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Response surface methodology (RSM) was employed to study the effects of various medium components on biomass production by Lactobacillus rhamnosus E/N. This strain is commonly used in the pharmaceutical and food industries due to its beneficial effect on the human gut and general health. The best medium composition derived from RSM regression was (in g/l) glucose 15.44, sodium pyruvate 3.92, meat extract 8.0, potassium phosphate 1.88, sodium acetate 4.7, and ammonium citrate 1.88. With this medium composition biomass production was 23 g/l of dry cell weight after 18 h of cultivation in bioreactor conditions, whereas on MRS the yield of biomass was 21 g/l of dry cell weight. The cost of 1 g of biomass obtained on MRS broth was calculated at the level of 0.44 € whereas on the new optimal medium it was 25% lower. It may be concluded then, that the new medium, being cheaper than the control MRS allows large scale commercial cultivation of the L. rhamnosus strain. This study is of relevance to food industry because the possibility to obtain high yield of bacterial biomass is necessary step in manufacturing of probiotic food.