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Journal of Culture Collections,
Volume 2, 1997-1998, pp.
3-9
GENERAL CHARACTERISTICS OF TWO XYLANOLYTIC BACTERIAL STRAINS ISOLATED FROM BULGARIAN HOT SPRINGS Rossica Mandeva, Plamen Dimitrov and Elka Emanuilova* Institute of Microbiology, Bulgarian Academy of Sciences "Acad. G. Bonchev" str., bl. 26, 1113 Sofia, Bulgaria Code Number: cc98001 Summary A comparison of freeze-drying and vacuum-drying of plant pathogenic bacteria showed no short-term differences in survival. Storage long term with vacuum-drying suggests that this simpler method offers similar survival rates to freeze-drying. A modification of the procedure in which the condenser is by-passed in the latter stages of drying offers the prospect of achieving lower pressures which are not limited by the vapour pressure of ice. Introduction Application of xylanases for pre-treatment of paper pulp to remove xylan decreasing consumption of chlorine chemicals requires a thermostable xylanase, active in alkaline pH. Thermostable xylanases are isolated from different thermophilic bacteria but most of them are not alkali-resistant [1, 6, 12]. On the other hand bacterial xylanases, active at alkaline pH, are not stable at high temperatures [7, 9, 10, 15]. In our previous work we have isolated two alkali-tolerant thermophilic strains (SP and BC) with xylanolitic activity by continuous cultivation from samples collected near Bulgarian hot springs. It is established that the xylanases have been thermostable at 70°C for 30 min and resistant to pH 5.5-8.0 (strain SP) and 6.0 -7.5 (strain BC) [2]. The present investigation is undertaken with a view to characterise and identify the strains, as well as the xylanase production in shaken flask cultivation. Materials and Methods Methods described by Gerhard et al., Gordon et al., and Sneath [3, 4, 13] were used for morphological, cultural and biochemical characteristics of the strains. For xylanase production, the strains were cultivated in medium containing (g/l) birch wood xylan (Fluka) -2.0; yeast extract (Difco) - 1.0; bacto peptone -2.0; pH - 8.0-8.5. The cultivation was carried out in 500 ml flasks containing 200 ml medium, at 60°C, using a platform shaker (New Brunsweek) at 240 rpm. Xylanase activity was assayed by mixing 0.05 ml culture supernatant with 0.05 ml of 1.0% birch wood xylan in phosphate buffer (pH-7.0), and was incubated for 5 min at 70°C. The reducing sugars were determined by dinitrosalicylic acid method, using D-xylose as a standard. The supernatant, mixed with the substrate solution without incubation was used as a control. Samples were measured at 540 nm. One unit (U) of xylanase activity was defined as the amount of enzyme that produced 1 µmol xylose for 1 min, at pH 7 and temperature 70°C. The cell concentration was expressed as the optical density (OD) at 660 nm. The cell dry weight of the cultures was estimated from OD using calibration accounting that one unit of OD was approximately 1.0 mg dry cell wt/ml for strain SP, and 0.850 mg dry cell wt/ml for strain BC. Results and Discussion The results for the morphological, cultural and biochemical properties of the isolated strains SP and BC are summarised in Table 1 and Table 2. They showed that the strains were closely related to each other. Comparing the characteristics of both of the strains with the description of different thermophilic Bacillus species [14], as well as the description of heterogeneous species Bacillus stearothermophilus [4, 13], the isolated strains were assigned to belong to the last one. The strains SP and BC showed some differences from the species B. stearothermophilus concerning growth at higher pH value, negative reaction for indol, utilisation of raffinose, arabinose and manitol, lack of gelatine hydrolysis. In Fig. 1 the growth and xylanase production of strain SP and BC are shown. The xylanase activity of the culture supernatants appeared after the 4th hour and increased to the 12-14th hour of cultivation (0.3 - 0.4 U/ml), when the cultures were in the stationary phase of growth and the concentration of the reducing sugars was very low (0.01-0.02 mg/ml). The decrease of the cell concentration of strain SP after the 10th hour was due to the cell lysis .The results indicated that the xylanase production by both of the strains was not growth associated and was carbon-source-repressed. Among the numerous xylanase producers from the Bacillus species two strains of B.stearothermophilus were described: B. stearothermophilus 4125 [5, 6] and B. stearothermophilus T-6 [8]. The comparison between the conditions for the production and properties of the xylanases of strain SP and strain BC [2] and these for the above mentioned strains is shown in Table 3. Considering this comparison, the relatively high xylanase activity achieved for a short period of cultivation (12-14 h), the enzyme thermostability and pH stability, we can conclude that strains SP and BC are potentially perspective B.stearothermophilus producers of thermostable xylanases and they are of interest for the future studies for improvement on the cultivation conditions and respectively on the enzyme yield. Acknowledgements This research was supported by a grant from the Ministry of Education, Science and Technologies of Bulgaria, Project K 509. References
Copyright 1998 - National Bank for Industrial Microorganisms and Cell Cultures - Bulgaria The following images related to this document are available:Photo images[cc98001t3.jpg] [cc98001f1.jpg] [cc98001t1.jpg] [cc98001t2.jpg] |
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