A PUBLISHED KINETIC MODEL EXPLAINS THE VARIATION IN NITROGEN CONTENT OF Pichia guilliermondii DURING ITS BATCH CULTIVATION ON DIESEL OIL

A PUBLISHED KINETIC MODEL EXPLAINS THE VARIATION IN NITROGEN CONTENT OF Pichia guilliermondii DURING ITS BATCH CULTIVATION ON DIESEL OIL

W. BORZANI

Instituto Mauá de Tecnologia, Escola de Engenharia Mauá, Estrada das Lágrimas 2035, São Caetano do Sul – SP – Brazil, CEP 09580-900

Phone: [phone], FAX: [phone]

Received: August 14, 1998; Accepted: December 10, 1998

Abstract - Variation in nitrogen content of Pichia guilliermondii during its batch cultivation on media containing diesel oil as the main carbon source may be explained by means of a kinetic model proposed earlier to interpret the kinetics of nitrogen consumption during the process.

Keywords: Pichia guilliermondii, batch cultivationkinetic model.

INTRODUCTION

During batch cultivation of Pichia guilliermondii (Candida guilliermondii) on media containing diesel oil and ammonium sulfate as the main sources of carbon and nitrogen respectively, the cellular nitrogen content significantly increases at the beginning (10-20h) of the process, and after reaching a maximum value that depends on the experimental conditions, slowly decreases towards its initial value as cultivation proceeds (Concone, 1976; Doin, 1976). Thus, the kinetic study of nitrogen consumption in the above processes (Borzani and Hiss, 1984) leads to the following model

(1)

where S and X are the concentrations of ammonium sulfate and yeast cells (dry matter), respectively, k is the rate constant, and t is time.

The above model was proposed to explain the empirical equations that correlate the values of S and X obtained in experiments carried out by Hiss et al. (1977) with the main purpose to study the influence of the pH level on the growth of Pichia guillierondii on diesel oil. Similar empirical equations also correlate the values of S and X obtained by Concone (1976) and Doin (1976) to study the influence of the initial concentration of diesel oil on the above cultivation. When the pH level increased from 3.0 to 7.0, the value of k increased from 0.023 L/g to 0.108 L/g. When the initial concentration of diesel oil increased from 19.3 g/L to 159.1 g/L, the value of k decreased from 0.141 L/g to 0.043 L/g. Obviously, other experimental conditions as, for instance, temperature, dissolved oxygen concentration and aqueous medium composition, affect k. The first practical consequence of equation (1) was an increase (93%) in process productivity (Borzani and Hiss, 1984). The main purpose of this paper is to show that the above model also explains the observed variation in cellular nitrogen content.

NITROGEN CONTENT OF THE YEAST CELLS CALCULATED FROM EQUATION (1)

Equation (1) results in

(2)

where , and S₀ and X₀ are the initial values of S and X, respectively. Consequently, remembering that the main nitrogen source was ammonium sulfate, we may write where

(3)

(4)

where N is the nitrogen concentration calculated from S (N = 0.2121S). Therefore, the decrease in N is equal to the increase in cellular nitrogen calculated by n = g × X, where g is the nitrogen content of the yeast cells. We may then write

(5)

Equations (4) and (5) result in

(6)

The nitrogen content of the yeast cells will then be

(7)

Assuming that the model represented by equation (1) is applicable, it is then possible to calculate g .

APPLICATION TO AN ACTUAL TEST

Table 1 shows the results obtained in a typical test carried out by Concone (1976), where g_e is the measured value of g .

Table 1: Variations in yeast cell concentration (X), ammonium sulfate concentration (S), and the nitrogen content of the yeast cells (g

t(h)	X(g/l)	S(g/l)	100g e(%)
0	0.11	10.27	8.4
4	0.21	10.20	---
8	0.43	10.12	---
12	1.13	9.70	9.2
16	2.13	8.81	9.5
20	3.82	7.98	9.1
24	6.09	6.89	8.9
28	8.28	5.82	8.6
32	10.72	5.03	8.3
36.2	12.97	4.43	7.9
40	14.06	4.22	7.8

Figure 1 shows that equation (8) correlates the experimental values of S and X from Table 1. The relative differences between the values of S calculated by equation (8) and the corresponding experimental values vary from 0.49% to 2.84% (average, 1.23%; standard deviation, 0.94%).

Figure 1:
Correlation between the ammonium sulfate concentration (S) and the yeast cell concentration (X). See Table 1.

(8)

As shown in Figure 1, we may conclude that equation (1) has been satisfied.

Equation (8) results in

(9)

Equation (9) shows that in this particular test b = 2.183 g/L and k = 0.0654 L/g. Therefore, as X₀ = 0.11 g/L and g _eo = 0.084 (initial value of g _e), the values of n₀ and will be 0.00924 g/L and 0.9928, respectively.

Equation (7) will then be

(10)

where g _c is the calculated value of g .

Figure 2 shows that equation (10) leads to values of g _c significantly greater than the corresponding experimental values g _e. The following facts may be considered in order to explain the relatively high differences between g _c and g _e: #1. during implementation of the method used to measure X (Concone et al., 1972), the yeast cells were maintained in contact with aqueous solutions for a relatively long time; #2. water soluble compounds are leached-out when cells are washed (Mallette, 1969). Let us then assume that the observed differences between g _c and g _e are due to the transport of soluble nitrogen compounds from the yeast cells to the aqueous phase during the measurement of X. Equation (11) must then be satisfied (Geankoplis, 1983).

Figure 2
: Comparison of the nitrogen content of the yeast cells experimentally measured (g _e) and calculated by equation (10) at various yeast cell concentrations (X).

(11)

where C is the concentration of leached-out nitrogen compounds at time t, C_s is the maximum value of C, k_L is the mass transport coefficient, A is the area of the aqueous phase/yeast cells interface, and V is the volume of the aqueous phase. Calling v the volume of fermenting medium sample used to measure X, the mass of yeast cells in the sample will be v.X and consequently

(12)

Equations (11) and (12) lead to

(13)

where

(14)

Thus, as g _c and g _e are known, we may calculate the mass of water-soluble nitrogen compounds assumed to be transported from the yeast cells to the aqueous phase, í.e.,

(15)

Consequently, we may write

(16)

Combining equations (13) and (16)

(17)

where

(18)

(19)

Figure 3 shows that equation (20) correlates C’ and X. The relative differences between the values of C’ calculated by equation (20) and the corresponding values calculated by (g _c - g _e)X varied from 0% to 8.0% (average, 3.2%; standard deviation, 2.5%).

Figure 3:
Comparison of the concentrations of leached-out nitrogen compounds (C’) calculated from equation (18) (· ) and by equation (20) at various yeast cell concentrations (X).

(20)

It is then possible to explain the differences between g _c and g _e as a consequence of the leaching-out of nitrogen compounds in the yeast cells during the experimental measurement of X.

The values of K and are certainly affected by uncontrollable factors as the permeability of the cell envelopes, the specific area of the cells/aqueous medium interface, and the types and concentrations of water-soluble nitrogen compounds within the cells. The above factors may be affected by the experimental conditions, leading to values of K and different from those represented in equation (20). Values of K as low as 0.089 L/g, and of as high as 0.500 g/L were derived from other tests carried out by Concone (1976). It must be pointed out that, in spite of the possibility to explain the differences between g _c and g _e as proposed in this paper, another assumption could be considered, i.e., the production of nitrogen compouds undetectable by the adopted method to measure the amounts of nitrogen within the cells and/or in the fermenting medium.

CONCLUSIONS

The kinetic model proposed by Borzani and Hiss (1984) explains the experimentally observed profile of the curve which represents the variation in nitrogen content of the yeast cells during the batch cultivation of Pichia guilliermondii on diesel oil. The differences between the nitrogen contents calculated from the above model (equation (10)) and the corresponding measured values (Table 1) are due to the transport of water-soluble nitrogen compounds from the yeast cells to the aqueous phase during the measurement of X.

NOMENCLATURE

A Area of the aqueous phase/yeast cells interface, m² C Concentration of leached-out nitrogen compounds concentration, g/L C_s Maximum value of C, g/L C’ See equation (18), g/L C_S’ See equation (19), g/L k Rate constant, L/g k₁ See equation (12), m²/g k_L Mass transport coefficient, L/m².h K See equation (14), L/g m Mass of leached-out nitrogen compounds, calculated by equation (15), g n Cellular nitrogen concentration calculated by g X, g/L n₀ Initial value of n, g/L N Nitrogen concentration calculated by 0.2121 S, g/L S Ammonium sulfate concentration, g/L S₀ Initial value of S, g/L t Time, h v Volume of the fermenting medium sample, L V Volume of the aqueous phase in the method used to measure X, L X Yeast cell concentration (dry matter), g/L X_o Initial value of X, g/L a See equation (2), g/L b 0.2121a , g/L g Nitrogen content of the yeast cells g _c Value of g calculated by equation (10) g _e Measured value of g g _eo Initial value of_{g e}

REFERENCES

Borzani, W. and Hiss, H., Kinetics of Nitrogen Consumption During the Batch Growth of Candida guilliermondii on Diesel Oil and on Molasses, Biotechnol. Lett., 6, Nº 8, 511 (1984).

Concone, B.R.V., Hiss, H., Paz, M.P.G., Patricio, C.G. and Borzani, W., Measurement of Yeast Concentrations in Liquid Hydrocarbon Fermentations: Influence of Experimental Conditions and Statistical Significance of the Results Obtained, Biotechnol. Bioeng., 14, Nº 4, 609 (1972).

Concone, B.R.V., Contribuição ao Estudo do Cultivo de Candida guilliermontii em meio contendo óleo Diesel como fonte de carbono: influência da concentração inicial do óleo em alguns parâmetros do processo descontínuo de cultivo realizado em fermentador de 30 litros. Ph. D. thesis. University of São Paulo, Brazil (1976).

Doin, P.A., Contribuição ao estudo do cultivo de Candida guilliermontii em meio contendo óleo Diesel como fonte de carbono: influência da concentração inicial do óleo em alguns parâmetros do processo descontínuo de cultivo realizado em fermentador de 15 litros. Ph. D. thesis. University of São Paulo, Brazil (1976).

Geankoplis, C.J., Transport processes and unit operations, pp. 714-715, Prentice Hall, New Jersey (1983).

Hiss, H., Finguerut, J. and Borzani, W., The Influence of pH on the Growth of Candida guilliermondii in Media Containing Diesel Oil as the Main Carbon Source, J. Ferment. Technol., 55, Nº 4, 405 (1977).

Mallette, M.F., Evaluation of growth by physical and chemical methods. In: Methods in Microbiology, J.R. Norris and D.W. Ribbons, eds., vol.1, pp. 541-542, Academic Press, London (1969).

• Borzani, W. and Hiss, H., Kinetics of Nitrogen Consumption During the Batch Growth of Candida guilliermondii on Diesel Oil and on Molasses, Biotechnol. Lett., 6, Nş 8, 511 (1984).
• Concone, B.R.V., Hiss, H., Paz, M.P.G., Patricio, C.G. and Borzani, W., Measurement of Yeast Concentrations in Liquid Hydrocarbon Fermentations: Influence of Experimental Conditions and Statistical Significance of the Results Obtained, Biotechnol. Bioeng., 14, Nş 4, 609 (1972).
• Concone, B.R.V., Contribuiçăo ao Estudo do Cultivo de Candida guilliermontii em meio contendo óleo Diesel como fonte de carbono: influęncia da concentraçăo inicial do óleo em alguns parâmetros do processo descontínuo de cultivo realizado em fermentador de 30 litros. Ph. D. thesis. University of Săo Paulo, Brazil (1976).
• Doin, P.A., Contribuiçăo ao estudo do cultivo de Candida guilliermontii em meio contendo óleo Diesel como fonte de carbono: influęncia da concentraçăo inicial do óleo em alguns parâmetros do processo descontínuo de cultivo realizado em fermentador de 15 litros. Ph. D. thesis. University of Săo Paulo, Brazil (1976).
• Geankoplis, C.J., Transport processes and unit operations, pp. 714-715, Prentice Hall, New Jersey (1983).
• Hiss, H., Finguerut, J. and Borzani, W., The Influence of pH on the Growth of Candida guilliermondii in Media Containing Diesel Oil as the Main Carbon Source, J. Ferment. Technol., 55, Nş 4, 405 (1977).

Publication Dates

• Publication in this collection
  23 Apr 1999
• Date of issue
  Mar 1999

History

• Accepted
  10 Dec 1998
• Received
  14 Aug 1998