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Sludge Amendment Accelerating Reclamation Process of Reconstructed Mining Substrates Dan Li1, Ningning Yin1,2, Ruiwei Xu3, Liping Wang1,4*, Zhen Zhang5 & Kang Li1

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Sludge Amendment Accelerating Reclamation Process of Reconstructed Mining Substrates Dan Li1, Ningning Yin1,2, Ruiwei Xu3, Liping Wang1,4*, Zhen Zhang5 & Kang Li1 www.nature.com/scientificreports OPEN Sludge amendment accelerating reclamation process of reconstructed mining substrates Dan Li1, Ningning Yin1,2, Ruiwei Xu3, Liping Wang1,4*, Zhen Zhang5 & Kang Li1 We constructed a mining soil restoration system combining plant, complex substrate and microbe. Sludge was added to reconstructed mine substrates (RMS) to accelerate the reclamation process. The efect of sludge on plant growth, microbial activity, soil aggregate stability, and aggregation- associated soil characteristics was monitored during 10 years of reclamation. Results show that the height and total biomass of ryegrass increases with reclamation time. Sludge amendment increases the aggregate binding agent content and soil aggregate stability. Soil organic carbon (SOC) and light-fraction SOC (LFOC) in the RMS increase by 151% and 247% compared with those of the control, respectively. A similar trend was observed for the glomalin-related soil protein (GRSP). Stable soil aggregate indexes increase until the seventh year. In short, the variables of RMS determined after 3–7 years insignifcantly difer from those of the untreated sample in the tenth-year. Furthermore, signifcant positive correlations between the GRSP and SOC and GRSP and soil structure-related variables were observed in RMS. Biological stimulation of the SOC and GRSP accelerates the recovery of the soil structure and ecosystem function. Consequently, the plant–complex substrate–microbe ecological restoration system can be used as an efective tool in early mining soil reclamation. Te ecological restoration of coal mining areas is strongly limited by soil nutrient defciencies, unstable soil structures, and slow growth rates of the vegetation. Soil reconstruction is the core component of land reclamation, and thus the quality of reconstructed soil directly determines the quality of land reclamation 1. Coal gangue and fy ash are produced during coal mining, processing, and use. In recent years, an increasing amount of research has begun to explore the use of industrial solid waste in mining areas to build reconstructed mine substrates (RMS), such as coal gangue 2,3. On this basis, sewage sludge is one of the amendments used to enrich RMS and speed up the reclamation process. Positive efects of sludge amendment have been reported in many studies. For example, sludge amendment enriches the soil nutrients and promotes plant growth 4–6. Sludge amendment is an important factor stimulating the microbial activity and development of microbial communities in the soil. Soil microbial communities, such as bacteria and fungi, play vital roles in nutrient cycling, reduction in plant stresses, and formation of soil aggregates 7,8. Arbuscular mycorrhizal fungi (AMF) are abundant in various types of soils and are particularly critical for the improvement of the plant growth and adaptation to stressed environments9. AMF transfer mineral nutrients to the host plants. In return, plants provide lipids and sugars to their fungal partners10. Glomalin is a typically heat resilient soil glycoprotein, which is produced by AMF dur- ing symbiotic association with the roots of about 70% of all plant families 11,12. Te quantifcation of glomalin is ofen tagged as glomalin-related soil protein (GRSP)13,14. To follow AMF development over longer time scales, the determination of GRSP as a stable, specifc biomarker has been suggested 15. It has been hypothesised that the glomalin acts as “glue” during the formation of the aggregate and substantially contributes to the soil organic carbon (SOC)14. As an essential indicator of the soil quality, the SOC and its diferent labile fractions play important roles in determining the chemical, physical, and biological properties of RMS. Most previous studies focused on total carbon sequestration and the results showed that the SOC increases with the reclamation time 16,17. Te SOC consists of various components with diferent physicochemical properties, degrees of stabilisation, and turnover rates. Te light-fraction SOC (LFOC) content is primarily derived from plant residues, roots, and fungal hyphae 1School of Environment and Spatial Informatics, China University of Mining and Technology, Xuzhou 221116, China. 2School of Chemistry and Environmental Engineering, Jiujiang University, Jiujiang 332005, China. 3College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China. 4Engineering Research Center of Mine Ecological Construction, Ministry of Education, Xuzhou 221116, China. 5College of Natural Resources and Environment, Joint Institute for Environmental Research and Education, South China Agricultural University, Guangzhou 510642, China. *email: [email protected] Scientifc Reports | (2021) 11:2905 | https://doi.org/10.1038/s41598-021-81703-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ Ryegrass 2 years 4 years 6 years 8 years 10 years CK2 CK10 Height (cm) 54 (1.05) 55 (0.72) 58 (0.58) 60 (1.35) 61 (1.29) 41 (1.24) 56.12 (1.01) Aboveground biomass (*10– 17.41 (2.52) 18.01 (1.02) 18.55 (1.01) 19.53 (1.04) 19.58 (1.21) 5.79 (0.78) 18.32 (0.58) 2 g m−2) Root biomass (*10–2 g m−2) 1.49 (0.22) 1.61 (1.03) 2.07 (0.12) 2.14 (0.1) 2.15 (0.11) 0.65 (0.09) 2.05 (0.12) Table 1. Height and biomass of ryegrass in reconstructed mine substrates (RMS). Parentheses show standard deviations. Figure 1. Average well colour development (AWCD) of the microbial community in reconstructed mine substrates (RMS) for diferent years. in diferent decomposition stages and is generally sensitive to land-use changes, plant types, and soil depths 18. Te distribution of the diferent functional SOC pools plays a key role in the SOC accumulation19,20. Soil organic matter is a source of energy and carbon for soil microorganisms, such as bacteria and fungi, which in turn enhance the formation of soil micro- and macroaggregates through mucilage21,22. Terefore, the aggregate stability is a widely used indicator for the evaluation of the physical soil quality and susceptibility to erosion23. Researchers have emphasised the interrelation between the GRSP, SOC, and diferent sources of compost and sewage sludge24. However, there is a lack of data regarding the accumulation of the GRSP and SOC during reclamation. Te strong interrelationship among the SOC, GRSP, and soil aggregation might be due to organic amendment. Long-term monitoring of the soil quality during reclamation is vital for gaining insights into the soil improvement level. We reconstructed a plant–complex substrate–microbe ecological restoration system in the Pangzhuang coal mine in the northeast of Xuzhou, China, and sludge was added to the RMS to accelerate the reclamation process. Te objectives during the ten-year reconstruction were as follows: (i) investigate the response of the plant devel- opment and soil microbial activity to sludge amendment in RMS; (ii) assess the efect of sludge amendment on SOC and SOC fraction (LFOC) and GRSP (easily extractable GRSP, EE-GRSP; total GRSP, T-GRSP); and (iii) identify the interrelationship among the SOC, GRSP, and soil aggregation. Results Growth of ryegrass. Table 1 presents the height and biomass of ryegrass in RMS. Signifcant diferences were observed between the tested treatments. Te height increases with the reclamation time and eventually is 1.5 times higher than that of CK2 (i.e. control group 2; see “Materials and methods”) in the tenth-year reclama- tion. Te changes of the aboveground biomass and root biomass of ryegrass are similar to that of the height in RMS; they reach a maximum of 19.58 × 10–2 g m−2 and 2.15 × 10–2 g m−2, respectively, in the tenth year. Te growth of ryegrass in RMS exceeds that of CK10 (i.e. control group 10; see “Materials and methods”) in the sixth year. Height and biomass of ryegrass remain increasing throughout the whole experiment period while the total biomass of the ryegrass progressively increases. Efect of sludge addition on the functional diversity of the microbial community in RMS. Fig- ure 1 shows that the average well colour development (AWCD) of the microbial community in the reclaimed mine areas is of “S” type, with the culture time both in the frst and ffh years. In the initial stage of reclamation, the AWCD values of the four groups are below 0.3 and insignifcantly change within 24 h. Te AWCD values then rapidly increase, indicating that the microorganisms have an enhanced ability to utilise the C sources dur- Scientifc Reports | (2021) 11:2905 | https://doi.org/10.1038/s41598-021-81703-9 2 Vol:.(1234567890) www.nature.com/scientificreports/ Treatment H D U CK1 3.06 (0.18) a 0.95 (0.01) a 6.08 (1.09) a CK5 3.07 (0.18) a 0.95 (0.01) a 8.34 (0.95) b 1 year 3.32 (0.02) b 0.96 (0.01) a 10.08 (0.19) c 5 years 3.23 (0.06) ab 0.96 (0.01) a 11.18 (0.59) c Table 2. Functional diversity (i.e. Shannon (H), Simpson (D), and McIntosh (U)) indices of the soil microbial community in reconstructed mine substrates (RMS). Parentheses show standard deviations. Diferent small letters indicate signifcant diferences among diferent reclaimed soil treatments at a signifcance level of p < 0.05. ing 24–48 h. Finally, the growth almost stops afer 120 h. Te AWCD values of the four reclaimed substrates change as follows: slow growth, rapid growth, and stabilisation. Tis indicates that the microorganisms exhibit strong metabolic activities afer a period of adaptation. Both in the frst and ffh years, the AWCD values are signifcantly higher than those of CK1 (i.e. control group 1; see “Materials and methods”)and CK5 (i.e. control group 5; see “Materials and methods”) and stabilise at 1.61 and 1.73, respectively. Tis study shows that the microbial activity in the ffh year is higher than that in the frst year.
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