chlorella pyrenoidosa growth and chemical composition.

Literature

1. LEM, N.W. , GLICK, B.R.(1985): Biotechnological uses of cya-

nobacteria. Biotech.Adv. J3 195-208

2. VENKETARAMAN, L.V. , BECKER, E.W.(1985): Biotechnology and

utilization of algae. The Indián experience. Sharada

Press, India 1-11 , 32-33 , 89 pp.

3. TCHORBANOV, B. , B0ZKH0VA, M.(l988): Enzymatic hydrolysis of

cell proteins in green algae Chlorella and Scenedesmus

after extraction with organic solvents.

Enzyme Microbiol.Technoi. 10 233-238

4. BERLINER, M.0.(1986): Proteins in Chlorella vulgáris.

Microbios. 46 199-203

5. RODRIGUEZ-LŐPEZ, M.(1964): Influence of the inoculum and

the médium on the growth of Chlorella pyrenoidosa.

Natúré 203 666-667

6 . FABREGAS, J. , HERRER0, C. , ABALDE, J. , CABEZAS, B.(1986):

The marine microalga Chlorella stigmatophora as a po-

tential source of single cell protein: enhancement of

the protein content in response to nutrient enrichment.

J. Industr.Microbiol. 1 251-257

7. MEISCH, H.W. , BIELIG, H.J.(1975): Effect of vanadium on

growth,chlorophyll formation and iron metabolism in

unicellular green algae. Arch.Microbiol. 105 77-82

8 . WELCH, R.M.(1981): The biological significance of nickel.

J. Plánt Nutr. 3 345-356

9. BALOGH, Á. , KISS, F. , SZABOLCSI, L.(1987): Influence of

gallium on growth of green and blue-green algae.

Proc. Internat.Tr.El.Symp., Budapesti Ed.: PAIS, I. )

127-132

10. BRADFORD, M.M.(1976): A rapid and sensitive method fór the

quantation of microgram quantities of protein utili-

zing the principle of protein-dye binding.

Anal. Biochem. 72 248-254

11. STAUBER, J.L. , FLORENCE, T.M.(1987): Mechanism of toxicity

of ionic copper and copper complexes to algae.

Marine Biology 94 511-519

- 86 -

- 87 -

EFFECT OF TITANIUM ON GROWTH AND PHOTOSYNTHETIC PIGMENT COMPO-

SITION OF CHLORELLA PYRENOIDOSA( green alga ). II. EFFECT OF

TITANIUM ASCORBATE ON PIGMENT CONTENT AND CHLOROPHYLL META-

BOLISM OF CHLORELLA

L. SIMON , F. HAJDÚ*, Á. BALOGH , I. PAIS**

Biological and Chemical Department of Teacher’s Training Colle­

ge, Nyíregyháza, Hungary

Central Chemical Research Institute of Hungárián Academy of

Sciences, Budapest

X XChemical Department of University of Horticulture and Food

Industry, Budapest

Introduction

Titanium is one of the most widely distributed elements in

natúré. In spite of the large amount of titanium present in the

soil this element is unable to produce readily soluble compo-

unds. Its concentration in the soil solution is very low

( 7.10~^ % ). High concentration of titanium in algae have been

reported. The alga Lithothamia sp. concentrates titanium to le-

vels thousand times greater than the level of titanium in the_ o

sea water is. The highest concentration found was 2.10 %( 1 ).

There are certain evidences that titanium may play a role

on photosynthetic processes of microalgae. Considerable amount

- 88 -

of titanium as a compound with substance similar to phospho-

pantotein was isolated from red alga Ahnfeltia plicata, brown

alga Laminaria japonica, green alga Ulva fenestrata and from

the flowering plánt Zostera marina. This titanium compound may

be involved in reductive processes of living cells, particularly

under conditions of reduced illumination( 2 , 3 , 4 ) . Titanium

ascorbate treatment enhanced oxygen-production and chlorophyll

A content of blue-green alga Anacystis nidulans, activating of

photosynthesis was supposed( 5 , 6 ).

At present, in spite of the employment of modern separa-

tion method( high-performance liquj.d chromatography, HPLC ) in

pigment analysis, very little is known about the catabolism of

chlorophylls in biological systems in vivő and in vitro. Besi-

des physicochemical processes, enzymatic and non-enzymatic fac-

tors may play role in biodegradation of chlorophylls. A number

of enzymes have been described with such possible roles bút the-

re is no known enzymatic reaction about which it is possible to

say with certainty that it has specific function in the degra-

dation of chlorophylls in vivo( 7 ).

Different oxidizing enzymes such as lipoxygenase, chloro-

phylloxidase and peroxidase have been described to have chlo­

rophyll degrading effect in vitro. Peroxidase is capable of

degrading both chlorophyll and chlorophyllide in the presence

of í^Og and monophenols( 8 , 9 ) . Action of non-oxidizing en-

zymes may alsó cause chlorophyll degradation. The major degra-

dation products in chlorophyll A metabolism are chlorophyllide

A, pheophytin A and pheophorbide A, which results from the loss2+ 2+

of phytol, Central bound Mg and both phytol and Mg , respéc-

- 89 -

tively. Chloroplast membrane bound chlorophyllase catalyses both

the hydrolysis( dephytolating of chlorophyll ) and transesterifi-

cation of chlorophylls and their derivates. This en2yme is pre-

sent in Chlorella sp. and is activated by cell disruption( 10 ).

There are certain evidences fór existence of an enzyme ca-

pable of catalysing removal of magnesium from chlorophyll and/or

chlorophyllide. Membrane bound magnesium-releasing enzyme( MRE )

in conjunction with chlorophyllase formed all the major chloro­

phyll A degradation products in marine diatoms( 11 ). MRE was

activated by desintegration of cells and its activity was asso-

ciated with pigmented membranes. The enzymatic reaction was inhi­

bited by M g C ^ and proved completely heat labile. The reaction

occurs at neutral and basic pH which exclude the non-enzymatic-

removal of Mg2+( 11 ). Chlorophyllide A degrading enzyme showing

Mg-releasing activity catalyzing the formation of pheophorbide A

and pyropheophorbide A was observed in Chlorella mutant( 12 ).

The aim of our work was to investigate the effect of tita­

nium ascorbate treatment on photosynthetic pigment content, pig­

ment composition and chlorophyll metabolism of Chlorella pyre­

noidosa green alga.

Materials and methods

Solvents

All organic solvents used fór pigment extraction were ana-

lytical grade, all chromatographic solvents were HPLC grade.

Culture conditions

Chlorella pyrenoidosa green algae were propagated in 2000

- 90 -

cm Erlenmeyer flasks fór 72 hours under authotrophic conditions.

Treatment of algae with 1 ^uM amount of titanium( as titanium

ascorbate ) and culture conditions were detailed in PART I.

Extraction of pigments

3Fór extraction of Chlorella pigments 50 cm of cell suspen-

sion was harvested by centrifugation( 1000 g , 15 minutes ). The

harvested cells were washed with distilled water and used fór

pigment analysis. The pigments were extracted with a mixture of

chloroform-acetone-isopropyl alcohol( 2 :1 :1 , v/v ). 0 .2-0.5 g

of frozen algal cells were ground in a mortar with quartz sand

at 4 °C using 100 cm^ of the solvent mixture. The pigment filt-

rates were collected and dryed over anhydrous Na^SO^. Absorption

spectra were recorded between 350-750 nm using BECKMAN 26 spec-

trophotometer.

HPLC analysis and identification of pigments

The solvent of extracted pigments was evaporated to dryness

under reduced pressure at 35 °C. The dry pigments residue was 3

redissolved in 2—4 cm of isopropyl alcohol-acetonitrile-water

( 60:35:5, v/v ). 20 ^ul of pigment solution was injected fór

HPLC analysis.

ISC0 HPLC system was used fór the analysis with the follo-

wing parameters:

Column: C^g.10 ^um , 250 x 4.6 mm( Labor MIM )

Mobile phase: isopropyl alcohol-acetonitril-water

( 60:35:5 v/v )3

Flow rate: 1 cm /min

3

- 91 -

Detection wawelength: 440 , 660 nm

Chart speed: 0.5 cm/min

Multiple channel detector enabled us to detect pigments at

440 and 660 nm at the same time. Since carotenoids and xantho-

phylles do nőt make sign at 660 nm, we can distinguish that two

groups of photosynthetic pigments from chlorophylls and their

derivates easily. Individual pigments were identified according

to their spectral characteristics by HPLC scanning technique

( fór details see 13 ).

Enzyme preparation

Method of SHI0I( 10 ) was followed by modifications to pre-

pare membrane bound protein extract showing chlorophyll degra-3ding enzyme activity. 2000 cm of well multiplied untreated al­

gal suspension was harvested by centrifuging( 2500 g , 10 minu-3

tes ). Cell paste( 15 g wet weight ) was suspended in 60 cm of

potassium-phosphate buffer( 0.01 M , pH 7.2 ) and ground thoro-

ughly in a mortar with quartz sand fór 60 minutes at 4 °C. 1 %

of NaCl and 60 cm^ of ice-cold n-butanol( - 18 °C ) was added

to the homogenate which was further rnixed fór 2 minutes. The

aqueus solution and the butanol fraction was then collected and

centrifuged again at 12000 g fór 10 minutes. (NH^^SO^ was added

to the butanol fraction to give 30 % saturation. The solution

was centrifuged( 10000 g , 20 minutes ) and the protein paste

formed was collected. The precipitate was dissolved in a small

volume of 0.01 M potassium-phosphate buffer( pH 7.2 ) and clari-

fied by centrifugation at 10000 g fór 10 minutes. The (NH^^SO^

preparation was desalted using Sephadex G-15 column( 42 x 1.5

cm ). The protein containing activate fractions were pooled and

*

concentrated with alysis against 50 % sucrose containing po-

tassium-phosphate buffer( 0.01 M , pH 7.2 ). Each procedure was

carried out at 4 °C.

HPLC assay fór chlorophyll degrading activity

350 cm solution of extracted pigments was taken and the

solvent was evaporated under vacuum at 35 °C. The dry residue3 3

was redissolved in 3 cm of chloroform and 0.2 cm of Triton

X-100. The solvent was evaporated again and the pigment residue

was suspended in 9.3 cm of potassium-phosphate buffer( 0.04 M,

pH 7.0 ). The mixture was placed in ultrasonic bath fór 1 minu-

te. The chlorophyll degrading activity was started by adding3

0.5 cm of protein extract with vigorous shaking. The reaction

mixture was placed in dark and incubated at 25 °C. 20 ^ul sam-

ples were injected intő HPLC column at 1, 25, 50 and 75 minutes

of reaction time. The chlorophyll degrading activity was ex-

pressed as the decrease in the peak area of chlorophyll A and B

measured by HPLC at 660 nm. The area of peaks was quantified by

computing integrátor.

Results and discussion

Chlorophylls, carotenoids and their derivates are compo-

nents of photosynthetic membranes and constitute 3-5 % of the

dry algal biomass( 14 ). Chlorella sp. contains 3.77 mg/l chlo­

rophyll A and 0.79 mg/l chlorophyll B as determined by HPLC

( 15 ). The analysis of the pigment composition of green algae

is rather difficult considering the diverse polar characteris-

tic of the pigments. Applying new solvent mixture( chloroform-

acetone-isoprop.yl alcohol, 2:1:1 v/v ) fór extraction of Chlo-

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- 93 -

rella pigments we got good extraction of xanthophylls with re-

latively high polarity, chlorophylls with médium polarity,

pheophytins, carotenes and their derivatives, which are of low

polarity. Ou t new HPLC method elaborated fór separation of pig­

ments of Chlorella took only 25 minutes and sufficient resolu-

tion of 9 pigments was achieved. By this method the main xantho­

phylls of green algae such as neoxanthin, violaxanthin, lutein-

epoxide and luteine were separated without applying gradient

elution together with other photosynthetic pigments of Chlo-

rella( 13 ) ( Figure 3- )•

1 ,uM quantity of titanium added as titanium ascorbate in

nutrient médium significantly enhanced pigment amount of Chlo-

rella( Figure 1. and 3- )• Comparing peak areas of individual

pigments and quantities of algal cells used fór HPLC analysis

we found that chlorophyll A and B content was enhanced by 26 %

and 20 %, totál amount of xanthophylls and carotenoids was en­

hanced by 20 % in titanium treated cells.

Fór better understanding chlorophyll metabolism in tita­

nium treated and control pigments of algae a model experiment

was done. We have prepared a protein extract containing chlo-

rophyll-degrading enzyme which - to all probability - contains2+

magnesium-releasing enzyme. MRE remove Central bound Mg from

chlorophylls and form pheophytins. Absorption spectra of Ti-

treated and untreated pigment extracts are shown in Figure 1.

Peaks at 431 nm and 663 nm are characteristic fór chlorophyll A.

After 75 minutes of enzymatic degradation the absorption spectra

were shifted, peaks at 410 nm and 667 nm are characteristic fór

pheophytin A( 16 ). Degradation of Ti-treated and untreated

ra0*öo mM mC-Krf • • 1 d 3 OjjO ro

>o O,& SSi (8 -o o13«-g">CL B «t■ m (0 < t í O rt- 3 O «3 •.

c f O ® r f

^2 -0 0U1

•-3 _ °íz a -% >-*•

\ o ^ fi =rcf M <D O CO HJ

®O M I *-* M

o >-*.*0 ’TÚ

FIG. 3.: Reveraed-phaae HPLC chromatograma of titanium treated/1 uM/ and control pigmentB of Chlorella green alga.Coluon:Cbromail C 10 , Eluent: iaopropyl-«le.obol-acetonltril-H,o«60:35: 5 y/v,Flow rate:l ml/min Detectlon: 440 nm.bbO nm. ' *

- 96 -

algal pigments incubated with chlorophyll-degrading enzyme was

detected by HPLC technique at 660 nm. The reaction mixture con-

sists of extracted pigments, solubilizator, buffer and chloro­

phyll degrading protein extract. New derivatives of chlorophyll

A and B occurs during enzymatic degradation of Ti-treated pig-

ments( Figure k . ), diverse mechanism of chlorophyll metabolism

is therefore supposed. Relative peak areas of chlorophyll A and

B determined by HPLC were compared during enzymatic degradation

of Ti-treated and control pigments( Figure 5. and 6 . ). While

control chlorophyll A was almost totally degraded, 68 % of ti­

tanium treated chlorophyll remained unaffected( Figure 5. ).

Clorophyll B was unaffected by protein extract containing chlo-

rophyll-degrading enzyme, and there was no significant differen-

ce between treated and untreated pigments( Figure 6 . ).

Since photosynthetic pigment composition of algae and hig-

her plants is very similar( 16 ) this experiment is suitable fór

modelling chlorophyll metabolism in leaves and crops of higher

plants treated with titanium ascorbate( TITAVIT ). Probably bet-

ter utilization of magnesium and iron stimulated pigment bio-

synthesis in titanium-ascorbate treated Chlorella cells and

increased amount of carotenoids or higher amount of Mg present

in algae cells, and protected chlorophylls from enzymatic des-

truction in vitro. Verification of this hypothesis needs further

investigations.

- 97 -

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Literature

1. SKHOLNIK; M.Y.Y.(1984): Developments in crop Science.

Trace elements in plants. Elsevier , 284— 286 pp.

2. GRYZHANKOVA, L.N. , B0ICHENK0, E.A.(1975): Study of titanium

compounds in plants( in Russian ).

Fiziol.Rastjenij 22 1177-1181

3. GRYZHANKOVA, L.N. , LAKTIONOVA, N.V. , BOICHENKO, E.A. ,

KARYAKIN, A.V.(1973): Distribution of polyvalent metals

in different types of algae( in Russian ).

Okeanolog. 13 611-614

4. GRYZHANKOVA, L.N. , UDJELNOVA, T.M. , BOICHENKO, E.A.(1975 ):

Iron, manganese and titanium compounds in marine

plants( in Russian ).

Izv.Akad.Nauk SSSR, Ser.Bioi. 1_ 76-82

5. KISS, F. , DEÁK, Gy. , FEHÉR, M. , BALOGH, Á. , SZABOLCSI,

L. , PAIS, 1.(1985): The effect of titanium and galli­

um on photosynthetic rate of algae.

J. Plánt Nutr. 8 825-831

6 . KISS, F. , BALOGH, k . , SZABOLCSI, L. , PAIS, 1.(1987):

Effect of titanium ascorbate on Anacystis nidulans

( in Hungárián ). Acta Acad.Ped. ( in press )

7. HENDRY, G.A.F. , HOUGHTON, J.D. , BROWN, S.B.(1987): The

degradation of chlorophyll - a biological enigma.

New Phytol. 107(11) 270-272

- 100 -

8 . MATILE, P.H.(1980): Catabolism of chlorophyll: involvement

of peroxidase? Z. Pflanzenphys. 99 475—478

9. KATÓ, M. , SHIMIZU, S.(1985): Chlorophyll metabolism in hig­

her plants VI. Involvement of peroxidase in chloro-

phyll-degradation. Plánt Cell Physiol. 26 1291-1301

10. SHIOI, Y. , SASA, T.(1986): Purification of solubilised

chlorophyllase from Chlorella protothecoides.

Meth. Enzymol. 123 421-427

11. OWENS, T.G. , FALKOWSKI, P.G.(1982): Enzymatic degradation

of chlorophyll-A by marine phytoplankton in vitro.

Phytochem. 21_ 979-964

12. ZIEGLER, R. , BLAHETA, A. , GUHA, N. , SCHÖNEGGE, B.(1988):

Enzymatic formation of pheophorbide and pyropheophor-

bide during chlorophyll degradation in a mutant of

Chlorella fusca. J. Plánt Physiol. 132 327-332

13. SIMON, L. , HAJDÚ, F. , BIACS, P.(1988): Separation and de-

termination of Chlorella photosynthetic pigment by

HPLC. Intemat.Conf.Biochem.Separation, Keszthely,

Hungary( in press ).

14. VENKATARAN, L.V. , BECKER, E.W.(1982): Biotechnology and

utilisation of algae, The Indián experience.

Sharada Press, India 102 pp.

15- SART0RY, D.P.(1985): The determination of algal chlorophyl-

lous pigment by HPLC and spectrophotometry.

Water Rés. 19 605-610

- 101 -

16. LICHTENTHALER, H.K.(1987): Chlorophylls and carotenolds

Pigments of photosynthetic biomembranes.

Meth. Enzymol. 148 350-382