FREEZE RECOVERY AND NITROGENASE ACTIVITY IN ANTARCTIC CYANOBACTERIUM NOSTOC COMMUNE Rajan Kumar Gupta 1 Department of Botany, Pt. L.M.S. Govt. P.G. College, Rishikesh 249201 (Dehradun), Uttarakhand, India e-mail: rajankgupta1@rediffmail.com Abstract. Cyanobacteria, especially terrestrial forms, are exposed frequently to alternating freeze-thaw cycles during the early spring and late fall seasons in Antarctic regions. However, the question of how cyanobacteria respond to freezing has received little attention. In order to study the freezing recovery and its impact on Nitrogenase activity in the dominant cyanobacterium in the region Nostoc commune was cultured and propagated on sand with aqueous N-free BG-11 medium. Laboratory experiments were conducted to characterize the in vivo freeze recovery physiology of nitrogenase activity. Nitrogenease activity was monitored by the acetylene reduction activity. Frozen Nostoc mats were thawed and warmed to 2,4,6,8,10,20, or 25 o C, nitrogenase activity was detected within 6h after thawing. Optimum thawing temperature with respect to the recovery of nitrogenase activity was 20 o C. In Subsiquent experiments, laboratory grown Nostoc mats were used along with the following conditions : prefreezing treatment of 3d of exposure to light or darkness, freezing and then thawing to 20 o C in light or darkness, with or without DCMU or chloramphenicol. Approximately 25% of the energy in the initial recovery of nitrogenase activity (up to 12 h after thawing) appeard to be supplied via the utilization of carbon compounds stored before freezing. Photosynthetic condition (i.e., light and without DCMU) were necessary for maximum recovery of nitrogenase activity. In the presence of protein synthesis inhibitor chlormphenicol, nitrogenase activity was still detected at 12 to 48 h after thawing. Although damage may occur to nitrogenase, some of the enzyme was capable of surviving the freeze-thaw period in vivo. However, complete recovery of nitrogenase activity (equal to prefreezing activity) may entail some de novo synthesis of nitrogenase. This work is supported by the Department of Ocean Development, New Delhi and NCAOR, Goa, India by providing me an opportunity to visit the Antarctic field during XIth and XIVth Indian Scientific Expeditions to Antarctica and collect the samples. Key words: Cyanobacteria, Antarctica, , Nitrogenase, Nostoc 1. Introduction Cyanobacteria, especially terrestrial forms, are exposed frequently to alternating freeze-thaw cycles during the early spring and late fall seasons in Antarctic regions. However, the question of how cyanobacteria respond to freezing has received little attention. Ono and Murata (1981 a, b) investigated the effect of chilling on the photosynthetic activities of Anacystis nidulans. They noted that both the dark reactions and primary photochemical reaction are damaged by chilling treatment. They correlated the chilling susceptibility with the fluidity of membrane lipids. Increased membrane permeability, as measured by leakage of potassium ions and other ions from the cytoplasm to the surrounding medium, was attributed to an alterd state of cytoplasmic lipids at chilling temperatures (Ono and Murata 1982). This loss of ions then resulted in an inactivation of photosynthesis. The effects of freezing on nitrogenase activity has been only studied with nitrogenase extract preparation . Haystead et al (1970) showed that nitrogenase activity in extracts of Anabaena cylindrica reapidly decreased when incubated at 0 o C. After 12d at 0 o C, there was 61.7% inhibition of nitrogenase activity when compared with control samples (20 o C). In initial studies on crude extracts of Clostridium sp, Dua and Burris (1963) reported an inactivation of approximately 85% after 12 at 0 o . Zumft and Mortenson (1975) have the data on the cold lability of nitrogenae (specifically the azoferodoxin protein component). 116 2011 International Conference on Nanotechnology and Biosensors IPCBEE vol.25(2011) © (2011) IACSIT Press, Singapore