Leners in Applled Mlcrobiology 1995, 20, 34H52 Effects of glyphosate on nitrogen fixation of free-living heterotrophic bacteria A. Santos and M. Flores Department o, Microbi%gy, Facu/ty o, 810/ogy, Complutense University o, Madrid, Madrid, $pain OST/65: received 12 Oecember 1994 and accepted 13 Oecember 1994 A. SANTOS ANO M. FLORES. 1995. The effect ofthe herbicide glyphosate (N- (phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vine/andii was studied. Azotobacter vine/andii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha - 1 is needed to find sorne inhibitory effect. Specific respiration rates were 19·17 mmol O 2 h- 1 g-l dry weight for A. chroococcum and 12·09 mmol h - 1 g - 1 for A. vine/andii. When 20 kg ha - 1 was used with A. vine/andii, respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha -1. Nitrogen fixation dropped drastically 80% with 20 kg ha -1 in A. vine/and# and 98% with 28 kg ha -1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis. INTRODUCTION Members of the genus Azolobacter play an important role in nitrogen soil cycle (Ab-del-Malek 1971). They have been found in soils throughout the world, the predominant species depending upon the pH and moisture content of the soil. Soil of the rhizosphere of certain plants may contain larger numbers of azotobacters (10 6 -10 7 g-1 of soil) than soils without roots. Cyst formation contributes to main- tenance of this bacteria under suboptimal conditions. Azotobacter chroococcum appears to be the most widespread species, occurring mainly in neutral and alkaline soils. An organism that can reduce dinitrogen (N 2) to ammonia (NH 3 ) has the task of replenishing the biologically unavail- able nitrogen on the planet and the reward of being able to overcome the limitation imposed by a nitrogen-deficient environment. In addition, ecological studies show that free- living nitrogen fixation micro-organisms fix N 2 in all of the seasons of the year, giving a considerable amount of organic nitrogen to agricultural soils. Usually, an estimate of 10--15 kg N ha - 1 annum - 1 is given. Many factors affect soil ecology, such as herbicides and other chemicals used in agriculture. One of these herbicides is glyphosate (N-(phosphonomethyl)glycine) which is a broad spectrum herbicide that is very effective on deep- rooted perennial species, annual and biennial species of grasses, sedges and broadleaved weeds. Co"espo"¿t/lct lo,' Dr A. Sanlos, Deparlmt/ll o[ Microbiology, Facully o[ Biology, Complulltlse Universi/y o[ Madrid, Madrid, Spain. ([) 1995 The Society for Applied Bacteriology Mechanisms of action are not well known but a dis- ruption of phenolic metabolism has been implicated. Glyphosate appears to inhibit the aromatic amino acid bio- synthetic pathway, and accumulation of chlorophylls and carotenoids producing ultrastructural alterations and damages. The damage observed was a partial disruption of the chloroplast envelope and swelling of the rough endo- plasmic reticulum (RER) (Cañal el al. 1985; Kitchen el al. 1981). Glyphosate is degraded by micro-organisms in soils, therefore, various metabolites or degradation products of glyphosate have been identified. Aminomethylphosphonic acid is the principal product of glyphosate degradation in soils. Sarcosine, glycine and even CO 2 are possible non- phytotoxic products of glyphosate degradation in soils (Moshier and Penner 1978; Pipke and Amrhein 1988; Fitz- gibbon and Braymer 1988; Liu el al. 1991). MATERIALS AND METHODS Organlama, medlum and herblclde Azolobacter chroococcum (CECT* 203) and Azotobacter v;ne- landii (CECT 204) were grown in a modified Burk culture media, containing (g 1- 1 ): glucose, 20; K 2 HP0 4 , 0,64; KH 2 P0 4 , 0,16; MgS0 4 . 7H 2 0, 0,2; FeS04, 0'003; dis- tilled water, 1000 mi; pH 7·2.