Assessing Plant Growth-Promoting Activities of Bacillus pumilus: Quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC)

Assessing Plant Growth-Promoting Activities of Bacillus pumilus: Quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC) | 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 Assessing Plant Growth-Promoting Activities of Bacillus pumilus: Quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC) Shambhu Swarnakar, Arka Pratim Chakraborty This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4729095/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background Microbial-mediated plant growth promotion has gained significant attention in agriculture due to its potential to improve crop yield and sustainability. Indole-3-acetic acid (IAA) and tryptophan are key compounds synthesized by certain bacteria, known to positively influence plant development. High-performance liquid chromatography (HPLC) is a robust analytical technique capable of accurately quantifying these compounds, enabling a deeper understanding of plant-microbe interactions. Results In this study, bacterial isolates designated as MUSRH-5 were investigated for their plant growth-promoting activities. Cultured bacterial strains were assessed for their efficacy in promoting plant growth. Through HPLC analysis, varying concentrations of IAA and tryptophan were observed among the bacterial isolates, indicating differing metabolic capabilities. Significant variations in plant growth-promoting activities were also noted, with certain strains demonstrating notable effectiveness. Conclusion The findings of this study highlight the potential of selected bacterial isolates, particularly MUSRH-5 strains, to enhance plant growth through the production of growth-promoting compounds. The utilization of HPLC for precise quantification provided valuable insights into the metabolic capabilities of these bacteria and their impact on plant development. This research underscores the importance of microbial-mediated plant growth promotion in sustainable agriculture and environmental management, emphasizing the role of bacterial isolates as valuable assets in enhancing agricultural productivity and mitigating environmental challenges. Plant growth-promoting bacteria indole-3-acetic acid tryptophan HPLC microbial interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction In modern agriculture and environmental science, harnessing the potential of PGPR has become increasingly crucial for sustainable crop production and ecosystem health. These beneficial bacteria, through various mechanisms, enhance plant growth, improve nutrient uptake, and confer resistance to environmental stresses. Among the myriad of compounds produced by these bacteria, indole-3-acetic acid (IAA) and tryptophan play pivotal roles in regulating plant growth and development (Chen et al. 2024 ). Indole-3-acetic acid (IAA), a naturally occurring auxin, is a key phytohormone involved in numerous physiological processes, including cell division, elongation, and differentiation. Tryptophan, an essential amino acid and precursor to IAA biosynthesis, serves as a substrate for microbial production of this hormone. Consequently, the ability of bacteria to synthesize and release IAA into the rhizosphere can profoundly influence plant growth and productivity (Fathy et al. 2023 ). The quantification of IAA and tryptophan, therefore, serves as a valuable tool for assessing the plant growth-promoting potential of microbial isolates. High-performance liquid chromatography (HPLC) stands out as a reliable method for accurately measuring the concentrations of these compounds. By employing HPLC, researchers can elucidate the metabolic capabilities of bacterial isolates and evaluate their effectiveness in enhancing plant growth (Huang et al. 2023 ). This study aims to investigate the plant growth-promoting activities of isolated bacterial strains and quantify the levels of IAA and tryptophan using HPLC. By characterizing the interactions between bacteria and plants at the molecular level, we can gain insights into the mechanisms underlying plant-microbe associations and their implications for sustainable agriculture and environmental management. This research contributes to the ongoing efforts to harness the potential of beneficial microbes in promoting plant growth and mitigating environmental challenges (Paes MDCRP 2023 ). Materials and Methods Bacterial Isolation and Culture Sample Collection Soil samples were taken from some distinct mustard-growing regions in Uttar Dinajpur in order to identify rhizospheric PGPR. Sterile polythene bags were used to gather soil samples from the field and soil samples were placed in the lab, labeled, and stored at 4ºC in a refrigerator until use (Clare et al. 2022 ). Isolation Procedure Serial dilution method was employed to obtain dilutions of the soil samples. These dilutions were then plated on Nutrient agar media such as Nutrient Agar supplemented. Incubation Plates were incubated at the appropriate temperature (usually 28–30°C) for 24–48 hours to allow bacterial growth. Pure Culture Selection Well-isolated colonies with distinct morphologies were selected and streaked onto fresh agar plates to obtain pure cultures (Rychshanova et al. 2022 ). Screening for Indole-3-Acetic Acid (IAA) Production Inoculum Preparation Pure bacterial cultures were inoculated into tryptophan-supplemented broth and incubated under optimal conditions for IAA production. Extraction of IAA After incubation, bacterial cultures were centrifuged, and the supernatant was collected for IAA extraction using ethyl acetate solvent. Sample Preparation Extracted samples were filtered to remove any particulate matter and stored at appropriate temperatures until HPLC analysis (Rychshanova et al. 2022 ). HPLC Analysis for IAA Quantification Instrument Setup High-Performance Liquid Chromatography (HPLC) system equipped with a suitable column (e.g., C18 column) and UV-Vis detector was used. Standard Preparation Standard solutions of pure IAA at known concentrations were prepared to generate a calibration curve. Chromatographic Conditions Optimal chromatographic conditions including mobile phase composition, flow rate, and detection wavelength were established. Sample Injection and Analysis Extracted samples along with standard solutions were injected into the HPLC system, and IAA concentrations were determined by comparing peak areas with the calibration curve (Zhang et al. 2024 ). Tryptophan Quantification Tryptophan Extraction Tryptophan was extracted from bacterial cultures using appropriate solvents such as acidified ethanol. Sample Preparation Extracted tryptophan samples were filtered and diluted as necessary for HPLC analysis. HPLC Analysis Similar to IAA analysis, HPLC was utilized to quantify tryptophan concentrations using appropriate chromatographic conditions and calibration curves generated from standard solutions (Girgin et al. 2024 ). Statistical Analysis Data Processing Quantitative data obtained from HPLC analysis were compiled and subjected to statistical analysis using Agilent software. Descriptive Statistics Mean values, standard deviations, and other relevant statistical parameters were calculated for IAA and tryptophan concentrations. Inferential Statistics Statistical tests such as Student's t-test were performed to assess significant differences among bacterial isolates (Gharsallah et al. 2024 ). Quality Control Measures Instrument Calibration The HPLC system was calibrated regularly using standard solutions to ensure the accuracy and precision of measurements. Control Samples Positive and negative control samples were included in each batch of analysis to monitor assay performance and detect any systematic errors. Reproducibility Experiments were repeated independently to confirm the reproducibility of results (Mohammadi et al. 2024 ). Ethical Considerations Compliance The study adhered to ethical guidelines for research involving microbial cultures and laboratory safety protocols. Approval Any necessary approvals from institutional or regulatory bodies were obtained before conducting the research. Results and Discussion Isolation and Identification of Bacterial Isolates The isolation and identification of bacterial isolates represent fundamental steps in elucidating their potential roles in promoting plant growth. Soil samples collected from Uttar Dinajpur diverse agricultural sites were subjected to a rigorous isolation process to obtain pure cultures of bacterial strains (Fig. 1& Fig. 2). Serial dilutions of soil samples were plated on Nutrient Agar supplemented with 50 µg/mL kanamycin. After 48 hours of incubation at 30°C, distinct bacterial colonies were observed on the plates(Fig. 3). Colonies exhibited diverse morphologies, including creamy white, yellow, and pink colonies, varying in size and texture (Mustafa et al. 2024 ). Preliminary identification of bacterial isolates was conducted based on morphological characteristics such as colony morphology, Preliminary examination revealed the majority of isolates to be Gram-positive rods with varying motility. Biochemical tests including catalase, IAA production, andPhosphate solubilization assays provided additional insights into the metabolic capabilities of the isolates (Table 1)(Fig. 4). Furthermore, molecular techniques such as Polymerase Chain Reaction (PCR) and sequencing of the 16S ribosomal RNA gene were employed for accurate taxonomic classification of representative isolates (Figs. 5 and 6). Sequence analysis facilitated the comparison of genetic sequences with databases to determine the phylogenetic relationships and taxonomic affiliations of the bacterial isolates Bacillus pumilus (Aubier et al. 2024 ). Overall, the isolation and identification process revealed the taxonomic diversity and functional potential of the bacterial isolates Bacillus pumilus , laying the foundation for subsequent investigations into their plant growth-promoting activities. These findings contribute to our understanding of soil microbial ecology and hold promise for the development of sustainable agricultural practices. Production of Indole-3-Acetic Acid (IAA) and Tryptophan The production of Indole-3-Acetic Acid (IAA) and Tryptophan by bacterial isolates Bacillus pumilus was assessed as key indicators of their plant growth-promoting potential and disease control. Bacterial cultures were inoculated into tryptophan-supplemented broth and incubated under controlled conditions conducive to IAA and tryptophan production. Following incubation, bacterial cultures Bacillus pumilus were subjected to extraction procedures to isolate IAA and tryptophan from the culture medium. Extraction methods typically involved the use of organic solvents such as ethyl acetate to efficiently recover the target compounds from the aqueous phase (Calatrava et al. 2024 ). Quantification of IAA and tryptophan was performed using High-Performance Liquid Chromatography (HPLC), a sensitive analytical technique capable of separating and quantifying compounds in complex mixtures. Samples were injected into the HPLC system, and peak areas corresponding to IAA and tryptophan were determined (Figs. 7 & 8). Calibration curves generated from standard solutions of known concentrations of IAA and tryptophan were used to quantify the amounts of these compounds present in Bacillus pumilus bacterial cultures. The concentrations of IAA and tryptophan produced by bacterial isolate MUSRH-05 were calculated respectively RT 3.131 min and 2.644 based on peak area i.e., 5704.8125 and 2027.7877, Height 399.2582 and 157.6192, RF 0.05317& 0.07465 and expressed in 303.333 ng/ul and 151.383 ng/ul. The assessment of IAA and tryptophan production provided insights into the metabolic capabilities of bacterial isolates MUSRH-5 and their potential to influence plant growth through phytohormone synthesis. Variations in the production levelsAnnotations indicate different levels (e.g., Level 1, Level 2, Level 3) on the calibration curves of IAA and tryptophan among isolates highlighted the functional diversity within the bacterial community and underscored the importance of characterizing individual isolates for their specific plant growth-promoting traits. HPLC Analysis High-Performance Liquid Chromatography (HPLC) Make- Agilent and Model - Infinity 1260 the principal analytical technique for quantifying Indole-3-Acetic Acid (IAA) and Tryptophan produced by bacterial isolates. HPLC is a powerful tool capable of separating, identifying, and quantifying components within complex mixtures with high sensitivity and precision. Instrumentation: The HPLC DAD system utilized in this study comprised a solvent delivery system, an injector for sample introduction Flow rate- 1ml/min a chromatographic column for separation, a detector for compound detection, and a data acquisition system for analysis (Adil et al. 2024 ). Chromatographic Column: A suitable chromatographic column, such as a reverse-phase C18 column, was selected based on its ability to efficiently separate IAA and Tryptophan from other components present in the sample matrix. Mobile Phase: A mobile phase consisting of a solvent or solvent mixture was carefully chosen to elute the analytes of interest from the chromatographic column. The composition and gradient of the mobile phase is Channel A- Acetonitrile 60%, Channel B- Water 40%, and Flow rate- 1ml/min to achieve optimal separation and resolution of IAA and Tryptophan peaks (Terzi et al. 2024 ). Detection: UV-Vis spectroscopy was employed as the detection method, with the wavelength 254nm(405, 470, 400, etc.) likely correspond to the detection wavelengths used during the analysisselected to detect the absorbance of IAA and Tryptophan. The detector recorded the absorbance of eluting compounds as they passed through the column, generating chromatograms that represented the concentration of analytes over time (Fig. 9) Calibration: Before sample analysis, calibration curves were constructed using standard solutions of Injection Volume- 10ul of IAA and Tryptophan. These calibration curves enabled the quantification of analytes in the samples by comparing their peak areas or heights with those of standards (Strieder et al. 2024 ). Sample Injection and Analysis: Extracted samples containing IAA and Tryptophan were injected Volume- 10ul into the HPLC system using an auto-sampler. The samples were eluted through the chromatographic column under optimized conditions, and the wavelengths (405, 470, 400, etc.) likely correspond to the detection wavelengths used during the analysis ( MixCALM ) (Fig. 8). Regression Equation o The calibration curves follow the equation: y = ax + b Here, y represents the instrument response (area or height). x represents the amount of the compound. a and b are coefficients determined from the calibration data. Data Processing: The chromatographic data obtained from HPLC analysis were processed using specialized software. Peak integration Agilent Technologies software was used to determine the peak areas or heights corresponding to IAA and Tryptophan peaks. The concentrations of these compounds in the samples were calculated based on the calibration curves and expressed in appropriate units (e.g., µg/mL). In summary, HPLC analysis provided a robust and reliable method for quantifying Indole-3-Acetic Acid (IAA) and Tryptophan produced by bacterial isolates, facilitating the assessment of their plant growth-promoting activities with high sensitivity and accuracy. Conclusion In conclusion, the assessment of plant growth-promoting activities of isolated Bacillus pumilus through the quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC) represents a significant stride towards understanding the intricate dynamics between soil microbiota and plant health. Through meticulous isolation and cultivation techniques, diverse bacterial strains were successfully identified, showcasing the richness of microbial diversity within agricultural soils. The findings of this study underscore the pivotal role of bacterial communities in promoting plant growth through the production of phytohormones such as IAA and Tryptophan. The variation observed in the production of these compounds among Bacillus pumilus emphasizes the functional diversity within soil microbial populations. The implications of these findings extend beyond agricultural productivity to encompass broader environmental sustainability goals. By harnessing the plant growth-promoting activities of Bacillus pumilus , we have the potential to reduce dependency on synthetic fertilizers and pesticides, mitigating environmental degradation associated with conventional agricultural practices. Looking ahead, future research endeavors should delve deeper into the mechanisms underlying plant-microbe interactions and their impact on crop health and productivity. Moreover, exploring the potential synergistic effects of microbial consortia and their application in field trials could pave the way for the development of novel biofertilizers and biostimulants. In conclusion, this study contributes to the growing body of knowledge on the beneficial roles of rhizospheric bacteria in sustainable agriculture. By unraveling the intricate relationships between microbial communities and plant physiology, we can pave the way for innovative solutions that promote agricultural resilience, environmental stewardship, and food security in a rapidly changing world. Abbreviations PGPR: Plant Growth-Promoting Rhizobacteria, IAA: Indole-3-Acetic Acid, HPLC: High-performance liquid chromatography. Declarations Acknowledgements Heredity Life sciences Pvt Ltd, Bhubaneswar, Odisha and Edison Life Science Laboratory, Kolkata, West Bengal, India are thankfully acknowledged for the molecular identification of bacterial strain. Authors also acknowledged Environcheck for HPLC analysis of IAA, tryptophan . Conflict of Interest Authors declare that there are no conflict of interest. Compliance with Ethical Standards Disclosure of potential conflicts of interest The authors declare that they have no conflicts of interest. Research involving human participants and/or animals This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent No informed consent is required. Data Availability Statement No datasets were generated or analyzed during the current study. Funding No funding support is availed. Consent of publication I, Shambhu Swarnakar performed the experiment and surveyed field for soil collection. Photo belings to me. I have no objection if image is used for publication purposes in the article. References Adil M, Filimban F Z, AmbrinQuddoos A, Sher AA, Naseer M (2024). Phytochemical screening, HPLC analysis, antimicrobial and antioxidant effect of Euphorbia parviflora L.(Euphorbiaceae Juss.). Scientific Reports , 14 (1), 5627. 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Microchemical Journal , 199 , 110025. Zhang H, Rong Z, Li Y, Yin Z, Lu C, Zhao H, Ding X (2024). NIT24 and NIT29‐mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice. Molecular Plant Pathology , 25 (1), e13409. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 31 Jul, 2024 Submission checks completed at journal 30 Jul, 2024 First submitted to journal 12 Jul, 2024 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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06:30:18","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":76802,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4729095/v1/597b2043abb2b817f4ded73b.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessing Plant Growth-Promoting Activities of Bacillus pumilus: Quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn modern agriculture and environmental science, harnessing the potential of PGPR has become increasingly crucial for sustainable crop production and ecosystem health. These beneficial bacteria, through various mechanisms, enhance plant growth, improve nutrient uptake, and confer resistance to environmental stresses. Among the myriad of compounds produced by these bacteria, indole-3-acetic acid (IAA) and tryptophan play pivotal roles in regulating plant growth and development (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIndole-3-acetic acid (IAA), a naturally occurring auxin, is a key phytohormone involved in numerous physiological processes, including cell division, elongation, and differentiation. Tryptophan, an essential amino acid and precursor to IAA biosynthesis, serves as a substrate for microbial production of this hormone. Consequently, the ability of bacteria to synthesize and release IAA into the rhizosphere can profoundly influence plant growth and productivity (Fathy et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe quantification of IAA and tryptophan, therefore, serves as a valuable tool for assessing the plant growth-promoting potential of microbial isolates. High-performance liquid chromatography (HPLC) stands out as a reliable method for accurately measuring the concentrations of these compounds. By employing HPLC, researchers can elucidate the metabolic capabilities of bacterial isolates and evaluate their effectiveness in enhancing plant growth (Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aims to investigate the plant growth-promoting activities of isolated bacterial strains and quantify the levels of IAA and tryptophan using HPLC. By characterizing the interactions between bacteria and plants at the molecular level, we can gain insights into the mechanisms underlying plant-microbe associations and their implications for sustainable agriculture and environmental management. This research contributes to the ongoing efforts to harness the potential of beneficial microbes in promoting plant growth and mitigating environmental challenges (Paes MDCRP \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Isolation and Culture\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eSample Collection\u003c/strong\u003e \u003cp\u003eSoil samples were taken from some distinct mustard-growing regions in Uttar Dinajpur in order to identify rhizospheric PGPR. Sterile polythene bags were used to gather soil samples from the field and soil samples were placed in the lab, labeled, and stored at 4\u0026ordm;C in a refrigerator until use (Clare et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIsolation Procedure\u003c/strong\u003e \u003cp\u003eSerial dilution method was employed to obtain dilutions of the soil samples. These dilutions were then plated on Nutrient agar media such as Nutrient Agar supplemented.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIncubation\u003c/strong\u003e \u003cp\u003ePlates were incubated at the appropriate temperature (usually 28\u0026ndash;30\u0026deg;C) for 24\u0026ndash;48 hours to allow bacterial growth.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePure Culture Selection\u003c/strong\u003e \u003cp\u003eWell-isolated colonies with distinct morphologies were selected and streaked onto fresh agar plates to obtain pure cultures (Rychshanova et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eScreening for Indole-3-Acetic Acid (IAA) Production\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eInoculum Preparation\u003c/strong\u003e \u003cp\u003ePure bacterial cultures were inoculated into tryptophan-supplemented broth and incubated under optimal conditions for IAA production.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExtraction of IAA\u003c/strong\u003e \u003cp\u003eAfter incubation, bacterial cultures were centrifuged, and the supernatant was collected for IAA extraction using ethyl acetate solvent.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSample Preparation\u003c/strong\u003e \u003cp\u003eExtracted samples were filtered to remove any particulate matter and stored at appropriate temperatures until HPLC analysis (Rychshanova et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHPLC Analysis for IAA Quantification\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eInstrument Setup\u003c/strong\u003e \u003cp\u003eHigh-Performance Liquid Chromatography (HPLC) system equipped with a suitable column (e.g., C18 column) and UV-Vis detector was used.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStandard Preparation\u003c/strong\u003e \u003cp\u003eStandard solutions of pure IAA at known concentrations were prepared to generate a calibration curve.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eChromatographic Conditions\u003c/strong\u003e \u003cp\u003eOptimal chromatographic conditions including mobile phase composition, flow rate, and detection wavelength were established.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSample Injection and Analysis\u003c/strong\u003e \u003cp\u003eExtracted samples along with standard solutions were injected into the HPLC system, and IAA concentrations were determined by comparing peak areas with the calibration curve (Zhang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTryptophan Quantification\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eTryptophan Extraction\u003c/strong\u003e \u003cp\u003eTryptophan was extracted from bacterial cultures using appropriate solvents such as acidified ethanol.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSample Preparation\u003c/strong\u003e \u003cp\u003eExtracted tryptophan samples were filtered and diluted as necessary for HPLC analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHPLC Analysis\u003c/strong\u003e \u003cp\u003eSimilar to IAA analysis, HPLC was utilized to quantify tryptophan concentrations using appropriate chromatographic conditions and calibration curves generated from standard solutions (Girgin et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eData Processing\u003c/strong\u003e \u003cp\u003eQuantitative data obtained from HPLC analysis were compiled and subjected to statistical analysis using Agilent software.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDescriptive Statistics\u003c/strong\u003e \u003cp\u003eMean values, standard deviations, and other relevant statistical parameters were calculated for IAA and tryptophan concentrations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInferential Statistics\u003c/strong\u003e \u003cp\u003eStatistical tests such as Student's t-test were performed to assess significant differences among bacterial isolates (Gharsallah et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuality Control Measures\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eInstrument Calibration\u003c/strong\u003e \u003cp\u003eThe HPLC system was calibrated regularly using standard solutions to ensure the accuracy and precision of measurements.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eControl Samples\u003c/strong\u003e \u003cp\u003ePositive and negative control samples were included in each batch of analysis to monitor assay performance and detect any systematic errors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eReproducibility\u003c/strong\u003e \u003cp\u003eExperiments were repeated independently to confirm the reproducibility of results (Mohammadi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthical Considerations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCompliance\u003c/strong\u003e \u003cp\u003eThe study adhered to ethical guidelines for research involving microbial cultures and laboratory safety protocols.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eApproval\u003c/strong\u003e \u003cp\u003eAny necessary approvals from institutional or regulatory bodies were obtained before conducting the research.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and Identification of Bacterial Isolates\u003c/h2\u003e \u003cp\u003eThe isolation and identification of bacterial isolates represent fundamental steps in elucidating their potential roles in promoting plant growth. Soil samples collected from Uttar Dinajpur diverse agricultural sites were subjected to a rigorous isolation process to obtain pure cultures of bacterial strains (Fig.\u0026nbsp;1\u0026amp; Fig.\u0026nbsp;2). Serial dilutions of soil samples were plated on Nutrient Agar supplemented with 50 \u0026micro;g/mL kanamycin. After 48 hours of incubation at 30\u0026deg;C, distinct bacterial colonies were observed on the plates(Fig.\u0026nbsp;3). Colonies exhibited diverse morphologies, including creamy white, yellow, and pink colonies, varying in size and texture (Mustafa et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePreliminary identification of bacterial isolates was conducted based on morphological characteristics such as colony morphology, Preliminary examination revealed the majority of isolates to be Gram-positive rods with varying motility. Biochemical tests including catalase, IAA production, andPhosphate solubilization assays provided additional insights into the metabolic capabilities of the isolates (Table\u0026nbsp;1)(Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eFurthermore, molecular techniques such as Polymerase Chain Reaction (PCR) and sequencing of the 16S ribosomal RNA gene were employed for accurate taxonomic classification of representative isolates (Figs.\u0026nbsp;5 and 6). Sequence analysis facilitated the comparison of genetic sequences with databases to determine the phylogenetic relationships and taxonomic affiliations of the bacterial isolates \u003cem\u003eBacillus pumilus\u003c/em\u003e (Aubier et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, the isolation and identification process revealed the taxonomic diversity and functional potential of the bacterial isolates\u003cem\u003eBacillus pumilus\u003c/em\u003e, laying the foundation for subsequent investigations into their plant growth-promoting activities. These findings contribute to our understanding of soil microbial ecology and hold promise for the development of sustainable agricultural practices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProduction of Indole-3-Acetic Acid (IAA) and Tryptophan\u003c/h2\u003e \u003cp\u003eThe production of Indole-3-Acetic Acid (IAA) and Tryptophan by bacterial isolates \u003cem\u003eBacillus pumilus\u003c/em\u003e was assessed as key indicators of their plant growth-promoting potential and disease control. Bacterial cultures were inoculated into tryptophan-supplemented broth and incubated under controlled conditions conducive to IAA and tryptophan production.\u003c/p\u003e \u003cp\u003eFollowing incubation, bacterial cultures \u003cem\u003eBacillus pumilus\u003c/em\u003e were subjected to extraction procedures to isolate IAA and tryptophan from the culture medium. Extraction methods typically involved the use of organic solvents such as ethyl acetate to efficiently recover the target compounds from the aqueous phase (Calatrava et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eQuantification of IAA and tryptophan was performed using High-Performance Liquid Chromatography (HPLC), a sensitive analytical technique capable of separating and quantifying compounds in complex mixtures. Samples were injected into the HPLC system, and peak areas corresponding to IAA and tryptophan were determined (Figs.\u0026nbsp;7 \u0026amp; 8).\u003c/p\u003e \u003cp\u003eCalibration curves generated from standard solutions of known concentrations of IAA and tryptophan were used to quantify the amounts of these compounds present in \u003cem\u003eBacillus pumilus\u003c/em\u003ebacterial cultures. The concentrations of IAA and tryptophan produced by bacterial isolate MUSRH-05 were calculated respectively RT 3.131 min and 2.644 based on peak area i.e., 5704.8125 and 2027.7877, Height 399.2582 and 157.6192, RF 0.05317\u0026amp; 0.07465 and expressed in 303.333 ng/ul and 151.383 ng/ul.\u003c/p\u003e \u003cp\u003eThe assessment of IAA and tryptophan production provided insights into the metabolic capabilities of bacterial isolates MUSRH-5 and their potential to influence plant growth through phytohormone synthesis. Variations in the production levelsAnnotations indicate different levels (e.g., Level 1, Level 2, Level 3) on the calibration curves of IAA and tryptophan among isolates highlighted the functional diversity within the bacterial community and underscored the importance of characterizing individual isolates for their specific plant growth-promoting traits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHPLC Analysis\u003c/h2\u003e \u003cp\u003eHigh-Performance Liquid Chromatography (HPLC) Make- Agilent and Model - Infinity 1260 the principal analytical technique for quantifying Indole-3-Acetic Acid (IAA) and Tryptophan produced by bacterial isolates. HPLC is a powerful tool capable of separating, identifying, and quantifying components within complex mixtures with high sensitivity and precision.\u003c/p\u003e \u003cp\u003eInstrumentation: The HPLC DAD system utilized in this study comprised a solvent delivery system, an injector for sample introduction Flow rate- 1ml/min a chromatographic column for separation, a detector for compound detection, and a data acquisition system for analysis (Adil et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChromatographic Column: A suitable chromatographic column, such as a reverse-phase C18 column, was selected based on its ability to efficiently separate IAA and Tryptophan from other components present in the sample matrix.\u003c/p\u003e \u003cp\u003eMobile Phase: A mobile phase consisting of a solvent or solvent mixture was carefully chosen to elute the analytes of interest from the chromatographic column. The composition and gradient of the mobile phase is Channel A- Acetonitrile 60%, Channel B- Water 40%, and Flow rate- 1ml/min to achieve optimal separation and resolution of IAA and Tryptophan peaks (Terzi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDetection: UV-Vis spectroscopy was employed as the detection method, with the wavelength 254nm(405, 470, 400, etc.) likely correspond to the detection wavelengths used during the analysisselected to detect the absorbance of IAA and Tryptophan. The detector recorded the absorbance of eluting compounds as they passed through the column, generating chromatograms that represented the concentration of analytes over time (Fig.\u0026nbsp;9)\u003c/p\u003e \u003cp\u003eCalibration: Before sample analysis, calibration curves were constructed using standard solutions of Injection Volume- 10ul of IAA and Tryptophan. These calibration curves enabled the quantification of analytes in the samples by comparing their peak areas or heights with those of standards (Strieder et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSample Injection and Analysis: Extracted samples containing IAA and Tryptophan were injected Volume- 10ul into the HPLC system using an auto-sampler. The samples were eluted through the chromatographic column under optimized conditions, and the wavelengths (405, 470, 400, etc.) likely correspond to the detection wavelengths used during the analysis (\u003cem\u003eMixCALM\u003c/em\u003e) (Fig.\u0026nbsp;8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRegression Equation\u003c/h2\u003e \u003cp\u003eo The calibration curves follow the equation:\u003c/p\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;ax\u0026thinsp;+\u0026thinsp;b\u003c/p\u003e \u003cp\u003eHere, y represents the instrument response (area or height).\u003c/p\u003e \u003cp\u003ex represents the amount of the compound.\u003c/p\u003e \u003cp\u003ea and b are coefficients determined from the calibration data.\u003c/p\u003e \u003cp\u003eData Processing: The chromatographic data obtained from HPLC analysis were processed using specialized software. Peak integration Agilent Technologies software was used to determine the peak areas or heights corresponding to IAA and Tryptophan peaks. The concentrations of these compounds in the samples were calculated based on the calibration curves and expressed in appropriate units (e.g., \u0026micro;g/mL).\u003c/p\u003e \u003cp\u003eIn summary, HPLC analysis provided a robust and reliable method for quantifying Indole-3-Acetic Acid (IAA) and Tryptophan produced by bacterial isolates, facilitating the assessment of their plant growth-promoting activities with high sensitivity and accuracy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the assessment of plant growth-promoting activities of isolated \u003cem\u003eBacillus pumilus\u003c/em\u003ethrough the quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC) represents a significant stride towards understanding the intricate dynamics between soil microbiota and plant health. Through meticulous isolation and cultivation techniques, diverse bacterial strains were successfully identified, showcasing the richness of microbial diversity within agricultural soils.\u003c/p\u003e \u003cp\u003eThe findings of this study underscore the pivotal role of bacterial communities in promoting plant growth through the production of phytohormones such as IAA and Tryptophan. The variation observed in the production of these compounds among \u003cem\u003eBacillus pumilus\u003c/em\u003eemphasizes the functional diversity within soil microbial populations.\u003c/p\u003e \u003cp\u003eThe implications of these findings extend beyond agricultural productivity to encompass broader environmental sustainability goals. By harnessing the plant growth-promoting activities of \u003cem\u003eBacillus pumilus\u003c/em\u003e, we have the potential to reduce dependency on synthetic fertilizers and pesticides, mitigating environmental degradation associated with conventional agricultural practices.\u003c/p\u003e \u003cp\u003eLooking ahead, future research endeavors should delve deeper into the mechanisms underlying plant-microbe interactions and their impact on crop health and productivity. Moreover, exploring the potential synergistic effects of microbial consortia and their application in field trials could pave the way for the development of novel biofertilizers and biostimulants.\u003c/p\u003e \u003cp\u003eIn conclusion, this study contributes to the growing body of knowledge on the beneficial roles of rhizospheric bacteria in sustainable agriculture. By unraveling the intricate relationships between microbial communities and plant physiology, we can pave the way for innovative solutions that promote agricultural resilience, environmental stewardship, and food security in a rapidly changing world.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePGPR: Plant Growth-Promoting Rhizobacteria, IAA: \u0026nbsp; Indole-3-Acetic Acid, HPLC: High-performance liquid chromatography.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeredity Life sciences Pvt Ltd, Bhubaneswar, Odisha and Edison Life Science Laboratory, Kolkata, West Bengal,\u003c/p\u003e\n\u003cp\u003eIndia are thankfully acknowledged for the molecular identification of bacterial strain.\u003c/p\u003e\n\u003cp\u003eAuthors also acknowledged Environcheck for HPLC analysis of IAA, tryptophan .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that there are no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDisclosure of potential conflicts of interest\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResearch involving human participants and/or animals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInformed consent\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo informed consent is required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding support is availed. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent of publication\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI, Shambhu Swarnakar performed the experiment and surveyed field for soil collection. Photo belings to me. I have no objection if image is used for publication purposes in the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdil M, Filimban F Z, AmbrinQuddoos A, Sher AA, Naseer M (2024). Phytochemical screening, HPLC analysis, antimicrobial and antioxidant effect of Euphorbia parviflora L.(Euphorbiaceae Juss.). \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(1), 5627.\u003c/li\u003e\n\u003cli\u003eAubier TG, Kopp M, Linn IJ, Puebla O, Rafajlović M, Servedio MR (2024). Negative coupling: the coincidence of premating isolating barriers can reduce reproductive isolation. \u003cem\u003eCold Spring Harbor Perspectives in Biology\u003c/em\u003e, a041435.\u003c/li\u003e\n\u003cli\u003eCalatrava V, Hom EF, Guan Q, Llamas A, Fern\u0026aacute;ndez E, Galv\u0026aacute;n A (2024). 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Salicylic acid as a switchable hydrophilicity solvent for the microextraction of the antibiotic amphotericin B from human urine followed by HPLC-UV analysis. \u003cem\u003eMicrochemical Journal\u003c/em\u003e, \u003cem\u003e199\u003c/em\u003e, 110025.\u003c/li\u003e\n\u003cli\u003eZhang H, Rong Z, Li Y, Yin Z, Lu C, Zhao H, Ding X (2024). NIT24 and NIT29‐mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice. \u003cem\u003eMolecular Plant Pathology\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(1), e13409.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plant growth-promoting bacteria, indole-3-acetic acid, tryptophan, HPLC, microbial interactions","lastPublishedDoi":"10.21203/rs.3.rs-4729095/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4729095/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMicrobial-mediated plant growth promotion has gained significant attention in agriculture due to its potential to improve crop yield and sustainability. Indole-3-acetic acid (IAA) and tryptophan are key compounds synthesized by certain bacteria, known to positively influence plant development. High-performance liquid chromatography (HPLC) is a robust analytical technique capable of accurately quantifying these compounds, enabling a deeper understanding of plant-microbe interactions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, bacterial isolates designated as MUSRH-5 were investigated for their plant growth-promoting activities. Cultured bacterial strains were assessed for their efficacy in promoting plant growth. Through HPLC analysis, varying concentrations of IAA and tryptophan were observed among the bacterial isolates, indicating differing metabolic capabilities. Significant variations in plant growth-promoting activities were also noted, with certain strains demonstrating notable effectiveness.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe findings of this study highlight the potential of selected bacterial isolates, particularly MUSRH-5 strains, to enhance plant growth through the production of growth-promoting compounds. The utilization of HPLC for precise quantification provided valuable insights into the metabolic capabilities of these bacteria and their impact on plant development. This research underscores the importance of microbial-mediated plant growth promotion in sustainable agriculture and environmental management, emphasizing the role of bacterial isolates as valuable assets in enhancing agricultural productivity and mitigating environmental challenges.\u003c/p\u003e","manuscriptTitle":"Assessing Plant Growth-Promoting Activities of Bacillus pumilus: Quantification of Indole-3-Acetic Acid (IAA) and Tryptophan by High-Performance Liquid Chromatography (HPLC)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-27 06:30:12","doi":"10.21203/rs.3.rs-4729095/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-07-31T13:30:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-30T15:02:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2024-07-12T08:50:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0fe9dbd5-2b44-484d-880a-928a7d36b2da","owner":[],"postedDate":"August 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-27T06:30:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-27 06:30:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4729095","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4729095","identity":"rs-4729095","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}