Replacement of acetic acid with ammonium acetate boosts triacylglycerol productivity without significant growth retardation in the green alga Chlamydomonas reinhardtii

Replacement of acetic acid with ammonium acetate boosts triacylglycerol productivity without significant growth retardation in the green alga Chlamydomonas reinhardtii | 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 Replacement of acetic acid with ammonium acetate boosts triacylglycerol productivity without significant growth retardation in the green alga Chlamydomonas reinhardtii Wattanapong Sittisaree, Tanayos Berkban, Chotika Yokthongwattana, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4341488/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 Microalgal cultivation is one of the main factors restricting biomass production as well as energy fuel production. It is widely known that nitrogen starvation condition triggers triacylglycerol accumulation in many green algae. Yet, such a condition suppresses growth of the algae. It is of our interest to develop culture conditions and cultivation systems for TAGs induction that does not hamper growth. We report in this study that the substitution of acetic acid with ammonium acetate in the modified TAP medium could trigger significant amount of TAG in Chlamydomonas reinhardtii while not drastically reducing growth. Application of ammonium acetate in semi-continuous cultivation showed that microalgal growth and TAG productivity could be maintained for up to 5 rounds. This finding could lead to further studies and optimizations to apply ammonium acetate for microalgal cultivation for TAG production. ammonium acetate biodiesel Chlamydomonas green algae TAG Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Demands for food and energy consumption are increasing albeit diminishing supply of fossil fuels. Thus, searching for new sources of fuel has been considered by a number of scientists globally. Biomass and biodiesel from many organisms have been recognized as one of the sources for renewable energy (Widjaja et al., 2009 ). Biodiesel refers to a plant or animal oil that consists of long-chain alkyl (methyl, ethyl, or propyl) esters. It was achieved by trans-esterifying triglyceride oil and monohydric alcohol (Moser, 2009 ). Among the natural sources for biodiesel, microalgae have many advantages in comparison with the other sources. Microalgae cultivation is easy, inexpensive, and has low nutrient requirements (Taparia et al., 2016 ). In addition, microalgae have a higher growth rate and biomass production than conventional biodiesel production from crops and forestry (Chisti, 2007 ). Biodiesel production in several microalgal species have often been reported. The common algae ( Chlorella, Dunaliella , and Tetraselmis ) can accumulate the oil content in the range of 20–50% (Mata et al., 2010 ). Although the production of biofuel from microalgae has many advantages, this process still has limitations. First, large-scale production is still not cost-optimized (in terms of cost per yield). Second, lipid extraction and purification are also energy intensive. Finally, monitoring microbial community contamination is difficult (Remmers et al., 2018 ). To overcome these limitations, the characteristics of microalgae for biofuel production have been proposed including high lipid production, fast-growing, and high biomass production. The induction of triglyceride and fatty acid content in microalgae is suitable for improving microalgal biofuel production. Nutrient depletion conditions can increase triglyceride and fatty acid content (Tan and Lee, 2016 ). Under nitrogen deprivation, microalgae accumulated high lipid droplets and triglyceride content. However, this condition reduced the growth and biomass of microalgae. In addition, this condition also affects chlorophylls and total protein content (Ikaran et al., 2015 ). Therefore, new lipid induction strategies have been challenged. Many researchers have considered new strategies to induce lipid content in microalgae, such as screening high lipid production species, optimization of stresses and culture conditions, and genetic engineering (Guiheneuf et al., 2016). Among the cultivation strategies, mixotrophic cultivation is an excellent strategy to obtain large biomass and high growth rates of microalgae. They grow with light and organic carbon to generate organic matter and cellular energy (Wang, J., et al. 2014 ). The popular organic source in a mixotrophic condition is acetate. Fan and their collage showed that excess acetate supply resulted in a significant accumulation of TAG rather than the other conditions (Fan et al. 2012 ). Their research found that increasing acetate from 20 to 60 mM caused significant increase production of TAG. Not only in the normal condition, but acetate also induced TAG accumulation under nitrogen starvation which is a lipid-inducing condition (Ramanan et al. 2013 ). The transcriptomic analysis reveals that the coordination of nitrogen starvation and acetate addition suggests an up-regulated increase in acetate use and carbon flux for the generation of lipid storage (Goodenough et al. 2014 ). While the advantages of acetate supply on TAG production is clear, the appropriate acetate source can still be questioned. The common acetate source for microalgal culture is acetic acid. This compound is the component of Tris-acetate phosphate medium common for several microalgal cultures. In addition to acetic acid, sodium acetate is the second common acetate source. Many studies have reported that the addition of sodium acetate can increase microalgal growth and biomass (Li et al. 2020 , Lacroux et al. 2021 ). Moreover, supplementation of potassium acetate under nitrogen starvation has been reported to induce TAG accumulation in microalgae (Pan-utai et al.2017). In this study, we compare the use of 4 common acetate compounds, including acetic acid, sodium acetate, potassium acetate, and ammonium acetate for cultivation of a model unicellular green alga Chlamydomonas reinhardtii . Growth and biomass productivity as well as TAG production have been investigated. The results showed that using ammonium acetate as the main acetate source can enhance TAG production even under normal growth condition, albeit not as high compared to nitrogen starvation. Results presented in this paper demonstrated that ammonium acetate is the potential carbon source for increasing microalgal growth and TAG production. Material and method Algal strains and growth conditions Chlamydomonas reinhardtii strain CC-125 wild type mt+ [137c] was obtained from the Chlamydomonas Resource Center ( http://www.chlamycollection.org ). Stock culture of the C. reinhardtii was grown in Tris-acetate phosphate (TAP) medium (Gorman et al. 1965) under constant light intensity of 50 µmol photons m − 2 s − 1 at 25°C. Prior to any experiment explained in the result section, the stock cells were inoculated into the specified growth medium at initial cell density of about 3x10 5 cell ml − 1 . Stock and experimental cultures were grown without stirring or shaking. To remove dissolved oxygen in the culture, slight agitation were applied twice a day. Growth and biomass determination Cell numbers of all culture conditions were measured daily by hemocytometer. Microalgal biomass was measured as dry weight. PVDF membrane filter was dried at 60 ºC in overnight and pre-weighed before use. Ten milliliters of microalgal culture were filtered through the filter. This cell-pellet-containing filter was dried in the oven at 60 ºC for three days before being weighted. Biomass concentration and productivity were calculated as following: $$Biomass Productivity \left(mg {L}^{-1} {d}^{-1}\right)= \frac{Final Weight \left(mg\right)-Filter Weight \left(mg\right)}{\left(Algal Volume \left(L\right)x Days of Growth \right(d)}$$ Total lipid and triglyceride content (TAG) measurement For total lipid content determination, the microalgal cell pellet containing ~ 1.75 × 10 8 cells was extracted by 3 ml of chloroform and methanol (1:2 v/v) according to the modified Bligh and Dyer method (Breil et al. 2017 ) followed by centrifugation. The supernatant containing extracted lipid was transferred to a pre-weighed 10 ml test tube. This supernatant was evaporated by airflow for three days in a chemical hood. The amount of lipid was measured gravimetrically and calculated as following: $$Lipid Productivity \left(mg {L}^{-1} {d}^{-1}\right)= \frac{Final Weight \left(mg\right)-Initial Tube Weight \left(mg\right)}{\left(Algal Volume \left(L\right)x Days of Growth \right(d)}$$ TAG content was analyzed by thin-layer chromatography (TLC). Crude dried lipid from total lipid extraction above was dissolved in 200 µl chloroform: methanol (1:2 v/v). Dried soybean oil (1 µg) was used as a standard TAG by dissolving into the same amount of the chloroform: methanol solvent. Four microliters of each dissolved lipid sample and the standard were spotted on the TLC plate (Silica gel 60 F254, Merck). Solvent containing hexane:diethyl ether:acetic acid (70:30:1) was used as a mobile phase for the TLC (Pugkaew et al. 2017). Visualization of lipid band was carried out by iodine vapor staining for 30 minutes. Approximate amount of TAG in each of the samples was calculated by comparing the band intensities with that of the standard soybean oil. Results Effects of acetate sources on cellular TAG content Although acetate is the most common organic carbon source for microalgal cultivation under heterotrophic and mixotrophic conditions, there can be several forms of acetate sources to constitute the growth medium. In this study, various sources of acetate were tested to determine their effects on the cellular TAG content of C. reinhardtii . The acetate sources used included sodium acetate, potassium acetate, and ammonium acetate, all substituted for acetic acid in the standard TAP medium. Each modified TAP medium maintained a final concentration of 17 mM of acetate ion, matching the concentration of acetic acid in the regular TAP medium. The growth media were labeled as follows: Tris for Tris minimal medium (containing no organic carbon source), AcOH for the regular TAP medium, NaOAc for the modified TAP medium with sodium acetate substitution, KOAc for the modified TAP medium with potassium acetate substitution, and NH4OAc for the modified TAP medium with ammonium acetate substitution. C. reinhardtii cells were initially inoculated at a density of approximately 3x10 5 cells ml − 1 and allowed to grow for 4 days before harvesting. It's important to note that under our growth conditions using the TAP medium, C. reinhardtii cultures typically reach the late-logarithmic phase by day 4 and enter the stationary phase from day 5 onwards. The growth characteristics of C. reinhardtii under each acetate source are illustrated in Fig. 1 A. When no organic carbon source (Tris) was present, minimal growth was observed compared to that of the standard TAP medium (AcOH), which could reach a cell density of about 3.9x10 6 cells ml − 1 . Substituting acetic acid with NaOAc or KOAc resulted in higher growth rates and significantly higher cell densities, reaching about 5x10 6 cells ml − 1 . On the other hand, C. reinhardtii cultured in the presence of NH 4 OAc showed early growth saturation by day 3, reaching a cell density of about 3x10 6 cells ml − 1 compared to the other acetate sources tested. We further investigated the impact of these different acetate sources on the cellular TAG content of the algae. Algal cells were harvested on the 4th day of growth, and total lipids were extracted from individual samples for analysis using TLC (thin-layer chromatography). Commercial soybean oil served as the standard for the migration of TAG on the TLC plate. Figure 1 B displays the iodine-vapor-stained TLC profile showing the separated lipids extracted from algal cells cultivated under various acetate sources. The migration position of the standard TAG on the TLC plate is easily discerned (Fig. 1 B, Std lane). C. reinhardtii cultured in Tris, NaOAc, and KOAc exhibited minor amounts of TAG, manifesting as faint bands on the TLC plate. Intriguingly, cells grown in the presence of NH 4 OAc accumulated a substantial amount of TAG, as observed in the TLC profile (NH 4 OAc lane). Densitometric analysis confirmed that the TAG contents in Tris, AcOH, NaOAc, and KOAc were not significantly different, whereas the TAG content in NH 4 OAc was nearly three times higher. Figure 1 also demonstrates that the presence of acetic acid (AcOH) (regular TAP), sodium acetate (NaOAc), and potassium acetate (KOAc) in the growth medium results in a 12–40% increase in cellular TAG content compared to that of the Tris-minimal medium. Substituting acetic acid with NH 4 OAc leads to a remarkable 150% and 75% enhancement over the Tris-minimal medium and regular TAP medium, respectively. Effects of ammonium acetate concentration on TAG accumulation The results displayed in Fig. 1 indicate that NH 4 OAc has the potential to enhance TAG accumulation in C. reinhardtii . Subsequently, we explored deeper into identifying the optimum concentration of NH 4 OAc for enhancing TAG accumulation. In this experiment, C. reinhardtii cells were initially inoculated at a cell density of approximately 3x10 5 cells ml − 1 in modified TAP medium with varying acetic acid substitutions: 0 (Tris minimal medium), 10, 17, 30, and 60 mM of NH 4 OAc. The inoculated cells were allowed to grow for a period of 4 days. Cells cultured in Tris minimal medium (0 mM) showed minimal growth similar to the observation in Fig. 1 A. The substitution of acetic acid with NH 4 OAc at 10 and 17 mM resulted in the highest growth rates among all the concentrations tested, culminating in a final cell density of nearly 3x10 6 cells ml − 1 by day 4 (Fig. 2 A). However, increasing NH 4 OAc concentrations led to diminished growth rates in C. reinhardtii . Cells cultivated in the modified TAP medium with NH 4 OAc concentrations of 30 and 60 mM exhibited slower growth and reached a final density of approximately 1.5x10 6 cells ml − 1 (Fig. 2 A). Cellular TAG content was determined after a 4-day period of growth. Interestingly, in contrast to the slower growth rate, an increase in NH 4 OAc concentration in the media resulted in enhanced TAG content. C. reinhardtii cultivated in Tris minimal medium (0 mM NH4OAc) contained only approximately 1 pg cell − 1 (Fig. 2 B, 0 mM). A rise in NH 4 OAc concentrations in the media to 10, 17, 30, and 60 mM led to an increase in cellular TAG content to around 1.5, 2.3, 2.9, and 4.5 pg cell − 1 , respectively (Fig. 2 B). Even though the modified TAP medium with 60 mM NH 4 OAc appeared to promote the highest TAG content per cell, the final cell density in the culture on day 4 was lower than in the other conditions. Consequently, we calculated and compared TAG productivity among all culture conditions. In the absence of any acetate source (Fig. 2 C, 0 mM), TAG productivity was only approximately 0.15 mg L − 1 day − 1 . The presence of NH 4 OAc in the growth medium increased TAG productivity to about 1–2 mg L − 1 day − 1 (Fig. 2 C), showing statistical significance over the condition without any acetate (0 mM). Nevertheless, TAG productivity in all NH 4 OAc replacement media showed no significant difference statistically. As the medium containing 17 mM and 60 mM NH 4 OAc resulted in equally high TAG productivity, we focused on the 17 mM NH 4 OAc for further studies due to the fact that it promoted a better algal growth rate. Effects of NH 4 OAc and N-starvation under batch cultivation In the literature, it is widely recognized that nitrogen starvation induces significant accumulation of triacylglycerols (TAG) in microalgae. Our findings presented so far that substituting NH 4 OAc in the modified TAP medium also enhances TAG accumulation prompts interest in comparing these two conditions. Given that the N-free TAP medium (TAP-N) does not support growth, we conducted an experiment with Chlamydomonas reinhardtii cells initially cultured in the standard TAP medium for 2 days prior to harvesting. The harvested cell pellets underwent two washes with nitrogen-free TAP growth medium (TAP-N) before being resuspended in either the standard TAP medium (TAP), the modified TAP medium with 17 mM NH 4 OAc substitution (NH 4 OAc), or TAP-N. These resuspended cultures were allowed to grow for an additional 4 days. Over this 4-day period, C. reinhardtii cells in the TAP medium exhibited continuous growth, doubling their initial cell density of ~ 3x10 6 to approximately 6.5x10 6 cells ml − 1 (Fig. 3 A). Conversely, cells resuspended in NH 4 OAc showed lag period during the initial 2 days. By day 3, cell density in NH 4 OAc culture slightly increased to about 4x10 6 cells ml − 1 . On day 4, the NH 4 OAc culture maintained a stable cell density of 4x10 6 cells ml − 1 (Fig. 3 A). For TAP-N culture, cell density exhibited slight increase from day 0 to day 3, from ~ 2.4x10 6 cells ml − 1 to about 3.4x10 6 cells ml − 1 (Fig. 3 A). Transition from day 3 to day 4 in TAP-N culture resulted in significant decrease of cell density down to 2.5x10 6 cells ml − 1 (Fig. 3 A). At the end of the 4-day growth period, cultures were harvested and subjected to TAG analyses. The cellular TAG content was quantitated, revealing approximately 1.2 pg cell − 1 for cultures resuspended in the TAP medium, compared to approximately 4.5 pg cell − 1 for those in NH 4 OAc medium and approximately 7.5 pg cell − 1 for TAP-N medium. These findings unequivocally confirm the phenomenon of TAP-N being able to enhance TAG accumulation in microalgal cells. Furthermore, they reaffirm the results presented so far, which indicate NH 4 OAc's ability to increase TAG content in C. reinhardtii cells. To comprehensively evaluate the efficacy of these 2-step batch cultivation methods across different growth media, we further assessed biomass productivity. Cells suspended in TAP medium exhibited the highest dry-weight biomass productivity at approximately 150 mg L − 1 day − 1 , followed by NH 4 OAc (130 mg L − 1 day − 1 ) and TAP-N (82 mg L − 1 day − 1 ) (Table 1 ). These biomass productivity results correlate with the relative growth rates observed in Fig. 3 A. In addition, TAG productivity mirrored cellular TAG content trends, with TAP-N yielding the highest output at approximately 6.4 mg L − 1 day − 1 , compared to approximately 4.3 mg L − 1 day − 1 for NH 4 OAc and approximately 1.3 mg L − 1 day − 1 for TAP (Table 1 ). Interestingly, one could also note that the TAG contents per cell in this experiment were slightly higher than the results shown in Fig. 1 B and Fig. 2 C. It could be due to the difference in culture conditions. In the experiments for the results in Fig. 1 B and Fig. 2 C, C. reinhardtii cells were inoculated in the medium at very low cell density of about 3x10 5 cells ml − 1 . In this experiment, on the other hand, cells were collected and resuspended in the growth medium at about 10 times higher density. Table 1 Comparative biomass and TAG productivities of C. reinhardtii grown under regular TAP medium, modified TAP medium containing NH3OAc and nitrogen-free TAP medium. Growth Medium Biomass Productivity (mg L − 1 d − 1 ) TAG Productivity (mg L − 1 d − 1 ) TAP 154 ± 11.9 1.36 ± 0.808 NH 4 OAC 130 ± 14.4 4.33 ± 0.264 ** TAP-N 82 ± 19.1 ** 6.37 ± 1.52 *** Effects of NH 4 OAc and TAP-N under semi-continuous cultivation The results from batch cultivation, as depicted in Fig. 3 and Table 1 , suggest the potential application of NH 4 OAc in enhancing TAG production in microalgae. Given that NH 4 OAc does not entirely impede growth as TAP-N does, our interest was to assess both media under semi-continuous cultivation. In the initial round, C. reinhardtii cells underwent treatment identical to that of the aforementioned batch cultivation. At the end of the 4-day growth period, rather than harvesting the entire cultures, only 70% of the samples were collected, while the remaining 30% were replenished with fresh corresponding growth medium (either NH 4 OAc or TAP-N) to restore the original volume. The replenished cultures were permitted to grow for an additional 4 days before undergoing a repeat harvest of 70% and media replenishment. This process was iterated for up to 6 rounds, denoted as rounds 1 through 6. The harvested cells were subjected to TAG analysis and productivity calculation. In round 1, the TAG productivity exhibited a pattern similar to that of the batch cultivation, with TAP-N yielding higher TAG productivity (approximately 8.4 mg L − 1 day − 1 in TAP-N and 6.5 mg L − 1 day − 1 in NH 4 OAc) (Fig. 4 , R1). By round 2, the TAG productivities of both cultures had declined to a similar value of approximately 4 mg L − 1 day − 1 (Fig. 4 , R2). Given TAP-N medium’s inability to sustain algal growth, the TAP-N culture failed to proliferate in round 3, whereas the NH 4 OAc cells continued to grow and complete the semi-continuous cultivation through round 6. TAG productivity values for NH 4 OAc cultures in rounds 3, 4, 5, and 6 were 2.2, 1.8, 1.9, and 3.0 mg L − 1 day − 1 , respectively (Fig. 4 , R3-R6). Discussion We show in this paper that substitution of acetic acid with ammonium acetate while maintaining the normal concentration of acetate anion could significantly boost TAG accumulation in the model alga Chlamydomonas reinhardtii . Acetate has been reported as one of the important components in the growth medium for TAG induction. It has been suggested that acetate acts as a direct substrate for lipid biosynthesis precursors (Goodson et al. 2011 ). Excess acetate supply could lead to significant accumulation of TAG over than the other nutrient component (Ma et al. 2022 ). Our results in Fig. 1 suggest that although acetate alone could help boost up TAG accumulation, combination of acetate and ammonium further enhances the effects to another level. Nevertheless, this finding is without precedence. Lin et al. ( 2017 ) reported that neutral lipid content of Nannochloropsis oculata was enhanced when ammonium was used as a nitrogen source, especially in the presence of acetate. In addition, a study in Spirulina platensis lipid content was significantly decreased by ammonium under photoautotrophic condition while being significantly increased in the mixotrophic growth (Li et al. 2019 ). Chlorella pyrenoidosa cultivation can also accumulate high content of neutral lipid after addition of sodium acetate to the growth medium containing ammonium (Liu et al. 2018 ). Optimization of ammonium acetate concentration for TAG induction is crucial for further large-scale applications. Concentration of acetate is one of the factors to consider because too high acetate concentration could result in declined microalgal cell density (Bogaert et al. 2019 ). More importantly, high level of ammonium is known to be toxic to the algal cells (Wang et al. 2019 ). Our results showed that ammonium acetate concertation in the medium between 10–17 mM could still support growth while boosting the TAG productivity. Higher concentrations of NH 4 OAc of 30 and 60 mM, although promoting higher TAG content, diminished culture growth. TAG productivity in the 2-step batch culture of C. reinhardtii in the modified TAP medium containing NH 4 OAc was about 40% lower than that of the TAP-N. This productivity gap became irrelevant under semi-continuous cultivation. This is because TAP-N medium could not sustain microalgal proliferation, leading to failure of cells growth after 2 rounds. NH 4 OAc, on the other hand, could sustain C. reinhardtii growth in the semi-continuous cultivation up to at least 6 rounds demonstrated in this study. In summary, we demonstrate in this work an initial finding for a potential application of this synergistic effect of acetate and ammonium for microalgal cultivation using semi-continuous system. Additional testing of this concept in other microalgae as well as further optimizations of this system, i.e. fine-tuning the NH 4 OAc concentration, ratios of cell collection and medium reconstitution, may lead to real practical large-scale cultivation. Declarations Acknowledgment: WS thanks the 2020 Graduate Development Scholarship support from the National Research Council of Thailand. Ethical Approval : N/A Funding : This work was financially supported by Mahidol University. Availability of data and materials : Raw data for all figures are available upon request. Conflicts of interest : The authors declare no conflict of interest. Author Contribution WS planned the whole project and performed most of the experiments as well as writing the first draft.TB assisted WS in several experiments, especially TLC analysis.CY cosupervised WS and TB for the whole project and helped edit the manuscript.KY is the PI and corresponding author responsible for the whole project and approved the final manuscript. References Bogaert KA, Perez E, Rumin J, Giltay A, Carone M, Coosemans N, et al. Metabolic, physiological, and transcriptomics analysis of batch cultures of the green microalga Chlamydomonas grown on different acetate concentrations. Cells 2019;8(11):1367. Breil C, Abert Vian M, Zemb T, Kunz W, Chemat F. "Bligh and Dyer" and Folch methods for solid-liquid-liquid extraction of lipids from microorganisms. Comprehension of solvatation mechanisms and towards substitution with alternative solvents. Int J Mol Sci 2017;18(4):708. Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25(3):294-306. 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Mixotrophic cultivation of microalgae for biodiesel production: status and prospects. Appl Biochem Biotechnol 2014;172(7):3307-29. Wang J, Zhou W, Chen H, Zhan J, He C, Wang Q. Ammonium nitrogen tolerant Chlorella strain screening and its damaging effects on photosynthesis. Front Microbiol 2019;9. Widjaja A, Chien C-C, Ju Y-H. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris . J Taiwan Inst Chem Engrs 2009;40(1):13-20. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4341488","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299457661,"identity":"e540cafe-b988-4320-a0c4-79e827892e23","order_by":0,"name":"Wattanapong Sittisaree","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Wattanapong","middleName":"","lastName":"Sittisaree","suffix":""},{"id":299457662,"identity":"7444c313-f92b-4d31-87c8-e1e71766a1f3","order_by":1,"name":"Tanayos Berkban","email":"","orcid":"","institution":"Kasetsart University","correspondingAuthor":false,"prefix":"","firstName":"Tanayos","middleName":"","lastName":"Berkban","suffix":""},{"id":299457663,"identity":"1929eeaf-63cc-45ed-bf2f-88af282fb754","order_by":2,"name":"Chotika Yokthongwattana","email":"","orcid":"","institution":"Kasetsart University","correspondingAuthor":false,"prefix":"","firstName":"Chotika","middleName":"","lastName":"Yokthongwattana","suffix":""},{"id":299457665,"identity":"ccdc9644-f6ea-4398-8cbf-ece463961dfa","order_by":3,"name":"Kittisak Yokthongwattana","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIie3PMUvEMBTA8VcC6RI8xxwn9xmuFA4ErV8l4YFTz8XxhCsIuUldu/ktMucI2NHVsZPzuYhCEdP2FIfGWwXzH9pA34++AIRCfzBO3EMAzNyLwNadKYjI9N98hETFF4nKnkBHmI+4wQJ2hLDuj3vIeD2q6rrJUuBo09M8uziI0Ri4yuBsUgySSbuYVDgHfo640HhJ2bMw8IDAjswgmXakICfA89QuNJGK5zMD1LjFhJ+IZtWTY73akQ8/6RYT1M5bgpG2PYmUn4yvSVJKVaXuCpjc6Eqq9i7yFhl7Gib8cVO/vDXL5C5Gy9/1Ut6vcVNvX7NpXA6T7+jhzwF3Zr/Pt43M/plQKBT6n30CZGZXxigWTUEAAAAASUVORK5CYII=","orcid":"","institution":"Mahidol University","correspondingAuthor":true,"prefix":"","firstName":"Kittisak","middleName":"","lastName":"Yokthongwattana","suffix":""}],"badges":[],"createdAt":"2024-04-29 08:48:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4341488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4341488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56271672,"identity":"7b059251-a55f-4b2b-9e2b-3cd7846dfb66","added_by":"auto","created_at":"2024-05-10 18:22:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":523374,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of acetate sources on \u003cem\u003eC. reinhardtii\u003c/em\u003e growth and TAG accumulation. Cells were inoculated at initial density of 3x10\u003csup\u003e5\u003c/sup\u003e cells ml\u003csup\u003e-1\u003c/sup\u003e in the medium containing various acetate source as following: Tris – Tris minimal medium containing no acetate source, AcOH – regular TAP medium with acetic acid as the carbon source, NaOAc – modified TAP medium with substitution of acetic acid with sodium acetate, KOAc – modified TAP medium with substitution of acetic acid with potassium acetate and NH4OAc – modified TAP medium with substitution of acetic acid with ammonium acetate. All cultures were allowed to grow for 4 days. (A) Growth profile of the algal cells in each medium. (B) Iodine-stain TLC showing lipid profile of the cells after 4-day growth in each medium. (C) TAG content of each algal sample calculated from the TLC band intensities normalized to that of the soybean oil standard. Data are average of 3 independent experiments ± SE. Asterisk ** represents sample with statistical significance over that of the Tris (P value \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4341488/v1/bdff864181dc7b738b7637cb.jpg"},{"id":56271722,"identity":"0b32dbfb-8003-4974-94aa-355352322040","added_by":"auto","created_at":"2024-05-10 18:23:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":702768,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ammonium acetate concentrations on \u003cem\u003eC. reinhardtii\u003c/em\u003e growth and TAG accumulation. Experiments were the same as that described in Fig. 1 except with varied concentrations of ammonium acetate. (A) Growth profile of the algal cells in each medium. (B) TAG content of each algal sample calculated from the TLC band intensities. (C) Calculated TAG productivity. Data are average of 3 independent experiments ± SE. Asterisks *, **, *** represents sample with statistical significance over that of the Tris, P value \u0026lt; 0.05, 0.01 and 0.001, respectively.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4341488/v1/62bcb87e267ce21515fb3701.jpg"},{"id":56271684,"identity":"26073974-b7fe-4525-8b80-aa60c7fdf767","added_by":"auto","created_at":"2024-05-10 18:22:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":470786,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of 2-step TAG induction batch cultivations using regular TAP medium, modified TAP with 17 mM NH\u003csub\u003e4\u003c/sub\u003eOAc substitution and N-free TAP medium (TAP-N). Cells were pre-grown in regular TAP medium before being harvested and resuspended in the specified growth medium. After the 4-day growth, cells were collected and subjected to TAG analysis. (A) Growth profile of individual algal cultures. (B) TAG content of each algal sample calculated from the TLC band intensities. Data are average of 3 independent experiments ± SE. Asterisks **, *** represents sample with statistical significance over that of the Tris, P value \u0026lt; 0.01 and 0.001, respectively.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4341488/v1/abdec2e669236d412f92bb8d.jpg"},{"id":56271773,"identity":"d87e6592-caa2-4f7f-a45a-ca090d6fa15b","added_by":"auto","created_at":"2024-05-10 18:24:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":390991,"visible":true,"origin":"","legend":"\u003cp\u003eComparative TAG productivity in semi-continuous cultivation system using modified TAP medium with 17 mM NH\u003csub\u003e4\u003c/sub\u003eOAc substitution and TAP-N. Experiments were conducted similarly to that specified in Fig. 3 except that at the end of each round (4-day growth) only 70% of the culture volume was collected and the remaining cultures were reconstituted with fresh corresponding medium to the original volume. The collected cells were subjected to TAG analysis. Repeats were performed for rounds 2-6. Data are average of 3 independent experiments ± SE.\u003c/p\u003e","description":"","filename":"Fig.4new.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4341488/v1/5476d19f5c19bc2a5d7728fd.jpg"},{"id":59706891,"identity":"4995b1b2-8be5-4bc1-88b9-c7e8cbbbc464","added_by":"auto","created_at":"2024-07-05 06:07:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2593008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4341488/v1/1690aa3d-d141-4803-9c0e-aade384704cb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Replacement of acetic acid with ammonium acetate boosts triacylglycerol productivity without significant growth retardation in the green alga Chlamydomonas reinhardtii","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDemands for food and energy consumption are increasing albeit diminishing supply of fossil fuels. Thus, searching for new sources of fuel has been considered by a number of scientists globally. Biomass and biodiesel from many organisms have been recognized as one of the sources for renewable energy (Widjaja et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Biodiesel refers to a plant or animal oil that consists of long-chain alkyl (methyl, ethyl, or propyl) esters. It was achieved by trans-esterifying triglyceride oil and monohydric alcohol (Moser, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Among the natural sources for biodiesel, microalgae have many advantages in comparison with the other sources. Microalgae cultivation is easy, inexpensive, and has low nutrient requirements (Taparia et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, microalgae have a higher growth rate and biomass production than conventional biodiesel production from crops and forestry (Chisti, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Biodiesel production in several microalgal species have often been reported. The common algae (\u003cem\u003eChlorella, Dunaliella\u003c/em\u003e, and \u003cem\u003eTetraselmis\u003c/em\u003e) can accumulate the oil content in the range of 20\u0026ndash;50% (Mata et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the production of biofuel from microalgae has many advantages, this process still has limitations. First, large-scale production is still not cost-optimized (in terms of cost per yield). Second, lipid extraction and purification are also energy intensive. Finally, monitoring microbial community contamination is difficult (Remmers et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To overcome these limitations, the characteristics of microalgae for biofuel production have been proposed including high lipid production, fast-growing, and high biomass production. The induction of triglyceride and fatty acid content in microalgae is suitable for improving microalgal biofuel production. Nutrient depletion conditions can increase triglyceride and fatty acid content (Tan and Lee, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Under nitrogen deprivation, microalgae accumulated high lipid droplets and triglyceride content. However, this condition reduced the growth and biomass of microalgae. In addition, this condition also affects chlorophylls and total protein content (Ikaran et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, new lipid induction strategies have been challenged. Many researchers have considered new strategies to induce lipid content in microalgae, such as screening high lipid production species, optimization of stresses and culture conditions, and genetic engineering (Guiheneuf et al., 2016).\u003c/p\u003e \u003cp\u003eAmong the cultivation strategies, mixotrophic cultivation is an excellent strategy to obtain large biomass and high growth rates of microalgae. They grow with light and organic carbon to generate organic matter and cellular energy (Wang, J., et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The popular organic source in a mixotrophic condition is acetate. Fan and their collage showed that excess acetate supply resulted in a significant accumulation of TAG rather than the other conditions (Fan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Their research found that increasing acetate from 20 to 60 mM caused significant increase production of TAG. Not only in the normal condition, but acetate also induced TAG accumulation under nitrogen starvation which is a lipid-inducing condition (Ramanan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The transcriptomic analysis reveals that the coordination of nitrogen starvation and acetate addition suggests an up-regulated increase in acetate use and carbon flux for the generation of lipid storage (Goodenough et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the advantages of acetate supply on TAG production is clear, the appropriate acetate source can still be questioned. The common acetate source for microalgal culture is acetic acid. This compound is the component of Tris-acetate phosphate medium common for several microalgal cultures. In addition to acetic acid, sodium acetate is the second common acetate source. Many studies have reported that the addition of sodium acetate can increase microalgal growth and biomass (Li et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Lacroux et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, supplementation of potassium acetate under nitrogen starvation has been reported to induce TAG accumulation in microalgae (Pan-utai et al.2017). In this study, we compare the use of 4 common acetate compounds, including acetic acid, sodium acetate, potassium acetate, and ammonium acetate for cultivation of a model unicellular green alga \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. Growth and biomass productivity as well as TAG production have been investigated. The results showed that using ammonium acetate as the main acetate source can enhance TAG production even under normal growth condition, albeit not as high compared to nitrogen starvation. Results presented in this paper demonstrated that ammonium acetate is the potential carbon source for increasing microalgal growth and TAG production.\u003c/p\u003e"},{"header":"Material and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAlgal strains and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e strain CC-125 wild type mt+ [137c] was obtained from the Chlamydomonas Resource Center (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.chlamycollection.org\u003c/span\u003e\u003cspan address=\"http://www.chlamycollection.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Stock culture of the \u003cem\u003eC. reinhardtii\u003c/em\u003e was grown in Tris-acetate phosphate (TAP) medium (Gorman et al. 1965) under constant light intensity of 50 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 25\u0026deg;C. Prior to any experiment explained in the result section, the stock cells were inoculated into the specified growth medium at initial cell density of about 3x10\u003csup\u003e5\u003c/sup\u003e cell ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Stock and experimental cultures were grown without stirring or shaking. To remove dissolved oxygen in the culture, slight agitation were applied twice a day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGrowth and biomass determination\u003c/h2\u003e \u003cp\u003eCell numbers of all culture conditions were measured daily by hemocytometer. Microalgal biomass was measured as dry weight. PVDF membrane filter was dried at 60 \u0026ordm;C in overnight and pre-weighed before use. Ten milliliters of microalgal culture were filtered through the filter. This cell-pellet-containing filter was dried in the oven at 60 \u0026ordm;C for three days before being weighted. Biomass concentration and productivity were calculated as following:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$Biomass Productivity \\left(mg {L}^{-1} {d}^{-1}\\right)= \\frac{Final Weight \\left(mg\\right)-Filter Weight \\left(mg\\right)}{\\left(Algal Volume \\left(L\\right)x Days of Growth \\right(d)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTotal lipid and triglyceride content (TAG) measurement\u003c/h2\u003e \u003cp\u003eFor total lipid content determination, the microalgal cell pellet containing\u0026thinsp;~\u0026thinsp;1.75 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells was extracted by 3 ml of chloroform and methanol (1:2 v/v) according to the modified Bligh and Dyer method (Breil et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) followed by centrifugation. The supernatant containing extracted lipid was transferred to a pre-weighed 10 ml test tube. This supernatant was evaporated by airflow for three days in a chemical hood. The amount of lipid was measured gravimetrically and calculated as following:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$Lipid Productivity \\left(mg {L}^{-1} {d}^{-1}\\right)= \\frac{Final Weight \\left(mg\\right)-Initial Tube Weight \\left(mg\\right)}{\\left(Algal Volume \\left(L\\right)x Days of Growth \\right(d)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTAG content was analyzed by thin-layer chromatography (TLC). Crude dried lipid from total lipid extraction above was dissolved in 200 \u0026micro;l chloroform: methanol (1:2 v/v). Dried soybean oil (1 \u0026micro;g) was used as a standard TAG by dissolving into the same amount of the chloroform: methanol solvent. Four microliters of each dissolved lipid sample and the standard were spotted on the TLC plate (Silica gel 60 F254, Merck). Solvent containing hexane:diethyl ether:acetic acid (70:30:1) was used as a mobile phase for the TLC (Pugkaew et al. 2017). Visualization of lipid band was carried out by iodine vapor staining for 30 minutes. Approximate amount of TAG in each of the samples was calculated by comparing the band intensities with that of the standard soybean oil.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffects of acetate sources on cellular TAG content\u003c/h2\u003e \u003cp\u003eAlthough acetate is the most common organic carbon source for microalgal cultivation under heterotrophic and mixotrophic conditions, there can be several forms of acetate sources to constitute the growth medium. In this study, various sources of acetate were tested to determine their effects on the cellular TAG content of \u003cem\u003eC. reinhardtii\u003c/em\u003e. The acetate sources used included sodium acetate, potassium acetate, and ammonium acetate, all substituted for acetic acid in the standard TAP medium. Each modified TAP medium maintained a final concentration of 17 mM of acetate ion, matching the concentration of acetic acid in the regular TAP medium. The growth media were labeled as follows: Tris for Tris minimal medium (containing no organic carbon source), AcOH for the regular TAP medium, NaOAc for the modified TAP medium with sodium acetate substitution, KOAc for the modified TAP medium with potassium acetate substitution, and NH4OAc for the modified TAP medium with ammonium acetate substitution. \u003cem\u003eC. reinhardtii\u003c/em\u003e cells were initially inoculated at a density of approximately 3x10\u003csup\u003e5\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and allowed to grow for 4 days before harvesting. It's important to note that under our growth conditions using the TAP medium, \u003cem\u003eC. reinhardtii\u003c/em\u003e cultures typically reach the late-logarithmic phase by day 4 and enter the stationary phase from day 5 onwards. The growth characteristics of \u003cem\u003eC. reinhardtii\u003c/em\u003e under each acetate source are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. When no organic carbon source (Tris) was present, minimal growth was observed compared to that of the standard TAP medium (AcOH), which could reach a cell density of about 3.9x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Substituting acetic acid with NaOAc or KOAc resulted in higher growth rates and significantly higher cell densities, reaching about 5x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. On the other hand, \u003cem\u003eC. reinhardtii\u003c/em\u003e cultured in the presence of NH\u003csub\u003e4\u003c/sub\u003eOAc showed early growth saturation by day 3, reaching a cell density of about 3x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compared to the other acetate sources tested.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further investigated the impact of these different acetate sources on the cellular TAG content of the algae. Algal cells were harvested on the 4th day of growth, and total lipids were extracted from individual samples for analysis using TLC (thin-layer chromatography). Commercial soybean oil served as the standard for the migration of TAG on the TLC plate. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB displays the iodine-vapor-stained TLC profile showing the separated lipids extracted from algal cells cultivated under various acetate sources. The migration position of the standard TAG on the TLC plate is easily discerned (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Std lane). \u003cem\u003eC. reinhardtii\u003c/em\u003e cultured in Tris, NaOAc, and KOAc exhibited minor amounts of TAG, manifesting as faint bands on the TLC plate. Intriguingly, cells grown in the presence of NH\u003csub\u003e4\u003c/sub\u003eOAc accumulated a substantial amount of TAG, as observed in the TLC profile (NH\u003csub\u003e4\u003c/sub\u003eOAc lane). Densitometric analysis confirmed that the TAG contents in Tris, AcOH, NaOAc, and KOAc were not significantly different, whereas the TAG content in NH\u003csub\u003e4\u003c/sub\u003eOAc was nearly three times higher. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also demonstrates that the presence of acetic acid (AcOH) (regular TAP), sodium acetate (NaOAc), and potassium acetate (KOAc) in the growth medium results in a 12\u0026ndash;40% increase in cellular TAG content compared to that of the Tris-minimal medium. Substituting acetic acid with NH\u003csub\u003e4\u003c/sub\u003eOAc leads to a remarkable 150% and 75% enhancement over the Tris-minimal medium and regular TAP medium, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffects of ammonium acetate concentration on TAG accumulation\u003c/h2\u003e \u003cp\u003eThe results displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that NH\u003csub\u003e4\u003c/sub\u003eOAc has the potential to enhance TAG accumulation in \u003cem\u003eC. reinhardtii\u003c/em\u003e. Subsequently, we explored deeper into identifying the optimum concentration of NH\u003csub\u003e4\u003c/sub\u003eOAc for enhancing TAG accumulation. In this experiment, \u003cem\u003eC. reinhardtii\u003c/em\u003e cells were initially inoculated at a cell density of approximately 3x10\u003csup\u003e5\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in modified TAP medium with varying acetic acid substitutions: 0 (Tris minimal medium), 10, 17, 30, and 60 mM of NH\u003csub\u003e4\u003c/sub\u003eOAc. The inoculated cells were allowed to grow for a period of 4 days. Cells cultured in Tris minimal medium (0 mM) showed minimal growth similar to the observation in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The substitution of acetic acid with NH\u003csub\u003e4\u003c/sub\u003eOAc at 10 and 17 mM resulted in the highest growth rates among all the concentrations tested, culminating in a final cell density of nearly 3x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, increasing NH\u003csub\u003e4\u003c/sub\u003eOAc concentrations led to diminished growth rates in \u003cem\u003eC. reinhardtii\u003c/em\u003e. Cells cultivated in the modified TAP medium with NH\u003csub\u003e4\u003c/sub\u003eOAc concentrations of 30 and 60 mM exhibited slower growth and reached a final density of approximately 1.5x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCellular TAG content was determined after a 4-day period of growth. Interestingly, in contrast to the slower growth rate, an increase in NH\u003csub\u003e4\u003c/sub\u003eOAc concentration in the media resulted in enhanced TAG content. \u003cem\u003eC. reinhardtii\u003c/em\u003e cultivated in Tris minimal medium (0 mM NH4OAc) contained only approximately 1 pg cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, 0 mM). A rise in NH\u003csub\u003e4\u003c/sub\u003eOAc concentrations in the media to 10, 17, 30, and 60 mM led to an increase in cellular TAG content to around 1.5, 2.3, 2.9, and 4.5 pg cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Even though the modified TAP medium with 60 mM NH\u003csub\u003e4\u003c/sub\u003eOAc appeared to promote the highest TAG content per cell, the final cell density in the culture on day 4 was lower than in the other conditions. Consequently, we calculated and compared TAG productivity among all culture conditions. In the absence of any acetate source (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, 0 mM), TAG productivity was only approximately 0.15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The presence of NH\u003csub\u003e4\u003c/sub\u003eOAc in the growth medium increased TAG productivity to about 1\u0026ndash;2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), showing statistical significance over the condition without any acetate (0 mM). Nevertheless, TAG productivity in all NH\u003csub\u003e4\u003c/sub\u003eOAc replacement media showed no significant difference statistically. As the medium containing 17 mM and 60 mM NH\u003csub\u003e4\u003c/sub\u003eOAc resulted in equally high TAG productivity, we focused on the 17 mM NH\u003csub\u003e4\u003c/sub\u003eOAc for further studies due to the fact that it promoted a better algal growth rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffects of NH\u003csub\u003e4\u003c/sub\u003eOAc and N-starvation under batch cultivation\u003c/h2\u003e \u003cp\u003eIn the literature, it is widely recognized that nitrogen starvation induces significant accumulation of triacylglycerols (TAG) in microalgae. Our findings presented so far that substituting NH\u003csub\u003e4\u003c/sub\u003eOAc in the modified TAP medium also enhances TAG accumulation prompts interest in comparing these two conditions. Given that the N-free TAP medium (TAP-N) does not support growth, we conducted an experiment with \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e cells initially cultured in the standard TAP medium for 2 days prior to harvesting. The harvested cell pellets underwent two washes with nitrogen-free TAP growth medium (TAP-N) before being resuspended in either the standard TAP medium (TAP), the modified TAP medium with 17 mM NH\u003csub\u003e4\u003c/sub\u003eOAc substitution (NH\u003csub\u003e4\u003c/sub\u003eOAc), or TAP-N. These resuspended cultures were allowed to grow for an additional 4 days. Over this 4-day period, \u003cem\u003eC. reinhardtii\u003c/em\u003e cells in the TAP medium exhibited continuous growth, doubling their initial cell density of ~\u0026thinsp;3x10\u003csup\u003e6\u003c/sup\u003e to approximately 6.5x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Conversely, cells resuspended in NH\u003csub\u003e4\u003c/sub\u003eOAc showed lag period during the initial 2 days. By day 3, cell density in NH\u003csub\u003e4\u003c/sub\u003eOAc culture slightly increased to about 4x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. On day 4, the NH\u003csub\u003e4\u003c/sub\u003eOAc culture maintained a stable cell density of 4x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). For TAP-N culture, cell density exhibited slight increase from day 0 to day 3, from ~\u0026thinsp;2.4x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to about 3.4x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Transition from day 3 to day 4 in TAP-N culture resulted in significant decrease of cell density down to 2.5x10\u003csup\u003e6\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the end of the 4-day growth period, cultures were harvested and subjected to TAG analyses. The cellular TAG content was quantitated, revealing approximately 1.2 pg cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for cultures resuspended in the TAP medium, compared to approximately 4.5 pg cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for those in NH\u003csub\u003e4\u003c/sub\u003eOAc medium and approximately 7.5 pg cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for TAP-N medium. These findings unequivocally confirm the phenomenon of TAP-N being able to enhance TAG accumulation in microalgal cells. Furthermore, they reaffirm the results presented so far, which indicate NH\u003csub\u003e4\u003c/sub\u003eOAc's ability to increase TAG content in \u003cem\u003eC. reinhardtii\u003c/em\u003e cells. To comprehensively evaluate the efficacy of these 2-step batch cultivation methods across different growth media, we further assessed biomass productivity. Cells suspended in TAP medium exhibited the highest dry-weight biomass productivity at approximately 150 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, followed by NH\u003csub\u003e4\u003c/sub\u003eOAc (130 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and TAP-N (82 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These biomass productivity results correlate with the relative growth rates observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. In addition, TAG productivity mirrored cellular TAG content trends, with TAP-N yielding the highest output at approximately 6.4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, compared to approximately 4.3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003efor NH\u003csub\u003e4\u003c/sub\u003eOAc and approximately 1.3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for TAP (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Interestingly, one could also note that the TAG contents per cell in this experiment were slightly higher than the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. It could be due to the difference in culture conditions. In the experiments for the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, C. \u003cem\u003ereinhardtii\u003c/em\u003e cells were inoculated in the medium at very low cell density of about 3x10\u003csup\u003e5\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In this experiment, on the other hand, cells were collected and resuspended in the growth medium at about 10 times higher density.\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\u003eComparative biomass and TAG productivities of C. reinhardtii grown under regular TAP medium, modified TAP medium containing NH3OAc and nitrogen-free TAP medium.\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrowth Medium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiomass Productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAG Productivity\u003c/p\u003e \u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\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\u003eTAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e154\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.808\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eOAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e130\u0026thinsp;\u0026plusmn;\u0026thinsp;14.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.264\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTAP-N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e82\u0026thinsp;\u0026plusmn;\u0026thinsp;19.1\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffects of NH\u003csub\u003e4\u003c/sub\u003eOAc and TAP-N under semi-continuous cultivation\u003c/h2\u003e \u003cp\u003eThe results from batch cultivation, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, suggest the potential application of NH\u003csub\u003e4\u003c/sub\u003eOAc in enhancing TAG production in microalgae. Given that NH\u003csub\u003e4\u003c/sub\u003eOAc does not entirely impede growth as TAP-N does, our interest was to assess both media under semi-continuous cultivation. In the initial round, \u003cem\u003eC. reinhardtii\u003c/em\u003e cells underwent treatment identical to that of the aforementioned batch cultivation. At the end of the 4-day growth period, rather than harvesting the entire cultures, only 70% of the samples were collected, while the remaining 30% were replenished with fresh corresponding growth medium (either NH\u003csub\u003e4\u003c/sub\u003eOAc or TAP-N) to restore the original volume. The replenished cultures were permitted to grow for an additional 4 days before undergoing a repeat harvest of 70% and media replenishment. This process was iterated for up to 6 rounds, denoted as rounds 1 through 6. The harvested cells were subjected to TAG analysis and productivity calculation. In round 1, the TAG productivity exhibited a pattern similar to that of the batch cultivation, with TAP-N yielding higher TAG productivity (approximately 8.4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in TAP-N and 6.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in NH\u003csub\u003e4\u003c/sub\u003eOAc) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, R1). By round 2, the TAG productivities of both cultures had declined to a similar value of approximately 4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, R2). Given TAP-N medium\u0026rsquo;s inability to sustain algal growth, the TAP-N culture failed to proliferate in round 3, whereas the NH\u003csub\u003e4\u003c/sub\u003eOAc cells continued to grow and complete the semi-continuous cultivation through round 6. TAG productivity values for NH\u003csub\u003e4\u003c/sub\u003eOAc cultures in rounds 3, 4, 5, and 6 were 2.2, 1.8, 1.9, and 3.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, R3-R6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe show in this paper that substitution of acetic acid with ammonium acetate while maintaining the normal concentration of acetate anion could significantly boost TAG accumulation in the model alga \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. Acetate has been reported as one of the important components in the growth medium for TAG induction. It has been suggested that acetate acts as a direct substrate for lipid biosynthesis precursors (Goodson et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Excess acetate supply could lead to significant accumulation of TAG over than the other nutrient component (Ma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e suggest that although acetate alone could help boost up TAG accumulation, combination of acetate and ammonium further enhances the effects to another level. Nevertheless, this finding is without precedence. Lin et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that neutral lipid content of \u003cem\u003eNannochloropsis oculata\u003c/em\u003e was enhanced when ammonium was used as a nitrogen source, especially in the presence of acetate. In addition, a study in Spirulina platensis lipid content was significantly decreased by ammonium under photoautotrophic condition while being significantly increased in the mixotrophic growth (Li et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e cultivation can also accumulate high content of neutral lipid after addition of sodium acetate to the growth medium containing ammonium (Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOptimization of ammonium acetate concentration for TAG induction is crucial for further large-scale applications. Concentration of acetate is one of the factors to consider because too high acetate concentration could result in declined microalgal cell density (Bogaert et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). More importantly, high level of ammonium is known to be toxic to the algal cells (Wang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our results showed that ammonium acetate concertation in the medium between 10\u0026ndash;17 mM could still support growth while boosting the TAG productivity. Higher concentrations of NH\u003csub\u003e4\u003c/sub\u003eOAc of 30 and 60 mM, although promoting higher TAG content, diminished culture growth. TAG productivity in the 2-step batch culture of \u003cem\u003eC. reinhardtii\u003c/em\u003e in the modified TAP medium containing NH\u003csub\u003e4\u003c/sub\u003eOAc was about 40% lower than that of the TAP-N. This productivity gap became irrelevant under semi-continuous cultivation. This is because TAP-N medium could not sustain microalgal proliferation, leading to failure of cells growth after 2 rounds. NH\u003csub\u003e4\u003c/sub\u003eOAc, on the other hand, could sustain \u003cem\u003eC. reinhardtii\u003c/em\u003e growth in the semi-continuous cultivation up to at least 6 rounds demonstrated in this study.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrate in this work an initial finding for a potential application of this synergistic effect of acetate and ammonium for microalgal cultivation using semi-continuous system. Additional testing of this concept in other microalgae as well as further optimizations of this system, i.e. fine-tuning the NH\u003csub\u003e4\u003c/sub\u003eOAc concentration, ratios of cell collection and medium reconstitution, may lead to real practical large-scale cultivation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e WS thanks the 2020 Graduate Development Scholarship support from the National Research Council of Thailand.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEthical Approval\u003c/u\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eFunding\u003c/u\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Mahidol University.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e:\u003c/p\u003e\n\u003cp\u003eRaw data for all figures are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConflicts of interest\u003c/u\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWS planned the whole project and performed most of the experiments as well as writing the first draft.TB assisted WS in several experiments, especially TLC analysis.CY cosupervised WS and TB for the whole project and helped edit the manuscript.KY is the PI and corresponding author responsible for the whole project and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBogaert KA, Perez E, Rumin J, Giltay A, Carone M, Coosemans N, et al. 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J Taiwan Inst Chem Engrs 2009;40(1):13-20.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"ammonium acetate, biodiesel, Chlamydomonas, green algae, TAG","lastPublishedDoi":"10.21203/rs.3.rs-4341488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4341488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroalgal cultivation is one of the main factors restricting biomass production as well as energy fuel production. It is widely known that nitrogen starvation condition triggers triacylglycerol accumulation in many green algae. Yet, such a condition suppresses growth of the algae. It is of our interest to develop culture conditions and cultivation systems for TAGs induction that does not hamper growth. We report in this study that the substitution of acetic acid with ammonium acetate in the modified TAP medium could trigger significant amount of TAG in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e while not drastically reducing growth. Application of ammonium acetate in semi-continuous cultivation showed that microalgal growth and TAG productivity could be maintained for up to 5 rounds. This finding could lead to further studies and optimizations to apply ammonium acetate for microalgal cultivation for TAG production.\u003c/p\u003e","manuscriptTitle":"Replacement of acetic acid with ammonium acetate boosts triacylglycerol productivity without significant growth retardation in the green alga Chlamydomonas reinhardtii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 18:05:32","doi":"10.21203/rs.3.rs-4341488/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":"ef161617-cff8-455a-b4e1-658b097c73ea","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-05T05:59:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-10 18:05:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4341488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4341488","identity":"rs-4341488","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}