Alkalinity control in sludge propels the conversion of concrete slurry waste into micro- and nano-sized biogenic CaCO₃

During the concrete production process, a large amount of concrete slurry waste (CSW) is generated, attributed to the washing of concrete trucks, dust reduction, and sphere cleaning operations. Characterized by a pH level exceeding 11.5, CSW is rich in calcium silicate (Xuan et al. 2016). CSW is classified as a corrosive hazardous substance. If disposed of in landfills, it would cause detrimental effects on our surrounding environment and ecosystems due to its high pH value. In addition, sedimentation and post-treatment processes lead to the accumulation of waste slurry particles, contributing to dust pollution in the concrete production environment and overlooking the potential recycling and utilization of significant mineral resources. However, the Chinese national standard (GB/T 14902-2012) mandates the recycling of CSW. Previous research has demonstrated that Bacillus sp. can utilize calcium sources in concrete to synthesize CaCO₃ bio-nanoparticles, which are beneficial for repairing concrete cracks and filling pores (Qin et al. 2021b). Consequently, microbial-induced calcium carbonate precipitation (MICP) offers a viable alternative for the recycling of calcium resources in CSW.

CSW is recognized as a potential source of calcium carbonate. Upon introducing CO₂ into the aqueous phase, up to 30% of calcium can be extracted from CSW, yielding CaCO₃ with a purity exceeding 97% (Iizuka et al. 2012). However, CO₂′s low water solubility constrains CaCO₃ production (Hui et al. 2022). The adjustment of pH enhances CO₂ capture, promotes production, and facilitates the generation of abundant CaCO₃ nanoparticles. More importantly, bio-nano-CaCO₃, synthesized by urease-producing bacteria and enriched from waste-activated sludge via the MICP method, has various applications including repairing concrete voids, cementing soil, improving soil strength, and suppressing coal dust in coal mines (Yang et al. 2020; Liu et al. 2022). Utilizing urease-producing bacteria to produce micro- and nano-CaCO₃ from carbonized sludge and urine for the production of sustainable bio-cement has been explored (Yang et al. 2022). The use of Bacillus pasteurii to produce bio-CaCO₃ has been demonstrated to enhance the strength of expansive soils (Tian et al. 2022). Sporosarcina ureilytica ML-2 produces bio-CaCO₃ for treating waste sludge and immobilizing heavy metals (Zeng et al. 2023). Nonetheless, it is important to consider that using a single strain and adding calcium sources and urea to generate bio-CaCO₃ can significantly increase the costs involved. Breaking down urea can lead to the excessive production and accumulation of ammonia nitrogen, which, in turn, can acidify the water and promote eutrophication (Kumar et al. 2023). There remains a lack of direct research on producing micro- and nano-sized biogenic CaCO₃ (bio-CaCO₃) using microbial communities from activated sludge and CSW. The biological metabolic mechanisms underlying MICP for nanoparticle production in CSW are not well understood, particularly the impact of pH on biochemical reaction pathways and alkalinity regulation.

For this, a comprehensive investigation was conducted on the system that harnesses CSW as a valuable resource through the bio-metabolism process of activated sludge. Additionally, the impact of pH on the efficiency of carbon capture and the production of CaCO₃ was thoroughly examined. At varying pH levels, analyses were conducted based on the bacterial community structure, the relative cell count, and the proportions of polysaccharides and proteins produced. Scanning electron microscopy (SEM) was used to determine the particle size, while X-ray diffraction (XRD) was used to identify the composition and crystalline form of the products. Inductively coupled plasma mass spectrometry (ICP-MS) was employed to verify the heavy metal residues in the supernatant following sludge settling and CaCO₃ precipitates. These findings offer novel insights into the recycling of CSW calcium resources and the generation of bio-CaCO₃, potentially benefiting the early strength of concrete.

CSW was obtained from Shaanxi Future Venture Construction Technology Co., Ltd, Xi'an and stored at 4°C before use. Sludge, possessing an 80% moisture content, was procured from the No. 4 Wastewater Treatment Plant (WWTP). Chemical reagents for the experiments were procured from Sigma-Aldrich (MO, USA). Ca²⁺, pH, suspended solids (SS), and chemical oxygen demand (COD) were determined according to the following national standards (GB7476-1987), (GB11901-89) and (HJ828-2017). Results are presented in Table 1.

To better adapt microbes to the carbon source in actual wastewater, the hydrothermal pretreatment of sludge organisms was employed as a simulated carbon source. This process breaks down lipids into long-chain fatty acids and volatile fatty acids, and proteins into ammonia and volatile fatty acids (Qin et al. 2021c). There is minimal degradation of long-chain fatty acids in sludge subjected to 90 °C hydrolysis (Charuwat et al. 2018). Consequently, Table 1 shows the biochemical oxygen demand (BOD₅) and COD during pyrolysis for 1 h at a pH of 11 and a temperature of 90 °C. The BOD₅/COD ratio exceeding 0.6 indicates its bioavailability.

About 4 L of sludge microbes were introduced into an 8 L reactor. Intermittent aeration was performed at a rate of 7 L min⁻¹ to maintain dissolved oxygen levels between 2 and 5 mg L⁻¹. Mechanical stirring ensures the uniform distribution of sludge in the reactor. Furthermore, an automatic NaOH syringe pump was utilized to maintain an alkaline pH. The reactor is placed in a water bath system to ensure that a consistent temperature of 25 °C is maintained. Sludge pyrolysate was added to maintain an input COD of 300 mg L⁻¹. The reaction cycle spanned 24 h, comprising 23 h of aerobic reaction. After that, there was 30 min of settling and the supernatant was then combined with CSW to produce nanoparticles, followed by 30 min dedicated to CaCO₃ nanoparticle generation. The nanoparticle precipitates were enriched through centrifugation, with the supernatant subsequently discarded. The enriched precipitates were then dried and weighed. After the aerobic reaction, the supernatant following sludge settling was collected, and here and content were analyzed adhering to standards (DZ/T 0064.49—2021). Heavy metals, such as Cr, Cu, Zn, Cd and Pb, were measured in the supernatant following sludge settling and CaCO₃ precipitates using ICP-MS (Thermo Fisher iCPA Q USA). Before measurement, the samples undergo acid dissolution treatment. In addition, to fully utilize the CSW and reduce operating costs, a reflux experiment was set up, which differs from the former in that the CSW reflux reactor, after generating calcium carbonate, provides alkalinity.

The generated nanoparticles were analyzed using SEM (SU-8082, Japan) to assess their structure and particle size. XRD analysis (Ultima IV, Japan) was employed to determine the composition and crystalline structure of the nanoparticles. Next-generation sequencing based on 16S rRNA was utilized to analyze the microbial community structure. Sludge microorganisms with pH ranges of 7.5–8.0, 8.5–9.0, and 9.5–10.0 were entrusted to Beijing Kinko Biotechnology Co for testing.

The sludge was immersed in 10 g L⁻¹ of fluorescein isothiocyanate isomer I (FITC) to stain proteins, then in 0.1 g L⁻¹ of concanavalin A-alexa fluor (Con A) to label polysaccharides, and in 1 mg L⁻¹ of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) to stain nucleic acids. Staining of the sludge was conducted by methodologies previously described (Chen et al. 2007; Yang et al. 2023). Subsequently, the samples were paraffin-embedded, sectioned using a microtome (Leica RM2126RT, Germany), and examined and imaged with a fluorescence microscope (Olympus BX61 + DP72, Japan). The excitation wavelengths for Con A, FITC, and DAPI were 555, 488, and 358 nm, respectively.

To confirm the reproducibility of the experimental results, all experiments were performed in triplicate. The graphs were drawn using Origin (OriginLab, USA, 2018). Values are expressed as mean values ± standard deviations. Image J (NIH, USA) was utilized to calculate the fluorescence area.

As illustrated in Table 2, following the carbon source calculation resulting from the transformation of sewage organics into CO₂, a mass balance pertaining to carbon transformation and capture has been established in reactors with a pH of 9.5–10. The conversion ratio of COD to CO₂ has been derived from the existing literature (Bao et al. 2015). During this process, the CO₂ concentration supplemented by air in aerobic aeration amounts to 1,467.79 mg L⁻¹, markedly surpassing the carbon capture quantity of 148.63 mg L⁻¹ in the control water and 726.31 mg L⁻¹ in the bio-CaCO₃ generation reactor. This demonstrates that a substantial portion of the CO₂ entering the reactor during the aeration is released into the air. Additionally, the carbon capture quantity in the reactor exceeds the combined carbon of the control water and the incoming source. This implies that the alkali addition not only enhances the conversion of nearly all soluble COD into CO₂ via bio-metabolism but also aids in dissolving some insoluble COD in the sludge pyrolysate, thereby enabling more efficient utilization by microbial aerobic respiration. In addition, an alkaline environment favors carbon capture. The alkali addition facilitates microbial carbon transformation, leading to CO₂ production, which is advantageous for storing the generated CO₂ in water.

Owing to the high alkalinity of CSW, which exhibits a pH of 12–13.5 (Xuan et al. 2016), its return to the reactor following CaCO₃ production can offer an alkaline environment, facilitating waste resource utilization and diminishing the operational costs of the reactor. As depicted in Figure 2(a) and 2(c), with pH levels of 8.5–9, the carbon capture effectiveness stabilized between 700 and 800 mg L⁻¹ following 3 days of rapid increase. In the no-reflux approach, carbon capture effectiveness nearly peaked at 702.5 mg L⁻¹ on the 2nd day, in contrast to a near peak of 742.6 mg L⁻¹, while reflux reached only 591.2 mg L⁻¹, suggesting a more expedited startup period. During days 3–8, reflux progressively increased from 704.3 mg L⁻¹ to a maximum of 789.7 mg L⁻¹. This increase is attributed to the additional minor carbon source from the reflux CSW (refer to Table 1), while the subsequent decline in carbon capture effectiveness is likely due to the inhibition of cellular respiration from accumulated heavy metals and salts (Hong et al. 2013; Ma et al. 2015; He et al. 2017). In the no-reflux reactor depicted in Figure 2(c), carbon capture effectiveness reached its apex at 742.58 mg L⁻¹ on the 3rd day. Despite a minor decline from days 4 to 10, it remained relatively stable at 650–750 mg L⁻¹, marginally lower than the reflux reactor. Figure 2(b) and 2(d) illustrates that under reflux and no-reflux operation way, the peak production of bio-CaCO₃ was 1,376.4 and 1,204 mg L⁻¹, respectively, aligning closely with theoretical projections. Reflux's CSW does not impact the efficiency of carbon capture or the production of bio-CaCO₃. In addition, Figure 2(a) and 2(c) exhibits distinct peaks in levels and corresponding valleys in levels on day 5. This observed trend can be attributed to the bulking of sludge that occurred around day 5, coinciding with a sludge settling velocity of approximately 90% (refer to Figure S5). Consequently, certain microorganisms present in the bulking sludge experienced mortality in the highly alkaline environment, leading to the leaching of intracellular lipids. These lipids were subsequently converted into fatty acids (Wilson & Novak 2009), thereby contributing to the reduction in pH levels. Moreover, this conversion process also served as an additional carbon source for the system. In summary, for effective pH adjustment of the reactor and reduced operational costs, the adoption of the CSW reflux way is recommended, as it is more conducive to the production of bio-CaCO₃.

Activated sludge is known to contain heavy metals such as Pb, Cr, Cd, Ni, Zn, and Cu (Dewil et al. 2007), while CSW may accumulate Pb, Cr, and Cd (Xuan et al. 2016). Consequently, assessing the risk of heavy metal leaching during the reactor operation is crucial. Given that activated sludge can immobilize heavy metals, mainly through adsorption from surface functional groups, most residual heavy metals in CSW are likely to be adsorbed by the sludge and eliminated through sedimentation and discharge (Commenges-Bernole & Marguerie 2008; Ramrakhiani et al. 2016; Zhou et al. 2016). ICP-MS analysis (Table 3) reveals negligible amounts of Cr, Cd, and Pb in the supernatant following sludge settling in the no-reflux way, with Zn below 20 μg L⁻¹ and Cu at 36.20 μg L⁻¹. These low concentrations of metal ions should be absorbed by the sludge microbes and removed from the reactor by increasing the hydraulic retention time (HRT) and reducing the sludge retention time (SRT). In the reflux way, concentrations of Cr, Cd, and Pb following sludge settling are negligible in the supernatant. Post-day 9, Zn levels in the supernatant following CaCO₃ precipitation drop to just 0.05 μg L⁻¹. A contrast to 5.59 ± 0.28 μg L⁻¹, seen following sludge settling, suggests minimal Zn incorporation into bio-CaCO₃. The supernatant after sludge settling still contains a significant concentration of Cu, with levels reaching as high as 53.51 μg L⁻¹. It is worth noting that this concentration falls significantly below the groundwater quality standard (GB/T 14848-2017) stipulated by the Chinese national standard. However, the concentration of Cu in the supernatant significantly decreases to only 19.10 μg L⁻¹ after CaCO₃ precipitation. These findings indicate a limited uptake of Cu into the bio-CaCO₃. This could be attributed to the mixing of CSW and sludge supernatant in approximately a 1:1 ratio during the production process of CaCO₃, resulting in the Cu concentration in the supernatant after CaCO₃ precipitation being approximately half that present in the supernatant after sludge settling. Based on this, it can be inferred that the mobility of Cu bound within the activated sludge solution is restricted.

Furthermore, the supernatant following CaCO₃ precipitation in the reflux way contained slightly more heavy metals than that in the no-reflux way. This increase is potentially attributed to the salt accumulation within microbial cells due to the reflux of the supernatant in the reactor, which inhibits biological activity and diminishes the subsequent adsorptive ability of heavy metals (Hong et al. 2013; He et al. 2017). Nevertheless, these heavy metal concentrations remain well below the thresholds established by the comprehensive sewage discharge standard (GB8978-1996). In summary, the reflux way results in concentrations of Cr, Cd, and Pb in the supernatant that are below 3 μg L⁻¹, while Cu and Zn are somewhat higher, reaching maximum values of 53.51 and 17.4 μg L⁻¹, respectively. By increasing the HRT and reducing the SRT, these heavy metals can be primarily concentrated within the sludge microbes and effectively removed from the system.

Utilizing CSW as a calcium source and sludge β-oxidation products as a carbon source enabled the successful production of bio-CaCO₃ under alkaline conditions. The sludge pyrolysate, as a simulated COD, has a BOD₅/COD ratio that exceeds 0.6 with good biodegradability.

The ideal pH for CaCO₃ production was determined to be 8.5–9, achieving a maximum carbon capture of 742.6 mg L⁻¹ and a CaCO₃ yield of up to 1,190.7 mg L⁻¹. Mass balance analysis indicated that the addition of alkali enhances microbial β-oxidation, which degrades COD to produce CO₂ and aids in CO₂ storage. In addition, the reflux of supernatant in the reactor, providing an alkalinity environment and reducing costs, is recommended. As the pH value increases, the number of microorganisms increases. Concurrently, a decrease in the relative protein content per microorganism was observed, suggesting that protein plays an important role in β-oxidation. Here, polysaccharides initially increase the relative content, followed by a decrease, with the elevation at pH 8.5–9, which serves as an adaptive response to adverse conditions. Simultaneously, Proteobacteria and Bacteroidota emerge as dominant phyla, with Thauera being a prevalent genus adept in β-oxidation. SEM disclosed that the majority of CaCO₃ precipitate particles exhibit a sphere shape, with a size of 0.7–10 μm in the range. XRD analysis demonstrated that the primary crystalline form of the precipitate is calcite. ICP-MS analysis indicated minimal heavy metal in the supernatant following sludge setting, and these trace heavy metals could be further removed by sludge discharge.

This methodology presents a novel strategy for waste sludge minimization, CSW reclamation, and the generation of bio-CaCO₃. The micro-/nano-bio-CaCO₃ production has a promising future, which can be used for concrete crack repair and strength enhancement. The discovery of alkaline regulation has expanded the application of MICP in CSW. Nevertheless, as there may be residual organic matter in the production process of bio-CaCO₃, further investigation is warranted to confirm the durability and mechanical properties of concrete integrated with this form of CaCO₃. The new operation should involve feeding a small amount of CSW into the reactor to maintain pH and continuously produce bio-CaCO₃ for slurry recharge in concrete. Furthermore, it is important to investigate whether the existing CaCO₃ in the CSW may agglomerate during prolonged placement, which will influence the particle size of MICP bio-CaCO₃.

This work was financially supported by the Natural Science Foundation of China (Grant No. 51808044) and the Qinchuangyuan ‘Scientist + Engineer’ Team Construction Project of Shaanxi Province (2022KXJ-119). Supplementary data associated with this article can be found in the online version.

Jinbo Zhao: conceptualization, writing – original draft, writing – review and editing, methodology, software. Jiacheng Feng: data curation, writing – review and editing, methodology, visualization. Yifan Du: formal analysis, investigation, writing – review and editing. Zhiyang Yan: methodology, supervision, resources, writing – review and editing. Xiaoguang Li: conceptualization, project administration, writing – review and editing. Jinyi Qin: conceptualization, validation, funding acquisition, project administration, resources. Ming Su: supervision, validation. Min Yang: supervision, writing – review and editing.

Data cannot be made publicly available; readers should contact the corresponding author for details.

The authors declare there is no conflict.