Biocrust-phosphogypsum interactions enhance microbial nutrient cycling and surface properties in phosphogypsum stockpiles

Abstract

The accumulation of phosphogypsum (PG), a solid byproduct of phosphoric acid production, poses a significant ecological risk to the surrounding environment. Biologically driven in situ remediation represents an eco-friendly approach. Biocrusts play an irreplaceable role in the ecological restoration of degraded ecosystems. However, their potential in PG systems has received limited attention. To explore the interactions between PG and biocrusts, this study sampled PG stockpiles of different ages (0, 2, and 12 years), including the biocrust layer (natural thickness: 2–20 mm) and the underlying PG layer (0–5 cm below the biocrusts). The results showed that the fine particle size of PG (< 100 µm), together with its abundant available phosphorus (AP) and exchangeable calcium ions, provided favorable conditions for biocrust formation and development. After 12 years, total chlorophyll (Tchl), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) in the biocrust layer increased by 71.00-fold, 81.37-fold, and 108.37-fold, respectively. Moreover, the PG – biocrust interface interactions enhanced microbial nutrient cycling, particularly the carbon cycle (e.g., inorganic carbon fixation genes increased by 29.11-fold, intracellular polymer degradation by 17.52-fold, and fermentation by 25.59-fold). These biological processes ultimately led to marked improvements in the physicochemical properties of the surface PG layer (0–5 cm). Specifically, soil organic carbon (SOC) increased by 175.28%, pH rose from 3.39 to 5.61, and soluble fluoride content decreased to 14.20% of the original level in PG layer (0–5 cm). Overall, this study provides a potential pathway and a theoretical basis for the in situ green remediation of PG stockpiles.

Keywords

phosphogypsum / biocrusts / metagenomic analysis / carbon cycle / calcium-bound soil organic carbon

References

[1] Bounaga A, Alsanea A, Lyamlouli K, et al.. Microbial transformations by sulfur bacteria can recover value from phosphogypsum: A global problem and a possible solution [J]. Biotechnology Advances, 2022, 57: 107949

[2] Akfas F, Elghali A, Aboulaich A, et al.. Exploring the potential reuse of phosphogypsum: A waste or a resource? [J]. Science of the Total Environment, 2024, 908: 168196

[3] Kijjanapanich P, Annachhatre A P, Lens P N L. Biological sulfate reduction for treatment of gypsum contaminated soils, sediments, and solid wastes [J]. Critical Reviews in Environmental Science and Technology, 2014, 44(10): 1037-1070

[4] Xiang J-c, Qiu J-p, Zheng P-k, et al.. Usage of biowashing to remove impurities and heavy metals in raw phosphogypsum and calcined phosphogypsum for cement paste preparation [J]. Chemical Engineering Journal, 2023, 451: 138594

[5] Bilal E, Bellefqih H, Bourgier V, et al.. Phosphogypsum circular economy considerations: A critical review from more than 65 storage sites worldwide [J]. Journal of Cleaner Production, 2023, 414: 137561

[6] Wu H, Zhu X-z, Tang C-j, et al.. Variation of humic carbon and microbial communities in bauxite residue following co-application of straw and phosphogypsum [J]. Journal of Central South University, 2024, 31(2): 460-476

[7] Singh A, Yadav V K, Gautam H, et al.. The role of plant growth promoting rhizobacteria in strengthening plant resistance to fluoride toxicity: A review [J]. Frontiers in Microbiology, 2023, 14: 1271034

[8] Barrow N J, Hartemink A E. The effects of pH on nutrient availability depend on both soils and plants [J]. Plant and Soil, 2023, 487(1): 21-37

[9] Zheng S, Bai D-s, Yang X, et al.. Changes in soil microecology of gangue reclamation areas after 10 years of in situ restoration with herbaceous plants (Artemisia sacrorum and Imperata cylindrica) and trees (Populus spp.) [J]. Ecological Engineering, 2022, 182: 106719

[10] Bai D-s, Yang X, Lai J-l, et al.. In situ restoration of soil ecological function in a coal gangue reclamation area after 10 years of elm/poplar phytoremediation [J]. Journal of Environmental Management, 2022, 305: 114400

[11] Liu Z-x, Gu H-d, Yao Q, et al.. Soil pH and carbon quality index regulate the biogeochemical cycle couplings of carbon, nitrogen and phosphorus in the profiles of Isohumosols [J]. Science of the Total Environment, 2024, 922: 171269

[12] Dai Z-m, Xiong X-q, Zhu H, et al.. Association of biochar properties with changes in soil bacterial, fungal and fauna communities and nutrient cycling processes [J]. Biochar, 2021, 3(3): 239-254

[13] Bekchanova M, Kuppens T, Cuypers A, et al.. Biochar’s effect on the soil carbon cycle: A rapid review and meta-analysis [J]. Biochar, 2024, 6(1): 88

[14] Wang S-c, Yuan X-q, Li T, et al.. Changes in soil microbe-mediated carbon, nitrogen and phosphorus cycling during spontaneous succession in abandoned Pb Zn mining areas [J]. Science of the Total Environment, 2024, 920: 171018

[15] Sun J-y, Yu K-l, Chen N, et al.. Biocrusts modulate carbon losses under warming across global drylands: A Bayesian meta-analysis [J]. Soil Biology and Biochemistry, 2024, 188: 109214

[16] Monus B D, Nghalipo E N, Marufu V J, et al.. Contributions of hypolithic communities to surface soil organic carbon across a hyperarid-to-arid climate gradient [J]. Geoderma, 2023, 433: 116428

[17] Weber B, Belnap J, Büdel B, et al.. What is a biocrust? A refined, contemporary definition for a broadening research community [J]. Biological Reviews, 2022, 97(5): 1768-1785

[18] Kidron G J, Lichner L, Fischer T, et al.. Mechanisms for biocrust-modulated runoff generation – A review [J]. Earth-Science Reviews, 2022, 231: 104100

[19] Wang Q, Zhang Q-y, Han Y-c, et al.. Carbon cycle in the microbial ecosystems of biological soil crusts [J]. Soil Biology and Biochemistry, 2022, 171: 108729

[20] Belnap J, Phillips S L, Witwicki D L, et al.. Visually assessing the level of development and soil surface stability of cyanobacterially dominated biological soil crusts [J]. Journal of Arid Environments, 2008, 72(7): 1257-1264

[21] Havrilla C A, Chaudhary V B, Ferrenberg S, et al.. Towards a predictive framework for biocrust mediation of plant performance: A meta-analysis [J]. Journal of Ecology, 2019, 107(6): 2789-2807

[22] Leong Y K, Chang J S. Bioremediation of heavy metals using microalgae: Recent advances and mechanisms [J]. Bioresource Technology, 2020, 303: 122886

[23] Yang K, Zhao Y-g, Gao L-qian. Biocrust succession improves soil aggregate stability of subsurface after “Grain for Green” Project in the Hilly Loess Plateau, China [J]. Soil and Tillage Research, 2022, 217: 105290

[24] Wu L, Zhang C, Vadiveloo A, et al.. Efficient nutrient recycling from wastewater to deserts: A comparative study on biocrust cyanobacteria performance [J]. Chemical Engineering Journal, 2024, 491: 151927

[25] Cheng C, Gao M, Zhang Y-d, et al.. Effects of disturbance to moss biocrusts on soil nutrients, enzyme activities, and microbial communities in degraded karst landscapes in southwest China [J]. Soil Biology and Biochemistry, 2021, 152: 108065

[26] Geng Y-c, Zhou P-p, Wang Z-c, et al.. The roles of rare and abundant microbial species in the primary succession of biological soil crusts are differentiated in metal tailings ponds with different states [J]. Journal of Hazardous Materials, 2024, 472: 134577

[27] Kuang X-l, Peng L, Chen S-n, et al.. Immobilization of metal(loid)s from acid mine drainage by biological soil crusts through biomineralization [J]. Journal of Hazardous Materials, 2023, 443: 130314

[28] Kuang X-l, Hu Y-l, Peng L, et al.. Application of biological soil crusts for efficient cadmium removal from acidic mine wastewater [J]. Journal of Hazardous Materials, 2024, 465: 133524

[29] Aboal J R, Couto J A, Fernández J A, et al.. Physiological responses to atmospheric fluorine pollution in transplants of Pseudoscleropodium purum [J]. Environmental Pollution, 2008, 153(3): 602-609

[30] Gobbie K G, Pietrasiak N, Jusko B M, et al.. Climate and gypsum parent material shape biocrust communities and moss ecology in the Chihuahuan and Mojave Deserts [J]. Geoderma, 2025, 453: 117131

[31] Belobrov V P, Lebedeva M P, Abrosimov K N, et al.. Microdiagnostics of initial pedogenesis on a phosphogypsum dump [J]. Spanish Journal of Soil Science, 2018, 8: 2601

[32] Nieland D L, Wilson C A, Fleeger J W. Preliminary evaluation of the use of phosphogypsum for reef substrate in the gulf of Mexico [J]. Gulf of Mexico Science, 1998, 16(1): 9

[33] Tayibi H, Choura M, Lopez F A, et al.. Environmental impact and management of phosphogypsum [J]. Journal of Environmental Management, 2009, 90(8): 2377-2386

[34] Cao Y-m, Cui Y, Yu X-k, et al.. Bibliometric analysis of phosphogypsum research from 1990 to 2020 based on literatures and patents [J]. Environmental Science and Pollution Research, 2021, 28(47): 66845-66857

[35] Zhang C, Chen X-r, Zhou K-q, et al.. Synergistic effects of clays and cyanobacteria on the accumulation dynamics of soil organic carbon in artificial biocrusts [J]. Journal of Environmental Management, 2025, 374: 124110

[36] HJ 1415-2025. Technical specification for pollution control of utilization and harmless storage of phosphogypsum [S], 2025(in Chinese)

[37] Deiss L, Sall A, Demyan M S, et al.. Does crop rotation affect soil organic matter stratification in tillage systems? [J]. Soil and Tillage Research, 2021, 209: 104932

[38] Frohloff D, Rytz A, Clare M, et al.. Design-based soil sampling strategy for unbiased and precise soil characteristics in arable fields [J]. Biology and Fertility of Soils, 2026, 62(6): 929-934

[39] Lan S-b, Ouyang H-l, Wu L, et al.. Biological soil crust community types differ in photosynthetic pigment composition, fluorescence and carbon fixation in Shapotou region of China [J]. Applied Soil Ecology, 2017, 111: 9-16

[40] Ritchie R J. Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents [J]. Photosynthetica, 2008, 46(1): 115-126

[41] Shabtai I A, Wilhelm R C, Schweizer S A, et al.. Calcium promotes persistent soil organic matter by altering microbial transformation of plant litter [J]. Nature Communications, 2023, 14: 6609

[42] Qian L, Ye X-w, Xiao J-s, et al.. Nitrogen concentration acting as an environmental signal regulates cyanobacterial EPS excretion [J]. Chemosphere, 2022, 291: 132878

[43] Walkley A, Black I A. An examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method [J]. Soil Science, 1934, 37(1): 29-38

[44] Wang Y, Chen L, Li Z-h, et al.. The role of iron-rich organic fertilizer in promoting the growth of Chinese cabbage and inhibiting the transformation of cadmium [J]. Science of the Total Environment, 2024, 908: 168430

[45] Tan J-q, Wang X-z, Zhang M, et al.. Chlorella sorokiniana FK-montmorillonite interaction enhanced remediation of heavy metals in tailings [J]. Science of the Total Environment, 2023, 876: 163208

[46] Ciesielski H, Sterckeman T, Santerne M, et al.. A comparison between three methods for the determination of cation exchange capacity and exchangeable cations in soils [J]. Agronomie, 1997, 17(1): 9-16

[47] Cui S-f, Fu Y-z, Zhou B-q, et al.. Transfer characteristic of fluorine from atmospheric dry deposition, fertilizers, pesticides, and phosphogypsum into soil [J]. Chemosphere, 2021, 278: 130432

[48] Wan D, Ma M-k, Peng N, et al.. Effects of long-term fertilization on calcium-associated soil organic carbon: Implications for C sequestration in agricultural soils [J]. Science of the Total Environment, 2021, 772: 145037

[49] Wang Z-h, Zhang H, Xiong Y, et al.. Remediation mechanism of high concentrations of multiple heavy metals in contaminated soil by Sedum alfredii and native microorganisms [J]. Journal of Environmental Sciences, 2025, 147: 179-188

[50] Yang Y, Zhang Y-e, Yu X-x, et al.. Soil microorganism regulated aggregate stability and rill erosion resistance under different land uses [J]. Catena, 2023, 228: 107176

[51] Guo G, Kong Y, Xu Y-y, et al.. Soil organic matter components and sesquioxides integrally regulate aggregate stability and size distribution under erosion and deposition conditions in Southern China [J]. Journal of Hydrology, 2024, 639: 131588

[52] Xue P, Fu Q, Li T-x, et al.. Effects of biochar and straw application on the soil structure and water-holding and gas transport capacities in seasonally frozen soil areas [J]. Journal of Environmental Management, 2022, 301: 113943

[53] van Genuchten M T. A closed-form equation for predicting the hydraulic conductivity of unsaturated soils [J]. Soil Science Society of America Journal, 1980, 44(5): 892-898

[54] Langmead B, Salzberg S L. Fast gapped-read alignment with bowtie 2 [J]. Nature Methods, 2012, 9(4): 357-359

[55] Brum J R, Ignacio-Espinoza J C, Roux S, et al.. Patterns and ecological drivers of ocean viral communities [J]. Science, 2015, 348(6237): 1261498

[56] Franzosa E A, McIver L J, Rahnavard G, et al.. Species-level functional profiling of metagenomes and metatranscriptomes [J]. Nature Methods, 2018, 15(11): 962-968

[57] Finstad K M, Nuccio E E, Grant K E, et al.. Radiocarbon analysis of soil microbial biomass via direct chloroform extraction [J]. Radiocarbon, 2024, 66(5): 854-862

[58] Rakhsh F, Golchin A, Beheshti Al Agha A, et al.. Mineralization of organic carbon and formation of microbial biomass in soil: Effects of clay content and composition and the mechanisms involved [J]. Soil Biology and Biochemistry, 2020, 151: 108036

[59] Gao L-q, Bowker M A, Xu M-x, et al.. Biological soil crusts decrease erodibility by modifying inherent soil properties on the Loess Plateau, China [J]. Soil Biology and Biochemistry, 2017, 105: 49-58

[60] Rawls W J, Pachepsky Y A, Ritchie J C, et al.. Effect of soil organic carbon on soil water retention [J]. Geoderma, 2003, 116(1–2): 61-76

[61] Zhou H, Chen C, Wang D-z, et al.. Effect of long-term organic amendments on the full-range soil water retention characteristics of a Vertisol [J]. Soil and Tillage Research, 2020, 202: 104663

[62] King A E, Amsili J P, Córdova S C, et al.. A soil matrix capacity index to predict mineral-associated but not particulate organic carbon across a range of climate and soil pH [J]. Biogeochemistry, 2023, 165(1): 1-14

[63] Liu Y-d, Huang Y-n, Ndzelu B S, et al.. Carbon saturation level regulates the stability of mineral-associated organic carbon in forest soils [J]. Catena, 2025, 259: 109391

[64] Zielinska S, Radkowski P, Ossowski T, et al.. First insight into microbial community composition in a phosphogypsum waste heap soil [J]. Acta Biochimica Polonica, 2017, 64(4): 693-698

[65] Ge H-m, Li C-c, Huang C-h, et al.. Bacterial community composition and metabolic characteristics of three representative marine areas in northern China [J]. Marine Environmental Research, 2025, 204: 106892

[66] Cui S, Wang Z-r, Kong L-c, et al.. Dynamics of microbial communities and functional genes under the interactions between phosphorus inactivation agents and phosphorus-solubilizing bacteria in sediments [J]. Journal of Water Process Engineering, 2025, 77: 108395

[67] Arteaga-Mejia M, Velázquez-Guadalupe A, Castillo-Villanueva E, et al.. Sulfur oxidation by new and non-canonical bacteria in a subsurface flow constructed wetland treating domestic wastewater [J]. Microorganisms, 2026, 14(3): 565

[68] Liao J-x, Huang Y, Su Y-j, et al.. Biochar effects on bacterial communities in recycled concrete aggregates [J]. Frontiers of Environmental Science & Engineering, 2025, 19(4): 47

[69] Krysenko S, Wohlleben W. Role of carbon, nitrogen, phosphate and sulfur metabolism in secondary metabolism precursor supply in Streptomyces spp [J]. Microorganisms, 2024, 12(8): 1571

[70] Liao J-x, Hao G-c, Guo H-w, et al.. Effects of biochar on plant and microbial communities in landfill soil [J]. Applied Soil Ecology, 2024, 204: 105749

[71] Manici L M, Caputo F, De Sabata D, et al.. The enzyme patterns of Ascomycota and Basidiomycota fungi reveal their different functions in soil [J]. Applied Soil Ecology, 2024, 196: 105323

[72] Liao j, Bordoloi S, Wang y, et al.. Field study of biochar effects on vegetation and bacterial communities in a restored mine [J]. Applied Soil Ecology, 2025, 211: 106141

[73] Zhang J, Elser J J. Carbon: Nitrogen: Phosphorus stoichiometry in fungi: A meta-analysis [J]. Frontiers in Microbiology, 2017, 8: 1281

[74] Lauro F M, DeMaere M Z, Yau S, et al.. An integrative study of a meromictic lake ecosystem in Antarctica [J]. The ISME Journal, 2011, 5(5): 879-895

[75] Chen L-z, Rossi F, Deng S-q, et al.. Macromolecular and chemical features of the excreted extracellular polysaccharides in induced biological soil crusts of different ages [J]. Soil Biology and Biochemistry, 2014, 78: 1-9

[76] Coats E R, Mockos A, Loge F J. Post-anoxic denitrification driven by PHA and glycogen within enhanced biological phosphorus removal [J]. Bioresource Technology, 2011, 102(2): 1019-1027

[77] Zhao T, Tashiro Y, Sonomoto K. Construction and metabolic analysis of acetone-butanol-ethanol fermentation using mixed acetic acid and lactic acid in wastewater [J]. Industrial Crops and Products, 2022, 187: 115503

[78] Zhou R, Zuo W-l, Xie Y-w, et al.. Sodium bisulfite boosted exopolysaccharide production by Auxenochlorella protothecoides: Potential mechanisms harnessing H₂O₂ signaling and carbon reallocation [J]. Bioresource Technology, 2025, 420: 132121

[79] Tu Q-c, Lin L, Cheng L, et al.. NCycDB: A curated integrative database for fast and accurate metagenomic profiling of nitrogen cycling genes [J]. Bioinformatics, 2019, 35(6): 1040-1048

[80] Llorens-Marès T, Yooseph S, Goll J, et al.. Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics [J]. The ISME Journal, 2015, 9(7): 1648-1661

[81] Dai Z-m, Liu G-f, Chen H-h, et al.. Long-term nutrient inputs shift soil microbial functional profiles of phosphorus cycling in diverse agroecosystems [J]. The ISME Journal, 2020, 14(3): 757-770

[82] Chaudhary S, Sindhu S S, Dhanker R, et al.. Microbesmediated sulphur cycling in soil: Impact on soil fertility, crop production and environmental sustainability [J]. Microbiological Research, 2023, 271: 127340

[83] Zhao Y, Xu W-w, Wang N. Effects of covering sand with different soil substrates on the formation and development of artificial biocrusts in a natural desert environment [J]. Soil and Tillage Research, 2021, 213: 105081

[84] Xiao J-s, Lan S-b, Zhang Z-l, et al.. Physical disturbance reduces cyanobacterial relative abundance and substrate metabolism potential of biological soil crusts on a gold mine tailing of Central China [J]. Frontiers in Microbiology, 2022, 13: 811039

[85] Wang B-r, Huang Y-m, Li N, et al.. Initial soil formation by biocrusts: Nitrogen demand and clay protection control microbial necromass accrual and recycling [J]. Soil Biology and Biochemistry, 2022, 167: 108607

[86] Deng S-q, Zhang D-y, Wang G-h, et al.. Biological soil crust succession in deserts through a 59-year-long case study in China: How induced biological soil crust strategy accelerates desertification reversal from decades to years [J]. Soil Biology and Biochemistry, 2020, 141: 107665

[87] Li J-y, Jin X-y, Zhang X-c, et al.. Comparative metagenomics of two distinct biological soil crusts in the Tengger Desert, China [J]. Soil Biology and Biochemistry, 2020, 140: 107637

[88] Berney M, Greening C, Conrad R, et al.. An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia [J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(31): 11479-11484

[89] Tran P Q, Bachand S C, McIntyre P B, et al.. Depth-discrete metagenomics reveals the roles of microbes in biogeochemical cycling in the tropical freshwater Lake Tanganyika [J]. The ISME Journal, 2021, 15(7): 1971-1986

[90] Iamjud K. The effect of fallow season cover crops on nutrient cycling and soil health in row crop production in the mid-south[D], 2021

[91] De Tender C, Mesuere B, Van der Jeugt F, et al.. Peat substrate amended with chitin modulates the N-cycle, siderophore and chitinase responses in the lettuce rhizobiome [J]. Scientific Reports, 2019, 9: 9890

[92] Chalchissa F B, Kuris B K. Modelling soil organic carbon dynamics under extreme climate and land use and land cover changes in Western Oromia Regional state, Ethiopia [J]. Journal of Environmental Management, 2024, 350: 119598

[93] Arwenyo B, Varco J J, Dygert A, et al.. Contribution of modified P-enriched biochar on pH buffering capacity of acidic soil [J]. Journal of Environmental Management, 2023, 339: 117863

[94] Wang J-c, Qiu M, Shen Z-y, et al.. Microbial modulation of soil pH: A self-benefiting mechanism exemplified by Bacillus [J]. Soil Biology and Biochemistry, 2025, 210: 109949

[95] Wu L, Hu J, Shaaban M, et al.. Long-term manure amendment enhances N₂O emissions from acidic soil by alleviating acidification and increasing nitrogen mineralization [J]. Journal of Integrative Agriculture, 2026, 25(1): 262-272

[96] Larsen S, Widdowson A E. Soil fluorine [J]. Journal of Soil Science, 1971, 22(2): 210-221

[97] Liu S-f, Gao B, Qin H-h, et al.. Concentration mechanism of fluorine, arsenic, and uranium in groundwater of the Hailar Basin, China [J]. Environmental Earth Sciences, 2022, 81(18): 444

[98] Cao S-q, Li Y-b, Wu X-y, et al.. Efficient recovery of highly pure CaF₂ from fluorine-containing wastewater using an icy lime solution [J]. Water Science & Technology, 2024, 90(1): 32-44

[99] Cui L-l, Liu Z-l, Suo J, et al.. Filamentous cyanobacterium Spirulina platensis mediates phosphate biomineralization for efficient fluoride immobilization and drives metabolic shift toward stress resistance and biomineralization [J]. Journal of Hazardous Materials, 2025, 500: 140454

[100] Xie Y-c, Wen X-m, Tu Y-l, et al.. Mechanisms of artificial biological soil crusts development for anti-desertification engineering on the Qinghai-Tibetan Plateau [J]. Environmental Technology & Innovation, 2024, 33: 103542

[101] Zhao Y, Zhao Y-q, Xu W-w, et al.. Acquiring high-quality and sufficient propagules/fragments for cyanobacteria crust inoculation and restoration of degraded soils in a sandy desert [J]. Land Degradation & Development, 2023, 34(5): 1593-1597

[102] Zhang C, Zhou K-q, Wang Z, et al.. Montmorillonite as an “accelerator” for the microbial carbon pump during artificial biocrust construction [J]. Plant and Soil, 2025, 513(1): 1363-1379

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