JOURNALOF BIOSCIENCEAND BIOENGINEERING Vol. 95, No. 3,298-301. 2003
Optimization of Aeration and Agitation Rates to Improve Cellulase-Free Xylanase Production by Thermotolerant Streptomyces sp. Ab 106 and Repeated Fed-Batch Cultivation Using Agricultural Waste CHARIN TECHAPUN, 1NAIYATAT POOSARAN, 1 M A S A N O R I W A T A N A B E , 2 AND K E N S A S A K I 2.
Department of Biotechnology, Faculty of Agro-Industry, Chiangmai University, Chiangmai 50100, Thailand and Materials Science and Engineering, Graduate School of Engineering, Hiroshima Kokusai Gakuin University, 6-20-1 Nakano, Akiku, Hiroshima 739-0321, Japan ~ Received 13 September 2002/Accepted 17 October 2002
Thermostable cellulase-free xylanase was produced by Streptomyces sp. A b l 0 6 using agricultural waste, sugar cane bagasse, as the substrate at 50°C and pH 7.0. The central composite facecentered experimental design was applied to evaluate the optimal agitation and aeration rates in a 5-1 fermentor. The highest activity (16.0+0.5 IU/ml) was obtained at an aeration rate of 1 vvm and an agitation rate of 150 rpm (kLa=351 h-l). Using the repeated fed-batch cultivation technique, the maximum xylanase activity of 32+1 IU/ml was obtained during the second cycle of repeated fedbatch culture. [Key words: aeration, agitation, cellulase-free xylanase, fed-batch]
The chemical bleaching process in the pulp industry uses large amounts of chlorine and chlorine-based chemicals. The by-products formed during chemical processing are toxic, mutagenic, persistent, bioaccumulating, and cause numerous harmful disturbances to biological systems (1). New bleaching procedures, termed "biobleaching processes", can replace chemical processes (2). Biobleaching involves the use of microorganisms and enzymes in bleaching pulp. Cellulase-free xylanases (1,4-[3-D-xylanohydrolase; EC 3.2.1.8) depolymerize lignin directly by attacking hemicellulose and altering the interface between the cellulose and lignin, thereby facilitating the removal of the lignin-associated hemicellulosic fraction with minimum damage to the pulp. This process is less severe, less expensive, and importantly, less toxic than conventional chemical treatment (1). In our previous paper (3), it was reported that the newly isolated thermotolerant Streptomyces sp. Abl06 has the ability to produce thermotolerant xylanase that has no cellulolytic and mannanase activities. This Ab 106 produced a relatively thermostable and alkaline-tolerant xylanase for which more than 70% activity remained at pH 9.0, and 70°C with a half-life of more than 6 h which is desirable for practical biobleaching processes (4). The combined experimental design method was applied to xylanase production by Streptomyces sp. Ab106 to optimize the medium composition (5). Temperature and pH were also optimized by the central composite design method using a stirred-type fermentor. The maximum concentration of
xylanase, 15 1U/ml, was produced at 50°C and pH 7.0 (6). However, for large-scale xylanase production, it seems that the aeration and agitation rates were additional factors that influence cell growth and xylanase production. Optimization of the agitation and aeration rates in the xylanase production by this strain has not yet been investigated. In this study, optimization of aeration and agitation rates was investigated to determine the conditions suitable for xylanase production in a stirred tank reactor. In addition, xylanase production by repeated fed-batch culture was also investigated to determine the highest limit of xylanase production by this organism. Streptomyces sp. Ab106 was used. This strain was isolated from the soil in Thailand and selected as a potent thermotolerant cellulase-free xylanase producer (3). For the isolation, used an optimized medium as described previously (5). Preculture was carried out in a 1000-ml conical flask (250 ml of medium) on a rotary shaker (100 rpm) at 50°C for 6 d (6). A stirred tank fermentor (5/, medium 4 l, Biostat-B5; B. Braun, Melsungen, Germany) containing a 5% (v/v) inoculum in preculmre broth was used to culture strain Abl06. The culture was subjected to a variety of aeration (0-2 vvm) and agitation rates (0-300 rpm) at 50__+0.2°C, and the pH was controlled at 7.0_+0.1. An aeration rate of 0 w m represented a static culture, but it was mixed to give a homogeneous culture broth before sampling, once a day. To improve the enzyme productivity, the use of repeated fed-batch culture was investigated. When the cell growth had reached the stationary phase, fresh medium (10%, v/v) was added. Three cycles of medium feeding were used in
* Corresponding author, e-mail:
[email protected] phone: +81-(0)82-820-2570 fax: +81-(0)82-820-2560 298
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TABLE 1. Maximum and minimum levels of aeration and agitation rates used in the CCF design
NOTES
299
a
14
Independent variable
Level 0 1.0 150
-1 0 0
X 1, aeration rate (vvm) X2, agitation rate (rpm)
+1 2.0 300
1
,
TABLE 2. Xylanase production with various aeration and agitation rates determined by the CCF design for the two independent variables Treatment 1
X~
X2
-1
-1
Aeration Agitation Xylanase rate rate (vvm) (rpm) (IU/mt) 0
2 +1 -1 2 3 -1 +1 0 4 +l +l 2 5 0 0 1 6 0 0 1 7 -1 0 0 8 +1 0 2 9 0 -1 1 10 0 +1 1 11 0 0 1 12 0 0 1 Xylanase was measured after 6 d of culture.
0
0 300 300 150 150 150 150 0 300 150 150
3.4 1.1 0.58 4.82 15.1 16.0 10.1 t2.3 6.0 1.16 15.3 16.5
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this experiment. The crude enzyme concentration was measured following the methods outlined in our previous paper (4). One unit of enzyme activity (IU, international unit) was used. The KLa after fermentation was measured by the dynamic method (7). Aeration and agitation rates were optimized using a central composite face-centered (CCF) experimental design (8) to determine the optimal conditions by a method other than an experimental one. The use o f a CCF design is a convenient way to optimize a particular process. The CCF matrix was considered within these limits. This method requires three levels for each factor, the factor settings of which are the true limit. In this study, we need this design to evaluate the effects and interaction o f the two variables, the aeration rate and agitation rate. A CCF matrix with a center point was designed. Table 1 shows the maximum and minimum values o f the aeration and agitation rates, as chosen for the trials in this study. Table 2 shows the xylanase production with various aeration and agitation rates determined using the CCF design. The variables were the aeration and agitation rates, and the responses were the xylanase activities. A second-degree quadratic model was established, as represented by Eq. 1, using the method of least squares (Sigma Plot program, ver. 3.0; Jandel Scientific, Erkrath, Germany) as described previously (6). Z = 1.829+0.134X+6.372Y-0.0005X2-2.841Y 2 (1) Here Z is the xylanase yield (1U/ml), X is the agitation rate (rpm), and Y is the aeration rate (vvm). Streptomyces sp. Ab 106 was cultured under various aeration and agitation rates. First, with a fixed agitation rate of 150 rpm (Fig. la), it was found that an increase in the aera-
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500
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0
50
100
150
200
250
300
Agitation rate (rpm) FIG. 1. Xylanase production under various conditions of aeration and agitation rates in a 5-I stirred-type fermentor containing of 4-I medium, at pH7.0+0.1 and at 50_+0.2°C. (a) Time course of xylanase
production under various aeration rates, at a fixed agitation rate of 150 rpm. Triangles, Aaeration rate of 0 vvm; squares, aeration rate of 1.0 vvm; and circles, aeration rate of 2.0 vvm. (b) Linear response plot of aeration and agitation rates with respect to xylanase production. Closed circles, Aeration rate of 0 vvm; squares, aeration rate of 1.0 vvm; and triangles, aeration rate of 2.0 vvm. Open circles, KLa.Data, except KLa, represent those complied after 6 d of culture. tion rate from 0 to 1 w m increased the xylanase production. However, when the aeration rate was increased to more than 1 vvm, xylanase production gradually decreased. This result agrees well with the results o f Reddy et al. (9), whereby, at an aeration rate higher than 0.75 vvm, there was decreased xylanase production by Thermomyces lanuginosus SSBR However, in present study xylanase production reached the maximum level after 6 d of culturing in every cultivation. Second, the effect o f the aeration and agitation rates on xylanase production is shown in Fig. lb. It is shown that high aeration and agitation rates reduced xylanase yields. An increase in the agitation rate alone reduced xylanase production (Fig lb). From microscopic analysis (data not shown), it was found that a high agitation rate altered the morphology of Streptomyces sp. A b l 0 6 due to the high shear rate, thus resulting in the reduction o f xylanase productivity at an agitation rate of 250 rpm. These results are in good agreement with the results o f Palma et al. (10), and Cho et al. (11), who reported that high agitation rates affect the morphology o f Penicillium janthinellum and Paecilomyces sinelairii, respectively. The KLa increased with an increase in the agitation rate,
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Culture time (d) FIG. 2. Xylanase production in repeated fed-batch culture of
Streptomyces Abl06 with sugar cane bagasse medium. Temperature, pH and agitation were kept at 50_+0.2°C, 7.0_+0.1 and 200+5 rpm, respectively. (a) Circles, squares and triangles show xylanase production without aeration, and at aeration rates of 0.5 vvm and 1 vvm, respectively. (b) Closed circles, closed squares and closed triangles show cell growth without aeration, and at aeration rates of 0.5 vvm and 1 vvm, respectively. Open squares and open triangles show the dissolved oxygen tension at aerations of 0.5 vvm and 1 vvm, respectively. Arrows, Medium feeding. but plateaued rates at higher than 200 rpm. Cellulase and mannanase were not detected in any culture. However, to separately optimize the aeration and agitation rates for xylanase production, the CCF design was applied to design the experiments and to check the applicability of the experimented results. By using the CCF design, it was also confirmed that a maximum xylanase production of 16.0_+0.5 IU/ml could be achieved at an agitation rate of 150 rpm and an aeration rate of 1.0 w m as observed in the experimental results shown in Fig. 1. The KLa was 351 h -1 under the above conditions which is desirable for practical applications. Xylanase production by Streptomyces sp. Abl06 using fed-batch culture is shown in Fig. 2. The optimized agitation rate was 150 rpm. However, considering the high cell density of the culture, the agitation rate was slightly increased up to 200 rpm to supply more oxygen to the cells. Medium feeding supported the cell growth and xylanase production in the first and second cycles. High xylanase productivity (32_+ 1 IU/ml) was achieved in the second cycle compared to the original batch culture (15_+ l 1U/ml). Such high production of a thermostable cellulase-free xylanase has not been observed in other microorganisms. Two cycles of medium feeding resulted in a xylanase yield two fold that observed in batch cultivation. However, following the third
cycle of medium feeding, the cell growth and xylanase production were gradually decreased. Long term operation is not beneficial for cell growth or xylanase production due to cell lysis. Supplying oxygen was effective for cell growth and xylanase production and an increase in the aeration rate increased both. Due to the high cell density in the last cycle, the level amount of aeration used seemed to be inadequate for cell growth. However, we observed that too much agitation (a rate higher than 250 rpm) led to shearing of the cells and a reduction of the xylanase yield (from microscopic observation, data not shown). Therefore, effective aeration with medium agitation rate such as in an airlift bioreacter seems to be essential for such xylanase production and investigation of this is continuing. However, the value of 32_+ 1 IU/ml was considerably higher than that reported to date for most other mesophilic and thermophilic actinomycetes that produce cellulase-free xylanase: 12 IU/ml xylanase produced by Streptomyces albus (12), and 22 IU/ml from Streptomyces cuspidosporous (13). In addition, among thermostable and alkaline-tolerant xylanase producers, 22.4 IU/ml was the highest value obtained compared with 19 iU/ml by Bacillus circulans AB 16 (14), 18 IU/ml by Thermomyces thalophilus (15), and 9.9 IU/ml by Clostridium absonum CFR-702 (16). REFERENCES 1. Beg, Q. K., Bhushan, B., Kapoor, M., and Hoondal, G. S.: Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp. Enzyme Microb. Technol., 27, 459-466 (2000). 2. Jimenez, L., Martinez, C., Perez, I., and Lopez, F.: Biobleaching procedures for pulp from agricultural residues using Phaneroehaete chrysosporium and enzymes. Process Biochem., 32, 297-304 (1997). 3. Techapun, C., Sinsuwongwat, S., Poosaran, N., Watanabe, M., and Sasaki, K.: Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Sterptomyces sp. Biotechnol. Lett., 23, 1685-1689 (2001). 4. Teehapun, C., Chareonrat, T., Watanabe, M., Poosaran, N., and Sasaki, K.: Thermostable and alkaline-tolerant cellulase-free xylanase produced from thermotolerant Streptomyces sp. Abl06. J. Biosci. Bioeng., 93, 431-433 (2002). 5. Teehapun, C., Sinsuwongwat, S., Watanabe, M., Sasaki, K., and Poosaran, N.: Production of cellulase-free xylanase by a thermotolerant Streptomyces sp. from agricultural waste and media optimization using mixture design and PlackketBurman experimental design methods. Biotechnol. Lett., 24, 1437-1442 (2002). 6. Techapnn, C., Chareonrat, T., Watanabe, M., Poosaran, N., and Sasaki, K.: Optimization of thermostable and alkaline-tolerant cellulase-free xylanase production from agricultural waste by thermotolerant Streptomyces sp. Abl06, using the central composite experimental design. Biochem. Eng. J., 12, 99-105 (2002). 7. Taguehi, H. and Humphrey, A.E.: Dynamic measurement of volumetric oxygen transfer coefficient in fermentation systems. J. Ferment. Technol., 44, 881-889 (1966). 8. Box, G. E. P., Hunter, W. G., and Hunter, S. J.: Statistics for experiments, p. 600-653. Wiley, New York, USA (1978). 9. Reddy, V., Reddy, P., Pflay, B., and Singh, S.: Effect of aeration on the production of hemicellulases by T. lanuginosus SSBP in a 30 l bioreactor. Process Biochem., 37, 12211228 (2002).
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10. Palma, M.B., Milagres, A.M.E, Prata, A.M.R., and Mancilha, I.M.: Influence of aeration and agitation rate on the xylanase activity from Penicillium janthinellum. Process Biochem., 31, 141-145 (1996).
11. Cho, ¥. J., Hwang, H. J., Kim, S. W., Song, C. H., and Yun, J.W.: Effect of carbon source and aeration rate on broth rheology and fungal morphology during red pigment production by Paecilomyces sinclairii in a batch bioreactor. J. Biotechnol., 95, 13-23 (2002).
12. Antanopoulos, V.T., Hernandez, M., Arias, M.E., Mavrakos, E., and Ball, A.S.: The use of exla-acellularenzyme from Streptomyces albus ATCC3005 for the bleaching of eucalyptus kraft pulp. Appl. Microbiol. Biotechnol., 54, 92-97 (2001). 13. Maheswari, M.U. and Chandra, T. S.: Production and po-
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tential applications of xylanase from a new strain of Streptomyces cuspidosporous. World J. Microbiol. Biotechnol., 16, 257-263 (2000). 14. Dhillon, A., Gupta, J.K., and Khanna, S.: Enhanced production, purification, and characterization of a novel cellulase-poor thermostable, alkalotolerant xylanase from Bacillus circulans AB16. Process Biochem., 35, 849-856 (2000). 15. Kohli, U., Nigam, P., Singh, D., and Chaudlaary, K.: Thermostable, alkalophflic and cellulase-free xylanase production by Thermomyces thaloFhilus subgroup C. Enzyme Microb. Technol., 28, 606-610 (2001). 16. Rani, D. S. and Nand, K.: Production ofthermostable cellulase-free xylanase by Clostridium absonum CFR-702. Process Biochem., 36, 355-362 (2000).
