(Laboratory of Organic Chemistry, The University, Leiden, the Netherlands).

SOME GROWTH EXPERIMENTS W I T H P O L Y T O M A UVELLA ON S Y N T H E T I C MEDIA x) by

j. LINKS 2), A. VERLOOP and E. HAVINGA (Received October 15, 1960). INTRODUCTION. The nutrition of the phytoflagellates belonging to the genus Polytom.a has been studied by several authors (e.g. PRINGSHEI~.I, 1937; PROVASOLI, 1937; LwoI~I~and DusI, 1937). In growth experiments with Polyloma uvella, strain 1, kindly given to us by Prof. PRINGSHEII~Lwe obtained good growth in a synthetic medimn with asparagine as sole source of nitrogen but no development occurred when glycine was substituted for asparagine. Since such a differential behaviour towards these nitrogen sources had not been reported earlier for Polytoma species, the phenomenon was further investigated. In table 1 the composition of some media used by LwovI: (1932) and by PRINGSHEI~.I (1937) for Polytoma obt,u,s~l,m and Po~ytoma uvella se,nsu stricto respectively, is compared with that of two media used in the present investigation. LwoI~F and PRINGSHEII~fobtained with glycine "good growth" and with asparagine "very good growth". As to asparagine compare ROTTIER, 1936. When we omitted the yeast extract and the glucose {caramel} from the non-synthetic medium, growth became very poor. Replacing glycine by asparagine always resulted in excellent growth with1) The contents of this article has been earlier published as part of a doctor's thesis, written in the Dutch language (LINKS, 1955). 2) Present address: Laboratory of Physiological Chemistry, University of Amsterdam, J.D. Meyerplein 3, Amsterdam.

Growth experiments with Polytoma uvella.

77

TABLE 1. Composition of media for Polytoma uvella and Polytoma obtusum used by various investigators.

Components N a-acetate asparagine or : glycine ammonium salts glucosamine cysteine peptones K oHPO~ MgSO,t KC1 CaC1._, FeC1a NaoC03 glucose- caramel yeast extract

LWOFF1) (1932) 0.1 0.1 0.1 0.l 0.1 0.1 0.1 0.02 0.01 O.01 0.0052) 0.0052)

Amount in % used by: PRINGSHEIM L I N K S (1937) 3) (1948) 0.2 0.2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.01 0.02 0.01 0.01

LINKS (1952) 0.2 0.2

0.02 0.01

0.00001 0.1

0.02 0.2 10 (v/v)

0.02

All media had the same p H of 7.5 and were made up with distilled water. 1) Compare LWOFF and Dusl (1937 and 1941). 2) Added after sterilization. 3) This non-synthetic medium was based on data of PRINGSHEIM and MAINX (1926) and a personal communication from PRINGSHEIM (1948). o u t a n y a d d e d "caramel", y e a s t e x t r a c t , CaC12 or FeC13. Conseq u e n t l y w e d e c i d e d t o i n v e s t i g a t e t h e i n f l u e n c e of t r a c e e l e m e n t s , t h i a m i n a n d c y a n o c o b a l a m i n ( v i t a m i n Br, ). Glycine and asparagine do not serve as carbon-sources (c[. PRINGSHEIM, 1937). These substances as well as several yeast constituents, may, however, influence heavy metal ion concentrations by chelate formation (HUTNER and PROVASOLI, 1951). Vitamins are reported not to be required for Polytoma uvella (PROVASOLI, 1937 ; LWOFF, 1947 ; HALL, 1943 ; LWO~F and DosI, 1937). But, as the need of thiamin varies strongly within the species of the phytofiagellates (LwoFF, 1947; LWOFF and DusI, 1937), one could think of a thiamin requiring mutant in the present case. Also it might be conceivable t h a t for a certain phytoflagellate the need for thiamin is greater in a medium with glycine than with asparagine (c/. SMYTH, 1940). Further, thiamin stimulates carotenogenesis in Polytoma uvella (LINKS el al., 1960). Yeast extract (HuTNER and PROVASOLI, 1951), asparagine (ROBBINS el al., 1950) and metabolic products of microbial infections of the cultures (Pr~INOS-

78

J. Links, A. Verloop and E. Havinga,

HEI.~t, 1946) can show B,~ activity and several phytoflagellates are known to need or to be stimulated by extremely small concentrations of this vitamin (LITTLE el al.. 1951; HUTNER el a,l., 1956). The initial diverging results with the two different N-sources could also have been due to accidental contamination of the medium with cotton plug material which may contain thiamin (ScI-IOPFI'~R and RVTZ, 1937) and other growth factors {HuTNI~Rel a.l., 1958). EXPERIMENTAL. Cultivation. Cultures were grown in 10 ml medium, made up with distilled water, in 30 ml culture tubes provided with cotton plugs. The tubes had been cleaned successively with a hot soda. solution, diluted HC1, tapwater and distilled water. Sterilization : 120 ~C. for 20 rain. I n c u b a tion t e m p e r a t u r e : 23 ~ • 1~ C. Daylight was excluded. Two successive subcultures were made 7 days after each inoculation with 0.03 ml per 10 ml. Microbial

vitamin

B,2 a s s a y .

Vitamin B,2 activity was determined with L a c l o b a c i l h , . s l e i c h m a n n i l A.T.C.C. 31-3 in the m e d i u m of SKEGGS (SKEGGS el al., 1950; AUSSEN, 1954). Found (in m?, Bv_,/g): asparagine (Hoffmann-La Roche) 1..35 -k 0.02 (in duplicate) asparagine (B.D.H.) 9.03 • 0.03 (in duplicate) glycine (Brocades, Stheeman and Pharmacia) 0.0 Na-acetate (Hopkins and Willialns. Analar) 0.0 RESULTS .aND CONCLUSIONS. Replacing glycine hy asparagine in the simple m e d i u m (Na-aeerate, 3 inorganic salts) always restored good ~ o w t h . The difference between growth on the two media remained upon : a) Reerystallization of the asparagine (5 • from H20 ) and ot the glycine (6 • from H20 ). b) Addition of c o m b i n a t i o n s of "caramel", CaCI~ a n d FeC1 a after sterilization. c) Addition of 10-6-10-3% ZnC12; according to polarographic determination, the H o f f m a n n - L a Roche asparagine contained between 10-6 and 10-4 % ZnC12 and no acetate or glycine.

Growth experiments with Polyloma 2wella.

79

d) Addition of the ash constituents of asparagine and yeast extract (dry substance 2~/o). e) Addition of the trace element mixture of HUTNER et al. (1950) (inconclusive results). /) Addition of thiamin (1.3 mg/1). g) Addition of vitamin B12, folic acid, CoCI~ or p-amino-benzoic acid (10-'~-2.10 .3 %). Some Polytoma cultures with the basic acetate-glycine medium, without "caramel", CaC12 or FeCla, showed good growth after accidental infection with bacteria. Other Polytoma cultures on the same medium at first showed no sign of growth but started to grow quite well about 2 months after inoculation. The above mentioned results do not give an indication of any influence of trace elements or of the vitamins studied that might offer a clue to understanding why replacing of glycine by asparagine effects such a remarkable improvement of growth. Apparently asparagine does not only act as a nitrogen source but may have an additional function, probably by its, usually easy, in vivo convertibility into the Krebs cycle intermediate, oxaIacetate. Accidental infection with bacteria, yeast extract and long adaptation times of pure Polyloma cultures on the glycine-acetate medium probably provide small amounts of Krebs cycle intermediates, required for good growth. These could be succinate or malate. A parallel with our findings of the usually poor ~ o w t h of Polyloma ,~tvella (strain 1) on the glycine-acetate medium and its excellent growth on the asparagine-acetate medium is found in the growth experiments of HUTNER,B:\cI-I and Ross with another phytoflagellate, the Z strain of E uglen,a gracilis, published, independently and almost sinmltaneously with ours (HuTNER el al., 1956; LINKS, 1955). These investigators found that their organism utilized its C-substrate (sugar) vigorously if readily available nitrogen and a Krebs cycle intermediate (or auxiliary substrates readily convertible to such an intermediate) were present: these requirements were met by glutamic acid and aspartic acid, or asparagine. Summary. 1) Polyloma ,uvella (strain l) grows excellently on the following basic medium : sodium acetate, 0.2 % ; asparagine, 0.2 ~ ; K2HPO 4,

80

J. Links, A. Verloop and E. Havinga.

0 . 0 2 % ; M g S Q . 7 H 2 0 , 0 . 0 1 % ; Na2COa, 0 . 0 2 % ; p H = 7.5 (in d i s t i l l e d w a t e r ) . C a r a m e l , CaC12, FeCla or o t h e r a d d i t i o n a l t r a c e m e t a l s are n o t r e q u i r e d . 2) R e p l a c e m e n t of a s p a r a g i n e b y g l y c i n e r e s u l t e d i n v e r y p o o r gxowth. A d d i t i o n of t h i a m i n , v i t a m i n B~2 or t r a c e e l e m e n t s d i d n o t i m p r o v e t h e g l y c i n e m e d i u m . I t is s u g g e s t e d t h a t Polytoma uvella n e e d s s m a l l a m o u n t s of a K r e b s cy'cle i n t e r m e d i a t e for g o o d growth. Acknowledgements. The authors wish to t h a n k Professor E. G. PRINGSHEI~I who kindly provided

Polylon'~a. uvella, strain 1, Mrs. Dr P. Y. F. PRINS-VAN DER MEULEN for valuable technical assistance, Dr B. G. AUSSEN (Nederlands Instituut voor Volksvoeding, Amsterdam) for performing the vitamin B~, determinations and Dr D. DE NOBEL and Dr J. HEE~ISKERK of the Laboratory of Physical Chemistry for performing the polarographic determinations. References. AUSSEN, B. S. 1954. Thesis, Amsterdam. HALL, R. P. 1948. Vitamins and Hormones 1, 252. HUTNER, S. H., PROVASOLI, L., SCHA'rZ, A. and HASKINS, C. P. 1950. Proc. Am. Phil. Soc. 94, 152. HUTNEP,, G. H, and PROVASOLI, L. 1951. Chapter II "The Phytoflagellates" in A. LWOFF (ed.) Biochemistry and Physiology of Protozoa I; New York, HU'rNEIL S. H., BACH, M. K. and Ross, G. J. M. 1956. ]. Protozool. 3, 101. HUTNER, S. H., CURY, A., BAKER, H. 1958. Anal. Chem. 30, 849. LINKS, J. 1955. Thesis, Leiden. LINKS, J., VERLOOP, A. and HAWNGA, E. 1960. Archiv. Mikrobioh 36, 306, LIT'rLE, P. A., OLESON, ]. J. and WILLIA~IS, J. H. i951. Proc. Soc. Exp. Biol. Med. 78, 510. LwoI~v, A. 1932. Recherches biochimiques sur la nutrition des Protozoaires. Monographie de l ' I n s t i t u t Pasteur, Paris. Lwol~F, A. and DusI, H. 1937. Comptes rend. 205, 630, 756, 882. LWOFF, A. and DusL H. 1941. Ann. Inst. Pasteur 67, 230. LWOFF, A. 1947. Aml. Rev. Microbiol. 1, 101. PR1NGSHEIM, F~. G. and ~'IAINX,F. 1926. Planta 1, 621. PRINGSm:.IM, E. G. 1937. Planta 26, 665. PRINGSHEI,~I, E. G. 1946. Pure Culture of Algae. Cambridge Univ. Press PROWXSOLI, L. 1937. C.R. Soc. Biol. 126, 280. ROBmNS, W. ]., HERVEY, A. and S'rEI~BENS, M. E. 1950. Science 112, 455. Ro'rTIER, P. B. 1936. C.R. Soc. Biol. 122, 65 and 776. SCHOPI:ER, W. H. and RVTZ, W. 1937. Arch. Mikrobiol. 8, 244. SI':EGGS, H. e., NEPPLE, H. M., \fALENTIK, K. A., HUFF, J. H. and \VR1GII'r, L. D. 1950. ]. Biol. Chem. 184, 211. SMYrH, D. H. 1940. Biochem. J. 34, 1598.