Production of ceHulolytic enzymes by immobilized Sporotrichum thermophile Alka Singh, Reeta Goel and B.N. Johri

Department of Microbiology, C.B.S.H., G.B.Pant University of Agriculture & Technology, Pantnagar, India

Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes. Immobilized mycelium was consid- erably less effective than free cells for cellulase productivity. Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium. Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency.

Keywords: Celluloytic enzymes; Sporotrichum thermophile; immobilized mycelia

Introduction

Thermophilic microorganisms have received consider- able attention lately for the production of a variety of enzymes, j-4 This is largely due to the fact that en- zymes produced by thermophiles exhibit optimal ac- tivity at temperatures higher than their mesophilic counterparts. 5 Among thermophilic fungi, cellulase systems of Chaetomium thermophile, Myceliophthora thermophilum, Sporotrichum thermophile, Talaro- myces emersoni, and Thermoascus aurantiacus have been examined exhaustively. 6-8 However , S. ther- mophile has been studied in the most detail, as it syn- thesizes a complete set of enzymes necessary for the breakdown of crystalline ce l lu lose)

There have been intensive efforts to improve cellu- lase productivity of the microbial systems through mu- tation and strain improvement , as well as by develop- ment of new bioreactors. 1°,11 The use of immobilized cell technology has opened up newer avenues for achieving improved reactor performance. Several re- ports on immobilization of Penicillium funiculosum and Trichoderma reesei for cellulase production have now appeared.i2.13 While both spores and mycelia can be immobilized and cultivated in situ, the former would bet ter withstand cell damage during immobiliza- tion. One of the major advantages of cell immobiliza- tion for cellulase would appear to be multiple inoculum use so that the cost of enzyme production could be reduced.

Address reprint requests to Dr. Goel at the Department of Microbi- ology, C.B.S.H., G.B.Pant University of Agriculture & Technol- ogy, Pantnagar 263 145, India Received 8 June 1988; revised 17 April 1989

The present work was aimed at immobilization of spores of S. thermophile in agar, polyacrylamide, and alginate to generate in situ mycelia for cellulase pro- duction and to examine the reuse of the immobilized inoculum.

Materials and methods

Organism

A wild-type strain of Sporotrichum (Chrysosporium) thermophile Apinis isolated from decomposing rice straw was used. It was maintained in Emerson YpSs agar at 45°C, stored at 4°C, and transferred every month?

Culture conditions

Shake flask cultures were used for evaluating cellulo- lytic activity of free and immobilized cells. The me- dium used for enzyme production contained (g l-J): casamino acids, 0.5; KHzPO4, 1.0; KC1, 0.5; MgSO4 • 7H20, 0.2; CaCI2 • 2H20, 0.1; yeast extract, 0.5; mi- crocrystalline cellulose, 10.0; trace element, I0 ml; pH, 7.0. The trace element solution contained (mg l-I): ZnSO4 • 7H20, 30.0; MnCI2,3.0; H3BO3, 30.0; COC12 • 6H20, 20.0; CuCI2 • 2H20, 1.0; NiCI2 • 6H20, 2.0; NazMoO4 • 2H20, 3.0; ferric ammonium citrate, 10.0.

One hundred milliliters of lhe medium were dis- pensed in 250-ml conical flasks and sterilized at 15 psi for 15 min at 121°C. Each flask was inoculated with 1 ml suspension to provide approximately | 0 6 spores ml -I. Cultures were incubated at 45°C up to 6 days on a rotary shaker at 200 rev rain -1.

464 Enzyme Microb. Technol., 1990, vol. 12, June © 1990 Butterworth Publishers

Enzyme activity determination

Cultures were harvested after 1, 2, 3, 4, 5, and 6 days and the filtrate centrifuged at 10,000 rev min -~ for 15 min. Cellulase activity was determined in the superna- tant for exoglucanase (EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and beta-glucosidase (EC 3.2.1.21) as re- lease of reducing sugars according to IUPAC.~4 En- zyme activity was expressed in terms of International Units (IU) per milliliter of the culture filtrate; an IU refers to mg glucose ml-~ rain -~. Specific enzyme ac- tivity denotes cellulase units per milligram protein. Protein was estimated by Lowry's method.J5

Cell immobilization

Spores of S. thermophile were entrapped in three dif- ferent matrices by established techniques. The matri- ces used were agar) 6 alginate) 7 and polyacrylamide. J8 Since performance of S. thermophile in agar beads was superior to other supports, extended :zxperimentation was conducted only in this system. Ten milliliter spore suspension containing approximately 106 spores ml -~ were mixed with 20 ml of 2.5% agar (w/v) for the preparation of beads. Flasks inoculated with agar beads were incubated under culture conditions similar to free cells.

Optimization of cellulase production by immobilized cells

Culture flasks were withdrawn at 3, 4, and 5 days after inoculation, and cellulase activity in the culture filtrate was determined. The pH optima for enzyme produc- tion were determined in culture broths adjusted in the range 5.0 to 8.0. Temperature optima for cellulase pro- duction were studied by incubating a series of flasks at 40°C, 45°C, 50°C, and 55°C. To determine the optimum substrate level, microcrystalline cellulose was added to the culture medium in the concentration range 1.0% to 5.0% (w/v).

Reusability

The reusability of agar beads was tested in batch cul- ture by replacing the old culture broth every 24 h by fresh medium. Other cultural conditions were similar to those described above. All three components of the cellulase system were assayed from the same sample to avoid variation.

Results and discussion

Cellulase activity of the native cells

The ceUulolytic activity of the culture filtrate of S. thermophile on 1% microcrystalline cellulose powder is shown in Figure 1. Maximum cellulase was pro- duced in 3 days at 45°C; the enzyme levels for exoglu- canase, endoglucanase, and beta-glucosidase were 0.33, 0.14, and 1.02 IU m1-1, respectively.

A detailed account of cellulase production by S. thermophile was recently presented by Bhat and Ma-

Production of cellulolytic enzymes: A. Singh et al.

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heshwari, j9 Although exoglucanase and endoglu- canase activities were low, it degraded cellulose at a faster rate than T. reesei. Extracellular beta-glucosi- dase was the principal variable component of the cellu- lase system in S. thermophile. The results of the present study conform more closely to those reported by Canevascini and Cosar. 2°

Choice of support for immobilized system

Three immobilization supports were used: agar beads, alginate beads, and polyacrylamide gel. Among these, agar beads performed better than the other supports. Assuming the enzyme activity of free native cells as 100%, the relative cellulase activity of immobilized cells was extrapolated. The data presented in Figure 2 demonstrate that maximum cellulase production oc- curred in 5 days: the levels of exoglucanase, endoglu- canase, and beta-glucosidase were 0.15, 0.04, and 0.28 IU m1-1, respectively.

Linko et al. J2 have reported extracellular produc- tion of cellullolytic enzymes by Penicillium funiculo- sum entrapped in carrageenan beads and in cubes pre- pared either from hydrophobic (PU3) or hydrophilic (PU6) urethane prepolymers. These workers obtained particularly high beta-glucosidase activity for P. funi- culosum. Turker and Mavituna ~3 immobilized the my- celia of T. reesei QM 9123 within the open porous network of reticulated polyurethane foam matrices and compared cellulolytic activity with freely sus- pended cells. According to them, the method of immo- bilization had no detrimental effect on cell activity and

Enzyme Microb. Technol., 1990, vol. 12, June 465

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Figure 2

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Screening of various supports for immobil ization of S. thermophile and cellulase production

similar results were obtained with both freely sus- pended and immobilized cells. Earlier work on immo- bilization of T. reesei for cellulase production con- forms to these observations. 21,22

Optim&ation of conditions for cellulase production

Cellulase production in immobilized S. thermophile was maximum at pH 6.0 (Figure 3) for all three en- zyme components. There was a 15% to 20% loss in activity at either pH 5 or 7. Beta-glucosidase activity dropped sharply at pH 8 but endoglucanase activity was the same at pH 7 and 8. For exoglucanase, the

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decline in cellulase level from pH 7 to 8 was not ~s sharp. Bhat and Maheshwari 19 have also reported pH 6.0 as optimum for cellulase production by free cells of S. thermophile. In a recent report, Grajek 23 has re- ported that the optimum pH for extracellular aryl-beta- glucosidase production for his isolate of S. ther- mophile from wood waste material was around 6.5 at 43°C. This value corresponded to the pH favoring my- celial growth.

Temperature dependence for cellulase produced by immobilized S. thermophile was investigated in the range 40°C to 55°C (Figure 4). The exoglucanase activ-

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466 Enzyme Microb. Technol., 1990, vol. 12, June

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ity varied from 0.12 to 0.14, endoglucanase from 0.12 to 0.13, and beta-glucosidase from 0.21 to 0.42 IU ml -]. The temperature optima for all three compo- nents, however, was 45°C. According to Bhat and Ma- heshwari, ~9 the optimum temperature for cellulase pro- duction by their isolate of S. thermophile was 50°C. Grajek, 23 on the other hand, obtained maximum aryl- beta-glucosidase for a wood isolate at 40°C; for this isolate the activity determined in free cells growing at 38°C and 46°C was only 14% and 8% below the maxi- mum value. Coutts and Smith 5 have also reported that the best temperature for cellulase production by S. thermophile was 40°C. In another thermophile, Thiela- via terrestris, however, maximum cellulase was pro- duced at 44°C. 24

The influence of substrate concentration on cellu- lase production by immobilized cells was studied by incorporating microcrystalline cellulose (1-4%) in the medium (Figure 5). Maximum levels of exoglucanase, endoglucanase, and beta-glucosidase were achieved at 2% substrate; enzyme activity was of the order of 0.15, 0.05, and 0.62 IU m1-1. At higher substrate concentra- tions, the activity of beta-glucosidase dropped much more rapidly than the other two components of the cellulolytic system. Grajek 23 examined the effect of various carbon sources on aryl-beta-glucosidase pro- duction by free cells of S. thermophile and found that heterogeneous cellulosic substrates such as wheat bran, wheat straw, and sugar beet pulp were better inducers than pure cellulosics. In the present study, microcrystalline cellulose supported greater beta-glu- cosidase production than exoglucanase or endoglu- canase at the 2% level, suggesting that the behavior of immobilized mycelia can be different from that of the free cells.

Production of cellulolytic enzymes: A. Singh et al.

Reuse efficiency

In order to evaluate the reusability of the agar beads, growth was permitted for 6 days and the old culture broth was decanted. Beads were now allowed a 24-h reactivation period before addition of fresh medium. Three such batch culture replacements were made with the same immobilized inocula. The activity for exoglucanase and endoglucanase after the second re- use dropped by about 50%; beta-glucosidase activity declined from 0.28 to 0.04 IU ml -~ during this period. The cellulase activity of immobilized S. thermophile after the third reuse was almost negligible. The reuse efficiency of the agar beads was thus not very satisfac- tory. In their study involving production of cellulolytic enzymes by immobilized P. funiculosum, Linko et al. ]2 were successful in using the same carrageenan beads in at least three continuous batches.

Conclusion

Spores of S. thermophile were immobilized in agar and alginate beads and polyacrylamide gel to generate in situ mycelia for production of cellulolytic enzymes. Maximum cellulase by free cells was produced on day 3 at 45°C, but immobilized cells required 5 days to reach the maximum level. Among three supports, agar beads gave better results and were therefore chosen to determine optimal conditions for cellulase production. A pH optimum of 6.0 and temperature of 45°C allowed maximal release of endoglucanase, exoglucanase, and beta-glucosidase by immobilized S. thermophile. Mi- crocrystalline cellulose at the 2% level permitted opti- mal release of cellulase from immobilized agar beads. The reusability of agar beads in batch culture was not very encouraging, because the level of exoglucanase and endoglucanase dropped by nearly 50% after the second reuse. It appears that the diffusion of the sub- strate into immobilized beads may be a barrier for reusability. Alternatively, the release of the enzyme from the support matrix may be a limitation. Further work is necessary to examine these points so that the viability of the immobilized mycelia for cellulase pro- duction can be profitably exploited.

Acknowledgements

The authors thank the Dean, C.B.S.H., G.B.Pant Uni- versity for the facilities provided. One of us (A.S.) acknowledges award of an assistantship by the Uni- versity during the course of this work.

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