Appl Microbiol Biotechnol (1984) 20:281-283 Short contribution Aprr . o.d Microbiology Biotechnology © Springer-Verlag 1984 Development of an optimal heterotrophic growth medium for Chlorella vuigaris Robert E. Burrell, William E. Inniss, and Colin I. Mayficld Department of Biology, University of Waterloo, Waterloo, Ontario, Canada, N2L 3G1 Summary ~inal biomass yields of Chlorella vulgaris cultured heterotrophically in bristol medium amended with 0.1% (w/v) yeast extract (Difco) or 0.5% glucose (w/v) were 26 and 5b times higher, respectively, than yields obtained for autotrophically grown cells in the light. Similarly, final biomass increases were 35 and 138 fold for these organic substrates in the dark. The mixture of 0.1% yeast extract and 0.5% glucose was optimal and produced increases in final biomass of 70 and 140 times in the light and dark, respectively. Introduction Studies of algal heterotrophy are extensive and have included representative organisms from virtually all groups of algae (Droop 1974; Neilson and Lewin 1974). An equally large number of organic compounds have been examined as potential substrates including sugars and amino acids (Pearsall and Bengry 1940; heilson and Lewin 1974; Coughlan 1977; Vilicic 1979) and urea (Carpenter et al 1972). ~any recent studies have involved high rate algal ponds and their potential in treating waste water and producing biomass (ADeliovich and ~eisman 197b; Goldman 1979a, 1979b; Oron et al 19UI). However, there does not appear to have been any attempts to aevelop a standard medium for optimal heterotrophic growth of' green algae. Future biotechnological studies involving physiological aspects of algal heterotrophy and algal biomass production require an optimal heterotrophic medium for standardization and comparison of results. In the present paper, the development of heterotrophic medium for the optimal heterotrophic growth and biomass production of the green alga Chlorella vulgaris is reported. Materials and aethoas Chlorella vulgaris (ATCC 9765) was grown in 25 different media. Twenty-four media were preparea by adding yeast extract (0.01, 0.03, 0.05, 0.07 and 0.1% w/v) or glucose (0.1, 0.3, 0.5, 0.7, 1.0, 3.0 and 5.0% w/v) or all combinations of yeast extract (0.01, 0.05 and 0.1% w/v) and glucose (0.1, 0.3, 0.5 and 1.0% w/v) to Bristol solution. The remaining medium was unamended Bristol solution (Nichols 1973). Seventy-five mL of each medium were equally distributed amongst a group of 5 test tubes, inoculated with 1.5 x 10-- cells of ~. vulgaris and incubated on a slant board either under Agrolite (~estinghouse) fl_u2re~ ~ent tubes continuously providing 30 ~E m s of energy or in complete darkness at 25°C ± 1.0 C° . Growth was measured spectrophotometrically (678 nm) every other day for 30 days. Student's two-tailed t-test (Zar 1974) was used for comparison of experimental means. Results and Discussion Growth of ~. vulgaris in Bristol solution amended with varying amounts of yeast extract was much better than growth in Bristol solution alone. The response was proportional, with the greatest amount of growth occurring in the highest concentration of yeast extract (0.1%). Maximum growth in the yeast extract-amended media, relative to that which occurred in Bristol solution, increased 26 and 35 times in the light and dark, respectively. A comparison of growth in the light and dark showed that in the light a 6-fold increase at the highest concentration Offprint requests to: W. E. Inniss