Plant Cell Reports (1983) 2:59-62

Plant Cell Reports © Springer-Verlag 1983

Continuous Culture Growth of Photoautotrophic Cell Suspensions from Chenopodium rubrum W. Ht~semann Lehrstuhl fiir Biochemie der Pflanzen, Universit~t Mttnster, Hindenburgplatz 55, D-4400 Mt~nster, FRG Received February 3, 1983

Abstract The c o n s t r u c t i o n and o p e r a t i o n o f a continuous c u l t u r e system f o r the p r o p a g a t i o n o f c e l l susp e n s i o n s from Chenopodium rubrum under p h o t o a u t o t r o p h i c c o n d i t i o n s has been d e s c r i b e d . A dilution r a t e o f O . 1 6 / d a y gave an equilibrium c u l t u r e d e n s i t y o f 1 , 1 0 0 , 0 0 0 c e l l s / m l and a mean d o u b l i n g t i m e o f 150 h o u r s . D u r i n g cont i n u o u s c u l t u r e s t e a d y s t a t e c o n d i t i o n s with r e s p e c t t o n u t r i e n t u p t a k e , c e l l p r o t e i n and c h l o r o p h y l l c o n t e n t , s t a r c h a c c u m u l a t i o n , in vitro activities of enzymes r e l a t e d t o d i f f e r e n t m e t a b o l i c pathways could be e s t a b l i s h e d . P h o t o s y n t h e t i c CO9 a s s i m i l a t i o n o f steady state c e i l s was a b o u t ~ 100 pmol C02/mg chlorophyll x h o u r . Dark CO2 f i x a t i o n was 3~ of th~ l i g h t v a l u e s .

r e g u l a t i o n o f t h e p h o t o s y n t h e t i c carbon metabolism. Meanwhile t h e r e are two r e p o r t s on t h e c o n t i nuous g r o w t h o f p h o t o a u t o t r o p h i c c e l l cultures from S p i n a c e a o l e r a c e a ( D a l t o n 1980) and Asparagus (Peel 1982). For both c u l t u r e s , long-term s t e a d y - s t a t e g r o w t h c o n d i t i o n s c o u l d be e s t a blished. The p r e s e n t c o m m u n i c a t i o n d e s c r i b e s f u r t h e r p r o g r e s s in t h e a p p l i c a t i o n of an op~n c n n t i n u o u s c u l t u r e system, o p e r a t i n g on t h e a i r l i f t p r i n c i p l e , f o r t h e p h o t o a u t o t r o p h i c growth o f c e l l s u s p e n s i o n s from C. rubrum. For i l l u s t r a t i o n , d a t a on c e l l g r o w t h , n u t r i e n t s u p t a k e , cell composition and equilibrium enzyme activities o f t h e c e l l s d u r i n g s t e a d y s t a t e g r o w t h c o n d i t i o n s are p r e s e n t e d .

Abbreviations

Materials

C. rubrum = Chenopodium rubrum, DTE = d i t h i o erythritol, EDTA = e t h y l e n e - d i a m i n e - t e t r a a c e t i c a c i d , HEPES = N-2-hydrox_y-ethylpiperazine-N2-ethansulfonic acid, S.E.x = standard error o f t h e mean. Introduction Photoautotrophic cell suspension culturesfrom C. r u b r u m , p r o p a g a t e d in s m a l l b a t c h e s u s i n g the two-tier c u l t u r e method (H~semann 1981) or grown a t l a r g e - s c a l e c u l t u r e volumes in a 2-i airlift f e r m e n t e r (H~semann 1982), pass t h r o u g h a sequence o f d i f f e r e n t physiological stages. The l i m i t a t i o n s in t h e use o f batch c u l t u r e s f o r s t u d i e s on t h e r e g u l a t i o n o f c e l l metabol i s m because o f t h e i r t r a n s i e n t c o n d i t i o n s may p a r t i a l l y be overcome by u s i n g open cont i n u o u s c u l t u r e systems (Fowler 1977; Wilson 1980). In such systems, t h e a d d i t i o n o f f r e s h c u l t u r e medium is b a l a n c e d by the w i t h d r a w a l of e q u a l volumes of c e l l s u s p e n s i o n . The c e l l s can be m a i n t a i n e d in a s t e a d y s t a t e o f b a l a n c e d g r o w t h in a r e l a t i v e l y constant cell environment. U n t i l r e c e n t l y c o n t i n u o u s c u l t u r e systems were o n l y used f o r c e l l growth under heterotrophic conditions. Thus t h e c o n t i n u o u s g r o w t h o f h i g h e r p l a n t c e l l s in s u s p e n s i o n under s t r i c t l y photoautotrophic conditions w i l l o f f e r a new approach f o r s t u d i e s on t h e d e v e l o p m e n t and m a i n t a i n a n c e of photoautotrophy in a c t i v e l y d i v i d i n g c e i l s and on t h e

and Methods

C o n s t r u c t i o n a l and o p e r a t i o n a l c o n t i n u o u s c u l t u r e system.

details

of t h e

The b a s i c V-shaped c u l t u r e v e s s e l , made from an E r l e n m e y e r f l a s k used in t h e i n v e r t e d pos i t i o n , t h e mode o f a e r a t i o n and m i x i n g t h e c e l l s by t h e a i r l i f t principle, the various i n l e t and o u t l e t p o r t s made o f ground g l a s s j o i n t s , r u b b e r t u b i n g c o n n e c t i o n s , t h e f l o w of tempered w a t e r f o r t e m p e r a t u r e c o n t r o l as w e l l as t h e mode o f k e e p i n g the c u l t u r e u n i t aseptic by s e v e r a l f i l t e r d e s i g n s and p r e v e n t i n g l o s ses o f m o i s t u r e by t h e use o f w a t e r - c o o l e d c o n d e n s o r s have been d e s r i b e d in d e t a i l r e c e n t l y (H~semann 1982). For a c o n t i n u o u s o p e r a t i o n t h e f e r m e n t e r system was e q u i p p e d w i t h two o i l - f r e e diaphragm pumps (FE 211, B . B r a u n , M e l s u n g e n , Germany) for simultaneous addition of fresh culture medium and w i t h d r a w a l o f e q u a l amounts of c e l l suspension (Fig.l). The pumps f o r a d d i n g t h e medium were c o n n e c t e d by v i t o n t u b i n g s (2 mm i n n e r d i a m e t e r ) t o t h e c u l t u r e s a m p l i n g flask and t o a I 0 - i c u l t u r e medium s t o r a g e f l a s k . In a d d i t i o n , two s t e r i l e filters made o f c o t ton w o o l , e n c l o s e d in g l a s s t u b e s ( 20 cm i n l e n g t h , 2 cm i n n e r d i a m e t e r ) were f i t t e d to t h e medium s t o r a g e f l a s k t o f u n c t i o n as a i r pressure equilizing t u b e s . Both medium pumps were c o n t r o l l e d by a t i m e r ( W. J ~ r s c h , Nordw a l d e , Germany) f o r s y n c h r o n o u s o p e r a t i o n at distinct time intervals.

6O

air sferilisingfilter design

!

C021air ci.~.rc" - c~lalation

mediuminput

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mediumpumps

,l~

wifhd-"~awal \ cell suspension

No-~ " - - w a f e r ~

L~ medium-

sforage flask

Plant material

jack~f '

water cooled condenser

diaphragm pump

and growth c o n d i t i o n s

Stock c u l t u r e s from p h o t o a u t o t r o p h i c , v i t a m i n and hormone-independent c e l l suspensions from C. rubrum were r o u t i n e l y propagated in 30-ml batches as p r e v i o u s l y d e s r i b e d (HOsemann 1981 ). The c u l t u r e medium (MS-medium) c o n t a i n e d the m i n e r a l s a l t s a c c o r d i n g to the f o r m u l a of Murashige and Skoog ( 1 9 6 2 ) w i t h o u t any o r g a n i c constituents. C e l l s from the s t o c k c u l t u r e s , r e s t i n g in t h e phase of s t a t i o n a r y g r o w t h , were t r a n s f e r r e d i n t o 1.5 1 f r e s h MS-medium of t h e f e r m e n t e r v e s s e l . An i n i t i a l c u l t u r e d e n s i t y of about 600,000 c e l l s / m l was e s t a b l i s h e d . The c e l l s were f i r s t batch propagated u n t i l a c u l t u r e d e n s i t y of 900,000 c e l l s / m l was achieved and c o n t i n u o u s c u l t u r e growth could be s t a r t e d . For c o n t i n u o u s o p e r a t i o n of t h e c u l t u r e u n i t , e v e r y 30 minutes 5 ml of f r e s h MS-medium was added and equal amounts of c e l l suspension were w i t h d r a w n . A d i l u t i o n r a t e of O.16/day was c a l c u l a t e d by taken 1.5 1 as t h e mean w o r k i n g volume of the c u l t u r e u n i t . Continuous w h i t e l i g h t was p r o v i d e d by 3 lamps, HQL 125 W (Osram, Germany), a d j u s t i n g t h e l i g h t intensity to 10,000 l u x at t h e o u t e r g l a s s w a l l of the c u l t u r e v e s s e l . The c e l l s were grown at 25°C. A 2% ( v / v ) C 0 2 c o n c e n t r a t i o n in the gaseous atmosphere of the c u l t u r e u n i t was p r o v i d e d by a 2 M KHCO3/K2CO3-buffer s o l u t i o n a c c o r d i n g to Warburg et a l . ( 1961).The a i r / C O 2 - m i x t u r e was set into circulation t h r o u g h the c l o s e d c u l t u r e system at a f l o w r a t e of 300 m l / m i n by an oil-free diaphragm pump (WISA 300 ASK, W.Sauer, W u p p e r t a l , Germany). Analytical

3= inoculationport

p

S

flow of tempered water

1= pressure equiIizingtube 2: thermometer

Measurements

Q u a n t i t a t i v e d e t e r m i n a t i o n s of t h e ammonianitrogen, nitrate-nitrogen and i n o r g a n i c phosphate c o n t e n t of the c u l t u r e medium, t h e p r o cedure of c e l l c o u n t i n g , e x t r a c t i o n and d e t e r m i n a t i o n of c h l o r o p h y l l , p r o t e i n , s t a r c h and sugar were performed as d e s c r i b e d p r e v i o u s l y (HOsemann 1981). P h o t o s y n t h e t i c C02 rd a s ing s i mtiol at ht ie° n was measured u s i n g 14-C02 acco method of HOsemann e t a l . ( 1 9 7 9 ) .

Fig.

I

S e c t i o n a l view of a continuous culture system w o r k i n g on the airlift principle for the photoautot r o p h i c growth of C. rubrum c e l l suspensions.

C%-reservoir KHCOJK2CO3buffer

For enzyme e x t r a c t i o n , u s u a l l y I g c e l l s (one p a r t ) were ground in a c h i l l e d m o r t a r w i t h 2 p a r t s ( w / v ) of t h e e x t r a c t i o n medium. The r e s u l t i n g homogenate was c e n t r i f u g e d f o r 20 minutes at 25,000 x g a t 4°C. The c l e a r supern a t a n t was d e s a l t e d by Sephadex G 25 chromatography. R i b u l o s e b i s p h o s p h a t e c a r b o x y l a s e (EC 4 . 1 . 1 . 3 1 ) and p h o s p h o e n o l p y r u v a t e c a r b o x y l a s e ( EC 4.1. 1.31) were e x t r a c t e d and assayed as d e s r i b e d earlier (HOsemann 1981). N i t r a t e r e d u c t a s e (EC 1 . 6 . 6 . 1 ) and n i t r i t e r e d u c t a s e (EC 1 . 6 . 6 . 4 ) were e x t r a c t e d in a 100 mM phosphate b u f f e r , pH 7 . 5 , c o n t a i n i n g I mM EDTA and I mM DTE. Enzyme a c t i v i t i e s were assayed a c c o r d i n g t o Jordan and Fletcher (1979).

Pyruvate kinase (EC 2.7.1.40), NADH-linked malate dehydrogenase (EC 1.1.1.37) and NADPHlinked i s o c i t r a t e dehydrogenase (EC 1.1.1.41) were extracted with a 50 mM HEPES-buffer, pH 7.6, containing I mM EDTA and I mM DTE. Enzyme a c t i v i t i e s were determined spectrophotometrically by the procedure of Bergmeyer (1970). StatLstical

Methods

The e x p e r i m e n t a l data were o b t a i n e d from t h r e e independent runs of t h e c o n t i n u o u s c u l t u r e system. The r e s u l t s were expressed as t h e mean (~) of at l e a s t 5 s i n g l e measurements, c o r r e c t e d by the s t a n d a r d e r r o r ( S . E . ) The per cent s t a n d a r d e r r o r (%S.E.~) of the mean was taken as a c r i t e r i o n for steadiness. Any p a r a m e t e r which had a v a l u e f o r t h e %S.E.~ below 10%, was c o n s i d e r e d s t e a d y . Results Establishment

of s t e a d y s t a t e

growth

P h o t o a u t o t r o p h i c , v i t a m i n - and phytohormonei n d e p e n d e n t c e l l s u s p e n s i o n s from C. rubrum were f i r s t batch propagated in MS-medium until the cells divided exponentially ( 20% i n c r e a s e in c e l l number w i t h i n 2 d a y s ) . A c u l t u r e d e n s i t y of about 900,000 c e l l s / m l was reached at day 8 of t h e c u l t u r e p e r i o d ,

61 Fig.

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2

Development of a s t e a d y s t a t e f o r sev e r a l g r o w t h parameters in continuous photoautotrophic cell c u l t u r e s from C. r u brum. The c e i l s were propagated at 2% (v/v) CO2 under 10,000 lux continuous i l l u m i n a t i o n at 25°C. Continuous operat i o n of the c u l t u r e system was started at day 8. The values are the means of 3 r e p l i c a t e s . Steadiness is documented by a S.E.~ below 10%.

E

- 0.? I

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so t h a t t h e c o n t i n u o u s o p e r a t i o n of t h e c u l t u r e system c o u l d be started ( F i g . 2 . A d i l u t i o n rate of O . 1 6 / d a y was e s t a b l i s h e d and equil i b r i u m c u l t u r e d e n s i t i e s of 1,100,000 c e l l s / m l were a c h i e v e d w i t h i n 4 days and remalned cons t a n t ( 2% S . E . ~ ) o v e r a range o f 3 weeks. At steady state cell densities, the specific g r o w t h r a t e was c a l c u l a t e d t o be O . 1 6 / d a y , since the dilution rate equals the specific growth rate during steady state conditions. The c o r r e s p o n d i n g mean d o u b l i n g t i m e of t h e c e l l p o p u l a t i o n was a p p r o x i m a t e l y 100 h o u r s .

M o r e o v e r , C. rubrum c e l l s from s t e a d y s t a t e cultures stLll a c c u m u l a t e d s t a r c h and sugar as can be seen from T a b l e I . Small f l u c t u a t i o n s met t h e c r i t e r i o n for steadiness.

The s t e a d y s t a t e v a l u e s f o r c e l l p r o t e i n and chlorophyll c o n t e n t , and t h e e q u i l i b r i u m lev e l s o f p h o s p h a t e , ammonia and n i t r a t e in the c u l t u r e medium are g i v e n i n F i g . 2. The i n i tial b a t c h p r o p a g a t i o n o f t h e c e i l s f o r 8 days r e s u l t e d in a marked r e d u c t i o n of t h e c o n t e n t of p h o s p h a t e (50%) and ammonia as w e l l as nitrate (25%) in t h e medium. D u r i n g f o l l o w i n g c o n t i n u o u s c u l t u r e g r o w t h , s t e a d y s t a t e nutrient l e v e l s of t h e c u l t u r e medium were a c h i e v e d . The f l u c t u a t i o n s I were always below t h e 10% range of t h e S . E . x .

C02-assimi lation :

The t r a n s f e r o f c e l l s from t h e s t a t L o n a r y phase of g r o w t h i n t o t h e l a r g e - s c a l e culture volume of t h e f e r m e n t e r v e s s e l and s u b s e q u e n t b a t c h p r o p a g a t i o n f o r 8 days was accompanied by a s i g n i f i c a n t d e c r e a s e in t h e c h l o r o p h y l l and p r o t e i n c o n t e n t of t h e c e i l s by 50% and 25%, r e s p e c t i v e l y ( Fig. 2 ). During following c o n t i n u o u s c u l t u r e g r o w t h , s t e a d y s t a t e concentrations o f a v e r a g e l y 233 ug p r o t e i n and 11.2 ug c h l o r o p h y l l per 1 , 1 0 0 , 0 0 0 c e l l s were b u i l t up r a p i d l y . S t e a d i n e s s i s documented by a v a l u e of t h e S . E . 7 below I 0 % . Photosynthetic C02-assimilation t i o n of s t a r c h and s u g a r .

and a c c u m u l a -

Continuously dividing photoautotrophic cel! s u s p e n s i o n s from C r u b r u a s s i m i l a t e d a b o u t 100 umol CO2 / mg c h l o r o p h y l l x hour, equival e n t t o 1.1 umol CO2 / 1 , 1 0 0 , 0 0 0 c e l l s x h o u r . The dark incorporation o f CO2 was 3% o f t h e iight values ( Table I ).

Table

1

C02-assimilation, s t a r c h and s u g a r co n t e n t i n p h o t o a u t o t r o p h i c cell c u l t u r e s from C. rubrum d u r i n g steady state growth.

light

1.100+-0.070

umol C02/I06 c e l l s x h

dark

0.035 + 0.003

umo] C02/I06 c e l l s x h

5.0

+-0.5

ug / 106 c e l l s

Glucose:

0.9

+0.1

ug / 106 c e l l s

Sucrose:

2.1

+-0.2

ug / 106 c e l l s

Starch

:

C02-assimilation was determined by the incorporation of 14-C02 under standard c u l t u r e conditions. The data presented here are the means of 5 replicates corrected by the S.E. Steady s t a t e c o n d i t i o n s c o u l d a l s o be conf i r m e d from t h e l e v e l of i n v i t r o a c t i v i t i e s of a v a r i e t y of enzymes r e l a t e d to d i f f e r e n t m e t a b o l i c p a t h w a y s , such as CO - a s s i m i l a t i o n nitrate reduction, glycolysis ~nd c i t r i c acid c y c l e ( T a b l e 2 ). The a c t i v i t i e s of enzymes in s t e a d y s t a t e e x p e r i m e n t s are e x p r e s s e d on a per c e l l b a s i s with regard to the metabolic properties of the cells. During continuous culture growth, equilibrium enzymatic activities, possessing a h i g h degree o f s t e a d i n e s s , were p r e s e n t . The S . E . x a l w a y s ranged below t h e 10% v a l u e .

62 Table 2

Steady s t a t e a c t i v i t i e s of enzymes from d i f f e r e n t m e t a b o l i c pathways in photoautotrophic c e l l s from C. rubrum m a i n t a i n e d i n c o n t i n u o u s - culture.

Type o f enzyme

nkat

/

106 c e i l s

Ribulosebisphosphate carboxylase

0 . 2 8 7 ± 0.020

Phosphoenolpyruvate carboxylase

0.333 ± 0.030

Nitrate

reductase

0 . 1 6 2 ± 0.010

Nitrite

reductase

0.088 ± 0.008

Isocitrate dehydrogenase (NADPH)

0.650 ± 0.032

Malate dehydrogenase (NADH)

28.800 ± 2.300

Pyruvate kinase

0 . 1 8 0 ± 0.018

rate, thus

documenting balanced growth.

Continuous (chemostat) cultures allow the transition of c e l l s from one s t e a d y s t a t e t o a n o t h e r by c h a n g i n g t h e c o n c e n t r a t i o n o f t h e growth-limiting nutrient.ln photoautotrophic c e l l s u s p e n s i o n s from C. rubrum s t r i k i n g changes in t h e in v i t r o a c t i v i t i e s of t h e r i bulosebisphosphate and p h o s p h o e n o l p y r u v a t e c a r b o x y l a s e ~ a s w e l l as in t h e pathway o f photosynthetically f i x e d carbon can be a c h i e v e d by a l t e r i n g t h e r a t i o of a m m o n i a - n i t r o g e n t o nitrate-nitrogen i n t h e c u l t u r e medium (HUsemann, u n p u b l i s h e d r e s u l t s ) . Therefore, photoa u t o t r o D h i c c h e m o s t a t c u l t u r e s from C. rubrum would p r o v i d e an i d e a l system f o r s t u d y i n g t h e r e g u l a t i n g e f f e c t s of d i f f e r e n t nutrients, e s p e c i a l l y o f n i t r o g e n , on p h o t o s y n t h e s i s . Chemostat c u l t u r e s would a l s o a i d in s t u d i e s on t h e i n t e r d e p e n d e n c e of a s s i m i l a t i v e and dissimilative m e t a b o l i c pathways in p h o t o s y n t h e s i z i n g c e l l s from h i g h e r p l a n t s . Acknowledgements

The data presented are the means ± S.E. of at least 5 independent determinations. Discussion The e x p e r i m e n t a l data p r e s e n t e d i n t h i s comm u n i c a t i o n have been s e l e c t e d t o i l l u s t r a t e the satisfactory o p e r a t i o n of t h e c o n t i n u o u s airlift c u l t u r e system, a p p l i e d f o r t h e f i r s t t i m e t o t h e c o n t i n u o u s g r o w t h o f C. rubrum c e l l s under p h o t o a u t o t r o p h i c conditions. Some e x p e r i m e n t a l d i f f i c u l t i e s arose from t h e t e n d e n c y f o r t h e c e l l s to s t i c k t o t h e g l a s s w a l l s of t h e c u l t u r e v e s s e l . D a i l y r o u t i n e of w i p i n g t h e c e i l s back i n t o t h e c u l t u r e medium was n e c e s s a r y . C u l t u r e d e n s i t i e s h i g h e r t h a n 1,500,000 c e l l s / m l increased the risk of b l o c k a g e of t h e c e l l h a r v e s t i n g system. T h e r e f o r e , i t was n e c e s s a r y t o e s t a b l i s h t h e c u l t u r e d e n s i t y below t h i s c r i t i c a l l e v e l by a p p r o p r i a t e s e t t i n g of t h e d i l u t i o n rate. In c o n t r a s t t o t h e method used f o r t h e p h o t o a u t o t r o p h i c g r o w t h of C. rubrum c e l l s u s p e n s i o n s , c e l l s from Spinacea o l e r a c e a ( D a l t o n 1980) and Asparagus o f f i c i n a l i s (Peel 1982) were grown p h o t a u t o t r o o h i c a l l y in continuous c u l t u r e by p a s s i n g a stream of a i r , e n r i c h e d i n COt but reduced in 09 c o n t e n t , t h r o u g h t h e cell ~uspension. The data f o r s t e a d y s t a t e g r o w t h i n p h o t o a u t o t r o D n i c c e l l c u l t u r e s from t h o s e t h r e e d i f f e r e n t p l a n t s p e c i e s m e n t i o n e d above d o c u ment t h a t m u l t i p l i c a t i o n and d i f f e r e n t i a t i o n of chloroplasts can keep pace w i t h t h e c e l l division rate. Electron microscopic studies c o n f i r m w e l l d e v e l o p e d t h y l a k o i d s and grana stacks in the chloroplasts from a c t i v e l y d i v i d i n g c e l l s from C. rubrum (H~semann, unpub l i s h e d r e s u l t s ) . Thus, t h e h y p o t h e s i s of L a e t s c h and S t e t l e r (1965) t h a t c e l l g r o w t h and c h l o r o p l a s t differentiation will possibly e x c l u d e one a n o t h e r , o b v i o u s l y c a n n o t be applied to those cells that exclusively meet t h e i r e n e r g y and carbon s u p p l y by p h o t o s y n thesis. The s t e a d y s t a t e v a l u e s f o r p r o t e i n , chlorop h y l l and s t a r c h c o n c e n t r a t i o n s as w e l l as the activities o f enzymes from d i f f e r e n t m e t a b o l i c pathways show t h a t m e t a b o l i c a c t i vities are p r o p o r t i o n a l to the cell division

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