(Instituto "Jaime Ferritn" de Microbiologia C. S. I. C. - Madrid)

The influence of growth rate on cellular size of Erwinia carotovora

P. FERNANDEZ, A. PORTOL~S and M. T. IRIARTE

(Eingegangen am 11.12.1967)

In previous papers (PIRT 1957, PERRET 1958, HERBERT 1959) variations in the cellular size of several bacteria growing in continuous culture at, different rates were observed. A study on Erwinia carotovora growing in chemostat with two different sources of energy was carried out. Variations in t,he growth yield constants with glucose and pectin as limiting factors are shown. In addition, the influence of growth conditions on cellular size, pH variations and utilization of hydrocarbonated sources have been observed.

Materiul and methods I n all experiments, the organisms used were Eiwinia carotovora KCPPB 312. Stock cultures

were kept on slants of phytopathogen medium a t 28 "C. The basic medium used in the chemostat was: Na,HPO,. 12 H,O 28,4 mg"& (NH,),HP04

50 mg%, L-asparagine - H,O 52,s mgo{, and yeast extract 40 mg%. Either 276 glucose or 2"/, pectin was added to the basic medium as carbon source (limiting

factor). The continuous culture apparatus used was similar to that described by ROSENBERGER

and ELSDEN (1960); the operational steady-state procedure was carried out a t 27 "C 0,5. Samples of the culture were obtained a t different times, optical density (OD), dry weight, and the p H values, and sugar were determined.

Direct determinations of dry weight on washed cells were made a t 105 "C and the optical density was measured in a Spectronic 20 a t 610 mp.

Glucose and other reducing substances were determined by the SOMOGY-KELSON method. p H values of the culture were controlled in a COLEMAN Metnon I1 a t 20 "C.

Results The steady-state growth of E . carotovora, a t different dilution rates, with 2 yo

glucose and 2% pectin as limiting factors are shown in Fig. 1. In the buffered media, with pectin, the pH values dropped from 7,2 to 6,4 at the higher dilution rates while in the glucose series the pH values dropped from 7,O to 6,O for the intermediate dilution rates and reached 5,6-5,s in the higher ones.

The ratios between bacterial dry weights and optical density in the culture a t different rates were determined in the chemostat and the results presented in Fig. 2 show variations in the cellular sizes of E . carotovora in close relationship with the dilution rate.

A comparative examination of the variations in pH, sugar contents, OD and dry weight in the chemostat a t different rates is shown in Fig. 3 and results for the two sources of energy a t two different dilution rates (0,179 and 0,516) are compared.

366

20

Q Z6- b14. a z2 p a

h; ?GBr

,a4- .- S06-

02

Glucose Pectin - -

I - = 4

" 0 8 Y - c 0 0 0 "

. a a 40

-

c c

0

I , a iii az 03 a 4 QS 0.6

Dilution rate f h r )

Fig. '7. Rclat ionships between optical densit.j-/dry weight in E. curo~oz~oru on chemostat n.it.h glucose (0) or pect.in ( 0 ) as limiting fact.ors

3Pr Glucose r Pectin

EB mg dry wt bacteria/4 - rng. N cellular 0 Cell production W Total carbohydrate content in medurn

Fig. 3. Results of two continuous cultore runs corresponding to two rates with glucose or pectin as limiting nutriens

Growth rate and cellular size of Brwiitin cnrotororu 267

Discussion

E . carotovora growing in a chemostat a t different flow rates can nearly always use either glucose or pectin as the only source of energy.

The comparative study of Fig. 1 shows that variations of the different steady- state levels at several dilution rates were lower when pectin was used as a source of energy than that in which glucose was added. This could be regarded as the result of the production of an adaptative exoenzyrne which can improve the utilization of the sourcc of energy.

Regarding the fact that the light scattering phenomenon is a function of particle size and the ratios between OD/dry weight a t several flow rates, cell sizes decrease when dilution rates grow greater. This phenomenon was cmphasiz- ed when pectin was the source of energy used. Prom this we can consider that infectivity has a proportionate relation with doubling time arid an unproporti- onate relation to cellular size.

Cell yields, pH, OD and glucose variations a t differcnt rates show that the "cell multiplication rate" was higher when pectin was used instead of glucose as a source of energy. I n spite of i t , the protein ratio, which always decreases when the dilution rate increases, was lower when pectin was addcd.

Summary

A preliminary study of the continuous cult'ure of E. carotoaorn is reported by using glucose Under these condit'ions i t was possible

This phenomenon was enhanced in t'he presence of pect'in and, on t'he other hand, i t was

and pect'in as limiting factors a t different flow rates. to observe a cellular size decrease in proportion to the increase of the diiut.ion rat,e.

also found that pectin shows a higher production constant,.

R e f e r e n c e s

HERBERT, D., 1959. Some principles of continuous culture. In : Recent Progress in Micro- biology. Symp. 7tll Congr. Int. Microbiol.

PERRET, C. J., 1958. The effect of growth rate on the anatomy of Escherichln coli. J. gen. Microbiol., 18, vii.

PIRT, S. J., 1957. The oxygen requirements of growing cultures of Aerobacter species de- termined by means of t'he continuous cult'ure t'echnique. J. gen. Microbiol, 16, 59.

ROSENBERGER, R. F. and ELSUEN. S. R., 1960. The yields of Streptococcus faecnlia groivn in continuous culture. J. gen. Microbiol., 22, 726-739.

SONOCWI, M., 1952. Notes on sugar determination. J. biol. Chemistry, 196, 19-23.