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The dairy industry is facing a problem associated with 1.6 billion tons of acid whey per year as a waste stream. The extended amount of acid whey has encouraged studies for novel approaches of acid whey utilization. The production of lactobionic acid (LBA) using dairy waste has been in rapid demand as an economically feasible and environmentally friendly approach. The composition of acid whey makes lactose conversion into LBA by Pseudomonas taetrolens complicated. Therefore, the aim of the current research was to evaluate factors (quality of whey (salts, protein concentration, pH), volume of inoculum, and cultivation time) with the purpose of increasing the suitability of acid whey for biotechnological LBA production. LBA production was performed in a 4L bioreactor, which was equipped with a pH electrode and a dissolved oxygen electrode. The whole experiment was performed at a temperature of 30 °C under 350 rpm agitation. The continuous aeration was set at 0.5 L/min. The current study presents the study of acid and sweet whey combinations in different ratios (100:0; 50:50; 60:40, 70:30, 80:20, respectively) inoculated with 10% or 30% v/v of fresh P. taetrolens inoculum reaching up to 59.9 ± 1% LBA yield during cultivation. Increasing protein and pH in a substrate slows down the lactose converting ability of P. taetrolens. Results demonstrated that increasing the acid whey amount in a substrate can affect the LBA yield, and a combination of sweet and acid whey could be a good solution for biotechnological LBA production using dairy waste. Doi: 10.28991/HEF-SP2022-01-04 Full Text: PDF