A two-phase aerobic batch culture is shown in Fig. 1. The initial expo­nential growth phase on glucose lasted until about 26 h. The specific growth rate (determined from the logarithm of the CO2 evolution rate [CER]) was 0.22 h1. After 25.4 h, DNS measurements of reducing sugars indicated that the glucose was exhausted from the medium (Fig. 2). Although the CO2 evolution rapidly decreased, there was a residual CO2 evolution, which only gradually decreased to zero. This was accompanied by a reduction in the OD (Fig. 2), indicating degradation of biomass. The measured cellulase activity remained constant after the depletion of glucose at a level of about 0.3 FPU/mL.

Second-Stage Batch Cultivation on Cellulose

After 67 h, cellulose in the form of Solka-floc was added as described above. There was an immediate increase in the CER at this point. The increase continued until t = 73 h, at which point there was a sharp decrease in CER. This did not coincide with complete depletion of glucose from the medium (Fig. 3). The enzyme activity increased continually up to a value of about 2.6 FPU/mL. This maximum coincided with the depletion of glucose and occurred at about t = 100 h. From the integrated area of the CO2 evolution (Fig. 1), one can estimate that the CO2 evolved on cellulose was about 80% of the value obtained from glucose.

Fig. 1. CO2 concentration in outlet gas (——- ) and cellulase activity (■, expressed as FPU/mL) vs time for aerobic batch cultivation of T. reesei Rut-C30. The initial growth medium was a Mandels medium with 10 g/L of glucose as the carbon source. At t = 67 h, Solka-floc was added to a concentration of 10 g/L.

Fig. 2. Reducing sugars (■) determined by DNS method and OD (A) vs time for aerobic batch cultivation of T. reesei Rut-C30. The initial growth medium was a Mandels medium with 10 g/L of glucose as the carbon source. At t = 67 h, Solka Floc was added to a concentration of 10 g/L. (There were no measurements of OD after the addition of cellulose.)

Fig. 3. CO2 concentration in outlet gas (———————————————————— ) and glucose concentration (■) vs time for second aerobic batch phase in which T. reesei Rut-C30 grew on Solka-floc as carbon source.

Fig. 4. Concentration of glucose (—□—) and cellobiose (— ■ —) during enzymatic hydrolysis of Solka-floc. The enzyme was prepared from a culture of T. reesei and the initial enzyme loading corresponded to 27.4 FPU/g substrate. Hydrolysis was carried out at 28°C and pH 5.0.

Fig 5. Concentration of glucose (———- ) and cellobiose (——— ) during enzymatic

hydrolysis of Solka-floc. Two different enzymes were used; enzyme was prepared from a culture of T. reesei (□) and commercially available Celluclast (A). Enzyme loading was 91.9 FPU/g substrate. Hydrolysis was carried out at 28°C and pH 5.0.

In Vitro Enzymatic Hydrolysis Rates

The initial, rather rapid increase in CER found in the second stage of two-phase batch cultivation was somewhat unexpected. We decided to compare this value to the initial rates of glucose and cellobiose formation in in vitro enzymatic hydrolysis experiments (Figs. 4 and 5). The enzyme loadings were chosen to represent the actual enzyme-to-substrate ratio relevant to the point of cellulose addition and the point of maximum CER.

For the enzyme solution prepared using T. reesei, the approximate forma­tion rate of glucose was in the former case 0.18 g/(L-h) and in the second case 0.5 g/(L-h). The formation rate of cellobiose was 0.33 g/(L-h) in the first case and <0.2 g/(L-h) in the second case. Using Celluclast in a loading revevant to the point of maximum CER, a similar value was found for glucose, but a higher value was found for cellobiose (Fig. 5).

With a typical yield of CO2 on sugar, Ysc, of 0.4 (C-mol/C-mol), one can estimate that the sugar formation rate would give a CER of 0.018 mol of CO2/h for a 2-L culture as in Fig. 1. This corresponds to a CO2 concentration in the outlet gas of 1%. Within 0.5 h after addition of cellulose (Fig. 3), the measured value was in fact 1%, in good agreement with the estimated value. Calculations for the higher enzyme activity (91.9 FPU/mL) indicate that the formed glucose and cellobiose would give a CER of 0.018 mol of CO2/h, corresponding to a CO2 concentration in the outlet gas of 1.4%, which is a bit lower than the actual observed value.

Discussion

The final cellulase activity obtained from 10 g/L of cellulose was 2.6 FPU/mL. This is in good agreement with previously reported yields for the strain Rut-C30. For example, Persson et al. (6) quote a yield of 233 FPU/g of substrate in batch cultures. There was a steady increase in cellulase activity throughout the cultivation on cellulose in the current work, despite the fact that the free glucose concentration reached a value as high as 1 g/L. How­ever, at that point of maximum glucose concentration, a sharp decrease in CO2 evolution occurred, and the glucose concentration started to decrease after that point. The reason for this may be depletion of a medium compo­nent, or it may also be related to the regulation of enzyme expression. This is supported by the fact that the rate of activity increase changes at that point.

On depletion of glucose in the initial growth phase, there was a rapid decrease in CO2 evolution, but it did not decrease to zero. Measurements of OD660 showed a decrease in biomass, suggesting that the residual CO2 evolution is the result of endogenous metabolism. A higher volumetric enzyme productivity could therefore potentially be obtained if cellulose had been added earlier, provided that there was no remaining glucose repression effect.

The main point of making a two-stage culture was to enable the study of a pulse addition of cellulose. However, separating an initial biomass formation on glucose (or on other monosaccharides) from cellulase pro­duction with cellulose as substrate has advantages also from a process point of view. By using a two-stage process, a basal cellulase activity can be obtained before the addition of cellulose. This level allows the utilization of cellulose to commence rather quickly as shown by the CO2 evolution. By contrast, a one-phase batch process starting directly from cellulose will initially be very slow owing to a very low hydrolysis rate. As has been pointed out previously, a key question is, to what extent will the cellulase expression be repressed by the glucose liberated in the hydrolysis.

Acknowledgment

This work was supported in part by a fellowship from the Marie Curie Training Site QCIM. We also acknowledge the National Research Fund of Hungary (OTKA T029382) and the National Research and Development Program (NKFP-OM-00231/2001) for financial support.

References

1. Wyk, J. P. H. (1999), Biomass Bioenergy 16, 239-242.

2. Kheshgi, H. S., Prince, R. C., and Marland, G. (2000), Annu. Rev. Energ. Environ. 25, 199-244.

3. Sun, Y. and Cheng, J. (2002), Bioresour. Technol. 83, 1-11.

4. Bhat, M. K. (2000), Biotechnol. Adv. 18, 355-383.

5. Himmel, M. E., Ruth, M. F., and Wyman, C. E. (1999), Curr. Opin. Biotechnol. 10, 358-364.

6. Persson, I., Tjerneld, F., and Hahn-Hagerdal, B. (1991), Process Biochem. 26, 65-74.

7. Beguin, P. and Aubert, J. P. (1994), FEMS Microbiol. Rev. 13, 25-28.

8. Tolan, J. S. and Foody, B. (1999), in Advances in Biochemical Engineering/Biotechnology, vol. 65, Scheper, T., ed., Springer-Verlag, Berlin, Germany, pp. 40-67.

9. Yu, X. B., Hyun, S. Y., and Yoon-Mo, K. (1998), J. Microbiol. Biotechnol. 8, 208-213.

10. Ilmen, M., Saloheimo, A., Onnela, M.-L., and Pentilla, M. E. (1997), Appl. Environ. Microbiol. 63, 1298-1306.

11. Kubicek, C. P., Messner, R., Cruber, F., Mach, R. L., and Kubicek-Pranz, E. M. (1993), Enzyme Microb. Technol. 15, 90-99.

12. Suto, M. and Tomita, F. (2001), J. Biosci. Bioeng. 92, 305-311.

13. Mandels, M. and Weber, J. (1969), Adv. Chem. Ser. 95, 391-414.

14. Bigelow, M. and Wyman, C. E. (2002), Appl. Biochem. Biotechnol. 98/100, 921-934.

15. Miller, G. (1959), Anal. Chem. 31, 426-28.

16. Mandels, M., Andreotti, R., and Roche, C. (1976), Biotechnol. Bioeng. Symp. 6, 21-23.

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