Influence of pH and light intensity on the morphology of the nutritious green microalga Monoraphidium littorale

Influence of pH and light intensity on the morphology of the nutritious green microalga Monoraphidium littorale | 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 Influence of pH and light intensity on the morphology of the nutritious green microalga Monoraphidium littorale Arifa Sultana, Saleha Khan, Md. Mahfuzul Haque, Zoarder Faruque Ahmed, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8605830/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Morphological plasticity in Monoraphidium littorale —the transition from fusiform to oval and spherical cells—affects growth staging, autosporulation, and potential product yields. The relative contributions and kinetics of two key drivers, pH and light intensity, remain insufficiently resolved. Methods Replicated batch cultures were maintained in standard medium under controlled temperature and photoperiod. Independent experiments tested pH (6.5–9.0) and light intensity (10, 30, 50, 70 µmol m⁻² s⁻¹). Cell morphotypes (fusiform, oval, spherical) were enumerated from day 20 to day 70 using a consistent microscopy protocol. Results All treatments remained uniformly fusiform until ~ day 20. Thereafter, alkaline pH (≥ 8.5) advanced morphotype switching and produced earlier dominance of oval/spherical forms, indicating a graded, state-setting effect. Light intensity showed a delayed, threshold-like signature: divergence among treatments became pronounced only after ~ day 20, with ≥ 50–70 µmol m⁻² s⁻¹ compressing the timeline to oval/spherical dominance, while 10–30 µmol m⁻² s⁻¹ preserved fusiform cells longer into stationary phase. Integrating both experiments, pH acts as a continuous baseline regulator, whereas light functions as a phase-gated accelerator. Conclusions pH and light exert complementary control over M. littorale morphogenesis. A practical, two-stage strategy emerges—grow under near-neutral pH and sub-threshold light to build biomass (fusiform dominance), then shift to alkaline pH and supra-threshold irradiance to expedite remodeling/autosporulation. Future factorial (pH × light) designs and image-based morphometrics should quantify interactions and refine time-indexed set-points for energy-aware, reproducible bioprocess control. Monoraphidium littorale morphological plasticity pH light intensity autosporulation culture control Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 1. Introduction Microalgae are increasingly central to the bio-economy because they synthesize high-value products including carotenoids, polyunsaturated fatty acids, proteins, and diverse bioactive metabolites—relevant to nutraceutical, pharmaceutical, food, cosmetic, and aquaculture industries (Villaro et al., 2021; Saha & Murray, 2018 ; Novoveská et al., 2019 ; Bhalamurugan et al., 2018 ). Among these, the keto-carotenoid astaxanthin is commercially prominent for its potent antioxidant capacity and for enhancing pigmentation and health performance in aquaculture species, thereby improving product acceptance as well as conferring immunological and physiological benefits (Villaro et al., 2021; Saha & Murray, 2018 ). Growing demand for natural pigments and functional ingredients has, in turn, intensified interest in microalgal biomass as a sustainable feed additive and as a partial substitute for conventional feedstocks such as fishmeal and soymeal, with reported gains in pigmentation, growth, and immunity, and reduced reliance on synthetic additives (Berchielli-Morais et al., 2015 ; Batista et al., 2017 ; Dixit et al., 2021 ; Silva et al., 2022 ; Kratzer & Murkovic, 2021 ). Within this broader context, Monoraphidium littorale has emerged as a promising species owing to its robust growth and favorable biochemical profile. Reports indicate that its fatty-acid composition encompasses nutritionally relevant omega-3 and omega-6 fractions, supporting potential applications in aquaculture feeds and health-oriented products; preliminary evidence further suggests that metabolites from M. littorale may enhance overall performance in cultured organisms (Silva et al., 2022 ; Dixit et al., 2021 ; Sahni et al., 2019 ; Margenat et al., 2023 ). Although M. littorale has been proposed as a candidate for natural pigment production, including putative astaxanthin pathways, such claims require cautious evaluation and optimization under controlled cultivation regimes. A central challenge is that the physiology and morphology of microalgae are tightly coupled to environmental conditions. Nutrient concentration, temperature, and irradiance all modulate cellular function, yet pH is often a dominant determinant of growth, photosynthetic performance, nutrient uptake, and cell-wall dynamics (Salbitani et al., 2021 ; Bensalem et al., 2020 ; Hu et al., 2023 ). Studies in chlorophytes (e.g., Scenedesmus, Coccomyxa) show that pH shifts can drive changes in morphotypes, pigment biosynthesis, and growth rates—highlighting the capacity for morphological plasticity as a strategic response to environmental pressure (Akgül & Akgül, 2022 ; Widyaningrum et al., 2024 ). While lower pH may increase the availability of certain nutrients, it can also suppress pigment production and alter cell shape; conversely, alkaline conditions can facilitate remodeling toward more stress-tolerant forms (Sahni et al., 2019 ; Min et al., 2023 ). Consequently, deliberate pH control has become integral to maximizing biomass and metabolite yields across several commercially relevant taxa (Uebel et al., 2019 ; Bhalamurugan et al., 2018 ). Light intensity constitutes a second, equally important lever, governing energy supply for photosynthesis and shaping redox balance and carbon assimilation. However, for M. littorale the combined influence of pH and irradiance on morphological trajectories remains poorly resolved. This gap is consequential: morphological state in chlorophytes is not merely descriptive but often indexes physiological status, stress tolerance, and, by extension, prospects for productivity and biochemical composition. To address this, the present study investigates the time-dependent morphological responses of M. littorale to controlled pH (6.5–9.0) and light intensity (10, 30, 50, 70 µmol m⁻² s⁻¹) across culture phases. We test the working hypothesis that alkaline pH imposes a graded, state-setting bias toward remodeling, whereas higher irradiance acts as a phase-gated accelerator once cultures exit exponential growth. By mapping the transitions among fusiform, oval, and spherical morphotypes under each driver, we aim to establish practical set-points for culture staging and to provide a basis for energy-aware, scalable bioprocess control in aquaculture and nutraceutical applications (Merlo et al., 2021 ; Chen et al., 2015 ). Objectives of the Study To describe the life cycle and main cell shapes (fusiform, oval, spherical) of Monoraphidium littorale during culture. To measure how pH (6.5–9.0) changes cell shape over time. To measure how light intensity (10, 30, 50, 70 µmol m⁻² s⁻¹) changes cell shape over time. To compare the effects of pH and light and identify which factor acts earlier and more strongly. To determine the time points when fusiform cells start to decline under each condition. To suggest simple culture settings (pH and light) for biomass growth and for triggering remodeling for practical use. 2. Materials and methods M. littorale was isolated from the freshwater ponds adjacent to the Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh. Through random sampling, various microalgal samples were collected those were kept in sterilized nutrient solutions (Bold Basal Medium). Streaked agar plating and serial dilution were frequently used for isolation. Stock cultures were maintained using Bold Basal Medium (BBM) (Table 1 ) at ambient temperature of 28–30°C, light intensity 60 µmol m − 2 s − 1 , and photoperiod 12:12 h, L:D. The pH of the medium was adjusted over the culture period by addition of NaOH and HCL. Culture media were autoclaved for 15 min at 121 o C, and aged for several days prior to inoculation. The study was performed in triplicate for each pH level (treatment) using an inoculum for the culture in logarithmic phase having cell density of 2‧74 \(\:\times\:\) 10 4 cells mL − 1 . Morphology was monitored immediately after collection of cells from cultures in exponential and stationary phases. In order to prepare BBM as a culture medium, 10 mL solution from each of the listed chemicals (Table 1 ) from serial nos. 1 to 6 were poured into a 1 L conical flask. Then 1 mL of the solution of each of the listed (Table 1 ) chemicals from serial nos. 7 to 10 were poured into the flask, and distilled water was added to make the volume 1.0 L. The solution into the flask was mixed well and sterilized at 121°C for 15 minutes with moist heat using an autoclave (Model SS-V35HD, WINCOM, China). For growth studies, M. littorale cells were grown in a 500-mL flask containing BBM. The determination of the cell concentration was done by direct counting using a Sedgewick-Rafter chamber. Morphological observation was performed right after the inoculation and then after every 10 days up to 70 days of culture. Samples were shaken well before being collected for counting. Table 1 Stock solutions of the chemicals for the preparation of BBM for M. littorale culture. * No. Stocks of Chemicals gL − 1 1 NaNO 3 25.00 2 MgSO 4 . 7H 2 O 7.50 3 NaCl 2.50 4 K 2 HPO 4 7.50 5 KH 2 PO 4 17.50 6 CaCl 2 . 2H 2 O 2.50 7 Trace Elements ZnSO 4 . 7H 2 O 4.42 MnCl 2 . 4H 2 O 1.44 MoO 3 0.71 CuSO 4 . 5H 2 O 1.57 Co (NO 3 ) 2 . 6H 2 O 0.49 8 H 3 BO 3 11.40 9 EDTA-KOH solution EDTA Na 2 50.00 KOH 31.00 10 FeSO 4 .7H 2 O with 1.0 ml Concentrated H 2 SO 4 4.98 * The chemicals were bought from Z.H. Scientific and Chemicals Mart, Bangladesh. 2.1 Morphological observations Morphological changes of M. littorale was observed by using a light microscope (B-510BT OPTIKA, Italy). The morphological study of M. littorale was done for 70 days. 2.2 Statistical Analysis of the Data The data are expressed as mean ± standard deviation (SD) of the three replicates. To analyze the data on the morphological shapes of the microalgae in different treatments (pH levels) using SPSS 25. Significant differences (at P ˂ 0.05) among the means have been determined using Duncan’s multiple range test (DMRT) [25]. 2.3 Light-intensity experiment Cultures in late-log phase were inoculated into BBM and exposed to four light treatments: 10, 30, 50, and 70 µmol m⁻² s⁻¹ under 12:12 h L:D at 28–30°C. Each treatment used n = 3 flasks (500 mL) with an identical inoculum density as in the pH experiment. Morphotypes (fusiform, oval, spherical) were enumerated at day 20, 30, 40, 50, 60, 70 using the same microscopy and counting procedures. 3. Results 3.1 Life cycle of M. littorale This study primarily summarized the morphology of M. littorale in response to six different pH levels (pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0). The shape of M. littorale cells varied with different pH levels along with the culture period, and morphological observation revealed three types of shapes: fusiform, oval, and spherical (Fig. 1 , Fig. 2 ). In acidic environment, at the early stationary phase, M. littorale showed only fusiform cells. On the other hand, in alkaline environment M. littorale gradually changed into oval and spherical cells along with fusiform cells. Firstly, fusiform cells turned into oval-shaped cells, and then oval-shaped cells turned into spherical-shaped cells. 3.1.1 Auto-sporulation of M. littorale from fusiform to fusiform At the initial stage of the culture age, during the logarithmic phase (6–18 days), M. littorale reproduced through auto-sporulation and produced only 4, 8, or 16 fusiform autospores, like the mother cell, in the presence of enough nutrients in the culture medium. This time, no morphological alteration was observed at varying pH values (6.5–9.0; Fig. 3 ). 3.1.2 Auto-sporulation of M. littorale from fusiform to oval and oval to spherical During the stationary phase at different pH levels (6.5–9.0), M. littorale reproduced by auto-sporulation along with some additional morphological changes and produced 4, 8, or 16 oval and spherical autospores. This time, fusiform cells first turned into oval-shaped cells, and these oval-shaped cells again produced oval-shaped cells. Finally, the oval-shaped cells of M. littorale turned into spherical cells. These spherical cells can again go through cell division and produced 4, 8, or 16 spherical autospores. Under favorable culture conditions, these autospores were also able to transform their spherical shape into a fusiform shape by consuming sufficient nutrients, as they are a resilient species that can recoil or spring back into their previous shape after being bent, stretched, or compressed (Fig. 4 ). 3.2 Effects of pH on morphology of M. littorale Morphological alterations of M. littorale in response to varying pH levels ranging from 6.5 to 9.0 with intervals of 0.5 were observed during the culture period, particularly started on the 20th day and continued on every 10 days up to the 70th day of the culture period. In the early stationary phase in an acidic to slightly alkaline environment, only fusiform cells of M. littorale were observed, whereas a small proportion of oval and spherical cells were observed under alkaline conditions. As the culture age progressed into the stationary phase, fusiform cells began to convert into oval and spherical cells, which occurred simultaneously with an increase in pH. In the late stationary phase, oval and spherical cells became dominant over fusiform cells in alkaline conditions. 3.2.1 Morphological changes of M. littorale on 20th and 30th day In the present investigation it was found that transformations in morphology, from fusiform to oval, subsequently followed by spherical, were related to changes in pH and the length of culture period. The morphological alterations of M. littorale during the early stationary phase (20th and 30th day) are shown in Fig. 5 and Fig. 6 . On the 20th day, all the cells of M. littorale were found fusiform (100%) across all tested pH levels ranging from 6.5 to 9.0. Fusiform cells were found at lower pH values (6.5 and 7.0) for the entire duration of the early stationary phase, particularly on the 20th and 30th days. As the pH increased and culture age continued for the next 10 days, fusiform-shaped cells began to transform their shape into oval-shaped as well as spherical-shaped at a significantly lower rate (5 − 10%) for the pH levels of 7.5 to 9.0 on the 30th day. 3.2.2 Morphological changes of M. littorale on 40th and 50th day At mid-stationary phase (on 40th and 50th day), morphological changes of M. littorale at different pH levels (from 6.5 to 9.0) are shown in Fig. 7 and Fig. 8 . On the 40th day, oval and spherical-shaped cells were observed at neutral pH (7.0) to alkaline pH (9.0), increasing from 5% (oval and spherical) to 25% (oval) and 15% (spherical). In contrast, only fusiform cells were observed in an acidic (pH 6.5) environment. Significantly more oval and spherical-shaped cells were found in higher alkaline (pH 8.0 to 9.0) conditions than in neutral (pH 7.0) and mildly alkaline (pH 7.5) conditions. On the 50th day, cells with oval, spherical, and fusiform shapes were observed at all pH levels investigated. Moreover, a significantly higher percentage of oval and spherical-shaped cells were observed in the alkaline conditions (pH 8.0 to 9.0) than in the acidic to slightly alkaline conditions (pH 6.5 to 7.5). 3.2.3 Morphological changes of M. littorale on 60th and 70th day At the late stationary phase (60th and 70th day), morphological changes of M. littorale at pH 6.5 to 9.0 are presented in Fig. 9 and Fig. 10 . On the 60th day, a higher number of oval (50%) and spherical (35%) shaped cells were observed in higher alkaline conditions (pH 8.0 to 9.0), and a moderate number of oval (30%) and spherical (20%) shaped cells were found in slightly alkaline conditions (pH 7.5). Besides, a comparatively lower number of oval (15%) and spherical-shaped (10%) cells were observed in acidic to neutral conditions (pH 6.5 to 7.0). On the 70th day, the number of fusiform-shaped cells exhibited a significant decrease (P < 0.05) from acidic to alkaline conditions (pH 6.5–9.0) (Fig. 9 ). The highest number of M. littorale cells exhibited morphological changes in higher alkaline conditions (pH 8.0–9.0), as 95% of the cells turned into oval and spherical-shaped cells (45% oval and 50% spherical, respectively). On the 70th day, the highest number of fusiform-shaped cells (60%–50%) and a comparatively lower number of oval and spherical cells (25%–30% oval and 15%–20% spherical) were observed in acidic to neutral conditions (pH 6.5 and 7.0, respectively). Moreover, a moderate number of M. littorale cells showed morphological alterations in a slightly alkaline condition (pH 7.5), with nearly equal proportions (35% fusiform, 35% oval, and 30% spherical) of all types of cells observed. 3.3 Effects of Light Intensity on Morphology of M. littorale We evaluated four irradiance levels (10, 30, 50, and 70 µmol m⁻² s⁻¹) to determine when, and how strongly, light intensity alters the time-course of morphotype change in Monoraphidium littorale . Across all treatments, cultures remained uniformly fusiform during the first ~ 20 days; therefore, morphological scoring was performed from day 20 onward at 10-day intervals until day 70. 3.3.1 Morphological Changes of M. littorale at 20th & 30th Day in Different Light Intensities At day 20, all light treatments showed exclusively fusiform cells (100%). By day 30, divergence was evident only at higher irradiance: at 70 µmol m⁻² s⁻¹, ~ 20% of cells were oval and ~ 10% spherical (≈ 70% fusiform), and at 50 µmol m⁻² s⁻¹, ~ 15% were oval and ~ 5% spherical (≈ 80% \ fusiform). In contrast, 30 and 10 µmol m⁻² s⁻¹ maintained 100% fusiform populations in (Fig. 11 ) and (Fig. 12 ). 3.3.2 Morphological Changes of M. littorale at 40th & 50th Day in Different Light Intensities Morphological changes of M. littorale at mid stationary phase (40th & 50th day) for 10 µmol m − 2 s − 1 , 30 µmol m − 2 s − 1 , 50 µmol m − 2 s − 1 and 70 µmol m − 2 s − 1 are shown in Fig. 48. At 40th day spherical and oval shaped cells were found comparatively higher number in 70 µmol m − 2 s − 1 than 30 µmol m − 2 s − 1 and 10 µmol m − 2 s − 1 . 50% & 70% fusiform shaped cells were observed in 70 µmol m − 2 s − 1 and 50 µmol m − 2 s − 1 , respectively and rest were oval and spherical shaped cells. On the other hand, 90% & 95% fusiform-shaped cells were found in 30 µmol m − 2 s − 1 and 10 µmol m − 2 s − 1 , respectively. Only 5% oval and 5% spherical shaped cells were found in 30 µmol m − 2 s − 1 but no spherical cells were observed in 10 µmol m − 2 s − 1 . At 50th day 30% & 50% fusiform shaped cells were observed in 70 µmol m − 2 s − 1 and 50 µmol m − 2 s − 1 , respectively but 70% & 85% fusiform shaped cells were observed in 30 µmol m − 2 s − 1 and 10 µmol m − 2 s − 1 and rest of all cells were oval and spherical shaped cells (Fig. 13 ) and (Fig. 14 ). 3.3.3 Morphological Changes of M. littorale at 60th & 70th Day in Different Light Intensities Morphological changes of M. littorale at late stationary phase 60th & 70th day of culture period for 10 µmol m − 2 s − 1 , 30 µmol m − 2 s − 1 , 50 µmol m − 2 s − 1 and 70 µmol m − 2 s − 1 are shown in Fig. 50. At 60th day, only 10% fusiform shaped cells were found in 70 µmol m − 2 s − 1 and 30% found in 50 µmol m − 2 s − 1 but 50% and 70% fusiform cells were observed in 30 µmol m − 2 s − 1 and 10 µmol m − 2 s − 1 , respectively. At 70th day only spherical and oval shaped cells were found in 70 µmol m − 2 s − 1 and very little number of spindle shaped cells were found in 50 µmol m − 2 s − 1 . On the contrary, 40% & 50% fusiform shaped cells were observed in 30 µmol m − 2 s − 1 and 10 µmol m − 2 s − 1 , respectively (Fig. 15 ) and (Fig. 16 ). 3.4 Morphological Changes of M. littorale in Different Light Intensities In the present study growth and morphology of M. littorale were seen to be markedly affected by different light intensities. No oval or spherical cells were observed before 20 days of culture period of M. littorale for all light intensities. From the inoculation day to 20 days of culture period for all light intensities M. littorale produced only spindle shaped cells. But after 20 days of culture period M. littorale started to show morphological changes gradually. At early stationary phase (20th − 30th day), no morphological changes observed at 20th day and at 30th day spherical and oval-shaped cells were found in 70 µmol m −2 s − 1 (spherical 10%, oval 20%) and 50 µmol m −2 s − 1 (spherical 5%, oval 15%) intensity but that time no oval or spherical shaped cells were found in low light and natural light intensity. At mid stationary phase (40th − 50th day), at 40th day only 5% and 10% oval shaped cells were found in 10 µmol m −2 s − 1 and 30 µmol m −2 s − 1 respectively but there were no spherical shaped cells and that time, comparatively higher amount of oval and spherical shaped (30% oval, 20% spherical) cells found in 70 µmol m −2 s − 1 and lower amount of oval (20%) and spherical (10%) shaped cells found in 50 µmol m −2 s − 1 . At 50th day highest morphological changes observed at 70 µmol m −2 s − 1 and lowest morphological changes observed at 10 µmol m −2 s − 1 . Spherical and oval-shaped cells were dominant in 70 µmol m −2 s − 1 and 50 µmol m −2 s − 1 and spindle-shaped cells were dominant in 30 µmol m −2 s − 1 and 10 µmol m −2 s − 1 . Finally, at late stationary phase (60th − 70th day), spindle shaped cells were completely disappeared from 70 µmol m −2 s − 1 and fully turn into spherical and oval-shaped cells (70th day). Only 10% spindle shaped cells were observed in 70 µmol m −2 s − 1 but at that time 40% and 50% spindle shaped cells were observed in 30 µmol m −2 s − 1 and 10 µmol m −2 s − 1 respectively (70th day). Low light and natural light intensity will take more time to turn into completely spherical and oval shaped cells. In this study, good morphological changes of M. littorale were observed in high light intensity (70 µmol m-2s-1), and very slow morphological changes were observed in natural light intensity (70 µmol m-2s-1). This may be due to the presence of nutrient availability and a slower growth rate than high light intensity. Discussion This study shows that Monoraphidium littorale follows a reproducible morphogenetic sequence—fusiform → oval → spherical—yet the timing and completeness of that sequence differ systematically under pH versus light intensity. Alkaline pH acts as a tonic, baseline regulator that biases cellular physiology toward earlier and more complete morphotype switching soon after cultures exit exponential growth, consistent with pH-linked plasticity and wall remodeling reported for green microalgae (e.g., Salbitani et al., 2021 ; Bensalem et al., 2020 ; Akgül & Akgül, 2022 ). By contrast, light intensity behaves as a phase-gated accelerator with threshold-like effects: little divergence is evident before ~ day 20, after which higher irradiance (≥ 50–70 µmol m⁻² s⁻¹) compresses the timeline to oval/spherical dominance, in line with the role of irradiance as an energy-flux variable that modulates photochemistry and redox state once cultures are physiologically primed (Hu et al., 2023 ). Practically, the pH response is graded and monotonic (increasing alkalinity → predictably faster remodeling), whereas the light response is nonlinear and concentrated around an effective threshold, yielding minimal morphological payoff until that threshold is crossed. The morphology of Fibrocapsa japonica markedly influenced by temperature and salinity, at 15-25 o C and 20–30 ppt the cells were found mostly as oval shaped in logarithmic phase whereas all the oval shaped cells changed to spherical within 10 days at higher temperature (30 o C) and lower salinity and oval-shaped cells was highest at 60–140 µE/m 2 /s. pH did not influence the morphology of the cells under the range of pH (6.5-9.0) reported by Khan et al. (1996) which is not similar to our studies. Khan et al. (1998) also reported the morphology of C. marina cells was strongly affected by temperature, at 20 o C and 25 o C, the population occurred mostly in a spindle like form, whereas at 10 o C, 90% of the cells became spherical within 10 days of inoculation and also reported that light intensity and pH did not influence morphology of C. marina markedly under the range of light intensities (20–180 µE m −2 s − 1 ) and pH (6.5–8.5) which is also not similar to our studies and in our studies M. littorale showed comparatively slower morphological changes than C. marina . Mechanistically, external pH redefines the chemical milieu—altering proton gradients and inorganic-carbon speciation—thereby shifting allocation from vegetative elongation to remodeling/autosporulation, a pattern congruent with reports of pH-conditioned plasticity and pigment regulation in chlorophytes (Salbitani et al., 2021 ; Min et al., 2023 ). Light’s influence emerges later because it depends on biomass accumulation and resource competition; beyond this point, supra-threshold irradiance rapidly accelerates the same morphogenetic program that alkaline pH has already biased (Hu et al., 2023 ). These differences translate into distinct process levers: pH offers a precise, low-cost control for predictable staging, while light is best deployed as a timed trigger. Accordingly, a two-stage protocol—grow under near-neutral pH and sub-threshold light to prolong fusiform dominance, then shift to alkaline pH (≥ 8.5) and ≥ 50–70 µmol m⁻² s⁻¹ to drive remodeling—aligns morphology with downstream objectives such as biomass or stress-linked products (Saha & Murray, 2018 ; Novoveská et al., 2019 ; Kratzer & Murkovic, 2021 ; Silva et al., 2022 ; Uebel et al., 2019 ). Two caveats frame interpretation. First, pH and light were tested in separate runs; a full factorial (pH × light) is now needed to quantify interaction terms and test the prediction that alkaline conditions lower the effective light threshold and that high light amplifies the alkaline bias. Second, categorical morphology should be complemented by image-based morphometrics (e.g., aspect-ratio distributions) and concurrent biochemical profiling (chlorophylls, carotenoids, lipids) so that morphotype proportions become a predictive control variable for productivity landscapes (Merlo et al., 2021 ; Chen et al., 2015 ). In sum, pH is a state-setting, continuous controller that establishes the developmental bias early, whereas light is a thresholded, phase-gated trigger that accelerates remodeling once the culture is primed. Leveraging this complementarity yields an energy-aware, time-scheduled route to reproducible morphotype control in M. littorale , improving staging, harvest timing, and—pending biochemical validation—alignment of culture states with target productivity. Conclusion Monoraphidium littorale exhibits a robust, phase-dependent morphogenetic program that can be steered predictably by external pH and light intensity. Alkaline conditions exert an early, graded bias toward remodeling, while high irradiance acts later as a thresholded trigger that accelerates the same trajectory once cultures are primed beyond ~ day 20. This division of labor between a state-setting variable (pH) and a phase-gated accelerator (light) explains the observed differences in sensitivity, onset, and completeness of switching from fusiform to oval/spherical cells. The resulting control logic is operationally straightforward: prolong vegetative growth and biomass accumulation under near-neutral pH with sub-threshold light, then induce rapid remodeling by shifting to alkaline pH and ≥ 50–70 µmol m⁻² s⁻¹ irradiance. While our independent experiments delineate clear roles for each factor, future work should adopt factorial pH × light designs, higher-resolution image morphometrics, and concurrent biochemical profiling to quantify interaction terms and link morphotype proportions to productivity. Recognizing pH as the continuous regulator and light as the timed trigger provides a principled basis for energy-aware, time-scheduled cultivation protocols and more reproducible harvest timing in M. littorale. Declarations The authors have no Competing Interests. Funding The author didn’t get any funding for this study. Author Contribution Arifa Sultana: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Visualization; Writing—original draft; Writing—review & editing; Project administration; Supervision; Corresponding author.Saleha Khan: Methodology; Investigation; Data curation; Visualization; Writing—review & editing.Md. Mahfuzul Haque: Formal analysis; Validation; Visualization; Writing—review & editing.Zoarder Faruque Ahmed: Conceptualization; Resources; Supervision; Funding acquisition (if applicable); Writing—review & editing.Md. Nayeem Khan: Methodology; Formal analysis; Validation; Writing—review & editing.Correspondence: Arifa Sultana, [email protected] Data Availability 1) Primary (raw) datasets generatedA. Morphotype enumeration dataset (core quantitative data) manuscript final (1)For each sampling timepoint and replicate flask, the following were recorded from microscopy counts:Experiment: pH experiment or light-intensity experimentTreatment level:pH: 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 manuscript final (1)Light intensity: 10, 30, 50, 70 µmol m⁻² s⁻¹ manuscript final (1)Sampling day(s): morphological observation conducted at 10-day intervals up to day 70; quantitative morphotype scoring is reported from day 20 to day 70 (day 20, 30, 40, 50, 60, 70). manuscript final (1)Replicate ID: R1, R2, R3 (triplicates) manuscript final (1)Raw morphotype counts (per sample):Fusiform cell countOval cell countSpherical cell countTotal counted cells per sample (sum of the three morphotypes)B. Microscopy image dataset (core qualitative/visual data) manuscript final (1)Light-microscope micrographs documenting fusiform, oval, spherical morphotypes and autosporulation stagesImages include scale bars (10 µm) and were taken under standardized microscopy conditions (OPTIKA light microscope). manuscript final (1)2) Derived datasets/outputs generated from raw dataA. Percentage composition (per sample and per treatment-time mean) manuscript final (1)From the raw counts, the study generated:% Fusiform = (fusiform / total) × 100% Oval = (oval / total) × 100% Spherical = (spherical / total) × 100B. Summary statistics and inferential outputs (SPSS) manuscript final (1)Mean ± SD for each morphotype (and/or for fusiform proportion) across triplicatesSignificance testing using DMRT at p < 0.05, producing letter-group outputs used in figures. manuscript final (1)3) Recommended “source data” table format (ready to archive / share)A single spreadsheet (CSV/XLSX) can store all quantitative records with these columns:experiment (pH / light)treatment_value (e.g., 8.5 or 70)treatment_unit (pH units or µmol m⁻² s⁻¹)day (20, 30, 40, 50, 60, 70)replicate (1–3)fusiform_noval_nspherical_ntotal_nfusiform_pctoval_pctspherical_pct(optional metadata columns) temperature_C, photoperiod, medium, microscope_model References Akgül, F. and Akgül, R. (2022). Combined effect of nitrogen and phosphorus on growth and biochemical composition of tetradesmus obliquus (turpin) m.j. wynne. International Journal of Secondary Metabolite, 9(4), 525–537. https://doi.org/10.21448/ijsm.1102592 Batista, A., Niccolai, A., Fradinho, P., Fragoso, S., Bursic, I., Rodolfi, L., … Raymundo, A. (2017). Microalgae biomass as an alternative ingredient in cookies: sensory, physical and chemical properties, antioxidant activity and in vitro digestibility. Algal Research, 26, 161–171. https://doi.org/10.1016/j.algal.2017.07.017 Bensalem, S., Pareau, D., Cinquin, B., Français, O., Pioufle, B., & Lopes, F. (2020). Impact of pulsed electric fields and mechanical compressions on the permeability and structure of chlamydomonas reinhardtii cells. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-59404-6 Berchielli-Morais, F., Fernandes, J., & Sipaúba-Tavares, L. (2015). Diets supplemented with microalgal biomass: effects on growth, survival and colouration of ornamental fishhyphessobrycon eques (steindacher 1882). Aquaculture Research, 47(10), 3061–3069. https://doi.org/10.1111/are.12756 Bhalamurugan, G., Orsat, V., & Mark, L. (2018). Valuable bioproducts obtained from microalgal biomass and their commercial applications: a review. Environmental Engineering Research, 23(3), 229–241. https://doi.org/10.4491/eer.2017.220 Chen, J., Wang, Y., Benemann, J., Zhang, X., Hongjun, H., & Qin, S. (2015). Microalgal industry in china: challenges and prospects. Journal of Applied Phycology, 28(2), 715–725. https://doi.org/10.1007/s10811-015-0720-4 Dixit, R., Sagaram, U., Gocher, C., Kumar, G., & Dasgupta, S. (2021). Biomolecular characterisation of marine microalga in comparison to fishmeal and soymeal as an alternative feed ingredient. Phytochemical Analysis, 33(3), 365–372. https://doi.org/10.1002/pca.3094 Hu, J., Meng, W., Su, Y., Qian, C., & Fu, W. (2023). Emerging technologies for advancing microalgal photosynthesis and metabolism toward sustainable production. Frontiers in Marine Science, 10. https://doi.org/10.3389/fmars.2023.1260709 Kratzer, R. and Murkovic, M. (2021). Food ingredients and nutraceuticals from microalgae: main product classes and biotechnological production. Foods, 10(7), 1626. https://doi.org/10.3390/foods10071626 Margenat, A., Fabregat, C., & Jorba, M. (2023). Microwave-assisted extraction combined with enzymatic pre-treatment for chlorella vulgaris protein solubilisation… https://doi.org/10.21203/rs.3.rs-2763204/v1 Merlo, S., Gabarrell, X., Tonon, Â., & Rossi, S. (2021). Marine microalgae contribution to sustainable development. Water, 13(10), 1373. https://doi.org/10.3390/w13101373 Min, S., Bastiaens, L., Verspreet, J., & Hayes, M. (2023). Applications of microalgae in foods, pharma and feeds and their use as fertilizers and biostimulants: legislation and regulatory aspects for consideration. Foods, 12(20), 3878. https://doi.org/10.3390/foods12203878 Novoveská, L., Ross, M., Stanley, M., Pradelles, R., Wasiolek, V., & Sassi, J. (2019). Microalgal carotenoids: a review of production, current markets, regulations, and future direction. Marine Drugs, 17(11), 640. https://doi.org/10.3390/md17110640 Saha, S. and Murray, P. (2018). Exploitation of microalgae species for nutraceutical purposes: cultivation aspects. Fermentation, 4(2), 46. https://doi.org/10.3390/fermentation4020046 Sahni, P., Aggarwal, P., Sharma, S., & Singh, B. (2019). Nuances of microalgal technology in food and nutraceuticals: a review. Nutrition & Food Science, 49(5), 866–885. https://doi.org/10.1108/nfs-01-2019-0008 Salbitani, G., Prete, F., Carfagna, S., Sansone, G., & Barone, C. (2021). Enhancement of pigments production by nannochloropsis oculata cells in response to bicarbonate supply. Sustainability, 13(21), 11904. https://doi.org/10.3390/su132111904 Silva, M., Kamberovic, F., Uota, S., Ismael-Mohammed, K., Viegas, C., Simes, D., … Barreira, L. (2022). Microalgae as potential sources of bioactive compounds for functional foods and pharmaceuticals. Applied Sciences, 12(12), 5877. https://doi.org/10.3390/app12125877 Uebel, L., Costa, J., Olson, A., & Morais, M. (2019). Industrial plant for production of spirulina sp. leb 18. Brazilian Journal of Chemical Engineering, 36(1), 51–63. https://doi.org/10.1590/0104-6632.20180361s20170284 Villaró, S., Ciardi, M., Morillas-España, A., Sánchez‐Zurano, A., Acién, G., & Lafarga, T. (2021). Microalgae derived astaxanthin: research and consumer trends and industrial use as food. Foods, 10(10), 2303. https://doi.org/10.3390/foods10102303 Widyaningrum, D., Sadek, N., Cecilia, D., Oktafika, R., & Tedjakusuma, F. (2024). The effect of temperature and encapsulation on the stability of microalgae pigments. Iop Conference Series Earth and Environmental Science, 1352(1), 012090. https://doi.org/10.1088/1755-1315/1352/1/012090 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8605830","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629611200,"identity":"534fe5b3-f3a7-493f-96ac-1572599bf06a","order_by":0,"name":"Arifa Sultana","email":"data:image/png;base64,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","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Arifa","middleName":"","lastName":"Sultana","suffix":""},{"id":629611201,"identity":"b68c78a7-a337-40c3-a8cf-ded67ba2c027","order_by":1,"name":"Saleha Khan","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Saleha","middleName":"","lastName":"Khan","suffix":""},{"id":629611202,"identity":"c6531106-94e3-445c-a3ad-fa1f9e3a25d7","order_by":2,"name":"Md. Mahfuzul Haque","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Mahfuzul","lastName":"Haque","suffix":""},{"id":629611203,"identity":"bb3aa8d0-7148-4866-aefe-116d4c4d9bfc","order_by":3,"name":"Zoarder Faruque Ahmed","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zoarder","middleName":"Faruque","lastName":"Ahmed","suffix":""},{"id":629611204,"identity":"982ae8ea-5971-49dc-80ef-8d1c786e3126","order_by":4,"name":"MD Nayeem Khan","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"MD","middleName":"Nayeem","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2026-01-15 01:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8605830/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8605830/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108097470,"identity":"856150f7-299c-4ce1-b047-f8741beb5e85","added_by":"auto","created_at":"2026-04-29 10:14:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58447,"visible":true,"origin":"","legend":"\u003cp\u003eThree morphological types of cells (Fusiform, Oval and Spherical) of \u003cem\u003eM. littorale \u003c/em\u003e. Scale bar: 10\u003cem\u003eμ\u003c/em\u003em.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/90c4b93cd1ce329ccc61583e.jpg"},{"id":108182303,"identity":"2edcb778-2274-4704-be85-56bebc2d51e7","added_by":"auto","created_at":"2026-04-30 08:59:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121799,"visible":true,"origin":"","legend":"\u003cp\u003eAll morphological stages of \u003cem\u003eM. littorale . \u003c/em\u003eScale bar: 10\u003cem\u003eμ\u003c/em\u003em.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/3577c846786a26612513c35c.jpg"},{"id":108181607,"identity":"9e0d452e-c738-4a23-868d-54ab29af7f83","added_by":"auto","created_at":"2026-04-30 08:58:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37216,"visible":true,"origin":"","legend":"\u003cp\u003eFusiform to fusiform auto-sporulation of \u003cem\u003eM. littorale.\u003c/em\u003e(1) a matured mother cell with a single nucleus; (2) division of the nucleus; (3) appearance of at least four nuclei; (4) thickening of the mother cell wall, with the four nuclei becoming more visible and newer cell wall beginning to form around each nucleus; (5) beginning of cross-wall cell division within the mother cell; (6) initiation of the disappearance of the mother cell wall; (7) disappearance of the mother cell wall and the appearance of four daughter cells which are attached to each other in a way appearing like a single cell; and (8) separation of four daughter cells from the mother cell and from each other, looking like a bunch of fresh green paddy seeds.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/15ba265b5c5987889919857f.jpg"},{"id":108181726,"identity":"0f77e14f-fca5-4c2e-8dac-289f4095902b","added_by":"auto","created_at":"2026-04-30 08:58:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39748,"visible":true,"origin":"","legend":"\u003cp\u003eAuto-sporulation of \u003cem\u003eM. littorale \u003c/em\u003efrom fusiform to oval (1−6) and oval to spherical (7−10). (1) a mature mother cell containing a single nucleus; (2) sporulation initiates with the division of the nucleus; (3) appearance of four nuclei; (4) beginning of a new spore wall to develop around each nucleus while the mother cell gradually undergoes breakdown; (5) disintegration of the mother cell wall, leading to the division of the fragmented portion, ultimately generating four spores; (6) initially, these spores exhibit an oval shape; (7) the oval cells subsequently give rise to teardrop-shaped oval cells; (8, 9) again, oval cells undergo a transformation into spherical cells, which possess the capacity to reproduce and go through self-division; (10) eventually, a total of eight spherical spores are formed.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/4c4be9c96a4a747d45c12957.jpg"},{"id":108097474,"identity":"18c5618d-e0a7-48ec-9527-881db6309a3e","added_by":"auto","created_at":"2026-04-29 10:14:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121741,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eM. littorale \u003c/em\u003e\u0026nbsp;at early stationary phase, on 20\u003csup\u003eth\u003c/sup\u003e and 30\u003csup\u003eth\u003c/sup\u003e day of the culture period at pH of 6.5 to 9.0. Means of fusiform cells with different letters (bold black letters for 20\u003csup\u003eth \u003c/sup\u003eday and red letters for 30\u003csup\u003eth\u003c/sup\u003e day) are significantly (P\u0026lt;0:05) different from one another.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/ca92bdb99b02c868ab8b77ac.jpg"},{"id":108181908,"identity":"aa8a8fd8-21ec-466f-a82f-a3d0690a4825","added_by":"auto","created_at":"2026-04-30 08:59:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":157177,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological conditions of \u003cem\u003eM. littorale \u003c/em\u003eat early stationary phase. (a)–(f) showed the changed morphotype observed on the 30\u003csup\u003eth\u003c/sup\u003e day of the culture period in six different pH media. Each scale bar: 10 \u003cem\u003eµ\u003c/em\u003em; 40× magnification (B-510BT OPTIKA, Italy).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/0cd5c9efcc63c602dfdab492.jpg"},{"id":108181577,"identity":"69f9426b-0462-4bbe-9229-2621aa4ab4c8","added_by":"auto","created_at":"2026-04-30 08:58:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":137931,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eM. littorale \u003c/em\u003eat mid stationary phase, 40\u003csup\u003eth\u003c/sup\u003e and 50\u003csup\u003eth\u003c/sup\u003e day of the culture period in different treatments having pH 6.5 to 9.0. Means of fusiform cells with different letters (bold black letters for 40\u003csup\u003eth\u003c/sup\u003e day and red letters for 50\u003csup\u003eth\u003c/sup\u003e day) are significantly (P\u0026lt;0:05) different from one another.z\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/186e0ba7295d0cf9eea1fd37.jpg"},{"id":108182862,"identity":"60c4ab76-f75f-4ed0-a356-9db8a0b74dc1","added_by":"auto","created_at":"2026-04-30 08:59:38","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":165516,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological conditions of \u003cem\u003eM. littorale \u003c/em\u003eat mid-stationary phase. (a) – (f) showed the changed morphotype observed on the 50\u003csup\u003eth\u003c/sup\u003e day of the culture period in six different pH media. Each scale bar: 10 \u003cem\u003eµ\u003c/em\u003em; 40× magnification (B-510BT OPTIKA, Italy).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/95e0b609ea84f5cdfb42861e.jpg"},{"id":108097478,"identity":"5bf4d5ff-e1b7-4d86-92a6-ae41e103790f","added_by":"auto","created_at":"2026-04-29 10:14:15","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":168307,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eM. littorale \u003c/em\u003eat late stationary phase, 60\u003csup\u003eth\u003c/sup\u003e and 70\u003csup\u003eth\u003c/sup\u003e day of the culture period, in different treatments having pH 6.5 to 9.0. Means of fusiform cells with different letters (bold black letters for 60\u003csup\u003eth\u003c/sup\u003e day and brown letters for 70\u003csup\u003eth\u003c/sup\u003e day) are significantly (P\u0026lt;0:05) different from one another.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/82b2bd2991fe8eed3bb54f47.jpg"},{"id":108182100,"identity":"3a8b3216-fd99-4e69-a6cf-b083a0c6c763","added_by":"auto","created_at":"2026-04-30 08:59:08","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":212119,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological alteration of \u003cem\u003eM. littorale \u003c/em\u003eat late stationary phase. (a) – (f) showed the changed morphotype observed on the 70\u003csup\u003eth\u003c/sup\u003e day of the culture period in six different pH media. Each scale bar: 10 \u003cem\u003eµ\u003c/em\u003em; 40× magnification (B-510BT OPTIKA, Italy).\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/36a4899e1e9c8d35785a4725.jpg"},{"id":108182858,"identity":"d5e2656c-f3fc-42a6-8cc4-8c180648f4fb","added_by":"auto","created_at":"2026-04-30 08:59:38","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":48180,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eM. littorale\u003c/em\u003e at early stationary phase, 20\u003csup\u003eth \u003c/sup\u003e\u0026amp; 30\u003csup\u003eth\u003c/sup\u003e day of culture period in different light intensities. Means with different letters are significantly different from one another.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/ee1fe12192d7a0aad22e0f1b.jpg"},{"id":108182290,"identity":"9c887e4f-6c55-4bfd-8476-db4397540c45","added_by":"auto","created_at":"2026-04-30 08:59:18","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":162375,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological conditions at early stationary phase (20\u003csup\u003eth\u003c/sup\u003e \u0026amp; 30\u003csup\u003eth\u003c/sup\u003e day) of \u003cem\u003eM. littorale. \u003c/em\u003eat different light intensity; a, b, c, d, 20\u003csup\u003eth\u003c/sup\u003e day of culture period at 10, 30, 50 \u0026amp; 70 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively and e, f, g, h, 30\u003csup\u003eth\u003c/sup\u003e day of culture period at 10, 30, 50 \u0026amp; 70 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/ccdfa38b1a15931ae7ce14fa.jpg"},{"id":108181841,"identity":"6ba45055-17ca-46c9-b90f-1bd7b6f8cac6","added_by":"auto","created_at":"2026-04-30 08:58:58","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":42031,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eM. littorale \u003c/em\u003eat mid stationary phase, 40\u003csup\u003eth\u003c/sup\u003e \u0026amp; 50\u003csup\u003eth\u003c/sup\u003e day of culture period in different light intensities. Means with different letters are significantly different from one another.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/5a445a39de6efb06c492905b.jpg"},{"id":108803622,"identity":"c0f5d4b1-0a03-4490-a84b-1baef824cab6","added_by":"auto","created_at":"2026-05-08 15:01:36","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":132484,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological conditions at mid stationary phase (40\u003csup\u003eth\u003c/sup\u003e \u0026amp; 50\u003csup\u003eth\u003c/sup\u003e day) of \u003cem\u003eM. littorale. \u003c/em\u003eat different light intensity; a, b, c, d, 40\u003csup\u003eth\u003c/sup\u003e day of culture period at 10, 30, 50 \u0026amp; 70 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively and e, f, g, h, 50\u003csup\u003eth\u003c/sup\u003e day of culture period at 10, 30, 50 \u0026amp; 70 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/aaedc137ae50ff126841fb53.jpg"},{"id":108097483,"identity":"c0246bc0-c1b7-4b5d-8ba7-b1f364b106d3","added_by":"auto","created_at":"2026-04-29 10:14:15","extension":"jpg","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":47481,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eM. littorale \u003c/em\u003eat late stationary phase, 60\u003csup\u003eth\u003c/sup\u003e \u0026amp; 70\u003csup\u003eth\u003c/sup\u003e day of culture period in different light intensities. Means with different letters are significantly different from one another.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/364c19527bf0110f2d01b542.jpg"},{"id":108182282,"identity":"9198a422-7775-4867-9fb5-828b44ee2587","added_by":"auto","created_at":"2026-04-30 08:59:18","extension":"jpg","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":223717,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological conditions at late stationary phase (60\u003csup\u003eth\u003c/sup\u003e \u0026amp; 70\u003csup\u003eth\u003c/sup\u003e day) of \u003cem\u003eM. littorale. \u003c/em\u003eat different light intensity; a, b, c, d, 60\u003csup\u003eth\u003c/sup\u003e day of culture period at 10, 30, 50 \u0026amp; 70 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively and e, f, g, h, 70\u003csup\u003eth\u003c/sup\u003e day of culture period at 10, 30, 50 \u0026amp; 70 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/d802a25d5865d49a704c668a.jpg"},{"id":108809013,"identity":"7bdc0f2a-6263-47d8-9b0c-3dc7fa2f8dcb","added_by":"auto","created_at":"2026-05-08 15:48:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2276249,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8605830/v1/c7290e01-fcd1-41fe-8376-9765bbdd7fbb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of pH and light intensity on the morphology of the nutritious green microalga Monoraphidium littorale","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicroalgae are increasingly central to the bio-economy because they synthesize high-value products including carotenoids, polyunsaturated fatty acids, proteins, and diverse bioactive metabolites\u0026mdash;relevant to nutraceutical, pharmaceutical, food, cosmetic, and aquaculture industries (Villaro et al., 2021; Saha \u0026amp; Murray, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Novovesk\u0026aacute; et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bhalamurugan et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Among these, the keto-carotenoid astaxanthin is commercially prominent for its potent antioxidant capacity and for enhancing pigmentation and health performance in aquaculture species, thereby improving product acceptance as well as conferring immunological and physiological benefits (Villaro et al., 2021; Saha \u0026amp; Murray, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Growing demand for natural pigments and functional ingredients has, in turn, intensified interest in microalgal biomass as a sustainable feed additive and as a partial substitute for conventional feedstocks such as fishmeal and soymeal, with reported gains in pigmentation, growth, and immunity, and reduced reliance on synthetic additives (Berchielli-Morais et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Batista et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dixit et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kratzer \u0026amp; Murkovic, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWithin this broader context, Monoraphidium littorale has emerged as a promising species owing to its robust growth and favorable biochemical profile. Reports indicate that its fatty-acid composition encompasses nutritionally relevant omega-3 and omega-6 fractions, supporting potential applications in aquaculture feeds and health-oriented products; preliminary evidence further suggests that metabolites from \u003cem\u003eM. littorale\u003c/em\u003e may enhance overall performance in cultured organisms (Silva et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dixit et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sahni et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Margenat et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although \u003cem\u003eM. littorale\u003c/em\u003e has been proposed as a candidate for natural pigment production, including putative astaxanthin pathways, such claims require cautious evaluation and optimization under controlled cultivation regimes.\u003c/p\u003e \u003cp\u003eA central challenge is that the physiology and morphology of microalgae are tightly coupled to environmental conditions. Nutrient concentration, temperature, and irradiance all modulate cellular function, yet pH is often a dominant determinant of growth, photosynthetic performance, nutrient uptake, and cell-wall dynamics (Salbitani et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bensalem et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies in chlorophytes (e.g., Scenedesmus, Coccomyxa) show that pH shifts can drive changes in morphotypes, pigment biosynthesis, and growth rates\u0026mdash;highlighting the capacity for morphological plasticity as a strategic response to environmental pressure (Akg\u0026uuml;l \u0026amp; Akg\u0026uuml;l, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Widyaningrum et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While lower pH may increase the availability of certain nutrients, it can also suppress pigment production and alter cell shape; conversely, alkaline conditions can facilitate remodeling toward more stress-tolerant forms (Sahni et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Min et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, deliberate pH control has become integral to maximizing biomass and metabolite yields across several commercially relevant taxa (Uebel et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bhalamurugan et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLight intensity constitutes a second, equally important lever, governing energy supply for photosynthesis and shaping redox balance and carbon assimilation. However, for \u003cem\u003eM. littorale\u003c/em\u003e the combined influence of pH and irradiance on morphological trajectories remains poorly resolved. This gap is consequential: morphological state in chlorophytes is not merely descriptive but often indexes physiological status, stress tolerance, and, by extension, prospects for productivity and biochemical composition.\u003c/p\u003e \u003cp\u003eTo address this, the present study investigates the time-dependent morphological responses of \u003cem\u003eM. littorale\u003c/em\u003e to controlled pH (6.5\u0026ndash;9.0) and light intensity (10, 30, 50, 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) across culture phases. We test the working hypothesis that alkaline pH imposes a graded, state-setting bias toward remodeling, whereas higher irradiance acts as a phase-gated accelerator once cultures exit exponential growth. By mapping the transitions among fusiform, oval, and spherical morphotypes under each driver, we aim to establish practical set-points for culture staging and to provide a basis for energy-aware, scalable bioprocess control in aquaculture and nutraceutical applications (Merlo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eObjectives of the Study\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo describe the life cycle and main cell shapes (fusiform, oval, spherical) of \u003cem\u003eMonoraphidium littorale\u003c/em\u003e during culture.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo measure how pH (6.5\u0026ndash;9.0) changes cell shape over time.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo measure how light intensity (10, 30, 50, 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) changes cell shape over time.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo compare the effects of pH and light and identify which factor acts earlier and more strongly.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the time points when fusiform cells start to decline under each condition.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo suggest simple culture settings (pH and light) for biomass growth and for triggering remodeling for practical use.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003cem\u003eM. littorale\u003c/em\u003e was isolated from the freshwater ponds adjacent to the Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh. Through random sampling, various microalgal samples were collected those were kept in sterilized nutrient solutions (Bold Basal Medium). Streaked agar plating and serial dilution were frequently used for isolation. Stock cultures were maintained using Bold Basal Medium (BBM) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at ambient temperature of 28\u0026ndash;30\u0026deg;C, light intensity 60 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and photoperiod 12:12 h, L:D. The pH of the medium was adjusted over the culture period by addition of NaOH and HCL. Culture media were autoclaved for 15 min at 121\u003csup\u003eo\u003c/sup\u003e C, and aged for several days prior to inoculation. The study was performed in triplicate for each pH level (treatment) using an inoculum for the culture in logarithmic phase having cell density of 2‧74 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Morphology was monitored immediately after collection of cells from cultures in exponential and stationary phases.\u003c/p\u003e \u003cp\u003eIn order to prepare BBM as a culture medium, 10 mL solution from each of the listed chemicals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) from serial nos. 1 to 6 were poured into a 1 L conical flask. Then 1 mL of the solution of each of the listed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) chemicals from serial nos. 7 to 10 were poured into the flask, and distilled water was added to make the volume 1.0 L. The solution into the flask was mixed well and sterilized at 121\u0026deg;C for 15 minutes with moist heat using an autoclave (Model SS-V35HD, WINCOM, China). For growth studies, \u003cem\u003eM. littorale\u003c/em\u003e cells were grown in a 500-mL flask containing BBM. The determination of the cell concentration was done by direct counting using a Sedgewick-Rafter chamber. Morphological observation was performed right after the inoculation and then after every 10 days up to 70 days of culture. Samples were shaken well before being collected for counting.\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\u003eStock solutions of the chemicals for the preparation of BBM for \u003cem\u003eM. littorale\u003c/em\u003e culture. *\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStocks of Chemicals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003egL\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\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e. 2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrace Elements\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZnSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMnCl\u003csub\u003e2\u003c/sub\u003e. 4H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCuSO\u003csub\u003e4\u003c/sub\u003e. 5H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCo (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. 6H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEDTA-KOH solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEDTA Na\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO with 1.0 ml Concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.98\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\u003e* The chemicals were bought from Z.H. Scientific and Chemicals Mart, Bangladesh.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Morphological observations\u003c/h2\u003e \u003cp\u003eMorphological changes of \u003cem\u003eM. littorale\u003c/em\u003e was observed by using a light microscope (B-510BT OPTIKA, Italy). The morphological study of \u003cem\u003eM. littorale\u003c/em\u003e was done for 70 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Statistical Analysis of the Data\u003c/h2\u003e \u003cp\u003eThe data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of the three replicates. To analyze the data on the morphological shapes of the microalgae in different treatments (pH levels) using SPSS 25. Significant differences (at \u003cem\u003eP\u003c/em\u003e ˂ 0.05) among the means have been determined using Duncan\u0026rsquo;s multiple range test (DMRT) [25].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Light-intensity experiment\u003c/h2\u003e \u003cp\u003eCultures in late-log phase were inoculated into BBM and exposed to four light treatments: 10, 30, 50, and 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; under 12:12 h L:D at 28\u0026ndash;30\u0026deg;C. Each treatment used n\u0026thinsp;=\u0026thinsp;3 flasks (500 mL) with an identical inoculum density as in the pH experiment. Morphotypes (fusiform, oval, spherical) were enumerated at day 20, 30, 40, 50, 60, 70 using the same microscopy and counting procedures.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Life cycle of M. littorale\u003c/h2\u003e \u003cp\u003eThis study primarily summarized the morphology of \u003cem\u003eM. littorale\u003c/em\u003e in response to six different pH levels (pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0). The shape of \u003cem\u003eM. littorale\u003c/em\u003e cells varied with different pH levels along with the culture period, and morphological observation revealed three types of shapes: fusiform, oval, and spherical (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In acidic environment, at the early stationary phase, \u003cem\u003eM. littorale\u003c/em\u003e showed only fusiform cells. On the other hand, in alkaline environment \u003cem\u003eM. littorale\u003c/em\u003e gradually changed into oval and spherical cells along with fusiform cells. Firstly, fusiform cells turned into oval-shaped cells, and then oval-shaped cells turned into spherical-shaped cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Auto-sporulation of M. littorale from fusiform to fusiform\u003c/h2\u003e \u003cp\u003eAt the initial stage of the culture age, during the logarithmic phase (6\u0026ndash;18 days), \u003cem\u003eM. littorale\u003c/em\u003e reproduced through auto-sporulation and produced only 4, 8, or 16 fusiform autospores, like the mother cell, in the presence of enough nutrients in the culture medium. This time, no morphological alteration was observed at varying pH values (6.5\u0026ndash;9.0; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Auto-sporulation of M. littorale from fusiform to oval and oval to spherical\u003c/h2\u003e \u003cp\u003eDuring the stationary phase at different pH levels (6.5\u0026ndash;9.0), \u003cem\u003eM. littorale\u003c/em\u003e reproduced by auto-sporulation along with some additional morphological changes and produced 4, 8, or 16 oval and spherical autospores. This time, fusiform cells first turned into oval-shaped cells, and these oval-shaped cells again produced oval-shaped cells. Finally, the oval-shaped cells of \u003cem\u003eM. littorale\u003c/em\u003e turned into spherical cells. These spherical cells can again go through cell division and produced 4, 8, or 16 spherical autospores. Under favorable culture conditions, these autospores were also able to transform their spherical shape into a fusiform shape by consuming sufficient nutrients, as they are a resilient species that can recoil or spring back into their previous shape after being bent, stretched, or compressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects of pH on morphology of M. littorale\u003c/h2\u003e \u003cp\u003eMorphological alterations of \u003cem\u003eM. littorale\u003c/em\u003e in response to varying pH levels ranging from 6.5 to 9.0 with intervals of 0.5 were observed during the culture period, particularly started on the 20th day and continued on every 10 days up to the 70th day of the culture period. In the early stationary phase in an acidic to slightly alkaline environment, only fusiform cells of \u003cem\u003eM. littorale\u003c/em\u003e were observed, whereas a small proportion of oval and spherical cells were observed under alkaline conditions. As the culture age progressed into the stationary phase, fusiform cells began to convert into oval and spherical cells, which occurred simultaneously with an increase in pH. In the late stationary phase, oval and spherical cells became dominant over fusiform cells in alkaline conditions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Morphological changes of M. littorale on 20th and 30th day\u003c/h2\u003e \u003cp\u003eIn the present investigation it was found that transformations in morphology, from fusiform to oval, subsequently followed by spherical, were related to changes in pH and the length of culture period. The morphological alterations of \u003cem\u003eM. littorale\u003c/em\u003e during the early stationary phase (20th and 30th day) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. On the 20th day, all the cells of \u003cem\u003eM. littorale\u003c/em\u003e were found fusiform (100%) across all tested pH levels ranging from 6.5 to 9.0. Fusiform cells were found at lower pH values (6.5 and 7.0) for the entire duration of the early stationary phase, particularly on the 20th and 30th days. As the pH increased and culture age continued for the next 10 days, fusiform-shaped cells began to transform their shape into oval-shaped as well as spherical-shaped at a significantly lower rate (5\u0026thinsp;\u0026minus;\u0026thinsp;10%) for the pH levels of 7.5 to 9.0 on the 30th day.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Morphological changes of M. littorale on 40th and 50th day\u003c/h2\u003e \u003cp\u003eAt mid-stationary phase (on 40th and 50th day), morphological changes of \u003cem\u003eM. littorale\u003c/em\u003e at different pH levels (from 6.5 to 9.0) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. On the 40th day, oval and spherical-shaped cells were observed at neutral pH (7.0) to alkaline pH (9.0), increasing from 5% (oval and spherical) to 25% (oval) and 15% (spherical). In contrast, only fusiform cells were observed in an acidic (pH 6.5) environment. Significantly more oval and spherical-shaped cells were found in higher alkaline (pH 8.0 to 9.0) conditions than in neutral (pH 7.0) and mildly alkaline (pH 7.5) conditions. On the 50th day, cells with oval, spherical, and fusiform shapes were observed at all pH levels investigated. Moreover, a significantly higher percentage of oval and spherical-shaped cells were observed in the alkaline conditions (pH 8.0 to 9.0) than in the acidic to slightly alkaline conditions (pH 6.5 to 7.5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Morphological changes of M. littorale on 60th and 70th day\u003c/h2\u003e \u003cp\u003eAt the late stationary phase (60th and 70th day), morphological changes of \u003cem\u003eM. littorale\u003c/em\u003e at pH 6.5 to 9.0 are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. On the 60th day, a higher number of oval (50%) and spherical (35%) shaped cells were observed in higher alkaline conditions (pH 8.0 to 9.0), and a moderate number of oval (30%) and spherical (20%) shaped cells were found in slightly alkaline conditions (pH 7.5). Besides, a comparatively lower number of oval (15%) and spherical-shaped (10%) cells were observed in acidic to neutral conditions (pH 6.5 to 7.0). On the 70th day, the number of fusiform-shaped cells exhibited a significant decrease (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from acidic to alkaline conditions (pH 6.5\u0026ndash;9.0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The highest number of \u003cem\u003eM. littorale\u003c/em\u003e cells exhibited morphological changes in higher alkaline conditions (pH 8.0\u0026ndash;9.0), as 95% of the cells turned into oval and spherical-shaped cells (45% oval and 50% spherical, respectively). On the 70th day, the highest number of fusiform-shaped cells (60%\u0026ndash;50%) and a comparatively lower number of oval and spherical cells (25%\u0026ndash;30% oval and 15%\u0026ndash;20% spherical) were observed in acidic to neutral conditions (pH 6.5 and 7.0, respectively). Moreover, a moderate number of \u003cem\u003eM. littorale\u003c/em\u003e cells showed morphological alterations in a slightly alkaline condition (pH 7.5), with nearly equal proportions (35% fusiform, 35% oval, and 30% spherical) of all types of cells observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of Light Intensity on Morphology of M. littorale\u003c/h2\u003e \u003cp\u003eWe evaluated four irradiance levels (10, 30, 50, and 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) to determine when, and how strongly, light intensity alters the time-course of morphotype change in \u003cem\u003eMonoraphidium littorale\u003c/em\u003e. Across all treatments, cultures remained uniformly fusiform during the first\u0026thinsp;~\u0026thinsp;20 days; therefore, morphological scoring was performed from day 20 onward at 10-day intervals until day 70.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Morphological Changes of M. littorale at 20th \u0026amp; 30th Day in Different Light Intensities\u003c/h2\u003e \u003cp\u003eAt day 20, all light treatments showed exclusively fusiform cells (100%). By day 30, divergence was evident only at higher irradiance: at 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;, ~\u0026thinsp;20% of cells were oval and ~\u0026thinsp;10% spherical (\u0026asymp;\u0026thinsp;70% fusiform), and at 50 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;, ~\u0026thinsp;15% were oval and ~\u0026thinsp;5% spherical (\u0026asymp;\u0026thinsp;80% \\ fusiform). In contrast, 30 and 10 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; maintained 100% fusiform populations in (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Morphological Changes of M. littorale at 40th \u0026amp; 50th Day in Different Light Intensities\u003c/h2\u003e \u003cp\u003eMorphological changes of \u003cem\u003eM. littorale\u003c/em\u003e at mid stationary phase (40th \u0026amp; 50th day) for 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are shown in Fig.\u0026nbsp;48. At 40th day spherical and oval shaped cells were found comparatively higher number in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e than 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. 50% \u0026amp; 70% fusiform shaped cells were observed in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively and rest were oval and spherical shaped cells. On the other hand, 90% \u0026amp; 95% fusiform-shaped cells were found in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Only 5% oval and 5% spherical shaped cells were found in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e but no spherical cells were observed in 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. At 50th day 30% \u0026amp; 50% fusiform shaped cells were observed in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively but 70% \u0026amp; 85% fusiform shaped cells were observed in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and rest of all cells were oval and spherical shaped cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Morphological Changes of M. littorale at 60th \u0026amp; 70th Day in Different Light Intensities\u003c/h2\u003e \u003cp\u003eMorphological changes of \u003cem\u003eM. littorale\u003c/em\u003e at late stationary phase 60th \u0026amp; 70th day of culture period for 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are shown in Fig.\u0026nbsp;50. At 60th day, only 10% fusiform shaped cells were found in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 30% found in 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e but 50% and 70% fusiform cells were observed in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. At 70th day only spherical and oval shaped cells were found in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and very little number of spindle shaped cells were found in 50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. On the contrary, 40% \u0026amp; 50% fusiform shaped cells were observed in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Morphological Changes of M. littorale in Different Light Intensities\u003c/h2\u003e \u003cp\u003eIn the present study growth and morphology of \u003cem\u003eM. littorale\u003c/em\u003e were seen to be markedly affected by different light intensities. No oval or spherical cells were observed before 20 days of culture period of \u003cem\u003eM. littorale\u003c/em\u003e for all light intensities. From the inoculation day to 20 days of culture period for all light intensities \u003cem\u003eM. littorale\u003c/em\u003e produced only spindle shaped cells. But after 20 days of culture period \u003cem\u003eM. littorale\u003c/em\u003e started to show morphological changes gradually.\u003c/p\u003e \u003cp\u003eAt early stationary phase (20th \u0026minus;\u0026thinsp;30th day), no morphological changes observed at 20th day and at 30th day spherical and oval-shaped cells were found in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (spherical 10%, oval 20%) and 50 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (spherical 5%, oval 15%) intensity but that time no oval or spherical shaped cells were found in low light and natural light intensity. At mid stationary phase (40th \u0026minus;\u0026thinsp;50th day), at 40th day only 5% and 10% oval shaped cells were found in 10 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 30 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively but there were no spherical shaped cells and that time, comparatively higher amount of oval and spherical shaped (30% oval, 20% spherical) cells found in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and lower amount of oval (20%) and spherical (10%) shaped cells found in 50 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. At 50th day highest morphological changes observed at 70 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and lowest morphological changes observed at 10 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Spherical and oval-shaped cells were dominant in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and spindle-shaped cells were dominant in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, at late stationary phase (60th \u0026minus;\u0026thinsp;70th day), spindle shaped cells were completely disappeared from 70 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and fully turn into spherical and oval-shaped cells (70th day). Only 10% spindle shaped cells were observed in 70 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e but at that time 40% and 50% spindle shaped cells were observed in 30 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively (70th day). Low light and natural light intensity will take more time to turn into completely spherical and oval shaped cells.\u003c/p\u003e \u003cp\u003eIn this study, good morphological changes of \u003cem\u003eM. littorale\u003c/em\u003e were observed in high light intensity (70 \u0026micro;mol m-2s-1), and very slow morphological changes were observed in natural light intensity (70 \u0026micro;mol m-2s-1). This may be due to the presence of nutrient availability and a slower growth rate than high light intensity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study shows that \u003cem\u003eMonoraphidium littorale\u003c/em\u003e follows a reproducible morphogenetic sequence\u0026mdash;fusiform \u0026rarr; oval \u0026rarr; spherical\u0026mdash;yet the timing and completeness of that sequence differ systematically under pH versus light intensity. Alkaline pH acts as a tonic, baseline regulator that biases cellular physiology toward earlier and more complete morphotype switching soon after cultures exit exponential growth, consistent with pH-linked plasticity and wall remodeling reported for green microalgae (e.g., Salbitani et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bensalem et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Akg\u0026uuml;l \u0026amp; Akg\u0026uuml;l, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By contrast, light intensity behaves as a phase-gated accelerator with threshold-like effects: little divergence is evident before ~\u0026thinsp;day 20, after which higher irradiance (\u0026ge;\u0026thinsp;50\u0026ndash;70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) compresses the timeline to oval/spherical dominance, in line with the role of irradiance as an energy-flux variable that modulates photochemistry and redox state once cultures are physiologically primed (Hu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Practically, the pH response is graded and monotonic (increasing alkalinity \u0026rarr; predictably faster remodeling), whereas the light response is nonlinear and concentrated around an effective threshold, yielding minimal morphological payoff until that threshold is crossed.\u003c/p\u003e \u003cp\u003eThe morphology of \u003cem\u003eFibrocapsa japonica\u003c/em\u003e markedly influenced by temperature and salinity, at 15-25\u003csup\u003eo\u003c/sup\u003eC and 20\u0026ndash;30 ppt the cells were found mostly as oval shaped in logarithmic phase whereas all the oval shaped cells changed to spherical within 10 days at higher temperature (30\u003csup\u003eo\u003c/sup\u003eC) and lower salinity and oval-shaped cells was highest at 60\u0026ndash;140 \u0026micro;E/m\u003csup\u003e2\u003c/sup\u003e/s. pH did not influence the morphology of the cells under the range of pH (6.5-9.0) reported by Khan \u003cem\u003eet al.\u003c/em\u003e (1996) which is not similar to our studies. Khan \u003cem\u003eet al.\u003c/em\u003e (1998) also reported the morphology of \u003cem\u003eC. marina\u003c/em\u003e cells was strongly affected by temperature, at 20\u003csup\u003eo\u003c/sup\u003eC and 25\u003csup\u003eo\u003c/sup\u003eC, the population occurred mostly in a spindle like form, whereas at 10\u003csup\u003eo\u003c/sup\u003eC, 90% of the cells became spherical within 10 days of inoculation and also reported that light intensity and pH did not influence morphology of \u003cem\u003eC. marina\u003c/em\u003e markedly under the range of light intensities (20\u0026ndash;180 \u0026micro;E m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and pH (6.5\u0026ndash;8.5) which is also not similar to our studies and in our studies \u003cem\u003eM. littorale\u003c/em\u003e showed comparatively slower morphological changes than \u003cem\u003eC. marina\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMechanistically, external pH redefines the chemical milieu\u0026mdash;altering proton gradients and inorganic-carbon speciation\u0026mdash;thereby shifting allocation from vegetative elongation to remodeling/autosporulation, a pattern congruent with reports of pH-conditioned plasticity and pigment regulation in chlorophytes (Salbitani et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Min et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Light\u0026rsquo;s influence emerges later because it depends on biomass accumulation and resource competition; beyond this point, supra-threshold irradiance rapidly accelerates the same morphogenetic program that alkaline pH has already biased (Hu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These differences translate into distinct process levers: pH offers a precise, low-cost control for predictable staging, while light is best deployed as a timed trigger. Accordingly, a two-stage protocol\u0026mdash;grow under near-neutral pH and sub-threshold light to prolong fusiform dominance, then shift to alkaline pH (\u0026ge;\u0026thinsp;8.5) and \u0026ge;\u0026thinsp;50\u0026ndash;70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; to drive remodeling\u0026mdash;aligns morphology with downstream objectives such as biomass or stress-linked products (Saha \u0026amp; Murray, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Novovesk\u0026aacute; et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kratzer \u0026amp; Murkovic, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Uebel et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo caveats frame interpretation. First, pH and light were tested in separate runs; a full factorial (pH \u0026times; light) is now needed to quantify interaction terms and test the prediction that alkaline conditions lower the effective light threshold and that high light amplifies the alkaline bias. Second, categorical morphology should be complemented by image-based morphometrics (e.g., aspect-ratio distributions) and concurrent biochemical profiling (chlorophylls, carotenoids, lipids) so that morphotype proportions become a predictive control variable for productivity landscapes (Merlo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In sum, pH is a state-setting, continuous controller that establishes the developmental bias early, whereas light is a thresholded, phase-gated trigger that accelerates remodeling once the culture is primed. Leveraging this complementarity yields an energy-aware, time-scheduled route to reproducible morphotype control in \u003cem\u003eM. littorale\u003c/em\u003e, improving staging, harvest timing, and\u0026mdash;pending biochemical validation\u0026mdash;alignment of culture states with target productivity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eMonoraphidium littorale\u003c/em\u003e exhibits a robust, phase-dependent morphogenetic program that can be steered predictably by external pH and light intensity. Alkaline conditions exert an early, graded bias toward remodeling, while high irradiance acts later as a thresholded trigger that accelerates the same trajectory once cultures are primed beyond ~\u0026thinsp;day 20. This division of labor between a state-setting variable (pH) and a phase-gated accelerator (light) explains the observed differences in sensitivity, onset, and completeness of switching from fusiform to oval/spherical cells. The resulting control logic is operationally straightforward: prolong vegetative growth and biomass accumulation under near-neutral pH with sub-threshold light, then induce rapid remodeling by shifting to alkaline pH and \u0026ge;\u0026thinsp;50\u0026ndash;70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; irradiance. While our independent experiments delineate clear roles for each factor, future work should adopt factorial pH \u0026times; light designs, higher-resolution image morphometrics, and concurrent biochemical profiling to quantify interaction terms and link morphotype proportions to productivity. Recognizing pH as the continuous regulator and light as the timed trigger provides a principled basis for energy-aware, time-scheduled cultivation protocols and more reproducible harvest timing in \u003cem\u003eM. littorale.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no Competing Interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe author didn\u0026rsquo;t get any funding for this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eArifa Sultana: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Visualization; Writing\u0026mdash;original draft; Writing\u0026mdash;review \u0026amp; editing; Project administration; Supervision; Corresponding author.Saleha Khan: Methodology; Investigation; Data curation; Visualization; Writing\u0026mdash;review \u0026amp; editing.Md. Mahfuzul Haque: Formal analysis; Validation; Visualization; Writing\u0026mdash;review \u0026amp; editing.Zoarder Faruque Ahmed: Conceptualization; Resources; Supervision; Funding acquisition (if applicable); Writing\u0026mdash;review \u0026amp; editing.Md. Nayeem Khan: Methodology; Formal analysis; Validation; Writing\u0026mdash;review \u0026amp; editing.Correspondence: Arifa Sultana, [email protected]\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003e1) Primary (raw) datasets generatedA. Morphotype enumeration dataset (core quantitative data) manuscript final (1)For each sampling timepoint and replicate flask, the following were recorded from microscopy counts:Experiment: pH experiment or light-intensity experimentTreatment level:pH: 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 manuscript final (1)Light intensity: 10, 30, 50, 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; manuscript final (1)Sampling day(s): morphological observation conducted at 10-day intervals up to day 70; quantitative morphotype scoring is reported from day 20 to day 70 (day 20, 30, 40, 50, 60, 70). manuscript final (1)Replicate ID: R1, R2, R3 (triplicates) manuscript final (1)Raw morphotype counts (per sample):Fusiform cell countOval cell countSpherical cell countTotal counted cells per sample (sum of the three morphotypes)B. Microscopy image dataset (core qualitative/visual data) manuscript final (1)Light-microscope micrographs documenting fusiform, oval, spherical morphotypes and autosporulation stagesImages include scale bars (10 \u0026micro;m) and were taken under standardized microscopy conditions (OPTIKA light microscope). manuscript final (1)2) Derived datasets/outputs generated from raw dataA. Percentage composition (per sample and per treatment-time mean) manuscript final (1)From the raw counts, the study generated:% Fusiform = (fusiform / total) \u0026times; 100% Oval = (oval / total) \u0026times; 100% Spherical = (spherical / total) \u0026times; 100B. Summary statistics and inferential outputs (SPSS) manuscript final (1)Mean \u0026plusmn; SD for each morphotype (and/or for fusiform proportion) across triplicatesSignificance testing using DMRT at p \u0026lt; 0.05, producing letter-group outputs used in figures. manuscript final (1)3) Recommended \u0026ldquo;source data\u0026rdquo; table format (ready to archive / share)A single spreadsheet (CSV/XLSX) can store all quantitative records with these columns:experiment (pH / light)treatment_value (e.g., 8.5 or 70)treatment_unit (pH units or \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;)day (20, 30, 40, 50, 60, 70)replicate (1\u0026ndash;3)fusiform_noval_nspherical_ntotal_nfusiform_pctoval_pctspherical_pct(optional metadata columns) temperature_C, photoperiod, medium, microscope_model\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkg\u0026uuml;l, F. and Akg\u0026uuml;l, R. (2022). 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Foods, 10(10), 2303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods10102303\u003c/span\u003e\u003cspan address=\"10.3390/foods10102303\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWidyaningrum, D., Sadek, N., Cecilia, D., Oktafika, R., \u0026amp; Tedjakusuma, F. (2024). The effect of temperature and encapsulation on the stability of microalgae pigments. Iop Conference Series Earth and Environmental Science, 1352(1), 012090. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/1755-1315/1352/1/012090\u003c/span\u003e\u003cspan address=\"10.1088/1755-1315/1352/1/012090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Monoraphidium littorale, morphological plasticity, pH, light intensity, autosporulation, culture control","lastPublishedDoi":"10.21203/rs.3.rs-8605830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8605830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMorphological plasticity in \u003cem\u003eMonoraphidium littorale\u003c/em\u003e\u0026mdash;the transition from fusiform to oval and spherical cells\u0026mdash;affects growth staging, autosporulation, and potential product yields. The relative contributions and kinetics of two key drivers, pH and light intensity, remain insufficiently resolved.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eReplicated batch cultures were maintained in standard medium under controlled temperature and photoperiod. Independent experiments tested pH (6.5\u0026ndash;9.0) and light intensity (10, 30, 50, 70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;). Cell morphotypes (fusiform, oval, spherical) were enumerated from day 20 to day 70 using a consistent microscopy protocol.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll treatments remained uniformly fusiform until ~\u0026thinsp;day 20. Thereafter, alkaline pH (\u0026ge;\u0026thinsp;8.5) advanced morphotype switching and produced earlier dominance of oval/spherical forms, indicating a graded, state-setting effect. Light intensity showed a delayed, threshold-like signature: divergence among treatments became pronounced only after ~\u0026thinsp;day 20, with \u0026ge;\u0026thinsp;50\u0026ndash;70 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; compressing the timeline to oval/spherical dominance, while 10\u0026ndash;30 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; preserved fusiform cells longer into stationary phase. Integrating both experiments, pH acts as a continuous baseline regulator, whereas light functions as a phase-gated accelerator.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003epH and light exert complementary control over \u003cem\u003eM. littorale\u003c/em\u003e morphogenesis. A practical, two-stage strategy emerges\u0026mdash;grow under near-neutral pH and sub-threshold light to build biomass (fusiform dominance), then shift to alkaline pH and supra-threshold irradiance to expedite remodeling/autosporulation. Future factorial (pH \u0026times; light) designs and image-based morphometrics should quantify interactions and refine time-indexed set-points for energy-aware, reproducible bioprocess control.\u003c/p\u003e","manuscriptTitle":"Influence of pH and light intensity on the morphology of the nutritious green microalga Monoraphidium littorale","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 10:14:03","doi":"10.21203/rs.3.rs-8605830/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d65d0efe-13c6-4388-ad27-06ab2d481ca7","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T10:14:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 10:14:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8605830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8605830","identity":"rs-8605830","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}