Photosynthetic and morphological responses of Chaetoceros sp. to nutrient limitation over culture age

Photosynthetic and morphological responses of Chaetoceros sp. to nutrient limitation over culture age | 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 Photosynthetic and morphological responses of Chaetoceros sp. to nutrient limitation over culture age Sashenka Fierro, Roberto Cruz-Flores, M. del Pilar Sánchez-Saavedra This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7652804/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract This study investigated the physiological and morphological responses of the marine diatom Chaetoceros sp. strain CHX1 when cultured under two contrasting nutrient conditions: nutrient-rich f medium and nutrient-limited f/5 medium. Growth curves showed no significant differences in specific growth rate during the exponential phase; however, final cell densities were higher in f medium. Over culture time, cells exhibited increased biovolume and changes in shape, particularly in f/5 medium, where a subpopulation of spherical cells emerged during the late stationary phase. Pigment content, including chlorophylls a and c and total carotenoids, varied significantly across growth phases and media, with nutrient limitation promoting pigment accumulation as a potential stress response. Photosynthetic performance—assessed via relative electron transport rate (rETR), efficiency (α), saturation irradiance (I k ), and maximum quantum yield (Fv/Fm)—was highest during exponential growth and declined under nutrient limitation. Fv/Fm was particularly sensitive to nutrient stress, showing consistently lower values in nutrient-limited (f/5) cultures compared to the full-strength (f) medium across all growth phases. Chaetoceros sp. exhibited pronounced morphological and photophysiological plasticity in response to nutrient availability and culture duration. Under nutrient limitation (f/5), cells developed larger biovolume, spherical morphology, elevated carotenoid levels, and reduced photosynthetic efficiency—traits associated with stress acclimation and the onset of dormancy. The formation of resting cells represents a novel response for this strain, suggesting that controlled nutrient limitation may serve as a tool for diatom preservation, with applications in aquaculture and biotechnology. Photosynthesis Chaetoceros sp. growth rate cell size pigments Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Diatoms are key contributors to marine primary production and play a central role in global biogeochemical cycles. Their ability to adapt to fluctuating environmental conditions, such as changes in light and nutrient availability, has allowed them to colonize diverse aquatic habitats (Falkowski et al. 1998 ; Armbrust 2009 ). Nutrient limitation, particularly nitrogen and phosphorus deficiency, is a common feature of marine systems and strongly influences phytoplankton physiology, cellular morphology, and biochemical composition (Arrigo 2005 ; Hockin et al. 2012 ). Previous studies have shown that under nutrient-depleted conditions, diatoms may exhibit physiological adjustments such as altered pigment composition, reduced photosynthetic efficiency, changes in cell size, and shifts in carbon allocation (Masojídek et al. 2013 ; Liu et al. 2024 ). These traits are particularly relevant for biotechnological applications, where the modulation of biochemical content (e.g., pigments, lipids, essential fatty acids) under stress conditions may be used to enhance the productivity or nutritional quality of microalgal biomass (Masojídek et al. 2013 ). Nutrient limitation, especially of nitrogen and phosphorus, is a common condition in natural marine systems and a critical factor influencing phytoplankton productivity and composition (Moore et al. 2013 ). Coastal environments, in particular, often experience strong nutrient gradients due to upwelling, river inputs, or anthropogenic activity, resulting in variable nutrient regimes across spatial and temporal scales (Cloern 2001 ; Lomas and Glibert 1999 ). To simulate this natural variability under controlled conditions, laboratory studies frequently use diluted nutrient media such as f/2, f/4, or f/5 as analogues of nutrient-limited environments, while full-strength f medium is used to represent eutrophic or nutrient-replete conditions (Rhee 1978 ; Pacheco-Vega et al. 2010 ; Bhattacharjya et al. 2024 ). The use of f/5 medium in particular enables researchers to reproduce nutrient conditions closer to those found in oligotrophic or seasonally depleted coastal waters, where diatom growth is constrained, and physiological stress responses may be triggered. Comparing the growth, pigment composition, and photosynthetic efficiency of microalgae under f and f/5 media can therefore provide valuable insights into their adaptive plasticity and potential for biotechnological applications under variable nutrient regimes (Flynn et al. 2012 ; Masojídek et al. 2013 ; Bhattacharjya et al. 2024 ; Brenes-Monje and Sánchez-Saavedra 2025). Chaetoceros spp. are among the most abundant and ecologically successful marine diatoms and are widely used in aquaculture due to their high growth rate and rich fatty acid profiles (Thompson et al. 1990 ; Brown et al. 1997 ). However, species- and strain-specific responses to environmental factors remain poorly characterized, especially under nutrient limitation. Understanding these responses is crucial for optimizing their cultivation in applied settings and for predicting their ecological behavior in nutrient-variable marine systems. In this context, the use of Chaetoceros sp. strain CHX1 is particularly relevant due to its origin from local coastal waters of Baja California, México and its ecological prevalence in nutrient-variable environments. As one of the most cosmopolitan diatom genera, Chaetoceros species have been widely recognized for their high growth rates, physiological plasticity, and favorable biochemical composition for aquaculture feed (Ma and Hu 2024 ). Evaluating the performance of native strains under controlled nutrient regimes provides valuable insights into their potential for biomass production and resilience under environmentally realistic conditions, supporting future biotechnological or aquaculture applications. This study investigates the physiological and morphological responses of Chaetoceros sp. (strain CHX1) to varying nutrient conditions (f and f/5 media) across distinct growth phases. We analyzed changes in growth rate, cell morphology, pigment composition, and photosynthetic performance to uncover the species' adaptive strategies under nutrient limitation. Our results provide valuable insights into the phenotypic plasticity of diatoms and underscore the potential of Chaetoceros sp. as a resilient and versatile candidate for applications in sustainable aquaculture and applied phycology. Materials and Methods Microalgae culture conditions The diatom Chaetoceros sp. strain CHX1 was acquired from the Algal Biotechnology Laboratory collection at CICESE. Strain CHX1 was isolated as a single clone from Todos Santos Bay, Mexico (Trujillo-Valle 1993 ). Non-axenic batch cultures were grown in triplicate one-liter flasks containing 800 mL of filtered and sterilized seawater enriched with either f medium (Guillard and Ryther 1962 ) or f/5 medium to simulate nutrient-limited conditions. Each flask was inoculated with 5×10 4 cells mL -1 using cells previously acclimated for 40 days to the corresponding growth medium (f or f/5). Cultures were maintained under continuous irradiance of 100 µmol photons m -2 s -1 provided by white fluorescent lamps (Phillips® F40T12/DX) at 26 ± 1°C. Flasks were manually shaken once daily. Irradiance was measured at the bottom surface of the flasks using a 4π QSL-100 quantum radiometer (Biospherical Instruments, USA). Growth and cell size Cell density was recorded daily by counting with a hemocytometer and a compound microscope (Olympus CX31). The specific growth rate (µ) was calculated during the exponential growth phase using the equation described by Fogg and Thake ( 1987 ): $$\:\mu\:=\frac{{log}_{2}N-{log}_{2}{N}_{0}}{t}$$ where N 0 and N are the cell densities at the beginning and end of the exponential growth phase, t is the time interval in days, and µ is expressed in divisions per day, representing the average number of cell divisions per day. The pervalvar axis (PA) and apical axis (AA) of diatoms were measured during the exponential growth phase (day 1 for both f and f/5 media), stationary growth phase (day 9 for f medium and day 7 for f/5 medium), and late stationary growth phase (day 22 for both media). Images of 100 randomly selected cells from each culture were captured using a compound microscope (Olympus CX31) with 40x magnification coupled with a digital camera (Zeiss Axiocam 208). Linear cell dimensions were measured using Zeiss ZEN software (version 3.11). Biovolume (setae excluded) was estimated assuming an ellipsoid or prism on elliptic base shape for Chaetoceros sp. cells (Sun and Liu 2003 ), except for a subpopulation of spherical cells observed in the late stationary growth phase in f and f/5 medium, which were modeled as spheres. Pigment and photosynthesis For pigment quantification, 5 mL aliquots from each replicate were collected during the exponential, stationary, and late stationary growth phases. Samples were filtered through 24 mm VWR GF-C glass fiber filters (1 µm pore size). Chlorophylls a and c , as well as total carotenoids were extracted using 90% acetone and quantified with a HACH model 6000 spectrophotometer, following the protocol described by Parsons et al. ( 1984 ). Pigment concentrations were calculated using the equations proposed by Jeffrey and Humphrey ( 1975 ). Photosynthetic activity was evaluated via chlorophyll a fluorescence using a pulse-amplitude modulated (PAM) fluorometer (Walz, Junior PAM), following the methodology by (Pérez-Varillas and Sánchez-Saavedra 2025 ). Aliquots of 10 mL from each replicate were taken during the exponential, stationary, and late stationary growth phases in cultures grown in two media: f and f/5. Prior to measurement, samples were dark-adapted for 20 minutes to ensure reopening of photosystem II (PSII) reaction centers. The photosynthetic parameters —including maximum relative electron transport rate (ETRmax), electron transport efficiency (α), saturation irradiance (Ik), and maximum quantum yield of PSII (Fv/Fm)—were calculated from rapid light-response curves using the equations described by Eilers and Peeters ( 1988 ). All measurements were conducted in triplicate for each treatment and phase. The use of PAM fluorometry allows for a rapid and non-invasive assessment of PSII functionality, providing valuable insight into photosynthetic performance and potential photoinhibition in response to environmental and nutritional conditions. Statistical Analysis Experimental results are presented as mean values ± standard deviation. Normality and homoscedasticity assumptions were verified prior to statistical testing. When these assumptions were met, analysis of covariance (ANCOVA) was applied to cell density and ETR data, while two-way analysis of variance (ANOVA) was used to pigment content and photosynthetic parameter data. Post hoc comparisons between group means were conducted using Tukey’s test. All statistical analyses were performed using Statistica® software version 8.0 (StatSoft, Inc.), with a significance level of p < 0.05. Results Growth and cell size Growth curves for Chaetoceros sp. displayed a two-day exponential growth phase in both full-strength (f) and nutrient-limited (f/5) media (Fig. 1 ). No significant differences were detected in specific growth rate or generation time between the two media during this phase. A significant interaction between culture medium and time was nevertheless observed across the entire experiment ( p = 0.003). Cultures grown in f medium continued to increase until day 22, when they reached their maximum cell density, whereas those grown in f/5 medium peaked on day 16 and declined thereafter (Table 1 ). Table 1 Growth rate (µ), generation time (Gt) at exponential growth phase, exponential phase duration, maximum cell density and final cell density of Chaetoceros sp. cultivated in f and f/5 growth media. Mean values ± SD, n = 3 Growth medium µ (divisions day − 1 ) Gt (days) Exponential phase duration (days) Maximum cell density (×10 6 cells mL − 1 ) Final cell density (×10 6 cells mL − 1 ) f 1.97 ± 0.11 0.52 ± 0.03 2 1.76 at day 22 1.76 f/5 1.84 ± 0.03 0.59 ± 0.02 2 1.02 at day 16 0.92 High variability was observed in both the apical axis (AA) and pervalvar axis (PA) across all growth phases in cultures grown in both f and f/5 media, with a tendency toward cell enlargement over cultivation time (Fig. 2 ). During the exponential growth phase, the AA ranged between 4.1–10.5 µm and the PA ranged between 4.0-10.3 µm. In the late stationary growth phase, the AA ranged between 4.2–9.9 and the PA ranged between 4.9–13.8 µm. The PA/AA ratio indicated a tendency for cells to change morphology depending on the culture medium. In f medium, diatoms exhibited a steady increase in PA over time, leading to a PA/AA ratio that rose from 1.09 in the exponential growth phase to 1.53 in the late stationary growth phase. In the late stationary growth phase under f/5 medium conditions, cells exhibited a marked morphological transformation, adopting a nearly spherical shape. This morphological shift was corroborated by an increase in AA and the PA/AA ratio over time. Biovolume showed a slight increase over time in both f and f/5 media; however, cells consistently exhibited higher values in f/5 medium across all growth phases (Fig. 2 ). In the late stationary growth phase, a distinct subpopulation was observed, comprising 81% of the cells in f/5 medium and 28% in f medium, characterized by nearly spherical morphology, while the remaining cells retained the typical prism on elliptic base or ellipsoid shape. The highest biovolume recorded (307.8 µm 3 ) corresponded to cells in f/5 medium during the late stationary growth phase, surpassing that of all other growth conditions. Pigments and photosynthesis The content of photosynthetic pigments—chlorophyll a , chlorophyll c , and carotenoids—did not differ significantly ( p > 0.05) between culture media during the exponential growth phase. However, significant differences were observed during both the stationary and late stationary growth phases ( p < 0.05) (Fig. 3 ). Chlorophyll a content was significantly influenced by growth phase ( p < 0.0001) and culture medium ( p < 0.001) (Fig. 3 a). Cultures grown in f medium exhibited higher chlorophyll a content during the late stationary growth phase ( p < 0.0001), while cultures in f/5 medium had significant higher chlorophyll a level during the stationary growth phase ( p = 0.004). Chlorophyll c content was significantly affected by growth phase ( p < 0.0001), culture medium ( p = 0.023), and their interaction ( p = 0.0402) (Fig. 3 b). Higher chlorophyll c concentrations were observed in f-medium cultures during both the stationary ( p = 0.0051) and late stationary growth phases ( p = 0.0081). In both media, chlorophyll c content in cultures peaked during the stationary growth phase ( p < 0.0001). Carotenoid content was significantly affected by growth phase ( p = 0.0011), with highest values recorded in the late stationary growth phase in f medium ( p = 0.0024) and in the stationary growth phase in f/5 medium ( p = 0.0014) (Fig. 3 c). A significant effect of culture medium on carotenoid content was observed only during the stationary growth phase, with higher values in f/5-grown cultures ( p = 0.0009). No significant differences were observed in other growth phases. Photosynthesis-irradiance curves (rETR) curves showed significant variations across growth phases, with maximum values observed during the exponential growth phase for both f ( p = 1.46×10 − 8 ) and f/5 media ( p = 2.08×10 − 11 ) (Fig. 4 ). Photosynthetic parameters also varied significantly with growth phase and culture medium (Fig. 5 ). Photosynthetic efficiency (α), saturation irradiance (I k ), maximum relative electron transport rate (ETR max ), and maximum quantum yield of PSII (Fv/Fm) were all higher in the exponential growth phase across both treatments. Culture medium had a significant effect on α ( p = 0.0009) and Fv/Fm ( p < 0.0001), with lower values observed in f/5 medium during the late stationary growth phase (Fig. 5 a and 5 d). Discussion Nutrient-limiting conditions are common in marine environments, and phytoplankton species display diverse physiological responses to such stressors. Diatoms, in particular, have shown remarkable adaptability to environmental fluctuations, allowing them to thrive and persist across a wide range of marine habitats (Ambrust 2009). The use of f/5 medium in this study was guided not only by experimental design but also by ecological relevance, as it simulates the nutrient conditions characteristic of the Pacific Ocean off the coast of Ensenada, Baja California—the natural habitat where the Chaetoceros sp. strain used in this research was originally isolated. Studies in coastal waters of the Gulf of California have reported typical surface concentrations of phosphates (~ 0.85 µM) and nitrates (~ 2.35 µM) (Bustos-Serrano et al. 2006), while nearby coastal channels show nitrate-nitrite levels around 4.9 µM and phosphorus ~ 2.5 µM (Soto-Balderas and Alvarez-Borrego 1991 ; Linacre et al. 2017 ). These values closely match the nutrient composition of the f/5 medium, supporting its use as a realistic representation of the natural habitat conditions for Chaetoceros sp. No significant differences were observed between f and f/5 media in the growth rate or generation time of Chaetoceros sp. during the exponential growth phase of our experiments. However, by the end of the cultivation period, cultures grown in f/5 medium reached significantly lower maximum cell densities compared to those grown in full-strength f medium. This reduction was primarily attributed to nutrient limitation, which became increasingly pronounced during the stationary and late stationary growth phases. The growth rate of Chaetoceros sp. in both culture media was similar to that previously reported for this strain by Pérez-Varillas and Sánchez-Saavedra ( 2025 ). However, it was higher than those reported for other Chaetoceros species cultured under similar nutrient conditions (Liang et al. 2006 ; Pacheco-Vega et al. 2010 ; Chen et al. 2023 ). This enhanced growth performance highlights the strain’s potential for aquaculture applications. Microalgae with intermediate cell sizes, around 100 µm³, exhibit the highest photosynthetic productivity per biomass unit and the fastest growth rates (Borowitzka 2016 ). The maximum cell density in f medium cultures (1.76×10⁶ cells mL⁻¹) was lower than that found in the study by Sánchez-Saavedra and Voltolina ( 2006 ) for the same species (5.43×10⁶ cells mL⁻¹). These discrepancies may be attributed to differences in cultivation conditions, such as culture volume, inoculum size, and the light intensity employed. The results show that the pervalvar axis (PA) and biovolume of Chaetoceros sp. increased progressively through the growth phases in both f and f/5 media. These findings are consistent with Pérez-Varillas and Sánchez-Saavedra ( 2025 ), who reported larger biovolumes during the stationary phase compared to the exponential phase for the same strain. In centric diatoms, vegetative cell enlargement serves as an alternative mechanism for restore cell size when conditions for sexual reproduction are not met, providing an adaptive advantage (Mills and Kaczmarska 2006 ; Kaczmarska et al. 2022 ). Stress factors such as nutrient limitation or temperature fluctuations can induce changes in cell morphology and carbon content, as reported in species like Thalassiosira pseudonana (O'Donnell et al. 2021 ). In the present study, the larger biovolume observed under nutrient-limited (f/5) conditions across all growth phases may enhance survival or promote vertical migration, particularly under stress (Fu et al. 2022 ). This response aligns with previous reports showing that nutrient stress can significantly increase cell size in diatoms (Peter and Sommer 2015 ). For instance, phosphorus limitation induced elongation of the pervalvar axis and thickening of setae in Chaetoceros peruvianus resulting in increased cell volume (Smodlaka Tankovic et al. 2018 ). Silica limitation also led to increased cell volume in C. calcitrans and T. pseudonana (Laing 1985 ; Li et al. 2021 ). Cell enlargement under high irradiance or Si limitation promotes sinking, potentially allowing cells to reach deeper, less stressful environments where photo-oxidative damage is reduced (Li et al. 2021 ). Large cells exhibit superior adaptation to fluctuating environmental conditions, largely due to their lower respiration rates and greater capacity for nutrient storage (Geider et al. 1986 ; Litchman et al. 2009 ). The content of photosynthetic pigments—chlorophylls a and c , and carotenoids—was similar in both culture media during the exponential growth phase, when nutrients were not limiting. In contrast, significant differences emerged during the stationary and late stationary phases. Notably, pigment levels in cultures grown in f medium during the late stationary phase resembled those in f/5 medium during the stationary phase, suggesting that nutrient concentration exerts a stronger influence than growth phase alone on the physiological response of diatoms. Cells in both f and f/5 media exhibited higher levels of chlorophylls a and c during the stationary and late stationary phases than during the exponential phase, in response to reduced light availability and nutrient depletion in the medium. These findings suggest that under suboptimal conditions, Chaetoceros sp. increases its chlorophyll a content to enhance light-harvesting capacity—a strategy previously reported in diatoms and other microalgae to sustain photosynthetic activity (Sigaud-Kutner et al. 2002 ; Masojídek et al. 2013 ; López-Sandoval et al. 2014 ). Carotenoid content was significantly affected by both growth phase and nutrient availability. The highest concentrations were recorded during the stationary phase in f/5 medium and during the late stationary phase in both media, aligning with periods of pronounced nutrient depletion. This suggests a photoprotective response to nutritional stress. Under such conditions, the absorbed light energy may exceed the metabolic capacity for its utilization, causing an imbalance between photochemical energy input and downstream metabolic processes—potentially resulting in photoinhibition or oxidative stress (Li et al. 2021 ). Carotenoids are known to quench reactive oxygen species (ROS), and their accumulation under nutrient stress has been widely reported in marine diatoms as a defense mechanism (Dimier et al. 2007 ; Latowski et al. 2014 ; Kuczynska et al. 2015 ). Therefore, the elevated carotenoid levels in f/5-grown cultures likely reflect an adaptive strategy to mitigate photo-oxidative damage under nutrient-deficient conditions (Lepetit and Dietzel 2015 ). Photosynthetic performance, as indicated by rETR curves and associated parameters, declined as Chaetoceros sp. cultures advanced through the growth phases. This trend likely reflects metabolic stress due to progressive nutrient depletion. A balanced flow between photosynthetic energy input and carbon allocation for biosynthesis depends on optimal environmental conditions, including light, nutrient availability and, temperature (Yan et al. 2018 ). Under nitrogen-limited conditions, phytoplankton exhibit reduced photosynthetic efficiency, primarily due to impaired development and functioning of the light-harvesting complexes (Young and Beardall 2003 ). The culture medium and growth phase significantly affected photosynthetic parameters, including α, ETRmax and the maximum quantum yield of PSII (Fv/Fm), with lower values observed in f/5 medium. This agrees with previous studies, for instance, Liang et al. ( 2006 ) reported declines in α, ETRmax, I k , and Fv/Fm in Phaeodactylum tricornutum and Chaetoceros muellerii during batch culture aging, indicating that nutrient limitation progressively impairs the photosynthetic apparatus. In our study, Fv/Fm was particularly sensitive to nutrient conditions, showing differences between media even in the exponential phase, unlike other photosynthetic traits such as pigment content. This supports previous findings by Tan et al. ( 2019 ), who observed reductions in Fv/Fm under nitrogen or phosphorus limitation across several microalgal groups, including diatoms, and proposed this parameter as a reliable indicator of phytoplankton physiological status. High I k values during the exponential growth phase of Chaetoceros sp. in both f and f/5 media indicate a high capacity to tolerate elevated light intensities (> 2 × 10³ µmol photons m⁻² s⁻¹). Similarly, higher I k values during the exponential phase (~ 1–3 × 10³ µmol photons m⁻² s⁻¹), compared to the stationary phase, have been reported for P. tricornutum , C. muelleri and C. gracilis (Pérez-Varillas and Sánchez-Saavedra 2025 ). In the study by Liang et al. ( 2006 ), I k decreased with increasing culture age in P. tricornutum and C. muelleri , although their values were considerably lower (80–200 µmol photons m⁻² s⁻¹). These differences may reflect variations in culture conditions, methodologies used for fluorescence measurements, and intrinsic species-specific traits. Due to its tolerance to high irradiance, Chaetoceros sp. exhibits a high potential for aquaculture and other biotechnological applications (Pérez-Varillas and Sánchez-Saavedra 2025 ). Chaetoceros sp. cells during the exponential growth phase exhibited a higher metabolic rate, reflected in high growth rates, greater photosynthetic efficiency (α and Fv/Fm), and smaller cell size—features typical of an energy-intensive strategy under favorable conditions. As culture aged, growth slowed or ceased, pigment content increased, photosynthetic efficiency declined, and cell enlarged, particularly under nutrient-limited (f/5) conditions. This transition indicates a reallocation of resources from biomass production to maintenance, stress tolerance, and photoprotection—a survival strategy under nutrient shortage (Geider et al. 1993 ) and fluctuating light conditions typical of coastal environment (Lavaud 2007 ). The photosynthetic apparatus dynamically regulates light harvesting and energy dissipation through thylakoid-associated complexes, which undergo highly adaptive responses to environmental cues (Lepetit and Dietzel 2015 ). Photosynthetic regulation in strains from coastal habitats—characterized by strong fluctuations in nutrients and light—exhibits greater flexibility than that of strains from open-ocean ecosystems (Lavaud et al. 2007 ). Our results show that nutrient limitation in f/5 medium induced significant morphological and physiological changes in Chaetoceros sp., including increased biovolume, a shift toward spherical cells, and reduced photosynthetic activity during the late stationary phase. These findings are consistent with previous reports describing the development of resting stages in diatoms under unfavorable conditions and vegetative senescence (McQuoid and Hobson 1996 ). The decline in photosynthetic pigments (chlorophylls a and c , and carotenoids), relative electron transport rate (rETR), and key photosynthetic parameters—particularly photosynthetic efficiency (α) and the maximum quantum yield of PSII (Fv/Fm)—in cells grown in f/5 medium during the late stationary phase is associated with reduced metabolic activity. In a study by Kuwata et al. ( 1993 ), resting cells of Chaetoceros pseudocurvisetus exhibited a decrease in chlorophyll a content and photosynthetic rate of up to 17% compared to vegetative cells. Nutrient-limiting conditions can trigger dormant-like stages in diatoms, enabling them to survive until favorable environmental conditions are restored—a key survival strategy in dynamic marine ecosystems (Ellegaard and Ribeiro 2018 ). The formation of resting cells observed in Chaetoceros sp. cultured in f/5 medium represents a novel finding, as this response has not been previously described for this strain isolated from Todos Santos Bay, Mexico. This nutrient-limitation-induced response offers a new methodological approach for the controlled induction of resting cells, with potential applications in aquaculture and biotechnology, particularly for cell preservation. Conclusions This study demonstrates that Chaetoceros sp. displays notable physiological and morphological plasticity in response to variations in nutrient availability and the duration of cultivation. Growth, cell morphology, pigment composition, and photosynthetic parameters varied significantly between full-strength (f) and nutrient-limited (f/5) media, particularly during the stationary and late stationary growth phases. Notably, cells grown under nutrient limitation (f/5) exhibited increased biovolume, spherical morphology, elevated carotenoid content, and reduced photosynthetic performance—features consistent with stress responses and the onset of dormancy. The formation of putative resting cells under f/5 nutrient-limited conditions reveals a novel and ecologically meaningful adaptive response in this Chaetoceros strain, previously unreported for isolates from Todos Santos Bay, Mexico. These results suggest that controlled nutrient limitation may serve as a practical tool for inducing dormancy in diatoms, with implications for the preservation of ecologically or economically important strains in aquaculture. The ability of Chaetoceros sp. to modulate its cellular architecture and energy metabolism under suboptimal conditions highlights its adaptive capacity and supports its potential for sustainable use in marine biotechnological applications. Declarations Acknowledgments S. Fierro acknowledges their Doctoral scholarship from Secretaría de Ciencias, Humanidades, Tecnología e Innovación (SECIHTI). To PhD. C.A. Molina-Cárdenas and F.Y. Castro-Ochoa for their technical assistance with photosynthesis analysis. To PhD. B. Barón-Sevilla and PhD. M.D.R. Zacarías-Soto for providing the equipment used for the diatom measurements. Authors’ contributions S.F.: Conceptualization, methodology, validation, formal analysis, investigation, writing - original draft. R.C.F.: Review and editing, supervision. M.P.S.S.: Resources, writing – review and editing, supervision, project administration, funding acquisition. All authors read and approved the final manuscript Funding: This work was funded by Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE, project: 623-108). Data availability Data will be available upon reasonable request. 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Limnol Oceanogr 23(1):10–25 Sánchez-Saavedra MP, Voltolina D (2006) The growth rate, biomass production and composition of Chaetoceros sp. grown with different light sources. Aquacult Eng 35:161-165 Sigaud-Kutner TCS, Pinto E, Okamoto OK, Latorre LR, Colepicolo P (2002) Changes in photsynthetic pigment content during growth. Physiol Plant 114:566-571 Smodlaka Tankovic M, Baricevic A, Ivancic I, Kuzat N, Medic N, Pustijanac E, Novak T, Gasparovic B, Maric Pfannkuchen D, Pfannkuchen M (2018) Insights into the life strategy of the common marine diatom Chaetoceros peruvianus Brightwell. PLoS One 13:e0203634 Soto-Balderas MG, Alvarez-Borrego S (1991) Inorganic nutrients in the inundation channels of tidal marshes of a coastal lagoon of northwestern Baja California. Cienc Mar 17(3):1-20 Sun J, Liu D (2003) Geometric models for calculating cell biovolume and surface area for phytoplankton. J Plankton Res 25:1331-1346 Tan L, Xu W, He X, Wang J (2019) The feasibility of Fv/Fm on judging nutrient limitation of marine algae through indoor simulation and in situ experiment. Estuar, Coast Shelf Sci 229:106411 Thompson PA, Guo MX, Harrison PJ (1990) Effects of variation in temperature and nutrient availability on the biochemical composition of marine phytoplankton. J Phycol 28:481-488 Trujillo-Valle L (1993) La colección de microalgas del CICESE. Comunicaciones Académicas, Serie Acuicultura. Centro de Investigación Científica y de Educación Superior de Ensenada, Ensenada, Mexico. CIACT9301 pp 1-38 Yan D, Beardall J, Gao K (2018) Variation in cell size of the diatom Coscinodiscus granii influences photosynthetic performance and growth. Photosynth Res 137:41-52 Young EB, Beardall J (2003) Photosynthetic function in Dunaliella tertiolecta (Chlorophyta) during a nitrogen starvation and recovery cycle. J Phycol 39:897–905 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 Dec, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviews received at journal 26 Oct, 2025 Reviewers agreed at journal 06 Oct, 2025 Reviewers invited by journal 24 Sep, 2025 Editor assigned by journal 24 Sep, 2025 Submission checks completed at journal 24 Sep, 2025 First submitted to journal 18 Sep, 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. 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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-7652804","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":525280280,"identity":"576812b3-e5d5-44c2-96d1-cf273d3d31b7","order_by":0,"name":"Sashenka Fierro","email":"","orcid":"","institution":"Center for Scientific Research and Higher Education at Ensenada","correspondingAuthor":false,"prefix":"","firstName":"Sashenka","middleName":"","lastName":"Fierro","suffix":""},{"id":525280281,"identity":"ac8d697d-84af-4072-a369-013197f05bdd","order_by":1,"name":"Roberto Cruz-Flores","email":"","orcid":"","institution":"Center for 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08:50:25","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120115,"visible":true,"origin":"","legend":"","description":"","filename":"9c1e4edae25649d2b756a0a9bb990f741structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/88512975203c82a4d7de011a.xml"},{"id":92929609,"identity":"a54521d9-5097-41a7-9cbf-588620d177e3","added_by":"auto","created_at":"2025-10-07 08:50:25","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126837,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/4777cd8cff2d2e9d1b5c724d.html"},{"id":92929592,"identity":"0c2907f4-9912-46a6-bb1c-574975ca29e3","added_by":"auto","created_at":"2025-10-07 08:50:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51034,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of \u003cem\u003eChaetoceros\u003c/em\u003esp. cultivated in f and f/5 media. Different letters indicate significant differences between treatments (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Mean values ± SD, \u003cem\u003en\u003c/em\u003e=3\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/1342d7a8565cf282520fbbc2.png"},{"id":92930543,"identity":"d2b6b880-0cd8-46c4-bbad-92ff0e2a3ee4","added_by":"auto","created_at":"2025-10-07 08:58:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61729,"visible":true,"origin":"","legend":"\u003cp\u003eBox and whisker plots for cell dimensions: apical axis (AA), pervalvar axis (PA), PA/AA ratio and biovolume for \u003cem\u003eChaetoceros \u003c/em\u003esp.\u003cem\u003e \u003c/em\u003egrown in f (black) and f/5 (grey) media at different growth phases. For each treatment 100 cells were measured.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/c5899157f1b5f81f9a18f78e.png"},{"id":92929594,"identity":"c79d0ee3-32f9-4b58-8995-d789f7b8aea3","added_by":"auto","created_at":"2025-10-07 08:50:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59712,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll \u003cem\u003ea \u003c/em\u003e(a), chlorophyll \u003cem\u003ec \u003c/em\u003e(b), and carotenoids (c) (µg mL\u003csup\u003e-1\u003c/sup\u003e) in \u003cem\u003eChaetoceros\u003c/em\u003e sp. grown in f and f/5 media at three growth phases. Different lowercase letters indicate statistically significant differences among growth phases (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05: a\u0026gt;b\u0026gt;c), while different uppercase letters denote significant differences between culture media (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05: A\u0026gt;B), as determined by two-way ANOVA followed by Tukey’s HSD test. Mean values ± SD, \u003cem\u003en\u003c/em\u003e=3\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/51d4c6c9261f906edf2844fc.png"},{"id":92930544,"identity":"66fee279-1725-402f-b732-52ee29134637","added_by":"auto","created_at":"2025-10-07 08:58:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97804,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between electron transport rate (ETR) and irradiance in \u003cem\u003eChaetoceros\u003c/em\u003e sp. at three growth phases, cultivated in f and f/5 media. Different lowercase letters indicate statistically significant differences among growth phases (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05: a\u0026gt;b\u0026gt;c). Mean values ± SD, \u003cem\u003en\u003c/em\u003e=3\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/84b9e6c47aec4f333f8efb7d.png"},{"id":92930833,"identity":"9c2821e3-1567-453f-851a-0841b211ec6b","added_by":"auto","created_at":"2025-10-07 09:06:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66546,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthetic parameters: electron transport efficiency, α, (a); maximum electron transport rate, ETR\u003csub\u003emax\u003c/sub\u003e (b); light saturation points, E\u003csub\u003ek\u003c/sub\u003e (c) and maximum quantum yield of PS II, Fv/Fm (d), for \u003cem\u003eChaetoceros\u003c/em\u003e sp. at three growth phases, cultured in f and f/5 media. Different lowercase letters indicate statistically significant differences among growth phases (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05: a\u0026gt;b\u0026gt;c), while different uppercase letters denote significant differences between culture media (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05: A\u0026gt;B), as determined by two-way ANOVA followed by Tukey’s HSD test. Mean values ± SD, \u003cem\u003en\u003c/em\u003e=3\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/116dc981f5a0870468c8c826.png"},{"id":92931842,"identity":"70a24b79-6207-49ae-ad59-e1b1ba49405f","added_by":"auto","created_at":"2025-10-07 09:14:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":931158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7652804/v1/d45f109f-5a4b-4c78-a4da-8adf15adc781.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Photosynthetic and morphological responses of Chaetoceros sp. to nutrient limitation over culture age","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiatoms are key contributors to marine primary production and play a central role in global biogeochemical cycles. Their ability to adapt to fluctuating environmental conditions, such as changes in light and nutrient availability, has allowed them to colonize diverse aquatic habitats (Falkowski et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Armbrust \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Nutrient limitation, particularly nitrogen and phosphorus deficiency, is a common feature of marine systems and strongly influences phytoplankton physiology, cellular morphology, and biochemical composition (Arrigo \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hockin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies have shown that under nutrient-depleted conditions, diatoms may exhibit physiological adjustments such as altered pigment composition, reduced photosynthetic efficiency, changes in cell size, and shifts in carbon allocation (Masoj\u0026iacute;dek et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These traits are particularly relevant for biotechnological applications, where the modulation of biochemical content (e.g., pigments, lipids, essential fatty acids) under stress conditions may be used to enhance the productivity or nutritional quality of microalgal biomass (Masoj\u0026iacute;dek et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNutrient limitation, especially of nitrogen and phosphorus, is a common condition in natural marine systems and a critical factor influencing phytoplankton productivity and composition (Moore et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Coastal environments, in particular, often experience strong nutrient gradients due to upwelling, river inputs, or anthropogenic activity, resulting in variable nutrient regimes across spatial and temporal scales (Cloern \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Lomas and Glibert \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). To simulate this natural variability under controlled conditions, laboratory studies frequently use diluted nutrient media such as f/2, f/4, or f/5 as analogues of nutrient-limited environments, while full-strength f medium is used to represent eutrophic or nutrient-replete conditions (Rhee \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Pacheco-Vega et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bhattacharjya et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe use of f/5 medium in particular enables researchers to reproduce nutrient conditions closer to those found in oligotrophic or seasonally depleted coastal waters, where diatom growth is constrained, and physiological stress responses may be triggered. Comparing the growth, pigment composition, and photosynthetic efficiency of microalgae under f and f/5 media can therefore provide valuable insights into their adaptive plasticity and potential for biotechnological applications under variable nutrient regimes (Flynn et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Masoj\u0026iacute;dek et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bhattacharjya et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Brenes-Monje and S\u0026aacute;nchez-Saavedra 2025).\u003c/p\u003e\u003cp\u003e\u003cem\u003eChaetoceros\u003c/em\u003e spp. are among the most abundant and ecologically successful marine diatoms and are widely used in aquaculture due to their high growth rate and rich fatty acid profiles (Thompson et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Brown et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). However, species- and strain-specific responses to environmental factors remain poorly characterized, especially under nutrient limitation. Understanding these responses is crucial for optimizing their cultivation in applied settings and for predicting their ecological behavior in nutrient-variable marine systems.\u003c/p\u003e\u003cp\u003eIn this context, the use of \u003cem\u003eChaetoceros\u003c/em\u003e sp. strain CHX1 is particularly relevant due to its origin from local coastal waters of Baja California, M\u0026eacute;xico and its ecological prevalence in nutrient-variable environments. As one of the most cosmopolitan diatom genera, \u003cem\u003eChaetoceros\u003c/em\u003e species have been widely recognized for their high growth rates, physiological plasticity, and favorable biochemical composition for aquaculture feed (Ma and Hu \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Evaluating the performance of native strains under controlled nutrient regimes provides valuable insights into their potential for biomass production and resilience under environmentally realistic conditions, supporting future biotechnological or aquaculture applications.\u003c/p\u003e\u003cp\u003eThis study investigates the physiological and morphological responses of \u003cem\u003eChaetoceros\u003c/em\u003e sp. (strain CHX1) to varying nutrient conditions (f and f/5 media) across distinct growth phases. We analyzed changes in growth rate, cell morphology, pigment composition, and photosynthetic performance to uncover the species' adaptive strategies under nutrient limitation. Our results provide valuable insights into the phenotypic plasticity of diatoms and underscore the potential of \u003cem\u003eChaetoceros\u003c/em\u003e sp. as a resilient and versatile candidate for applications in sustainable aquaculture and applied phycology.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMicroalgae culture conditions\u003c/h2\u003e\u003cp\u003eThe diatom \u003cem\u003eChaetoceros\u003c/em\u003e sp. strain CHX1 was acquired from the Algal Biotechnology Laboratory collection at CICESE. Strain CHX1 was isolated as a single clone from Todos Santos Bay, Mexico (Trujillo-Valle \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Non-axenic batch cultures were grown in triplicate one-liter flasks containing 800 mL of filtered and sterilized seawater enriched with either f medium (Guillard and Ryther \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) or f/5 medium to simulate nutrient-limited conditions. Each flask was inoculated with 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e using cells previously acclimated for 40 days to the corresponding growth medium (f or f/5). Cultures were maintained under continuous irradiance of 100 \u0026micro;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e provided by white fluorescent lamps (Phillips\u0026reg; F40T12/DX) at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Flasks were manually shaken once daily. Irradiance was measured at the bottom surface of the flasks using a 4π QSL-100 quantum radiometer (Biospherical Instruments, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGrowth and cell size\u003c/h3\u003e\n\u003cp\u003eCell density was recorded daily by counting with a hemocytometer and a compound microscope (Olympus CX31). The specific growth rate (\u0026micro;) was calculated during the exponential growth phase using the equation described by Fogg and Thake (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1987\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\mu\\:=\\frac{{log}_{2}N-{log}_{2}{N}_{0}}{t}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eN\u003c/em\u003e are the cell densities at the beginning and end of the exponential growth phase, \u003cem\u003et\u003c/em\u003e is the time interval in days, and \u0026micro; is expressed in divisions per day, representing the average number of cell divisions per day.\u003c/p\u003e\u003cp\u003eThe pervalvar axis (PA) and apical axis (AA) of diatoms were measured during the exponential growth phase (day 1 for both f and f/5 media), stationary growth phase (day 9 for f medium and day 7 for f/5 medium), and late stationary growth phase (day 22 for both media). Images of 100 randomly selected cells from each culture were captured using a compound microscope (Olympus CX31) with 40x magnification coupled with a digital camera (Zeiss Axiocam 208). Linear cell dimensions were measured using Zeiss ZEN software (version 3.11). Biovolume (setae excluded) was estimated assuming an ellipsoid or prism on elliptic base shape for \u003cem\u003eChaetoceros\u003c/em\u003e sp. cells (Sun and Liu \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), except for a subpopulation of spherical cells observed in the late stationary growth phase in f and f/5 medium, which were modeled as spheres.\u003c/p\u003e\n\u003ch3\u003ePigment and photosynthesis\u003c/h3\u003e\n\u003cp\u003eFor pigment quantification, 5 mL aliquots from each replicate were collected during the exponential, stationary, and late stationary growth phases. Samples were filtered through 24 mm VWR GF-C glass fiber filters (1 \u0026micro;m pore size). Chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e, as well as total carotenoids were extracted using 90% acetone and quantified with a HACH model 6000 spectrophotometer, following the protocol described by Parsons et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Pigment concentrations were calculated using the equations proposed by Jeffrey and Humphrey (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1975\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhotosynthetic activity was evaluated via chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence using a pulse-amplitude modulated (PAM) fluorometer (Walz, Junior PAM), following the methodology by (P\u0026eacute;rez-Varillas and S\u0026aacute;nchez-Saavedra \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Aliquots of 10 mL from each replicate were taken during the exponential, stationary, and late stationary growth phases in cultures grown in two media: f and f/5. Prior to measurement, samples were dark-adapted for 20 minutes to ensure reopening of photosystem II (PSII) reaction centers.\u003c/p\u003e\u003cp\u003eThe photosynthetic parameters \u0026mdash;including maximum relative electron transport rate (ETRmax), electron transport efficiency (α), saturation irradiance (Ik), and maximum quantum yield of PSII (Fv/Fm)\u0026mdash;were calculated from rapid light-response curves using the equations described by Eilers and Peeters (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). All measurements were conducted in triplicate for each treatment and phase.\u003c/p\u003e\u003cp\u003eThe use of PAM fluorometry allows for a rapid and non-invasive assessment of PSII functionality, providing valuable insight into photosynthetic performance and potential photoinhibition in response to environmental and nutritional conditions.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eExperimental results are presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Normality and homoscedasticity assumptions were verified prior to statistical testing. When these assumptions were met, analysis of covariance (ANCOVA) was applied to cell density and ETR data, while two-way analysis of variance (ANOVA) was used to pigment content and photosynthetic parameter data. Post hoc comparisons between group means were conducted using Tukey\u0026rsquo;s test. All statistical analyses were performed using Statistica\u0026reg; software version 8.0 (StatSoft, Inc.), with a significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGrowth and cell size\u003c/h2\u003e\u003cp\u003eGrowth curves for \u003cem\u003eChaetoceros\u003c/em\u003e sp. displayed a two-day exponential growth phase in both full-strength (f) and nutrient-limited (f/5) media (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant differences were detected in specific growth rate or generation time between the two media during this phase. A significant interaction between culture medium and time was nevertheless observed across the entire experiment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). Cultures grown in f medium continued to increase until day 22, when they reached their maximum cell density, whereas those grown in f/5 medium peaked on day 16 and declined thereafter (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eGrowth rate (\u0026micro;), generation time (Gt) at exponential growth phase, exponential phase duration, maximum cell density and final cell density of \u003cem\u003eChaetoceros\u003c/em\u003e sp. cultivated in f and f/5 growth media. Mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrowth medium\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026micro;\u003c/p\u003e\u003cp\u003e(divisions day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGt\u003c/p\u003e\u003cp\u003e(days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExponential phase duration (days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMaximum cell density\u003c/p\u003e\u003cp\u003e(\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFinal cell density\u003c/p\u003e\u003cp\u003e(\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ef\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.76 at day 22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ef/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.02 at day 16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHigh variability was observed in both the apical axis (AA) and pervalvar axis (PA) across all growth phases in cultures grown in both f and f/5 media, with a tendency toward cell enlargement over cultivation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During the exponential growth phase, the AA ranged between 4.1\u0026ndash;10.5 \u0026micro;m and the PA ranged between 4.0-10.3 \u0026micro;m. In the late stationary growth phase, the AA ranged between 4.2\u0026ndash;9.9 and the PA ranged between 4.9\u0026ndash;13.8 \u0026micro;m. The PA/AA ratio indicated a tendency for cells to change morphology depending on the culture medium. In f medium, diatoms exhibited a steady increase in PA over time, leading to a PA/AA ratio that rose from 1.09 in the exponential growth phase to 1.53 in the late stationary growth phase. In the late stationary growth phase under f/5 medium conditions, cells exhibited a marked morphological transformation, adopting a nearly spherical shape. This morphological shift was corroborated by an increase in AA and the PA/AA ratio over time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBiovolume showed a slight increase over time in both f and f/5 media; however, cells consistently exhibited higher values in f/5 medium across all growth phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the late stationary growth phase, a distinct subpopulation was observed, comprising 81% of the cells in f/5 medium and 28% in f medium, characterized by nearly spherical morphology, while the remaining cells retained the typical prism on elliptic base or ellipsoid shape. The highest biovolume recorded (307.8 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e) corresponded to cells in f/5 medium during the late stationary growth phase, surpassing that of all other growth conditions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePigments and photosynthesis\u003c/h3\u003e\n\u003cp\u003eThe content of photosynthetic pigments\u0026mdash;chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003ec\u003c/em\u003e, and carotenoids\u0026mdash;did not differ significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between culture media during the exponential growth phase. However, significant differences were observed during both the stationary and late stationary growth phases (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Chlorophyll \u003cem\u003ea\u003c/em\u003e content was significantly influenced by growth phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and culture medium (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Cultures grown in f medium exhibited higher chlorophyll \u003cem\u003ea\u003c/em\u003e content during the late stationary growth phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while cultures in f/5 medium had significant higher chlorophyll \u003cem\u003ea\u003c/em\u003e level during the stationary growth phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eChlorophyll \u003cem\u003ec\u003c/em\u003e content was significantly affected by growth phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), culture medium (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023), and their interaction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0402) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Higher chlorophyll \u003cem\u003ec\u003c/em\u003e concentrations were observed in f-medium cultures during both the stationary (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0051) and late stationary growth phases (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0081). In both media, chlorophyll \u003cem\u003ec\u003c/em\u003e content in cultures peaked during the stationary growth phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eCarotenoid content was significantly affected by growth phase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0011), with highest values recorded in the late stationary growth phase in f medium (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0024) and in the stationary growth phase in f/5 medium (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0014) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). A significant effect of culture medium on carotenoid content was observed only during the stationary growth phase, with higher values in f/5-grown cultures (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0009). No significant differences were observed in other growth phases.\u003c/p\u003e\u003cp\u003ePhotosynthesis-irradiance curves (rETR) curves showed significant variations across growth phases, with maximum values observed during the exponential growth phase for both f (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.46\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e) and f/5 media (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.08\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Photosynthetic parameters also varied significantly with growth phase and culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Photosynthetic efficiency (α), saturation irradiance (I\u003csub\u003ek\u003c/sub\u003e), maximum relative electron transport rate (ETR\u003csub\u003emax\u003c/sub\u003e), and maximum quantum yield of PSII (Fv/Fm) were all higher in the exponential growth phase across both treatments. Culture medium had a significant effect on α (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0009) and Fv/Fm (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with lower values observed in f/5 medium during the late stationary growth phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNutrient-limiting conditions are common in marine environments, and phytoplankton species display diverse physiological responses to such stressors. Diatoms, in particular, have shown remarkable adaptability to environmental fluctuations, allowing them to thrive and persist across a wide range of marine habitats (Ambrust 2009).\u003c/p\u003e\u003cp\u003eThe use of f/5 medium in this study was guided not only by experimental design but also by ecological relevance, as it simulates the nutrient conditions characteristic of the Pacific Ocean off the coast of Ensenada, Baja California\u0026mdash;the natural habitat where the \u003cem\u003eChaetoceros\u003c/em\u003e sp. strain used in this research was originally isolated. Studies in coastal waters of the Gulf of California have reported typical surface concentrations of phosphates (~\u0026thinsp;0.85 \u0026micro;M) and nitrates (~\u0026thinsp;2.35 \u0026micro;M) (Bustos-Serrano et al. 2006), while nearby coastal channels show nitrate-nitrite levels around 4.9 \u0026micro;M and phosphorus\u0026thinsp;~\u0026thinsp;2.5 \u0026micro;M (Soto-Balderas and Alvarez-Borrego \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Linacre et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These values closely match the nutrient composition of the f/5 medium, supporting its use as a realistic representation of the natural habitat conditions for \u003cem\u003eChaetoceros\u003c/em\u003e sp.\u003c/p\u003e\u003cp\u003eNo significant differences were observed between f and f/5 media in the growth rate or generation time of \u003cem\u003eChaetoceros\u003c/em\u003e sp. during the exponential growth phase of our experiments. However, by the end of the cultivation period, cultures grown in f/5 medium reached significantly lower maximum cell densities compared to those grown in full-strength f medium. This reduction was primarily attributed to nutrient limitation, which became increasingly pronounced during the stationary and late stationary growth phases. The growth rate of \u003cem\u003eChaetoceros\u003c/em\u003e sp. in both culture media was similar to that previously reported for this strain by P\u0026eacute;rez-Varillas and S\u0026aacute;nchez-Saavedra (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, it was higher than those reported for other \u003cem\u003eChaetoceros\u003c/em\u003e species cultured under similar nutrient conditions (Liang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pacheco-Vega et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This enhanced growth performance highlights the strain\u0026rsquo;s potential for aquaculture applications. Microalgae with intermediate cell sizes, around 100 \u0026micro;m\u0026sup3;, exhibit the highest photosynthetic productivity per biomass unit and the fastest growth rates (Borowitzka \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The maximum cell density in f medium cultures (1.76\u0026times;10⁶ cells mL⁻\u0026sup1;) was lower than that found in the study by S\u0026aacute;nchez-Saavedra and Voltolina (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) for the same species (5.43\u0026times;10⁶ cells mL⁻\u0026sup1;). These discrepancies may be attributed to differences in cultivation conditions, such as culture volume, inoculum size, and the light intensity employed.\u003c/p\u003e\u003cp\u003eThe results show that the pervalvar axis (PA) and biovolume of \u003cem\u003eChaetoceros\u003c/em\u003e sp. increased progressively through the growth phases in both f and f/5 media. These findings are consistent with P\u0026eacute;rez-Varillas and S\u0026aacute;nchez-Saavedra (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), who reported larger biovolumes during the stationary phase compared to the exponential phase for the same strain. In centric diatoms, vegetative cell enlargement serves as an alternative mechanism for restore cell size when conditions for sexual reproduction are not met, providing an adaptive advantage (Mills and Kaczmarska \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kaczmarska et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStress factors such as nutrient limitation or temperature fluctuations can induce changes in cell morphology and carbon content, as reported in species like \u003cem\u003eThalassiosira pseudonana\u003c/em\u003e (O'Donnell et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, the larger biovolume observed under nutrient-limited (f/5) conditions across all growth phases may enhance survival or promote vertical migration, particularly under stress (Fu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This response aligns with previous reports showing that nutrient stress can significantly increase cell size in diatoms (Peter and Sommer \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For instance, phosphorus limitation induced elongation of the pervalvar axis and thickening of setae in \u003cem\u003eChaetoceros peruvianus\u003c/em\u003e resulting in increased cell volume (Smodlaka Tankovic et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Silica limitation also led to increased cell volume in \u003cem\u003eC. calcitrans\u003c/em\u003e and \u003cem\u003eT. pseudonana\u003c/em\u003e (Laing \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cell enlargement under high irradiance or Si limitation promotes sinking, potentially allowing cells to reach deeper, less stressful environments where photo-oxidative damage is reduced (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Large cells exhibit superior adaptation to fluctuating environmental conditions, largely due to their lower respiration rates and greater capacity for nutrient storage (Geider et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Litchman et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe content of photosynthetic pigments\u0026mdash;chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e, and carotenoids\u0026mdash;was similar in both culture media during the exponential growth phase, when nutrients were not limiting. In contrast, significant differences emerged during the stationary and late stationary phases. Notably, pigment levels in cultures grown in f medium during the late stationary phase resembled those in f/5 medium during the stationary phase, suggesting that nutrient concentration exerts a stronger influence than growth phase alone on the physiological response of diatoms.\u003c/p\u003e\u003cp\u003eCells in both f and f/5 media exhibited higher levels of chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e during the stationary and late stationary phases than during the exponential phase, in response to reduced light availability and nutrient depletion in the medium. These findings suggest that under suboptimal conditions, \u003cem\u003eChaetoceros\u003c/em\u003e sp. increases its chlorophyll \u003cem\u003ea\u003c/em\u003e content to enhance light-harvesting capacity\u0026mdash;a strategy previously reported in diatoms and other microalgae to sustain photosynthetic activity (Sigaud-Kutner et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Masoj\u0026iacute;dek et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; L\u0026oacute;pez-Sandoval et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCarotenoid content was significantly affected by both growth phase and nutrient availability. The highest concentrations were recorded during the stationary phase in f/5 medium and during the late stationary phase in both media, aligning with periods of pronounced nutrient depletion. This suggests a photoprotective response to nutritional stress. Under such conditions, the absorbed light energy may exceed the metabolic capacity for its utilization, causing an imbalance between photochemical energy input and downstream metabolic processes\u0026mdash;potentially resulting in photoinhibition or oxidative stress (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Carotenoids are known to quench reactive oxygen species (ROS), and their accumulation under nutrient stress has been widely reported in marine diatoms as a defense mechanism (Dimier et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Latowski et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kuczynska et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, the elevated carotenoid levels in f/5-grown cultures likely reflect an adaptive strategy to mitigate photo-oxidative damage under nutrient-deficient conditions (Lepetit and Dietzel \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhotosynthetic performance, as indicated by rETR curves and associated parameters, declined as \u003cem\u003eChaetoceros\u003c/em\u003e sp. cultures advanced through the growth phases. This trend likely reflects metabolic stress due to progressive nutrient depletion. A balanced flow between photosynthetic energy input and carbon allocation for biosynthesis depends on optimal environmental conditions, including light, nutrient availability and, temperature (Yan et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Under nitrogen-limited conditions, phytoplankton exhibit reduced photosynthetic efficiency, primarily due to impaired development and functioning of the light-harvesting complexes (Young and Beardall \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The culture medium and growth phase significantly affected photosynthetic parameters, including α, ETRmax and the maximum quantum yield of PSII (Fv/Fm), with lower values observed in f/5 medium. This agrees with previous studies, for instance, Liang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported declines in α, ETRmax, I\u003csub\u003ek\u003c/sub\u003e, and Fv/Fm in \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e and \u003cem\u003eChaetoceros muellerii\u003c/em\u003e during batch culture aging, indicating that nutrient limitation progressively impairs the photosynthetic apparatus.\u003c/p\u003e\u003cp\u003eIn our study, Fv/Fm was particularly sensitive to nutrient conditions, showing differences between media even in the exponential phase, unlike other photosynthetic traits such as pigment content. This supports previous findings by Tan et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who observed reductions in Fv/Fm under nitrogen or phosphorus limitation across several microalgal groups, including diatoms, and proposed this parameter as a reliable indicator of phytoplankton physiological status. High I\u003csub\u003ek\u003c/sub\u003e values during the exponential growth phase of \u003cem\u003eChaetoceros\u003c/em\u003e sp. in both f and f/5 media indicate a high capacity to tolerate elevated light intensities (\u0026gt;\u0026thinsp;2 \u0026times; 10\u0026sup3; \u0026micro;mol photons m⁻\u0026sup2; s⁻\u0026sup1;). Similarly, higher I\u003csub\u003ek\u003c/sub\u003e values during the exponential phase (~\u0026thinsp;1\u0026ndash;3 \u0026times; 10\u0026sup3; \u0026micro;mol photons m⁻\u0026sup2; s⁻\u0026sup1;), compared to the stationary phase, have been reported for \u003cem\u003eP. tricornutum\u003c/em\u003e, \u003cem\u003eC. muelleri\u003c/em\u003e and \u003cem\u003eC. gracilis\u003c/em\u003e (P\u0026eacute;rez-Varillas and S\u0026aacute;nchez-Saavedra \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the study by Liang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), I\u003csub\u003ek\u003c/sub\u003e decreased with increasing culture age in \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. muelleri\u003c/em\u003e, although their values were considerably lower (80\u0026ndash;200 \u0026micro;mol photons m⁻\u0026sup2; s⁻\u0026sup1;). These differences may reflect variations in culture conditions, methodologies used for fluorescence measurements, and intrinsic species-specific traits. Due to its tolerance to high irradiance, \u003cem\u003eChaetoceros\u003c/em\u003e sp. exhibits a high potential for aquaculture and other biotechnological applications (P\u0026eacute;rez-Varillas and S\u0026aacute;nchez-Saavedra \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eChaetoceros\u003c/em\u003e sp. cells during the exponential growth phase exhibited a higher metabolic rate, reflected in high growth rates, greater photosynthetic efficiency (α and Fv/Fm), and smaller cell size\u0026mdash;features typical of an energy-intensive strategy under favorable conditions. As culture aged, growth slowed or ceased, pigment content increased, photosynthetic efficiency declined, and cell enlarged, particularly under nutrient-limited (f/5) conditions. This transition indicates a reallocation of resources from biomass production to maintenance, stress tolerance, and photoprotection\u0026mdash;a survival strategy under nutrient shortage (Geider et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and fluctuating light conditions typical of coastal environment (Lavaud \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe photosynthetic apparatus dynamically regulates light harvesting and energy dissipation through thylakoid-associated complexes, which undergo highly adaptive responses to environmental cues (Lepetit and Dietzel \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Photosynthetic regulation in strains from coastal habitats\u0026mdash;characterized by strong fluctuations in nutrients and light\u0026mdash;exhibits greater flexibility than that of strains from open-ocean ecosystems (Lavaud et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur results show that nutrient limitation in f/5 medium induced significant morphological and physiological changes in \u003cem\u003eChaetoceros\u003c/em\u003e sp., including increased biovolume, a shift toward spherical cells, and reduced photosynthetic activity during the late stationary phase. These findings are consistent with previous reports describing the development of resting stages in diatoms under unfavorable conditions and vegetative senescence (McQuoid and Hobson \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The decline in photosynthetic pigments (chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e, and carotenoids), relative electron transport rate (rETR), and key photosynthetic parameters\u0026mdash;particularly photosynthetic efficiency (α) and the maximum quantum yield of PSII (Fv/Fm)\u0026mdash;in cells grown in f/5 medium during the late stationary phase is associated with reduced metabolic activity. In a study by Kuwata et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), resting cells of \u003cem\u003eChaetoceros pseudocurvisetus\u003c/em\u003e exhibited a decrease in chlorophyll \u003cem\u003ea\u003c/em\u003e content and photosynthetic rate of up to 17% compared to vegetative cells. Nutrient-limiting conditions can trigger dormant-like stages in diatoms, enabling them to survive until favorable environmental conditions are restored\u0026mdash;a key survival strategy in dynamic marine ecosystems (Ellegaard and Ribeiro \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The formation of resting cells observed in \u003cem\u003eChaetoceros\u003c/em\u003e sp. cultured in f/5 medium represents a novel finding, as this response has not been previously described for this strain isolated from Todos Santos Bay, Mexico. This nutrient-limitation-induced response offers a new methodological approach for the controlled induction of resting cells, with potential applications in aquaculture and biotechnology, particularly for cell preservation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eChaetoceros\u003c/em\u003e sp. displays notable physiological and morphological plasticity in response to variations in nutrient availability and the duration of cultivation. Growth, cell morphology, pigment composition, and photosynthetic parameters varied significantly between full-strength (f) and nutrient-limited (f/5) media, particularly during the stationary and late stationary growth phases. Notably, cells grown under nutrient limitation (f/5) exhibited increased biovolume, spherical morphology, elevated carotenoid content, and reduced photosynthetic performance\u0026mdash;features consistent with stress responses and the onset of dormancy. The formation of putative resting cells under f/5 nutrient-limited conditions reveals a novel and ecologically meaningful adaptive response in this \u003cem\u003eChaetoceros\u003c/em\u003e strain, previously unreported for isolates from Todos Santos Bay, Mexico. These results suggest that controlled nutrient limitation may serve as a practical tool for inducing dormancy in diatoms, with implications for the preservation of ecologically or economically important strains in aquaculture. The ability of \u003cem\u003eChaetoceros\u003c/em\u003e sp. to modulate its cellular architecture and energy metabolism under suboptimal conditions highlights its adaptive capacity and supports its potential for sustainable use in marine biotechnological applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e S. Fierro acknowledges their Doctoral scholarship from Secretar\u0026iacute;a de Ciencias, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (SECIHTI). To PhD. C.A. Molina-C\u0026aacute;rdenas and F.Y. Castro-Ochoa for their technical assistance with photosynthesis analysis. To PhD. B. Bar\u0026oacute;n-Sevilla and PhD. M.D.R. Zacar\u0026iacute;as-Soto for providing the equipment used for the diatom measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.F.: Conceptualization, methodology, validation, formal analysis, investigation, writing - original draft. R.C.F.: Review and editing, supervision. M.P.S.S.: Resources, writing \u0026ndash; review and editing, supervision, project administration, funding acquisition. \u0026nbsp;All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was funded by Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE, project: 623-108).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eData will be available upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e The authors followed all applicable international, national, and/or institutional guidelines for the care and use of microalgae cultures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArmbrust EV (2009) The life of diatoms in the world\u0026apos;s oceans. 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Environmental and Experimental Botany 114:30-47\u003c/li\u003e\n \u003cli\u003eLi Z, Li W, Zhang Y, Hu Y, Sheward R, Irwin AJ, Finkel ZV (2021) Dynamic photophysiological stress response of a model diatom to ten environmental stresses. J Phycol 57:484-495\u003c/li\u003e\n \u003cli\u003eLinacre L, Lara-Lara JR, Mirabal-G\u0026oacute;mez U, Durazo R, Baz\u0026aacute;n-Guzm\u0026aacute;n C (2017) Microzooplankton grazing impact on the phytoplankton community at a coastal upwelling station off northern Baja California, Mexico. Cienc Mar 43(2):93-108\u003c/li\u003e\n \u003cli\u003eLitchman E, Klausmeiera A, Yoshiyama K (2009) Contrasting size evolution in marine and freshwater diatoms. PNAS 106:2665-2670\u003c/li\u003e\n \u003cli\u003eLiu H, Yuan Z, Gosnell KJ, Liu T, Tammen JK, Wen Z, Engel A, Liu X, Huang B, Kao SJ, Achterberg EP (2024) Patterns of (micro) nutrient limitation across the South Pacific Ocean. Commun Earth Environ 5(1):596\u003c/li\u003e\n \u003cli\u003eLiang Y, Heraud P, Beardall J (2006) Changes in growth, chlorophyll fluorescence and fatty acid composition with culture age in batch cultures of \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e and \u003cem\u003eChaetoceros muelleri\u0026nbsp;\u003c/em\u003e(Bacillariophyceae). Bot Mar 49:165-173\u003c/li\u003e\n \u003cli\u003eLomas MW, Glibert PM (1999) Interactions between NH₄⁺\u0026nbsp;and NO₃⁻\u0026nbsp;uptake and assimilation: comparison of diatoms and dinoflagellates at several growth temperatures. Mar Biol 133:541\u0026ndash;551\u003c/li\u003e\n \u003cli\u003eL\u0026oacute;pez-Sandoval DC, Rodr\u0026iacute;guez-Ramos T, Cerme\u0026ntilde;o P, Sobrino C, Mara\u0026ntilde;\u0026oacute;n E (2014) Photosynthesis and respiration in marine phytoplankton: Relationship with cell size, taxonomic affiliation, and growth phase. 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J Phycol 39:897\u0026ndash;905\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photosynthesis, Chaetoceros sp., growth rate, cell size, pigments","lastPublishedDoi":"10.21203/rs.3.rs-7652804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7652804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the physiological and morphological responses of the marine diatom \u003cem\u003eChaetoceros\u003c/em\u003e sp. strain CHX1 when cultured under two contrasting nutrient conditions: nutrient-rich f medium and nutrient-limited f/5 medium. Growth curves showed no significant differences in specific growth rate during the exponential phase; however, final cell densities were higher in f medium. Over culture time, cells exhibited increased biovolume and changes in shape, particularly in f/5 medium, where a subpopulation of spherical cells emerged during the late stationary phase. Pigment content, including chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e and total carotenoids, varied significantly across growth phases and media, with nutrient limitation promoting pigment accumulation as a potential stress response. Photosynthetic performance—assessed via relative electron transport rate (rETR), efficiency (α), saturation irradiance (I\u003csub\u003ek\u003c/sub\u003e), and maximum quantum yield (Fv/Fm)—was highest during exponential growth and declined under nutrient limitation. Fv/Fm was particularly sensitive to nutrient stress, showing consistently lower values in nutrient-limited (f/5) cultures compared to the full-strength (f) medium across all growth phases. \u003cem\u003eChaetoceros sp.\u003c/em\u003e exhibited pronounced morphological and photophysiological plasticity in response to nutrient availability and culture duration. Under nutrient limitation (f/5), cells developed larger biovolume, spherical morphology, elevated carotenoid levels, and reduced photosynthetic efficiency—traits associated with stress acclimation and the onset of dormancy. The formation of resting cells represents a novel response for this strain, suggesting that controlled nutrient limitation may serve as a tool for diatom preservation, with applications in aquaculture and biotechnology.\u003c/p\u003e","manuscriptTitle":"Photosynthetic and morphological responses of Chaetoceros sp. to nutrient limitation over culture age","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 08:50:20","doi":"10.21203/rs.3.rs-7652804/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-12-16T01:55:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231522821646009535645752555380243533988","date":"2025-11-20T08:25:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192337355684587549587103181222567159942","date":"2025-10-31T07:02:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T10:09:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188504308556717856227710532407763679373","date":"2025-10-06T09:38:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T00:50:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-25T00:47:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-24T16:52:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2025-09-18T20:39:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bd234987-0ae5-497e-b85d-a28ade39a2d7","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-16T02:08:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-07 08:50:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7652804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7652804","identity":"rs-7652804","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}