Transactions of the Illinois State Academy of Science (1995), Volume 88, 3 and 4, pp. 97-107 Factors Affecting Nitrogen Fixation in Azolla caroliniana William D. Hechler and Jeffrey O. Dawson Forestry Department University of Illinois Urbana, IL 61801 ABSTRACT The effects of environmental variables on nitrogen fixation and growth of Azolla caroliniana were studied. Nitrogenase activity was highest at ~25˚C. Nitrogenase activity was highest at photosynthetic photon flux densities of 400-1000 μmol·m -2 ·s -1 and decreased sharply at lower levels. Azolla showed great sensitivity to drying in that nitrogenase activity was approximately halved as moisture concentration decreased from 95% to 87% of total fresh plant mass. A strong diurnal pattern of nitrogenase activity was detected; this pattern was not due to an endogenous rhythm. High plant density decreased specific nitrogenase activity per unit plant mass and per unit area; the optimal plant density for nitrogen fixation on an area basis is approximately 50-100 g dry ·m -2 . Two or 10.0 mg NH 4 -N·l -1 decreased nitrogenase activity; 10.0 mg NO 3 -N·l -1 decreased nitrogenase activity while 10.0 mg urea-N·l -1 was associated with an increase in nitrogenase activity. Ten mg NH 4 -N·l -1 caused a decline in Azolla growth; 10.0 mg NO 3 - N·l -1 resulted in an increased growth rate. INTRODUCTION The genus Azolla comprises seven species of symbiotic nitrogen-fixing aquatic ferns. Azolla caroliniana is found in rivers and cypress swamps of southern Illinois. The symbiosis consists of an association between the fern Azolla and the cyanobacterium (blue-green alga) Anabaena azollae, which inhabits cavities in the leaves of the fern (Lin and Watanabe 1988, Hill 1975). The cyanobacterium carries out the process of nitrogen fixation, i.e., the enzymatic reduction of atmospheric dinitrogen to ammonia (Oes 1913, Olson 1970, Venkataraman 1962). Azolla is economically important as a natural source of nitrogen fertilizer for agriculture in many areas of the world where industrially manufactured nitrogen fertilizers are expensive or unavailable (Lumpkin and Plucknett 1982). Azolla has the potential to become important in the industrialized countries for nitrogen fertilization and soil organic matter management. Its role in the nitrogen economy of natural wetlands in North America has not been established. Physiologically, Azolla species and strains differ in their responses to environmental variables (Vu et al. 1986, Subudhi and Watanabe 1981). The purpose of this study was to determine the effects of temperature, photosynthetic photon flux density (PPFD), time of day, moisture, plant density, and combined nitrogen on nitrogen fixation by Azolla caroliniana of southern Illinois. This information could be useful in the cultivation of Azolla for use in