respiration rates in heterotrophic, free-living protozoa

Microb Ecol (1983) 9:99-122 MICROBIAL ECOLOGY �9 1983 Springer-Verlag

Respiration Rates in Heterotrophic, Free-living Protozoa

T. Fenchel I and B. J. Finlay 2

tInstitute of Ecology and Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark; and 2Freshwater Biological Association, Windermere Laboratory, The Ferry House, Ambleside, Cumbria LA22 0LP, England

Abstract. Published estimates of pro tozoan respiratory rates are reviewed with the object o f clarifying their value in ecological studies. The data show a surprisingly large variance when similarly sized cells or individual species are compared. This is a t t r ibuted to the range o f physiological states in the cells concerned. The concept o f basal metabol ism has little meaning in protozoa. During balanced growth, energy metabol i sm is nearly linearly propor t ional to the growth rate constant; at the init iation o f starvation, metabol ic rate rapidly declines. Moti l i ty requires an insignificant fraction o f the energy budget o f protozoans. For growing cells, metabolic rate is approximate ly propor t ional to weight o.75 and the data fall nearly exactly on a curve extrapolated from that describing the respiration rates of poi- k i lotherm metazoans as a function o f body weight. It is conceivable that p ro tozoan species exist with lower m a x i m u m potential growth and metabolic rates than those predicted from cell vo lume and the equations der ived f rom the available data. However , the lack o f informat ion concerning the state of the cells studied prevents verification of this idea. Laboratory measurements o f pro tozoan respiratory rates have no predict ive value for p ro tozoa in nature other than delimiting a potential range. For small protozoans, this range may, on an individual basis, represent a factor o f 50.

Introduction

Labora tory measurements o f respiratory rates are assumed to represent basal metabol ic rates and are carried out in order to evaluate the role o f individual species in energy flow through natural ecosystems. They are widely quoted in the literature. While a t tempting to apply such data in various contexts, we have found that published values for species of similar size, or even for individual species, show an enormous scatter, up to a factor of about 50. We have also found that certain basic bioenergetic propert ies o f small organisms are not widely understood. The reason for this is probably that mos t protozoologists have a background o f zoology rather than microbiology, for in certain respects pro tozoa resemble bacteria more than macroscopic animals.

Hemmingsen [33, 34] and Zeuthen [101] discussed metabol ic rates o f protozoa and other types o f organisms as a function o f body weight in terms o f Huxley 's al lometric equation: R = aW b. These authors agreed that unicellular

100 T. Fenchel and B. J. Finlay

organisms, like metazoans, have a value o f the exponent b o f a round 0.75, and also that the constant a takes a value some 7 t imes lower in protozoa compared with poiki lo therm animals. This point of view is still widely reproduced in textbooks o f compara t ive physiology. However , the data were presented on graphs in which the vertical and horizontal axes span some 12 and 14 decades, respectively. This obscures the fact that the then available data on "basal metabolic rates" of protozoa show a wide scatter. Since the papers o f Hem- mingsen and Zeuthen appeared, a large body o f isolated measurements o f pro tozoan respiration has been published, but only Klekowski [47] and Fenchel [24] have a t tempted an analysis along the lines presented here.

The purpose o f the present paper is to review the available data on protozoan respiration, to analyze the informat ion they contain, and to suggest more frtiitful approaches to the study o f ecological bioenergeties o f protozoa.

Mater ia ls and Methods

All available published data concerning the whole-cell respiration of free-living heterotrophic protozoan species were used (Table 1 and Fig. 1). Some of the data were excluded if considered unrealistic or if the sterility of the protozoa was questionable. Cell volumes were taken from the reference if stated; otherwise they were estimated by us from linear dimensions or based on other published or unpublished measurements. Where necessary, values of respiratory rates were cor- rected to 20~ assuming a value of Q~o of 2. All available information in the references concerning the physiological state of the cells (i.e., whether they were growing, taken from growing or stationary cultures, or starved for a stated period of time) was noted. Data on growth rates were taken from references 2, 21, 22, 24, 26, 54, 78, and 88. To render all the data from the literature in a comparable form and for theoretical considerations on the relation between growth efficiency, respiratory rates and growth rate constants, we have used the following conversions throughout: 1 g wet weight (or 1 ml cell volume) is equivalent to 0.15 g dry weight [78, 95], 0.071 g C and 0.0185 g N [27]. The respiration of 1 ml O2 is assumed to generate 20.2 J with a respiratory quotient of 1.0. All of these figures, of course, are subject to variations corresponding to different species and physiological states. However, when comparing organisms spanning a size range of 6 orders of magnitude, these minor variations will be effectively lost. Additional estimates of the respiration rate of Tetrahymena pyriformis in a wide range of physiological states were obtained using methods already described [28]. Starving cells were supported in phosphate buffer [11].

Results

Figure 1 shows all data on pro tozoan respirat ion plot ted logarithmically against log cell volume. For any 1 cell volume, the data span a factor o f about 50. I f only values for organisms der ived f rom growing cultures are included (Fig. 2), the variance is considerably reduced. Values for large amoebae seem to be systematically lower than those for other, similarly sized protozoans. Included in Fig. 2 are Hemmingsen ' s [34] line for metazoan poikilotherms:

log10 R (nl 02 cell -~ h 1) = lOgloVOl (#m 3) X 0.74 - 3.83,

and a line in which a only takes the value - 4 . 6 9 , assumed by Hemmingsen to describe protozoan respiration. The regression line for the data on ciliates and flagellates in Fig. 2 is log~oR = log~ovol X 0.75 - 4.09 (correlation coefficient 0.97).

Respiration Rates in Protozoa 101

0

- 2

- 3

- 4

- 5

-IogloR(nl 02h "1)

�9 o c i l i a t e s

A .", f l a g e l l a t e s

�9 V a m o e b a e

// A

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. r o . J/ .l o o O

,,~._t1~ t . "

Ioglo cell volume ~m 3) I I I I I I I I I I 0 1 2 3 4 5 6 7 8 9

Fig. 1. Published data on protozoan respiration rate per cell from the references listed in Table 1. Filled symbols (0 �9 v) represent measurements on growing cells or cells taken from growing cultures. Data points connected by lines ( ) represent extremes reported for single species in individual studies, usually representing growing and + starving ceils, respectively; open symbols (O Lx ~7) represent + starving cells or cells in undefined physiological state. Broken line represents one set of data which deviates strongly from the remaining data.

Discuss ion

Evaluation of the Data

It is apparent f rom Figs. 1 and 2 that variat ion in physiological state contr ibutes most to the overall variance in the data. When cells o f similar physiological state are considered, the predictabili ty o f respiration rate f rom cell vo lume is considerably improved. Even so, "growing cells" is in most cases an imprecise term since the different species may grow at different rates, according to food resources and other conditions. It is conceivable that i f more detailed informat ion had been generally available (viz. growth rate constants), the data would allow for an even higher degree of predictability.

Estimates o f cell vo lume consti tute another source of error. Several authors base these on l inear dimensions and assume some idealized geometrical shape (e.g., cylinders, ellipsoids, 2 cones, etc). This procedure may lead to a considerable error. Fur thermore , the size o f individual pro tozoa varies with the phys-

Tab

le 1

. D

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d fr

om 2

3 ~

Car

tesi

an d

iver

s [4

7]

Dio

phry

s sp

. 0.

132

21,0

00

Rec

alcu

late

d fr

om 2

3 ~

Car

tesi

an d

iver

s [4

7]

Eup

Iote

s va

nnus

0.

0074

[7

,500

] R

ecal

cula

ted

from

25 ~

G

ilso

n di

ffer

enti

al

[79]

re

spir

omet

er

Fro

nton

ia l

euca

s 0.

051

614,

000

Uns

peci

fied

C

arte

sian

div

ers

[52[

e~

Tab

le 1

. C

onti

nued

Res

pira

tion

ra

te

Vol

ume"

S

peci

es

(nl

O5

cell

-~ h

-~)

(/~m

0 C

ondi

tion

C

omm

ents

M

etho

d R

efer

ence

O

lntr

anst

ylum

0.

168

26,0

00

Uns

peci

fied

st

eini

Ope

rcul

aria

0.

447

120,

000

Uns

peci

fied

nu

lans

Par

amec

ium

0.

354

100,

500

Tak

en f

rom

au

reli

a gr

owin

g cu

l-

ture

s

P.

aure

lia

0.22

2 [2

00,0

00]

Uns

peci

fied

P.

aure

lia

0.78

0 [2

00,0

00]

Tak

en f

rom

gr

owin

g cu

l-

ture

s

P.

aure

lia

0.03

3 15

8,00

0 U

nspe

cifi

ed

P.

calk

insi

0.

106

I135

,000

] S

tarv

ed

P.

caud

atum

0.

490

[500

,000

]

P.

caud

atum

2.

111

640,

000

P.

caud

atum

1.

24--

4.44

20

0,00

0-

1.2

• 10

6

Uns

peci

fied

Tak

en f

rom

gr

owin

g cu

l-

ture

s

Tak

en f

rom

gr

owin

g cu

l-

ture

s

Mea

n of

figs

in

Tab

le 2

; re

calc

ulat

ed f

rom

27*

Res

p. i

s m

ean

of r

ange

gi

ven;

rec

alcu

late

d fr

om

26*

Res

p. i

s m

ean

valu

e fo

r st

arvi

ng c

ells

of

2 m

at-

ing

type

s

Rec

alcu

late

d fr

om 2

1.20

Rec

alcu

late

d fr

om 2

2.7

~

Car

tesi

an d

iver

s [4

7]

Car

tesi

an d

iver

s [4

7]

War

burg

[6

4]

Car

tesi

an d

iver

s [8

3]

Car

tesi

an d

iver

s [8

6]

Car

tesi

an d

iver

s [5

2]

Car

tesi

an d

iver

s [6

]

Nov

el c

apil

lary

[3

8]

met

hod

War

burg

[6

4]

Cap

illa

ry r

espi

ro-

[ 16]

m

eter

,-0

O

Tab

le 1

. C

onti

nued

c

o

Res

pira

tion

ra

te

Vol

ume a

S

peci

es

(nl

02 c

ell -

~ h

-t)

(gm

3)

Con

diti

on

Com

men

ts

Met

hod

Ref

eren

ce

P.

caud

atum

1.

8-0.

19

950,

000-

- C

ells

in

vari

ous

Rec

alcu

late

d fr

om 2

5 ~

Car

tesi

an d

iver

s [8

1]

170,

000

phys

. st

ates

ta

ken

from

cu

ltur

es

P.

caud

atum

0.

75

530,

000

Tak

en f

rom

C

arte

sian

div

ers

(?)

[46]

gr

owin

g cu

l-

ture

s

Pla

cus

sp.

I 0.

956

383,

000

Uns

peci

fied

R

ecal

cula

ted

from

28.

8 ~

Cla

rk-t

ype

02 e

lec-

[4

2]

trod

e

Pla

cus

sp.

2 0.

782

712,

000

Rec

alcu

late

d fr

om 2

8.8

~ C

lark

-typ

e 02

ele

c-

[42]

tr

ode

Pod

ophr

yafi

xa

0.02

1;

49,5

00;

Tak

en f

rom

C

arte

sian

div

ers

[50]

0.

0077

14

,900

gr

owin

g cu

l-

ture

; st

arve

d 96

h

Spir

osto

mum

1.

77

[8 X

10 6

] C

eils

tak

en

Res

p. t

aken

fro

m T

able

2;

War

burg

[8

5]

ambi

guum

fr

om n

on-

reca

lcul

ated

fro

m 2

5 ~

grow

ing

cul-

tu

re

S. a

mbi

guum

12

.7

[12

• 1

06

] T

aken

fro

m

Car

tesi

an d

iver

s (?

) [4

6]

grow

ing

cul-

tu

res

~-

t~

S. i

nter

med

ium

0.

27

[500

,000

] S

tarv

ed

Car

tesi

an d

iver

s [7

2]

S. m

inus

0.

31

[500

,000

] S

tarv

ed

Car

tesi

an d

iver

s [7

2]

S. t

eres

0.

041

349,

000

Uns

peci

fied

C

arte

sian

div

ers

[50]

Sten

tor

1.5

1.33

•

106

Sta

rved

3-5

d

Car

tesi

an d

iver

s [9

6]

coer

uleu

s

Uns

peci

fied

Tab

le 1

. C

onti

nued

Res

pira

tion

~"

ra

te

Vol

ume ~

Sp

ecie

s (h

i O

2 ce

ll -l

h-0

0t

m 3)

C

ondi

tion

C

omm

ents

M

etho

d R

efer

ence

~'

S. c

oeru

leus

0.

075-

-0.3

5 1

• 10

6-

Cel

ls o

f va

riou

s C

arte

sian

div

ers

[49]

7.

3 •

106

size

s ta

ken

from

gro

win

g ~'

cu

ltur

es

Stro

mbi

dium

sp.

0.

111

1,50

0 U

nspe

cifi

ed

Rec

alcu

late

d fr

om 2

2 ~

Car

tesi

an d

iver

s [4

7]

Styl

onyc

hia

2.33

90

0,00

0 U

nspe

cifi

ed

Car

tesi

an d

iver

s [4

7]

myt

ilus

Tet

rahy

men

a 0.

0595

22

,449

C

ells

fro

m a

R

ecal

cula

ted

from

21

";

Dro

ppin

g m

ercu

ry

gele

ii

nong

row

ing

figs

. quo

ted

are

for

a el

ectr

ode

cult

ure

6-da

y cu

ltur

e T.

gel

eii

0.27

0;

54,0

00;

Gro

win

g;

Rec

alcu

late

d fr

om 2

6.8~

W

arbu

rg

[62]

0.

0873

[3

2,00

0]

star

ved

high

er r

ate

is f

or e

xpo.

ph

ase

cell

s (T

able

4)-

- lo

wer

rat

e is

for

sam

e ce

lls

in p

hosp

hate

buf

fer

T. g

elei

i 0.

471

23,6

00

Gro

win

g R

esp.

is

max

. va

lue

from

W

arbu

rg

[66]

T

able

3;

reca

lcul

ated

fr

om 2

5 ~

T. p

yrif

orm

is

0.04

57

[10,

000]

U

nspe

cifi

ed

Rec

alcu

late

d fr

om 2

2*

War

burg

[5

8]

T. p

yrif

orm

is

0.01

99

[10,

000]

S

tarv

ed

Res

p. i

s en

doge

nous

rat

e W

arbu

rg

[80]

re

calc

ulat

ed f

rom

25*

T.

pyr

i.for

mis

0.

524;

50

,000

; G

row

ing;

R

ecal

cula

ted

from

28*

W

arbu

rg

[31]

0.

048

[ 15,

000]

st

arve

d

T. p

yrif

orm

is

0.00

707;

[5

,000

];

Star

ved;

gro

w-

Rec

alcu

late

d fr

om 2

5*

War

burg

[5

9]

0.06

3 [ 1

5,00

0]

ing

T. p

yrif

orm

is

0.19

8 [1

0,00

0]

Gro

win

g R

is

mea

n va

lue

for

divi

d-

Am

pull

a di

vers

[5

7]

ing

cell

s, r

ecal

cula

ted

from

26*

[l]

O

~D

Tab

le 1

. C

onti

nued

o

Res

pira

tion

ra

te

Vol

ume*

S

peci

es

(nl

02 c

ell -~

h

')

(tzm

3)

Con

diti

on

Com

men

ts

Met

hod

Ref

eren

ce

T.

pyri

form

is

0.07

63

[10,

000]

U

nspe

cifi

ed

R r

ecal

cula

ted

from

27 ~

W

arbu

rg

[94]

T. p

yrif

orm

is

0.03

7 [1

0,00

0]

Sta

rved

R

esp.

is

mea

n of

con

trol

s W

arbu

rg

[14]

in

Tab

les

1 an

d 2;

rec

al-

cula

ted

from

28 ~

T. p

yrif

orm

is

0.10

4 [1

0,00

0]

Gro

win

g R

is

mea

n va

lue

for

2 W

arbu

rg

[12]

st

rain

s gr

owin

g on

4 e

n-

ergy

sou

rces

T. p

yrif

orm

is

0.02

1 8,

000

Car

tesi

an d

iver

s [5

0]

T. p

yrif

orm

is

0.19

I

1,00

0 G

row

ing

Car

tesi

an d

iver

s (?

) [4

6]

T. p

yr~o

rmis

0.

119

21,5

00

Tak

en f

rom

W

arbu

rg

[3]

grow

ing

cul-

tu

res

T. p

yrif

orm

is

0.02

2 21

,500

T

aken

fro

m

Car

tesi

an d

iver

s [3

] gr

owin

g cu

l-

ture

s

T. p

yrif

orm

is

0.67

2 6,

000

Gro

win

g R

ecal

cula

ted

from

30 ~

C

lark

-typ

e 02

ele

c-

D.

Llo

yd (

pets

. tr

ode

com

m.,

[56

1)

T. p

yrif

orm

is

0.06

42

13,0

00

Gro

win

g G

radi

ent

dive

rs

A.

Cow

ling

(p

ers.

com

m.)

T. p

yr~o

rmis

0.

0038

0;

! ,73

8;

Sta

rved

3 d

; R

ecal

cula

ted

from

30 ~

C

lark

-typ

e O

2 el

ec-

Thi

s st

udy

0.14

7 14

,791

gr

owin

g tr

ode

Tia

rina

fusu

s 0.

147

27,0

00

Uns

peci

fied

R

ecal

cula

ted

from

24 ~

C

arte

sian

div

ers

[47]

Tra

chel

orap

his

sp.

1.06

1 34

0,00

0 N

atur

al s

am-

Rec

alcu

late

d fr

om 2

5 ~

Car

tesi

an d

iver

s [9

3]

pies

--as

- su

med

to

be

grow

ing

cell

s

Tab

le 1

. C

onti

nued

Res

pira

tion

ra

te

Vol

ume"

=.

O

Sp

ecie

s (n

l 02

cel

l -~

h -~

) (u

rn 3)

C

ondi

tion

C

omm

ents

M

etho

d R

efer

ence

Uro

nem

a m

arin

um

0.01

36

[500

] U

nspe

cifi

ed

Rec

alcu

late

d fr

om 2

5 ~

Gil

son

diff

eren

tial

[7

9]

resp

irom

eter

U

rost

yla

gran

dis

0.15

; 1.

7 [ 1

66,0

00]

Cys

t; s

tarv

ed

Car

tesi

an d

iver

s [7

2]

Vor

ticel

la

0.84

0 55

0,00

0 U

nspe

cifi

ed

Car

tesi

an d

iver

s [4

7]

O

conv

alla

ria

V. e

phem

erae

0.

112

19,0

00

Uns

peci

fied

C

arte

sian

div

ers

[47]

V.

mic

rost

oma

0.00

98

26,0

00

Car

tesi

an d

iver

s [5

0]

V. s

imil

is

0.79

5 10

2,00

0 U

nspe

cifi

ed

Car

tesi

an d

iver

s [4

7]

Zoo

tham

nium

0.

359

49,0

00

Uns

peci

fied

C

arte

sian

div

ers

[47]

ca

rino

gam

mar

i

Figu

res

in b

rack

ets

are

our

esti

mat

es,

othe

rwis

e th

ey d

eriv

e fr

om t

he r

efer

ence

.


3

-3

- 4

IogloR(nl 02h -I) /

/ �9 c i l i a t e s /

. / /

fl o Ilat~..~ / V _ .a~e . .____ / / / /

v amoebae / Vv// / �9 V/

/ /

. s "~

/

/ / t / /~ V /V

/ V / /

7 � 9 / / /

/ / - - /

/ / / /

/ / /

/ /

/ /

I o

log cel l volume(iJm 3) I I I I I I I I I 1 2 3 4 5 6 7 8 9

Fig. 2. Same as Fig. 1, but only rates for growing cells are included. The upper and lower lines represent Hemmingsen's [34] regression lines for poikilotherm metazoa and for unicellular organisms, respectively.

iological state and env i ronmen ta l condit ions. Dur ing balanced growth, cell vo lume increases with increasing growth rate constant , and growing cells are larger at lower t empera tu res [24, 26, 43, 54]. When starved, m a n y species respond with addi t ional cell d ivis ions producing cells which are only 50 or 25% of the vo lume o f growing cells [24, 89]. However , in mos t o f the l i terature on respirat ion, such p h e n o m e n a are ignored and only 1 vo lume is stated which m a y not cor respond to that o f the cells in the respi rometer .

These 2 factors, viz., insufficient in fo rmat ion on growth rates and in part , unreliable es t imates on cell vo lumes , p robab ly together account for mos t o f the devia t ions f rom a line o f the fo rm log R = log vol • b + a. This is suppor ted by the fact that in cases where there are m o r e independent es t imates for a single species, the data show a var iance which is as large as that for different species o f s imilar size. For example , 3 authors working with growing individuals o f the ciliate Tetrahymena pyriformis stated cell vo lumes o f 5.01, 1.10, and 1.48 • 104 # m 3 and found respirat ion rates o f 0.524, 0.190, and 1.148 nl 02 cell -1 h - ' , respectively; the weight specific respirat ion rates calculated for these data span a factor o f abou t 2. Four m e a s u r e m e n t s for Paramecium caudatum in the l i terature give weight specific respirat ion rates ranging f rom 1.43-5.79 • 10 -6


nl 02 ~tm -3 h-L These data, in fact, represent the maximum deviation from the regression line in Fig. 2.

Several authors have discussed the significance of methodology of respiration measurements; these comprise Cartesian or gradient diver techniques [32, 102], Warburg respirometry [62], or oxygen electrodes [24, 25, 56]. If the purpose of a study is to follow changes in 02 consumption during the cell cycle, diver techniques (or synchronized cultures) are needed. When the objective is to examine the bioenergetics of cell populations, however, it is unclear why any of the techniques mentioned should show special advantages or yield more accurate results, as inferred by some authors [3, 53].

The essential problem in the bulk of the literature is ignorance of the physiological state of the cells in the respiration chamber and in particular, ignorance of the fact that the cells change rapidly as a response to deprivation of food. This effect, o f course, is aggravated by increasing the duration of the experiment. In this sense, Warburg respirometry or oxygen electrodes used in conjunction with a large number of cells are preferable since reliable readings can be made over a period of only 10-15 min or so.

Interpretation of the Data and General Considerations

We have assumed that the data on flagellates and ciliates in Fig. 2 can be fitted to the allometric equation R = Wb'a with 1 set of values for the constants a and b, whereas amoebae represent a lower value of a. Other interpretations are possible, for example, that the exponent b takes a lower value for large protozoa. The available data do not permit an objective choice, particularly since respiration measurements for large protozoa are scarce. It is suggestive, however, that if the values of a and b are considered invariant with cell size, they take values extremely close to those established for poikilotherm metazoa. This interpretation is also consistent with the available data on growth rates, as discussed below.

In prokaryotic microorganisms, it has been established that during the balanced growth of carbon limited cells, growth efficiency is nearly invariant with the growth rate constant, so that for each tool of adenosine triphosphate (ATP) generated, approximately 10 g d.w. cells are produced. If the C atoms of the substrate have an oxidation level such as that in glucose, and the energy metabolism is aerobic, this corresponds to a growth efficiency of 0.67. This is so, since for every g C dissimilated, 0.5 mol ATP is generated. To produce 1 g cell carbon, the cell needs to assimilate 1 g C and at the same time to dissimilate another 0.25/0.5 = 0.5 g C (with the above assumption that 4 g C is produced for every mol ATP generated). Net growth efficiency therefore becomes l/(1 + 0.5) = 0.67. Metabolic rate is nearly proportional to the growth rate, and the energy budget requirements for maintenance constitute an extremely small fraction of that involved in growth [5, 70, 87]. There is now good evidence that this picture also holds for eukaryotic microorganisms. Thus, Curds and Cockburn [17] and Fenchel [24] showed that the growth efficiency of Tetra- hymena and of various microflagellates in continuous and batch cultures is constant within the range of rates of balanced growth that can be maintained


I

0

-i

-2

-3

I

0

Fig, 3.

IOglO JJm(h -I )

�9 cilicttes f logel lo tes omoeboe

logl, 0 cell ,votume,(vm3! I I I I

I 2 3 4 5 6 7 8 9

Assumed maximum growth rate constants as a function of cell volume for various protozoans. Included are lines derived from eq. (2) for different values of net growth efficiency.

in the laboratory. Calow [10] has, in accordance with the avai lable data, gen- eralized that in p ro tozoa (and in growing tissue of po ik i lo therm metazoa) the net growth efficiency in t e rms o f C is a round 0.6.

With these assumpt ions , the relat ionship between respirat ion rate, R (rag 02 cell -~ h-~), and growth rate constant , ~(h-~), during balanced growth is given by

R = # X ( 1 - E ) / E •

where E is the net growth efficiency (product ion over p roduc t ion + respiration) and body weight is expressed as mg C per cell. I f R is expressed in nl 02 cell -~ h -~ and body weight (W) in units o f pg w.w. ( ~ 1 #m3), this becomes:

R = # X W X 1 .325X 10 - 4 X ( 1 - E ) / E (1)

since R = W b • a, we have that

log~o~ = (b - 1)lOglo W + log~oa - log~0[(1 - E)/E] - 3.878 (2)

This gives the expected relat ion between the potential cell growth rate constant and cell vo lume.

In Fig. 3, avai lable data on what has been assumed to approach m a x i m u m growth rates are plot ted against cell vo l um e for a n u m b e r of p ro tozoan species. The reservat ions with respect to the reliability o f the respirat ion da ta discussed


above also apply here. In this case also, several independent estimates o f individual species show a variance o f nearly the same magni tude as when different, similarly sized species are compared. Shown in the figure are a number o f lines der ived from eq. (2), assuming Hemmingsen ' s [34] values of a and b for poiki lotherm metazoa and for different values of net growth efficiency. The data are not inconsistent with a value o f (b - 1) o f - 0 , 2 5 and conversion efficiencies in the range 0.4-0.6. The analysis o f these data cannot be stretched much further. However , it is likely that the data points falling in the lower range ei ther represent cultures that were not growing at their m a x i m u m rate (and hence had a lower respiratory rate than predicted) or represent species which in fact cannot grow as fast as suggested by eq. (2) and correspondingly cannot realize a respiratory rate predicted by the Hemmingsen line. This may apply to the species o f amoebae.

Two o f the species o f heterotrophic flagellates represented in Fig. 3, studied by Fenchel [24], have a conversion efficiency of C of nearly 0.6. However , the m a x i m u m growth and respiration rates recorded were both slightly lower than predicted from the constant values o f Hemmingsen so they fall on the line corresponding to E = 0.5 in Fig. 3. In conclusion, it is likely that net growth efficiency in general is somewhat higher than suggested by Fig. 3, which does not necessarily contradict Calow's [10] generalization o f 0.6.

Metabolic Rates in Starving Protozoa

At least all free-living pro tozoa can display balanced growth rates below a certain m a x i m u m value. There is also evidence to suggest the existence of a m i n i m u m growth rate. At resource levels below that sustaining this m i n i m u m growth rate, the cells undergo various physiological changes corresponding to a state o f s tarvat ion [24, 25, 91]. These changes may be considered adaptive in terms o f survival, dispersal, and the ability to resume growth rapidly when food resources become available again. So far, there are few systematic studies with respect to respiratory rates, al though reduction in respiratory rate as a response to starvation has been described [25, 32]. It is obvious that a small pro tozoan with a generation t ime of, say, 3 h (~t = 0.23 h -t) respires an amount o f C corresponding to its own cell-C in about 4.5 h (by arguments similar to those leading to eq. (1)). Such a cell could only survive for a very short t ime i f suddenly starved (e.g., in a Cartesian diver) unless it could decrease its respiratory rate quickly.

It is typical for starving cells to be smaller than growing ones. This is in part achieved through 1 or more successive cell divisions following the onset o f starvation and in part through the util ization of parts o f the cytoplasm for energy metabol ism [25]. The weight specific metabol ic rate also falls. In small protozoa, the respiratory rate per cell may eventually decrease to 2--4% o f that in growing cells. Figure 4 and Table 2 show examples for 4 differently sized protozoa. The data suggest that the ability to decrease weight specific respiratory rate is much more p ronounced in small protozoa; this may not be surprising i f selective p remium is for survival in an absolute timescale.


Table 2, Cell volumes and respiratory rates for growing and starving cells of four species of protozoa, based on the extreme values of Fig. 4

Weight specific Respiration respiralioa

Volume (~nr 31 nl 02 cell ~ h ~ nl 02 tam -~ • 10 ~

Species Growth Starvation Growth Starvation Growth Starvation Reference

Chaos carolinensis 5 • 10 v 1,25 X 10' I6.9 4.2 0.34 0.34 36 Paramecium caudatum 9.5 X 10' 1.7 X I 05 1.8 0.19 1.89 I. 12 82 Tetrahymena pyr~fbrmig 1.5 • ID a 1.7 • 10 ~ 0.145 3.8 X 10 J 9.7 2.24 this study Ochromonar sp. 240 50 3.2 X 10 -J 7.1 X 10 5 13.3 1.4 25

Energy Expenditure of Motility and Other Factors Influencing Bioenergetic Parameters

By analogy to larger animals it is often assumed that in protozoa, moti l i ty may account for a measurable or even considerable fraction o f the entire energy budget. Laybourn [51] at t r ibuted a high respiratory rate in Didinium nasutum (this species does not, in fact, have an exceptionally high respiratory rate) to the "ve ry high level o f swimming." Similar views are expressed by Klekowski [47]. However , small organisms spend an extremely small fraction o f their energy budget on moti l i ty (for a general discussion, see ref. 76).

Crude estimates o f the energy expendi ture for moti l i ty in protozoa are readily calculated [41 ]. For example, take spherically shaped pro tozoa like Ochromonas and Didinium. The former has a d iameter of about 8 ta and swims with a velocity of about 60 / i r a sec-~; for the latter species the corresponding values are about 120 tam and 1,000 tam sec -t [84]. F rom Stoke's law we have that the power required to pull a sphere with a diameter , D, through water with the viscocity n (assumed here to be 0.01 Poise) and with the velocity, v, is 3 7rD v z 77- For the 2 organisms in quest ion this works out to be about 2.7 • 10 -9 and 1.1 • 10 -s erg see -~, respectively. I f we assume that the overall efficiency (hydrodynamical efficiency X efficiency o f t ransforming chemical work into mechanical work) o f the ciliary propulsion mechanism is 1%, power required for Ochromonas and Didinium is 2.7 X 10 7 and 1.1 • 10 -3 erg sec -~, respectively. The 02 consumpt ion o f growing cells o f the 2 species is 4.5 • 10 -3 and 2.7 nl 02 cell -1 h-t; this corresponds to the generation o f 2.5 • 10 4 and 1.5 X 10 -t erg sec -t, respectively. An est imate o f the fraction o f energy spent on moti l i ty is thus 0.1-0.7% o f the entire energy budget. This est imate o f the efficiency o f flagellar propuls ion is supported by empirical evidence. It has been est imated that an active flagellum generates 1-2 X 10 -7 erg see -t in order to overcome viscous and elastic forces and the chemical power input o f a flagellum has been found to be 2-8 X 10 -7 erg sec -t [35, 99, and references therein]. Ochromonas owes its moti l i ty to a single flagellum whereas Didinium has some 2 • 103 cilia [84]. These arguments render it highly improbable that moti l i ty in protozoa can be detected at all in terms o f 02 consumption.

Although moti l i ty can be ignored in the present context, certain environ- mental factors may influence ei ther o f the parameters: respiration, growth rate, and net growth efficiency. Parker [71 ] found that the growth rate o f the marine ciliate Uronema marinum decreased significantly when grown at salinities de- viating f rom an op t imum value. It is possible that this reflects a decrease in


-I

- 2

- 3

- 4

I I 2 -

LOg+oR (nl 02 h'l)

1

o

I I 0 1

I I I I I I I

.if

� 9

Oe �9 Tetrahymena

Ochromonas

=lq~b e e C h a ~

,r

P a r a m e c i u m

Loglo cell vol. Igm 31 I I I I I I I 2 3 4 5 6 7 8

Fig. 4. Relationships between respiration rate and cell size in 4 protozoan species, each represented by a range of physiological states. The Ochromonas and Paramecium data are taken from population growth cycles (Fenchel [25] and Scholander et al. [811 respectively). Growth cycle data were obtained for Tetrahymena (this study together with values from cells starved for up to 4 days in phosphate buffer). The Chaos data were obtained from the starvation experiment (27 d duration) of Holter and Zeuthen [36].

net growth efficiency since an increased a m o u n t o f energy is needed for osmot ic work. Since nei ther yields nor respirat ion rates are reported, this cannot be evaluated.

Several workers have found that growth rates o f phagot rophic pro tozoa depend on the quali ty o f the food organisms: Tay lo r and Berger [90] found this to be so for a n u m b e r o f ciliate species fed var ious bacterial strains. Again, too little in fo rmat ion is given to evaluate the reason for this and several expla- nat ions are possible: (1) differential efficiencies o f capture which is highly de- pendent on the d imens ions o f food particles [23]; (2) differential gross growth efficiency and respira tory rate (due to, e.g., different thicknesses o f cell walls or similar), but unal tered net growt.h efficiency; (3) certain bacter ia contain l imit ing amoun t s o f necessary componen t s for p ro tozoan growth or are slightly toxic leading to a decreased net growth efficiency but an unal tered respiratory rate. This latter effect is possibly demons t r a t ed by Tetrahymena pyriformis growing in axenic (peptone) laboratory med ia in which net growth efficiency is lower than when the ciliates are reared on bacteria. In the axenic med ium, some essential growth substance is apparent ly limiting, so that in batch cultures growth stops long before the organic C is exhausted [ 17].


Significance o f the Allometric Scaling with Size

When organisms f rom a wide size range are compared , weight specific metabol ic rate and potent ial growth rate both decrease with an exponent o f abou t - 0 . 2 5 . This re la t ionship mus t reflect some basic physiological constraint on how rapidly ceils can grow and divide. The actual values o f the constants describing this relat ion have s t imulated much speculat ion in the literature, but this topic will not be discussed here. The point we would stress is that it is conceivable that the m a x i m u m growth rate constants o f m a n y individual species or entire t axonomic groups m a y deviate f rom the a l lometr ic equat ion and that these devia t ions could be adapt ive . Most o f the species o f p ro tozoa grown in the laboratory , and for which data on growth and respiratory rates exist, are " o p - por tun is t s" in nature. They obvious ly depend on the pa tchy occurrence o f high concentra t ions o f food (main ly bacteria associated with decaying organic m a - terial) and the adapt ive value o f a potent ia l to max imize growth rates to an upper l imit set by some still unknown physiological constra int is clear. Other species, however , m a y not be adapted to such a "feas t and f amine" existence since in nature, they are exposed to a more constant , but low level o f food resources. Such fo rms are likely to exhibi t a smal ler range o f potent ia l growth and respiratory rates. A m o n g bacteria, such a spec t rum o f potent ia l growth rates is well establ ished [92].

The s tudy o f such features a m o n g pro tozoa would be o f interest to evolu- t ionary ecology and c o m p a r a t i v e physiology. Unfor tunate ly , the data avai lable for p ro tozoa are o f l imi ted value in this context. Systematic studies o f respira t ion rates of cells with an exactly measured b iomass and f rom cultures during balanced growth at known rates are necessary. Such data are scarce and future studies o f this sort would be valuable. However , the cont inued accumula t ion o f data on respiratory rates o f p ro tozoa o f undefined physiological state is o f very restricted interest.

A final word should be given concerning the appl icat ion o f data on respiratory rates in the predict ion o f carbon flow through natural populat ions. In this type o f inquiry, the real p rob lem is one o f quant ifying growth rates, which again are roughly propor t iona l to the entire energy flow. However , growth rates o f natural popula t ions are not likely to be cons tan t ove r t ime. Rather , they will fluctuate between zero and some m a x i m u m rate characterist ic for the species in question. Respi ra t ion and growth rates ob ta ined in the labora tory prov ide no in format ion regarding this level o f fluctuation; they mere ly serve to del imit a potent ial range. As shown for some species, this range m a y approach a factor o f 50.

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