Cell density, biomass production and chlorophyll a content of P. tricornutum cultivated using DMW
The difference in P. tricornutum harvested biomass and chlorophyll a content observed across conditions is attributed to variation in the nitrogen content of the diluted DMW and synthetic media sources used throughout the study. The 10%, 30%, and 60% DMW dilutions contain approximately -49%, 35%, and 244% of the nitrogen present in the synthetic medium, respectively (Fig. 1c). A positive relationship between nitrogen content, chlorophyll content, and growth has been observed across all major evolutionary lineages of microalgae including P. tricornutum and other diatom species (Qiao et al. 2016; Yodsuwan et al. 2017). Nitrogen limitation reduces the cellular chlorophyll content in P. tricornutum (Valenzuela et al. 2013) and is considered part of a nitrogen-recycling mechanism that repurposes the nitrogen contained within the chlorophyll porphyrin rings and chlorophyll binding proteins for other cellular processes (Hörtensteiner and Kräutler 2011). Discrepancies observed between cell density, chlorophyll a content, and biomass measurements (Fig. 1a–c) in the 30% and 60% DMW conditions compared to the F2 control may be in part due to the complex composition of the dairy wastewater medium source. The increase in biomass and chlorophyll a content observed following cultivation on 30% and 60% DMW dilutions is consistent with the higher starting nitrogen content in these conditions compared to the F2 control, yet no difference in the final cell density was observed at the end of the culture period. Microscopic observation of cultures throughout the culture period (Fig. 1d) rules out microbial contamination but revealed the presence of cellular debris in the 30% and 60% DMW conditions compared to the F2 and 10% DMW conditions. Therefore, these results present the possibility that an unidentified component of the wastewater may promote cell degradation in the higher DMW concentrations. These results informed our design of the proteomic experiments to include a “nitrogen equivalent” dilution of DMW compared to the F2 media (20%), as well as a “nitrogen deficient” dilution (10%).
Neutral lipid content of P. tricornutum grown on DMW dilutions
Differences in the neutral lipid content of P. tricornutum grown on 10%, 30%, and 60% were observed when compared to cultures grown on F2 medium using two Nile Red staining methods (Fig. 2a,b). The first Nile Red method (Chen et al. 2009; Franz et al. 2013) measured the relative intracellular lipid content throughout the 15-day culture period and revealed an increase in the relative intracellular lipid content of 10% DMW compared to all other conditions by day 10 (Fig. 2a). The neutral lipid content of P. tricornutum cultured in the 60% DMW condition remained relatively unchanged over time, whereas the neutral lipid content gradually increased throughout the culture period in the F2, 10%, and 30% DMW conditions. An increase in the neutral lipid content was observed in the 10% DMW condition (0.36 mg neutral lipids/mg biomass (mg mg-1)) when compared to synthetic medium (F2) control (0.15 mg mg-1), 30% DMW (0.10 mg mg-1), and 60% DMW (0.034 mg mg-1) (Fig. 2b). The increase in neutral lipid content following cultivation on the 10% DMW condition is most likely a response to nitrogen depletion conditions (Burch and Franz 2016; Yang et al. 2014; Yodsuwan et al. 2017) and is supported by the differences in growth, biomass, chlorophyll a content, and initial nitrogen concentrations detected between the conditions tested (Fig. 1a–c, Fig. 2b).
Fatty acid profile of P. tricornutum grown on DMW dilutions
Cultivation of P. tricornutum using more DMW generally decreased the levels of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) while increasing docosapentaenoic acid (DPA, C22:5 n-6) relative to cultivation in synthetic medium (Table 2, Supplementary Table 1). Consistent with previous reports for P. tricornutum and other diatoms (Siron et al. 1989; Yang et al. 2017; Zhao et al. 2014), fatty acid analysis using GC-MS revealed that palmitic acid (16:0), palmitoleic acid (16:1), and eicosapentaenoic acid (EPA, 20:5 n-3) were the most abundant species detected when using 10% and 30% DMW for cultivation. In addition, the relative abundance of DPA to DHA is consistent with previous reports (Yang et al. 2017). Cultivation using 30% and 60% DMW shifted the fatty acid profile where an increase in DPA content was especially notable compared to F2 and 10% DMW conditions.
This shift to produce more DPA using DMW correlates with the higher initial nitrogen content when more DMW is utilized for cultivation, but the combined data (Supplementary Table 1) suggest that nitrogen content is not the only factor contributing to this effect. Phaeodactylum tricornutum and other diatoms are known to increase SFA and MUFA levels in response to nutrient limitation (Liang et al. 2006; Liang and Mai 2005), and the modification of the initial nitrogen concentration in media has been shown to influence fatty acid composition in P. tricornutum (Qiao et al. 2016). The accumulation of DPA may also be a physiological response to stressors present in the DMW that enhances the resilience in P. tricornutum. As membrane fluidity increases with chain length and degree of unsaturation (Harwood 1988; Jiang and Gao 2004), DPA may contribute to the resilience of P. tricornutum through the relief of turgor pressure, protein recruitment, and nutrient uptake (for review, see Juneja et al. (2013) and Sayanova et al. (2017)). Given that DPA is a commercially valuable omega-3 fatty acid with well-documented human health benefits (Byelashov et al. 2015), increased DPA production using a cheap and abundant alternative nutrient source may have important commercial applications.
Overview of proteomic data comparing P. tricornutum growth on DMW dilutions to growth on synthetic medium
Biochemical and physiological analysis of P. tricornutum cultured in DMW revealed similarities to results observed following nitrogen limitation in this species, with increased lipid contents associated with lower environmental nitrogen concentrations. Therefore, proteomic analysis was performed on P. tricornutum cultures grown on two DMW concentrations that were either nitrogen-limited or nitrogen-equivalent when compared to the nitrogen content of synthetic medium. P. tricornutum was grown on either 10% DMW (nitrogen-limited), 20% DMW (nitrogen-equivalent), or synthetic medium (F2) and sampled for proteomic analysis after 5 days of cultivation (see the “Overview of proteomic data comparing P. tricornutum growth on DMW dilutions to growth on synthetic medium” section and Fig. 3a,b).
Nitrogen metabolism
Proteins involved in nitrogen metabolism were upregulated in the nitrogen-limited 10% DMW condition but not with nitrogen-equivalent 20% DMW compared to F2 (Fig. 4a). P. tricornutum responded to the low environmental nitrogen levels in 10% DMW by increasing its nitrogen uptake and assimilation abilities, as evidenced by the upregulation of a urea transporter (B7FZW5, 10%/F2 + 1.54), nitrate reductase (B7G997, 10%/F2 + 1.35) and glutamate dehydrogenase (B7G3X3, 10%/F2 + 1.30). Upregulation of a xanthine/uracil permease (B7FP05, 10%/F2 + 4.07), purine permease (B7GBV6, 10%/F2 + 2.04), and xanthine dehydrogenase (B7GAV1, 10%/F2 + 1.28) may also provide P. tricornutum with an additional source of nitrogen containing compounds during nitrogen limitation. Cultivation on 10% DMW also led to upregulation of proteins whose functions include the release of ammonia from various macromolecules, including a formidase (B7FYS6, 10%/F2 + 1.89), a putative amidase (B7GE02 + 4.15), two putative cytidine deaminases (B7GCB8, 10%/F2 + 2.08, B7FYE0, 10%/F2 + 2.45), and a protein with hydrolase activity (B7G1X3, 10%/F2 + 2.22) involved in the NAD salvage pathway that catalyzes the release ammonia from nicotinamide. Collectively, upregulation of these proteins supports the conclusion of Longworth and colleagues that P. tricornutum cells remodel their proteome to internally scavenge and recycle nitrogen sources during nitrogen-limited conditions (Longworth et al. 2016).
Iron response
When compared to the F2 control, P. tricornutum grown on either 10% and 20% DMW conditions led to upregulation of multiple proteins associated with an iron limitation response (Fig. 4b) (Allen et al. 2008; Lommer et al. 2012; Morrissey et al. 2015; Morrissey and Bowler 2012). The upregulation of proteins associated with iron limitation suggested that the DMW study was lacking in iron. Subsequent analytical testing of the soluble iron content confirmed the iron-limited nature of the DMW, with the 20% and 10% DMW conditions containing only 3.4% (0.014 mg L-1) and 1.7% (0.007 mg L-1) of the iron found in the F2 medium (0.414 mg L-1), respectively. Diatoms as a group have historically been utilized in environmental monitoring studies (Dixit and Smol 1994) and can survive during periods of low environmental iron availability, with P. tricornutum being particularly tolerant to conditions of low environmental iron (Kustka et al. 2007). The response to low iron levels was especially dramatic in the 20% DMW condition, with four of the top five most upregulated proteins (9–16-fold) identified matching proteins previously linked to an iron starvation response (Allen et al. 2008). These highly induced proteins include iron starvation-induced protein ISIP2A (B7FYL2, 20%/F2 + 4.74), ISIP1 (B7GA90, 20%/F2 + 7.93), ISIP3 (B7G4H8, 20%/F2 + 6.25), and flavodoxin (B7GCM3, 20%/F2 + 6.52). ISIP2A is a cell surface protein whose function in the concentration and uptake of Fe(III) was recently characterized (Morrissey et al. 2015), whereas ISIP1 and ISIP3 are cell surface proteins whose functions during iron limitation remain to be determined. Flavodoxin is an electron-transfer protein that substitutes for ferredoxin in the photosynthetic electron transport during periods of iron limitation.
The expression patterns of iron-responsive proteins in P. tricornutum cultured on 10% DMW conditions suggests that the proteomic response is less robust in 10% DMW compared to 20% DMW condition. Expression of multiple iron response proteins were significantly higher in the 20% DMW condition (Fig. 4b), and the muted iron limitation response observed in the 10% DMW dataset may be in part due to P. tricornutum reacting to the additional stress of low environmental nitrogen that was not present in the 20% DMW condition. Other data from this study support this hypothesis, as P. tricornutum grown in 10% DMW showed a robust response to nitrogen limitation and most likely diverted substantial amounts of energy and resources to synthesize proteins necessary for survival in this environment. These differences are attributed in part to temporal aspects of the iron response and are the result of when sampling occurred for proteomic analysis.
Amino acid metabolism
Differences in the abundance of several proteins involved with amino acid metabolism were observed in P. tricornutum cultures for both 10% and 20% DMW, but meaningful trends in the data were not observed. The upregulation of a vitamin B12-independent methionine synthase (B7G1X4, 10%/F2 + 3.38, 20%/F2 + 2.89) was observed in both 10% DMW and 20% DMW conditions, and this protein is upregulated in cobalamin-limited environments (Bertrand et al. 2012). Additional downregulation of a vitamin B12-dependent methionine synthase (B7GBG7, 10%/F2 -1.21) was observed in the 10% DMW condition. Taken together, differential regulation of these proteins indicates that this DMW source may also be limiting in other micronutrients besides iron.
Photosynthesis
Photosynthesis-related proteins were generally downregulated when comparing the nitrogen-deplete 10% DMW condition to both the nitrogen-equivalent 20% DMW and F2 conditions (Fig. 4c, Supplementary Fig. 1). The most prominent downregulation occurred in pathways controlling biosynthesis of photosynthetic pigments and their precursors. Downregulations of glutamyl-tRNA synthase (B7GCT6, 10%/F2 -1.69, 20%/10% -2.02) and hemL (B7G134, 10%/F2 -1.20) indicate a reduction in the biosynthesis of tetrapyrroles, compounds with important functions as cofactors in chlorophyll pigments, as well as heme-containing proteins. Decreased expressions of proteins hemE (B7GB77, 10%/F2 -1.74), PPO (B7GDU9, 10%/F2 -2.00, 10%/20% -1.67), CHLD (B7FTA2, 10%/F2 -1.75, 10%/20% -1.43), and DVR (B7GC47, 10%/F2 -1.75) indicate that chlorophyll biosynthesis was also downregulated. Biosynthesis of carotenoids was also downregulated, as evidenced by the downregulation of proteins involved in biosynthesis of isoprenoids (HDS, B7FV10 10%/F2 -1.26; GGPS, B7FU89, 10%/F2 -1.74, 10%/20%, -1.59) and carotenoids (PDS1, B7FZL9, 10%/F2 -1.48; ZDS, B7FPC4, 10%/F2 -1.18; CRTISO5, B7FQF7, 10%/20% -1.67). No evidence of downregulation of chlorophyll and carotenoid biosynthesis was observed when comparing the 20% DMW condition to the F2 control.
Multiple light-harvesting complex (LHCs) fucoxanthin-chlorophyll a/c binding proteins (FCPs) were observed upregulated in the 10% and 20% DMW conditions compared to the F2 control (Fig. 4c). Unlike what has been observed in other proteomic experiments investigating nitrogen limitation in P. tricornutum, cultivation in 10% DMW culture did not lead to a general decrease in the expression of chlorophyll binding light-harvesting protein, and the pattern of regulation in these proteins resembles the 20% DMW condition. Iron deficiency has been shown to cause a rearrangement of the photosynthetic apparatus in diatoms (Allen et al. 2008) and may influence light-harvesting proteins in an effort to increase photoprotection mechanisms. Indeed, LHCX2 (B7FR60, 20%/F2 + 2.85, 10%/F2 + 2.51), an important antenna protein involved in nonphotochemical quenching (NPQ) in P. tricornutum (Lepetit et al. 2017; Nymark et al. 2009), was upregulated in both 10% and 20% DMW conditions. Flavodoxin (B7GCM3, 20%/F2 + 6.52, 10%/F2 + 6.70), as described above, was upregulated in both 10% and 20% DMW conditions when compared to the F2 control and was the only differentially regulated protein specific to the photosynthetic apparatus.
Rubisco activase (A0T0M5, 10%/F2 -1.14) and the carbonic anhydrase isoform CA4 (B7FNU0, 10%/F2 -1.92) were downregulated in the 10% DMW condition, suggesting the decreased role of photosynthetic carbon fixation during periods of nitrogen limitation. A candidate bicarbonate transporter part of the SLC4 family (B7S437, 20%/F2 -2.65, 10%/F2 -2.47) was also downregulated in both 10% and 20% DMW conditions. Finally, FBA5C (B7GE67, 20%/F2 + 4.66, 10%/F2 + 3.12), a plastidic fructose bisphosphate aldolase isoform, was upregulated in both 10% and 20% DMW conditions. It has been hypothesized that during times of low carbon assimilation, a condition observed during iron limitation in P. tricornutum (Allen et al. 2008), the reaction FBAC5 catalyzes may run in the reverse direction, consequently supplying carbon skeletons for anabolic reactions (Allen et al. 2012).
Protein metabolism
After 5 days of cultivation on 10% and 20% DMW, differential regulation of multiple proteins involved in protein synthesis and degradation was observed (Fig. 5). Compared to the F2 control, P. tricornutum cultured on 10% DMW led to downregulation of 19 ribosomal structural proteins, indicating an overall decrease in protein synthesis. Four proteins with annotated proteolytic functions were upregulated (all 10%/F2: B7GDQ0, + 1.60; B7FSD0, + 3.25; B7FUY6, + 1.25; B7G5X7, + 2.36), while one protease (B5Y4I2, 10%/F2 -1.89) and a ubiquitin extension protein (B7G5H7, 10%/F2 -2.26) were downregulated. P. tricornutum grown in 20% DMW also exhibited mixed regulation of proteins involved in protein degradation with two proteases upregulated (all 20%/F2: B7FSD0, + 3.07; B7FVG8, + 1.73) and one protease downregulated (B5Y4I2, 20%/F2 -2.85). However, in contrast to the 10% DMW condition, the downregulation of ribosomal structural proteins was not observed, and five ribosomal structural proteins (all 20%/F2: A0T0J5, + 1.45; B7FP95, + 2.85; A0T0C7, + 1.80; A0T0I4, + 1.91; B7FQC3, + 2.22) were upregulated when compared to the F2 control.
Proteins involved in regulating protein synthesis, including those involved in translation initiation (B7G3T4, 20%/F2 + 1.58, 10%/F2 + 1.56; B7FV44, 20%/F2 2.23, 10%/F2 2.91) and elongation (B7G3C4, 20%/F2 + 1.53, 10%/F2 + 1.44), were upregulated in both 10% and 20% DMW conditions, with an additional protein with functions in translation initiation (B7G5G1, 10%/F2 + 1.78) upregulated in the 10% DMW condition only. Taken together, this regulation pattern supports the conclusion that P. tricornutum grown in 10% DMW decreases protein synthesis while simultaneously enhancing targeted protein degradation. This response may function as part of a mechanism that reduces protein synthesis as a general sink for nitrogen while also increasing nitrogen availability for the biosynthesis of specific proteins during periods of low environmental nitrogen availability. Organisms exert a tight control on protein synthesis and degradation in response to fluctuating environmental conditions, and the mixed regulation of proteases combined with the upregulation of ribosomal structural proteins and of proteins with translation initiation and elongation factor activity suggest the importance of controlling protein synthesis during cultivation on this DMW source.
Central carbon metabolism and oxidative phosphorylation
A broad upregulation of proteins involved in central carbon metabolism and oxidative phosphorylation were observed in P. tricornutum cultivated on 10% DMW, including enzymes associated with glycolysis (B7GC94, 10%/F2 + 1.27; B7G8B7, 10%/F2 + 1.73), the TCA cycle (all 10%/F2: B7FZD7, + 1.39; B7G228, + 1.27; B7FWX5, + 2.35), and oxidative phosphorylation (all 10%/F2 B7GBT9, + 2.14; B7FXG3, + 1.19; B7G964, + 2.11; B7G360, + 1.61) (Fig. 4d). Upregulation of dihydroorotate dehydrogenase (B7G8S8, + 1.91) during cultivation on 10% DMW was also observed, which functions in pyrimidine biosynthesis and energy generation at the mitochondrial membrane. The additional upregulation of two putative mitochondrial phosphate transporters observed following cultivation on 10% DMW (B7G6E4, 10%/F2 + 1.27) or 10% and 20% DMW conditions (B7G5H6, 20%/F2 + 1.86, 10%/F2 + 1.67), respectively, and may function to supply inorganic phosphate to the mitochondria for ATP biosynthesis.
Interestingly, cultivation on 10% and 20% DMW dilutions resulted in an upregulation of two short chain dehydrogenase/reductases (SDR) (B7G0Q2, 20%/F2 + 1.99, 10%/F2 + 1.54; B7GBR2, 20%/F2 + 2.11, 10%/F2 + 2.61) and an aldo-keto reductase (AKR) (B7GDN9, 20%/F2 + 1.78, 10%/F2 + 1.84) when compared to the F2 control. Although SDR and AKR protein families are very diverse in their structure and functions, both families contain NAD+/NADP+ binding domains and can participate in oxidoreductase reactions that reduce aldehydes and ketones to primary and secondary alcohols, respectively (Penning 2015). The downregulation of glyoxalase II (B7GDI1, 20%/F2 -2.20) in the 20% DMW condition could mean that the observed upregulation of AKR and SDR proteins with unknown specific functions may be involved in the detoxification of methylglyoxal (Grant et al. 2003; Yamauchi et al. 2011), a toxic byproduct of carbon metabolism. SDR and AKR protein families also have known roles in redox sensing (Kavanagh et al. 2008) and stress tolerance (Kanayama et al. 2014), and three additional alcohol dehydrogenases (all 10%/F2: B7GEB0, + 1.44; B7GEA6, + 1.44; B7S4B2, + 1.35) of unknown specificity that were upregulated following 10% DMW cultivation may have been upregulated in response to low levels of environmental nitrogen.
Redox/stress
Analysis of the proteome suggests that P. tricornutum responds to DMW conditions by increasing the abilities to regulate oxidative stress levels and maintain redox homeostasis (Fig. 4e). The increased expression of glutathione reductase (GSR_1, B7G9Q1, 10%/F2 + 2.01) in 10% DMW and glutathione synthase (B5Y5J7, 20%/F2 + 1.78) in 20% DMW suggests that P. tricornutum attempts to increase or replenish reduced glutathione (GSH) levels in response to DMW conditions. Maintaining high levels of reduced glutathione pools have been shown to increase abiotic stress tolerance in Arabidopsis (Cheng et al. 2015) and may enhance the ROS detoxification ability of P. tricornutum. Thioredoxin h (B7G7L6, 20%/F2 + 2.10, 10%/F2 + 2.18) was upregulated in both 10% and 20% DMW conditions. H-type thioredoxins have known functions in oxidative stress management (Serrato and Cejudo 2003) and may be involved in the redox regulation of glutathione reductases as seen in Triticum aestivum (Gelhaye et al. 2003). B7G7L6 shares a 40.4% identity with Trxh1 in Oryza sativa, which is induced during abiotic stress, including an elucidated function in managing reactive oxygen species balance in the rice apoplast during salt stress (Zhang and Guo 2012). Increased expression of a Mpv17/PMP-like protein (B7G8P1, 20%/F2 + 1.46) targeting to the peroxisomal membrane provides further evidence of ROS management in the 20% DMW condition. Nitrogen starvation produces oxidative microenvironments in P. tricornutum (Rosenwasser et al. 2014), and additional upregulation of a peroxiredoxin (B7GCV6, 10%/F2 + 3.43), an NADP+ transhydrogenase (B5Y5A6, 10%/F2 + 1.79), and putative glutathione-s-transferase (B5Y512, 10%/F2 + 1.44) in the 10% DMW condition may be required to combat oxidative stress during nitrogen limitation.
Cellular signaling and gene expression
P. tricornutum grown on both 10% and 20% DMW for 5 days exhibited alterations in expression profiles for multiple proteins involved in cellular signaling (Fig. 4f). Compared to the F2 control, hCDK3 (B7FUX2, 10%/F2 + 145) was upregulated in 10% DMW, and CDKA2 (B7GDW6, 20%/F2 -1.41) was downregulated in 20% DMW, suggesting altered regulation of the cell cycle during cultivation on DMW. The upregulations of two calcium-dependent kinases (B7GBS5, 20%/F2 + 1.66, 10%/F2 + 2.86; B7G8H4, 20%/F2 + 2.82, 10%/F2 + 3.33) in both 10% and 20% DMW, along with the upregulation of phospholipase C (B7S3T2, 10%/F2 + 1.78) and copine 1 (B7FSV2, 10%/F2 + 1.30) in 10% DMW, suggest that calcium signaling plays an important role in acclimatizing to culture on this DMW source. Culture on both DMW conditions also downregulated a P-type ATPase (B7GCD8 20%/F2 -1.84, 10%/F2 -1.54) identified as a calcium ion carrier, providing further evidence of modulated calcium signaling pathways. It must be noted, however, that P. tricornutum utilizes calcium signaling as part of its iron limitation response (Allen et al. 2008; Falciatore et al. 2000), and given the iron-limited composition of the DMW used in this study, further studies must be performed to confirm and elucidate the importance of calcium-based signaling during acclimatization to culture on DMW sources.
Three proteins with roles that function as molecular chaperones were downregulated in the 20% DMW condition, including a DnaJ (hsp40) homolog (B7G4P7, 20%/F2 -4.17), an hsp90 homolog (B7GES3, 20%/F2 -4.52), and the molecular chaperone cdc37 (B7S3L1, 20%/F2 -1.60). These proteins are components within a multi-protein complex that function to chaperone various targets, including protein kinases (Arlander et al. 2006). The functional consequence of their downregulation in the 20% DMW condition is unknown but signify further evidence of alterations in signal transduction.
Phaeodactylum tricornutum grown on DMW exhibited differences in proteins involved in transcriptional regulation when compared to culture on synthetic medium. Two transcription factors, one identified as containing an HMG box domain (B7FQD6, 20%/F2, -4.25) and another identified Myb1R2 (B7G7Q5, 20%/F2 -2.32) (Rayko et al. 2010), were downregulated in 20% DMW. An additional protein identified as splicing factor 3B subunit 3 (B7FP73, 20%/F2 -1.97), which functions in pre-mRNA splicing, was also downregulated in the 20% DMW. The CO2 responsive bZIP11 (B7G9T9, 10%/F2 + 1.34) transcription factor was upregulated in 10% DMW, while CSF4 (B7G5B5, 20%/F2 + 2.62, 10%/F2 + 2.77), a DNA-binding protein containing a cold shock domain, was upregulated in both 10% and 20% DMW. Although this study cannot clearly implicate the functional consequences that result from altering transcriptional regulation and cellular signaling pathways, it may contribute to the proteomic re-organization observed in P. tricornutum in response to DMW cultivation.
Membrane transport and trafficking
Proteomic analysis after 5 days of growth on DMW suggests that P. tricornutum responds to DMW conditions by controlling small molecule and protein transport throughout various cellular compartments. Upregulation of small GTPases belonging to the Ras superfamily of proteins were observed in both 10% and 20% conditions, including proteins identified as RabX1 (B7S3S7, 20%/F2 + 1.92, 10%/F2 + 2.29) and Arf1 (B7FR50, 20%/F2 + 3.97, 10%/F2 + 4.32), with the Rab GTPase Sec4 (B7G9L9, 10%/F2 + 1.79) observed upregulated in the 10% DMW condition only (Supplementary Fig. 2). Both RabX1, which shares a 78.9% sequence identify with the Ras-related protein Rab-8A in Fragilariopsis cylindrus, and Arf1 are involved in endocytosis. Additional support of increased endocytic pathways was observed in the 10% DMW condition, where upregulation of adaptor protein complex components AP1/2beta (B7S4C6, 10%/F2 + 1.23) and AP2M1 (B7FSB1, 10%/F2 + 1.48) was observed (Supplementary Fig. 3). The increased expression of a V-type H+ transporting ATPase (B7G360, 10%/F2 + 1.61) in 10% DMW suggests an increased role for phagocytosis in this condition and, taken together, could provide P. tricornutum grown on the DMW an additional scavenging mechanism to acquire limiting nutrients such as iron and nitrogen from the outside environment.
Protein trafficking was generally upregulated during DMW culture. The nuclear transport factor NFT2 (B7G8V7, 20%/F2 + 1.57, 10%/F2 + 2.21) was upregulated in both conditions, and the Ran-GTPase importin-7 (B7FVE6, 10%/F2 + 1.21) was upregulated in 10% DMW. Nuclear export by exportin-1 (B7GEG3, 20%/F2 -1.68) was downregulated in 20% DMW. The alpha subunit of sec61 (B7GD39, 20%/F2 + 1.79, 10%/F2 + 1.35), sec31 (B7G255, 20%/F2 + 1.63, 10%/F2 + 2.20), and a protein identified as part of the SRP-independent protein family (B7FUG7, 20%/F2 + 1.44, 10%/F2 + 1.57) were upregulated in 10% and 20% DMW conditions, suggesting an increase in protein translocation through the endoplasmic reticulum (Supplementary Fig. 2), and provide support for the importance of controlling protein synthesis during DMW culturing.
Cultivation on DMW increased the expression of a MFS superfamily transport protein (B7FTZ4, 20%/F2 + 1.46, 10%/F2 + 1.60) that putatively functions in the transport of molybdate ions across membranes. In its biologically active form, molybdenum acts as a cofactor in specific enzymes, including purine catabolism such as xanthine dehydrogenase (B7GAV1, 10%/F2 + 1.28) and nitrogen assimilation nitrate reductase (B7G997, 10%/F2 + 1.35), both of which were upregulated in the 10% DMW condition. Two additional putative phosphate transporters (B7GBF0, 20%/F2 -3.90 and B7G4H1, 20%/F2 -2.48) were also downregulated in 20% DMW.
Lipid metabolism
Phaeodactylum tricornutum cultivated on either 10% or 20% DMW upregulated an acyl carrier protein (ACP) (B7FRX6, 20%/F2 + 2.50, 10%/F2 + 2.46) and downregulated a stearoyl-ACP desaturase (B7FQK1, 20%/F2 -2.01, 10%/F2 -1.92). ACPs perform important functions as cofactors in fatty acid biosynthesis, and overexpressions of specific ACP isoforms have been demonstrated to alter FA composition (Branen et al. 2001) and modulate salt stress tolerance in Arabidopsis (Huang et al. 2017). Stearoyl-ACP desaturase requires iron as a cofactor, and iron limitation in this medium source may explain the observed downregulation. A decrease in FA desaturase is expected to alter the fatty acid profile towards increased saturation, and in C. reinhardtii, iron starvation downregulates expression of multiple FA desaturase genes, increasing the saturated to unsaturated FA ratio while altering the FA composition of membrane lipids (Urzica et al. 2013). Interestingly, a recent study where proteomic analysis was performed on of P. tricornutum grown on iron-limited or iron-starved F2 medium did not cause downregulation of FA desaturases (Zhao et al. 2018), highlighting microalgae species-specific differences in responding to iron limitation. Indeed, the FA composition of P. tricornutum cultured on DMW did not increase FA saturation when compared to culture on F2, and FA unsaturation actually increased in all DMW dilution used besides the nitrogen-limited 10% DMW condition (Table 2).