1. Introduction
The transition from biofuels to bio-product based economy, demands a suitable feedstock with capability of producing multiple high-value renewables (HVRs). Microalgae are by far the most abundant primary producers responsible for photosynthetic conversion of light energy and carbon dioxide (CO2) into sustainable renewables[1]. Recent advances in bioprocess technology supports the development of microalgal cell factories for establishing environmentally sustainable manufacturing of HVRs. In this regard Phaeodactylum tricornutum , a unicellular, marine pennate diatom, is considered as a potential feedstock for the production of biofuel and HVRs such as[2, 3], eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)[4], chrysolaminarin[5], fucoxanthin[6, 7], etc., and can be considered as a suitable microalgal cell factory for sustainable biorefinery processes[8].
One of the major fatty acid in P. tricornutum is the ω-3 fatty acid EPA (C20:5), having a significant commercial importance in pharmaceutical and nutraceutical industries[9, 10]. These long chain polyunsaturated fatty acids (LC-PUFAs) have a bioactive role against a variety of disorders, including coronary heart disease, thrombosis, and recently in prospective adjuvant therapy in COVID-19-related cardiovascular problems[11-14]. EPA content in P. tricornutum is reported upto 3-5 % of DCW (dry cell weight) with an average productivity of 56 mg L-1D-1, highlighting a potential alternate vegan source of ω-3 fatty acid production[15]. Commercially EPA from P. tricornutum is available in global market viabrand name SIMRIS® ALGAE OMEGA-3, containing 50 mg of EPA per capsule (www.simris.com/pages/ingredients).
Additionally, fucoxanthin (1% - 6% DCW) an important HVR, is the primary carotenoid produced in P.tricornutum[16][6]. During photosynthesis, xanthophylls acts as a light harvesting pigment connected to fucoxanthin-chlorophyll a/c-proteins (FCP), which are an integral part of the thylakoids[17]. Furthermore, due to its unique structure, fucoxanthin has various major bioactivities such as anti-oxidant, anti-obesity, anti-cancer properties, and it has been found to be an effective treatment for chronic disorders such as Alzheimer’s[18-21]. Commercial source of fucoxanthin is primarily brown seaweeds, which are difficult to meet market demands due to low productivity, low quality, and high cost. The amount of fucoxanthin generated by P. tricornutum is substantially higher than brown seaweeds, making it a potential choice for commercial production[22].
Media engineering strategies for microalgae cultivation have recently acquired appeal as feasible techniques for achieving high HVR output[7, 23]. Though the high costs of substrates, is a major impediment for generating commercially viable product [24]. Mixotrophy, on the other hand, represents an innovative methodology for HVRs production in P. tricornutum[25, 26]. It appears challenging to attain high biomass, EPA, and fucoxanthin content simultaneously; hence, designing an appropriate strategy for the mixotrophic cultivation ofP. tricornutum is critical for the commercial co-production of HVRs.
P. tricornutum grows mixotrophically and has been reported to grow efficiently on glucose, fructose, mannose, lactose and glycerol[27]. Furthermore, various nitrogen sources such as nitrate, nitrite, ammonia, and urea has been employed for its cultivation[28]. It has been reported that cultivation of P. tricornutum on glycerol supplemented medium yields 0.4 g L-1D-1 of biomass and 8.5 mg L-1D-1 of EPA[29].Furthermore, utilizing urea as sole nitrogen source resulted in a considerable enhancement in EPA content (26 mg g-1)[28-30]. Compared to the conventional nitrogen source i.e. sodium nitrate (NaNO3), urea is the cost effective and environmental friendly substrate for the cultivation of microalgae and thus the production of HVRs[28, 31]. The nutrient costs for the large scale cultivation of P. tricornutum on F/2 medium (with NaNO3) approximately to be USD 0.15 kg-1 biomass, whereas its cost decreased to half i.e. USD 0.07, kg-1 biomass when grown on a modified medium (with urea) (Cui et al., 2021). Whereas crude glycerol, a by-product of biodiesel processing, reported for the production of β-carotene and DHA from Schizochytrium limanicumand Blakeslea trispora[32]. As a result, using these substrates might be a viable method for sustainable production of biomass, and HVRs from P. tricornutum .
The aim of this study is to examine the effect of glycerol, urea, NaNO3 and their various combinations for the production of HVRs in P. tricornutum , with a primary focus on EPA along with fucoxanthin. Our preliminary screening revealed that glycerol (0.1M) and urea (441 M) could be used as low-cost substrate for the generation of biomass, lipids, carbohydrates and EPA. Moreover, feeding additional urea to the culture supplemented with glycerol led to significant enhancement of biomass, EPA, and fucoxanthin production. In this context, we highlight an alternate strategy, beneficial for the sustainable co-production of various HVRs from P. tricornutum .