ISOLATION, GROWTH, AND RESPIRATION OF A THERMOPHILIC BLUE‐GREEN ALGA

HOLTON, RAYMOND W. (U. Texas, Austin.) Isolation, growth, and respiration of a thermophilic blue-green alga. Amer. Jour. Bot. 49(1): 1-6. Illus. 1962.-The isolation of a bacteria-free culture of Hapalosiphon laminosus (= Mastigocladus laminosus) from an algal mass obtained from Liard Hot Springs, British Columbia, is described. Seven different bacterial media at 2 incubation temperatures were used to test for bacteria in suspensions of partially broken up algae. The algal growth rate was obtained by calculating the slope of the straight line obtained in a plot of the cube root of the dry wt against time. In cultures aerated with air the growth was optimal between 35 and 50 C, with an upper limit at 55 and a lower limit at 25. Aeration with 1% CO2 in air significantly increased growth rates and the optimum was at 45-50 C. Unaerated flasks grew more slowly than aerated ones. The endogenous Qo2 (in ild/hr-mg dry wt) at 45 was 8.0 for growing cells and 2.8 for starved cells kept in darkness 18 hr. The Qo0 was maximal between pH 7.0 and 8.5. The concentration of phosphate buffer affected the Qo9 and rates in 0.02 M were slightly greater than those in distilled water and much greater than those in 0.4 M. The Qo2 of growing cells was slightly stimulated by 2,4-dinitrophenol (DNP), whereas glucose or glucose plus DNP did not affect it. In contrast, the endogenous Qo2 of starved cells was nearly doubled in glucose and tripled in DNP plus glucose. THERMOPHILIC microorganisms have been of interest to biologists for nearly a century and a half (Gaughran, 1947), but it has not been until recent years that research has been carried out regarding the physiological and biochemical basis of their thermophily. The ultimate aim of such research is to explain at the molecular level why thermophiles can exist at temperatures at which many protoplasmic constituents of mesophiles are destroyed. At least 2 general types of explanations have been offered to explain thermophily and experimental data have been advanced in support of each of these theories (Koffler, 1957). While several experimental differences between protoplasmic constituents of mesophiles and thermophiles have been observed, complete understanding of thermophily is not yet possible. Most of the experimental work has been carried out using thermophilic bacteria. Among the few physiological studies with thermophilic algae are the experiments of Marre and co-workers (1958) and Kubin (1959) on algae at the biochemical level. Therefore, it was with the ultimate aim of trying to understand the nature of thermophily in the blue-green algae as well as to study the general biochemistry of these organisms that experiments on 1 Received for publication February 27, 1961. I am grateful to Dr. Francis Drouet, University of Arizona, for identification of the alga and for comments concerning its taxonomy. To Mr. Gerald L. Schneberger and Mr. Earl C. Renshaw go my thanks for their competent technical assistance. The research was supported by grants from the Horace H. Rackham School of Graduate Studies of The University of Michigan: Faculty Research-Research Equipment Fund Project #140 and Horace H. Rackham Fund Project R #437. The work reported on was conducted in the Department of Biology, Flint College, UJniversity of Michigan, Flint, Michigan. the thermophile, Hapalosiphon laminosus, were initiated. This paper presents the procedure used for the isolation of H. laminosus, studies on the effect of temperature and aeration on its growth rate, and data on its respiration. A preliminary account of part of this work has appeared (Holton, 19.59). METHODS AND MATERIALS-Isolation of the organism-The organism was isolated from a mass of algal material taken from the Liard Hot Springs, Mile 496, Alaska Highway, British Columbia, Canada, in July, 1958. When a portion of this algal mass was placed in liquid mineral media and incubated in the light at 45 C, at least 1 species of algae grew well. Pour plates of a dilute suspension of the algal mixture were prepared and individual filaments were picked out with an inoculating needle with the aid of a dissecting microscope. These individual filaments were cultured in liquid media and a resulting culture was replated and individual filaments sub-cultured. The sub-culturing was repeated a third time to insure that only a single algal species was present. Application of bacterial staining techniques to the unialgal cultures revealed an occasional gram-positive coccus in the sheath of the alga. This coccus grew well at 37 C in thioglycollate broth and in brain heart infusion but showed no visible growth at 55 C after 72 hr, although it remained viable. The bacterium appeared to be a micrococcus but was not further identified. Attempts to rid the alga of this bacterium by the use of several different antibiotics failed, asexpected, as the alga appeared to be as sensitive as the bacterium to penicillin, tetracycline, and chloromycetin. Then ultraviolet light treatments (Gerloff, Fitzgerald, and Skoog, 1950) were tried, and a 10-min exposure was found to kill the bacterium, but not the alga. I The Journal for November-December (48: 859-951) was issued December 6, 1961] AMERICAN JOURNAL OF BOTANY, Vol. 49, No. 1, 1962