Natural weathering promoted the recovery of microbial community in bauxite residues

Natural weathering promoted the recovery of microbial community in bauxite residues | 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 Natural weathering promoted the recovery of microbial community in bauxite residues Yang Gao, Liping Zhang, Huitong Li, Huarang Sun, Ran Mu, Zehua Pi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7133212/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 Purpose Natural weathering process increased microbial diversity and in bauxite residue. However, the its effect on the microbial metabolic activities is still poorly understood. Methods In this study, residue samples with different weathering time were collected to evaluate the microbial metabolic activities and community structure in bauxite residue. Results Community level physiological profiles (CLPP) results indicated that natural weathering process significantly increased the microbial metabolic activities and changed microbial carbon resources utilization pattern. The microbial metabolic diversity index increased from 2.68 to 3.29 and the average well color development increased from 0.41 to 1.47. The Shannon diversity significantly increased from 5.79 to 6.26 and the observed OTU numbers increased from 1009 to 1331, respectively. The relative abundance of Proteobacteria, Bacteroidota, Acidobacteria, Actinobacteria and Chloroflexi significantly increased following long term weathering process. Random forest model showed that Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae were the core species in the promotion of microbial metabolic activities. Redundancy analysis (RDA) indicated that the development of microbial communities was signifianltly affected by MWD followed by pH, TOC, AP, TN, EC and AN. Conclusions Natural weathering process could increase microbial metabolic activities and diversity, thus promote the recovery of microbial community in bauxite residue. Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae played vital roles in the promotion of microbial carbon metabolism in bauxite residue. Our results emphasize the significance of specific functional microbial species in the recovery of microbial communities in bauxite residue disposal areas. Bauxite residue Natural weathering process Microbial metabolic activities Microbial composition Soil formation in bauxite residue Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Highlights Long-term natural weathering increased microbial metabolic activities in bauxite residue. Microbial groups including Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae played important roles in the recovery of microbial metabolic in bauxite residue. The shifts in the bacterial community were mainly driven by MWD followed by pH, TOC, AP, TN, EC and AN. Introduction Bauxite residue is the by-product produced during the alumina production process (Power et al. 2011 ). Currently, the utilization of bauxite residue is still limited and it is mainly stored in large-scale dam, which pose potential ecological risks to the surrounding environment (Gelencser et al. 2011 ; Ruyters et al. 2011 ). Recently, the improvement of bauxite residue from tailing to soil has been advocated as the promising way in the management of bauxite residue (Xue et al. 2019 ). However, due to the adverse properties including high alkalinity/salinity, poor structure and limited nutrient conditions in bauxite residue, successful vegetation case is limited. The natural weathering process can improve the physicochemical properties and promote microbial activities in bauxite residue (Kong et al. 2017a ; Santini and Fey 2013 ; Zhu et al. 2016a ). Firstly, natural weathering process such as wind and water erosion promote the alkaline anion leaching and alkaline minerals dissolution, which largely decreased alkalinity and salinity in bauxite residue (Kong et al. 2017a ; Kong et al. 2018 ). Secondly, natural weathering process improves aggregate formation in bauxite residue, which benefit to the improvement of the water, fertilizer, air and heat conditions in bauxite residue (Zhu et al. 2016a ). Thirdly, during natural weathering process, organic carbon derived from plant tissue and root exudates promote the development of plant community (Zhu et al. 2023 ). However, the currently studies mainly focus on the improvement of the physicochemical properties in bauxite residue, and neglect the establishment of biogeochemical process, such as carbon/nitrogen fixation, carbon/nitrogen mineralization and organic matter humification in bauxite residue. Therefore, the investigation of dynamics of microbial metabolic activities during the natural weathering process may provide us a comprehensive understanding of the soil formation process in bauxite residue. Microbial community are the main driver for diverse ecological processes such as greenhouse gas emissions, organic carbon degradation and nitrogen fixation, and aggregates formation (Bardgett and van der Putten 2014 ; Crowther et al. 2019 ). For example, some specific functional microorganisms, such as nitrogen-fixing bacteria and phosphorus-solubilizing bacteria, can increase the availability of nitrogen and phosphorus in soil through biological nitrogen fixation and phosphate solubilization process (Hartmann and Six 2023 ). Phosphate-solubilizing microorganisms could produce organic acids to promote the dissolution of insoluble phosphate in soil, which may benefit to plant growth (Raymond et al. 2020 ). Arbuscular mycorrhizal fungi (AMF) could entrap soil particles and force them together along s along the expanding hyphae to form aggregates (Rashid et al. 2016 ). In addition, the AMF could also secrete diverse extracellular polymeric substance, glomalin, hydrophobins and related soil proteins as cementing agent to promote the aggregate formation in soil (Rashid et al. 2016 ). Therefore, the role of microbial community should be taken into consideration in ecological remediation in bauxite residue, not merely investigate the changes of physiochemical properties in bauxite residue. Currently, several studies investigated the dynamic changes of microbial community in bauxite residue. During natural weathering process, the microbial diversity significantly increased, and microbial composition changed from those dominated by alkalophilic and halophilic assemblages to those dominated neutrophilic assemblages (Wu et al. 2020 ). Weathering for over 50 years, the microbial communities in bauxite residue changed to the assemblages that was similar to those of the natural soil microbial community around the mining area (Wu et al. 2021 ). Moreover, long term restoration enriched the abundance of some specific microbial groups, which may participate in nitrogen fixation or phosphate solubilization process, and largely increased the network stability and complexity of microbial communities in bauxite residue (Deng et al. 2024 ). By reshaping the microbial community, long-term vegetation restoration significantly improved the multifunctional of bauxite residue (Jiang et al. 2023 ). However, current studies mainly focused on the development of microbial communities during restoration, whereas neglected the changes in microbial metabolic activities. Therefore, to obtained comprehensive knowledge on the soil formation of bauxite residue, the development of microbial metabolic activities must be taken into consideration. A typical Bauxite Residue Disposal Area in Central China was chosen in this study. The objectives of this research were: 1) to analyze the effect of natural weathering process on the development of the salinity/alkalinity and nutrients condition in bauxite residue. 2) to monitor the dynamic changes of microbial activities and microbial structure in bauxite residue. 3) to establish the relationships between microbial carbon metabolic characteristics and physiochemical properties in bauxite residue. This study aims to provide theoretical support for the field-scale ecological remediation of bauxite residue. Materials and methods Study sites and soil sampling Residue samples were selected from a Bauxite Residue Disposal Area (BRDA), which belongs to Henan Branch China Aluminum Corporation CO, LTD. The average annual precipitation was 380 mm and annual temperature was 16.0 ℃ in the sampling area. On the surface of Bauxite Residue Disposal Area, there were obvious plant encroachment, which dominated by herbaceous plant including Cynodon, Artemisia, Festuca arundinacea etc. According to the disposal history, four sites, which representing different years since restoration (5, 10, 20 and 30 years since disposal) were chosen to collect samples. In each site, five samples were collected randomly. Totally, twenty bauxite residue samples were collected in this study. All samples were placed in polyethylene plastic bags and transported to the laboratory under 4℃ conditions. When transported back to the laboratory, the samples were filtered with the 2-mm mesh and divided into four parts. One part was naturally dried for the determination of physicochemical properties. One part was stored under 4℃ for Biolog test. One part was stored under -80℃ for further determination of microbial community. Community-Level Physiological Profiling Biolog EcoPlates were employed to assess the microbial carbon metabolic capacity (Garland and Mills 1991). Each EcoPlate contains thirty-two types carbon source and one control, which in total thirty-three treatments. Three were three replicates for each carbon source treatments, in total ninety-six wells in one EcoPlate. The thirty-two types carbon source could be classified to six main carbon type such as amino acids, amines, carbohydrates, carboxylic acids, phenolic acids and polymers. The detailed information was listed in Table S1. In brief, five-gram sample was dissolved with sterile 0.05M phosphate buffer solution in 100 mL plastic bottle. The sample-water mixture solution was shaken for 30 min under the 180 r/min condition. 1 ml the mixture solution was taken and added to a test tube containing 9 ml sterile phosphate buffer, and a 10 -2 dilution was obtained. Repeat the above step one more time, and 10 -3 dilution was obtained. 150 μL of the obtained dilution (10 -3 ) was added into each microplate well in the plate and then incubated at 25 ± 1 ℃ in dark in a thermostatic incubator. The light absorption of each well was read by using a BIOLOG plate reader (BioTek ELx800, BioTek, USA) at 750 nm and 590 nm wavelengths every 24 hours. High-throughput sequencing The DNA was extracted by using the PowerSoil® DNA Isolation Kit. In 16S rRNA gene, the V4 region was chosen as the marker gene and then amplified. The primer was 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′- GGACTACHVGGGTWTCTAAT-3′). The PCR amplification program was described in Jiang’ report (Jiang et al. 2023). The amplified PCR product was extracted by using agarose (2%) and then purified by using DNA Gel Extraction Kit. The concentration of purified DNA was tested using Nanodrop 2000 spectrophotometer. Finally, the qualified DNA was sequenced on the Illumina MiSeq PE300 platform in Majorbio company. Data processing Biolog data: The average well color development (AWCD) was calculated as the difference value of OD590 in carbon well and control well. (1) In the above equation, A i : the absorbance reading of OD 590 in the carbon well i; A 0 : the absorbance reading of OD 590 in the blank well. n: the numbers of carbon source; When the difference value of OD 590 in plate well were negative or under 0.06, they are coded as zeroes for subsequent data analysis. The Shannon index (H), Simpson index (D) and Mclntosh index (U) of microbial carbon metabolism were calculated as follows: (2) (3) (4) In the above equation, , . DNA Sequencing data: The raw sequences were firstly analyzed on the QIIME pipeline and then filtered those low-quality sequences (the quality scores < 20 and sequences length < 150 bp) (Caporaso et al. 2010). Secondly, the chimeras within sequences were eliminated by using UCHIME software (Edgar et al. 2011). Thirdly, the remained sequences were clustered into operational taxonomic units (OTUs) based on 97% similarity by using UPARSE version7.1 (Edgar 2013). Finally, RDP Classifier was applied to annotate the taxonomy of representative OTU in 16S rRNA database (Silva v138). The alpha diversity indices were calculated by using “vegan” package in R platform (Dixon 2003). Principal Coordinates Analysis (PCoA) was conducted by using “vegan” R package in R platform (Dixon 2003). Redundancy analysis (RDA) was conducted by using “radcca.hp” R package in R platform (Dixon 2003). Random forest analysis was performed by using the “RandomForest” package in R platform (Dixon 2003). Results Variation of physicochemical properties in bauxite residue The alkalinity and salinity significantly decreased, whereas nutrients condition and physical significantly improved following long term natural weathering process in bauxite residue (Table 1 ). Compared to 5Y sites, the TOC, TN, AN and AP were significantly increased in 30Y sites ( P < 0.05). In addition, the MWD also significantly increased in 30Y sites when compared with that in 5Y sites ( P < 0.05). Contrary to the improvement of nutrients condition and physical properties, he alkalinity and salinity index such as pH, EC and Na + content all significantly decreased in 30Y sites when compared with that in 5Y sites ( P < 0.05). Table 1 Physicochemical properties in bauxite residue. Site pH EC Na TOC TN AN TP AP MWD cmol/kg g/kg g/kg mg/kg g/kg mg/kg 5Y 10.18a 0.65a 11.73a 4.75d 0.42d 39.81d 0.82a 8.83d 0.49d (0.09) (0.04) (0.94) (0.45) (0.04) (12.07) (0.06) (0.66) (0.04) 10Y 9.32b 0.36b 6.03b 9.23c 0.73c 69.25c 0.77ab 25.89c 0.72c (0.1) (0.04) (0.63) (0.6) (0.09) (9.62) (0.06) (4.66) (0.07) 20Y 8.52c 0.27bc 5.72b 13.19b 0.89b 95.49b 0.75b 40.72b 0.86b (0.26) (0.02) (0.91) (1.15) (0.05) (8.01) (0.01) (4.33) (0.06) 30Y 8.28c 0.24c 4.12c 15.11a 0.97a 120.33a 0.72b 53.36a 0.89a (0.2) (0.02) (0.58) (1.34) (0.08) (17.94) (0.03) (6.95) (0.05) Notes: 5Y, weathered for 5 years; 10Y, weathered for 10 years; 20Y, weathered for 20 years; 30Y, weathered for 30 years; TOC, organic carbon; TN, total nitrogen; AN, available nitrogen; TP, total phosphorous; AP, available phosphorous; MWD, mean weight diameter; Values with the same lower case were not significant at P < 0.05 among different sites. Microbial carbon metabolism in the process of natural weathering in bauxite residue Long term natural weathering process significantly changed the microbial carbon utilization patterns in bauxite residue (Fig. 1 A). The principal coordinates analysis showed that the PC1 and PC2 explained 85.36% and 8.74% of the variance in microbial carbon utilization patterns, respectively (Fig. 1 A). Specifically, in 5Y sites, the carbon source with highest utilization intensity was amine, followed by carboxylic acid, amino acids, phenolic acid, polymer and carbohydrate (Fig. 1 B). The utilization intensity of amino acids, carbohydrate, carboxylic acid, phenolic acid and polymer significantly increased, whereas that of amine significantly decreased in microbial carbon utilization patterns during the natural weathering process (Fig. 1 B). In 30Y sites, the carbon source with highest utilization intensity was carbohydrate and amino acids, followed by polymer, carboxylic acid, phenolic acid and amine (Fig. 1 B). The microbial carbon metabolism activities and diversity were promoted during natural weathering process (Fig. 2 ). The maximum AWCD and diversity index were all observed in the 30Y sites. The AWCD value increased from 0.41 (5Y) to 0.83 (10Y), 1.14 (20Y) and 1.47 (30Y), respectively (Fig. 2 A). In addition, the microbial carbon metabolism diversity index (Shannon index and Simpson index) significantly increased in 30Y sites when compared to 5Y sites (Fig. 2 B-D). These results suggested that long term natural weathering process might stimulate the recovery of microbial communities and enhanced the carbon metabolism in bauxite residue. Microbial communities in the process of natural weathering in bauxite residue Long term natural weathering process significantly increased the diversity and richness of microbial communities in bauxite residue (Fig. 3 ). The highest observed species number was observed in 30Y sites, which significantly higher than that in all sites ( P < 0.05). The highest Shannon index was observed in 20Y sites, which significantly higher than that in 10Y and 5Y sites ( P 0.05). The composition of microbial community significantly varied during natural weathering processes in bauxite residue (Fig. 4 ). At the phylum level, the dominant microbial groups were Firmicutes (39.88%), Proteobacteria (16.27%), Chloroflexi (9.49%), Cyanobacteria (6.9%), Actinomycetota (6.3%) and Bacteroidota (5.93%) in the 5Y sites (Table. S2). Other abundant groups (the relative abundance > 1%) were Planctomycetota (4.42%), Verrucomicrobiota (3.86%), Crenarchaeota (2.24%), Acidobacteriota (1.49%) and Nitrospirota (1.07%) (Table. S2). During the long-term natural weathering processes, the relative abundance of Firmicutes, Cyanobacteria, Verrucomicrobiota and Crenarchaeota significantly decreased ( P < 0.05), whereas the relative abundance of Proteobacteria, Bacteroidota, Acidobacteria, Nitrospirota significantly increased ( P < 0.05) (Fig. 4 ). In 30Y sites, the dominant groups changed to Proteobacteria (43.69%), Bacteroidota (13.65%), Acidobacteria (12.24%), Actinobacteria (5.42%) and Chloroflexi (5.07%) (Table. S2). Other relatively abundant groups were Firmicutes (4.63%), Planctomycetota (4.84%) and Nitrospirota (3.55%) (Table. S2). The relative abundance of all the microbial groups showed significantly difference between 5Y and 30Y sites, except for Planctomycetota and Actinobacteria ( P < 0.05). At the family level, the microbial community was dominated by Bacillaceae (22.39%), Halomonadaceae (7.51%), Clostridiaceae (6.53%) and Chloroflexaceae (5.49%) in the 5Y sites (Table. S3). Natural weathering processes decreased the abundance of Bacillaceae, Halomonadaceae and Clostridiaceae, whereas increased the abundance of Acidobacteriaceae, Beijerinckiaceae, Blastocatellaceae, Caulobacteraceae, Chitinophagaceae, Devosiaceae, Enterobacteriaceae, Microscillaceae, Nitrospiraceae, Nitrosomonadaceae, Planctomycetaceae, Rhizobiaceae, Sphingobacteriaceae and Sphingomonadaceae in bauxite residue (Fig. 5 ). Differed with those microbial groups in 5Y sites, the microbial community were dominated by Sphingomonadaceae (7.53%) and Beijerinckiaceae (5.92%) in 30Y sites (Table. S3). In addition, the increased microbial groups induced by natural weathering process such as Acidobacteriaceae, Beijerinckiaceae, Blastocatellaceae, Caulobacteraceae, Chitinophagaceae, Devosiaceae also exhibit relatively high abundances (1%-5%) in 30Y sites. Interestingly, all the increased microbial groups showed significantly difference between 5Y and 30Y sites ( P < 0.05). Random forest model showed the microbial diversity and composition both significantly affect the carbon metabolism activities in bauxite residue (Fig. 6 ). The importance value of Chao 1 and ACE index were both higher than diversity index Shannon and Simpson index, which indicated that microbial richness rather than diversity was more important to the recovery of microbial carbon metabolism activities in bauxite residue (Fig. 6 A). Among the top 10 increased microbials groups, Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae significantly promoted the microbial carbon metabolism activities in bauxite residue (Fig. 6 B). The linear regression analysis showed that the AWCD was significantly correlated with Beijerinckiaceae (0.874), Rhizobiaceae (0.898), Microscillaceae (0.851), Caulobacteraceae (0.845), Blastocatellaceae (0.889), Acidobacteriaceae (0.888), Sphingomonadaceae (0.827), Nitrosomonadaceae (0.741) and Chitinophagaceae (0.702) (Fig. 7 ). Correlation between microbial community and bauxite residue In this study, mantel test and redundancy analysis were employed to identify the key environmental factors in affecting the development of microbial communities during long term weathering process. Mantel test indicated that the 16S gene abundance showed negative correlation with pH, EC, Na and TP ( P < 0.05), whereas showed positive correlation with TOC, TN, AN, AP and MWD ( P < 0.05) (Fig. 8 ). The Shannon diversity index, Chao 1 index and ACE index were all showed significantly negative correlations with pH, EC, Na and TP ( P < 0.05), whereas showed positive correlations with TOC, TN, AN, AP and MWD ( P < 0.05) (Fig. 8 ). Redundancy analysis (RDA) indicated that MWD, followed by pH, TOC, AP, TN, EC and AN were the mainly driving factor in the development of microbial community, while TP had little significant influence (Table S4). These environment factors could explain 94.79% of the total variation of microbial community, indicated that the development of microbial communities mainly driven by the change of physiochemical properties in bauxite residue (Fig. 9 ). Thus, the long-term natural weathering process could promote the recovery of microbial communities by improving properties in bauxite residue. Discussion Improvement of natural weathering process on the environmental conditions of bauxite residues The long-term natural weathering processes improved the physiochemical properties in bauxite residue, which expressed as alkalinity/ salinity reduction, nutrients accumulation and physical structure improvement. The alkalinity reduction in bauxite residue may be induced by rainwater leaching. The natural weathering processes induced alkaline anion (e.g OH − , CO 3 2− /HCO 3 − , and aluminates) leaching and the alkalinity solids (e.g sodalite, hydrogarnet and calcite) dissolving, which may cause the alkalinity reduction in bauxite residue (Kong et al. 2017a ). In addition, the secretion of organic acids in plant rhizosphere metabolism or its release during litter decomposition may be another reason for the alkalinity reduction in bauxite residue. The microbial decomposition of biomass residues would produce numerous organic acid (e.g., acetic acid, citric acid and isocitric acid) and neutralized alkalinity in bauxite residue (You et al. 2019 ). The development of plant communities including plant rhizosphere metabolism and litter decomposition process not only neutralized alkalinity in bauxite residue, but also induced nutrients accumulation in bauxite residue. On one hand, plant rhizosphere metabolism would release nutrients compounds including carbohydrate, organic acids, and amino acids, and thus increased nutrients elements in bauxite residue. On the other hand, the dead plant tissue would be decomposed and release nutrients into bauxite residue (Pereira et al. 2023 ). After 30 years of natural weathering, significant improvement of physical structure was observed in bauxite residue, which expressed as the increase in MWD (from 0.51 to 0.91 mm). The improvement of physical structure may be related to the development of plant communities. On one hand, Plant roots could align soil particles along its expanding roots to form aggregates (Xiao et al. 2024 ). On the other hand, plant exudes such as mucilages, polysaccharides and extracellular compound could bind particles together through cation bridge, polysaccharide -OH group and H proton (Xiao et al. 2024 ). The improvement of physical structure not only affected by plant activities, also be affect by the reduction of salinity in bauxite residue. The increased MWD in long term weathered bauxite residue (30Y) may related to the decreased ESP, as excessive Na + would cause the expansion of lattice and disintegration of clay particles in bauxite residue (Xue et al. 2021 ). In addition, increased organic carbon may benefit to the formation of organic-mineral complexes, thus improved structural texture in bauxite residue (Zhu et al. 2016b ). Improvement of natural weathering process on microbial activity in bauxite residues The recovery of microbial activity is critical to the rehabilitation in bauxite residue. However, the high salinity and alkalinity in bauxite residue commonly restrict the microbial activities in bauxite residue, as the high salinity/alkalinity was toxic to microbial cells and enzyme catalytic sites (Raiesi and Sadeghi 2019 ). In the present study, natural weathering process significantly increased microbial metabolic capacity and diversity on carbon source. This may be related to the improvement of physiochemical properties in bauxite residue. In the early stage of weathering process, the alkalinity and salinity were extreme high in bauxite residue, which large restrict the microbial activities. High salinity may lead to cell dehydration, which damage the integrity of cell membrane, and then affect the activity of enzymes and the normal operation of metabolic pathways. The excessive accumulation of Na + , Cl − may interfere with the activity of enzymes in microbial cells, by binding to key active sites in enzyme molecules, resulting in structural inactivation (Oren 2011 ). In high alkaline environment, most of the neutral and acidic environment microorganisms are difficult to survive, and only alkali-resistant bacteria (such as some actinomycetes) are left, with poor carbon metabolism capacity (Banda et al. 2020 ; Zaitseva et al. 2018 ). On the other hand, the accumulation of organic carbon and nutrients induced by natural weathering process would positively stimulate microbial growth and enhance metabolic activity by providing more available carbon sources and energy. For example, exudates derived from plant root, that mainly consist of carbohydrates, amino acids, organic acids and other carbon-based compounds are the main carbon and energy sources of microorganisms, which could directly promote their growth and metabolic activities (Yuan et al. 2017 ). In addition, organic acids (such as acetic acid, oxalic acid) can reduce soil pH and create an environment suitable for acidic microbial growth, while dissolving mineral-bound carbon and increasing the availability of carbon, which provide carbon source for microbial growth and indirectly increase microbial carbon metabolic activities (Kong et al. 2017b ). Improvement of natural weathering process on microbial communities in bauxite residues The natural weathering process not only promoted the recovery of microbial activity, but also changed the microbial community’s composition. It had been shown that the quality of bauxite residue was significantly correlated with microbial diversity and composition (Deng et al. 2024 ). In this study, the natural weathering process significantly increased microbial diversity in bauxite residue, which were similar to the previous works (Wu et al. 2020 ; Wu et al. 2021 ). Except for the increase in microbial diversity, natural weathering process also dramatically changed the microbial composition in bauxite residue, which expressed as a dramatically change from alkalophilic and halophilic assemblages to neutrophilic assemblages. In 5Y bauxite residue, the microbial communities were dominated by Streptococcaceae, Bacillaceae. The adverse geochemical conditions (e.g., extreme pH, salinity, metal toxicity) and nutrient deficiencies (e.g., limited nitrogen and phosphorus) in bauxite residue only allowed several microorganisms groups with high saline-alkali tolerance (Bacillaceae and Actinobacteria) survived, which coincide with low diversity in 5Y bauxite residue (Krishna et al. 2014 ; Santini et al. 2015 ). Bacillaceae could produce spore dormant structures, which were highly resistant to desiccation, heat, UV radiation, and chemical stressors (Dittmann et al. 2015 ). They could persist in poor nutrient and extreme pH biotopes (such as dry soils, mineral rock or acid mine) and be reactivated when nutrients and water become available. During long term natural weathering process, the properties significantly improved, which significantly affected the microbial composition in bauxite residue (Wu et al. 2020 ; Wu et al. 2021 ). After weathering for 30 years, the abundance of Bacillaceae, Chloroflexaceae, Halomonadaceae and Clostridiaceae significantly decreased, whereas the abundance of Sphingomonadaceae, Beijerinckiaceae, Blastocatellaceae, Microscillaceae, Anaerolineaceae, Nitrospiraceae, Enterobacteriaceae, Chitinophagaceae, Acidobacteriaceae, Nitrosomonadaceae, Rhizobiaceae, Devosiaceae and Caulobacteraceae significantly increased in bauxite residue (Fig. 4 , Tables S1). On one hand, the increase of organic carbon derived from litter decomposition and root exudates following natural weathering process may provide more metabolic substrate and promote the growth of organotrophic microorganism such as Sphingomonadaceae, Acidobacteriaceae, Rhizobiaceae (You et al. 2019 ). On the other hand, the improvement of microenvironment in bauxite residue (such as pH reduction and formation of aggregate structure) also provided a suitable habitat for this family of microorganisms (You et al. 2019 ). The relative abundance of Acidobacteria commonly showed negative correlations with soil pH (Lauber et al. 2009 ). Thus, the pH decreased in bauxite residue would induce the enrichment of Acidobacteria. In addition, the formation of aggregates may enhance water and oxygen permeability, which promoted aerobic microorganisms growth (including Beijerinckiaceae) (Qian et al. 2022 ). Moreover, the increased organic carbons may induce the enrichment of R-strategy groups such as Proteobacteria and Bacteroidetes in bauxite residue (Yang et al. 2023 ). Plant root exudate may be another critical reason for the development of microbial communities in bauxite residue. Root exudates are rich in organic compounds (such as sugars, organic acids, amino acids, etc.), accounting for 20%-40% of plant photosynthetic products, which could provide carbon source and energy for microorganisms (Zhalnina et al. 2018 ). Interestingly, some increased microbial groups in restored bauxite residue were capable with nitrogen fixation such as Beijerinckiaceae, Rhizobiaceae and Devosiaceae (Fig. 5 , Tables S2). This was coincided with Deng’s work (Deng et al. 2024 ), that plant development enriched microbial groups with nitrogen fixing ability in bauxite residue. Root exudates could promote nitrogen fixation in mining soil by stimulating the development of azotobacter (Li et al. 2021 ). Some specific root exudates metabolites in the rhizosphere, such as flavonoids and coumarins, have a facilitation effect on the root bacterial community, especially nitrogen fixing bacteria (Qiao et al. 2024 ). The main driving factors in the development of microbial communities Soil microorganisms play vital roles in the ecosystem by regulating biochemical processes. However, microorganisms were sensitive to environmental changes, which affect biochemical processes including organic carbon degradation, carbon mineralization and nitrogen fixation. Previous studies showed that the natural weathering processes improved geochemical properties in bauxite residue, which provided an opportunity for the recovery of microbial community (Wu et al. 2020 ; Wu et al. 2021 ). In this study, the Shannon diversity of microbial community was negative correlated with pH and EC, whereas was positive correlated with TOC, TN, AP, and AN. This conincide with the previous findings, which suggest that alkalinity reduction and nutrients accumulation promoted the microbial diversity in bauxite residue (Wu et al. 2020 ; Wu et al. 2021 ). Similar to microbial diversity, the environment factors had comprehensive effects on microbial communities. RDA analysis showed that variations in microbial composition were correlated with pH, EC, Na + , TOC, TN, AN, TP, AP and MWD, which indicated that alkalinity/salinity and nutrients availability strongly affect microbial development in bauxite residue (Fig. 9 , Tables S3). Commonly, pH condition and nutrients availability were the mainly driving factors in the development of microbial communities in degraded ecosystem. For instance, Santini et al. ( 2018 ) found that pH controlled the microbial community assembly in gold tailings (Santini et al. 2018 ). In an abandoned REE mine site, the bacterial communities were mainly driven by soil nutrients (Chao et al. 2016 ). Deng et al. ( 2020 ) also found that microbial communities were dramatically affected by soil pH and available nutrients in an open-cut iron mining area (Deng et al. 2020 ). Conclusion This study investigated the effect of natural weathering process on microbial carbon metabolisms and associated microbial community in bauxite residue. Firstly, natural weathering process effectively decreased alkalinity, whereas increased nutrients content and improved the physical structure in bauxite residue. These improvements became more obvious as the weathering time increased. Secondly, natural weathering process induced an obvious increase in the activity of microbial carbon metabolism and significant development in microbial community, expressed as the increase in microbial diversity and changes in microbial composition. During natural weathering process, the microbial community changed from alkalophilic and halophilic assemblages to neutrophilic assemblages in bauxite residue, particularly enriched functional microbial groups include acid-producing, organic carbon decomposition and nitrogen fixing. Thirdly, the improvement of physiochemical properties (e.g., pH, ESP, and MWD) in bauxite residue, were important to the development of microbial community. These results suggested that natural weathering process could effectively promote the recovery of microbial community by improving geochemical conditions in bauxite residues. Declarations CRediT authorship contribution statement Yang Gao : Conceptualization, Investigation, Formal analysis, Writing – original draft. Liping Zhang : Writing – review & editing, Project administration, Funding acquisition. Huitong Li : Investigation. Yiqing Gao : Investigation. Huarang Sun : Methodology, Investigation. Ran Mu : Visualization, Investigation. Zehua Pi : Visualization, Investigation. Data availability Data will be made available on request. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was funded by the National Natural Science Foundation of China (No. 52474161); Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article. References Banda, J.F., Lu, Y.C., Hao, C.B., Pei, L.X., Du, Z.R., Zhang, Y., Wei, P.F., Dong, H., 2020. The Effects of Salinity and pH on Microbial Community Diversity and Distribution Pattern in the Brines of Soda Lakes in Badain Jaran Desert, China. Geomicrobiol J. 37, 1-12. https://doi.org/10.1080/01490451.2019.1654568 . Bardgett, R.D., van der Putten, W.H., 2014. Belowground biodiversity and ecosystem functioning. Nature. 515, 505-511. https://doi.org/10.1038/nature13855 . Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Tumbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods. 7, 335-336. https://doi.org/10.1038/nmeth.f.303 . Chao, Y.Q., Liu, W.S., Chen, Y.M., Chen, W.H., Zhao, L.H., Ding, Q.B., Wang, S.Z., Tang, Y.T., Zhang, T., Qiu, R.L., 2016. Structure, Variation, and Co-occurrence of Soil Microbial Communities in Abandoned Sites of a Rare Earth Elements Mine. Environ. Sci. Technol. 50, 11481-11490. https://doi.org/10.1021/acs.est.6b02284 . Crowther, T.W., van den Hoogen, J., Wan, J., Mayes, M.A., Keiser, A.D., Mo, L., Averill, C., Maynard, D.S., 2019. The global soil community and its influence on biogeochemistry. Science. 365, eaav0550. https://doi.org/10.1126/science.aav0550 . Deng, D.D., Sun, W., Wu, H., Yang, X.Y., Zhu, F., Jiang, Y.F., Huang, S.W., Xue, S.G., Jiang, J., 2024. Plant encroachment increase multifunctionality in bauxite residue by constructing diverse and stable microbial communities. Plant Soil. https://doi.org/10.1007/s11104-024-06860-y . Deng, J.J., Yin, Y., Zhu, W.X., Zhou, Y.B., 2020. Response of soil environment factors and microbial communities to phytoremediation with Robinia pseudoacacia in an open-cut magnesite mine. Land Degrad. Dev. 31, 2340-2355. https://doi.org/10.1002/ldr.3599 . Dittmann, C., Han, H.M., Grabenbauer, M., Laue, M., 2015. Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress. J. Struct. Biol. 191, 156-164. https://doi.org/10.1016/j.jsb.2015.06.019 . Dixon, P., 2003. VEGAN, a package of R functions for community ecology. J. Veg. Sci. 14, 927-930. https://doi.org/10.1111/j.1654-1103.2003.tb02228.x . Edgar, R.C., 2013. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods. 10, 996-998. https://doi.org/10.1038/Nmeth.2604 . Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 27, 2194-2200. https://doi.org/10.1093/bioinformatics/btr381 . Garland, J.L., Mills, A.L., 1991. Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl. Environ. Microbiol. 57, 2351-2359. https://doi.org/10.1128/aem.57.8.2351-2359.1991 . Gelencser, A., Kovats, N., Turoczi, B., Rostasi, A., Hoffer, A., Imre, K., Nyiro-Kosa, I., Csakberenyi-Malasics, D., Toth, A., Czitrovszky, A., Nagy, A., Nagy, S., Acs, A., Kovacs, A., Ferincz, A., Hartyani, Z., Posfai, M., 2011. The Red Mud Accident in Ajka (Hungary): Characterization and Potential Health Effects of Fugitive Dust. Environ. Sci. Technol. 45, 1608-1615. https://doi.org/10.1021/es104005r . Hartmann, M., Six, J., 2023. Soil structure and microbiome functions in agroecosystems. Nat. Rev. Earth. Env. 4, 4-18. https://doi.org/10.1038/s43017-022-00366-w . Jiang, Y.F., Huang, S.W., Zhu, F., Guo, X.Y., Zhang, X.C., Zhu, M.X., Zhang, Y.F., Xue, S.G., 2023. Long-term weathering difference in soil-like indicators of bauxite residue mediates the multifunctionality driven by microbial communities. Sci. Total Environ. 890, 164377. https://doi.org/10.1016/j.scitotenv.2023.164377 . Kong, X.F., Guo, Y., Xue, S.G., Hartley, W., Wu, C., Ye, Y.Z., Cheng, Q.Y., 2017a. Natural evolution of alkaline characteristics in bauxite residue. J. Clean Prod. 143, 224-230. https://doi.org/10.1016/j.jclepro.2016.12.125 . Kong, X.F., Li, M., Xue, S.G., Hartley, W., Chen, C.R., Wu, C., Li, X.F., Li, Y.W., 2017b. Acid transformation of bauxite residue: Conversion of its alkaline characteristics. J. Hazard. Mater. 324, 382-390. https://doi.org/10.1016/j.jhazmat.2016.10.073 . Kong, X.F., Tian, T., Xue, S.G., Hartley, W., Huang, L.B., Wu, C., Li, C.X., 2018. Development of alkaline electrochemical characteristics demonstrates soil formation in bauxite residue undergoing natural rehabilitation. Land Degrad. Dev. 29, 58-67. https://doi.org/10.1002/ldr.2836 . Krishna, P., Babu, A.G., Reddy, M.S., 2014. Bacterial diversity of extremely alkaline bauxite residue site of alumina industrial plant using culturable bacteria and residue 16S rRNA gene clones. Extremophiles. 18, 665-676. https://doi.org/10.1007/s00792-014-0647-8 . Lauber, C.L., Hamady, M., Knight, R., Fierer, N., 2009. Pyrosequencing-Based Assessment of Soil pH as a Predictor of Soil Bacterial Community Structure at the Continental Scale. Appl. Environ. Microbiol. 75, 5111-5120. https://doi.org/10.1128/aem.00335-09 . Li, Y., Yuan, L., Xue, S., Liu, B.J., Jin, G., 2021. Artificial root exudates excite bacterial nitrogen fixation in the subsurface of mine soils. Appl. Soil Ecol. 157, 103774. https://doi.org/10.1016/j.apsoil.2020.103774 . Oren, A., 2011. Thermodynamic limits to microbial life at high salt concentrations. Environ. Microbiol. 13, 1908-1923. https://doi.org/10.1111/j.1462-2920.2010.02365.x . Pereira, D.G.C., Portugal, A.F., Giustolin, T.A., Maia, V.M., Megda, M.X.V., Kondo, M.K., 2023. Litter decomposition and nutrient release in different land use systems in the Brazilian semi-arid region. Catena. 231, 107345. https://doi.org/10.1016/j.catena.2023.107345 . Power, G., Grafe, M., Klauber, C., 2011. Bauxite residue issues: I. Current management, disposal and storage practices. Hydrometallurgy. 108, 33-45. https://doi.org/10.1016/j.hydromet.2011.02.006 . Qian, Z.Z., Zhuang, S.Y., Gao, J.S., Tang, L.Z., Harindintwali, J.D., Wang, F., 2022. Aeration increases soil bacterial diversity and nutrient transformation under mulching-induced hypoxic conditions. Sci. Total Environ. 817, 153017. https://doi.org/10.1016/j.scitotenv.2022.153017 . Qiao, M.J., Sun, R.B., Wang, Z.X., Dumack, K., Xie, X.G., Dai, C.C., Wang, E.T., Zhou, J.Z., Sun, B., Peng, X.H., Bonkowski, M., Chen, Y., 2024. Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification. Nat. Commun. 15, 2924. https://doi.org/10.1038/s41467-024-47159-x . Raiesi, F., Sadeghi, E., 2019. Interactive effect of salinity and cadmium toxicity on soil microbial properties and enzyme activities. Ecotox. Environ. Safe. 168, 221-229. https://doi.org/10.1016/j.ecoenv.2018.10.079 . Rashid, M.I., Mujawar, L.H., Shahzad, T., Almeelbi, T., Ismail, I.M.I., Oves, M., 2016. Bacteria and fungi can contribute to nutrients bioavailability and aggregate formation in degraded soils. Microbiol. Res. 183, 26-41. https://doi.org/10.1016/j.micres.2015.11.007 . Raymond, N.S., Gómez‐Muñoz, B., van der Bom, F.J.T., Nybroe, O., Jensen, L.S., Müller‐Stöver, D.S., Oberson, A., Richardson, A.E., 2020. Phosphate‐solubilising microorganisms for improved crop productivity: a critical assessment. New Phytol. 229, 1268-1277. https://doi.org/10.1111/nph.16924 . Ruyters, S., Mertens, J., Vassilieva, E., Dehandschutter, B., Poffijn, A., Smolders, E., 2011. The Red Mud Accident in Ajka (Hungary): Plant Toxicity and Trace Metal Bioavailability in Red Mud Contaminated Soil. Environ. Sci. Technol. 45, 1616-1622. https://doi.org/10.1021/es104000m . Santini, T.C., Fey, M.V., 2013. Spontaneous Vegetation Encroachment upon Bauxite Residue (Red Mud) As an Indicator and Facilitator of In Situ Remediation Processes. Environ. Sci. Technol. 47, 12089-12096. https://doi.org/10.1021/es402924g . Santini, T.C., Raudsepp, M., Hamilton, J., Nunn, J., 2018. Extreme Geochemical Conditions and Dispersal Limitation Retard Primary Succession of Microbial Communities in Gold Tailings. Front Microbiol. 9, 2785. https://doi.org/10.3389/fmicb.2018.02785 . Santini, T.C., Warren, L.A., Kendra, K.E., 2015. Microbial Diversity in Engineered Haloalkaline Environments Shaped by Shared Geochemical Drivers Observed in Natural Analogues. Appl. Environ. Microbiol. 81, 5026-5036. https://doi.org/10.1128/Aem.01238-15 . Wu, H., Chen, L., Zhu, F., Hartley, W., Zhang, Y.F., Xue, S.G., 2020. The dynamic development of bacterial community following long-term weathering of bauxite residue. J. Environ. Sci. 90, 321-330. https://doi.org/10.1016/j.jes.2019.12.001 . Wu, H., Tang, T., Zhu, F., Wei, X.M., Hartley, W., Xue, S.G., 2021. Long term natural restoration creates soil-like microbial communities in bauxite residue: A 50-year filed study. Land Degrad. Dev. 32, 1606-1617. https://doi.org/10.1002/ldr.3728 . Xiao, T., Li, P., Fei, W.B., Wang, J.D., 2024. Effects of vegetation roots on the structure and hydraulic properties of soils: A perspective review. Sci. Total Environ. 906, 167524. https://doi.org/10.1016/j.scitotenv.2023.167524 . Xue, S.G., Ke, W.S., Zhu, F., Fan, J.R., Wang, Q.L., Liu, Z., Hartley, W., 2021. Evaluating aggregate stability, surface properties and disintegration behaviour of bauxite residue induced by Ca/Na. Land Degrad. Dev. 32, 803-816. https://doi.org/10.1002/ldr.3703 . Xue, S.G., Wu, Y.J., Li, Y.W., Kong, X.F., Zhu, F., William, H., Li, X.F., Ye, Y.Z., 2019. Industrial wastes applications for alkalinity regulation in bauxite residue: A comprehensive review. J. Cent. South. Univ. 26, 268-288. https://doi.org/10.1007/s11771-019-4000-3 . Yang, Y., Chai, Y.B., Xie, H.J., Zhang, L., Zhang, Z.M., Yang, X., Hao, S.L., Gai, J.P., Chen, Y.L., 2023. Responses of soil microbial diversity, network complexity and multifunctionality to three land-use changes. Sci. Total. Environ. 859, 160255. https://doi.org/10.1016/j.scitotenv.2022.160255 . You, F., Zhang, L.P., Ye, J., Huang, L.B., 2019. Microbial decomposition of biomass residues mitigated hydrogeochemical dynamics in strongly alkaline bauxite residues. Sci. Total. Environ. 663, 216-226. https://doi.org/10.1016/j.scitotenv.2019.01.317 . Yuan, Y.S., Zhao, W.Q., Xiao, J., Zhang, Z.L., Qiao, M.F., Liu, Q., Yin, H.J., 2017. Exudate components exert different influences on microbially mediated C losses in simulated rhizosphere soils of a spruce plantation. Plant Soil. 419, 127-140. https://doi.org/10.1007/s11104-017-3334-6 . Zaitseva, S.V., Abidueva, E.Y., Radnagurueva, A.A., Bazarov, S.M., Buryukhaev, S.P., 2018. Structure of Microbial Communities of the Sediments of Alkaline Transbaikalia Lakes with Different Salinity. Microbiology. 87, 559-568. https://doi.org/10.1134/s0026261718040185 . Zhalnina, K., Louie, K.B., Hao, Z., Mansoori, N., da Rocha, U.N., Shi, S., Cho, H., Karaoz, U., Loque, D., Bowen, B.P., Firestone, M.K., Northen, T.R., Brodie, E.L., 2018. Dynamic root exudate chemistry and microbial substrate preferences drive patterns in rhizosphere microbial community assembly. Nat. Microbiol. 3, 470-480. https://doi.org/10.1038/s41564-018-0129-3 . Zhu, F., Li, X.F., Xue, S.G., Hartley, W., Wu, C., Han, F.S., 2016a. Natural plant colonization improves the physical condition of bauxite residue over time. Environ. Sci. Pollut. R. 23, 22897-22905. https://doi.org/10.1007/s11356-016-7508-1 . Zhu, F., Li, Y.B., Xue, S.G., Hartley, W., Wu, H., 2016b. Effects of iron-aluminium oxides and organic carbon on aggregate stability of bauxite residues. Environ. Sci. Pollut. R. 23, 9073-9081. https://doi.org/10.1007/s11356-016-6172-9 . Zhu, F., Zhang, X.C., Guo, X.Y., Yang, X.W., Xue, S.G., 2023. Root architectures differentiate the composition of organic carbon in bauxite residue during natural vegetation. Sci. Total. Environ. 883, 163588. https://doi.org/10.1016/j.scitotenv.2023.163588 . Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7133212","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488847639,"identity":"0f45a135-20c6-4a4c-9424-f0dac04890c7","order_by":0,"name":"Yang Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie2RMUsDMRTHXzm5LrnqmKPVfIUU4RYLuvk1IgfX5brfoOWKkFsqXZ38GOL4QqB1yAeo6ODUqYWCSx1ajKPDxY6C+Q0ZHv8ff94LgMfzhyGHzVGJ62J3EjYr3EvpxGOt1L3B0xYxYi+lx+dZqiOJVw/0gjuj7FJP6efTDeHznCMJ3/qSgoBN8VirdE2WxXdmRmKz5EjJYiDbI2yMzWu9UuYJjeSUtGa2hdNgIDsogoZ0KJNlEm+tAmgVwYN+SO3rUhjNk3Ykr8nRcyYQhRa/Kpwu0rNjicQeGVWJWVfaIyvXLmySqpeVHJ7br7z92O56jFWVet8UjhaEAwqgf06xNv/dUkKwBhi6Mh6Px/Pf+QJPAWRCnEPe7AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0000-2664-390X","institution":"China University of Mining and Technology Beijing Campus School of Chemical and Environmental Engineering","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Gao","suffix":""},{"id":488847640,"identity":"32657f22-5279-406e-903e-febe0e5441bf","order_by":1,"name":"Liping Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Zhang","suffix":""},{"id":488847641,"identity":"a753a50d-12f7-488c-a0bd-8ae5d07ed33d","order_by":2,"name":"Huitong Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huitong","middleName":"","lastName":"Li","suffix":""},{"id":488847642,"identity":"7da17e63-9662-4178-a99c-b6345c7b7901","order_by":3,"name":"Huarang Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huarang","middleName":"","lastName":"Sun","suffix":""},{"id":488847643,"identity":"8c054363-7623-4153-a76a-135d9ad05f8c","order_by":4,"name":"Ran Mu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Mu","suffix":""},{"id":488847644,"identity":"c6b6909d-760c-4761-9fe4-4b0d55ba5459","order_by":5,"name":"Zehua Pi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zehua","middleName":"","lastName":"Pi","suffix":""},{"id":488847645,"identity":"dfb21c04-dd31-4ba2-9bb1-745072e2b395","order_by":6,"name":"Yiqing Gao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yiqing","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2025-07-15 18:06:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7133212/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7133212/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87478370,"identity":"5dce038b-84d6-4c47-851c-692129d1f342","added_by":"auto","created_at":"2025-07-24 09:26:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85114,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial utilization characteristic of 31 carbon substrates on Eco-plate™ (A, NMDS of the carbon utilization patterns; B, AWCD of 31 carbon substrates).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/028c7afef046f0ce294b192b.png"},{"id":87478371,"identity":"5fa674f1-2879-4484-a33f-6d949b734f9e","added_by":"auto","created_at":"2025-07-24 09:26:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54859,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial carbon metabolism activities and diversity in bauxite residue (A, AWCD index; B, Shannon index; C, Simpson index; D, Mclntosh index).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/62671dbb1c7377f81cc2a2c5.png"},{"id":87478368,"identity":"9533e8e3-2b59-4ba2-9653-0f51a9129fa9","added_by":"auto","created_at":"2025-07-24 09:26:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35651,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity of bacterial community in bauxite residue during natural restoration (A, Shannon index; B, OTU number).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/4886f19380313aac78806aaa.png"},{"id":87478868,"identity":"7b6cfc04-4215-4185-b095-a560b5b84c9c","added_by":"auto","created_at":"2025-07-24 09:34:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51460,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of the relative abundance of microbial community in bauxite residue during natural restoration on phylum level.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/f0f26e56c48eb7cd9fe01be6.png"},{"id":87478384,"identity":"fe3b06c0-7b4d-4752-8d7a-31ffdcb6d9a0","added_by":"auto","created_at":"2025-07-24 09:26:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138041,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of the top 20 microbial groups in bauxite residue under different restoration level on family level.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/8bf3f1ed54f0e8f580b39355.png"},{"id":87478381,"identity":"e65f5dbc-e9ed-41e6-a8c6-68e1e07e66a2","added_by":"auto","created_at":"2025-07-24 09:26:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23035,"visible":true,"origin":"","legend":"\u003cp\u003eRandom forest analysis of (A) microbial diversity index and (B) key microbial taxa to carbon metabolism activity in bauxite residue.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/283aadb6385ed0ab724cc52d.png"},{"id":87478869,"identity":"37e6c538-f31b-473a-8be7-c2eb9a125a6c","added_by":"auto","created_at":"2025-07-24 09:34:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":128045,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between carbon metabolism activity and key microbial taxa in bauxite residue.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/d458a97cdb2d59f329f35bd4.png"},{"id":87478874,"identity":"6ae4336d-8365-4d16-857e-0e22e2f9f2fa","added_by":"auto","created_at":"2025-07-24 09:34:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":98464,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between community diversity index and richness index with soil chemical properties.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/2eeb4f9b7210149e2bb1c743.png"},{"id":87479853,"identity":"a53b61a5-303e-45ff-a614-ec12134c4872","added_by":"auto","created_at":"2025-07-24 09:42:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":86722,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis (RDA) between residue properties and microbial communities in bauxite residue.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/501835a81000b62e9c06aa1b.png"},{"id":91332442,"identity":"f894d80a-192e-4025-93f7-bdaf11e8d8b5","added_by":"auto","created_at":"2025-09-15 11:11:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1540802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/953e15cf-1601-4a17-b18b-6f3ad4e7fdae.pdf"},{"id":87478382,"identity":"397e7dd3-4d4f-4bea-a03e-7878007ecd68","added_by":"auto","created_at":"2025-07-24 09:26:08","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":45879,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7133212/v1/d2c5f3d0aac8da6b4f2cad82.docx"}],"financialInterests":"","formattedTitle":"Natural weathering promoted the recovery of microbial community in bauxite residues","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eLong-term natural weathering increased microbial metabolic activities in bauxite residue.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Microbial groups including Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae played important roles in the recovery of microbial metabolic in bauxite residue.\u003c/li\u003e\n \u003cli\u003eThe shifts in the bacterial community were mainly driven by MWD followed by pH, TOC, AP, TN, EC and AN.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eBauxite residue is the by-product produced during the alumina production process (Power et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Currently, the utilization of bauxite residue is still limited and it is mainly stored in large-scale dam, which pose potential ecological risks to the surrounding environment (Gelencser et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ruyters et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Recently, the improvement of bauxite residue from tailing to soil has been advocated as the promising way in the management of bauxite residue (Xue et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, due to the adverse properties including high alkalinity/salinity, poor structure and limited nutrient conditions in bauxite residue, successful vegetation case is limited.\u003c/p\u003e\u003cp\u003eThe natural weathering process can improve the physicochemical properties and promote microbial activities in bauxite residue (Kong et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Santini and Fey \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e). Firstly, natural weathering process such as wind and water erosion promote the alkaline anion leaching and alkaline minerals dissolution, which largely decreased alkalinity and salinity in bauxite residue (Kong et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Kong et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Secondly, natural weathering process improves aggregate formation in bauxite residue, which benefit to the improvement of the water, fertilizer, air and heat conditions in bauxite residue (Zhu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e). Thirdly, during natural weathering process, organic carbon derived from plant tissue and root exudates promote the development of plant community (Zhu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the currently studies mainly focus on the improvement of the physicochemical properties in bauxite residue, and neglect the establishment of biogeochemical process, such as carbon/nitrogen fixation, carbon/nitrogen mineralization and organic matter humification in bauxite residue. Therefore, the investigation of dynamics of microbial metabolic activities during the natural weathering process may provide us a comprehensive understanding of the soil formation process in bauxite residue.\u003c/p\u003e\u003cp\u003eMicrobial community are the main driver for diverse ecological processes such as greenhouse gas emissions, organic carbon degradation and nitrogen fixation, and aggregates formation (Bardgett and van der Putten \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Crowther et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, some specific functional microorganisms, such as nitrogen-fixing bacteria and phosphorus-solubilizing bacteria, can increase the availability of nitrogen and phosphorus in soil through biological nitrogen fixation and phosphate solubilization process (Hartmann and Six \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phosphate-solubilizing microorganisms could produce organic acids to promote the dissolution of insoluble phosphate in soil, which may benefit to plant growth (Raymond et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Arbuscular mycorrhizal fungi (AMF) could entrap soil particles and force them together along s along the expanding hyphae to form aggregates (Rashid et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, the AMF could also secrete diverse extracellular polymeric substance, glomalin, hydrophobins and related soil proteins as cementing agent to promote the aggregate formation in soil (Rashid et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the role of microbial community should be taken into consideration in ecological remediation in bauxite residue, not merely investigate the changes of physiochemical properties in bauxite residue.\u003c/p\u003e\u003cp\u003eCurrently, several studies investigated the dynamic changes of microbial community in bauxite residue. During natural weathering process, the microbial diversity significantly increased, and microbial composition changed from those dominated by alkalophilic and halophilic assemblages to those dominated neutrophilic assemblages (Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Weathering for over 50 years, the microbial communities in bauxite residue changed to the assemblages that was similar to those of the natural soil microbial community around the mining area (Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, long term restoration enriched the abundance of some specific microbial groups, which may participate in nitrogen fixation or phosphate solubilization process, and largely increased the network stability and complexity of microbial communities in bauxite residue (Deng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). By reshaping the microbial community, long-term vegetation restoration significantly improved the multifunctional of bauxite residue (Jiang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, current studies mainly focused on the development of microbial communities during restoration, whereas neglected the changes in microbial metabolic activities. Therefore, to obtained comprehensive knowledge on the soil formation of bauxite residue, the development of microbial metabolic activities must be taken into consideration.\u003c/p\u003e\u003cp\u003eA typical Bauxite Residue Disposal Area in Central China was chosen in this study. The objectives of this research were: 1) to analyze the effect of natural weathering process on the development of the salinity/alkalinity and nutrients condition in bauxite residue. 2) to monitor the dynamic changes of microbial activities and microbial structure in bauxite residue. 3) to establish the relationships between microbial carbon metabolic characteristics and physiochemical properties in bauxite residue. This study aims to provide theoretical support for the field-scale ecological remediation of bauxite residue.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eStudy sites and soil sampling\u003c/h2\u003e\n\u003cp\u003eResidue samples were selected from a Bauxite Residue Disposal Area (BRDA), which belongs to Henan Branch China Aluminum Corporation CO, LTD. The average annual precipitation was 380 mm and annual temperature was 16.0 ℃ in the sampling area.\u003c/p\u003e\n\u003cp\u003eOn the surface of Bauxite Residue Disposal Area, there were obvious plant encroachment, which dominated by herbaceous plant including Cynodon, Artemisia, Festuca arundinacea etc. According to the disposal history, four sites, which representing different years since restoration (5, 10, 20 and 30 years since disposal) were chosen to collect samples. In each site, five samples were collected randomly. Totally, twenty bauxite residue samples were collected in this study. All samples were placed in polyethylene plastic bags and transported to the laboratory under 4℃ conditions.\u003c/p\u003e\n\u003cp\u003eWhen transported back to the laboratory, the samples were filtered with the 2-mm mesh and divided into four parts. One part was naturally dried for the determination of physicochemical properties. One part was stored under 4℃\u0026nbsp;for Biolog test. One part was stored under -80℃ for further determination of microbial community.\u003c/p\u003e\n\u003ch2\u003eCommunity-Level Physiological Profiling\u003c/h2\u003e\n\u003cp\u003eBiolog EcoPlates were employed to assess the microbial carbon metabolic capacity (Garland and Mills 1991). Each EcoPlate contains thirty-two types carbon source and one control, which in total thirty-three treatments. Three were three replicates for each carbon source treatments, in total ninety-six wells in one EcoPlate. The thirty-two types carbon source could be classified to six main carbon type such as amino acids, amines, carbohydrates, carboxylic acids, phenolic acids and polymers. The detailed information was listed in Table S1.\u003c/p\u003e\n\u003cp\u003eIn brief, five-gram sample was dissolved with sterile 0.05M phosphate buffer solution in 100 mL plastic bottle. The sample-water mixture solution was shaken for 30 min under the 180 r/min condition. 1 ml the mixture solution was taken and added to a test tube containing 9 ml sterile phosphate buffer, and a 10\u003csup\u003e-2\u003c/sup\u003e dilution was obtained. Repeat the above step one more time, and 10\u003csup\u003e-3\u003c/sup\u003e dilution was obtained. 150 \u0026mu;L of the obtained dilution (10\u003csup\u003e-3\u003c/sup\u003e) was added into each microplate well in the plate and then incubated at 25 \u0026plusmn; 1 ℃ in dark in a thermostatic incubator. The light absorption of each well was read by using a BIOLOG plate reader (BioTek ELx800, BioTek, USA) at 750 nm and 590 nm wavelengths every 24 hours.\u003c/p\u003e\n\u003ch2\u003eHigh-throughput sequencing\u003c/h2\u003e\n\u003cp\u003eThe DNA was extracted by using the PowerSoil\u0026reg; DNA Isolation Kit. In 16S rRNA gene, the V4 region was chosen as the marker gene and then amplified. The primer was 338F (5\u0026prime;-ACTCCTACGGGAGGCAGCAG-3\u0026prime;) and 806R (5\u0026prime;- GGACTACHVGGGTWTCTAAT-3\u0026prime;). The PCR amplification program was described in Jiang\u0026rsquo; report (Jiang et al. 2023). The amplified PCR product was extracted by using agarose (2%) and then purified by using DNA Gel Extraction Kit. The concentration of purified DNA was tested using Nanodrop 2000 spectrophotometer. Finally, the qualified DNA was sequenced on the Illumina MiSeq PE300 platform in Majorbio company.\u003c/p\u003e\n\u003ch2\u003eData processing\u003c/h2\u003e\n\u003cp\u003eBiolog data: The average well color development (AWCD) was calculated as the difference value of OD590 in carbon well and control well.\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"127\" height=\"45\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (1)\u003c/p\u003e\n\u003cp\u003eIn the above equation, A\u003csub\u003ei\u003c/sub\u003e: the absorbance reading of OD\u003csub\u003e590\u003c/sub\u003e in the carbon well i; \u0026nbsp;A\u003csub\u003e0\u003c/sub\u003e: the absorbance reading of OD\u003csub\u003e590\u003c/sub\u003e in the blank well. n: the numbers of carbon source; When the difference value of OD\u003csub\u003e590\u003c/sub\u003e in plate well were negative or under 0.06, they are coded as zeroes for subsequent data analysis.\u003c/p\u003e\n\u003cp\u003eThe Shannon index (H), Simpson index (D) and Mclntosh index (U) of microbial carbon metabolism were calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"131\" height=\"27\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (2)\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"89\" height=\"27\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(3)\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"80\" height=\"31\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (4)\u003c/p\u003e\n\u003cp\u003eIn the above equation, \u003cimg width=\"95\" height=\"45\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e, \u003cimg width=\"76\" height=\"25\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e.\u003c/p\u003e\n\u003cp\u003eDNA Sequencing\u0026nbsp;data: The raw sequences were firstly analyzed on the QIIME pipeline\u0026nbsp;and then filtered those low-quality sequences (the quality scores \u0026lt; 20 and sequences length \u0026lt; 150 bp) (Caporaso et al. 2010). Secondly, the chimeras within sequences were eliminated by using UCHIME software (Edgar et al. 2011). Thirdly, the remained sequences were clustered into operational taxonomic units (OTUs) based on 97% similarity by using UPARSE version7.1 (Edgar 2013). Finally, RDP Classifier was applied to annotate the taxonomy of representative OTU in 16S rRNA database (Silva v138).\u003c/p\u003e\n\u003cp\u003eThe alpha diversity indices were calculated by using \u0026ldquo;vegan\u0026rdquo; package in R platform (Dixon 2003). Principal Coordinates Analysis (PCoA) was conducted by using \u0026ldquo;vegan\u0026rdquo; R package in R platform (Dixon 2003). Redundancy analysis (RDA) was conducted by using \u0026ldquo;radcca.hp\u0026rdquo; R package in R platform (Dixon 2003). Random forest analysis was performed by using the \u0026ldquo;RandomForest\u0026rdquo; package in R platform (Dixon 2003).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eVariation of physicochemical properties in bauxite residue\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe alkalinity and salinity significantly decreased, whereas nutrients condition and physical significantly improved following long term natural weathering process in bauxite residue (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to 5Y sites, the TOC, TN, AN and AP were significantly increased in 30Y sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the MWD also significantly increased in 30Y sites when compared with that in 5Y sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Contrary to the improvement of nutrients condition and physical properties, he alkalinity and salinity index such as pH, EC and Na\u003csup\u003e+\u003c/sup\u003e content all significantly decreased in 30Y sites when compared with that in 5Y sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysicochemical properties in bauxite residue.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSite\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTOC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eAP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eMWD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ecmol/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003emg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003emg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.18a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.65a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.73a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.75d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.42d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e39.81d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.82a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8.83d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.49d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(0.09)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(0.04)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(0.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(0.04)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(12.07)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(0.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(0.66)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e(0.04)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.32b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.36b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.03b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.23c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.73c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e69.25c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.77ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e25.89c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.72c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(0.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(0.04)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(0.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(0.09)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(9.62)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(0.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(4.66)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e(0.07)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.52c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.27bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.72b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.19b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.89b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e95.49b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.75b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e40.72b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.86b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(0.26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(0.02)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(1.15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(0.05)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(8.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(0.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(4.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e(0.06)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.28c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.24c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.12c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.11a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.97a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e120.33a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.72b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e53.36a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.89a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(0.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(0.02)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.58)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(1.34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(0.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(17.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(0.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(6.95)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e(0.05)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eNotes: 5Y, weathered for 5 years; 10Y, weathered for 10 years; 20Y, weathered for 20 years; 30Y, weathered for 30 years; TOC, organic carbon; TN, total nitrogen; AN, available nitrogen; TP, total phosphorous; AP, available phosphorous; MWD, mean weight diameter;\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eValues with the same lower case were not significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 among different sites.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobial carbon metabolism in the process of natural weathering in bauxite residue\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLong term natural weathering process significantly changed the microbial carbon utilization patterns in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The principal coordinates analysis showed that the PC1 and PC2 explained 85.36% and 8.74% of the variance in microbial carbon utilization patterns, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Specifically, in 5Y sites, the carbon source with highest utilization intensity was amine, followed by carboxylic acid, amino acids, phenolic acid, polymer and carbohydrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The utilization intensity of amino acids, carbohydrate, carboxylic acid, phenolic acid and polymer significantly increased, whereas that of amine significantly decreased in microbial carbon utilization patterns during the natural weathering process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In 30Y sites, the carbon source with highest utilization intensity was carbohydrate and amino acids, followed by polymer, carboxylic acid, phenolic acid and amine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe microbial carbon metabolism activities and diversity were promoted during natural weathering process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The maximum AWCD and diversity index were all observed in the 30Y sites. The AWCD value increased from 0.41 (5Y) to 0.83 (10Y), 1.14 (20Y) and 1.47 (30Y), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, the microbial carbon metabolism diversity index (Shannon index and Simpson index) significantly increased in 30Y sites when compared to 5Y sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). These results suggested that long term natural weathering process might stimulate the recovery of microbial communities and enhanced the carbon metabolism in bauxite residue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobial communities in the process of natural weathering in bauxite residue\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLong term natural weathering process significantly increased the diversity and richness of microbial communities in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest observed species number was observed in 30Y sites, which significantly higher than that in all sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest Shannon index was observed in 20Y sites, which significantly higher than that in 10Y and 5Y sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but showed no significant difference between 20Y and 30Y sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe composition of microbial community significantly varied during natural weathering processes in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At the phylum level, the dominant microbial groups were Firmicutes (39.88%), Proteobacteria (16.27%), Chloroflexi (9.49%), Cyanobacteria (6.9%), Actinomycetota (6.3%) and Bacteroidota (5.93%) in the 5Y sites (Table. S2). Other abundant groups (the relative abundance\u0026thinsp;\u0026gt;\u0026thinsp;1%) were Planctomycetota (4.42%), Verrucomicrobiota (3.86%), Crenarchaeota (2.24%), Acidobacteriota (1.49%) and Nitrospirota (1.07%) (Table. S2). During the long-term natural weathering processes, the relative abundance of Firmicutes, Cyanobacteria, Verrucomicrobiota and Crenarchaeota significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the relative abundance of Proteobacteria, Bacteroidota, Acidobacteria, Nitrospirota significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In 30Y sites, the dominant groups changed to Proteobacteria (43.69%), Bacteroidota (13.65%), Acidobacteria (12.24%), Actinobacteria (5.42%) and Chloroflexi (5.07%) (Table. S2). Other relatively abundant groups were Firmicutes (4.63%), Planctomycetota (4.84%) and Nitrospirota (3.55%) (Table. S2). The relative abundance of all the microbial groups showed significantly difference between 5Y and 30Y sites, except for Planctomycetota and Actinobacteria (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the family level, the microbial community was dominated by Bacillaceae (22.39%), Halomonadaceae (7.51%), Clostridiaceae (6.53%) and Chloroflexaceae (5.49%) in the 5Y sites (Table. S3). Natural weathering processes decreased the abundance of Bacillaceae, Halomonadaceae and Clostridiaceae, whereas increased the abundance of Acidobacteriaceae, Beijerinckiaceae, Blastocatellaceae, Caulobacteraceae, Chitinophagaceae, Devosiaceae, Enterobacteriaceae, Microscillaceae, Nitrospiraceae, Nitrosomonadaceae, Planctomycetaceae, Rhizobiaceae, Sphingobacteriaceae and Sphingomonadaceae in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Differed with those microbial groups in 5Y sites, the microbial community were dominated by Sphingomonadaceae (7.53%) and Beijerinckiaceae (5.92%) in 30Y sites (Table. S3). In addition, the increased microbial groups induced by natural weathering process such as Acidobacteriaceae, Beijerinckiaceae, Blastocatellaceae, Caulobacteraceae, Chitinophagaceae, Devosiaceae also exhibit relatively high abundances (1%-5%) in 30Y sites. Interestingly, all the increased microbial groups showed significantly difference between 5Y and 30Y sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRandom forest model showed the microbial diversity and composition both significantly affect the carbon metabolism activities in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The importance value of Chao 1 and ACE index were both higher than diversity index Shannon and Simpson index, which indicated that microbial richness rather than diversity was more important to the recovery of microbial carbon metabolism activities in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Among the top 10 increased microbials groups, Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae significantly promoted the microbial carbon metabolism activities in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe linear regression analysis showed that the AWCD was significantly correlated with Beijerinckiaceae (0.874), Rhizobiaceae (0.898), Microscillaceae (0.851), Caulobacteraceae (0.845), Blastocatellaceae (0.889), Acidobacteriaceae (0.888), Sphingomonadaceae (0.827), Nitrosomonadaceae (0.741) and Chitinophagaceae (0.702) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCorrelation between microbial community and bauxite residue\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, mantel test and redundancy analysis were employed to identify the key environmental factors in affecting the development of microbial communities during long term weathering process. Mantel test indicated that the 16S gene abundance showed negative correlation with pH, EC, Na and TP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas showed positive correlation with TOC, TN, AN, AP and MWD (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The Shannon diversity index, Chao 1 index and ACE index were all showed significantly negative correlations with pH, EC, Na and TP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas showed positive correlations with TOC, TN, AN, AP and MWD (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRedundancy analysis (RDA) indicated that MWD, followed by pH, TOC, AP, TN, EC and AN were the mainly driving factor in the development of microbial community, while TP had little significant influence (Table S4). These environment factors could explain 94.79% of the total variation of microbial community, indicated that the development of microbial communities mainly driven by the change of physiochemical properties in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Thus, the long-term natural weathering process could promote the recovery of microbial communities by improving properties in bauxite residue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eImprovement of natural weathering process on the environmental conditions of bauxite residues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe long-term natural weathering processes improved the physiochemical properties in bauxite residue, which expressed as alkalinity/ salinity reduction, nutrients accumulation and physical structure improvement. The alkalinity reduction in bauxite residue may be induced by rainwater leaching. The natural weathering processes induced alkaline anion (e.g OH\u003csup\u003e\u0026minus;\u003c/sup\u003e, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, and aluminates) leaching and the alkalinity solids (e.g sodalite, hydrogarnet and calcite) dissolving, which may cause the alkalinity reduction in bauxite residue (Kong et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). In addition, the secretion of organic acids in plant rhizosphere metabolism or its release during litter decomposition may be another reason for the alkalinity reduction in bauxite residue. The microbial decomposition of biomass residues would produce numerous organic acid (e.g., acetic acid, citric acid and isocitric acid) and neutralized alkalinity in bauxite residue (You et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The development of plant communities including plant rhizosphere metabolism and litter decomposition process not only neutralized alkalinity in bauxite residue, but also induced nutrients accumulation in bauxite residue. On one hand, plant rhizosphere metabolism would release nutrients compounds including carbohydrate, organic acids, and amino acids, and thus increased nutrients elements in bauxite residue. On the other hand, the dead plant tissue would be decomposed and release nutrients into bauxite residue (Pereira et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAfter 30 years of natural weathering, significant improvement of physical structure was observed in bauxite residue, which expressed as the increase in MWD (from 0.51 to 0.91 mm). The improvement of physical structure may be related to the development of plant communities. On one hand, Plant roots could align soil particles along its expanding roots to form aggregates (Xiao et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the other hand, plant exudes such as mucilages, polysaccharides and extracellular compound could bind particles together through cation bridge, polysaccharide -OH group and H proton (Xiao et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The improvement of physical structure not only affected by plant activities, also be affect by the reduction of salinity in bauxite residue. The increased MWD in long term weathered bauxite residue (30Y) may related to the decreased ESP, as excessive Na\u003csup\u003e+\u003c/sup\u003e would cause the expansion of lattice and disintegration of clay particles in bauxite residue (Xue et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, increased organic carbon may benefit to the formation of organic-mineral complexes, thus improved structural texture in bauxite residue (Zhu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImprovement of natural weathering process on microbial activity in bauxite residues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe recovery of microbial activity is critical to the rehabilitation in bauxite residue. However, the high salinity and alkalinity in bauxite residue commonly restrict the microbial activities in bauxite residue, as the high salinity/alkalinity was toxic to microbial cells and enzyme catalytic sites (Raiesi and Sadeghi \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the present study, natural weathering process significantly increased microbial metabolic capacity and diversity on carbon source. This may be related to the improvement of physiochemical properties in bauxite residue. In the early stage of weathering process, the alkalinity and salinity were extreme high in bauxite residue, which large restrict the microbial activities. High salinity may lead to cell dehydration, which damage the integrity of cell membrane, and then affect the activity of enzymes and the normal operation of metabolic pathways. The excessive accumulation of Na\u003csup\u003e+\u003c/sup\u003e, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e may interfere with the activity of enzymes in microbial cells, by binding to key active sites in enzyme molecules, resulting in structural inactivation (Oren \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In high alkaline environment, most of the neutral and acidic environment microorganisms are difficult to survive, and only alkali-resistant bacteria (such as some actinomycetes) are left, with poor carbon metabolism capacity (Banda et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zaitseva et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, the accumulation of organic carbon and nutrients induced by natural weathering process would positively stimulate microbial growth and enhance metabolic activity by providing more available carbon sources and energy. For example, exudates derived from plant root, that mainly consist of carbohydrates, amino acids, organic acids and other carbon-based compounds are the main carbon and energy sources of microorganisms, which could directly promote their growth and metabolic activities (Yuan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, organic acids (such as acetic acid, oxalic acid) can reduce soil pH and create an environment suitable for acidic microbial growth, while dissolving mineral-bound carbon and increasing the availability of carbon, which provide carbon source for microbial growth and indirectly increase microbial carbon metabolic activities (Kong et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImprovement of natural weathering process on microbial communities in bauxite residues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe natural weathering process not only promoted the recovery of microbial activity, but also changed the microbial community\u0026rsquo;s composition. It had been shown that the quality of bauxite residue was significantly correlated with microbial diversity and composition (Deng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, the natural weathering process significantly increased microbial diversity in bauxite residue, which were similar to the previous works (Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExcept for the increase in microbial diversity, natural weathering process also dramatically changed the microbial composition in bauxite residue, which expressed as a dramatically change from alkalophilic and halophilic assemblages to neutrophilic assemblages. In 5Y bauxite residue, the microbial communities were dominated by Streptococcaceae, Bacillaceae. The adverse geochemical conditions (e.g., extreme pH, salinity, metal toxicity) and nutrient deficiencies (e.g., limited nitrogen and phosphorus) in bauxite residue only allowed several microorganisms groups with high saline-alkali tolerance (Bacillaceae and Actinobacteria) survived, which coincide with low diversity in 5Y bauxite residue (Krishna et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Santini et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Bacillaceae could produce spore dormant structures, which were highly resistant to desiccation, heat, UV radiation, and chemical stressors (Dittmann et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). They could persist in poor nutrient and extreme pH biotopes (such as dry soils, mineral rock or acid mine) and be reactivated when nutrients and water become available.\u003c/p\u003e\u003cp\u003eDuring long term natural weathering process, the properties significantly improved, which significantly affected the microbial composition in bauxite residue (Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After weathering for 30 years, the abundance of Bacillaceae, Chloroflexaceae, Halomonadaceae and Clostridiaceae significantly decreased, whereas the abundance of Sphingomonadaceae, Beijerinckiaceae, Blastocatellaceae, Microscillaceae, Anaerolineaceae, Nitrospiraceae, Enterobacteriaceae, Chitinophagaceae, Acidobacteriaceae, Nitrosomonadaceae, Rhizobiaceae, Devosiaceae and Caulobacteraceae significantly increased in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Tables S1). On one hand, the increase of organic carbon derived from litter decomposition and root exudates following natural weathering process may provide more metabolic substrate and promote the growth of organotrophic microorganism such as Sphingomonadaceae, Acidobacteriaceae, Rhizobiaceae (You et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the other hand, the improvement of microenvironment in bauxite residue (such as pH reduction and formation of aggregate structure) also provided a suitable habitat for this family of microorganisms (You et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The relative abundance of Acidobacteria commonly showed negative correlations with soil pH (Lauber et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Thus, the pH decreased in bauxite residue would induce the enrichment of Acidobacteria. In addition, the formation of aggregates may enhance water and oxygen permeability, which promoted aerobic microorganisms growth (including Beijerinckiaceae) (Qian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the increased organic carbons may induce the enrichment of R-strategy groups such as Proteobacteria and Bacteroidetes in bauxite residue (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePlant root exudate may be another critical reason for the development of microbial communities in bauxite residue. Root exudates are rich in organic compounds (such as sugars, organic acids, amino acids, etc.), accounting for 20%-40% of plant photosynthetic products, which could provide carbon source and energy for microorganisms (Zhalnina et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Interestingly, some increased microbial groups in restored bauxite residue were capable with nitrogen fixation such as Beijerinckiaceae, Rhizobiaceae and Devosiaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Tables S2). This was coincided with Deng\u0026rsquo;s work (Deng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), that plant development enriched microbial groups with nitrogen fixing ability in bauxite residue. Root exudates could promote nitrogen fixation in mining soil by stimulating the development of azotobacter (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Some specific root exudates metabolites in the rhizosphere, such as flavonoids and coumarins, have a facilitation effect on the root bacterial community, especially nitrogen fixing bacteria (Qiao et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe main driving factors in the development of microbial communities\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSoil microorganisms play vital roles in the ecosystem by regulating biochemical processes. However, microorganisms were sensitive to environmental changes, which affect biochemical processes including organic carbon degradation, carbon mineralization and nitrogen fixation. Previous studies showed that the natural weathering processes improved geochemical properties in bauxite residue, which provided an opportunity for the recovery of microbial community (Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, the Shannon diversity of microbial community was negative correlated with pH and EC, whereas was positive correlated with TOC, TN, AP, and AN. This conincide with the previous findings, which suggest that alkalinity reduction and nutrients accumulation promoted the microbial diversity in bauxite residue (Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilar to microbial diversity, the environment factors had comprehensive effects on microbial communities. RDA analysis showed that variations in microbial composition were correlated with pH, EC, Na\u003csup\u003e+\u003c/sup\u003e, TOC, TN, AN, TP, AP and MWD, which indicated that alkalinity/salinity and nutrients availability strongly affect microbial development in bauxite residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e, Tables S3). Commonly, pH condition and nutrients availability were the mainly driving factors in the development of microbial communities in degraded ecosystem. For instance, Santini et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that pH controlled the microbial community assembly in gold tailings (Santini et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In an abandoned REE mine site, the bacterial communities were mainly driven by soil nutrients (Chao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Deng et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also found that microbial communities were dramatically affected by soil pH and available nutrients in an open-cut iron mining area (Deng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study investigated the effect of natural weathering process on microbial carbon metabolisms and associated microbial community in bauxite residue. Firstly, natural weathering process effectively decreased alkalinity, whereas increased nutrients content and improved the physical structure in bauxite residue. These improvements became more obvious as the weathering time increased. Secondly, natural weathering process induced an obvious increase in the activity of microbial carbon metabolism and significant development in microbial community, expressed as the increase in microbial diversity and changes in microbial composition. During natural weathering process, the microbial community changed from alkalophilic and halophilic assemblages to neutrophilic assemblages in bauxite residue, particularly enriched functional microbial groups include acid-producing, organic carbon decomposition and nitrogen fixing. Thirdly, the improvement of physiochemical properties (e.g., pH, ESP, and MWD) in bauxite residue, were important to the development of microbial community. These results suggested that natural weathering process could effectively promote the recovery of microbial community by improving geochemical conditions in bauxite residues.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYang Gao\u003c/strong\u003e: Conceptualization, Investigation, Formal analysis, Writing – original draft. \u003cstrong\u003eLiping Zhang\u003c/strong\u003e: Writing – review \u0026amp; editing, Project administration, Funding acquisition. \u003cstrong\u003eHuitong Li\u003c/strong\u003e: Investigation. \u003cstrong\u003eYiqing Gao\u003c/strong\u003e: Investigation. \u003cstrong\u003eHuarang Sun\u003c/strong\u003e: Methodology, Investigation. \u003cstrong\u003eRan Mu\u003c/strong\u003e: Visualization, Investigation. \u003cstrong\u003eZehua Pi\u003c/strong\u003e: Visualization, Investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (No. 52474161);\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBanda, J.F., Lu, Y.C., Hao, C.B., Pei, L.X., Du, Z.R., Zhang, Y., Wei, P.F., Dong, H., 2020. The Effects of Salinity and pH on Microbial Community Diversity and Distribution Pattern in the Brines of Soda Lakes in Badain Jaran Desert, China. Geomicrobiol J. 37, 1-12. \u003cu\u003ehttps://doi.org/10.1080/01490451.2019.1654568\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eBardgett, R.D., van der Putten, W.H., 2014. Belowground biodiversity and ecosystem functioning. Nature. 515, 505-511. \u003cu\u003ehttps://doi.org/10.1038/nature13855\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eCaporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Tumbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods. 7, 335-336. \u003cu\u003ehttps://doi.org/10.1038/nmeth.f.303\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eChao, Y.Q., Liu, W.S., Chen, Y.M., Chen, W.H., Zhao, L.H., Ding, Q.B., Wang, S.Z., Tang, Y.T., Zhang, T., Qiu, R.L., 2016. Structure, Variation, and Co-occurrence of Soil Microbial Communities in Abandoned Sites of a Rare Earth Elements Mine. Environ. Sci. Technol. 50, 11481-11490. \u003cu\u003ehttps://doi.org/10.1021/acs.est.6b02284\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eCrowther, T.W., van den Hoogen, J., Wan, J., Mayes, M.A., Keiser, A.D., Mo, L., Averill, C., Maynard, D.S., 2019. The global soil community and its influence on biogeochemistry. Science. 365, eaav0550. \u003cu\u003ehttps://doi.org/10.1126/science.aav0550\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eDeng, D.D., Sun, W., Wu, H., Yang, X.Y., Zhu, F., Jiang, Y.F., Huang, S.W., Xue, S.G., Jiang, J., 2024. Plant encroachment increase multifunctionality in bauxite residue by constructing diverse and stable microbial communities. Plant Soil. \u003cu\u003ehttps://doi.org/10.1007/s11104-024-06860-y\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eDeng, J.J., Yin, Y., Zhu, W.X., Zhou, Y.B., 2020. Response of soil environment factors and microbial communities to phytoremediation with Robinia pseudoacacia in an open-cut magnesite mine. Land Degrad. Dev. 31, 2340-2355. \u003cu\u003ehttps://doi.org/10.1002/ldr.3599\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eDittmann, C., Han, H.M., Grabenbauer, M., Laue, M., 2015. Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress. J. Struct. Biol. 191, 156-164. \u003cu\u003ehttps://doi.org/10.1016/j.jsb.2015.06.019\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eDixon, P., 2003. VEGAN, a package of R functions for community ecology. J. Veg. Sci. 14, 927-930. \u003cu\u003ehttps://doi.org/10.1111/j.1654-1103.2003.tb02228.x\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eEdgar, R.C., 2013. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods. 10, 996-998. \u003cu\u003ehttps://doi.org/10.1038/Nmeth.2604\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eEdgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 27, 2194-2200. \u003cu\u003ehttps://doi.org/10.1093/bioinformatics/btr381\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eGarland, J.L., Mills, A.L., 1991. Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl. Environ. Microbiol. 57, 2351-2359. \u003cu\u003ehttps://doi.org/10.1128/aem.57.8.2351-2359.1991\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eGelencser, A., Kovats, N., Turoczi, B., Rostasi, A., Hoffer, A., Imre, K., Nyiro-Kosa, I., Csakberenyi-Malasics, D., Toth, A., Czitrovszky, A., Nagy, A., Nagy, S., Acs, A., Kovacs, A., Ferincz, A., Hartyani, Z., Posfai, M., 2011. The Red Mud Accident in Ajka (Hungary): Characterization and Potential Health Effects of Fugitive Dust. Environ. Sci. Technol. 45, 1608-1615. \u003cu\u003ehttps://doi.org/10.1021/es104005r\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eHartmann, M., Six, J., 2023. Soil structure and microbiome functions in agroecosystems. Nat. Rev. Earth. Env. 4, 4-18. \u003cu\u003ehttps://doi.org/10.1038/s43017-022-00366-w\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eJiang, Y.F., Huang, S.W., Zhu, F., Guo, X.Y., Zhang, X.C., Zhu, M.X., Zhang, Y.F., Xue, S.G., 2023. Long-term weathering difference in soil-like indicators of bauxite residue mediates the multifunctionality driven by microbial communities. Sci. Total Environ. 890, 164377. \u003cu\u003ehttps://doi.org/10.1016/j.scitotenv.2023.164377\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eKong, X.F., Guo, Y., Xue, S.G., Hartley, W., Wu, C., Ye, Y.Z., Cheng, Q.Y., 2017a. Natural evolution of alkaline characteristics in bauxite residue. J. Clean Prod. 143, 224-230. \u003cu\u003ehttps://doi.org/10.1016/j.jclepro.2016.12.125\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eKong, X.F., Li, M., Xue, S.G., Hartley, W., Chen, C.R., Wu, C., Li, X.F., Li, Y.W., 2017b. Acid transformation of bauxite residue: Conversion of its alkaline characteristics. J. Hazard. Mater. 324, 382-390. \u003cu\u003ehttps://doi.org/10.1016/j.jhazmat.2016.10.073\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eKong, X.F., Tian, T., Xue, S.G., Hartley, W., Huang, L.B., Wu, C., Li, C.X., 2018. Development of alkaline electrochemical characteristics demonstrates soil formation in bauxite residue undergoing natural rehabilitation. Land Degrad. Dev. 29, 58-67. \u003cu\u003ehttps://doi.org/10.1002/ldr.2836\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eKrishna, P., Babu, A.G., Reddy, M.S., 2014. Bacterial diversity of extremely alkaline bauxite residue site of alumina industrial plant using culturable bacteria and residue 16S rRNA gene clones. Extremophiles. 18, 665-676. \u003cu\u003ehttps://doi.org/10.1007/s00792-014-0647-8\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eLauber, C.L., Hamady, M., Knight, R., Fierer, N., 2009. Pyrosequencing-Based Assessment of Soil pH as a Predictor of Soil Bacterial Community Structure at the Continental Scale. Appl. Environ. Microbiol. 75, 5111-5120. \u003cu\u003ehttps://doi.org/10.1128/aem.00335-09\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eLi, Y., Yuan, L., Xue, S., Liu, B.J., Jin, G., 2021. Artificial root exudates excite bacterial nitrogen fixation in the subsurface of mine soils. Appl. Soil Ecol. 157, 103774. \u003cu\u003ehttps://doi.org/10.1016/j.apsoil.2020.103774\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eOren, A., 2011. Thermodynamic limits to microbial life at high salt concentrations. Environ. Microbiol. 13, 1908-1923. \u003cu\u003ehttps://doi.org/10.1111/j.1462-2920.2010.02365.x\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003ePereira, D.G.C., Portugal, A.F., Giustolin, T.A., Maia, V.M., Megda, M.X.V., Kondo, M.K., 2023. Litter decomposition and nutrient release in different land use systems in the Brazilian semi-arid region. Catena. 231, 107345. \u003cu\u003ehttps://doi.org/10.1016/j.catena.2023.107345\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003ePower, G., Grafe, M., Klauber, C., 2011. Bauxite residue issues: I. Current management, disposal and storage practices. Hydrometallurgy. 108, 33-45. \u003cu\u003ehttps://doi.org/10.1016/j.hydromet.2011.02.006\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eQian, Z.Z., Zhuang, S.Y., Gao, J.S., Tang, L.Z., Harindintwali, J.D., Wang, F., 2022. Aeration increases soil bacterial diversity and nutrient transformation under mulching-induced hypoxic conditions. Sci. Total Environ. 817, 153017. \u003cu\u003ehttps://doi.org/10.1016/j.scitotenv.2022.153017\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eQiao, M.J., Sun, R.B., Wang, Z.X., Dumack, K., Xie, X.G., Dai, C.C., Wang, E.T., Zhou, J.Z., Sun, B., Peng, X.H., Bonkowski, M., Chen, Y., 2024. Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification. Nat. Commun. 15, 2924. \u003cu\u003ehttps://doi.org/10.1038/s41467-024-47159-x\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eRaiesi, F., Sadeghi, E., 2019. Interactive effect of salinity and cadmium toxicity on soil microbial properties and enzyme activities. Ecotox. Environ. Safe. 168, 221-229. \u003cu\u003ehttps://doi.org/10.1016/j.ecoenv.2018.10.079\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eRashid, M.I., Mujawar, L.H., Shahzad, T., Almeelbi, T., Ismail, I.M.I., Oves, M., 2016. Bacteria and fungi can contribute to nutrients bioavailability and aggregate formation in degraded soils. Microbiol. Res. 183, 26-41. \u003cu\u003ehttps://doi.org/10.1016/j.micres.2015.11.007\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eRaymond, N.S., G\u0026oacute;mez‐Mu\u0026ntilde;oz, B., van der Bom, F.J.T., Nybroe, O., Jensen, L.S., M\u0026uuml;ller‐St\u0026ouml;ver, D.S., Oberson, A., Richardson, A.E., 2020. Phosphate‐solubilising microorganisms for improved crop productivity: a critical assessment. New Phytol. 229, 1268-1277. \u003cu\u003ehttps://doi.org/10.1111/nph.16924\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eRuyters, S., Mertens, J., Vassilieva, E., Dehandschutter, B., Poffijn, A., Smolders, E., 2011. The Red Mud Accident in Ajka (Hungary): Plant Toxicity and Trace Metal Bioavailability in Red Mud Contaminated Soil. Environ. Sci. Technol. 45, 1616-1622. \u003cu\u003ehttps://doi.org/10.1021/es104000m\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eSantini, T.C., Fey, M.V., 2013. Spontaneous Vegetation Encroachment upon Bauxite Residue (Red Mud) As an Indicator and Facilitator of In Situ Remediation Processes. Environ. Sci. Technol. 47, 12089-12096. \u003cu\u003ehttps://doi.org/10.1021/es402924g\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eSantini, T.C., Raudsepp, M., Hamilton, J., Nunn, J., 2018. Extreme Geochemical Conditions and Dispersal Limitation Retard Primary Succession of Microbial Communities in Gold Tailings. Front Microbiol. 9, 2785. \u003cu\u003ehttps://doi.org/10.3389/fmicb.2018.02785\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eSantini, T.C., Warren, L.A., Kendra, K.E., 2015. Microbial Diversity in Engineered Haloalkaline Environments Shaped by Shared Geochemical Drivers Observed in Natural Analogues. Appl. Environ. Microbiol. 81, 5026-5036. \u003cu\u003ehttps://doi.org/10.1128/Aem.01238-15\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eWu, H., Chen, L., Zhu, F., Hartley, W., Zhang, Y.F., Xue, S.G., 2020. The dynamic development of bacterial community following long-term weathering of bauxite residue. J. Environ. Sci. 90, 321-330. \u003cu\u003ehttps://doi.org/10.1016/j.jes.2019.12.001\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eWu, H., Tang, T., Zhu, F., Wei, X.M., Hartley, W., Xue, S.G., 2021. Long term natural restoration creates soil-like microbial communities in bauxite residue: A 50-year filed study. Land Degrad. Dev. 32, 1606-1617. \u003cu\u003ehttps://doi.org/10.1002/ldr.3728\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eXiao, T., Li, P., Fei, W.B., Wang, J.D., 2024. Effects of vegetation roots on the structure and hydraulic properties of soils: A perspective review. Sci. Total Environ. 906, 167524. \u003cu\u003ehttps://doi.org/10.1016/j.scitotenv.2023.167524\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eXue, S.G., Ke, W.S., Zhu, F., Fan, J.R., Wang, Q.L., Liu, Z., Hartley, W., 2021. Evaluating aggregate stability, surface properties and disintegration behaviour of bauxite residue induced by Ca/Na. Land Degrad. Dev. 32, 803-816. \u003cu\u003ehttps://doi.org/10.1002/ldr.3703\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eXue, S.G., Wu, Y.J., Li, Y.W., Kong, X.F., Zhu, F., William, H., Li, X.F., Ye, Y.Z., 2019. Industrial wastes applications for alkalinity regulation in bauxite residue: A comprehensive review. J. Cent. South. Univ. 26, 268-288. \u003cu\u003ehttps://doi.org/10.1007/s11771-019-4000-3\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eYang, Y., Chai, Y.B., Xie, H.J., Zhang, L., Zhang, Z.M., Yang, X., Hao, S.L., Gai, J.P., Chen, Y.L., 2023. Responses of soil microbial diversity, network complexity and multifunctionality to three land-use changes. Sci. Total. Environ. 859, 160255. \u003cu\u003ehttps://doi.org/10.1016/j.scitotenv.2022.160255\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eYou, F., Zhang, L.P., Ye, J., Huang, L.B., 2019. Microbial decomposition of biomass residues mitigated hydrogeochemical dynamics in strongly alkaline bauxite residues. Sci. Total. Environ. 663, 216-226. \u003cu\u003ehttps://doi.org/10.1016/j.scitotenv.2019.01.317\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eYuan, Y.S., Zhao, W.Q., Xiao, J., Zhang, Z.L., Qiao, M.F., Liu, Q., Yin, H.J., 2017. Exudate components exert different influences on microbially mediated C losses in simulated rhizosphere soils of a spruce plantation. Plant Soil. 419, 127-140. \u003cu\u003ehttps://doi.org/10.1007/s11104-017-3334-6\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eZaitseva, S.V., Abidueva, E.Y., Radnagurueva, A.A., Bazarov, S.M., Buryukhaev, S.P., 2018. Structure of Microbial Communities of the Sediments of Alkaline Transbaikalia Lakes with Different Salinity. Microbiology. 87, 559-568. \u003cu\u003ehttps://doi.org/10.1134/s0026261718040185\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eZhalnina, K., Louie, K.B., Hao, Z., Mansoori, N., da Rocha, U.N., Shi, S., Cho, H., Karaoz, U., Loque, D., Bowen, B.P., Firestone, M.K., Northen, T.R., Brodie, E.L., 2018. Dynamic root exudate chemistry and microbial substrate preferences drive patterns in rhizosphere microbial community assembly. Nat. Microbiol. 3, 470-480. \u003cu\u003ehttps://doi.org/10.1038/s41564-018-0129-3\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eZhu, F., Li, X.F., Xue, S.G., Hartley, W., Wu, C., Han, F.S., 2016a. Natural plant colonization improves the physical condition of bauxite residue over time. Environ. Sci. Pollut. R. 23, 22897-22905. \u003cu\u003ehttps://doi.org/10.1007/s11356-016-7508-1\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eZhu, F., Li, Y.B., Xue, S.G., Hartley, W., Wu, H., 2016b. Effects of iron-aluminium oxides and organic carbon on aggregate stability of bauxite residues. Environ. Sci. Pollut. R. 23, 9073-9081. \u003cu\u003ehttps://doi.org/10.1007/s11356-016-6172-9\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003eZhu, F., Zhang, X.C., Guo, X.Y., Yang, X.W., Xue, S.G., 2023. Root architectures differentiate the composition of organic carbon in bauxite residue during natural vegetation. Sci. Total. Environ. 883, 163588. \u003cu\u003ehttps://doi.org/10.1016/j.scitotenv.2023.163588\u003c/u\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bauxite residue Natural weathering process Microbial metabolic activities Microbial composition Soil formation in bauxite residue","lastPublishedDoi":"10.21203/rs.3.rs-7133212/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7133212/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eNatural weathering process increased microbial diversity and in bauxite residue. However, the its effect on the microbial metabolic activities is still poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, residue samples with different weathering time were collected to evaluate the microbial metabolic activities and community structure in bauxite residue.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCommunity level physiological profiles (CLPP) results indicated that natural weathering process significantly increased the microbial metabolic activities and changed microbial carbon resources utilization pattern. The microbial metabolic diversity index increased from 2.68 to 3.29 and the average well color development increased from 0.41 to 1.47. The Shannon diversity significantly increased from 5.79 to 6.26 and the observed OTU numbers increased from 1009 to 1331, respectively. The relative abundance of Proteobacteria, Bacteroidota, Acidobacteria, Actinobacteria and Chloroflexi significantly increased following long term weathering process. Random forest model showed that Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae were the core species in the promotion of microbial metabolic activities. Redundancy analysis (RDA) indicated that the development of microbial communities was signifianltly affected by MWD followed by pH, TOC, AP, TN, EC and AN.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eNatural weathering process could increase microbial metabolic activities and diversity, thus promote the recovery of microbial community in bauxite residue. Beijerinckiaceae, Rhizobiaceae, Microscillaceae, Caulobacteraceae, Sphingomonadaceae, Nitrospiraceae and Chitinophagaceae played vital roles in the promotion of microbial carbon metabolism in bauxite residue. Our results emphasize the significance of specific functional microbial species in the recovery of microbial communities in bauxite residue disposal areas.\u003c/p\u003e","manuscriptTitle":"Natural weathering promoted the recovery of microbial community in bauxite residues","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 09:26:03","doi":"10.21203/rs.3.rs-7133212/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":"9950b309-c0be-400c-a157-371e3b014f2b","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-13T05:06:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 09:26:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7133212","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7133212","identity":"rs-7133212","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}