Tannic acid enhanced biomass accumulation in a dose-dependent manner without affecting growth and photosynthesis
Chemical modulator at sub-optimum concentration could elicit adverse effects in algal cells [9], thus, we attempted to scrutinize the optimum tannic acid concentration for algal cellular physiological properties. As shown in Fig. 1, cellular biomass accumulation was gradually increased with the concentration of tannic acid. Biomass content was found to be slightly decreased under 50 mg/L of tannic acid treatment than that of 10 mg/L. Among the various concentrations assessed (0.5, 1, 10 and 50 mg/L), 10 mg/L of tannic acid was observed to be the optimum concertation to stimulate biomass production in P. tricornutum. Tannic acid notably increased cell growth rate throughout the growth phase.
On the other hand, no significant difference was found in photosynthetic performance as measured by Fv/Fm, ETR, and NPQ between treated and control cells. However, a slight decrease in Fv/Fm value was observed (Fig. 1). We also investigated the distinct photosynthetic pigment profile, including Chla, Chlc, and fucoxanthin, which are the light- harvesting pigments and composition of chlorophyll-protein complexes. They played an important role in light harvesting for photosynthetic light utilization efficiency. Despite the alterations imparted by tannic acid on photosynthesis, the growth was not significantly retarded, which is similar to the previous report that provision of 1 mM melatonin slightly reduced photosynthesis without any adverse impact on cell growth in Monoraphidium sp. [10]. The photosynthesis parameter observed was also in line with the determination of photosynthetic pigments, which showed that chlorophyll a, chlorophyll c and fucoxanthin content was slightly decreased (Fig. 1). This finding is consistent with the previous report that tannic acid did not influence growth and photosynthesis enhancement in Desmodesmus armatus, while exhibit impairment in cyanobacteria Microcystis aeruginosa [11].
The diadinoxanthin cycle is an important approach in photoprotection, including enhanced dissipation of excess light energy, stress signaling and antioxidant activity [12]. The ZEP and VER genes were differentially regulated as a response to tannic acids treatment (Fig. 2), and activation of the de-epoxidation reaction in the diadinoxanthin circles. This process is thought to be respond to the oxidative stress and ROS scavenging. It is demonstrated that diadinoxanthin cycles pathway avoids additional damage in photosynthesis process caused by tannic acid, which do not influence growth consequently.
The hydrolysis capability of tannic acid enhanced lipid accumulation
Thereafter, we assessed the impact of tannic acid on lipid accumulation on day 9 of the cultivation period using qualitative Nile-red fluorometric analysis. As shown in Fig. 1, tannic acid significantly increased the lipid accumulation in a dose-dependent manner, which was similar to its impact on cellular biomass content. The lipid fluorometric data showed it reached its peak at 10 mg/L tannic acid treated cells. With the increasing concentration from 0.5 mg/L to 10 mg/L, the relative lipid content was gradually increased, but at 50 mg/L the lipid content was slightly decreased than 10 mg/L TA treated cells. The tannic acid (10 mg/L) significantly enhanced the lipids by 1.31-fold than that of control cells (Fig. 3). Further to examine whether the tannic acid induced any morphological aberrations, we stained the treated and control cells with lipophilic Nile-red stain and observed the stained cells under the confocal microscope. Tannic acid treatment enhanced lipid droplets in terms of volume and numbers than control cells, whereas no other morphological abnormalities were observed (Fig. 3). These data denoted that tannic acid specifically regulated metabolite production without directly impacting microalgal cellular physiological properties. Aromatic compounds such as polyphenols could be biodegradation by microbial metabolism potentials. Glucose and gallic acid as the major metabolites of tannic acid degradative pathway [13]. We determined the glucose content in the culture. Glucose content was significantly increased in treated cells during the log phase and then decreased on day 7 of cultivation (Fig. 3). Zheng et al. showed that P. tricornutum could consume and utilize glucose [14]. The hydrolysis capability of tannic acid increase glucose produces in the log phase, resulting in uncompromised cellular biomass despite a modest loss in photosynthesis.
Tannic acid enhanced antioxidant potential and regulated synthesis of primary metabolites in a growth-phase dependent manner
Inspired by the unaltered physiological factors and simultaneous increment of biomass and lipid content in response to tannic acid treatment, we further sought to unravel the intrinsic mechanism. Tannic acid has been considered the cheaper and natural compound that exhibits strong antioxidant potential in various systems [15,16]; however, its antioxidant role is yet to be explored in microalgae. We attempted to outline the antioxidant property of tannic acid by estimating the MDA contents and determined the enzymatic activity of SOD, CAT, and GSH-Px. Interestingly, tannic acid significantly decreased MDA content (Fig.4), meanwhile, tannic acid also increased the activity of SOD, CAT and GSH-Px (Fig. 4).
Given the growth-phase dependent lipid enhancement under tannic acid treatment, we determined total protein, carbohydrate and lipid content in the log phase (5th day) and stationary phase (7th day). During the log phase, total carbohydrates content was significantly increased. However, its content decreased and no significant difference was found between control and treated cells on day 7 of cultivation (Fig. 4). On the other hand, no notable change was observed in protein level between the treated and WT cells on day 5 and 7 of cultivation, implying that tannic acid predominantly regulated the lipogenic circuits so that the energy and carbon molecules for amino acid synthesis could be redirected to energy metabolic pathways. Particularly, during the stationary phase, these carbon precursors could be reallocated into lipid biosynthesis rather than carbohydrate metabolism as denoted by the reduced carbohydrates and increased lipids, respectively [2,17].
Tannic acid altered the metabolic properties of P. tricornutum and facilitated PUFA synthesis
The economic and functional characteristics of algal lipids are determined by their lipids and fatty acids composition [6]. We further investigated the lipid classes and fatty acid profile upon tannic acid treatment. Total lipids were fractionated into neutral-, glyco- and phospho-lipids. Tannic acid treatment significantly increased neutral and glycolipids; however, no change was found for phospholipids between treated and control cells (Fig. 5), suggesting that tannic acid enhanced neutral and glycolipids, consistent with the previous finding which reported that quercetin carried out the similar mechanism in C. vulgaris [7]. Tannic acid-mediated lipid alteration in the treated cells exemplify a crucial strategy to concurrently overproduce neutral- and glycol-lipids, which have received huge interest as biofuel feedstock and functional food ingredients, respectively [18]. It is worth noting that the increased glycolipid could alleviate the photosynthetic stress associated with the increased glucose, which might result in the unimpaired overall photosynthetic efficiency of the treated cells [19]. Hyperaccumulation of industrially relevant fatty acids, particularly increasing the long-chain polyunsaturated fatty acids which has been regarded crucial since the previous attempts to overproduce algal PUFAs had little success [20]. Therefore, we determined total fatty acids and also PUFAs including eicosapentaenoic acid (EPA), arachidonic acid (ARA), and docosahexaenoic acid (DHA). GC-MS analysis showed that total saturated- and monounsaturated-fatty acids were decreased, whereas total polyunsaturated fatty acid was increased, with the particular increment of EPA and DHA. Notably, DHA was increased by 2.25-fold (Fig. 5).
Tannic acid precisely orchestrated the lipid and polyunsaturated fatty acid metabolic pathways
In order to uncover the possible molecular mechanisms, we examined the impact of tannic acid on regulating the expression of key genes involved in lipid and PUFA synthesis. We observed that tannic acid provision up-regulated the expression of key genes associated with triacylglyceride (TAG) synthesis such as GPAT1, GPAT2, GPAT3, LPAT1, LPAT3, DGAT2A and DGAT2D (Fig. 6). It is well known that TAG is synthesized by the sequential acylation of glycerol backbone by GPAT, LPAT and DGAT [21], the increased expression of these genes is in line with the increased neutral lipids content in the treated cells. Besides, availability of reducing equivalent NADPH is crucial for lipogenesis as lipids are reduced biomolecules [22]. As shown in Fig. 6, expression of malic enzyme was significantly increased in treated cells. Interestingly, we found that expression levels of D5b, E6b and FAD2 were increased remarkably, which demonstrated the role of tannic acid in upregulating these genes and corroborated the high PUFA content (Fig. 7) [20].
Though there are increasing reports that have identified potential chemical modulators to enhance valuable biochemicals in microalgae, there are very few reports available that uncover the role of natural polyphenol tannic acid in microalgal physiological and biochemical properties. It is reported that tannic acid provision affected growth and photosynthesis in a concentration-dependent manner in D. armatus [11]. Nevertheless, the role of tannic acid in regulating key metabolic circuits remains largely unexplored. Our present report advances our understanding to the lipogenic and physiological role of tannic acid that unprecedentedly upregulates key genes involved in NADPH, TAG and PUFA biogenesis in the industrially suitable microalgae.