Improvement effects of calcium silicate hydrate and humic acid-encapsulated Alcaligenes faecalis on acidic farmland soil and alterations in microbial community structure

Improvement effects of calcium silicate hydrate and humic acid-encapsulated Alcaligenes faecalis on acidic farmland soil and alterations in microbial community structure | 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 Improvement effects of calcium silicate hydrate and humic acid-encapsulated Alcaligenes faecalis on acidic farmland soil and alterations in microbial community structure Luhua Jiang, Manjun Miao, Ziwen Guo, Jiejie Yang, Yulong Peng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7738393/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Soil acidification poses a critical threat to agricultural safe production by compromising soil fertility. Conventional remediation strategies for acidic soils face persistent challenges including short-lived efficacy, structural deterioration, and secondary pollution risks. This study presents an innovative bioremediation approach utilizing Alcaligenes faecalis ( A. faecalis ), a potent alkaline-metabolizing microorganism, coated by calcium silicate hydrate (CSH) and humic acid (HA) as a biocarrier for acidic farmland soil restoration. Through microcosm experiments and high-throughput sequencing analysis, we demonstrated that the remediation approach achieved remarkable soil pH elevation from an initial 4.66 to 6.41 within 21 d. Soil available nitrogen (AN) and silicon (ASi) contents increased to 252.83–595.70 mg/kg and 89.24-133.57 mg/kg at 21 d, respectively; and available phosphorus (AP) content increased from 15.59–39.88 mg/kg to 20.44–55.98 mg/kg at 7 d. There was a positive correlation between pH and all nutrient indicators for soil samples. Moreover, the restoration could also change the microbial diversity and structure. This led to an increase in the relative abundance of functional genera including Bacillus (from 0.44–0.65% to 4.92–21.89%), Arthrobacter (from 0.13–0.30% to 0.17–16.31%), and Paenochrobactrum (from 0.02–0.04% to 0.75–17.43%) at 5 d. These microorganisms were related to soil nitrogen, phosphorus, and silicon cycling, which were positively correlated with an increase in AN, ASi and AP content in soils. Results highlight the application potential of Alcaligenes faecalis as a biomaterial for soil improvement in acidic farmland, providing a new option for the improvement of acidic soil. Farmland soil Fertility improvement Reduce acidity Alcaligenes faecalis Calcium silicate hydrate Microbial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction With nitrogen nitrification and base cations loss, soil acidification is becoming seriously, which has become a global environmental issue (Dong et al., 2022). Globally, about 50% of arable soils are classified as acidic (pH < 5.5) and suffer from soil acidification (Dai et al., 2017), especially in East Asia such as China and South Korea (Duan et al., 2016). The acidity of soils has been reported to directly affect available nutrient contents, resulting in a decrease in soil fertility (Hagh-Doust et al., 2023 ). Meantime, soil acidification has also largely led to the decline in the abundance and diversity of bacterial community (Li et al., 2023). Therefore, exploring effective ways to reduce soil acidity is of great significance for improving soil nutrient contents and restoring the health of acidic soil microbial community. Acidic soils can be ameliorated by applying alkaline amendments such as lime, biochar, and steel slag (Uwiringiyimana et al., 2024 ). These alkaline amendments directly neutralized H + in the soil by dissolving OH − and HCO 3 − . And it also supplied base cations (such as Ca 2+ , Mg 2+ , and K + ) to replace Al 3+ in the soil, reducing soil acidity. Among these amendments, lime remained the most traditional and common practice to reduce soil acidity. It could increase soil pH and yields for most crops, particularly legumes, which were sensitive to soil pH (Li et al., 2019 ). Similarly, biochar is regarded as an effective soil amendment for ameliorating acidic soils. It could also increase soil pH due to its alkaline nature (pH 8.8–10.3) and the carbonates in the ash (Bolan et al., 2023 ; Lee et al., 2024). And some studies have found that biochar also contributed to acidic soil fertility by suppling large amounts of nutrients, such as C, N, and P, for plant growth. However, these traditional alkaline amendments still face serious challenges. For example, these applications might induce soil structural degradation, nutrient imbalance and reacidification, and even lead to environmental pollution risks (Dong et al., 2025). Recently, microbial-mediated remediation offers a more sustainable approach and has received increasing attention in acid soil improvement due to its green environment and low cost. Microorganisms could not only continuously regulate soil pH through metabolic activities, but also synergistically drive nutrient cycling and microbial community restoration (Oro et al., 2024 ). Thus, it could be considered as an alternative to traditional alkaline amendments. Alcaligenes faecalis ( A. faecalis ), a common bacterium in soils, exhibited unique advantages in acid soil improvement as a bioremediating material with the function of nitrogen fixation and secreting alkaline metabolites (Yang et al., 2024; Zhong et al., 2020 ). However, being added as exogenous microorganisms to acidified soils had many limitations, such as unfavorable survival and low adaptability due to the competition with native microbes (Sun et al., 2020 ). This greatly limited its application in acid soil improvement. Therefore, to improve their survival rates, it was possible to immobilize them on carrier materials as initial habitat after entering the soil. Calcium silicate hydrate (CSH) was a porous material rich in silicon and trace elements, which could be considered an ideal carrier for immobilization. Meanwhile, CSH as an alkaline amendment, could increase pH when applied to the soil (Qing et al., 2022) and provide a favorable environment for the survival of A. faecalis . Besides, the elements contained in CSH provided nutrients for bacteria reproduction, while A. faecalis also promoted the release of silicon contained in CSH to increase acidic soil silicon content. Until now, there have been no studies on the application of CSH as a bacterial carrier for the improvement of acidic soils. Although A. faecalis loaded on CSH could improve the pH and microbial activity, this approach still did not compensate for the deficiency of organic matter in acidic soil. The lack of organic matter not only affected soil nutrients but also limited the growth and structure of microbial community (Bashir et al., 2021 ). Humic acid (HA) could enhance soil acid buffering capacity and increase the abundance of beneficial microorganisms in acidic soil (Xu et al., 2021). Meanwhile, HA could complex with calcium ions in CSH to form stable humic-Ca complexes, which was conducive to the accumulation of soil organic matter (Tang et al., 2021 ). In addition, it could be recognized as a carrier material for microorganisms to colonize soil due to its large surface area and high cation exchange capacity (Hao et al., 2025). Therefore, using CSH and HA as carrier materials can enhance the adaptability of A. faecalis in acidic farmland soils and synergistically improve soil improvement performances. In this study, we focused on the effects of CSH and HA-encapsulated A. faecalis on the acidic soils improvement. Thus, the aims of the present study were to (i) analyze the effects of different treatments on the improvement of soil pH and available nutrient contents in acidic soil; (ii) explore the changes of soil microbial community diversity and structure after amendment; (iii) unlock the underlying relationships between soil physicochemical properties and the composition and structure of microbial community. 2. Materials and Methods 2.1 Experimental Materials Synthesis of CSH was prepared by directly mixing Na 2 SiO 3 solution (0.5 mol/L) with CaCl 2 solution (1 mol/L) at Ca/Si molar ratio of 1:1 under microwave hydrothermal conditions. Firstly, the obtained solution was put into a microwave ablator (MDS-6G, Xinyi, China) and reacted at 160 ℃ for 6 h under microwave power of 400 W. After that, it was naturally cooled to room temperature. The slurry was then centrifuged and washed with deionized water. After drying in an oven at 60 ℃ for 24 hours, the product was ground through a 100-mesh sieve. This process yielded CSH with pH values ranging from 10.9 to 11.6, and a silicon content of approximately 10% (Yang et al., 2023). The A. faecalis (CCTCC No: M 2020786) was cultured in NB medium for enrichment culture. The density of live bacteria was 1×10 8 cells/mL. And the pH value of the bacterial solution was 8.05–8.85. HA suspension was provided by Shandong Linker Agritec Co., Ltd., with organic matter content ≥ 100 g/L and the concentration of HA being 40 g/L. The surface morphology of CSH, CSH coated by A. faecalis , and CSH and HA-encapsulated A. faecalis was examined by scanning electron microscope (SEM, JSM-IT500, JEOL Japan). The chemical composition of CSH was determined by using an X-ray fluorescence spectrometer (XRF, Axios, PANalytical, Netherlands). 2.2 Microcosm Experiments Soil samples were collected using a diagonal sampling method, air-dried, and ground through a 100-mesh sieve for soil physicochemical properties analysis. The remaining soil was placed in sterile self-sealing bags and stored in the refrigerator at -80 ℃ for 16S rDNA analysis experiments. The soil samples used for this study were collected from paddy in Nanxiong City and Taishan City, Guangdong Province. They were named as S1, S2 for Nanxiong, and S3, S4 for Taishan, respectively. After removing large stones and plant debris, soil samples (500 g) were mixed well and packed in polythene plastic pots and then tested at room temperature (25 ℃). Four treatment groups were set up in this experiment. No amendment was added into soils set as a control group (CK); CSH was applied at 1.0‰ addition (T1); The application of 1.0‰ CSH was combined with a 5% addition rate of A. faecalis liquid (T2); and the application included 1.0‰ CSH, 5% A. faecalis liquid, and 150 µL of HA (T3). Three replicates were set up for each treatment. The microcosm experiments were conducted in an incubator at a constant temperature of 25 ℃ and a relative humidity of 60%. The water-to-soil ratio was fixed at 1:4 (v/m). The experiment lasted for 21 days, and soil samples were collected on the 1st, 3rd, 5th, 7th, 14th, and 21st days for subsequent analysis. 2.3 Sample Analysis Soil pH was measured in water extracts at a 2.5:1 water-soil ratio (v/w) using a digital pH meter, according to NY/T 1377–2007. Soil organic matter (SOM) content was determined by the potassium dichromate (K 2 Cr 2 O 7 ) digestion method according to NY-T 1121.6–2006. The soil ammonium nitrogen (NH 4 -N) and nitrate nitrogen (NO 3 -N) were determined by extraction with potassium chloride solution-spectrophotometric method (HJ 634–2012). The content of available phosphorus (AP) was extracted with 0.5 mol/L sodium bicarbonate solution at pH 8.5 and then measured using the molybdenum antimony anti-colorimetric method (Olsen, 1954 ). The determination of available silicon (ASi) content was analyzed by using the silicon molybdate spectrophotometric method (NY/T 1121.15–2006). Soil samples collected on 0, 5, and 21 d after improvement were analyzed by using 16S rDNA amplicon sequencing. Total soil genomic DNA was extracted by using the IlluminaMiSeq kit, and primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3') were used to amplify the hypervariable region V3-V4 of the bacterial 16S rDNA gene by polymerase chain reaction (PCR). The amplification reaction included an initial denaturation step at 98 ℃ for 2 minutes, followed by 28 cycles of denaturation at 98 ℃ for 15 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds. This was concluded with a final extension step at 72 ℃ for 5 minutes. The amplified PCR products were detected by agarose gel electrophoresis at a concentration of 1.2%, purified by using the Agarose Gel Recovery Kit (SOMEGA Biotek Inc., Doraville, GA, USA), and finally quantified by using Qubit (Invitrogen, USA). Amplicon libraries were constructed for sequencing, and the size and concentration of the amplicon libraries were assessed by using an Agilent 2100 Bioanalyser (Agilent, USA) equipped with the Illumina Library Quantification Kit (Kapa Biosciences, Woburn, MA, USA). Libraries were sequenced on the NovaSeq-PE250 sequencing platform (Illumina Inc., Carlsbad, CA, USA). 2.4 Data Analysis The DNA was sequenced using the Illumina NovaSeq platform. To obtain high-quality clean data, it was necessary to use overlap to splice, quality control and chimera filter the double-ended data. Primer sequences introduced from sequence databases were removed using FLASH (v 1.2.8) software, and chimeric sequences were filtered using Vsearch (v 2.3.4) after merging sequences. After using DADA2 (Divisive Amplicon Denoising Algorithm) in QIIME2 to perform denoising and replication clustering, the OTUs table, feature tables, and feature sequences were obtained. Alpha diversity indexes including Chao1 richness, Simpson and Shannon index were analyzed using the R package ggplot2 (v. 4.1.3). Redundancy analysis (RDA) was performed using the R package vegan (v. 3.6.3). The heatmap of Pearson correlation between environmental factors and dominant bacteria was plotted using the R package stats (v. 3.6.3). Significance analysis was calculated by SPSS software (IBM SPSS Statistics 24). The remaining graphs were plotted using OriginPro 2024b software. 3. Results and Discussion 3.1 Material Characterizations and Soil Physicochemical Properties Analysis The SEM images provided significant insights into the surface morphology of CSH and CSH-HA before and after loaded with A. faecalis . As shown in Fig. 1 a, the surface of CSH exhibited prominent ripples and wrinkles with a large number of irregular pore structures. This morphology could be attributed to the amorphous structure of CSH (Guo et al., 2022). The SSA BET value of CSH was 35.79 m 2 /g and the PV value was 0.258 cm 3 /g. This indicated that CSH had a large specific surface area and a well-developed pore structure, both of which were crucial in providing sites for microbial attachment. As shown in Fig. 1 b, the rod-shaped feature of A. faecalis was clearly visible and these strains appeared to adhere at the CSH surface. And the surface structure of CSH became looser and more porous after loading A. faecalis . It could be speculated that A. faecalis metabolically synthesized extracellular polymeric substances (EPS) to corrode the surface of CSH, which further promoted the loosening of its structure (Wang et al., 2023). Besides, the porous structure of CSH might provide a habitat for microbial growth, which served as a carrier that could promote the survival of A. faecalis after entering the soil environment. The SEM of CSH-HA carrier after being loaded with A. faecalis was shown in Fig. 1 c. CSH-HA carrier had a richer surface structure, which increased the contactable area with A. faecalis and favored its adherence. As reflected by Fig. 1 d, the main element composition of CSH was O (39.90%), Ca (34.89%), Si (21.34%), and Na (2.81%). Ca could enhance the metabolic activity of A. faecalis and was important for the synthesis of EPS (Ibrahim et al., 2015 ). This would promote the formation of biofilms on the CSH surface, making it easy for the attachment of A. faecalis . Additionally, the Ca and Na elements in CSH could enhance the acid buffering capacity of the soil. The cations such as Ca 2+ and Na + could easily exchange with H + in acidic soil (Bernard et al., 2021 ), resulting in soil pH increasing. Besides, Si in CSH was mainly amorphous silicate(Guo et al., 2022), which could replenish the ASi content in the soils when applied. This would help to improve soil fertility and increase the drought stress tolerance of plants as well as their resistance to heavy metal toxicity, pests and diseases (Kovács et al., 2022; Thorne et al., 2021 ). The physicochemical properties of soil samples are shown in Table 1 . As seen, soil samples were strongly acidic with pH values of 5.13 (S1), 5.46 (S2), 5.35 (S3) and 4.66 (S4), respectively. This difference in acidity might be associated with soil properties, microbial communities, and organic matter contents (Hong et al., 2018 ). The NH 4 -N content of S4 (143.93 mg/kg) was higher than that of S1 (81.42 mg/kg), S2 (71.69 mg/kg), and S3 (82.38 mg/kg). NO 3 -N content varied across the samples; and S3 exhibited the highest content (20.19 mg/kg), while S4 had the lowest (4.57 mg/kg). NO 3 -N was easily lost by leaching, while NH 4 -N was not easily lost due to soil colloid adsorption, which favored soil nitrogen conservation in S4 (Dal Molin et al., 2018). Besides, the soil NH 4 -N content was all higher than the NO 3 -N content from S1 to S4. This might be due to the low pH value reduced the activity of ammonia-oxidizing bacteria, which was unfavorable to its nitrification process and thus inhibited the conversion of NH 4 -N to NO 3 -N (Lin et al., 2021). The AP content of S1 and S2 was significantly higher than that of S3 and S4. And the content of S4 was the lowest with the value of 15.59 mg/kg. This was ascribed to soil phosphate would be sorbed onto the surfaces of silicate minerals and Al- and Fe-oxides when the soil pH value below 5 in S4. And then phosphate was converted from the adsorbed fraction to the precipitated Al- and Fe-phosphates in acidic conditions (Kruse et al., 2015). The SOM content in soil samples ranged from 3.18% to 4.03%. This indicated that all the soil samples could be classified as Level II (3–4%, richness) according to China’s second soil census nutrient classification standards (Liu et al., 2023). S4 had the highest SOM content at 4.03% and the lowest pH value at 4.66. Previous studies have shown that soil pH exhibited a significant negative correlation with SOM; and the accumulation of organic acids produced from organic matter decomposition or soil microbial metabolism caused low pH in S4 (Adeleke et al., 2017 ). The ASi content showed a trend of S4 > S1 > S3 > S2, which was exactly opposite to the trend of pH. The main source of silicon was the weathering of soil minerals, especially the decomposition of silicon-rich minerals such as feldspar and mica. Silicate weathering could be strongly modulated by soil pH and increase an order of magnitude for each unit decrease in pH (Bufe et al., 2021). Table 1 Physicochemical properties of S1, S2, S3 and S4 Sample pH NH 4 -N (mg/kg) NO 3 -N (mg/kg) AP (mg/kg) SOM (%) ASi(mg/kg) S1 5.13 ± 0.01 c 81.42 ± 6.46 b 16.03 ± 0.95 b 38.47 ± 1.81 a 3.18 ± 0.49 b 63.04 ± 0.79 b S2 5.46 ± 0.03 a 71.69 ± 3.65 b 12.76 ± 2.25 c 39.88 ± 2.07 a 3.47 ± 0.09 b 38.91 ± 1.39 d S3 5.35 ± 0.02 b 82.38 ± 4.55 b 20.19 ± 1.87 a 30.32 ± 1.10 b 3.22 ± 0.12 b 47.83 ± 1.91 c S4 4.66 ± 0.01 d 143.93 ± 9.12 a 4.57 ± 0.23 d 15.59 ± 0.81 c 4.03 ± 0.09 a 67.83 ± 1.00 a Note: Different case letters in the same column indicate significant differences between soil samples ( P < 0.05) 3.2 Changes in Soil Fertility Characteristics After Amendment 3.2.1 pH As shown in Fig. 2 , the soil pH values showed an increasing trend in the initial period. Specifically, S1 exhibited a peak pH value after 5 d of treatment, whereas S2, S3, and S4 samples reached their peak pH values at 3 d. This rapid pH increase was primarily because the applied amendments were strongly alkaline (pH values 10.9–11.6). After 5 d, pH values of all treatments fluctuated considerably and began to decline. In terms of specific soil samples, the pH values of S1, S2, and S4 under T3 were significantly higher than other treatments (T1 and T2) at 21 d. For S1, the improvement of pH value under T1 and T2 showed a similar trend, but T2 was slightly better than T1. This slight superiority in T2 might be partly attributed to the presence of A. faecalis . Its metabolic activities might have generated additional alkaline substances that enhanced the increasing of pH compared to T1. Besides, the pH value of S1 increased to 6.59 under T3 at 21 d, which was higher than that of T2. While HA in T3 treatment, with its abundant oxygen-containing functional groups such as carboxyl, phenolic, and hydroxyl (Tiwari et al., 2023), could absorb protons through association reactions at low pH conditions and then increase the soil pH (Xu et al., 2012 ). The pH improvement of S2 under different treatment groups exhibited a trend of T3 ๥ T2 ๥ T1. T3 obviously increased the pH value of S2, and the value reached up to 7.28 and 7.16 at 3 d and 21 d, respectively. It showed less fluctuation in pH value during 3–21 d. This might be attributed to the fact that the higher SOM content of S2 (Table 1 ), which had stronger acid buffering capacity. Additional added HA also contributed substantial organic matter, further increasing the SOM content of S2 and suppressing soil acidification. And Ca ion from CSH could prevent SOM from being degraded. It remained SOM at a high level through mechanisms such as adsorption and complexation (Gruba and Mulder, 2015 ; Shabtai et al., 2023), thereby maintaining pH stability. The pH improvement of S3 under these treatments (T1, T2, and T3) was the best in the samples of S1-S4. And the differences in interval of pH improvement between the treatments (T1, T2, and T3) were slight in S3. Although the differences among treatments were not as obvious as in other samples, the metabolic activities of A. faecalis in T2 and T3 might still have a subtle effect on soil acid-base balance. Besides, the pH value of S4 peaked on 3 d and then dropped sharply on 7 d under all treatments. The sharp decrease might be ascribed to the rapid consumption of alkali in these amendments. Moreover, the content of SOM and NH 4 -N in S4 was 4.03% and 143.93 mg/kg, respectively, as shown in Table 1 . As reported, high level of SOM (≥ 3.0%) and excessive NH 4 -N could aggravate soil acidification (Zhang et al., 2022), due to the nitrification of NH 4 -N and the decomposition of SOM (Zhao et al., 2022). Both of these effects would lead to the sharp decrease of soil pH value on 7 d. In addition, the pH value was back up to 6.41 under T3 at 21 d, which was close to the peak pH value of 3 d (6.46). This could contribute to the addition of HA in T3 treatment which could improve soil pH buffering capacity. In the view of the changes of pH value in S1, S2, and S4 under different treatments (T1, T2, and T3), the T3 treatment (CSH + A. faecalis + HA) had the best performance in increasing the pH value for the studied soil samples. However, it's crucial to note that this study was based on 21-day short-term microcosm experiments in the laboratory, and the amelioration effect of the system might deteriorate under field conditions. 3.2.2 SOM SOM content can serve as a vital indicator of soil health, which determines soil nutrients and microbial activity (Xu et al., 2018). As shown in Fig. 3 a, the content of SOM exhibited comparable trends under these treatments (T1, T2, and T3) in S1. Specifically, the SOM content showed a decreasing trend from 5–7 d and then increased gradually from 7–21 d. Furthermore, T3 treatment led to a significantly decrease of SOM content at 7 d in S1 when compared to T1 and T2, while SOM content increased rapidly at 7–21 d. When HA was added into soils, microbial activity would increase and the decomposition of SOM was accelerated (Bingeman et al., 1953 ). This might lead to the decrease of SOM content at 7 d in S1 when T3 treated. The SOM content increased 36.79% at 21 d in S1 in comparison to the initial soil when T3 treated. This change might be due to the interactions of HA with mineral components in soils, leading to greater organic matter accumulation under the T3 treatment. This was because the presence of metal cations in the soil and CSH, such as Ca and Al cations, induced the formation of cationic bridges. These bridges mostly formed between HA and mineral surfaces. Therefore, this process would promoted HA aggregation and higher SOM content. In S2 (Fig. 3 b), the SOM content decreased significantly during 0–7 d and then it was even lower than that of CK group from 7–21 d under T1, T2, and T3. The greatest decline in SOM in S2 in comparison to S1, S3, and S4 might be due to limited N content in S2. As shown in Table 1 , the available nitrogen content of S2 was only 84.45 mg/kg. As reported, the expression of genes (Cellulase/Amylase) associated with recalcitrant SOM degradation was enhanced under these conditions (Cui et al., 2022 ). Moreover, N limitation was more favorable for the growth of bacteria such as Acidobacteria and Chloroflexi, which were involved in the degradation process of recalcitrant or complex SOM (Dai et al., 2018; Kiem and Kögel-Knabner, 2003 ). Together, these factors contributed to the decrease of SOM in S2. For S3 (Fig. 3 c), SOM content continuously increased during 0–21 d and the increase of SOM content was basically similar under these treatments. As shown in Fig. 3 d, the SOM content of S4 showed an increasing trend from 0–21 d under T2 treatment. The fluctuation of SOM content was the largest (1.41%) under T1 and slightly (0.44%) under T3. 3.2.3 AN As shown in Fig. 4 , the available nitrogen (AN) content of S2, S3, and S4 exhibited a consistent increasing trend under treatments (T1, T2, and T3) during the observation period. For S1, S2, and S3, the addition of A. faecalis (T2) resulted in a significantly higher AN content than the other treatment groups at 5 d and 7 d. This might be due to A. faecalis was a bacterium with nitrogen-fixing capability, which might convert atmospheric nitrogen (N 2 ) into AN (Vermeiren et al., 1999). Specially, S2 showed high AN content (231.75 mg/kg) and low SOM content (1.81%) after T2 treatment for 7 d. It was possible that A. faecalis could decompose more recalcitrant SOM to obtain N. Furthermore, the biological nitrogen fixation capacity of A. faecalis was limited by the low initial pH of S4 (4.66), resulting in a lower AN (T2) than the other groups at 5 d and 7 d. After the addition of HA (T3) to soil samples, the AN content of S1, S2, S3, and S4 increased slowly in the early stages (5–7 d) but faster in the later stage (21 d). The main reason might be that HA had the ability to store and slowly release N (Chen et al., 2017 ). As reported, HA could easily bind with NH 4 + to form more stable ammonium humate due to its strong complexation and absorption capabilities (Xu et al., 2021). In S1, S2, and S4, the AN content under T3 treatment showed a significant increase compared with other treatments (T1 and T2). This was because that HA contained N, which could also be a N source when applied into soils. Besides, HA was also used as a carbon source, and its rich C and N content could increase diazotroph community abundance and its soil N-fixing rate (Zheng et al., 2023 ), thus enhancing the AN content. The above results showed that the enhancement of T3 outperformed T1 and T2 in most cases by the 21 d observation across S1 to S4. This demonstrated that T3 treatment seemed to be the most effective treatment in providing a sustainable increase in AN content. 3.2.4 AP and ASi As shown in Fig. 3 – 5 , there were different degrees of improvement effects on C, N, and P contents in the soil under different treatment groups. Among them, the increase in N content was more significant, whereas the change in P content was relatively stable. P was the key growth-limiting nutrient when N was sufficient (Jin et al., 2019 ). As shown in Fig. 5 , the AP value of S1, S2, and S3 showed the similar change trends under treatments (T1, T2, and T3), which increased during 0–7 d and then decreased. And the T3 treatment on AP enhancement was superior to the other treatment groups. It was possible that humus competed with phosphate for the same binding sites on Fe(Al)-(hydr)oxides to form humic-Fe(Al) complexes, which hindered the formation of precipitated Fe(Al)-phosphates, thus increasing the solubility of P in soils (Gerke, 2021 ). In addition, T1 and T2 might be more volatile in maintaining AP over time compared to T3, while the improvement effect of T3 was more stable. This might be ascribed to the formation of humic-metal-P complexes through metal bridges after combined application of HA and CSH, allowing for a slow release of AP (Urrutia et al., 2013 ). As shown in Fig. 6 , the increase of ASi content was T2 > T1 > T3 in S1 and S2 after treatment. This was probably because A. faecalis accelerated the dissolution and release of silicon from the CSH and then increased the ASi content in soils. For S3 and S4, the ASi content exhibited an increase in the order of T1 > T2 > T3. It could be speculated that use of HA might have a negative effect on the enhancement of ASi content. It had been reported that HA could bind to dissolved Si in acidic soils via bridging Al to form HA-Al-Si complexes (Merdy et al., 2022 ). This interaction between HA and Si allowed ASi to be immobilized, resulting in lower ASi levels under the T3 treatment. The ASi content of S1, S3, and S4 decreased obviously at 0–7 d, which might be associated with the decrease in soil pH (Fig. 2 ). As reported, Al(OH) 3 precipitate at lower pH (around 6) was amorphous (Du et al., 2009 ). And silicic acid in soils would form aluminum silicate precipitates with amorphous Al(OH) 3 (Spinthaki et al., 2021), which in turn led to the decrease of ASi content. 3.2.5 Correlation Between Soil pH and Nutrient Availability The availability of nutrients is directly affected by soil pH (Barrow and Hartemink, 2023 ). Clarifying the effect of soil pH on nutrient availability is important for assessing the effectiveness of acidic soil improvement measures. The relationship between soil pH and nutrients was further explored by using Pearson correlation analysis in this study. Correlation analysis of S1 at 5 d revealed a strongly significant positive correlation between pH and ASi ( r = 0.905, P < 0.001). And soil pH exhibited a significant positive correlation with ASi, SOM, AN and AP at 21 d ( P < 0.001). This suggested that the alkaline soil environment was more favorable for the release of silicon. This was because silicates bound to metals (such as Ca and Mg) required the increase in pH to solubilize under alkaline soil environments (Vasanthi et al., 2018 ). In addition, silicate solubilizing bacteria (SSB) could produce ammonia and amines through metabolic activities, which could shift the pH of the soil towards alkaline and in turn promote the solubilization of silicates (Raturi et al., 2021). As illustrated in Fig. S1 (b), soil pH showed a highly significant negative correlation ( P < 0.01) with SOM in S2 compared to S1. And this could be considered due to the differences between soil physicochemical properties (Table 1 ). Meanwhile, SOM was a negative effect on the ASi content. It could co-precipitate with silicate minerals to form stable organo-mineral complexes, thus reducing the content of ASi (Tamrat et al., 2019 ). For S3, pH had a significant positive correlation with ASi and AN ( P < 0.001). It was attributed to higher pH improved the availability of nutrients. The positive correlation of SOM on ASi, AN, and AP gradually strengthened from 5–21 d. This indicated that SOM also strongly affected nutrient contents and availability in soils. A significant positive correlation was shown between soil pH and all nutrient indicators for S4 soil samples. Overall, the combined application of CSH, A. faecalis , and HA exhibited excellent acidity regulation capacity, which increased soil pH and also enhanced nutrient availability. Among them, CSH was rich in base cations with strong ion exchange capacity, which could effectively increase the soil pH. Combined A. faecalis application for acid soil, it could effectively release the Si in CSH to increase the content of ASi. This could help reduce crop exposure to drought stress and pests (Wang et al., 2021 ). Combined application of CSH, A. faecalis , and HA, the carboxyl and phenolic groups in HA and the Ca 2+ of CSH could form stable clay-humic aggregates via cationic bridges to increase soil soluble phosphate (H 2 PO 4 − ) content (Tiwari et al., 2023). Meantime, HA was rich in organic carbon, allowing to improve the acid buffering capacity of soil. The functional groups such as carboxyl, carbonyl, phenolic hydroxyl, and amino groups in HA could form humic acid ammonium salts with NH 4 + in the soil to reduce soluble N loss (Sun et al., 2023 ). 3.3 Changes of Soil Microbial Community After Amendment 3.3.1 Microbial Community Diversity Microbial communities played a critical role in nutrient cycling (Li et al., 2021), which strongly determined soil health and quality. A total of 120 soil samples from S1-S4 were subjected to 16S rDNA sequencing, and was clustered into 47,405 OTUs. The number of OTUs shared by the microbial community of four treatment groups on 5 d and 21 d was 381 and 270, respectively (Fig. S2(a)). The OTUs showed a decreasing trend under T1 and T2 treatment and increasing trend under T3 from 5–21 d. Furthermore, Venn diagrams revealed that a total of 10,557, 10,589, and 11,508 OTUs were detected under treatments (T1, T2, and T3) after 21 d of amendment period, respectively. The number of OTUs significantly increased in all treatments (T1, T2, and T3) compared to the CK group. The number of unique OTUs was 2278, 5817, 5472, and 6544 in control, T1, T2, and T3 treatments (21 d), respectively. These indicated that a large number of unique microorganisms were found in the amended soil under T1, T2, and T3. These results showed that different treatments affected soil microbial community α diversities, including Chao1 richness, Simpson and Shannon indices. Among them (Fig. S2(b)), the α diversity index after T2 and T3 was significantly lower than that of CK. This was probably because the exogenously added A. faecalis competed with indigenous microbes to improve their survival in the new environment (Yang et al., 2024). This result might lead to the reduction of some microbial species, thereby decreasing the species richness. Moreover, the addition of A. faecalis in T2 and T3 also led to the dominance of exogenous microorganisms in the soil. This single dominant microbial community usually caused a decrease in soil microbial diversity (Simpson and Shannon indices), altering soil multifunctionality (Romdhane et al., 2022). The phenomenon observed was a decrease in the Chao1, Simpson, and Shannon indices, but an increase in the number of OTUs under T1, T2, and T3 compared to the CK group. It indicated that although the number of microbial species increased, the species evenness and community balance might have been disrupted. This could be related to large changes in soil fertility characteristics (pH, SOM, AN, AP and ASi) after amendment (Fig. 2 – 6 ). These changes could exacerbate the instability of microbial community. Meanwhile, competitive imbalances and unequal distribution of resources were also one of the reasons for the reduction of species diversity and microecosystems instability (Zhang et al., 2025 ). Therefore, the number of OTUs increased, but diversities (Chao1 richness, Simpson and Shannon indices) decreased. 3.3.2 Microbial Community Structure Soil microbial communities involved in phylum included Actinobacteriota, Proteobacteria, Firmicutes, Planctomycetota, Chloroflexi, Acidobacteriota, Myxococcota, Bacteroidota, Verrucomicrobiota, and Gemmatimonadota (Fig. 7 a). The dominant phyla were Planctomycetota (20.96%), Proteobacteria (20.03%) and Actinobacteriota (13.90%) in the S1 soil samples. The relative abundance of Actinobacteriota was most remarkable in T3 (43.29%) followed by T1 (30.82%) and T2 (26.05%) at 5 d, while it was 34.53% in control group. We found that the relative abundance of Actinobacteriota tended to increase from 26.05–43.29% to 37.60-47.06%, while Planctomycetota tended to decrease from 13.79–21.14% to 8.15–10.37%. This could be influenced by the soil pH from acidic (around pH 5) to neutral (around pH 6.5). As reported, Actinobacteriota existed optimally in soil environments around pH 7 and its activity was inhibited in acidic soils (Kurtbӧke, 2017 ). Planctomycetota was recognized as a mildly acidophilic microorganism (Kaboré et al., 2020 ) and its activity was negatively correlated with pH. For S2, the relative abundance of Proteobacteria increased, while that of Acidobacteria decreased in the T2 and T3 compared to the CK and T1. And the increase in the abundance of these beneficial microorganism, Proteobacteria and Actinobacteriota, favoured the restoration of soil fertility. They played an important role in nitrogen fixation and phosphate solubilization (Kim et al., 2021; Shivlata and Satyanarayana, 2017 ). Planctomycetota (24.38%), Proteobacteria (21.43%), and Actinobacteriota (19.71%) were the dominant bacterial phyla in the S3 soil microbial community (5 d). There were significant changes in abundance between these phyla after 21 d of succession. Specifically, the relative abundance of Acidobacteriota decreased, whereas Actinobacteriota (36.43–51.20%) and Proteobacteria (22.56–36.60%) were in absolute dominance. It was reported that Acidobacteria was positively related with soil pH, while Proteobacteria and Actinobacteriota were negatively related with this factor (Wang et al., 2018). The pH of the amended acidic soil increased in our study (Fig. 2 ). Therefore, it could be speculated that the different responses of the major phyla to pH might be responsible for the changes in soil microbial community. The application of amendments increased the abundance of some species favourable to soil nutrient cycling. For example, the phyla Firmicutes (25.88–37.33%) of S4 was most abundant under the three treatments in comparison to the CK treatment. The increase in the abundance of the phyla Firmicutes had a direct positive effect on soil organic matter mineralization (Xiao et al., 2022). The dominant genera (top 10) between different succession stage (5 d and 21 d) were shown in Fig. 7 b. For S1, the abundance of Arthrobacter was higher in the T3 than in the other groups, accounting for 11.93% (5 d). This genus plays an important role in soil carbon and nitrogen cycling to improve soil functionality (Aguilera-Huertas et al., 2023). The genera Nocardioides and Oryzihumus increased in abundance with 21 d of succession. And these genus are members of the phylum Actinobacteria and have excellent pollutant degradation ability (Ma et al., 2023 ) and drought tolerant (Metze et al., 2023). The changes in soil microbial structure reflected somehow the succession of functional microorganisms. The genera Paenochrobactrum (17.43–21.44%) and Bacillus (6.16–8.71%) of S2 had the highest abundance in T2 and T3 compared to the CK and T1 (5 d). And then Massilia (12.67–13.24%) and Terrabacter (5.42–7.02%) became the dominant bacterial genera in T2 and T3 with the successional stage. Many bacteria in the soil have the ability to solubilize silicon and phosphorus, such as Bacillus and Arthrobacter (Bist et al., 2020; Zheng et al., 2017). Bacillus (13.08–17.16%) and Arthrobacter (9.13–16.31%) of S3 had the highest abundance in the T1-T3, while they were 4.63% and 8.96% in CK, respectively. It was further observed that the abundance of plant growth promoting microorganism increased in the T1-T3 treatments. For example, the abundance of Massilia and Terrabacter increased by 7.10-17.71% and 1.32–5.96%, respectively. These changes contributed to enhancing nitrogen cycling and reducing heavy metal toxicity in acidic soil (He et al., 2024). The microbial community in S4 was dominated by Bacillus (15.35–21.89%) at 5 d, while it was 3.47% in control group. Among the unclassified genera, the highest abundance was Isosphaeraceae_unclassified , representing 5.76–7.63% (5 d). This genus contained encoded hao with N fixation capacity, which could increase soil nutrient availability (Camargo et al., 2023). The relative abundance of these two dominant genera ( Bacillus and Isosphaeraceae_unclassified ) tended to decrease and Terrabacter tended to increase with succession stage. On the whole, the T2 and T3 treatments promoted the positive changes of soil microbial communities, and the abilities related to soil nitrogen fixation, silicon dissolution, and phosphorus dissolution were improved. Nonetheless, the exogenous A. faecalis introduced in the T2 and T3 treatments exerted positive effects on the soil microbial community structure. Long-term field experiments are still warranted to monitor their potential impacts on human health. 3.4 The Correlation of Soil Microbial Community and Environmental Factors The redundancy analysis (RDA) of soil samples, environmental factors, and microbial community structure (genus level) is shown in Fig. S3. As seen, when compared the microbial communities of T1, T2 and T3 groups in S1, soil pH was positively related with groups T2 and T3 and negatively related with group T1 (5 d). And the dominant genus Oryzihumus in group T2 was positively correlated with NO 3 -N, ASi, NH 4 -N, SOM and AP (21 d). This showed that the relative abundance of microbial communities shifted with increasing soil pH and nutrient availability. This is consistent with previous studies which indicated that soil pH and nutrient availability would directly influence the structure and diversity of microbial communities (Sarkodie et al., 2024). As illustrated by Fig. S3(b), microbial communities in the group T1 clustered separately from groups T2 and T3. The dominant genera Paenochrobactrum of S2 in groups T2 and T3 were negatively correlated with SOM and positively correlated with NO 3 -N. And the dominant genus Actinobacteriota_unclassified in T1 was positively associated with SOM and positively associated with NO 3 -N (5 d). These data indicated differences in the soil microbial communities between treatment groups regulated by environmental factors. The RDA of S2 at 21 d explained 77.79% of the total variation in the microbial community structure, which 51.82% (RDA1) and 25.97% (RDA2) was explained by the first two axes. As shown in Fig. S3(c), AP significantly influenced the variation of soil microbial abundance. The dominant Paenochrobactrum in the T3 was negatively correlated with AP (5 d). But after 21 d, the dominant genus Paenochrobactrum in T3 was positively related with AP, NO 3 -N, SOM, ASi, and NH 4 -N. This suggested that the increase content of soil nutrients favored microbial growth, providing C, N, P, and Si sources for these microbes. The soil pH, NH 4 -N, ASi, AP, SOM, and NO 3 -N of S4 were positively correlated with the first constrained axis (Fig. S3(d)). Soil pH was significantly correlated with the dominant microbial genus both before and after community succession. It confirmed the role of soil pH for the growth of dominant microorganisms and the importance of regulating the structure of microbial communities (Hong et al., 2022 ). And the composition ratio and dose of the combined amendments were also the main influencing factors (Chen et al., 2019 ). In addition, the differences in soil physicochemical properties (pH and nutrient contents) between soil samples could also affect the diversity of bacterial communities (Li et al., 2020). Soil pH and available nutrients have a direct influence on the balance of soil ecosystem and microbial diversity. Based on the Pearson correlation heatmap between soil bacterial abundance and environmental factors (Fig. 8 ), it could be seen that pH, ASi, AP, SOM, NH 4 -N, and NO 3 -N might be the main drivers of soil microbial community succession. As the dominant phylum, Actinobacteriota exhibited a significant positive correlation with pH (Fig. 8 a). And the phylum Acidobacteriota, Planctomycetota, Chloroflexi and Myxococcota showed negative correlations with pH, showing acidophilic characteristics. This indicated that pH could have an important impact on the microbial community structure. pH has been demonstrated to be a key factor influencing the structure and function of microbial communities in acidic soil (Wan et al., 2020 ). Acidobacteria, Planctomycetes, and Chloroflex are slow-growing oligotrophic bacteria that typify the K-strategy (Yang et al., 2023). They exhibited strongly negatively correlated with NH 4 -N and ASi. The phylum Actinobacteriota showed significant positive correlations with AP and NO 3 -N. It is reported that Actinobacteriota have multifunctional traits in soil health, such as N fixation (Swarnalakshmi et al., 2016 ), P solubilization (Zeng et al., 2024 ), SOM decomposition (Liu et al., 2023), and alleviation of drought stress (Kumar et al., 2020 ), which demonstrated the importance of the phyla in soil. And Firmicutes showed significantly positively correlated with SOM, ASi and NH 4 -N. Furthermore, Firmicutes was a typical eutrophic bacterium that showed a progressive increase in correlation with NH 4 -N and SOM from 5 d to 21 d. The above analysis exhibited that the relative abundance of most copiotrophic taxa increased with increasing pH value and nutrient contents. Conversely, oligotrophic taxa were negatively relationship with pH and nutrients. Therefore, it could be revealed that amendment measures would promote the shifts of soil microbial communities in a positive direction by increasing soil pH and available nutrient contents. The correlations between the environmental factors and microbial community structure at the genus level are presented in Fig. 8 b. The relative abundance of Massilia , Terrabacter , Arthrobacter , Nocardioides , Oryzihumus , Bacillus , and Paenochrobactrum was significantly positively correlated with pH ( P < 0.01). Whereas Isosphaeraceae_ unclassified and Aquisphaera were negatively correlated with pH ( P < 0.01). This could be explained by their different abilities to adapt to changes in soil pH. Meantime, the genus Arthrobacter and Bacillus were reported to have alkalophilic traits, with an optimum at pH 7.0–8.0 (Fritze, 1996 ; Lee et al., 2003 ). And the increase of soil pH after the application of amendments (T3) was more favorable to their survival (Fig. 7 b and Fig. 8 b). These results revealed that the application of the soil amendments might increase the abundance of genera preferring both neutral and alkaline soil environments (5.5 < pH < 7.5). Bacillus showed a significant positive association with ASi content at 5 d ( r = 0.781, P < 0.001). As reported, this genus could solubilize silicates, which belonged to SSB (Vasanthi et al., 2018 ). This could be speculated that Bacillus might be involved in the Si cycling. Besides, Bacillus as a well-known genus of beneficial soil bacteria, and some species of them could effectively inhibit the growth of pathogenic bacteria such as Ralstonia solanacearum (Raza et al., 2016 ). Thus, multifunctionality of the Bacillus revealed its importance in soil amendment. Arthrobacter is a typical phosphate solubilizing bacterium (PSB) (Chen et al., 2006 ), showing a significant positive correlation with NO 3 -N and AP. The soil pH was reported to be majorly responsible for the abundance of PSB community and its capacity to solubilize P (Zheng et al., 2019). This demonstrated that soil pH played a dominant role in the participation of Arthrobacter in soil P cycling. 4. Conclusions This study revealed that the application of A. faecalis coated by CSH and HA was more efficient in increasing the pH of acidic soil. It not only significantly increased the soil pH, but also enhanced the contents of AN, AP, and ASi. The correlation analysis found that pH was positively correlated with all nutrient indicators (SOM, AN, AP, and ASi) of most soil samples. This indicated that improving soil acidity is crucial for enhancing soil fertility. Regarding the microbial community, the application of A. faecalis coated by CSH and HA triggered distinct responses. The relative abundances of beneficial microorganisms like Bacillus , Arthrobacter , and Paenochrobactrum notably increased. These microorganisms were not only associated with the elevation of soil pH, underscoring the role of pH in shaping the microbial community composition, but also positively correlated with the increase in nutrient contents. This suggested their active participation in soil nutrient cycling processes, such as N, P, and Si cycling. Declarations Acknowledgements The authors are grateful to the School of Minerals Processing and Bioengineering, Central South University and Key Laboratory of Biometallurgy of Ministry of Education for providing the infrastructure and facilities to carry out this research. The authors have immense gratitude to the editors for their comments which helped a lot to improve the quality of the paper and standardize the format. Author information Authors and Affiliations School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China Luhua Jiang, Manjun Miao, Ziwen Guo, Jiejie Yang, Yulong Peng, Junzhao Wu, Bo Miao, Xueduan Liu Key Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha 410083, China Luhua Jiang, Manjun Miao, Ziwen Guo, Jiejie Yang, Yulong Peng, Junzhao Wu, Bo Miao, Xueduan Liu Yuelushan Laboratory, Changsha 410128, China Luhua Jiang, Xueduan Liu Corresponding author Correspondence to Xueduan Liu. Ethics approval and consent to participate This study does not involve animal or human subjects. Consent for publication All authors have consent to publish this paper with this journal. Availability of data and material All data included in this study are available upon request by contact with the corresponding author. Competing interests The authors declare no competing interests. Funding This study was supported by the Yuelushan Laboratory Breeding Program (No. YLS-2025-ZY02040), the National Natural Science Foundation of China (Grant No. 52174341), the Hunan Provincial Key Research and Development Plan (Grant No. 2022WK2017 and 2023NK2030), and the Natural Science Foundation of Hunan Province of China (Grant No. 2022JJ40583). Author contributions Luhua Jiang: Conceptualization, Writing - review and editing, Supervision, Project administration, Funding acquisition; Manjun Miao: Writing - original draft preparation, Methodology, Validation, Formal analysis, Investigation; Ziwen Guo and Jiejie Yang: Formal analysis; Yulong Peng: Investigation; Junzhao Wu: Formal analysis; Bo Miao: Resources; Xueduan Liu: Conceptualization, Resources, Supervision, Project administration, Funding acquisition. References Adeleke, R., Nwangburuka, C. and Oboirien, B.: 2017, 'Origins, roles and fate of organic acids in soils: A review', S AFR J BOT 108, 393-406. 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10:20:56","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":401467,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/4d89734785d19149b9a2bade.png"},{"id":94184240,"identity":"7018e913-a67a-4ea4-a57f-1f38afbb98d1","added_by":"auto","created_at":"2025-10-23 10:20:56","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":221143,"visible":true,"origin":"","legend":"","description":"","filename":"cfd7d147a8f34dda83e6b30a2de721191structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/c3a7a14502d9df3467846027.xml"},{"id":94184834,"identity":"6e2f7e5e-dbd2-46c2-a3ef-ad0ad041419c","added_by":"auto","created_at":"2025-10-23 10:28:55","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":234163,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/6a3f37aadaf6b10abcb6959f.html"},{"id":94184217,"identity":"8fdd3a16-eb45-4242-a932-4e8855b1f4d6","added_by":"auto","created_at":"2025-10-23 10:20:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1407972,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images of CSH (a), CSH coated by \u003cem\u003eA. faecalis\u003c/em\u003e(b), and CSH and HA-encapsulated \u003cem\u003eA. faecalis\u003c/em\u003e (c); the elemental components of CSH determined by XRF (d)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/bf0a0ee036db529e5c25ab4a.png"},{"id":94184218,"identity":"c3a058f4-cff9-403b-8861-b8c9a4e3159e","added_by":"auto","created_at":"2025-10-23 10:20:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1656195,"visible":true,"origin":"","legend":"\u003cp\u003eSoil sample pH under different treatments. (a) S1, (b) S2, (c) S3, and (d) S4. Different-colored lines indicate the different treatment groups\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/841d38cdcb4f80b374408c0f.png"},{"id":94184833,"identity":"e0c01c97-af36-4c2c-a7a8-af16449705e8","added_by":"auto","created_at":"2025-10-23 10:28:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4907100,"visible":true,"origin":"","legend":"\u003cp\u003eSOM content under different treatments. (a) S1, (b) S2, (c) S3, and (d) S4. Different-colored lines indicate the different treatment groups\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/7cd1ff1461abcd2355029b58.png"},{"id":94184220,"identity":"31bdc82d-ae72-45ae-8e6a-4fdba0255a35","added_by":"auto","created_at":"2025-10-23 10:20:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5066946,"visible":true,"origin":"","legend":"\u003cp\u003eSoil AN content under different treatments. (a) S1, (b) S2, (c) S3, and (d) S4. Different-colored lines indicate the different treatment groups\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/9dfac23d1f6a3b942f09f17b.png"},{"id":94184835,"identity":"9ceabaa2-6fea-4891-a578-4cfdf85689f5","added_by":"auto","created_at":"2025-10-23 10:28:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4959263,"visible":true,"origin":"","legend":"\u003cp\u003eSoil AP content under different treatments. (a) S1, (b) S2, (c) S3, and (d) S4. Different-colored lines indicate the different treatment groups\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/c2734333880ca57726f3a248.png"},{"id":94184225,"identity":"0490e722-c0a3-4496-a357-452a2202e51a","added_by":"auto","created_at":"2025-10-23 10:20:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5141148,"visible":true,"origin":"","legend":"\u003cp\u003eSoil ASi content under different treatments. (a) S1, (b) S2, (c) S3, and (d) S4. Different-colored lines indicate the different treatment groups\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/64e4af17c26c5f94985a3f4c.png"},{"id":94184840,"identity":"7f782367-4c9e-49be-a3b9-d65fa9157970","added_by":"auto","created_at":"2025-10-23 10:28:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1586925,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of dominant microorganisms (top 10) at the phyla level (a) and genus level (b) under different treatments\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/86a88749cb971379f9ff6488.png"},{"id":94184248,"identity":"7a5ec8e0-68fd-4a3d-ad50-2b54a4e2c95e","added_by":"auto","created_at":"2025-10-23 10:20:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2193564,"visible":true,"origin":"","legend":"\u003cp\u003ePearson correlation between the dominant soil bacterial phyla at 5 d (a) and 21 d (b), genus at 5 d (c) and 21 d (d) and soil environmental factors. Note: the horizontal and vertical axes represent environmental factors and microorganisms, respectively; red color indicates positive correlations and blue color indicates negative correlations; * \u003cem\u003eP\u003c/em\u003e＜0.05; ** \u003cem\u003eP\u003c/em\u003e＜0.01; *** \u003cem\u003eP\u003c/em\u003e＜0.001\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/960c2835f05b63964988e97f.png"},{"id":94185967,"identity":"24ca5b5f-b329-40f6-b1a0-be2430ed8d46","added_by":"auto","created_at":"2025-10-23 10:45:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27998526,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/4ef4e24b-6099-4aa8-bf4b-c987ac203a5d.pdf"},{"id":94184221,"identity":"35340fec-7b78-4e9c-bcf2-419652d59fae","added_by":"auto","created_at":"2025-10-23 10:20:55","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":940598,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7738393/v1/730940a8d5982629d4f0f938.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improvement effects of calcium silicate hydrate and humic acid-encapsulated Alcaligenes faecalis on acidic farmland soil and alterations in microbial community structure","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWith nitrogen nitrification and base cations loss, soil acidification is becoming seriously, which has become a global environmental issue (Dong et al., 2022). Globally, about 50% of arable soils are classified as acidic (pH\u0026thinsp;\u0026lt;\u0026thinsp;5.5) and suffer from soil acidification (Dai et al., 2017), especially in East Asia such as China and South Korea (Duan et al., 2016). The acidity of soils has been reported to directly affect available nutrient contents, resulting in a decrease in soil fertility (Hagh-Doust et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Meantime, soil acidification has also largely led to the decline in the abundance and diversity of bacterial community (Li et al., 2023). Therefore, exploring effective ways to reduce soil acidity is of great significance for improving soil nutrient contents and restoring the health of acidic soil microbial community.\u003c/p\u003e\u003cp\u003eAcidic soils can be ameliorated by applying alkaline amendments such as lime, biochar, and steel slag (Uwiringiyimana et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These alkaline amendments directly neutralized H\u003csup\u003e+\u003c/sup\u003e in the soil by dissolving OH\u003csup\u003e\u0026minus;\u003c/sup\u003e and HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. And it also supplied base cations (such as Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, and K\u003csup\u003e+\u003c/sup\u003e) to replace Al\u003csup\u003e3+\u003c/sup\u003e in the soil, reducing soil acidity. Among these amendments, lime remained the most traditional and common practice to reduce soil acidity. It could increase soil pH and yields for most crops, particularly legumes, which were sensitive to soil pH (Li et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, biochar is regarded as an effective soil amendment for ameliorating acidic soils. It could also increase soil pH due to its alkaline nature (pH 8.8\u0026ndash;10.3) and the carbonates in the ash (Bolan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al., 2024). And some studies have found that biochar also contributed to acidic soil fertility by suppling large amounts of nutrients, such as C, N, and P, for plant growth. However, these traditional alkaline amendments still face serious challenges. For example, these applications might induce soil structural degradation, nutrient imbalance and reacidification, and even lead to environmental pollution risks (Dong et al., 2025). Recently, microbial-mediated remediation offers a more sustainable approach and has received increasing attention in acid soil improvement due to its green environment and low cost. Microorganisms could not only continuously regulate soil pH through metabolic activities, but also synergistically drive nutrient cycling and microbial community restoration (Oro et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, it could be considered as an alternative to traditional alkaline amendments.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAlcaligenes faecalis\u003c/em\u003e (\u003cem\u003eA. faecalis\u003c/em\u003e), a common bacterium in soils, exhibited unique advantages in acid soil improvement as a bioremediating material with the function of nitrogen fixation and secreting alkaline metabolites (Yang et al., 2024; Zhong et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, being added as exogenous microorganisms to acidified soils had many limitations, such as unfavorable survival and low adaptability due to the competition with native microbes (Sun et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This greatly limited its application in acid soil improvement. Therefore, to improve their survival rates, it was possible to immobilize them on carrier materials as initial habitat after entering the soil. Calcium silicate hydrate (CSH) was a porous material rich in silicon and trace elements, which could be considered an ideal carrier for immobilization. Meanwhile, CSH as an alkaline amendment, could increase pH when applied to the soil (Qing et al., 2022) and provide a favorable environment for the survival of \u003cem\u003eA. faecalis\u003c/em\u003e. Besides, the elements contained in CSH provided nutrients for bacteria reproduction, while \u003cem\u003eA. faecalis\u003c/em\u003e also promoted the release of silicon contained in CSH to increase acidic soil silicon content. Until now, there have been no studies on the application of CSH as a bacterial carrier for the improvement of acidic soils.\u003c/p\u003e\u003cp\u003eAlthough \u003cem\u003eA. faecalis\u003c/em\u003e loaded on CSH could improve the pH and microbial activity, this approach still did not compensate for the deficiency of organic matter in acidic soil. The lack of organic matter not only affected soil nutrients but also limited the growth and structure of microbial community (Bashir et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Humic acid (HA) could enhance soil acid buffering capacity and increase the abundance of beneficial microorganisms in acidic soil (Xu et al., 2021). Meanwhile, HA could complex with calcium ions in CSH to form stable humic-Ca complexes, which was conducive to the accumulation of soil organic matter (Tang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, it could be recognized as a carrier material for microorganisms to colonize soil due to its large surface area and high cation exchange capacity (Hao et al., 2025). Therefore, using CSH and HA as carrier materials can enhance the adaptability of \u003cem\u003eA. faecalis\u003c/em\u003e in acidic farmland soils and synergistically improve soil improvement performances.\u003c/p\u003e\u003cp\u003eIn this study, we focused on the effects of CSH and HA-encapsulated \u003cem\u003eA. faecalis\u003c/em\u003e on the acidic soils improvement. Thus, the aims of the present study were to (i) analyze the effects of different treatments on the improvement of soil pH and available nutrient contents in acidic soil; (ii) explore the changes of soil microbial community diversity and structure after amendment; (iii) unlock the underlying relationships between soil physicochemical properties and the composition and structure of microbial community.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental Materials\u003c/h2\u003e\u003cp\u003eSynthesis of CSH was prepared by directly mixing Na\u003csub\u003e2\u003c/sub\u003eSiO\u003csub\u003e3\u003c/sub\u003e solution (0.5 mol/L) with CaCl\u003csub\u003e2\u003c/sub\u003e solution (1 mol/L) at Ca/Si molar ratio of 1:1 under microwave hydrothermal conditions. Firstly, the obtained solution was put into a microwave ablator (MDS-6G, Xinyi, China) and reacted at 160 ℃ for 6 h under microwave power of 400 W. After that, it was naturally cooled to room temperature. The slurry was then centrifuged and washed with deionized water. After drying in an oven at 60 ℃ for 24 hours, the product was ground through a 100-mesh sieve. This process yielded CSH with pH values ranging from 10.9 to 11.6, and a silicon content of approximately 10% (Yang et al., 2023). The \u003cem\u003eA. faecalis\u003c/em\u003e (CCTCC No: M 2020786) was cultured in NB medium for enrichment culture. The density of live bacteria was 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/mL. And the pH value of the bacterial solution was 8.05\u0026ndash;8.85. HA suspension was provided by Shandong Linker Agritec Co., Ltd., with organic matter content\u0026thinsp;\u0026ge;\u0026thinsp;100 g/L and the concentration of HA being 40 g/L. The surface morphology of CSH, CSH coated by \u003cem\u003eA. faecalis\u003c/em\u003e, and CSH and HA-encapsulated \u003cem\u003eA. faecalis\u003c/em\u003e was examined by scanning electron microscope (SEM, JSM-IT500, JEOL Japan). The chemical composition of CSH was determined by using an X-ray fluorescence spectrometer (XRF, Axios, PANalytical, Netherlands).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Microcosm Experiments\u003c/h2\u003e\u003cp\u003eSoil samples were collected using a diagonal sampling method, air-dried, and ground through a 100-mesh sieve for soil physicochemical properties analysis. The remaining soil was placed in sterile self-sealing bags and stored in the refrigerator at -80 ℃ for 16S rDNA analysis experiments. The soil samples used for this study were collected from paddy in Nanxiong City and Taishan City, Guangdong Province. They were named as S1, S2 for Nanxiong, and S3, S4 for Taishan, respectively. After removing large stones and plant debris, soil samples (500 g) were mixed well and packed in polythene plastic pots and then tested at room temperature (25 ℃). Four treatment groups were set up in this experiment. No amendment was added into soils set as a control group (CK); CSH was applied at 1.0\u0026permil; addition (T1); The application of 1.0\u0026permil; CSH was combined with a 5% addition rate of \u003cem\u003eA. faecalis\u003c/em\u003e liquid (T2); and the application included 1.0\u0026permil; CSH, 5% \u003cem\u003eA. faecalis\u003c/em\u003e liquid, and 150 \u0026micro;L of HA (T3). Three replicates were set up for each treatment. The microcosm experiments were conducted in an incubator at a constant temperature of 25 ℃ and a relative humidity of 60%. The water-to-soil ratio was fixed at 1:4 (v/m). The experiment lasted for 21 days, and soil samples were collected on the 1st, 3rd, 5th, 7th, 14th, and 21st days for subsequent analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Sample Analysis\u003c/h2\u003e\u003cp\u003eSoil pH was measured in water extracts at a 2.5:1 water-soil ratio (v/w) using a digital pH meter, according to NY/T 1377\u0026ndash;2007. Soil organic matter (SOM) content was determined by the potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) digestion method according to NY-T 1121.6\u0026ndash;2006. The soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e-N) and nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e-N) were determined by extraction with potassium chloride solution-spectrophotometric method (HJ 634\u0026ndash;2012). The content of available phosphorus (AP) was extracted with 0.5 mol/L sodium bicarbonate solution at pH 8.5 and then measured using the molybdenum antimony anti-colorimetric method (Olsen, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1954\u003c/span\u003e). The determination of available silicon (ASi) content was analyzed by using the silicon molybdate spectrophotometric method (NY/T 1121.15\u0026ndash;2006).\u003c/p\u003e\u003cp\u003eSoil samples collected on 0, 5, and 21 d after improvement were analyzed by using 16S rDNA amplicon sequencing. Total soil genomic DNA was extracted by using the IlluminaMiSeq kit, and primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3') were used to amplify the hypervariable region V3-V4 of the bacterial 16S rDNA gene by polymerase chain reaction (PCR). The amplification reaction included an initial denaturation step at 98 ℃ for 2 minutes, followed by 28 cycles of denaturation at 98 ℃ for 15 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds. This was concluded with a final extension step at 72 ℃ for 5 minutes. The amplified PCR products were detected by agarose gel electrophoresis at a concentration of 1.2%, purified by using the Agarose Gel Recovery Kit (SOMEGA Biotek Inc., Doraville, GA, USA), and finally quantified by using Qubit (Invitrogen, USA). Amplicon libraries were constructed for sequencing, and the size and concentration of the amplicon libraries were assessed by using an Agilent 2100 Bioanalyser (Agilent, USA) equipped with the Illumina Library Quantification Kit (Kapa Biosciences, Woburn, MA, USA). Libraries were sequenced on the NovaSeq-PE250 sequencing platform (Illumina Inc., Carlsbad, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Data Analysis\u003c/h2\u003e\u003cp\u003eThe DNA was sequenced using the Illumina NovaSeq platform. To obtain high-quality clean data, it was necessary to use overlap to splice, quality control and chimera filter the double-ended data. Primer sequences introduced from sequence databases were removed using FLASH (v 1.2.8) software, and chimeric sequences were filtered using Vsearch (v 2.3.4) after merging sequences. After using DADA2 (Divisive Amplicon Denoising Algorithm) in QIIME2 to perform denoising and replication clustering, the OTUs table, feature tables, and feature sequences were obtained. Alpha diversity indexes including Chao1 richness, Simpson and Shannon index were analyzed using the R package ggplot2 (v. 4.1.3). Redundancy analysis (RDA) was performed using the R package vegan (v. 3.6.3). The heatmap of Pearson correlation between environmental factors and dominant bacteria was plotted using the R package stats (v. 3.6.3). Significance analysis was calculated by SPSS software (IBM SPSS Statistics 24). The remaining graphs were plotted using OriginPro 2024b software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Material Characterizations and Soil Physicochemical Properties Analysis\u003c/h2\u003e\u003cp\u003eThe SEM images provided significant insights into the surface morphology of CSH and CSH-HA before and after loaded with \u003cem\u003eA. faecalis\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the surface of CSH exhibited prominent ripples and wrinkles with a large number of irregular pore structures. This morphology could be attributed to the amorphous structure of CSH (Guo et al., 2022). The SSA\u003csub\u003eBET\u003c/sub\u003e value of CSH was 35.79 m\u003csup\u003e2\u003c/sup\u003e/g and the PV value was 0.258 cm\u003csup\u003e3\u003c/sup\u003e/g. This indicated that CSH had a large specific surface area and a well-developed pore structure, both of which were crucial in providing sites for microbial attachment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, the rod-shaped feature of \u003cem\u003eA. faecalis\u003c/em\u003e was clearly visible and these strains appeared to adhere at the CSH surface. And the surface structure of CSH became looser and more porous after loading \u003cem\u003eA. faecalis\u003c/em\u003e. It could be speculated that \u003cem\u003eA. faecalis\u003c/em\u003e metabolically synthesized extracellular polymeric substances (EPS) to corrode the surface of CSH, which further promoted the loosening of its structure (Wang et al., 2023). Besides, the porous structure of CSH might provide a habitat for microbial growth, which served as a carrier that could promote the survival of \u003cem\u003eA. faecalis\u003c/em\u003e after entering the soil environment. The SEM of CSH-HA carrier after being loaded with \u003cem\u003eA. faecalis\u003c/em\u003e was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. CSH-HA carrier had a richer surface structure, which increased the contactable area with \u003cem\u003eA. faecalis\u003c/em\u003e and favored its adherence. As reflected by Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, the main element composition of CSH was O (39.90%), Ca (34.89%), Si (21.34%), and Na (2.81%). Ca could enhance the metabolic activity of \u003cem\u003eA. faecalis\u003c/em\u003e and was important for the synthesis of EPS (Ibrahim et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This would promote the formation of biofilms on the CSH surface, making it easy for the attachment of \u003cem\u003eA. faecalis\u003c/em\u003e. Additionally, the Ca and Na elements in CSH could enhance the acid buffering capacity of the soil. The cations such as Ca\u003csup\u003e2+\u003c/sup\u003e and Na\u003csup\u003e+\u003c/sup\u003e could easily exchange with H\u003csup\u003e+\u003c/sup\u003e in acidic soil (Bernard et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), resulting in soil pH increasing. Besides, Si in CSH was mainly amorphous silicate(Guo et al., 2022), which could replenish the ASi content in the soils when applied. This would help to improve soil fertility and increase the drought stress tolerance of plants as well as their resistance to heavy metal toxicity, pests and diseases (Kov\u0026aacute;cs et al., 2022; Thorne et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe physicochemical properties of soil samples are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As seen, soil samples were strongly acidic with pH values of 5.13 (S1), 5.46 (S2), 5.35 (S3) and 4.66 (S4), respectively. This difference in acidity might be associated with soil properties, microbial communities, and organic matter contents (Hong et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The NH\u003csub\u003e4\u003c/sub\u003e-N content of S4 (143.93 mg/kg) was higher than that of S1 (81.42 mg/kg), S2 (71.69 mg/kg), and S3 (82.38 mg/kg). NO\u003csub\u003e3\u003c/sub\u003e-N content varied across the samples; and S3 exhibited the highest content (20.19 mg/kg), while S4 had the lowest (4.57 mg/kg). NO\u003csub\u003e3\u003c/sub\u003e-N was easily lost by leaching, while NH\u003csub\u003e4\u003c/sub\u003e-N was not easily lost due to soil colloid adsorption, which favored soil nitrogen conservation in S4 (Dal Molin et al., 2018). Besides, the soil NH\u003csub\u003e4\u003c/sub\u003e-N content was all higher than the NO\u003csub\u003e3\u003c/sub\u003e-N content from S1 to S4. This might be due to the low pH value reduced the activity of ammonia-oxidizing bacteria, which was unfavorable to its nitrification process and thus inhibited the conversion of NH\u003csub\u003e4\u003c/sub\u003e-N to NO\u003csub\u003e3\u003c/sub\u003e-N (Lin et al., 2021). The AP content of S1 and S2 was significantly higher than that of S3 and S4. And the content of S4 was the lowest with the value of 15.59 mg/kg. This was ascribed to soil phosphate would be sorbed onto the surfaces of silicate minerals and Al- and Fe-oxides when the soil pH value below 5 in S4. And then phosphate was converted from the adsorbed fraction to the precipitated Al- and Fe-phosphates in acidic conditions (Kruse et al., 2015). The SOM content in soil samples ranged from 3.18% to 4.03%. This indicated that all the soil samples could be classified as Level II (3\u0026ndash;4%, richness) according to China\u0026rsquo;s second soil census nutrient classification standards (Liu et al., 2023). S4 had the highest SOM content at 4.03% and the lowest pH value at 4.66. Previous studies have shown that soil pH exhibited a significant negative correlation with SOM; and the accumulation of organic acids produced from organic matter decomposition or soil microbial metabolism caused low pH in S4 (Adeleke et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The ASi content showed a trend of S4\u0026thinsp;\u0026gt;\u0026thinsp;S1\u0026thinsp;\u0026gt;\u0026thinsp;S3\u0026thinsp;\u0026gt;\u0026thinsp;S2, which was exactly opposite to the trend of pH. The main source of silicon was the weathering of soil minerals, especially the decomposition of silicon-rich minerals such as feldspar and mica. Silicate weathering could be strongly modulated by soil pH and increase an order of magnitude for each unit decrease in pH (Bufe et al., 2021).\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 of S1, S2, S3 and S4\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\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\u003eNH\u003csub\u003e4\u003c/sub\u003e-N (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e-N (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAP (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSOM (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eASi(mg/kg)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.42\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e63.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.76\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e38.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.55 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e143.93\u0026thinsp;\u0026plusmn;\u0026thinsp;9.12 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e67.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Different case letters in the same column indicate significant differences between soil samples (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Changes in Soil Fertility Characteristics After Amendment\u003c/h2\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 pH\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the soil pH values showed an increasing trend in the initial period. Specifically, S1 exhibited a peak pH value after 5 d of treatment, whereas S2, S3, and S4 samples reached their peak pH values at 3 d. This rapid pH increase was primarily because the applied amendments were strongly alkaline (pH values 10.9\u0026ndash;11.6). After 5 d, pH values of all treatments fluctuated considerably and began to decline. In terms of specific soil samples, the pH values of S1, S2, and S4 under T3 were significantly higher than other treatments (T1 and T2) at 21 d. For S1, the improvement of pH value under T1 and T2 showed a similar trend, but T2 was slightly better than T1. This slight superiority in T2 might be partly attributed to the presence of \u003cem\u003eA. faecalis\u003c/em\u003e. Its metabolic activities might have generated additional alkaline substances that enhanced the increasing of pH compared to T1. Besides, the pH value of S1 increased to 6.59 under T3 at 21 d, which was higher than that of T2. While HA in T3 treatment, with its abundant oxygen-containing functional groups such as carboxyl, phenolic, and hydroxyl (Tiwari et al., 2023), could absorb protons through association reactions at low pH conditions and then increase the soil pH (Xu et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The pH improvement of S2 under different treatment groups exhibited a trend of T3 ๥ T2 ๥ T1. T3 obviously increased the pH value of S2, and the value reached up to 7.28 and 7.16 at 3 d and 21 d, respectively. It showed less fluctuation in pH value during 3\u0026ndash;21 d. This might be attributed to the fact that the higher SOM content of S2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which had stronger acid buffering capacity. Additional added HA also contributed substantial organic matter, further increasing the SOM content of S2 and suppressing soil acidification. And Ca ion from CSH could prevent SOM from being degraded. It remained SOM at a high level through mechanisms such as adsorption and complexation (Gruba and Mulder, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shabtai et al., 2023), thereby maintaining pH stability. The pH improvement of S3 under these treatments (T1, T2, and T3) was the best in the samples of S1-S4. And the differences in interval of pH improvement between the treatments (T1, T2, and T3) were slight in S3. Although the differences among treatments were not as obvious as in other samples, the metabolic activities of \u003cem\u003eA. faecalis\u003c/em\u003e in T2 and T3 might still have a subtle effect on soil acid-base balance. Besides, the pH value of S4 peaked on 3 d and then dropped sharply on 7 d under all treatments. The sharp decrease might be ascribed to the rapid consumption of alkali in these amendments. Moreover, the content of SOM and NH\u003csub\u003e4\u003c/sub\u003e-N in S4 was 4.03% and 143.93 mg/kg, respectively, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As reported, high level of SOM (\u0026ge;\u0026thinsp;3.0%) and excessive NH\u003csub\u003e4\u003c/sub\u003e-N could aggravate soil acidification (Zhang et al., 2022), due to the nitrification of NH\u003csub\u003e4\u003c/sub\u003e-N and the decomposition of SOM (Zhao et al., 2022). Both of these effects would lead to the sharp decrease of soil pH value on 7 d. In addition, the pH value was back up to 6.41 under T3 at 21 d, which was close to the peak pH value of 3 d (6.46). This could contribute to the addition of HA in T3 treatment which could improve soil pH buffering capacity. In the view of the changes of pH value in S1, S2, and S4 under different treatments (T1, T2, and T3), the T3 treatment (CSH\u0026thinsp;+\u0026thinsp;\u003cem\u003eA. faecalis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;HA) had the best performance in increasing the pH value for the studied soil samples. However, it's crucial to note that this study was based on 21-day short-term microcosm experiments in the laboratory, and the amelioration effect of the system might deteriorate under field conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 SOM\u003c/h2\u003e\u003cp\u003eSOM content can serve as a vital indicator of soil health, which determines soil nutrients and microbial activity (Xu et al., 2018). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the content of SOM exhibited comparable trends under these treatments (T1, T2, and T3) in S1. Specifically, the SOM content showed a decreasing trend from 5\u0026ndash;7 d and then increased gradually from 7\u0026ndash;21 d. Furthermore, T3 treatment led to a significantly decrease of SOM content at 7 d in S1 when compared to T1 and T2, while SOM content increased rapidly at 7\u0026ndash;21 d. When HA was added into soils, microbial activity would increase and the decomposition of SOM was accelerated (Bingeman et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1953\u003c/span\u003e). This might lead to the decrease of SOM content at 7 d in S1 when T3 treated. The SOM content increased 36.79% at 21 d in S1 in comparison to the initial soil when T3 treated. This change might be due to the interactions of HA with mineral components in soils, leading to greater organic matter accumulation under the T3 treatment. This was because the presence of metal cations in the soil and CSH, such as Ca and Al cations, induced the formation of cationic bridges. These bridges mostly formed between HA and mineral surfaces. Therefore, this process would promoted HA aggregation and higher SOM content. In S2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), the SOM content decreased significantly during 0\u0026ndash;7 d and then it was even lower than that of CK group from 7\u0026ndash;21 d under T1, T2, and T3. The greatest decline in SOM in S2 in comparison to S1, S3, and S4 might be due to limited N content in S2. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the available nitrogen content of S2 was only 84.45 mg/kg. As reported, the expression of genes (Cellulase/Amylase) associated with recalcitrant SOM degradation was enhanced under these conditions (Cui et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, N limitation was more favorable for the growth of bacteria such as Acidobacteria and Chloroflexi, which were involved in the degradation process of recalcitrant or complex SOM (Dai et al., 2018; Kiem and K\u0026ouml;gel-Knabner, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Together, these factors contributed to the decrease of SOM in S2. For S3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), SOM content continuously increased during 0\u0026ndash;21 d and the increase of SOM content was basically similar under these treatments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the SOM content of S4 showed an increasing trend from 0\u0026ndash;21 d under T2 treatment. The fluctuation of SOM content was the largest (1.41%) under T1 and slightly (0.44%) under T3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 AN\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the available nitrogen (AN) content of S2, S3, and S4 exhibited a consistent increasing trend under treatments (T1, T2, and T3) during the observation period. For S1, S2, and S3, the addition of \u003cem\u003eA. faecalis\u003c/em\u003e (T2) resulted in a significantly higher AN content than the other treatment groups at 5 d and 7 d. This might be due to \u003cem\u003eA. faecalis\u003c/em\u003e was a bacterium with nitrogen-fixing capability, which might convert atmospheric nitrogen (N\u003csub\u003e2\u003c/sub\u003e) into AN (Vermeiren et al., 1999). Specially, S2 showed high AN content (231.75 mg/kg) and low SOM content (1.81%) after T2 treatment for 7 d. It was possible that \u003cem\u003eA. faecalis\u003c/em\u003e could decompose more recalcitrant SOM to obtain N. Furthermore, the biological nitrogen fixation capacity of \u003cem\u003eA. faecalis\u003c/em\u003e was limited by the low initial pH of S4 (4.66), resulting in a lower AN (T2) than the other groups at 5 d and 7 d. After the addition of HA (T3) to soil samples, the AN content of S1, S2, S3, and S4 increased slowly in the early stages (5\u0026ndash;7 d) but faster in the later stage (21 d). The main reason might be that HA had the ability to store and slowly release N (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As reported, HA could easily bind with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e to form more stable ammonium humate due to its strong complexation and absorption capabilities (Xu et al., 2021). In S1, S2, and S4, the AN content under T3 treatment showed a significant increase compared with other treatments (T1 and T2). This was because that HA contained N, which could also be a N source when applied into soils. Besides, HA was also used as a carbon source, and its rich C and N content could increase diazotroph community abundance and its soil N-fixing rate (Zheng et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), thus enhancing the AN content. The above results showed that the enhancement of T3 outperformed T1 and T2 in most cases by the 21 d observation across S1 to S4. This demonstrated that T3 treatment seemed to be the most effective treatment in providing a sustainable increase in AN content.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4 AP and ASi\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, there were different degrees of improvement effects on C, N, and P contents in the soil under different treatment groups. Among them, the increase in N content was more significant, whereas the change in P content was relatively stable. P was the key growth-limiting nutrient when N was sufficient (Jin et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the AP value of S1, S2, and S3 showed the similar change trends under treatments (T1, T2, and T3), which increased during 0\u0026ndash;7 d and then decreased. And the T3 treatment on AP enhancement was superior to the other treatment groups. It was possible that humus competed with phosphate for the same binding sites on Fe(Al)-(hydr)oxides to form humic-Fe(Al) complexes, which hindered the formation of precipitated Fe(Al)-phosphates, thus increasing the solubility of P in soils (Gerke, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, T1 and T2 might be more volatile in maintaining AP over time compared to T3, while the improvement effect of T3 was more stable. This might be ascribed to the formation of humic-metal-P complexes through metal bridges after combined application of HA and CSH, allowing for a slow release of AP (Urrutia et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the increase of ASi content was T2\u0026thinsp;\u0026gt;\u0026thinsp;T1\u0026thinsp;\u0026gt;\u0026thinsp;T3 in S1 and S2 after treatment. This was probably because \u003cem\u003eA. faecalis\u003c/em\u003e accelerated the dissolution and release of silicon from the CSH and then increased the ASi content in soils. For S3 and S4, the ASi content exhibited an increase in the order of T1\u0026thinsp;\u0026gt;\u0026thinsp;T2\u0026thinsp;\u0026gt;\u0026thinsp;T3. It could be speculated that use of HA might have a negative effect on the enhancement of ASi content. It had been reported that HA could bind to dissolved Si in acidic soils via bridging Al to form HA-Al-Si complexes (Merdy et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This interaction between HA and Si allowed ASi to be immobilized, resulting in lower ASi levels under the T3 treatment. The ASi content of S1, S3, and S4 decreased obviously at 0\u0026ndash;7 d, which might be associated with the decrease in soil pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As reported, Al(OH)\u003csub\u003e3\u003c/sub\u003e precipitate at lower pH (around 6) was amorphous (Du et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). And silicic acid in soils would form aluminum silicate precipitates with amorphous Al(OH)\u003csub\u003e3\u003c/sub\u003e (Spinthaki et al., 2021), which in turn led to the decrease of ASi content.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5 Correlation Between Soil pH and Nutrient Availability\u003c/h2\u003e\u003cp\u003eThe availability of nutrients is directly affected by soil pH (Barrow and Hartemink, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Clarifying the effect of soil pH on nutrient availability is important for assessing the effectiveness of acidic soil improvement measures. The relationship between soil pH and nutrients was further explored by using Pearson correlation analysis in this study. Correlation analysis of S1 at 5 d revealed a strongly significant positive correlation between pH and ASi (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.905, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). And soil pH exhibited a significant positive correlation with ASi, SOM, AN and AP at 21 d (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This suggested that the alkaline soil environment was more favorable for the release of silicon. This was because silicates bound to metals (such as Ca and Mg) required the increase in pH to solubilize under alkaline soil environments (Vasanthi et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, silicate solubilizing bacteria (SSB) could produce ammonia and amines through metabolic activities, which could shift the pH of the soil towards alkaline and in turn promote the solubilization of silicates (Raturi et al., 2021). As illustrated in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(b), soil pH showed a highly significant negative correlation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) with SOM in S2 compared to S1. And this could be considered due to the differences between soil physicochemical properties (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, SOM was a negative effect on the ASi content. It could co-precipitate with silicate minerals to form stable organo-mineral complexes, thus reducing the content of ASi (Tamrat et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For S3, pH had a significant positive correlation with ASi and AN (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). It was attributed to higher pH improved the availability of nutrients. The positive correlation of SOM on ASi, AN, and AP gradually strengthened from 5\u0026ndash;21 d. This indicated that SOM also strongly affected nutrient contents and availability in soils. A significant positive correlation was shown between soil pH and all nutrient indicators for S4 soil samples. Overall, the combined application of CSH, \u003cem\u003eA. faecalis\u003c/em\u003e, and HA exhibited excellent acidity regulation capacity, which increased soil pH and also enhanced nutrient availability. Among them, CSH was rich in base cations with strong ion exchange capacity, which could effectively increase the soil pH. Combined \u003cem\u003eA. faecalis\u003c/em\u003e application for acid soil, it could effectively release the Si in CSH to increase the content of ASi. This could help reduce crop exposure to drought stress and pests (Wang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Combined application of CSH, \u003cem\u003eA. faecalis\u003c/em\u003e, and HA, the carboxyl and phenolic groups in HA and the Ca\u003csup\u003e2+\u003c/sup\u003e of CSH could form stable clay-humic aggregates via cationic bridges to increase soil soluble phosphate (H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) content (Tiwari et al., 2023). Meantime, HA was rich in organic carbon, allowing to improve the acid buffering capacity of soil. The functional groups such as carboxyl, carbonyl, phenolic hydroxyl, and amino groups in HA could form humic acid ammonium salts with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the soil to reduce soluble N loss (Sun et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Changes of Soil Microbial Community After Amendment\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Microbial Community Diversity\u003c/h2\u003e\u003cp\u003eMicrobial communities played a critical role in nutrient cycling (Li et al., 2021), which strongly determined soil health and quality. A total of 120 soil samples from S1-S4 were subjected to 16S rDNA sequencing, and was clustered into 47,405 OTUs. The number of OTUs shared by the microbial community of four treatment groups on 5 d and 21 d was 381 and 270, respectively (Fig. S2(a)). The OTUs showed a decreasing trend under T1 and T2 treatment and increasing trend under T3 from 5\u0026ndash;21 d. Furthermore, Venn diagrams revealed that a total of 10,557, 10,589, and 11,508 OTUs were detected under treatments (T1, T2, and T3) after 21 d of amendment period, respectively. The number of OTUs significantly increased in all treatments (T1, T2, and T3) compared to the CK group. The number of unique OTUs was 2278, 5817, 5472, and 6544 in control, T1, T2, and T3 treatments (21 d), respectively. These indicated that a large number of unique microorganisms were found in the amended soil under T1, T2, and T3. These results showed that different treatments affected soil microbial community α diversities, including Chao1 richness, Simpson and Shannon indices. Among them (Fig. S2(b)), the α diversity index after T2 and T3 was significantly lower than that of CK. This was probably because the exogenously added \u003cem\u003eA. faecalis\u003c/em\u003e competed with indigenous microbes to improve their survival in the new environment (Yang et al., 2024). This result might lead to the reduction of some microbial species, thereby decreasing the species richness. Moreover, the addition of \u003cem\u003eA. faecalis\u003c/em\u003e in T2 and T3 also led to the dominance of exogenous microorganisms in the soil. This single dominant microbial community usually caused a decrease in soil microbial diversity (Simpson and Shannon indices), altering soil multifunctionality (Romdhane et al., 2022). The phenomenon observed was a decrease in the Chao1, Simpson, and Shannon indices, but an increase in the number of OTUs under T1, T2, and T3 compared to the CK group. It indicated that although the number of microbial species increased, the species evenness and community balance might have been disrupted. This could be related to large changes in soil fertility characteristics (pH, SOM, AN, AP and ASi) after amendment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These changes could exacerbate the instability of microbial community. Meanwhile, competitive imbalances and unequal distribution of resources were also one of the reasons for the reduction of species diversity and microecosystems instability (Zhang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, the number of OTUs increased, but diversities (Chao1 richness, Simpson and Shannon indices) decreased.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Microbial Community Structure\u003c/h2\u003e\u003cp\u003eSoil microbial communities involved in phylum included Actinobacteriota, Proteobacteria, Firmicutes, Planctomycetota, Chloroflexi, Acidobacteriota, Myxococcota, Bacteroidota, Verrucomicrobiota, and Gemmatimonadota (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The dominant phyla were Planctomycetota (20.96%), Proteobacteria (20.03%) and Actinobacteriota (13.90%) in the S1 soil samples. The relative abundance of Actinobacteriota was most remarkable in T3 (43.29%) followed by T1 (30.82%) and T2 (26.05%) at 5 d, while it was 34.53% in control group. We found that the relative abundance of Actinobacteriota tended to increase from 26.05\u0026ndash;43.29% to 37.60-47.06%, while Planctomycetota tended to decrease from 13.79\u0026ndash;21.14% to 8.15\u0026ndash;10.37%. This could be influenced by the soil pH from acidic (around pH 5) to neutral (around pH 6.5). As reported, Actinobacteriota existed optimally in soil environments around pH 7 and its activity was inhibited in acidic soils (Kurtbӧke, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Planctomycetota was recognized as a mildly acidophilic microorganism (Kabor\u0026eacute; et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and its activity was negatively correlated with pH. For S2, the relative abundance of Proteobacteria increased, while that of Acidobacteria decreased in the T2 and T3 compared to the CK and T1. And the increase in the abundance of these beneficial microorganism, Proteobacteria and Actinobacteriota, favoured the restoration of soil fertility. They played an important role in nitrogen fixation and phosphate solubilization (Kim et al., 2021; Shivlata and Satyanarayana, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Planctomycetota (24.38%), Proteobacteria (21.43%), and Actinobacteriota (19.71%) were the dominant bacterial phyla in the S3 soil microbial community (5 d). There were significant changes in abundance between these phyla after 21 d of succession. Specifically, the relative abundance of Acidobacteriota decreased, whereas Actinobacteriota (36.43\u0026ndash;51.20%) and Proteobacteria (22.56\u0026ndash;36.60%) were in absolute dominance. It was reported that Acidobacteria was positively related with soil pH, while Proteobacteria and Actinobacteriota were negatively related with this factor (Wang et al., 2018). The pH of the amended acidic soil increased in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, it could be speculated that the different responses of the major phyla to pH might be responsible for the changes in soil microbial community. The application of amendments increased the abundance of some species favourable to soil nutrient cycling. For example, the phyla Firmicutes (25.88\u0026ndash;37.33%) of S4 was most abundant under the three treatments in comparison to the CK treatment. The increase in the abundance of the phyla Firmicutes had a direct positive effect on soil organic matter mineralization (Xiao et al., 2022).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dominant genera (top 10) between different succession stage (5 d and 21 d) were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb. For S1, the abundance of \u003cem\u003eArthrobacter\u003c/em\u003e was higher in the T3 than in the other groups, accounting for 11.93% (5 d). This genus plays an important role in soil carbon and nitrogen cycling to improve soil functionality (Aguilera-Huertas et al., 2023). The genera \u003cem\u003eNocardioides\u003c/em\u003e and \u003cem\u003eOryzihumus\u003c/em\u003e increased in abundance with 21 d of succession. And these genus are members of the phylum Actinobacteria and have excellent pollutant degradation ability (Ma et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and drought tolerant (Metze et al., 2023). The changes in soil microbial structure reflected somehow the succession of functional microorganisms. The genera \u003cem\u003ePaenochrobactrum\u003c/em\u003e (17.43\u0026ndash;21.44%) and \u003cem\u003eBacillus\u003c/em\u003e (6.16\u0026ndash;8.71%) of S2 had the highest abundance in T2 and T3 compared to the CK and T1 (5 d). And then \u003cem\u003eMassilia\u003c/em\u003e (12.67\u0026ndash;13.24%) and \u003cem\u003eTerrabacter\u003c/em\u003e (5.42\u0026ndash;7.02%) became the dominant bacterial genera in T2 and T3 with the successional stage. Many bacteria in the soil have the ability to solubilize silicon and phosphorus, such as \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eArthrobacter\u003c/em\u003e (Bist et al., 2020; Zheng et al., 2017). \u003cem\u003eBacillus\u003c/em\u003e (13.08\u0026ndash;17.16%) and \u003cem\u003eArthrobacter\u003c/em\u003e (9.13\u0026ndash;16.31%) of S3 had the highest abundance in the T1-T3, while they were 4.63% and 8.96% in CK, respectively. It was further observed that the abundance of plant growth promoting microorganism increased in the T1-T3 treatments. For example, the abundance of \u003cem\u003eMassilia\u003c/em\u003e and \u003cem\u003eTerrabacter\u003c/em\u003e increased by 7.10-17.71% and 1.32\u0026ndash;5.96%, respectively. These changes contributed to enhancing nitrogen cycling and reducing heavy metal toxicity in acidic soil (He et al., 2024). The microbial community in S4 was dominated by \u003cem\u003eBacillus\u003c/em\u003e (15.35\u0026ndash;21.89%) at 5 d, while it was 3.47% in control group. Among the unclassified genera, the highest abundance was \u003cem\u003eIsosphaeraceae_unclassified\u003c/em\u003e, representing 5.76\u0026ndash;7.63% (5 d). This genus contained encoded \u003cem\u003ehao\u003c/em\u003e with N fixation capacity, which could increase soil nutrient availability (Camargo et al., 2023). The relative abundance of these two dominant genera (\u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eIsosphaeraceae_unclassified\u003c/em\u003e) tended to decrease and \u003cem\u003eTerrabacter\u003c/em\u003e tended to increase with succession stage. On the whole, the T2 and T3 treatments promoted the positive changes of soil microbial communities, and the abilities related to soil nitrogen fixation, silicon dissolution, and phosphorus dissolution were improved. Nonetheless, the exogenous \u003cem\u003eA. faecalis\u003c/em\u003e introduced in the T2 and T3 treatments exerted positive effects on the soil microbial community structure. Long-term field experiments are still warranted to monitor their potential impacts on human health.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 The Correlation of Soil Microbial Community and Environmental Factors\u003c/h2\u003e\u003cp\u003eThe redundancy analysis (RDA) of soil samples, environmental factors, and microbial community structure (genus level) is shown in Fig. S3. As seen, when compared the microbial communities of T1, T2 and T3 groups in S1, soil pH was positively related with groups T2 and T3 and negatively related with group T1 (5 d). And the dominant genus \u003cem\u003eOryzihumus\u003c/em\u003e in group T2 was positively correlated with NO\u003csub\u003e3\u003c/sub\u003e-N, ASi, NH\u003csub\u003e4\u003c/sub\u003e-N, SOM and AP (21 d). This showed that the relative abundance of microbial communities shifted with increasing soil pH and nutrient availability. This is consistent with previous studies which indicated that soil pH and nutrient availability would directly influence the structure and diversity of microbial communities (Sarkodie et al., 2024). As illustrated by Fig. S3(b), microbial communities in the group T1 clustered separately from groups T2 and T3. The dominant genera \u003cem\u003ePaenochrobactrum\u003c/em\u003e of S2 in groups T2 and T3 were negatively correlated with SOM and positively correlated with NO\u003csub\u003e3\u003c/sub\u003e-N. And the dominant genus \u003cem\u003eActinobacteriota_unclassified\u003c/em\u003e in T1 was positively associated with SOM and positively associated with NO\u003csub\u003e3\u003c/sub\u003e-N (5 d). These data indicated differences in the soil microbial communities between treatment groups regulated by environmental factors. The RDA of S2 at 21 d explained 77.79% of the total variation in the microbial community structure, which 51.82% (RDA1) and 25.97% (RDA2) was explained by the first two axes. As shown in Fig. S3(c), AP significantly influenced the variation of soil microbial abundance. The dominant \u003cem\u003ePaenochrobactrum\u003c/em\u003e in the T3 was negatively correlated with AP (5 d). But after 21 d, the dominant genus \u003cem\u003ePaenochrobactrum\u003c/em\u003e in T3 was positively related with AP, NO\u003csub\u003e3\u003c/sub\u003e-N, SOM, ASi, and NH\u003csub\u003e4\u003c/sub\u003e-N. This suggested that the increase content of soil nutrients favored microbial growth, providing C, N, P, and Si sources for these microbes. The soil pH, NH\u003csub\u003e4\u003c/sub\u003e-N, ASi, AP, SOM, and NO\u003csub\u003e3\u003c/sub\u003e-N of S4 were positively correlated with the first constrained axis (Fig. S3(d)). Soil pH was significantly correlated with the dominant microbial genus both before and after community succession. It confirmed the role of soil pH for the growth of dominant microorganisms and the importance of regulating the structure of microbial communities (Hong et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). And the composition ratio and dose of the combined amendments were also the main influencing factors (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, the differences in soil physicochemical properties (pH and nutrient contents) between soil samples could also affect the diversity of bacterial communities (Li et al., 2020).\u003c/p\u003e\u003cp\u003eSoil pH and available nutrients have a direct influence on the balance of soil ecosystem and microbial diversity. Based on the Pearson correlation heatmap between soil bacterial abundance and environmental factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), it could be seen that pH, ASi, AP, SOM, NH\u003csub\u003e4\u003c/sub\u003e-N, and NO\u003csub\u003e3\u003c/sub\u003e-N might be the main drivers of soil microbial community succession. As the dominant phylum, Actinobacteriota exhibited a significant positive correlation with pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). And the phylum Acidobacteriota, Planctomycetota, Chloroflexi and Myxococcota showed negative correlations with pH, showing acidophilic characteristics. This indicated that pH could have an important impact on the microbial community structure. pH has been demonstrated to be a key factor influencing the structure and function of microbial communities in acidic soil (Wan et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Acidobacteria, Planctomycetes, and Chloroflex are slow-growing oligotrophic bacteria that typify the K-strategy (Yang et al., 2023). They exhibited strongly negatively correlated with NH\u003csub\u003e4\u003c/sub\u003e-N and ASi. The phylum Actinobacteriota showed significant positive correlations with AP and NO\u003csub\u003e3\u003c/sub\u003e-N. It is reported that Actinobacteriota have multifunctional traits in soil health, such as N fixation (Swarnalakshmi et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), P solubilization (Zeng et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), SOM decomposition (Liu et al., 2023), and alleviation of drought stress (Kumar et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which demonstrated the importance of the phyla in soil. And Firmicutes showed significantly positively correlated with SOM, ASi and NH\u003csub\u003e4\u003c/sub\u003e-N. Furthermore, Firmicutes was a typical eutrophic bacterium that showed a progressive increase in correlation with NH\u003csub\u003e4\u003c/sub\u003e-N and SOM from 5 d to 21 d. The above analysis exhibited that the relative abundance of most copiotrophic taxa increased with increasing pH value and nutrient contents. Conversely, oligotrophic taxa were negatively relationship with pH and nutrients. Therefore, it could be revealed that amendment measures would promote the shifts of soil microbial communities in a positive direction by increasing soil pH and available nutrient contents.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe correlations between the environmental factors and microbial community structure at the genus level are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb. The relative abundance of \u003cem\u003eMassilia\u003c/em\u003e, \u003cem\u003eTerrabacter\u003c/em\u003e, \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003eNocardioides\u003c/em\u003e, \u003cem\u003eOryzihumus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003ePaenochrobactrum\u003c/em\u003e was significantly positively correlated with pH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Whereas \u003cem\u003eIsosphaeraceae_ unclassified\u003c/em\u003e and \u003cem\u003eAquisphaera\u003c/em\u003e were negatively correlated with pH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This could be explained by their different abilities to adapt to changes in soil pH. Meantime, the genus \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e were reported to have alkalophilic traits, with an optimum at pH 7.0\u0026ndash;8.0 (Fritze, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). And the increase of soil pH after the application of amendments (T3) was more favorable to their survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). These results revealed that the application of the soil amendments might increase the abundance of genera preferring both neutral and alkaline soil environments (5.5\u0026thinsp;\u0026lt;\u0026thinsp;pH\u0026thinsp;\u0026lt;\u0026thinsp;7.5). \u003cem\u003eBacillus\u003c/em\u003e showed a significant positive association with ASi content at 5 d (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.781, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As reported, this genus could solubilize silicates, which belonged to SSB (Vasanthi et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This could be speculated that \u003cem\u003eBacillus\u003c/em\u003e might be involved in the Si cycling. Besides, \u003cem\u003eBacillus\u003c/em\u003e as a well-known genus of beneficial soil bacteria, and some species of them could effectively inhibit the growth of pathogenic bacteria such as \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (Raza et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, multifunctionality of the \u003cem\u003eBacillus\u003c/em\u003e revealed its importance in soil amendment. \u003cem\u003eArthrobacter\u003c/em\u003e is a typical phosphate solubilizing bacterium (PSB) (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), showing a significant positive correlation with NO\u003csub\u003e3\u003c/sub\u003e-N and AP. The soil pH was reported to be majorly responsible for the abundance of PSB community and its capacity to solubilize P (Zheng et al., 2019). This demonstrated that soil pH played a dominant role in the participation of \u003cem\u003eArthrobacter\u003c/em\u003e in soil P cycling.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study revealed that the application of \u003cem\u003eA. faecalis\u003c/em\u003e coated by CSH and HA was more efficient in increasing the pH of acidic soil. It not only significantly increased the soil pH, but also enhanced the contents of AN, AP, and ASi. The correlation analysis found that pH was positively correlated with all nutrient indicators (SOM, AN, AP, and ASi) of most soil samples. This indicated that improving soil acidity is crucial for enhancing soil fertility. Regarding the microbial community, the application of \u003cem\u003eA. faecalis\u003c/em\u003e coated by CSH and HA triggered distinct responses. The relative abundances of beneficial microorganisms like \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eArthrobacter\u003c/em\u003e, and \u003cem\u003ePaenochrobactrum\u003c/em\u003e notably increased. These microorganisms were not only associated with the elevation of soil pH, underscoring the role of pH in shaping the microbial community composition, but also positively correlated with the increase in nutrient contents. This suggested their active participation in soil nutrient cycling processes, such as N, P, and Si cycling.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the School of Minerals Processing and Bioengineering, Central South University and Key Laboratory of Biometallurgy of Ministry of Education for providing the infrastructure and facilities to carry out this research. The authors have immense gratitude to the editors for their comments which helped a lot to improve the quality of the paper and standardize the format.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Author information\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchool of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuhua Jiang, Manjun Miao, Ziwen Guo, Jiejie Yang, Yulong Peng, Junzhao Wu, Bo Miao, Xueduan Liu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKey Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha 410083, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuhua Jiang, Manjun Miao, Ziwen Guo, Jiejie Yang, Yulong Peng, Junzhao Wu, Bo Miao, Xueduan Liu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYuelushan Laboratory, Changsha 410128, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuhua Jiang, Xueduan Liu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xueduan Liu.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not involve animal or human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have consent to publish this paper with this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available upon request by contact with the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Yuelushan Laboratory Breeding Program (No. YLS-2025-ZY02040), the National Natural Science Foundation of China (Grant No. 52174341), the Hunan Provincial Key Research and Development Plan (Grant No. 2022WK2017 and 2023NK2030), and the Natural Science Foundation of Hunan Province of China (Grant No. 2022JJ40583).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuhua Jiang: Conceptualization, Writing - review and editing, Supervision, Project administration, Funding acquisition; Manjun Miao: Writing - original draft preparation, Methodology, Validation, Formal analysis, Investigation; Ziwen Guo and Jiejie Yang: Formal analysis; Yulong Peng: Investigation; Junzhao Wu: Formal analysis; Bo Miao: Resources; Xueduan Liu: Conceptualization, Resources, Supervision, Project administration, Funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdeleke, R., Nwangburuka, C. and Oboirien, B.: 2017, \u0026apos;Origins, roles and fate of organic acids in soils: A review\u0026apos;, S AFR J BOT 108, 393-406.\u003c/li\u003e\n\u003cli\u003eAguilera-Huertas, J., Cuartero, J., Ros, M., Pascual, J.A., Parras-Alc\u0026aacute;ntara, L., Gonz\u0026aacute;lez-Rosado, M., \u0026Ouml;zbolat, O., Zornoza, R., Egea-Cortines, M., Hurtado-Navarro, M. and Lozano-Garc\u0026iacute;a, B.: 2023, \u0026apos;How binomial (traditional rainfed olive grove-Crocus sativus) crops impact the soil bacterial community and enhance microbial capacities\u0026apos;, J ENVIRON MANAGE 345, 118572.\u003c/li\u003e\n\u003cli\u003eBarrow, N.J. and Hartemink, A.E.: 2023, \u0026apos;The effects of pH on nutrient availability depend on both soils and plants\u0026apos;, PLANT SOIL 487, 21-37.\u003c/li\u003e\n\u003cli\u003eBashir, O., Ali, T., Baba, Z.A., Rather, G.H., Bangroo, S.A., Mukhtar, S.D., Naik, N., Mohiuddin, R., Bharati, V. and Bhat, R.A.: 2021, \u0026apos;Soil Organic Matter and Its Impact on Soil Properties and Nutrient Status\u0026apos;, in G.H. 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122760.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Farmland soil, Fertility improvement, Reduce acidity, Alcaligenes faecalis, Calcium silicate hydrate, Microbial community","lastPublishedDoi":"10.21203/rs.3.rs-7738393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7738393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoil acidification poses a critical threat to agricultural safe production by compromising soil fertility. Conventional remediation strategies for acidic soils face persistent challenges including short-lived efficacy, structural deterioration, and secondary pollution risks. This study presents an innovative bioremediation approach utilizing \u003cem\u003eAlcaligenes faecalis\u003c/em\u003e (\u003cem\u003eA. faecalis\u003c/em\u003e), a potent alkaline-metabolizing microorganism, coated by calcium silicate hydrate (CSH) and humic acid (HA) as a biocarrier for acidic farmland soil restoration. Through microcosm experiments and high-throughput sequencing analysis, we demonstrated that the remediation approach achieved remarkable soil pH elevation from an initial 4.66 to 6.41 within 21 d. Soil available nitrogen (AN) and silicon (ASi) contents increased to 252.83\u0026ndash;595.70 mg/kg and 89.24-133.57 mg/kg at 21 d, respectively; and available phosphorus (AP) content increased from 15.59\u0026ndash;39.88 mg/kg to 20.44\u0026ndash;55.98 mg/kg at 7 d. There was a positive correlation between pH and all nutrient indicators for soil samples. Moreover, the restoration could also change the microbial diversity and structure. This led to an increase in the relative abundance of functional genera including \u003cem\u003eBacillus\u003c/em\u003e (from 0.44\u0026ndash;0.65% to 4.92\u0026ndash;21.89%), \u003cem\u003eArthrobacter\u003c/em\u003e (from 0.13\u0026ndash;0.30% to 0.17\u0026ndash;16.31%), and \u003cem\u003ePaenochrobactrum\u003c/em\u003e (from 0.02\u0026ndash;0.04% to 0.75\u0026ndash;17.43%) at 5 d. These microorganisms were related to soil nitrogen, phosphorus, and silicon cycling, which were positively correlated with an increase in AN, ASi and AP content in soils. Results highlight the application potential of \u003cem\u003eAlcaligenes faecalis\u003c/em\u003e as a biomaterial for soil improvement in acidic farmland, providing a new option for the improvement of acidic soil.\u003c/p\u003e","manuscriptTitle":"Improvement effects of calcium silicate hydrate and humic acid-encapsulated Alcaligenes faecalis on acidic farmland soil and alterations in microbial community structure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 10:20:50","doi":"10.21203/rs.3.rs-7738393/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":"cb4f8251-b5de-4284-9bfb-83316ed4e786","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T17:39:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 10:20:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7738393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7738393","identity":"rs-7738393","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}