This study shows that strain of thermotolerant Kluyveromyces marxianus was used for the production of ethanol and invertase (fructofuranosiadse, Ffase). This strain (D-67283) was collected from Shakkar Gunj Sugar Mills, Jhang, selected through Gamma rays on 1.5 % (w/v) deoxy-Dglucose (DG) in liquid medium after growth at 60 °C for 5 days and designated as K. marxianus M15. The selected mutant strain produced maximum ethanol and Ffase at 48 h of cultivation on different substrates including glucose, sucrose, and molasses each at 10, 12, 15 and 17% total sugars in 23 L fermentor (working volume 15 L). Optimized studies on different carbon sources displayed that product formation rate (Qp) was greater on glucose- followed by molasses- medium but was found to be lower on sucrose medium. Product yield (Yp/s) and specific product yield (Yp/x) were also significantly higher on glucose (15%) whereas found as lower on sucrose medium. Specific product formation rate (qp) was also recorded higher on glucose medium and remained lower on sucrose medium. Nitrogen sources like ammonium sulphate, corn steep liquor (CSL), and urea were added to the growth medium to enhance growth and ethanol formation. All these sources were used at the rate to contain 0.11, 0.16 and 0.21 % nitrogen in the growth medium. The best results were observed for fermentation kinetic parameters of growth and product formation by ammonium sulphate using 0.75% (w/v). In further studies, temperature of fermentation was optimized for maximum ethanol production, and substrate utilization, for both wild and mutant strains of K. marxianus. For this purpose, they were grown at different temperatures ranging from 20- 65 oC. The study further revealed that maximum ethanol production on molasses medium supplemented with ammonium sulphate was used 0.75% at pH 5.5 after 48 h at 40 oC. xx Effect of various agitation rates from 250-450 rpm on production of ethanol by K. marxianus cells was carried out and the maximum amount of ethanol produced on the sugar based used was more than 89.96 %. Further increase in agitation intensity did not increase ethanol production in both organisms. Hence, agitation rate of 300 rpm was optimized. The production of ethanol is an anaerobic fermentation process; therefore supply of oxygen to the yeast culture is of great importance as it is needed to support an initial amount of cell mass for maximum ethanol production. At a constant rate of oxygen supply, agitation rate supports uniform distribution of cells and maintain a constant temperature by uniformly stirring the media to dissipate excess heat. Effect of supplying air to the fermentor at different aeration rates (0.25 - 1.5 LL-1min-1) on ethanol fermentation, amount of ethanol produced was 72.5-74.8 gL-1 when the aeration rate was kept at 1.0 LL-1min-1 for 8 h and then between 0.25 - 0.50 LL-1min-1. Further increase in aeration rate, resulted in lower production of ethanol and greater amount of cell mass in the fermented broth but up to a certain extent. The maximum amount of ethanol (75 gL-1) produced when the aeration rate was kept at 0.30 LL -1min-1. Maximum ethanol specific or volumetric productivity increased with the increased temperature up to 40 oC and 45 °C in the case of wild and mutant respectively. The activation enthalpy for ethanol formation (DH* = 55.6 KJ/mol) pathway was lower than that for phytase production ((DH*70-80 KJ mol-1).The estimated values of enthalpy of Ffase formation (13.1 kJ mo-1l) network was lower as compared with the product inactivation (12.0 kJ mol-1 ) network. This usually happens in the case of thermotolerant organisms as reported earlier. Over all these studies revealed that mutant strain acquired significantly better changes in the genetic make up and qualifies for its evaluation at industrial scale ethanol production.