The present work reports the nutritional requirements and environmental conditions for submerged culture of Streptomyces clavuligerus for clavulanic acid production using orthogonal matrix method (Taguchi L-16 design) and also fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. Clavulanic acid production was increased by 18% with the span of feeding glycerol and reached a maximum at 1.30 mg/ml with 120 h glycerol feeding as compared to 1.10 mg/ml in the control. The production also increased with the span of feeding amino acids and reached a maximum of 1.31 and 1.86 mg/ml with feeding arginine and threonine, respectively in 120 h. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls (1.10 and 1.70 mg/ml, respectively). (c) 2006 Elsevier Ltd. All rights reserved.