APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1999, p. 4292–4293 0099-2240/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 65, No. 9
Association of Bacteria and Yeasts in Hot Springs EUGENE G. RIKHVANOV,* NINA N. VARAKINA, DMITRI YU. SOZINOV, AND VIKTOR K. VOINIKOV Siberian Institute of Plant Physiology and Biochemistry, Siberian Division, Russian Academy of Sciences, Irkutsk 664033, Russia Received 6 October 1998/Accepted 18 June 1999
The thermophilic bacterium Bacillus sp. strain TB-1 was isolated in association with the yeast Debaryomyces vanriji from hot springs at 46°C. It was shown that TB-1 excreted thiamine into the culture broth, which not only promoted D. vanriji growth in mixed culture but also increased the maximal temperature for yeast growth. same hot spring, were also checked for the promotion of yeast growth under the same conditions but no stimulatory effect was observed. Pure and mixed suspensions of Bacillus sp. strain TB-1 and S. cerevisiae were also plated on solid minimal medium and cultured for 72 h at 30°C. TB-1 strongly stimulated the growth of S. cerevisiae on minimal solid medium; in pure culture this yeast could not grow on this medium without the addition of vitamins. Thus, the stimulating factor can diffuse in the agar and affect yeast growth even when there is no direct contact between microorganisms. We failed to obtain similar results with the D. vanriji growth at 30°C but demonstrated the stimulation of its growth by TB-1 after incubation at 41°C. After 48 h at 41°C, only bacterial colonies formed and yeast growth was suppressed, although yeast cells remained viable. However, after 36 h of incubation at room temperature, stimulation of yeast growth again became apparent. Bacillus sp. strain TB-1 was grown on minimal medium to a density of 107 CFU/ml, cells were removed by centrifugation, and the supernatant was filter sterilized. TB-1 cultural liquid possessed the same promoting effect on D. vanriji growth at 30°C as had live bacterial cells (Fig. 2a). Similar results were obtained with S. cerevisiae. Since the bacterial cells inactivated by heat did not affect yeast growth and since the promoting effect was preserved in the case of addition of TB-1 cultural liquid, the possibility that this action is conditioned by the lysis of dead bacterial cells can be ruled out.
Temperature is one of the most important factors that governs species distribution. Mineral springs of the Baikal region, with temperatures ranging from 2 to 80°C, provide a unique experimental range for studies of the adaptation of organisms to extreme environmental factors. Yeasts are ubiquitous organisms and can be found in all environments. The temperature limits for the growth of yeast species vary between ⫺2 and 45°C (2), and the yeast cell is thus an attractive system to study the mechanism of thermal adaptation. All organisms have evolved a variety of strategies to adapt to harsh temperature conditions (8, 12, 14, 15), but temperature adaptation may occur not only at the level of species but also at the level of communities (10). The thermophilic bacterium Bacillus sp. strain TB-1 was isolated in association with the yeast Debaryomyces vanriji from hot springs. The aim of the present work was to study the effect of TB-1 and its extracellular metabolites on the growth of D. vanriji at various incubation temperatures. All experiments were repeated a minimum of three times and produced consistent results. D. vanriji GK46-2 and Bacillus sp. strains TB-1, TB-2, and TB-3, isolated from a hot spring in the vicinity of Goryachinsk (eastern Siberia) at a temperature of 46°C, and Saccharomyces cerevisiae 4AP2445 were used in this study. Bacteria were grown at 30°C in YEPD medium (15) to a density 5 ⫻ 108 CFU/ml on a water bath shaker (130 rpm). Cells were harvested by centrifugation at 6,000 rpm for 10 min, washed, and suspended in minimal medium [per liter: 0.9 g of KH2SO4, 0.1 g of K2HSO4, 2 g of MgSO4 䡠 7H2O, 1 g of (NH4)2SO4, 20 g of dextrose]. Cultures of D. vanriji were grown in minimal medium at 30°C to a density of 5 ⫻ 106 CFU/ml and used to inoculate control and test (i.e., with the addition of bacterial cells) flasks. The number of yeast cells formed was determined by spreading the cells on agar medium. The counting of yeast colonies was performed after 72 h of incubation at 22°C, when bacterial growth was suppressed. Bacillus sp. strain TB-1 strongly stimulated the growth of D. vanriji in mixed culture in liquid minimal medium at 30°C (Fig. 1). The significance of the TB-1 effect on yeast growth depended on the number of bacterial cells introduced. The stimulatory effect on yeast growth was absent if bacterial cells were inactivated by boiling before inoculation. Two other strains of bacteria (Bacillus sp. strains TB-2 and TB-3), isolated from the
* Corresponding author. Mailing address: Siberian Institute of Plant Physiology and Biochemistry, Siberian Division, Russian Academy of Sciences, Lermontov St. 132, Irkutsk 664033, Russia. Phone: (3952) 460721. Fax: (3952) 510754. E-mail:
[email protected].
FIG. 1. Effect of mixed inoculation with Bacillus sp. strain TB-1 on the growth of D. vanriji in liquid minimal medium at 30°C. Symbols: ⫻, yeast ⫹ TB-1 (105 CFU/ml); Œ, yeast ⫹ TB-1 (5 ⫻ 104 CFU/ml); ■, yeast ⫹ TB-1 (104 CFU/ml); F, yeast ⫹ TB-1 (105 CFU/ml inactivated by heat); }, yeast (control).
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FIG. 2. Effect of Bacillus sp. strain TB-1 cultural liquid (CL) on the growth of D. vanriji in liquid minimal medium at 30°C (a), at 41°C (b), and at 41°C in the presence of thiamine (c). Symbols: Œ, 25% CL; ■, 5% CL; }, control.
The maximum growth temperature of D. vanriji in rich nutrient medium (YEPD) is 41°C, but in minimal medium this strain can grow only at 37°C. So, in the control there was no growth of D. vanriji on solid minimal medium at 41°C. The addition of TB-1 cultural liquid in ratio of 1:1 restored the ability of yeast cells to grow under these conditions. Similarly, yeast growth in liquid medium at 41°C practically was not observed but was clearly evident after the addition of TB-1 cultural liquid in a ratio of 1:4 (Fig. 2b). However, the addition of thiamine to the liquid minimal medium (200 g/liter) not only sharply increased the growth of D. vanriji at 41°C but also practically nullified the effect of TB-1 cultural liquid on yeast growth (Fig. 2c). Accordingly, we determined thiamine content in the cultural liquid of TB-1 by oxidation with potassium ferrocyanide in alkaline medium (5) and have shown that thiamine was present at a concentration of 0.15 g/ml. Conclusion. Integrated associations among different species occur in all environments. One member of such an association can promote the growth rate of the other (11, 16–18). There are examples of relationships among various species allowing one of them to tolerate adverse exposure to high temperature (1, 3, 4, 6, 9). Thus far, however, relatively little is known about the biochemical and molecular mechanisms of this phenomenon. The results obtained in this study indicate that the Bacillus sp. strain TB-1, which in nature occurs in association with D. vanriji, excretes thiamine, which not only promotes yeast growth but also increases its optimal temperature for growth. It was shown previously that the activity of maize mitochondria inactivated after incubation at 27°C can be reconstituted by the addition of thiamine pyrophosphate (13). Thiamine increased the resistance of Yersinia kristensenii cells to antibiotics and heavy metals (7). We conclude that yeasts may exist in hot springs in association with bacteria and that this may influence the resistance of the yeasts to extreme temperatures. We are very grateful to J. Sutton for checking the English language of the manuscript. This work was supported by the Russian Foundation of Basic Research (RFBR-Baikal, project 97-04-96174) and the State Science Technique Program “Integration” (5.193). REFERENCES 1. Alayse-Danet, A. M., D. Desbruye´res, and F. Gail. 1987. The possible nutritional or detoxification role of the epibiotic bacteria of Alvenellid polychaetes:
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