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On-line version ISSN 0104-6632
Braz. J. Chem. Eng. vol. 14 no. 3 São Paulo Sept. 1997
INFLUENCE OF SURFACE-ACTIVE AGENT CONCENTRATION ON THE SPECTROPHOTOMETRIC BIOMASS ESTIMATION DURING SUBMERGED GROWTH OF
M.C. Sakai and H. Hiss*
*Instituto Butantan (Laboratório de Desenvolvimento de Processos II- Centro de Biotecnologia)
Av. Vital Brasil, 1500 - CEP 05503-900, São Paulo, SP, Brazil - Phone (011) 813-7222 - Fax (011) 815-1505
(Received: January 3, 1997; Accepted: June 6, 1997)
Abstract: The influence of the surface-active agent Tween 80 (polyoxyethylene 20 sorbitan monoleate) on the dispersed growth of Mycobacterium bovis - BCG - was studied. The slopes of the bacterial dry mass / absorbance curves, were significantly reduced, by the increase of the Tween 80 concentration in the medium. Such an influence was probably due to the effect Tween 80 had on the average size of bacterial clumps during the culture carried out in a bench fermenter.
Keywords: BCG, Mycobacterium bovis, submerged culture, Tween 80.
The simplicity and quickness of the turbidimetric method, makes it very suitable for the quantitative study of the microorganisms growth (Borzani, 1972). One restriction of the method, however, consists on the low reproducibility of the standard curves, obtained with the same kind of microorganism, but previously cultivated in different type of media (Borzani, 1972); depending on the medium composition, a different mean organism size may be obtained, which in turn influences the light scattering. The second disadvantage lies on the case of microorganisms that tend to form clumps, which mean size depends strongly on the cultivation conditions. For this reason, and also to enhance the growth, it is necessary to promote the dispersion of the bacterial clumps by means of high speed agitation in the fermenter, or by adding a surface-active agent that does not retard or inhibit the growth.
Dubos (1945) observed that the addition of small amounts of long chain fatty acids esters of polyhydric alcohols permitted submerged and rapid growth of tubercle bacilli.
No references were found in the literature that describe the influence of the surface-active agent concentration on the standard-curve coefficient. This subject is the focus of this paper.
MATERIALS AND METHODS
Mycobacterium bovis - BCG - was grown on autoclaved Sauton medium (Cassagne, 1961) and cultivated in a Biolafitte fermenter with a 20 liter capacity, during 8 to10 days, under the following conditions: initial inoculated volume=12 L; air flow rate=15 L/min; constant agitation speed = 960 rpm; temperature= 37± 1 oC; initial pH=7.2 .
Tween 80, sterilized by filtration, was added to the medium:
- at the beginning of cultivation to concentrations of 0.1 (experiment 7), 0.25 (experiment 6) and 0.5 (experiments 2 and 4) kg of Tween 80 per m3 of culture (Table1);
- after a certain fermentation time (experiments 1, 3 and 5) to a concentration of 0.5 kg of Tween 80 per m3 of culture.
The inoculum was prepared as follows: the liophilized microorganism was transferred to a 300 mL-Erlenmeyer flask containing 100 mL of Sauton liquid medium and cultivated in a shaker at 37oC and 198 rpm. After 14 days of cultivation, the cells were transferred to ten 500 mL-Erlenmeyer flasks each containing 200 mL of fresh medium, and left to grow under the same conditions for 8 days. The contents of these flasks were used as inoculum for the fermenter.
Samples of the cultured medium were taken at specified times for pH, cell concentration and absorbance measurements. No previous centrifugation of the sample were necessary for the absorbance measurements, provided that the medium had the same absorbance effect as distilled water. So, the dilutions (whenever the absorbance exceeded 0.5) were made with such liquid and the result extrapolated, dividing the absorbance value by the correspondent dilution. The readings were made at 540 nm, with a Micronal spectrophotometer mod. B382.
The cell concentrations were expressed as dry biomass and determined by filtration and drying of the cells to constant weight in an oven at 60oC.
RESULTS AND DISCUSSION
Figure 1 shows the linear relation between cell concentrations and absorbance for one of the experiments (experiment no 5). Each value of X corresponds to a sample taken at a different fermentation time. Linearity is maintained during growth for the same fermentation run, indicating a constant mean particle size, or an insignificant change.
The initial pH medium after inoculation was 7.4 and reached 7.8 at the final of growth.
Initial conditions have a striking effect on the size, however, as can be verified by the following results.
The regression results obtained from bacterial dry weight versus absorbance curves and concentration and time of addition of Tween are presented in Table 1.
The interpretation for the different slopes of the standard curves is illustrated in Figure 2. The traced line indicates that two samples of a bacterial culture (1 and 2) having the same mass concentration may have different absorbance readings because the higher average particle size (sample 1) gives rise to a lower light dispersion than the smaller particles (sample 2). For this reason, the slopes of these curves will be associated with the average size of cell clumps.
This comparison was applied to experimental values of b (Table 1):
Comparing the linear regressions in experiments 2 and 4, with Tween added at the beginning of cultivation (t= 0), with the same concentration of 0.5 kg/m3 very close values of b can be observed (considered equal at the 5 % level). Therefore, no significant differences exist between the average size of cell clumps in either case.
In experiments 1, 3 and 5 where Tween was added after inoculation (t= 3.7, 2.7 and 3.7 days, respectively) with the same concentration of 0.5 kg/m3 the b values were greater than those from experiments 2 and 4. So, a further addition of Tween was not efficient in promoting the dispersion of cell clumps.
In experiments 2, 4, 6 and 7, all with Tween added at the beginning of cultivation (t= 0) but in different concentrations, the greatest b value was obtained in experiment 7, where the Tween concentration was the lowest (0.1 kg/m3). This implied that the average size value of cell clumps was the greatest.
The influence of surface-active agent concentration on the b value is clear in Figure 3; the influence is more pronounced at concentrations of Tween between 0.1 and 0.25 kg/m3.
Likewise, Schaefer et al. observed that in this region a good dispersion of cell clumps was reached, and that the addition of Tween in a concentration above 0.25 kg/m3 did not enhance the growth rate.
Table 1: Regression values obtained from bacterial dry weight measurements versus absorbances at 540 nm. The experiments are listed according to the concentration of Tween and the fermentation time of its addition to the medium
|Experiment||addition of Tween||b||r|
|concentration (*)||time (days)|
* kg of Tween 80 per m3 of culture
Figure 1: Cells concentrations (X) as a function of absorbances at 540 nm, and respective linear regression from experiment 5.
Figure 2: Schematic representation of two spectrophotometric standard curves at a given wavelength.
Figure 3: b coefficient values (Table 1) as a function of the concentration of Tween 80 in the medium at the beginning of cultivation.
Abs Absorbance at 540 nm
r Correlation coefficient
b Bacterial dry weight / absorbance units, kg/m3
t Fermentation time, days
X Cell concentrations (dry weight), kg/m3
Borzani, W. O Método Turbidimétrico na Medida
de Concentrações e de Velocidades de Reprodução de Microrganismos. Revista de Microbiologia, 3(3): 155-156 (1972).
Cassagne, H. Milliex de Culture. Tome 2. Collection Techniques de Base, p. 242 (1961).
Dubos, R.J. Rapid and Submerged Growth of Mycobacteria in Liquid Media. Proceedings of the Society for Experimental Biology and Medicine, 58:361-362 (1945).
Schaefer, W.B. ; Marsahk, A. and Burkhart, B. The Growth of Mycobacterium tuberculosis as a Function of Its Nutrients, Journal of Bacteriology, 58:549-563 (1949).