Microbial carbon fixation is strongly regulated by microclimates: temperature shaped elevational variations of CO₂ fixation pathways and potential in warm-temperate forests

Abstract

Soil microbial CO₂ fixation plays a pivotal role in the global terrestrial carbon cycle. In forest ecosystems, the changes in the pathways and potential of microbial CO₂ fixation as well as their underlying mechanisms along environmental gradients remain unclear, particularly considering the effects of variations in forest microclimates. To address this gap, this study employed metagenomic sequencing and ¹³C stable isotope labeling to investigate CO₂-fixing microorganisms, pathways, potential, and their relationships with environmental factors across elevations from 894 to 2200 m. The results showed that the dominant CO₂-fixing microorganisms were Proteobacteria, Actinobacteria and Acidobacteria in this study, while their compositional proportions showing clear disparities, ranging from 30.88 to 48.17%, 16.02 to 30.75% and 13.55 to 23.52% with elevations, respectively. Among the 6 pathways detected, the dicarboxylate–hydroxybutyrate (DC/4-HB) and 3-hydroxypropionate (3-HP) cycles dominated CO₂ fixation at low elevations, and the reductive citrate (rTCA) cycle prevailed at high elevations. Soil microbial CO₂ fixation rates ranged from 22.46 to 78.99 mg m^–2 d^–1, with significantly higher values at high elevations than that at middle–low elevations. As proxies for forest microclimates, air and soil temperatures were the main drivers of elevational changes in CO₂ fixation pathways and potential. Moreover, they indirectly inhibited the abundance of CO₂-fixing microorganisms and pathways by curbing the accumulation of soil nutrients (microbial biomass carbon, ammonium nitrogen, reactive organic carbon and available potassium), thereby weakening microbial CO₂ fixation potential (path coefficient = 0.78). Microclimates also directly and significantly reduced the microbial CO₂ fixation potential (path coefficient = –0.67). Our results revealed the elevational variations and microclimate-driven mechanisms of microbial CO₂ fixation, with implications for soil carbon cycling and ecosystem responses to climate warming.

Keywords

Forest microclimates / Soil microorganisms / CO₂ fixation pathways / CO₂ fixation potential / Elevational variation

References

[1] AbdelRahman MAE. An overview of land degradation, desertification and sustainable land management using GIS and remote sensing applications. Rend Fis Acc Lincei, 2023, 34(3): 767-808

[2] AbdelRahman MAE. Reimagining soil stewardship in the anthropocene: nature-positive pathways, pedological perspectives, and land use innovations for soil health and security. Soil Secur, 2025, 21: 100206

[3] Akinyede R, Taubert M, Schrumpf M, Trumbore S, Küsel K. Rates of dark CO₂ fixation are driven by microbial biomass in a temperate forest soil. Soil Biol Biochem, 2020, 150: 107950

[4] Akinyede R, Taubert M, Schrumpf M, Trumbore S, Küsel K. Temperature sensitivity of dark CO₂ fixation in temperate forest soils. Biogeosciences, 2022, 19(17): 4011-4028

[5] Ananyeva K, Wang W, Smucker A, Kravchenko A. Can intra-aggregate pore structures affect the aggregate's effectiveness in protecting carbon?. Soil Biol Biochem, 2013, 57: 868-875

[6] Bárcenas-Moreno G, Gómez-Brandón M, Rousk J, Bååth E. Adaptation of soil microbial communities to temperature: comparison of fungi and bacteria in a laboratory experiment. Glob Change Biol, 2009, 15: 2950-2957

[7] Bar-Even A, Noor E, Milo R. A survey of carbon fixation pathways through a quantitative lens. J Exp Bot, 2012, 63(6): 2325-2342

[8] Becker A, Körner C, Brun J-J, Guisan A, Tappeiner U. Ecological and land use studies along elevational gradients. Mt Res Dev, 2007, 27(1): 58-65

[9] Berg IA, Kockelkorn D, Ramos-Vera WH, Say RF, Zarzycki J, Hügler M, Alber BE, Fuchs G. Autotrophic carbon fixation in archaea. Nat Rev Microbiol, 2010, 8(6): 447-460

[10] Chen YJ, Neilson JW, Kushwaha P, Maier RM, Barberán A. Life-history strategies of soil microbial communities in an arid ecosystem. ISME J, 2020, 15(3): 649-657

[11] Claassens NJ, Sousa DZ, dos Santos VAPM, de Vos WM, van der Oost J. Harnessing the power of microbial autotrophy. Nat Rev Microbiol, 2016, 14: 692-706

[12] De Frenne P, Lenoir J, Luoto M, Scheffers BR, Zellweger F, Aalto J, Ashcroft MB, Christiansen DM, Decocq G, De Pauw K, Govaert S, Greiser C, Gril E, Hampe A, Jucker T, Klinges DH, Koelemeijer IA, Lembrechts JJ, Marrec R, Meeussen C, Ogée J, Tyystjärvi V, Vangansbeke P, Hylander K. Forest microclimates and climate change: importance, drivers and future research agenda. Glob Change Biol, 2021, 27(11): 2279-2297

[13] Dong ZM, Layzell D. H₂ oxidation, O₂ uptake and CO₂ fixation in hydrogen treated soils. Plant Soil, 2001, 229: 1-12

[14] Erb TJ. Carboxylases in natural and synthetic microbial pathways. Appl Environ Microbiol, 2011, 77(24): 8466-8477

[15] Fa KY, Liu Z, Zhang YQ, Qin SG, Wu B, Liu JB. Abiotic carbonate dissolution traps carbon in a semiarid desert. Sci Rep, 2016, 6: 23570

[16] Fan W, Tang F, Wang J, Dong J, Xing J, Shi F. Drought-induced recruitment of specific root-associated bacteria enhances adaptation of alfalfa to drought stress. Front Microbiol, 2023, 14: 1114400

[17] Fröberg M, Grip H, Tipping E, Svensson M, Strömgren M, Kleja DB. Long-term effects of experimental fertilization and soil warming on dissolved organic matter leaching from a spruce forest in Northern Sweden. Geoderma, 2013, 200–201: 172-179

[18] Gao E-B, Wu JH, Ye PL, Qiu HY, Chen HY, Fang Z. Rewiring carbon flow in Synechocystis PCC 6803 for a high rate of CO₂-to-ethanol under an atmospheric environment. Front Microbiol, 2023, 14: 1211004

[19] Hopkinson BM, Dupont CL, Allen AE, Morel FMM. Efficiency of the CO₂-concentrating mechanism of diatoms. Proc Natl Acad Sci U S A, 2011, 108(10): 3830-3837

[20] Huang JM, Baker Brett J, Li JT, Wang Y. New microbial lineages capable of carbon fixation and nutrient cycling in deep-sea sediments of the northern South China Sea. Appl Environ Microbiol, 2019, 85(15): e00523-e1519

[21] Huang Q, Huang YM, Wang BR, Dippold MA, Li HH, Li N, Jia PH, Zhang HX, An SS, Kuzyakov Y. Metabolic pathways of CO₂ fixing microorganisms determined C-fixation rates in grassland soils along the precipitation gradient. Soil Biol Biochem, 2022, 172: 108764

[22] Huber H, Gallenberger M, Jahn U, Eylert E, Berg IA, Kockelkorn D, Eisenreich W, Fuchs G. A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis. Proc Natl Acad Sci U S A, 2008, 105(22): 7851-7856

[23] Hügler M, Huber H, Molyneaux SJ, Vetriani C, Sievert SM. Autotrophic CO₂ fixation via the reductive tricarboxylic acid cycle in different lineages within the phylum Aquificae: evidence for two ways of citrate cleavage. Environ Microbiol, 2007, 9(1): 81-92

[24] Jansson JK, Hofmockel KS. Soil microbiomes and climate change. Nat Rev Microbiol, 2020, 18: 35-46

[25] Jørgensen BB, Findlay AJ, Pellerin A. The biogeochemical sulfur cycle of marine sediments. Front Microbiol, 2019, 10: 849

[26] Kemppinen J, Lembrechts JJ, Van Meerbeek K, Carnicer J, Chardon NI, Kardol P, Lenoir J, Liu D, Maclean I, Pergl J, Saccone P, Senior RA, Shen T, Słowińska S, Vandvik V, von Oppen J, Aalto J, Ayalew B, Bates O, Bertelsmeier C, Bertrand R, Beugnon R, Borderieux J, Brůna J, Buckley L, Bujan J, Casanova-Katny A, Christiansen DM, Collart F, De Lombaerde E, De Pauw K, Depauw L, Di Musciano M, Díaz Borrego R, Díaz-Calafat J, Ellis-Soto D, Esteban R, de Jong GF, Gallois E, Garcia MB, Gillerot L, Greiser C, Gril E, Haesen S, Hampe A, Hedwall P-O, Hes G, Hespanhol H, Hoffrén R, Hylander K, Jiménez-Alfaro B, Jucker T, Klinges D, Kolstela J, Kopecký M, Kovács B, Maeda EE, Máliš F, Man M, Mathiak C, Meineri E, Naujokaitis-Lewis I, Nijs I, Normand S, Nuñez M, Orczewska A, Peña-Aguilera P, Pincebourde S, Plichta R, Quick S, Renault D, Ricci L, Rissanen T, Segura-Hernández L, Selvi F, Serra-Diaz JM, Soifer L, Spicher F, Svenning J-C, Tamian A, Thomaes A, Thoonen M, Trew B, Van de Vondel S, van den Brink L, Vangansbeke P, Verdonck S, Vitkova M, Vives-Ingla M, von Schmalensee L, Wang R, Wild J, Williamson J, Zellweger F, Zhou X, Zuza EJ, De Frenne P. Microclimate, an important part of ecology and biogeography. Glob Ecol Biogeogr, 2024, 33(6): e13834

[27] Körner C. The use of ‘altitude’ in ecological research. Trends Ecol Evol, 2007, 22(11): 569-574

[28] Li D, Liu C-M, Luo R, Sadakane K, Lam T-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, 2015, 31(10): 1674-1676

[29] Li Z, Kravchenko AN, Cupples A, Guber AK, Kuzyakov Y, Philip Robertson G, Blagodatskaya E. Composition and metabolism of microbial communities in soil pores. Nat Commun, 2024, 15(1): 3578

[30] Liang C, Schimel JP, Jastrow JD. The importance of anabolism in microbial control over soil carbon storage. Nat Microbiol, 2017, 2(8): 17105

[31] Liang YJ, Fu R, Sailike A, Hao HJ, Yu ZC, Wang R, Peng N, Li SC, Zhang W, Liu YY. Soil labile organic carbon and nitrate nitrogen are the main factors driving carbon-fixing pathways during vegetation restoration in the Loess Plateau, China. Agric Ecosyst Environ, 2025, 378(8): 109283

[32] Liao H, Qin F, Wang K, Zhang YC, Hao XL, Chen WL, Huang QY. Long-term chemical fertilization-driving changes in soil autotrophic microbial community depresses soil CO₂ fixation in a Mollisol. Sci Total Environ, 2020, 748: 141317

[33] Liao H, Hao XL, Qin F, Delgado-Baquerizo M, Liu YR, Zhou JZ, Cai P, Chen WL, Huang QY. Microbial autotrophy explains large-scale soil CO₂ fixation. Glob Change Biol, 2023, 29(1): 231-242

[34] Liu ZF, Klatt CG, Ludwig M, Rusch DB, Jensen SI, Kühl M, Ward DM, Bryant DA. Candidatus Thermochlorobacter aerophilum: an aerobic chlorophotoheterotrophic member of the phylum Chlorobi defined by metagenomics and metatranscriptomics. ISME J, 2012, 6(10): 1869-1882

[35] Liu JB, Kong WD, Zhang GS, Khan A, Guo GX, Zhu CM, Wei XJ, Kang SC, Morgan-Kiss RM. Diversity and succession of autotrophic microbial community in high-elevation soils along deglaciation chronosequence. FEMS Microbiol Ecol, 2016, 92(10 ArticleID: fiw160

[36] Liu Z, Sun YF, Zhang YQ, Feng W, Lai ZR, Fa KY, Qin SG. Metagenomic and ¹³C tracing evidence for autotrophic atmospheric carbon absorption in a semiarid desert. Soil Biol Biochem, 2018, 125: 156-166

[37] Liu ZH, Wang K, Chen Y, Tan TW, Nielsen J. Third-generation biorefineries as the means to produce fuels and chemicals from CO₂. Nat Catal, 2020, 3(3): 274-288

[38] Liu BL, Hou LJ, Zheng YL, Zhang ZX, Tang XF, Mao TQ, Du JZ, Bi QQ, Dong HP, Yin GY. Dark carbon fixation in intertidal sediments: controlling factors and driving microorganisms. Water Res, 2022, 216 ArticleID: 118381

[39] Liu BL, Qi L, Zheng YL, Zhang C, Zhou J, An ZR, Wang B, Lin ZK, Yao C, Wang YX, Yin GY, Dong HP, Li XF, Liang X, Han P, Liu M, Zhang GS, Cui Y, Hou LJ. Four years of climate warming reduced dark carbon fixation in coastal wetlands. ISME J, 2024, 18(1 ArticleID: wrae138

[40] Ma LW, Liu L, Lu YS, Chen L, Zhang ZC, Zhang HW, Wang XR, Shu L, Yang QP, Song QN, Peng QH, Yu ZP, Zhang J. When microclimates meet soil microbes: temperature controls soil microbial diversity along an elevational gradient in subtropical forests. Soil Biol Biochem, 2022, 166: 108566

[41] Maxim R-B, Nicole D, Manuel K. Genetic evidence for two carbon fixation pathways (the Calvin-Benson-Bassham cycle and the Reverse Tricarboxylic Acid cycle) in symbiotic and free-living bacteria. Msphere, 2019, 4(1): e00394-e1318

[42] McKinley DC, Ryan MG, Birdsey RA, Giardina CP, Harmon ME, Heath LS, Houghton RA, Jackson RB, Morrison JF, Murray BC, Pataki DE, Skog KE. A synthesis of current knowledge on forests and carbon storage in the United States. Ecol Appl, 2011, 21(6): 1902-1924

[43] Melillo JM, Frey SD, DeAngelis KM, Werner WJ, Bernard MJ, Bowles FP, Pold G, Knorr MA, Grandy AS. Long-term pattern and magnitude of soil carbon feedback to the climate system in a warming world. Science, 2017, 358(6359): 101-105

[44] Miltner A, Richnow H, Kopinke F-D, Kästner M. Assimilation of CO₂ by soil microorganisms and transformation into soil organic matter. Org Geochem, 2004, 35(9): 1015-1024

[45] Mohamed ES, Abu-hashim M, AbdelRahman MAE, Schütt B, Lasaponara R. Evaluating the effects of human activity over the last decades on the soil organic carbon pool using satellite imagery and GIS techniques in the Nile Delta area, Egypt. Sustainability, 2019, 11(9): 2644

[46] Munda S, Nayak AK, Shahid M, Bhaduri D, Chatterjee D, Mohanty S, Tripathi R, Kumar U, Kumar A, Khanam R, Jambhulkar N. Soil quality assessment of lowland rice soil of eastern India: implications of rice husk biochar application. Heliyon, 2023, 9(7): e17835

[47] Naylor D, Sadler N, Bhattacharjee A, Graham EB, Anderton CR, McClure R, Lipton M, Hofmockel KS, Jansson JK. Soil microbiomes under climate change and implications for carbon cycling. Annu Rev Environ Resour, 2020, 45: 29-59

[48] Peng XQ, Wang W. Stoichiometry of soil extracellular enzyme activity along a climatic transect in temperate grasslands of northern China. Soil Biol Biochem, 2016, 98: 74-84

[49] Philippot L, Chenu C, Kappler A, Rillig MC, Fierer N. The interplay between microbial communities and soil properties. Nat Rev Microbiol, 2024, 22(4): 226-239

[50] Pieter DF, Lenoir J, Luoto M, Scheffers BR, Zellweger F, Aalto J, Ashcroft MB, Christiansen DM, Decocq G, De Pauw K, Govaert S, Greiser C, Gril E, Hampe A, Jucker T, Klinges DH, Koelemeijer IA, Lembrechts JJ, Marrec R, Meeussen C, Ogée J, Tyystjärvi V, Vangansbeke P, Hylander K. Forest microclimates and climate change: Importance, drivers and future research agenda. Glob Change Biol, 2021, 27(11): 2279-2297

[51] Qi L, Zheng Y, Hou L, Liu B, Zhou J, An Z, Wu L, Chen F, Lin Z, Yin G, Dong H, Li X, Liang X, Liu M. Potential response of dark carbon fixation to global warming in estuarine and coastal waters. Glob Change Biol, 2023, 29(13): 3821-3832

[52] Qiu S, Fang MZ, Yu Q, Niu T, Liu HJ, Wang F, Xu CL, Ai MS, Zhang JY. Study of spatialtemporal changes in Chinese forest eco-space and optimization strategies for enhancing carbon sequestration capacity through ecological spatial network theory. Sci Total Environ, 2023, 859: 160035

[53] Shi JY, Gong JR, Li XB, Zhang ZH, Zhang WY, Li Y, Song LY, Zhang SQ, Dong JJ, Baoyin T-t. Plant–microbial linkages regulate soil organic carbon dynamics under phosphorus application in a typical temperate grassland in northern China. Agric Ecosyst Environ, 2022, 335: 108006

[54] SidAhmed A, Holecz F, Gatti L, Barbieri M, Ahmed A, Gafar A, AbdelRahman MAE, Yassin A. Inference of forest tree volume using synthetic aperture in central Sudan. Lett Spat Resour Sci, 2025, 18(1): 1

[55] Spohn M, Müller K, Höschen C, Mueller CW, Marhan S. Dark microbial CO₂ fixation in temperate forest soils increases with CO₂ concentration. Glob Change Biol, 2020, 26(3): 1926-1935

[56] Sun YF, Yang MQ, Ding Y, Deng DT, Lai ZR, Long WX. The contributions of dark microbial CO₂ fixation to soil organic carbon along a tropical secondary forest chronosequence on Hainan Island, China. CATENA, 2024, 247: 108556

[57] Tao Y, Wang Y, Cui YH, Sun R, Zhang B, Qu JH, Cai HG, Zhang Y. Bioenhanced remediation of dibutyl phthalate contaminated black soil by immobilized biochar microbiota. J Environ Manage, 2025, 373: 123317

[58] Tolli J, King G. Diversity and structure of bacterial chemolithotrophic communities in pine forest and agroecosystem soils. Appl Environ Microbiol, 2005, 71(12): 8411-8418

[59] Walker TW, Kaiser C, Strasser F, Herbold CW, Leblans NI, Woebken D, Janssens IA, Sigurdsson BD, Richter A. Microbial temperature sensitivity and biomass change explain soil carbon loss with warming. Nat Clim Chang, 2018, 8(10): 885-889

[60] Wang XX, Zhang W, Liu Y, Jia ZJ, Li H, Yang YF, Wang DM, He HB, Zhang XD. Identification of microbial strategies for labile substrate utilization at phylogenetic classification using a microcosm approach. Soil Biol Biochem, 2021, 153: 107970

[61] Wang XY, Wang YR, Wang Y, Yang JY, He HY, Ren YC, Wang CL, Li HB, Zhang XZ, Zhang HF, Wang JJ, Yang XQ, Song HJ. Tree mycorrhizal associations strongly mediate soil microbial β-diversity along an elevational gradient in a warm-temperate forest. Appl Soil Ecol, 2025, 205(2): 105776

[62] Wild J, Kopecky M, Macek M, Sanda M, Jankovec J, Haase T. Climate at ecologically relevant scales: A new temperature and soil moisture logger for long-term microclimate measurement. Agric for Meteorol, 2019, 268: 40-47

[63] Willing CE, Pellitier PT, Van Nuland ME, Alvarez-Manjarrez J, Berrios L, Chin KN, Villa LM, Yeam JJ, Bourque SD, Tripp W, Leshyk VO, Peay KG. A risk assessment framework for the future of forest microbiomes in a changing climate. Nat Clim Chang, 2024, 14(5): 448-461

[64] Witt A, Pozzi R, Diesch S, Hädicke O, Grammel H. New light on ancient enzymes – in vitro CO₂ fixation by pyruvate synthase of Desulfovibrio africanus and Sulfolobus acidocaldarius. FEBS J, 2019, 286(22): 4494-4508

[65] Wu LW, Zhang Y, Guo X, Ning DL, Zhou XS, Feng JJ, Yuan MM, Liu S, Guo JJ, Gao ZP, Ma J, Kuang JL, Jian SY, Han S, Yang ZF, Ouyang Y, Fu Y, Xiao NJ, Liu XD, Wu LY, Zhou AF, Yang YF, Tiedje JM, Zhou JZ. Reduction of microbial diversity in grassland soil is driven by long-term climate warming. Nat Microbiol, 2022, 7(7): 1054-1062

[66] Wu HW, Cui HL, Fu CX, Li R, Qi FY, Liu ZL, Yang G, Xiao KQ, Qiao M. Unveiling the crucial role of soil microorganisms in carbon cycling: a review. Sci Total Environ, 2024, 909: 168627

[67] Xiao KQ, Ge T, Wu XH, Peacock CL, Zhu ZK, Peng JJ, Bao P, Wu JS, Zhu YG. Metagenomic and ¹⁴C tracing evidence for autotrophic microbial CO₂ fixation in paddy soils. Environ Microbiol, 2021, 23(2): 924-933

[68] Xu X, Sherry RA, Niu SL, Li DJ, Luo YQ. Net primary productivity and rain-use efficiency as affected by warming, altered precipitation, and clipping in a mixed-grass prairie. Glob Change Biol, 2013, 19(9): 2753-2764

[69] Yamamoto M, Arai H, Ishii M, Igarashi Y. Role of two 2-oxoglutarate: ferredoxin oxidoreductases in Hydrogenobacter thermophilus under aerobic and anaerobic conditions. FEMS Microbiol Lett, 2006, 263(2): 189-193

[70] Yang Y, Dou YX, Wang BR, Xue ZJ, Wang YQ, An SS, Chang SX. Deciphering factors driving soil microbial life‐history strategies in restored grasslands. iMeta, 2023, 2(1): e66

[71] Yang YX, Yang XD, Gong L, Ding ZL, Zhu HQ, Tang JH, Li XZ. Changes in soil microbial carbon fixation pathways along the oasification process in arid desert region: a confirmation based on metagenome analysis. CATENA, 2024, 239: 107955

[72] Zhang JT, Zhang F. Ecological relations between forest communities and environmental variables in the Lishan Mountain Nature Reserve. China Afr J Agric Res, 2011, 6(2): 248-259

[73] Zhang MG, Zhou ZK, Chen WY, Cannon CH, Raes N, Slik JF. Major declines of woody plant species ranges under climate change in Yunnan, China. Divers Distrib, 2014, 20(4): 405-415

[74] Zheng ZC, Liu BY, Fang X, Fa KY, Liu Z. Dryland farm soil may fix atmospheric carbon through autotrophic microbial pathways. CATENA, 2022, 214(5): 106299

RIGHTS & PERMISSIONS

Northeast Forestry University