ORIGINAL PAPER Use of Response Surface Methodology to Study the Combined Effect of Salt, pH, and Temperature on the Growth of Food-Spoiling Halotolerant Yeast, Debaryomyces nepalensis NCYC 3413 Pradeep Lal & Sawan Kumar & Sathyanarayana N. Gummadi Received: 29 September 2008 / Accepted: 14 January 2009 / Published online: 5 February 2009 # Springer Science + Business Media, LLC 2009 Abstract This paper reports the interaction of salt (NaCl and KCl), initial pH, and temperature and their effects on the specific growth rate and lag phase of food spoiling halotolerant yeast, Debaryomyces nepalensis. The optimi- zation of salt, initial pH, and temperature was carried out using response surface methodology based on central composite design. The mathematical model showed that salt has a significant effect on specific growth rate and lag phase of D. nepalensis. The optimal conditions of salt concentration, pH, and temperature of growth were found to be 0.3 M NaCl, 7.1, 26 °C and 0.6 M KCl, 5.6, 25°C, respectively. Under these conditions, a maximum specific growth rate of 0.41 and 0.5 h -1 was observed in medium containing NaCl and KCl, respectively. Lag phase can be increased most effectively either by increasing salt concen- trations or both by decreasing (≤20°C) or increasing the temperature (≥40°C) with moderate (1.5 M) or low salt concentration (0.5 M), respectively. Results show that D. nepalensis need to generate weak acids to maintain the intracellular pH under pH and saline stress conditions. The results obtained in this study will be helpful in using optimal conditions for the maximum growth of the strain for the production of certain metabolites like organic acids and glycerol and designing food preservation procedures. Keywords Yeast . Optimization . Response surface methodology . Microbial growth . Halotolerance . Intracellular pH . Organic acid Introduction Yeast can be found in a wide variety of environments due to their ability to withstand different environmental stress and utilize a variety of substrates. Different yeasts like Debaryomyces nepalensis and Debaryomyces hansenii, which survive under salt stress condition, have been reported to be common contaminant in several food products like cheese, milk, yogurt, bread, spices, and beverages which are at various pH and contain various levels of salt, sugar, and water (Deak 1991; Fleet 1992; Smits and Brul 2005; Almagro et al. 2000). Usually, the combination of temperature, salt, and pH are the important factors affecting the shelf life of these food products (Papouskova and Sychrova 2007). Thus, it is imperative that shelf life of these edibles be ensured with respect to such halotolerant yeast under different environmental conditions of salt, pH, and temperature. To withstand salt stress condition, these yeasts develop unique strategies such as intracellular accumulation of osmolytes and salt-adapted enzymes. These unique properties of halotolerance are of considerable biotechnological significance (Ventosa and Nieto 2005). Previously, we have isolated a yeast strain which could utilize pectin as the sole carbon source. Based on the 26s rDNA sequence analysis, this yeast strain was identified to be D. nepalensis (Gummadi and Kumar 2006a). D. nepal- ensis was reported to tolerate 2 M NaCl, 3 M KCl, and 0.5 M LiCl with the specific growth rate higher as compared to D. hansenii (Gummadi et al. 2007). The maximum and minimum temperature for complete inhibi- tion of growth was found to be 42 °C and 8 °C, respectively. The strain showed synergistic effect for growth at 35–40 °C with 0.5 M NaCl and 30–40 °C with Food Bioprocess Technol (2011) 4:723–730 DOI 10.1007/s11947-009-0183-1 P. Lal : S. Kumar : S. N. Gummadi (*) Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India e-mail: gummadi@iitm.ac.in