a photoelectric colorimeter at 610 mm. The turbidity value was converted to dry biomass concentration by referring to a standard calibration curve of biomass.
J. Biosci., Vol. 15, Number 4, December 1990, pp. 313–322. © Printed in India.
Uptake of volatile n-alkanes by Pseudomonas PG-I S. S. CAMEOTRA* † and H. D. SINGH Biochemistry Division, Regional Research Laboratory, Jorhat 785 006, India *Present address: Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India. MS received 12 May 1990 Abstract. Using a bacterial species Pseudomonas PG-1, evidence has been obtained which indicates that uptake of n-pentane to n-octane by microbial cells takes place primarily from the gas phase either directly or via the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0·3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes, n-nonane and n-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth on n-pentane to n-octane. Keywords. Uptake; volatile n-alkanes; Pseudomonas species.
Introduction There are a few reports available on the microbial utilization of volatile liquid hydrocarbons. Cagler et al. (1969) and Hamer et al. (1980) demonstrated the possibility of using volatile liquid alkanes for the production of single cell protein. Lukins and Foster (1963) reported that mycobacterium strains which utilized npropane and n-butane would not utilize n-pentane and n-hexane as growth substrates but in exceptional cases. Senez and Konovaltshikoff-Mazoyer (1956) reported growth of a Pseudomonas aeruginosa strain on n-heptane. Calma-Calamite et al (1971) reported utilization of n-octane vapours by Candida maltosa. Merdinger and Merdinger (1970) showed that Pullularia pullens was capable of growing on nhexane, n-heptane and n-octane. Zawdzki and Langowska (1983) reported utilization of high n-octane concentrations by mixed cultures. Leavitt (1969) reported that the ability of an organism to grow on volatile liquid alkanes is strongly related to the partial pressure. Uemura et al. (1969) showed that transfer rate of pentane increased with the increase in partial pressure of pentane. The specific growth rate also increased with the increase in the partial pressure of pentane till an optimum level, beyond which growth rate was inhibited. In this communication, results of studies conducted to ascertain the mode of transfer of short chain liquid alkanes ranging from n-pentane to n-decane to the cells are presented. In the case of the more volatile alkanes (n-pentane to n-octane) mode of substrate transfer to the cells is primarily from the gas phase while with less volatile alkanes (n-nonane and n-decane), substrate transfer is predominantly through a substrate solubilization system elaborated by the cells.
†
To whom all the correspondence should be addressed. Abbreviations used: ESF, Emulsifying and solubilizing factor.
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Materials and methods Alkanes n-pentane to n-decane were obtained from Koch-Light Laboratories Ltd., England. Liquid petroleum gas (LPG) was obtained from Indian Oil Corporation, Guwahati (Assam). The characteristics and culture conditions of Pseudomonas PG-1 have been described previously (Reddy et al., 1983; Cameotra et al., 1983). Isolation of n-nonane and n-decane, emulsifying and solubilizing factor (ESF) from the cellfree culture broth and estimation of the alkane solubilization rate was carried out as described previously (Cameotra et al., 1983). Specially designed 500 ml conical flask (figure 1) were used for the growth of organism on n-C6, n-C 7 and n-C8. These
Figure 1. Fermentation flask (500 ml) used for microbial cultivation on volatile alkanes.
flasks had a central well in which the alkane was placed, a side arm closed with a rubber bung through which substrate was added and samples withdrawn with the help of a syringe, and a gas tight stop cock. At different time intervals, 1 ml of the culture broth was withdrawn and after suitable dilution, turbidity was measured in a photoelectric colorimeter at 610 mm. The turbidity value was converted to dry biomass concentration by referring to a standard calibration curve of biomass concentration against turbidity. In the case of n-nonane and n-decane direct measurement of dry biomass was made as described earlier (Singh et al., 1972). The partial pressure of alkanes in the incubation flasks were estimated by referring to the calibration curves of partial pressure (Pg) against alkane concentrations using the same capacity flasks and same volume of mineral medium (figure 2). Results and discussion Growth of Pseudomonas PG-1 on n-pentane, n-hexane and n-heptane Specially designed fermentation flasks (figure 1) were used for the cultivation of Pseudomonas PG-1 on these volatile alkanes. The effect of addition of n-pentane, nhexane and n-heptane (1% v/v) directly to the culture medium and in the central well on growth of Pseudomonas PG-1 in gas tight flasks is illustrated in figure 3.
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Figure 2. Calibration curve of partial pressure plotted against alkane concentration added to gas tight flask containing 50 ml mineral medium at 34°C.
Figure 3. Effect of addition of volatile alkanes (1% v/v) to the aqueous medium and in the central well of the gas tight flasks on the growth of Pseudomonas PG-1.
Inhibition of growth was observed when these alkanes were added directly to the aqueous medium. The inhibition was primarily in the form of a lag phase of 2–6 h duration (figure 3, log plot), n-Hexane and n-heptane showed longer, log phase (6 h) than n-pentane. Specific growth rates were also reduced with these two alkanes
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when substrate was added to the medium. With n-pentane however, the specific growth rates were not much different in both modes of substrate addition These effects could be due to higher volatility of n-pentane in comparison to n-hexane and n-heptane with the result that the added liquid pentane was very quickly vaporised so that practically no contact with the cells occurred. With n-hexane and n-heptane contact time might be comparatively longer resulting in stronger inhibition. The effect of 2·5 mM EDTA on growth characteristics of Pseudomonas PG-I on n-pentane, n-hexane and n-heptane (1 % vol) added to the central well of the gas tight flasks is shown in figure 4. It is seen that EDTA caused only slight inhibition
Figure 4. Effect of 2·5 µm EDTA on the growth of Pseudomonas PG-1 on n-pentane, nhexanc and n-heptane (1 % v/v) with substrates, added to the central well of the gas tight flasks.
of growth on these alkanes which is negligible in comparison to the strong inhibitory effect of EDTA observed during growth of this organism on n-nonane and n-decane, EDTA at this concentration was previously shown to be a strong inhibitor of alkane solubilizing system elaborated by this organism (Reddy et al., 1982). These results suggest that substrate transfer from n-pentane, n-hexane and n-heptane to the cells is by diffusion from the gaseous phase. In order to study the problem of substrate transport in depth, the effect of a wide range of n-hexane and n-heptane concentrations on the growth characteristics were studied. Hexane concentrations ranging from 0·06% to 2% by volume were added to the central well of the gas tight flasks by a syringe through the neoprene bung. The Pg of the alkane inside the flask as calculated from the calibration curve (figure 2) ranged from 3·3 to 110 mm Hg which were far below its vapour pressure of 220 mm Hg at 34°C indicating little possibility of the alkane vapour being
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condensed on the liquid mineral medium. As shown in table 1, specific growth rate increased from 0·104 to 0·214 h–1 along with the increase in alkane concentration from 0·396 g/1 to 1·584 g/1 but specific growth rate decreased along with the increase in alkane concentration above 1·98 g/1 (0·3% v/v) indicating substrate inhibition of growth. A lag phase of growth was observed with alkane concentrations of 6·6 g/1 (1 % v/v) and 13·2 g/1 (2% v/v). However, the duration of the exponential growth was longer with higher concentration of alkane. Biomass yield on alkane ranged from 0·61 to 0·73 at lower concentrations of the substrate giving an average value of 0·66. With the increase, in substrate concentration biomass yield decreased and at the highest substrate concentration of 13·2 g/1 (2% v/v) studied, biomass yield on alkane was a negligible value of 0·05. Table 1. Effect of different concentrations of n-hexane on the growth characteristics of Pseudomonas PG-1 in gas-tight flasks with the substrate added to the central well.
Table 2 shows the growth characteristics of the organism on n-heptane concentrations ranging from 0·408 g/1 (0·06% v/v) to 13·6 g/1 (2% v/v) added to the central well. The partial pressure inside the flask ranged from 3 to 70 mm Hg. At 2% by volume of n-heptane, the flask was saturated with heptane vapour which has a vapour pressure of 70 mm Hg at 34°C. Beyond this concentration, part of the added heptane would remain in liquid phase and condensation of the vapour on liquid mineral medium would occur. As in the case of growth on n-hexane, specific growth rate increased with the increase in substrate concentration until an alkane concentration of 2·04 g/1 was reached. Beyond this, specific growth rate decreased along with the increase of substrate concentration. An average biomass yeild of 0·655 was obtained at low substrate concentrations of alkane. Unlike the growth on n-hexane, the duration of the exponential growth was not improved at higher substrate concentrations of heptane in comparision to that at lower alkane concentration probably due to stronger inhibition caused by n-heptane.
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Table 2. Effect of different concentrations of n-heptane on the growth characteristics of Pseudomonas PG-1 in gas-tight flasks with the substrate added to the central well.
The findings of the above experiments are illustrated in figure 5 which shows a plot of the specific growth rate against substrate concentration and in figure 6 which shows the double reciprocal plot of specific growth rate against substrate concentration. As shown in figure 5, the specific growth rate reached the peak at substrate concentrations of 0·24–0·3% by volume corresponding to partial pressures of 13·2–16·5 mm Hg for n-hexane and 12–15 mm Hg for n-heptane, specific growth rate sharply declined at 0·4% by volume of substrate concentration above which
Figure 5. Effect of substrate concentration on the specific growth rate of Pseudomonas PG-1 growing on n-hexane and n-heptane in gas tight flasks.
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Figure 6. Growth of Pseudomonas PG-1 on n-hexane and n-heptane. Double reciprocal plot of specific growth rate (µ) against substrate concentration S (g/1).
the decline was gradual. It appears that substrate inhibition was maximum at substrate concentration around 0·4% by volume. An attempt was made to explain the above relationship by applying the substrate inhibition equation of Andrews (1968): 1
where,µ = specific growth rate (h–1), = maximum specific growth in the absence of inhibition (h–1), K s=substrate saturation constant; lowest substrate concentration at one-half the maximum specific growth rate in the absence of inhibition (g/1), Κi=substrate inhibition constant; highest substrate concentration at which the specific growth rate is equal to one-half the maximum specific growth rate in the absence of inhibition (g/1) and S = limiting substrate concentration (g/1). Rearranging equation 1 to the following multilinear equation: 2 and applying it to data in tables 1 and 2, the following values of the constants were obtained through multiple regression analysis:
Table 3 shows a comparision of the specific growth rates calculated from equation 1 using the above growth constants with the experimentally observed specific growth rates at different n-hexane and n-heptane concentrations. The data
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Cameotra and Singh Table 3. Calculated specific growth rates (µ) through application of Andrew’s equation as compared with observed specific growth rates at different concentrations of n-hexane and n-heptane.
reveal though there is a broadly similar pattern of substrate inhibition in the observed and calculated values of specific growth rates but the correlation between the two sets of data is unsatisfactory. This suggests that Andrew’s equation of substrate inhibition function is not applicable in the case of volatile alkanes. Figure 6 shows the double reciprocal plot of specific growth rate against concentrations of n-hexane and n-heptane. It was found that at low substrate concentrations, a linear relationship between 1/µ and 1/S was obtained. This indicates that growth followed a Monod-type of relationship. Uemura et al. (1969) also reported Monod-type of growth at low concentrations of n-pentane and n-heptane. In the experiments described here, as the substrates were segregated in the central well of the gas tight flasks, the substrate transfer must be taking place solely from the gas phase. Correspondingly, inhibitory effect of high substrate concentration must also be solely due to high partial pressure of the gases. Growth on n-octane Figure 7 shows the growth pattern of Pseudomonas PG-1 on 0·4% by volume of noctane. However, the growth was similar in both modes of substrate addition (directly to the medium or segregated in central well) indicating that substrate uptake is primarily from the gas phase. Reddy (1982) showed that even a high level of substrate concentration when added to the central well of the gas tight flask promoted a good growth of the organism. This level of alkane when added directly to the medium was strongly inhibitory to growth. This suggests that unlike n-pentane to n-heptane, inhibitory effect of n-octane may be mainly due to the damaging action of the liquid phase on the cells rather than the high partial pressure of the alkane. The addition of 2·5 mM EDTA in the medium did not affect the growth of the organism on 0·4% by volume n-octane added directly to the medium. This suggests that alkane solubilizing mechanism which plays a predominant role in substrate transfer in case of higher chain length alkanes does not play any role in the uptake
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Figure 7. Growth pattern of Pseudomonas PG-I on 0·4% by volume of n-octane.
Table 4. Formation of hydrocarbon ESF from volatile and semi-volatile älkanes and the hydrocarbon solubilizing activity of the factors.
*Solubilization was done with the alkane used as growth substrate. **Growth was done in conventional cotton-plugged conical flasks.
of n-octane by microbial cells. n-Octane uptake takes place primarily from the gas phase either directly or via the aqueous medium. Growth on n-nonane and n-decane Normal nonane and decane have higher boiling points and much lower vapour pressures than their lower homologues and therefore they are much less volatile at the temperature of cultivation. As listed in table 4, high level of these alkanes
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(2 % v/v) promoted formation of good biomass of the organism in the conventional cotton-plugged conical flasks. This indicates that substrate uptake from the gas phase is negligible and also that the liquid alkanes had very little toxic effect on the cells. EDTA (2·5 mM) strongly inhibited the growth of the organism on these two alkanes. Further evidence that uptake of n-nonane and n-decane is mediated by substrate solubilization was obtained by actual isolation of the alkane solubilizing factor from the cell-free broths (table 4). As shown in the table good yeilds of the hydrocarbon ESFs were obtained from n-nonane and n-decane cultures while cultures grown on n-pentane to n-octane did not produce any hydrocarbon ESF. High solubilization rates of 2·1 g·1–1 h–1 and 1·86g 1–1 h–1 were obtained with n-nonane and n-decane ESF respectively. These high rates of solubilization would be able to account fully the substrate uptake and growth of the organism during the growth on n-nonane and n-decane. References Andrews, J. F. (1968) Biotechnol. Bioeng., 10, 707. Cagler, Μ. Α., Thompson, A. R., Houston, C. W. and Rose, W. C. (1969) Biotechnol. Bioeng., 11, 417. Calma-Calamite, E., Arntz, P. and Bos, P. (1971) An. Inst. Nac. Invest. Agrar. (Spain) Ser. Gen., 1, 165. Cameotra, S. S.s Singh, H. D., Hazarika, A. K. and Baruah, J. Ν. (1983) Biotechnol. Bioeng., 25, 2945. Hamer, G., Hamden, I. Y., Khamis, A. S. and Baroon, Z. H. (1980) Biotechnol. Bioeng., 22, 995. Leavitt, R. L. (1969) Bacterial. Proc., 118, 166. Lukins, H. B. and Foster, J. W. (1963) J. Bacteriol., 85, 1074. Merdinger, E. and Merdinger, R. P. (1970) Appl. Microbiol., 20, 651. Reddy, P. G. (1982) Studies on the utilization of aliphatic hydrocarbons by microorganisms, Ph.D. thesis, Gauhati University, Gauhati. Reddy, P. G., Singh, H. D., Roy, P. K. and Baruah, J. Ν. (1982) Biotechnol. Bioeng., 24, 1241. Reddy, P. G., Singh, H. D., Pathak, M. G., Bhagat, S. D. and Baruah, J. Ν. (1983) Biotechnol. Bioeng., 25, 387. Senez, J. C. and Konovaltshikoff-Mazoyer, M. (1956) C. R. Acad. Sci., 242, 2873. Singh, Η. D., Barua, P. K., Pillai, K. R., Baruah, J. Ν. and Iyengar, Μ. S. (1972) Indian J. Biochem. Biophys., 9, 315. Uemura, N., Takahashi, J. and Veda, K. (1969) J. Ferment. Technol., 47, 220. Zawdzki, Z. and Langowska, I. (1983) Acta. Microbiol. Pol., 32, 191.
