P l a n t a n d Soil X X V I I I , no.

1 F e b r u a r y 1968

SHORT COMMUNICATION

A p p a r a t u s f o r t h e M e a s u r e m e n t of O x y g e n C o n s u m p t i o n by
t h e R o o t S y s t e m of a P l a n t

Introduction
R e s e a r c h o n t h e p h y s i o l o g y of t h e r o o t s y s t e m of a p l a n t m a y m a k e it
d e s i r a b l e to s t u d y t h e o x y g e n c o n s u m p t i o n of t h e w h o l e r o o t s y s t e m of t h e
intact plant.
F o r t h i s p u r p o s e s e v e r a l m e t h o d s are a v a i l a b l e 2 P l a n t s g r o w n in w a t e r
c u l t u r e c a n b e t r a n s f e r r e d w i t h t h e i r whole r o o t s y s t e m to a vessel w i t h a n
o x y g e n - s a t u r a t e d n u t r i e n t solution. D u r i n g a s h o r t p e r i o d (e.g. 20 m i n u t e s )
t h e r o o t s t a u s t h a v e a free s u p p l y of t h e o x y g e n in t h i s n u t r i e n t solution. As
diffusion of o x y g e n t h r o u g h t h e w a t e t p h a s e is v e r y poor, t h e r e m a y b e a
c h a n c e of o x y g e n deficiency n e a r t h e roots. To p r e v e n t t h i s t h e n u t r i e n t
s o l u t i o n was m i x e d b y stirring. T h i s h a d to b e d o n e v e r y c a r e f u l l y to p r e v e n t
u n d e s i r e d a e r a t i o n f r o m t h e a t m o s p h e r e i n t o t h e n u t r i e n t solution. A f t e r 20
m i n u t e s t h e o x y g e n c o n t e n t of t h e s o l u t i o n c a n b e m e a s u r e d b y t h e W i n k l e r
m e t h o d 3. So it is possible t o c a l c u l a t e t h e o x y g e n c o n s u m p t i o n . T h i s a n d
s i m i l a r t e c h n i q u e s are i n a d e q u a t e ; it is impossible t o e s t i m a t e c o n s m n p t i o n
c o n t i n u o u s l y a n d t h e metho4ls are n o t e x a c t e n o u g h .
"vVith t h e C 1a r k e l e c t r o d e 1 it is possible t o m a k e e x a c t m e a s u r e m e n t s now.
W e d e v e l o p e d a simple a p p a r a t u s w h e r e p l a n t s c a n b e g r o w n in a w e l l - a e r a t e d
w a t e r c u l t u r e for a l o n g p e r i o d (e.g. t h r e e m o n t h s or longer). I n t h e s a m e
a p p a r a t u s it is possible t o m e a s u r e t h e o x y g e n c o n s u m p t i o n c o n t i n u o u s l y
for 10 m i n u t e s in e v e r y h o u r w i t h o u t m o v i n g t h e p l a n t s . D u r i n g r e a d i n g s
t h e r e is n o c h a n c e of u n d e s i r e d a e r a t i o n f r o m outside. Y e t t h e n u t r i e n t
s o l u t i o n still h a s a s t r o n g c u r r e n t a r o u n d t h e roots t o g u a r a n t e e t h e s a m e
oxygen concentration everywhere.

The Clark electrode

T h e C l a r k o x y g e n - s e n s o r is a p o l a r o g r a p h i c s y s t e m 1 4. T h e c a t h o d e is a
gold r i n g e m b e d d e d in a l u c i t e b l o c k ; t h e a n o d e is a silver coit, c o a t e d w i t h
AgC1, recessed in a c e n t r a t well of t h e block. A t e f l o n m e m b r a n e (0.025 m m )
s t r e t c h e d across t h e e n d of t h e sensor isolates t h e sensor e l e m e n t s f r o m t h e i r
e n v i r o n m e n t . T h e i n t e r i o r is filled w i t h a 5 0 % a q u e o u s s o l u t i o n of KC1
T h e sensor is p l a c e d in a m o v i n g n u t r i e n t solution. O n surIace of t h e gold
c a t h o d e e l e c t r o r e d u c t i o n of o x y g e n t o w a t e r occurs. N e a r t o t h e c a t h o d e t h e
o x y g e n p r e s s u r e b e c o m e s zero b e c a u s e o x y g e n is c o n s u m e d as i t r e a c h e s t h e

- - 173 - -
174 SHORT COMMUNICATION

c a t h o d e . T h e a m b i e n t o x y g e n p r e s s u r e to be m e a s u r e d is outside t h e m e m -
b r a n e . Because of t h e difference in o x y g e n pressure, o x y g e n diffuses t h r o u g h
t h e m e l n b r a n e . T h e flow of o x y g e n is d i r e c t l y p r o p o r t i o n a l to t h e difference
in o x y g e n p r e s s u r e across t h e m e m b r a n e . T h e c a t h o d e , w h i c h is m a i n t a i n e d
a t 0.8 volts, is depolarized b y t h i s oxygen, allowing a c u r r e n t to ftow. This
c u r r e n t is d i r e c t l y p r o p o r t i o n a l to t h e o x y g e n pressure to w h i c h t h e sensor
is exposed.
T h e a m b i e n t n u t r i e n t s o l u t i o n h a s to b e s t i r r e d a n d m i x e d c o n t i n u o u s l y
d u r i n g r e a d i n g to g u a r a n t e e t h a t t h e a v e r a g e o x y g e n e o n c e n t r a t i o n in t h e
n u t r i e n t s o l u t i o n c o n s t a n t l y occurs o n t h e o u t e r surface of t h e m e m b r a n e .

The c~ppamlus /or growth and measurement

A s c h e m e of t h e a p p a r a t u s is g i v e n in Fig. 1. I t consists of 4 p l a s t i c vessels
(A, B, C, a n d D) a n d a t a n k u n d e r n e a t h (E). T h e t a n k is large to g u a r a n t e e a
c o n s t a n t c o m p o s i t i o n of t h e n u t r i e n t solution.

Fig. 1. S c h e m e of t h e a p p a r a t u s (longitudinal section).

The solution is p u m p e d out of the tank by a small p u m p (F) to a flow-

meter (G). This flowmeter isused to control the volume of solution circulating.
The solution fallsfree into vessel A, /illingit up to the outlet pipe a. It flows
through valve 2 to vessel B, which is filledup to outlet pipe b. B y valves 5
and 7 it goes to vessel C, also fillingthis up to outlet pipe c. After flowing
through valve I0, the solution enters vessel D by stand pipe d. It overflows
around the stand pipe and then it drains back into the tank by outlet pipe e.
In this circuit teere are three places of intense aeration of the nutrient
solution: the free fallat the inletin vessel A, the overflow d o w n in a thin layer
around stand pipe d and where the solution drains back into the tank (E).
Vessels B a n d C Eave a c o v e r i n g disk of plastic inside as s h o w n in Fig. 2,
p l a c e d j u s t a b o v e t h e outlet. This disk is loose a n d t e s t s o n a p l a s t i c r i n g
a t t a c h e d t o t h e wall of t h e vessel. B e t w e e n t h e disk a n d t h e r i n g t e e r e is a
r u b b e r ring.
S t a n d pipe d h a s a v a r i a b l e h e i g h t a n d acts as a n overflow, h e n c e t h e level
of t h e s o l u t i o n in t h e vessel A, B a n d C c a n b e a d j u s t e d a few c m a b o v e t h e
disk b y t h e s t a n d pipe in vessel D, T h e s o l u t i o n a b o v e t h e disk is a w a t e r
 SHORT COMMUNICATION 175

seal to p r e v e n t undesired aeration from the air into the m l t r i e n t solution

b e n e a t h the disk. This is only i m p o r t a n t during the m e a s u r e m e n t (see below).
I n the middle of t e e disk there is a hole on which a t u b e is placed (/). The
s t e m of a p l a n t can be placed in this tube, preferably free, hence w i t h o u t
any supporting m a t e r i a l such as foam plastic. The p l a n t can be k e p t in
position b y hanging w i t h string. Contact of fresh air w i t h the n u t r i e n t
solution in this t u b e m u s t be p r e v e n t e d b y a small rubber or w e a k plastic
cap around the stem and around the top of the t u b e (g).
Near this wide t u b e there is a sma]] hole w i t h a small pipe u n d e r n e a t h t h e
disk (t~). This gives an open connection between the n u t r i e n t solution under-
n e a t h and a b o v e t e e disk. This pipe is a buffer to p r e v e n t aeration of the
solution in the vessel during readings. I t has to be long enough to p r e v e n t
solution a b o v e the disk m i x i n g w i t h solution below, if there are small fluctu-
ations in w a t e r v o l u m e t h r o u g h fluctuations in p u m p capacity. This is also
only i m p o r t a n t during t h e m e a s u r e m e n t (see below).

Fig. 2. Detail of a growing vessel (longitudinal section).

P l a n t s grow v e r y weil in this apparatus. W e used it successfully for t o m a t o e s

and cucumbers.
The o x y g e n c o n s u m p t i o n of a p l a n t (e.g. in vessel B) can be measured b y
isolating it in a k n o w n v o l u m e of n u t r i e n t solution, whose o x y g e n dep]etion
can be m e a s u r e d o v e r a certain period. During this t i m e aeration from outside
taust be excluded.
Valves 1 and 6 h a v e to be opened and v a l v e 2 and 5 to be closed. The
circulation of aerated n u t r i e n t solution t h r o u g h vessel C will be continued,
bu~ there is a bypass for vessel B.
A pipe connects v a l v e 3 and 4 (Fig. 1). A small p u m p (H) is placed in this
pipe and also a hollow block of polished m e t h y l acrylate (K). I n this block a
hole w i t h a screw t h r e a d is m a d e for a t t a c h i n g the o x y g e n sensor (k) and a
 176 SHORT COMMUNICATION

smaller one to h o l d a t h e r m i s t o r (l) for t e m p e r a t u r e e s t i m a t i o n . B o t h are

a t t a c h e d to t h e m e t e r for o x y g e n d e t e r m i n a t i o n .
W e used a polished m e t h y l a c r y l a t e b l o c k b e c a u s e it is essential to e x a m i n e
t h e t e f l o n m e m b r a n e of t h e o x y g e n sensor. If t h e r e is a n air b u b b l e u n d e r -
n e a t h , n e w KC1 a n d a n e w m e m b r a n e h a v e t o be p l a c e d o n it.
B y o p e n i n g t h e v a l v e s 3 a n d 4 a n d s w i t c h i n g o n t h e m o t o r of p u m p H,
t h e n u t r i e n t s o l u t i o n in vessel B will b e m i x e d a n d c i r c u l a t e d p a s t t h e o x y g e n
sensor. T h e w a t e t seal a b o v e t h e disk i n vessel ]3 p r e v e n t s a e r a t i o n .
T h e o x y g e n m e t e r gives t h e o x y g e n c o n t e n t d i r e c t l y in p p m O2 so we c a n
r e a d off c o n s u m p t i o n b y t h e whole r o o t s y s t e m a g a i n s t time. D u r i n g t h i s
t i m e t h e r e is n o a e r a t i o n of t h e s o l u t i o n in vessel B. O x y g e n deficiency
p r e v e n t s r e a d i n g s b e i n g t a k e n o v e r a p e r i o d ionger t h a n e.g. 10 nlinutes. This
d e p e n d s of course on t h e size a n d k i n d of p l a n t a n d Oll t h e v o l u m e of t h e vessel.
I n o u r case vessel B, t o g e t h e r w i t h t h e c o n n e c t i n g pipes c o n t a i n e d 3.2 litre
of n u t r i e n t solution. A m a t u r e t o m a t o p l a n t c a n c o n s u m e 50 p e r c e n t of t h e
o x y g e n in t h i s v o l u m e w i t h i n 15 m i n u t e s .
K n o w i n g t h e v o l u m e of s o l u t i o n in vessel t3 a n d in c o n n e c t i n g pipes, we
c a n c a l c u l a t e t h e o x y g e n c o n s u m p t i o n of t h e whole r o o t s y s t e m .
I t is easiest to d e t e r m i n e o x y g e n c o n t e n t a t c o n s t a n t t e m p e r a t u r e b u t i t is
also possible iI t e m p e r a t u r e c h a n g e s d u r i n g d e t e r m i n a t i o n . Details of t h i s
are g i v e n in t h e i n s t r u c t i o n s for t h e o x y g e n m e t e r 4
I t is possible to h a v e m o r e r e p l i c a t e vessels like B a n d C in one a p p a r a t u s ,
b u t care m u s t b e t a k e n t h a t t h e pipes are wide e n o u g h to p r e v e n t a n u n e q u a l
level of s o l u t i o n in t h e vessels.
ABRAM A. STEINER,
Centre for Plant Physiologieal Researeh,
Wageningen, Netherlands
Reeeived July 4, 1967

Literature
1 C l a r k , L. C. Jr., Trans. Am. Soc. ArtificiM InterIlal Organs 2, 41-65 (1956).
2 S t e i n e r , A. A., Jaarversl. Proefsta. Groente- en Fruitteelt onder Glas Naaldwijk
1964, p. 107-103 (1965).
3 T i l l m a n s , J., Die chemische Untersuchung von Wasser und Abwasser. 2e Aufl.
Knapp, Halle (1932).
4 Yellow Spring Instruments, Ohio, Instruetions for Y.S.I. Model 51 Oxygen Meter
(1964).