Anaukwu, C.G., Ezemba, C.C., Anakwenze, V.N.and Ekwealor, I.A. ABSTRACT Background: Biosurfactants are secondary metabolites produced by microorganisms. Owing to their unique features which includes low toxicity and biodegradability, biosurfactant have gained importance as emulsifiers, foaming agents,wetting agents, food ingredients, detergents and biostimulants. The composition and emulsifying activity of the biosurfactant not only depends on the producer strain but also on the fermentation conditions such as the carbon source, the nitrogen source as well as the C:N ratio and other physical parameters. The effects of fermentation conditions on biosurfactant production by Pseudomonas monteilii AF064458 and Citrobacter murliniae AF025369 were studied. Methods: Pseudomonas monteilii AF064458 and Citrobacter murliniae AF025369, isolated from soil contaminated with spent-engine oil were used for biosurfactant production. Shake flask fermentation experiments were conducted in mineral salt medium and the effects of medium/fermenter volume ratio, carbon and nitrogen sources, growth factors, pH and agitation speed on biosufactant production by the isolates were examined. Results: An increase in biosurfactant production by P. monteilii AF064458 was observed when 40% medium/fermenter volume ratio was used, while 50% ratio improved biosurfactant accumulation in the broth culture of C. murliniae AF025369. Maltose at 1% and glucose at 2% concentrations enhanced biosurfactant production by P.monteilii AF064458 and C. murliniae AF025369 respectively. Sodium nitrate was the nitrogen of choice for both isolates and at 1% and 0.5% , biosurfactant production by P. monteilii AF064458 and C. murliniae AF025369 respectively were improved. At a pH of 7.5, P. monteilii AF064458 gave high biosurfactant, while C. murliniae AF025369 stimulated an increase at a pH of 8.0.The effect of agitation speed revealed that biosurfactant production in P.monteilii AF064458 and C. murliniae AF025369 was optimal at 200rpm. Conclusions: This study has shown that optimizing the fermentation conditions in mineral salt medium improved biosurfactant yield by Pseudomonas monteilii AF064458 and Citrobacter murliniae AF025369. Optimization of nutritional and physical parameters is recommended for substantially higher biosurfactant production by microorganisms. Keywords: Biosurfactants, surface tension, emulsification index, critical micelle concentrations, bacteria, soil   Optimization of Fermentation Conditions for Improved Biosurfactant Production by Pseudomonas monteilii AF064458 and Citrobacter murliniae AF025369 By Anaukwu, C.G., Ezemba, C.C., Anakwenze, V.N.and Ekwealor, I.A. Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka,Nigeria E-mail: chika.Gladys@ymail.com; cg.anaukwu@unizik.edu.ng METHODOLGY Fermentation: Fermentation process was carried out following method described by Anaukwu et al. (2015). A mineral salt medium consisting of basal medium (g/L): KCl, 1.1; NaCl, 1.1; FeSO4.7H2O, 0.00028; KH2PO4, 3.4; K2HPO4, 4.4; MgSO4.7H2O, 0.5; Yeast extract, 0.5]; 2ml of Trace element solution (ZnSO4.7H2O, 0.29 ; CaCl2.4H2O, 0.24; CuSO4.5H2O, 0.25; MnSO4. 7H2O, 0.17); H2O. Effects of varying concentration of carbon sources(Glucose, Glycerol, Maltose, Mannitol, Sucrose), varying concentrations of nitrogen sources (KNO3, NH4Cl, NaNO3, NH4SO4), different medium /volume ratio( 10 -50%), various growth promoting factors at 0.2% concentration(Yeast extract, Casein, Peptone, a mixture of peptone and casein, a mixture of peptone and yeast extract, a mixture of yeast extract and casein ), different pH values (5-8), different agitation speed (100-250) were studied. Level of biosurfactant production was assessed measuring the emulsification index after 24hr. RESULTS INTRODUCTION Biosurfactants are surface active agents produced extracellularly or as part of the cell membrane by bacteria, yeasts and fungi (Mulligan and Gibbs, 2004), and are known to offer several benefits over their chemically synthesized counterparts. They are biodegradable, biocompatable and digestible, availability of raw materials and acceptable production economics, environmentally friendly and specific in their actions (Kapadia and Yagnik, 2013). Biosurfactants have several applications in agriculture, medicine, petroleum and industry. In agriculture, they are used for hydrophilization of heavy soils to obtain good wettability and to achieve even distribution of fertilizer in the soil. They also prevent the caking of certain fertilizer during storage and promote spreading and penetration of the toxicants in pesticides (Makkar and Rockne, 2003). Biosurfactants such as Fengycins have been reported to possess antifungal activity, and therefore, may be employed in biocontrol of plant diseases (Kachholz and Schlingmann, 1987). Some microorganisms, such as Bacillus subtilis, Pseudomonas aeruginosa, Torulopsis bombicola have also been reported to utilize crude oil and hydrocarbons by producing biosurfactant and thus can be used for oil spill clean-ups (Ristau and Wanger, 1983). This study was conducted to isolate bacterial species with good potentials for biosurfactant production, and to determine the tenso-active characteristics of the active producers. CONCLUSION This study has shown that optimizing the fermentation conditions in mineral salt medium improved biosurfactant yield by Pseudomonas monteilii AF064458 and Citrobacter murliniae AF025369. Optimization of nutritional and physical parameters is recommended for substantially higher biosurfactant production by microorganisms