Borowitzka, Huisman, Osborn - 1991 - Culture of the Astaxanthin-producing Green Alga Haematococcus Pluvialis1. Effects of Nutrients on g

8/17/2019 Borowitzka, Huisman, Osborn - 1991 - Culture of the Astaxanthin-producing Green Alga Haematococcus Pluvialis1.…

1/10

Journal

of

Applied Phycology 3: 295-304, 1991.

© 1991

Kluwer

Academic

Publishers.

Printed in Belgium. 295

Culture of

the astaxanthin-producing green alga Haematococcus

pluvialis

1.

Effects of nutrients

on

growth and cell type

Michael

A.

Borowitzka,

John M. Huisman

& Ann

Osborn

Algal Biotechnology Laboratory, School

of

Biological

and Environmental

Sciences, Murdoch University,

Murdoch,

W.A. 6150, Australia

Received 5 July 1991; revised 25 July 1991; accepted 26 July 1991

Key words:

Haematococcaceae,

palmella,

aplanospore,

acetate, temperature, nitrogen,

phosphate

Abstract

The freshwater green alga

Haematococcus

pluvialis

(Strain

Vischer

1923/2)

grows best at high nitrate

concentrations

(about 0.5 to 1.0 g 1-1 KNO

3

), intermediate

phosphate

concentration (about 0.1

g -

1

K

2

HPO

4

) and

over

a

wide

range of Fe concentrations. Low nitrate or high

phosphate

induce the for-

mation

of

reddish

palmella

cells and aplanospores.

Mixotrophic growth

with

acetate improves growth

rate and

final

cell

yield,

and

also stimulates

the formation

of

the

astaxanthin-containing

palmella

cells

and aplanospores.

H.

pluvialis cannot

grow

above about 28

°

C,

or

above

a

salinity of approximately

1

w/v NaCl.

An increase

in temperature

or

the addition of NaCl

also

stimulates the formation

of palmella

cells and

aplanospores.

Introduction

Several species of

algae

accumulate high

concen-

trations

of carotenoids such

as

f-carotene, astax-

anthin and canthaxanthin under certain

condi-

tions (Borowitzka,

1988a). Of

these, the green

halophilic flagellate Dunaliellasalina,

which

accu-

mulates > 10

of

dry

weight

as f-carotene, is

the

best known and is used as

a

commercial source

of this carotenoid (Borowitzka & Borowitzka,

1988a).

Recently,

there

has been

increased

inter-

est in microalgae

which

accumulate the ketocar-

otenoid astaxanthin;

these

include species

of

Chlamydomonas, Euglena

and

Haematococcus

(Viala,

1966;

Borowitzka, 1988a; Grung etal.,

1990).

Haematococcus (Chlorophyta, Haemato-

coccaceae)

is

of most interest

since it

can

be

cul-

tured easily and contains > 1% of

dry

weight

astaxanthin,

mainly

in the

form

of the

3S,3 S

mono- and

di-esters

(Renstrom

etal.,

1981;

Grung

et

al., 1990). This

astaxanthin

is

accumu-

lated

in

the

perinuclear

region

of

the

cytoplasm

in

the palmella cells and aplanospores

(Sprey,

1970;

Santos

& Mesquita,

1984).

Astaxanthin is the

preferred

pigment for use

in

the feed of salmonid fish

such as trout and salmon

(Foss

et al.,

1984; Storebakken et

al.,

1987), and

Haematococcus

is being

considered as a

possible

natural

source

of

this pigment (Sommer et

al.,

1991).

There

have, however,

been

few

studies

of

the nutrient and

culture

condition

requirements of

Haematococcus,with

particular emphasis

on

those

requirements which result

in

optimal

production

of

astaxanthin (Pringsheim,

1914, 1966; Droop,


2/10

296

1961). This

paper

is the first

of a series

which

systematically examines

the requirements for

growth

and

carotenogenesis

in

this

alga,

with the

aim

of

determining

the optimum

conditions for

mass

culture

and astaxanthin

production.

contains

some

astaxanthin

in

the

perinuclear re-

gion, and (iii) non-motile,

thick-walled

aplanos-

pores, which become

completely reddened due

to

the accumulation

of

astaxanthin

in the cytoplasm

(Elliot, 1931).

These

three cell

types

were counted

separately.

Materials and

methods

A

culture

of

Haematococcuspluvialis (Strain Vis-

cher

1923/2) was

obtained from the culture col-

lection

of he

Czechoslovak

Academy of Sciences,

Institute of

Botany,

Tr6bon.

This

strain was iso-

lated originally by

Vischer in Switzerland

and

is

now

lodged in

the

Murdoch

University Microal-

gal

Culture collection

as strain

MUR-

1.

A

second

isolate, isolated originally

by Droop

from

a rock-

pool

in

Finland, was

obtained from the

Gottingen

Algal Collection (their

number 34-1h) and is

now

incorporated

in our

collection

as MUR-64.

Experiments were

carried out in MCM me-

dium

in

50 ml cultures in

a temperature

controlled

cabinet with

a 10:14h day:night

cycle and

a

20

C: 15

C

day:night

temperature

range.

Light

was

provided by cool-white fluorescent

lamps at

an

irradiance

of approximately

120 mol

photons m-

2

sec-

'

(PAR). MCM

medium con-

tains

the following

(mgl- ): KNO

3

,

200;

K

2

HPO

4

, 20; MgSO

4

.7H

2

0,

100; CaC1

2

.6H

2

0,

80; Vitamin

B

12

,

0.004;

EDTA, 0.0198;

FeCI3.6H

2

0O,

.0244.

1ml of trace

element

mix

(containing

in mgl- l: ZnC12, 4.1; H

3

BO

3

,

61;

CoC1

3

.6H

2

0, 5.1; CuSO

4

.5H

2

0, 6.0;

MnC1

2

.4H

2

0, 4.1;

(NH

4

)

6

Mo70

2 4

.4

2

0, 38.0)

was

added

and the

pH adjusted

to

pH

7.0 after

autoclaving.

For

the

experiments

reported

here

the KNO

3

and/or K

2

HPO

4

concentration

of the

medium

was varied

as

required

and Fe

concen-

trations

were

modified by different

additions

of

EDTA-chelated FeC13.6H

2

0. In

some

experi-

ments

the

KNO

3

was

replaced with

either NH

4

C1,

NH

4

NO

3

or urea to examine the effect

of

the

different N sources.

Cell counts were carried out

microscopically

using a

haemacytometer.

Haematococcuspluvialis

can exist

in

any one

of three

cell

types: (i)

green,

flagellated macrozooids, (ii)

palmella

stage

which

Results

Nitrogen andphosphate

Over

the range of KNO

3

concentrations

tested

(0.01 to 1.0

g 1-

1

; 0.123

to 12.3 mM)

growth

was

best

between

0.05

and

0.5

gl-

1

(Fig.

1).

At

1.0 g 1-

1 the

final cell yield

was

reduced

com-

pared

with

the lower

KNO

3

concentrations.

The

lowest nitrate culture

(0.01

g

l-')

reached the

maximum

cell

number at about

7 days and had

completely

reddened (palmella)

by

10

days. In the

0.05

g 1-

1

culture,

visible

carotenoid

accumula-

tion began after 21 days whereas

in the other

cultures

only

isolated reddish palmella

cells

and

aplanospores

were observed.

In the 0.01gl-

KNO

3

culture a few

isolated aplanospores

were

observed after 38 days.

The

effects of

phosphate concentration

were

studied over

the range of 0.001

to 0.2

g 1K

2

HPO

4

(0.0048 to

0.975 mM)

(Fig. 2).

Growth rate and

cell yield were similar

at all concentrations used.

In general, the

maximum

cell

number

was reached

after

10 days

of

growth, by

which time the high-

est

phosphate culture (0.2 g 1-

1) consisted com-

pletely

of

reddish palmella

cells.

Visible

carote-

noid formation and palmella

formation

in

the 0.1

and

0.05

g 1-

cultures began

at about

day

18,

however by

day 21

these

cultures still contained

significant

amounts of

green

motile

cells; at lower

phosphate concentrations no reddish palmella

cells nor aplanospores

had

formed

by

day 21.

In order

to

examine whether

there

was any

synergism

between NO

3

and

PO4 concentrations,

a

factorial

experiment,

varying the

concentration

of

both of

these

nutrients, was carried out. Four

treatments

were used (see Fig.

3).

Growth

rate

and final

cell yield were

similar

in

all treatments

(Fig. 3), with the exception

of

the high

N/high

P


3/10

0.01 g.1

-

l

i

0O5

a.1-1

1000glE1310lll~l13

°

I I I I

I I

I

I I

I I I I I

I I

I

I

a

1-1

.

[ 1n 01

I H In]

3 1 0 0 0

I I I

Il

0 5

10

16 20 060 36 40 46

1.0 g.l

Time (Days)

0

100

n~~1

4

m

j

o

lu

0 I I 20 2 6 40 4

o 1 1 2 25

3o

5

o 4

Time (Days)

Fig.

1.

Effect of nitrate concentration

on

growth and cell

type in H.

pluvialis MUR-1. Open bars

=

green

macrozooids;

Dashed

bars = reddish palmella cells; solid bars = aplanospores.

culture

where

the

final cell yield

was

slightly

higher

(Fig. 3D).

Accumulation of

carotenoid and

apl-

anospore formation

was first observed

in the

low

N/low

P

culture

at

day 17 (Fig.

3A)

and

in the low N/high P culture

at

day 21 (Fig. 3B).

The high-NO

3

cultures contained

only

very

few

palmella cells and aplanospores

by day

21

(Figs.

3C,D).

In order to

examine

the

effects

of different ni-

trogen

sources,

we

grew both

MUR-1

and MUR-

64 with

0.01 M ofeither

KNO

3

,

NH

4

Cl,

NH

4

NO

3

or urea (Figs.

4 and

5).

Both MUR-1

and

MUR-

100000

297

u

10000

1000

100

1ooooo 0.1 g.1 I

N

1z

U

U

0

01

U

0

0

10000

1000

100

100000

10000

1000

100

As

------

.

.


4/10

0.001 g 1

d

jo:u0hUw

l

0.005 g.l1

/

-[ 1 11B

Blti

:

36

TI C

K

I I I I I I

I I I I I

I I

I

I I I I

I

1-

XU

6 c

IU

M~ I

inn

~H

I

I

I I I I I

I i-- I

-I I

I- I I

_,-~ ~0 6

10 15

20 25 0 05

Time

(Days)

100

6o0

5 1

O5

0

6

so 6 40 6

Time Days)

Fig.

2.

Effect

of phosphate concentration on

growth

and

cell

type

in

H.

pluvialis

MUR-1.

(Bars as

in Fig.

1).

64

grew fastest

on

urea and slightly slower

on

ammonium

chloride

or ammonium

nitrate.

Am-

monium

nitrate,

however, was

the least

suitable

N

source for growth for

both strains,

resulting

in a

reduced

maximum cell

number; all the

other N-

sources had

similar

final cell yields

by day 21. The

formation

of red palmella

cells

and

aplanospores

was

stimulated when KNO

3

was the N

source,

followed

by

urea; ammonium chloride inhibited

the formation

of palmella

cells and aplanospores.

Iron

Three concentrations of iron (0.0244,

0.122,

0.244

mg

1- ), added

as EDTA-chelated

FeCl

3

.6H

2

0, were

tested. The

results are shown

in Fig. 6. There were

no significant differences

between the three treatments and,

after

21 days,

none of the cultures showed any major degree of

reddening.

298

IVUUUU

N

m

='

~3

10000

1000

10 0

100000

0.05

I-L

/

0000

1000

100

100000

a

IN

U

O

10000

1000

10 0

I

i.

A

.

0 .2 gl'~

r

_

inn


5/10

299

A

feg*

IO t

B

10rf

11 10 Kt

I I

I I I I I I I I I I

I I I I I

-

1

flfll

a

1111

to

111 t

-

_ _ i I I I I- i

- I I I I I I

I I I

0 6 1 1O

0

25 5

40

45 0 6

1 20 as O S6 40 45

Time Days)

Time

(Days)

Fig. 3. Effect of nitrate

and phosphate concentration on growth

and cell

type in H.

pluvialis

MUR-1. (A) 0.05 g

1- '

KNO

3

+

0.005

g I-' K

2

PO

4

; (B)

0.05

g l-

KNO

3

+

0.05 gl-

K2PO

4

; (C)

0.15 g

- KNO

3

+0.005 gl-

1

K

2

PO

4

;

(D)

0.15 gl-l KNO

3

+

0.05

g 1- ' K

2

P0

4

. (Bars as in Fig. 1).

Acetate

andpH

Haematococcus

has been shown to be able to grow

better mixotrophically on

acetate (Pringsheim,

1966). The effect of the addition of 0.1 g 1-1 ac-

etate

was therefore examined

at

two

pH

values.

The

medium was not

strongly

buffered so that

by

the end of the experiment the pH had risen mark-

edly in all cultures; i.e. in the acetate-containing

cultures the medium

pH at

day 30 was between

9.4 and 9.8, and in the acetate-free cultures

it

was

between

pH

10.9

and 11.1. Acetate markedly

en-

hanced the growth rate

and also

induced

the for-

mation

of red palmella

cells and

aplanospores

(Fig.

7).

Cell yield was slightly higher in

the

pH

6.5

+

acetate

culture,

however at this pH pal-

mella

formation

was slower

than

in

the

pH

7.5 + acetate culture; i.e. by day 9 the pH 7.5

culture

containing acetate

consisted only

of

pal-

mella

cells,

whereas this took 20 days in the

pH 6.5 culture

(Fig. 7). The

cultures

without ac-

etate had formed almost

no

palmella cells

by

day

30

when

the experiment

was

terminated

(Fig.

7)

and they

also

grew

slower;

the pH 7.5

culture,

however, eventually reached a final cell

density similar

to that

of the

pH 6.5

culture

con-

taining acetate.

Temperature

and

salinity

The temperature

tolerance

of both

strains

was

examined in order to determine the optimum

tem-

perature and the

lethal

temperature.

Figure

8

100000

10000

1000

U

100

100000 I

I

10000

1000

0

U

100


6/10

SA

0~~~~~

:1

10

00C:

[j

I I I I

NH Cl

Loo

1

.

Din Ureu

4NU

urea

0~~~~

I~~~~~~~~~~~~~~

jn n lfl It Hamfl

1l

0

*1°

I I I I I I

0

5 10

15

20

20

0

5 t10

15 0

35

Time

(Days) Time (Days)

Fig.4. Effect of nitrogen source on

growth

and

cell

type in H. pluvialis

MUR-1. Growth curves for

duplicate cultures

are

shown

and the

%

cell type

is

a

mean value of these duplicates (Bars

as in Fig.

1).

shows

the effects of temperature

on growth and

cell

type

in MUR-1.

The

alga grew best at the

lowest

temperature tested,

15

C, and

also

grew

well at 25 C,

however

at

28 C growth was

strongly

inhibited and

at 35 C

the culture

died.

Increasing

temperature

also

induced

aplanospore

formation

and,

at

28 C,

all the

cells

had become

aplanospores

by

day

21

(Fig.

8).

Strain

MUR-64

gave

almost

identical results.

Both

strains

of H.pluvialis

had a

very low sa-

linity tolerance;

1

w/v NaCl, when added to

the

medium, reduced growth and enhanced aplano-

spore formation,

and

higher salinities proved le-

thal.

Discussion

Previous studies on the

nutrition

of Haemato-

coccus pluvialis

have

shown the

complex

relation-

ships between nutrients,

growth,

cell

yield, cell

type and

astaxanthin formation

(e.g. Jacobsen,

1912;

Lwoff & Lwoff,

1929; Droop,

1954,

1961;

Pringsheim, 1966).

The

concentrations of

nitrate and

phosphate

could

be varied over a

wide range

without

having

a significant effect

on growth rate

or

cell yield.

However,

the

results

reported

here show

that

low

nitrogen

concentrations

or high phosphate con-

centrations

stimulate the formation

of

red

pal-

mella cells in H.

pluvialis.

The

factorial

experi-

ment, in

which

both

nitrate and phosphate

concentrations

were varied,

shows

that the main

factor

leading

to carotenoid

accumulation is

ni-

trogen

starvation

(i.e.

when protein

synthesis is

reduced)

and

this is

stimulated

by low

phosphate

concentrations. This

is similar

to

the

observa-

tions of

Droop

(1955)

on H. pluvialis, and is

also

similar to Dunaliella

salina where

nitrogen star-

300

100000

10000

1000

S

1-

a

c

100

100000

10000

1000

.

i)

U

10 0

WIn

~ ^ "-


7/10

an NH CI

.

VS

_l.u

a

5 O

Ig'flflfl UHOU~fl 1

H

I .I I

I

I

I

I

.

,~fl

£IU

1

501

U

H

did

0 :

n 5 *

I I

I I

I I I

I I

I I I

0

5 10 15 20 25 0

10

15

2

25

Time (Days)

Fig.

5. Effect

of nitrogen

source on growth

and cell

types

of H.

and

the %

cell

type is a

mean

value

of these duplicates.

(Bars

vation accelerates

the

rate of

-carotene forma-

tion (Semenenko

& Adullayev,

1980;

Ben-Amotz,

1987) and to many other

algae where

N-starvation

induces

the

formation

of

lipids (Piorreck etal.,

1984; Borowitzka,

1988b).

The

best

nitrogen source

for

carotenoid

forma-

tion and

cell yield in

H.

pluvialis

is

nitrate,

al-

though growth

rate is reduced

compared

to other

N-sources;

however, the

final cell

yield is similar

to that reached

with other N-sources.

Ammo-

nium

chloride

gave good

growth rates but

inhib-

ited

carotenogenesis,

and ammonium

nitrate was

a less

effective

N source

for both

growth

and

carotenogenesis.

H. pluvialis

also grows well

on

urea;

however,

carotenogenesis

was slightly

in-

hibited

in

the urea-grown cultures

compared

to

the nitrate-grown

cultures.

Previous

studies of

Time (Days)

pluvialis

MUR-64. Growth

curves

for duplicate

cultures are

shown

as in Fig.

1).

H.

pluvialis

have

also shown that nitrate-N

is pre-

ferred to

ammonium-N

(Proctor,

1957), although

Stross

(1963)

noted that

exponentially

growing

cells

at acid pH preferred

ammonium.

H. pluvialis

has

been reported to differ

from most other mi-

croalgae in preferring

nitrate

to ammonium, at

least

in dilute laboratory

culture

(Syrett, 1962).

Our

results

do

not

support

this,

and this

may il-

lustrate that

there are

strain differences

in

the

mechanism

of nitrogen

utilization in H. pluvialis

(cf. also

Stross, 1963).

However,

for

high cell

density,

rapidly

growing

algal

cultures, nitrate

is

probably

the better N source

since under

these

conditions

ammonium

may

lead

to

cell death

be-

cause of

the rapid

acidification

of

the

medium

resulting

from

ammonium

uptake and metabo-

lism

(Borowitzka

& Borowitzka,

1988b).

Like

100000

3 1

N

a

a

u

10000

1000

100

lUUUoo

10000

1000

N

U

100

------


8/10

0.0244

mg.l

LN

-I .. 1

.

I....

~~IW000 [188~~~0000000 O BB~~~t0000000

O B n 10

I I I I

I I I I

I

I I .I I

I

I

I I I

I I

I

p

~~~o

102 S54 450

0 1 10000244

S

1

10o

so0Z5

3 0

aIst

0

0

10 120 25 -q 35 40 45 0 0 10 1520

25 0 *q 40 45 0 § 10 120 25 30 35 40 45

Time

(Days)

Time

(Days)

Time

(Days)

Fig.

6. Effect

of EDTA-chelated

FeC13

concentration on growth

0.122

mgI- ; (c)

0.244 m g- . (Bars

as in Fig.

1).

Dunaliella

salina, carotenoid formation

is inhib-

ited

in ammonium grown cultures (Goldman

etal., 1982; Borowitzka

&

Borowitzka,

1988b).

Haematococcus is able to grow over

a

wide

range

of iron concentrations,

and

low concentra-

tions of iron

do

not

stimulate

carotenogenesis.

None

of the concentrations of

iron

tested were

high enough

to

be

toxic.

As observed previously

by Droop

(1961)

and

Pringsheim

(1966),

acetate

appears

to be an

im-

portant carbon

source,

enhancing both growth

and

carotenogenesis. Acetate

has

also been

shown to enhance

growth

of

other freshwater

algae,

such

as Chlorella, in the light (Endo

et

al.,

1977). The

effect of

acetate

is,

however, influ-

enced by

pH and, at

pH 7.5, palmella and apl-

anospore

formation is delayed. In many algae, the

light-dependent

carbon assimilation from acetate

begins

with

an

ATP-dependent

activation

by

ei-

ther acetyl-coenzyme

A synthethase as in

Euglena

gracilis,

or

an acetate

kinase as in Chlorellafusca

and Scenedesmus

(Wiessner, 1979).

The acetate

is

then

incorporated

predominantly

into lipids.

A

higher rate of lipid

formation

could therefore

ac-

count

for the more rapid

reddening of the acetate-

grown

Haematococcus

cells.

The slower pH

rise of

the medium

of the acetate-grown

cells can

also be

explained

by the fact

that CO

2

is liberated

during

acetate

oxidation.

This

would

reduce the require-

ment for exogenous

CO

2

for

photosynthesis

and

and

cell

type

in

H.

pluvialis

MUR-1. (a)

0.0244

mg

1-1;

(b)

thus

would lead to

reduced

alkalinization

of the

medium

as less CO

2

would be

taken

up.

The

differences between the pH

6.5 culture and the

pH 7.5

culture are more difficult

to

account

for,

but

may relate to different

rates

of acetate

uptake

at the two

initial

pH values (cf. Syrett, 1962).

The results

presented here suggest

that nitrate

is

the best

N source

for commercial culture of

H.

pluvialis for

the production of astaxanthin, with

the

yield

of astaxanthin amenable to

manipula-

tion by

altering

the nitrogen

concentration.

The

addition

of acetate

further stimulates caroteno-

genesis without

apparently

affecting growth rate

and

final cell yield

significantly.

Although the

ad-

dition

of vitamins such

as

thiamine

has also been

shown to stimulate growth (Pringsheim, 1966),

this would

not be feasible

in

large-scale

open

air

cultures,

although it could

be

envisaged

for

fermenter-grown cultures. Large-scale

culture

must

also carefully

control the

maximum

temper-

ature

reached by the

culture

since growth rate

is

reduced

at higher

temperatures.

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Documents

Borowitzka, Huisman, Osborn - 1991 - Culture of the Astaxanthin-producing Green Alga Haematococcus Pluvialis1. Effects of Nutrients on g