Isolation and lipid production of thraustochytrids from fishing village in Tangkolak Indonesia

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The study specifically focuses on thraustochytrids isolated from a fishing village in the northern coastal area of Java, Indonesia, known for its significant organic content. Eight isolates were obtained from this coastal environment, demonstrating robust growth and lipid production capabilities. Notably, isolate BML-38 exhibited superior biomass and lipid production compared to commercial thraustochytrid ATCC strains, particularly in crude glycerol-based media. This positions it as a strong candidate for sustainable and cost-effective lipid production. BML-38 also produced a higher concentration of pentadecanoic acid (C15:0) and a similar concentration of heptadecanoic acid (C17:0), in addition to DHA. The outcomes of this investigation open new avenues, as thraustochytrids from the coastal area exhibit the capacity to utilize waste materials while competitively producing valuable compounds such as odd-chain fatty acids and DHA. This dual capability positions these strains as noteworthy contributors to sustainable lipid production and waste remediation strategies. thraustochytrids microalgae DHA Odd-chain Fatty Acids Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Microalgae have long been acknowledged for their capacity to synthesize valuable compounds, including docosahexaenoic acids (DHA) and odd-chain fatty acids, which are essential unsaturated fatty acids vital for human health. While humans necessitate DHA, our bodies do not naturally manufacture it, compelling us to obtain it from external sources, typically deep-sea fishes. Meanwhile, odd chain fatty acids has been found to be able to alleviate comorbidities, reduced chances of cancer and dementia (To et al. 2020; Venn-Watson et al. 2020; da Rocha et al. 2023). Currently, the growing emphasis on healthier lifestyles has spurred an increased demand for these beneficial fatty acids. These challenges underscore the imperative to explore alternative sources of DHA and odd chain fatty acids, such as thraustochytrids. The cultivation of thraustochytrids provides a more sustainable and accessible means of procuring DHA and odd chain fatty acids for human consumption compared to the extraction of rare deep-sea fish or sharks (Hadley et al. 2017). Thraustochytrids are marine single-celled eukaryotic protists that hold substantial promise as reservoirs of valuable substances with applications in medicine, pharmacy, food alternatives, and bioenergy (Hadley et al. 2017; Abdel-Wahab et al. 2021). Previous investigations have identified these microorganisms in coastal areas adjacent to Mangrove trees. For example, Gupta et al. (2013) effectively isolated these microorganisms in Australia, while similar research in the Arabian Sea and Thailand has yielded analogous outcomes. In these studies, researchers isolated thraustochytrids from clear coastal regions rich in mangrove trees, utilizing pollen baiting techniques. By dispersing pine pollen on the water surface of collected samples, they observed the attraction of thraustochytrids to the pollen. Subsequently, the gathered pollen was placed on growth media containing glucose, yeast extract, and peptone. Thraustochytrids possess the remarkable ability to generate oil or lipids, constituting up to 70–80\% w/w of their biomass. These lipids encompass beneficial fatty acids, including DHA and odd chain fatty acids, primarily located intracellularly. Therefore, the emphasis on harnessing thraustochytrids as fatty acids-producing microorganisms should prioritize augmenting biomass production and refining lipid extraction methods. Given that carbon is the primary precursor for lipids, enhancing carbon sources for thraustochytrids and exploring alternative materials to reduce production expenses becomes pivotal. While glucose has traditionally been the predominant carbon source for fermenting thraustochytrids, its cost presents a barrier to large-scale production. Previous studies have exhibited promising outcomes by employing glycerol as a carbon source for DHA and odd chain fatty acids production (Kujawska et al. 2021). Additionally, Indonesia, a major producer of Palm Oil, generates a substantial quantity of glycerol waste from biodiesel production, providing an abundant and cost-effective resource. In this research, we highlight the potential nutritional value inherent in the coastal areas of fishing villages, which thraustochytrids could leverage as a resource for lipid production. Exploring these approaches offers opportunities to discover novel organisms with the capacity to yield unique lipid contents. The objective of this study is to identify native thraustochytrids and assess their growth and lipid production using crude glycerol from biodiesel waste as an alternative growth medium. In total, eight strains were collected through pollen baiting methods, and among them, BML-38 demonstrated exceptional biomass and lipid production in glycerol waste-based medium. This ability to efficiently utilize waste materials holds the promise of transforming them into valuable resources, contributing to the sustainable management of waste. Material and methods Thraustochytrids isolation The isolation of thraustochytrids was carried out following the method outlined by Gupta et al. (2013) with some modifications. Seawater samples were collected from fishing village in Tangkolak, the northern coastal area of Java, Indonesia. The samples are then stored at a temperature of 10°C. To prepare the samples, 50 mL of seawater was transferred to a Falcon tube, and a combination of streptomycin (500 mg/L), rifampicin (50 mg/L), and chloramphenicol (50 mg/L) was added. After 3 days, 100 mL of the samples was plated onto GYP (Glucose, Yeast Extract, Peptone) medium, with the composition per litre as follows: Glucose (2 g), Peptone (1 g), Yeast extract (1 g), Sterile seawater (1 L or 50% seawater/half-strength), streptomycin (500 mg/L), rifampicin (50 mg/L), and chloramphenicol (50 mg/L). Culture extract of Paraburkholderia sp. CP01 containing antifungal compound were also added to the agar media to selectively inhibit yeast growth (Prihatna et al. 2022). The samples were then observed under a microscope for possible thraustochytrids identification. Isolate identification and phylogenetic analysis The total genomic DNA of thraustochytrids strains were isolated with a DNAeasy Mini Kit (Qiagen, Netherlands). The 18sRNA analysis were performed using primer LabyF (5’-CCATCAGTTGTCGACCGTA-3’) and LabyR (5’-GTTAAGACTACGATGGTATCTAA-3’) (Hong et al. 2011). The amplification system, with a total volume of 25 µL, consisted of 12.5 µL GoTaq PCR Master-Mix (Promega), 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 1 µL DNA template, and 9.5 µL sterile water. The PCR amplification conditions were as follows: 95°C for 3 min; 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min; and a final extension at 72°C for 7 min. The amplification products were sequenced using the ABI Prism 3130 Genetic Analyzer (Applied Biosystems). The obtained sequences were analyzed using Geneious software version 11.1.5. The phylogenetic tree of the auxin-degrading loci regulator genes was generated using MAFFT v7.450 and Fasttree 2.1.11 implemented in the Geneious software. Thraustochytrids biomass production in glucose and glycerol based medium Thraustochytrids were initially cultivated in GYP agar media for 2–3 days before being transferred to 10 mL of a growth medium containing 3% glucose and 0.1% yeast extract for an additional 3 days. This was followed by transferring the culture to 100 mL of the same medium with the same concentrations. To enhance production and explore alternative nutritional sources for thraustochytrids, these microalgae were grown in glucose based medium (9% glucose + 2.25% yeast extract) and glycerol based medium (9% crude glycerol obtained from oil palm biodiesel production and 2.25% nitrogen sources). After incubation for 4 days, the samples were centrifuged, and the supernatant was discarded. The resulting pellet was weighed to obtain the wet cell mass. Subsequently, the samples were freeze-dried for 2 days before being weighed again for dry weight. The results of biomass and lipid production are analysed with One-way ANOVA using R statistical program (4.2.1 version. R Core Team, 2021), and the significance between each isolate are tested with HSD significance test. Lipid extraction and analysis For lipid extraction and analysis, the samples underwent osmotic shock treatment, disrupting the cells by exploiting the osmotic pressure differences between the interior and exterior of the cells. Subsequently, the samples were shaken in a 10% NaCl solution at 200 rpm for 24 hours. This was followed by sonication at 20 kHz with 40% amplitude, using a 40-second on and 20-second off cycle, for a total working time of 20 minutes. The lipid was extracted following the monophasic lipid extraction protocol, known as the Methanol-Chloroform method (Fu et al. 2017). This method utilized Methanol-chloroform interaction to separate the lipid. The lipid extraction process began with approximately 150 µg of powdered thraustochytrids, each placed in a 1.5 mL Eppendorf tube. To ensure accuracy, a blank sample tube was prepared for every five samples. Subsequently, 50 µL of glass beads were added to both the sample and blank tubes, followed by the introduction of 200 µL of ice-cold 60% methanol containing 0.01% Butylated Hydroxytoulene (BHT) and 50 µL of a lipid standard mixture with the same BHT content. After lysis through a tissuelyser at 30 s-1 for 15 minutes, 120 µL of H2O and 470 µL of methanol containing 0.01% BHT were added. If further lysis was needed, an additional 10 minutes of lysis was conducted. Then, 270 µL of chloroform with 0.01% BHT was introduced, followed by vigorous vortexing and shaking using the tissuelyser for 30 minutes at 16.7 s-1. After centrifugation for phase separation, the supernatant was transferred into 2 mL Eppendorf tubes. Subsequently, 100 µL of H 2 O and 400 µL of chloroform:methanol (1:2, v:v) with 0.01% BHT were added and vortexed. A second round of shaking, centrifugation, and supernatant collection followed. The combined supernatant was vacuum-evaporated until dry, and the lipids were resuspended in 500 µL of isopropanol:methanol:chloroform (4:2:1, v:v:v) with 0.01% BHT. After centrifugation, the supernatant (~ 400 µL) was transferred to glass vials, saturated with N2 gas, and stored at -80°C for subsequent mass spectrometry analysis. The GC analysis used 37 FAME Mix Supelco as standard (Sigma Aldrich, Germany) and following GC analysis settings from previous study (Kumar Aiswarya et al. 2014). Results Isolation and identification of thraustochytrids The isolation of thraustochytrids from our intricate environmental matrix posed formidable challenges, primarily due to their necessity to outcompete contaminants, which often displayed robust growth rates. After a ten-day incubation period, we collected several samples, subjecting them to careful examination under a light microscope. Thraustochytrids are characterized by a distinct nucleus, clearly discernible at 40x magnification, rendering them distinguishable from empty bacterial cells (Fig. 1 ). In total, we succeeded in isolating eight samples using a modified approach based on the method described by Gupta et al. (2013). To precisely determine the taxonomic identity of the eight isolates and position them within the broader phylogenetic context among other eukaryotic organisms, a thorough identification process was conducted. All samples exhibited a close association with Auranthiochytrium acetophilum, displaying substantial genetic similarities exceeding 97% (Fig. 2 ). Utilizing the NCBI BLAST analysis, the isolates were categorized into three groups. Group 1, demonstrating the highest similarity to A. acetophilum, comprises BML-19, BML-40, BML-42, and BML-43. Strains BML-37 and BML-38 are closely related and grouped together, as are strains BML-44 and BML-45, indicating a noteworthy degree of similarity between these specific isolates. Growth and lipid production of thraustochytrids isolates The strains isolated from Tangkolak have exhibited superior biomass and lipid production in comparison to the well-known ATCC 20889 isolate, a Schizochytrium strain approved by the European Commission for use as an additive in infant food (EFSA Panel on Nutrition et al. 2022). Among the Tangkolak isolates, BML-38 displayed the highest biomass production, reached 45 grams per liter (g/L), while ATCC 20889 exhibited significantly lower growth, 10 g/L (Fig. 3 A). Furthermore, Tangkolak strains have also demonstrated higher lipid production compared to ATCC 20889, with BML-38 surpassing 6 g/L, while ATCC 20889 recorded just under 1 g/L (Fig. 3 B). The higher lipid production observed in BML-38 suggests the potential for increased DHA and Odd-chain fatty acids production, which could be of significant importance in various applications, including in infant nutrition. Utilizing crude glycerol as an alternative carbon source for thraustochytrids All eight isolates obtained from Tangkolak were tested in a 50 mL medium containing crude glycerol. In general, all isolates grown in crude glycerol exhibited higher biomass compared to when grown in glucose-based medium. In crude glycerol-based medium, BML-38 and BML-45 displayed the highest growth rate among the isolates, reaching ~ 60 g/L (Fig. 3 C). For direct comparison between different media in larger scale (500 mL), we used only isolates BML-38. The biomass production in glycerol-based media reached 48 g/L, while glucose-based media yielded 36 g/L and technical glycerol yielded 22 g/L (Fig. 3 D). Consistent findings across various scales suggest that crude glycerol, a byproduct of biodiesel production from oil palm waste, holds significant promise as an economical alternative substance for producing fatty acids by thraustochytrids. Fatty acids components of thraustochytrids isolates Despite their biomass production, it is crucial to conduct lipid profile analysis on these strains. BML-38 exhibits a higher pentadecanoic acid (C15:0) content compared to commercial isolate ATCC 20889, but lower in DHA content (Fig. 4 ). Interestingly, BML-38 and ATCC 20889 share similar C17:0 levels. This finding showed that BML-38 emerges as particularly promising due to its capability to produce both odd-chain fatty acids and DHA. Discussion This study has successfully isolated eight strains of thraustochytrids capable of producing biomass and lipid using a crude glycerol-based medium. This capability may be attributed to their origin in a coastal area near a fishing village, where human activities generate more waste compared to open seas or oceans, common sources of thraustochytrid isolates like ATCC 20889 and ATCC 1381 (Okino et al. 2018). Furthermore, despite originating from the same location, we identified three groups of thraustochytrid isolates based on their 18S rRNA diversity, indicating significant microalgae diversity. These findings underscore the potential of coastal areas near fishing villages as valuable hotspots for discovering novel thraustochytrids for sustainable biomass and lipid production, offering an innovative approach to utilizing local environments for biotechnological applications. Future studies involving multiple locations throughout coastal area in Indonesia could unveil a more comprehensive understanding of the country's thraustochytrid biodiversity. One isolate, named BML-38, has been shown to produce more biomass and odd-chain fatty acids than the commercial isolates ATCC 20889. While our bodies require odd-chain fatty acids, we do not naturally synthesize them, necessitating external sources such as thraustochytrids. Moreover, odd-chain fatty acids have been demonstrated to exhibit anti-cancer effects, particularly in breast cancer (To et al. 2020). Lower concentrations of C17:0, which are also present in the fatty acid profile of BML-38, may be associated with the progression from GAD65Ab positivity to adult-onset diabetes (Lampousi et al. 2023). The remarkable capacity of the isolate BML-38 to produce substantial biomass and a higher proportion of beneficial odd-chain fatty acids make it a promising candidate for biotechnology application. The application of crude glycerol as an alternative carbon source holds considerable promise, particularly for companies operating in the biotechnology and agriculture sectors. The use of glucose as the primary carbon source often results in significantly increased production costs, especially when technical-grade glucose, a common choice in thraustochytrid studies, is employed. Previously, Ye et al. (2020) highlighted the potential of thraustochytrids to grow in a mixture of glucose and glycerol to reduce production costs. Furthermore, thraustochytrids have been demonstrated to utilize waste cooking oil as a carbon source for growth and to produce DHA and odd-chain fatty acids from waste cooking oil (Patel et al. 2022). Similarly, our thraustochytrid isolates demonstrated the ability to thrive on crude glycerol as a carbon source. Crude glycerol contains a variety of compounds, including soap, methanol, fatty acids, triglycerides, and metals, all of which appear to play pivotal roles in enhancing microbial growth (Kumar et al. 2019). The underperformance of BML-38 in technical glycerol underscores the presence of other beneficial compounds in crude glycerol that bolster biomass production. These findings lend further support to the notion that thraustochytrids from Tangkolak hold potential for the bioremediation of biodiesel waste. Moreover, if these strains could effectively utilize palm oil waste as a carbon source, it could offer a sustainable solution to address palm oil meal effluent challenges. It is noteworthy that pine pollen proved to be an effective bait for attracting thraustochytrids amid the diverse array of cohabiting organisms. Pine pollen analysis revealed a substantial abundance of saccharides, with starch and pectin constituting the majority at levels ranging from 30–44%, complemented by the presence of simple sugars within the range of 3–10%. Furthermore, protein, peptone, glutamic acid, and flavonoids were detected (Gupta et al. 2016). Of particular interest, peptic and glutamic acids were identified as potential chemotactic inducers for Thraustochytrids zoospores, implying that the release of these compounds could guide zoospore movement (Gupta et al. 2016). A similar hypothesis was put forth by Hayakawa et al. (1991), suggesting that the pine pollen's chemical composition might be responsible for instigating zoospore motility. Conclusion In summary, thraustochytrids from fishing village in Tangkolak, Indonesia, hold significant promises as a source of valuable compounds, such as odd chain fatty acids, with potential applications in various industries. Their ability to utilize waste materials and alternative carbon sources underscores their potential for sustainable and cost-effective lipid production. Further research and development in this field can unlock numerous opportunities for these microorganisms in biotechnology and beyond. Declarations Funding: Not applicable Competing interests: The authors declare that they have no competing interests. Availability of data and material: All data generated or analyzed during this study are included in this published article [and its supplementary information files]. Code availability : The custom code used in the current study is available from the corresponding author upon reasonable request. Authors' contributions : AE performed the experiments, analysed the data and wrote the paper, MIP analyzed the data and wrote the paper, MIS performed the HPLC analysis, CP conceived and designed the study, AS conceived and designed the study. References Abdel-Wahab MA, El-Samawaty AERMA, Elgorban AM, Bahkali AH (2021) Fatty acid production of thraustochytrids from Saudi Arabian mangroves. 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Nutrients. https://doi.org/10.3390/nu12061663 Venn-Watson S, Lumpkin R, Dennis EA (2020) Efficacy of dietary odd-chain saturated fatty acid pentadecanoic acid parallels broad associated health benefits in humans: could it be essential? Sci Rep. https://doi.org/10.1038/s41598-020-64960-y Ye H, He Y, Xie Y, Sen B, Wang G (2020) Fed-batch fermentation of mixed carbon source significantly enhances the production of docosahexaenoic acid in Thraustochytriidae sp. PKUMn16 by differentially regulating fatty acids biosynthetic pathways. Bioresour Technol. https://doi.org/https://doi.org/10.1016/j.biortech.2019.122402 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. 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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-3834275","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265702786,"identity":"4a716017-931a-457f-95e6-38a239be703e","order_by":0,"name":"Axel Emdi","email":"","orcid":"","institution":"PT. Wilmar Benih Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Axel","middleName":"","lastName":"Emdi","suffix":""},{"id":265702787,"identity":"9425c41f-aee9-41e9-a138-713bc83a8e28","order_by":1,"name":"Maria Indah Purnamasari","email":"","orcid":"","institution":"PT. 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Isolates of thraustochytrids shown in (a), and mycelia of contaminants depicted in (b).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3834275/v1/3e36adc677c71e81c754b323.png"},{"id":49328393,"identity":"bbe10985-27ed-49a5-95ef-f3c64f07154b","added_by":"auto","created_at":"2024-01-08 18:04:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79326,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogenetic tree of thraustochytrid isolates from the fishing village in Tangkolak, Indonesia, compared with other related taxa. Branch distances represent nucleotide substitution rates. The bolded isolates indicate those isolated by this study.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3834275/v1/e50d776d8cc175483686a0ba.png"},{"id":49328391,"identity":"a85979fa-1761-486f-ba09-cf08c359d91d","added_by":"auto","created_at":"2024-01-08 18:04:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131426,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth and lipid production of thraustochytrid isolates grown in media with different carbon sources. (A) Wet cell weight of thraustochytrids isolates grown in glucose-based media. (B) Crude lipid production of thraustochytrids grown in glucose-based media. (C) Wet cell weight of thraustochytrids isolates grown in crude glycerol-based media. (D) Comparison between glucose, crude glycerol, and technical glycerol-based media for the growth of thraustochytrid isolate BML-38 in 500 mL.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3834275/v1/d7cace08379a92e6a23ff59f.png"},{"id":49328395,"identity":"a5cef3d9-4474-4a95-987d-6509c2086e24","added_by":"auto","created_at":"2024-01-08 18:04:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":247079,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of fatty acids produced by isolate BML-38 and commercial isolate ATCC 20889. Fatty acids identities for chromatographic peaks: 1. Lauric Acid (C12); 2. Pentadecanoic Acid (C15:1); 3. Palmitoleic Acid (C16:2); 4. Heptadecanoic Acid (C17:1); 5. Docosahexaenoic Acid (DHA)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3834275/v1/90f34e126f73df1265fdb2d0.png"},{"id":66251406,"identity":"d6ffdce8-df6e-4a69-9214-a8590ffc88c3","added_by":"auto","created_at":"2024-10-09 08:54:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1422690,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3834275/v1/8a73aabd-55d3-44a2-baa7-30f247cbc797.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and lipid production of thraustochytrids from fishing village in Tangkolak Indonesia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroalgae have long been acknowledged for their capacity to synthesize valuable compounds, including docosahexaenoic acids (DHA) and odd-chain fatty acids, which are essential unsaturated fatty acids vital for human health. While humans necessitate DHA, our bodies do not naturally manufacture it, compelling us to obtain it from external sources, typically deep-sea fishes. Meanwhile, odd chain fatty acids has been found to be able to alleviate comorbidities, reduced chances of cancer and dementia (To et al. 2020; Venn-Watson et al. 2020; da Rocha et al. 2023). Currently, the growing emphasis on healthier lifestyles has spurred an increased demand for these beneficial fatty acids. These challenges underscore the imperative to explore alternative sources of DHA and odd chain fatty acids, such as thraustochytrids. The cultivation of thraustochytrids provides a more sustainable and accessible means of procuring DHA and odd chain fatty acids for human consumption compared to the extraction of rare deep-sea fish or sharks (Hadley et al. 2017).\u003c/p\u003e \u003cp\u003eThraustochytrids are marine single-celled eukaryotic protists that hold substantial promise as reservoirs of valuable substances with applications in medicine, pharmacy, food alternatives, and bioenergy (Hadley et al. 2017; Abdel-Wahab et al. 2021). Previous investigations have identified these microorganisms in coastal areas adjacent to Mangrove trees. For example, Gupta et al. (2013) effectively isolated these microorganisms in Australia, while similar research in the Arabian Sea and Thailand has yielded analogous outcomes. In these studies, researchers isolated thraustochytrids from clear coastal regions rich in mangrove trees, utilizing pollen baiting techniques. By dispersing pine pollen on the water surface of collected samples, they observed the attraction of thraustochytrids to the pollen. Subsequently, the gathered pollen was placed on growth media containing glucose, yeast extract, and peptone.\u003c/p\u003e \u003cp\u003eThraustochytrids possess the remarkable ability to generate oil or lipids, constituting up to 70\u0026ndash;80\\% w/w of their biomass. These lipids encompass beneficial fatty acids, including DHA and odd chain fatty acids, primarily located intracellularly. Therefore, the emphasis on harnessing thraustochytrids as fatty acids-producing microorganisms should prioritize augmenting biomass production and refining lipid extraction methods. Given that carbon is the primary precursor for lipids, enhancing carbon sources for thraustochytrids and exploring alternative materials to reduce production expenses becomes pivotal. While glucose has traditionally been the predominant carbon source for fermenting thraustochytrids, its cost presents a barrier to large-scale production. Previous studies have exhibited promising outcomes by employing glycerol as a carbon source for DHA and odd chain fatty acids production (Kujawska et al. 2021). Additionally, Indonesia, a major producer of Palm Oil, generates a substantial quantity of glycerol waste from biodiesel production, providing an abundant and cost-effective resource.\u003c/p\u003e \u003cp\u003eIn this research, we highlight the potential nutritional value inherent in the coastal areas of fishing villages, which thraustochytrids could leverage as a resource for lipid production. Exploring these approaches offers opportunities to discover novel organisms with the capacity to yield unique lipid contents. The objective of this study is to identify native thraustochytrids and assess their growth and lipid production using crude glycerol from biodiesel waste as an alternative growth medium. In total, eight strains were collected through pollen baiting methods, and among them, BML-38 demonstrated exceptional biomass and lipid production in glycerol waste-based medium. This ability to efficiently utilize waste materials holds the promise of transforming them into valuable resources, contributing to the sustainable management of waste.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThraustochytrids isolation\u003c/h2\u003e \u003cp\u003eThe isolation of thraustochytrids was carried out following the method outlined by Gupta et al. (2013) with some modifications. Seawater samples were collected from fishing village in Tangkolak, the northern coastal area of Java, Indonesia. The samples are then stored at a temperature of 10\u0026deg;C. To prepare the samples, 50 mL of seawater was transferred to a Falcon tube, and a combination of streptomycin (500 mg/L), rifampicin (50 mg/L), and chloramphenicol (50 mg/L) was added. After 3 days, 100 mL of the samples was plated onto GYP (Glucose, Yeast Extract, Peptone) medium, with the composition per litre as follows: Glucose (2 g), Peptone (1 g), Yeast extract (1 g), Sterile seawater (1 L or 50% seawater/half-strength), streptomycin (500 mg/L), rifampicin (50 mg/L), and chloramphenicol (50 mg/L). Culture extract of \u003cem\u003eParaburkholderia sp.\u003c/em\u003e CP01 containing antifungal compound were also added to the agar media to selectively inhibit yeast growth (Prihatna et al. 2022). The samples were then observed under a microscope for possible thraustochytrids identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolate identification and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe total genomic DNA of thraustochytrids strains were isolated with a DNAeasy Mini Kit (Qiagen, Netherlands). The 18sRNA analysis were performed using primer LabyF (5\u0026rsquo;-CCATCAGTTGTCGACCGTA-3\u0026rsquo;) and LabyR (5\u0026rsquo;-GTTAAGACTACGATGGTATCTAA-3\u0026rsquo;) (Hong et al. 2011). The amplification system, with a total volume of 25 \u0026micro;L, consisted of 12.5 \u0026micro;L GoTaq PCR Master-Mix (Promega), 1 \u0026micro;L forward primer (10 \u0026micro;M), 1 \u0026micro;L reverse primer (10 \u0026micro;M), 1 \u0026micro;L DNA template, and 9.5 \u0026micro;L sterile water. The PCR amplification conditions were as follows: 95\u0026deg;C for 3 min; 30 cycles of 95\u0026deg;C for 30 sec, 55\u0026deg;C for 30 sec, and 72\u0026deg;C for 1 min; and a final extension at 72\u0026deg;C for 7 min. The amplification products were sequenced using the ABI Prism 3130 Genetic Analyzer (Applied Biosystems). The obtained sequences were analyzed using Geneious software version 11.1.5. The phylogenetic tree of the auxin-degrading loci regulator genes was generated using MAFFT v7.450 and Fasttree 2.1.11 implemented in the Geneious software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThraustochytrids biomass production in glucose and glycerol based medium\u003c/h2\u003e \u003cp\u003eThraustochytrids were initially cultivated in GYP agar media for 2\u0026ndash;3 days before being transferred to 10 mL of a growth medium containing 3% glucose and 0.1% yeast extract for an additional 3 days. This was followed by transferring the culture to 100 mL of the same medium with the same concentrations. To enhance production and explore alternative nutritional sources for thraustochytrids, these microalgae were grown in glucose based medium (9% glucose\u0026thinsp;+\u0026thinsp;2.25% yeast extract) and glycerol based medium (9% crude glycerol obtained from oil palm biodiesel production and 2.25% nitrogen sources). After incubation for 4 days, the samples were centrifuged, and the supernatant was discarded. The resulting pellet was weighed to obtain the wet cell mass. Subsequently, the samples were freeze-dried for 2 days before being weighed again for dry weight. The results of biomass and lipid production are analysed with One-way ANOVA using R statistical program (4.2.1 version. R Core Team, 2021), and the significance between each isolate are tested with HSD significance test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLipid extraction and analysis\u003c/h2\u003e \u003cp\u003eFor lipid extraction and analysis, the samples underwent osmotic shock treatment, disrupting the cells by exploiting the osmotic pressure differences between the interior and exterior of the cells. Subsequently, the samples were shaken in a 10% NaCl solution at 200 rpm for 24 hours. This was followed by sonication at 20 kHz with 40% amplitude, using a 40-second on and 20-second off cycle, for a total working time of 20 minutes. The lipid was extracted following the monophasic lipid extraction protocol, known as the Methanol-Chloroform method (Fu et al. 2017). This method utilized Methanol-chloroform interaction to separate the lipid. The lipid extraction process began with approximately 150 \u0026micro;g of powdered thraustochytrids, each placed in a 1.5 mL Eppendorf tube. To ensure accuracy, a blank sample tube was prepared for every five samples. Subsequently, 50 \u0026micro;L of glass beads were added to both the sample and blank tubes, followed by the introduction of 200 \u0026micro;L of ice-cold 60% methanol containing 0.01% Butylated Hydroxytoulene (BHT) and 50 \u0026micro;L of a lipid standard mixture with the same BHT content. After lysis through a tissuelyser at 30 s-1 for 15 minutes, 120 \u0026micro;L of H2O and 470 \u0026micro;L of methanol containing 0.01% BHT were added. If further lysis was needed, an additional 10 minutes of lysis was conducted. Then, 270 \u0026micro;L of chloroform with 0.01% BHT was introduced, followed by vigorous vortexing and shaking using the tissuelyser for 30 minutes at 16.7 s-1. After centrifugation for phase separation, the supernatant was transferred into 2 mL Eppendorf tubes. Subsequently, 100 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO and 400 \u0026micro;L of chloroform:methanol (1:2, v:v) with 0.01% BHT were added and vortexed. A second round of shaking, centrifugation, and supernatant collection followed. The combined supernatant was vacuum-evaporated until dry, and the lipids were resuspended in 500 \u0026micro;L of isopropanol:methanol:chloroform (4:2:1, v:v:v) with 0.01% BHT. After centrifugation, the supernatant (~\u0026thinsp;400 \u0026micro;L) was transferred to glass vials, saturated with N2 gas, and stored at -80\u0026deg;C for subsequent mass spectrometry analysis. The GC analysis used 37 FAME Mix Supelco as standard (Sigma Aldrich, Germany) and following GC analysis settings from previous study (Kumar Aiswarya et al. 2014).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and identification of thraustochytrids\u003c/h2\u003e \u003cp\u003eThe isolation of thraustochytrids from our intricate environmental matrix posed formidable challenges, primarily due to their necessity to outcompete contaminants, which often displayed robust growth rates. After a ten-day incubation period, we collected several samples, subjecting them to careful examination under a light microscope. Thraustochytrids are characterized by a distinct nucleus, clearly discernible at 40x magnification, rendering them distinguishable from empty bacterial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In total, we succeeded in isolating eight samples using a modified approach based on the method described by Gupta et al. (2013).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo precisely determine the taxonomic identity of the eight isolates and position them within the broader phylogenetic context among other eukaryotic organisms, a thorough identification process was conducted. All samples exhibited a close association with Auranthiochytrium acetophilum, displaying substantial genetic similarities exceeding 97% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Utilizing the NCBI BLAST analysis, the isolates were categorized into three groups. Group 1, demonstrating the highest similarity to A. acetophilum, comprises BML-19, BML-40, BML-42, and BML-43. Strains BML-37 and BML-38 are closely related and grouped together, as are strains BML-44 and BML-45, indicating a noteworthy degree of similarity between these specific isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGrowth and lipid production of thraustochytrids isolates\u003c/h2\u003e \u003cp\u003eThe strains isolated from Tangkolak have exhibited superior biomass and lipid production in comparison to the well-known ATCC 20889 isolate, a \u003cem\u003eSchizochytrium\u003c/em\u003e strain approved by the European Commission for use as an additive in infant food (EFSA Panel on Nutrition et al. 2022). Among the Tangkolak isolates, BML-38 displayed the highest biomass production, reached 45 grams per liter (g/L), while ATCC 20889 exhibited significantly lower growth, 10 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, Tangkolak strains have also demonstrated higher lipid production compared to ATCC 20889, with BML-38 surpassing 6 g/L, while ATCC 20889 recorded just under 1 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The higher lipid production observed in BML-38 suggests the potential for increased DHA and Odd-chain fatty acids production, which could be of significant importance in various applications, including in infant nutrition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eUtilizing crude glycerol as an alternative carbon source for thraustochytrids\u003c/h2\u003e \u003cp\u003eAll eight isolates obtained from Tangkolak were tested in a 50 mL medium containing crude glycerol. In general, all isolates grown in crude glycerol exhibited higher biomass compared to when grown in glucose-based medium. In crude glycerol-based medium, BML-38 and BML-45 displayed the highest growth rate among the isolates, reaching\u0026thinsp;~\u0026thinsp;60 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). For direct comparison between different media in larger scale (500 mL), we used only isolates BML-38. The biomass production in glycerol-based media reached 48 g/L, while glucose-based media yielded 36 g/L and technical glycerol yielded 22 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Consistent findings across various scales suggest that crude glycerol, a byproduct of biodiesel production from oil palm waste, holds significant promise as an economical alternative substance for producing fatty acids by thraustochytrids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFatty acids components of thraustochytrids isolates\u003c/h2\u003e \u003cp\u003eDespite their biomass production, it is crucial to conduct lipid profile analysis on these strains. BML-38 exhibits a higher pentadecanoic acid (C15:0) content compared to commercial isolate ATCC 20889, but lower in DHA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, BML-38 and ATCC 20889 share similar C17:0 levels. This finding showed that BML-38 emerges as particularly promising due to its capability to produce both odd-chain fatty acids and DHA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study has successfully isolated eight strains of thraustochytrids capable of producing biomass and lipid using a crude glycerol-based medium. This capability may be attributed to their origin in a coastal area near a fishing village, where human activities generate more waste compared to open seas or oceans, common sources of thraustochytrid isolates like ATCC 20889 and ATCC 1381 (Okino et al. 2018). Furthermore, despite originating from the same location, we identified three groups of thraustochytrid isolates based on their 18S rRNA diversity, indicating significant microalgae diversity. These findings underscore the potential of coastal areas near fishing villages as valuable hotspots for discovering novel thraustochytrids for sustainable biomass and lipid production, offering an innovative approach to utilizing local environments for biotechnological applications. Future studies involving multiple locations throughout coastal area in Indonesia could unveil a more comprehensive understanding of the country's thraustochytrid biodiversity.\u003c/p\u003e \u003cp\u003eOne isolate, named BML-38, has been shown to produce more biomass and odd-chain fatty acids than the commercial isolates ATCC 20889. While our bodies require odd-chain fatty acids, we do not naturally synthesize them, necessitating external sources such as thraustochytrids. Moreover, odd-chain fatty acids have been demonstrated to exhibit anti-cancer effects, particularly in breast cancer (To et al. 2020). Lower concentrations of C17:0, which are also present in the fatty acid profile of BML-38, may be associated with the progression from GAD65Ab positivity to adult-onset diabetes (Lampousi et al. 2023). The remarkable capacity of the isolate BML-38 to produce substantial biomass and a higher proportion of beneficial odd-chain fatty acids make it a promising candidate for biotechnology application.\u003c/p\u003e \u003cp\u003eThe application of crude glycerol as an alternative carbon source holds considerable promise, particularly for companies operating in the biotechnology and agriculture sectors. The use of glucose as the primary carbon source often results in significantly increased production costs, especially when technical-grade glucose, a common choice in thraustochytrid studies, is employed. Previously, Ye et al. (2020) highlighted the potential of thraustochytrids to grow in a mixture of glucose and glycerol to reduce production costs. Furthermore, thraustochytrids have been demonstrated to utilize waste cooking oil as a carbon source for growth and to produce DHA and odd-chain fatty acids from waste cooking oil (Patel et al. 2022). Similarly, our thraustochytrid isolates demonstrated the ability to thrive on crude glycerol as a carbon source. Crude glycerol contains a variety of compounds, including soap, methanol, fatty acids, triglycerides, and metals, all of which appear to play pivotal roles in enhancing microbial growth (Kumar et al. 2019). The underperformance of BML-38 in technical glycerol underscores the presence of other beneficial compounds in crude glycerol that bolster biomass production. These findings lend further support to the notion that thraustochytrids from Tangkolak hold potential for the bioremediation of biodiesel waste. Moreover, if these strains could effectively utilize palm oil waste as a carbon source, it could offer a sustainable solution to address palm oil meal effluent challenges.\u003c/p\u003e \u003cp\u003eIt is noteworthy that pine pollen proved to be an effective bait for attracting thraustochytrids amid the diverse array of cohabiting organisms. Pine pollen analysis revealed a substantial abundance of saccharides, with starch and pectin constituting the majority at levels ranging from 30\u0026ndash;44%, complemented by the presence of simple sugars within the range of 3\u0026ndash;10%. Furthermore, protein, peptone, glutamic acid, and flavonoids were detected (Gupta et al. 2016). Of particular interest, peptic and glutamic acids were identified as potential chemotactic inducers for Thraustochytrids zoospores, implying that the release of these compounds could guide zoospore movement (Gupta et al. 2016). A similar hypothesis was put forth by Hayakawa et al. (1991), suggesting that the pine pollen's chemical composition might be responsible for instigating zoospore motility.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, thraustochytrids from fishing village in Tangkolak, Indonesia, hold significant promises as a source of valuable compounds, such as odd chain fatty acids, with potential applications in various industries. Their ability to utilize waste materials and alternative carbon sources underscores their potential for sustainable and cost-effective lipid production. Further research and development in this field can unlock numerous opportunities for these microorganisms in biotechnology and beyond.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e All data generated or analyzed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e: The custom code used in the current study is available from the corresponding author upon reasonable request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e: AE performed the experiments, analysed the data and wrote the paper, MIP analyzed the data and wrote the paper, MIS performed the HPLC analysis, CP conceived and designed the study, AS conceived and designed the study.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Wahab MA, El-Samawaty AERMA, Elgorban AM, Bahkali AH (2021) Fatty acid production of thraustochytrids from Saudi Arabian mangroves. Saudi J Biol Sci 28:855-864. \u003c/li\u003e\n\u003cli\u003eda Rocha LS, Mendes CB, Silva JS, Alcides RLGF, Mendon\u0026ccedil;a IP, Andrade da Costa BLS, Machado SS, Ximenes da Silva A (2023) Triheptanoin, an odd-medium-chain triglyceride, impacts brain cognitive function in young and aged mice. Nutr Neurosci. https://doi.org/10.1080/1028415X.2023.2178096\u003c/li\u003e\n\u003cli\u003eEFSA Panel on Nutrition , Turck D, Bohn T, Castenmiller J, De Henauw S, Hirsch-Ernst KI, Maciuk A, Mangelsdorf I, McArdle HJ, Naska A, Pelaez C, Pentieva K, Siani A, Thies F, Tsabouri S, Vinceti M, Cubadda F, Frenzel T, Heinonen M, Knutsen HK (2022) Safety of oil from \u003cem\u003eSchizochytrium sp.\u003c/em\u003e (strain ATCC 20889) for use in infant and follow-on formula as a novel food pursuant to Regulation (EU) 2015/2283. EFSA J. https://doi.org/https://doi.org/10.2903/j.efsa.2022.7083\u003c/li\u003e\n\u003cli\u003eFu C, Chai YR, Ma LJ, Wang R, Hu K, Wu JY, Li JN, Liu X, Lu JX (2017) Evening primrose (\u003cem\u003eOenothera biennis\u003c/em\u003e) omega 6 fatty acid desaturase gene family: cloning, characterization, and engineered GLA and SDA production in a staple oil crop. Mol Breed. https://doi.org/10.1007/s11032-017-0682-0\u003c/li\u003e\n\u003cli\u003eGupta A, Singh D, Byreddy AR, Thyagarajan T, Sonkar SP, Mathur AS, Tuli DK, Barrow CJ, Puri M (2016) Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity. Biotechnol J 11:345\u0026ndash;355.\u003c/li\u003e\n\u003cli\u003eGupta A, Wilkens S, Adcock JL, Puri M, Barrow CJ (2013) Pollen baiting facilitates the isolation of marine thraustochytrids with potential in omega-3 and biodiesel production. J Ind Microbiol Biotechnol 40:1231-1240.\u003c/li\u003e\n\u003cli\u003eHadley KB, Bauer J, Milgram NW (2017) The oil-rich alga \u003cem\u003eSchizochytrium \u003c/em\u003esp. as a dietary source of docosahexaenoic acid improves shape discrimination learning associated with visual processing in a canine model of senescence. Prostaglandins Leukot Essent Fatty Acids 118:10\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eHayakawa M, Tamura T, Iino H, Nonomura H (1991) Pollen-baiting and drying method for the highly selective isolation of \u003cem\u003eActinoplanes\u003c/em\u003e spp. from soil. J Ferment Bioeng 72:433\u0026ndash;438. \u003c/li\u003e\n\u003cli\u003eHong DD, Anh HTL, Thu NTH (2011) Study on biological characteristics of heterotrophic marine microalga-\u003cem\u003eSchizochytrium mangrovei\u003c/em\u003e pq6 isolated from Phu Quoc Island, Kien Giang province, Vietnam. J Phycol 47:944\u0026ndash;954. \u003c/li\u003e\n\u003cli\u003eKujawska N, Talbierz S, Dębowski M, Kazimierowicz J, Zieliński M (2021) Cultivation method effect on \u003cem\u003eSchizochytrium\u003c/em\u003e sp. Biomass growth and docosahexaenoic acid (dha) production with the use of waste glycerol as a source of organic carbon. Energies. https://doi.org/10.3390/en14102952\u003c/li\u003e\n\u003cli\u003eKumar A, Lindley MR, Mastana S (2014) A time efficient adaptation of GC FID method for the analysis of pbmc lipid composition. J Biochem Tech 5:760\u0026ndash;764.\u003c/li\u003e\n\u003cli\u003eKumar LR, Yellapu SK, Tyagi RD, Zhang X (2019) A review on variation in crude glycerol composition, bio-valorization of crude and purified glycerol as carbon source for lipid production. 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Bioresour Technol. https://doi.org/https://doi.org/10.1016/j.biortech.2019.122402\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":"thraustochytrids, microalgae, DHA, Odd-chain Fatty Acids","lastPublishedDoi":"10.21203/rs.3.rs-3834275/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3834275/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis research explores thraustochytrids, microorganisms with promising applications in sustainable docosahexaenoic acid (DHA) and odd-chain fatty acid production. The study specifically focuses on thraustochytrids isolated from a fishing village in the northern coastal area of Java, Indonesia, known for its significant organic content. Eight isolates were obtained from this coastal environment, demonstrating robust growth and lipid production capabilities. Notably, isolate BML-38 exhibited superior biomass and lipid production compared to commercial thraustochytrid ATCC strains, particularly in crude glycerol-based media. This positions it as a strong candidate for sustainable and cost-effective lipid production. BML-38 also produced a higher concentration of pentadecanoic acid (C15:0) and a similar concentration of heptadecanoic acid (C17:0), in addition to DHA. The outcomes of this investigation open new avenues, as thraustochytrids from the coastal area exhibit the capacity to utilize waste materials while competitively producing valuable compounds such as odd-chain fatty acids and DHA. This dual capability positions these strains as noteworthy contributors to sustainable lipid production and waste remediation strategies.\u003c/p\u003e","manuscriptTitle":"Isolation and lipid production of thraustochytrids from fishing village in Tangkolak Indonesia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 18:04:12","doi":"10.21203/rs.3.rs-3834275/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":"3685ca46-4db2-47c8-aa9f-b97494eea5f6","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-09T08:54:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 18:04:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3834275","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3834275","identity":"rs-3834275","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}