Apatites

RNA was extracted and purified from biofilms at distinct stages of microcolonies development (21, 24, 30, 31, and 34 h) using standard protocols [22] (link). The developmental stages of S. mutans biofilms were characterized previously [21] (link), [23] (link), which varies from initial microcolonies assembly across the apatite surface (at 20 h) to vertical growth and merging process followed by further increase in size and thickness (from 20 to 30 h and beyond). All purified RNAs presented RNA integrity number (RIN) ≥8.5 (Agilent 2100 electrophoresis bioanalyzer, Agilent Technologies, Santa Clara, CA, USA). The reverse transcriptase PCR, and quantitative amplification conditions were similar to those described previously [20] (link). The primers were designed using Beacon Designer 2.0 software (Premier Biosoft International, Palo Alto, CA) (see Table 1).
Briefly, cDNAs were synthesized using the BioRad iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., CA). To check for DNA contamination, purified total RNA without reverse transcriptase served as a negative control. The resulting cDNA and negative controls were amplified by a MyiQ qPCR detection system with iQ SYBR Green supermix (Bio-Rad Laboratories, Inc., CA, USA) and specific primers. A standard curve was plotted for each primer set, as described elsewhere [20] (link). The standard curves were used to transform the quantification cycle (Cq) values to the relative number of cDNA molecules. Relative expression was calculated by normalizing each gene of interest to the reference gene 16S rRNA [20] (link).

Dendrocalamus farinosus potted plants were grown at the experimental base (location: 31°32 ‘44 “N, 104°41′ 402″ E, altitude 480 m) of the College of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan Province. Topsoil (0–20 cm depth) was obtained from the test site and screened to remove rock and plant litter. The soil contained 37% silt, 36% clay, and 27% sand. The soil physical and chemical properties were as follows: pH 6.49 ± 0.01, electrical conductivity 112.93 ± 1.06 μS·cm⁻¹, total carbon content: 1220 ± 102 mg kg⁻¹, total organic carbon content: 511 ± 12 mg kg⁻¹, total nitrogen content: 353 ± 11 mg kg⁻¹, total phosphorus content: 180 ± 9 mg kg⁻¹, available nitrogen content: 142 ± 11 mg kg⁻¹, available phosphorus content: 22.4 ± 7.3 mg kg⁻¹.
Three fertilizers were used in this study. The organic fertilizer (organic matter ≥45%, N + P₂O₅ + K₂O ≥ 5%, N [1.4%], P [4.5%], K [1.7%]) was purchased from Henan Lotus Environmental Technology Fertilizer Corporation (Henan, China). The B. mucilaginosus microbial fertilizer (microbial agent, 10 billion colony forming units [CFU]/g) was purchased from Huanwei Biology; B. mucilaginosus decomposes K, Si, and P from soil minerals such as feldspar, mica, and apatite, thus increasing K and P supply to the soil and ultimately crop yields (Liu et al., 2006 (link)). The B. amyloliquefaciens microbial fertilizer (microbial agent, 100 billion CFU/g) was purchased from Nongbao Biology; B. amyloliquefaciens is a typical plant-growth-promoting rhizobacterium (PGPR), which promotes plant growth and improves nitrogen use efficiency (Xue et al., 2021 (link)).

The raw materials of cast-in-situ phosphogypsum are mainly phosphogypsum, phosphorus slag powder, hydrated lime, cement, a retarder and a water reducer. Through preliminary investigations, it was found that the gypsum phase of phosphogypsum produced by different phosphoric acid plants is slightly different. Therefore, the gypsum phase of phosphogypsum raw materials should be tested before application. Figure 1 shows the construction photos of cast-in-situ phosphogypsum walls and treated phosphogypsum. The phosphogypsum and phosphorous slag micropowder were taken from Guizhou Wengfu Phosphorus Industry Group. The gypsum phase composition of phosphogypsum was determined and is shown in Table 1. The specific surface area of the phosphorus slag powder was 380~420 m²/kg and its chemical composition is shown in Table 2. The hydrated lime is commercially available, and the mass fraction of effective CaO was no less than 60%. The cement is P.O325 ordinary Portland cement and the water-reducing agent was polycarboxylic acid, with a concentration of no less than 10%. The retarder was sodium citrate. A JMS-6490LV scanning electron microscope was used to analyze the morphology of the phosphogypsum and phosphorus slag powder. The results are shown in Figure 2, where the scale bar in Figure 2a is $100 \mathsf{\mu }\mathrm{m}$ , and the scale bar in Figure 2b is $10 \mathsf{\mu }\mathrm{m}$ . The crystals of hemihydrate gypsum in phosphogypsum are generally parallelogram plates, and the gap between crystals is large. The phosphorus slag micro powder particles are in the shape of “gravel”, with clear edges and corners but no fixed cleavage surface. The results of the X-ray diffraction (SIEMENS D5000 XRD diffractometer, Munich, Germany ) analysis are shown in Figure 3. The phosphorus slag powder is mainly composed of glass, containing a small amount of pseudowollastonite, gunite and apatite. It is a volcanic material with potential activity and can be filled in the gap between hemihydrate gypsum crystals after casting. The acidity of phosphogypsum is neutralized by hydrated lime, the anhydrite is consumed by cement, the water reducer is used to reduce the water consumption, and the retarder is used to delay the setting time after being cast-in-situ.
Taking the total mass mix proportion of phosphogypsum, phosphorous slag micropowder and hydrated lime as 100%, the cement consumption was calculated according to the mass of the mixture of phosphogypsum, phosphorous slag micropowder and hydrated lime. The mixed dry materials were obtained after uniform mixing. The consumption of water reducer and retarder was calculated according to the mass of the mixed dry materials. Considering the possible construction deviation, the material mix proportion shown in Table 3 is proposed, and the water cement ratio is 0.43.

Every experiment was conducted with the first step of cultivating the relevant bacterial strains overnight. The reproducible results were ensured using the same optical density (OD) of 1. Of note, even with the same OD, the absolute cell number somewhat differs within the strains due to the cell size and OD is not strictly linearly related to the cell count except within a limited range. Thus, the more reliable way to obtain reproducible conditions is by using cfu counting. Yet, precedent experiments of the correlation of OD and cfu showed that, despite the differences in size and shape of the bacterial strains, the number of cfu are within the same range. This bacterial solution was applied on the apatite pellets placed in an MTP. An incubation period of 24 h at 37 °C followed. The experiments were performed in triplicate (n = 3) and the results are provided in terms of mean ± standard deviation.