Viola odorata plant extracts are extensively used in indigenous medicine to treat infectious diseases. V. odorata's limited availability in nature, due to geographical conditions and adulteration, makes continuous supply of high-quality plant raw material challenging. This results in low-quality plant extracts that can have inconsistent and sometimes adverse effects when used in medicine. As an effective solution, in vitro plant biomass cultivation conserves plant diversity, ensuring high-quality raw materials with uniform quality and quantity. Toward this, the current study focuses on establishing bioprocess strategy to achieve maximum V. odorata biomass productivity up to bioreactor level. In this context, in vitro culture conditions were rationally optimised, focusing upon both statistical optimization and one-factor-at-a-time (OFAT) approach, for improved biomass productivity. In particular, basal medium, carbon and nitrogen source, inoculum density, macronutrient concentration, shaking speed, temperature, pH, light intensity, and plant growth regulators were systematically optimized in order to achieve the maximum biomass. This led to increased biomass production from 10.2 ± 0.8 g DW L-1 to 21.68 ± 0.82 g DW L-1 within 12 days of the batch cultivation period. Optimized culture conditions were implemented in three different types of bioreactors, namely, stirred tank, airlift, and bubble column, to cultivate V. odorata biomass. Of these three methods, cultivation in a stirred tank reactor produced ~19.7 g DW L-1 of maximum biomass within a 12-day batch cultivation period. Further, the metabolite profile of the bioreactor cultivated biomass (alcoholic extract) was compared with that of the natural plant biomass using gas chromatography-mass spectrometry based metabolomics, yielding around 18 key principles (metabolites) that exhibit resemblance with that in the natural source. The antibacterial activity of the plant extract was tested against three bacteria responsible for respiratory tract infections, and their antioxidant activity was compared with that of Vitamin E, wherein the results demonstrated comparable activity. The novelty of this study is in the fact that the V. odorata plant cells were cultivated in bioreactors for the first time for potential medicinal applications. It provides an alternative method for sustainable production of plant biomass, thereby conserving the natural plant sources.
