Microbial and Biogeochemical Responses to Drought in Soil Carbon Cycling Systems

Microbial and Biogeochemical Responses to Drought in Soil Carbon Cycling Systems | 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 Microbial and Biogeochemical Responses to Drought in Soil Carbon Cycling Systems Abdullah Al Mamun, Md Mushfiqur Rahaman Chowdhury, Saad Bin Islam, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6475985/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Soil is the largest terrestrial carbon reservoir, with microorganisms playing a pivotal role in organic matter decomposition and carbon stabilization. Drought, intensified by climate change, alters microbial dynamics and soil carbon cycling. This study investigates the effects of drought-induced stress on microbial community composition, carbon allocation, and soil biochemical properties in agricultural and forest soils in Bangladesh. Soils from a drought-prone maize field and a semi-natural forest site in Kushtia, Bangladesh, were incubated in mesocosms under control (25% WHC) and drought (10% WHC) conditions, with and without carbon amendments. Microbial activity and structure were assessed through PLFA analysis and 16S rRNA sequencing. Soil CO₂ emissions, microbial biomass carbon (MBC), dissolved organic carbon (DOC), and soil nutrients were also analyzed. Drought significantly reduced microbial biomass (45.3 to 32.3 nmol PLFA g⁻¹, p = 0.021), MBC (215.7 ± 12.4 to 128.5 ± 10.7 µg C g⁻¹, p < 0.01), and CO₂ emissions (3.85 ± 0.21 to 2.16 ± 0.18 µg CO₂-C g⁻¹ day⁻¹). DOC decreased from 12.4 ± 1.2 to 7.1 ± 0.9 mg C g⁻¹ (p = 0.012). Gram-negative bacteria declined by 41.2%, while Gram-positive taxa and fungi increased, indicating a shift to drought-tolerant communities. Carbon use efficiency slightly increased (56.0–59.5%). Drought suppresses microbial activity and alters community structure, reducing soil carbon and nutrient availability. These findings underscore the need for drought-resilient soil management in climate-sensitive regions like Bangladesh. Biogeography General Microbiology Agroecology Renewable Resources Applied Biochemistry Biogeochemical Carbon cycling Microbial biomass Drought Climate change Phospholipid fatty acid Figures Figure 1 Figure 2 1. Introduction Soil serves as the largest terrestrial carbon reservoir and plays a crucial role in regulating global carbon cycles (Bertini and Azevedo 2022 ; Prajapati et al., 2023 ). Microorganisms drive key processes such as organic matter decomposition, nutrient mineralization, and carbon stabilization (Raza et al., 2023 ). Their metabolic activity directly influences the balance between carbon storage and loss, primarily through carbon use efficiency (CUE) (Iven et al., 2023 ), which is the fraction of assimilated carbon retained as biomass versus respired as CO 2 (Butcher 2023 ). Drought, an increasingly frequent consequence of climate change, profoundly affects soil microbial dynamics (Bogati and Walczak 2022 ). Reduced moisture alters microbial community structure, enzyme activity, and carbon turnover, thereby impacting long-term soil fertility and atmospheric CO 2 emissions (Amante and Wedajo 2024 ). In air-sdried conditions, microbial communities often shift toward stress-tolerant taxa such as Actinobacteria and Firmicutes , while overall metabolic efficiency declines (Griffin-LaHue et al., 2023 ). This study investigates how drought-induced stress influences microbial community composition, carbon allocation strategies, and biogeochemical carbon fluxes in Bangladesh agricultural and forest soils. By combining biochemical and molecular tools, we aim to unravel microbial mechanisms driving soil carbon cycling under moisture limitation. Understanding these processes is essential for predicting ecosystem feedbacks to climate change and informing sustainable soil management. 2. Materials and Methods 2.1. Sample Collection Soil samples were collected from two different ecosystems in the Kushtia district of Bangladesh. One sample was taken from drought-affected agricultural soil in a maize field with minimal irrigation history. And another sample collected from a semi-natural deciduous agricultural area with dense vegetation cover. From each site, five soil cores (0–15 cm depth) were collected using a sterilized soil auger. These samples were pooled, mixed thoroughly, sieved through a 2 mm mesh to remove debris, and stored at 4°C for further analysis. 2.2. Experimental Design: Soil Mesocosms To study the effect of drought, soil samples were placed in a container and and subjected to two treatments: a control condition (25% WHC) and a drought condition (10% WHC). A control with 25% water holding capacity (WHC), and 10% WHC which represent drought conditions. A total of 12 mesocosms, each containing 500 grams of dry soil, were set up with the following treatments: control (25% WHC, no amendment), control + carbon (25% WHC with glucose and plant litter extract), drought (10% WHC, no amendment), and drought + carbon (10% WHC with glucose and plant litter extract). Each treatment had three biological replicates, totaling 12 mesocosms. The carbon input consisted of 1% glucose, which was made by dissolving 5 grams of glucose in 100 mL distilled water and mixing it into the 500 g of soil, and plant litter extract, which was prepared by steeping 50 grams of air-dried forest leaf litter in 1 liter of distilled water for 24 hours, then autoclaving and filtering the solution through a No. 1 Whatman filter paper. The mesocosms were incubated in the dark at 25°C for 14 days, and soil moisture was checked every two days by weighing the pots and adding sterile distilled water to maintain the intended WHC. 2.3. Microbial Biomass and Phospholipid Fatty Acid (PLFA) Analysis For microbial biomass and PLFA analysis, 5 g of fresh soil from each mesocosm were used for PLFA extraction according to Bligh and Dyer method (Teixeira et al., 2024 ). The fatty acids were methylated and analyzed by Gas Chromatography–Flame Ionization Detection (GC-FID). The biomarkers used for the analysis included i15:0 and a15:0 for Gram-positive bacteria, 16:1ω7c and cy17:0 for Gram-negative bacteria, 18:2ω6,9c for fungi, and 10Me16:0 for actinobacteria. 2.4. Soil Chemical Properties The pH of the soil was measured in a 1:2.5 soil-to-water suspension using a digital pH meter. Soil organic carbon was determined using the Walkley–Black dichromate oxidation method (El Mouridi et al., 2023 ). Total nitrogen was measured by Kjeldahl digestion, and available phosphorus was determined using Olsen’s method (Ibrahim et al., 2021 ). 2.5. DNA Extraction and 16S rRNA Sequencing 0.5 g of soil was used for DNA extraction using the DNeasy PowerSoil Kit (Qiagen). The V3–V4 region of the 16S rRNA gene was amplified using primers 341F and 805R. Sequencing was done using an Illumina MiSeq platform. Reads were quality-filtered, clustered into OTUs (97% similarity), and taxonomically assigned using the SILVA database. 2.6. Dissolved Organic Carbon (DOC) and Microbial Biomass Carbon (MBC) DOC was measured by shaking 10 grams of soil in 50 mL of distilled water for 1 hour, followed by filtration through a 0.45 µm filter, and measuring the absorbance at 254 nm using a UV-Vis spectrophotometer. MBC was determined using the chloroform fumigation-extraction method, where 10 grams of soil were fumigated with ethanol-free chloroform for 24 hours, extracted with 0.5 M K 2 SO 4 , oxidized with K 2 Cr 2 O 7 , and the absorbance was measured at 600 nm. 2.7. Statistical Analysis All measurements were performed in triplicates. Data were tested for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test. One-way or two-way ANOVA was applied to assess significant differences among treatments, followed by Tukey’s HSD post-hoc test for pairwise comparisons. A p < 0.05 was considered statistically significant. Principal Component Analysis (PCA) was performed on PLFA and microbial community data to explore treatment-related patterns. All statistical analyses were carried out using R programing and OriginPro. 3. Results and Discussion 3.1. Soil Mesocosms analysis The soil mesocosm experiment showed a marked difference in soil properties under drought stress versus control conditions (Table 1). At the end of the incubation period, the drought-stressed mesocosms had significantly lower moisture content (10% water holding capacity) compared to control mesocosms (25% water holding capacity), which was expected. Soil moisture loss under drought conditions caused a decrease in microbial biomass and altered microbial community structure, confirming that drought stress significantly impacts microbial function and diversity. 3.2. Microbial Biomass and Phospholipid Fatty Acid Analysis PLFA profiling showed that total microbial biomass significantly decreased under drought conditions (from 45.3 to 32.3 nmol PLFA g⁻¹ soil; p = 0.021), indicating reduced microbial activity and growth (Table 2). Notably, Gram-negative bacterial abundance declined by 41.2% under drought (p = 0.015), while Gram-positive bacteria increased by 18.4% (p = 0.042), reflecting a shift in community structure toward drought-tolerant groups. The fungal community exhibited a modest increase (12.6%, p = 0.058), and the fungi-to-bacteria ratio rose by 60%, suggesting that fungi may be better equipped to survive drought through efficient osmoregulation and stress tolerance (Fig. 1). These changes suggest that microbes shift towards stress tolerance mechanisms, potentially reducing their efficiency in carbon processing under drought. 3.3. Soil Respiration analysis Drought significantly reduced soil microbial activity and carbon dynamics (Table 3). Soil CO₂ emissions decreased from 3.85 ± 0.21 µg CO 2 -C g − 1 day − 1 under control conditions (25% WHC) to 2.16 ± 0.18 µg CO 2 -C g − 1 day − 1 under drought (10% WHC), indicating a 43.9% reduction ( p < 0.01). Similarly, microbial biomass carbon declined from 215.7 ± 12.4 µg C g − 1 to 128.5 ± 10.7 µg C g − 1 , a 40.5% decrease ( p < 0.01). This demonstrates a substantial loss of microbial biomass under moisture-limited conditions, which is consistent with previous studies showing that drought limits microbial growth by restricting substrate diffusion and enzyme activity. Interestingly, carbon use efficiency (CUE), calculated as biomass C/CO 2 -C, increased slightly from 56.0 to 59.5 (+ 6.2%), though the change was not statistically significant. This suggests that microbes under drought may invest more in biomass production relative to respiration, possibly reallocating resources to maintain biomass rather than metabolic activity. However, the biological significance of this slight shift in CUE remains uncertain without long-term data. The Pearson correlation between microbial biomass and CO 2 -C emissions was strong under control conditions (r = 0.81), but diminished under drought (r = 0.31), indicating that drought decouples microbial biomass from respiration rates. This disruption implies altered metabolic strategies or shifts in microbial community composition. Overall, drought not only suppresses microbial activity but also alters microbial functionality, with potential consequences for soil carbon storage and ecosystem resilience in agricultural systems. 3.4. Soil Chemical Properties Soil chemical properties were measured to assess changes in key parameters such as pH, soil organic carbon (SOC), total nitrogen (TN), and available phosphorus (AP), under both control and drought conditions (Table 4). The analysis was performed using standard laboratory techniques, including a pH meter for soil pH, a Walkley-Black method for SOC, Kjeldahl digestion for TN, and a colorimetric method for AP. Soil pH showed a slight decrease from 6.48 ± 0.09 under control conditions to 6.37 ± 0.08 under drought conditions (p = 0.05). This minor decline could indicate increased acidity due to microbial activity or changes in cation exchange in drought conditions. However, the difference is not large enough to suggest a major shift in pH that could drastically affect soil fertility. The SOC content in soils decreased significantly under drought conditions, from 2.12 ± 0.15 g kg − 1 to 1.89 ± 0.13 g kg − 1 (p = 0.01), indicating a decrease of approximately 10.8% under drought. This decrease might be linked to the lower microbial activity observed under drought, as microbes are less active in breaking down organic material when water is limited. Consequently, the decomposition of plant material and soil organic matter might slow down, reducing carbon input into the soil. Total nitrogen in soil also decreased significantly from 0.21 ± 0.02 g kg − 1 in the control to 0.18 ± 0.02 g kg − 1 under drought (p = 0.03). The reduction could be due to lower nitrogen fixation by soil microbes under water stress or reduced organic matter decomposition, which typically supplies nitrogen in the form of ammonium and nitrate. Available phosphorus (AP) in the soil was reduced from 15.3 ± 1.2 mg kg − 1 under control conditions to 13.8 ± 1.0 mg kg − 1 under drought (p = 0.04). This reduction could be attributed to changes in microbial community composition (e.g., decreased phosphorus-solubilizing microbes) and decreased microbial activity, which directly impacts nutrient availability in soils under drought. The significant reduction in soil organic carbon, nitrogen, and phosphorus suggests that drought has a negative impact on soil fertility, likely due to reduced microbial activity, slower organic matter decomposition, and changes in microbial community structure. These effects can further impair soil health and ecosystem functioning, leading to impaired nutrient cycling. While the slight pH decrease suggests possible changes in microbial metabolic processes, the impact on soil chemical properties is moderate in the context of this study. Nonetheless, the overall nutrient reduction emphasizes the vulnerability of soil ecosystems to prolonged drought, which could have long-term implications for soil fertility and agricultural productivity. 3.5. Microbial diversity analysis DNA sequencing of the 16S rRNA gene revealed distinct microbial community compositions between control and drought treatments. The control soil had a more diverse bacterial community with 342 OTUs, while the drought soil exhibited reduced diversity (287 OTUs), indicating that drought stress selects for a subset of bacteria capable of surviving under water-limited conditions. The decrease in OTUs under drought is consistent with other findings, suggesting that environmental stress, such as drought, can lead to microbial community shifts, favoring drought-tolerant taxa. At the taxonomic level, via PCA analysis (Fig. 2), drought treatment resulted in an increase in the relative abundance of Gram-positive bacteria, particularly Firmicutes and Actinobacteria, known for their drought-resilient characteristics. Conversely, Proteobacteria and Bacteroidetes, which typically dominate in well-watered conditions, decreased significantly under drought. These results suggest that drought-induced environmental stress shapes bacterial community composition by promoting species that are more adapted to stress, thereby affecting the functional capacity of the microbial community in soil, potentially influencing soil carbon cycling. 3.6. Dissolved Organic Carbon (DOC) and Microbial Biomass Carbon (MBC) The DOC concentration was significantly reduced under drought conditions (7.1 ± 0.9 mg C g − 1 soil) compared to control conditions (12.4 ± 1.2 mg C g − 1 soil, p = 0.012). This reduction suggests that drought conditions may limit the breakdown of organic matter and decrease the availability of labile carbon sources for microbial consumption (Table 5). The decrease in DOC may also reflect reduced microbial activity under water-limited conditions, as less substrate is available for microbial processing. A significant reduction in MBC under drought conditions (0.42 ± 0.03 mg C g − 1 soil) compared to the control (0.65 ± 0.05 mg C g − 1 soil, p = 0.019) suggests that drought not only reduces microbial biomass but also inhibits microbial growth. The decline in MBC can be attributed to the reduced water availability, which affects microbial metabolic processes and biomass production. the reductions in both DOC and MBC under drought conditions indicate that drought stress can disrupt microbial activity and growth, thereby influencing carbon cycling processes in the soil. This underscores the importance of understanding microbial responses to drought for predicting long-term changes in soil carbon storage and flux. 4. Conclusions Drought stress significantly alters soil microbial communities, reduces microbial biomass, and suppresses soil carbon and nutrient cycling in both agricultural and forest soils. Our findings indicate a shift toward drought-tolerant taxa, particularly Gram-positive bacteria and fungi, under moisture-limited conditions. These microbial changes are accompanied by lower CO₂ emissions, reduced microbial biomass carbon, and altered soil chemistry, including decreases in organic carbon, nitrogen, and phosphorus availability. While microbial carbon use efficiency increased slightly, the overall decline in microbial activity suggests long-term implications for soil fertility and carbon storage under future drought scenarios. This study underscores the need for drought-resilient soil management strategies in climate-vulnerable regions like Bangladesh. Declarations Acknowledgment The authors wish to thank the department of Biotechnology and Genetic Engineering for supporting this research Ethics declarations This article does not include any studies by any of the authors that used human or animal participants. All authors are conscious and accept responsibility for the manuscript. No part of the manuscript content has been published or accepted for publication elsewhere. Funding There was no fund available. Conflict of interest The authors declare no competing interests. Data availability The corresponding author Abdullah Al Mamun is responsible for all data and materials. Code availability There was no code available. Authors Contributions A.A.M. comprehended and planned the study, carried out the analysis, wrote the manuscript; and prepared the graphs and illustrations; M.R.C., S.B.I., S.H., S.A.Z, M.M., R.M., and S.K. contributed to the critical revision of the manuscript and wrote the manuscript; A.A.M. supervised the whole work, and all authors approved the final manuscript. References Bertini, S. C. B., & Azevedo, L. C. B. (2022). Soil microbe contributions in the regulation of the global carbon cycle. In Microbiome under changing climate (pp. 69-84). Woodhead Publishing. https://doi.org/10.1016/B978-0-323-90571-8.00003-1 Prajapati, S. K., Kumar, V., Dayal, P., Gairola, A., Borate, R. B., & Srivastava, R. (2023). THE ROLE OF CARBON IN LIFE'S BLUEPRINT AND CARBON CYCLE UNDER-STANDING EARTH'S ESSENTIAL CYCLING SYSTEM: BENEFITS AND HARMS TO OUR PLANET. International Journal , 1 (1), 21-32. https://doi.org/10.5281/zenodo.8385430 Raza, T., Qadir, M. F., Khan, K. S., Eash, N. S., Yousuf, M., Chatterjee, S., ... & Oetting, J. N. (2023). Unraveling the potential of microbes in decomposition of organic matter and release of carbon in the ecosystem. Journal of Environmental Management , 344 , 118529. https://doi.org/10.1016/j.jenvman.2023.118529 Iven, H., Walker, T. W., & Anthony, M. (2023). Biotic interactions in soil are underestimated drivers of microbial carbon use efficiency. Current Microbiology , 80 (1), 13. https://doi.org/10.1007/s00284-022-02979-2 Butcher, K. R. (2023). Edaphic and Climatic Regulation of Microbial Carbon-Use Efficiency in Managed Semi-Arid Systems. https://doi.org/10.26076/d8eb-cf83 Bogati, K., & Walczak, M. (2022). The impact of drought stress on soil microbial community, enzyme activities and plants. Agronomy , 12 (1), 189. https://doi.org/10.3390/agronomy12010189 Amante, G., & Wedajo, M. (2024). Impacts of Climate change on soil microbial diversity, distribution and abundance. International Journal on Food, Agriculture and Natural Resources , 5 (2), 158-168. https://doi.org/10.46676/ij-fanres.v5i2.342 Griffin-LaHue, D., Wang, D., Gaudin, A. C., Durbin-Johnson, B., Settles, M. L., & Scow, K. M. (2023). Extended soil surface drying triggered by subsurface drip irrigation decouples carbon and nitrogen cycles and alters microbiome composition. Frontiers in Soil Science , 3 , 1267685. https://doi.org/10.3389/fsoil.2023.1267685 Teixeira, P. P., Vidal, A., Teixeira, A. P., Souza, I. F., Hurtarte, L. C., Silva, D. H., ... & Silva, I. R. (2024). Decoding the rhizodeposit-derived carbon's journey into soil organic matter. Geoderma , 443 . https://doi.org/10.1016/j.geoderma.2024.116811 El Mouridi, Z., Ziri, R., Douaik, A., Bennani, S., Lembaid, I., Bouharou, L., ... & Moussadek, R. (2023). Comparison between Walkley-Black and loss on ignition methods for organic matter estimation in different Moroccan soils. Ecological Engineering & Environmental Technology , 24 . https://doi.org/10.12912/27197050/163121 Ibrahim, K. H., Wang, L., Wu, Q., Duan, Y., Ma, C., & Zhang, S. (2021). Soil Phosphorus management based on changes in Olsen P and P budget under Long-term fertilization experiment in fluvo-aquic soil. https://doi.org/10.15159/AR.21.006 Additional Declarations The authors declare no competing interests. Supplementary Files GraphicalAbstract.png Graphical abstract of overall study. 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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-6475985","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445773768,"identity":"11dd6fbd-183c-4d3d-a7a1-a55f71a58114","order_by":0,"name":"Abdullah Al Mamun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBCDBCBmfAAkePiI1sLDwMBsANLCRooWNgkQi6AW/vbTiZ9utt3Js+dffKzya46dDBsD88NHN/BokTiTu1k6t+1ZMY/Es7TbstuSgQ5jMzbOwWfNgdwNQC2HE3skzpjdltzGDNTCwyaNT4v8+bebf0O0nP9WLLmtnrAWgxu52yC28PewMX7cdpiwFsMbb7dZ55w7XMxzg81YmnHbcR42ZgJ+kTufu/l2TtnhPPb+ww8//txWbc/P3vzwMV7vw4FEAgMzD4jBTJRyEOA/wMD4g2jVo2AUjIJRMJIAADgVSyVlArbwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0007-2840-759X","institution":"Islamic University","correspondingAuthor":true,"prefix":"","firstName":"Abdullah","middleName":"Al","lastName":"Mamun","suffix":""},{"id":445773769,"identity":"58bad798-6cb2-498d-9180-1b08f1f61f4c","order_by":1,"name":"Md Mushfiqur Rahaman Chowdhury","email":"","orcid":"https://orcid.org/0000-0003-1041-5229","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Mushfiqur Rahaman","lastName":"Chowdhury","suffix":""},{"id":445773770,"identity":"46f9822d-4ef3-46db-9582-c5927f8008ca","order_by":2,"name":"Saad Bin Islam","email":"","orcid":"https://orcid.org/0000-0001-5972-7050","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Saad","middleName":"Bin","lastName":"Islam","suffix":""},{"id":445773771,"identity":"dd77923c-8c60-4325-a8bc-8be47075b5c9","order_by":3,"name":"Md. Sakhawat Hossain","email":"","orcid":"https://orcid.org/0009-0007-0941-759X","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Sakhawat","lastName":"Hossain","suffix":""},{"id":445773772,"identity":"939f24ea-aa16-4b4c-8b33-6a0c82fb715f","order_by":4,"name":"Sabbir Alam Zia","email":"","orcid":"","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Sabbir","middleName":"Alam","lastName":"Zia","suffix":""},{"id":445773773,"identity":"12371556-074e-4ece-8354-42cd4b9b7f8a","order_by":5,"name":"Mukur Mondal","email":"","orcid":"https://orcid.org/0009-0006-7589-3028","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Mukur","middleName":"","lastName":"Mondal","suffix":""},{"id":445773774,"identity":"d59239f2-2605-4e49-af6a-b8c860ccc57c","order_by":6,"name":"Md Rakib Mia","email":"","orcid":"","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Rakib","lastName":"Mia","suffix":""},{"id":445773775,"identity":"61d0d536-573b-4477-bad1-c4515c83c7c1","order_by":7,"name":"Salman Khan","email":"","orcid":"https://orcid.org/0009-0007-5668-2126","institution":"Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Salman","middleName":"","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2025-04-18 04:44:08","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6475985/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6475985/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81109865,"identity":"916da0c7-6f5a-4dc0-a006-2d1ae1c5618a","added_by":"auto","created_at":"2025-04-22 10:21:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16467,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of drought on soil microbial community composition as determined by PLFA analysis. Bar plots show the mean ± standard deviation (n = 3) of total microbial biomass and specific microbial groups (Gram-negative bacteria, Gram-positive bacteria, fungi, and fungi:bacteria ratio) under control and drought conditions. Drought treatment significantly reduced total microbial biomass and Gram-negative bacteria, while increasing Gram-positive bacteria and the fungi:bacteria ratio, indicating a shift in microbial community structure.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6475985/v1/0b4e10f53abb0ac1faeee4e1.png"},{"id":81109375,"identity":"9161f6c1-dca4-4393-ba88-492a2636464d","added_by":"auto","created_at":"2025-04-22 10:13:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35477,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) biplot illustrating differences in soil microbial community composition based on 16S rRNA gene sequencing data under control and drought conditions. Each point represents a sample grouped by dominant bacterial phyla associated with either control (e.g., Proteobacteria, Bacteroidetes) or drought (e.g., Actinobacteria, Firmicutes) treatments. The axes represent the first two principal components (PC1 and PC2), explaining XX% and YY% of the total variation, respectively. Colored arrows indicate the direction and strength of influence of key phyla on the ordination pattern. Bold lines and labeled vectors highlight phyla most responsive to drought-induced environmental change.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6475985/v1/5de8d00cfb18ea4167c0c0cb.png"},{"id":81110897,"identity":"2cc8878b-0c0a-495b-bea1-1b39fdff11d7","added_by":"auto","created_at":"2025-04-22 10:29:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":704949,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6475985/v1/d252d1e1-ee35-4e62-8443-6488cfd8c20f.pdf"},{"id":81109378,"identity":"2b01f0fe-fe1e-43ec-8c08-e8c9ae6dddad","added_by":"auto","created_at":"2025-04-22 10:13:50","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1219983,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract of overall study.\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6475985/v1/b4d3d2bc30c52b9fbf9435bf.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eMicrobial and Biogeochemical Responses to Drought in Soil Carbon Cycling Systems\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSoil serves as the largest terrestrial carbon reservoir and plays a crucial role in regulating global carbon cycles (Bertini and Azevedo \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Prajapati et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microorganisms drive key processes such as organic matter decomposition, nutrient mineralization, and carbon stabilization (Raza et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Their metabolic activity directly influences the balance between carbon storage and loss, primarily through carbon use efficiency (CUE) (Iven et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which is the fraction of assimilated carbon retained as biomass versus respired as CO\u003csub\u003e2\u003c/sub\u003e (Butcher \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Drought, an increasingly frequent consequence of climate change, profoundly affects soil microbial dynamics (Bogati and Walczak \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Reduced moisture alters microbial community structure, enzyme activity, and carbon turnover, thereby impacting long-term soil fertility and atmospheric CO\u003csub\u003e2\u003c/sub\u003e emissions (Amante and Wedajo \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In air-sdried conditions, microbial communities often shift toward stress-tolerant taxa such as \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e, while overall metabolic efficiency declines (Griffin-LaHue et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This study investigates how drought-induced stress influences microbial community composition, carbon allocation strategies, and biogeochemical carbon fluxes in Bangladesh agricultural and forest soils. By combining biochemical and molecular tools, we aim to unravel microbial mechanisms driving soil carbon cycling under moisture limitation. Understanding these processes is essential for predicting ecosystem feedbacks to climate change and informing sustainable soil management.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample Collection\u003c/h2\u003e \u003cp\u003eSoil samples were collected from two different ecosystems in the Kushtia district of Bangladesh. One sample was taken from drought-affected agricultural soil in a maize field with minimal irrigation history. And another sample collected from a semi-natural deciduous agricultural area with dense vegetation cover. From each site, five soil cores (0\u0026ndash;15 cm depth) were collected using a sterilized soil auger. These samples were pooled, mixed thoroughly, sieved through a 2 mm mesh to remove debris, and stored at 4\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental Design: Soil Mesocosms\u003c/h2\u003e \u003cp\u003eTo study the effect of drought, soil samples were placed in a container and and subjected to two treatments: a control condition (25% WHC) and a drought condition (10% WHC). A control with 25% water holding capacity (WHC), and 10% WHC which represent drought conditions. A total of 12 mesocosms, each containing 500 grams of dry soil, were set up with the following treatments: control (25% WHC, no amendment), control\u0026thinsp;+\u0026thinsp;carbon (25% WHC with glucose and plant litter extract), drought (10% WHC, no amendment), and drought\u0026thinsp;+\u0026thinsp;carbon (10% WHC with glucose and plant litter extract). Each treatment had three biological replicates, totaling 12 mesocosms. The carbon input consisted of 1% glucose, which was made by dissolving 5 grams of glucose in 100 mL distilled water and mixing it into the 500 g of soil, and plant litter extract, which was prepared by steeping 50 grams of air-dried forest leaf litter in 1 liter of distilled water for 24 hours, then autoclaving and filtering the solution through a No. 1 Whatman filter paper. The mesocosms were incubated in the dark at 25\u0026deg;C for 14 days, and soil moisture was checked every two days by weighing the pots and adding sterile distilled water to maintain the intended WHC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Microbial Biomass and Phospholipid Fatty Acid (PLFA) Analysis\u003c/h2\u003e \u003cp\u003eFor microbial biomass and PLFA analysis, 5 g of fresh soil from each mesocosm were used for PLFA extraction according to Bligh and Dyer method (Teixeira et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The fatty acids were methylated and analyzed by Gas Chromatography\u0026ndash;Flame Ionization Detection (GC-FID). The biomarkers used for the analysis included i15:0 and a15:0 for Gram-positive bacteria, 16:1ω7c and cy17:0 for Gram-negative bacteria, 18:2ω6,9c for fungi, and 10Me16:0 for actinobacteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Soil Chemical Properties\u003c/h2\u003e \u003cp\u003eThe pH of the soil was measured in a 1:2.5 soil-to-water suspension using a digital pH meter. Soil organic carbon was determined using the Walkley\u0026ndash;Black dichromate oxidation method (El Mouridi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Total nitrogen was measured by Kjeldahl digestion, and available phosphorus was determined using Olsen\u0026rsquo;s method (Ibrahim et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. DNA Extraction and 16S rRNA Sequencing\u003c/h2\u003e \u003cp\u003e0.5 g of soil was used for DNA extraction using the DNeasy PowerSoil Kit (Qiagen). The V3\u0026ndash;V4 region of the 16S rRNA gene was amplified using primers 341F and 805R. Sequencing was done using an Illumina MiSeq platform. Reads were quality-filtered, clustered into OTUs (97% similarity), and taxonomically assigned using the SILVA database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Dissolved Organic Carbon (DOC) and Microbial Biomass Carbon (MBC)\u003c/h2\u003e \u003cp\u003eDOC was measured by shaking 10 grams of soil in 50 mL of distilled water for 1 hour, followed by filtration through a 0.45 \u0026micro;m filter, and measuring the absorbance at 254 nm using a UV-Vis spectrophotometer. MBC was determined using the chloroform fumigation-extraction method, where 10 grams of soil were fumigated with ethanol-free chloroform for 24 hours, extracted with 0.5 M K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, oxidized with K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, and the absorbance was measured at 600 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll measurements were performed in triplicates. Data were tested for normality using the Shapiro\u0026ndash;Wilk test and for homogeneity of variance using Levene\u0026rsquo;s test. One-way or two-way ANOVA was applied to assess significant differences among treatments, followed by Tukey\u0026rsquo;s HSD post-hoc test for pairwise comparisons. A \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Principal Component Analysis (PCA) was performed on PLFA and microbial community data to explore treatment-related patterns. All statistical analyses were carried out using R programing and OriginPro.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Soil Mesocosms analysis\u003c/h2\u003e \u003cp\u003eThe soil mesocosm experiment showed a marked difference in soil properties under drought stress versus control conditions (Table\u0026nbsp;1). At the end of the incubation period, the drought-stressed mesocosms had significantly lower moisture content (10% water holding capacity) compared to control mesocosms (25% water holding capacity), which was expected. Soil moisture loss under drought conditions caused a decrease in microbial biomass and altered microbial community structure, confirming that drought stress significantly impacts microbial function and diversity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Microbial Biomass and Phospholipid Fatty Acid Analysis\u003c/h2\u003e \u003cp\u003ePLFA profiling showed that total microbial biomass significantly decreased under drought conditions (from 45.3 to 32.3 nmol PLFA g⁻\u0026sup1; soil; p\u0026thinsp;=\u0026thinsp;0.021), indicating reduced microbial activity and growth (Table\u0026nbsp;2). Notably, Gram-negative bacterial abundance declined by 41.2% under drought (p\u0026thinsp;=\u0026thinsp;0.015), while Gram-positive bacteria increased by 18.4% (p\u0026thinsp;=\u0026thinsp;0.042), reflecting a shift in community structure toward drought-tolerant groups. The fungal community exhibited a modest increase (12.6%, p\u0026thinsp;=\u0026thinsp;0.058), and the fungi-to-bacteria ratio rose by 60%, suggesting that fungi may be better equipped to survive drought through efficient osmoregulation and stress tolerance (Fig.\u0026nbsp;1). These changes suggest that microbes shift towards stress tolerance mechanisms, potentially reducing their efficiency in carbon processing under drought.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Soil Respiration analysis\u003c/h2\u003e \u003cp\u003eDrought significantly reduced soil microbial activity and carbon dynamics (Table\u0026nbsp;3). Soil CO₂ emissions decreased from 3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;g CO\u003csub\u003e2\u003c/sub\u003e-C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under control conditions (25% WHC) to 2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;g CO\u003csub\u003e2\u003c/sub\u003e-C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under drought (10% WHC), indicating a 43.9% reduction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, microbial biomass carbon declined from 215.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4 \u0026micro;g C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 128.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7 \u0026micro;g C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a 40.5% decrease (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This demonstrates a substantial loss of microbial biomass under moisture-limited conditions, which is consistent with previous studies showing that drought limits microbial growth by restricting substrate diffusion and enzyme activity. Interestingly, carbon use efficiency (CUE), calculated as biomass C/CO\u003csub\u003e2\u003c/sub\u003e-C, increased slightly from 56.0 to 59.5 (+\u0026thinsp;6.2%), though the change was not statistically significant. This suggests that microbes under drought may invest more in biomass production relative to respiration, possibly reallocating resources to maintain biomass rather than metabolic activity. However, the biological significance of this slight shift in CUE remains uncertain without long-term data. The Pearson correlation between microbial biomass and CO\u003csub\u003e2\u003c/sub\u003e-C emissions was strong under control conditions (r\u0026thinsp;=\u0026thinsp;0.81), but diminished under drought (r\u0026thinsp;=\u0026thinsp;0.31), indicating that drought decouples microbial biomass from respiration rates. This disruption implies altered metabolic strategies or shifts in microbial community composition. Overall, drought not only suppresses microbial activity but also alters microbial functionality, with potential consequences for soil carbon storage and ecosystem resilience in agricultural systems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Soil Chemical Properties\u003c/h2\u003e \u003cp\u003eSoil chemical properties were measured to assess changes in key parameters such as pH, soil organic carbon (SOC), total nitrogen (TN), and available phosphorus (AP), under both control and drought conditions (Table\u0026nbsp;4). The analysis was performed using standard laboratory techniques, including a pH meter for soil pH, a Walkley-Black method for SOC, Kjeldahl digestion for TN, and a colorimetric method for AP. Soil pH showed a slight decrease from 6.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 under control conditions to 6.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 under drought conditions (p\u0026thinsp;=\u0026thinsp;0.05). This minor decline could indicate increased acidity due to microbial activity or changes in cation exchange in drought conditions. However, the difference is not large enough to suggest a major shift in pH that could drastically affect soil fertility. The SOC content in soils decreased significantly under drought conditions, from 2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.01), indicating a decrease of approximately 10.8% under drought. This decrease might be linked to the lower microbial activity observed under drought, as microbes are less active in breaking down organic material when water is limited. Consequently, the decomposition of plant material and soil organic matter might slow down, reducing carbon input into the soil. Total nitrogen in soil also decreased significantly from 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the control to 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under drought (p\u0026thinsp;=\u0026thinsp;0.03). The reduction could be due to lower nitrogen fixation by soil microbes under water stress or reduced organic matter decomposition, which typically supplies nitrogen in the form of ammonium and nitrate. Available phosphorus (AP) in the soil was reduced from 15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under control conditions to 13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under drought (p\u0026thinsp;=\u0026thinsp;0.04). This reduction could be attributed to changes in microbial community composition (e.g., decreased phosphorus-solubilizing microbes) and decreased microbial activity, which directly impacts nutrient availability in soils under drought. The significant reduction in soil organic carbon, nitrogen, and phosphorus suggests that drought has a negative impact on soil fertility, likely due to reduced microbial activity, slower organic matter decomposition, and changes in microbial community structure. These effects can further impair soil health and ecosystem functioning, leading to impaired nutrient cycling. While the slight pH decrease suggests possible changes in microbial metabolic processes, the impact on soil chemical properties is moderate in the context of this study. Nonetheless, the overall nutrient reduction emphasizes the vulnerability of soil ecosystems to prolonged drought, which could have long-term implications for soil fertility and agricultural productivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Microbial diversity analysis\u003c/h2\u003e \u003cp\u003eDNA sequencing of the 16S rRNA gene revealed distinct microbial community compositions between control and drought treatments. The control soil had a more diverse bacterial community with 342 OTUs, while the drought soil exhibited reduced diversity (287 OTUs), indicating that drought stress selects for a subset of bacteria capable of surviving under water-limited conditions. The decrease in OTUs under drought is consistent with other findings, suggesting that environmental stress, such as drought, can lead to microbial community shifts, favoring drought-tolerant taxa. At the taxonomic level, via PCA analysis (Fig.\u0026nbsp;2), drought treatment resulted in an increase in the relative abundance of Gram-positive bacteria, particularly Firmicutes and Actinobacteria, known for their drought-resilient characteristics. Conversely, Proteobacteria and Bacteroidetes, which typically dominate in well-watered conditions, decreased significantly under drought. These results suggest that drought-induced environmental stress shapes bacterial community composition by promoting species that are more adapted to stress, thereby affecting the functional capacity of the microbial community in soil, potentially influencing soil carbon cycling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Dissolved Organic Carbon (DOC) and Microbial Biomass Carbon (MBC)\u003c/h2\u003e \u003cp\u003eThe DOC concentration was significantly reduced under drought conditions (7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mg C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soil) compared to control conditions (12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mg C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soil, p\u0026thinsp;=\u0026thinsp;0.012). This reduction suggests that drought conditions may limit the breakdown of organic matter and decrease the availability of labile carbon sources for microbial consumption (Table\u0026nbsp;5). The decrease in DOC may also reflect reduced microbial activity under water-limited conditions, as less substrate is available for microbial processing. A significant reduction in MBC under drought conditions (0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soil) compared to the control (0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg C g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soil, p\u0026thinsp;=\u0026thinsp;0.019) suggests that drought not only reduces microbial biomass but also inhibits microbial growth. The decline in MBC can be attributed to the reduced water availability, which affects microbial metabolic processes and biomass production. the reductions in both DOC and MBC under drought conditions indicate that drought stress can disrupt microbial activity and growth, thereby influencing carbon cycling processes in the soil. This underscores the importance of understanding microbial responses to drought for predicting long-term changes in soil carbon storage and flux.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eDrought stress significantly alters soil microbial communities, reduces microbial biomass, and suppresses soil carbon and nutrient cycling in both agricultural and forest soils. Our findings indicate a shift toward drought-tolerant taxa, particularly Gram-positive bacteria and fungi, under moisture-limited conditions. These microbial changes are accompanied by lower CO₂ emissions, reduced microbial biomass carbon, and altered soil chemistry, including decreases in organic carbon, nitrogen, and phosphorus availability. While microbial carbon use efficiency increased slightly, the overall decline in microbial activity suggests long-term implications for soil fertility and carbon storage under future drought scenarios. This study underscores the need for drought-resilient soil management strategies in climate-vulnerable regions like Bangladesh.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank the department of Biotechnology and Genetic Engineering for supporting this research\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not include any studies by any of the authors that used human or animal participants. All authors are conscious and accept responsibility for the manuscript. No part of the manuscript content has been published or accepted for publication elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no fund available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author Abdullah Al Mamun is responsible for all data and materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no code available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.A.M. comprehended and planned the study, carried out the analysis, wrote the manuscript; and prepared the graphs and illustrations; M.R.C., S.B.I., S.H., S.A.Z, M.M., R.M., and S.K. contributed to the critical revision of the manuscript and wrote the manuscript; A.A.M. supervised the whole work, and all authors approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBertini, S. C. B., \u0026amp; Azevedo, L. C. B. (2022). Soil microbe contributions in the regulation of the global carbon cycle. In \u003cem\u003eMicrobiome under changing climate\u003c/em\u003e (pp. 69-84). Woodhead Publishing. https://doi.org/10.1016/B978-0-323-90571-8.00003-1\u003c/li\u003e\n\u003cli\u003ePrajapati, S. K., Kumar, V., Dayal, P., Gairola, A., Borate, R. B., \u0026amp; Srivastava, R. (2023). THE ROLE OF CARBON IN LIFE\u0026apos;S BLUEPRINT AND CARBON CYCLE UNDER-STANDING EARTH\u0026apos;S ESSENTIAL CYCLING SYSTEM: BENEFITS AND HARMS TO OUR PLANET. \u003cem\u003eInternational Journal\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(1), 21-32. https://doi.org/10.5281/zenodo.8385430\u003c/li\u003e\n\u003cli\u003eRaza, T., Qadir, M. F., Khan, K. S., Eash, N. S., Yousuf, M., Chatterjee, S., ... \u0026amp; Oetting, J. N. (2023). Unraveling the potential of microbes in decomposition of organic matter and release of carbon in the ecosystem. \u003cem\u003eJournal of Environmental Management\u003c/em\u003e, \u003cem\u003e344\u003c/em\u003e, 118529. https://doi.org/10.1016/j.jenvman.2023.118529\u003c/li\u003e\n\u003cli\u003eIven, H., Walker, T. W., \u0026amp; Anthony, M. (2023). Biotic interactions in soil are underestimated drivers of microbial carbon use efficiency. \u003cem\u003eCurrent Microbiology\u003c/em\u003e, \u003cem\u003e80\u003c/em\u003e(1), 13. https://doi.org/10.1007/s00284-022-02979-2 \u003c/li\u003e\n\u003cli\u003eButcher, K. R. (2023). Edaphic and Climatic Regulation of Microbial Carbon-Use Efficiency in Managed Semi-Arid Systems. https://doi.org/10.26076/d8eb-cf83\u003c/li\u003e\n\u003cli\u003eBogati, K., \u0026amp; Walczak, M. (2022). The impact of drought stress on soil microbial community, enzyme activities and plants. \u003cem\u003eAgronomy\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 189. https://doi.org/10.3390/agronomy12010189\u003c/li\u003e\n\u003cli\u003eAmante, G., \u0026amp; Wedajo, M. (2024). Impacts of Climate change on soil microbial diversity, distribution and abundance. \u003cem\u003eInternational Journal on Food, Agriculture and Natural Resources\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(2), 158-168. https://doi.org/10.46676/ij-fanres.v5i2.342\u003c/li\u003e\n\u003cli\u003eGriffin-LaHue, D., Wang, D., Gaudin, A. C., Durbin-Johnson, B., Settles, M. L., \u0026amp; Scow, K. M. (2023). Extended soil surface drying triggered by subsurface drip irrigation decouples carbon and nitrogen cycles and alters microbiome composition. \u003cem\u003eFrontiers in Soil Science\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, 1267685. https://doi.org/10.3389/fsoil.2023.1267685\u003c/li\u003e\n\u003cli\u003eTeixeira, P. P., Vidal, A., Teixeira, A. P., Souza, I. F., Hurtarte, L. C., Silva, D. H., ... \u0026amp; Silva, I. R. (2024). Decoding the rhizodeposit-derived carbon\u0026apos;s journey into soil organic matter. \u003cem\u003eGeoderma\u003c/em\u003e, \u003cem\u003e443\u003c/em\u003e. https://doi.org/10.1016/j.geoderma.2024.116811\u003c/li\u003e\n\u003cli\u003eEl Mouridi, Z., Ziri, R., Douaik, A., Bennani, S., Lembaid, I., Bouharou, L., ... \u0026amp; Moussadek, R. (2023). Comparison between Walkley-Black and loss on ignition methods for organic matter estimation in different Moroccan soils. \u003cem\u003eEcological Engineering \u0026amp; Environmental Technology\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e. https://doi.org/10.12912/27197050/163121\u003c/li\u003e\n\u003cli\u003eIbrahim, K. H., Wang, L., Wu, Q., Duan, Y., Ma, C., \u0026amp; Zhang, S. (2021). Soil Phosphorus management based on changes in Olsen P and P budget under Long-term fertilization experiment in fluvo-aquic soil. https://doi.org/10.15159/AR.21.006\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Islamic University","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":"Biogeochemical, Carbon cycling, Microbial biomass, Drought, Climate change, Phospholipid fatty acid","lastPublishedDoi":"10.21203/rs.3.rs-6475985/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6475985/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoil is the largest terrestrial carbon reservoir, with microorganisms playing a pivotal role in organic matter decomposition and carbon stabilization. Drought, intensified by climate change, alters microbial dynamics and soil carbon cycling. This study investigates the effects of drought-induced stress on microbial community composition, carbon allocation, and soil biochemical properties in agricultural and forest soils in Bangladesh. Soils from a drought-prone maize field and a semi-natural forest site in Kushtia, Bangladesh, were incubated in mesocosms under control (25% WHC) and drought (10% WHC) conditions, with and without carbon amendments. Microbial activity and structure were assessed through PLFA analysis and 16S rRNA sequencing. Soil CO₂ emissions, microbial biomass carbon (MBC), dissolved organic carbon (DOC), and soil nutrients were also analyzed. Drought significantly reduced microbial biomass (45.3 to 32.3 nmol PLFA g⁻\u0026sup1;, p\u0026thinsp;=\u0026thinsp;0.021), MBC (215.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4 to 128.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7 \u0026micro;g C g⁻\u0026sup1;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and CO₂ emissions (3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 to 2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;g CO₂-C g⁻\u0026sup1; day⁻\u0026sup1;). DOC decreased from 12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 to 7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mg C g⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.012). Gram-negative bacteria declined by 41.2%, while Gram-positive taxa and fungi increased, indicating a shift to drought-tolerant communities. Carbon use efficiency slightly increased (56.0\u0026ndash;59.5%). Drought suppresses microbial activity and alters community structure, reducing soil carbon and nutrient availability. These findings underscore the need for drought-resilient soil management in climate-sensitive regions like Bangladesh.\u003c/p\u003e","manuscriptTitle":"Microbial and Biogeochemical Responses to Drought in Soil Carbon Cycling Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 10:13:45","doi":"10.21203/rs.3.rs-6475985/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":"8fc779c5-af9b-4fdb-8293-cfabc2d7f616","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47450564,"name":"Biogeography"},{"id":47450565,"name":"General Microbiology"},{"id":47450566,"name":"Agroecology"},{"id":47450567,"name":"Renewable Resources"},{"id":47450568,"name":"Applied Biochemistry"}],"tags":[],"updatedAt":"2025-04-22T10:13:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-22 10:13:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6475985","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6475985","identity":"rs-6475985","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}