SURVIVAL OF BACTERIA AT LOW pH AND HIGH TEMPERATURE1

In most acid hot springs examined Sulfolobus was the predominant organism above 75°C at pH 2-3. It was found from 65-93°C at pH values from 1.6 to 3. The temperature optimum for growth was 70-75°C whereas the pI1 optimum for 14C-glut,anlatc uptake was from 2 to 5. Survival and growth were demonstrated by slide immersion studies and electron microscopy of natural samples. Rod-shaped bacteria live in the same habitat at 7%80°C and arc distinguished from Sulfolobus by their morphology and cell wall structure. Sulfolobus acidocaldarius (Brock et al. 1972) is a facultative autotroph found in nature in acid hot springs and hot acid soils. It grows optimally at pH 2-3 with a range from 0.9-5.8. This, in combination with the ability to grow at temperatures over 80°C makes Sulfolobus the most thermophilic acidophile known, since ThiobaCUUS (Schwartz and Schwartz 1965) and Bacillus acidocaldarius (Darland and Brock 1971) have upper temperature limits of 55 and 70°C. An extensive survey of bacteria in acid thermal springs (Brock and Darland 1970) showed that as the pH of the habitat dccreased, the upper temperature limit at which bacteria appeared also decreased. Several kinds of bacteria are found in acid springs (pH 2-3) at temperatures above 6O”C, but above 80°C Sulfolobus is the only one. This study was undertaken to establish the upper temperature limit at which SzllfoZobus grew, to investigate its temperature optimum, and to study bacterial growth rates in nature at temperatures above 70°C. MATERIALS AND METHODS Temperature was measured with a thermistor (YSI model 42SC) with a large “banjo” probe ( model 408). The response of the ’ This work was supported by U.S. Public Health Service Grant STl-GM-503 from the National Institute of General Medical Sciences. T. D. Brock provided laboratory facilities, a Present address: Department of Botany, University of California, Berkeley 94720. probe was checked periodically with a standard mercury thermometer. pH was measured in the field with a portable meter ( Orion model 401) with a combination glass electrode and in the laboratory with a Corning meter equipped with a similar glass electrode The meters were standardized with two buffers below pH 5. Growth rates were measured in natural habitats by immersing cleaned glass slides in the water, either plain or sulfur coated. For sulfur coating, slides were dipped in a 0.4% (w/v) solution of Parlodion in amyl acetate to form a moist thin film, immediately immersed in a 15% (w/v) solution of sulfur in carbon disulfide, and then heated for about 15 min at 65°C. The slides were placed in the outflow channels of springs and organisms were allowed to attach and grow for selected periods of time; they were then removed and sealed by ringing coverslips with nail polish. The growth rate was calculated by counting the number of cells in a microcolony (Bott and Brock 1969). After 24-48 hr the number of cells that attached near developing microcolonies was small and it was assumed that microcolonies neither gained nor lost cells as a result of passive attachment or detachment. Samples were observed in a microscope using a 40 X phase objective with a 12.5 x ocular. Cells growing in nature were counted with a Petroff-I-Iausser chamber after the sample was thoroughIy mixed. Methods used for electron microscopy of samples have been dcscribcd by Brock et al. (1972). LIMNOLOGY AND OCEANOGRAPIIY 877 NOVEMBER 1973, V. 18( 6) 878 R. L. s----^_ WkLSS