The interaction between nitrogen source and light intensity affects the biomass and phenotypic plasticity of Scenedesmus obliquus

The interaction between nitrogen source and light intensity affects the biomass and phenotypic plasticity of Scenedesmus obliquus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The interaction between nitrogen source and light intensity affects the biomass and phenotypic plasticity of Scenedesmus obliquus Jiyan Long, Yiqi Feng, Decai Huang, Yulu Lei, Xuexia Zhu, Zhou Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6808862/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2025 Read the published version in Biotechnology Letters → Version 1 posted 5 You are reading this latest preprint version Abstract As critical environmental factors, nitrogen and light not only regulate phytoplankton growth but also influence their phenotypic plasticity. Scenedesmus obliquus , an alga which is famous for its remarkable phenotypic plasticity, was studied to understand its response to varying combinations of nitrogen source and light intensity. It was cultured in media containing different nitrogen sources (NaNO 3 , NH 4 Cl, CO(NH 2 ) 2 ) under a range of light intensities (25, 50, 75, 100, 150 µmol photons m − 2 s − 1 ). Results showed that growth rates increased with higher light intensities across all nitrogen sources. Photosynthetic efficiency ( Fv/Fm and Φ PSII ) remained stable in NaNO 3 treatments, but declined with rising light intensity in NH 4 Cl and CO(NH 2 ) 2 treatments. The highest proportions of multicellular colonies were observed at 150 µmol photons m − 2 s − 1 for NH 4 Cl and NaNO 3 treatments, while colonies in CO(NH 2 ) 2 treatments peaked at 100 µmol photons m − 2 s − 1 , with colony size stabilized at approximately 2.1, 4.0, and 1.0 cells per particle under NaNO 3 , NH 4 Cl, and CO(NH 2 ) 2 treatments, respectively. Nitrogen removal efficiency improved with increasing light intensity across all treatments, though S. obliquus exhibited varying capacities to remove nitrogen depending on the sources. These findings demonstrated how S. obliquus adapts its growth, photosynthesis, and morphology to varying nitrogen sources and light intensities, and providing insights into its ecological versatility. This study provided a theoretical foundation for optimizing culture conditions in applications such as wastewater treatment and bioenergy production. Colony formation Light intensity Nitrogen resource Phenotypic plasticity Photosynthetic efficiency Scenedesmus obliquus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Microalgae are integral to aquatic ecosystems, serving as primary producers that underpin the structure and functioning of food webs and biogeochemical cycles. In particular, nitrogen is a key elemental constituent of many cellular macromolecules and also a crucial nutrient for organisms (Zhang et al. 2018 ). In natural waters, nitrogen is a critical factor influencing algal population dynamics, community compositions, and ecosystem functions. Microalgae can utilize various forms of nitrogen sources, including inorganic (e.g. nitrate, ammonia) and organic nitrogen (e.g. urea, nucleic acids, amino acids) (Zhang et al. 2023 ). Among them, nitrate and ammonium are primary nitrogen sources for most phytoplankton. Nitrate is initially reduced to nitrite by nitrate reductase (NR) in cytoplasm, and then nitrite is converted to ammonia by nitrite reductase in chloroplast (Hou et al. 2017 ). The energy cost of ammonia assimilation is lower than that of nitrate, leading to preferential use of ammonium in many autotrophic species, but nitrate is the most favored nitrogen source because it is more abundant in natural environment (Bloom et al. 1992 ). Different nitrogen sources can lead to distinct physiological and metabolic responses in microalgae, affecting their suitability for various applications. In the case of Scenedesmus bijugatus , nitrate performs better for biomass growth, and urea results in almost equal biomass as nitrate (Arumugam et al. 2013 ). Ammonium can be toxic to cells at elevated concentrations and can cause growth inhibition or even cell death, despite mixotrophic cultivation with ammonium could greatly promotes the growth, biomass accumulation, total lipid yield, and notably the triacylglycerol content in microalgae (Sittisaree et al. 2024 ). Therefore, careful selection of the nitrogen source is crucial for optimizing microalgal cultures to meet specific research or industrial objectives. Light is the dominant factor determining the photosynthesis, productivity and metabolic pathways of phytoplankton. The quality, intensity, and period of light all have significant effects on the amount of light energy received by microalgae, and these effects are species-dependent (Latsos et al. 2021 ). Increasing light intensity impacts microalgal growth through four phases—lag, limitation, saturation, and inhibition—with growth ceasing below critical intensity or diminishing at very high intensities due to photodamage to photosynthetic proteins (Ogbonna and Tanaka 2000 ). Light is not only essential for photosynthesis but also influences various signaling pathways that regulate nutrient uptake, cell division, and morphological changes (Dickman et al. 2006 ). In general, the optimal light intensity range for the growth of microalgae is 26–400 µmol m − 2 s − 1 , depending on the species (Maltsev et al. 2021 ). For example, for Ankistrodesmus convolutus and Chlorella vulgaris , the phosphate uptake rates increase during the daytime and decrease at night (Ahn et al. 2002 ). Trabelsi et al. ( 2008 ) showed light intensity had a positive influence on growth rate and extracellular polymeric substances concentration of cyanobacterium Arthrospira platensis. With increasing light intensity from 10 to 100 µE m − 2 s − 1 , the maximum nitrate uptake rate of Prorocentrum micans increase from 3.6 to 10.8 pM cell − 1 d − 1 , and the half saturation constant increase from 4.1 to 6.9 µM (Lee et al. 2017 ). In Chlorella pyrenoidosa , elevated light intensity induces cell division, whereas reduced light intensity leads to growth without division, and under continuous lighting, cell volume expands as light intensity diminishes (Wanka 1959 ). Scenedesmus obliquus , a freshwater microalga, is well documented for its phenotypic plasticity, a trait that is critical for its survival and adaptation to varying environmental conditions (Zhu et al. 2015 ). This phenotypic plasticity is largely manifested as the ability of organisms to alter their physiology, morphology, or development in response to environmental changes (Sultan 2000 ). S. obliquus mainly exists as unicells or two-, four- or eight-celled coenobia and varies in morphological phenotype (Pancha et al. 2014 ). Morphological changes in S. obliquus are mainly induced by changes in nutrient concentration, pH, surfactant or allochemicals released from grazers (Lürling 2006 ; Yang et al. 2016 ; Zhu et al. 2024b ). Morphological responses of S. obliquus to competitive pressures are closely related to the mode of competition, with unicellular and small colonies dominating when there is direct resource competition with macroalgae such as Chara and Myriophyllum (Lürling et al. 2006 ), but multicellular morphology dominates when there is competition for chemotaxis from algae such as Stratiotes aloides (Mulderij et al. 2005 ). In the field, unicellular S. obliquus are easily captured by zooplankton, therefore, the typical eight-celled particle is considered to resist herbivorous zooplankton (Hessen and van Donk 1993 ). However, compared with unicells, the multicellular colonies have a smaller surface-to-volume ratio, which is less conducive to absorbing light and nutrient. The inherent plasticity in morphological adaptions confers competitive growth advantages in S. obliquus that make them promising candidates for biodiesel production and wastewater treatment. Furthermore, the phenotypical plasticity in S. obliquus is of significant interest for both basic ecological research and biomass production for biofuel. In aquatic ecosystems, nitrogen availability and light intensity are two pivotal environmental factors that influence algal growth and function. Understanding how these factors interact can provide deeper insights into the ecological dynamics of algae and contribute to the optimization of algae-based technologies in biofuel production and wastewater treatment. Based on the above research background, it is necessary to study the differentiated response of S. obliquus to multiple nitrogen sources under varying light intensities. We formulated the scientific hypotheses: (1) Nitrogen source and light intensity can synergistically promote the growth of S. obliquus ; (2) Photosynthetic activity of S. obliquus declines with light intensity for NH 4 Cl and CO(NH 2 ) 2 as nitrogen sources, but remains stable in NaNO 3 ; (3) With the increase of light intensity, NaNO 3 and NH 4 Cl, as inorganic nitrogen sources, augment S. obliquus to form colonies, while CO(NH 2 ) 2 as an organic nitrogen source diminishes colony formation. To test our hypothesis, we conducted an experiment to analyze the response of S. obliquus to three type of nitrogen sources (NaNO 3 , NH 4 Cl, CO(NH 2 ) 2 ) and five light intensities (25, 50, 75, 100, 150 µmol photons m − 2 s − 1 ), and recorded the cell densities, colony distributions, photosynthetic efficiency and environmental nitrogen concentration during cultivation period. 2. Materials and methods 2.1. Microalgal cultivation The microalga S. obliquus FACHB-416 was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, China (Wuhan). The alga was axenically maintained in BG-11 medium at 25 ℃ under fluorescent light at 40 µmol photons m − 2 s − 1 with a light-dark period of 14 h: 10 h. Before the experiment, a portion of S. obliquus population was inoculated into axenic BG-11 medium to maintain their exponential phase. The entire transfer process was conducted in a sterile environment. 2.2. Experimental protocol In this experiment, five light intensities were set at 25, 50 ,75, 100, 150 µmol photons m − 2 s − 1 with light: dark = 14 h: 10 h, and three nitrogen resource were selected: NaNO 3 , NH 4 Cl, CO(NH 2 ) 2 . The concentration of each nitrogen was 18 mg·L − 1 ). The S. obliquus was cultured in 250 mL autoclaved Erlenmeyer flasks containing 150 mL of modified BG-11 under the condition as described above. Each treatment had three replicates, and the experiment lasted for 14 days. During the experiment, the flasks were shaken manually twice a day. The samples (5 mL) were taken daily: 1 mL was preserved with 2% Lugol’s solution for counting algal cells, 2 mL was used to detect photosynthetic activity of living cells, and the remaining 2 mL was used to measure the residual nitrogen concentration. 2.3. Determination of morphology and biomass production The numbers of cells, including unicells and cells in different colonies (i.e., two-celled, four-celled, and eight-celled colonies), were counted by using a hemocytometer under a microscope. Cells per particle (including unicells and different colonies) were determined to indicate the mean colony size. The algal population biomass and cells per particle were calculated based on the cell counts. The specific growth rates ( µ ) were calculated as: \(\mu =\left[ {ln\left( {{N_t}} \right) - ln\left( {{N_0}} \right)} \right]/\left( {t - {t_0}} \right)\) , where N t and N 0 are the cell density at instant time ( t ) and initial time ( t 0 ), respectively. The cells per particle ( C ) were calculated as: \(C=\left( {{n_1} \times 1+{n_2} \times 2+{n_4} \times 4+{n_8} \times 8} \right)/\left( {{n_1}+{n_2}+{n_4}+{n_8}} \right)\) , where n m are the numbers of unicells, or two-celled, four-celled, eight-celled colonies, respectively. The population density over time was fitted using the Logistic model: \({N_t}=K/\left[ {1+\left( {K/{N_0} - 1} \right){e^{ - rt}}} \right]\) , where N t represents the population density on day t , N 0 represents the initial population density, K represents the carrying capacity, r represents the instantaneous growth rate, and t represents the culture time. 2.4. Photosynthesis measurement Photosynthetic activities of S. obliquus were measured using a Phyto-PAM (Walz, Germany), based on samples (2 mL) collected daily in “ 2.2. Experimental protocol ”. Photosynthetic parameters included maximal efficiency of PSII photochemistry ( Fv/Fm ), effective quantum yield of PSII photosynthetic efficiency ( Φ PSII ), and maximum relative electron rate ( ETR max ). Before measurement, algal suspensions needed to undergo 3–5 minutes of dark adaptation. 2.5. Data analyses All values were presented as mean ± standard error. The combined effects of light intensity and different nitrogen sources on photosynthetic efficiency and the number of cells per colony of S. obliquus were analyzed by two-way RM ANOVA, followed by a Tukey test. Afterwards, specific growth rate was analyzed by one-way ANOVA and two-way ANOVA, then followed by Tukey test. All statistical analyses were performed using SigmaPlot 14.0, Origin 2021. The significance level was set at P < 0.05. 3. Results 3.1. Biomass production During the experiment, the biomass production of S. obliquus cultured in three nitrogen sources increased substantially, and all treatments were maintained proliferation until they reached carrying capacity (Fig. 1 ). Evidently, when NaNO 3 and CO(NH 2 ) 2 were used as nitrogen sources, the growth of S. obliquus performed better than that in NH 4 Cl (Fig. 1 a-c). One-way ANOVA showed that the growth of S. obliquus cultured in NH 4 Cl was not influenced by different light intensities (Fig. 1 d). When NaNO 3 and CO(NH 2 ) 2 were set as nitrogen sources, there was no significant difference in the specific growth rates under the light intensity ranging from 25 to 75 µmol photons m − 2 s − 1 ; however, when the light intensities were increased to 100 and 150 µmol photons m − 2 s − 1 , the growth rates were significantly enhanced (Fig. 1 d). Two-way ANOVA suggested that the nitrogen source type ( F = 36.224, P < 0.001) and light intensity ( F = 23.030, P < 0.001) had significant effects on the growth of S. obliquus , but there was no interaction between them ( F = 1.447, P = 0.218). Generally, higher light intensities led to earlier attainment of carrying capacities (Fig. 1 a-c, e). However, the theoretical maximums of carrying capacities were achieved at 75 µmol photons m − 2 s − 1 for both the NaNO 3 and CO(NH 2 ) 2 treatments. 3.2. Photosynthetic efficiency Generally, the photosynthetic parameters, including Fv/Fm , Φ PSII and ETR max , exhibited substantial variations in response to diverse nitrogen resources under a constant light intensity (Fig. 2 ). Two-way RM ANOVA showed that the Fv/Fm , Φ PSII and ETR max were significantly affected by light intensity and nitrogen source type, and there was a highly significant interaction between the two factors (Table 1 ). In NH 4 Cl treatments, increased light intensities were correlated with reduced Fv/Fm and Φ PSII , with values of both parameters being obviously lower at a light intensity of 150 µmol photons m − 2 s − 1 compared to the other conditions. In NaNO 3 treatments, the Fv/Fm was similar under different light intensities, and basically maintained stable at around 0.72 from the 9th day onwards, while the Φ PSII was lower under the light intensity of 150 µmol photons m − 2 s − 1 than under other light intensities. Nitrogen source CO(NH 2 ) 2 induced a rapid decline in Fv/Fm and Φ PSII , stabilizing by the 7th day. For ETR max , the maximum value occurred at 150 µmol photons m − 2 s − 1 , whereas the minimum value occurred at 100 µmol photons m − 2 s − 1 across all three nitrogen source treatments. Table 1 Two-way RM ANOVA for the effects of nitrogen source type and light intensity on Fv/Fm , Ф PSII , ETR max , and cells per particle of S. obliquus . Source of variation Fv/Fm Ф PSII ETR max Cells per particle F P F P F P F P Nitrogen source (A) 21.991 < 0.001 15.836 < 0.001 4.174 0.042 46.901 < 0.001 Light intensity (B) 35.625 < 0.001 46.222 < 0.001 7.962 < 0.001 3.47 0.023 A × B 11.972 < 0.001 14.414 < 0.001 4.031 0.001 4.833 < 0.001 3.3. Algal morphology The cells per particle provided an overall evaluation on the morphology of the algal population (Fig. 3 ). At the onset of the experiment, the number of cells per particle of S. obliquus was relatively consistent among all groups that underwent preliminary treatment, maintaining around 2. The cells per particle induced by different types of nitrogen sources and light intensities exhibited fluctuations during the early phase, followed by stabilization in the later phase (9-14th days). Two-way RM ANOVA revealed that both nitrogen source and light intensity significantly influenced the number of cells per particle, and there was a significant interaction between the light intensity and nitrogen source type (Table 1 ). Ultimately, the cells per particle of each treatment cultured with NH 4 Cl, NaNO 3 and CO(NH 2 ) 2 as nitrogen sources were maintained at approximately 2.1, 4, and 1.1, respectively. Varied colonial types also reflected the phenotypic plasticity of S. obliquus in response to different environmental conditions (Fig. 4 ). Initially, the proportion of cells in different colonies was similar across all groups. However, from third day, Among the four primary morphotypes, the unicells, two-, four- and eight-celled colonies were significantly changed with the different nitrogen sources and light intensity. In NaNO 3 treatment, the proportion of four-celled colony increased sharply. Compared with the first day, when NH 4 Cl and CO(NH 2 ) 2 were used as the nitrogen source, the number of unicells increased under five light intensities, rising by approximately 20% and 50%, respectively. After the 13-day culture period, the proportion of cells in different colonies in each treatment group reached a relatively stable level. Specifically, two-celled (~ 20%) and four-celled (~ 70%) colonies grew rapidly in response to NH 4 Cl. Meanwhile, four-celled colony (~ 90%) were predominant in NaNO 3 . In contrast, the high proportion of unicells (~ 75%) grew in CO(NH 2 ) 2 treatment. 3.4. Nitrogen content in the test solution Based on the changes of the nitrogen content of test solution during the experiment, nitrogen contents in NH 4 Cl, NaNO 3 , and CO(NH 2 ) 2 treatment groups decreased about 5.4–9.4, 14.0-17.9 and 12.8–16.9 mg·L − 1 , respectively, until the end of the experiment (Fig. 5 ). Thus, among the three types of nitrogen sources, S. obliquus has stronger removal capacity for NaNO 3 and CO(NH 2 ) 2 , but less for NH 4 Cl. Under consistent nitrogen sources, increased light intensity facilitated the uptake of nitrogen by S. obliquus , aligning with population dynamics. Moreover, the remaining total nitrogen content in the test solutions showed a significant negative correlation with algal cell densities (Fig. 6 ), indicating that there were differences in the growth of S. obliquus and their nitrogen consumption among different nitrogen sources. 4. Discussion Our study showed there was a positive correlation between increased light intensity and the growth rate of S. obliquus in all three nitrogen source treatments, with the NH 4 Cl treatments exhibiting a statistically non-significant increase, potentially due to the lower uptake of NH 4 Cl by S. obliquus. Additionally, NH 4 Cl and CO(NH 2 ) 2 presence leads to a marked decrease in S. obliquus photosynthesis with increasing light intensity, contrasting with the stability observed under NaNO 3 nitrogen conditions. During the early stage of the experiment, S. obliquus under various nitrogen treatments generally showed that increased unicells were correlated with higher light intensity. Subsequently, with the increase in light intensity, NaNO 3 facilitated the formation of colonies, whereas the CO(NH 2 ) 2 tended to promote the proliferation of unicells. These results generally confirmed our previous hypotheses. 4.1. Population dynamics of S. obliquus under different light intensities with different nitrogen sources Light intensity and nitrogen source were found to synergistically promote algal growth in our experiment. Specifically, higher cell density and growth rates were correlated with increased light intensity across all nitrogen sources. Some studies have shown an increase in specific growth rate with light intensity in Chlamydomonas reinhardtii , Chlorella vulgaris , Cryptomonas sp. , Dunaliella viridis , Neochloris oleoabundans , Scenedesmus sp., etc., coinciding with the maximum lipid production (Hwang and Maier 2019 ; Li et al. 2023 ; Liu et al. 2012 ; Weng et al. 2009 ). Interestingly, the difference in growth rate caused by light intensity was significant in the NaNO 3 and CO(NH 2 ) 2 treatments, but not in the NH 4 Cl treatments. As previously reported that NaNO 3 is the preferred nitrogen source for microalgal growth of Scenedesmus at the same light intensity, and CO(NH 2 ) 2 results in almost equal biomass as NaNO 3 (Arumugam et al. 2013 ). The influence of NH 4 Cl on microalgal growth is intricate, as it exhibits dual physiological effects. NH 4 Cl supplies nitrogen for microalgal growth but may be toxic at high concentrations, thus inhibiting proliferation. For example, Ellipsoidion sp. grows faster with NH 4 Cl than NaNO 3 during the logarithmic phase, but the trend reverses in the post-logarithmic phase (Xu et al. 2001 ). In present study, we also found that the cell abundances of all treatments were similar in early period, however, growth of S. obliquus in NH 4 Cl was markedly suppressed in later period (Fig. 1 E). The results indicated that, in the initial phase, ammonium enters directly into the algal cells mainly in the form non-ionic NH 4 + and accumulates abundantly with minimal energy consumption. However, excessive ammonium ions cannot be rapidly transferred to synthesize proteins. Consequently, this induces ammonium intoxication of the algal cells, thus affecting the growth of microalgae (Kim et al. 2016 ). In addition, the influx of NH 4 + into microalgal cells might lead to acidification, which is a condition detrimental to the growth of S. obliquus (Kleiner 1981 ; Miura et al. 2021 ). Microalgae transport CO(NH 2 ) 2 into cells via specific transporters and then convert it to ammonium nitrogen through urease, producing carbon dioxide (Gutierrez et al. 2016 ). Thus, CO(NH 2 ) 2 supplies both carbon and nitrogen to S. obliquus , potentially leading to ammonium ion accumulation in later stages, yet carbon supplementation can alleviate the associated negative impact (Liu et al. 2019 ). Correlation analysis indicated that lower residual nitrogen content in test solution was associated with higher algal cell density on the final day (Fig. 6 ). Therefore, the nitrogen removal efficiency of S. obliquus under different nitrogen sources may be mainly governed by population growth, such as treatments with NaNO 3 under high light intensities. The lower population growth in ammonium treatments ultimately leads to reduced nitrogen uptake and removal efficiency (Fig. 1 , 5 ). Algal cultivation in wastewater systems was has emerged as a sustainable strategy for removing the nutrients form the wastewaters and generating energy from the biomass (Bhattacharjee and Siemann 2015 ). Currently, S. abundans has been proven to effectively remove pollutants from domestic wastewater, which contains as much as 90.73% dissolved inorganic nitrogen (SundarRajan et al. 2020 ). Previous research also found that Chlorococcum sp . GD, Parachlorella kessleri TY and Scenedesmus sp. LX1 exhibited higher growth potential and nitrogen removal ability in wastewater with NaNO 3 , which was superior to wastewater with NH 4 Cl, and they emphasized that this phenomenon can be attributed to the suppressive impact of acidic pH during algal cultivation process (Lv et al. 2019 ; Xin et al. 2010 ). Another study also provided evidence that organic carbon increased algal biomass and nitrogen removal capacity (Ji et al. 2024 ), which may be the reason for the highest urea removal efficiency in this experiment. Consistent with previous findings, increasing light intensity is conducive for algae to rapid removal of nitrogen from the environment at the same time (Ouyang et al. 2015 ). In general, the nitrogen removal ability of S. obliquus varied for different nitrogen sources. 4.2. Effects of nitrogen sources on photosynthetic system of S. obliquus under different light intensities Varying nitrogen source conditions triggered distinct responses of algal cells, leading to heterogeneous response levels to light. Our data revealed that Fv/Fm and Φ PSII values were stable under various light intensities in NaNO 3 treatments, whereas in NH 4 Cl and CO(NH 2 ) 2 treatments, these values declined with prolonged culture time. Several researches have shown that NH 4 Cl and CO(NH 2 ) 2 can exert inhibitory effects on various phytoplankton species, including but not limited to C. vulgaris , Microcystis aeruginosa , Nannochloropsis oceanica , and S. quadricauda . Furthermore, the inhibitory impact is not confined to microscopic phytoplankton; it extends to larger aquatic macrophytes as well. For instance, species such as Cabomba caroliniana , Elodea nuttallii , and Sargassum thunbergii have been documented to exhibit reduced growth and biomass when subjected to similar treatments (Huang et al. 2017 ; Li et al. 2014 ; Liu et al. 2020 ; Peng et al. 2016 ; Zhu et al. 2024a ). A reduction in Fv/Fm and Φ PSII suggests a decrease in PSII photochemistry efficiency or a disorder in or damage to the photosynthetic apparatus (Gao et al. 2024 ). The changes are likely due to ammonium-induced disruption of the thylakoid proton gradient, in other words, ammonia ions enter thylakoid, creating an acidic environment, which decreases the transmembrane proton gradient necessary to drive ATP synthesis from ADP (Kikeri et al. 1989 ; Tikhonov 2013 ). This disruption of the thylakoid proton gradient can lead to a reduction in photosynthetic efficiency, as the proton gradient is a crucial driving force for ATP production, which is essential for the Calvin cycle and overall plant energy metabolism (Höhner et al. 2016 ). Another hypothesis is that ammonia ions may ligate to the PSII oxygen evolution reaction core, thereby increasing photosensitivity and consequently enhancing photosystem damage (Cazzaniga et al. 2020 ). Although CO(NH 2 ) 2 also supplies ammonia nitrogen to the system, hydrolysis of CO(NH 2 ) 2 produces CO 2 , potentially enhancing the carbon concentrating mechanism as a new carbon source and optimizing nitrogen supply, which promotes microalgae to adjust their photosynthetic carbon fixation strategy. Thus, when CO(NH 2 ) 2 is used as the nitrogen source, S. obliquus can maintain normal growth despite a decline in photosynthetic efficiency. In the present study, under various nitrogen source conditions, the maximum value of ETR max occurred at 150 µmol photons m − 2 s − 1 , while the ETR max values within the range of 25–100 µmol photons m − 2 s − 1 were closely. It is widely known that the ETR max of plants with different genotypes is similar under low light conditions (Howard et al. 2019 ). The impact of light intensity on the ETR max appears to be more pronounced. However, compared with ETR max under the three nitrogen sources, the efficiency of S. obliquus in utilizing light is obviously affected, which once again proves that NH 4 Cl causes stress on the S. obliquus . 4.3. Morphological plasticity of S. obliquus in response to nitrogen sources and light intensities Due to its distinctive biological traits, S. obliquus has been extensively utilized across various domains, including animal feed, energy production, and sewage disposal. The multicellular colonies of S. obliquus enhance sedimentation rates and confers greater stress tolerance, which not only facilitates the efficiency of resource harvesting but also augments the effectiveness of algal removal during biological water purification process. However, the diminutive size of the unicellular S. obliquus renders it an ideal food source for zooplankton. Based on these applications, understanding the morphological dynamics of S. obliquus is crucial for optimizing its use in these fields. We observed that under elevated light intensities, the multicellular colonies of S. obliquus escalated in cultures supplemented with NaNO 3 . In contrast, urea led to a progressive predominance of unicellular forms within the culture medium. Previously, the researchers emphasized that treatment with nitrate or nitrogen starvation can induce a transition from unicells to multicellular colonies (Pancha et al. 2014 ). Additionally, when a low concentration of ammonium nitrogen is utilized as the nitrogen source, the algae remain unicellular (Pancha et al. 2014 ). Tukaj et al. ( 1996 ) observed that the formation of S. armatus in eight-celled and sixteen-celled colonies increased with elevated light intensity, which was potentially driven by the accelerated division rate of algal cells under high light intensity. And following incubation under both low (10 and 30 µmol m⁻² s⁻¹) and high (120 µmol m⁻² s⁻¹) light intensities, Nostoc sphaeroides colonies softened and ultimately lost their spherical aggregation (Ma et al. 2015 ). In another study, 70–80% of S. obliquus existed as unicells at light intensities of 50 and 60 µmol photons m − 2 s − 1 , as well as at below 25 µmol photons m − 2 s − 1 , however, at 30 and 35 µmol photons m − 2 s − 1 , the proportion of unicells decreased, and the proportion of four-celled colonies and above was approximately 40% (Li et al. 2016 ). In aquatic ecosystems, the microalgal morphology significantly influences their migratory velocities, resistance to predation pressures, and specific surface area (Pančić and Kiørboe 2018 ). The formation of algal colonies primarily occurs through two pathways: (1) aggregation due to the failure of dividing cells to detach, and (2) adhesion of existing individual cells. It is generally believed that colonies of S. obliquus are formed via the first pathway, as the arrangement of cells in these colonies is orderly and regular, whereas pathway two would likely result in irregular cell arrangements (Bišová and Zachleder 2014 ; Dong et al. 2017 ; Yang et al. 2010 ). In the present study, S. obliquus showed optimal growth in NaNO 3 treatments, possibly because the increased light intensity promoted cell division and NaNO 3 stimulated polysaccharide secretion (Moreira et al. 2022 ), thus the most multicellular colonies were harvested. Ammonium salts have been reported to enhance the biosynthesis of polysaccharides in algae, albeit to a lesser extent compared to KNO 3 (Arad et al. 1988 ), but the high levels of ammonium will rapidly accumulate within the algal cells, causing toxicity and alterations in cellular biochemical composition, thereby changing colonial morphology. Therefore, we obtained an only moderately multicellular colony when NH 4 Cl as the nitrogen source. These two types of nitrogen sources seem to be quite different to the mechanism of CO(NH 2 ) 2 . Although CO(NH 2 ) 2 , serving as an organic nitrogen source, enhance the growth of S. obliquus by supplementing with additional carbon source, we found that the CO(NH 2 ) 2 treatments were dominated by unicells under five light intensities across the experiment. Empirical evidence indicates that CO(NH 2 ) 2 leads to a significant reduction in the extracellular polysaccharide content of microalgae, particularly at the stable phase of microalgae (Lupi et al. 1994 ). Consequently, in treatments employing CO(NH 2 ) 2 , the population of S. obliquus is predominantly composed of unicells. On the other hand, multicellular colonies show greater resistance to dark loss and high light intensity than unicells, with higher Fv/Fm and NPQ values (Zhang et al. 2011 ). Our photosynthetic efficiency data suggest that urea may impair the carbon concentrating mechanism, reducing the need for colony formation to capture light energy. 5. Conclusions The growth of S. obliquus across all treatments was significantly enhanced by the combined effects of sufficient nitrogen source and high light intensity. In the case of NH 4 Cl and CO(NH 2 ) 2 as nitrogen sources, photosynthetic activity decreased with increasing light intensity, whereas it remained relatively stable under NaNO 3 condition. Meanwhile, NaNO 3 facilitated colony formation in S. obliquus with increasing light intensity, whereas the CO(NH 2 ) 2 had the opposite effect, promoting the formation of unicells. In addition, the nitrogen removal ability of S. obliquus varied for different nitrogen sources. This study elucidated the combined impact of nitrogen sources and light intensity on the growth and phenotypic plasticity of S. obliquus , providing a foundation for optimizing these factors to enhance algal growth efficiency and obtain different colonies of S. obliquus . Declarations CRediT authorship contribution statement Jiyan Long: Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Yiqi Feng: Validation, Investigation. Decai Huang: Validation, Investigation. Yulu Lei: Investigation, Data curation, Formal analysis . Xuexia Zhu: Methodology, Conceptualization, Writing – review & editing. Zhou Yang: Conceptualization, Supervision, Funding acquisition, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We acknowledge the anonymous reviewers for their constructive comments. This study was supported by the National Natural Science Foundation of China (32271626) and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China. Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. Data availability Data will be made available on request. References Ahn C-Y, Chung A-S, Oh H-M (2002) Diel Rhythm of Algal Phosphate Uptake Rates in P-Limited Cyclostats and Simulation of Its Effect on Growth and Competition. J Phycol 38: 695-704. doi.org/10.1046/j.1529-8817.2002.01232.x Arad SM, Friedman OD, Rotem A (1988) Effect of Nitrogen on Polysaccharide Production in a Porphyridium sp. Appl Environ Microbiol 54: 2411-2414. doi.org/10.1128/aem.54.10.2411-2414.1988 Arumugam M, Agarwal A, Arya MC, Ahmed Z (2013) Influence of nitrogen sources on biomass productivity of microalgae Scenedesmus bijugatus . Bioresour Technol 131: 246-249. doi.org/10.1016/j.biortech.2012.12.159 Bhattacharjee M, Siemann E (2015) Low algal diversity systems are a promising method for biodiesel production in wastewater fed open reactors. Algae 30: 67-79. doi.org/10.4490/algae.2015.30.1.067 Bišová K, Zachleder V (2014) Cell-cycle regulation in green algae dividing by multiple fission. J Exp Bot 65: 2585-2602. doi.org/10.1093/jxb/ert466 Bloom AJ, Sukrapanna SS, Warner RL (1992) Root respiration associated with ammonium and nitrate absorption and assimilation by barley. Plant Physiol 99: 1294-1301. doi.org/10.1104/pp.99.4.1294 Cazzaniga S, Kim M, Bellamoli F, Jeong J, Lee S, Perozeni F, Pompa A, Jin E, Ballottari M (2020) Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii . Plant Cell Environ 43: 496-509. doi.org/10.1111/pce.13680 Dickman EM, Vanni MJ, Horgan MJ (2006) Interactive effects of light and nutrients on phytoplankton stoichiometry. Oecologia 149: 676-689. doi.org/10.1007/s00442-006-0473-5 Dong J, Gao Y, Chang M, Ma H, Han K, Tao X, Li Y (2017) Colony formation by the green alga Chlorella vulgaris in response to the competitor Ceratophyllum demersum . Hydrobiologia 805: 177-187. doi.org/10.1007/s10750-017-3294-0 Gao Y, Bernard O, Fanesi A, Perré P, Lopes F (2024) The effect of light intensity on microalgae biofilm structures and physiology under continuous illumination. Sci Rep 14: 1151. doi.org/10.1038/s41598-023-50432-6 Gutierrez J, Kwan TA, Zimmerman JB, Peccia J (2016) Ammonia inhibition in oleaginous microalgae. Algal Res 19: 123-127. doi.org/10.1016/j.algal.2016.07.016 Hessen DO, van Donk E (1993) Morphological changes in Scenedesmu s induced by substances released from Daphnia . Arch Hydrobiol 127: 129-140. doi.org/10.1127/archiv-hydrobiol/127/1993/129 Höhner R, Aboukila A, Kunz H-H, Venema K (2016) Proton Gradients and Proton-Dependent Transport Processes in the Chloroplast. Front Plant Sci 7: 218. doi.org/10.3389/fpls.2016.00218 Hou L-l, Liu F, Zang X, Zhang X, He B, Ding Y, Song X, Xiao D, Wang H (2017) Cloning and transcription analysis of the nitrate reductase gene from Haematococcus pluvialis . Biotechnol Lett 39: 589-597. doi.org/10.1007/s10529-016-2283-0 Howard MM, Bae A, Königer M (2019) The importance of chloroplast movement, nonphotochemical quenching, and electron transport rates in light acclimation and tolerance to high light in Arabidopsis thaliana . Am J Bot 106: 1444-1453. doi.org/10.1002/ajb2.1378 Huang W, Shao H, Li W, Jiang H, Chen Y (2017) Effects of urea on growth and photosynthetic metabolism of two aquatic plants ( Cabomba caroliniana A. Gray and Elodea nuttallii (Planch.) H. St. John). Aquat Bot 140: 69-77. doi.org/10.1016/j.aquabot.2016.04.003 Hwang J-H, Maier N (2019) Effects of LED-controlled spatially-averaged light intensity and wavelength on Neochloris oleoabundans growth and lipid composition. Algal Res 41: 101573. doi.org/10.1016/j.algal.2019.101573 Ji M, Gao H, Zhang J, Hu Z, Liang S (2024) Environmental impacts on algal–bacterial-based aquaponics system by different types of carbon source addition: water quality and greenhouse gas emission. Environ Sci Pollut Res 31: 26665-26674. doi.org/10.1007/s11356-024-32717-z Kikeri D, Sun A, Zeidel ML, Hebert SC (1989) Cell membranes impermeable to NH 3 . Nature 339: 478-480. doi.org/10.1038/339478a0 Kim G, Mujtaba G, Lee K (2016) Effects of nitrogen sources on cell growth and biochemical composition of marine chlorophyte Tetraselmis sp. for lipid production. Algae 31: 257-266. doi.org/10.4490/algae.2016.31.8.18 Kleiner D (1981) The transport of NH 3 and HN 4 + across biological membranes. Biochim Biophys Acta, Rev Bioenerg 639: 41-52. doi.org/10.1016/0304-4173(81)90004-5 Latsos C, van Houcke J, Blommaert L, Verbeeke GP, Kromkamp J, Timmermans KR (2021) Effect of light quality and quantity on productivity and phycoerythrin concentration in the cryptophyte Rhodomonas sp. J Appl Phycol 33: 729-741. doi.org/10.1007/s10811-020-02338-3 Lee KH, Jeong HJ, Kim HJ, Lim AS (2017) Nitrate uptake of the red tide dinoflagellate Prorocentrum micans measured using a nutrient repletion method: effect of light intensity. Algae 32: 139-153. doi.org/10.4490/algae.2017.32.5.20 Li M, Gao L, Lin L (2016) Specific growth rate, colonial morphology and extracellular polysaccharides (EPS) content of Scenedesmus obliquus grown under different levels of light limitation. Ann Limnol, Int J Limnol 51: 329-334. doi.org/10.1051/limn/2015033 Li X, Huff J, Crunkleton DW, Johannes TW (2023) Light intensity and spectral quality modulation for improved growth kinetics and biochemical composition of Chlamydomonas reinhardtii . J Biotechnol 375: 28-39. doi.org/10.1016/j.jbiotec.2023.08.007 Li XM, Zhang QS, Tang YZ, Yu YQ, Liu HL, Li LX (2014) Highly efficient photoprotective responses to high light stress in Sargassum thunbergii germlings, a representative brown macroalga of intertidal zone. J Sea Res 85: 491-498. doi.org/10.1016/j.seares.2013.08.004 Liu J, Yuan C, Hu G, Li F (2012) Effects of Light Intensity on the Growth and Lipid Accumulation of Microalga Scenedesmus sp. 11-1 Under Nitrogen Limitation. Appl Biochem Biotechnol 166: 2127-2137. doi.org/10.1007/s12010-012-9639-2 Liu N, Zhang H, Zhao J, Xu Y, Ge F (2020) Mechanisms of cetyltrimethyl ammonium chloride-induced toxicity to photosystem II oxygen evolution complex of Chlorella vulgaris F1068. J Hazard Mater 383: 121063. doi.org/10.1016/j.jhazmat.2019.121063 Liu X, Wang K, Wang J, Zuo J, Peng F, Wu J, San E (2019) Carbon dioxide fixation coupled with ammonium uptake by immobilized Scenedesmus obliquus and its potential for protein production. Bioresour Technol 289: 121685. doi.org/10.1016/j.biortech.2019.121685 Lupi FM, Fernandes HML, Tomé MM, Sá-Correia I, Novais JM (1994) Influence of nitrogen source and photoperiod on exopolysaccharide synthesis by the microalga Botryococcus braunii UC 58. Enzyme Microb Technol 16: 546-550. doi.org/10.1016/0141-0229(94)90116-3 Lürling M (2006) Effects of a surfactant (FFD-6) on Scenedesmus morphology and growth under different nutrient conditions. Chemosphere 62: 1351-1358. doi.org/10.1016/j.chemosphere.2005.07.031 Lürling M, van Geest G, Scheffer M (2006) Importance of Nutrient Competition and Allelopathic Effects in Suppression of the Green Alga Scenedesmus obliquus by the Macrophytes Chara , Elodea and Myriophyllum . Hydrobiologia 556: 209-220. doi.org/10.1007/s10750-005-1168-3 Lv J, Wang X, Feng J, Liu Q, Nan F, Jiao X, Xie S (2019) Comparison of growth characteristics and nitrogen removal capacity of five species of green algae. J Appl Phycol 31: 409-421. doi.org/10.1007/s10811-018-1542-y Ma R, Lu F, Bi Y, Hu Z (2015) Effects of light intensity and quality on phycobiliprotein accumulation in the cyanobacterium Nostoc sphaeroides Kützing. Biotechnol Lett 37: 1663-1669. doi.org/10.1007/s10529-015-1831-3 Maltsev Y, Maltseva K, Kulikovskiy M, Maltseva S (2021) Influence of Light Conditions on Microalgae Growth and Content of Lipids, Carotenoids, and Fatty Acid Composition. Biology 10: 1060. doi.org/10.3390/biology10101060 Miura R, Furuhashi K, Hasegawa F, Kaizu Y, Imou K (2021) Calcium carbonate prevents Botryococcus braunii growth inhibition caused by medium acidification. J Appl Phycol 34: 177-183. doi.org/10.1007/s10811-021-02622-w Moreira JB, Vaz BD, Cardias BB, Cruz CG, Almeida AC, Costa JA, Morais MG (2022) Microalgae Polysaccharides: An Alternative Source for Food Production and Sustainable Agriculture. Polysaccharides 3: 441-457. doi.org/10.3390/polysaccharides3020027 Mulderij G, Mooij WM, Van Donk E (2005) Allelopathic growth inhibition and colony formation of the green alga Scenedesmus obliquus by the aquatic macrophyte Stratiotes aloides . Aquat Ecol 39: 11-21. doi.org/10.1007/s10452-004-1021-1 Ogbonna JC, Tanaka H (2000) Light requirement and photosynthetic cell cultivation - Development of processes for efficient light utilization in photobioreactors. J Appl Phycol 12: 207-218. doi.org/10.1023/a:1008194627239 Ouyang Y, Zhao Y, Sun S, Hu C, Ping L (2015) Effect of light intensity on the capability of different microalgae species for simultaneous biogas upgrading and biogas slurry nutrient reduction. Int Biodeterior Biodegrad 104: 157-163. doi.org/10.1016/j.ibiod.2015.05.027 Pancha I, Chokshi K, George B, Ghosh T, Paliwal C, Maurya R, Mishra S (2014) Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077. Bioresource Technology 156: 146-154. doi.org/10.1016/j.biortech.2014.01.025 Pančić M, Kiørboe T (2018) Phytoplankton defence mechanisms: traits and trade‐offs. Biol Rev 93: 1269-1303. doi.org/10.1111/brv.12395 Peng G, Fan Z, Wang X, Chen C (2016) Photosynthetic response to nitrogen source and different ratios of nitrogen and phosphorus in toxic cyanobacteria, Microcystis aeruginosa FACHB-905. J Limnol 75: 560-570. doi.org/10.4081/jlimnol.2016.1458 Sittisaree W, Yokthongwattana K, Aonbangkhen C, Yingchutrakul Y, Krobthong S (2024) Effect of NH 4 Cl supplementation on growth, photosynthesis, and triacylglycerol content in Chlamydomonas reinhardtii under mixotrophic cultivation. J Appl Microbiol 135: lxae233. doi.org/10.1093/jambio/lxae233 Sultan SE (2000) Phenotypic plasticity for plant development, function and life history. Trends Plant Sci 5: 537-542. doi.org/10.1016/S1360-1385(00)01797-0 SundarRajan P, Gopinath KP, Arun J, GracePavithra K, Pavendan K, AdithyaJoseph A (2020) An insight into carbon balance of product streams from hydrothermal liquefaction of Scenedesmus abundans biomass. Renewable Energy 151: 79-87. doi.org/10.1016/j.renene.2019.11.011 Tikhonov AN (2013) pH-Dependent regulation of electron transport and ATP synthesis in chloroplasts. Photosynth Res 116: 511-534. doi.org/10.1007/s11120-013-9845-y Trabelsi L, Ben Ouada H, Bacha H, Ghoul M (2008) Combined effect of temperature and light intensity on growth and extracellular polymeric substance production by the cyanobacterium Arthrospira platensis . J Appl Phycol 21: 405-412. doi.org/10.1007/s10811-008-9383-8 Tukaj Z, Kubínová A, Zachleder V (1996) Effect of Irradiance on Growth and Reproductive Processes During the Cell Cycle In Scenedesmus Armatus (Chlorophyta) J Phycol 32: 624-631. doi.org/10.1111/j.0022-3646.1996.00624.x Wanka F (1959) Untersuchungen über die Wirkung des Lichts auf die Zellteilung von Chlorella pyrenoidosa . Arch Mikrobiol 34: 161-188. doi.org/10.1007/BF00412750 Weng H-X, Qin Y-C, Sun X-W, Chen X-H, Chen J-F (2009) Effects of light intensity on the growth of Cryptomonas sp. (Cryptophyceae). Environ Geol 57: 9-15. doi.org/10.1007/s00254-008-1277-1 Xin L, Hong-ying H, Ke G, Jia Y (2010) Growth and nutrient removal properties of a freshwater microalga Scenedesmus sp. LX1 under different kinds of nitrogen sources. Ecol Eng 36: 379-381. doi.org/10.1016/j.ecoleng.2009.11.003 Xu N, Zhang X, Fan X, Han L, Zeng C (2001) Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta). J Appl Phycol 13: 469. doi.org/10.1023/A:1012537219198 Yang J, Li B, Zhang C, Luo H, Yang Z (2016) pH-associated changes in induced colony formation and growth of Scenedesmus obliquus . Fundam Appl Limnol 187: 241-246. doi.org/10.1127/fal/2016/0846 Yang Z, Liu Y, Ge J, Wang W, Chen Y, Montagnes D (2010) Aggregate formation and polysaccharide content of Chlorella pyrenoidosa Chick (Chlorophyta) in response to simulated nutrient stress. Bioresour Technol 101: 8336-8341. doi.org/10.1016/j.biortech.2010.06.022 Zhang M, Shi X, Yu Y, Kong F (2011) The Acclimative Changes in Photochemistry after Colony Formation of the Cyanobacteria Microcystis Aeruginosa . J Phycol 47: 524-532. doi.org/10.1111/j.1529-8817.2011.00987.x Zhang Y, Wang Q, Liu X, Zheng H, Li A (2023) Two types of growth pattern of the five microalgal species under different nitrogen supplies. Biomass Bioenergy 171: 106720. doi.org/10.1016/j.biombioe.2023.106720 Zhang Y, Wu H, Sun M, Peng Q, Li A (2018) Photosynthetic physiological performance and proteomic profiling of the oleaginous algae Scenedesmus acuminatus reveal the mechanism of lipid accumulation under low and high nitrogen supplies. Photosynth Res 138: 73-102. doi.org/10.1007/s11120-018-0549-1 Zhu H, Ye Z, Xu Z, Wei L (2024a) Transcriptomic Analysis Reveals the Effect of Urea on Metabolism of Nannochloropsis oceanica . Life 14: 797. doi.org/10.3390/life14070797 Zhu X, Sun Y, Huang Y, Wang J, Yang Z (2024b) Long-term continuous mismatch between grazing cues and real grazing losses causes attenuation of induced morphological defense in Scenedesmus . J Appl Phycol 36: 1353-1362. doi.org/10.1007/s10811-023-03174-x Zhu X, Wang J, Lu Y, Chen Q, Yang Z (2015) Grazer-induced morphological defense in Scenedesmus obliquus is affected by competition against Microcystis aeruginosa . Sci Rep 5: 12743. doi.org/10.1038/srep12743 Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2025 Read the published version in Biotechnology Letters → Version 1 posted Editorial decision: Major revisions 13 Jul, 2025 Reviewers agreed at journal 23 Jun, 2025 Reviewers invited by journal 23 Jun, 2025 Editor assigned by journal 04 Jun, 2025 First submitted to journal 03 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6808862","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475271266,"identity":"f2fc398d-65ff-4369-b634-28091957aae8","order_by":0,"name":"Jiyan Long","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jiyan","middleName":"","lastName":"Long","suffix":""},{"id":475271267,"identity":"2a078891-2068-4e1b-bcd8-807414ce207e","order_by":1,"name":"Yiqi Feng","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yiqi","middleName":"","lastName":"Feng","suffix":""},{"id":475271268,"identity":"86f34704-c637-4941-b2e8-559c38e82496","order_by":2,"name":"Decai Huang","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Decai","middleName":"","lastName":"Huang","suffix":""},{"id":475271269,"identity":"663fc39b-7990-4324-b2d5-061b14f2cd55","order_by":3,"name":"Yulu Lei","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yulu","middleName":"","lastName":"Lei","suffix":""},{"id":475271270,"identity":"853b96ed-4879-4232-b09a-75c9e6451377","order_by":4,"name":"Xuexia Zhu","email":"","orcid":"","institution":"Yangzhou University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xuexia","middleName":"","lastName":"Zhu","suffix":""},{"id":475271271,"identity":"9a751c6e-7093-4542-9ea1-8fecb9d97002","order_by":5,"name":"Zhou Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYLACxgYGBn4GhgQYm0gtkg0kazE4gGDjBwbHzx5++XOHTeLm2w3PHvMw2MhuOMD87AFeLWfy0iwkz6QlbrtzIN2YhyHNeMMBNnMDvFoO5JgZGLYdTtx2IyFNmofhcOKGAzxsEni1nH9jZpAI1LJ5BljLfyK03MgxfnAQqGWDBFjLAcJaJG+8MWNsbEszngF0mOQcg2TjmYfZzPBq4TufY/zxZ5uNbP+MnDSJNxV2sn3Hm5/h1aJwgAHmDJ4EoDuBNDM+9UAg38DA/AHCZD9AQO0oGAWjYBSMVAAA5VRPcDgv0oYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9664-7105","institution":"Nanjing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-06-03 08:28:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6808862/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6808862/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10529-025-03637-w","type":"published","date":"2025-09-05T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85385581,"identity":"1dd45757-ad06-453c-b2a5-b5d4e0bc8c39","added_by":"auto","created_at":"2025-06-25 09:49:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36250,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation dynamics, carrying capacities and specific growth rates of \u003cem\u003eScenedesmus obliquus\u003c/em\u003e. (a-c) Population dynamics in NH\u003csub\u003e4\u003c/sub\u003eCl, NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, respectively; (d) specific growth rates; (e) carrying capacities\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/4fb95d800fdfad9e32869c5e.png"},{"id":85382930,"identity":"92d47b60-9ff9-4202-8e50-d31cd8af563f","added_by":"auto","created_at":"2025-06-25 09:33:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34127,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthetic efficiency (\u003cem\u003eFv/Fm\u003c/em\u003e, \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e) of \u003cem\u003eScenedesmus obliquus\u003c/em\u003e. (a-c) NH\u003csub\u003e4\u003c/sub\u003eCl; (c-d) NaNO\u003csub\u003e3\u003c/sub\u003e; (e-f) CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/e7ccd4d90809fc30143398db.png"},{"id":85385079,"identity":"5bb851eb-584b-478c-87da-7a1532692fc8","added_by":"auto","created_at":"2025-06-25 09:41:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75067,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of cells per particle of \u003cem\u003eScenedesmus obliquus\u003c/em\u003e. (a) 150 µmol photons m\u003csup\u003e-2 \u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (b) 100 µmol photons m\u003csup\u003e-2 \u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (c) 75 µmol photons m\u003csup\u003e-2 \u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (d) 50 µmol photons m\u003csup\u003e-2 \u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (e) 25 µmol photons m\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/4da1b85d1a3b657088d71152.png"},{"id":85385081,"identity":"65d98890-c021-4fce-8507-8bbfda126d9e","added_by":"auto","created_at":"2025-06-25 09:41:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54124,"visible":true,"origin":"","legend":"\u003cp\u003eThe proportion of cells in different colonies in \u003cem\u003eScenedesmus obliquus\u003c/em\u003e under different conditions\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/9fc7d8fec3187e33b84f5215.png"},{"id":85382934,"identity":"27b33f44-25bf-4c58-a013-c34fa772b56f","added_by":"auto","created_at":"2025-06-25 09:33:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":19643,"visible":true,"origin":"","legend":"\u003cp\u003eChange of the nitrogen contents in test solution during the experiment. (a) 25 µmol photons m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (b) 50 µmol photons m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (c) 75 µmol photons m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (d) 100 µmol photons m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e; (e) 150 µmol photons m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/916e7bc0980ec23b64ea988e.png"},{"id":85382924,"identity":"95bbda34-bedd-4ad3-835c-e8acb1427f59","added_by":"auto","created_at":"2025-06-25 09:33:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21796,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of population density and the nitrogen contents in test solution on the 14th day in different conditions\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/8f9cb76e00949e9042d861d5.png"},{"id":90827961,"identity":"4fbbfa1d-05d6-4192-8713-6d5bc774c7c5","added_by":"auto","created_at":"2025-09-08 16:04:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1299450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6808862/v1/045af57b-b4a7-4092-b8d6-93041212fedd.pdf"}],"financialInterests":"","formattedTitle":"The interaction between nitrogen source and light intensity affects the biomass and phenotypic plasticity of Scenedesmus obliquus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicroalgae are integral to aquatic ecosystems, serving as primary producers that underpin the structure and functioning of food webs and biogeochemical cycles. In particular, nitrogen is a key elemental constituent of many cellular macromolecules and also a crucial nutrient for organisms (Zhang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In natural waters, nitrogen is a critical factor influencing algal population dynamics, community compositions, and ecosystem functions. Microalgae can utilize various forms of nitrogen sources, including inorganic (e.g. nitrate, ammonia) and organic nitrogen (e.g. urea, nucleic acids, amino acids) (Zhang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among them, nitrate and ammonium are primary nitrogen sources for most phytoplankton. Nitrate is initially reduced to nitrite by nitrate reductase (NR) in cytoplasm, and then nitrite is converted to ammonia by nitrite reductase in chloroplast (Hou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The energy cost of ammonia assimilation is lower than that of nitrate, leading to preferential use of ammonium in many autotrophic species, but nitrate is the most favored nitrogen source because it is more abundant in natural environment (Bloom et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Different nitrogen sources can lead to distinct physiological and metabolic responses in microalgae, affecting their suitability for various applications. In the case of \u003cem\u003eScenedesmus bijugatus\u003c/em\u003e, nitrate performs better for biomass growth, and urea results in almost equal biomass as nitrate (Arumugam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Ammonium can be toxic to cells at elevated concentrations and can cause growth inhibition or even cell death, despite mixotrophic cultivation with ammonium could greatly promotes the growth, biomass accumulation, total lipid yield, and notably the triacylglycerol content in microalgae (Sittisaree et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, careful selection of the nitrogen source is crucial for optimizing microalgal cultures to meet specific research or industrial objectives.\u003c/p\u003e \u003cp\u003eLight is the dominant factor determining the photosynthesis, productivity and metabolic pathways of phytoplankton. The quality, intensity, and period of light all have significant effects on the amount of light energy received by microalgae, and these effects are species-dependent (Latsos et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Increasing light intensity impacts microalgal growth through four phases\u0026mdash;lag, limitation, saturation, and inhibition\u0026mdash;with growth ceasing below critical intensity or diminishing at very high intensities due to photodamage to photosynthetic proteins (Ogbonna and Tanaka \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Light is not only essential for photosynthesis but also influences various signaling pathways that regulate nutrient uptake, cell division, and morphological changes (Dickman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In general, the optimal light intensity range for the growth of microalgae is 26\u0026ndash;400 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, depending on the species (Maltsev et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, for \u003cem\u003eAnkistrodesmus convolutus\u003c/em\u003e and \u003cem\u003eChlorella vulgaris\u003c/em\u003e, the phosphate uptake rates increase during the daytime and decrease at night (Ahn et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Trabelsi et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) showed light intensity had a positive influence on growth rate and extracellular polymeric substances concentration of cyanobacterium \u003cem\u003eArthrospira platensis.\u003c/em\u003e With increasing light intensity from 10 to 100 \u0026micro;E m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the maximum nitrate uptake rate of \u003cem\u003eProrocentrum micans\u003c/em\u003e increase from 3.6 to 10.8 pM cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the half saturation constant increase from 4.1 to 6.9 \u0026micro;M (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e, elevated light intensity induces cell division, whereas reduced light intensity leads to growth without division, and under continuous lighting, cell volume expands as light intensity diminishes (Wanka \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1959\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eScenedesmus obliquus\u003c/em\u003e, a freshwater microalga, is well documented for its phenotypic plasticity, a trait that is critical for its survival and adaptation to varying environmental conditions (Zhu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This phenotypic plasticity is largely manifested as the ability of organisms to alter their physiology, morphology, or development in response to environmental changes (Sultan \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). \u003cem\u003eS. obliquus\u003c/em\u003e mainly exists as unicells or two-, four- or eight-celled coenobia and varies in morphological phenotype (Pancha et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Morphological changes in \u003cem\u003eS. obliquus\u003c/em\u003e are mainly induced by changes in nutrient concentration, pH, surfactant or allochemicals released from grazers (L\u0026uuml;rling \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Morphological responses of \u003cem\u003eS. obliquus\u003c/em\u003e to competitive pressures are closely related to the mode of competition, with unicellular and small colonies dominating when there is direct resource competition with macroalgae such as \u003cem\u003eChara\u003c/em\u003e and \u003cem\u003eMyriophyllum\u003c/em\u003e (L\u0026uuml;rling et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), but multicellular morphology dominates when there is competition for chemotaxis from algae such as \u003cem\u003eStratiotes aloides\u003c/em\u003e (Mulderij et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the field, unicellular \u003cem\u003eS. obliquus\u003c/em\u003e are easily captured by zooplankton, therefore, the typical eight-celled particle is considered to resist herbivorous zooplankton (Hessen and van Donk \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). However, compared with unicells, the multicellular colonies have a smaller surface-to-volume ratio, which is less conducive to absorbing light and nutrient. The inherent plasticity in morphological adaptions confers competitive growth advantages in \u003cem\u003eS. obliquus\u003c/em\u003e that make them promising candidates for biodiesel production and wastewater treatment. Furthermore, the phenotypical plasticity in \u003cem\u003eS. obliquus\u003c/em\u003e is of significant interest for both basic ecological research and biomass production for biofuel.\u003c/p\u003e \u003cp\u003eIn aquatic ecosystems, nitrogen availability and light intensity are two pivotal environmental factors that influence algal growth and function. Understanding how these factors interact can provide deeper insights into the ecological dynamics of algae and contribute to the optimization of algae-based technologies in biofuel production and wastewater treatment. Based on the above research background, it is necessary to study the differentiated response of \u003cem\u003eS. obliquus\u003c/em\u003e to multiple nitrogen sources under varying light intensities. We formulated the scientific hypotheses: (1) Nitrogen source and light intensity can synergistically promote the growth of \u003cem\u003eS. obliquus\u003c/em\u003e; (2) Photosynthetic activity of \u003cem\u003eS. obliquus\u003c/em\u003e declines with light intensity for NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e as nitrogen sources, but remains stable in NaNO\u003csub\u003e3\u003c/sub\u003e; (3) With the increase of light intensity, NaNO\u003csub\u003e3\u003c/sub\u003e and NH\u003csub\u003e4\u003c/sub\u003eCl, as inorganic nitrogen sources, augment \u003cem\u003eS. obliquus\u003c/em\u003e to form colonies, while CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e as an organic nitrogen source diminishes colony formation. To test our hypothesis, we conducted an experiment to analyze the response of \u003cem\u003eS. obliquus\u003c/em\u003e to three type of nitrogen sources (NaNO\u003csub\u003e3\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e) and five light intensities (25, 50, 75, 100, 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and recorded the cell densities, colony distributions, photosynthetic efficiency and environmental nitrogen concentration during cultivation period.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Microalgal cultivation\u003c/h2\u003e \u003cp\u003eThe microalga \u003cem\u003eS. obliquus\u003c/em\u003e FACHB-416 was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, China (Wuhan). The alga was axenically maintained in BG-11 medium at 25 ℃ under fluorescent light at 40 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a light-dark period of 14 h: 10 h. Before the experiment, a portion of \u003cem\u003eS. obliquus\u003c/em\u003e population was inoculated into axenic BG-11 medium to maintain their exponential phase. The entire transfer process was conducted in a sterile environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental protocol\u003c/h2\u003e \u003cp\u003eIn this experiment, five light intensities were set at 25, 50 ,75, 100, 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with light: dark\u0026thinsp;=\u0026thinsp;14 h: 10 h, and three nitrogen resource were selected: NaNO\u003csub\u003e3\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. The concentration of each nitrogen was 18 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The \u003cem\u003eS. obliquus\u003c/em\u003e was cultured in 250 mL autoclaved Erlenmeyer flasks containing 150 mL of modified BG-11 under the condition as described above. Each treatment had three replicates, and the experiment lasted for 14 days. During the experiment, the flasks were shaken manually twice a day. The samples (5 mL) were taken daily: 1 mL was preserved with 2% Lugol\u0026rsquo;s solution for counting algal cells, 2 mL was used to detect photosynthetic activity of living cells, and the remaining 2 mL was used to measure the residual nitrogen concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of morphology and biomass production\u003c/h2\u003e \u003cp\u003eThe numbers of cells, including unicells and cells in different colonies (i.e., two-celled, four-celled, and eight-celled colonies), were counted by using a hemocytometer under a microscope. Cells per particle (including unicells and different colonies) were determined to indicate the mean colony size. The algal population biomass and cells per particle were calculated based on the cell counts. The specific growth rates (\u003cem\u003e\u0026micro;\u003c/em\u003e) were calculated as: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu =\\left[ {ln\\left( {{N_t}} \\right) - ln\\left( {{N_0}} \\right)} \\right]/\\left( {t - {t_0}} \\right)\\)\u003c/span\u003e\u003c/span\u003e, where \u003cem\u003eN\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e and \u003cem\u003eN\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e are the cell density at instant time (\u003cem\u003et\u003c/em\u003e) and initial time (\u003cem\u003et\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e), respectively. The cells per particle (\u003cem\u003eC\u003c/em\u003e) were calculated as: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(C=\\left( {{n_1} \\times 1+{n_2} \\times 2+{n_4} \\times 4+{n_8} \\times 8} \\right)/\\left( {{n_1}+{n_2}+{n_4}+{n_8}} \\right)\\)\u003c/span\u003e\u003c/span\u003e, where \u003cem\u003en\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e are the numbers of unicells, or two-celled, four-celled, eight-celled colonies, respectively. The population density over time was fitted using the Logistic model: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({N_t}=K/\\left[ {1+\\left( {K/{N_0} - 1} \\right){e^{ - rt}}} \\right]\\)\u003c/span\u003e\u003c/span\u003e, where \u003cem\u003eN\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e represents the population density on day \u003cem\u003et\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e represents the initial population density, \u003cem\u003eK\u003c/em\u003e represents the carrying capacity, \u003cem\u003er\u003c/em\u003e represents the instantaneous growth rate, and \u003cem\u003et\u003c/em\u003e represents the culture time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Photosynthesis measurement\u003c/h2\u003e \u003cp\u003ePhotosynthetic activities of \u003cem\u003eS. obliquus\u003c/em\u003e were measured using a Phyto-PAM (Walz, Germany), based on samples (2 mL) collected daily in \u0026ldquo;\u003cem\u003e2.2. Experimental protocol\u003c/em\u003e\u0026rdquo;. Photosynthetic parameters included maximal efficiency of PSII photochemistry (\u003cem\u003eFv/Fm\u003c/em\u003e), effective quantum yield of PSII photosynthetic efficiency (\u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e), and maximum relative electron rate (\u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e). Before measurement, algal suspensions needed to undergo 3\u0026ndash;5 minutes of dark adaptation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Data analyses\u003c/h2\u003e \u003cp\u003eAll values were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. The combined effects of light intensity and different nitrogen sources on photosynthetic efficiency and the number of cells per colony of \u003cem\u003eS. obliquus\u003c/em\u003e were analyzed by two-way RM ANOVA, followed by a Tukey test. Afterwards, specific growth rate was analyzed by one-way ANOVA and two-way ANOVA, then followed by Tukey test. All statistical analyses were performed using SigmaPlot 14.0, Origin 2021. The significance level was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Biomass production\u003c/h2\u003e \u003cp\u003eDuring the experiment, the biomass production of \u003cem\u003eS. obliquus\u003c/em\u003e cultured in three nitrogen sources increased substantially, and all treatments were maintained proliferation until they reached carrying capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Evidently, when NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e were used as nitrogen sources, the growth of \u003cem\u003eS. obliquus\u003c/em\u003e performed better than that in NH\u003csub\u003e4\u003c/sub\u003eCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). One-way ANOVA showed that the growth of \u003cem\u003eS. obliquus\u003c/em\u003e cultured in NH\u003csub\u003e4\u003c/sub\u003eCl was not influenced by different light intensities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). When NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e were set as nitrogen sources, there was no significant difference in the specific growth rates under the light intensity ranging from 25 to 75 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; however, when the light intensities were increased to 100 and 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the growth rates were significantly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Two-way ANOVA suggested that the nitrogen source type (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;36.224, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and light intensity (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;23.030, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) had significant effects on the growth of \u003cem\u003eS. obliquus\u003c/em\u003e, but there was no interaction between them (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.447, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.218). Generally, higher light intensities led to earlier attainment of carrying capacities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c, e). However, the theoretical maximums of carrying capacities were achieved at 75 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for both the NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Photosynthetic efficiency\u003c/h2\u003e \u003cp\u003eGenerally, the photosynthetic parameters, including \u003cem\u003eFv/Fm\u003c/em\u003e, \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e,\u003c/sub\u003e exhibited substantial variations in response to diverse nitrogen resources under a constant light intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Two-way RM ANOVA showed that the \u003cem\u003eFv/Fm\u003c/em\u003e, \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e were significantly affected by light intensity and nitrogen source type, and there was a highly significant interaction between the two factors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In NH\u003csub\u003e4\u003c/sub\u003eCl treatments, increased light intensities were correlated with reduced \u003cem\u003eFv/Fm\u003c/em\u003e and \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e, with values of both parameters being obviously lower at a light intensity of 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compared to the other conditions. In NaNO\u003csub\u003e3\u003c/sub\u003e treatments, the \u003cem\u003eFv/Fm\u003c/em\u003e was similar under different light intensities, and basically maintained stable at around 0.72 from the 9th day onwards, while the \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e was lower under the light intensity of 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e than under other light intensities. Nitrogen source CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e induced a rapid decline in \u003cem\u003eFv/Fm\u003c/em\u003e and \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e, stabilizing by the 7th day. For \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e, the maximum value occurred at 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, whereas the minimum value occurred at 100 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e across all three nitrogen source treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTwo-way RM ANOVA for the effects of nitrogen source type and light intensity on \u003cem\u003eFv/Fm\u003c/em\u003e, \u003cem\u003eФ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e, and cells per particle of \u003cem\u003eS. obliquus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eFv/Fm\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cem\u003eФ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eCells per particle\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrogen source (A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e46.901\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLight intensity (B)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA \u0026times; B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Algal morphology\u003c/h2\u003e \u003cp\u003eThe cells per particle provided an overall evaluation on the morphology of the algal population (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At the onset of the experiment, the number of cells per particle of \u003cem\u003eS. obliquus\u003c/em\u003e was relatively consistent among all groups that underwent preliminary treatment, maintaining around 2. The cells per particle induced by different types of nitrogen sources and light intensities exhibited fluctuations during the early phase, followed by stabilization in the later phase (9-14th days). Two-way RM ANOVA revealed that both nitrogen source and light intensity significantly influenced the number of cells per particle, and there was a significant interaction between the light intensity and nitrogen source type (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Ultimately, the cells per particle of each treatment cultured with NH\u003csub\u003e4\u003c/sub\u003eCl, NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e as nitrogen sources were maintained at approximately 2.1, 4, and 1.1, respectively.\u003c/p\u003e \u003cp\u003eVaried colonial types also reflected the phenotypic plasticity of \u003cem\u003eS. obliquus\u003c/em\u003e in response to different environmental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Initially, the proportion of cells in different colonies was similar across all groups. However, from third day, Among the four primary morphotypes, the unicells, two-, four- and eight-celled colonies were significantly changed with the different nitrogen sources and light intensity. In NaNO\u003csub\u003e3\u003c/sub\u003e treatment, the proportion of four-celled colony increased sharply. Compared with the first day, when NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e were used as the nitrogen source, the number of unicells increased under five light intensities, rising by approximately 20% and 50%, respectively. After the 13-day culture period, the proportion of cells in different colonies in each treatment group reached a relatively stable level. Specifically, two-celled (~\u0026thinsp;20%) and four-celled (~\u0026thinsp;70%) colonies grew rapidly in response to NH\u003csub\u003e4\u003c/sub\u003eCl. Meanwhile, four-celled colony (~\u0026thinsp;90%) were predominant in NaNO\u003csub\u003e3\u003c/sub\u003e. In contrast, the high proportion of unicells (~\u0026thinsp;75%) grew in CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Nitrogen content in the test solution\u003c/h2\u003e \u003cp\u003eBased on the changes of the nitrogen content of test solution during the experiment, nitrogen contents in NH\u003csub\u003e4\u003c/sub\u003eCl, NaNO\u003csub\u003e3\u003c/sub\u003e, and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatment groups decreased about 5.4\u0026ndash;9.4, 14.0-17.9 and 12.8\u0026ndash;16.9 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, until the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, among the three types of nitrogen sources, \u003cem\u003eS. obliquus\u003c/em\u003e has stronger removal capacity for NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, but less for NH\u003csub\u003e4\u003c/sub\u003eCl. Under consistent nitrogen sources, increased light intensity facilitated the uptake of nitrogen by \u003cem\u003eS. obliquus\u003c/em\u003e, aligning with population dynamics. Moreover, the remaining total nitrogen content in the test solutions showed a significant negative correlation with algal cell densities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that there were differences in the growth of \u003cem\u003eS. obliquus\u003c/em\u003e and their nitrogen consumption among different nitrogen sources.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study showed there was a positive correlation between increased light intensity and the growth rate of \u003cem\u003eS. obliquus\u003c/em\u003e in all three nitrogen source treatments, with the NH\u003csub\u003e4\u003c/sub\u003eCl treatments exhibiting a statistically non-significant increase, potentially due to the lower uptake of NH\u003csub\u003e4\u003c/sub\u003eCl by \u003cem\u003eS. obliquus.\u003c/em\u003e Additionally, NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e presence leads to a marked decrease in \u003cem\u003eS. obliquus\u003c/em\u003e photosynthesis with increasing light intensity, contrasting with the stability observed under NaNO\u003csub\u003e3\u003c/sub\u003e nitrogen conditions. During the early stage of the experiment, \u003cem\u003eS. obliquus\u003c/em\u003e under various nitrogen treatments generally showed that increased unicells were correlated with higher light intensity. Subsequently, with the increase in light intensity, NaNO\u003csub\u003e3\u003c/sub\u003e facilitated the formation of colonies, whereas the CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e tended to promote the proliferation of unicells. These results generally confirmed our previous hypotheses.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Population dynamics of S. obliquus under different light intensities with different nitrogen sources\u003c/h2\u003e \u003cp\u003eLight intensity and nitrogen source were found to synergistically promote algal growth in our experiment. Specifically, higher cell density and growth rates were correlated with increased light intensity across all nitrogen sources. Some studies have shown an increase in specific growth rate with light intensity in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e, \u003cem\u003eChlorella vulgaris\u003c/em\u003e, \u003cem\u003eCryptomonas sp.\u003c/em\u003e, \u003cem\u003eDunaliella viridis\u003c/em\u003e, \u003cem\u003eNeochloris oleoabundans\u003c/em\u003e, \u003cem\u003eScenedesmus\u003c/em\u003e sp., etc., coinciding with the maximum lipid production (Hwang and Maier \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Weng et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Interestingly, the difference in growth rate caused by light intensity was significant in the NaNO\u003csub\u003e3\u003c/sub\u003e and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments, but not in the NH\u003csub\u003e4\u003c/sub\u003eCl treatments. As previously reported that NaNO\u003csub\u003e3\u003c/sub\u003e is the preferred nitrogen source for microalgal growth of \u003cem\u003eScenedesmus\u003c/em\u003e at the same light intensity, and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e results in almost equal biomass as NaNO\u003csub\u003e3\u003c/sub\u003e (Arumugam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The influence of NH\u003csub\u003e4\u003c/sub\u003eCl on microalgal growth is intricate, as it exhibits dual physiological effects. NH\u003csub\u003e4\u003c/sub\u003eCl supplies nitrogen for microalgal growth but may be toxic at high concentrations, thus inhibiting proliferation. For example, \u003cem\u003eEllipsoidion\u003c/em\u003e sp. grows faster with NH\u003csub\u003e4\u003c/sub\u003eCl than NaNO\u003csub\u003e3\u003c/sub\u003e during the logarithmic phase, but the trend reverses in the post-logarithmic phase (Xu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In present study, we also found that the cell abundances of all treatments were similar in early period, however, growth of \u003cem\u003eS. obliquus\u003c/em\u003e in NH\u003csub\u003e4\u003c/sub\u003eCl was markedly suppressed in later period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The results indicated that, in the initial phase, ammonium enters directly into the algal cells mainly in the form non-ionic NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and accumulates abundantly with minimal energy consumption. However, excessive ammonium ions cannot be rapidly transferred to synthesize proteins. Consequently, this induces ammonium intoxication of the algal cells, thus affecting the growth of microalgae (Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, the influx of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e into microalgal cells might lead to acidification, which is a condition detrimental to the growth of \u003cem\u003eS. obliquus\u003c/em\u003e (Kleiner \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Miura et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Microalgae transport CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e into cells via specific transporters and then convert it to ammonium nitrogen through urease, producing carbon dioxide (Gutierrez et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e supplies both carbon and nitrogen to \u003cem\u003eS. obliquus\u003c/em\u003e, potentially leading to ammonium ion accumulation in later stages, yet carbon supplementation can alleviate the associated negative impact (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCorrelation analysis indicated that lower residual nitrogen content in test solution was associated with higher algal cell density on the final day (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Therefore, the nitrogen removal efficiency of \u003cem\u003eS. obliquus\u003c/em\u003e under different nitrogen sources may be mainly governed by population growth, such as treatments with NaNO\u003csub\u003e3\u003c/sub\u003e under high light intensities. The lower population growth in ammonium treatments ultimately leads to reduced nitrogen uptake and removal efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Algal cultivation in wastewater systems was has emerged as a sustainable strategy for removing the nutrients form the wastewaters and generating energy from the biomass (Bhattacharjee and Siemann \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Currently, \u003cem\u003eS. abundans\u003c/em\u003e has been proven to effectively remove pollutants from domestic wastewater, which contains as much as 90.73% dissolved inorganic nitrogen (SundarRajan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Previous research also found that \u003cem\u003eChlorococcum sp\u003c/em\u003e. GD, \u003cem\u003eParachlorella kessleri\u003c/em\u003e TY and \u003cem\u003eScenedesmus sp.\u003c/em\u003e LX1 exhibited higher growth potential and nitrogen removal ability in wastewater with NaNO\u003csub\u003e3\u003c/sub\u003e, which was superior to wastewater with NH\u003csub\u003e4\u003c/sub\u003eCl, and they emphasized that this phenomenon can be attributed to the suppressive impact of acidic pH during algal cultivation process (Lv et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xin et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Another study also provided evidence that organic carbon increased algal biomass and nitrogen removal capacity (Ji et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which may be the reason for the highest urea removal efficiency in this experiment. Consistent with previous findings, increasing light intensity is conducive for algae to rapid removal of nitrogen from the environment at the same time (Ouyang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In general, the nitrogen removal ability of \u003cem\u003eS. obliquus\u003c/em\u003e varied for different nitrogen sources.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Effects of nitrogen sources on photosynthetic system of S. obliquus under different light intensities\u003c/h2\u003e \u003cp\u003eVarying nitrogen source conditions triggered distinct responses of algal cells, leading to heterogeneous response levels to light. Our data revealed that \u003cem\u003eFv/Fm\u003c/em\u003e and \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e values were stable under various light intensities in NaNO\u003csub\u003e3\u003c/sub\u003e treatments, whereas in NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments, these values declined with prolonged culture time. Several researches have shown that NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e can exert inhibitory effects on various phytoplankton species, including but not limited to \u003cem\u003eC. vulgaris\u003c/em\u003e, \u003cem\u003eMicrocystis aeruginosa\u003c/em\u003e, \u003cem\u003eNannochloropsis oceanica\u003c/em\u003e, and \u003cem\u003eS. quadricauda\u003c/em\u003e. Furthermore, the inhibitory impact is not confined to microscopic phytoplankton; it extends to larger aquatic macrophytes as well. For instance, species such as \u003cem\u003eCabomba caroliniana\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eSargassum thunbergii\u003c/em\u003e have been documented to exhibit reduced growth and biomass when subjected to similar treatments (Huang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). A reduction in \u003cem\u003eFv/Fm\u003c/em\u003e and \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e suggests a decrease in PSII photochemistry efficiency or a disorder in or damage to the photosynthetic apparatus (Gao et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The changes are likely due to ammonium-induced disruption of the thylakoid proton gradient, in other words, ammonia ions enter thylakoid, creating an acidic environment, which decreases the transmembrane proton gradient necessary to drive ATP synthesis from ADP (Kikeri et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Tikhonov \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This disruption of the thylakoid proton gradient can lead to a reduction in photosynthetic efficiency, as the proton gradient is a crucial driving force for ATP production, which is essential for the Calvin cycle and overall plant energy metabolism (H\u0026ouml;hner et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Another hypothesis is that ammonia ions may ligate to the PSII oxygen evolution reaction core, thereby increasing photosensitivity and consequently enhancing photosystem damage (Cazzaniga et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e also supplies ammonia nitrogen to the system, hydrolysis of CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e produces CO\u003csub\u003e2\u003c/sub\u003e, potentially enhancing the carbon concentrating mechanism as a new carbon source and optimizing nitrogen supply, which promotes microalgae to adjust their photosynthetic carbon fixation strategy. Thus, when CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e is used as the nitrogen source, \u003cem\u003eS. obliquus\u003c/em\u003e can maintain normal growth despite a decline in photosynthetic efficiency. In the present study, under various nitrogen source conditions, the maximum value of \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e occurred at 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while the \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e values within the range of 25\u0026ndash;100 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were closely. It is widely known that the \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of plants with different genotypes is similar under low light conditions (Howard et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The impact of light intensity on the \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e appears to be more pronounced. However, compared with \u003cem\u003eETR\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e under the three nitrogen sources, the efficiency of \u003cem\u003eS. obliquus\u003c/em\u003e in utilizing light is obviously affected, which once again proves that NH\u003csub\u003e4\u003c/sub\u003eCl causes stress on the \u003cem\u003eS. obliquus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Morphological plasticity of S. obliquus in response to nitrogen sources and light intensities\u003c/h2\u003e \u003cp\u003eDue to its distinctive biological traits, \u003cem\u003eS. obliquus\u003c/em\u003e has been extensively utilized across various domains, including animal feed, energy production, and sewage disposal. The multicellular colonies of \u003cem\u003eS. obliquus\u003c/em\u003e enhance sedimentation rates and confers greater stress tolerance, which not only facilitates the efficiency of resource harvesting but also augments the effectiveness of algal removal during biological water purification process. However, the diminutive size of the unicellular \u003cem\u003eS. obliquus\u003c/em\u003e renders it an ideal food source for zooplankton. Based on these applications, understanding the morphological dynamics of \u003cem\u003eS. obliquus\u003c/em\u003e is crucial for optimizing its use in these fields. We observed that under elevated light intensities, the multicellular colonies of \u003cem\u003eS. obliquus\u003c/em\u003e escalated in cultures supplemented with NaNO\u003csub\u003e3\u003c/sub\u003e. In contrast, urea led to a progressive predominance of unicellular forms within the culture medium.\u003c/p\u003e \u003cp\u003ePreviously, the researchers emphasized that treatment with nitrate or nitrogen starvation can induce a transition from unicells to multicellular colonies (Pancha et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, when a low concentration of ammonium nitrogen is utilized as the nitrogen source, the algae remain unicellular (Pancha et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Tukaj et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) observed that the formation of \u003cem\u003eS. armatus\u003c/em\u003e in eight-celled and sixteen-celled colonies increased with elevated light intensity, which was potentially driven by the accelerated division rate of algal cells under high light intensity. And following incubation under both low (10 and 30 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) and high (120 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) light intensities, \u003cem\u003eNostoc sphaeroides\u003c/em\u003e colonies softened and ultimately lost their spherical aggregation (Ma et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In another study, 70\u0026ndash;80% of \u003cem\u003eS. obliquus\u003c/em\u003e existed as unicells at light intensities of 50 and 60 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, as well as at below 25 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, however, at 30 and 35 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the proportion of unicells decreased, and the proportion of four-celled colonies and above was approximately 40% (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn aquatic ecosystems, the microalgal morphology significantly influences their migratory velocities, resistance to predation pressures, and specific surface area (Pančić and Ki\u0026oslash;rboe \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The formation of algal colonies primarily occurs through two pathways: (1) aggregation due to the failure of dividing cells to detach, and (2) adhesion of existing individual cells. It is generally believed that colonies of \u003cem\u003eS. obliquus\u003c/em\u003e are formed via the first pathway, as the arrangement of cells in these colonies is orderly and regular, whereas pathway two would likely result in irregular cell arrangements (Bišov\u0026aacute; and Zachleder \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dong et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the present study, \u003cem\u003eS. obliquus\u003c/em\u003e showed optimal growth in NaNO\u003csub\u003e3\u003c/sub\u003e treatments, possibly because the increased light intensity promoted cell division and NaNO\u003csub\u003e3\u003c/sub\u003e stimulated polysaccharide secretion (Moreira et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), thus the most multicellular colonies were harvested. Ammonium salts have been reported to enhance the biosynthesis of polysaccharides in algae, albeit to a lesser extent compared to KNO\u003csub\u003e3\u003c/sub\u003e (Arad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), but the high levels of ammonium will rapidly accumulate within the algal cells, causing toxicity and alterations in cellular biochemical composition, thereby changing colonial morphology. Therefore, we obtained an only moderately multicellular colony when NH\u003csub\u003e4\u003c/sub\u003eCl as the nitrogen source. These two types of nitrogen sources seem to be quite different to the mechanism of CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. Although CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, serving as an organic nitrogen source, enhance the growth of \u003cem\u003eS. obliquus\u003c/em\u003e by supplementing with additional carbon source, we found that the CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments were dominated by unicells under five light intensities across the experiment. Empirical evidence indicates that CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e leads to a significant reduction in the extracellular polysaccharide content of microalgae, particularly at the stable phase of microalgae (Lupi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Consequently, in treatments employing CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, the population of \u003cem\u003eS. obliquus\u003c/em\u003e is predominantly composed of unicells. On the other hand, multicellular colonies show greater resistance to dark loss and high light intensity than unicells, with higher \u003cem\u003eFv/Fm\u003c/em\u003e and \u003cem\u003eNPQ\u003c/em\u003e values (Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Our photosynthetic efficiency data suggest that urea may impair the carbon concentrating mechanism, reducing the need for colony formation to capture light energy.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe growth of \u003cem\u003eS. obliquus\u003c/em\u003e across all treatments was significantly enhanced by the combined effects of sufficient nitrogen source and high light intensity. In the case of NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e as nitrogen sources, photosynthetic activity decreased with increasing light intensity, whereas it remained relatively stable under NaNO\u003csub\u003e3\u003c/sub\u003e condition. Meanwhile, NaNO\u003csub\u003e3\u003c/sub\u003e facilitated colony formation in \u003cem\u003eS. obliquus\u003c/em\u003e with increasing light intensity, whereas the CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e had the opposite effect, promoting the formation of unicells. In addition, the nitrogen removal ability of \u003cem\u003eS. obliquus\u003c/em\u003e varied for different nitrogen sources. This study elucidated the combined impact of nitrogen sources and light intensity on the growth and phenotypic plasticity of \u003cem\u003eS. obliquus\u003c/em\u003e, providing a foundation for optimizing these factors to enhance algal growth efficiency and obtain different colonies of \u003cem\u003eS. obliquus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJiyan Long:\u003c/strong\u003e Data curation, Formal analysis, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eYiqi Feng:\u003c/strong\u003e Validation, Investigation. \u003cstrong\u003eDecai Huang:\u003c/strong\u003e Validation, Investigation. \u003cstrong\u003eYulu Lei:\u003c/strong\u003e Investigation, Data curation, Formal analysis\u003cstrong\u003e. Xuexia Zhu:\u003c/strong\u003e Methodology, Conceptualization, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eZhou Yang:\u003c/strong\u003e Conceptualization, Supervision, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the anonymous reviewers for their constructive comments. This study was supported by the National Natural Science Foundation of China (32271626) and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of informed consent, human/animal rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicts, informed consent, human or animal rights applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhn C-Y, Chung A-S, Oh H-M (2002) Diel Rhythm of Algal Phosphate Uptake Rates in P-Limited Cyclostats and Simulation of Its Effect on Growth and Competition. J Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e38: 695-704. doi.org/10.1046/j.1529-8817.2002.01232.x\u003c/li\u003e\n \u003cli\u003eArad SM, Friedman OD, Rotem A (1988) Effect of Nitrogen on Polysaccharide Production in a \u003cem\u003ePorphyridium\u0026nbsp;\u003c/em\u003esp. Appl Environ Microbiol\u003cem\u003e\u0026nbsp;\u003c/em\u003e54: 2411-2414. doi.org/10.1128/aem.54.10.2411-2414.1988\u003c/li\u003e\n \u003cli\u003eArumugam M, Agarwal A, Arya MC, Ahmed Z (2013) Influence of nitrogen sources on biomass productivity of microalgae \u003cem\u003eScenedesmus bijugatus\u003c/em\u003e. Bioresour Technol\u003cem\u003e\u0026nbsp;\u003c/em\u003e131: 246-249. doi.org/10.1016/j.biortech.2012.12.159\u003c/li\u003e\n \u003cli\u003eBhattacharjee M, Siemann E (2015) Low algal diversity systems are a promising method for biodiesel production in wastewater fed open reactors. Algae\u003cem\u003e\u0026nbsp;\u003c/em\u003e30: 67-79. doi.org/10.4490/algae.2015.30.1.067\u003c/li\u003e\n \u003cli\u003eBi\u0026scaron;ov\u0026aacute; K, Zachleder V (2014) Cell-cycle regulation in green algae dividing by multiple fission. J Exp Bot\u003cem\u003e\u0026nbsp;\u003c/em\u003e65: 2585-2602. doi.org/10.1093/jxb/ert466\u003c/li\u003e\n \u003cli\u003eBloom AJ, Sukrapanna SS, Warner RL (1992) Root respiration associated with ammonium and nitrate absorption and assimilation by barley. Plant Physiol\u003cem\u003e\u0026nbsp;\u003c/em\u003e99: 1294-1301. doi.org/10.1104/pp.99.4.1294\u003c/li\u003e\n \u003cli\u003eCazzaniga S, Kim M, Bellamoli F, Jeong J, Lee S, Perozeni F, Pompa A, Jin E, Ballottari M (2020) Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. Plant Cell Environ\u003cem\u003e\u0026nbsp;\u003c/em\u003e43: 496-509. doi.org/10.1111/pce.13680\u003c/li\u003e\n \u003cli\u003eDickman EM, Vanni MJ, Horgan MJ (2006) Interactive effects of light and nutrients on phytoplankton stoichiometry. Oecologia\u003cem\u003e\u0026nbsp;\u003c/em\u003e149: 676-689. doi.org/10.1007/s00442-006-0473-5\u003c/li\u003e\n \u003cli\u003eDong J, Gao Y, Chang M, Ma H, Han K, Tao X, Li Y (2017) Colony formation by the green alga \u003cem\u003eChlorella vulgaris\u0026nbsp;\u003c/em\u003ein response to the competitor\u003cem\u003e\u0026nbsp;Ceratophyllum demersum\u003c/em\u003e. Hydrobiologia\u003cem\u003e\u0026nbsp;\u003c/em\u003e805: 177-187. doi.org/10.1007/s10750-017-3294-0\u003c/li\u003e\n \u003cli\u003eGao Y, Bernard O, Fanesi A, Perr\u0026eacute; P, Lopes F (2024) The effect of light intensity on microalgae biofilm structures and physiology under continuous illumination. Sci Rep\u003cem\u003e\u0026nbsp;\u003c/em\u003e14: 1151. doi.org/10.1038/s41598-023-50432-6\u003c/li\u003e\n \u003cli\u003eGutierrez J, Kwan TA, Zimmerman JB, Peccia J (2016) Ammonia inhibition in oleaginous microalgae. Algal Res\u003cem\u003e\u0026nbsp;\u003c/em\u003e19: 123-127. doi.org/10.1016/j.algal.2016.07.016\u003c/li\u003e\n \u003cli\u003eHessen DO, van Donk E (1993) Morphological changes in \u003cem\u003eScenedesmu\u003c/em\u003es induced by substances released from \u003cem\u003eDaphnia\u003c/em\u003e. Arch Hydrobiol\u003cem\u003e\u0026nbsp;\u003c/em\u003e127: 129-140. doi.org/10.1127/archiv-hydrobiol/127/1993/129\u003c/li\u003e\n \u003cli\u003eH\u0026ouml;hner R, Aboukila A, Kunz H-H, Venema K (2016) Proton Gradients and Proton-Dependent Transport Processes in the Chloroplast. Front Plant Sci\u003cem\u003e\u0026nbsp;\u003c/em\u003e7: 218. doi.org/10.3389/fpls.2016.00218\u003c/li\u003e\n \u003cli\u003eHou L-l, Liu F, Zang X, Zhang X, He B, Ding Y, Song X, Xiao D, Wang H (2017) Cloning and transcription analysis of the nitrate reductase gene from \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e. Biotechnol Lett\u003cem\u003e\u0026nbsp;\u003c/em\u003e39: 589-597. doi.org/10.1007/s10529-016-2283-0\u003c/li\u003e\n \u003cli\u003eHoward MM, Bae A, K\u0026ouml;niger M (2019) The importance of chloroplast movement, nonphotochemical quenching, and electron transport rates in light acclimation and tolerance to high light in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Am J Bot\u003cem\u003e\u0026nbsp;\u003c/em\u003e106: 1444-1453. doi.org/10.1002/ajb2.1378\u003c/li\u003e\n \u003cli\u003eHuang W, Shao H, Li W, Jiang H, Chen Y (2017) Effects of urea on growth and photosynthetic metabolism of two aquatic plants (\u003cem\u003eCabomba caroliniana\u003c/em\u003e A. Gray and \u003cem\u003eElodea nuttallii\u003c/em\u003e (Planch.) H. St. John). Aquat Bot\u003cem\u003e\u0026nbsp;\u003c/em\u003e140: 69-77. doi.org/10.1016/j.aquabot.2016.04.003\u003c/li\u003e\n \u003cli\u003eHwang J-H, Maier N (2019) Effects of LED-controlled spatially-averaged light intensity and wavelength on \u003cem\u003eNeochloris oleoabundans\u0026nbsp;\u003c/em\u003egrowth and lipid composition. Algal Res\u003cem\u003e\u0026nbsp;\u003c/em\u003e41: 101573. doi.org/10.1016/j.algal.2019.101573\u003c/li\u003e\n \u003cli\u003eJi M, Gao H, Zhang J, Hu Z, Liang S (2024) Environmental impacts on algal\u0026ndash;bacterial-based aquaponics system by different types of carbon source addition: water quality and greenhouse gas emission. Environ Sci Pollut Res\u003cem\u003e\u0026nbsp;\u003c/em\u003e31: 26665-26674. doi.org/10.1007/s11356-024-32717-z\u003c/li\u003e\n \u003cli\u003eKikeri D, Sun A, Zeidel ML, Hebert SC (1989) Cell membranes impermeable to NH\u003csub\u003e3\u003c/sub\u003e. Nature\u003cem\u003e\u0026nbsp;\u003c/em\u003e339: 478-480. doi.org/10.1038/339478a0\u003c/li\u003e\n \u003cli\u003eKim G, Mujtaba G, Lee K (2016) Effects of nitrogen sources on cell growth and biochemical composition of marine chlorophyte Tetraselmis sp. for lipid production. Algae\u003cem\u003e\u0026nbsp;\u003c/em\u003e31: 257-266. doi.org/10.4490/algae.2016.31.8.18\u003c/li\u003e\n \u003cli\u003eKleiner D (1981) The transport of NH\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eand HN\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e across biological membranes. Biochim Biophys Acta, Rev Bioenerg\u003cem\u003e\u0026nbsp;\u003c/em\u003e639: 41-52. doi.org/10.1016/0304-4173(81)90004-5\u003c/li\u003e\n \u003cli\u003eLatsos C, van Houcke J, Blommaert L, Verbeeke GP, Kromkamp J, Timmermans KR (2021) Effect of light quality and quantity on productivity and phycoerythrin concentration in the cryptophyte \u003cem\u003eRhodomonas\u0026nbsp;\u003c/em\u003esp. J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e33: 729-741. doi.org/10.1007/s10811-020-02338-3\u003c/li\u003e\n \u003cli\u003eLee KH, Jeong HJ, Kim HJ, Lim AS (2017) Nitrate uptake of the red tide dinoflagellate \u003cem\u003eProrocentrum micans\u003c/em\u003e measured using a nutrient repletion method: effect of light intensity. Algae\u003cem\u003e\u0026nbsp;\u003c/em\u003e32: 139-153. doi.org/10.4490/algae.2017.32.5.20\u003c/li\u003e\n \u003cli\u003eLi M, Gao L, Lin L (2016) Specific growth rate, colonial morphology and extracellular polysaccharides (EPS) content of \u003cem\u003eScenedesmus obliquus\u003c/em\u003e grown under different levels of light limitation. Ann Limnol, Int J Limnol\u003cem\u003e\u0026nbsp;\u003c/em\u003e51: 329-334. doi.org/10.1051/limn/2015033\u003c/li\u003e\n \u003cli\u003eLi X, Huff J, Crunkleton DW, Johannes TW (2023) Light intensity and spectral quality modulation for improved growth kinetics and biochemical composition of \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. J Biotechnol\u003cem\u003e\u0026nbsp;\u003c/em\u003e375: 28-39. doi.org/10.1016/j.jbiotec.2023.08.007\u003c/li\u003e\n \u003cli\u003eLi XM, Zhang QS, Tang YZ, Yu YQ, Liu HL, Li LX (2014) Highly efficient photoprotective responses to high light stress in \u003cem\u003eSargassum thunbergii\u003c/em\u003e germlings, a representative brown macroalga of intertidal zone. J Sea Res\u003cem\u003e\u0026nbsp;\u003c/em\u003e85: 491-498. doi.org/10.1016/j.seares.2013.08.004\u003c/li\u003e\n \u003cli\u003eLiu J, Yuan C, Hu G, Li F (2012) Effects of Light Intensity on the Growth and Lipid Accumulation of Microalga \u003cem\u003eScenedesmus\u003c/em\u003e sp. 11-1 Under Nitrogen Limitation. Appl Biochem Biotechnol\u003cem\u003e\u0026nbsp;\u003c/em\u003e166: 2127-2137. doi.org/10.1007/s12010-012-9639-2\u003c/li\u003e\n \u003cli\u003eLiu N, Zhang H, Zhao J, Xu Y, Ge F (2020) Mechanisms of cetyltrimethyl ammonium chloride-induced toxicity to photosystem II oxygen evolution complex of \u003cem\u003eChlorella vulgaris\u0026nbsp;\u003c/em\u003eF1068. J Hazard Mater\u003cem\u003e\u0026nbsp;\u003c/em\u003e383: 121063. doi.org/10.1016/j.jhazmat.2019.121063\u003c/li\u003e\n \u003cli\u003eLiu X, Wang K, Wang J, Zuo J, Peng F, Wu J, San E (2019) Carbon dioxide fixation coupled with ammonium uptake by immobilized \u003cem\u003eScenedesmus obliquus\u0026nbsp;\u003c/em\u003eand its potential for protein production. Bioresour Technol\u003cem\u003e\u0026nbsp;\u003c/em\u003e289: 121685. doi.org/10.1016/j.biortech.2019.121685\u003c/li\u003e\n \u003cli\u003eLupi FM, Fernandes HML, Tom\u0026eacute; MM, S\u0026aacute;-Correia I, Novais JM (1994) Influence of nitrogen source and photoperiod on exopolysaccharide synthesis by the microalga \u003cem\u003eBotryococcus braunii\u003c/em\u003e UC 58. Enzyme Microb Technol\u003cem\u003e\u0026nbsp;\u003c/em\u003e16: 546-550. doi.org/10.1016/0141-0229(94)90116-3\u003c/li\u003e\n \u003cli\u003eL\u0026uuml;rling M (2006) Effects of a surfactant (FFD-6) on \u003cem\u003eScenedesmus\u0026nbsp;\u003c/em\u003emorphology and growth under different nutrient conditions. Chemosphere\u003cem\u003e\u0026nbsp;\u003c/em\u003e62: 1351-1358. doi.org/10.1016/j.chemosphere.2005.07.031\u003c/li\u003e\n \u003cli\u003eL\u0026uuml;rling M, van Geest G, Scheffer M (2006) Importance of Nutrient Competition and Allelopathic Effects in Suppression of the Green Alga \u003cem\u003eScenedesmus obliquus\u003c/em\u003e by the Macrophytes\u003cem\u003e\u0026nbsp;Chara\u003c/em\u003e, \u003cem\u003eElodea\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMyriophyllum\u003c/em\u003e. Hydrobiologia\u003cem\u003e\u0026nbsp;\u003c/em\u003e556: 209-220. doi.org/10.1007/s10750-005-1168-3\u003c/li\u003e\n \u003cli\u003eLv J, Wang X, Feng J, Liu Q, Nan F, Jiao X, Xie S (2019) Comparison of growth characteristics and nitrogen removal capacity of five species of green algae. J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e31: 409-421. doi.org/10.1007/s10811-018-1542-y\u003c/li\u003e\n \u003cli\u003eMa R, Lu F, Bi Y, Hu Z (2015) Effects of light intensity and quality on phycobiliprotein accumulation in the cyanobacterium Nostoc sphaeroides K\u0026uuml;tzing. Biotechnol Lett\u003cem\u003e\u0026nbsp;\u003c/em\u003e37: 1663-1669. doi.org/10.1007/s10529-015-1831-3\u003c/li\u003e\n \u003cli\u003eMaltsev Y, Maltseva K, Kulikovskiy M, Maltseva S (2021) Influence of Light Conditions on Microalgae Growth and Content of Lipids, Carotenoids, and Fatty Acid Composition. Biology\u003cem\u003e\u0026nbsp;\u003c/em\u003e10: 1060. doi.org/10.3390/biology10101060\u003c/li\u003e\n \u003cli\u003eMiura R, Furuhashi K, Hasegawa F, Kaizu Y, Imou K (2021) Calcium carbonate prevents \u003cem\u003eBotryococcus braunii\u003c/em\u003e growth inhibition caused by medium acidification. J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e34: 177-183. doi.org/10.1007/s10811-021-02622-w\u003c/li\u003e\n \u003cli\u003eMoreira JB, Vaz BD, Cardias BB, Cruz CG, Almeida AC, Costa JA, Morais MG (2022) Microalgae Polysaccharides: An Alternative Source for Food Production and Sustainable Agriculture. Polysaccharides\u003cem\u003e\u0026nbsp;\u003c/em\u003e3: 441-457. doi.org/10.3390/polysaccharides3020027\u003c/li\u003e\n \u003cli\u003eMulderij G, Mooij WM, Van Donk E (2005) Allelopathic growth inhibition and colony formation of the green alga \u003cem\u003eScenedesmus obliquus\u0026nbsp;\u003c/em\u003eby the aquatic macrophyte \u003cem\u003eStratiotes aloides\u003c/em\u003e. Aquat Ecol\u003cem\u003e\u0026nbsp;\u003c/em\u003e39: 11-21. doi.org/10.1007/s10452-004-1021-1\u003c/li\u003e\n \u003cli\u003eOgbonna JC, Tanaka H (2000) Light requirement and photosynthetic cell cultivation - Development of processes for efficient light utilization in photobioreactors. J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e12: 207-218. doi.org/10.1023/a:1008194627239\u003c/li\u003e\n \u003cli\u003eOuyang Y, Zhao Y, Sun S, Hu C, Ping L (2015) Effect of light intensity on the capability of different microalgae species for simultaneous biogas upgrading and biogas slurry nutrient reduction. Int Biodeterior Biodegrad\u003cem\u003e\u0026nbsp;\u003c/em\u003e104: 157-163. doi.org/10.1016/j.ibiod.2015.05.027\u003c/li\u003e\n \u003cli\u003ePancha I, Chokshi K, George B, Ghosh T, Paliwal C, Maurya R, Mishra S (2014) Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077. Bioresource Technology\u003cem\u003e\u0026nbsp;\u003c/em\u003e156: 146-154. doi.org/10.1016/j.biortech.2014.01.025\u003c/li\u003e\n \u003cli\u003ePančić M, Ki\u0026oslash;rboe T (2018) Phytoplankton defence mechanisms: traits and trade‐offs. Biol Rev\u003cem\u003e\u0026nbsp;\u003c/em\u003e93: 1269-1303. doi.org/10.1111/brv.12395\u003c/li\u003e\n \u003cli\u003ePeng G, Fan Z, Wang X, Chen C (2016) Photosynthetic response to nitrogen source and different ratios of nitrogen and phosphorus in toxic cyanobacteria, \u003cem\u003eMicrocystis aeruginosa\u0026nbsp;\u003c/em\u003eFACHB-905. J Limnol\u003cem\u003e\u0026nbsp;\u003c/em\u003e75: 560-570. doi.org/10.4081/jlimnol.2016.1458\u003c/li\u003e\n \u003cli\u003eSittisaree W, Yokthongwattana K, Aonbangkhen C, Yingchutrakul Y, Krobthong S (2024) Effect of NH\u003csub\u003e4\u003c/sub\u003eCl supplementation on growth, photosynthesis, and triacylglycerol content in \u003cem\u003eChlamydomonas reinhardtii\u0026nbsp;\u003c/em\u003eunder mixotrophic cultivation. J Appl Microbiol\u003cem\u003e\u0026nbsp;\u003c/em\u003e135: lxae233. doi.org/10.1093/jambio/lxae233\u003c/li\u003e\n \u003cli\u003eSultan SE (2000) Phenotypic plasticity for plant development, function and life history. Trends Plant Sci\u003cem\u003e\u0026nbsp;\u003c/em\u003e5: 537-542. doi.org/10.1016/S1360-1385(00)01797-0\u003c/li\u003e\n \u003cli\u003eSundarRajan P, Gopinath KP, Arun J, GracePavithra K, Pavendan K, AdithyaJoseph A (2020) An insight into carbon balance of product streams from hydrothermal liquefaction of \u003cem\u003eScenedesmus abundans\u003c/em\u003e biomass. Renewable Energy\u003cem\u003e\u0026nbsp;\u003c/em\u003e151: 79-87. doi.org/10.1016/j.renene.2019.11.011\u003c/li\u003e\n \u003cli\u003eTikhonov AN (2013) pH-Dependent regulation of electron transport and ATP synthesis in chloroplasts. Photosynth Res\u003cem\u003e\u0026nbsp;\u003c/em\u003e116: 511-534. doi.org/10.1007/s11120-013-9845-y\u003c/li\u003e\n \u003cli\u003eTrabelsi L, Ben Ouada H, Bacha H, Ghoul M (2008) Combined effect of temperature and light intensity on growth and extracellular polymeric substance production by the cyanobacterium \u003cem\u003eArthrospira platensis\u003c/em\u003e. J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e21: 405-412. doi.org/10.1007/s10811-008-9383-8\u003c/li\u003e\n \u003cli\u003eTukaj Z, Kub\u0026iacute;nov\u0026aacute; A, Zachleder V (1996) Effect of Irradiance on Growth and Reproductive Processes During the Cell Cycle In \u003cem\u003eScenedesmus Armatus\u0026nbsp;\u003c/em\u003e(Chlorophyta) J Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e32: 624-631. doi.org/10.1111/j.0022-3646.1996.00624.x\u003c/li\u003e\n \u003cli\u003eWanka F (1959) Untersuchungen \u0026uuml;ber die Wirkung des Lichts auf die Zellteilung von \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e. Arch Mikrobiol\u003cem\u003e\u0026nbsp;\u003c/em\u003e34: 161-188. doi.org/10.1007/BF00412750\u003c/li\u003e\n \u003cli\u003eWeng H-X, Qin Y-C, Sun X-W, Chen X-H, Chen J-F (2009) Effects of light intensity on the growth of \u003cem\u003eCryptomonas\u003c/em\u003e sp. (Cryptophyceae). Environ Geol\u003cem\u003e\u0026nbsp;\u003c/em\u003e57: 9-15. doi.org/10.1007/s00254-008-1277-1\u003c/li\u003e\n \u003cli\u003eXin L, Hong-ying H, Ke G, Jia Y (2010) Growth and nutrient removal properties of a freshwater microalga \u003cem\u003eScenedesmus\u003c/em\u003e sp. LX1 under different kinds of nitrogen sources. Ecol Eng\u003cem\u003e\u0026nbsp;\u003c/em\u003e36: 379-381. doi.org/10.1016/j.ecoleng.2009.11.003\u003c/li\u003e\n \u003cli\u003eXu N, Zhang X, Fan X, Han L, Zeng C (2001) Effects of nitrogen source and concentration on growth rate and fatty acid composition of \u003cem\u003eEllipsoidion\u0026nbsp;\u003c/em\u003esp. (Eustigmatophyta). J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e13: 469. doi.org/10.1023/A:1012537219198\u003c/li\u003e\n \u003cli\u003eYang J, Li B, Zhang C, Luo H, Yang Z (2016) pH-associated changes in induced colony formation and growth of \u003cem\u003eScenedesmus obliquus\u003c/em\u003e. Fundam Appl Limnol\u003cem\u003e\u0026nbsp;\u003c/em\u003e187: 241-246. doi.org/10.1127/fal/2016/0846\u003c/li\u003e\n \u003cli\u003eYang Z, Liu Y, Ge J, Wang W, Chen Y, Montagnes D (2010) Aggregate formation and polysaccharide content of\u003cem\u003e\u0026nbsp;Chlorella pyrenoidosa\u0026nbsp;\u003c/em\u003eChick (Chlorophyta) in response to simulated nutrient stress. Bioresour Technol\u003cem\u003e\u0026nbsp;\u003c/em\u003e101: 8336-8341. doi.org/10.1016/j.biortech.2010.06.022\u003c/li\u003e\n \u003cli\u003eZhang M, Shi X, Yu Y, Kong F (2011) The Acclimative Changes in Photochemistry after Colony Formation of the Cyanobacteria \u003cem\u003eMicrocystis Aeruginosa\u003c/em\u003e. J Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e47: 524-532. doi.org/10.1111/j.1529-8817.2011.00987.x\u003c/li\u003e\n \u003cli\u003eZhang Y, Wang Q, Liu X, Zheng H, Li A (2023) Two types of growth pattern of the five microalgal species under different nitrogen supplies. Biomass Bioenergy\u003cem\u003e\u0026nbsp;\u003c/em\u003e171: 106720. doi.org/10.1016/j.biombioe.2023.106720\u003c/li\u003e\n \u003cli\u003eZhang Y, Wu H, Sun M, Peng Q, Li A (2018) Photosynthetic physiological performance and proteomic profiling of the oleaginous algae \u003cem\u003eScenedesmus acuminatus\u003c/em\u003e reveal the mechanism of lipid accumulation under low and high nitrogen supplies. Photosynth Res\u003cem\u003e\u0026nbsp;\u003c/em\u003e138: 73-102. doi.org/10.1007/s11120-018-0549-1\u003c/li\u003e\n \u003cli\u003eZhu H, Ye Z, Xu Z, Wei L (2024a) Transcriptomic Analysis Reveals the Effect of Urea on Metabolism of \u003cem\u003eNannochloropsis oceanica\u003c/em\u003e. Life\u003cem\u003e\u0026nbsp;\u003c/em\u003e14: 797. doi.org/10.3390/life14070797\u003c/li\u003e\n \u003cli\u003eZhu X, Sun Y, Huang Y, Wang J, Yang Z (2024b) Long-term continuous mismatch between grazing cues and real grazing losses causes attenuation of induced morphological defense in\u003cem\u003e\u0026nbsp;Scenedesmus\u003c/em\u003e. J Appl Phycol\u003cem\u003e\u0026nbsp;\u003c/em\u003e36: 1353-1362. doi.org/10.1007/s10811-023-03174-x\u003c/li\u003e\n \u003cli\u003eZhu X, Wang J, Lu Y, Chen Q, Yang Z (2015) Grazer-induced morphological defense in \u003cem\u003eScenedesmus obliquus\u0026nbsp;\u003c/em\u003eis affected by competition against\u003cem\u003e\u0026nbsp;Microcystis aeruginosa\u003c/em\u003e. Sci Rep\u003cem\u003e\u0026nbsp;\u003c/em\u003e5: 12743. doi.org/10.1038/srep12743\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Colony formation, Light intensity, Nitrogen resource, Phenotypic plasticity, Photosynthetic efficiency, Scenedesmus obliquus","lastPublishedDoi":"10.21203/rs.3.rs-6808862/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6808862/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs critical environmental factors, nitrogen and light not only regulate phytoplankton growth but also influence their phenotypic plasticity. \u003cem\u003eScenedesmus obliquus\u003c/em\u003e, an alga which is famous for its remarkable phenotypic plasticity, was studied to understand its response to varying combinations of nitrogen source and light intensity. It was cultured in media containing different nitrogen sources (NaNO\u003csub\u003e3\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e) under a range of light intensities (25, 50, 75, 100, 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Results showed that growth rates increased with higher light intensities across all nitrogen sources. Photosynthetic efficiency (\u003cem\u003eFv/Fm\u003c/em\u003e and \u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePSII\u003c/em\u003e\u003c/sub\u003e) remained stable in NaNO\u003csub\u003e3\u003c/sub\u003e treatments, but declined with rising light intensity in NH\u003csub\u003e4\u003c/sub\u003eCl and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments. The highest proportions of multicellular colonies were observed at 150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for NH\u003csub\u003e4\u003c/sub\u003eCl and NaNO\u003csub\u003e3\u003c/sub\u003e treatments, while colonies in CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments peaked at 100 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with colony size stabilized at approximately 2.1, 4.0, and 1.0 cells per particle under NaNO\u003csub\u003e3\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, and CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatments, respectively. Nitrogen removal efficiency improved with increasing light intensity across all treatments, though \u003cem\u003eS. obliquus\u003c/em\u003e exhibited varying capacities to remove nitrogen depending on the sources. These findings demonstrated how \u003cem\u003eS. obliquus\u003c/em\u003e adapts its growth, photosynthesis, and morphology to varying nitrogen sources and light intensities, and providing insights into its ecological versatility. This study provided a theoretical foundation for optimizing culture conditions in applications such as wastewater treatment and bioenergy production.\u003c/p\u003e","manuscriptTitle":"The interaction between nitrogen source and light intensity affects the biomass and phenotypic plasticity of Scenedesmus obliquus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 09:33:25","doi":"10.21203/rs.3.rs-6808862/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-07-13T04:29:04+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-24T00:44:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-23T15:06:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-04T15:21:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2025-06-03T04:27:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7f3053ae-40a0-45d4-8f6a-7de4bd4721ec","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:00:44+00:00","versionOfRecord":{"articleIdentity":"rs-6808862","link":"https://doi.org/10.1007/s10529-025-03637-w","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2025-09-05 15:57:05","publishedOnDateReadable":"September 5th, 2025"},"versionCreatedAt":"2025-06-25 09:33:25","video":"","vorDoi":"10.1007/s10529-025-03637-w","vorDoiUrl":"https://doi.org/10.1007/s10529-025-03637-w","workflowStages":[]},"version":"v1","identity":"rs-6808862","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6808862","identity":"rs-6808862","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}