Aurantiochytrium can produce significant amounts of omega-3 fatty acids, specifically docosahexaenoic acid and docosapentaenoic acid. Euglena is a useful microorganism producing 59 different nutrients including paramylon related to human immune system. Sterilization within the bioreactor system is indispensable for a reliable cultivation of our target microorganisms: it can be achieved either by applying heat or chemicals. Heating is the most reliable method to eliminate contamination risk by fungi, bacteria, viruses, and spore forms. However, heat sterilization for reliable cultivation is not appropriate to heat-sensitive materials and cause a conversion of glucose via browning (Maillard) reactions. Thus, the present study investigated the use of a direct degradation of Peracetic acid (PAA) for cultivation of Aurantiochytrium and Euglena. Polymer-based bioreactor and glucose-containing media were chemically co-sterilized by higher than 0.04% peracetic acid (PAA) and neutralized through a reaction with ferric ion. Mono-cultivation was achieved without the need for washing steps and filtration, thereby avoiding the heat- induced degradation and dehydration of glucose. Use of chemically sterilized and neutralized medium, rather than heat-sterilized medium, led to a two-fold faster growth rate and greater productivity of omega-3 fatty acids in the cultivation of Aurantiochytrium. In addition, the chemical sterilization method was further optimized by using reducing sugars (glucose and sucrose) as well as aeration process. Surprisingly, a new method related with the photometric determination of peracetic acid was developed in the present study.