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# Optimizing nitrate and tryptone to enhance growth and triacylglycerol accumulation in Phaeodactylum tricornutum

Yufang Pan, Zhaowen Hu, Eric Marechal, Hanhua Hu

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Yufang Pan, Zhaowen Hu, Eric Marechal, Hanhua Hu. Optimizing nitrate and tryptone to enhance growth and triacylglycerol accumulation in Phaeodactylum tricornutum. 2024. hal-04708795

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<footnote>*Corresponding author</footnote>
19 Abstract

20 *Phaeodactylum tricornutum*, a unicellular diatom, is considered a potential feedstock
21 for the production of biofuel and a promising producer for high-value products
22 eicosapentaenoic acid and fucoxanthin. However, a high-efficient cultivating strategy to
23 achieve commercial production of triacylglycerol (TAG) from the diatom is an urgent
24 demand. In this study, we optimized the content and ratio of nitrate and tryptone in the
25 medium to enhance biomass and TAG accumulation simultaneously. Growth with
26 tryptone as the sole nitrogen gave rise to the lowest cell density but the highest TAG
27 content in *P. tricornutum* relative to nitrate, nitrite, ammonium or urea cultures. In 500
28 µM NaNO3 cultures, the growth of *P. tricornutum* increased with the increasing
29 concentration (from 294 to 7056 µM nitrogen) of supplemented tryptone, however
30 supplementation of high tryptone (≥882 µM nitrogen) decreased the neutral lipid
31 content. Elevating nitrogen concentration from 294 to 882 µM via tryptone addition in
32 250 µM nitrate culture increased cell densities from day 6 to 10 and neutral lipid content
33 on day 10. In particular, supplementing 588 µM nitrogen of tryptone in the 250 µM
34 nitrate culture gave rise to the highest neutral lipid content on days 8 and 10 (increased
35 by 109% and 62% relative to 500 µM nitrate-sole) with a comparable growth to that in
36 500 µM nitrate-sole culture from day 2 to 8. In conclusion, we optimized
37 nitrate/tryptone ratio and found that a suitable tryptone addition to a relatively low
38 nitrate culture was favourable to the biomass and TAG accumulation simultaneously in
39 *P. tricornutum*.

40

41 Introduction

42 *Phaeodactylum tricornutum*, a unicellular marine pennate diatom, is a common
43 experimental model system for diatom biology study [1]. It is also considered as a
potential feedstock for the production of biofuel because of its relatively high triacylglycerol (TAG) productivity [2], and its fatty acid composition meets the requirements of international biodiesel standards [3]. *P. tricornutum* has been shown to dominate and outcompete other microalgae species in outdoor mass-culture systems due to its tolerant to high pH, and growth ability under low light [4,5]. In addition, *P. tricornutum* is a promising producer for high-value products such as eicosapentaenoic acid (EPA), chrysolaminarin, and fucoxanthin, and can serve as a suitable microalgal cell factory [6,7].

Nitrogen is the indispensable nutrient in the biosynthesis of chlorophylls and proteins. *P. tricornutum* can utilize not only inorganic nitrogen sources like nitrate, nitrite and ammonium but also organic nitrogen sources like urea, tryptone and most of the amino acids [8–10]. *P. tricornutum* was shown to acclimate to environments with variable nitrogen sources by specific signaling pathways. For instance, when *P. tricornutum* is transferred from a high nitrate and low nitrite medium to a low nitrate and high nitrite one, nitrite serves as a substrate for nitric oxide (NO) production by nitrate reductase; this labile gaseous signal then triggers the expression of nitrite reductase to cope with the new condition [11]. This diatom is a successful photoautotroph but is also capable of mixotrophic growth on organic carbon sources like glucose, acetate, fructose and glycerol [12–16]. Conflicting lines of evidence showed that *P. tricornutum* could not natively consume glucose but can heterotrophically grow with glucose by introducing the human glucose transporter gene *Glut1* [17] or the *Chlorella kessleri* hexose uptake protein (HUP1) gene [18]. Numerous researchers have been devoted to the studies of biomass and high-value product improvement in *P. tricornutum* by medium optimization [9,19–21].
Tryptone provides a complex nutrition (free amino acids, peptides, vitamins and growth factors) and has been used for the cultivation of *P. tricornutum* as early as 1958 [22]. Compared to the conventional nitrogen source, sodium nitrate (NaNO₃), the mixture of tryptone and urea as the organic nitrogen source can induce a higher biomass and fucoxanthin production in *P. tricornutum* [21]. Enriching the f/2 medium with tryptone and yeast extract increases biomass, fucoxanthin production and TAG content up to 3.48-fold, 1.7-fold and 2.13-fold, respectively in *P. tricornutum* [23].

Neutral lipids (mainly TAGs) increase in nitrogen-deprived cultures while the photosynthetic capacity and chlorophyll content decrease in *P. tricornutum* [24], indicating that it is hard to accumulate biomass and TAGs simultaneously. The small (27 Mb) and well-characterized genome, available engineering tools, comprehensive knowledge of lipids biosynthetic pathways and their regulation, robust growth in mass-culture systems, tolerance of low-light levels and high pH suggest that *P. tricornutum* has great potentials of application [6]. Therefore, there is an urgent demand for the development of a high-efficient cultivating strategy to achieve commercial production of TAGs from the diatom. Glycerol and urea are often used as organic carbon and nitrogen sources, respectively, in mixotrophic growth of *P. tricornutum* [13–16], and tryptone containing both organic nitrogen and organic carbon has received little attention. In this study, we optimized the content of NaNO₃ and tryptone in the medium, and the results could provide the basis for the mixotrophic cultivation strategy that significantly increases neutral lipid production without reducing biomass of *P. tricornutum*.

## Materials and Methods

### Strain and Growth Conditions

*P. tricornutum* (CCMP2561), a gift from Chris Bowler, originated from the
93 Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA, USA).
94 For growth experiments in different nitrogen sources, algal cells were cultivated in the
95 250-mL Erlenmeyer flask containing 120 mL artificial seawater enriched with N-free
96 f/2 [25], and 500 µM nitrogen was added to the culture with NaNO₃, NaNO₂, NH₄Cl,
97 urea or tryptone as the sole nitrogen source. The cultures were incubated in a
98 shaker-incubator at 130 rpm with the initial cell density of 2 × 10⁵ cells mL⁻¹ under a
99 continuous light irradiation of 70 µmol photons m⁻² s⁻¹ at 22 °C. To eliminate the effect
100 of intracellular nitrogen sources, nitrogen-starved cells [26] were used as the inoculum.
101 For the medium optimization experiments, nitrate and tryptone of various
102 concentrations were used as the nitrogen source, and algal cells were cultivated in the
103 150-mL Erlenmeyer flask containing 60 mL medium incubated in a shaker-incubator at
104 100 rpm. For the fatty acid analysis, 200 mL cultures were grown in the 500-mL
105 Erlenmeyer flask shaken at 130 rpm or in the glass tubes (36-cm long and 3.5-cm
106 diameter) bubbled with filtered air at 0.4 vvm (air volumes per volume of reactor per
107 minute). For the growth experiment with amino acids, NaNO₃ in f/2 was replaced with
108 an individual out of the 20 amino acids at the nitrogen concentration of 882 µM,
109 cultivation was performed in 50-mL Erlenmeyer flask containing 35 mL medium and
110 shaken manually three times a day. For the growth analysis, 3 biological replication was
111 conducted and cell numbers were counted using a Countstar automated algae counter
112 (Countstar algae, Shanghai, China).

113 **Nitrogen Concentration Measurement**

114 Concentrations of nitrate and nitrite in the cultures were measured according to
115 Collos et al. [27]. In brief, the culture supernatant was collected and the absorbance was
116 measured at 220 nm, and sodium nitrate at a concentration range of 0~882 µM was used
117 as the standard. Ammonium nitrogen concentration was determined by standard
118 Nessler's reagent spectrophotometric method. In details, 2 mL of culture was
119 centrifuged at 10,000 rpm for 10 min, and then 0.5 mL of supernatant was added into a
120 colorimetric tube and diluted to 5 mL with ultrapure water. 0.05 mL potassium sodium
121 tartrate solution (500 g/L) was added to the colorimetric tube and mixed. 0.1 mL of
122 Nessler's reagent was added to the tube and mixed. After 10 min, the absorbance of the
123 solution was measured at a wavelength of 420 nm with ultrapure water as a reference.
124 Concentration of urea was measured at the wavelength of 426 nm after chromogenic
125 reaction of urea with 4-dimethylaminobenzaldehyde [28]. To be specific, a solution (2
126 mL) containing 2% (w/v) of 4-dimethylaminobenzaldehyde and 6% (v/v) hydrochloric
127 acid in absolute ethanol was added to 3 mL of the supernatant. After 20 min, the
128 absorbance of the solution was measured at 426 nm.

129 Relative Quantification of Triacylglycerol by Thin-Layer Chromatography and
130 Nile Red Staining

131 Total lipids were extracted from dry algal biomass using chloroform/methanol (1:1,
132 v/v) [29], and then were developed using one-dimensional thin-layer chromatography
133 (TLC) in hexane:diethyl ether:acetic acid (70:30:1, v/v/v) on silica gel plates 60 F254
134 (Merck KgaA, Darmstadt, Germany). TAG bands on the plates were identified by
135 staining with iodine, and triolein was used as the standard (Sigma-Aldrich). TAG
136 relative abundance was subjected to densitometric scanning using Image J software.
137 Relative abundance of the neutral lipid (mainly TAGs) was estimated by fluorescence
138 spectroscopy (Perkin-Elmer, LS 55) with the fluorescent dye Nile red (Sigma-Aldrich)
139 according to procedures described before [30]. Briefly, equal numbers of cells (6 ×10⁶
140 cells) were harvested and resuspended to 3 mL, and then 30 µL (100 µg/mL) Nile red
staining solution was added at 37°C for 30 min and subsequently excited at 531 nm, and
the fluorescent emission was measured at 572 nm. Background fluorescence for this
filter set was subtracted prior to the addition of Nile red.

144 Fatty Acid and Free Amino Acid Analysis

145
TAGs fractionated from the TLC plate and dry algal cells were esterified directly
146
by heating in 5% sulfuric acid in methanol. Then the fatty acid methyl esters were
147
analyzed by gas chromatography (GC9790II, Fuli instruments, China) as reported before
148
[31].

149
Free amino acids in the sterilized tryptone medium were analyzed using A300 amino
150
acid analyzer (membraPure Bodenheim, Germany) with the ion-exchange
151
chromatographic column (650-0042, membraPure Bodenheim, Germany). After
152
reacting with ninhydrin (20 g/L) for an hour, the resultant derivatives were measured by
153
UV detection at wavelengths of 440 and 570 nm, and the derivative of proline (Pro) was
154
only detected at 440 nm [32].

155

156 Results and Discussion

157 Nitrogen Source Affects Growth and TAG Accumulation

158
*P. tricornutum* can utilize both inorganic (nitrate, nitrite, and ammonium) and
159
organic nitrogen (urea and amino acids) as the sole nitrogen sources [10,33]. Inorganic
160
nitrogen incorporation operates via stepwise reductions, i.e. nitrate (by nitrate reductase)
161
to nitrite (by nitrite reductase) and to ammonium. Ammonium is then the entry point in
162
*P. tricornutum* intracellular carbon metabolism via two competing routes: on the one
163
hand the glutamine synthase (GS)/glutamate synthase (GOGAT) route, and on the other
164
hand the carbamoyl-phosphate route producing arginine (Arg) [11]. In this study, five
165
nitrogen sources were added individually to the medium to obtain a concentration of
166 500 µmol of nitrogen per liter. During the 10-day cultivation period (Fig. 1A),
167 ammonium chloride gave rise to the highest cell density of 1.47 × 10⁷ cells mL⁻¹,
168 consistent with the role of this reduced inorganic nitrogen source as the entry point in
169 intracellular carbon metabolism, and the cell densities were 10% and 6% lower when
170 nitrate and urea was used respectively as the nitrogen source. Although the growth was
171 not different from day 0 to day 6 with nitrate and nitrite as the sole nitrogen source, the
172 cell density decreased by 15% and 21% respectively on days 8 and 10 under nitrite
173 condition relative to nitrate condition. Contreras and Gillard show that the growth seems
174 to be almost the same in *P. tricornutum* grown with 200 µM NH₄Cl or NaNO₃ as the
175 sole nitrogen source before day 14 [10]. According to Fidalgo et al. [33], growth with 4
176 mM nitrogen of NaNO₃, NaNO₂ and urea was not much different before day 8 while the
177 highest cell density of 0.88 × 10⁷ cells mL⁻¹ was achieved on day 11 in nitrate cultures;
178 however, the growth of *P. tricornutum* cultured with 4 mM nitrogen of ammonia ceased
179 on day 4. Collos and Harrison indicate that the mean optimal ammonium concentration
180 was 340 µM and the tolerance to high toxic ammonium level was 3,600 µM in diatoms
181 [34]. In our study, cultivation was performed at 500 µM ammonium with the initial pH
182 8.0, and the highest cell density was observed in cultures with ammonium as the sole
183 nitrogen source, indicating that even if pH values changed, no negative effect on the
184 growth was noticed. Furthermore, pH values only change slightly based on the findings of
185 Collos and Harrison [34] at 500 µM ammonium condition.

186 Supplementing tryptone in the medium containing urea or nitrate enhances the
187 growth of *P. tricornutum* [21,23], while the effect of tryptone as the sole nitrogen on its
188 growth is unknown. We found the growth of *P. tricornutum* was the slowest in medium
189 with tryptone as the sole nitrogen and the lowest cell density of 3.6 × 10⁶ cells mL⁻¹ was
190 observed on day 10. The absorption of nitrate and nitrite was fast in the culture of *P*.
191 *tricornutum*, and they were almost exhausted on day 4, while nitrogen concentration
192 maintained a level of about 100 µM in the culture with urea or ammonium as the sole
193 nitrogen source on day 10. This supports that, although ammonium is the key entry in
194 intracellular carbon metabolism, nitrate and nitrite import were the most efficient. Cell
195 density decrease observed on day 8 and 10 with nitrite instead of nitrate further supports
196 that *P. tricornutum* nitrogen incorporation is best adapted to a natural supply of nitrate.
197 TLC analysis showed a 43%, 38%, 47%, and 52% increase of TAG content in *P.
198 tricornutum* grown with tryptone compared those with nitrate, nitrite, ammonium, and
199 urea on day 10, respectively (Fig. 1B).

200 Optimizing Tryptone Concentration in the Medium with Nitrate

201 Although cultivation with tryptone alone resulted in the highest TAG content, the
cell density was only about 1/4 of that in cultivation with nitrate, indicating that
202 tryptone as a single nitrogen source is not suitable for the cultivation of *P. tricornutum*.
203 To obtain the optimal ratio of inorganic and organic nitrogen sources, different ratios
204 (ranging from 0.07 to 2.33) of NaNO3 and tryptone were used to cultivate the diatom. In
205 500 µM NaNO3 cultures, supplementing more tryptone (from 294 to 7056 µM nitrogen)
206 enhanced the growth of *P. tricornutum*, and cell density on day 10 increased by 8%,
207 15%, 32%, 56%, 78%, 106%, and 107% in 294, 588, 882, 1764, 3528, 5292, and 7056
208 µM nitrogen of tryptone (namely 1/3, 2/3, 1, 2, 4, 6, and 8-fold of 882 µM nitrogen of
209 tryptone respectively) relative to 500 µM NaNO3 alone (Fig. 2A,B). In addition,
210 supplementing 294 and 588 µM nitrogen of tryptone increased the neutral lipid content
211 by 7% and 11%, however supplementation of higher tryptone decreased the neutral lipid
212 content by 32%, 56%, 78%, 106%, and 107% respectively in 882, 1764, 3528, 5292,
213 and 7056 µM nitrogen of tryptone relative to 500 µM nitrate cultures (Fig. 2C,D).
214 Furthermore, the neutral lipid content was still lower in 294, 441 and 588 µM nitrogen
215
216 of tryptone cultures when compared with 500 µM nitrogen of tryptone culture alone
217 (Fig. 2C), which indicated that supplemented nitrogen of tryptone should not beyond
218 882 µM considering the TAG content.

219 Since supplementing 294~588 µM nitrogen of tryptone only slightly enhanced the
220 growth as well as TAG content in *P. tricornutum* (Fig. 2A,C) and further elevating
221 tryptone increased biomass with a huge sacrifice of TAG accumulation, it seemed that a
222 total of about 500 µM nitrogen might be suitable for the 10 days' cultivation. Therefore,
223 further experiments were carried out for the optimization of ratio between nitrate and
224 tryptone with a total of 500 µM nitrogen. Adding 150 µM nitrogen of tryptone to the
225 culture with 350 µM nitrate brought about the growth comparable to that in cultures
226 with 500 µM nitrate (Fig. 3A), while increasing the neutral lipid content by 56%, 17%,
227 32%, and 43% respectively on day 4, 6, 8, and 10 (Fig. 3B). Supplementing 250 µM
228 nitrogen of tryptone to the culture with 250 µM nitrate decreased the cell densities by
229 8%, 8%, and 12% respectively on day 6, 8, and 10 but increased the neutral lipid content
230 by 106%, 55%, 67%, and 44% respectively on day 4, 6, 8, and 10 (Fig. 3B). Although
231 cultivation with 150 µM nitrate and 350 µM nitrogen of tryptone gave rise to the highest
232 neutral lipid content on day 6 and 8, cell density was much lower from day 4 to 10
233 compared with the nitrate culture and the other two supplemented cultures (Fig. 3A,B).

234 Based on the further optimization of the ratio between nitrate and tryptone,
235 supplementing 250 µM nitrogen of tryptone to the culture with 250 µM nitrate could
236 accumulate more neutral lipids without intense inhibition of growth. Thus, 250 µM
237 nitrate was used in the subsequent study. Elevating the concentration of nitrogen of
238 tryptone from 294 to 882 µM in the culture with 250 µM nitrate increased cell densities
239 from 9.6 to 12.2 × 10⁷ cells mL⁻¹ on day 10. To be specific, on day 10 cell density was
240 1%, 2%, 13%, and 28% higher respectively in 294, 441, 588, and 882 µM nitrogen of
241 tryptone-supplemented cultures relative to 250 µM nitrate culture alone (Fig. 3C). In
242 particular, supplementing 588 µM nitrogen of tryptone in the 250 µM nitrate culture
243 even gave rise to the highest neutral lipid content on days 8 and 10 (increased by 109%
244 and 62% relative to 500 µM nitrate) (Fig. 3D) with a comparable growth to that in 500
245 µM nitrate-sole culture from day 2 to 8 (Fig. 3C). Lower nitrate in the medium resulted
246 in higher neutral lipid accumulation in the culture of *P. tricornutum* regardless of
247 tryptone (no more than 882 µM nitrogen) addition (Fig. 3B,D), however
248 supplementation of tryptone could enhance the neutral lipid accumulation at the end of
249 cultivation. The previous study has reported that supplementation of 1 g L⁻¹ tryptone
250 (equivalent to 9 mM nitrogen), much higher than that used in our study, to f/2 medium
251 containing 882 µM nitrate significantly increased cell growth, TAG and fucoxanthin
252 content in *P. tricornutum* [23]. Our present study optimized the ratio of nitrate and
253 tryptone and found that a suitable tryptone addition to a relatively low nitrate culture
254 was favourable to the biomass and TAG accumulation simultaneously.

255 **Fatty Acid Composition at the Optimizing Tryptone Concentration**

256 To investigate whether tryptone supplementation has an impact on fatty acid
257 composition, algal cells were grown for 7 days (aeration cultures) or 10 days (shaking
258 cultures) in 500 µM nitrate-sole, 500 µM nitrogen of tryptone-sole, and 250 µM nitrate
259 supplemented with 250 µM or 588 µM nitrogen of tryptone cultures, and fatty acids
260 were analyzed by GC. Nitrogen sources did not much affect the fatty acid composition
261 and content, and the main fatty acids C14:0, C16:0, C16:1 and EPA accounted for
262 94.4~95.9% and 94.7~96.0% of the total fatty acids respectively in total lipid (Fig. 4A)
263 and TAG (Fig. 4B). Likewise, when the diatom *Nitzschia laevis* was cultured with 7.3
264 mM nitrate supplemented with 4.5~36 mM nitrogen of tryptone, the EPA contents were
265 similar to that of the control (7.3 mM nitrate-sole) [35]. However, cells with aeration
gave rise to about 21~30% higher EPA in total lipid and slightly decreased EPA in TAG
than shaking conditions (Fig. 4A), indicating that the increased EPA was mainly in
polar lipids rather than in TAG. Elevation of CO₂ has been shown to raise EPA content
in *Nannochloropsis* sp. [36]. Maximum EPA production was observed in *P. tricornutum*
when the air was supplemented with 1% CO₂ [37]. Apparently, increased supply of CO₂
is favourable to EPA accumulation.

## 272 Effects of Sole Amino Acid on Growth and TAG Content

Tryptone is a pancreatic digest of casein containing all amino acids found in casein
as well as larger peptide fractions. As a conventional source of nitrogen in medium for
bacteria and fungi, tryptone contributes amino acids, nitrogen and vitamins. Utilizing
different amino acids as nitrogen sources has different effects on growth of *P.*
*tricornutum* [10] and thus TAG content probably. Free amino acid composition in the
autoclaved medium containing 882 µM nitrogen of tryptone was detected by amino acid
analyzer (Fig. 5). The total nitrogen concentration of free amino acids detected was
136.7 µM, and Arg, lysine (Lys), leucine (Leu) and histidine (His) were the four most
abundant free amino acids in tryptone medium with the nitrogen concentration of 42.1,
33.9, 16.5 and 12.1 µM respectively. Contreras and Gillard [10] showed that growth of
*P. tricornutum* with Arg, glutamate (Glu), Glutamine (Gln), Leu, isoleucine (Ile), and
asparagine (Asn) as the sole nitrogen source (200 µM) exhibited comparable growth
with that in nitrate cultures before day 18~19, and then strong negative effects of Asn
were identified after day 19. The high impact on growth obtained with Gln and Glu is
consistent with the early incorporation of inorganic ammonium in carbon metabolism
via the GS/GOGAT route, whereas the impact of Arg relates to the early incorporation
of ammonium via the carbamoyl-phosphate route [11].
To evaluate the impact of amino acids in tryptone on *P. tricornutum*, the diatom was cultured in a medium with all the 20 individual amino acids as the sole nitrogen source (882 µM nitrogen). Except that *P. tricornutum* grew very slowly with Asn, His and Pro and even could not survive with tyrosine (Tyr), its cell density was comparable in aspartate (Asp), alanine (Ala), Glu, valine (Val) and serine (Ser) with that in nitrate, and was lower in Gln, Arg, glycine (Gly), phenylalanine (Phe), Lys, methionine (Met), threonine (Thr), cysteine (Cys) and tryptophan (Trp), but higher in Ile and Leu at lease on day 10 (Fig. 6A). Compared with the nitrate culture, more neutral lipids were detected by Nile red staining when the diatom was cultured with Asp, Gln or Trp as the sole nitrogen source (Fig. 6B), and accordingly these cultures were in a lighter brown color relative to the dark brown in the cultures of Asp, Ala, Glu, Val, Leu, Phe, and Met (Fig. 6C). It has been widely reported that the nitrogen limited cultures accumulate neutral lipids in *P. tricornutum* and the color of cultures was noticeably lighter [38,39], as the chlorophyll *a* and fucoxanthin content decreased under nitrogen limitation [40,41]. Likewise, in this study, we find the higher content of neutral lipids (detected by the Nile red staining fluorescence intensity) in cultures in a lighter brown. Interestingly, it was previously shown that nitrogen incorporation via the combined GS/GOGAT and carbamoyl-phosphate routes not only lead to Gln and Arg, but also to fumarate as a byproduct, feeding TAG production [11]. TLC analysis verified that growth with Lys, Trp, Arg and Gln produced more TAG in *P. tricornutum* (Fig. 6D).

Based on the results of amino acid composition and effects of individual amino acids on *P. tricornutum*, the promotion of growth by supplementing with tryptone to the medium might come from the additional nitrogen source provided by amino acids. However, at the same nitrogen concentration, growth with tryptone was worse than that with nitrate due to insufficient available nitrogen sources in tryptone. For example,
supplementing 882, 588, 441 and 294 µM nitrogen of tryptone to the 250 µM nitrate cultures achieved the available nitrogen of 387, 341, 318 and 296 µM if only the free amino acids in tryptone was considered. This can explain why supplementing 441 and 294 µM nitrogen of tryptone resulted in a final cell density equivalent to that in 250 µM nitrate cultures, but much lower than that in 500 µM nitrate cultures. In addition, as we know, during the photosynthesis, the linear electron flow generates ATP and NADPH at a ratio of about 1.3, whereas the Calvin-Benson-Bassham (CBB) cycle requires the ATP and NADPH ratio of 1.5, resulting in a deficiency of ATP or an excess of NADPH [42-44]. Nitrate reduction to ammonia is accompanied by eight electron transfers resulting in the consumption of NAD(P)H. Therefore, relative to the organic nitrogen, inorganic nitrogen nitrate as nitrogen source could consume the excessive NAD(P)H, and thus enhance the photosynthesis.

327

## 328 Conclusions

Although tryptone can serve as the sole nitrogen source for *P. tricornutum*, it should be of high concentrations because the available nitrogen concentration in the medium is too low. Furthermore, ammonium is the entry point to carbon metabolism, while nitrite and nitrate are incorporated with higher efficiency, nitrate being the more efficient inorganic nitrogen source. In addition, the use of nitrate can consume the excessive NAD(P)H and thus balance the ATP/NADPH ratio to meet the CBB cycle needs. In this study, we optimized nitrate/tryptone ratio and found that a suitable tryptone addition to a relatively low nitrate culture was favourable to the biomass and TAG accumulation simultaneously in *P. tricornutum*. Tryptone is one of the most commonly used organic nitrogen sources in the laboratory, however, its high cost and lower bioavailability limit the industrial-scale application. Organic wastewater contains
components similar to tryptone and the optimization strategy based on this study provides
potential application prospects for wastewater treatment.

342

343 Acknowledgments

This work was supported by the National Key R&D Program of China (grant nos.
2023YFA0914400 and 2023YFA0914402) and National Natural Science Foundation of
China (grant no. 31800302). E.M. and H.H. were supported by a CEA-CAS bilateral
program (grant no. 075GJHZ2022014MI).

348

349 Conflict of Interest

The authors have no financial conflicts of interest to declare.

351

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478

479
480 **Figures Legends**

481 **Fig. 1.** Effect of nitrogen sources on *Phaeodactylum tricornutum*: (A) Growth and
482 nitrogen utilization of algal cells grown in f/2 enriched artificial seawater medium
483 (nitrogen concentration was set at 500 µM with NaNO₃, NaNO₂, NH₄Cl, urea, or
484 tryptone as the sole nitrogen sources); (B) TAG content analyzed by thin-layer
485 chromatography in algal cells grown on day 10. S: 0.02 mg triolein as the standard for
486 triacylglycerol.

487 **Fig. 2.** Optimizing tryptone concentration at 500 µM NaNO₃ for *Phaeodactylum tricornutum*: (A) Growth curves of algal cells in the medium with tryptone of low concentration (≤588 µM nitrogen of tryptone); (B) Growth curves of algal cells in the medium with tryptone of high concentration (≥882 µM nitrogen of tryptone); (C) Accumulation of neutral lipid (fluorescence intensity normalized to cell number) detected by Nile red assay at low concentration tryptone; (D) Accumulation of neutral lipid at high concentration tryptone.

494 **Fig. 3.** Optimizing tryptone concentration at low NaNO₃ for *Phaeodactylum tricornutum*: (A) Growth curves of algal cells in the medium containing 500 µM nitrogen in total; (B) Growth curves of algal cells in the medium containing 250 µM NaNO₃ supplemented with tryptone of different concentrations; (C) Accumulation of neutral lipid (fluorescence intensity normalized to cell number) detected by Nile red assay at 500 µM nitrogen in total; (D) Accumulation of neutral lipid at 250 µM NaNO₃ supplemented with tryptone of different concentrations.

501 **Fig. 4.** Comparative analysis of fatty acid composition of *Phaeodactylum tricornutum* grown in the optimized medium in a shaker-incubator (shaking) or in the glass tubes
502 bubbled with air (air): (A) Fatty acid composition in total lipid (TL); (B) Fatty acid
503 composition in triacylglycerol (TAG).
504
505 **Fig. 5.** Concentration of free amino acids in the sterilized medium supplemented with
506 882 µM nitrogen of tryptone.

507 **Fig. 6.** Effects of single amino acids as the sole nitrogen sources on *Phaeodactylum*
508 *tricornutum*: (A) Cell density of algal cells grown in f/2 (nitrogen concentration was set
509 at 882 µM with different amino acids) enriched artificial seawater medium on days 8
510 and 10; (B) Neutral lipid detected by Nile red assay on days 8 and 10; (C) Culture flasks
511 at the end of the 10th day of growth; (D) TAG content analyzed by thin-layer
512 chromatography in algal cells grown on day 10. S: 0.02 mg triolein as the standard for
513 triacylglycerol.
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /><figcaption>Fig. 1.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 2.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 3.</figcaption></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Fig. 4.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Fig. 5.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>Fig. 6.</figcaption></figure>